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PMC4821137

small - cell lung cancer ( sclc ) , which comprises approximately 1015% of all lung cancers , is a neuroendocrine tumor , previously known as oat cell carcinoma , that is closely linked with tobacco smoking and characterized by an aggressive and rapid pattern of metastases , limited treatment options following progression , and overall poor prognosis . the disease is traditionally divided into two stages : limited - stage disease ( ld ) , confined to one hemithorax encompassable in a single radiation port , and extensive - stage disease ( ed ) , extending beyond those boundaries , which comprise the vast majority of patients at initial diagnosis [ 5 , 6 ] . for extensive - disease sclc , the standard first - line regimen is etoposide - platinum ( ep ) doublets followed by topotecan , approved for second - line treatment , since progression is the rule after first - line ep despite the fact that sclc is initially chemosensitive ( with response rates to first - line treatment on the order of 7090% in ls disease and 5060% in ed disease ) . in ed , despite a response rate of 1528% to topotecan , the median os rarely exceeds 6 months . according to the current nomenclature sensitive relapsed ( pfs <3 months ) , resistant ( pfs <3 months ) and refractory ( progression through first - line treatment ) . in general , subsequent readministration of ep doublets is contraindicated in resistant and refractory disease due to the likelihood of undue toxicities ( myelosuppression and nephrotoxicity , for example ) without benefit , since therapeutic resistance in these contexts is , with exceptions , construed as a stable and immutable trait . pretreatment or priming with epigenetic inhibitors , like the approved dna demethylators , e.g. , 5-azacitidine ( azacitidine , aza ) and 2-deoxy-5-azacitidine ( decitabine , dac ) and the histone deacetylases , e.g. , vorinostat and romidepsin as well as experimental rrx-001 , has been identified as a nascent strategy , still in its infancy , to attenuate or reverse resistance to chemotherapy and immunotherapy [ 12 , 13 , 14 ] , particularly in non - small cell and ovarian cancers . rrx-001 , in particular , has demonstrated evidence of episensitization to refractory irinotecan in the context of a phase ii clinical trial entitled rocket in metastatic colorectal cancer [ 16 , 17 , 18 ] . triple threat ( nct02489903 ) is an ongoing open - label phase ii trial in non - small lung cancer , sclc and high - grade neuroendocrine tumors , which investigates a resensitization strategy to previously administered , but now refractory , platinum doublets following treatment with and progression on ( by recist vs. 1.1 criteria ) single agent rrx-001 mixed ex vivo with 100 ml of autologous blood prior to infusion . the rationale for this unusual method of administration is related to the red blood cell partitioning of rrx-001 , which binds with high affinity to a cysteine residue in the -chain of hb ( cys 93 ) , displacing nitric oxide ( no ) in the process . when no is displaced in vivo via standard intravenous infusion , it stimulates venous nociceptors , with a mild to moderate sensation of pain or discomfort for patients ; however , in the infusion bag , mixed with blood , the no is reabsorbed by the red blood cells , which appears to completely abrogate the hyperalgesia , resulting in a well - tolerated and rapid ( ~30 min ) infusion . herein , we report a case of a patient with extensive refractory ed - sclc who was episensitized to cisplatin and etoposide , with a partial response , after treatment with weekly intravenous rrx-001 autologous blood mix . patient 001001 , a 49-year - old male veteran , former smoker , with a diagnosis of extensive - stage sclc , completed six cycles of carboplatin and etoposide in december 2012 . having progressed approximately 6 months later with sensitive relapsed ( pfs > 3 months ) disease , he was enrolled on a clinical trial at the nih in july 2013 where he received six cycles of cisplatin and etoposide followed by maintenance with the hdac inhibitor belinostat . approximately 1 year after completion of the nih trial , with symptoms of fatigue and inappetence due to disease progression as well as dyspnea and decreased exercise tolerance from a large unilateral pleural effusion , he enrolled as the first patient ( 001001 ) on the triple threat clinical trial at walter reed in june 2015 . after the first dose of rrx-001 , and continuing for the next 89 weeks , the patient reported a significantly increased appetite for and consumption of fried foods , specifically pork chops , and desserts , which led to dyspeptic symptoms . his fatigue , shortness of breath and exercise tolerance also significantly improved to the point that he resumed uphill running and lifting weights . a ct scan at 6 weeks ( fig . 1 ) , correlating with the symptomatic improvement , demonstrated near complete resolution of the unilateral malignant pleural effusion . in addition unfortunately , nearly 10 weeks after the start of triple threat , patient 001001 developed travelers thrombosis and pulmonary embolism presumably from a prolonged ( 10 h ) car ride , which prompted the pi to discontinue rrx-001 on suspicion of symptomatic progression since the patient began to complain of fatigue and inappetence as well as dyspnea associated to pulmonary embolism , even though repeat ct scans did not confirm radiologic progression . at this point , per protocol , since the patient still met all inclusion / exclusion criteria , he was rechallenged for a third time with ep doublets and , despite expected toxicities of fatigue and anorexia , he reported improved performance status . at 6 weeks after reintroduction of therapy , repeat ct scans demonstrated a partial response with an approximate 32% decrease in tumor size ( fig . one month later , a confirmatory scan showed a 50% decrease in tumor size , indicative of continued cytotoxic activity . to the best of our knowledge , triple threat is the first and only clinical trial to investigate resensitization or episensitization , since rrx-001 is an epigenetic inhibitor in sclc as well as nsclc and neuroendocrine tumors . as a highly aggressive tumor with a uniformly dismal prognosis and an unmet need for effective therapeutic options , sclc is extremely difficult to treat : the median survival time following recurrence rarely exceeds 6 months , the 2-year survival is < 10% , and there are virtually no 5-year survivors , requiring innovative strategies to address the difficult challenge of salvage treatment of relapsed sclc . dna methyltransferase and hdac inhibitors have demonstrated the potential to reverse therapeutic resistance in multiple tumor types because epigenetic alterations , an important driver of carcinogenesis and progression , are , in theory , reversible . paradoxically , these alterations are also stably maintained through mitotic cell division , leading to a kind of epigenetic memory , with priming of responses to temporally separate and subsequent , rather than simultaneous , therapy since the induced pattern of gene transcription persists . the clinical course of this sclc patient ( 001001 ) suggests , with the usual caveats about making generalizations from n - of-1 results , that reintroduction of etoposide - platinum after initial treatment with rrx-001 , leading to episensitization , is a safe and effective strategy for solid tumor types previously treated with platinum doublet - based chemotherapy . the phase ii triple threat clinical trial is ongoing , having enrolled 5 patients to date , none of whom , besides patient 001001 , have progressed on rrx-001 , and their data will also be reported , if episensitization , anticipated here as elsewhere , is demonstrated . any subjects have given their informed consent in the study and the study protocol has been approved by the relevant institute 's institutional review board , also known as an independent ethics committee , ethical review board , or research ethics board . the authors disclose that the clinical trial in which this case was observed is funded by epicentrx , inc .

rrx-001 is a pan - active , systemically nontoxic epigenetic inhibitor under investigation in advanced non - small cell lung cancer , small - cell lung cancer and high - grade neuroendocrine tumors in a phase ii clinical trial entitled triple threat ( nct02489903 ) , which reexposes patients to previously effective but refractory platinum doublets after treatment with rrx-001 . the purpose of this case study is first to report a partial response to carboplatin and etoposide in a patient with small - cell lung cancer pretreated with rrx-001 , indicating episensitization or resensitization by epigenetic mechanisms , and second to discuss the literature related to small - cell lung cancer and episensitization .

Introduction Case Report Discussion Statement of Ethics Disclosure Statement

PMC2868186

the availability of the mouse genome sequence and advances in genetic manipulation has resulted in multiple initiatives , both in the public and private sector , to produce mouse mutants on a large scale . coordinated efforts are on way to systematically knock out all mouse genes to provide researchers targeting constructs , vectors , live mice , and phenotypic data at a variety of level and detail . the relationship between the effect of genetic manipulation and the resulting phenotype is a key component to establishing a fundamental understanding of molecular and cellular process . parallel to the growth in mouse models to study disease patterns , rapid strides have been made in developing imaging instruments specifically targeted to the small animal . instruments like mri , pet , spect , ct , in vivo fluorescence , in vivo bioluminescence , and intravital imaging allow one to visually , and sometimes quantitatively , evaluate biological processes at the cellular and subcellular level in a living subject . however , the major challenge in successfully characterizing morphological features of transgenic mouse models has been the trade - off between resolution and field of view . researchers particularly want the ability to detect gross structural change or , in the absence thereof , localize on subtle variations at microscopic level in 3d in tissue and cell structures . thus the ideal system used in characterizing transgenics should offer multiscale 3d visualization at a very high native resolution . in this paper we describe the application of cryo - imaging system and the multiscale approach to characterize a transgenic mouse . our group has developed a cryo - imaging system , which provides contrast rich , bright field anatomical , and fluorescence cellular and molecular imaging of an entire mouse with micron - scale resolution . the system acquires three - dimensional ( 3d ) , very high - resolution , large field of view ( fov ) , brightfield anatomy and fluorescence molecular image volumes from sequential images of the tissue block face by alternately sectioning and imaging . in an earlier report , we have demonstrated few applications of cryo - imaging ranging from high - resolution anatomical imaging of whole mouse , vascular imaging , and molecular fluorescence imaging of fluorescent mouse models and embryos . the system has been used for validation of mr studies of human blood vessel lesions , ex vivo characterization of human atherosclerotic iliac plaque , and very recently for single cell detection of fluorescently labeled cancer and stem cells . true color enhanced volume rendering techniques provide fast 3d visualizations of cryo - imaged samples . cryo - imaging is unique among all 3d in vivo ( e.g. , micro - mri ) and microscopic techniques , because it allows micron resolution and information - rich color contrast mechanisms over very large 3d fields of view . mri imaging of whole mouse can produce gray - scale images after administering contrast agents with significantly less resolution . optical projection tomography is limited to small samples such as embryos and requires treatments to reduce scatter and increase transparency . diffuse optical tomography while tackling the light scatter problem with advanced algorithms can image a whole mouse albeit at limited resolution providing little anatomical detail . johnson et al . used very high - resolution ct and osmium tetroxide staining to image tissues and embryos . serial histology techniques to create a 3d volume involve serially cutting the sections , applying histological processing , mounting on glass slides , digitizing the slides , and then creating a 3d volume from the two - dimensional ( 2d ) images . such systems have been used for a variety of biomedical applications [ 13 , 14 ] including characterizing phenotypical change in large histological mouse model datasets . however , such processing suffers from tears , tissue shrinkage , errors in image alignment , and typically time - consuming manual editing of 3d reconstructions . systems have been mostly applied for imaging excised organs such as brains [ 16 , 17 ] and embryos [ 18 , 19 ] with adult whole mouse block - face imaging being limited to low - resolution photographs of sections taken for autoradiography studies and ct and block - face imaging for creating a mouse atlas . we have developed the robotic 3d cryo - imaging system and first applied it to high - resolution tiled imaging of a wild - type mouse . now we report , for the first time , morphological characterization of a transgenic mouse recently generated by researchers at case western reserve university . this mouse , dubbed supermouse by popular press , contains a chimeric gene in which the cdna for pepck - c was linked to the -skeletal actin gene promoter , expressing pepck - c in skeletal muscle . the transgenic pepck - c mice were seven times more active than their controls in their cages , ran for up to 5 km at a speed of 20 m / min on a mouse treadmill ( control animals run for 0.2 km at this speed ) . as reported earlier , the mice eat almost twice as much as controls but are half the body weight ; they also have an extended life span relative to the controls . in their seminal report , the researchers have detailed the study of biochemical pathways to better understand the repatterning of energy metabolism . the battery of tests involved assay of metabolites in blood and tissues , home cage activity testing , exercise capacity testing , respiratory quotient ( rq ) measurements during exercise , and histological and electron microscopy analysis of skeletal muscle . characterization of the pepck - c mouse with a cryo - imaging , multiscale approach involves detailed high - resolution imaging of a whole mouse and a control mouse , processing and manipulations of enormous giga - byte sized data volumes , and enhanced 3d visualizations . in the following sections we describe the cryo - imaging setup , the imaging techniques , and enhanced visualizations in 3d at multiscale and qualitative and quantitative comparison of organs . briefly , it consists of a cryo - microtome , a microscope imaging system , a robotic xyz positioner , and a computer control system . the cryo - microtome is a motorized , large section , whole body cryo - microtome with section thickness adjustable between 240 m and a maximum specimen dimension of 250 mm 110 mm 50 mm . an xyz robotic positioner carries the imaging system comprising of a stereo microscope , coaxial fluorescent attachment with multiple filter cubes , low light digital camera , and brightfield and fluorescent light sources . the computer control system automatically pans the positioner over the specimen for a high - resolution tiled image acquisition and controls the sectioning and image acquisition sequence through the custom developed programmable sectioning and cryo - imaging ( prosci ) software . the image processing and visualization system is a quad - core windows 64-bit pc with 32 gb of ram capable of handling large cryo - image volumes . a suite of matlab custom programs and custom amira scripts are used for image preprocessing tasks and 3d visualization . in order to acquire cryo - image data , mice covered under iacuc - approved projects , were euthanized in a method approved by case animal resource center which consists of either inhalation of carbon dioxide delivered from tank or anesthetization using an agent such as pentobarbital , at a dose prescribed by the case arc . a cryo - embedding compound , oct ( optimal cutting temperature , tissue - tek , terrance , ca ) , was used to completely immerse the mouse in an aluminum foil mould . the mould was then covered in more foil and placed inside a freezing chamber made of styrofoam and filled with liquid nitrogen . after about 15 minutes , the mould assembly was removed from the liquid nitrogen bath and placed inside the cryo - microtome chamber to equalize the specimen temperature to the cryo - microtome temperature . the mould was removed from the specimen after two to three hours and the frozen mouse block was mounted on the microtome stage using more oct . an initial facing - off then the section thickness was set to 40 m , the imaging system was readied for a pixel size of 15.6 m , and the system was started to alternately slice and acquire tiled brightfield images of the block - face . the system was run completely unattended with the user periodically alerted by the prosci program about imaging progress through email and cell phone text messages . details of the transgenic mice are available elsewhere . the transgenic animal used in this study weighed 25.8 gm and was 421 days old when sacrificed . once the mouse specimen was mounted on the stage , the operator selected the boundaries of the acquisition volume and the system calculated the number of tiles required per block - face taking into account the operator - entered minimum overlap zone between adjacent tiles , typically 15% . the robotic system computed the coordinates of each tile based on the pixel size and automatically sectioned and imaged the entire volume . brightfield images ( each 1360 by 1036 pixels ) were acquired in 6 4 tiling pattern to cover each block - face . a total of 586 sections were needed for the transgenic mouse and 616 for the control . a metadata file stored the global coordinates , imaging parameters , and experiment parameters . following acquisition of 2d images on the imaging workstation , nonuniform illumination pattern was compensated using a reference image of a white card and compensated images for each tiled block - face were composited together to yield a typical 2d whole mouse section image of 6500 by 3600 pixels . these stitched images were then automatically aligned to each other to correct for minor misalignments due to the repositioning error of the stage at the return point . the total stitched 2d image stacks amounted to a data size of > 60 gb . since such a large dataset can not be loaded fully in the memory of the visualization workstation , we converted the stack of images into a large data access ( lda ) volume inside our customized amira visualization software . lda allows us to subsample at various resolutions an entire mouse or organ , or to sample the data at full resolution within smaller cropped subvolumes . from a relatively low - resolution 3d mouse the whole mice were segmented from the oct blocks through our fully automated volume visualization . since cryo - image data is in color , unlike other gray - scale medical imaging data ( mri , ct , etc . ) we have designed color feature detectors which take into account the dominant color of a region to segment it . examples of red , green , and blue ratio feature detectors ( fr , fg , fb ) are shown below , where r , g , and b refer to the 8-bit data for red , green , and blue channels , respectively , ( 1)fr = rr+g+b , fg = gr+g+b , fb = br+g+b . for instance , regions containing a high proportion of red such as liver , lungs , and vasculature can be extracted using the red feature detector fr . for detecting stomach and intestinal regions , which are predominantly brown in color , we exploited the fact that brown is composed of one part of r , two parts of g , and zero ( 0 ) parts of b. a brown feature detector is therefore expressed as a weighted linear combination of red and green feature detectors : ( 2)fbr=(0.33fr+0.67fg ) . for segmenting out the embedding compound oct , which has a uniform white color a balanced color detector was used : ( 3)f=[abs(rg)+abs ( gb)+abs ( rb)]3imax , where imax = 255 . subsequent to feature detection , opacity transfer functions ( otfs ) were used to assign an appropriate opacity value to each voxel . below , we show an example of color - based step otf that we have employed in our visualizations : ( 4)={255,f > t,0,ft , where t is an empirically determined threshold . in other algorithms , we have optimized opacity by imposing several simultaneous constraints on an opacity function which maps each voxel to a custom opacity value . enhanced volume rendering techniques for high - resolution color cryo - imaging data have been described elsewhere . individual organs such as the heart , brain , kidney , spleen , and glands were delineated using a combination of feature detection for initial automatic segmentation and then manually correcting the results . in organs such as the ovaries , where lack of uniform color and lack of high contrast between neighboring tissues prevented automatic approaches , manual segmentation was carried out in selected sections and 2d interpolation volumes of organs and tissues were computed from pixel count of 2d segmentation , the 2d pixel dimension and section thickness . the gross animal weights and brain volumes were used as normalizing factors for comparing organ volumes between the transgenic and the control . for comparing specific bone lengths such as the femur , digital sections through the axis of the bone were created through interactive multiplanar reformatting of the original 3d data volume . measurement tools available in the software package were used to compute bone length and cross - sectional areas . the length of the animal ( nose to base of tail ) was used as the normalizing factor for comparison across animals . standard tools available in amira were used for volume presentation such as snapshot capture , and movie making . the image acquisition and multiscale volume visualization is illustrated in figure 1 . from the stitched 2d image stack ( figure 1(a ) ) , a 60 gb 3d volume was created and visualized at multiple resolutions . in the down - sampled whole mouse rendering , major organs were clearly seen through transparent skin ( figure 1(b ) ) . a bounding box was placed in the abdomen area to extract the region in highest resolution and organs such as the kidneys , adrenal glands , ovaries , and vasculature segmented and reconstructed in 3d ( figure 1(c ) ) . for example , a section through the left ovary shows the inner medulla consisting of loose connective tissue and large blood vessels and a peripheral cortex region containing a number of ovarian follicles in different stages of development as small as 50 m in diameter . a few fimbriae of the infundibulum of the uterine tube can also be seen . at this full resolution , these and other many small structures like 50 m villi of the small intestine , mesenteric blood vessels 20 m in diameter , thin walls of the vessels 30 m and skeletal muscle and nerve fibers were very clearly seen . coronal section images composited from 24 (= 4 6 ) individual tiles are shown in figure 2 . major organs like the heart , lungs , liver , salivary gland , stomach , spleen , pancreas , small intestine , colon and lymph node are visible . the very visible peritoneum of the control mouse encases a large amount of visceral adipose tissue as compared to the pepck - c transgenic mouse . this extraordinary difference in adipose tissue depots is a general finding for these animals . using semiautomatic segmentation techniques , we computed fractional vascularization in the visceral adipose tissue encased by the peritoneum in the lower abdomen area around the cervix . the gross adipose tissue volume , estimated from voxels in 86 sections , was 38% more in the younger control mouse compared to the pepck - c transgenic mouse . the percentage of tissue classified as vasculature within this limited adipose tissue mass was higher for the pepck mouse as compared to the control ( 2.5% vascularized in the transgenic animal ; 0.4% in control ) . the highly vascularized pancreas extending from the spleen to the duodenum can be also seen in both the section images . in figure 3 , the dermal layers were distinct with an 83% thinner hypodermis for the transgenic as compared to the control ( 502 mm for control and 84 mm thick for the pepck mouse ) . the epidermis of the control was 16% thinner than that of the transgenic ( 407 mm transgenic , 348 mm control ) . the dermis was similar in thickness ( 578 mm transgenic , 621 mm control ) . the parietal peritoneum , visible below the skin , was also measured and found to be same . cutaway through the 3d reconstructed hearts of the transgenic and control mice reveal the chambers of the heart , pulmonary trunk , and pulmonary veins . manual segmentation of the heart from the 2d images also enabled computation of the heart volume . the transgenic heart was 351.52 cubic mm in volume while the control heart was 366.87 cubic mm . multiplanar reformatting allowed bone length comparison of femur and tibia of the pepck mouse and the control animal ( figure 5 ) . the femur measured 11.46 mm ( transgenic ) and 12.41 mm ( control ) while the tibia measured 17.26 mm ( transgenic ) and 15.12 mm ( control ) . the body length from tip of nose to base of tail was estimated to be 87.6 mm for pepck mouse and 83.8 mm for the control mouse . when normalized to body length , the measures were similar for femur ( 13% for transgenic and 15% for control ) . however the tibia was longer for transgenic ( 20% for transgenic and 18% for control ) . the tibia to femur length ratio was calculated to be 24% higher for transgenic as compared to control . cortical area was measured at right midtibial and midfemoral diaphysis by extracting digital planes transverse to the bone axes . tibia cortical area was 0.74 mm for transgenic and 0.71 mm for control . femora cortical area was 1.35 mm for transgenic and 1.38 mm for control . the pericardial fat around the heart was segmented from the 2d section images and volume visualized in 3d ( figure 6 ) . the pericardial fat depots were more pronounced in the control mouse as compared to the pepck mouse which is visible readily in the 3d volume visualization . from the 2d segmentations , the major organs like brain , heart , kidney and spleen and glands such as pituitary , thymus and adrenal were segmented and volumes computed . the pericardial fat volume is also included . both absolute values as well normalized values by gross animal weights and brain volumes are presented . the high - resolution color data volumes from the whole mouse that were obtained using the case cryo - imaging system and multiscale viewing provide researchers the ability to seamlessly bridge the requirements of resolution and fov . most existing small animal imaging systems force the researchers either to scan the mouse at low - resolution to capture the entire animal in a single fov or to choose only a region of the whole mouse for investigation at higher - resolution . as we have shown in this application , with cryo - imaging , image data is acquired at a very high - resolution , and using multiscale viewing , it is possible to view in 3d or 2d at the whole body level , zoom to a region at higher - resolution , zoom to an organ , and finally zoom to examine tissues at the highest resolution . skeletal muscle fibers in the mouse , typically 45 m in diameter , were seen very clearly in cryo - images . in earlier work involving fluorescently labeled stem and cancer cells , we showed that one can zoom down to single cell level . the multiscale approach provides an efficient way of handling extremely large whole mouse data sets . a tiled color bright field acquisition with an adult whole mouse using a 6 4 tiling configuration and 40 m section thickness generates > 60 gb of color image data which is a prohibitively large size for volume rendering on a workstation with a conventional , single graphics processor . if fluorescence acquisition is added , it imposes an even greater burden due to increased data size . as a remedy to the extreme data problem , we designed the multiscale volume rendering approach which greatly improves the visualization experience . . some of the important criteria to consider during multiresolution volume rendering are data access time and graphics hardware limitations on the target machine . data access time can be greatly reduced by employing modern solid state disk ( ssd ) hard drives , which generally have higher data read and write speeds as compared to conventional hard drives . as for graphics hardware , the larger the amount of graphics ram , the smaller the decimation that needs to be applied to produce high - resolution renderings of subregions within the large data sets . first , it offers a convenient method of scanning an entire mouse since no special preparation involving stains are required . second , a low - resolution view of the entire mouse with volume visualization of internal organs is available through automatic segmentation of oct and automatic 3d rendering . third , the organs and tissues are displayed in true color providing a virtual necropsy experience . fourth , data can be seamlessly explored at every level through user - friendly select - and - view bounding boxes . semi - automatic segmentation through algorithmic extraction of features followed by interactive manual editing provides visualization and quantification options . while this application has only utilized brightfield imaging , the system is capable of obtaining molecular fluorescence image data as well , thereby rendering unique versatility to the system . the featuresets of the system make it useful for applications such as anatomical phenotyping . a particularly interesting approach for identifying mutant outliers was described by kovacevic et al . where an annotated 3d atlas of the average mouse brain with estimates of average and variability was created from mri images , against which mutants could be compared . given the ability to segment organs in color , cryo - imaging will be well suited for such analyses for transgenic mice . the published work on pepck - c transgenic mouse contained histology of excised skeletal muscle tissue and low - resolution whole body mr imaging . cryo - imaging of this transgenic strain and its control provides the first glimpse at characterizing some of the morphological difference between the pair . the high amount of total body fat in the control , clearly visible in the 2d sections , is consistent with the published mr results . however , unlike mri , the high - resolution of cryo - imaging allows distinction between various fat depots and color differences . as an example , we computed and compared the pericardial fat volumes and fractional vascularization in a region of the visceral fat depot in the abdomen . we were also able to detect various organs and separate the dermal layers similar to histology analysis . it has been observed that these transgenic mice live longer and are more energetic at an older age . we chose regions and organs of the two mice to compare few standard ageing biomarkers . we note that the older pepck mouse has less pericardial fat , less visceral and subcutaneous fat , thinner hypodermis and thicker epidermis as compared to the control animal . skin thickness measurements are widely accepted as a biomarker of ageing while visceral and pericardial fat deposits are biomarkers of potential metabolic disorders . the thymus and spleen were chosen as ageing biomarkers since the thymus is a primary lymphoid organ considered to be consistently sensitive to morphological effects and the spleen is a secondary lymphoid organ . characterization of thymic and splenic weights in age - related studies in nonhuman primates has shown that the thymus becomes smaller with age while the splenic changes were not statistically significant . in this study , we found the older pepck - c mouse has a larger thymus while the spleen is smaller than noted with the control animal . we also noted a difference in overall tissue coloration between the pepck mouse and control . in our experience from cryo - imaging wild type mice , younger mice have an overall brighter tissue color . in this regard , the pepck - c mouse looks adipose tissue is pinker , consistent with our observation of increased vascularity . through observations such as these , cryo - imaging can point researchers to examine certain tissues in more detail . a careful 3d volume measurement was carried out to compare the heart volume of this mouse with the control . while the absolute cubic volume was nearly the same , the transgenic heart volume was 40% greater when normalized by body weight . when normalized by brain volume this difference reduced . since cardiac volume estimates derived from the cryo - images are not correlated with the cardiac phase at the time of freezing , we estimated another measure of comparison . voxels belonging to the blood pool in the cardiac chambers and vessels in the hearts were semiautomatically identified through the color detector scheme and were excluded from cardiac volume measurement . the resulting cardiac tissue mass , when normalized by body weight , was found to be 22% more for pepck - c mouse than control . comparison of other organ volumes such as brain and kidney and glands such as pituitary and adrenal showed a general trend of larger organs for the pepck - c mouse . in order to reduce the variability due to animal size and weight , organ volume - to - animal weights were computed . to reduce the variability due to lean body mass , organ - to - brain volume normalization was used . in the case of bone lengths , digital resectioning along arbitrary planes allowed bone morphometric data to be computed from the cryo - image volume . measurements such as length and cortical area for tibia and femur were made for two comparisons . it has been reported that short - term induced exercise in mice leads to differences in bone properties such as length and cortical area for the femur and tibia [ 2628 ] . further , such changes have also been recorded in ageing studies [ 29 , 30 ] . the pepck - c mice are highly active and can endure sustained activity at maximal speed like running on mouse treadmills . the particular mouse we cryo - imaged had participated in numerous unrelated high - activity studies and was chronologically older than the control mouse . although no longitudinal study was performed on either specimen , nonetheless , we were interested in comparing the normalized values . the pepck - c mouse had a slightly higher value for tibia and lower for femora leading to a significantly higher tibia to femur ratio of 1.5 . the ratio of 1.2 for the control mouse is similar to ratios derived from published reports . the ratio of cortical area to bone length is considered to be indicative of bone growth . this ratio was similar for both the femora and tibia for both animals perhaps suggesting that ageing does not significantly deteriorate bone biomechanics for the transgenic . in this study , we have characterized the pepck - c supermouse and demonstrated that the case 3d cryo - imaging system can be adopted as a morphological phenotypic screen for transgenic animals . the multiscale approach offers researchers tissue information at the most appropriate resolution traversing down the scale from mouse to organ to tissue to single cells . the ability of being able to visualize structures in 3d and extract 2d sections along any arbitrary plane makes comparisons easy and focused . further , visualizations do not involve laborious manual segmentation and in most cases are achieved semiautomatically through the novel color feature detectors . the results of such phenotyping effort will provide a roadmap for adopting cryo - imaging for cataloging the plethora of transgenic mouse models .

we have developed , for the case 3d cryo - imaging system , a specialized , multiscale visualization scheme which provides color - rich volume rendering and multiplanar reformatting enabling one to visualize an entire mouse and zoom in to organ , tissue , and microscopic scales . with this system , we have anatomically characterized , in 3d , from whole animal to tissue level , a transgenic mouse and compared it with its control . the transgenic mouse overexpresses the cytosolic form of phosphoenolpyruvate carboxykinase ( pepck - c ) in its skeletal muscle and is capable of greatly enhanced physical endurance and has a longer life - span and reproductive life as compared to control animals . we semiautomatically analyzed selected organs such as kidney , heart , adrenal gland , spleen , and ovaries and found comparatively enlarged heart , much less visceral , subcutaneous , and pericardial adipose tissue , and higher tibia - to - femur ratio in the transgenic animal . microscopically , individual skeletal muscle fibers , fine mesenteric blood vessels , and intestinal villi , among others , were clearly seen .

1. Introduction 2. Cryo-Imaging System 3. Methods 4. Results 5. Discussion and Conclusion

PMC5341718

the 1970s and 1980s saw extensive research on microtubules and actin . during this period , the consensus developed that these cytoskeletal elements were unique to eukaryotes and that nothing related to tubulin or actin existed in bacteria or archaeans . this consensus was overthrown in the 1990s when a series of discoveries revealed that prokaryotes actually did have homologues of tubulin and actin and that these assembled into cytoskeletal filaments . it is now generally accepted that eukaryotic microtubules and actin filaments originated from these prokaryotic homologues . the key discoveries of bacterial tubulin and actin were published in 1992 and are reviewed here on their 25th anniversary . the discovery of ftsz as a bacterial homologue of tubulin came first and was made independently by three groups ( de boer et al . , 1992 ; raychaudhuri and park , 1992 ; mukherjee et al . , 1993 ) . these independent studies each purified ftsz protein from an escherichia coli expression system and demonstrated that it bound and hydrolyzed gtp . they noted that bacterial ftszs were missing the walker sequence motifs characteristic of g proteins but that they had a conserved short sequence , gggtgtg , that was almost identical to the g / aggtgsg sequence conserved in all - , - , and -tubulins . that sequence , known as the tubulin signature sequence , was believed to be involved in the binding of gtp in the tubulins . the three groups all concluded that the gtp - binding site of ftsz appeared to be related to that of tubulins . a year earlier , before any link to tubulin was known , bi and lutkenhaus ( 1991 ) were the first to propose that ftsz might be a cytoskeletal protein . electron microscopy to show that ftsz localized to the invaginating septum in dividing e. coli : in our model the role of ftsz is to form a cytoskeletal element that is functionally analogous to the role of actin in cytokinesis in animal cells . ( 1992 ) noted that the gtpase activity showed a dependence on ftsz concentration characteristic of the self - assembly of tubulin and actin . a subsequent study by mukherjee and lutkenhaus ( 1994 ) provided two major advances . first , they demonstrated that purified ftsz could assemble in vitro into filamentous polymers . second , they extended the sequence alignment to identify > 20 amino acids that were highly conserved in all tubulins and ftsz . they did this by starting the alignment at the gggtgtg sequence and inserting gaps in the ftsz to maximize identity . remarkably most of the gaps coincided with gaps already in the alignment of - , - , and -tubulins . i considered this alignment to be compelling evidence for full homology , but the editors of cell were more cautious , insisting on adding a question mark to my 1995 minireview , our laboratory took up the question of in vitro assembly and showed that ftsz assembled in vitro into sheets of protofilaments and mini - rings that were similar to tubulin polymers ( erickson et al . , 1996 ) . any question of homology was dramatically resolved when the structures of ftsz ( lwe and amos , 1998 ) and tubulin ( nogales et al . they had an identical complex fold , which is the ultimate test of homology . many archaeans have up to five ftsz homologues , some with very divergent sequences that likely serve functions other than cell division . the discovery of bacterial actins was complicated by the homology of actin to other protein families . sometimes , this is indicated by amino acid sequence identity , but often this sequence identity is too weak to recognize . the most definitive demonstration of homology is from protein structure . when the x - ray structure of actin was determined ( kabsch et al . , 1990 ) , it was seen to have a complex fold that was identical to that of hexokinase and also to hsp70 , a chaperonin . because this fold is so complex yet was shared so precisely , it was concluded that the three shared a common ancestry . actin , hexokinase , and hsp70 are homologues and are considered to be members of an actin superfamily . probably the original protein in this family was the sugar kinase , which underwent gene duplications that evolved into a chaperonin and separately into a protein that could assemble cytoskeletal filaments , actin . the first strong suggestion for actin homologues in bacteria was a theoretical study by bork et al . they used the recent x - ray structures of actin , hexokinase , and hsp70 to do a structure - based sequence alignment , which identified four short signature sequences that were conserved across the superfamily . they then looked for these signatures in bacterial proteins and found them in three : ftsa , mreb , and parm ( stba ) . these bacterial proteins were closest in sequence to hsp70 and actin rather than the sugar kinases and were therefore candidates for bacterial actin . ( 2001 ) studied the localization of mreb in bacteria by light microscopy , and found helical filaments running through the cell under the membrane . ( 2001 ) isolated mreb protein and showed by electron microscopy that it assembled thin filaments . their major discovery was x - ray crystallography , which showed that mreb had the actin fold and was assembled in the crystals into actin - like filaments . later work has discovered multiple prokaryotic actins with a variety of cytoskeletal functions , although the functions of most are unknown . it now seems clear that both tubulin and actin were invented in bacteria and/or archaeans and proliferated into diverse families of cytoskeletal filaments well before the emergence of eukaryotes ; for various perspectives on the evolution , see erickson ( 2007 ) , lwe and amos ( 2009 ) , and wickstead and gull ( 2011 ) . an interesting irony is that roles of tubulin and actin have somewhat switched from bacteria to eukaryotes . ftsz forms the cytokinetic ring in bacteria , whereas actin provides the major cytoskeletal framework in eukaryotes . some bacterial ( plasmid ) actins function for nucleoid segregation , a role performed by microtubules in eukaryotes . a global conclusion would be that once a protein has evolved the ability to assemble cytoskeletal filaments , these can be modified to perform a wide range of useful and sometimes overlapping cellular functions . the discovery of ftsz as a bacterial homologue of tubulin came first and was made independently by three groups ( de boer et al . , 1992 ; raychaudhuri and park , 1992 ; mukherjee et al . , 1993 ) . these independent studies each purified ftsz protein from an escherichia coli expression system and demonstrated that it bound and hydrolyzed gtp . they noted that bacterial ftszs were missing the walker sequence motifs characteristic of g proteins but that they had a conserved short sequence , gggtgtg , that was almost identical to the g / aggtgsg sequence conserved in all - , - , and -tubulins . that sequence , known as the tubulin signature sequence , was believed to be involved in the binding of gtp in the tubulins . the three groups all concluded that the gtp - binding site of ftsz appeared to be related to that of tubulins . a year earlier , before any link to tubulin was known , bi and lutkenhaus ( 1991 ) were the first to propose that ftsz might be a cytoskeletal protein . electron microscopy to show that ftsz localized to the invaginating septum in dividing e. coli : in our model the role of ftsz is to form a cytoskeletal element that is functionally analogous to the role of actin in cytokinesis in animal cells . ( 1992 ) noted that the gtpase activity showed a dependence on ftsz concentration characteristic of the self - assembly of tubulin and actin . a subsequent study by mukherjee and lutkenhaus ( 1994 ) provided two major advances . first , they demonstrated that purified ftsz could assemble in vitro into filamentous polymers . second , they extended the sequence alignment to identify > 20 amino acids that were highly conserved in all tubulins and ftsz . they did this by starting the alignment at the gggtgtg sequence and inserting gaps in the ftsz to maximize identity . remarkably most of the gaps coincided with gaps already in the alignment of - , - , and -tubulins . i considered this alignment to be compelling evidence for full homology , but the editors of cell were more cautious , insisting on adding a question mark to my 1995 minireview , our laboratory took up the question of in vitro assembly and showed that ftsz assembled in vitro into sheets of protofilaments and mini - rings that were similar to tubulin polymers ( erickson et al . , 1996 ) . any question of homology was dramatically resolved when the structures of ftsz ( lwe and amos , 1998 ) and tubulin ( nogales et al . , 1998 ) they had an identical complex fold , which is the ultimate test of homology . many archaeans have up to five ftsz homologues , some with very divergent sequences that likely serve functions other than cell division . the discovery of bacterial actins was complicated by the homology of actin to other protein families . sometimes , this is indicated by amino acid sequence identity , but often this sequence identity is too weak to recognize . the most definitive demonstration of homology is from protein structure . when the x - ray structure of actin was determined ( kabsch et al . , 1990 ) , it was seen to have a complex fold that was identical to that of hexokinase and also to hsp70 , a chaperonin . because this fold is so complex yet was shared so precisely , it was concluded that the three shared a common ancestry . actin , hexokinase , and hsp70 are homologues and are considered to be members of an actin superfamily . probably the original protein in this family was the sugar kinase , which underwent gene duplications that evolved into a chaperonin and separately into a protein that could assemble cytoskeletal filaments , actin . the first strong suggestion for actin homologues in bacteria was a theoretical study by bork et al . they used the recent x - ray structures of actin , hexokinase , and hsp70 to do a structure - based sequence alignment , which identified four short signature sequences that were conserved across the superfamily . they then looked for these signatures in bacterial proteins and found them in three : ftsa , mreb , and parm ( stba ) . these bacterial proteins were closest in sequence to hsp70 and actin rather than the sugar kinases and were therefore candidates for bacterial actin . ( 2001 ) studied the localization of mreb in bacteria by light microscopy , and found helical filaments running through the cell under the membrane . ( 2001 ) isolated mreb protein and showed by electron microscopy that it assembled thin filaments . their major discovery was x - ray crystallography , which showed that mreb had the actin fold and was assembled in the crystals into actin - like filaments . later work has discovered multiple prokaryotic actins with a variety of cytoskeletal functions , although the functions of most are unknown . it now seems clear that both tubulin and actin were invented in bacteria and/or archaeans and proliferated into diverse families of cytoskeletal filaments well before the emergence of eukaryotes ; for various perspectives on the evolution , see erickson ( 2007 ) , lwe and amos ( 2009 ) , and wickstead and gull ( 2011 ) . an interesting irony is that roles of tubulin and actin have somewhat switched from bacteria to eukaryotes . ftsz forms the cytokinetic ring in bacteria , whereas actin provides the major cytoskeletal framework in eukaryotes . some bacterial ( plasmid ) actins function for nucleoid segregation , a role performed by microtubules in eukaryotes . however , some tubz filaments also function for plasmid segregation . a global conclusion would be that once a protein has evolved the ability to assemble cytoskeletal filaments , these can be modified to perform a wide range of useful and sometimes overlapping cellular functions .

the year 2017 marks the 25th anniversary of the discovery of homologues of tubulin and actin in prokaryotes . before 1992 , it was largely accepted that tubulin and actin were unique to eukaryotes . then three laboratories independently discovered that ftsz , a protein already known as a key player in bacterial cytokinesis , had the tubulin signature sequence present in all - , - , and -tubulins . that same year , three candidates for bacterial actins were discovered in silico . x - ray crystal structures have since confirmed multiple bacterial proteins to be homologues of eukaryotic tubulin and actin . tubulin and actin were apparently derived from bacterial precursors that had already evolved a wide range of cytoskeletal functions .

INTRODUCTION Prokaryotic homologues of tubulin Prokaryotic homologues of actin

PMC4432531

the present study aims to evaluate the epidemiological profile of the 277 patients who suffered facial fractures and were treated at the hospital do trabalhador in curitiba in the state of parana , in 2010 , with an emphasis on variables such as gender , age , cause , and anatomical sites of fractures , and compared with clinical findings of other studies in the literature . maxillofacial trauma can be considered as a devastating assault found in trauma centers because of the emotional consequences and the possibility of deformity , in addition to its economic impact on a healthcare system1 . a maxillofacial injury involves not only the soft tissues and bones but also , by extension , can affect the brain , eyes , sinuses , and teeth . therefore , it is a trauma of a multidisciplinary approach , involving mainly the specialties of otolaryngology , ophthalmology , plastic surgery , maxillofacial , and neurosurgery6 . severe facial injuries may , in addition to psychological disorders , result in decreased productivity due to visual loss and damage during swallowing and phonation , increasing the costs arising from the trauma . the group most affected , men of working age , commonly found in many studies , partly explains the impact on productivity2 . in the last 4 decades , the incidence of facial trauma has increased , mainly due to increased traffic accidents and urban violence , especially in young individuals21 . they are very common in emergency rooms around the world and assume a prominent role in the care to polytrauma patients . the maxillofacial region is very prone to injury because of its prominence and little protection in the region . today the association of alcohol , drugs , car driving , and increased urban violence are increasingly present as causal factors of facial trauma , and what is worse , increasing its complexity7 . however , the epidemiology of craniofacial fractures can vary in type , frequency , severity , and cause , depending on the medical center studied and the period considered3 , because many factors influence the cause of maxillofacial injuries around the world , for example , cultural , economic , social , religious and geographical factors4 . therefore , epidemiological studies focused on facial injuries are of great interest as to the knowledge of the occurrence and quantity and severity of presentation , allowing the adoption of preventive measures for its control and management of patients . table 1 shows the summary of several epidemiological studies of facial fractures in the literature in several cities . the table was divided according to the author , publication year , study site , number of patients suffering from facial fractures , male prevalence , age more acomentida , main causes of fractures and other broken facial bones . we conducted a non - randomized retrospective study , of 277 patients with confirmed facial fractures diagnosed on radiographs and/or computed tomography , from the otolaryngology service at the hospital do trabalhador ( ht ) located in the city of curitiba in paran during the year 2010 . the study included all patients treated at the emergency room of ht , attended by the department of otolaryngology , who were victims of facial trauma in the period from january 1 to december 31 . we prepared a protocol for data collection performed by analysis of records and records of emergency in the sector of the medical file of the ht . this protocol included the following variables : medical record number , patient name , age , sex , origin , birth date , location of facial fracture , and cause . the cause of the fractures was studied according to : motor vehicle crashes ( combinations of collisions with cars , motorcycles , trucks / buses , and others ) , fall / bicycle accident , interpersonal violence with or without a firearm , drops own height or level falls , fractures arising from sports practices and impact / collisions with objects from accidental causes . the locations of the fractures were classified into facial : fractures of the nasal bone , mandible , orbital , maxillary , frontal , zygomatic bone fractures , and le fort i , ii , or iii . of the 277 patients treated in 2010 evaluated in this study , 207 were male ( 74.72% ) and 70 female ( 25.27% ) ; the male to female ration was 3:1 ( figure 1 ) . the patients ' ages ranged from 1 to 79 years with a mean of 33.57 years . distribution of patients with facial fractures according to sex in percentage ( n = 277 ) . the most affected age group was 20 to 39 years with 44.03% ( figure 2 ) . the extremes of age , younger than 10 years and older than 70 years , accounted for respectively 13 ( 4.69% ) and 8 ( 2.88% ) patients . distribution of patients with facial fractures according to age in percent ( n = 277 ) . the merits , 88.44% were residents of the city of curitiba , 10.83% of the metropolitan region of curitiba , and 0.72% from the interior the state of paran . the number of patients treated per month ranged from 16 to 27 , average of 23 , september being the month with the highest number of visits ( table 2 ) . regarding occupation , 55.23% had a professional activity , 16.96% were students , 5.77% were dependents , 5.41% were retirees , and 16.60% were unemployed or had indeterminate profession . regarding the cause of facial fractures , our chart review showed that the cause of fractures was interpersonal violence without firearm in 84 ( 30.32% ) cases , interpersonal violence with a firearm in 17 ( 6.31% ) , motor vehicle accidents ( car , truck , bus , motorcycle ) in 48 ( 17.32% ) , injuries while walking in 11 ( 3.97% ) , from falls from a height in 48 ( 17.32% ) , due to drop in level in 16 ( 5.77% ) , due to impact / collisions with objects in 35 ( 12.63% ) , fall / bicycle accidents in 10 ( 3.61% ) , and sports injuries in 8 ( 2.88% ) ( table 3 ) . the etiologic distribution by age showed a prevalence of violence without firearm in all age groups from 10 to 59 , with a peak incidence in the age group 3039 years with 41% . falls from height predominated in the age groups 010 and 6079 , representing 57.9% of the causes of facial fractures in the latter . the motor vehicle crashes were the second most common cause in the age group 2039 years with 22.13% of cases ( table 3 ) . the motorcycles were involved in 47% of automobile accidents . regarding the cause and gender , falls from height were the main cause of facial fractures in women , corresponding to 23 ( 32.85% ) cases in females followed by violence without firearm in 15 ( 21.42% ) cases , and third , vehicle accidents with 12 ( 17.14% ) cases . in the males , the main cause was violence without firearm in 69 ( 33.33% ) cases , followed by motor vehicle accidents in 36 ( 17.39% ) , and third , by impact / collision with objects in 26 ( 12.56% ) cases ( table 4 and figure 3 ) . distribution of patients with facial fractures by cause and gender ( n = 277 ) . patients had a total of 391 facial fractures , which were isolated in 205 ( 74% ) cases and associated with 2 or more locations in 72 ( 26% ) . mandibular fracture was the second most common , found in 56 ( 14.32 ) cases , followed by the orbit with 50 ( 12.78% ) . fracture of the maxilla was the fourth most frequent with 47 ( 12.02% ) , followed by the zygomatic bone with 39 ( 9.97 ) . the more complex fractures le fort i , ii , and iii corresponded to 11 ( 2.81% ) cases ( figure 4 ) . the face is susceptible to a variety of possible traumas , and it is important to note that aggression between facial injuries , especially fractures , plays a major role in emergency care worldwide . our study revealed a predominance of male patients with facial fractures corresponding to 74.72% , a 3:1 ratio , compatible with the literature including palma et al , 78% , falcon et al10 , 84% , and macedo et al , 72.8% . this higher incidence in males may be linked to cultural and social factors , considering that the males represent most of the economically active population , exhibit more abuse of alcohol and drugs , practice more contact sports , are involved in the majority in traffic , and thus are more exposed to the factors responsible for facial injuries . however , the incidence of trauma among women has increased in recent years due to the increased participation of women in the commercial workforce16 17 18 . the patients ' ages ranged from 1 to 79 years with a mean of 33.57 years . the most affected age group was 20 to 29 years , with 23.82% of cases . the age group also is in agreement with the findings of other authors such as silva et al13 . it is understandable that violence occurs more among young people by their restlessness and risk taking behaviors , including traffic risks influenced by extremely fast behavioral and moral changes16 . at the extremes of age , patients younger than 10 years and older than 70 years accounted for respectively 13 ( 4.69% ) and 8 ( 2.88% ) patients , consistent with the literature findings6 - 12 there are studies that show low incidence of facial trauma in children and the elderly due to the attention of family , stay at home , and care of children , as well as the characteristics of aging such as lessened social activity and sport , leaving little infrequently , and usually accompanied when they do.14 15 . with regard to professional activity , our study showed that 55.23% had jobs and 16.96% were students , similar to studies by macedo et al12 jcm junior et al in 20106 , and brazil et al 200616 , the latter , which showed 60.5% of patients and 16.9% economically active students . regarding the cause of facial fractures , our study showed the main causative agent of facial fractures was interpersonal violence without firearm with 84 ( 30.32% ) , and the second leading cause motor vehicle accidents ( car , truck , bus , motorcycle ) with 48 ( 17.32% ) cases , as well as falls from height with the same values . these data are compatible with the current literature , which shows a growing share of violence as a cause of facial fractures , surpassing automobile accidents . this confirms the tendency of most recent national studies to show an increased incidence of interpersonal violence and suggest that this is the main cause in facial trauma6 8 12 18 . this is mainly due to an increase in urban violence and a drop in severity of motor vehicle accidents due to public policies aimed at greater control speeding on the roads and encouraging the use of seat belts . moreover , the ban on drunk driving and the introduction of air bags and side protection bars have decreased the incidence of facial fractures , as well as the complexity17 20 . however , it calls attention to the involvement of 47% of motorcycles in traffic accidents , owing to the fact that unsafe vehicle speed abuse is practiced on the streets and that these vehicles are increasingly used because they are a means of low - cost transport . injurt from falls dominated as the cause in people aged under 10 and over 60 , as these are extremes of age , because locomotion and balance are directly proportional to age . the consciousness of appearance of the face and its social importance increases with age ( during a fall , older children and adults consider to protect the face)14 15 . in the case of elderly physiological mechanisms such as altered proprioception , weakness , tremor , and infeco urinary tract and lung , and alcohol are also referred to18 . with the number and location of the fracture , a total of 391 facial fractures , being isolated in 205 ( 74% ) cases , and 2 or more locations associated in 72 ( 26% ) mandibular fracture was the second most commonly found in 14.32% cases , followed by 12.78% of the fractures in the orbit ; maxilla fracture was the fourth most frequent with 12.02% followed by the zygomatic bone with 9.97% . the more complex fractures le fort i , ii , and iii accounted 2.81% of cases . these data differ widely in the literature , which show that in many cases , the jaw is the main bone fractured because it is the only mobile bone of the face and thus is more vulnerable to strong impact and fracture10 16 . on the other hand , studies like those by silva et al.13 and leite et al.19 corroborate our data demonstrating the nasal bone as the main fractured bones due to its prominent position , location on the mid - face , as well as the thin structures of the bones that constitute it . the most serious fractures such as le fort occurred in 2.81% of cases . of the total number of le fort fractures , 45% were due to interpersonal violence without firearm ; trauma and morbidity are high in this type of fractures , and the cause is easily avoidable . the study of the epidemiology of facial trauma is important for the studying the cause and effects of facial trauma , assist in the initial care of these patients , and publishing preventive policies . our study showed that males are more affected and the main causes of facial fractures are assaults , motor vehicle accidents , and falls . they are more common in young patients aged 2029 years followed by those aged 3039 years . in the vast majority are isolated fractures and the most affected bone is the nose , followed by the lower jaw , orbit , maxillary , and zygomatic . for the prevention of facial fractures , we must bear in mind the respect for traffic laws and routinely use seatbelts and helmets . furthermore , the incidence of facial fractures can be reduced by strategies for dealing with falls in children and the elderly , avoiding hostile situations , and creating stricter laws and public policies to reduce traffic accidents and reduce interpersonal violence .

summary introduction : epidemiological studies that focus on facial injuries are of great interest for the knowledge of occurrence and severity of presentation . aim : to study the epidemiological profile of 277 patients who suffered facial fractures at the hospital do trabalhador ( ht ) , with an emphasis on variables such as sex , age , cause , and anatomical sites of fractures , comparing the clinical findings with other studies . method : retrospective nonrandomized chart review of 277 patients who were treated at ht by the ent service during the full year 2010 , victims of facial fractures . results : of 277 patients , 74.72% were male and 25.27% female ( ratio 3:1 ) . according to age , the fractures were distributed as follows : 09 years : 4.69% , 1019 years : 17.32% , 2029 years : 23.82% , 3039 years : 20.21% , 4049 years : 16.24% , 5059 years : 10.83% , 6069 years : 3.97% , and 6079 years : 2.88% . the cause of trauma was most frequently interpersonal violence , 36.45% , followed by falls , 23.09% , and motor vehicle crashes with 17.32% . regarding location , nasal fracture was the most common , with 44.75% , followed by the mandible , 14.32% , orbit , 12.78% , maxillary , 12.02% , zygomatic , 9.97% , 3.32% and front le fort 2.88% . conclusion : the patients were mostly males , aged 2130 years , victims of aggression with the most commonly fractured bone being the nose . the adoption of personal and public strategies and measures may prevent facial fractures .

Introduction Review Method Results Discussion Conclusions

PMC3950745

world - wide , at least 2.8 million people die each year as a result of being overweight or obese and an estimated 35.8 million ( 2.3% ) of global dalys are caused by overweight or obesity . overweight and obesity lead to adverse metabolic effects on blood pressure , cholesterol , triglycerides ( tg ) and insulin resistance . some natural products have been considered to play a good role as an alternative to synthetic chemicals in this clinical condition . a wide variety of plants has been reported to have lipid lowering activity . among such plants nigella sativa ( n. sativa ) ( black seed ) in several studies has been investigated for its lipid lowering effects on animals . the seeds of the plant have been used in the southeast asia , middle and far east as a natural remedy to treat many diseases , including anti - microbial , anti - oxidant , anti - inflammatory , antitumor , hematologica , anti - hypertensive , anti - hypertensive , anti - diabetic , antilipidemic , hypophagic and anti - obesity effects and respiratory health . in fact , this plant has occupied a special place for its wide range of medicinal value in the islamic civilizations . due to the sayings of the holy prophet , mohammad ( peace be upon him ) that in the black seed there is healing for every illness except death . anwar and tayyab reported that n. sativa in the diet has a favorable effect on the lipid profile by lowering the tg , total cholesterol ( tc ) and low - density lipoprotein ( ldl)-cholesterol and increasing the high density lipoprotein ( hdl)-cholesterol in rats . exercise is most important for every living being ; in other words , we can also say that physical inactivity results in several types of diseases in the body . a sedentary life - style is associated with an increased risk for an acute myocardial infarction and death from coronary heart disease ( chd ) . the findings are consistent and show that sedentary people have about twice the risk of developing or dying from chd , compared to active people . physical inactivity is now recognized by the american heart association as an independent risk factor , comparable to the other established risk factors for chd . it is well - established that habitual physical activity improves physical fitness in middle - aged men and women . yar et al . had monitored the effect of n. sativa supplementation on cardiac reserve in rats . their results suggest that n. sativa generated homogenous cardiac hypertrophy similar to that provoked by exercise training . despite the aforementioned beneficial effects of exercise and n. sativa , thus , the purpose of this randomized , double blind , controlled trial was to investigate whether 8 weeks of aerobic training combined with n. sativa supplementation could improve lipid profile and maximal oxygen consumption ( vo2 max ) in sedentary overweight females . a total of 20 adult females with a mean age of 34.31 7.9 years , body mass index ( bmi ) 25 kg / m and sedentary life - style for at least 2 years participated in this study voluntarily . the main criterion for entry of adult females was a tc concentration more than 200 mg / dl . subjects taking lipid - altering medication within 8 weeks prior to the study were excluded . other exclusion criteria were pregnant , current smoking , lactation , having diabetes mellitus , cardiovascular disease ( cvd ) , renal or liver disease , thyroid dysfunction and myopathy . physical characteristics of the study participants * according to the subject 's profiles number and the random figures tables , they were randomized to receive either n. sativa capsules ( ns group ) or placebo ( pla group ) . each black seed capsule contained 500 10 mg n. sativa crushed seeds and subjects had to take 2 g of n. sativa per day for 8 weeks . they were instructed to take two capsules before breakfast and two capsule in the afternoon prior their food . subjects were also asked not to change their usual daily diet and physical activity during the study . subjects in two groups performed slow running ( 10 min ) , stretching muscles and loosening joints ( 10 min ) , aerobic training program ( 30 min ) and cool down ( 10 min ) . the intensity of training was 60 - 70% of target pulse rate calculated by karvonen et al . target heart rate ( hr ) zone = ( [ hr maximum hr rest ] { 60 - 70% } + hr rest ) hr maximum = 220 age . if the subjects are not able to take a measurement first thing in the morning , make sure subjects lie down for at least 10 min before taking a measurement . measurement was taken from the radial artery with forefinger and the middle finger of the right hand placed horizontally across the subject 's wrist while sitting on the chair . after that , the number of pulse beats multiplied by two to give the 1 min hr . cardio - respiratory fitness was determined by 1609 m ( 1 mi ) walk test using rockport fitness test . after a brief warm up , the subject walked as briskly as possible for 1609 m ( 1 mi ) with a hr monitor . the formula used to calculate vo2 max was : 132.853 ( 0.0349 mass [ kg ] ) ( 0.3877 age [ year ] ) + ( 6.315 gender ) ( 3.2649 time [ min ] ) ( 0.1565 hr [ bpm ] ) . the validity of the test was nearly high r = 0.88 and standard error of the test was 5 ml / kg / min . cardio - respiratory measurement was taken before and after 8 weeks of aerobic training program and supplementation . a total volume of 20 ml blood was drawn from the subjects in their forearm vein after a 12 h overnight fast immediately before and after 8 weeks of aerobic training program and supplementation in the sitting position after a 20-min rest between 8:00 and 9:00 a.m. plasma was separated by centrifugation and samples were stored at 10c until assays were determined ( within 48 h ) in all samples . the statistical analysis was initially performed using the changes in the one - sample kolmogorov - smirnov test . data was analyzed using paired t - test for within group 's comparison and unpaired t - test for between - groups comparison . a total of 20 adult females with a mean age of 34.31 7.9 years , body mass index ( bmi ) 25 kg / m and sedentary life - style for at least 2 years participated in this study voluntarily . the main criterion for entry of adult females was a tc concentration more than 200 mg / dl . subjects taking lipid - altering medication within 8 weeks prior to the study were excluded . other exclusion criteria were pregnant , current smoking , lactation , having diabetes mellitus , cardiovascular disease ( cvd ) , renal or liver disease , thyroid dysfunction and myopathy . according to the subject 's profiles number and the random figures tables , they were randomized to receive either n. sativa capsules ( ns group ) or placebo ( pla group ) . each black seed capsule contained 500 10 mg n. sativa crushed seeds and subjects had to take 2 g of n. sativa per day for 8 weeks . they were instructed to take two capsules before breakfast and two capsule in the afternoon prior their food . subjects were also asked not to change their usual daily diet and physical activity during the study . subjects in two groups performed slow running ( 10 min ) , stretching muscles and loosening joints ( 10 min ) , aerobic training program ( 30 min ) and cool down ( 10 min ) . the intensity of training was 60 - 70% of target pulse rate calculated by karvonen et al . target heart rate ( hr ) zone = ( [ hr maximum hr rest ] { 60 - 70% } + hr rest ) hr maximum = 220 age . resting hr was measured in the morning immediately after the participants were awake . if the subjects are not able to take a measurement first thing in the morning , make sure subjects lie down for at least 10 min before taking a measurement . measurement was taken from the radial artery with forefinger and the middle finger of the right hand placed horizontally across the subject 's wrist while sitting on the chair . after that , the number of pulse beats multiplied by two to give the 1 min hr . cardio - respiratory fitness was determined by 1609 m ( 1 mi ) walk test using rockport fitness test . after a brief warm up , the subject walked as briskly as possible for 1609 m ( 1 mi ) with a hr monitor . the formula used to calculate vo2 max was : 132.853 ( 0.0349 mass [ kg ] ) ( 0.3877 age [ year ] ) + ( 6.315 gender ) ( 3.2649 time [ min ] ) ( 0.1565 hr [ bpm ] ) . the validity of the test was nearly high r = 0.88 and standard error of the test was 5 ml / kg / min . cardio - respiratory measurement was taken before and after 8 weeks of aerobic training program and supplementation . a total volume of 20 ml blood was drawn from the subjects in their forearm vein after a 12 h overnight fast immediately before and after 8 weeks of aerobic training program and supplementation in the sitting position after a 20-min rest between 8:00 and 9:00 a.m. plasma was separated by centrifugation and samples were stored at 10c until assays were determined ( within 48 h ) in all samples . cardio - respiratory fitness was determined by 1609 m ( 1 mi ) walk test using rockport fitness test . after a brief warm up , the subject walked as briskly as possible for 1609 m ( 1 mi ) with a hr monitor . the formula used to calculate vo2 max was : 132.853 ( 0.0349 mass [ kg ] ) ( 0.3877 age [ year ] ) + ( 6.315 gender ) ( 3.2649 time [ min ] ) ( 0.1565 hr [ bpm ] ) . the validity of the test was nearly high r = 0.88 and standard error of the test was 5 ml / kg / min . cardio - respiratory measurement was taken before and after 8 weeks of aerobic training program and supplementation . a total volume of 20 ml blood was drawn from the subjects in their forearm vein after a 12 h overnight fast immediately before and after 8 weeks of aerobic training program and supplementation in the sitting position after a 20-min rest between 8:00 and 9:00 a.m. plasma was separated by centrifugation and samples were stored at 10c until assays were determined ( within 48 h ) in all samples . the statistical analysis was initially performed using the changes in the one - sample kolmogorov - smirnov test . all variables presented normal distribution . data was analyzed using paired t - test for within group 's comparison and unpaired t - test for between - groups comparison . statistical significance was accepted as p 0.05 . numbers of four subjects were excluded during the study , because of altering their usual aerobic training program and being irregular in taking capsules , so we did our statistical analysis on 16 subjects ( 8 subjects in ns group and 8 subjects in pla group ) who fully completed the study [ figure 1 ] . there were no differences among groups for age , bodyweight , height and bmi [ table 1 ] . changes of lipid profiles after 8 weeks for two groups are shown in table 2 . tc ( p < 0.001 ) and ldl - cholesterol ( p < 0.01 ) were reduced in pla group and vo2 max ( p < 0.01 ) increased . however , these changes were not statistically significant for tg , bmi and hdl - cholesterol ( p > 0.05 ) . the schematic diagram of the study changes in the health related physical fitness parameters and blood lipids in pla and ns group in the ns group tc ( p < 0.01 ) , tg ( p < 0.001 ) and ldl - cholesterol ( p < 0.001 ) levels were decreased by 4.79% , 7.97% and 5.02% respectively and bmi ( p < 0.01 ) was significantly reduced by 2.76% and hdl - cholesterol ( p < 0.01 ) and vo2 max ( p < 0.01 ) significantly increased by 5.76% and 2.53% respectively . furthermore , there were significant differences between the ns and pla groups for ldl - cholesterol ( p < 0.05 ) and hdl - cholesterol ( p < 0.05 ) values [ table 2 ] . the relationship between ldl - cholesterol and chd risk is continuous over a broad range of ldl - cholesterol levels : the higher the ldl - cholesterol level , the greater the chd risk . the increased permeability of the endothelium in the presence of elevated concentration of ldl promotes accumulation of ldl particles in the intima . these particles undergo oxidative modification , which may lead to inflammatory reactions and initiate atherosclerosis process . therefore , lowering ldl - concentration and improving antioxidant defense of the body are two important ways to prevent atheriovascular disease . the relationship between the dietary fats and cvd , especially chd , has been extensively investigated , with strong and consistent associations emerging from a wide body of evidence accrued from animal experiments , as well as observational studies , clinical trials and metabolic studies conducted in diverse human populations . the evidence shows that intake of saturated fatty acids is directly related to cardiovascular risk and raise total and ldl - cholesterol . when substituted for saturated fatty acids in metabolic studies with unsaturated fatty acids lower plasma total and ldl - cholesterol concentrations ; polyunsaturated fatty acids are somewhat more effective in this respect , especially linoleic acid . this double - blind , randomized placebo - control study examined the long - term n. sativa supplementation and aerobic training on lipid profile and vo2 max in sedentary overweight females . the main finding of this study was that 8 weeks of aerobic training with n. sativa supplementation improved hdl - cholesterol and impaired ldl - cholesterol . results of the present study are in consistent with other previous studies that found improvements in health related parameters of overweight and obesity females participants as a result of aerobic exercise program also n. sativa for the treatment of hyperlipidemia . since , we asked all subjects not to change their usual daily diet , it seems that this changes may be due to the result of consuming black seeds and regular aerobic training . furthermore , these results are also in agreement with the results obtained by anwar and tayyab who observed that polyunsaturated fat have hypotriglyceridemic effect . the choleretic function of n. sativa is either by reducing the synthesis of cholesterol by hepatocytes or by decreasing its fractional reabsorption from the small intestine . furthermore , it has been proved that volatile oil of n. sativa has two main constituents , i.e. nigellone and thymoquinone which play a key role in heart disease prevention . n. sativa is a rich source of unsaturated fatty acids , mainly linoleic acid ( 50 - 60% ) , oleic acid ( 20% ) , eicosadienoic acid ( 3% ) and dihomo linolenic acid ( 10% ) that cholesterol lowering effect of n. sativa may be attributed to the presence of phytosterols like beta - sitosterol , polyunsaturated fatty acids and its antioxidant activity . n. sativa may be able to reduce synthesis of cholesterol by hepatocytes and lower its absorption from the small intestine . it may also activate ldl - receptor by decreasing intracellular cholesterol , which leads to rapid clearance of ldl - cholesterol from blood circulation . the effect of n. sativa on increasing cholesterol secretion in the bile is another probable mechanisms , which can enhance its cholesterol lowering properties . furthermore , physical exercises performed regularly have effects on obesity , cardiovascular system , physical fitness and healthy life of the middle - aged individuals . exercise may promote reverse cholesterol transport by increasing pre- hdl - cholesterol recycling from hdl - cholesterol substrates that have been induced by exercise itself . this mechanism can explain the greater cholesterol efflux from cultured human fibroblasts when exposed to serum of activity people 's versus sedentary controls . several studies have confirmed this belief and the results of the present study are in agreement with the above statement . results of the present study is consistent with arazi et al . in pla group that applied at the end of 8-week of regular aerobic exercises on middle - aged sedentary women , found that decreased in ldl - cholesterol and increased in the hdl - cholesterol levels . in addition , bemelmans et al . applied at the end of 12-day of walking exercises on middle - aged sedentary men and women , found in their study that decreased in the ldl - cholesterol and tc levels and increased in the hdl - cholesterol levels . in addition , aerobic exercise training increases the percentage of skeletal slow - twitch fibers , which have a higher capacity to metabolize fatty acids liberated by lipoprotein lipase from tg - rich lipoproteins . also , exercise decreases the presence of small , dense ldl - cholesterol and increases that of larger , more buoyant ldl - cholesterol particles , in a relationship that appears dose - dependent with the amount and intensity of exercise . because over the last 10 years , there have been a growing number of studies that show the benefits of moderate intensity physical activity for cardiovascular health so in our study intensity of training was 60 - 70% of target pulse rate . despite the aforementioned results , n. sativa supplementation may facilitate additional lipid profile improvements in exercise by hypotriglyceridaemic effect and increasing cholesterol secretion in the bile . our results suggest that 2 g / day of n. sativa combined with an aerobic exercise program provides significant improvements in ldl - cholesterol and hdl - cholesterol that are known to influence cvd risk in sedentary overweight females . however , further studies with a larger sample size and diet assessment are still needed .

background : regular moderate intensity physical activity and lipid lowering effects of nigella sativa ( n. sativa ) supplementation may be appropriate management for sedentary overweight females . therefore , the aim of the present study was to examine the effects of long - term n. sativa supplementation and aerobic training on lipid profile and maximal oxygen consumption ( vo2 max ) in sedentary overweight females.methods:in this randomized , double - blind , controlled trial , which was conducted in kerman city ( iran ) , 20 sedentary overweight females were divided into two groups and assigned to n. sativa supplementation ( n. sativa capsules ) or a placebo for the 8 weeks , both groups participated in an aerobic training program ( 3 times / week ) . each n. sativa capsule contained 500 10 mg n. sativa crushed seeds and subjects had to take 2 g n. sativa per day for 8 weeks . blood lipids and vo2 max were determined at baseline and at the end of 8 weeks.results:n . sativa supplementation lowered total cholesterol ( tc ) ( p < 0.01 ) , triglyceride ( p < 0.001 ) , low - density lipoprotein ( ldl ) ( p < 0.001 ) and body mass index ( p < 0.01 ) and increased high density lipoprotein ( hdl ) and vo2 max ( p < 0.01 ) . aerobic training program lowered tc ( p < 0.001 ) and ldl ( p < 0.01 ) and increased vo2 max ( p < 0.01 ) . furthermore , we observed a significant effect of aerobic training program and n. sativa supplementation lowered ldl and hdl ( p < 0.05).conclusions : the present study demonstrates that 8-week aerobic training plus n. sativa supplementation have a synergistic effect in improve profile lipid parameters .

INTRODUCTION METHODS Subjects Supplementation Training program Resting HR Measurements Cardio-respiratory assessment Blood samples Statistical analysis RESULTS DISCUSSION CONCLUSIONS

PMC5248430

some cyclists use the bicycle as a means of transportation , others ride for recreation , and some are competitive cyclists . the repetitive movements of cycling put cyclists at a risk of overuse injuries , particularly to the leg joints ( knee and ankle ) . during 1985 , about 8.36% of causalities seen in the emergency room of king fahd hospital of the university were sports related , and knee injuries accounted for 24% of those injuries . in a cross - sectional study of 224 recreational cyclists in california in1995 using mailed surveys , 85% of the cyclists reported one or more overuse injuries , 36% of which required medical attention . miles / week , lower number of gears , and low level of experience were associated with increased prevalence . the study by zwingenberger et al . also suggested that low experience level was associated with increased prevalence of injury . in a study by silberman in new jersey in professional road cyclists , 94% of them had experienced at least one overuse injury in the preceding 12 months ; knee was the most common site . many injuries were mild , resulting in limited time off the bike . in another study by clarsen et al . among seven professional teams in the usa , involving 109 professional cyclists , the prevalence of knee overuse injury was 23% and most likely resulted in time loss . vleck and garbutt studied the prevalence of knee injury among elite triathletes in the uk . nearly 75% of them had overuse injury , and knee injuries accounted for 14.2%-21.9% of the injuries . almost 62.1% of the knee injuries occurred during running and 34.5% occurred during cycling . in a study conducted in london on military personnel undergoing a fitness test , it was found that obesity was associated with the increased incidence of musculoskeletal injury . many studies suggest that the bike fit and alignment , lack of training , and inappropriate equipment are major contributory factors and that efforts should be made to control these factors to reduce the prevalence of overuse injuries and reduce time off training . the outcome of a randomized controlled trial ( rct ) was favorable with respect to strengthening and stretching exercises in preventing anterior knee pain ( akp ) . however , a systematic review showed a marked reduction ( 50% ) in overuse injury by means of strengthening exercises , but , stretching exercises did not show any benefit . despite the increasing popularity of cycling , to the best of our knowledge , there have been no published epidemiological studies in saudi arabia , and indeed , few studies have been published worldwide that describe the prevalence of knee problems of cyclists and the associated risk factors . this study was conducted to assess the prevalence and the magnitude of knee pain and other knee problems of active cyclists in the eastern province and to determine the factors associated with knee pain in the study population . a cross - sectional study was conducted to assess the prevalence , magnitude , and associated factors of knee pain of active cyclists in the eastern province , saudi arabia , in 2015 . the study population included all active cyclists who were members of a recognized professional or amateur cycling club in the eastern province of saudi arabia . an online link to the questionnaire was sent to 513 cyclists , 311 of whom completed the questionnaire , giving a response rate of 60.62% . the following inclusion criteria were then used : ( 1 ) minimum age of 18 years ; ( 2 ) should have been bicycling for 6 months ; ( 3 ) and should ride the bicycle at least once per week . the data were collected using a structured , self - administered questionnaire which had been designed by the researchers after reviewing the literature and similar questionnaires based on the objectives of the study . the questionnaire consisted of seven parts : ( 1 ) sociodemographic data : age , gender , nationality , level of education , marital status , and club name ; ( 2 ) chronic medical illnesses that may affect the sport or predispose to knee symptoms , smoking , height and weight ; ( 3 ) participation in sport : goal , experience , frequency , duration , warm up , stretching exercises , strengthening exercises , and rest days ; ( 4 ) type of bicycle used , bicycle fitting , and clip - less pedal ; ( 5 ) current knee pain and pain in the past 12 months , frequency , precipitating event , site , and associated symptoms and management . a frequency scale comprising always , we tested six types of bicycles : road bicycle ( a bicycle built for traveling at speed on paved roads , time trial bicycle ( a racing bicycle designed for use by an individual or team time trials on roads ) , mountain bicycle ( a bicycle created for off - road cycling ) , hybrid bicycle ( a blend of characteristics of a road bicycle and a mountain bicycle ) , and the recumbent bicycle ( a bicycle on which the rider is in a laid - back position ) . the questionnaire was designed by the researchers , validated by four faculty , and piloted on forty cyclists ( different from the target group ) . the study was approved by the ethical committee of postgraduate saudi board program , eastern province . all participants were sent an invitation explaining the purpose of the study , reassuring them of confidentiality and a web link to the survey in october 2015 . sms , whatsapp application , and e - mail were used to deliver the invitations to the participants . completion and return of the questionnaire was considered as assent to participation in the study . returned questionnaire that did not meet the inclusion criteria were excluded , giving a total of 283 valid questionnaires . the data were coded , entered , and analyzed using statistical package for social sciences ( spss ) version 20.0 ( released in 2011 by ibm corp . , cyclists were classified into two groups : professional ( competitive cyclists who are registered in the saudi cycling federation ) and amateur ( unpaid cyclists not under the umbrella of the saudi cycling federation ) . the term bicycle fitting was used for a process whereby an expert adjusts the bicycle dimensions to suit those of the cyclist 's body . the term clip - less pedal was a system comprising special pedals and cleats , devices on the pedals that attach to the soles of clip - less cycling shoes . the term triathlon is used for a multiple - stage competition involving swimming , cycling , and running in immediate succession over various distances . body mass index ( bmi ) was classified into six categories according to the world health organization classification : < 18.5 as underweight , 18.524.9 as normal weight , 2529.9 as overweight , 3034.9 as obesity class i , 3539.9 as obesity class ii , and 40 as obesity class iii . the numeric rating scale ( 11 ) was used to categorize pain : mild ( 13 ) , moderate ( 46 ) , and severe ( 710 ) . age was classified into four groups : teenage ( 1819.99 ) , young adult ( 2039.99 ) , middle aged ( 4064.99 ) , and elderly ( 65 ) . data were presented using descriptive statistics in the form of frequencies and percentages for categorical variables and mean and standard deviation for quantitative variables . mann - whitney test was used to compare median ranks between different pain category groups . out of 513 , a total of 311 cyclists responded to the survey ( 60.6% response rate ) . majority of the participants were male ( 95.1% ) , and young adults ( 63.3% ) . teenagers constituted 45.4% of the professional teams and 2% of the amateur teams ( p = 0.0001 ) . almost two - thirds ( 69.3% ) were saudi , 12% were asians , while the remainder were of various other nationalities . half of them had a bachelor 's degree or higher , and about three - fourths ( 72.7% ) were married . concerning smoking , 13.6% of the participants smoked tobacco products , 21.1% had quit , and 65.2% had never smoked . amateur cyclists had higher smoking rates ( 15.7% ) compared to professional cyclists ( 2.3% ) ( p = 0.001 ) [ table 1 ] . distribution of active cyclists according to sociodemographic characteristics , eastern province , saudi arabia , 2015 . the overall prevalence of knee pain was 25.8% . the second most common symptom in relation to the knee joint was popping ( 6.7% ) followed by swelling of the knee ( 5.0% ) . the 12-month prevalence of knee pain was higher in amateur cyclists ( 27.6% ) compared to professional cyclists ( 15.9% ) , but that was not statistically significant [ figure 1 ] . distribution of knee symptoms among active cyclists by club type , eastern province , saudi arabia , 2015 the distribution did not statistically vary among the different studied sociodemographic categories or smoking status . as regards cyclists with knee pain , 39.7% of the cyclists seldom experienced knee pain , 39.7% experienced it sometimes , while almost 18.5% often or always experienced knee pain . knee pain occurred spontaneously with no obvious cause in 32.8% of cases , for 25% , it was precipitated by running , and for 17.2% , it was precipitated by cycling . anterior aspect of the knee was the most common location of the pain , accounting for 58.1% ; 21% were above the patella , 27.4% were below the patella , and 9.7% were behind the patella [ table 2 ] . characteristics of the knee pain among active cyclists , eastern province , saudi arabia , 2015 . ( n=75 ) the average duration of knee pain was 12 months that was higher than other knee symptoms such as swelling , locking , popping , and giving way . the average days - off training because of the knee problems was 2 days . swelling and the presence of a lump were associated with a higher average of days - off training . more than half ( 61.6% ) of knee pain was mild , 28.7% was moderate , and 9.5% was severe pain [ table 2 ] . of cyclists with knee pain , about half of those with knee pain ( 49.2% ) used medications for their pain , 28.6% underwent physiotherapy , and 12.7% underwent knee surgery . only two cases ( 3.17% ) the highest prevalence of knee pain was found in road racers ( 50% ) followed by cyclists who rode for fitness and weight loss ( 29.2% ) . the lowest prevalence of knee pain was found in those who used cycling as a mean of transportation ( 16.7% ) ( p = 0.020 ) [ table 3 ] . frequency distribution of various cycling and other sports - related factors among active cyclists by knee pain , eastern province , saudi arabia , 2015 . the distribution was much higher among cyclists who used to ride mountain bikes off road ( 80% ) , followed by those who used to ride time trial bikes ( 45.5% ) and cyclists who rode mountain bikes on road ( 34.8% ) . the lowest prevalence of knee pain was found in those who rode recumbent bikes ( 14.3% ) ( p = 0.042 ) [ table 3 ] . the prevalence was higher in underweight cyclists ( 62.5% ) and surprisingly lower in obese cyclists ( 23% ) , but this was not statistically significant ( p = 0.137 ) . the prevalence was higher in cyclists who participated in football games ( 40% ) compared to cyclists who did not ( 26.6% ) ( p = 0.368 ) [ table 3 ] . the average jogging hours was higher among cyclists with knee pain ( 2 h ) compared to cyclists who had no knee pain ( 1 h ) ( p = 0.007 ) . moreover , the average of swimming hours among cyclists with knee pain was higher as well ( 1 h ) ( p = 0.019 ) [ table 3 ] . when preventive measures were tested , there were no statistically significant differences between cyclists with knee pain and cyclists with no knee pain . the measures included a professional bike fit , coach supervision , clip - less pedal usage , warm up , strength training , and stretching exercises [ table 3 ] . there was no statistically significant difference in the average of years of cycling , cycling hours per week , riding frequency per week , kilometers ridden per week , and rest days between cyclists who had knee pain and cyclists who had no knee pain [ table 3 ] . knee pain , common in active cyclists in the eastern province of saudi arabia , has an overall 12-month prevalence of 25.8% . professional cyclists had a lower prevalence of knee pain than the amateur cyclists , suggesting that experience is protective . however , the results show no difference in the average years of the experience . professional cyclists undergo a high - quality bike fitting on a regular basis and get trained under direct coach supervision , in which an emphasis is put on the correct technique necessary in preventing injuries . berkovich et al . also posited the importance of coaching techniques in preventing knee injuries in cyclists . many researchers have also suggested that the appropriate bicycle configuration and saddle height are protective . however , the results of this study did not show any difference in the prevalence of injury between cyclists who had a professional bike fit and cyclists who did not . the 12-month prevalence in amateur cyclists of this study was higher than the 18-month prevalence in the uk adventure racing cyclists ( 21.9% ) . this is probably because the cyclists in the uk were more physically fit and experienced , based on years of cycling . the 12-month prevalence was higher in japanese triathletes ( 33.0% ) , but lower than what was found in recreational cyclists in california ( 41.7% ) . the prevalence of knee pain in this study was higher than the prevalence in the residents of al - qassim , the ksa , with clinical knee osteoarthritis ( 13% ) , but close to the prevalence of knee pain for non - hispanic and mexican - american males aged 6074 years ( 26.1% ) . most cyclists attributed their knee pain to causes other than bicycling itself ; only 17.2% thought that their pain was due to cycling . this is close to the conclusion drawn by deakon , which imputed 20% of injuries during triathlon to cycling . a large percentage of injuries occurred spontaneously ( 32.8% ) , suggesting insidious causes of knee pain rather than acute injuries . this conclusion is supported by the fact that the average running time was double in cyclists with knee pain . vleck and garbutt concluded that injuries that occur during running were almost twice than those of injuries that occur during the bicycling part of the triathlon events . akp affected 14.9% of cyclists in this study compared to 36% of the usa professional cyclists as described by clarsen et al . this suggests that patellofemoral pain syndrome , patellar tendenopathy , patella chondromalacia , fat pad impingement , or patellar bursitis are the possible etiologies of the knee pain in the studied population . by the same token , the average duration was 12 months , suggesting that the most likely causes were chronic . most knee pains were mild or moderate ( 62.1% and 28.8% ; respectively ) and were not frequent ; more serious injuries were most likely to be associated with more severe and frequent pain . needed surgery or an intra - articular injection ( 12.7% and 3.2% , respectively ) . the highest prevalence of knee pain ( 50.0% ) was in road racers , but the lowest was in commuters ( 16.7% ) , suggesting that the volume and intensity of training are the important risk factors for knee pain . however , there were no differences in the average bicycling hours per week , kilometers ridden per week , riding times per week , or rest days per week , which means that high - intensity training was a key factor . , there is a positive correlation between the hours spent cycling per week and rest days per week and the number of injuries . moreover , there was a relatively higher prevalence in cyclists who rode for fitness and weight loss . this may be explained by the low fitness level and high bmi of this group of cyclists as suggested by taanila et al . in this study , warm up , strength training , and stretching exercises however , an rct by coppack et al . showed that strength and stretching exercises reduced the incidence of akp by 75% . a systematic review by herman et al . also showed that proper neuromuscular warm - up strategies decreased the risk of injury . prevalence in underweight cyclists ( 62.5% ) was higher than obese and normal weight cyclists . this confirms the finding that being underweight was a risk factor of musculoskeletal overuse injuries . the rate of greater knee pain was not higher in the obese than in those with normal weight . smoking rate among amateur cyclists ( 15.7% ) was higher than what prevailed in the general population of saudi arabia ( 12.2% ) . though it was not associated with higher rate of knee pain in this study , it is well documented that smoking increased the risk for musculoskeletal injuries . we recommend clinical trials to evaluate the effect of the above - mentioned preventive measures on performance and injuries . there should be an annual survey to assess injury patterns , especially among professional cyclists . clinicians should be involved in increasing the awareness of the importance of bike fit , proper cycling and running techniques , and the right fit of footwear . emphasis should also be put on the importance of cessation of smoking , the maintenance of ideal body weight , and the avoidance of over training . the data were collected using a self - administered questionnaire , which may have been subject to recall bias . there may have been nonresponse bias as response rate was low ( 60.6% ) . a simple short survey was used for preventive measures , but a prospective study is necessary for a better assessment . . nevertheless , this first study of the population surveyed has highlighted the problem of knee pain in cyclists in saudi arabia and emphasized the need for health education about this problem and ways to prevent injuries . running , football participation , and being underweight were associated with higher rates of knee pain . the rate of knee pain in road racers and mountain cyclists was higher , probably because their knees were susceptible to overuse . there was no association with bicycle fitting , coaching , using clip - less pedals , warming up average years of cycling , average distance ridden in a week , resistance training , strength training , and average rest days in a week . the study underpins the importance of ensuring that athletes use the appropriate technique in cycling , running , and swimming , use appropriate footwear , and ensure that the increase in the training load is gradual in trial to prevent knee pain in cyclists , especially amateur cyclists .

introduction : bicycling is one of the most enjoyable aerobic exercises recommended for the promotion of an individual 's health . the eastern province of saudi arabia has seen a huge increase in the number of people who cycle . people have different goals for bicycling , but the injuries they sustain are common . most of them relate to overuse , particularly of lower body joints . this study was conducted to determine the prevalence of knee problems and factors associated with knee pain in cyclists.materials and methods : a cross - sectional study was conducted in october 2015 , using an online self - administered questionnaire . the questionnaire was based on pertinent literature , was piloted , and validated . a web link was sent to 513 cyclists ( professional and amateur ) using e - mail , whatsapp application , or sms . three hundred and eleven responses were received , 283 of which were included in the analysis.results:the overall prevalence of knee pain was 25.8% ; 27.6% for amateur cyclists and 15.9% for professional cyclists . only 17.2% knee pain was attributed to cycling , whereas in 32.8% it happened spontaneously and in 25% of cases it occurred while running . majority of the cyclists reported pain as mild ( 61.6% ) or moderate ( 28.7% ) ; anterior knee pain accounted for 58.1% knee pain . different goals of cycling and different bicycle types had statistically significant difference on the rate of knee pain . of underweight cyclists , 62.2% reported knee pain . cyclists who run more or participated in football had a higher rate of pain.conclusion:knee injuries are common with cyclists . factors such as the type of the bicycle , the goal of bicycling , club type , body mass index , and participation in other sports play a significant role in the rate of knee pain .

Introduction Materials and Methods Results Discussion Conclusion Financial support and sponsorship Conflicts of interest

PMC5034380

language can be counted as the first cognitive enhancement , and the invention of writing also dramatically improved our cognitive ability to remember , to learn , and to communicate. today , with the aid of recent phenomenal development of biomedical technology , we are witnessing the emergence of more direct biological cognitive enhancementschemical , physical , or electromagnetic intrusions into our physical brain. brain enhancement has been considered as one of the most important intersections of law and neuroscience . the idea of directly enhancing the brain with drugs , brain stimulation , or neurosurgery might seem frightening . but more than frightening , the use of these enhancement technologies raises crucial neuroethical and legal issues . for example , fairness can be a serious problem , if an effective enhancement technology is very costly and thus , available only to the rich . also , individuals could be coerced , explicitly or implicitly , to undergo cognitive enhancements for various reasons , such as criminal rehabilitation , medical treatment , or even academic success at school . despite these potential risks , professor hank greely has argued that safe and effective cognitive enhancement will benefit both individuals and society , and we should require better research on , and better regulation of , cognitive enhancement instead of banning the use of all cognitive enhancements : biomedicine will be creating more and more products and procedures that can be used for cognitive enhancement . some of them will be used in ways that will , on balance , improve human life and society . at the same time i am confident , though , that a knee - jerk rejection of all direct brain enhancement will be at least a missed opportunity and at worst an opening for a damaging underground and uncontrolled world of enhancement . in order to maximize the benefits and minimize the harms of these new technologies , we will need to look at particular enhancements rationally and to adopt , ban or regulate them carefully . one of the most eye - catching and readily accessible direct brain enhancement technologies is transcranial direct current stimulation ( tdcs ) . tdcs is a type of non - invasive neuromodulation , which delivers weak direct current ( dc ) ( 12 ma ) to the brain using small saline - soaked electrodes . this weak current is insufficient to cause neurons to fire , but it can alter the excitability of neurons by shifting their membrane potentials in a de- or hyper polarizing direction ; thus making them more or less likely to fire. anodal dc stimulation is known to increase neuronal excitability , while cathodal stimulation decreases it . application of electric currents to the human brain for therapeutic purposes is not a new idea . in ancient times , people placed an electric fish over the scalp to treat headache or epilepsy . later in 18th century , advances in the science of electrophysiology by galvani and volta inspired the use of direct currents for the treatment of mental disorders . nonetheless , erratic results and the advent of electroconvulsive therapy led to the decrease of interest in dc brain stimulation . however , reappraisal in the last decade has shed new light on the effects of dc stimulation on the human brain . a number of neurophysiological studies have demonstrated the efficacy of tdcs for the treatment of patients suffering from neuropsychiatric diseases , such as stroke , chronic pain , and depression . moreover , recent studies have reported that the use of tdcs on specific brain regions can improve cognitive functions , such as attention span , working and long - term memory , language , and mathematical ability of healthy subjects . for example , de vries and colleagues showed that anodal tdcs over broca 's area a region in the frontal lobe of the hemisphere of the brain improves artificial grammar learning . numerical learning was enhanced by anodal tdcs of the parietal cortex , and this effect was stable for at least 6 months after training. also , subjects who received anodal tdcs on the right anterior temporal lobe while naming pictures of famous people showed improved retrieval of proper names . in one study funded by the us defense advanced research projects agency , subjects who received anodal tdcs on right inferior frontal and right parietal cortex showed learning and performance improvement in a video game designed to train military personnel to accurately identify obscured and concealed objects in a complex environment . these interesting experiment results began to draw the media 's attention a new york times article in october 2013 described tdcs devices as jump starter kits for the mind. also , the number of tdcs articles published per year has been growing rapidly , and currently at least 224 clinical studies on tdcs are going on around the world . on top of the fact that it can be an effective tool for both treatment and cognitive enhancement , tdcs has two other especially appealing features most of the known side effects of tdcs are minor adverse effects , such as headache , or slight tingling or itching under the electrodes . at least nine tdcs devices for personal use can be purchased through websites as a complete unit with prices between approximately \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}200 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}400 . but for less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}40 for parts and thanks to these special features of tdcs , in recent years , a group of people who built or purchased the tdcs device for home use has appeared . researchers have expressed concerns about this so - called do - it - yourself ( diy ) use of tdcs . they have argued that even if there are no serious side effects , potential risks of misusing this device should not be ignored . for example , lay people who are diy users may not have sufficient knowledge on the structure of brain to place the electrodes accurately , causing unintended effects . moreover , reversing anodal and cathodal electrodes placement could lead to impairment of the brain by switching areas affected by excitatory and inhibitory stimulation . it is also possible that tdcs interacts with other treatment that diy users are undergoing , such as psychoactive medication , in detrimental ways . lastly , using tdcs to enhance some of the cognitive functions may adversely affect other functions of the brain , and potential long - term ( side ) effects of tdcs on the brain have not been fully explored . the fda 's own description of its regulatory regime for medical devices reads : if a product is labeled , promoted or used in a manner that meets the following definition in section 201(h ) of the federal food , drug , and cosmetic ( fd&c ) act , it will be regulated by the food and drug administration ( fda ) as a medical device and is subject to premarketing and postmarketing regulatory controls . an instrument , apparatus , implement , machine , contrivance , implant , in vitro reagent , or other similar or related article , including a component part , or accessory which is : ( 1 ) recognized in the official national formulary , or the united states pharmacopoeia , or any supplement to them , ( 2 ) intended for use in the diagnosis of disease or other conditions , or in the cure , mitigation , treatment , or prevention of disease , in man or other animals , or ( 3 ) intended to affect the structure or any function of the body of man or other animals , and which does not achieve its primary intended purposes through chemical action within or on the body of man or other animals and which is not dependent upon being metabolized for the achievement of any of its primary intended purposes . fda requires all medical products manufacturers to register their facilities , list their devices with fda , and follow general control requirements. medical devices are classified in three categories low ( class i ) , moderate ( class ii ) , and high - risk ( class iii ) devices according to the risks that the devices present . devices with low risk are considered as exempt from fda 's pre - market review and can be legally marketed upon registration . in terms of moderate- and high - risk devices , there are two administrative paths that manufacturers must take to bring the devices to markets . the first path is pre - market approval ( pma ) , which consists of conducting clinical studies , submitting pre - market approval ( pma ) application and requires evidence providing reasonable assurance that the device is safe and effective. usually novel and high - risk devices are subject to this process , and it is known to be very lengthy and expensive . the other path involves submitting a 510(k ) notification demonstrating that the devices is substantially equivalent to a device already on the market ( a predicate device) and results in fda clearance . this path , which tends to be less expensive and time - consuming than pma , is available for moderate - risk medical devices not exempt from pre - market review . once approved or cleared for marketing , manufacturers must comply with regulation on manufacturing , labeling , surveillance , device tracking , and adverse event reporting. some neurostimulation devices are already classified and regulated as medical devices by fda . for example , in 2011 , repetitive transcranial magnetic stimulation ( rtms ) , another non - invasive neuromodulation that generates electric currents using electromagnetic coils , was classified as a class ii medical device for treatment of major depressive disorder . also , cranial electrotherapy stimulation , whose mechanism is similar to tdcs except that it applies alternating current , not dc , to the brain , has been regulated as a medical device ( class iii ) for treatment of insomnia , depression , or anxiety since 1979 . fda guidance explicitly provides that there is no regulation for the therapeutic tdcs. a few clinics offer tdcs treatment to their patients with various medical conditions , such as depression and chronic pain , but these clinics apply tdcs as off - label use of the iontophoresis device a dc stimulator for introducing ions of soluble salts or other drugs into the body for medical purposes that fda classified as a medical device ( class ii / iii ) in 2004 . in addition , devices marketed for cognitive enhancement are not covered by any existing legislation in the usa . in their recent article , nick fitz and peter reiner stated that this regulatory vacuum is understandable given that the myriad applications of tdcs are fairly new. however , since tdcs devices are already on the market and being sold to the public , they argued that now we should begin the discussion on how to develop a regulatory policy for the diy use of tdcs . for example , foc.us , a fancy tdcs device marketed as a gamer headset to boost attention , completely sold out its first batch of 3000 units less than a month after its release in may 2013 . fitz and reiner urged all stakeholders regulators , scientists and the diy community to share in crafting policy proposals that ensure public safety while supporting diy innovation. they emphasized the importance of communication between policy makers and diy users to develop the ethos of responsible use based on the idea that people should have access to a diversity of opportunities created by enhancement technologies. responding to fitz and reiner 's call for a policy debate , maslen and colleagues proposed a regime that regulates cognitive enhancement devices such as tdcs by extending the existing legislation for medical devices . in line with the managed technological optimism that fitz and reiner advocate , they suggested the incorporation of a low - risk exemption for any cognitive enhancement devices falling below a given level of risk . in june 2013 , tdcs device kit , inc . , a tdcs device manufacturer based in california , voluntarily recalled its products after an inspection by california department of public health ( cdph ) . cdph determined that these products were not manufactured in compliance with good manufacturing practices for medical devices and that the devices lacked adequate labeling for directions for use and warnings against dangerous uses . this was a meaningful step taken by the government authorities , but it only had power in one state as there is no regulation on tdcs under the current fda regime for medical devices . despite the recent surge of attention to the potential risks and regulations of tdcs , there has been no attempt to systemically study what is really happening in the diy tdcs user community . discussions of issues related to the diy use of tdcs are mostly based on speculation , which may lead to importantly incorrect understanding of the diy community and its use of tdcs . this study is the first exploratory research on who diy tdcs users are and what is happening in this community . specifically , this study aims to answer following four questions on the diy use of tdcs through an online survey , a content analysis of web postings , and interviews with the diy users . what are the general demographic characteristics of diy users?why and how do they use this device?what are the perceived effects or side effects of this device?what are their concerns , if any , about the diy use of this device ? what are their concerns , if any , about the diy use of this device ? in particular , its survey was not of random users but of a set of self - selected diy tdcs users who responded to an invitation to participant the author posted on the two major diy tdcs internet web sites . also , only a small number of people interviewed , chosen from among those who , based on the websites , appeared to be influential in the diy tdcs user community . the unregulated , unregistered , and otherwise unreachable nature of the community of tdcs users made these the only feasible approaches , but they do undercut , to some extent , the confidence that can be placed in the findings . nonetheless , even if viewed as only preliminary data , these results provide some basic information on diy tdcs users and their attitudes and practices of diy tdcs which will help us to lay the groundwork for designing sound future policy and official guidelines on tdcs . section 1 describes the three research methodologies recruited in this study , and the findings on the diy tdcs user community are presented in section 2 . section 3 explores potential legal and regulatory implications of these findings considering possible regulatory options for the diy use of tdcs . finally , section 4 summarizes the paper 's major findings and briefly discusses future directions for research . there has been no official report on the size of the diy tdcs user population in the usa , in any other country , or worldwide . users do not need a prescription , do not need to register their use , and do not even need to purchase tdcs units . however , two major diy tdcs internet websites , www.reddit.com/r/tdcs and www.diytdcs.com , where diy users usually visit and share information about tdcs are potential resources for exploring diy tdcs users . the site , www.reddit.com/r/tdcs/ ( hereinafter subreddit tdcs ) , is an open forum for tdcs users and www.diytdcs.com ( hereinafter diytdcs ) is a personal blog by a famous lay expert on diy tdcs . the research subjects of this study are the registered users or visitors of these two websites ( 1 ) who built or purchased their personal tdcs devices and ( 2 ) who have used them for treatment or as a cognitive enhancer . the research methodology of this study has three parts : ( 1 ) an online questionnaire survey , ( 2 ) a content analysis of web postings on the diy use of tdcs , and ( 3 ) in - depth interviews . the survey was composed of a minimum of 21 to a maximum of 29 questions depending on the respondents answers to some questions on basic demographic information of diy users , their aims of practicing diy tdcs , current practices of tdcs ( including types of devices , stimulation protocols , and frequency of use ) , effects and side effects of tdcs , and their concerns about the diy tdcs ( see appendix a for the questionnaire ) . the survey was posted on subreddit tdcs from december 27 , 2013 to march 10 , 2014 and on diytdcs from december 29 , 2013 to march 10 , 2014 . the responses of this online survey do not form a random sample , and there is a potential bias because these respondents are self - selected to participate in this survey . however , since this study is the first preliminary attempt to learn about the diy tdcs user community , responses from this convenience sample should provide previously unknown but useful facts about this community . the second research methodology is a content analysis of web postings on subreddit tdcs . in this well - known and very active online forum , diy users ask and answer questions on the diy use of tdcs device , post interesting recent tdcs studies , and share their experiences of positive and adverse effects of tdcs devices . these web postings are an important source of information to answer some of the research questions . subreddit tdcs was established in april 2011 ; since then , there has been a significant increase in the number of postings ( fig . this study took a census of all 825 postings , and the comments to these postings were selectively included in the analysis in cases where the comments showed meaningful discussions on the issues raised in the postings . number of posting per quarter in www.reddit.com/r/t/dcs ( 20112013 ) . since there is no previously established theory or research study on the diy tdcs user community or subreddit tdcs , the codes were derived from an emergent process . through a preliminary examination of the postings , five major codes to categorize the main theme of each posting were developed : ( 1 ) building and operation of the tdcs device , ( 2 ) marketed devices , ( 3 ) effects / side effects of the device , ( 4 ) references ( external links ) to research papers , newspaper / magazine articles , or blog posts on tdcs , and ( 5 ) others . when there was more than one theme in a posting , this study coded the posting according to its primary purpose . for example , if a user began his or her posting with the side effect he or she has experienced , but then later asked about how to improve his or her diy circuit to prevent this side effect , this posting would be classified under the code , building and operation of the tdcs device. in addition to these major codes , two additional emergent codes the commenters reasons for using the tdcs device and their warnings about safety were prepared to describe recurrent topics that answer some of the research questions of this study but do not constitute the main theme of the postings . for example , diy tdcs users usually mentioned their purpose for using the tdcs device while asking questions about building and operation of the device ( see appendix b for detailed classification of the codes ) . after survey data analysis and content analysis of web postings were completed , five semistructured interviews were conducted to collect more detailed information on the user experience and users opinions on the official guidelines and potential government regulation of the tdcs device . first , this study conducted a brief analysis on the user ids and postings at subreddit tdcs to identify power users who could provide a general overview and informative facts on the diy tdcs user community . the analysis revealed five users who have made more than 200 postings and comments at subreddit tdcs ( appendix c ) . four of these five power users participated in the interviews : the moderator of subreddit tdcs , the webmaster of diytdcs , a user who has uploaded popular self - experimentation videos on his youtube channel , and a user who has been using tdcs for treatment of bipolar type ii and other medical conditions . the interviewees were also asked to give their thoughts on the major findings of the survey and content analysis as an active user or an observer who is familiar with the trend in the community . second , a physician who has been treating about 350 patients with tdcs for the last seven years was also recruited for the interview to provide a medical perspective on the diy use of tdcs . the interview with webmaster of diytdcs was conducted through email , and the moderator of subreddit tdcs and the physician was interviewed over skype . the phone and skype interviews were conducted for about 30 minutes to an hour and audiotaped with the consent of the interviewees . although the number of samples is very small , given the exploratory nature of this study , these interviews should provide a meaningful preliminary sketch of the diy tdcs community and current uses of tdcs along with the findings of the survey and content analysis of the web postings . in this section , the results of the survey , content analysis of web postings , and in - depth interviews will be presented and analysed under the four main research topics of this study : general demographic characteristics of diy users ; their reasons for and current uses of tdcs ; effects and side effects of tdcs ; and their concerns about the diy use of tdcs . there is no official estimate of the size of the diy tdcs user population . as of the end of 2013 , subreddit tdcs had about 2700 subscribers . according to the moderator of the website , there were 4000 individual hits on this forum per month in 2012 . the number has been gradually increasing , and this forum is now getting 6000 individual hits per month . although it is very hard to track whether a particular person is really using tdcs , the moderator estimated that 6000 might be the upper bound of the diy tdcs user population and the lower bound might be the number of subscribers to this website , about 3000 , assuming that an active , regular tdcs user will visit the website at least once a month . one of the power users who has posted self - experiment videos said that 800 people were subscribing his youtube channel , and another famous youtube channel on tdcs has 2700 subscribers . this power user estimated that the size of the diy tdcs user population is probably a few thousand , but not more than 10,000 . however , the webmaster of diytdcs , who is also one of the most active participants in subreddit tdcs since the establishment of that forum , questioned this estimate of the size of the diy tdcs community in the media and academic studies : i 'm not sure how large the community is just now . it seems people pop in for a period and then move on , though there are also committed diy tdcs users who are self - treating daily . my sense is that tdcs is a fringe interest that wo n't attain popularity unless some sort of yet to be seen benefit emerges . only half ( 62 ; 51% ) of the respondents reported that they are continuous and regular tdcs users and among these continuous and regular users , 61% of them have been using tdcs less than six months ( appendix d ) . the majority of the respondents who claimed that they are continuous and regular users began to use tdcs pretty recently . thus , although , based on evidence of internet uses , the diy tdcs user population seems to have been growing for the last few years , these rough indicators might suggest that the dedicated tdcs user population has been overstated . there seems to be a small core of dedicated diy tdcs users that enable a sustained presence at the tdcs subreddit . but apart from that a lot of people come in for a quick look and then wander off. whatever the community 's actual size , the results on the demographic characteristics of the surveyed community are analysed below . first , the survey respondents are overwhelmingly male114 respondents ( 94% ) are male , 5 respondents ( 4% ) are female . second , most of the respondents are in their 20s and 30s ( 86 ; 71% ) , but there are a significant number of respondents in their 50s and over ( 15 ; 12% ) ( fig . the physician who has been treating patients with tdcs reported that there are some elderly patients who want to restore their diminishing cognitive abilities . he argued that tdcs is an appealing brain stimulation technology not only for young self - experimenters or bio hackers but also for seniors suffering from deterioration of mental functioning . also , it is noteworthy that there are some minors using tdcs ( 7 ; 6% ) . most of the respondents ( 90 ; 74% ) are from north america the usa and canada , although the rest of them are widely spread around the world . in addition , the majority of the respondents have four - year undergraduate degree or masters degree ( 62 ; 51% ) , though a significant number of respondents only have a high - school degree or below ( 29 ; 24% ) ( appendix e ) . relatedly , most of the respondents are students ( 25 ; 21% ) or professionals ( 45 ; 37% ) ( appendix f ) . the reported annual income level of the respondents also seems to fall in line with their occupations the respondents either have reported income of less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}20,000(26 ; 21% ) or reported income of more than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}90,000 ( 26 ; 21% ) ( appendix g ) . however , making generalizations on respondents socio - economic status based on these data can be problematic given that the respondents are from all over the world . this part will delineate and analyse the data on diy users aims of using tdcs device , as well as their current use of tdcs such as types of tdcs devices , stimulation protocols , and frequency of use . according to the survey results , respondents first learned about tdcs through academic research studies on tdcs ( 43 ; 35.5% ) , tdcs websites ( 43 ; 35.5% ) , and newspaper or magazine articles on tdcs ( 30 ; 25% ) ( appendix h ) . this indicates that information on tdcs is mainly disseminated and shared through online media and forums , assuming that many of respondents may not have direct access to academic journals , and recent intriguing studies on the various effects of tdcs have been vigorously covered by the media . 3 presents the number and the percentage of subreddit tdcs postings classified under the five codes . this shows that this website functions as a forum for discussing how to build and use tdcs and sharing new scientific discoveries on the effects of tdcs featured in media and various online forums . postings at subreddit tdcs . a total of 13 ( 11% ) respondents use tdcs for treatment of medical conditions and 71 ( 59% ) respondents use it for cognitive enhancement . a total of 29 ( 24% ) respondents reported that they are using tdcs for both cognitive enhancement and treatment . the most common types of cognitive enhancement purpose for which the respondents use tdcs were attention ( 72 ) , learning ( motor , verbal , or numerical learning , etc . ) ( 68 ) , and working memory ( 67 ) ( fig . 4 ) . types of cognitive enhancement purpose for which respondents use tdcs . in addition , the most common medical condition for which the respondents use tdcs was depression ( fig . interviewees of this study also reported that tdcs is very effective for the treatment of depression , especially for treatment - resistant depression . according to the physician who has been treating patients with tdcs , most of his patients came to the clinic because they do not respond to psychoactive medications . based on his extensive clinical experiences , he claimed that tdcs is significantly beneficial for the treatment - refractory patient suffering from chronic depressionit takes only about five treatments before the effects of tdcs become noticeable. one of the power users who has bipolar type ii disorder also reported that the effect of tdcs is immediate and he can even tell when the machine is shutting off. types of medical condition for which respondents use tdcs . furthermore , this survey also asked , in cases where the respondents are using tdcs for treatment , whether these respondents are also taking other conventional medications . this question is intended to assess the risk of diy tdcs , assuming that there could be unknown adverse compounding effects of tdcs and other extant medications . the pharmacological status of the brain can have meaningful effect on the outcome of tdcs , and the variety of psychoactive agents that home users may employ is legion. in this survey , 18 respondents44% of respondents who are using tdcs for treatment of medical conditions reported that they are currently taking other medications for the same medical conditions . thus , a significant percentage of respondents who are using tdcs for treatment are exposed to the potential detrimental interactions with other medications . the survey result showed that among the 121 respondents , 47 ( 39% ) respondents built their own device and 58 ( 48% ) respondents bought out of the box tdcs devices . some of the respondents ( 11 ; 9% ) reported that they both built and bought their devices . in terms of home - built tdcs devices , the analysis of subreddit tdcs postings revealed that some open source tdcs circuit designs proposed by diy users had been available at this forum and improved through active discussions among users . openstim tdcs , by the moderator of subreddit tdcs , and another designed by a user named shawn nock , are the two most well - known open source tdcs circuits . the moderator of subreddit tdcs initially had wanted to buy one of the devices used at clinics or research labs but the prices of these devices were over his budget . after he found out that building a tdcs device does not require complicated knowledge of electrical engineering , he decided to design his own circuit , which is affordable ( costs less than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}50 ) and , he believes , safe , and to share it with other diy users . later , it has been evolved with the inputs from other users in the community in terms of what kind of features would be useful. in addition to these open source circuits , some users also compiled and posted basic schematics , tools and electrical components needed to build the tdcs device , and online or offline shops that one could get those tools and components , as well as their prices . thus , at subreddit tdcs , instructions on how to build an easy - to - use tdcs device at a very low cost are easily accessible to anyone who is interested in trying self - experimentation with tdcs . regarding the out of the box tdcs devices , currently about 10 devices are on the market . the survey result showed that the most prevalent device in this community is the foc.us headset . 6 ) types of out of the box tdcs device that respondents are using . however , both power users and the physician had unfavorable opinions on the foc.us headset , mostly because its placement of electrodes , which is called montage , is fixed . the physician argued that the montage of foc.us device is different from montages used in any of the clinical studies , and he did not think it has the same effects as the ordinary two electrodes system using iontophoresis devices that researchers have used in finding evidence of effectiveness . the webmaster of diytdcs also stated that unless some sort of tangible evidence of obvious benefit emerges , foc.us will turn out to be a fad and will most likely fade away. the fact that these commercial devices , such as foc.us , are gaining popularity among the diy users has caused a demographic shift in this diy tdcs community . the moderator of subreddit tdcs recalled that two years ago when the website was first established people were talking about designing electronics , designing techniques , and designing experiments. however , there is now a population of people who are basically end users and are not programmers , hackers or electronics people. this is also reflected in the recent sharp increase in the number of postings on these commercial devices at subreddit tdcs ( fig . the moderator argued that this has good and bad sides. it is good in the sense that many of these out of the box products are quite sophisticated. for example , the foc.us device can deliver custom waveforms , which is difficult with diy devices . if commercial devices become more popular , they can spur people 's interest in developing tdcs methods that can take advantage of the more advanced functionality . on the other hand , the moderator said we can not assume that these end users are knowledgeable enough to make informed decisions about their safety in regard to issues like whether a specific type of circuit is safe to use in these devices , and thus , three factors determine the safety of a tdcs protocol : dose of current , size of electrodes , and duration of stimulation . the first two factors determine current density ( current dose divided by electrode size ) , and the total current dosage is then measured by multiplying current density by the duration of stimulation . researchers have suggested that a stimulation protocol using 2535 cm electrodes with currents of 12 milliamperes ( ma ) for up to 2040 minutes is considered safe . the survey results showed that most of the respondents are following this safety guideline , though there are some exceptions . ( figs 810 ) for example , 43 respondents are using electrodes smaller than 10 cm . however , it seems that most of them are foc.us users : the size of electrodes of foc.us headset is about 46 cm , and there are 37 foc.us users among the respondents . when asked how they try to find out about these three factors that determine the safety of a stimulation protocol , most respondents answered that they find relevant information from academic research studies on tdcs or website postings , such as postings in subreddit tdcs ( appendix i ) . how often one can safely use tdcs per day or per week for an extended period of time is a question that does not have a clear answer yet . most of the tdcs studies applied tdcs to the subjects less than five to six times per week for a relatively short term ( ie few weeks ) . thus , these studies do not provide proper guidelines for people who are using tdcs at home more often for a longer period of time . a few postings on the frequency of use at subreddit tdcs led to users replying based on their own experiences and acknowledging the absence of proven guidelines by researchers . a total of 52 out of 56 respondents who are using tdcs regularly reported that they have stimulation sessions fewer than seven times per week ( on average less than once a day ) . this part presents , the data on the respondents self - reports about the effects and side effects of tdcs . in this exploratory study , it is not feasible to measure and test the actual effects of tdcs , such as to what extent tdcs has affected users cognitive abilities or medical conditions . the self - reports on the effects of tdcs posted at subreddit tdcs show that diy users themselves are having problems in assessing the effects of tdcs while using it at home . although the physician who has been treating his patients with tdcs described the effects of tdcs on chronic pain and depression as too good to be true , a power user who used tdcs mainly for cognitive enhancement reported that the effects of tdcs are extremely subtle. for example , there can be placebo effects and in cases where users take cognitive tests after or while simulating their brain ( ie lumosity , dual n - back game , or cambridge brain challenge ) , learning by repetition can also affect the test results . in addition , types of side effects which diy users are experiencing and the degree of these side effects will be analysed . a total of 54 ( 44% ) out of 121 responding users rated the effects of tdcs at four or five on the scale of one ( totally unsuccessful ) to five ( extremely successful ) ( appendix j ) . the quotes below are two examples of self - reports on the effects of tdcs posted at subreddit tdcs . the first quote is describing the effects of tdcs on cognitive abilities : a series of memory tests were administered by an electrical engineer who supervised the experiment , initially without tdcs . these tests demonstrated standard working memory results with a measured span of 58 components retained after a series of 15 terms were listed aloud to remember . with tdcs the data showed very promising results . apart from the subjective experience of heightened focus and a calmness that can be likened to being in the zone , the results of the memory test indicated a measurable increase in working memory span . for each subsequent test , all 15 words were remembered in correct order and recalled in correct order after tdcs was administered . [ t]he subjective experience combined with data reflecting very promising cognitive enhancements leads me to conclude tdcs overall effectiveness in increasing my learning potential during and post stimulation . the second quote describes the perceived effects of tdcs on a medical condition depression : from a subjective perspective , this is awesome . i actually went out jogging . mostly walking , actually , because there is n't enough electricity in the world to make me not be a fat ass who 's running for the first time since 2010 , but still way out of my usual . i initially questioned if this was a hypomanic episode or just what the release of 15 years of depression feels like , but i judge it to likely be hypomania . does n't matter , i feel like frankenstein 's monster . i was dead , if only in my mind , and then with a little bit of lightning , boom . subjectively perceived effects of tdcs , survey respondents were asked whether they are planning to use tdcs in the future . a total of 111 out of 121 respondents ( 92% ) replied that they would continue using tdcs in the future . moreover , 56 ( 46% ) of the respondents rated their willingness to recommend tdcs to family members or friends , who have the same aim(s ) or conditions(s ) as they do , at four or five on the scale of one ( will not recommend ) to five ( strongly recommend ) ( appendix k ) . a total of 56 out of 121 responding users ( 46% ) reported that they have experienced side effects while using tdcs . these respondents have experienced headache , discomforting changes such as pain , tingling , itching or burning under the electrodes , fatigue , nervousness , visual perceptual changes , acute mood changes , difficulties in concentrating , nausea , and sleeping disturbance ( appendix l ) . however , the degree of side effects was not severe the average severity of most types of side effects were rated at about one , on the scale of one ( not severe at all ) to five ( extremely severe ) , except discomforting changes , such as pain , tingling , itching or burning under the electrodes , where the average severity was about 2.3 ( appendix m ) . power users and the physician reported that the side effects of tdcs are almost negligible , and this result corresponds to previous empirical results showing that there are no severe short - term adverse or side effects . what concerns did diy users have , if any , about the use of tdcs are and what they think about the possibility of government regulation on tdcs or guidelines from government agencies , researchers , or physicians ? a total of 97 out of 121 survey respondents ( 80% ) reported that they have concerns about the diy use of tdcs . most of these respondents are worried about long - term potential impairment on the brain and the use of inaccurate position of electrodes ( fig . the most interesting finding in the analysis of web postings at subreddit tdcs was the users provided safety warnings . in subreddit tdcs , 100 safety warnings were found in the comments to the postings , and 77 out of these 100 warnings were the comments to the postings on building and operation of tdcs ( table 1 ) . if you have no idea what a 4 milliamp is , i suggest you stay away from this . a 9v battery can kill you!remember safety first before you try this.don't up the current , that is the wrong way around.seriously , if you are n't comfortable with your knowledge of currents and what effects different amperages can have on your body , do not attempt this. if you have no idea what a 4 milliamp is , i suggest you stay away from this remember safety first before you try this. do n't up the current , that is the wrong way around. seriously , if you are n't comfortable with your knowledge of currents and what effects different amperages can have on your body , do not attempt this. sometimes diy users also ask more specific questions on their own diy circuit at subreddit tdcs . they post pictures or detailed descriptions of their home - built circuit and ask whether it is safe to do self - experiment with the circuit . then , other users actively correct them if there are issues on the circuit that can cause danger . the general safety warnings and questions and comments about the safety of a specific diy circuit design show the existence of a self - regulating system within this online forum . the moderator of subedit tdcs stated that there is a lot of emphasis on individual responsibility a bio hacker 's mentality , but there is also a very much of a culture that values giving people information about dangerous things. the webmaster of diytdcs also acknowledged that there is definitely a self - regulating system in subreddit tdcs : those who do know about electronics easily recognize people who should n't be experimenting with self - built devices. other power users also agreed that there were a certain number of users in the community who have corrected and guided other users not to make dangerous mistakes : we are self - regulating community . everybody in reddit has to express some type of knowledge as he / she writes a posting and nobody in the community will let up on you understanding the dangers of messing around with electronic current . the reason that it happens is because i believe at least in the beginning most of the tdcs community on reddit were people who had a background in engineering such as building circuits . over the course of last three years , i have yet seen anybody post anything that tdcs has done some real damage . the majority of survey respondents ( 97 out of 121 respondents ) thought that it would be helpful to have an official guideline from government agency or experts ( researchers or physicians ) for the diy use of tdcs . all of the power user interviewees claimed that the part that most needs guidelines from the government or experts is placement of electrodes . they reported that getting reliable repeated placement of anodes and cathodes , and what montages should be used to treat or improve which things are the most confusing parts of practicing diy tdcs . moreover , the power users expressed concern that even though diy users locate the electrodes in the right place according to a specific montage , whether a person is left - handed or right - handed might affect the outcome because left - handed people 's brains may be organized differently from those of right - handed people . it is also possible that subtle differences in the neuroanatomy of individuals may alter the effects of the device. the physician also warned that the area under the anode that is being stimulated could be changed depending on where you locate the cathodes . some of the survey respondents also provided more detailed comments on the potential official guidelines or government regulation on tdcs . official guidelines would only be helpful if they were more than do n't do it . i think in many cases our society focuses too much on safety rather than encouraging curiosity.i believe that government regulation would be a detriment and stifle any new developments.tdcs has great potential . i 'd like guidelines to come out that are backed by proper and thorough research into the effects.while i think more information and studies being published from academic and medical sources would be great , i hope that the industry does not undergo any governmental regulation , and remains a practice individuals can perform on themselves and buy the related devices without legal issues.the use of these technologies will evolve and outpace the medically established research merely based on the network effect that the internet creates around anything it touches . we need to create a sort of social network for experimenters to share and teach each other . if they are smart , they will study and not interfere except to add useful insight from time to time from their own research.about official guidelines i'd welcome any helpful information , but such guidelines , in my experience tend to be over - cautious . i ca n't imagine any official agency ever advising the public to do what i do . my own experience has been very satisfactory , but i know that there are a lot of unknowns and that i 'm accepting an unknown measure of risk when i run an electrical current through my cranium . nobody should do that until they 've researched the matter thoroughly and assessed the risks for themselves . what i would like is an advisory agency for psychologists and psychiatrists to help them deliver tdcs treatment where it 's appropriate . i think it can be very beneficial , and not many professionals seem to be aware of it.i have great concerns with some of the equipment people are using to attempt this on their own brains . i 'm an electrical engineer so when i look at some of the devices people have designed it 's terrifying what these people are hooking up to themselves . it would be very , very useful for an official guideline to be available since some of these people are using simple voltage dividers with no understanding how current behaves when they apply these things to their heads. official guidelines would only be helpful if they were more than do n't do it . i think in many cases our society focuses too much on safety rather than encouraging curiosity. i believe that government regulation would be a detriment and stifle any new developments. tdcs has great potential . i 'd like guidelines to come out that are backed by proper and thorough research into the effects. while i think more information and studies being published from academic and medical sources would be great , i hope that the industry does not undergo any governmental regulation , and remains a practice individuals can perform on themselves and buy the related devices without legal issues. the use of these technologies will evolve and outpace the medically established research merely based on the network effect that the internet creates around anything it touches . we need to create a sort of social network for experimenters to share and teach each other . if they are smart , they will study and not interfere except to add useful insight from time to time from their own research. about official guidelines i'd welcome any helpful information , but such guidelines , in my experience tend to be over - cautious . i ca n't imagine any official agency ever advising the public to do what i do . my own experience has been very satisfactory , but i know that there are a lot of unknowns and that i 'm accepting an unknown measure of risk when i run an electrical current through my cranium . nobody should do that until they 've researched the matter thoroughly and assessed the risks for themselves . what i would like is an advisory agency for psychologists and psychiatrists to help them deliver tdcs treatment where it 's appropriate . i think it can be very beneficial , and not many professionals seem to be aware of it. i have great concerns with some of the equipment people are using to attempt this on their own brains . i 'm an electrical engineer so when i look at some of the devices people have designed it 's terrifying what these people are hooking up to themselves . it would be very , very useful for an official guideline to be available since some of these people are using simple voltage dividers with no understanding how current behaves when they apply these things to their heads. these comments suggest that although diy users acknowledge the need for guidelines for the diy tdcs ; many are opposed to and worried about blanket government regulations on tdcs devices , which would prevent them from continuing self - experiments . these blanket regulations could backfire and draw further attention to diy tdcs , and lead to the opening of uncontrollable underground markets for tdcs . according to the interviewees , one of the most serious concerns about government regulations seems to be the potential increase in the cost of practicing tdcs . the moderator of subreddit tdcs said that the community has a strong preference for devices that are extremely low cost . it is very difficult to convince people to have a current meter on the home - built device because it costs \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}5 more. criticizing the current us health care system that discourages physicians from reducing the costs of medical treatments , the physician also argued that other available options , comparable to tdcs , for treatment refractory patients are too expensive . for example , it costs \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}20,000\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}30,000 per year for the treatment of chronic depression using rtms , which is a classified medical device by fda , and insurance often does not cover the treatment . in contrast , his clinic offers a tdcs treatment program at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { \$ } $ \end{document}2500 , which includes four years of at - home supervision , as well as the initial evaluation , training , and a device although it still could be considered expensive for some patients . in other words , the costs could be the main reason why people with specific treatment refractory medical conditions , such as chronic pain or depression , opt to build or purchase a tdcs device and self - treat them . these people might view a strict regulatory oversight as a threat to deprive them of the only affordable last resort to keep their medical condition under control . as a last note , below quote from the interview with the moderator of subreddit tdcs suggests an interesting angle for a future regulatory regime regarding tdcs : if the goal of regulating tdcs device is to make sure that when people are getting tdcs device , it is safe and effective , i think labeling and regulating it as a medical device is not practical and that 's because they are so easy to make . the medical device testing is onerous and expensive and not something that many of the diy users are going to like to go through so , there are always going to be a supply of unregulated devices and you can not stop people from making this device . it would be better to adopt a wide - spread and easily understandable standard system for tdcs that is based on research on safety of tdcs and have a group to test tdcs devices to make sure that the devices meet the standard you would think twice about buying a car that has one star crash rating , and i would like to see something like that applies to tdcs devices as well . the findings of this study presented in the last section may provide preliminary empirical indicators to determine whether we need regulations and guidelines for diy tdcs and , if we do , how to design them to ensure a safe and responsible use of tdcs . this section will summarize some of the main findings and explore the tentative regulatory implications of these findings . first , unlike the implicit assumption made in media coverage on tdcs , the diy community does not comprise just risk takers with eccentric interests , such as young bio hackers or self - experimenters . the survey results showed some distinctive characteristics of the respondents clear male dominance and slight skew to ( a ) higher than average education level , ( b ) students and professionals , and ( c ) very low and high income . also , it should be noted that there was a wide distribution of ages among the respondents , which shows the potential of tdcs as an appealing technology satisfying various aims across generations . although the survey respondents did not necessarily form a representative sample , this demographic profile of diy tdcs users provides preliminary information on who will be the target subjects of potential regulations or official guidelines on the use of tdcs . second , according to the findings of the study , diy users interest in tdcs seems transient only half of the survey respondents are continuous and regular users , and the majority of these respondents ( 61% ) began to use tdcs less than six months ago . one of the interviewees also pointed out that there seem to be only a small number of committed diy users in the community . from this data , it can be inferred that there may be some hype around the estimation on the current size of the diy population . however , it is also true that findings on the perceived effects of tdcs appear to support more affirmative predictions on the future of the diy use of tdcs . a total of 54 ( 44% ) survey respondents reported that their use of tdcs was quite successful and satisfactory , and 111 respondents ( 92% ) replied that they would continue using tdcs in the future . also , 56 respondents ( 46% ) are willing to recommend tdcs to family members or friends , who have the same aim(s ) or condition(s ) as they do . interviewees expressed conflicting views on the future of the diy use of tdcs . some argued that unless more robust evidence on the effects of tdcs emerges in contrast , others who strongly believed in the potential of tdcs as treatment and cognitive enhancement predicted substantial increase in the diy tdcs user population . these somewhat contradictory findings suggest that there are a lot of ambiguities and mistaken assumptions about the use of tdcs . in general , it can be said that at this stage , diy use of tdcs is not currently widespread , that it does not seem to pose an imminent risk or danger to the public , and that there seems to be only a remote possibility of a dramatic increase of diy use of tdcs in the near future . nonetheless , more systematic and detailed investigations on the current state and prospect of diy tdcs are required so that regulatory agencies can carefully examine the need for and the appropriate timing of potential regulatory interventions . as discussed above , there is no regulation on therapeutic use of tdcs and devices intended for cognitive enhancement are not covered by any current regulations in the usa considering possible regulatory options , maslen et al . have expressed concerns about establishing a new separate regulatory regime for cognitive enhancement devices different from the current regime for medical devices . one of the reasons for their concern is that it is hard to categorically distinguish cognitive enhancement devices from medical devices . most of the current enhancing technologies , including tdcs , were originally developed for therapeutic purposes , such as treatments for illness or age - related mental deterioration , but the uses of these technologies have been subsequently expanded for enhancements . in other words , [ t]he very same device may be used both for therapeutic and enhancement purposes , in some cases using similar parameters. the types and levels of risks that are involved with therapeutic and enhancement use of a device also may not be meaningfully different . in addition , some scholars have argued that there is no philosophical difference between treatment and enhancement[b]oth therapy and enhancement aim to improve a human being 's biology and/or psychology. this study provides preliminary empirical support to the claim that the practical distinction between medical devices and enhancements can be muddy . the survey results showed the mixed and overlapping use of tdcs13 ( 11% ) respondents use tdcs for treatment of medical conditions , 71 ( 59% ) respondents for cognitive enhancement , and 29 ( 24% ) respondents for both cognitive enhancement and treatment . this suggests that if a regulatory agency decides to regulate tdcs either as a medical device or as a cognitive enhancement only , it will turn out to be an essentially underinclusive policy . according to the survey results , diy users claim to conform very well to safety guidelines for the current density ( current dose divided by electrode size ) , and the total current dosage ( current density multiplied by the duration of stimulation ) set by previous clinical studies . the analysis on the web postings in subreddit tdcs demonstrated that diy users are not only concerned about the safety of their own stimulation protocols but also cared about other users practice of tdcs . the general safety warnings by the users and the feedback process to verify the safety of individual diy circuit show the existence of an active self - regulating system in this online forum . according to previous literatures on self - regulation , self - regulation and state regulation can be intertwined and complement each other. the self - regulating system in the diy tdcs community suggests that it may be possible to build a partnership between the diy community and regulatory agencies to oversee the use of tdcs . in addition , the fact that many users consult website postings to get information on the three main elements ( dose of current , size of electrodes , and duration of stimulation ) of stimulation protocol reflects the users reliance on web postings to self - experiment with tdcs . thus , incorporating this bottom - up self - regulating system into a top - down state regulatory framework can increase the efficiency of the framework by self - motivating diy users to conform to the regulation and allowing the framework to more flexibly respond to unforeseen circumstances . fitz and reiner claimed that regulatory approach taken to diy by synthetic biologists ( diy bio ) could be a good example of a potential regulatory option for diy tdcs . synthetic biology is the design and construction of new biological parts , devices , and systems , and the re - design of existing , natural biological systems for useful purposes. in this case , the national science advisory board for biosecurity chose open communication and education through public events and conferences instead of traditional regulatory practice to develop a culture of responsibility by diy bio practitioners . fitz and reiner acknowledged that compared to diy bio , the costs of practicing diy tdcs are relatively low or sometimes even negligible , and thus , the risks associated with diy tdcs are greater than those of diy bio . however , they argued that the new regulatory approach for diy bio can be a practical option at least for the interim period until a regulatory agency comes up with a comprehensive regime for tdcs . the existence of the self - regulating system demonstrated in this study may provide support to fitz and reiner 's argument for the new regulatory approach . with voluntary participation and oversight by diy tdcs users , open communication and education can be a more viable and effective scheme for developing the culture of safe and responsible use . however , it is noteworthy that recently , there has been a demographic shift within the diy tdcs user community . the interviews with the power users of subreddit tdcs revealed that when this online forum was first established two years ago , most people in this forum had a background in engineering or did their homework on the effects and risks of tdcs by researching previous clinical studies on tdcs before their self - experiments . the self - regulating body was comprised of these people , and they were extremely helpful in giving real advices. however , the recent release of out of the box tdcs devices , such as foc.us , has increased interest in tdcs among the general public , and now there is a growing population of end users who do not know how tdcs circuits work or do not care to thoroughly investigate the effects and risks of tdcs . the increase in the size of the diy tdcs user community particularly the influx of nave end users may undermine the vitality of the self - regulating system in subreddit tdcs . if the regulatory agency wants to incorporate the self - regulating system into any future regulatory regime for tdcs , it should take into account the potential impacts of this demographic shift in the community on the self - regulating system . a total of 97 out of 121 survey respondents ( 80% ) reported that they had concerns about the diy use of tdcs . these respondents are mostly worried about long - term impairment on the brain and the use of inaccurate placement of electrodes . also , the same number of respondents answered that it would be helpful to have official guidelines from government agency or experts ( researchers or physicians ) for the diy use of tdcs . in other words , it can be said that diy users are waiting for the open communication and education by the authorities proposed in fitz and reiner 's article to ensure the safety of their self - experiments with tdcs . in addition , consistent with the survey results , all of the interviewed power user concurred that the issue where guidelines from the government or experts are most needed is placement of electrodes . as discussed above , locating the electrodes on the scalp to target a specific underlying brain region can be not only confusing but also very risky for lay users . therefore , in designing a guideline or education program for diy use of tdcs , the regulatory agency or expert groups may want to focus more on how to guide the placement of electrodes rather than other issues such as size of current or size of electrodes , for which there are already relatively clear safety standards . in contrast to their desire for official guidelines , the survey respondents took a very negative stance toward potential government regulation . some of the survey respondents answers showed that diy users would practice tdcs regardless of the government regulations , to wit : levying onerous requirements on diy users may drive the work further underground to the benefit of none. the significant financial advantages of diy tdcs compared to other treatments for chronic medical conditions seem to make this scenario more plausible . this raises a question whether it is legally or practically possible to enforce regulations on tdcs devices . however , for the fda to intervene and take enforcement actions , there need to be prohibited acts , such as the introduction of adulterated or misbranded drugs or devices into interstate commerce . building and using a tdcs device only for oneself does not seem to constitute any of the prohibited acts listed in the act . moreover , even if the fda may legally be able to outlaw the use of home - built tdcs devices through the broad interpretation or amendment of the act , how could it enforce such a ban , considering the ease and low cost of making such devices and the difficulty of detecting them , especially considering the limited resources that the fda has . in addition , regulations of marketing and sales of out of the box tdcs devices that would make device manufacturers subject to rigorous supervision , would likely lead to cost increases for the manufacturers that could increase the appeal of home - made tdcs devices . for example , arguing that enhancement devices in european union should be regulated through extending the existing legislations for medical devices , maslen et al . transparent , detailed , evidence - based information pertaining to the mechanisms , risks and effects that might be construed as benefits of the devices. providing such detailed information is currently not compulsory. this demanding requirement will inflict additional costs on the manufacturers and eventually , the consumers . however , given the ease of building tdcs devices and the diy community 's strong preference for low - cost devices , increasing the costs of devices may lead to uncontrollable self - manufacturer of the devices , causing users to hide out of reach of the fda . thus , further investigation is needed to analyse whether , in practice , the fda intervention and supervision can effectively ensure the safety and responsible use of tdcs devices . direct brain enhancement is not an imaginary story that exists only in the remote future . it was originally developed for treatment for medical conditions , such as depression , but clinical studies have also reported that it can improve various cognitive abilities among healthy people . this study is the first empirical attempt to investigate the diy tdcs user community . a questionnaire survey of diy users , interviews with some active power users , and a content analysis of web postings on tdcs showed distinctive demographic characteristics of the diy users , ambiguities and mistaken assumptions around the current state and future prospects of the diy use of tdcs , mixed use of tdcs for both treatment and cognitive enhancement , the existence of an active self - regulating system in the community , and users demands for official guidelines and their concerns about government regulations on tdcs . even with the limitation of a small non - random sample , the findings of this study provide a preliminary but useful empirical illustration of the diy tdcs user community , and increase our current understanding of the practice of diy tdcs however , more detailed subsequent studies are required to determine the need for and the timing of government interventions and to assess the strength and weakness of regulatory options proposed by researchers . furthermore , although this study just focused on one type of human biological cognitive enhancement there are numerous other enhancements , such as drugs , biologics , and dietary supplements , which could be used to improve the function of human bodies or brains . as discussed above , there have been growing discussions on whether and how to regulate these enhancements , including off - label uses of fda - approved drugs and devices for enhancement purposes . however , empirical understanding on the use of these enhancements is incomplete , as in the case of tdcs . owing to advances in biomedical science and increasing interest in human biological enhancements thus , for more meaningful and informed discussions of potential regulation , future studies on the actual practices of various human biological enhancements are required to clarify critical issues and factual assumptions regarding the use of these enhancements .

among currently available technologies , transcranial direct current stimulation ( tdcs ) is one of the most promising neuroenhancements because it is relatively effective , safe , and affordable . recently , lay people have begun to build or purchase the tdcs device to use it at home for treatment or as a cognitive enhancer . the tdcs device is currently not covered by the existing regulatory framework , but there are still significant potential risks of misusing this device , and its long - term effects on the brain have not been fully explored . thus , researchers have argued the need for regulations or official guidelines for the personal use of tdcs . however , until now , no systematic research on the do - it - yourself ( diy ) tdcs user community has been done . the present study explores the basic demographic characteristics of diy tdcs users as well as why and how they are using this device through a questionnaire survey , in - depth interviews , and a content analysis of web postings on the use of tdcs . this preliminary but valuable picture of the diy tdcs user community will shed light on future studies and policy analysis to craft sound regulations and official guidelines for the use of tdcs .

INTRODUCTION RESEARCH METHODOLOGIES FINDINGS TENTATIVE REGULATORY IMPLICATIONS OF THE FINDINGS CONCLUSION: SUMMARY AND FUTURE DIRECTIONS

PMC2257992

intellectual property ( ip ) is defined as property that can be protected under federal law , including copyrightable works , ideas , discoveries , and inventions.1 most research and publications on ip focus on technological development within companies and industries2,3 and on academic and government laboratories.4,5 this focus is both the result of and reinforcement for the misconception that modern technological innovation is the purview of industry and academia . in reality , many of the most influential and innovative medical and information technology ( it ) inventions have been created by independent inventors , including : wilson greatbatch , who patented the implantable cardiac pacemaker ; raymond damadian first described the concept of nuclear magnetic resonance and patented the first commercial unit ; raymond kurzweil patented and marketed the first omnifont optical character recognition system and numerous speech recognition innovations ; and linus torvalds wrote the program that became the linux kernel . despite the many remarkable achievements of independent innovators , numerous technical , economic , political , legal , and educational challenges confront independent invention . this article is written to serve as an educational resource and guide to the fledgling independent inventor in imaging informatics , with the hope of encouraging entrepreneurship , innovation , and creativity within the existing constraints of a field dominated by industry - initiated research and development ( r&d ) . the u.s . patent and trademark office ( uspto ) defines an independent inventor as one whose patent ( at the time of issuance ) is unassigned or assigned to an individual . the percentage of patents issued to independent inventors declined from 36.4% in 1987 to 26.8% in 1999.6 international patent data ( 1999 ) show great variability in the overall percentages of patents issued to independent inventors , with less industrialized countries having a higher percentage of independent invention ( 75% in hungary and 66% in brazil , for example ) , compared with more industrialized countries ( 26% in both the united kingdom and united states6 ) . although the relative percentage of patents issued to independent inventors has gradually decreased over time , the total number of patents awarded to independent inventors increased by approximately 30% from 1990 to 2000.7 these figures suggest that independent invention is alive and well in the united states and retains a vital role in innovation throughout industry and the marketplace . a recent survey published by weick and eakin8 explored the role and economic viability of independent inventors in the united states . eighty - four percent of inventor respondents reported developing working prototypes of their inventions within the past 5 years . the top invention categories were dominated by hardware / tools , household , and industrial / commercial products , and the most frequently cited scientific / technology inventions were categorized as electronics ( 13% ) and medical / therapeutic ( 12% ) . thirty - nine percent of respondent inventors reported generating sales from their inventions ( mean sales , $ 3.5 million ; median sales , $ 50,000 ) , and 22% reported profitability ( mean profit , $ 1.96 million ; median profit , $ 75,000 ) . the three principal means of ip commercialization included development of start - up companies ( 55% of respondents ) , licensing ip to another company ( 44% of respondents ) , and outright sales of ip ( 16% of respondents ) . inventors who elected to license their inventions to others were more likely to achieve a higher level of overall sales than those attempting to commercialize the ip themselves or who sold the ip to a third party . this corroborates previously reported findings by khan and sokoloff,9 who reported independent inventors commercial success to be greatest through licensing agreements . the prevalent paradigm of industry - sponsored r&d suffers from several limitations , some of which are outlined in table 1 . the various strengths that provide industry with a distinct ( and arguably unfair ) advantage over the independent inventor paradoxically provide a disincentive toward innovation . this can be explained in part by the life cycle of innovation embraced by many companies , particularly larger and more financially successful concerns . table 1existing limitations of industry - sponsored r&dlimitationa follow - the - pack ( lemming ) mentalitysmall , incremental approaches to new ipinternal corporate politics and constraintsshort - term outlook targeted at immediate gratificationproduct development done in usersreactive , not proactive , approach to market needs existing limitations of industry - sponsored r&d in the first stage ( infancy ) of this life cycle , innovation plays the dominant role in creating new ip , a process that provides the impetus and backbone for the original creation of many companies . the fledgling company is founded by a few energetic , industrious , and optimistic inventors who , through a combination of idealism , capitalism , and naivety , decide to embark on the development of a start - up company despite long odds against success . in the second stage ( childhood ) , the company grows through successful commercialization of their innovation and ip . as the company grows and achieves economic success , the culture begins to shift from optimism and innovation to pragmatism and risk aversity . the political and economic realities of the marketplace begin to surface , and the founders of the company often turn over operational control to financial and management professionals . if the company continues on a path of relative independence ( avoiding merger or acquisition ) and continues to achieve greater success in the marketplace , it may then enter stage 3 ( adulthood ) , in which it is often transformed from in entering this stage , the company often grows by acquiring smaller ( and more innovative ) companies , thereby expanding its product line and ip pipeline . the large company now responds to market economics , with product development largely dictated by the perceived needs of the customer base . invention is typically performed within the company ( in house ) by individuals who are often constrained by company politics and the economics of immediate gratification . , innovation is largely stifled and relegated to product line developments and incremental improvement . it is ironic that the very company that was initially created by innovation and creativity becomes transformed into one that strives to stifle ( and often crush ) the creativity of the independent ( and innovative ) inventor . in this life cycle , inventors themselves are no longer frontline users but , instead , are technicians and engineers . in the medical domain , these engineers are , for the most part , technology savvy , but clinically uninformed . as a result , refinements and new developments are based , at best , on minimal knowledge of how the product will truly function in the clinical environment . experts on a medical advisory board who have little practical experience outside of academia . as a result , even with a gloss of medical input , many products are developed within a rigid corporate culture by nonclinicians who are highly risk averse and focused on short - term horizons . the independent inventor , on the other hand , is not bound by the same corporate constraints and , instead , can bring a fresh and practical perspective to product innovation . this strategy of outsourcing innovation is uncommon within industry today but offers the potential to create new independent inventor industry synergies . the creation of an open market approach to innovation provides a cost - effective and practical means for companies to remove their self - induced ip constraints and seek out inventors from external sources to enhance innovation within new and existing product lines.10,11 table 2 outlines a ten - step iterative process for invention and product development . one example comes from my own work in private practice radiology , where one of my biggest concerns is the paucity of objective quality assurance ( qa ) metrics and standards within medical imaging and the lack of supporting technology . with the sole exception of mammography,12 no medical imaging modality has rigorous and mandatory qa standards , despite a broad agreement on the significance of the role of medical imaging qa in clinical and economic outcomes.13 simply stated , qa in its present form is largely idiosyncratic and often nonexistent . table 2stepwise approach to invention processapproachdefine an existing problem or questionreview the current technical solutionidentify existing limitations and inefficienciescollect data using existing technologydevise an alternative solution ( invention)develop a prototypecollect data using the prototyperefine the prototype based on data and end - user feedbackrepeat data collection ( and refinement as necessary)commercialize invention stepwise approach to invention process in attempting to objectively quantify existing qa practice , we performed several studies evaluating qa - related workflow , technology , and variability and the related effects on image quality.1417 the results have indicated that the current practice of medical imaging qa is fraught with error and inconsistencies , resulting in inefficient workflow , reduced technologist productivity , poor image quality , and suboptimal radiation dose to patients . the data call for a complete revamping in the clinical and technical ways in which qa activities are implemented . defining the solution becomes fairly straightforward when confronted with the paucity of data , lack of standards , wide inter- and intradepartmental variability in performance , and lack of supporting technology for qa assessment . the ideal solution to improve current inefficiencies would be an automated system to record , measure , track , analyze , and provide qa feedback to practitioners . after identifying and defining this deficiency , i proposed and described one such solution and submitted it for review by the uspto . although the idea may appear sound , it is only as good as the working prototype . as an independent inventor , i did not have the technical or economic resources required to create a working prototype and , therefore , elected to seek industry partnership through a licensing agreement . the project is far from complete , but the sequence of events outline in table 2 are those that i have come to recognize as requisite for creation and implementation of an invention , with the ultimate goal of creating independent inventor industry collaboration and a final product based on a data - driven engineering process.18 table 3 is a list of practical suggestions for the independent inventor . first and foremost is the reality that inventorship is equivalent to running a marathon , not a sprint . even under the best of circumstances , the patent review process takes 23 years and often longer , depending on the uspto backlog , the initial review of the patent examiner , and what once the review process has been completed , the inventor must begin the arduous task of marketing the invention and/or developing a prototype . because of the intensive resource requirements for prototype development , many inventors elect to pursue a licensing agreement with an established company that has the technical , financial , personnel , and legal resources to commercialize the patent and defend against potential future litigation . securing a patent the best deterrent is an ally with deep pockets and the combined economic and legal clout to punish any violators . table 3practical advice for inventorsadviceexpect success to take 510 yearsdo not quit your day jobengage professional legal services for prior art search patent preparation nondisclosure agreements commercializationdocument everything!do not market until you receive notice of allowancebe a little paranoid ; expect dishonestyfile a provisional patent asapconsider licensing as a means to commercializeleverage existing practical knowledge and experiencethink out of the box!leverage existing resources practical advice for inventors the patent application process in the united states typically costs in the range of $ 20,000$30,000 . the result is that many independent inventors are forced either to abandon their ip or seek external funding sources . although third parties exist in the form of independent investor and venture capitalist consortia , the inventor sometimes forfeits substantial economic and legal rights in entering such agreements . having sound legal advice with expertise in patents and licensing is critical in ensuring that the best interests of the inventor ( and the long - term security of his or her ip ) are protected new legislation is currently pending in congress ( hr 2795 ) that calls for major patent reforms . enactment of this legislation could have negative effects on the independent inventor by : changing the definition of inventor from first to invent to first to file;limiting a patent holder s rights to obtain a permanent injunction against an offending third party;creating a post - patent grant opposition proceeding ; andlimiting damages for infringement lawsuits changing the definition of inventor from first to invent to first to file ; limiting a patent holder s rights to obtain a permanent injunction against an offending third party ; creating a post - patent grant opposition proceeding ; and limiting damages for infringement lawsuits in the end , larger corporations and academic institutions have far more extensive resources and expertise to create , manage , and defend patents . these disproportionate resources should not preclude the independent inventor from seeking patents but should provide significant incentives to pursue symbiotic partnerships with industry that can ultimately benefit both parties . although do it yourself programs are widely advertised as ways for independent inventors to draft their own patent applications , investors should be wary of the long - term ramifications of even minor errors in the patent process . claim wording on patents is complex and most properly the province of specialized attorneys with extensive training and experience . the prior art search is another integral component of patent preparation that should be performed by skilled and experienced personnel . it is also essential to have the representation of a dedicated attorney in negotiating potential partnerships and collaborations , particularly in drafting a well - constructed nondisclosure agreement ( nda ) , which serves to protect the ip of both parties . trust and loyalty are admirable qualities , but the independent inventor must be cautious when discussing and exchanging ip . this takes on even greater importance with the impending congressional legislation that provides ip ownership to the first to file , as opposed to the traditional first to invent . ip until after a provisional patent has been submitted and then only with a mutually signed nda . to quote andy grove , former ceo of intel , only the paranoid survive . in the event that patent litigation occurs and two parties are vying for patent ownership , or one party is challenging the validity of a recently issued patent , the courts will look for dated documentation to determine which party was first to invent . as a result , it is imperative that the independent inventor document everything related to the invention from concept creation , to embellishment , to all forms of communication with third parties . most important , the intrinsic value of one s own creativity , practical knowledge , and experience should not be underestimated . having the luxury of being independent and unconstrained by corporate shackles can be a distinct advantage to the independent inventor . in - house patent applications must pass through serial scrutiny that often serves to squash innovation and unorthodox thinking . the independent inventor does not have these limitations and is , instead , free to explore any and all creative concepts , regardless of the existing product pipeline . although market viability and compatibility with a company s strategic planning remain critical points of analysis for potential inventor industry partnerships , the independent inventor should continuously strive to push the envelope and leverage his or her own unique insights and experience . the new independent inventor should take advantage of existing resources that can provide valuable insights , contact information , and educational programs . several of these internet - based resources are listed in table 4 and serve merely as starting points in the quest for inventor knowledge and experience . although the barrier to entry as an inventor is high , success can be achieved by those with insight , creativity , and perseverance . patent & trademark officehttp://www.uspto.govunited inventors associationhttp://www.uiausa.orginventor ed inc.http://www.inventored.orginvent nethttp://www.inventnet.comindex to u.s . patent classificationhttp://www.ibiblia.org / patents / index.htmllemelson foundationhttp://www.lemelson.org existing resources for the independent inventor the u.s . patent and trademark office ( uspto ) defines an independent inventor as one whose patent ( at the time of issuance ) is unassigned or assigned to an individual . the percentage of patents issued to independent inventors declined from 36.4% in 1987 to 26.8% in 1999.6 international patent data ( 1999 ) show great variability in the overall percentages of patents issued to independent inventors , with less industrialized countries having a higher percentage of independent invention ( 75% in hungary and 66% in brazil , for example ) , compared with more industrialized countries ( 26% in both the united kingdom and united states6 ) . although the relative percentage of patents issued to independent inventors has gradually decreased over time , the total number of patents awarded to independent inventors increased by approximately 30% from 1990 to 2000.7 these figures suggest that independent invention is alive and well in the united states and retains a vital role in innovation throughout industry and the marketplace . a recent survey published by weick and eakin8 explored the role and economic viability of independent inventors in the united states . eighty - four percent of inventor respondents reported developing working prototypes of their inventions within the past 5 years . the top invention categories were dominated by hardware / tools , household , and industrial / commercial products , and the most frequently cited scientific / technology inventions were categorized as electronics ( 13% ) and medical / therapeutic ( 12% ) . thirty - nine percent of respondent inventors reported generating sales from their inventions ( mean sales , $ 3.5 million ; median sales , $ 50,000 ) , and 22% reported profitability ( mean profit , $ 1.96 million ; median profit , $ 75,000 ) . the three principal means of ip commercialization included development of start - up companies ( 55% of respondents ) , licensing ip to another company ( 44% of respondents ) , and outright sales of ip ( 16% of respondents ) . inventors who elected to license their inventions to others were more likely to achieve a higher level of overall sales than those attempting to commercialize the ip themselves or who sold the ip to a third party . this corroborates previously reported findings by khan and sokoloff,9 who reported independent inventors commercial success to be greatest through licensing agreements . the prevalent paradigm of industry - sponsored r&d suffers from several limitations , some of which are outlined in table 1 . the various strengths that provide industry with a distinct ( and arguably unfair ) advantage over the independent inventor paradoxically provide a disincentive toward innovation . this can be explained in part by the life cycle of innovation embraced by many companies , particularly larger and more financially successful concerns . table 1existing limitations of industry - sponsored r&dlimitationa follow - the - pack ( lemming ) mentalitysmall , incremental approaches to new ipinternal corporate politics and constraintsshort - term outlook targeted at immediate gratificationproduct development done in usersreactive , not proactive , approach to market needs existing limitations of industry - sponsored r&d in the first stage ( infancy ) of this life cycle , innovation plays the dominant role in creating new ip , a process that provides the impetus and backbone for the original creation of many companies . the fledgling company is founded by a few energetic , industrious , and optimistic inventors who , through a combination of idealism , capitalism , and naivety , decide to embark on the development of a start - up company despite long odds against success . in the second stage ( childhood ) , the company grows through successful commercialization of their innovation and ip . as the company grows and achieves economic success , the culture begins to shift from optimism and innovation to pragmatism and risk aversity . the political and economic realities of the marketplace begin to surface , and the founders of the company often turn over operational control to financial and management professionals . if the company continues on a path of relative independence ( avoiding merger or acquisition ) and continues to achieve greater success in the marketplace , it may then enter stage 3 ( adulthood ) , in which it is often transformed from innovator to predator . in entering this stage , the company often grows by acquiring smaller ( and more innovative ) companies , thereby expanding its product line and ip pipeline . the large company now responds to market economics , with product development largely dictated by the perceived needs of the customer base . invention is typically performed within the company ( in house ) by individuals who are often constrained by company politics and the economics of immediate gratification . , innovation is largely stifled and relegated to product line developments and incremental improvement . it is ironic that the very company that was initially created by innovation and creativity becomes transformed into one that strives to stifle ( and often crush ) the creativity of the independent ( and innovative ) inventor . in this life cycle , inventors themselves are no longer frontline users but , instead , are technicians and engineers . in the medical domain , these engineers are , for the most part , technology savvy , but clinically uninformed . as a result , refinements and new developments are based , at best , on minimal knowledge of how the product will truly function in the clinical environment . experts on a medical advisory board who have little practical experience outside of academia . as a result , even with a gloss of medical input , many products are developed within a rigid corporate culture by nonclinicians who are highly risk averse and focused on short - term horizons . the independent inventor , on the other hand , is not bound by the same corporate constraints and , instead , can bring a fresh and practical perspective to product innovation . this strategy of outsourcing innovation is uncommon within industry today but offers the potential to create new independent inventor industry synergies . the creation of an open market approach to innovation provides a cost - effective and practical means for companies to remove their self - induced ip constraints and seek out inventors from external sources to enhance innovation within new and existing product lines.10,11 table 2 outlines a ten - step iterative process for invention and product development . one example comes from my own work in private practice radiology , where one of my biggest concerns is the paucity of objective quality assurance ( qa ) metrics and standards within medical imaging and the lack of supporting technology . with the sole exception of mammography,12 no medical imaging modality has rigorous and mandatory qa standards , despite a broad agreement on the significance of the role of medical imaging qa in clinical and economic outcomes.13 simply stated , qa in its present form is largely idiosyncratic and often nonexistent . table 2stepwise approach to invention processapproachdefine an existing problem or questionreview the current technical solutionidentify existing limitations and inefficienciescollect data using existing technologydevise an alternative solution ( invention)develop a prototypecollect data using the prototyperefine the prototype based on data and end - user feedbackrepeat data collection ( and refinement as necessary)commercialize invention stepwise approach to invention process in attempting to objectively quantify existing qa practice , we performed several studies evaluating qa - related workflow , technology , and variability and the related effects on image quality.1417 the results have indicated that the current practice of medical imaging qa is fraught with error and inconsistencies , resulting in inefficient workflow , reduced technologist productivity , poor image quality , and suboptimal radiation dose to patients . the data call for a complete revamping in the clinical and technical ways in which qa activities are implemented . defining the solution becomes fairly straightforward when confronted with the paucity of data , lack of standards , wide inter- and intradepartmental variability in performance , and lack of supporting technology for qa assessment . the ideal solution to improve current inefficiencies would be an automated system to record , measure , track , analyze , and provide qa feedback to practitioners . after identifying and defining this deficiency , i proposed and described one such solution and submitted it for review by the uspto . although the idea may appear sound , it is only as good as the working prototype . as an independent inventor , i did not have the technical or economic resources required to create a working prototype and , therefore , elected to seek industry partnership through a licensing agreement . the project is far from complete , but the sequence of events outline in table 2 are those that i have come to recognize as requisite for creation and implementation of an invention , with the ultimate goal of creating independent inventor industry collaboration and a final product based on a data - driven engineering process.18 first and foremost is the reality that inventorship is equivalent to running a marathon , not a sprint . even under the best of circumstances , the patent review process takes 23 years and often longer , depending on the uspto backlog , the initial review of the patent examiner , and what once the review process has been completed , the inventor must begin the arduous task of marketing the invention and/or developing a prototype . because of the intensive resource requirements for prototype development , many inventors elect to pursue a licensing agreement with an established company that has the technical , financial , personnel , and legal resources to commercialize the patent and defend against potential future litigation . securing a patent the best deterrent is an ally with deep pockets and the combined economic and legal clout to punish any violators . table 3practical advice for inventorsadviceexpect success to take 510 yearsdo not quit your day jobengage professional legal services for prior art search patent preparation nondisclosure agreements commercializationdocument everything!do not market until you receive notice of allowancebe a little paranoid ; expect dishonestyfile a provisional patent asapconsider licensing as a means to commercializeleverage existing practical knowledge and experiencethink out of the box!leverage existing resources practical advice for inventors the patent application process in the united states typically costs in the range of $ 20,000$30,000 . the result is that many independent inventors are forced either to abandon their ip or seek external funding sources . although third parties exist in the form of independent investor and venture capitalist consortia , the inventor sometimes forfeits substantial economic and legal rights in entering such agreements . having sound legal advice with expertise in patents and licensing is critical in ensuring that the best interests of the inventor ( and the long - term security of his or her ip ) are protected new legislation is currently pending in congress ( hr 2795 ) that calls for major patent reforms . enactment of this legislation could have negative effects on the independent inventor by : changing the definition of inventor from first to invent to first to file;limiting a patent holder s rights to obtain a permanent injunction against an offending third party;creating a post - patent grant opposition proceeding ; andlimiting damages for infringement lawsuits changing the definition of inventor from first to invent to first to file ; limiting a patent holder s rights to obtain a permanent injunction against an offending third party ; creating a post - patent grant opposition proceeding ; and limiting damages for infringement lawsuits in the end , larger corporations and academic institutions have far more extensive resources and expertise to create , manage , and defend patents . these disproportionate resources should not preclude the independent inventor from seeking patents but should provide significant incentives to pursue symbiotic partnerships with industry that can ultimately benefit both parties . although do it yourself programs are widely advertised as ways for independent inventors to draft their own patent applications , investors should be wary of the long - term ramifications of even minor errors in the patent process . claim wording on patents is complex and most properly the province of specialized attorneys with extensive training and experience . the prior art search is another integral component of patent preparation that should be performed by skilled and experienced personnel . it is also essential to have the representation of a dedicated attorney in negotiating potential partnerships and collaborations , particularly in drafting a well - constructed nondisclosure agreement ( nda ) , which serves to protect the ip of both parties . trust and loyalty are admirable qualities , but the independent inventor must be cautious when discussing and exchanging ip . this takes on even greater importance with the impending congressional legislation that provides ip ownership to the first to file , as opposed to the traditional first to invent . ip until after a provisional patent has been submitted and then only with a mutually signed nda . to quote andy grove , former ceo of intel , only the paranoid survive . in the event that patent litigation occurs and two parties are vying for patent ownership , or one party is challenging the validity of a recently issued patent , the courts will look for dated documentation to determine which party was first to invent . as a result , it is imperative that the independent inventor document everything related to the invention from concept creation , to embellishment , to all forms of communication with third parties . most important , the intrinsic value of one s own creativity , practical knowledge , and experience should not be underestimated . having the luxury of being independent and unconstrained by corporate shackles can be a distinct advantage to the independent inventor . in - house patent applications must pass through serial scrutiny that often serves to squash innovation and unorthodox thinking . the independent inventor does not have these limitations and is , instead , free to explore any and all creative concepts , regardless of the existing product pipeline . although market viability and compatibility with a company s strategic planning remain critical points of analysis for potential inventor industry partnerships , the independent inventor should continuously strive to push the envelope and leverage his or her own unique insights and experience . the new independent inventor should take advantage of existing resources that can provide valuable insights , contact information , and educational programs . several of these internet - based resources are listed in table 4 and serve merely as starting points in the quest for inventor knowledge and experience . although the barrier to entry as an inventor is high , success can be achieved by those with insight , creativity , and perseverance . despite the economic and legal impediments facing ip development in the current marketplace , many important innovations and technologic breakthroughs within medicine are the result of independent inventors . these independent inventors play a unique and vital role towards ip development in medical imaging and information technology through their practical first - hand experience , out of the box they are not restricted by many of the traditional constraints existing within an industry including the incremental approach to product development , internal politics , and a short - term focus on return on investment ( roi ) . the traditional paradigm of industry - sponsored r&d may be better served through the creation of a more collaborative approach of outsourcing innovation , thereby creating a synergy between independent inventors and industry sponsors . in the end , innovation can be better served by creating an atmosphere for open competition , creativity , and accountability .

while innovation and new product development is traditionally thought of as the exclusive domain of industry and academia , a large number of innovations in medicine and information technology have come from independent inventors , which account for almost 30% of new patents issued in the u.s . today . a large number of economic , political , and legal challenges exist within the current marketplace that serves as relative impediments to independent invention . this article explores the existing challenges facing the independent inventor and offers a number of recommendations and resources to facilitate independent inventors in their quest for innovation and entrepreneurship . the concept of outsourcing innovation is discussed as an alternative to the existing model of industry sponsored research and development ( r&d ) , with the goal of combining the unique attributes and strengths of independent inventors and industry sponsors .

Introduction Data on Independent Invention Existing Limitations of Industry-Sponsored R&D Defining the Invention Process Practical Advice for Inventors Conclusion

PMC2951100

based on the fact that computers are commonly used in hospital patient wards and operation theatres , it would be not surprising if they could be contaminated with nosocomial pathogens . cultures taken from the surface of computers keyboards yielded microorganisms such as coagulase - negative staphylococcus ( cns ) ( from 100% of all keyboards ) , diphtheroids ( 80% ) , micrococcus spp . ( 64% ) , methicillin - resistant staphylococcus aureus ( mrsa ; 4% ) , methicillin - sensitive staphylococcus aureus ( mssa ; 4% ) , vancomycin - sensitive enterococcus spp . these studies found all of the tested disinfection solutions were to be effective and compatible for use in disinfecting keyboards . it could be further demonstrated that contamination of the keyboards used by numerous persons was far higher than that seen in keyboards used by only one person . as a consequence to this unavoidable situation , anderson et al . also recommended routine cleaning and disinfection of the work station , especially in situations where keyboard usage involves numerous persons . on an intensive care unit it was discovered that the keyboard keys and mouse input devices of the ward station computers were contaminated up to 5.9% and 6.3% , respectively . interestingly , the telephone handles as well as intercoms were not contaminated due to the fact that they were regularly disinfected . analogously , keyboard keys used by anesthesiologists in the operating room were also shown to be contaminated ( most often with cns and bacillus spp . , but also with mrsa ) . consequent to these findings , a recommendation was made for routine hand disinfection along with daily wipe - disinfection of the computer contact surface . the general consensus from all of the studies examining computer keyboard and mouse devices is unanimous : the decisive preventative measure for the elimination of these infection sources is improved hand hygiene and routine disinfection / cleaning of the pc contact / touch surfaces , , , , , , , , . based upon the fact that most laptops are fitted with a cooling ventilation blower , it was decided to investigate whether the external air inlets into the vent and eventually blown back into the room actually culminates into contamination , thereby spreading potential pathogens into the surrounding area . in order to avoid a mixture of the emission from the laptop together with the surrounding contaminated environmental air , it had to be ascertained that the air exhausted from the laptop would be captured into a microbial air sampler , whereby a secure separation between contaminated room - air and laptop emission could be maintained . the laptops chosen for the study were examined within a laminar air flow safety workbench ( microflow , nunc gmbh , wiesbaden - biebrich ) . for the purpose of this study a box which could be disinfected as well as completely sealed was constructed and placed into the security workbench ( figure 1 ( fig . the suction vent of the microbial air sampler ( air deal 3 p , biomerieux deutschland gmbh , nrtigen ) was precisely positioned onto the opening punched out from the bottom of the box . the top cover of the box could be open in order to place the laptop within ( figure 2 ( fig . further course of the experiment involved the following steps : disinfection of all internal and external surfaces of the box using an alcohol - based surface disinfectant ( terralin liquid ; schlke and mayr gmbh , norderstedt ) possessing microbiocidal and virocidal efficacy ( declared max . effect time against non - sheathed viruses 2 min ) for a minimal duration of 5 minsimultaneous disinfection of the internal surfaces of the workbench with the same liquid solution also for 5 min with the safety workbench switched - ondisinfection of the surface of the microbial air sampler with the same liquid solution for 5 minsurgical , alcohol - based hand disinfection and disinfection of the lower arms according to the declared time of effect for duration of 1.5 min ( ahd 2000 , lysoform , dr . hans rosemann gmbh , berlin ) , sterile op gloves were subsequently put onplacement of the disinfected box into safety workbench , thereafter the box was not movedswitching - off of the safety workbench for the duration of time it took to carefully place the laptops into the box without causing any air - movement or disturbancesswitching - on of the safety workbench for a period of 20 secdisinfection of the gloved handsplacement of the blood agar culture plate into the microbial air sampler , unscrewing the collection head which for the first measurement of the day was initially sterile . for each new subsequent measurement the collection head was disinfected for a duration period of 5 min with a bottled disinfection solution.careful attachment of the microbial air sampler to the opening at the bottom of the box with special attention not to allow any space between the connection ( figure 3 ( fig . 3))switching - on the microbial air sampler for a time period of 5 min in order to determine the blank values ; that is the number of airborne bacteria released into the laminar air flow from the external body of the non - activated laptop removal of the agar plate from the microbial air sampler , disinfection of the collection head and placement of a new agar plateswitching - on the laptop and program start ; as soon as the ventilation fan is activated the microbial air sampler is immediately switched - on once again for a period of 5 min in order to register the air released by the ventilation blowerremoval of the laptop out of the box . disinfection of all internal and external surfaces of the box using an alcohol - based surface disinfectant ( terralin liquid ; schlke and mayr gmbh , norderstedt ) possessing microbiocidal and virocidal efficacy ( declared max . effect time against non - sheathed viruses 2 min ) for a minimal duration of 5 min simultaneous disinfection of the internal surfaces of the workbench with the same liquid solution also for 5 min with the safety workbench switched - on disinfection of the surface of the microbial air sampler with the same liquid solution for 5 min surgical , alcohol - based hand disinfection and disinfection of the lower arms according to the declared time of effect for duration of 1.5 min ( ahd 2000 , lysoform , dr . hans rosemann gmbh , berlin ) , sterile op gloves were subsequently put on placement of the disinfected box into safety workbench , thereafter the box was not moved switching - off of the safety workbench for the duration of time it took to carefully place the laptops into the box without causing any air - movement or disturbances switching - on of the safety workbench for a period of 20 sec disinfection of the gloved hands placement of the blood agar culture plate into the microbial air sampler , unscrewing the collection head which for the first measurement of the day was initially sterile . for each new subsequent measurement the collection head was disinfected for a duration period of 5 min with a bottled disinfection solution . careful attachment of the microbial air sampler to the opening at the bottom of the box with special attention not to allow any space between the connection ( figure 3 ( fig . 3 ) ) switching - on the microbial air sampler for a time period of 5 min in order to determine the blank values ; that is the number of airborne bacteria released into the laminar air flow from the external body of the non - activated laptop removal of the agar plate from the microbial air sampler , disinfection of the collection head and placement of a new agar plate switching - on the laptop and program start ; as soon as the ventilation fan is activated the microbial air sampler is immediately switched - on once again for a period of 5 min in order to register the air released by the ventilation blower removal of the laptop out of the box . in parallel , a smear was taken using cotton swabs at the site of the laptop exhaust vent where the cooled air is blown out . mller gmbh , eppelheim ) , subsequently placed into a casein - sojapepton - solution and vortexed . after 24 hr incubation of the csl , 50 l fractionated were spread onto columbia blood agar . the cultivated colonies derived from the collected air as well as those from the smear were biochemically differentiated in the same manner as those from enrichment cultures . the devices originated from a total of 7 different manufacturers and were drafted for use from various hospital stations without prior notice or disinfection ( table 1 ( tab . temperature pictures were performed at the blower exhaust vent and within the interior of the laptop using a thermo camera variocam high resolution ( infra tec gmbh dresden ) . for this the housing of the laptops were opened . in order to achieve a defined cpu - burden and the associated warmth development the software stress my pc the pre - values derived from the air circulated around the casing of the laptop stood at 40.117.9 colony forming units per m ( cfu / m ) ( table 2 ( tab . this was also found to be the case with molds whereby the factor was increased by only 1.2 ( 5.79.46 vs. 7.114.90 ) . with the exception of the aerobic sporulation , there was no differentiation observed between the blank values and the running blowers in terms of species distribution . in the case of the apathogenic sporulators , the figures were doubled from 4.86.1 cfu / m to 11.113.2 cfu / m whereby , however , this difference was not considered significant ( wilcoxon signed rank - test , p=0.211 ) . the spectrum of the freely released microorganisms encompassed mostly members of the localized flora of the skin ( coagulase - negative staphylococcus and micrococcus luteus ) , lacking pathogenic potential and therefore only capable of contaminating the body surface . from 2 laptops were 4 cfu of methicillin sensitive staphylococcus aureus released but each time only for the blank values ( laptops 2 & 12 ) . 1 laptop released 2 cfu of -hemolyzing streptococcus but in this case only after the blower was switched - on . a correlation between origin of the laptop , the manufacturer , the amount of freely - released germs as well as germ spectrum could not be developed . swab culture samples taken from the blower exit vents which were directly smeared demonstrated negative growth in 16 of the cases . the remaining 4 showed only members of the localized flora of the skin and/or aerobic spores . even following applied enrichment , 6 of the smears remained negative and the germ spectrum did not alter ( table 3 ( tab . the results were found to be so uniform that it was decided not to match the results to the specific laptop manufacturers . at the site of the blower exit vent of a laptop model travelmate 3000 following 10 min of running the software at maximum , measured temperature 56.4c ( figure 4 ( fig . 4 ) ) . suctioned cool air flowing over the internal surface of the laptop achieved a measured temperature of 73.2c ( figure 5 ( fig . the results of this study allow the assumption that the activated blowers within the investigated laptops did not result in an additional release of nosocomial pathogens into the surrounding environment . the exit vent of the blower was also not contaminated with nosocomial germs . due to the high temperatures in the inner parts of the laptop ( up to 73c ) , a biofilm could not be created within the blower space or surrounding surfaces from which the risk of disease activators could be emitted . even at the site of the blower vent the fact that spores were released with or without the activated blower leads to the assumption that they originated from the external surface of the laptop and perhaps from the air shaft of the blower . that spores and molds possess a high persistence against dry heat than vegetative bacteria ; it is possible that they could survive a short term within the airspace of the blower . this is , however , unlikely for nosocomial vegetative bacteria due to an intolerance of general temperatures 60c . before the point is reached where the processor has become heated - up and the blower is activated , the surrounding room air is sucked into the device . the air becomes immediately warm and dry , whereby bacteria have no possibility of clinging to the inner surface of the blower , reproducing and eventually building a biofilm . the explanation for this is that most disease activators are mesophilic ; meaning that they proliferate preferably at temperatures between 1545c and for the construction of biofilm require water in order to develop a completely hydrated eps - matrix . interpretation of the findings is thereby supported in that direct smears obtained from the airflow ventilated inner surface of the laptop were without exception negative . once the remaining warm air within the airspace of the blower is cooled after the laptop has been switched - off , it is possible through volume traction that contaminated external air finds its way into the blower compartment . that room air within ward stations is minimally burdened in a microbial sense ( e.g. 200400 cfu / m in a patient room and hall of an icu ward , it can be calculated that after a room air backflow of approximately 2 cm , the number of germs which had entered clearly stood at 1 cfu . even though the cooling blower was turned - off the conditions for biofilm development were not present .

inadequately performed hand hygiene and non - disinfected surfaces are two reasons why the keys and mouse - buttons of laptops could be sources of microbial contamination resulting consequently in indirect transmission of potential pathogens and nosocomial infections . until now the question has not been addressed whether the ventilation - blowers in laptops are actually responsible for the spreading of nosocomial pathogens . therefore , an investigational experimental model was developed which was capable of differentiating between the microorganisms originating from the external surfaces of the laptop , and from those being blown out via the ventilation - blower duct . culture samples were taken at the site of the external exhaust vent and temperature controls were collected through the use of a thermo - camera at the site of the blower exhaust vent as well as from surfaces which were directly exposed to the cooling ventilation air projected by the laptop . control of 20 laptops yielded no evidence of microbial emission originating from the internal compartment following switching - on of the ventilation blower . cultures obtained at the site of the blower exhaust vent also showed no evidence of nosocomial potential . high internal temperatures on the inner surfaces of the laptops ( up to 73c ) as well as those documented at the site of the blower exhaust vent ( up to 56c ) might be responsible for these findings .

Introduction Materials and methods Results Discussion

PMC2844817

male sprague - dawley rats were made diabetic with a single intraperitoneal injection of 75 mg / kg stz ( sigma , st . these diabetic rats were healthy and did not need insulin supplementation to maintain body weight . insulin implants ( two linplant implants ; linshin canada , toronto , on , canada ) were placed subcutaneously into a subgroup of stz - diabetic rats after 18 weeks of diabetes and remained in place for a further 4 weeks to test the impact of suboptimal insulin levels on mitochondrial function . animals were killed and tissue collected after 12 , 16 , or 22 weeks of diabetes . animal procedures followed guidelines laid down by the university of manitoba animal care committee using the canadian council of animal care guidelines . nonfasting blood glucose concentration was measured using the accu - chek compact plus glucometer ( roche , laval , qc , canada ) and blood a1c levels by the a1cnow+ system ( bayer healthcare , sunnyvale , ca ) . nerve sugar and polyol content was measured by gas chromatography of trimethylsilyl derivatives ( 17 ) and lipid peroxidation by spectrophotometric assay of total malondialdehyde ( mda ) and 4-hydroxyalkanal ( hae ) content ( lpo-586 kit , oxis , ca ) ( 20 ) . cohorts 1 , 2 , and 3 ( table 1 ) were used to prepare lumbar drg tissue homogenates for mitochondrial respirometry , and another group ( cohort 4 ) was used as a source of mitochondrial preparations from lumbar drg . lumbar drg were rinsed in ice - cold solution containing sucrose / tris / edta ( ste ) buffer ( 250 mmol / l sucrose , 10 mmol / l tris - hcl , 1 mmol / l edta , ph 7.4 ) and then homogenized with a polytron homogenizer ( kinematica , lucerne , switzerland ) using 3 7.5 s grinding pulses at 30-s intervals . in cohort 4 , mitochondria were isolated from lumbar drg using a differential centrifugation method ( 21 ) . the degree of integrity of the mitochondrial preparation was tested by adding exogenous cytochrome c ( 10 mol / l ) in the presence of pyruvate , malate , and adp . under these conditions , the respiration rate in lumbar drg was increased by 17 8% in control and 22 9% in 22 weeks stz - diabetic rats ( means sd , n = 4 ) . this demonstrates a low level of fragmentation of the mitochondria and the similarity of the preparation between the two groups . rat body weight , blood glucose levels , and glycated hemoglobin at end point starting body weight ranged between 275 and 325 g. cohorts were maintained for 1222 weeks . rats from cohort 3 and 4 were used for the preparation of lumbar drg tissue homogenate samples and isolated mitochondria samples from lumbar drg , respectively . values are means sd . * p < 0.05 vs. other groups ; * * p < 0.001 vs. diabetic ; * * * p < 0.05 vs. diabetic ( all by one - way anova with tukey 's post hoc test ) ; and * * * * p < 0.0001 vs. diabetic ( student 's t test ) . note that % a1c values in cohort 3 were derived using the dca 2000 + kit ( n = 45 ) and for cohort 4 were generated using the a1c now kit ( n = 811 ) . mitochondrial preparations were homogenized in ste buffer and 510 g protein resolved on a 10% sds - page gel and electroblotted onto nitrocellulose membrane . blots were then blocked in 5% nonfat milk containing 0.05% tween-20 , rinsed in pbs ( ph 7.4 ) , and incubated with the following antibodies : monoclonal anti cytochrome c oxidase subunit 4 ( cox iv , 1:1,000 ; mitosciences , eugene , or ) , monoclonal anti - nadh dehydrogenase ( ubiquinone ) iron - sulfur protein 3 ( ndufs3 , 1:1,000 ; mitosciences ) , and monoclonal anti - atp synthase subunit ( 1:2,000 dilution ; mitosciences ) . extracellular signal regulated kinase ( erk , 1:2,000 ; covance ) was probed as a loading control ( previous studies show this protein to not change level of expression in drg in diabetes ) . the blots were rinsed , incubated in western blotting luminol reagent ( santa cruz biotechnology ) , and imaged using a bio - rad fluor - s image analyzer ( bio - rad , hercules , ca ) . oxygen consumption was determined at 37c using the oroboros oxygraph-2k ( oroboros instruments , innsbruck , austria ) ( 22 ) . tissue homogenates or isolated mitochondria from lumbar drg were resuspended in kcl medium ( 80 mmol / l kcl , 10 mmol / l tris - hcl , 3 mmol / l mgcl2 , 1 mmol / l edta , 5 mmol / l potassium phosphate , ph 7.4 ) . various substrates and inhibitors for mitochondrial respiratory chain complexes were used as described in fig . all measurements of enzymatic activities in lumbar drg mitochondrial preparations were performed spectrophotometrically using a temperature - controlled ultrospec 2100 ultraviolet - visible spectrophotometer ( biopharmacia biotech , uppsala , sweden ) . complex i activity was measured as rotenone - sensitive nadh : cytochrome c reductase activity . purified mitochondria were thawed and subjected to three freeze - thaw cycles to disrupt mitochondrial membranes and permit access of substrates . freshly prepared assay buffer ( 50 mmol / l k - phosphate , ph 7.4 , 1 mmol / l potassium cyanide ( kcn ) , 100 mol / l nadh ) and 10 g protein of mitochondrial preparation were added to the cuvette and preincubated for 3 min at 25c . after addition of 100 mol / l oxidized cytochrome c , the reaction was followed for 2 min at 550 nm and then for 2 more minutes after addition of 25 mol / l rotenone to allow calculation of the rotenone - sensitive complex i activity . complex iv activity was measured at 25c by monitoring the absorbance decrease of reduced cytochrome c at 550 nm ( 23 ) . the reaction was started by addition of 40 mo / l reduced cytochrome c into 50 mmol / l phosphate buffer containing 5 g mitochondrial protein solubilized with 0.02% laurylmaltoside . activity of the krebs cycle enzyme , citrate synthase , was determined at 25c in medium containing 150 mmol / l tris - hcl ( ph 8.2 ) , 0.02% laurylmaltoside , 0.1 mmol / l dithionitrobenzoic acid , and 5 g mitochondrial protein ( 24 ) . the reaction was initiated by addition of 100 mol / l acetyl coa and changes in absorbance at 412 nm were measured for 1 min . this value was subtracted from the rate obtained after addition of 0.05 mmol / l oxaloacetic acid . adult lumbar drg sensory neuron cultures from age - matched control and 22-week stz - diabetic rats were prepared as described previously ( 17 ) . neurons were suspended in modified bottenstein and sato 's n2 medium ( 0.1 mg / ml transferrin , 20 nmol / l progesterone , 100 mol / l putrescine , 30 nmol / l sodium selenite , and 1 mg / ml bsa , 0.01 mmol / l cytosine arabinoside ; all from sigma ) in ham 's f12 . to mimic in vivo conditions , the following additions were made to the basal defined medium : control 10 nmol ros levels were imaged on a carl zeiss lsm510 inverted confocal microscope using two dyes : dihydrorhodamine 123 ( dhr123 ) or 5-(and-6)-chlromethyl-27-dichlorohydrofluorescein diacetate acetyl ester ( cm - h2dcfda ) . / l dhr 123 ( sigma , in 100% stock solution ) or 1.2 mol / l cm - h2dcfda ( invitrogen , carlsbad , ca ) for 30 or 15 min , respectively , at 37c . ros generation in lumbar drg mitochondria from control and 22-week stz - diabetic rats was evaluated by the detection of h2o2 . hydrogen peroxide production was determined fluorometrically by measuring oxidation of amplex red ( invitrogen ) ( 22 ) . fluorescence of the amplex red oxidation product was measured at 25c using spectramax gemini spectrofluorometer ( molecular devices , sunnyvale , ca ) . the assay was performed with 100 g mitochondrial protein per milliliter in kcl - based medium supplemented with 10 mmol / l pyruvate and 5 mmol / l malate in the presence or absence of antimycin a ( 10 g / ml ) . amplex red was used at 50 mol / l , with horseradish peroxidase at 0.5 units / ml . function of small sensory neurons was inferred by measuring hind paw thermal response latency using a modified hargreaves apparatus ( university anesthesia research and development , san diego , ca ) with a heating rate of 1c / s from a baseline of 30c and with a cutoff at 20 s ( 25 ) . the structural integrity of small sensory neurons was assessed by measuring intraepidermal nerve fiber ( ienf ) profiles in hind paw plantar skin that was immersion fixed in 4% paraformaldehyde and processed to paraffin blocks . sections ( 6 m ) were stained with the pan - neuronal marker pgp9.5 ( 1:1,000 ; biogenesis , u.k . ) and viewed under a light microscope to allow counting of ienf and subepidermal nerve profiles per unit length of the dermal : epidermal border ( 26 ) . structural integrity of large myelinated fibers was assessed by measuring mean axonal caliber in distal tibial nerves that were immersion fixed in 2.5% glutaraldehyde , postfixed in osmium tetroxide , and processed to araldite blocks before cutting 1.0-m sections . tissue sections were stained with p - phenylenediamine and viewed under a light microscope connected to a computer running image analysis software to allow calculation of axonal diameter ( 17 ) . where appropriate , data were subjected to one - way anova with post hoc comparison using tukey 's test or two - way anova with bonferroni 's post hoc tests ( graphpad prism 4 ; graphpad software , san diego , ca ) . in all other cases , male sprague - dawley rats were made diabetic with a single intraperitoneal injection of 75 mg / kg stz ( sigma , st . these diabetic rats were healthy and did not need insulin supplementation to maintain body weight . insulin implants ( two linplant implants ; linshin canada , toronto , on , canada ) were placed subcutaneously into a subgroup of stz - diabetic rats after 18 weeks of diabetes and remained in place for a further 4 weeks to test the impact of suboptimal insulin levels on mitochondrial function . animals were killed and tissue collected after 12 , 16 , or 22 weeks of diabetes . animal procedures followed guidelines laid down by the university of manitoba animal care committee using the canadian council of animal care guidelines . nonfasting blood glucose concentration was measured using the accu - chek compact plus glucometer ( roche , laval , qc , canada ) and blood a1c levels by the a1cnow+ system ( bayer healthcare , sunnyvale , ca ) . nerve sugar and polyol content was measured by gas chromatography of trimethylsilyl derivatives ( 17 ) and lipid peroxidation by spectrophotometric assay of total malondialdehyde ( mda ) and 4-hydroxyalkanal ( hae ) content ( lpo-586 kit , oxis , ca ) ( 20 ) . cohorts 1 , 2 , and 3 ( table 1 ) were used to prepare lumbar drg tissue homogenates for mitochondrial respirometry , and another group ( cohort 4 ) was used as a source of mitochondrial preparations from lumbar drg . lumbar drg were rinsed in ice - cold solution containing sucrose / tris / edta ( ste ) buffer ( 250 mmol / l sucrose , 10 mmol / l tris - hcl , 1 mmol / l edta , ph 7.4 ) and then homogenized with a polytron homogenizer ( kinematica , lucerne , switzerland ) using 3 7.5 s grinding pulses at 30-s intervals . in cohort 4 , mitochondria were isolated from lumbar drg using a differential centrifugation method ( 21 ) . the degree of integrity of the mitochondrial preparation was tested by adding exogenous cytochrome c ( 10 mol / l ) in the presence of pyruvate , malate , and adp . under these conditions , the respiration rate in lumbar drg was increased by 17 8% in control and 22 9% in 22 weeks stz - diabetic rats ( means sd , n = 4 ) . this demonstrates a low level of fragmentation of the mitochondria and the similarity of the preparation between the two groups . rat body weight , blood glucose levels , and glycated hemoglobin at end point starting body weight ranged between 275 and 325 g. cohorts were maintained for 1222 weeks . rats from cohort 3 and 4 were used for the preparation of lumbar drg tissue homogenate samples and isolated mitochondria samples from lumbar drg , respectively . * p < 0.05 vs. other groups ; * * p < 0.001 vs. diabetic ; * * * p < 0.05 vs. diabetic ( all by one - way anova with tukey 's post hoc test ) ; and * * * * p < 0.0001 vs. diabetic ( student 's t test ) . note that % a1c values in cohort 3 were derived using the dca 2000 + kit ( n = 45 ) and for cohort 4 were generated using the a1c now kit ( n = 811 ) . mitochondrial preparations were homogenized in ste buffer and 510 g protein resolved on a 10% sds - page gel and electroblotted onto nitrocellulose membrane . blots were then blocked in 5% nonfat milk containing 0.05% tween-20 , rinsed in pbs ( ph 7.4 ) , and incubated with the following antibodies : monoclonal anti cytochrome c oxidase subunit 4 ( cox iv , 1:1,000 ; mitosciences , eugene , or ) , monoclonal anti - nadh dehydrogenase ( ubiquinone ) iron - sulfur protein 3 ( ndufs3 , 1:1,000 ; mitosciences ) , and monoclonal anti - atp synthase subunit ( 1:2,000 dilution ; mitosciences ) . extracellular signal regulated kinase ( erk , 1:2,000 ; covance ) was probed as a loading control ( previous studies show this protein to not change level of expression in drg in diabetes ) . the blots were rinsed , incubated in western blotting luminol reagent ( santa cruz biotechnology ) , and imaged using a bio - rad fluor - s image analyzer ( bio - rad , hercules , ca ) . oxygen consumption was determined at 37c using the oroboros oxygraph-2k ( oroboros instruments , innsbruck , austria ) ( 22 ) . tissue homogenates or isolated mitochondria from lumbar drg were resuspended in kcl medium ( 80 mmol / l kcl , 10 mmol / l tris - hcl , 3 mmol / l mgcl2 , 1 mmol / l edta , 5 mmol / l potassium phosphate , ph 7.4 ) . various substrates and inhibitors for mitochondrial respiratory chain complexes were used as described in fig . all measurements of enzymatic activities in lumbar drg mitochondrial preparations were performed spectrophotometrically using a temperature - controlled ultrospec 2100 ultraviolet - visible spectrophotometer ( biopharmacia biotech , uppsala , sweden ) . complex i activity was measured as rotenone - sensitive nadh : cytochrome c reductase activity . purified mitochondria were thawed and subjected to three freeze - thaw cycles to disrupt mitochondrial membranes and permit access of substrates . freshly prepared assay buffer ( 50 mmol / l k - phosphate , ph 7.4 , 1 mmol / l potassium cyanide ( kcn ) , 100 mol / l nadh ) and 10 g protein of mitochondrial preparation were added to the cuvette and preincubated for 3 min at 25c . after addition of 100 mol / l oxidized cytochrome c , the reaction was followed for 2 min at 550 nm and then for 2 more minutes after addition of 25 mol / l rotenone to allow calculation of the rotenone - sensitive complex i activity . complex iv activity was measured at 25c by monitoring the absorbance decrease of reduced cytochrome c at 550 nm ( 23 ) . the reaction was started by addition of 40 mo / l reduced cytochrome c into 50 mmol / l phosphate buffer containing 5 g mitochondrial protein solubilized with 0.02% laurylmaltoside . activity of the krebs cycle enzyme , citrate synthase , was determined at 25c in medium containing 150 mmol / l tris - hcl ( ph 8.2 ) , 0.02% laurylmaltoside , 0.1 mmol / l dithionitrobenzoic acid , and 5 g mitochondrial protein ( 24 ) . the reaction was initiated by addition of 100 mol / l acetyl coa and changes in absorbance at 412 nm were measured for 1 min . this value was subtracted from the rate obtained after addition of 0.05 mmol / l oxaloacetic acid . adult lumbar drg sensory neuron cultures from age - matched control and 22-week stz - diabetic rats were prepared as described previously ( 17 ) . neurons were suspended in modified bottenstein and sato 's n2 medium ( 0.1 mg / ml transferrin , 20 nmol / l progesterone , 100 mol / l putrescine , 30 nmol / l sodium selenite , and 1 mg / ml bsa , 0.01 mmol / l cytosine arabinoside ; all from sigma ) in ham 's f12 . to mimic in vivo conditions , the following additions were made to the basal defined medium : control 10 nmol / l insulin and diabetic 25 mmol / l glucose . ros levels were imaged on a carl zeiss lsm510 inverted confocal microscope using two dyes : dihydrorhodamine 123 ( dhr123 ) or 5-(and-6)-chlromethyl-27-dichlorohydrofluorescein diacetate acetyl ester ( cm - h2dcfda ) . / l dhr 123 ( sigma , in 100% stock solution ) or 1.2 mol / l cm - h2dcfda ( invitrogen , carlsbad , ca ) for 30 or 15 min , respectively , at 37c . for dhr 123 and cm - h2dcfda , excitation / emission was 488/>505 nm . ros generation in lumbar drg mitochondria from control and 22-week stz - diabetic rats was evaluated by the detection of h2o2 . hydrogen peroxide production was determined fluorometrically by measuring oxidation of amplex red ( invitrogen ) ( 22 ) . fluorescence of the amplex red oxidation product was measured at 25c using spectramax gemini spectrofluorometer ( molecular devices , sunnyvale , ca ) . the assay was performed with 100 g mitochondrial protein per milliliter in kcl - based medium supplemented with 10 mmol / l pyruvate and 5 mmol / l malate in the presence or absence of antimycin a ( 10 g / ml ) . amplex red was used at 50 mol / l , with horseradish peroxidase at 0.5 units / ml . function of small sensory neurons was inferred by measuring hind paw thermal response latency using a modified hargreaves apparatus ( university anesthesia research and development , san diego , ca ) with a heating rate of 1c / s from a baseline of 30c and with a cutoff at 20 s ( 25 ) . the structural integrity of small sensory neurons was assessed by measuring intraepidermal nerve fiber ( ienf ) profiles in hind paw plantar skin that was immersion fixed in 4% paraformaldehyde and processed to paraffin blocks . sections ( 6 m ) were stained with the pan - neuronal marker pgp9.5 ( 1:1,000 ; biogenesis , u.k . ) and viewed under a light microscope to allow counting of ienf and subepidermal nerve profiles per unit length of the dermal : epidermal border ( 26 ) . structural integrity of large myelinated fibers was assessed by measuring mean axonal caliber in distal tibial nerves that were immersion fixed in 2.5% glutaraldehyde , postfixed in osmium tetroxide , and processed to araldite blocks before cutting 1.0-m sections . tissue sections were stained with p - phenylenediamine and viewed under a light microscope connected to a computer running image analysis software to allow calculation of axonal diameter ( 17 ) . where appropriate , data were subjected to one - way anova with post hoc comparison using tukey 's test or two - way anova with bonferroni 's post hoc tests ( graphpad prism 4 ; graphpad software , san diego , ca ) . in all other cases , stz - diabetic rats did not suffer weight loss during the study but showed reduced weight gain after 1222 weeks of stz - induced diabetes compared with age - matched controls ( table 1 ) . persistence of diabetes was also indicated by elevated nonfasting blood glucose and glycated hemoglobin levels ( table 1 ) . stz - diabetic rats of cohorts 3 and 4 that received insulin supplementation for the final 4 weeks of a 22-week period of diabetes showed a partial recovery of body weight accompanied by a partial decrease in blood glucose , but retained elevated glycated hemoglobin levels . insulin treatment also partially lowered nerve glucose content and corrected the deficit in myo - inositol ( table 2 ) . diabetes - induced increases in nerve fructose content and lipid peroxidation were not altered by insulin therapy . nerve protein content was not altered by diabetes , whereas insulin treatment significantly increased this parameter above in both controls and untreated diabetic rats . sugar , protein , and lipid peroxidation levels in sciatic nerve sciatic nerves were taken from cohort 4 . values are means sd ( n = 69 ) . * p < 0.05 vs. control ; * * p < 0.05 vs. diabetic + insulin . there was no overt evidence of degeneration in the distal tibial nerve of rats after 22 weeks of diabetes ( supplemental fig . 1a and , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1299/dc1 ) , and morphometric analysis indicated a mild trend toward decreased mean axonal diameter associated with a shift in the size - frequency distribution toward smaller fibers ( supplemental fig . paw thermal response latency was normal in rats after 22 weeks of diabetes , as were both ienf and subepidermal nerve profile counts ( supplemental fig . insulin supplementation for the last 4 weeks of diabetes significantly increased paw ienf profile number compared with controls ( supplemental fig . mitochondrial respiratory chain activity of drg was determined using a clarke type electrode in the presence of specific substrates and inhibitors of the mitochondrial respiratory chain . figure 1 demonstrates typical measurements of oxygen consumption in freshly isolated mitochondria of lumbar drg from age - matched control , diabetic , and insulin - treated diabetic rats . basal respiration in lumbar drg mitochondria is stated as respiration at state 4 with energetic substrates , pyruvate and malate ( p + m ) . coupled respiration at state 3 was induced by addition of adp . after this measurement , the atp - synthase specific inhibitor , oligomycin ( ol ) , was added to prevent reverse pumping of protons by the atpase , and then the uncoupled rate was determined by adding the uncoupling agent , carbonylcyanide p - trifluoromethoxyphenylhydrazone ( fccp ) . uncoupled respiration was inhibited at complex iii by antimycin a. ascorbate ( asc ) and n , n , n,n-tetramethyl - p - phenylenediamine ( tmpd ) were then added to determine the capacity of cytochrome c oxidase ( complex iv ) . tmpd is an artificial redox mediator that assists transfer of electrons from ascorbate to cytochrome c. complex iv respiration was calculated as the portion sensitive to kcn , a specific inhibitor of cytochrome c oxidase . representative measurements of oxygen consumption in isolated lumbar drg mitochondria from control , diabetic , and insulin - treated diabetic rats . freshly isolated lumbar drg mitochondria were assessed in control ( a ) , diabetic ( b ) , and insulin - treated ( c ) diabetic rats using oroboros oxygraph 2k as described in research design and methods . thick lines indicate the level of oxygen in the chamber of electrode and expressed in nanomoles per milliliter . thin lines indicate oxygen flux per mass ( picomoles o2 per second per milligram protein ) in the presence of specific substrates and inhibitors of the mitochondrial respiratory chain . mmol / l ) ; m , malate ( 5 mmol / l ) ; adp , adenosine diphosphate ( 2 mmol / l ) ; ol , oligomycin ( 1.0 mol / l ) ; aa , antimycin a ( 1 g / ml ) ; asc , ascorbate ( 5 mmol / l ) ; tmpd , n , n , n,n-tetramethyl - p - phenylenediamine dihydrochloride ( 0.5 mmol / l ) ; kcn , potassium cyanide ( 0.25 mmol / l ) . basal respiration with pyruvate and malate ( p + m ) , rates of coupled respiration at state 3 , and the respiration rate with ascorbate and tmpd ( asc + tmpd ; complex iv ) of lumbar drg tissue homogenates from control and stz - diabetic rats were not changed after 12 weeks of diabetes ( fig . , there was a statistically nonsignificant decrease in coupled respiration and the respiration rate with asc + tmpd in stz - diabetic rats . with progression of untreated diabetes to 22 weeks in both lumbar drg tissue homogenates ( fig . 3b ) , coupled respiration with p + m was decreased by 3144% in diabetic rats compared with control lumbar drg tissue . respiration rates with asc + tmpd were decreased by 2939% in diabetic lumbar drg tissues and mitochondrial preparations and protected by insulin ( figs . 2c and 3d ) . uncoupled respiration in mitochondria isolated from lumbar drg was also decreased in stz - diabetic rats compared with control and significantly improved by insulin supplementation ( fig . these deficits were specific for lumbar drg , since similar measurements in cortex revealed no effect after 22 weeks of diabetes ( supplemental table 1 ) . effect of 1222 weeks of stz - induced diabetes on the activity of the mitochondrial respiratory chain in freshly prepared lumbar drg tissue homogenate . measurements of oxygen consumption were performed with energetic substrates , pyruvate and malate , as described in fig . 1 . basal respiration ( a ) , pyruvate + malate + adp ( coupled oxidative phosphorylation ) ( b ) , and the respiration rate with asc + tmpd ( complex iv ) ( c ) were assessed in age - matched controls ( ctrl ) , stz - diabetic rats ( db ) , and stz - diabetic rats with insulin implant ( db + ins ) at 12 ( n = 5 ) , 16 ( n = 711 ) , and 22 ( n = 56 ) weeks of diabetes . values are means sd ; n = as indicated . * p < 0.05 vs. db + ins ; * * p < 0.001 vs. db ( one - way anova with tukey 's post hoc comparison ) . mitochondrial respiratory chain activity is impaired in freshly isolated mitochondria from lumbar drg of 22 weeks of stz - diabetic rats . after measurement of coupled respiration , the atp synthase specific inhibitor , oligomycin , was added to prevent reverse pumping of protons by the atpase , and then the uncoupled rate of mitochondrial respiration was generated by adding the uncoupling agent fccp . basal respiration ( a ) , pyruvate + malate + adp ( coupled oxidative phosphorylation ) ( b ) , fccp ( uncoupled respiration ) ( c ) , and the respiration rate with asc + tmpd ( complex iv ) ( d ) were measured in age - matched controls ( ctrl ) , stz - diabetic rats ( db ) , and stz - diabetic rats with insulin implant ( db + ins ) after 22 weeks of diabetes . values are means sd , n = 6 . * p < 0.05 vs. other groups ; * * p < 0.05 vs. db + ins ( one - way anova with tukey 's post hoc comparison ) . treatment of the mitochondrial preparation with the uncoupler fccp induced a four- to sixfold elevation in the rate of oxygen consumption over the basal rate confirming that the mitochondrial preparations were intact and of high quality in all groups . enzymatic activities of mitochondrial complexes and the krebs cycle enzyme , citrate synthase , were assessed in mitochondria isolated from lumbar drg of age - matched control and 22-week stz - diabetic rats . the activity of mitochondrial complex i was assessed as rotenone - sensitive nadh - cytochrome c reductase when cytochrome c is the acceptor of electrons . the enzymatic activities of rotenone - sensitive nadh - cytochrome c reductase ( complex i ) and cytochrome c oxidase ( complex iv ) as well as the krebs cycle enzyme , citrate synthase , enzymatic activities of mitochondrial respiratory chain and citrate synthase activity are decreased in isolated mitochondria from lumbar drg of stz - diabetic rats . enzymatic activity of complex i was assessed as rotenone - sensitive portion of nadh - cytochrome c reductase ( nccr ) ( n = 5 ) ( a ) , cytochrome c oxidase ( n = 67 ) ( b ) , and citrate synthase ( n = 5 ) ( c ) and was measured as described in research design and methods . values are means sd , n = as indicated . * p < 0.05 vs. control ( ctrl ) ( unpaired student 's t test ) . if decreased mitochondrial activity in diabetic animals was associated with alterations in ros generation , adult sensory neurons from lumbar drg were cultured from age - matched control and stz - diabetic rats and assessed within 2 h of plating for levels of ros using the fluorescent dyes dhr 123 and cm - h2dcfda . figure 5a c show that sensory neurons from all treatment groups exhibited similar levels of dhr 123 fluorescence emission from perikarya . in a separate group of stz - diabetic rats , ros levels in acutely isolated neurons were detected using cm - h2dcfda and confirmed the lack of any effect of diabetes on ros generation ( fig . f ) . ros generation ( h2o2 as detected by amplex red ) by mitochondria isolated from lumbar drg of age - matched control or 22-week stz - diabetic rats was detected fluorometrically using the amplex red kit . ros generation in lumbar drg mitochondria in the presence of the substrates , pyruvate and malate , was not elevated in stz - diabetic rats compared with control ( fig . however , when mitochondria samples were treated with antimycin a ( a specific inhibitor of complex iii ) , control rats significantly increased ros production compared with age - matched stz - diabetic rats ( fig . 5h ) . finally , quantitative western blotting showed diminished expression of some components of the mitochondrial electron transport chain ( fig . ndufs3 ( component of complex i ) and cox iv ( component of complex iv ) were significantly decreased by 50 and 20% , respectively , whereas levels of atp synthase -subunit and erk remained unaltered . ros were not elevated in acutely isolated drg sensory neurons or mitochondria from stz - diabetic rats . a f : ros levels were assessed using real - time fluorescence imaging of dhr 123 or cm - h2dcfda . a and b : dhr 123 loaded neurons from acutely isolated lumbar drg neurons of control or stz - diabetic rats . values are means sem ( n = 5080 neurons , observed in five independently assessed control ( ctrl ) and diabetic ( db ) rats ) . d and e : cm - h2dcfda based ros imaging of sensory neurons from control and stz - diabetic rats . values are means sem ( n = 200230 neurons , observed in two independently assessed control and diabetic rats ) . g : ros generation is not elevated in isolated mitochondria from lumbar drg of diabetic rats compared with control . mitochondrial ros generation was measured at state 4 with substrates pyruvate and malate ( p + m ) in the absence or presence of antimycin a ( aa ) . h2o2 was measured fluorometrically in 0.1 mg / ml mitochondrial suspension with amplex red kits ( 50 mol / l amplex red and 0.5 units / ml horseradish peroxidase ) significance level was determined by two - way anova with bonferroni 's post hoc tests . * * p < 0.01 for control ( p + m ) vs. control ( p + m + aa ) . ( a high - quality color representation of this figure is available in the online issue . ) expression of components of the electron transport chain are reduced in drg from stz - diabetic rats . shown are representative blots ( a d ) and charts in which ndufs3 ( e ) , cox iv ( f ) , and atp synthase subunit signal ( g ) have been presented relative to total erk level . values are the means sem , n = 6 . * * p < 0.005 vs. control ( ctr ) , * p < 0.05 vs. db + ins ( one - way anova with tukey 's post hoc test ) . in this study , we show , we believe for the first time , that mitochondrial respiratory function , activity , and expression of components of the respiratory chain and the krebs cycle enzyme , citrate synthase , are decreased in drg sensory neurons from rats with type 1 diabetes . this novel observation provides direct functional evidence of diabetes - induced alterations in mitochondrial performance at multiple sites along the mitochondrial respiratory chain . previous studies have used only indirect assessments of mitochondrial inner - membrane polarization to analyze mitochondrial dysfunction in drg sensory neurons in diabetes ( 1719 ) . these observations imply that the diabetes - induced impairment of mitochondrial function is not associated with generation of oxidative stress . mitochondrial dysfunction has been proposed to mediate development of diabetes complications in endothelial cells , skeletal muscle , cardiomyocytes , and neurons ( 1214,27 ) . studies of isolated endothelial cells demonstrate that a short period of exposure to high glucose concentrations induces hyperpolarization of the mitochondrial inner membrane , which is a consequence of glycolysis - driven enhancement of electron donation from the tricarboxylic acid pathway ( 5 ) . it was therefore proposed that the high electrochemical potential difference across the mitochondrial membrane , driven by excessive proton gradient , prolonged the life of superoxide - producing electron transfer intermediates such as ubi - semiquinone ( 5 ) . however , recent studies have revealed that respiration and enzymatic activities are decreased in mitochondria from hearts of stz - diabetic rats ( 12,13 ) , whereas several studies have reported decreased activity of the mitochondrial respiratory chain and the krebs cycle enzyme , citrate synthase , in skeletal muscle of patients with type 2 diabetes ( 1416 ) . our data are consistent with these findings and indicate that decreased rates of respiration and activities of mitochondrial complexes and citrate synthase occur in nervous tissues exposed to prolonged diabetes in vivo . diminished mitochondrial respiratory function caused by diabetes has been investigated by proteomic and gene array techniques ( 28,29 ) . reduced expression of oxidative phosphorylation genes have been found in type 2 diabetes ( 30 ) , and decreased expression of the transcriptional regulator nrf-1 and the transcriptional coactivator peroxisome proliferator activated receptor- coactivator 1 ( pgc-1 ) were observed in pre - diabetic and diabetic muscle ( 31 ) . 6 are consistent with these findings and support the suggestion that reduced activity of the mitochondrial respiratory chain could result from a proteome alteration leading to reduced expression / activity of a range of mitochondrial components . studies in lens ( 32 ) , retina ( 33 ) , and cardiac tissue ( 34 ) in experimental animal models of type 1 diabetes show that parts of glycolytic pathway function are depressed . in diabetic rats , activity of 6-phosphofructokinase is reduced in nerve ( 35 ) , and activity of hexokinase , the first rate - limiting step in glycolysis , is reduced in drg ( 36 ) . diminished activity of glycolytic pathway enzymes may combine to reduce delivery of pyruvate and other glucose metabolism - dependent products to the tricarboxylic acid pathway ( 3234,37 ) , and it has been shown that pool sizes of products of glycolysis , such as pyruvate , are either unchanged or reduced in diabetes ( 33,38 ) . it therefore appears that rates of electron donation to the electron transport chain are suboptimal in adult diabetic rats and may predispose to lower rates of mitochondrial respiratory chain activity and oxidative phosphorylation . finally , a diabetes - induced rise in resting intracellular calcium to 200 nmol / l ( 39 ) can trigger elevated mitochondrial ca buffering by entry of ca through the ca uniporter and other routes , which can cause partial or complete inner mitochondrial membrane depolarization ( 27 ) . there is evidence of aberrant mitochondrial buffering of calcium in neurons from diabetic rats . plasma membrane depolarization - induced ca transients are prolonged in diabetic neurons and blockade of mitochondrial uptake of ca using the mitochondrial uncoupler , cccp , prevented these abnormalities ( 40 ) . this implies , indirectly , that abnormal mitochondrial buffering of ca plays a role in shaping ca transients in diabetic neurons . in our present study , impaired mitochondrial respiratory chain activity was not accompanied by increased ros production in neuronal perikarya from stz - diabetic rats , whereas generation of ros in mitochondria from stz - diabetic rats treated with pyruvate and malate in the presence of antimycin a was significantly decreased compared with control . in contrast , studies using cultures of embryonic sensory neurons showed that a high concentration of glucose directly triggers oxidative stress and apoptosis ( 8) , suggesting differences in ros production or management between embryonic and adult neurons in culture . we have recently found that ros levels and adducts of 4-hydroxy-2-nonenal are elevated in the presence of high glucose in axons of adult sensory neurons isolated from 3- to 5-month stz - diabetic rats , with the perikarya being unaffected ( 41 ) . oxidative damage to lipids in the sciatic nerve was increased in the present study , supporting previous work ( 9,42,43 ) . these results suggest that mitochondrial function may differ between axons and perikarya or that oxidative stress in axons and other cells of the nerve trunk may derive from alternative sources . our studies were performed in rats that showed relatively mild stz - induced diabetes , as indicated by maintenance of starting body weight over a long period of time . marked hyperglycemia and increased a1c indicate that these rats were clearly diabetic and established biochemical consequences of hyperglycemia such as nerve polyol pathway flux ( indicated by nerve fructose accumulation ) and lipid peroxidation were present . this model contrasts with the acute and extreme stz - induced insulin - deficient diabetes frequently studied in rats and mice that may not adequately model long - term diabetes in well - managed patients . the absence of significantly reduced axonal caliber in myelinated fibers , altered thermal nociception , or loss of ienf after 22 weeks of diabetes confirms that stz lacked direct neurotoxic effects and emphasizes the relatively mild nature of the diabetes in our rats , since all of these disorders have been widely reported in previous stz - diabetic rodent studies . we do not suspect a lack of discrimination in our assays , as we have previously reported reduced axonal caliber ( 44 ) , thermal hypoalgesia ( 25 ) , and ienf loss ( 26 ) in stz - diabetic rodents . given that the rats in our present study showed marked hyperglycemia , polyol pathway flux , and lipid peroxidation , the data may suggest that additional risk factors may be required in concert with these established biochemical disorders to generate the indices of neuropathy described above . mitochondrial dysfunction appeared only after 16 weeks of diabetes and therefore represents a metabolic disorder of neurons that can emerge before onset of functional and structural manifestations of diabetic neuropathy . interestingly , abnormalities in calcium homeostasis also appear after 812 weeks of diabetes and could possibly preempt mitochondrial dysfunction ( 39 ) . the pathogenesis of this mitochondrial disorder may involve insulin deficiency per se , since our insulin treatment regime for the last month of diabetes had minimal effects on indices of systemic diabetes such as body weight , plasma , nerve sugar levels , lipid peroxidation , and a1c , but had marked effects on mitochondrial dysfunction . these findings agree with prior reports that insulin has direct effects on mitochondrial function ( 17,45 ) . insulin treatment also increased nerve total protein and ienf values above those of age - matched control rats , which may reflect the capacity of insulin to induce sensory neuron axonal sprouting ( 46,47 ) . the contribution of impaired insulin signaling to complications of both type 1 and type 2 diabetes , either independent of , or in concert with , hyperglycemia is clearly an area of emerging interest that deserves further study ( 11,4648 ) . in conclusion , our data show that mitochondrial respiratory chain function and activity / expression of mitochondrial complexes as well as the krebs cycle enzyme , citrate synthase , are decreased in mitochondria isolated from drg of stz - diabetic rats and can be normalized by insulin at levels that do not markedly alter the consequences of hyperglycemia . the appearance of mitochondrial dysfunction at a time before onset of overt functional or structural neuropathy indicates the possibility of a pathogenic role for insulin deficiency in diabetic neuropathy that is independent of hyperglycemia .

objectiveimpairments in mitochondrial physiology may play a role in diabetic sensory neuropathy . we tested the hypothesis that mitochondrial dysfunction in sensory neurons is due to abnormal mitochondrial respiratory function.research design and methodsrates of oxygen consumption were measured in mitochondria from dorsal root ganglia ( drg ) of 12- to- 22-week streptozotocin ( stz)-induced diabetic rats , diabetic rats treated with insulin , and age - matched controls . activities and expression of components of mitochondrial complexes and reactive oxygen species ( ros ) were analyzed.resultsrates of coupled respiration with pyruvate + malate ( p + m ) and with ascorbate + tmpd ( asc + tmpd ) in drg were unchanged after 12 weeks of diabetes . by 22 weeks of diabetes , respiration with p + m was significantly decreased by 3144% and with asc + tmpd by 2939% compared with control . attenuated mitochondrial respiratory activity of stz - diabetic rats was significantly improved by insulin that did not correct other indices of diabetes . activities of mitochondrial complexes i and iv and the krebs cycle enzyme , citrate synthase , were decreased in mitochondria from drg of 22-week stz - diabetic rats compared with control . ros levels in perikarya of drg neurons were not altered by diabetes , but ros generation from mitochondria treated with antimycin a was diminished compared with control . reduced mitochondrial respiratory function was associated with downregulation of expression of mitochondrial proteins.conclusionsmitochondrial dysfunction in sensory neurons from type 1 diabetic rats is associated with impaired rates of respiratory activity and occurs without a significant rise in perikaryal ros .

RESEARCH DESIGN AND METHODS Induction, treatment, and confirmation of type 1 diabetes. Preparation of tissue homogenates and isolated mitochondria from lumbar DRG. Western blotting for mitochondrial proteins. Mitochondrial respiration assay. Determination of enzymatic activities of mitochondrial complexes and citrate synthase. Analysis of ROS measurement in adult sensory neurons. Measurement of ROS in mitochondrial preparations from lumbar DRG. Nerve structure and function. Data analysis. RESULTS DISCUSSION Supplementary Material

PMC5282602

the establishment of the primary dna sequence of a species is a key point in modern research . it permits to obtain subsequently further insights on the functionality of the genes present in a genome . the first plant genome sequenced was that of the eudicot model plant arabidopsis thaliana , which was released in 2000 . from this date , and mainly after the appearance of next generation sequencing technology in 2005 , the number of sequenced plant species has exponentially increased . in recent years , several reviews had targeted the state of art of the plant sequencing projects [ 1 - 4 ] . among the sequenced species , several algae genomes , a moss , a lycophyte , three conifer and examples from the majority of the monocot and eudicot lineages have been sequenced [ 4 , 5 ] . interestingly , the vast majority of economical important crops have been sequenced and drafts of their genomes have been produced . once the genomic sequence has been parsed , assembled and annotated ; the corresponding data has been traditionally compiled in a database available for the scientific community . the importance of the creation of an open - access database comes from the rationale of the biological exploitation of the novel genomic resources generated by the researches . basic considerations on dealing with crop plant databases utilization in the genomic era has been recently reviewed . in this mini - review , the first step in the valorization of a genomic project is to know the availability of related projects completed or in progress . these searches can be done by two methods . by searching in the internet motor searches using key words or by exploring the specific databases related to genomic projects . although the information in these databases is not always up to date , second method is more adequate to obtain the best results . there are four main genomic projects databases that can be used when information on plant genomes is required , gold ( genomes online database ) , ncbi genomes , cogepedia and plabi ( table 1 ) . is a world wide web resource for comprehensive access to information regarding genome and metagenome sequencing projects , and their associated metadata . gold is now hosted by the jgi doe institute and the current release is the version 5 . the database currently hosts information for more than 20,000 studies , 60,000 biosamples , 60,000 sequencing projects and 50,000 analysis projects . one of the specific features of gold is that it includes nuclear and organelle genome projects , but also transcriptomic , methylation , exome and re - sequencing projects . around one hundred and more than 3,400 finished or ongoing projects involve species from the phyla chlorophyta and streptophyta , respectively . gold is manually curated , their stored metadata are quality - controlled , and fully supports and follows the genomic standards consortium ( gsc ) minimum information standards . ncbi genomes is a resource of the ncbi ( national center for biotechnology information ) which organizes information on genomes including sequences , maps , chromosomes , assemblies , and annotations . the ncbi genome database collects genomic sequencing projects for a given species and provides links to corresponding records in bioproject , assembly , nucleotide and protein databases . besides , a list of the current status of all genomes annotated at ncbi is provided . currently , more than 2,200 eukaryotic genomes have been deposited , and from that , more than 200 corresponds to plant species . the database provides a versioned assembly accession number that tracks changes to assemblies as they are updated by submitting groups over time . the assembly database reports metadata such as assembly names , simple statistical reports of the assembly as well as the assembly update history . links in the assembly resource allow users to easily download sequence and annotations for current versions of genome assemblies from the ncbi genomes ftp site . 2.3.cogepedia is the official wiki page of coge , a platform for performing comparative genomics research which provides a network of interconnected tools to manage , analyze , and visualize next generation sequenced data . their interest to plant genomics is the existence of a link in this page with much updated information on sequenced plant genomes . individual information on each species with links to the genome publication or the genome project main page is provided . 2.4.plabi is a plant genomic database with a very updated tool to know which plant species have been sequenced . links to the research articles where each plant genome has been published are also included . is a world wide web resource for comprehensive access to information regarding genome and metagenome sequencing projects , and their associated metadata . gold is now hosted by the jgi doe institute and the current release is the version 5 . the database currently hosts information for more than 20,000 studies , 60,000 biosamples , 60,000 sequencing projects and 50,000 analysis projects . one of the specific features of gold is that it includes nuclear and organelle genome projects , but also transcriptomic , methylation , exome and re - sequencing projects . around one hundred and more than 3,400 finished or ongoing projects involve species from the phyla chlorophyta and streptophyta , respectively . gold is manually curated , their stored metadata are quality - controlled , and fully supports and follows the genomic standards consortium ( gsc ) minimum information standards . ncbi genomes is a resource of the ncbi ( national center for biotechnology information ) which organizes information on genomes including sequences , maps , chromosomes , assemblies , and annotations . the ncbi genome database collects genomic sequencing projects for a given species and provides links to corresponding records in bioproject , assembly , nucleotide and protein databases . besides , a list of the current status of all genomes annotated at ncbi is provided . currently , more than 2,200 eukaryotic genomes have been deposited , and from that , more than 200 corresponds to plant species . the database provides a versioned assembly accession number that tracks changes to assemblies as they are updated by submitting groups over time . the assembly database reports metadata such as assembly names , simple statistical reports of the assembly as well as the assembly update history . links in the assembly resource allow users to easily download sequence and annotations for current versions of genome assemblies from the ncbi genomes ftp site . 2.3.cogepedia is the official wiki page of coge , a platform for performing comparative genomics research which provides a network of interconnected tools to manage , analyze , and visualize next generation sequenced data . their interest to plant genomics is the existence of a link in this page with much updated information on sequenced plant genomes . individual information on each species with links to the genome publication or the genome project main page is provided . 2.4.plabi is a plant genomic database with a very updated tool to know which plant species have been sequenced . links to the research articles where each plant genome has been published are also included . formerly , species specific databases were developed . the arabidopsis initiative resource , tair ( http://www.arabidopsis.org/ ) was the first database created to harbor the genomic sequence of a plant and to surround it with different tools to explore this sequence . these tools have been increasing with the subsequent releases of the genomic sequence and cover different aspects related not only with the arabidopsis genome , but with some other features such as germplasm , teaching , laboratory protocols or gene expression experiments . with the increasing of genome projects regarding various related species , databases from specific clades this database comprises the reference genomes of tomato , potato , pepper and solanum pennelli ; the draft genomes of solanum pimpinellifolium , nicotiana benthamiana and nicotiana tabaccum ; and the genomes of two inbred lines of solanum lycopersicum . as for individual databases , a set of tools have been implemented to analyze the genomic sequences . the individual / clade databases hosting the approximately 140 plant species sequenced to date can be easily found by writing the word genome and the species / clade in any internet search engine . at the same time , secondary databases have arisen to deal with features regarding comparative plant genomics research . these databases harbor different plant genomes and differ in both , the plant species they host , and the tools and resources they have . the aim of these projects is to explore how genomics is evolving from largely descriptive to highly predictive driven by quantitative measurements , with algorithms and computation as the domain - adapted language . several tools are common to all databases . for example , sequence similarity searches are the most reliable strategy to identify homologous proteins or genes by detecting statistically significant similarity . all databases include in their repertoire of bioinformatics tools a search engine to explore the annotated genome sequences , typically using the blast algorithm . likewise , genome browsers , such are the commonly used gbrowse or jbrowse , are key tools offered by all databases to provide a graphical interface for users to browse , search , retrieve and analyze genomic sequence and annotation data . because of their importance in modern research , several of the comparative genomic databases ensembl plants is an integrative resource presenting genome - scale information for 39 sequenced plant species , including 12 eudicots , 21 monocots , one moss , one pseudofern , two green algae , one red algae and the genome of amborella trichopoda , which belongs to a sister clade of the other angiosperm species . data provided includes genome sequence , gene models , functional annotation , and polymorphic loci . gene families , based in an all - versus - all blastp alignment , are provided . gene trees showing the evolutionary history of each gene family are available under the plant compara section . access to the data is provided through a genome browser with tracks displaying genome sequence and assembly information , additional gene model and variation datasets , and precomputed sequence alignments including ests , rnaseq experiments , repeat features , oligo - probe , and marker sets . ensembl plants is updated 45 times a year and is developed in collaboration with the gramene database and the transplant project ( http://www.transplantdb.eu ) that aims to facilitate the exchange and integration of plant genome data from distributed resources as well as the development of common standards and protocols . gramene is a curated online resource for comparative functional genomics in crops and model plant species , currently hosting 45 sequenced reference genomes in its build number 48 . since 2009 gramene has partnered with the plants division of ensembl genomes ( http://www.plants.ensemb.orgl/ ) to jointly produce the genome browser described above , which takes advantage of the ensembl infrastructure and provides an interface for exploration of genome features , functional ontologies , variation data and comparative phylogenomics . gramene includes a wide array of potentially useful tools such as genetic and physical maps with genes , ests and qtls locations , genetic diversity data sets , structure - function analysis of proteins , plant pathways databases ( biocyc and plant reactome platforms ) , and descriptions of phenotypic traits and mutations . plant gdb is a web resource devoted to develop robust genome annotation methods , tools , and standard training sets for plant genomes . from 2012 plant gdb also provides annotated transcript assemblies for more than 250 plant species , with transcripts mapped to their cognate genomic context where available , integrated with a variety of sequence analysis tools and web services . plant gdb hosts a plant genomics research outreach portal that facilitates access to a large number of resources for research and training . from july 2015 the plant gdb 's funding has ended and the website is no longer being updated . 3.4 . plants db is a database that has been developed by the plant genomics group of the pgsb ( plant genome and systems biology , formerly mips ) . plants db aims to provide a data and information resource for individual plant species , especially complex triticeae genomes , and currently hosts 13 monocot and dicot species . the database framework integrates genome data from both model and crop plants and facilitates knowledge transfer between them using state - of - the - art comparative genomics tools such as crowsnest , created to visualize and investigate syntenic relationships between monocot genomes , and the genome zipper concept for an ordered gene annotation in cereals . phytozome is the plant comparative genomics portal of the department of energy 's joint genome institute ( doe jgi ) . in the current release v10.3 , phytozome provides access to sixty - one sequenced and annotated green plant genomes , forty - seven of which have been clustered into gene families at 12 evolutionarily significant nodes . it harbors the individual species genomic sequences compiled in the doe jgi , and links to the individual pages of the other genomes it hosts . families of related genes representing the modern descendants of ancestral genes are available . they have been constructed from an all - versus - all blastp alignment used to compute the evolutionary distance between each two proteins , the identification of orthologs via reciprocal best hit or synteny analysis , and the accretion of paralogs using outgroup scores . these families allow easy access to clade - specific orthology / paralogy relationships as well as insights into clade - specific novelties and expansions . each gene has been annotated with available pfam , kog , kegg , panther and go assignments , and its evolutionary history at the level of sequence , gene structure , gene family and genome organization is provided . besides , phytozome provides access to the plant genomes it hosts using the jbrowse genome browsers available for all genomes . plaza is a platform designed to make comparative genomics in plants and developed in the university of ghent . the current version plaza 3.0 hosts 37 plant species covering a broad taxonomic range and includes 25 eudicot , 8 monocot , one moss and two algae species , as well as the genome of amborella trichopoda . plaza provides detailed structural and functional annotation of the genes , which has been expanded in the new version and now comprises data from gene ontology , mapman , uniprotkb / swiss - prot , plntfdb and planttfdb . from the more than one million genes annotated in the genomes it harbors , gene families and subfamilies have been delineated . first , by computing the protein sequence similarity through an all - against - all blast , and then by applying graph - based clustering methods implemented in tribemcl and orthomcl . other relevant data available in this database consist of phylogenetic trees to identify biologically relevant duplication and speciation events , and detailed information about genome organization to unveil small and large genome duplication events . furthermore , this database provides tools to transfer functional annotation from well - characterized plant genomes to other plant species . greenphyldb is a comparative genomics database jointly developed by biodiversity international and the international cooperation center for agricultural research for development ( cirad ) . the current version greenphyldb 4.0 hosts 37 species of the plantae kingdom including one red alga , two green algae , one moss , one lycophyte , one conifer , the ancestral angiosperm amborella , ten monocots and 20 eudicot species . this software uses different pairwise similarity matrices obtained by running protein - protein blast using increasing stringent thresholds . then , these matrices are used by a markov cluster algorithm to group proteins in families at different levels of clustering ( 1 to 4 ) . results of the automatic clustering are manually annotated using cross reference databases and analyzed by a phylogenetic - based approach to predict homologous relationships . currently , greenphyldb contains 8,347 clusters with more than 5 sequences at level 1 , from which 2,939 are annotated and 4,788 have available phylogenetic trees . for each gene cluster , this database offers an easy access to the gene composition by species , and provides protein domains , orthologous gene predictions and relevant external links . plantordb is a genome - wide database created to classify genes in families and to find orthologous genes clusters . the recently launched first version of plantordb hosts 41 species including six green algae , one moss , one lycophyte , six monocots and 27 eudicot species . gene families are available , which have been generated from an all - against - all blast search approach . the web interfaces provided by plantordb display information on the evolutionary features of an individual gene and its homolog gene family , which can be deduced from multiple sequence alignments and phylogenetics trees . tools to provide an accurate classification of a query sequence within a phylogenetic tree and a multiple sequence alignment are also available . salad is a comparative genomics database constructed from plant - genome - based proteome data sets . the version 3.0 , appeared in 2009 , hosts 10 species including a yeast species . the most important singularity of this database is the construction of dendrograms for protein families using the information derived from conserved motifs discovered using the meme software . protein clusters are determined from blastp searches and , then , meme motifs are used to select the proteins displayed in the dendrograms . a viewer to see microarray data sets of paralogous genes in the dendrograms is also provided . planttribes is a gene family resource for comparative genomics in plants that launched its current 2.0 version in 2007 . it harbors the proteome of ten plant species , from green algae to angiosperms , from which putative gene families were created by a blastp approach and the use of the mcl algorithm for clustering . the database is currently active and permits identify groups of related genes and their expression patterns , using the microarray information existing at the date the current version was released . plantgenie.org is a collection of web resources for searching , visualizing and analyzing genomics and transcriptomics data recently developed for different plant species . currently , it includes dedicated web portals for enabling in - depth exploration of poplar , norway spruce , and arabidopsis genomes . standard features , including genome browsers , gene list annotation , blast tools and gene information pages are provided . the aim of this database is to continue and develop the resource by inclusion of additional species , maintaining a focus on woody species . pogs2 is a relational database designed to facilitate cross - species inferences about gene functions and gene models in plants . in its current 2.0 version , the database integrates data from rice , maize , arabidopsis thaliana , and poplar by placing the complete predicted proteomes into putative orthologous groups each pog entry includes putative orthologs annotated with gene descriptions imported from species - specific databases , graphical representations of conserved protein domains and phylogenetic trees showing closely - related proteins . extant genomes of 16 eudicot and 10 monocot plants can be analyzed , as well as 23 ancestral reconstructed genomes . in the current 16.03 version , all the data on extant species come from the annotations of selected genomes available in ensembl plants and phytozome . a distinctive feature of this database is that the displayed genomic context of a gene is showed in parallel to the genomic context of all its orthologous and paralogous copies evolutionarily ordered in a phylogenetic tree . piece is a comparative genomics database focused in the comparison of exon - intron plant gene structures and their evolutionary and functional relationships . in the first and current version , annotated genes were extracted from 25 species , from green algae to angiosperms , and classified based on pfam motifs . phylogenetics trees are available for each gene family , which integrate exon - intron and protein motif information . both the sequences and gene structure information for each identified gene are also available . plantseed is a database created to support subsystems - based annotation and metabolic model reconstruction for plant genomes . annotations of protein families from ensembl plants for five eudicot and five monocot species have been used to construct subsystems , which are the integration of metabolic pathways or other biological processes with genome annotations . the manual curation of the subsystems permits a rapid reconstruction and modelling of primary metabolism for all plant genomes in the database . pgdbj is the plant genome database japan , a portal website that aims to integrate plant genome - related information from databases . the main tool developed in this web is the ortholog db , which comprises clusters of homologous sequences . clusters were obtained from reciprocal blast searches using the proteome of 20 plant species from the main clades of the viridiplantae kingdom . pgdbj also provides dna marker and qtl information of important agronomic traits for many plant species . the new high - throughput genomic methods implies the generation of massive data . computation capability is a major challenge for the handling of these data . the accessibility to computational tools easy to manage for a scientist without programming or informatics expertise is a solution to address this problem . in recent years , several web - based platforms have been created to store next - generation sequencing data and to host different applications to work with these data to extract biological relevant information . galaxy is an open web - based platform for genomic research with many tools to work with computation - reliant results . recently , an open - source project specific for plants , the iplant , has been launched . the iplant collaborative ( https://de.iplantcollaborative.org/ ) is a united states national science foundation ( nsf ) funded project created as an innovative , comprehensive , and foundational cyberinfrastructure in support of plant biology research . iplant includes the use of high - performance computing , use of large shared data storage , and the establishment of collaborations and virtual organizations around shared analysis tools and analyzed data . iplant hosts bioinformatics tools for most of the modern research challenges , such as data importers , sequence alignments and phylogenetic tree building , phylogenetic and evolutionary analyses , qtl mapping and genome - wide association studies , ultrahigh - throughput sequence processing , functional analyses , clustering and network analyses , variant detection and annotation , rnaseq analyses and chip - seq studies . in particular , to manage and serve published plant genome data , iplant has developed a workspace named dna subway . dna subway has been conceived to be a space for gene annotation and genome analysis that currently hosts tools to predict and annotate genes , prospect entire plant genomes for related genes and sequences , determine sequence relationships and analyze rna - seq reads to measure differential expression . to prospect genomes , it harbors the target program that permits a search for homologous sequences in 18 plant genomes , covering different clades from algae to angiosperms , and builds multiple sequence alignments and phylogenetic trees with the obtained sequences . bioinformatic programs and algorithms have arisen as key tools for modern research . in plants , the number and relevance of these tools have been increased in recent years , mainly those related to genomics aspects . new science implies the existence of databases where the genomes are hosted and exhaustive analysis can be performed . although these databases should be continuously updated , the assembly and annotation of many of the draft genomic sequences have not been improved from its first release . this is an actual challenge , since it is easy now to produce a new draft sequence but more complicated to improve its quality . for example , the pre - release of the version number 11 for the arabidopsis genome has appeared in araport ( https://www.araport.org/ ) in october 2015 with genomic sequences assembled in its five chromosomes . on the contrary , the first release of the genome of the lycophyte selaginella moellendorffii appeared in 2007 assembled into 768 scaffolds and has not been subsequently updated . as a next step in plant genomics , new databases have been developed to deal with the issue of comparing the increasing number of sequenced genomes . most comparative genomic databases are periodically updated and have become powerful tools to extract shared information coming from evolutionarily related plant species . however , the extremely high new information obtained in modern laboratories using computational approaches makes necessary the existence of new high - performance computational developments that uses the computational power of the internet and the storage of resources in the cloud . as a new challenge , actual projects in arabidopsis and rice are directed to the comparison of thousands of genomes obtaining from different genotypes of the same species . innovative solutions for storage and visualization , such as the rice snp - seek database ( http://oryzasnp.org/iric-portal/ ) , have to be developed to use efficiently the massive new information obtained . as an example of the continuous evolution of genomic databases , the iplant collaborative resource has evolved into the cyverse cyberinfrastructure , which provides tools not only for plant research ( http://www.cyverse.org/ ) .

in recent years , the genomic sequence of numerous plant species including the main crop species has been determined . computational tools have been developed to deal with the issue of which plant has been sequenced and where is the sequence hosted . in this mini - review , the databases for genome projects , the databases created to host species / clade projects and the databases developed to perform plant comparative genomics are revised . because of their importance in modern research , an in - depth analysis of the plant comparative genomics databases has been performed . this comparative analysis is focused in the common and specific computational tools developed to achieve the particular objectives of each database . besides , emerging high - performance bioinformatics tools specific for plant research are commented . what kind of computational approaches should be implemented in next years to efficiently analyze plant genomes is discussed .

OVERVIEW GENOMIC PROJECTS DATABASES Gold PLANT GENOMIC DATABASES EMERGING HIGH-PERFORMANCE COMPUTING WEB RESOURCES CONCLUDING REMARKS NOTE ADDED IN PROOF CONFLICT OF INTEREST

PMC4003750

organizational culture is defined as the ways in which people know and understand the values and beliefs of a specific group of people or an institution . these values and beliefs are established over time , are considered valid , and are taught to new members who enter into the culture [ 1 , 2 ] . organizational beliefs and values are guiding principles that influence the development of individuals ' attitudes towards the organization and how individuals within that culture make decisions or invest their time . academic nursing programs are situated within challenging and complex organizational cultures characterized by a combination of the traditional research , teaching , and service requirements of academia coupled with professional practice requirements . added to the complex culture of academic nursing is the integration of new technology in conjunction with a demand for diverse teaching strategies . simulation , specifically using mid- to high - fidelity equipment , which closely mimics real life experience without the real life risks , is considered new technology . although simulation has been used in nursing education programs since the early nineteen hundreds [ 7 , 8 ] , advances in the technological capabilities and the types of simulator equipment available today reinforce the notion of simulation being viewed by nursing faculty as a new teaching strategy . additionally , there have been inconsistent levels of adoption and incorporation of simulation among individual nurse educators and across nursing programs . individual factors such as faculty members ' attitudes and perceptions of simulation have been studied [ 1012 ] , but there is a gap in the literature related to how organizational culture shapes and contributes to attitudes , perceptions , and behaviors that impact the adoption and incorporation of simulation into nursing curricula . akhtar - danesh and colleagues conducted a q - methodology study in canada of nursing faculty members ' perceptions related to the adoption and integration of simulation . benefits included aligning with students ' positive perceptions about the use of simulation as a valuable teaching strategy , but challenges were identified about the usefulness and practicality of simulation in undergraduate education . other researchers have reported that having the time to learn about and use simulation as well as having the resources to coordinate simulation - based education are the most significant barriers related to adoption and incorporation [ 1214 ] . in the nursing education literature , identification of the individual perceptions , values , and concerns that influence the uptake of simulation have been well documented . while there has been some preliminary work to identify organizational influences on this issue such as the lack of faculty time and resources to implement this new technology [ 1214 ] , what has been consistently lacking is an examination of the organizational culture that leads to the development of attitudes , values , beliefs , and perceptions among nursing faculty as they respond to change or adopt an innovation . there is more to integrating simulation into nursing curricula than faculty buy - in . the choices people make and the activities they invest in are not always guided by personal choice but by the culture of the organization . therefore , it is important to examine how organizational factors impact the adoption and incorporation of simulation as a teaching strategy as it is becoming more prevalent in nursing education and as institutions look for ways to facilitate this change . schein 's theory of organizational culture was used to sensitize the researcher about concepts of organizational culture . this theory provided both a clear definition of organizational culture and distinguishable levels of culture that should be considered . although grounded theory research is not typically conducted using an existing theoretical framework , schein 's model was used to ensure exploration of core constructs associated with organizational culture . schein defines organizational culture as the ways of knowing within a specific group that have been established over time and have been shown to be effective in managing problems . they are considered valid , taught formally and informally to new members , and include both explicit and tacit knowledge . schein suggests there are three major tenets to consider when analyzing organizational culture : artifacts , espoused beliefs or values , and basic assumptions , each representing different levels of culture . artifacts are predominantly the observed characteristics of organizational culture such as building design and structure and dress norms ; these represent the most superficial level of organizational culture . schein suggests that the best way to understand organizational culture is to examine it at deeper levels . beliefs and values are considered legitimate and significant guiding principles of an institution [ 1 , 2 ] . they are the goals , aspirations , and ideologies that signify what is most important to an organization or for what the organization is ultimately striving . beliefs and values are often represented in public statements such as mission and vision statements . at the deepest level of schein 's model are the basic assumptions which are thought to guide actions , contribute to attitudes and behaviours , and impact thoughts and feelings . nursing programs in ontario , canada , provided ideal circumstances in which to consider this issue . in 2004 , the ontario ministry of health and long - term care demonstrated a commitment to nursing education through a $ 20 million investment for the purchase of simulation equipment for undergraduate nursing programs . monies were distributed to all 34 programs , ranging from $ 196,300 to $ 706,400 , with the average institution receiving $ 500,000 . simulation equipment was the only allowable expense and nursing programs were expected to secure funds to ensure the continued operation and use of the equipment . charged with this expectation , nursing programs had to undergo a process of adopting and incorporating simulation into the curricula . so many nursing programs dealing with this initiative at the same time provided a good opportunity to examine the organizational elements that impacted this change . as the focus of the funding was mid- to high - fidelity simulation equipment , virtual simulations and standardized patients were excluded from this study . charmaz 's approach served to answer the research question : how do the organizational cultures of ontario undergraduate programs of nursing shape the adoption and incorporation of simulation as a teaching and learning strategy within the curricula ? , colleges in ontario have worked in partnership with universities to offer collaborative nursing programs where students can obtain a baccalaureate degree . collaborative programs are offered as articulated or integrated models . in articulated collaborations , the first two years of the nursing program are offered at the college and the second two years are offered at the university . in integrated collaborations , the delivery of the program happens at all of the sites , and the faculty members from both the college and university are involved throughout each year of the program . nursing programs were selected based on the following criteria : ( a ) listed on the college of nurses of ontario web site as an accredited program , ( b ) offered a four - year baccalaureate degree in english either on its own or in collaboration with another educational institution located within ontario . nursing programs that met the inclusion criteria were separated into geographical regions and then selected by stratified randomization to increase variability and reduce bias . theoretical sampling as a form of purposeful sampling was used to ensure the core constructs and the deepest levels of organizational culture were examined . the examination of artifacts was excluded from this study since there was an absence of equal opportunities to examine the artifacts at all of the sites . knowledge about the beliefs and values was obtained by studying guiding documents such as the mission , vision , or philosophy statements at the level of the institution , the faculty , and the nursing program . sampling to investigate basic assumptions , the deepest level of organizational culture , occurred at the level of the nursing program through in - depth semistructured interviews with participants . participants from multiple roles within the program included nursing administrators , nursing faculty members , and key simulation leaders , thereby facilitating maximum variation sampling . snowball sampling was used to identify additional participants and documents [ 21 , 22 ] . theoretical sampling was used as data collection progressed and categories and themes started to emerge . this continued until data were sufficient to provide detailed descriptions of the experience from the participants ' perspective . that is , sampling continued until theoretical sufficiency was obtained [ 19 , 23 ] . this included up to two in - depth semistructured interviews with nursing administrators , nursing faculty members , and simulation leaders . first interviews lasted approximately 6075 minutes and explored organizational factors involved in the processes of adoption and incorporation of simulation . second interviews , lasting 3060 minutes , were conducted to further develop the categories emerging from the data . to gain a rich description of the organizational culture , these documents were compiled and sent to participants in advance of the interview . during the interviews , participants were asked if and how the documents shaped the adoption and incorporation of simulation into the curricula . participants were also asked if any other documents contributed to this process and to share those documents when possible . nvivo 9 qualitative software was used to store , organize , and manage all data . interviews were transcribed verbatim ; then , data were analyzed line - by - line and incident - by - incident resulting in multiple codes , many of which were unique terms used by the participants ( in vivo codes ) . the initial codes moved to focused codes that served to develop analytical categories for further examination and exploration . once the tentative categories were further explored and developed , they were raised to conceptual categories which are more abstract and led to the nascent structure of the theory . documents were analyzed for key concepts and compared to interviews to see if the concepts were captured implicitly , explicitly , or at all . during data analysis a seven - phase process of adoption and incorporation emerged which resulted in the differentiation of high- , mid- , and low - uptake sites . the constant comparative method was used to explore how organizational culture impacted or shaped the process . to facilitate this , creating memos and theoretical sampling were used to capture thoughts , define codes , illuminate properties , make connections , and explore emerging themes . during this process this allowed for further exploration and expansion of emerging categories until theoretical saturation was reached . participation was voluntary and participants could withdraw from the study at any time or choose not to answer questions . identifying information representatives of thirteen nursing programs across the province of ontario , including seven universities and six colleges , participated in this study . of these sites seven were identified as high - uptake , five as mid - uptake , and one as low - uptake . 55.5% of participants worked in the high - uptake sites , 40.7% at mid - uptake sites , and 3.7% at the low - uptake site . their roles included simulation leaders ( n = 6 ) , nursing administrators ( n = 5 ) , nursing faculty ( n = 12 ) , and those serving in a combined role ( n = 4 ) . all had a baccalaureate degree with the majority having a master 's degree ( 85.1% ) ; 14.8% had a phd . the majority ( 75% ) were between the ages of 41 and 60 years . refer to table 2 for a summary of the type of documents and the level of the institution they represent . to explain how educational organizational cultures influence the adoption and incorporation of simulation , a theoretical model identified as the organizational elements that shape simulation in nursing ( oessn ) model , figure 1 , was developed . a brief description of the full model will be followed by an in - depth explanation of each of the key organizational components . the model displays concentric circles that represent the different levels of the organization that impact the adoption and incorporation of simulation into nursing curricula . the next circle represents the faculty of health ( or otherwise named ) and the innermost circle represents the nursing program . each of these three levels has within it values and beliefs , which are termed guiding philosophies , and contributes to the organizational culture of the nursing program . within the level of the nursing program , five key elements of the organizational culture emerged . they represent what schein asserts are basic assumptions in that they influenced perceptions and attitudes and impacted behaviours and actions related to the adoption and incorporation of simulation . they are ( a ) nursing leaders , ( b ) information exchange , ( c ) physical locale , ( d ) shared motivators , and ( e ) scaffolding to manage change . the central organizational element among the five was the nursing leaders who were typically nursing administration and the key simulation person ( simulation leader ) . within the model , there is an arrow from the nursing administrator to simulation leader indicating the distribution of power required in the creation of a new role to manage the simulation initiative . the five key organizational elements impacted the seven - phase process of adoption and incorporation , represented in the model by the large downward arrow . the phases include ( a ) securing resources , ( b ) nursing leaders working in tandem , ( c ) getting it out of the box , ( d ) learning about simulation and its potential for teaching , ( e ) finding a fit , ( f ) trialing the equipment , and ( g ) integrating into the curriculum . the seven - phase process leads directly to the levels of uptake which occurred across a continuum . high - uptake was defined as nursing programs that moved through all of the seven phases of the process and successfully integrated simulation into all levels of the curricula in which nursing content is taught . institutions that met some , but not all , phases of the process were classified as mid - uptake and institutions that were not able to progress through any of the phases were classified as low - uptake . institutions that were classified as high - uptake experienced more outcomes than the other sites and the outcomes had the potential to cross all levels of the organization . the remainder of this paper will provide in - depth explanations of the organizational elements that shape the adoption and incorporation of simulation into nursing curricula and will , at times , be discussed in relation to the level of uptake . they were selected amongst all of the data to illustrate or provide an example of the broader context being described . before discussing each of the organizational components in detail , it is useful to discuss the relationship between the level of uptake and the guiding philosophies . the guiding philosophies , in the form of mission , vision , value , or philosophy statements , impacted on nursing programs ' capacity to adopt and incorporate change . these beliefs and values influence actions and decision making when managing change . the explicit knowledge and use of the organizational guiding philosophies varied considerably across the uptake continuum . many of the high - uptake sites consciously used the mission and vision statements of the institution , faculty , and nursing program as a method to make decisions and situate simulation within the curricula . when asked about adopting and incorporating simulation , one simulation leader indicated this by stating : [ simulation ] relates to the mission , it 's innovative being involved in things that are innovative is part of our mission ( 006 ) . specifically , the terms innovative , research focused , experiential learning , and quality of students ' experiences , as well as the institutional teaching and research components facilitated decision making with regard to simulation . nursing leaders used the guiding philosophies as a strategy for change but also as a tactic to acquire necessary resources . this is represented by the two - way arrow in the oessn model between the leaders and the concentric circles representing the guiding philosophies . one nursing simulation leader reinforced this when stating:so anything that we do has to somehow relate if we 're asking for resources , financial or human , it has to relate to the mission , vision , or values in order to be accepted . so any proposal that i bring forward , i ensure that it relates to those statements and i include rationale as to how this meets our mission , vision , or values ( 002 ) . if we 're asking for resources , financial or human , it has to relate to the mission , vision , or values in order to be accepted . so any proposal that i bring forward , i ensure that it relates to those statements and i include rationale as to how this meets our mission , vision , or values ( 002 ) . in the mid - uptake sites the impact of guiding assumptions was much more implicit . participants used language such as cutting edge , engaging students in learning , and evaluating simulation during the interviews that mirrored or resembled the key concepts of the institutional , faculty , or nursing program guiding documents . when questioned directly about the influence these documents had on the adoption and incorporation of simulation , participants needed to refer back to the guiding documents in order to make connections . one simulation leader emphasized this stating : i 'm sitting here looking at the two mission statements on the other end of the spectrum is the low - uptake site . when questioned about how the guiding philosophies shape the adoption and incorporation of simulation a nursing a faculty member responded saying there is nothing in our statements specific to simulation ( 027 ) . the findings show that it is was not necessarily the specific language of the underlying beliefs and values but the way in which leaders and nursing faculty members chose to use them that shaped the adoption and incorporation of simulation . with an initiative as extensive as this one , many nursing administrators had this insight realizing they would not be able to do it all themselves , so simulation leaders were appointed or volunteered . this was the beginning of the shared leadership that has shaped the adoption and incorporation of simulation in nursing curricula . this was noted by one simulation leader as follows : our former chair was instrumental in giving me . the green light to go ahead and take the lead on this ( 001 ) . this shared leadership became the driving organizational force that was required to implement simulation as a teaching and learning strategy in nursing curricula . they were the key organizational element and served as the axis on which the other essential organizational elements interacted . the leaders did not act as individual leaders working on separate tasks but shared strategic leadership working in tandem toward a common goal . one nursing administrator highlighted this sayingin any successful venture one person ca n't do it all it was a joint effort the ideas for the strategic plan were mine but without [ the simulation leader ] it would n't have gone anywhere and still would be an idea ( 012 ) . in any successful venture one person ca n't do it all it was a joint effort everybody brings their own unique knowledge and skill to the table . the ideas for the strategic plan were mine but without [ the simulation leader ] it would n't have gone anywhere and still would be an idea ( 012 ) . the nursing leader 's role is multifaceted and required the leader to engage multiple strategies to facilitate the simulation initiative . negotiating involves working with nursing faculty members within the nursing program as well as with groups or individuals at the level of the faculty and the institution to exchange ideas and secure necessary resources . navigating includes mapping out a certain course or plan for simulation within the nursing curriculum . networking consists of generating a support system within the institution or among community members who have similar interests in simulation or can assist with the navigating process . much of what drove the leaders to support the simulation initiative were the overarching guiding philosophies . these statements articulate what is significant or important to the institution , faculty , and nursing program and , in turn , imply value . for example , institutions that promoted innovation within their mission or vision statements had nursing leaders that used that value statement as a rationale to secure resources and promote the implementation of simulation into the curriculum because it ultimately aligned with and supported the mission of the institution . another driving factor among nursing leaders was the expectation of use and the shared motivators that are discussed in relation to the four remaining organizational elements . information about simulation , its potential for enhancing the teaching and educational experience , and information about the additional resource requirements needed to support it had to be shared between nursing leaders , nursing faculty members , collaborative partners , and among different levels within the institution . the exchange of information about simulation was required at the onset of adoption and incorporation to get it started , but ongoing communication was also required to maintain simulation as part of the nursing curriculum . information was communicated in a variety of ways and was significantly influenced by the nursing leaders who used formal and informal as well as written and spoken communication . one formal strategy used by several high - uptake sites was student evaluations of simulations . these evaluations included student feedback about the satisfaction with , or perceptions of , simulation as a teaching / learning strategy . the summaries of these evaluations were presented at formal meetings as a way to facilitate the formal exchange of information among individuals or groups that were using simulation and those who were considering using it or uncertain about using it . the nursing leaders who facilitated this type of formal communication placed value on simulation and provided ongoing opportunities for simulation to be considered as a teaching / learning opportunity . one nursing administrator explained this sayingwe've built in evaluation all along then twice a year we invite the sim coordinator to do a presentation to faculty about the events and activities that have occurred and i think what that does is keeping the thinking process alive and helping faculty members anticipate what they might consider in their own courses ( 025 ) . we 've built in evaluation all along then twice a year we invite the sim coordinator to do a presentation to faculty about the events and activities that have occurred and i think what that does is keeping the thinking process alive and helping faculty members anticipate what they might consider in their own courses ( 025 ) . all sites used some formal information exchange strategies such as introducing simulation at faculty or curricula meetings or learning about simulation at conferences . however , the majority of low and mid - uptake sites used fewer formal exchanges of information and used them less frequently than the high - uptake sites . this was articulated by one faculty member stating we do n't have a formal process ; it 's more informal discussions ( 023 ) . this informal exchange of information was also more prevalent and diverse in the high - uptake sites and occurred in a variety of ways . predominantly , it was centered on one - on - one or small group discussions . also included were email updates about simulation , discussions about research findings , or displays of the findings . one simulation leader maintained this saying our manager would just send something out via email and it was by word - of - mouth ( 021 ) . informal information exchanges strategies often served to promote interest and generate ideas about simulation . multiple and diverse ways of communication facilitated movement through the seven - phase process of adoption and incorporation . exchange of information was needed for all of the phases but was crucial in the phases of learning about simulation and its potential for teaching , finding and fit , and securing additional resources . the transfer of information about simulation between all levels of the organization was needed to secure a place for it in the curriculum . information exchange is not only influenced by the communication strategies of the leaders , but also shaped by the physical environment . one faculty member recognized this stating our offices were across the hall from each other so we were always bouncing ideas off of each other ( 024 ) . having people work in close proximity allowed for spontaneous conversations and sharing of ideas related to simulation . this concept of physical environment leads to the subsequent organizational element , physical locale that shaped the adoption and incorporation of simulation into nursing curricula . a significant amount of space is required to store , maintain , and utilize simulation equipment . when the initial request for funding was advertised , institutions were required to show the appropriate allocation of space or a renovation plan . this resulted in many nursing administrators having to fight for space and many simulation leaders having to deal with the space provided . space was rented that was located away from the nursing program on another campus , in a different part of the city , or on another campus . many participants claimed that the physical environment of the lab shaped the adoption and incorporation of simulation into nursing curricula . often the remote physical location of the lab was isolating for many simulation leaders and created disconnects between people making it difficult to share or generate ideas . one simulation leader stated the labs are completely separate from the nursing program and that also is a little bit tricky because sometimes people do n't want to come over here ( 007 ) . the isolated location of many simulation labs leads to feelings of frustration and the perception that simulation as a teaching strategy was undervalued or unsupported by the institution . this impeded the ability of some institutions to move successfully through all of the phases in the process . despite the isolation and disconnection that occurred in many new spaces , institutions which renovated spaces also had their own unique challenges . renovated spaces were often described as inadequate or inefficient because of lack of knowledge about simulation equipment during the planning of the renovations . one simulation leader summed this up stating the way our simulation area has been set up is not the best layout . it 's not even connected to the space that we 're in it was n't a well thought out design ( 027 ) . renovations completed during the early stages of this initiative before stakeholders had a full understanding of the complexity of the equipment often resulted in frustration among simulation leaders and other users and ineffective use of the equipment . the physical locale proved to be a significant organizational element that shaped the adoption and incorporation of simulation in shared nursing curricula being offered among collaborative partners . within the context of this study , nursing programs are offered in collaboration with college and university partners ; typically collaborations comprised two or three institutions . there is a physical distance between the institutions as most collaborative partners are not housed in the same location . despite sharing curricula , the physical locale of the lab in separate physical environments provided a greater organizational influence than the collaborative partnership . one faculty summed this up saying although we are a collaborative program , we are not really collaborative with regard to sim . i have no idea what is being done there and they have no idea what we do here ( 011 ) . the expectations and philosophies of the physical institution where the lab is housed took precedent over the collaborations , meaning that the institution where the simulation lab was located had greater influence in shaping the adoption and incorporation of simulation than collaborative partnerships delivering the same curricula . two participants provided insight to this ; one simulation leader stated the barrier is the collaboration the two philosophies are so different they do utilize simulation in some respect ; they certainly run their simulation much differently than we ( 022 ) ; while a nursing administrator added collaborative programs are extremely difficult to work with because there are substantial differences just between colleges and universities in terms of how they think but i mean programs take on the personalities of their people as well ( 015 ) . collective or shared social responses or common experiences shared by many proved to be motivating factors of the organizational culture that shaped the adoption and incorporation of simulation . the initial reaction experienced by most institutions was one wanting to take advantage of a rare opportunity . the reaction of some programs was to apply for the funding and then , later , figure out how to use the money . one simulation leader explained we 're going to take it [ the provincial simulation funding ] and we 'll learn what to do with it after ( 010 ) . wanting to take advantage of a onetime funding opportunity was a significant motivator experienced by all institutions . people were unsure of what to do with the equipment , of what the equipment was capable , and of how simulation might impact their workload and teaching responsibilities . this resulted in avoidance in many organizations , some not even opening the boxes until two to three years had passed . what helped some institutions move past the uncertainty was primarily the creation of the simulation leader role . once this person was employed , expectations of this role developed and included learning about the potential of the equipment , how to set it up , and how to use it . while the uncertainty dissipated over time in the high - uptake sites , the low - uptake site continues to be immobilized and indecisive about simulation . this was highlighted by one faculty member there who stated we got the equipment out of the boxes and put it in a closet ; we do not use it very often and there really is no one to show us how to use it ( 027 ) . concern , as a shared motivating factor , was related to not wanting to be left behind and to sustainability . many mid - uptake sites and some high - uptake sites that moved slower through the process of adoption and incorporation experienced uneasiness about not being as competitive as other institutions that were utilizing simulation to a greater level or with more consistency . they expressed concern over recruitment of students and felt that the slow or minimal uptake of simulation could be a deterrent for some students . several participants also shared the belief that simulation was an expectation in all health related education programs and if they were not able to implement simulation as a teaching strategy , they would be left behind . the angst about being left behind is driven by the concept of time as a measurement of success . while there was neither an expected timeline nor clearly articulated time sensitive goals required in the initial proposal , many institutions measured their success in terms of time . the high - uptake sites , because of the leadership and past experiences managing this type of change , navigated the adoption more rapidly than other sites and placed a value of success on this as noted by one nursing administrator expressing pride in the short time frame her nursing program took from conception to integration stating it was less than one year ( 012 ) . time was also emphasized by a nursing faculty member at a mid - uptake sight who stated i have shamed a few people into doing it because everybody else was doing it[simulation ] , so we better get going with it [ simulation ] ( 017 ) . on the far end of the uptake spectrum is the low - uptake site where the simulation leader expressed frustration and embarrassment stating despite being eight years into this initiative to have it only in one course it is just kind of weird we are so far behind in simulation ( 027 ) . the other concern common among all mid and high - uptake sites was the issue of sustainability . since the funding was a one - time nonsustainable resource , there was insecurity as to how to implement and sustain it once in place . there were also concerns about the equipment breaking or implementing it to a level within the curriculum where the demand would outweigh the availability , thus resulting in the need to purchase additional equipment but not having the funds . while the concern about being left behind has lessened for some institutions , it remains a concern for many , especially for the sites which have collaborative partners who outrival them with simulation . since nursing is a practice - based profession , much learning comes from interaction with and practice of psychomotor skills on patients . the adoption and incorporation of simulation does not negate nor replace the learning from and with patients but it provides an opportunity to practice and refine skills prior to them taking place with patients . this enables students to practice in a safe and secure setting without concern for patient safety . one faculty member summed this up stating[students ] need to demonstrate before they can do it [ on patients ] , [ simulation ] allows them to experiment , make mistakes , and figure it out for themselves . clinical teachers are out there to protect the patient so [ during simulation ] their mindset is n't this is n't a patient that needs protecting but a group of students who can learn from making mistakes ( 008 ) . [ students ] need to demonstrate before they can do it [ on patients ] , [ simulation ] allows them to experiment , make mistakes , and figure it out for themselves . clinical teachers are out there to protect the patient so [ during simulation ] their mindset is n't this is n't a patient that needs protecting but a group of students who can learn from making mistakes ( 008 ) . positive student responses to simulation provided the most significant motivating factor for moving forward with the implementation of simulation . participants consistently reported on students ' feelings of security working with simulated patients , relief that they could practice without harm , and that all students could benefit from the learning instead of the one lucky enough to have an experience during clinical training . participants said it reduced anxiety and competition between students because they were all provided with the same experience . one simulation leader clarified this statinginstead of having only one or two students have the learning experience in the clinical setting we can set up experiences with simulation we can make sure all of our students have experiences they may not always get ( 001 ) . instead of having only one or two students have the learning experience in the clinical setting we can set up experiences with simulation we can make sure all of our students have experiences they may not always get ( 001 ) . the students ' responses were a profound motivating factor for participants of this study consistently among all of the mid- and high - uptake sites . two statements by participants summed this up , one by a faculty member who stated i think what motivates us to do [ simulation ] is the student 's reaction ( 010 ) , and the other by a simulation leader who stated the feedback we get is so positive from the students and the instructors we feel like we can see the learning happening ( 020 ) . student responses appeared to energize the participants and spur them onto further use simulation as a teaching / learning strategy . the final motivating factor of the organizational culture stemmed from the overarching institutional expectations such as workload and the value of independence . workload expectations are a significant driving force when adopting a new innovation , particularly one as extensive as simulation . there is a considerable amount of learning , trialing , and time required to become proficient in running the equipment , developing learning experiences , and implementing them . some institutions have no way to accommodate this into workload assignments which results in people taking this on in their own time , aligning the work required with their own personal goals or research interests , or not participating at all . one simulation leader highlighted this sayingit 's hard to know [ if ] it 's an expectation of the organization . it seems like if you 're tenure track you would never do this kind of work load 70% teaching and they would have 40% teaching and 40% research their teaching work load allocation is really different because their research is way up ( 024 ) . it 's hard to know [ if ] it 's an expectation of the organization . it seems like if you 're tenure track you would never do this kind of work load 70% teaching and they would have 40% teaching and 40% research their teaching work load allocation is really different because their research is way up ( 024 ) . due to the different workload agreements , some of the college sites were able to adjust workloads to include simulation placing them at the higher end of the uptake continuum . independent organizational culture that holds the time - honoured practice of academic freedom above other values provides unique challenges for nursing programs wanting to adopt or integrate simulation . one nursing administrator confirmed this point stating the university culture is such that we all have to be individuals to get ahead , to get tenure and promotion , and do research which becomes a very competitive , isolating experience ( 005 ) . a faculty member shared her view of this stating if the expectation came from on high that thou shall implement [ simulation ] , i ca n't see that happening because we could always preach academic freedom ; i do n't see how they could ever tell us we had to ( 003 ) . this level of independence that is the standard in many academic nursing programs creates unique challenges when trying to adopt and incorporate an innovation that requires a significant amount of extra time and a high degree of teamwork . the shared dynamic is a key organizational element that drives people to act and make decisions . this element of organizational culture included support and structure as a framework to facilitate movement through the process of adoption and incorporation . it involved reaching outside the nursing program and linking with in - house partners to share resources . this proved to be beneficial in many high - uptake sites , especially when requesting resources that benefit more than one program within the same institution . it also included the sharing of personnel resources and expertise which in turn provided an institutional cost saving strategy . securing the framework to support simulation outside of the nursing program served to move simulation forward and to contribute to incorporation as it became a faculty wide initiative rather than just a nursing program initiative . as this option was not available to all institutions some chose to secure support by linking with community partners to share resources and expertise . essentially this provided the similar support as noted with the institutions that linked with their in - house partners ; it made the simulation initiative larger than just the nursing program . the benefit of connecting and linking with others for support in turn raises the profile of the initiative , as summarized by one nursing faculty member who stated a new building was built for the faculty there was space in that new building allocated for clinical education , and simulation for all the health sciences and we also rent it out to groups like the hospital educators ( 014 ) . sharing resources was a way to manage change , and using the same lab , equipment , or personnel proved to be beneficial . creating links beyond the nursing program strengthened the scaffolding and contributed to the level of uptake . institutions which chose not to link with community partners or did not have in - house partners to link with were at a disadvantage since the simulation initiative was supported primarily by just the nursing departments , and this led to a lower level of uptake . scaffolding to manage change intersects with all of the other organizational elements and is essential to accommodate initial and ongoing changes . it includes being creative with limited resources , providing recognition for extra work , and orienting new members . while links external to the nursing program provided extra support and facilitated movement through the process of adoption and incorporation , there were two strategies within the nursing programs that provided support and created a structure to promote simulation as a teaching strategy . consistently , participants stated that the work which was required to make simulation part of the curriculum was unparalleled to anything they had previously experienced . one nursing faculty member stated we 're actually supposed to do three hours a week for sim , which if you multiply by a 15 week semester , is 45 hours a semester , i do 45 hours in 3 days ( 010 ) . institutions that compensated or recognized the extra work involved were higher on the uptake continuum than those that did not . recognition was acknowledged in the form of education and travel that was paid for by the institution . one nursing administrator discussed recognition stating [ simulation leaders ] get a lot of rewards because these are the folks we are sending to conferences all over the world ( 015 ) . recognition also came in the form of role designation , giving the simulation leaders a new and specific title . what this did was to differentiate the simulation leaders and give them a new status within their institution . recognition provided incentive for those involved in simulation , thus strengthening the support system to facilitate the incorporation of simulation . while recognition is an astute place to start with the development of a new role , one aspect which could have strengthened the scaffolding and provided additional support would be the addition of a job description and annual appraisal documents that were reflective of the role and the work therein . most participants stated their annual evaluation had nothing to do with their work relating to simulation . few appraisal templates were shared with the researcher as participants voiced there was little to no connection between the work they do and their annual evaluation process . there 's nothing that comes out of the appraisal process that forces me to teach in a particular way ( 006 ) . one simulation leader from a mid - level uptake site interpreted this disconnection as a devaluation of her work and her teaching methods , stating there is a lack of associated respect or merit placed on any of [ simulation ] activities ( 009 ) . while recognition is one strategy to strengthen support and provide a framework for success , adding specific job descriptions and having people evaluated for the work they do are strategies that could be used to add an additional level of structure and support to a newly developing role in nursing . the last scaffolding strategy that was consistent among the majority of the high - uptake sites was the orientation of new members . orientation of current and new nursing faculty members and staff is a strategy to maintain change and assign value to the innovation . one nursing administrator stated we focused on everybody getting a baseline understanding of simulation so new faculty and staff goes to the [ simulation leader ] for training ( 012 ) . other examples of orienting new members included training modules for new faculty and the availability of individual training . orientation was a consideration in both of the rapid high and a couple of slower high - uptake sites , but most of the other sites had not given this much consideration as stated by one faculty member we do n't really have a process for [ orientation ] ( 023 ) . using strategies such as being creative with resources , providing recognition for extra work , and orienting new members proved to be effective scaffolding strategies that contributed to the adoption and incorporation of simulation . the organizational culture of nursing programs and the academic institutions in which they are situated contribute significantly toward the adoption and incorporation of simulation into the curriculum . this study revealed that five key organizational elements contribute to the adoption and incorporation of simulation into nursing curricula . the oessn model shows these organizational elements as bordered by the institutional , faculty , or nursing program guiding philosophies . the connection between the beliefs and values and the leaders in the oessn was portrayed as a two - way path . this dual direction concept adds to schein 's theory by suggesting that movement between and among these concepts exists and can facilitate the uptake of an innovation into an academic program . schein purports that beliefs and values impact actions and decisions , but this study reveals that movement from the beliefs and values is not unidirectional ; flowing downward , the guiding philosophies influence the decision making of leaders ; flowing upward , the leaders used the values and beliefs as rationale to support their requests for additional resources . organizational philosophies are an important aspect that should be used to guide the adoption and incorporation of simulation . despite funding constraints in academia , a recommendation resulting from this study is that when requesting funding support , nursing programs strategically use the guiding philosophies as tools to negotiate the needed resources when adopting or incorporating an innovation . an important consideration from the findings of this research is the shared emotional reaction to time as a driving force . the initial emotion of not wanting to be left behind and the ongoing emotion related to perceptions of success both denote time by the form of measurement . graham suggests that time is a concept of culture that influences people 's perceptions . he further suggests that the completion of tasks is directly related to time , and a task that is not completed or takes additional unanticipated time is often referred to as being behind graham further suggests that time spent completing tasks positions people or institutions for future progress . this is consistent with what was found in this study when participants expressed feelings of satisfaction with how far they had progressed with the adoption and incorporation of simulation over a short period of time . surprising because few institutions were able to articulate goals related to simulation therefore making it difficult to measure accurately their success or validate their progress or lack thereof . the concept of time as a measurement of success is simultaneously predictable within the context of this study . researchers have suggested that north america typically has a monochronic perception of time , where the focus is future oriented and concerned with completing tasks within set time frames . there is also the philosophy that time can be wasted and that time provides meaning and affects judgment [ 29 , 30 ] . the result of measuring progression and or success by time when there is no clear vision or goals related to the adoption and intergradations of simulation causes unnecessary emotional reactions which can become a barrier . one recommendation is to take lessons from polychronic time oriented cultures that focus on participation and achievement of milestones rather than fixed or unreal time frames . many aspects of adopting and incorporating an innovation are multifaceted and require many levels of cooperation within the organizations that takes time . recommendations from this study would be to set clear measurable goals related to the adoption and incorporation of simulation and the purpose that it will serve in the program . the quality and achievement of goals this research provides insight into the emotional and cultural concepts of time when adopting and incorporating simulation and could generate future research in this area . using schein 's theory sensitized the researchers to the concept of space as an organizational element . while space as a concept has the potential to be interpreted in a variety of ways , the findings of this research suggest that the physical locale is an essential driving force within the context of adopting and incorporating simulation into nursing curricula . while space is a commodity at many postsecondary institutions , this study found it was more than just the availability of space the physical location of the lab elicited emotional responses such as isolation or questions of value . nursing program members perceived simulation to be undervalued or not valued if the allocated space was not ideally located nor within close proximity to the nursing department . recommendations would be to encourage leaders to negotiate desired space close to the department if possible . if not , then leaders should communicate that space in academic settings is a scarce resource , a commodity , and that value exists even when the space is less than ideal . schein states that the orientation of new members can perpetuate the values and beliefs of an organization . this was noted in a few of the high - uptake sites in this study but , overall , was limited in the other sites . he further states that the culture of the organization is taught to new members so that they can learn how to think , feel , and act in relation to the work environment . not including simulation as part of the orientation of new members may lead to its devaluation , or new hires might view it as optional . this provides an opportunity to show the value of simulation as a teaching strategy and perpetuates it as part of the culture . the findings from this study indicate that there are many motivating factors involved at the level of the nursing program including the positive responses of students toward simulation as a teaching strategy . while there is not a substantial body of evidence supporting the effectiveness of simulation to positively influence the acquisition of new or sustained clinical skills [ 32 , 33 ] , organizations are highly motivated and focused on implementing simulation for the outcome of improved student satisfaction and student engagement with the learning environment . the connections and the fluidity between and among the key organizational elements were an important finding from this study . the five organizational elements link together and are interdependent . having this understanding of the interconnectedness of the organizational factors provides an opportunity for leaders to facilitate change . for example , the institutional expectations link directly to scaffolding . it is an expectation in most institutions that some form of annual appraisal is done . participants in this study shared that they felt their work related to simulation was not reflected in this process . this disconnection provides an ideal opportunity for leaders to communicate , share information , and align work contributions so they fit within the parameters of the appraisal process . other than the inherent link between leadership and communication [ 15 , 34 ] , the concept of interconnectedness adds to the literature on organizational culture . the dimensions of organizational culture are often presented in isolation of each other [ 15 , 34 , 35 ] . having an awareness of this would provide an advantage to nursing programs wanting to incorporate simulation because they could look at these aspects from a holistic viewpoint , knowing that one element of organizational culture impacts the other . being cognizant of this when creating strategies to enhance this synergy could facilitate the adoption and incorporation of simulation as well as other innovations into nursing curricula . the inclusion of multiple sites added to the transferability of the findings . an audit trail consisting of memos , reflexive journaling , and field notes was maintained throughout the study to maintain confirmability [ 37 , 38 ] . this study did not include an exploration of nursing program , faculty , or institutional finances , flow of funding within organizations or among collaborative programs , nor union agreements . inclusion of these aspects may have elicited additional findings related to the organizational elements that shape the adoption and incorporation of simulation into nursing curricula . the organizational cultures of nursing programs in ontario that shape the adoption and incorporation of simulation into curricula has been represented by the oessn model that depicts the five key organizational elements : leaders , information exchange , physical locale , shared motivators , and scaffolding as a strategy to manage change . this paper was conceptualized from interviews with nursing administrators , nursing faculty members , and simulation leaders as well as a review of key documents from 13 different nursing programs . this research has contributed to the literature on simulation and enhanced the literature on organizational culture . the oessn model provides a framework that nursing programs could use to initiate or further facilitate the adoption and incorporation of simulation into curricula .

purpose . to create a substantive mid - range theory explaining how the organizational cultures of undergraduate nursing programs shape the adoption and incorporation of mid - to high - level technical fidelity simulators as a teaching strategy within curricula . method . a constructivist grounded theory was used to guide this study which was conducted in ontario , canada , during 2011 - 12 . semistructured interviews ( n = 43 ) with participants that included nursing administrators , nursing faculty , and simulation leaders across multiple programs ( n = 13 ) informed this study . additionally , key documents ( n = 67 ) were reviewed . purposeful and theoretical sampling was used and data were collected and analyzed simultaneously . data were compared among and between sites . findings . the organizational elements that shape simulation in nursing ( oessn ) model depicts five key organizational factors at the nursing program level that shaped the adoption and incorporation of simulation : ( 1 ) leaders working in tandem , ( 2 ) information exchange , ( 3 ) physical locale , ( 4 ) shared motivators , and ( 5 ) scaffolding to manage change . conclusions . the oessn model provides an explanation of the organizational factors that contributed to the adoption and incorporation of simulation into nursing curricula . nursing programs that use the oessn model may experience a more rapid or broad uptake of simulation when organizational factors that impact adoption and incorporation are considered and planned for .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

PMC4847391

view confronted the notion that the history of healing is par excellence the history of doctors. antithetical to a physician - centred approach was porter s call to do at present , porter declared , we remain profoundly ignorant of how ordinary people in the past have actually regarded health and sickness. an alternative vision for the history of medicine would be written from the patient s point of view. thirty years on , there remains a concerted effort to elucidate the sufferer s role in the history of health and healing . recent developments , however , most notably in the history of psychiatry , reveal a groundswell of dissatisfaction among scholars who argue that the historian can no longer be content with describing the theories and practices of the psychiatrist , or , inversely , the subjective experience of the patient alone . rather , they claim , researchers need to explore the interactive dimensions of the doctor patient relationship and beyond . to facilitate this shift in focus from a history of patients to a history of these complex clinical dynamics , specifically , patient case notes have long been recognised as a potentially fruitful archival resource . in this paper i chart my own engagement with the patient records of glasgow s gartnavel mental hospital by focusing on superintendent dr david kennedy henderson and his introduction to gartnavel of a so - called dynamic approach to mental health care in the 1920s . in order to appreciate the significance of such records , the article begins with henderson s articulation of his psychotherapeutic method , before proceeding to an in - depth hermeneutic investigation into samples of gartnavel s case notes . in the process , i explore patient psychiatrist relationships , not as distinct entities , but as mutually dependent and interrelated subjects of historical enquiry . these materials reveal a complex and multi - faceted clinical picture that involves various strategies of negotiation deployed by all members of the clinical encounter . although henderson promoted sensitivity to the stories , patients compliance or resistance was based largely on their gender , social class and admission status . these elements , which often determined their willingness to tell their stories , directly or indirectly , made their way into the case notes . therefore , gartnavel s patient records involve processes of construction and reconstruction at various levels by patients , psychiatrists , medical staff and historians alike . dr david kennedy henderson ( 18841965 ) , gartnavel s superintendent during the period of interest ( 192132 ) , trained under some of the most eminent psychiatrists of his time . these included thomas clouston in edinburgh , adolf meyer in baltimore , august hoch in new york and emil kraepelin in munich . between 1908 and 1919 , he worked at the royal edinburgh asylum , the pathological laboratories of the new york state hospitals , the nussbaumstrasse clinic in munich , the henry phipps psychiatric clinic in baltimore , the lord derby war hospital in lancashire and gartnavel mental hospital in glasgow , publishing scholarly articles on such topics as cerebral syphilis , war psychosis , and catatonia . entering the profession at a time of scepticism regarding nascent psychoanalytic theories and kraepelinian classificatory systems , henderson s blend of kraepelinian , freudian , and meyerian teachings was in many ways pioneering . long before any other centre in great britain began to stir in response to the remarkable things which were being done on the north american continent , wrote henderson s colleague donald ewan cameron in 1965 , [ henderson ] began to send his young men to train with adolf meyer. the influence of meyer who by the inter - war years had become a leading figure of north american psychiatric thought played a particularly significant role in bringing new therapeutic practices to britain . specifically , it led to the introduction at gartnavel of what meyer and henderson called a dynamic psychotherapeutic approach . meyer , hailed during his lifetime as the dean of american psychiatry , was instrumental in shaping and promoting a new conception of the mind in early twentieth - century north america . a swiss - born migr , meyer came to reject the rigid somaticism of late nineteenth - century psychiatry as it was then practised in north america . rather , he saw mental illness as the cumulative result of unhealthy reactions of the individual mind to its [ physical and social ] environment ; he thus replaced the concept of mental illness as disease with that of mental illness as a reaction type that required both physical and psychological explanation. it was during henderson s early years of training under meyer , first in new york ( 190811 ) and later in baltimore ( 191215 ) , that he was taught to combine psychological studies of experience and individual subjectivity with pathological anatomy . surrounded by colleagues such as august hoch , who pioneered north american personality studies in the early twentieth century , henderson became engrossed in a field of research which took as its subject matter the relationship between patients thoughts , feelings and the external world . the focus of this interrelationship was often to be ascertained through conversation with the patient . according to meyer , [ t]he psychiatrist the user of biography must help the person himself to restore the capacity for self - regulation. here , the patient s own language and own understanding of his or her illness was used as the basis for any advice and further elucidation. in the wake of the psychoanalytic movement s increasing popularity both in the continent and in north america , personality studies , dream analyses , word association tests and talking therapies became part of these psychiatrists investigatory and therapeutic arsenal . unlike freud , however , meyer taught his students and colleagues to desist from transforming patient stories into complex , theory - laden narratives . rather , he advised that the psychiatrist use the patient s own story as the basis for therapeutic intervention . this would be achieved through trust and co - operation . to transform individual stories into clinically objective facts , meyer s long - lasting contribution to psychiatry was to standardise methods of examination and case note - taking procedures . all of the potentially relevant factors in a case were to be made objectively evident through longitudinal studies of an individual s life history , recorded in uniform and systemic fashion. as ruth leys writes : [ meyer ] provided his students with a basic outline to be followed , specifying the order of procedure and actual questions to be put to the patient , something that had been lacking in previous handbooks of psychiatry . [ meyer ] provided his students with a basic outline to be followed , specifying the order of procedure and actual questions to be put to the patient , something that had been lacking in previous handbooks of psychiatry . another novelty that meyer introduced to north american institutions was the practice of holding so - called staff meetings , the proceedings of which were recorded by clinical stenographers . the art of history - taking , henderson later observed when reflecting upon meyer s teachings , was invaluable in relation to prognosis , diagnosis and treatment. such methods became the cornerstone of north american psychiatric practice in the decades to come . upon henderson s promotion to physician - superintendent of gartnavel mental hospital in 1921 , he instigated new clinical practices based on meyer s methods . remarks , examinations , staff conferences , nursing reports , laboratory testing , diagnosis and prognosis ; each formed a constituent element of the meyerian - inspired dynamic case note at gartnavel . leaning heavily upon meyer s 1918 case - taking guides and those of his american colleague , dr george hughes kirby , henderson typically divided the gartnavel patient records in tripartite fashion , consisting of the anamnesis ( personal history ) , the physical examination and the mental examination . the duty of writing case notes fell to clinical clerks and stenographers as well as more senior medical officers . the marks these authors made on the page are highly revelatory as to the processes of selection and interpretation by which medical knowledge was produced . meyerian case - taking guides aimed to capture a live and continued account of examinatory procedures . case notes include inverted commas , used to denote verbatim recordings of speech , while colons , dashes and question marks were used to highlight pauses , rhythms and silences that punctuated patients spoken words . brackets , used to insert the writer s own observations of tone , emotional content or actions that accompanied speech , add another layer of interpretative depth . all of this was ultimately intended to enable the psychiatrist to better understand the organic , as well as psychological , components of a patient s ills . meyer s teachings motivated henderson to push scottish psychiatry away from its former emphasis on the description of symptoms , classification and brain pathology , and towards the study of the individual s personality as a reaction to his or her environment . gartnavel s resident medical officers were encouraged to embrace the clinical teachings of kraepelin , the personality studies of meyer , august hoch and george amsden , and the case notes reveal occasions where therapeutic interventions effectively took the form of talking and occupational therapies . rejecting the seeming rigidity of somatic - pathological interpretations then largely prevalent in the british psychiatric profession , henderson declared that general descriptions of clinical syndromes , while interesting , are not of first importance. what is wanted, he noted in the preface to his jointly authored text - book of psychiatry ( 1927 ) , is an understanding of the patient as a human being , and of the problems which he is meeting in a morbid way with his [ t]he physical and the psychological can not be divorced the individual must be treated as a whole. this philosophy , shared by meyer and henderson alike , guided henderson to pursue psychiatry as a living subject in relation not only to general medicine , but to the social problems of everyday life. it is in accordance with these principles , he wrote , and with what is called the dynamic view , that we have utilised clinical records so extensively. the resulting records , based on the practices of north american colleagues , reveal materials in which the doctor patient dialogues are preserved in an attempt to faithfully reproduce the patient s voice . these case notes capture , with remarkable clarity , some of the narratives , actions and anxieties exhibited by patients and psychiatrists as they interacted with one another . contextualised against a backdrop of waiting rooms , patient dormitories , examination and consultation spaces , gartnavel s dynamic case notes can be used by the historian to reconstruct a historical human geography of patient psychiatrist interactions within these clinical confines . yet , as we shall see , the question remains as to precisely how much construction and reconstruction is involved in the production and later interpretation of these records . upon henderson s promotion to physician - superintendent of gartnavel mental hospital in 1921 , he instigated new clinical practices based on meyer s methods . remarks , examinations , staff conferences , nursing reports , laboratory testing , diagnosis and prognosis ; each formed a constituent element of the meyerian - inspired dynamic case note at gartnavel . leaning heavily upon meyer s 1918 case - taking guides and those of his american colleague , dr george hughes kirby , henderson typically divided the gartnavel patient records in tripartite fashion , consisting of the anamnesis ( personal history ) , the physical examination and the mental examination . the duty of writing case notes fell to clinical clerks and stenographers as well as more senior medical officers . the marks these authors made on the page are highly revelatory as to the processes of selection and interpretation by which medical knowledge was produced . meyerian case - taking guides aimed to capture a live and continued account of examinatory procedures . case notes include inverted commas , used to denote verbatim recordings of speech , while colons , dashes and question marks were used to highlight pauses , rhythms and silences that punctuated patients spoken words . brackets , used to insert the writer s own observations of tone , emotional content or actions that accompanied speech , add another layer of interpretative depth . all of this was ultimately intended to enable the psychiatrist to better understand the organic , as well as psychological , components of a patient s ills . meyer s teachings motivated henderson to push scottish psychiatry away from its former emphasis on the description of symptoms , classification and brain pathology , and towards the study of the individual s personality as a reaction to his or her environment . gartnavel s resident medical officers were encouraged to embrace the clinical teachings of kraepelin , the personality studies of meyer , august hoch and george amsden , and the case notes reveal occasions where therapeutic interventions effectively took the form of talking and occupational therapies . rejecting the seeming rigidity of somatic - pathological interpretations general descriptions of clinical syndromes , while interesting , are not of first importance. what is wanted, he noted in the preface to his jointly authored text - book of psychiatry ( 1927 ) , is an understanding of the patient as a human being , and of the problems which he is meeting in a morbid way with his [ t]he physical and the psychological can not be divorced the individual must be treated as a whole. this philosophy , shared by meyer and henderson alike , guided henderson to pursue psychiatry as a living subject in relation not only to general medicine , but to the social problems of everyday life. it is in accordance with these principles , he wrote , and with what is called the dynamic view , that we have utilised clinical records so extensively. the resulting records , based on the practices of north american colleagues , reveal materials in which the doctor these case notes capture , with remarkable clarity , some of the narratives , actions and anxieties exhibited by patients and psychiatrists as they interacted with one another . contextualised against a backdrop of waiting rooms , patient dormitories , examination and consultation spaces , gartnavel s dynamic case notes can be used by the historian to reconstruct a historical human geography of patient psychiatrist interactions within these clinical confines . yet , as we shall see , the question remains as to precisely how much construction and reconstruction is involved in the production and later interpretation of these records . during the summer of 1930 , two male attendants accompanied a general practitioner to the house of a middle - aged male patient , mr patrick johnstone . upon their arrival , the attendants remained outside mr johnstone s front room and , after much talk and argument between doctor and patient , they were asked to enter . i looked round the room , an attendant later wrote , and noticed that lying near the door on the floor [ were ] a hatchet and splinters of glass. softly laying a hand on mr johnstone s arm , he led the patient out of the room , but having reached the front door of the house mr johnstone made to run away. while the attendant held him back , johnstone began to shout for the police and [ mentioned ] something about a warrant. the doctor looked for a constable , but after it was made clear to mr johnstone that his wife would accompany him to gartnavel , he was finally coerced into the awaiting motorcar . on the way to the hospital mr johnstone sat in silence . upon reaching the entrance of gartnavel , , he alighted the steps to the front door and , with a rapidity of pace , walked briskly into the male reception hall . he then stopped , pausing in order to light his pipe and , after having caught sight of one of the medical officers , johnstone strode forward , cordially offering his greetings to the medical officer , and stated his pleasure at meeting him . it is at this moment , when patient and practitioner met upon the threshold of the hospital , that the construction of a patient s case notes began . this was the point at which an individual became subsumed within the socially and diagnostically diverse yet somewhat amorphous mass of patients at gartnavel ( which reached an annual average of 450 predominantly middle - class residents ) . nursing attendants and general practitioners gave information as to the immediate actions of the person before admission . then , gradually , as he or she entered the hospital , family and friends were invited to give information about the patient s history , so that case notes built up a picture of the individual from the joint perspectives of medical professionals and lay informants . for gartnavel s psychiatrists , the point at which their attention shifted from the narrative of lay informants and medical certificates towards first - hand clinical observation marked a distinct turning point in the medical record . at this juncture , the temporality of the case note changed to the immediate observation of the patient as he or she adapted to hospital routines . during admission , when forms were presented and/or letters signed ( based on whether the individual was a certified or voluntary patient ) , the waiting room signalled an individual s legal , temporal and physical transition to institutional care . it was from within such a liminal space that gartnavel s practitioners were presented with their first opportunity to capture a live and continued account of the patient s story as he or she entered the hospital . meyer viewed this moment of transition as a valuable experimental phase which provided one of the most decisive opportunities to observe first - hand the individual s adaptive strategies to the demands of a new environment . in accordance with meyer s investigational techniques , the process of admission thus allowed gartnavel s medical officers to observe these adaptive strategies in a set of controllable , easily replicable environmental conditions . the journeys , waiting rooms and wards here became the backdrop to individual life histories . pathological laboratory to which the patient was observed to adapt , consigned in vignettes of the admission process which were thought to afford a natural picture of psychobiological maladaptation. while henderson similarly envisaged gartnavel as a pathological laboratory , he also saw it as a therapeutic space in which one could potentially cure patients allegedly dysfunctional behaviours . it was crucial to henderson s approach that a patient recognise his or her condition as a mental illness that necessitated treatment within a hospital by medical professionals . as such , conforming to the hospital regime and accepting the authority of the psychiatrist was considered one of the first steps to therapeutic success , requiring the patient to adapt to this new one of the most important factors affecting an individual s reaction to this new setting , as observed by henderson , was his or her status as a voluntary or certified patient . early in his career he stressed that the line of division between those patients predisposed to recovery and those who failed was often drawn between the cooperative and non - cooperative patients , not between any supposed standard of sanity or insanity. throughout the 1920s gartnavel s admission staff therefore diligently recorded whether patients showed fear and/or ambivalence upon admission , or , by way of contrast , demonstrated acceptance and eagerness to submit to the hospital s treatments . my analysis suggests that there was , indeed , a correspondence between level of compliance to the hospital regime and patients admission status . sources show numerous occasions on which certified patients were recorded as resisting , evading and distrusting the legitimacy and efficacy of hospital care , while voluntary patients more often took on the role of what would now be called service user by cooperating with , or even negotiating , the terms of their treatment . this crucial ( if under - studied ) dimension of hospital care distinguishes scottish patient stories from those of their english and especially continental counterparts . indeed , voluntary boarding was generally seen in the nineteenth century as a typically scottish feature . in england , on the other hand , voluntary admission to rate - aided mental hospitals was not widely practised until the 1930 mental health act . in gartnavel s annual report for the year 1923 , 45 per cent of patients admitted that year were classified as voluntary . this rose to an average of 50 per cent in 1924 , and 60 per cent in 1927 . ( further research into the background of voluntary patients may show that such admissions may have been related to social class . yet preliminary investigations suggest that voluntary patients ranged from the highest fee - paying boarders , paying 160 per annum for their treatment , to the lowest ones paying 1 10s per week . ) this contrast between voluntary and certified patients is illustrated in the case notes of harriet paterson , a young female patient who entered gartnavel as a certified patient in 1924 . after being transferred from a nursing home , she arrived dressed in what was described as a flimsy evening frock and , through a cascade of tears , she protested against her , it was reported that paterson regarded her father as having no legal right to force her into an asylum because she was a married woman . still crying and showing herself as very distressed to the medical officers physical marriage a scotch marriage , and that there was nothing immoral about her. annabel brown , another young female patient , is similarly recorded as having sat in the reception room : clinging to her father , very pale faced and distressed , not demanding to be taken away , but making no move to go to the ward with sister , and expressing fear doubt imploring her father to give her just another minute , kissing embracing him in a manner painful to observe . he , himself , showed much indecision of mind , encouraging her in her embraces , asking [ medical officer ] if he were sure this was the best place for her , enquiring how she would be treated etc . clinging to her father , very pale faced and distressed , not demanding to be taken away , but making no move to go to the ward with sister , and expressing fear doubt imploring her father to give her just another minute , kissing embracing him in a manner painful to observe . he , himself , showed much indecision of mind , encouraging her in her embraces , asking [ medical officer ] if he were sure this was the best place for her , enquiring how she would be treated etc . in the introduction to a social history of madness , roy porter called for in - depth investigation into the socio - historical contexts that inform the narratives of those considered mentally ill . focusing on the ways in which patients negotiated definitions of their illnesses as well as how they viewed themselves as patients and individuals , porter explored illness as a mutable , indefinite concept , aligned to changing notions of individual and social identity . the vividness of these descriptions in the gartnavel records is valuable in that it throws light on the social processes behind dynamic case note construction . these sources enable readers to engage with the wider socio - historical contexts as well as the subjective and interpersonal relationships that gave meaning to an individual s actions . harriet patterson s case , for example , illustrates that conflicting inter - war notions of morality , authority and legality played a role in her attitude . her behaviour suggests that her own perception of her admission was based upon ( and shaped by ) cultural norms rather than strictly medical notions of health and illness . therefore , factors such as class and gender are integral to our understanding of this story . these patient narratives shed light on what it meant to be classified as mentally ill in a period in which common cultural understandings of mental illness were interwoven with notions of sexual ( a)morality and transgressive gender roles . they also show that patients ( especially certified ones ) developed complex strategies of resistance to counter the medical authority and preserve their sense of agency . duncan barrowman , a voluntary boarder transferred from stirling district mental hospital , declared that he was glad to arrive at gartnavel as he was also voluntarily admitted in late november 1925 , he plunged straight into a long rambling discourse about his symptoms beginning with pimples on his anus ending with a heat in his throat all while signing the voluntary forms in the male reception room . haggling for his treatment , armstrong stated that he was willing to pay anything up to 20 weekly , but the clinical clerk noted that he insisted that he should have things done exactly as he wanted them. one of the most important factors affecting an individual s reaction to this new setting , as observed by henderson , was his or her status as a voluntary or certified patient . early in his career he stressed that the line of division between those patients predisposed to recovery and those who failed was often drawn between the cooperative and non - cooperative patients , not between any supposed standard of sanity or insanity. throughout the 1920s gartnavel s admission staff therefore diligently recorded whether patients showed fear and/or ambivalence upon admission , or , by way of contrast , demonstrated acceptance and eagerness to submit to the hospital s treatments . my analysis suggests that there was , indeed , a correspondence between level of compliance to the hospital regime and patients admission status . sources show numerous occasions on which certified patients were recorded as resisting , evading and distrusting the legitimacy and efficacy of hospital care , while voluntary patients more often took on the role of what would now be called service user by cooperating with , or even negotiating , the terms of their treatment . this crucial ( if under - studied ) dimension of hospital care distinguishes scottish patient stories from those of their english and especially continental counterparts . indeed , voluntary boarding was generally seen in the nineteenth century as a typically scottish feature . in england , on the other hand , voluntary admission to rate - aided mental hospitals was not widely practised until the 1930 mental health act . in gartnavel s annual report for the year 1923 , 45 per cent of patients admitted that year were classified as voluntary . this rose to an average of 50 per cent in 1924 , and 60 per cent in 1927 . ( further research into the background of voluntary patients may show that such admissions may have been related to social class . yet preliminary investigations suggest that voluntary patients ranged from the highest fee - paying boarders , paying 160 per annum for their treatment , to the lowest ones paying 1 10s per week . ) this contrast between voluntary and certified patients is illustrated in the case notes of harriet paterson , a young female patient who entered gartnavel as a certified patient in 1924 . after being transferred from a nursing home , she arrived dressed in what was described as a flimsy evening frock and , through a cascade of tears , she protested against her , it was reported that paterson regarded her father as having no legal right to force her into an asylum because she was a married woman . still crying and showing herself as very distressed to the medical officers legal marriage but rather a physical marriage a scotch marriage , and that there was nothing immoral about her. annabel brown , another young female patient , is similarly recorded as having sat in the reception room : clinging to her father , very pale faced and distressed , not demanding to be taken away , but making no move to go to the ward with sister , and expressing fear doubt imploring her father to give her just another minute , kissing embracing him in a manner painful to observe . he , himself , showed much indecision of mind , encouraging her in her embraces , asking [ medical officer ] if he were sure this was the best place for her , enquiring how she would be treated etc . clinging to her father , very pale faced and distressed , not demanding to be taken away , but making no move to go to the ward with sister , and expressing fear doubt imploring her father to give her just another minute , kissing embracing him in a manner painful to observe . he , himself , showed much indecision of mind , encouraging her in her embraces , asking [ medical officer ] if he were sure this was the best place for her , enquiring how she would be treated etc . in the introduction to a social history of madness , roy porter called for in - depth investigation into the socio - historical contexts that inform the narratives of those considered mentally ill . focusing on the ways in which patients negotiated definitions of their illnesses as well as how they viewed themselves as patients and individuals , porter explored illness as a mutable , indefinite concept , aligned to changing notions of individual and social identity . the vividness of these descriptions in the gartnavel records is valuable in that it throws light on the social processes behind dynamic case note construction . these sources enable readers to engage with the wider socio - historical contexts as well as the subjective and interpersonal relationships that gave meaning to an individual s actions . harriet patterson s case , for example , illustrates that conflicting inter - war notions of morality , authority and legality played a role in her attitude . her behaviour suggests that her own perception of her admission was based upon ( and shaped by ) cultural norms rather than strictly medical notions of health and illness . therefore , factors such as class and gender are integral to our understanding of this story . these patient narratives shed light on what it meant to be classified as mentally ill in a period in which common cultural understandings of mental illness were interwoven with notions of sexual ( a)morality and transgressive gender roles . they also show that patients ( especially certified ones ) developed complex strategies of resistance to counter the medical authority and preserve their sense of agency . duncan barrowman , a voluntary boarder transferred from stirling district mental hospital , declared that he was glad to arrive at gartnavel as he was fed up with his former co - patients . as for george armstrong , also voluntarily admitted in late november 1925 , he plunged straight into a long rambling discourse about his symptoms beginning with pimples on his anus ending with a heat in his throat all while signing the voluntary forms in the male reception room . haggling for his treatment , armstrong stated that he was willing to pay anything up to 20 weekly , but the clinical clerk noted that he insisted that he should have things done exactly as he wanted them. the physical examination played an important part in the hospital routine and , as such , featured prominently in case notes . here , records similarly capture vignettes of patients reactive strategies towards the rules , regulation and reality of their new status . typical of these examination records are statements attesting to the individual s general health , with observations made about pulse , cleanliness of tongue , physical build , distribution of hair , reflexes , and so on . it was in this part of the hospital routine , following admission , that patient stories began to emerge more clearly in records , leading to the more in - depth mental examination. however , as this section indicates , various factors determined the patients willingness to tell their stories . in cases where the reactions of a patient to the physical examination provoked him or her to enter into conversation with the psychiatrist to discuss symptoms and experiences , these dialogues could find their way into the case note . before discussing these processes of authorial selection by medical authorities , it is important to get a picture of the way in which an array of power structures , narrative conventions and spatial parameters shaped the dialogues that passed between patients and doctors . their experiences are recorded as ranging from compliance and passivity to fear , hostility and resistance : she was extremely unwilling to allow a physical examination to be done . she refused to allow the chest to be examined saying that she understood this to be a mental hospital and that she did not see what examination of a patient s chest had to do with mental disease . the necessity of making a physical examination was explained to her but she still persisted in her unreasonable attitude after a great deal of delay and persuasion she allowed the examination asking in a sarcastic tone as it proceeded if the mo [ medical officer ] could find any evidence of mental disease from his examination . she refused to allow the chest to be examined saying that she understood this to be a mental hospital and that she did not see what examination of a patient s chest had to do with mental disease . the necessity of making a physical examination was explained to her but she still persisted in her unreasonable attitude after a great deal of delay and persuasion she allowed the examination asking in a sarcastic tone as it proceeded if the mo [ medical officer ] could find any evidence of mental disease from his examination . patient narratives , whether elicited upon admission during the physical and mental examination or later in the open ward , sometimes held aetiological significance . one morning in july 1929 , a medical officer was conducting his rounds when , upon entering one of the female wards , he observed miss elizabeth murray , a newly admitted voluntary patient , acting in what he characterised as a high state . upon miss murray s admission to the hospital the previous day it had been recorded that she spoke to herself a great deal and at times got very impulsive. during the latter part of the morning she had lain in bed : her eyes , observed a clinical clerk , were flashing and she immediately made strange signs. the notes indicate that as the day went on [ s]he blew from her mouth and made movements of her arms which seemed to indicate that she was pushing or brushing away the medical officer. the next day miss murray remained in bed and reportedly became very antagonistic when a doctor came to sit next to her . speaking in a loud declamatory voice , she warned that if he remained seated in front of her she would spit on him . she spat on me , three times ; and then she said something like thank god , thank god , he does not flinch. as she calmed down , curtains were drawn around the bed and miss murray allowed the nursing sister to arrange her dress for the physical examination . again she showed momentary flashes of antagonism and proceeded to sing aloud danny boy , danny boy as the physician attempted to auscultate the thorax . gradually , this period of excitement subside[d] and he was no ( definite ) miss murray then made reference to the coming of heaven , and stated how when she was a child she had a fancy . she looked into a looking glass and saw another room ; and [ said ] that she had wished to get into that other room , the other side of the glass . she looked into a looking glass and saw another room ; and [ said ] that she had wished to get into that other room , the other side of the glass . miss murray smiled when the medical officer mentioned alice through the looking glass , but he remarked : she seemed to suggest we were now through the glass we were through the veil of death into heaven. in cases such as this , the psychiatrist primarily studied the patient for signs of disease and physical disorder . however , he also used common cultural discourses , metaphor and metonymy to engage with the patient s inner world . while the clinical encounter was recorded from the perspective of the psychiatrist , the narrative from below ( including non - verbal forms of communication , such as a glance or spit ) was accorded a high degree of significance and made its way into the case notes . to be sure , the organic origins of mental disorders were given etiological importance , as suggested by the above psychiatrist s attempt to auscultate the thorax ; yet records show that it was also crucial to let the patient tell her story . by forging a linguistic bridge between the reality of the patient s own world and the world jointly shared by patient and psychiatrist , henderson sought to promote an empathic , non - reductionist approach . for him , rather , it was considered of great value to get an exact description of the delusions and hallucinations [ if any were present ] , and how the patient has reacted to them. it is not the situation itself that matters , he noted , but what the subject feels about it , that was of greatest significance . yet precisely what psychiatrists did with the patient s story is made most evident in the ensuing mental examination . as mentioned above , by the early 1920s dynamic case - taking guides were being published in north america , and an analysis of gartnavel s case notes reveals a resemblance to these published procedures . these guides advised that medical officers begin the examination by assessing the patient s stream of mental activity and studying their emotional reaction ( affect) , their mental trend : content of thought as well as their insight and judgement. patients inner worlds , feelings and mode of communication were to be judged in relation to their appreciation of the shared several cases suggest , both patient and psychiatrist could fight to establish the reality of that shared world . indeed , as with the physical examination , the flow of the conversation could be resisted or disrupted by the patient s actions . thus robert norton , admitted in 1926 as a voluntary boarder , rather resented being asked questions. reflecting on the encounter , the examining psychiatrist wrote : he discussed the way in which the questions were put , was suspicious of my motives in questioning him , and he refused to allow me to take any notes . he said that if i took notes he would refuse to speak to me at all he constantly repeated the questions i asked him , he found fault with it in various ways , he complained about the manner in which it was asked , he took exception to the way i sat on a chair besides his bed , and eventually he refused to co - operate any further , finishing up by saying that he considered i was just as insane as he was . he discussed the way in which the questions were put , was suspicious of my motives in questioning him , and he refused to allow me to take any notes . he said that if i took notes he would refuse to speak to me at all he constantly repeated the questions i asked him , he found fault with it in various ways , he complained about the manner in which it was asked , he took exception to the way i sat on a chair besides his bed , and eventually he refused to co - operate any further , finishing up by saying that he considered i was just as insane as he was . in this case , the patient interpreted the act of note - taking as intrusive the record of harriet smith indicates that she : showed a natural anxiety that what she said was not overheard by anyone else in the ward . at one point in the examination she asked anxiously if anyone saw the report and said she did not like notes being taken of what she said . showed a natural anxiety that what she said was not overheard by anyone else in the ward . at one point in the examination she asked anxiously if anyone saw the report and said she did not like notes being taken of what she said . the spaces in which these initial examinations were conducted are important to understanding this resistance from below. case notes reveal that patients often remained within their dormitory beds , surrounded by other patients and ward nurses . the open dormitories that housed many of gartnavel s lower - class patients necessitated indeed , there is evidence to suggest that mental examinations , conducted in such a public place behind only the flimsiest of partitions , could prove an inconducive environment for the revelation of patient stories . without the establishment of trust , henderson and his colleagues were denied access to the patient s story and therefore deprived of the dialogue that was so central to their investigative methods . case notes highlight the fact that obtaining the illness narrative was often a thing to be hard won , not given over lightly or indeed entirely by the patient . it was not atypical , then , for medical officers to have to fight to establish such a dialogue . on a morning in may 1921 , a certain miss margaret beaton lay crying in bed as henderson and an accompanying clinical clerk entered the ward and sat beside her . a preliminary discussion between henderson and miss beaton ensued , followed by the formal mental examination . having been informed by a ward nurse that the patient had not slept since her arrival , henderson found her to be in a highly emotional state . frequently sobbing throughout the interview , her general sullen appearance was noted by the clinical clerk , while henderson began an analysis of his patient by systematically exploring the different mental fields ( grouped under henderson opened the conversation with a succession of simple questions , asking miss beaton to confirm her name and her general complaints , with the purpose of eliciting spontaneous speech . he obtained a perfectly lucid response from her and no evidence of organic disturbance was noted in the interview transcript . henderson therefore proceeded to enquire about the degree of insight miss beaton had into her condition . endeavouring to ascertain whether she considered herself mentally ill and whether she would co - operate or resist the examination process , he asked : i was foolish , selfish and stubborn and rather a bad temper and this is what they ve done.who ? i was foolish , selfish and stubborn and rather a bad temper and this is what they ve done. who ? your mother and brother ? yes . her home , she replied , was depressing , characterised by poverty and unhappiness. her mother was described as a woman who would never give into anything , was suspicious of everyone and whose old - fashioned ways clashed with her own . henderson suggested that her mother s suspicions may have been justified , as he reminded miss beaton that she had herself threatened her mother in various ways . i did nt mean it , she responded . as henderson drew the conversation towards the domestic sphere , miss beaton s present actions and emotions began to take meaning in the context of his psychotherapeutic model . he sought out a longitudinal history that would show whether her emotional outbursts and depressive states periodically reoccurred throughout her life history , or whether such incidents were more dependent upon present environmental factors ( and might therefore be more amenable to treatment ) . after he had asked miss beaton whether she could explain her general irritability i havent had anything that other girls have had.lifes hard? suggested henderson.yessick of the thing?not all together. [ sic ] i havent had anything that other girls have had. life s hard? suggested henderson . not all together. [ sic ] when asked about her friends , miss beaton replied that she was always one too many , that no one took her side and that everything was always blamed on her . as henderson reflected upon miss beaton s self - reported history of fraught familial relationships and social isolation , he began to analyse the depression and anger she exhibited within the mental examination from a longitudinal perspective . since she projected the source of her troubles on to others , henderson tried to reassure her that her family wanted to make her better . do many a thing for punishmentwe want to help youi do nt think it s the way to make me not people , the type of people.dr henderson said it worked out although not apparently likely on surface.that is nt so this has ruined me . they ll triumph over me ; they do nt care a bitthis idea was disagreed by dr henderson ; but she persisted it was so . i do nt think it s the way to make me not people , the type of people. dr henderson said it worked out although not apparently likely on surface. that is nt so this has ruined me . they ll triumph over me ; they do nt care a bit this idea was disagreed by dr henderson ; but she persisted it was so . here , we see the patient s narrative conflicting with henderson s , both aiming to establish the miss beaton insisted that her confinement to gartnavel was a form of punishment ; that her family had sullied her reputation and cast her from her home . the intensity of her emotions , depth of despair and strength of her antagonism flowed out in one long tirade . also highlighted in this case is the multi - vocal nature behind the production of such records . at one point , miss beaton momentarily turned her attention away from henderson to confront the attendant clinical clerk . i know you re taking notes , she stated , thus breaking away from the enclosure of a two - way dialogue . all at once we are reminded that this interview was not carried out within an enclosed and private space , but in an open ward , among medical staff of whose gaze she was fully aware . behind the overt content of the mental examination report , we are subtly alerted to her surroundings , to the recording mechanisms that actively shaped her dialogue and to an indistinct , yet pervasive , sense of the clinical atmosphere still infusing the transcript . these examples point to the complex physical and social conditions behind note production , which directly or indirectly made their way into patient narratives . on a formal level the various grammatical choices and narrative stylistics ( for example , quotation marks within quotation marks ) , while revealing something of the tempo and rhythm of speech , are not reproduced uniformly throughout case note records ; indeed they are also prone to inconsistencies in punctuation . my approach here is to replicate , as far as possible , the original layout , as well as any grammatical errors or inconsistencies that appear within the original sources . by retaining such inconsistencies , i retain layers of authorial agency to highlight that case notes can not , alone , be used to understand the subtleties of these narratives from below. in this respect , it also becomes important to draw a distinction between records produced at the time of a clinical encounter and those retrospectively compiled in an administrative environment . this is significant when considering the kinds of information recorded by the first - person observer and that which may have been revised and altered in the hours , days and months after clinical observations . for instance , upon the arrival of a new patient at gartnavel the task of the clinical clerk was to transpose admission data ( such as medical certificates ) into the case note record . the information was often typewritten and produced in an administrative , rather than a specialist clinical environment . other records of mental and physical examinations , however , were often penned by hand in the ward , in close proximity to the patient . meyer wrote , unequivocally , that notes should be written down at the time of observation ; for him , this offered a more immediate , unrevised investigation of events as they unfolded . however , mental and physical examination records show revisions and overlays of additional handwritten , sometimes typed , observations . different handwriting styles and typewritten sections attest to the thought development of one or more medical officers , showing the process of case note construction to be a fluid , sometimes unstable and often collaborative process . staff meeting was , for a number of patients , the next stage in their analysis , as gartnavel s medical officers endeavoured to construct clinically rigorous [ i]n order to get at the dynamic factors it is usually necessary to have repeated interviews and finally to make a careful analysis of the entire material. staff meeting records , which were reproduced in verbatim transcriptions , bear marks of the introduction of new recording technologies and further stylistic forms of narrative representation . the following section illustrates how the disparate parts of the physical and mental examination were drawn together in the staff meeting , highlighting the marriage of voices from below and from above in case note construction . staff meetings , which pre - dated the contemporary case conferences , were put into use at gartnavel from 1921 onwards . following meyer s practices in the united states , henderson instigated the routine by which gartnavel s staff gathered within a meeting room three mornings a week and were introduced to newly admitted patients to discuss their cases . used as an educational and training exercise , the staff meeting consisted of a single examining psychiatrist directing questions towards the patient , while a trained stenographer , who sat silently among the staff , recorded in verbatim fashion the conversation that passed between patient and practitioner . in accordance with meyer so , while the acts and utterances of patients were recorded , so too were the provoking actions of the psychiatrist . transcribed from the perspective of the stenographer who sat clicking away at their stenographic machine , these materials reveal , most clearly , the interactive dimensions of those clinical encounters . by preserving the raw dialogues that passed between the various actors , such sources allow an in - depth hermeneutic enquiry into the illness narrative as it was shared , modified and negotiated by both medical officer and patient . these transcripts are , in many ways , a more direct source of patient narratives than the case note record . for instance , they contain a more uniform application of grammar , with the dialogues of patients and medical officers recorded in straightforward question - and - answer format . they are also the product of single typists rather than a conglomerate of writers who recorded , word - for - word , the events of the staff meeting at the time it was conducted . finally , these materials reveal the presence of other members who also , directly or not , contributed to shaping these dialogues . several days , sometimes weeks , after arriving at the hospital , many of gartnavel s newly admitted patients were encouraged to attend the staff meeting . escorted by a nurse from their respective wards to the ground floor meeting room , the patient was greeted by a medical practitioner upon arrival . asked to sit down , he or she was directed to face the interviewing medical officer , while a gathering of gartnavel s resident staff were seated behind the patient . i usually try to see every person who comes here in this way , henderson stated to one patient during a staff meeting in 1925 , to have a talk over things with them. as within all mental examinations carried out in gartnavel during this period , the perceived efficacy of this approach depended a great deal upon the patient s willingness to co - operate . however , for a number of individuals , the social setting of this event was itself a significant factor in shaking their confidence . speaking in the presence of several members of staff created a further loss of intimacy . a certain miss mary smith , for example , was very agitated , and very averse to coming into the room. asked by henderson what makes you so frightened? , miss smith failed to respond and became very anxious that the nurse who accompanied her , and dr henderson and dr thomson , should be seated , while she herself insisted on standing throughout the interview. another staff meeting transcript highlights how the presence of multiple staff members could alter the tone and content of a patient s narrative . complaining about her hospital conditions , mrs mary cranford exclaimed : i am just kept like any common prisoner . the board of medical officers , who were sitting behind her , had gone unnoticed until this point ; she then turned around in her chair and said : excuse me , gentlemen , i did not know you were sitting there or i would have been a little bit more cautious. patients did not easily confide during these encounters . in order to establish confidence and encourage the patient to speak , henderson and meyer both advised that he or she be treated absolutely on an equal footing with the physician. if an adequate level of trust was established between the two parties , one of the first steps taken to understand the patient s supposedly dysfunctional behaviour was to develop a sensitivity to the stories , [ which ] the patient may just by chance tell. delusional statements , wrote meyer , must be accepted as one would accept the religious convictions of a person of a different denomination , while remarks made to other psychiatrists must show the same consideration ; no pressure must be used. moreover , in order not to disturb [ the patient s ] sense of safety or make [ her ] suspicious that confidences are being betrayed , the physician should as far as possible only engage in topics of conversation that have been worked out and discussed conjointly with the patient. rather than the medical officer s narrative running counter to the alleged delusional beliefs or rigidly conceived premises held by the patient , the practitioner engaged and indeed endorsed meyer advised that , through a process of compromise , ideas pertaining to this shared common reality gradually be introduced into the conversation . by encouraging their interlocutors to elucidate further the meaning of concepts and phrases particularly pertinent to the case within the staff meeting , medical officers endeavoured to comprehend a patient s own , sometimes unique , usage of language : patient . i can always do much as i like but i do nt function properly . you have heard of such cases before , but it is a freakish state ; you have heard of men who can do unnatural things with their bodies you have heard about the clincher ? it made my nerves worse . a man who is in proper condition has a proper spine i can always do much as i like but i do nt function properly . you have heard of such cases before , but it is a freakish state ; you have heard of men who can do unnatural things with their bodies you have heard about the clincher ? well , he does not work as any other natural man would dr henderson . it made my nerves worse . a man who is in proper condition has a proper spine it has turned the wrong way. contextualising and further clarifying the meaning of phrases such as working from his head , having one s spine twist or to get the wind up enabled gartnavel s practitioners to establish a shared , common language through which to establish confidence and understanding . this was considered an initial step in the process of correcting a patient s faulty adaptations . in this respect , it is interesting to note that while patients were encouraged to act as collaborator[s ] in the treatment endeavour , these stories could undergo revision as the meeting progressed . at the beginning of her interview , for example , a patient named helen davidson was determined to reveal nothing on the subject of spirituality . after having earlier intimated her belief that she was the virgin mary , she was interviewed by the medical practitioner : q. was there any intimation conveyed to you from any outside source in regard to this ? did you have a feeling as if there was some heavenly presence intimating this thing to you?a . no.q . as if there was a heavenly voice that said this thing to you ? did it seem to come from your own inner consciousness , or what ? was it something that seemed to grow up inside you?a . no.q . how was the information conveyed to you then?a . by reading.q . by reading what?a q. was there any intimation conveyed to you from any outside source in regard to this q. did you have a feeling as if there was some heavenly presence intimating this thing to you ? q. as if there was a heavenly voice that said this thing to you ? did it seem to come from your own inner consciousness , or what ? was it something that seemed to grow up inside you ? q. how was the information conveyed to you then ? q. what was it that specially seemed to convey that idea to you ? as tensions between this patient s narrative and that of the psychiatrists saw the patient contradict herself , denying , then confessing that these ideas sprang from reading the bible , the reader is made aware of the active re - negotiation of a patient s life history within this setting . we are reminded that we can not unearth an essential truth about a patient s history ; instead what appears is an active co - construction of the patient s narrative . the reader is confronted not with the meeting of two dichotomous narratives , but with an array of stories , being continually reshaped and revised in ( and beyond ) the meeting . amidst these processes of co - construction and reconstruction , new meanings and new identities emerge . how to interpret and compare the authorial presence behind the semi - verbatim transcriptions of the physical and mental examinations with the verbatim transcriptions of the staff meeting ? though barely considered by meyer and not at all by henderson , the presence of this significant player in the staff meeting ( which by then was becoming widely practised in north america due to meyer s influence and teaching ) was given some consideration in a 1937 article published in the american journal of psychiatry : presumably for the sake of the records a stenographer is present . it is a pity she can not be made invisible . upon the other hand , a clever one can be trained to make running comments and notes upon the patient s responses when these concern solely matters of fact , thus avoiding in part the long series of questions and answers which present such a hopeless array upon many staff conference records . a signal system can be readily devised for informing the stenographer when to make verbatim records , when to summarize and when to make no notes whatever . presumably for the sake of the records it is a pity she can not be made invisible . upon the other hand , a clever one can be trained to make running comments and notes upon the patient s responses when these concern solely matters of fact , thus avoiding in part the long series of questions and answers which present such a hopeless array upon many staff conference records . a signal system can be readily devised for informing the stenographer when to make verbatim records , when to summarize and when to make no notes whatever . this wish for invisibility of the stenographer is , in many ways , a reflection of the stenographer s discretion on the pages of gartnavel s staff meeting transcripts . unlike case note records , which intersperse verbatim samples of speech with clinical observation , gartnavel s stenographers leave only the barest of clues upon the transcript as to their authorial presence . only occasionally do the stenographer s own observations break through into the transcript , to offer summaries of staff members comments or give brief indications as to the actions and behaviours of patients . it is from these brief asides that we glean a sense of the pace and tone of the conversation , of the groups and individuals to which certain statements were directed , and of the movement and unspoken interactions within these enclosed meeting rooms . the third - person narrative of the stenographer contrasts sharply with the first - person authorial tone of the medical officer . indeed , verbatim samples of speech are clearly distinguished from the stenographer s own summaries . when examining the stenographer s use of punctuation and descriptive inserts , we notice , in many cases , that the pace and tempo of the patient s voice is preserved in the way of stage directions ( for example , very slowly and deliberately ) . indications are also given to mark those points where dialogue jars , with voices cutting across or over - spilling into one another . as for exclamation marks , although used sparingly , most appear within text that records henderson s speech ; and , although we can not be sure of the exact tone or pitch of his voice , it appears to have been raised on a number of occasions : patient . i will investigate that you will be punished for keeping me staying here.dr h. i am perfectly innocent i have nothing to do with your coming here , you must not punish me ! patient . i will investigate that you will be punished for keeping me staying here. dr h. i am perfectly innocent i have nothing to do with your coming here , you must not punish me ! despite the above , records show that patient narratives in the staff meeting were not always transcribed in fully verbatim fashion . when confronted with the colloquial use of words , stenographers tried to retain the characteristic phrases and mannerisms that marked patients language ; but on occasion a certain accent or dialect could present too difficult to replicate . ( this patient spoke with a marked brogue , and in a low voice at times , and it was difficult indeed impossible frequently to catch what he said). these notes , which endeavour to represent textually the structure , fluidity , tempo and intonations of spoken dialogue , not only highlight the interpersonal relations shaping one another s responses . they also emphasise the subjectivity of their authors . by casting light on the mediatory role of the stenographer , such passages interrogate the nature of ( purportedly transparent ) verbatim records in the making of patient stories from below. words are both crucially reflective of the goings - on in the human world , but also unavoidably generative of that world , write chris philo and cheryl mcgeachan . as such , one must explore how psychiatrists , clinical clerks and stenographers not only recorded but also actively shaped reconstructed the discursive happenings of the clinical encounters at gartnavel mental hospital . while these records allow glimpses into the patients subjective worlds , they also reveal a process by which patient narratives were collected , compared and classified ; that is , they highlight the epistemic virtue of such records in this rapidly changing context . as michel foucault theorised , the arrival of the case note facilitated the process by which patients were described , judged , measured , compared with others so they may be precisely understood through their very individuality , and yet simultaneously corrected , classified , normalized or excluded so that their identities became fixed by the medical gaze. with these case notes revealing the profoundly polyphonic nature of mental examinations at that time and place , we may , therefore , question precisely how such sources while rich in patient psychiatrist dialogues capture words that exposed , and were generative of , the world around them . unlike contemporary psychoanalytic case notes which were effectively used to verify specific theoretical models , dynamic case notes , as used by henderson and meyer , were intended to record the patient s verbally expressed wishes , desires and emotions as standing above or in implicit precedence to any particular interpretation. these materials , while infused with the authorial agency of their psychiatrists , unveil a history of patient psychiatrist dialogues in which the power to define the story of illness ebbs and flows between both parties . they show that physical as well as the interpersonal spaces in which the two parties spoke significantly shaped the content and delivery of their dialogues . moreover , while psychiatrists endeavoured to penetrate the more intimate , interior spaces of the patient s mind memory , thought , emotion and imagination it had to be given freely by patients themselves , whose stories were in many ways considered the key to unlocking the origins , meanings and possible treatments of their conditions . patients willingness to reveal their stories depended on a number of factors such as gender , social class and admission status . undoubtedly , dynamic case notes offer a rare window into the patient s view at that seminal period . wishing to distance himself from psychoanalytic case studies , which placed patients language within a rigid and specialised framework of understanding and interpretation , henderson went to considerable lengths to reproduce a faithful , coherent account of complex clinical events . but how far do these textual sources represent the reality they pertain to capture ? due to new techniques of transcription introduced at that period , the historian can get a fuller picture of the complex social interactions between all members of the medical encounter and , in the process , analyse the important role played by patients in shaping this history from below. yet the conditions of production must also be taken into account when interpreting such materials . as jonathan andrews has shown in his study of gartnavel s case notes in the nineteenth century , the influence of prevailing conceptualisations of mental illness has played a significant role in the changing degree to which physicians included the patient s voice in these records . some of the greatest challenges for the historian reside in the areas of incompleteness and inter - textual discrepancies. in the same vein , if we know little about the process of case note selection in the 1920s , we also tend to overlook the process of interpretation by contemporaneous psychiatrists and historians alike , adding yet more levels of ( re)construction . as such , when considering the incorporation of sources from below , historians ought to keep in mind the context in which these stories were generated . this becomes all the more relevant in a setting in which verbatim and semi - verbatim transcripts are presented as narrative truth. as i have shown , the analysis of institutional spaces , clinical practices and psychiatric forms of knowledge must also be taken into account when following porter s call to explore the

this article contextualises the production of patient records at glasgow s gartnavel mental hospital between 1921 and 1932 . following his appointment as asylum superintendent in 1921 , psychiatrist david kennedy henderson sought to introduce a so - called dynamic approach to mental health care . he did so , primarily , by encouraging patients to reveal their inner lives through their own language and own understanding of their illness . to this effect , henderson implemented several techniques devised to gather as much information as possible about patients . he notably established routine staff meetings in which a psychiatrist directed questions towards a patient while a stenographer recorded word - for - word the conversation that passed between the two parties . as a result , the records compiled at gartnavel under henderson s guidance offer a unique window into the various strategies deployed by patients , but also allow physicians and hospital staff to negotiate their place amidst these clinical encounters . in this paper , i analyse the production of patient narratives in these materials . the article begins with henderson s articulation of his dynamic psychotherapeutic method , before proceeding to an in - depth hermeneutic investigation into samples of gartnavel s case notes and staff meeting transcripts . in the process , patient psychiatrist relationships are revealed to be mutually dependent and interrelated subjects of historical enquiry rather than as distinct entities . this study highlights the multi - vocal nature of the construction of stories from below and interrogates their subsequent appropriation by historians .

The Patients View Henderson, Meyer and the Introduction of Dynamic Case Notes The Dynamic Case Note as Established at Gartnavel Setting the Stage: The Admission Compliance and Resistance Patient Stories: The Physical and Mental Examination Patient Stories: The Staff Meeting Concluding Remarks

PMC3597927

to establish occlusal plane while recording maxillomandibular relation during fabrication of complete denture , many methods have been tried in the past . needless to say that correct orientation of the occlusal plane plays a vital role in providing optimal esthetic and functional satisfaction.1 improper orientation of this plane jeopardizes the coordination between the components of oro - facial articulatory complex.2 the plane of occlusion is formed after joining the incisal edges of the mandibular central incisors to the disto - buccal cusps of the mandibular first molars . the occlusal plane is defined as the common plane established by the incisal and occlusal surfaces of the teeth ( glossary of prosthodontic terms-8 ) . it is not a plane in the true sense of the word , but represents the mean of the curvature of the inciso - occlusal surfaces of teeth . there is a controversy for the orientation of the occlusal plane during complete denture fabrication . various landmarks and techniques had been used over the years by clinicians and researchers for establishing this plane which include : establishing the occlusal plane according to aesthetic requirements anteriorly and parallel to the ala - tragus line posteriorly ( craddock,3 1951 ; schlosser & gehl,3 1953 ) , positioning the occlusal plane parallel to and midway between the residual ridges ( nagle and sears,3,4 1962 ; boucher et al.,5 boucher6 1964 ) , orientating the occlusal plane with the buccinator grooves and the commissure of the lips ( lundquist and luther,7 1970 ) , terminating the occlusal plane posteriorly at the middle or upper third of the retromolar pad ( ismail and bowman,8 1968 ) , positioning the occlusal plane on the same level as the lateral border of the tongue ( ismail and bowman,8 1968 ; yasaki,9 1961 ) and many more concepts are reported in literature . however , the most acceptable method of orienting the occlusal plane is to make it parallel to the ala - tragus or camper 's line as reported in several modern text books . in spite of its widespread acceptability , it is surprising to note that the exact location of the point on tragus while marking ala - tragus line is unclear . this has created lot of confusion while selecting a point for marking ala - tragus line . so this study was therefore undertaken to decide the most appropriate point on tragus to be used as a reference point while marking ala - tragus line . this study was carried out using simple and economical instruments and materials . for the study , following two devices 1 ) was developed to verify the parallelism of the occlusal plane of maxillary occlusal rim with ala - tragus line in posterior and interpupillary line in the anterior region . it comprises of lower plate having outer " u " shaped frame which will be located over the face and inner fork plate having a shape of average arch . three metal scales having holes at both the ends to pass through four threaded vertical rods - two lateral scale and one frontal scale and these scales can be adjusted and fixed at a desired height on vertical rods using nuts to simulate parallelism of the occlusal plane with ala - tragus line and interpupillary line . four threaded vertical rods with the corresponding nuts were used to fix the lower frame with the upper two lateral and frontal plate at a desirable vertical height to check parallelism of the occlusal plane with ala - tragus line which was marked using superior point on tragus with ala . the occlusal rim was positioned over the edentulous foundation and was supported by the fork plate of the lower frame . the maxillary occlusal rim was adjusted for : height , labial inclination and arch form . the occlusal plane was then adjusted to achieve parallelism - anteriorly with interpupillary , posteriorly with ala - tragus line ( fig . the metal foil was then adapted over the adjusted occlusal surface of the rim ( fig . the lateral scales were adjusted in line with the ala - tragus line and were fixed in position by tightening the nuts over the threaded vertical rods . the frontal scales was also adjusted at the level of interpupillary line and secured tightly in position using the nuts ( fig . the transpar ent plastic angle measuring device:- rectangular acrylic sheet ( transparent ) measuring 16 13 cm was used as a platform plate . over this plate 3 plastic ( transparent ) scales were fixed ( one horizontal at the bottom and two in lateral sides in such a way that the inner angles between these scales were exactly measured 90 ( fig . the protractor was modified to obtain reading between 0 - 90. this modified protractor has provided the opportunity to measure the angles between various planes used in this study . the transparency of this device offered clarity while measuring the angles by positioning it over the tracing line(s ) of the cephalograph accurately . data was collected for two groups of the subjects : ( a ) dentulous group , ( b ) edentulous group having sample size of 30 for each group with equal gender distribution . selection criteria for dentulous group includes -subjects aged between 18 - 30 years , having 28 to 32 teeth and ideal arch alignment with angle 's class i molar relationship , andno history of orthodontic treatment with normal profile . selection criteria for edentulous group includes subjects aged between 50 - 70 years , havingh no history of tmj disorder and associated pathology , neurological disorder and maxillofacial trauma with clinically healthy oral mucosa . three points were indentified on tragus of ear as superior ( s ) , middle ( m ) and inferior ( m ) . the stainless steel balls ( 3 mm in diameter ) were fixed at these selected points and also at the ala of the nose with fine quality double sided adhesive tape to ensure perfect placement to capture the recording on cephalographs ( fig . lateral cephalographs were then shooted , developed and fixed by using standard technique . for edentulous patients , the maxillary ( wax ) occlusal rim was adjusted forlabial inclination , arch form , midline and canine lines were marked and occlusal plane was adjusted using occlusal plane analyzer ( anteriorly parallel to the interpupillary and posteriorly parallel to the ala - tragus line ) . now this adjusted maxillary occlusal rim with adapted metal foil on the occlusal surface was positioned over the maxillary foundation . the ala - tragus line was marked by joining superior point of tragus with base of the ala of the nose . the points used for tracing were orbitale ( or ) , porion-(po ) , superior ( s ) , middle ( m ) , inferior ( i ) point of tragusand inferior point of the ala of nose ( a ) . various planes marked were frankfort horizontal plane ( fh ) , occlusal plane ( op ) and three planes formed by joining s to a ( sa plane ) , m to a ( ma plane ) and i to a ( ia plane ) . various angles evaluated in this study were ca nt of occlusal plane ( coo ) , angle between fh plane with sa plane ( sfh ) , ma plane ( mfh ) and ia plane ( ifh ) . the angles between op plane and sa plane ( sop ) ma plane ( mop ) and ia plane ( iop)were also measured ( fig . angle coo was used as a standard which is the angle formed between the fh plane and the occlusal plane . in downs analysis , the occlusal plane used was formed by marking a line bisecting the occlusal surfaces of permanent molars and incisal overbite . mean value of angle coo was found to be 9.3 and ranges between 1.5 to 14 in those samples used in downs study.10 - 12 this angle measures the slope of the occlusal plane relative to frankfort horizontal plane . over the traced cephalographs of dentulous patient , the values of angles coo , sfh , mfh , and ifh were measured , tabulated and compared with the value of average coo angle from downs analysis . along with this , the value of angles sop , mop , iop were also measured and tabulated . on the cephalographs of edentulous subjects , the angle between fhp and sa , ma and ia was measured , tabulated and compared with the value of average coo angle from downs analysis ( fig . the measurements recorded for dentulous and edentulous subjects were tabulated and subjected for the statistical analysis ( statistica- version 6 , stas.soft . , comparitive evaluation of the angles - coo , sfh , mfh , ifh , sop , mop , iop in dentulous subjects and sfh , mfh , ifh in edentulous subjects were carried out to find out the significance and its usefulness in deciding the most appropriate point on tragus to be used as a reference point while marking the ala - tragus line . statistical tests used were descriptive analysis , student 's unpaired t - test and pearson 's correlation coefficient . after statistical analysis the results were tabulated and following results were drawn:- table 1 ( dentulous group ) demonstrated mean value of coo in male and female was 9.93 and 9.60 , respectively . with average value of 9.76 . mean ifh in male and female was 11.20 and 9.60 , respectively with average value of 10.40 . the mean iop in male and female was 2.26 and 2.66 , respectively with average value of 2.46 . the mean ifh was closest to mean coo and mean iop was closest to 0 compared to the sop and mop . in table 2 , the edentulous group shows that the mean ifh in male and female was 10.93 and 10.20 , respectively with average value of 10.56 . the mean ifh was closest to average coo 9.3. correlation and probability of coo with sfh , mfh , and ifh of the dentulous group was calculated and positive correlation was observed between coo and sfh , mfh , ifh . student 's unpaired t test exhibits the mean difference in means of sfh , mfh , ifh with coo found to be statistically significant sfh ( p=.007 ) , mfh ( p=.005 ) and ifh ( p=.007 ) . the mean angle of ifh in male was 11.2 with standard deviation of 2.45 and ifh in female was 9.6 with standard deviation of 3.13 , which is closest to average coo mean 9.3 compared to sfh and mfh . in tables 3 and 4 , it is noticed that the mean difference in mean angle was found to be statistically significant for sfh ( t = 14.90 , p=.000 ) , mfh ( t = 9.65 , p=.000 ) , ifh ( t = 2.99 , p=.006 ) in male and for sfh ( t = 12.37 , p=.000 ) , mfh ( t = 5.88 , p=.000 ) in female but statistically insignificant for ifh in female(t = 0.37 , p=.714 ) . the mean angle of ifh in male was 10.93 with standard deviation of 2.89 and in female was 10.20 with standard deviation of 3.42 , which is closest to average coo 9.3 when compared to sfh and mfh . tables 5 and 6 demonstrate student 's unpaired t test which shows that the mean difference in mean angle was found to be statistically significant for sfh ( t = 13.24 , p=.000 ) , mfh ( t = 5.52 , p=.000 ) , ifh ( t = 2.18 , p=.037 ) in female and for sfh ( t = 11.56 , p=.000 ) , mfh ( t = 6.93 , p=.000 ) in female , but was statistically insignificant in female ( t = + + 1.01 , p=.318 ) . some of the existing concepts regarding orientation of the occlusal plane in the edentulous patients are of the opinion that the occlusal plane has relation with camper 's line . it would seem desirable to locate it accurately and propagate its use while establishing the occlusal plane.13 the definitions of the ala - tragus line has created confusion . the exact points of references on tragus and ala are not categorically specified.14 spratley14 described it as running from the center of the ala to the center ( middle ) of the tragus . boucher5,6 defines it as " the line running from the inferior border of the ala of the nose to the superior border of the tragus of the ear . " however , out of the seven texts that propound its use , only one has provided proper definition and it cites boucher's5,6 definition . two texts recommended the concept without defining or illustrating it , while three provided only pictorial representation . the latter are of immediate concern , for in each illustration ; camper 's line is clearly depicted as extending to a point , not at the superior border , but at the center of the tragus of the ear . this pictorial representation corresponds to the definition given by of ismail and bowman8 ( 1968 ) and evidently reflects an understanding of the concept , which predates boucher 's definition.15 camper 's plane is defined as ( i ) a plane established by the inferior border of the left or right ala of the nose and the superior border of both ears ; ( ii ) a plane passing from the acanthion to the centre of the bony external auditory meatus ( gpt 1999 ) . the occlusal plane is considered to be parallel to this plane.15 in this study , the widely accepted downs analysis10 was used as a base line for comparison . downs analysis on variations in facial relationship indicates that the ca nt of the occlusal plane ( coo ) , the angular relation between the occlusal plane and the frankfort plane , ranged from 1.5 to 14 with a mean value of 9.3.10 - 12 the frankfort horizontal plane is a fixed anatomical craniometrical landmark , thus being used as a skeletal guideline while locating occlusal plane posteriorly.16 it has no effect of the loss of teeth and it can be easily located with the help of lateral cephalograph and therefore fh plane was used in this study . the mean value of coo was observed to be 9.7 in this study which is within acceptable range of 1.5 to 14 and closest to mean value of 9.3 as demonstrated in the downs study . the difference of 0.4 degree is clinically insignificant and may be accredited to the variations in racial population . the mean angle ( total ) ifh found in dentulous subjects and edentulous subjects was 10.40 and 10.56 , respectively . thus , the mean value of ifh angle is comparable with the mean value of the angle established in downs analysis.10 - 12 the mean angular value of iop 2.46 2.48 , is least among sop and mop . this observation of the present study is similar to the observation of van niekerk et al.15 ( 1985 ) who recorded an angle between the occlusal plane of complete dentures and the ala tragus line ( inferior point on tragus was used ) as 2.45 ( sd = 3.24 ) . this means that the line marked from the inferior point of tragus to ala of nose was the most parallel line with the occlusal plane in dentulous subjects as compare to the lines from superior and middle point of tragus with ala of nose . this underlined the importance of using " inferior point " for marking ala - tragus line during recording of jaw relation for edentulous patient . the results of this study are in agreement with the previous studies by clapp13 ( 1910 ) , dalby13 ( 1912 and 1914 ) , wilson13 ( 1917 ) , giffen13 ( 1925 ) , gillis13 ( 1933 ) and hartono17 ( 1967 ) . the statistical analysis carried out for males and females for the various angles in dentulous and edentulous , males and females shows no significant difference ( p > .005 ) . this indicates that gender difference does not cause any variations in the angular relationship between the ala - tragus line and frankfort horizontal plane and alatragus line and occlusal plane . therefore it was concluded that inferior point on the tragus is the most valid point for males as well as female while marking ala - tragus line for establishing occlusal plane . in accordance to the results obtained in this study the order of preference out of three tragus point for marking ala - tragus line is inferior point ( i ) , middle point ( m ) and superior point ( s ) . it is also observed that when posterior occlusal plane was established using ala - tragus line marked from the superior or middle point , the occlusal plane was placed at a higher position which is in agreement with the results of the previous studies.2,5,6,18 - 22 but , it is contrary to the study of nissan et al.23 where superior point on tragus was recommended . the middle point on the tragus was also recommended as a point for establishing occlusal plane in some studies.13,21,24 - 26 these variation may be due to effect of racial and ethnic populations . the values of sfh , mfh , ifh this study for both dentulous and edentulous subjects confirms that the inferior point gives closest value of coo closer to downs analysis . statistically significant difference was observed between average coo and mean angular values of sfh , mfh , ifh for all groups , except for ifh values for female in both dentulous ( p=.714 ) , and edentulous ( p=.318 ) group where there was no significant difference found . therefore , as indicated by the results of this study , the inferior point in females gives closer angle with fhp , to average value of coo . the inferior point marked on tragus is the most appropriate point for marking ala - tragus line for establishing occlusal plane in edentulous subjects .

purposethe purpose of this study was to decide the most appropriate point on tragus to be used as a reference point at time of marking ala tragus line while establishing occlusal plane.materials and methodsthe data was collected in two groups of subjects : 1 ) dentulous 2 ) edentulous group having sample size of 30 for each group with equal gender distribution ( 15 males , 15 females each ) . downs analysis was used for base value . lateral cephalographs were taken for all selected subjects . three points were marked on tragus as superior ( s ) , middle ( m ) , and inferior ( i ) and were joined with ala ( a ) of the nose to form ala - tragus lines . the angle formed by each line ( sa plane , ma plane , ia plane ) with frankfort horizontal ( fh ) plane was measured by using custom made device and modified protractor in all dentulous and edentulous subjects . also , in dentulous subjects angle between frankfort horizontal plane and natural occlusal plane was measured . the measurements obtained were subjected to the following statistical tests ; descriptive analysis , student 's unpaired t - test and pearson 's correlation coefficient.resultsthe results demonstrated , the mean angle coo ( ca nt of occlusal plane ) as 9.76 , inferior point on tragus had given the mean angular value of ifh [ angle between ia plane ( plane formed by joining inferior point - i on tragus and ala of nose- a ) and fh plane ) as 10.40 and 10.56 in dentulous and edentulous subjects respectively which was the closest value to the angle coo and was comparable with the values of angle coo value in downs analysis . angulations of ala - tragus line marked from inferior point with occlusal plane in dentulous subject had given the smallest value 2.46 which showed that this ala - tragus line was nearly parallel to occlusal plane.conclusionthe inferior point marked on tragus is the most appropriate point for marking ala - tragus line .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC4210180

proteins are complex molecules of fundamental importance in biological processes . numerical simulation using molecular dynamics ( md ) has proven to be a powerful tool to predict many important properties such as the native state of the protein or its free energy . in this paper , we will focus on methods , based on md , to calculate reaction rates , which are defined as transition rate between metastable states or conformations of the protein . as a byproduct of our analysis , we will also calculate the main mechanisms of the reaction , i.e. , the transition pathways . for example , a cartoon model of the free energy for a biomolecule is shown in figure 1 . the left region represents the reactant states ( r ) , and the right the product states ( p ) . trajectories spend most of their time in r or p with infrequent transitions due to the energy barrier that separates the two states . molecular dynamics sampling of a one - dimensional free - energy surface between reactant ( r ) and product ( p ) states . by periodically recording the molecular conformation , energy , and other observables , various relevant properties of the protein can be computed . in 1977 mccammon et al . applied md to the bovine pancreatic trypsin inhibitor ( bpti ) . while the system was simple ( vacuum with a crude force field ) , the simulation nonetheless contributed to shifting the view of proteins as rigid since this initial simulation , md has been used to study a wide variety of topics , such as identification of integral motions such as hinge bending modes , trna flexibility , and the study of e. coli chaperone groel . the application of md to larger and more complicated molecules impacts development of force - field parameters such as the charmm and amber families , and solvent models ( generaly categorized as implicit or explicitly defined ) . recently , md has been used to study an hiv capsid and a complete satellite tobacco mosaic virus , as well as screening designed proteins . in fact , a large portion of the major supercomputers time is currently dedicated to this type of simulation . the main difficulty is that the time scales of interest are typically in the millisecond ( 10 s ) while a typical time step in md is on the order of the femtosecond ( 10 s ) . therefore , a brute force simulation would require on the order of 1000 billion time steps , which is impractical given the current hardware . high - performance computing infrastructures allow development of efficient parallel algorithms to speed up simulations . specialized hardware , such as mdgrape and anton , provide orders - of - magnitude speedup over traditional high - power computing ( hpc ) simulations . similarly , work with graphical processing units ( gpus ) have shown the that gpus achieved greater performance over a single cntral processing unit ( cpu ) , allowing a single gpu to simulate biological systems with comparable performance to a cluster . most approaches to date attempted to accelerate a single ( or a few ) very long simulations . due to the sequential nature of md simulations , this is clearly a challenging task , which limited scalability . , much work has gone into the development of fast and efficient algorithms to dedicate large resources to long simulations of molecules . for instance , namd and amber were among the first to achieve scaling to hundreds of nodes and microsecond - time scales using parallel implementations . improvements to constraint algorithms , particle interactions , and memory access patterns have resulted in significant performance improvements over the decades . at the other end , a recent class of methods is attempting to predict equilibrium properties , such as free energy , reaction rate and reaction pathways , from a large number of short trajectories . in fact , in this paper , we will demonstrate large - scale simulations using parallel computer grids , showing that scalability and performance can be achieved even on slow networks or when using geographically remote compute nodes . this class of approaches have been applied with great success by several groups , using a method called the markov state model ( msm ) . the entry ( i , j ) in the matrix stores the probability of a trajectory to go from cell i to cell j after some lag time . from the eigenvalues , eigenvectors , and other properties of the matrix , we can extract all relevant time scales and kinetics of interest . this type of approach requires running a large number of trajectories of length and as such is easily parallelizable . the accuracy of this method is independent of the presence of energy barriers or of the metastability of regions in conformational space . instead it relies on efficient local sampling to predict time scales that can be orders of magnitude longer than the aggregate simulation time . one limitation of msm is that its accuracy depends on the choice of . in fact , getting converged rates is very difficult . at short lag times , a significant bias is present in the prediction , that is even in the presence of infinite sampling the predicted rate is incorrect . this can be addressed by increasing the lag time but this has two limitations . , it is difficult to obtain converged results with respect to the lag time and sampling size . once a msm has been computed , one can obtain the implicit time scales from the diagonalization of the stochastic transition matrix . these implicit time scales are highly sensitive to the time lag , or the time between samples used to construct the transition matrix : at short time lags implicit time scales have a bias that makes them appear much faster than they really are . at large time lags the statistical error dominates , and it is hard to find a compromise that balances these two errors . in this paper , we considered a variant method , called the weighted ensemble ( we ) approach which has a higher computational cost but convergence is more robust and easier to monitor . the we method was introduced by huber and kim to address the problem of capturing infrequent reaction events under brownian dyanamics . further studies have applied it to simulated annealing ( si ) obtaining higher success rate ( order of magnitude ) for finding the global optimum as compared to traditional si , as well as in the investigation of super oxide dismutase and monoclonal antibody nc6.8 . have extended we and describe several key properties : ( i ) we produces unbiased results for both markovian and non - markovian dynamics , indicating that the method is much more general than previously understood and ( ii ) the method depends on the number of cells , which may be updated dynamically , allowing the system to discover unknown cells without loss of accuracy in the calculation of reaction rates . applications of we to toy models and alanine dipeptid1e , coarse - grained models of adenylate cyclase , and the sodium symport mhp2 transporter protein have yielded promising results . in we a large number of parallel walkers ( md trajectories ) are used . later , we will explain in more details the algorithm , but the basic process is to start a large number of trajectories from regions in a partitioning of the free - energy surface . after a small number of md integration steps we consider the population of walkers in each cell . then , using a statistically unbiased procedure , we are able to either remove walkers from overpopulated cells or , on the contrary , repopulate cells that are becoming depleted . this is done through an appropriate subselection ( killing walkers ) or duplication process . this ensures that when the simulation restarts the number of walkers in each cell is the same , and is equal to some target number . then , we resume the simulation and integrate forward in time each walker using md . this process is repeated until the quantities of interest , such as the reaction rates , which are computed through an appropriate postprocessing procedure , are converged . this method avoids the slow time scales found in md by ensuring that all cells are equally populated . this means for example that regions near the transition barrier between two metastable regions are sampled as often as minimum free energy basins . this method can be interpreted as a way of enhancing the sampling of conformational space . we note that its efficiency still depends on an appropriate choice of macro - states or cells , although the fact that these cells form a simple partition and can be constructed in a variety of ways ( e.g. , they can be simple voronoi cells ) gives a lot of flexibility to the method . some of the advantages of we is that it is easy to setup , is inherently massively parallel and scalable , and is unbiased . for example , unlike msm , we is guaranteed to converge to the exact result with enough samples . notwithstanding these properties , awe , like msm and other related methods , remains computationally expensive due to the large number of walkers that need to be run . this paper will present a software infrastructure we developed to facilitate running such calculations with accelerated convergence . this is achieved by an appropriate selection of the initial conditions as described in hence the method is known as accelerated weighted ensemble ( awe ) , an extension of we where the initial weights have been approximated to the steady state weights to accelerate the convergence . we compute them from a transition matrix obtained from the simulation , based on cells as they come from clustering , perhaps using msmbuilder . awe also uses a simpler and more accurate method for resampling and generating new walker populations through splitting and merging of walkers . awe allows one to eliminate the bias introduced in msm by generating the statistically exact distribution of walkers inside each cell . msm uses the equilibrium distribution for the walkers in each cell , which causes a bias . instead , awe uses an out - of - equilibrium distribution corresponding to walkers flowing from reactants to product states ( e.g. , folding / unfolding ) and vice versa , thereby producing exact statistics . we focused on the following main goals when designing the software:1.allow using any md code in a black - box manner , without having to make intrusive changes inside the code . this is important since many md codes are available and people are often required to use a particular md code because of some special required capability . for instance , the following md codes are usable with our framework : amber , charmm , gromacs , lammps , namd , mdynamix , orac , gromos , tinker , desmond , dl_poly.2.dynamically scale to available resources as they are added and removed as availability or funding allows and robustly handle resource failure . details are discussed later , but the main idea is to support , for example , starting awe using a dedicated cluster locally then adding cloud infrustructure such as amazon ec2.3.use a scripting language for quick prototyping , interactive simulations , and user - friendly codes . the use of this simple scripting interface also means that it is not difficult to reuse our framework for other methods that require running a large number of short trajectories with some appropriate postprocessing , e.g. , msm and other related methods . allow using any md code in a black - box manner , without having to make intrusive changes inside the code . this is important since many md codes are available and people are often required to use a particular md code because of some special required capability . for instance , the following md codes are usable with our framework : amber , charmm , gromacs , lammps , namd , mdynamix , orac , gromos , tinker , desmond , dl_poly . dynamically scale to available resources as they are added and removed as availability or funding allows and robustly handle resource failure . details are discussed later , but the main idea is to support , for example , starting awe using a dedicated cluster locally then adding cloud infrustructure such as amazon ec2 . use a scripting language for quick prototyping , interactive simulations , and user - friendly codes . the use of this simple scripting interface also means that it is not difficult to reuse our framework for other methods that require running a large number of short trajectories with some appropriate postprocessing , e.g. , msm and other related methods . in this paper , we present a distributed computing implementation of awe , based on workqueue ( wq ) , that is capable of using computing resources with different architectural properties ( e.g. , gpu , cpu ) , as well as different job execution semantics such as dedicated ( hpc grid ) or cycle - scavenging ( condor ) resources . at peak performance , for our validation , awe - wq simulated an aggregate 1.5 ms , with a peak performance of 1000 ns / h , showing that a large amount of resources can be efficiently managed by the system . similar software packages include the copernicus framework developed by pronk et al . , adaptive markov state models ( adaptive msms ) by bowman et al . , and the weighted ensemble simulation toolkit with parallelization and analysis ( westpa ) developed by zwier et al . copernicus is a framework for running ensemble molecular dynamics that allows multiple resources to be used , supports multiple project types , and adaptive sampling . adaptive msms iteratively build msm models to determine the contribution of each state to the slowest kinetic process . based on this information further simulations are run from states based proportionally to their contribution to the uncertainty . westpa is a software framework for running we simulations . currently under development by lillian chong and dan zuckermann , westpa has been used to study several systems , such as molecular association and the sodium symporter mhp1 . the fault - tolerance model , intelligent task scheduling , and caching distinguish awe - wq . these features allow the program to dynamically handle resource addition and removal , automatically handle worker failures , recover from machine failures , support clusters of heterogeneous computers , and minimize data transfer . we first describe the algorithm , design challenges , and implementation in the design and implementation section and provide a brief overview of usage . second , in the results section , we demonstrate that awe - wq meets the criteria described in the design and implementation section as well as provide a validation of the implementation on a nontrivial protein . finally , we provide public access to the software and the data sets used for the results as well as describe the current limitations and future directions in availability and future directions section . the we approach proceeds as follows and illustrated in figure 2.1.partition state - space into c cells.2.determine w number of simulations ( walkers ) to maintain in each cell.3.parallel step ( walk ) : run the n = w c short simulations , assign each of the n final conformations to a cell.4.synchronization barrier ( resample ) : once all the n simulations are assigned , merge and split the walkers to maintain w walkers in each cell and update the associated weights.5.go to step 3 unless weights converged . parallel step ( walk ) : run the n = w c short simulations , assign each of the n final conformations to a cell . synchronization barrier ( resample ) : once all the n simulations are assigned , merge and split the walkers to maintain w walkers in each cell and update the associated weights . go to step 3 unless weights converged . given a population of walkers with weights wi , we first calculate a mean weight wm = i(wi / ntargetwalkers ) , where ntargetwalkers is the desired number of walkers that we are trying to achieve . then , if a walker has a weight greater than wm , it is split into an integer number of walkers of weight wm , with a remainder that is reinserted into the list of walkers to process . walkers that have a weight less than wm are merged in a statistically exact way to create walkers with a weight greater or equal to wm . by repeating this process of splitting and merging walkers , we are able to generate a population of walkers with weights exactly equal to wm , in a statistically exact manner . this is different from the procedure in the work of huber and kim that generates walkers with weights between wm/2 and 2wm but not exactly equal to the target weight wm . the choice of method for defining cells is orthogonal to we . as long as a cell definition exists , in cells.dat currently , awe - wq is able to use it . this allows one to define cells arbitrarily to examine the effect of a cell definition method . we based the cells on the msm construction in order to accelerate the convergence of cell weights . as explained previously , the main reason to use we is to remove the bias present in msm models due to the lag time and the ( incorrect ) distribution of walkers inside each cell . upon reaching steady - state the weight of each cell converges to its probability , allowing this procedure to calculate free energy . each region represents a cell on the energy surface , the circles the walking simulations , the colors the association with the reactant ( r ) or product ( p ) regions . calculation of transition rates requires a further modification : define two sets of states , r and p , associated with the reactant and product regions ( figure 2 ) . each walker is assigned a color , such as blue for r and red for p , corresponding to the previously visited region . at each step in the simulation whenever a blue walker enters p , its color changes to red , and vice versa . the rate from r to p is then directly obtained by computing the flux of blue particles that change color , and similarly for the p to r rate . if extended to multiple colors and sets multiple kinetic rates can be determined . in practical terms , the r and p regions may correspond to the unfolded and folding regions and the fluxes to the folding and unfolding rates . by providing an initial approximation of the free energy the convergence to steady the method requires the following three steps ( as illustrated in figure 3 ) : ( 1 ) most of the work is done in parallel as a large number of independent short md trajectories . ( 2 ) the parallel barrier : wait for all md steps to complete then collect walker assignments and final positions , split and merge walkers , and update weights . each walker is then , in parallel , executed and assigned to a cell . the join waits for all walkers to finish , before the resample procedure is applied . if the system has converged then the program halts , others walkers are rerun . in order to use awe - wq one must define the cells from which to run the walkers . the general protocol , illustrated in figure 4 , is as follows : ( 1 ) sample the search space , ( 2 ) determine cell definitions , ( 3 ) prepare input files , ( 4 ) run awe - wq , ( 5 ) monitor progress . first some sampling must be done to explore state space . this can be accomplished with programs such as gromacs , charmm , amber , namd , or using infrastructure such as folding@home . for this instance , sampling of alanine dipeptide has been run previously , which can be extracted : to start , we have collected md data and stored them in the xtc directory . the directory structure is xtc / run#/frame0.xtc , where # is the trajectory number . the sampling data is imported into msmbuilder using the convertdatatohdf.py command , paramterized with a pdb file containing the system coordinates and the path to the md data : diagram of the workflow to run awe - wq with associated programs and descriptions . first a sampling must be run with some md software such as gromacs , charmm , amber , etc . prepare the input to awe - wq by converting the files and defining metastable states . run awe - wq by starting the master process and allocating workers with various resources . conformations are clustered with a hybrid k - centers / k - medoids algorithm using the rmsd between atoms as the distance metric . using a subset ( such as all non - hydrogens ) prevents too fine a granularity from overfitting the data . finally , we will cluster the data into 100 groups . by inspecting the implied time scales ( not shown ) extract a given number of conformations from each of the cells defined above ( savestructures.py ) and import the cell definitions from msmbuilder format to awe - wq format ( awe - import - gens ) and setup the initial files for the awe - wq run ( awe - prepare ) . regions of metastable states need to then be determined , which are ultimately given as a list of the cells belonging to each region ( e.g. , folded and unfolded ) and scripts are given to do so using rmsd to a reference structure ( awe - calc - gens - rmsd , awe - define - regions ) . we plan to maintain 10 simulations in the cells , so we need to extract conformations from the states using msmbuilder . in order to run awe - wq we must then import the cell definitions which were written by msmbuilder to gens.lh5 . in order to compute the fluxes we need specify regions of metastable states . awe - wq provides some commands to assist with this process : awe - calc - gens - rmsd and awe - define - regions . we use awe - calc - gens - rmsd to compute the rmsd of each cell to some reference conformation such as the native state . by plotting the distribution of values we can classify conformations with rmsd 2.3 as folded and those with rmsd 2.5 as unfolded . the two output files folded.dat and unfolded.dat now contain the integer indices of the states belonging to these regions . we can now prepare for awe - wq by checking dependencies and populating the directory with other necessary files by running awe - prepare . there are two components to consider when running awe - wq : the master process and the resources . the master is the driver of the algorithm , managing task definitions , scheduling , processing , and the resampling procedure . in order to run the walkers this process loads the initial conformations ( walkers ) for each cell , and is in charge of scheduling each task , processing the result , and the resampling procedure . this runs awe - wq maintaining 10 walkers in 100 cells , whose definition is provided in cells.dat with initial weights in data / populations.dat . the metastable regions are provided in folded.dat and unfolded.dat as a list of cell i d numbers belonging to each region . finally , we give a name to the master ( awe - wq ) to that workers can easily locate the host and port . resources can be allocated either directly using work_queue_worker or managed automatically using work_queue_pool . using work_queue_worker also allows the worker to operate as a foreman , enabling the hierarchical distribution of tasks . the work_queue_pool program maintains workers for the master based on need and is in charge of submission to various backends such as the local machine , condor , sge , pbs , etc . use work_queue_status to get the current resources runtime status ( number of workers , number of tasks waiting / completed , etc . ) . by using awe - plot - wq - stats the plot of the resource usage over the runtime of the program can be obtained . additionally , the current status of the master , such as workers busy and tasks complete can be viewed using the work_queue_status command . the awe - flux command allows the convergence of the calculated flux to be monitored . for instance , the energy surface for alanine dipeptide can be visualized as a function of its dihedral angles . as such , we can plot , as shown in figure 5 , the cell coordinates and the initial estimation of the weights as well as computed weights after several iterations of awe - wq . since the awe method follows a pattern of fan - out followed by fan - in we implement using a master / worker paradigm . awe - wq is designed to ( 1 ) support off - the - shelf md backend software , ( 2 ) dynamically scale according to resource availability , ( 3 ) support heterogeneous runtime environments , and ( 4 ) be robust in the face of system failure . application of awe to a nontrivial biological system may have on the order of 10 000 simulations per iteration . on one cpu , if each walker requires 30 min to simulate , 7 months would be required to run a single iteration of awe . since walkers are short , assuming 30 min per walker , 7 months would be required to run 10 000 walkers for each awe iteration using a single processor . with 1000 cpus , the expected time drops to 5 h , assuming uniform performance . additionally , the synchronization barrier allows straggling workers to slow down the entire application . since we expect to need hundreds of iterations to converge , the challenge is to implement awe such that it scales to 1000 + processors and can be sustained for months with no major slowdown due to stragglers . rather than reimplement the core md algorithms which are present in multiple software packages ( i.e. , gromacs , amber , charmm , etc . ) this flexibility presented several challenges by imposing a requirement of specifying the steps needed to run the backend , the transfer of files , the execution environment , and the interaction with the other steps of the procedure . the approach taken in the following : a walker is described as a task which consists of a program to run and the input and output files . once specified , this task is scheduled to run on an available worker , which is a process that accepts any input files , executes the task s command , and returns the results upon request . the awe - prepare script sets up files and scripts in the local directory necessary to run awe - wq . one of these scripts , execute-task.sh , executes the commands on the worker . in order to use a different md backend execute-task.sh needs to define how to run the md , and awe - wq needs to specify the files to send to the worker . our solution to the worker fault - tolerance problem implies the elasticity the ability to dynamically scale to available resources of the program . if a task fails during execution , it is rescheduled to run on the next available worker . as resources are added or removed , tasks are started or reschedule , respectively . this allows a project to be started with the campus cluster , then ec2 machines run until budget exhaustion , then continued with only the cluster . managing the resource pools are the interactions between the master , the catalog server ( cs ) , and workqueue pool ( wqp ) processes . the cs allows workers to dynamically determine the location of the master by mapping a project name ( e.g. , awe - wq ) to a hostname and port ( e.g. , fah.crc.nd.edu:1024 ) . the master registers this information when started , which allows it to be restarted in the event of machine failure without requiring the resources to be reallocated . in addition , the cs store information about the current resource requirements of a master , such as the number of tasks waiting to be scheduled . a wqp can thus use this information to automatically start workers based on the runtime needs . this is further enabled by the asynchronous model , which treats tasks as independent , arbitrarily executable entities . additionally , awe - wq can use heterogeneous resources , allowing systems with different properties and ( potentially ) operational semantics to be used . for instance , this supports using gpu - based as well as cpu - based md backend codes . this is accomplished by extending the task abstraction to dynamically resolve file location upon worker connection rather than statically . for example , an input file can be specified for a task as binaries/$os/$arch / mdrun . when a worker connects to the master it reports the operating system and architecture of the remote node in the $ os and $ arch variables , which may then be expanded to send binaries / linux / i686/mdrun and binaries / darwin / x86_64/mdrun to to workers running on a 32-bit linux and 64-bit mac os machine , respectively . finally , these variables can be overridden by the user to provide additional granularity . awe - wq is implemented with the assumption that machine failures are not only possible but in fact common , which is the opposite of the paradigm underlying mpi infrastructures . as such two broad categories of failures need to be accounted for : failure of the worker or master processes . in the first case , worker failure are handled transparently and tasks rescheduled to the next available worker . failure of the master node would normally result in the termination with an inability to resume . this is overcome by using a transactional logging mechanism : a combination of persistent and volatile logs . each result from a walker is added to a log and after each awe iteration the state of the system is checkpointed to disk and the log cleared . if the process fails , then the state is initialized from the checkpoint and the log used to replay and missed results from that iteration . the second involves task replication to remove the effect of straggling workers . by monitoring the state of the workers either busy or idle ( wi)and the number of tasks waiting to be assigned a worker , task replication can be engaged when wi > 0 . once a task returns , all its replicates are canceled , results are accepted in a first - come - first - serve fashion . by combining replication with preferential scheduling , workers are continuously used and slow workers allowed to contribute results until the fast machines are available , which then run the replicated tasks . the inner loop of awe - wq is an event - loop with asynchronous task execution . each iteration begins by translating the internal data model of the walkers into task , which are submitted to the wq scheduler . while tasks are executing , the program is idle until a result is returned . each result creates an event that must be handled , either by translation or resubmission . handlers are lightweight : read the final coordinates and cell assignment into the data models . since task executions are asynchronous and results are processed serially , the event model enforces load balancing by spreading out the calls to the event handlers . increasing scalability is further accomplished by minimizing the communication overhead and maximizing the parallel work . ideally , the master process will spend most of its time waiting . by categorizing files as those common to all tasks ( common ) or specific to the current one ( unique ) , the common files can be cached on the workers . since we expect the number of walkers to be larger than resource availability the caching mechanism shifts overhead to depend on resources rather than problem size . for instance , using a gromacs backend each task requires 34 mb in input files , binaries , and miscellaneous files . in the case of the ww domain simulation , each task required 34 mb of gromacs - related files as overhead and 100 kb of task specific data . assuming 10 000 tasks / iteration and 500 iterations , the total amount of data transferred is 34 ( mb / task ) 10 000 ( tasks / iteration ) 500 iterations 2 ( tb / mb ) = 162 tb . by caching input files , data transferred depends only on the number of new workers seen , rather than total number of tasks , which may be orders of magnitude greater . in this case , assuming 1000 workers are connected the total data transferred becomes 34 mb 1000 2 ( gb / mb ) = 33 gb . we have run awe - wq for several months where 3.5 million tasks have executed over 600 compute years in a heterogeneous environment simulating over 1.5 ms of time . in this section available resources consisted of the following : notre dame hpc grid with 6000 cores shared among campus users ; notre dame condor pool with variable ( usually around 1200 ) cores and flocking capabilities with purdue university and the university of wisconsin madison ; stanford university s institute for computational and mathematical engineering ( icme ) cluster with 200 cpus and 100 nvidia gpus , and cloud virtual machines via amazon ec2 and microsoft azure . the gpu cluster executes tasks within minutes , while the nd hpc grid and ec2 machines are the best performant cpu - based pools . the range of performance of condor machines is unsurprising and illustrative of the cycle - scavenging nature of the platform . the azure machines were the least performant resources , partly due to the necessity of running the tasks via cygwin . distribution of task execution times indicate several peaks associated with the different underlying resources being used . figure 7 demonstrates the fault - tolerance elasticity and scalability of the application as an expanded view of the number of busy resources over the runtime the inset shows the entire run . unexpected failures of the master ( months 4 , 6 ) initiated development and integration of features , bug fixes , and monitoring capabilities . due to power outages and infrastructure upgrades the master process restarted multiple times . in spite of these failures over 250 iterations were completed without data loss due to the transactional logging mechanism . the periodically persisting state additionally allowed us to integrate new features , such as task replication , and bug fixes over the course of the project s execution . focusing on the region between days 865 and 900 shows a steady decrease in the number of busy resources until a new pool of workers became available and awe - wq automatically started using them . it must be emphasized as well that minimal human input was needed to take advantage of the additional resources : due to the use of the catalog server all that was required was to start a wq pool process for the additional resources . the periodic dips , every 5 days in the first half and every 20 h for the second , correspond to the global synchronization barrier , each period requiring 12 000 tasks to complete . by scheduling the tasks based on resource availability and progression through the awe algorithm , resources are fully utilized and the long tail effect from straggling workers is eliminated . due to the use of the caching mechanism communication overhead is reduced : while the entire task payload ( common and unique files ) is 34 mb it is only for the first task a worker executes that his is transferred . each subsequent task only requires 100 kb of data to be transferred ( both in- and outbound traffic ) . the ww domain is a 34 residue protein domain with two conserved tryptophan ( w ) residues and comprised of two antiparallel -sheets as shown in figure 8 . simulation parameters were the amber96 force field with generalized born implicit solvent , a viscosity parameter ( ) of 1 ps , temperature of 395 k ( close to the optimal folding temperature ) , saving conformations every 1 ns . the simulation was run for 200 s during which multiple folding and unfolding events were observed . the conformations sampled by the simulation were then clustered using the and carbons using a cutoff of 3 . in order to reduce the time spent sampling the unfolded space we established that conformations with an rmsd to the folded structure less than 3 and 6 , and greater as folded , intermediate , and unfolded , respectively . using these definitions the number of unfolded states was reduced , for a total of 601 cells . the awe - wq parameters were thus 601 cells , 20 walkers per cell , and walker length of 500 ps using the same md parameters as the source data . expanded view of the entire run ( inset ) demonstrating elasticity , scalability , and fault - tolerance . in the inset , long gaps ( such as the sudden failures at month 4 and 6 ) in activity indicate times when major development was done on the code ( features added , bugs fixed ) . despite the troubles experienced by the master node , calcuation of the results resumed once the code was deployed . plotting the number of busy workers over many days shows the ability of the software to adapt to a changing environment . the periodic dips indicate the global synchronization barrier , each requiring 12 000 tasks to complete . a large increase after day 885 occurred when many nodes were suddenly available and were automatically incorporated . over the course of many months awe - wq completed 345 iterations , with an aggregate 2.3 ms of time simulated using 3.6 million tasks , and a peak performance of 1000 ns / h . analysis indicates that the awe - wq results converge within acceptable error tolerance . table 1 displays the forward and backward rate estimations computed using awe - wq and the 200 s simulation . the awe estimation lies within the confidence interval with smaller statistical error than the brute force simulation . figure 9 illustrates that forward and backward fluxes took 30 iterations to converge to the confidence interval built from brute force simulation and are stabilized within the interval afterward . the resampling procedure of awe introduces no bias in the estimation of transition rates while exhibiting smaller statistical error , showing an improvement over the brute force method . transitions among the states are examined with a time lag of = t = 0.5 ns . three significant pathways from extended to folded state are extracted from the network and shown in figure 10 . starting from an unfolded conformation , two pathways involve multiple helical intermediate structures before -sheet formation occurs via rearrangement . the third pathway occurs as a rapid collapse before the formation of loop 2 . loop 1 is then able to form , after which the molecule undergoes further refinement to arrive at the native state . average forward ( top ) and backward ( bottom ) flux from awe ( blue curve ) with error bar of one standard deviation estimated by the block averaging method , compared with confidence interval built from the brute - force simulation . the red solid line represents the mean flux from the brute - force simulation , and the dashed lines are mean flux plus / minus one standard deviation . first three significant pathways from extended to folded conformations . two pathways ( top , middle ) involve helix formation as an intermediary while the third pathway ( bottom ) consists of a collapse then refinement . awe - wq meets our criteria : allowing the use of off - the - shelf components for md backends , pooling of heterogeneous resources , scalability to over 1000 workers , and ability to cope with failures of both worker and master processes . the ww domain was used to validate awe - wq by applying the method to a long md simulation in which multiple folding events are observed . the forward and backward reaction rates computed by awe - wq are within the range and have smaller error , of the rates calculated from the reference simulation .

a limitation of traditional molecular dynamics ( md ) is that reaction rates are difficult to compute . this is due to the rarity of observing transitions between metastable states since high energy barriers trap the system in these states . recently the weighted ensemble ( we ) family of methods have emerged which can flexibly and efficiently sample conformational space without being trapped and allow calculation of unbiased rates . however , while we can sample correctly and efficiently , a scalable implementation applicable to interesting biomolecular systems is not available . we provide here a gplv2 implementation called awe - wq of a we algorithm using the master / worker distributed computing workqueue ( wq ) framework . awe - wq is scalable to thousands of nodes and supports dynamic allocation of computer resources , heterogeneous resource usage ( such as central processing units ( cpu ) and graphical processing units ( gpus ) concurrently ) , seamless heterogeneous cluster usage ( i.e. , campus grids and cloud providers ) , and support for arbitrary md codes such as gromacs , while ensuring that all statistics are unbiased . we applied awe - wq to a 34 residue protein which simulated 1.5 ms over 8 months with peak aggregate performance of 1000 ns / h . comparison was done with a 200 s simulation collected on a gpu over a similar timespan . the folding and unfolded rates were of comparable accuracy .

Introduction Design and Implementation Results Conclusions

PMC2609950

mouthrinses are a common adjunct to mechanical hygiene measures to facilitate the control of supragingival plaque , therefore dental caries and gingivitis.13 chlorhexidine ( chx ) is a bisbiguanide antiseptic active against gram - positive and gram - negative bacteria , facultative anaerobes and aerobes , moulds , yeasts and viruses . oral chx mouthrinses have been effective in decreasing plaque formation and controlling gingivitis4,5 and dental caries.6,7 its antibacterial activity arises from its positive charge at physiological ph , which produces nonspecific binding to the negatively - charged membrane phospholipids of bacteria ; this causes an alteration in bacterial osmotic equilibrium , with potassium and phosphorus leakage . as the chx concentration increases , several studies have shown that chx has toxic effects on a variety of eukaryotic cells , with the presumed cytotoxicity mechanism being related to electrostatic resulting in inhibition of membrane - bound na - k - atpase . release of lysosomal enzymes into the medium from rat peritoneal macrophages has also been described by knuuttila and sderling8 and an increase in permeability to ca accompanied by leakage of ldh , from human gingival cells exposed to chx by babich et al.9 this increased cell permeability due to the high affinity of chx for negatively - charged organic radicals does not appear to be the only toxicity mechanism . it has been reported that protein synthesis is also affected in different degrees by chx.10,11 more recently chx has been reported to inhibit the activities of two types of matrix metalloproteinases ( gelatinases a and b ) via a cation - chelating mechanism.12 it is a potent chemotherapeutic agent against streptococcus mutans and dental caries . the effectiveness of chlorhexidine containing gels , mouthwashes , and toothpastes in caries prevention was confirmed by some researchers.1316 benzydamine hcl is a unique inflammatory analgesic agent structurally unrelated to steroid group , but also differs chemically from other non - steroidal anti - inflammatory agents in that it is a base rather than an acid . similar to corticosteroids , it stabilizes the cell membrane preventing the release of arachidonic acid , which initiates the inflammatory process . in common with nsaid s , benzydamine inhibits cyclo - oxygenase , reducing synthesis of prostaglandin and related substances.17 biomarkers of genotoxicity can be used as indicators of environmental carcinogen exposure . micronucleus ( mn ) formation has been shown to be a reliable and sensitive biomarker for cytogenetic damage due to potential environmental mutagens . briefly , micronuclei are acentric chromosome fragments or whole chromosomes left behind during mitotic cellular division and appear in the cytoplasm of interphase cells as small additional nuclei.18,19 in light of the fact that over 90% of cancers are of epithelial origin20 the mn assay has also been used in many epidemiological studies as an effective indicator of chromosome damage in exfoliated epithelial cells from lung , bladder , nasal and buccal cavity and cervix.2125 in the present study , our aim was to determine the cytotoxicity of the mouthrinses klorhex ( 0.2% chlorhexidine gluconate ) , andorex ( 0.15% benzydamine hcl and 0.12% chlorhexidine gluconate ) and tanflex ( 0.15% benzydamine hcl ) on buccal epithelial cells by mn test . the study population included 28 patients , 13 males and 15 females with aged 1624 undergone three mouthrinses application were analyzed before and after one week exposure . dmft ( the proportion of the number of teeth decayed , missing / extracted or filled ) scores of patients were zero . the patients had gingivitis as evidenced by multiple sites with a probing depth of 3 mm or less and without bone loss by radiographs . all participants had not previously received non - surgical and surgical periodontal therapy and were drawn from the patients with gingivitis at the department of periodontology . subjects were medically healthy with no relevant medical or pharmacotherapy history that might influence the conduct of the study past 6 months and all of them were non - smokers and were not alcohol consumers . it was important that they had no caries or dental restorations ; because it is known that some dental materials increase the frequency of micronuclei.26 therefore , the patients were chosen carefully among whose dmft scores were zero . the criteria of patient selection were mentioned above and they were applied for all patients in this study . the patients were classified as three equal groups according to mouthrinses they used and the age differences among groups were not statistically significant ( p>.05 ) . all participants received primary phase of non - surgical treatment including oral hygiene instruction and scaling . after the treatment , mouthrinses were prescribed and the rinses were selected randomly . during one week , the patients used the prescribed mouthrinses rinsing twice a day . the rinses were klorhex ( 0.2% chlorhexidine gluconate ) ( drogsan , turkey ) , andorex ( 0.15% benzydamine hcl and 0.12% chlorhexidine gluconate ) ( delta vital , turkey ) and tanflex ( 0.15% benzydamine hcl ) ( abdi ibrahim , turkey ) . physiologic saline was used for the control group . at baseline and after one week plaque index27 ( pi ) ( 0=no plaque in the gingival area , 1=a film of plaque adhering to the free gingival margin , and adjacent area of the tooth . the plaque may be recognized only by running a probe across the tooth surface , 2=moderate accumulation of soft deposits within the gingival pocket and on the gingival margin and/or adjacent tooth surface that can be seen by the naked eye , 3=abundance of soft matter within the gingival margin and adjacent tooth surface ) and gingival index28 ( gi ) ( 0=normal gingiva , 1=mild inflammation , slight change in color , slight edema ; no bleeding on palpation , 2=moderate inflammation , redness , edema , and glazing ; bleeding on probing , 3=severe inflammation , marked redness and edema , ulcerations ; tendency to spontaneous bleeding ) and mn incidence were recorded . plaque and gingival indices were scored from buccal , lingual , mesial and distal points of each tooth . the slides were aged at 37c overnight and then stained with giemsa and screened and 3000 nucleated cells were analyzed for the presence of mn at a final 100x magnification for each participant . micronuclei were identified as dna - containing structures in the cytoplasm , separated form the main nucleus , and of an area less than 1/3 of the area of the main nucleus , non - refractivity , not touching and same the color as the nucleus or lighter.29 the difference in the pre- and post - treatment incidence of pi , gi and mn was compared by wilcoxon signed ranks test . changing of mn incidence was calculated as percentage for each group and compared with control by oneway anova and tukey hsd tests . the slides were aged at 37c overnight and then stained with giemsa and screened and 3000 nucleated cells were analyzed for the presence of mn at a final 100x magnification for each participant . micronuclei were identified as dna - containing structures in the cytoplasm , separated form the main nucleus , and of an area less than 1/3 of the area of the main nucleus , non - refractivity , not touching and same the color as the nucleus or lighter.29 the difference in the pre- and post - treatment incidence of pi , gi and mn was compared by wilcoxon signed ranks test . changing of mn incidence was calculated as percentage for each group and compared with control by oneway anova and tukey hsd tests . the difference in the pre- and post - treatment incidence of the mn , gi and pi values of this study at initial and the1 week are shown in table 1 . in groups of mouthrinses , incidence of the mn there was no difference between pre- and post - treatment period in pi scores in andorex and tanflex groups ( p>.05 ) . in the third group ( klorhex ) , the changing of mn incidence as percentage for each group and comparing with control are shown in table 2 . there was not any difference between andorex and control group ( p>.05 ) . in the other study groups , mn incidence was significantly increased after 7 days treatment of the mouthrinses ( p<.05 ) . increased frequency of micronucleated cells is a biomarker of genotoxic effects that can reflect exposure to agents with clastogenic and aneugenic modes of action.21 the micronucleus assay was applied to verify the effects of the three mouthrinses treatment . the results of this study showed that although the micronuclei incidence increased in klorhex , tanflex and andorex groups after exposure to mouthrinses , the micronuclei incidence of klorhex and tanflex groups increased , except andorex , when compared with the control group . chx is a chemical agent currently used as a local antiseptic in daily clinical practice . the cytotoxicity of chx has been assayed using cell lines or primary cultures of mammalian cells.8,11 eren et al30 evaluated the chx with comet assay ( single cell gel electrophoresis , or scge ) and suggested that a statistical increase was observed in the damaged buccal and blood cells after the chx application . they mentioned that detected dna damage after chx use might be the indication of an earlier effect , before dna repair begins , and could be reversible . wilken et al31 determined the in vitro cytotoxic effect of 0.2% chlorhexidine gluconate and 0.15% benzydamine - hcl and revealed that all the human gingival fibroblasts exposed to chlorhexidine gluconate and benzydamine - hcl were immediately fixated onto the tissue culture surfaces . they concluded that it should be ascribed to the activity of the active ingredients in the mouthrinses and the relative cytotoxicity of the active ingredients of all mouthrinses is very high for human gingival fibroblasts . various organisms and genetic endpoints , including the gene mutations as well as chromosomal damage in mammalian cells , comprise a test battery for analyzing the mutagenic activity of a chemical.32 among these assays , the micronucleus test in vitro is a multiple - endpoint test to indicate chromosomal aberrations.33,34 however , in literature there is not a study that evaluates the cytotoxicity of neither chx nor benzydamine - hcl by mn test . although our main aim was not to compare the measurements of plaque and/or gingivitis between the mouthrinses , the approach clearly revealed the expected result that chx was significantly more effective at preventing the development of plaque than the other two rinses . however , it might be doubtful whether chx is still the golden standard as mouthrinse for the prevention of plaque formation and development of gingivitis3537 when considering its cytotoxicity on human cells as shown in the present study . chx mouthrinses have been effective in decreasing plaque formation and controlling both gingivitis4,5 and dental caries.6,7 but , in this study the patients were chosen carefully among whose dmft scores were zero , because it is known that some dental materials increase the frequency of micronuclei,26 and this could affect the results of the study . mn test that we have applied to buccal epithelial cells of patients detects the possible cytotoxic and/or mutagenic effects of some mouthrinses in this tissue . this study suggests that the amount of absorbed mouthrinses may have been sufficient to induce mn frequencies in buccal epithelial cells of patients even in the range of the biological tolerance levels of these mouthrinses . but it was strange that although there were differences between both klorhex and the control groups and tanflex and the control groups , there were not any differences between andorex and the control groups in our study . hidalgo & dominguez38 showed that cytotoxicity mechanism could be produced in a time- and chx concentration- dependent manner . this reason can be contributed to this result , as lowered concentration of chx exists in andorex . in another point of view , the combinations of chx and benzydamine hcl can lessen the cytotoxicity of this mouthrinse . although there is not any study about the effects of this combination ( andorex ) on cytotoxicity , waaler et al39 and barkvoll & rolla40 noted that triclosan reduced the toxicity of sodium lauryl sulphate while used in combinations . the exposure to cytotoxic and/or mutagenic factors , host factors , methods and scoring data explain the 75% of total variance , with the largest contribution attributable to laboratory methods.41 regarding the host factors , bonassi et al41 suggested that the large majority data showed a higher mn frequency in females and a uniform increase in mn frequencies with age in both genders , an increase that is especially steep after 40 years of age . in our study , we could not find any differences in genders and all study subjects were young people , therefore we could not find also any differences in age . it is common in turkey to prescribe mouthrinses to the patients who suffer from periodontal diseases by the clinicians . mouthrinses are widely utilized in daily oral and dental hygiene to control plaque.4,7 patients should be alerted that mechanical plaque control is more important than chemical plaque control and warned about improper use of these products . the findings support only the prescription of agents or products with the highest proven clinical indication . in conclusion , our findings with the micronucleus test to indicate chromosomal mutations add valuable information to complete the overall elucidation of the cytotoxic activity of some mouthrinses . the formation of micronuclei to various extents by these chemicals was occurred in the present investigation .

objectivesto determine the cytotoxicity of three commercial mouthrinses klorhex , andorex and tanflex on buccal epithelial cells using micronucleus ( mn ) test.materials and methods28 patients with aged 1624 undergone three mouthrinses application were analyzed before and after one week exposure . physiologic saline was used for the control group . the mn incidence was scored in the buccal epithelial of each participants . the difference in pre- and post - treatment after one week incidence of mn and plaque ( pi ) and gingival indices ( gi ) was compared by non - parametric statistical tests.resultsthe micronuclei incidence increased in klorhex , tanflex and andorex groups after exposure to mouth rinses ( p<.05 ) . but when compared with the control group , there was not any difference between andorex and control group ( p>.05 ) . in the other study groups , mn incidence was significantly increased after 7 days treatment ( p<.05 ) . gi scores of all groups were decreased significantly ( p<.05 ) . pi scores were decreased only in the klorhex group ( p<.05).conclusionsour primary findings support the presence of possible cytotoxic effects of the mouthrinses on gingival epithelial cells .

INTRODUCTION MATERIALS AND METHODS Micronucleus test Statistical analysis RESULTS DISCUSSION CONCLUSIONS

PMC4988435

its import is common sense : heat will flow from hot to cold , never the reverse . the second law can also be stated alternatively : entropy can not but increase . the irrevocable increase in entropy , however , is not as tangible a notion as the irreversible motion of energy down from height . so , what does entropy mean ? on one hand entropy s is quite frequently equated with disorder . on the other hand , when the entropy change ds is multiplied with temperature t , the result tds is a perceptible change in energy . so , it is pertinent to ask : how does increasing disorder relate to decreasing free energy ? this question has remained open despite many studies , and hence continues to be right in the forefront of scientific inquiry of life given in terms of physics . on one hand life , by its numerous processes that consume free energy in insolation , food , etc . , is no different from any other process , for instance , inanimate processes that level off temperature gradients . on the other hand life 's tendency to organize true enough , plants , animals and other kingdoms of life do display amazing complexity . then again , some minerals may crystallize to gigantic monoliths with astounding degrees of order . are n't these contrasting examples alone implying that neither disorder nor order is an end in itself , but only an outcome of the irreversible consumption of free energy ? boltzmann was impressed by darwin 's tenet of evolution by natural selection , and wanted to see evolution as a manifestation of the natural law . boltzmann realized that complicated systems comprising numerous particles are best described in statistical terms , though he considered only an ideal gas . according to his kinetic theory of gases entropy s = kb ln w is the logarithm of probability ( wahrscheinlichkeit ) multiplied by boltzmann 's constant kb . the probability meant for boltzmann the number of possible ways the state of a system could be realized from various positions and momenta of its copious constituents . the logarithm of w served to give a convenient additive measure , known as entropy s. boltzmann associated increasing entropy with increasing disorder by reasoning that an orderly ensemble of gas molecules has a low number of possible positions and momenta , whereas when dispersing to macroscopic uniformity , the number of possible permutations will be high , corresponding to the maximum microscopic disorder . noteworthy , boltzmann did not consider dispersal in energetic terms , because the ideal gas , unlike any real substance , was imagined to become disordered without any change in energy , i.e. , without dissipation . in other words thereby , his definition of entropy became conceptually detached from energy , and hence it deviates from reality . thus , it remains for us to formulate how the irrevocable decrease in free energy relates to the probable evolution of a system toward thermodynamic balance in its surroundings . to this end we are guided by common observations that the surrounding density in energy dictates the course of a system . for example , water molecules on a cold surface , when warmed up , will vaporize and disperse . conversely when the gas cools down , the molecules will condense back on the cold surface . likewise , seedlings will organize their growth toward sunshine , whereas when left in darkness their growth disperses aimlessly . thus , the probable course of a system from one state to another is driven by the energy difference between the system and its surroundings , not by disorder or order that are merely consequences of free energy consumption . eventually , when all free energy has been consumed , the system and its surroundings have attained common density in energy , i.e. , the most probable state . although it is next to impossible to know exactly how a complex system comprises of its entities , we may nevertheless formally depict the state of a system exactly by placing its constituents on levels of an energy diagram . in this scale - free manner we can express in energetic terms the probability pj for a population of entities , labeled with j , to exist . then , by considering all populations , we can formulate the total probability p = pj for the entire system to exist in its surrounding density in energy . we begin by assigning each j - entity with a distinct energy attribute gj , given relative to the average energy kbt of the system at temperature t. the j - entities that populate a distinct energy level in numbers nj ( fig . its logarithm is known as the chemical potential j = kbt ln nj + gj.figure 1.the system is depicted in terms of an energy level diagram . at each level , indexed by k , there is a population of nk individuals each with energy gk . when an entity in the population nk transforms to an entity in the population nj , horizontal arrows indicate paths of transformations which are available for changes in the potential energy bound in matter and vertical wavy arrows denote concurrent changes driven by energy in light . the system evolves , step - by - step , via absorptive or emissive jk - transformations that are mediated or catalyzed by the entities themselves , toward a more probably partition of entities eventually arriving at a stationary - state balance where the levels are populated so that the average energy kbt equals that in the system 's surroundings . a sufficiently statistical system will evolve gradually because a single step of absorption or emission is a small perturbation of the average energy . hence at each step of evolution the outlined skewed quasi - stationary partition does not change much . this maximum - entropy distribution accumulates along a sigmoid curve ( dotted ) which is on a log - log scale ( insert ) a straight line of entropy s vs. [ chemical ] potential energy . the system is depicted in terms of an energy level diagram . at each level , indexed by k , there is a population of nk individuals each with energy gk . when an entity in the population nk transforms to an entity in the population nj , horizontal arrows indicate paths of transformations which are available for changes in the potential energy bound in matter and vertical wavy arrows denote concurrent changes driven by energy in light . the system evolves , step - by - step , via absorptive or emissive jk - transformations that are mediated or catalyzed by the entities themselves , toward a more probably partition of entities eventually arriving at a stationary - state balance where the levels are populated so that the average energy kbt equals that in the system 's surroundings . a sufficiently statistical system will evolve gradually because a single step of absorption or emission is a small perturbation of the average energy . hence at each step of evolution the outlined skewed quasi - stationary partition does not change much . this maximum - entropy distribution accumulates along a sigmoid curve ( dotted ) which is on a log - log scale ( insert ) a straight line of entropy s vs. [ chemical ] potential energy . the probability(1)pj=[k=1nkegjk / kbte+iqjk / kbt]nj / nj!for the population nj to exist depends on the density in energy nk exp(gk / kbt ) that is bound in its surrounding substrates , labeled with k , in numbers nk , each with energy gk , as well as on the flux of photons whose energy matches the energy difference gjk = gk gj per entity between the k - substrates and j - products . this influx or efflux of energy to the system , i.e. , dissipation , is denoted by iqjk . the imaginary part merely indicates that the vector potential from the surroundings to the system or vice versa is orthogonal to the scalar [ chemical ] potential . the division by factorial nj ! enumerates the inconsequential exchange of identical entities ( fig . 1 ) . the indexing includes transformation stoichiometry by running from k = 1 to an unknown upper limit that will be eventually reached when the system attains thermodynamic balance with its surroundings . 1 ensures that if any one vital k - ingredient is missing altogether , the j - population can not exist , i.e. , pj = 0 , as well as , if no flux of energy couples from the surroundings to the system , the jk - transformation can not take place . the total probability for the system comprising the diverse populations , indexed with j , is simply the product of pj : s(2)p=j=1pj=j=1[k=1nkegjk / kbte+iqjk / kbt]nj / nj ! again the product form j ensures that if any one vital j - population is missing altogether , the system can not exist , i.e. , p = 0 . the equation 2 is the complete formal account of a system , but due to its product forms it is not particularly amenable for analysis . the logarithm of p when multiplied with kb , will return a convenient additive measure , known as entropy(3)s = kblnp = kbln[j=1(k=1nkegjk / kbte+iqjk / kbt)nj / nj!]=j=1njkb+nj(k=1jk+iqjk)/tobtained by denoting the potential difference jk= k j using stirling 's approximation lnnj ! 3 ) is easy to understand in tangible energetic terms when multiplied with temperature t. then it is apparent that the first term jnjkbt means energy that is bound in the diverse populations nj . the second term jnj(kjk + iqjk ) means free energy that is still present between the system and its surroundings , and hence available for consumption by various jk - transformation mechanisms . the open system will evolve from one state to another by consuming free energy via various jk - transformations . the associated flux of energy carriers , e.g. , photons , from the system to its surroundings or vice versa leads to the increase in entropy , until all energy differences have leveled off . when all energy is bound , there are no longer driving forces and the system is stationary . at the maximum entropy state there is no net flow of quanta between the system and its surroundings , and hence at the thermodynamic balance there is no gain or loss of energy either . the equation of motion from one state to another is obtained by differentiating entropy ( eq . 3)(4)dsdt=j=1dsdnjdnjdt=1tj=1dnjdt(k=1jk+iqjk)0using the chain rule . the two - term product shows that when there are resources , i.e. , free energy , aj = k(jk + iqjk ) > 0 , the population nj will increase , i.e. , dtnj > 0 by consuming it . conversely , when the resources have been over - depleted , i.e. , free energy ( known also as chemical affinity ) aj = k(jk + iqjk ) < 0 , the population will downsize , i.e. , dtnj < 0 . thus , the product is always non - negative , i.e. , the second law ds 0 holds always without any distinction between animate and inanimate . the flow of time couples inherently with flow of energy because both time and energy are attributes of the force carriers . when evolution has consumed all forms of free energy , thermodynamic balance is attained and ds = 0 . the free energy minimum state is lyaponov - stable so that any perturbation nj away from a steady - state population nj will cause decrease in s(nj ) < 0 and concurrently increase in dts(nj ) > 0 . in other words , the further away nj is from nj , the larger is the restoring force aj at times it is claimed that entropy of an animate system could possibly decrease at the expense of an entropy increase elsewhere . such an assertion would entail that the force carries , i.e. , quanta of actions would emerge from nothing or that they would vanish to nothing . the conservation of quanta requires that the population change dtnj = kjkajk / kbt is proportional to free energy by various mechanisms , denoted by jk , that facilitate free energy consumption via the jk - transformations . for example , a digestive track in its entirety is a mechanism to consume free energy in food . it is worth emphasizing that the evolutionary equation ( eq . 4 ) can not be solved because the variables of motion , e.g. , the population changes dnj / dt , can not be separated from their driving forces , aj . this is to say that the natural processes are path - dependent . in accord with observations the evolutionary equation ( eq . 4 ) shows that the notions of entropy and its increase do not express anything more than that which can be expressed in energetic terms . explicitly , entropy ( eq . this unnatural and ill - founded interpretation follows from boltzmann 's idealistic derivation of entropy . when energy of a system is defined to be constant then the system can not change its state , i.e. , evolve by acquiring or losing quanta . the evolutionary equation ( eq . 4 ) is only implicitly that entropy will increase in least time . to see clearly that nature abhors gradients we take a convenient continuum approximation of the evolutionary equation using the continuum definition of chemical potential j = ( u/nj ) in terms of the scalar potential u and likewise of dissipation qj = ( q/nj ) in terms of the vector potential q , to obtain(5)tdsdt=dudt+idqdt=dvdtd2kdt=vu+idqdtwhere definitions of velocity v = dtx and spatial gradient = d / dx have been used as well as a shorthand notation v for the total potential that combines both the scalar u and vector q potentials . the equality tds = d2k between the change in entropy and the change in kinetic energy is obtained by taking projection of the force f = dtp along with velocity v , i.e. , dt(2k ) = vdtp = vf = tdts . when dividing eq . 5 by velocity , the resulting equation reveals that the force that directs down along the potential energy gradient f = v is equivalent to the path 's direction up along the entropy gradient , f = dtp = ts . thus , we conclude that the second law of thermodynamics ( eqs . 4 and 5 ) and newton 's second law of motion are equivalent expressions for transformations from one state to another in the continuum limit , as they should be . moreover , the 2 law of thermodynamic is recognized as equivalent to the principle of least action , in its original form , where kinetic energy is the integrand of action 2kdt = pvdt to be minimized . our derivation of the principle of increasing entropy clearly differs from that of boltzmann , and hence also our reasoning that natural processes consume free energy in the least time departs from those deductions where entropy is associated with disorder . in the end , according to the thermodynamics of open systems , all systems evolve to attain energetic balance with their surroundings in least time , rather than seeking some absolute equilibrium . therefore , there is nothing perplexing about non - equilibrium thermodynamics and equilibrium thermodynamics is nothing but the dynamics of a free energy minimum state , i.e. , at a stasis . namely , skewed nearly lognormal distributions and logarithmic spirals that accumulate along sigmoid growth and decline curves , and hence follow mostly straight lines on log - log plots , i.e. , comply with power laws . there is no profound puzzle about self - organization and no true mystery about emergence either . moreover , the non - deterministic evolutionary equation complies with observations that natural processes are path - dependent . it is of interest to comment on an opinion article that this journal carried last year about a view of life based on the indian philosophy , vednta , as it maintained that abiogenesis is not possible . the rationale presented is that life and consciousness are not separable and life only comes from life . it is not our purpose to argue against this philosophical view , except to say that as it is presented , as an alternative to the ontological view ( darwinian biology ) , which the authors allege regards the organism as a complex machine [ and ] presumes life as just a chance occurrence , without any inner purpose , it is not the only alternative . we take a holistic view of life based on physics and chemistry serving the purpose to eliminate disequilibria in energy deposition in the locally available most efficient way with this view we see no impediments to consciousness , an ambiguous notion itself being part of this process , or to it having been initiated by abiogenesis . the application of thermodynamics in non - equilibrium , non - isolated systems has been an active area of research over many decades . we would argue that the assumption of increasing entropy being synonymous with disorder has had a major disruptive effect on biological thought . among the most notable work in this area has been that of ilya prigogine and co - workers . recognizing that some evolving systems produce ordered dissipative structures spontaneously , apparently in violation of the second law , i.e. , ds / dt < 0 , prigogine and colleagues proposed that in such systems the change in entropy was given by the sum of 2 terms , one for the entropy generated within the system ( dsi / dt ) and the other accounting for the entropy of exchange of energy across the boundary of the open system ( dse / dt ) . the second law , ds / dt > 0 , would hold if dse / dt was sufficiently negative to offset increases in dsi / dt due to dissipation . in other words , despite the appearance of ds / dt < 0 another un - measurable entropy term was acting in compensation . under this interpretation , in the practical case of bnard cells , where ordered dissipative structures occur spontaneously , dse / dt < 0 would be assumed to offset the obligatory increase in internal entropy . in fact , bnard cells are a direct demonstration of increased entropy appearing as order , rather than disorder and the term dse / dt is unnecessary and in this relatively simple case it is difficult to see what exactly it would represent . others have explored the thermodynamic implications of non - equilibrium systems from a perspective similar to the least action approach taken here . for example , it has been proposed that if a stationary state ( or equilibrium state ) of a system is perturbed , then the system will respond by trying to restore the system by the most efficient means . this approach is based upon an earlier re - statement of the laws of thermodynamics to the effect that an isolated system , e.g. , gas molecules , in which internal constraints , typically compartments , are removed , will acquire a unique state of equilibrium characterized by maximum entropy . however , the authors resolve what they call the schrdinger paradox , the ability of organisms to retain their highly organized state against the supposed degrading effects of the second law by invoking the importation of high - quality energy by organisms at the expense of creating disorganization in their environments . interestingly , schneider and sagan provide empirical evidence against penrose 's proposed resolution of the same supposed paradox , namely that organisms effectively feed on the products , e.g. vegetation , of low entropy high energy photons and emit high entropy low energy photons back to space . however , measurements of energy input and output for ecosystems show that the more mature an ecosystem the more free energy dissipated corresponds to a lower emission of long wave radiation and a lower surface temperature which is partly contributed to by the latent heat of evaporation as a result of transpiration . avery addresses the same problem , but proposes that the free energy that drives the life process contains information , coined thermodynamic information that can act to enhance molecular order . the gibbs free energy at any time in a system as a result of its chemical species represents the amount of available thermodynamic information and that which is not dissipated as heat can be retained in the complex internal chemistry of the system as order . utilization of this information internally offsets the supposed disordering effects due to increases by driving the molecular machinery to build up statistically unlikely , e.g. , information containing , structures . what avery proposes is in fact a circuitous route to the conclusion we have drawn without invoking the novel concept of thermodynamic information : the dissipation of free energy can lead to complex and ordered molecular structures which are , in fact , not unlikely , but the most probable states of the system . similar arguments have been given also by others , but more recently , the physicist jeremy england has claimed a breakthrough that implies that the origin of life was as inevitable as a rock falling under gravity . he proposes that if a group of energy driven particles hops over a barrier to adopt one of 2 more ordered states , each emitting low grade heat in the process , the one that emits the most heat , or concomitantly , falls to the lower internal energy state , will be favored . this favored state will be predisposed to dissipate more of the external driving energy and thus undergo further transitions to even more ordered states , by this route , he argues , life would inevitably arise from inanimate material given an external energy driving source , the sun . the dynamic basis of this model is textbook newtonian mechanics f = ma , which is time reversible , in contrast to eq . 5 divided by velocity . the dissipated heat serves to increase the barrier to the reverse transition , thus favoring the transition to more ordered states and eventually the self - assembly of life . this argument omits 2 important points : 1 ) that life is based on metabolism and , therefore , chemistry and 2 ) that a dissipative system can , under the right conditions , self - assemble simply because the self - assembled entity is the more probable , as described herein . finally , it is important to realize that entropy , although a macro state variable , can not be directly measured but has to be inferred . however , the consistent and comprehensive definition in equation 3 makes entropy easy to understand in energetic terms by multiplying with t. specifically entropy multiplied by t of one system can be free energy for another , whether embedded or not . so , for example , stored energy from one organism can be nutrient for another and information , which is also a form of free energy , generated in one system can be applied in another . thus , we conclude that the alleged qualitative distinction between animate and inanimate is without substantiation as well as that the contest between order and disorder is contrived . according to the 2 law of thermodynamics there are only quantitative differences between diverse means and mechanisms to consume free energy , as well as between outcomes of natural processes .

abstractthe second law of thermodynamics is on one hand understood to account for irrevocable flow of energy from the top down , on the other hand it is seen to imply irreversible increase of disorder . this tension between the 2 stances is resolved in favor of the free energy consumption when entropy is derived from the statistical mechanics of open systems . the change in entropy is shown to map directly to the decrease in free energy without any connotation attached to disorder . increase of disorder , just as order , is found to be merely a consequence of free energy consumption . the erroneous association of disorder with entropy stems from an unwarranted assumption that a system could undergo changes of state without concomitant dissipation , i.e. , a change in energy .

Introduction On the origin of misconception Probability in energetic terms The probable evolution Conclusions Disclosure of potential conflicts of interest

PMC3633797

the emerging phenomenon of so - called health tourism has been in europe since the time of ancient greece and rome ( 12 ) . the term health tourism was introduced in 1987 by goodrich ( 3 ) . this multi - faceted topic has been considered as an academic one , from mid-1990s . in addition to some books introducing the tourist targets , there are papers criticized the heath tourism and its different aspects ( 48 ) . gun et al . therefore , to achieve a complete understanding of health tourism , there is a need to study changes over time . the whole process starts from 16 century , which meant a trip to use mineral water supplies , and then use the newly established spa to improve health . currently , we have expanded its meaning which embraces not only the previous meaning but also pre - arranged trip to the hospital for treatment objectives ( 1013 ) . today , health tourism , embraces three separate categories including medical tourism , curative tourism , and wellness tourism . this type of tourism can be seen in all ages , but mostly in older and retired tourists from western countries . on average , women of 45 years age are clients of this type of tourism . these customers typically have high incomes , high levels of education and a lot of time for tourism and personal needs ( 14 ) . currently , health tourism is a major topic of the combination of two components which requires the cooperation of several services running simultaneously , and surveillance organization monitoring and coordinating an enterprise network in the country ( 15 ) . for the first time medical tourism , was considered in iran by the ministry of health in 2002 . islamic countries health tourism congress in mashhad ( 2010 ) considered iran after the jordan of the highest potential in health tourism in these countries . there are several medical centers ; specialty and subspecialty , relatively low cost , enjoyment of natural resources , proximity to arab markets and similarity of culture and language with the some neighboring countries are including advantages in attracting foreign patients and medical tourists in iran . despite the relatively good state of skilled practitioners and efficient health and wellness in the country , attracting tourists for health services hardly had been in the center of authorities thoughts . only in recent years , iranian cultural heritage and tourism organization and the ministry of health , have planned and studied in this context . previous studies conducted in iran shows that this country encompasses the most abilities to attract tourists looking for treatment and enjoy sightseeing simultaneously ( 16,17 ) . because of the lack of comprehensive planning system for health tourism , all related studies have tried to understand the different aspects of this process and provide practical solutions and suggestions which are constructive in terms of planning and strategy of medical tourism ( 1821 ) . shiraz , as a famous city not only in iran , but throughout the world , although has established itself as a high rank place as for having the most pleasant attractive monuments and sightseeing , but unfortunately has not gone through deep survey in terms of absorbing patients . annually hundreds of patients from neighboring countries enter shiraz in hope of seeking health and attractive places . nemazee and khodadoost hospitals , in fact , are famous names among hospitals for many residents of adjacent countries . so far , scientific appraisal could not make clear all positive and negative aspects of this arena . this study aimed to clarify all aspects of health tourism in shiraz city due to studying the demand situation that means health tourists needs in last years . furthermore , this study would like to fill the gap of functional data necessary for health authorities and policy makers . shiraz as the capital of fars province is the fourth most populous city in iran and has a temperate weather . northwest axis of the town because of the natural landscape and temperate climate has been introduced as the health tourism route by the tourism and cultural heritage organization . shiraz has one of the oldest and greatest archaic places in the world , persepolis , which caused this city to be very famus for tourists around the world . geographical and cultural proximity to the persian gulf area countries , foreign direct flights , in addition to be confidence to physicians in shiraz , have provided a favorable matrix for the promotion of health and medical tourism planning . in addition to shiraz university of medical sciences as a prominent unit , there are 12 private and 22 governmental hospitals . this city has been known for liver transpiration operations from almost half a century ago . the efficient authorities on the topic were identified through the hospitals engaging in medical tourism . the research was based on theoretical sampling through which the experienced people of extensive knowledge on medical tourism were interviewed . then the first subjects were asked to introduce more people engaging in that topic based on a snowballing method . accordingly , the questions were categorized based on the research objectives . altogether , 50 respondents were interviewed . for the uniformity of the results , all replies were included in delphi questionnaire , and then it was sent to 30 of respondents , which 25 of them replied it . the process of recognizing the demand for health tourism in shiraz was based on the three principles including ( i ) gathering the number of foreigner health tourists ; ( ii ) identification of the destination country and ( iii ) knowledge on the nature of occupational therapy . determining the supply sector of health tourism has been considered more difficult due to its different parts and tools ( 22 ) . therefore , due to scattering angles presented in supply section , the demand and supply model ( 9 ) was utilized to categorize the supply services ( fig . given the importance of cultural factors in this research , the socio - cultural index was considered as a control and efficient indicator in all steps of the recognizing the supply . the model provides conditions that extensive discussion can be analyzed in relation to the product , services or related schedule . then the output of this chart was designed in ( swat ) based questionnaire and was sent to top ten experts on medical tourism for scoring . shiraz as the capital of fars province is the fourth most populous city in iran and has a temperate weather . northwest axis of the town because of the natural landscape and temperate climate has been introduced as the health tourism route by the tourism and cultural heritage organization . shiraz has one of the oldest and greatest archaic places in the world , persepolis , which caused this city to be very famus for tourists around the world . geographical and cultural proximity to the persian gulf area countries , foreign direct flights , in addition to be confidence to physicians in shiraz , have provided a favorable matrix for the promotion of health and medical tourism planning . in addition to shiraz university of medical sciences as a prominent unit , there are 12 private and 22 governmental hospitals . this city has been known for liver transpiration operations from almost half a century ago . the efficient authorities on the topic were identified through the hospitals engaging in medical tourism . the research was based on theoretical sampling through which the experienced people of extensive knowledge on medical tourism were interviewed . then the first subjects were asked to introduce more people engaging in that topic based on a snowballing method . accordingly , the questions were categorized based on the research objectives . altogether , 50 respondents were interviewed . for the uniformity of the results , all replies were included in delphi questionnaire , and then it was sent to 30 of respondents , which 25 of them replied it . the process of recognizing the demand for health tourism in shiraz was based on the three principles including ( i ) gathering the number of foreigner health tourists ; ( ii ) identification of the destination country and ( iii ) knowledge on the nature of occupational therapy . determining the supply sector of health tourism has been considered more difficult due to its different parts and tools ( 22 ) . therefore , due to scattering angles presented in supply section , the demand and supply model ( 9 ) was utilized to categorize the supply services ( fig . 2 ) . given the importance of cultural factors in this research , the socio - cultural index was considered as a control and efficient indicator in all steps of the recognizing the supply . the model provides conditions that extensive discussion can be analyzed in relation to the product , services or related schedule . then the output of this chart was designed in ( swat ) based questionnaire and was sent to top ten experts on medical tourism for scoring . using interviews conducted in this study , it was found that active hospitals on attraction of foreign patients averagely admitted 15 and 50 foreign patients monthly and annually , respectively . arab countries in the persian gulf were detected as the main marketing for shiraz medical tourism . accordingly oman encompassed the highest rate with 20% of admitted patients . according to fig . 3 , eye treatments with 30% and orthopedic therapies with 6% were demonstrated as the highest and lowest rates in terms of foreign patients needs . the tourist attractions of shiraz , according to health experts were divided into two parts : physical and non - physical . each part of the table 1 shows the importance of each section for arrived patients . these items have been numbered and inserted in the table based on their importance . the most important factor in physical attractiveness it was found that world - famous historical attractions of shiraz were not in the center of consideration for arabs residing in the persian gulf countries and only religious places were welcomed by them . regarding non - physical attractions , 80% of replies determined that closeness of cultural , and religious beliefs and familial relationships on one hand and trusting to iranian physicians on the other hand were amongst the most reasons for selecting iran as a destination for medical tourism by these patients . besides , we found that economical and financial factors had the minimum effect on their decision to select a destination . in the field of transportation , the high quality services were detected as the main pillar for attraction of health tourists especially for patients coming from arab countries settled nearby the persian gulf . table 2 shows that providing a complete package including all aspects of tourism services constituted an important point for improving the outcome with 72% rate . moreover , regarding to accommodation , 84% of patients preferred to stay in luxury hotels ; instead , some of them coming with family preferred the hotel apartments or furnished hotels . ancillary services in this study the need for tourists to spend recovering in the garden area with fresh air , with ease in visa issue was of the same importance ( table 2 ) . among the most effective advertising and marketing methods that were selected by experts , media activities , with 96 percent of respondents encompassed the most important factor in attracting foreign patients . in addition , a sales office in the country of origin with the language of that country , with 88% of comments was identified as the second most effective marketing method . furthermore among the advertising methods , the existence of information and advertising office in origin countries showed the greatest impact on attraction of the city s tourists . based on a thorough understanding of the strengths and weaknesses of health tourism in shiraz , demand and supply conditions were incorporated using the target network model and were included in four methods of create , maintain , abstinence and review ( table 3 ) . this study was conducted to verify the potential aspects of health tourism in shiraz city , southern iran . due to a variety of factors although the outputs extracted from this study are specific to the region , yet , planning and general methods presented in the paper is applicable to other regions of the country as well . the results showed that among the presented facilities in this city , eye care had the highest rank in the context of this demand , while the country of oman could get the top rank in terms of countries of the highest medical tourists . tribal sheikhs and residents of oman from long time ago have had constructive relations with iran . persisted sand storms caused people of oman to travel to iran many times per year . successful reputation in the field of infertility and treatment by a muslim physician caused arabs to trust an iranian physician which justifies traveling 16% of them to iran for medical tourism affairs . from collected data of our study , we can conclude that although geographical distance has a significant impact on the choice of destination therapy , yet this factor is affected by economical , environmental , cultural and social factors . the realities of social and cultural differences have persuaded muslim countries to manage their important care necessities within another muslim country . in the context of intercultural adaptation on tourism , one out of every 10 people is able to adapt to foreign conditions ( 23 ) . preparation and planning , provides tourist trips in the face of possible problems ( 24 ) . another study showed that the patients were looking for medical care of high quality and reasonable price ( 25 ) . our results showed that patients who traveled to shiraz had no concern on the latter fact but mostly considered the prominent muslim physicians , similar cultural affairs and close distance as the most standing factors in selecting this city . our results showed that despite the reputation of nemazee hospital , tourists nowadays prefer to seek treatment in private hospitals in shiraz . the survey also found that tourists are looking for luxury accommodation and prefer hospitals so called 5 stars ( 26 , 27 ) . this study showed that the patients were looking for medical care of high quality and reasonable price . according to kaufman and mueller ( 22 ) , the conditions for health tourism hotels are as follows : 35 star accommodations , health information , health care , personal and cultural programs and a variety of amenities . destination promotion is one of the marketing and manufacturing of marketing mix , it is important in recognizing and developing destinations ( 28 ) . evidence suggests that an office of information and related advertisements in destination media would have an important affect on attracting the patients to shiraz . a research showed that among the variables of notification mechanism , just the advertising media had a significant correlation with attracting the medical tourists ( 29 ) . this implies that the advertising opportunities in the international media in the field of health care , physicians , high quality services , and technology can lead to attracting the medical tourists ( 30 ) . for example , prepare pictures of health centers and putting them up at the airports and terminals ( 31 ) . in this study , the activities of middlemen , so called brokers , were identified as the main attraction factor . due to the familiarity in language and having cultural relations with applicants , these channels show great ability in marketing , attracting and encouraging people to select shiraz for health tourism affairs . all outputs of this study are included in table 3 which contains functional solutions to authorities . in creation section , a bird s eye view on it implies that health tourism in shiraz , although successful in meeting many requirement in this arena , is in a situation which demands different infrastructures to not only satisfy the present set of tourists , but to promote the attraction more numbers of them as much as possible . in the section of maintenance , previous results have been confirmed and it might be concluded that health tourism in shiraz and iran , has its base on close relation between origination and destination , as well as the popular fame of iranian physicians . some studies imply that neglecting the proper planning , not only will result to stop the process , but will cause to demolish even this small package and considering the competitive situation of present period , consequently the patients will be guided to other islamic countries . factors mentioned in restrain section not only have no indication in health tourism system of shiraz but are not requested by applicants . although , these factors might improve the rate of attraction of health tourists but considering that demand section does not need them and executive power should be implemented for other parts , investment on this category only waste the power and energy of the organizations . eventually , revision section , although named as deletion in the main model of targets network , but due to importance of all points in planning for health tourism , nothing should be neglected . deep looking into the case shows that this category has not been considered properly by demand section , so not only it should not be omitted but needs more attention in programming . our findings demonstrated that implementing 4 strategies in planning for medical tourism in shiraz would result into significant improvement of attraction more patients as follows : allocation the responsibility of the executive affairs to specific section to remove the weak points of managing- legislation.proper monitoring of convalescence period and presenting variable packages of tourism.policy making on absorbing the foreign investments to establish the project of shiraz health town.propaganda expands based on cultural closeness between supply and target bazaar , in addition to branding shiraz medical ability . allocation the responsibility of the executive affairs to specific section to remove the weak points of managing- legislation . policy making on absorbing the foreign investments to establish the project of shiraz health town . propaganda expands based on cultural closeness between supply and target bazaar , in addition to branding shiraz medical ability . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .

background : the aim of the present study was to appraisal the prose and cone of shiraz city in terms of its potential in the context of health tourism.methods:the statistical sample included medical and health tourism sector in the city of shiraz in the northwest of fars province , south of iran . the efficient authorities on the topic were identified through the hospitals engaging in medical tourism . the research was based on theoretical sampling through which the experienced people of extensive knowledge on medical tourism were interviewed.results:active hospitals on attraction foreign patients averagely admit 15 and 50 foreign patients monthly and annually , respectively . arab countries in the persian gulf were detected as the main marketing for shiraz medical tourism . oman encompassed the highest rate with 20% of admitted patients . eye treatments with 30% and orthopedic with 6% were demonstrated as the highest and lowest rates in terms of foreign patients needs , respectively . closeness of cultural and religious beliefs and familial relationships on one hand and trusting to iranian physicians on the other hand were amongst the most reasons for selecting iran as a destination for medical tourism by patients.conclusion:implementing 4 strategies on monitoring medical tourism would result into significant improvement of attracting more foreigner patients not only into shiraz but all around iran . these items have been discussed in the text .

Introduction Materials and Methods Study area Collecting data Analyzing data Results Discussion Conclusion Ethical considerations

PMC1566587

prior studies reported an association between ambient air concentrations of total suspended particles and so2 during pregnancy and adverse pregnancy outcomes . we examined the possible impact of particulate matter up to 10 microm ( pm10 ) and up to 2.5 microm ( pm2 . 5 ) in size on intrauterine growth retardation ( iugr ) risk in a highly polluted area of northern bohemia ( teplice district ) . the study group includes all singleton full - term births of european origin over a 2-year period in the teplice district . information on reproductive history , health , and lifestyle was obtained from maternal questionnaires . the mean concentrations of pollutants for each month of gestation were calculated using continuous monitoring data . three intervals ( low , medium , and high ) were constructed for each pollutant ( tertiles ) . odds ratios ( ors ) for iugr for pm10 and pm2.5 levels were generated using logistic regression for each month of gestation after adjustment for potential confounding factors . adjusted ors for iugr related to ambient pm10 levels in the first gestational month increased along the concentration intervals : medium 1.62 [ 95% confidence interval ( ci ) , 1.07 - 2.46 ] , high 2.64 ( ci , 1.48 - 4.71 ) . ors for pm2.5 were 1.26 ( ci , 0.81 - 1.95 ) and 2.11 ( ci , 1 . 20 - 3.70 ) , respectively . no other associations of iugr risk with particulate matter were found . influence of particles or other associated air pollutants on fetal growth in early gestation is one of several possible explanations of these results . timing of this effect is compatible with a current hypothesis of iugr pathogenesis . seasonal factors , one of the other possible explanations , is less probable . more investigation is required to examine these findings and alternative explanations.imagesfigure 1

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PMC3303860

the cytoskeletal components of endothelial cells play important roles in maintaining the fundamental structure of the thin inner layer of the blood vessels called the endothelium . endothelial cells form a single cell layer on the surfaces of blood vessels and are therefore constantly subjected to both fluid shear stress and periodic strain caused by blood pressure induced by the pulsatile flow . endothelial cells have been shown to possess many stress fibers both in vitro and in situ , and these cells are known to be capable of responding to the level of fluid shear stress by changing their shape [ 1 , 2 ] , distribution of cytoskeletal components [ 35 ] , expression of signal transduction - associated proteins [ 6 , 7 ] , and expression of various genes [ 8 , 9 ] . endothelial cells in culture are known to respond to cyclic stretching and hyperosmotic shock . on exposure to uniaxial cyclic stretching , endothelial cells become aligned perpendicular to the axis of stretching [ 10 , 11 ] . cyclic stretching applied to cells in culture is a model of pulsatile stretching induced by blood pressure in vivo , and the effects differ from those of fluid shear stress generated by the blood flow . hemodynamic shear stress caused by blood flow occurs in combination with cyclic stretching caused by the pressure of pulsatile flow generated by blood pressure . previous in vivo experiments indicated that stress fibers in endothelial cells respond to fluid shear stress and show increases in both number and thickness in a manner related to the magnitude of shear stress ( for review , see katoh et al . , previously , we reported increases in number and thickness of stress fibers and focal adhesions in both the apical and basal portions of endothelial cells in an artificial coarctation zone in the abdominal aorta where fluid shear stress is significantly high in comparison to endothelial cells subjected to averaged shear stress . the plaque - like vinculin - containing spots detected at the ends of stress fibers were enlarged in the coarctation area , especially in the apical portions of the cells . in addition , stress fibers and their sites of association with the plasma membrane are closely attached to both the apical and basal portions of endothelial cells , and we suggested that they may play key roles in force transfer by fluid stress [ 13 , 14 ] . our findings also suggested that even in the case of endothelial cells in situ , the apical plaques ( i.e. , stress fiber - plasma membrane attachment sites with accumulation of focal adhesion - associated proteins ) and their associated stress fibers are candidates for sensing and/or transferring mechanical signals of fluid shear stress applied to the laminar surface of endothelial cells [ 6 , 14 ] . apical plaques are enlarged in the apical portion of endothelial cells in the coarctation zone reflecting the response of the apical plaque and its associated stress fibers to mechanical stimuli generated by blood flow . such responses increase according to the magnitude of applied shear stress , in agreement with the observations in traditional in vitro cell culture systems [ 2 , 1518 ] . stress fibers are major higher - order structural components of the cytoskeleton in nonmuscle cells , which are composed of actomyosin filaments and show contractility both in vitro and in situ . we reported previously that stress fibers could be isolated from fibroblasts without loss of morphological or functional characteristics and that they represent a major part of the contractile apparatus within the cell . we also reported that the stress fibers are located not only in the basal portion of the cell , but also in the apical portion in both cultured fibroblasts and in guinea pig aortic endothelial cells , and we called these apically located stress fibers apical stress fibers . some apical stress fibers not only connect to the apical plaques but also make direct connections with focal adhesions in the basal portion of endothelial cells , and the apical stress fibers have the ability to transfer mechanical forces from the apical to the basal portion of the cell . apical stress fibers in endothelial cells in situ are directly subjected to fluid shear stress , and the mechanical stimuli generated by this fluid shear stress are applied directly to the apical stress fibers . blood vessels in the living animal are subjected to pulsatile stretches generated by the heart via the circulatory system . these pulsatile stretches seem to induce changes in endothelial cell shape and the formation of cytoskeletal components . previous in vitro experiments showed that cyclic stretching applied to cells in culture causes the cells to become oriented perpendicular to the direction of stretching [ 10 , 21 , 22 ] consistent with in vivo results . on the other hand , in cells exposed to unidirectional tension , the stress fibers become organized along the axis of tension . in situ experiments indicated that guinea pig venous endothelial cells were elongated in the direction of blood flow to a greater extent than unperturbed aortic endothelial cells . moreover , thick stress fibers located at the basal side of venous endothelial cells were fewer in number than in aortic endothelial cells . the morphological differences between venous and aortic endothelial cells seem to be due to the sustained exposure of the former cell type to significantly lower levels of fluid shear stress than the latter . however , cell culture conditions preclude accurate observations because the cells have been artificially removed from the living animal . therefore , analyses of the fundamental mechanisms involved in the responses to mechanical stimuli , such as fluid shear stress , pulsatile enlargement of blood vessel diameter , and/or stretching , should be performed in living intact blood vessels . in the basal portion of endothelial cells , stress fibers generally run along the axis of blood flow in typical aortic and venous endothelial cells . however , we reported previously that stress fibers in the apical portion of venous endothelial cells run perpendicular to the direction of blood flow . these observations raised questions regarding whether the mechanism by which stress fibers run is independent of the direction of blood flow . both the right and left renal arteries branch off from the abdominal aorta at an angle of 90 and carry blood to the kidneys . blood in the renal artery is filtered by the kidneys , and so the resistance to blood flow applied to the surface of the renal artery should be higher than that in most other arteries . mechanical stress applied to the endothelial cells in the renal artery should be different from the straight portion of the abdominal aorta in situ , and therefore the distribution of cytoskeletal components , such as stress fibers and focal adhesions , should differ considerably between renal artery endothelial cells and endothelial cells experiencing unidirectional flow in situ . the observations outlined above prompted us to examine the detailed distributions of cytoskeletal components and associated proteins . here , we carefully compared the cytoskeletal components of endothelial cells in the renal artery with those of unperturbed arterial and venous endothelial cells . the results indicated that the cytoskeletal components of endothelial cells in the renal artery showed quiet different distribution patterns from the stress fibers in unperturbed aortic and venous endothelial cells . aortae were obtained from adult guinea pigs weighing 250400 g. some animals were treated as described below . after perfusion with 0.85% nacl containing 200 units of heparin sodium via the left ventricle , the abdominal aortae were excised and fixed with 1% or 2% paraformaldehyde using the microwave irradiation method . louis , mo ) , antipaxillin ( bd transduction laboratories , san jose , ca ) , anti - alpha - actinin ( sigma ) , and antiphosphorylated tyrosine - containing protein antibody ( clone py-20 ; icn pharmaceuticals , aurora , oh ) were purchased from the sources indicated . fitc - labeled secondary antibody against rabbit igg was purchased from zymed ( san francisco , ca ) . fixed aortae were rinsed several times with phosphate - buffered saline ( pbs ) and cut into small pieces . after permeabilization with 0.5% triton x-100 in pbs for 5 min , specimens were incubated with antibodies ; some specimens were double - stained with rhodamine - labeled phalloidin . specimens were mounted in 90% glycerol in pbs containing 2.5% 1,4-diazabicyclo[2.2.2]octane ( dabco ; sigma ) and examined by confocal laser scanning microscopy ( clsm ) ( fv-1000 ; olympus , tokyo , japan ) with a plan apochromat 60 ( n.a . stereo pair images were reconstructed from 20 to 30 serial optical sections obtained by clsm using image j software ( nih , bethesda , ml ) . for ultrasound microimaging , renal arteries were obtained from adult guinea pigs weighing approximately 380420 g. ultrasound images of the renal artery were obtained using a high - frequency ultrasound imaging system ( vevo 770 ; visualsonics , toronto , on , canada ) . animals were anesthetized by intraperitoneal injection of pentobarbital sodium at a dose of 10 mg/400 g body weight ( abbott laboratories , abbott park , il ) . after perfusion , the abdomen was surgically opened , and living ultrasound images of the renal artery and abdominal aorta were obtained , and blood flow speed was measured using an ultrasound imaging system . the distributions of stress fibers and focal adhesions were determined by confocal laser scanning microscopy in whole - mount preparations of the guinea pig renal artery . specimens were double stained with rhodamine - labeled phalloidin for visualization of stress fibers and with antivinculin antibody for visualization of focal adhesions ( figure 1 ) . phalloidin - positive actin - containing filaments in the renal artery also showed a punctate linear staining pattern with anti - alpha - actinin antibody , so they were classified as well - developed stress fibers ( data not shown ) . when the focal plane was adjusted at the basal portion of the cell , stress fibers were shown to run perpendicular to the direction of blood flow ( figure 1(b ) ; arrowheads ) . on the other hand , when the focal plane was adjusted to the apical portion of the endothelial cells , many stress fibers were observed running along the direction of blood flow ( figure 1(a ) ; flow ) . some stress fibers were also shown to run perpendicular to the direction of flow ( figure 1(a ) ; arrowheads ) . both ends of the stress fibers were associated with vinculin ( figure 1(d ) ) or paxillin ( data not shown ) , confirming the identity of vinculin - positive spots as focal adhesions . focal adhesions revealed by staining with antivinculin antibody were closely associated with the stress fibers in the basal portion of the cell in a dot - like configuration ( figure 1(d ) ) . significant immunofluorescent staining with antivinculin antibody was not detected at the apical surface of the cells . however , intense staining was also detected at the sites of cell - to - cell association , presumably adherens junctions of the endothelial cells ( figure 1(c ) ) . stress fibers in the unperturbed aortic endothelial cells ran along the direction of blood flow in the basal portion of the cell [ 14 , 28 ] . in the basal portion of the cell , the stress fibers in the renal artery were shown to run perpendicular to the direction of blood flow . in the apical portion of unperturbed endothelial cells located in the abdominal aorta , stress fibers were only observed along the direction of blood flow in both the apical and basal portions . to determine the distribution of stress fibers in renal arterial endothelial cells in greater detail , three - dimensional stereo images stained with rhodamine - labeled phalloidin were reconstructed from confocal serial optical sections ( figure 2(c ) for stereo image ) . many stress fibers running both parallel and perpendicular to the direction of flow were observed in the apical portion of the cell ( figure 2(a ) for a single confocal image ) . only thick stress fibers running perpendicular to the direction of blood flow were observed in the basal portion of the cell ( figure 2(b ) for a single confocal image ; figure 2(c ) for stereo image ) . as certain proteins undergo tyrosine phosphorylation during intracellular signaling events , we next examined the tyrosine phosphorylation levels of endothelial cells in the renal artery ( figure 3 ) . en face preparations stained with an antibody against phosphotyrosine - containing proteins or an antibody against vinculin were examined by confocal laser scanning microscopy . positive staining with antiphosphotyrosine antibody was observed in a uniform pattern at the apical surface of the endothelial cells ( figure 3(a ) ) . when the focal plane was adjusted to the middle level of the cells , staining was detected at sites of cell - to - cell apposition ( figure 3(b ) ) . the focal plane was adjusted to the basal portion of the cell , and staining with antiphosphotyrosine antibody was mainly detected at cell - to - cell adhesion sites , but not along stress fibers or focal adhesions ( figure 3(c ) ) . on the other hand , staining with antivinculin was detected at both sites of cell - to - cell apposition ( figure 3(e ) ) and focal adhesions at the basal side of the cell ( figure 3(f ) ) . no significant staining was detected at the apical surface of the cell ( figure 3(d ) ) . staining with antivinculin and antiphosphotyrosine antibodies did not show complete colocalization at the basal portion of cells ( figures 3(c ) and 3(f ) ) . the renal artery was also double - stained for f - actin with rhodamine - labeled phalloidin and with antiphosphotyrosine antibody . figure 4 shows a stereo view of a cell double - stained with rhodamine - labeled phalloidin to visualize actin filaments and with antiphosphotyrosine antibody , and the results indicated no colocalization of tyrosine - phosphorylated proteins and stress fibers . positive staining for phosphotyrosine was detected on the plasma membrane over endothelial cells , especially along the cell - to - cell adhesion sites . however , the specific distribution of staining for phosphotyrosine was not detected along the stress fibers in the basal or apical portions of the cell . to visualize and measure blood flow in the renal artery , we measured biological parameters using an ultrasound microimaging system . living images of the abdominal aorta and the renal artery are shown in figure 5(a ) . doppler blood flow recordings were also made for the abdominal aorta ( figure 5(b ) ) and renal artery ( figure 5(c ) ) . retrograde blood flow was detected in the renal artery in cyclic diastolic phases ( figure 5(c ) ) , indicating that the renal arterial endothelial cells are subjected to forward and backward force caused by the circulatory pulse . it is difficult to calculate the precise shear stress applied to endothelial cells in the renal artery because of the complicated forward and backward flow , pulsatile pressure , and resistance pressure from the kidney . however , pulsatile flow in the renal artery results in significant fluctuations in the magnitude of shear stress and/or pressure , which are not predicted to occur in unperturbed aortic endothelial cells . further studies are required to precisely determine the fluid shear stress and pressure applied to the endothelial cells in the renal artery . fluctuating and retrograde flow in the renal artery seem to have an influence on stress fiber localization , as stress fibers run perpendicular to the direction of blood flow in the basal portion of the cells ( see figures 1 and 2 ) . endothelial cells respond to fluid shear stress and show changes in morphology and organization of cytoskeletal components . unperturbed aortic endothelial cells , such as those in the straight portion of the abdominal aorta , show elongation along the direction of blood flow . in this region , cytoskeletal components , such as stress fibers and focal adhesions , are also enhanced in a manner corresponding to the magnitude of blood flow [ 6 , 14 ] . we reported previously that cytoskeletal components , such as stress fibers and focal adhesions , are significantly increased in both number and size in the surgical coarctation zone in the straight portion of the abdominal aorta where fluid shear stress is expected to be high . confocal laser scanning microscopy indicated the presence of thick stress fibers in the basal portion of the cells in the artificial coarctation zone . stress fibers were also observed in the apical portion along the direction of blood flow , where they are continually exposed to mechanical stimulation generated by the blood flow . on the other hand , venous endothelial cells had fewer thick basal stress fibers than unperturbed aortic endothelial cells . moreover , many apical stress fibers running perpendicular to the direction of blood flow were seen in the apical region of venous epithelial cells . the results of the present study indicated that stress fibers run perpendicular to the direction of blood flow in both the apical and basal regions of renal endothelial cells . although the renal arterial endothelial cells were elongated along the direction of flow , stress fibers were localized perpendicular to the axis of elongation in the basal portion of these cells . the complex nature of the different environmental conditions seemed to induce alterations in the cytoskeletal components and associated morphological changes in these cells , compared to unperturbed aortic and venous endothelial cells . high fluid shear stress is generally thought to result in increases in both the number and thickness of stress and focal adhesions . however , the results of the present study indicated that the arrangement of stress fibers and cell shape along the direction of blood flow were independently regulated by mechanical stimuli . fluid shear stress induced orientation of the stress fibers and associated focal adhesions along the direction of blood flow , while oscillatory flow caused the stress fibers and focal adhesions to be oriented perpendicular to the direction of blood flow . table 1 summarizes the directions of stress fibers in aortic , venous , and renal arterial endothelial cells . , we showed that tyrosine - phosphorylated proteins are localized at the endothelial cell surface and sites of cell - to - cell apposition in the renal artery . these observations were consistent with those in unperturbed aortic endothelial cells , so that signaling by mechanical stimuli may occur in the same position in renal arterial and other arterial endothelial cells in situ . we and other groups have previously identified several proteins that undergo tyrosine phosphorylation induced by fluid shear stress . pecam-1 , which is mainly localized at the sites of endothelial cell - to - cell apposition , is tyrosine phosphorylated when endothelial cells are subjected to high levels of shear stress . src family proteins are also tyrosine phosphorylated when cells are subjected to high shear stress . moreover , expression of c - src is increased in the artificial coarctation zone in the abdominal aorta . these observations suggested that pecam-1 and c - src are primary candidates for signal transduction of mechanical stimuli in endothelial cells in the renal artery . the bidirectionality of the oscillating blood flow itself may be the key to the perpendicular orientation , or some unique protein(s ) made only by renal endothelial cells may interact to perpendicularly reorient the basal stress fibers . we are currently engaged in studies of the unique molecules expressed in renal arterial endothelial cells . stress fibers running perpendicular to the direction of blood flow were shown to be present at ( 1 ) the apical surface of venous endothelial cells , ( 2 ) the apical surface of aortic endothelial cells in the artificial coarctation zone , ( 3 ) the apical side of renal arterial endothelial cells ( as demonstrated in the present study ) , and ( 4 ) the basal side of renal arterial endothelial cells ( as demonstrated in the present study ) . the directions of stress fibers in endothelial cells in aortic , venous , and renal arteries are summarized in table 1 . venous endothelial cells also possess apical stress fibers that run perpendicular to the direction of blood flow . however , in the basal portion of venous endothelial cells , stress fibers were found to run along the direction of flow . although the reasons for the above discrepancy related to blood flow are still unclear , it is likely that the stress fiber direction is determined not only by the direction of blood flow but also by other mechanical forces , such as pressure and/or stretching force to which the endothelial cells are exposed . it is of interest that endothelial cells in the renal artery were aligned along the direction of blood flow . in the basal portions of the cell , all of the stress fibers ran perpendicular to the direction of blood flow . moreover , focal adhesions in this region were also aligned along the perpendicular stress fibers . the alignments of stress fibers and focal adhesions in this region were completely independent of the direction of blood flow . on the other hand , in the apical portion of the renal arterial endothelial cells , the mechanism underlying the organization of stress fibers that run perpendicular to the direction of blood flow is unclear , but it is likely that these perpendicular stress fibers respond to stretching force generated by blood pressure applied to the endothelial cells . in mesothelial cells subjected to single - direction stretching , stress fibers were aligned in the direction of stretch . on the other hand , cells subjected to cyclic stretch - activated ( sa ) ion channels play a role in reorientation of endothelial cells that is not dependent on fluid shear stress force . an inhibitor of sa channels was shown to block sa - induced reorientation of endothelial cells , but no changes were observed in stress fiber organization by the inhibitor . these observations suggest that the stress fibers running perpendicular to the direction of flow may be organized by the stretching force generated by blood flow . in the apical portion of the cell , stress fibers were found to run along the direction of blood flow as observed in unperturbed aortic endothelial cells . these observations suggested that the orientation of apical stress fibers was primarily induced by the fluid shear stress , because the endothelial cell surface is directly exposed to fluid shear stress together with pulsatile pressure . the organization of cytoskeletal components in endothelial cells on the inner surface of blood vessels in the renal artery is controlled by local mechanical stimuli , consisting of a complex combination of bidirectional fluid shear stress , and oscillatory longitudinal and/or circumferential pressure . in this study , we found a difference between the directionality of stress fibers in renal endothelial cells compared to the unperturbed aortic endothelial cells . the renal artery branches off from the abdominal aorta at an angle of almost 90 and is inserted directly into the kidney . this geometry may result in special physiological blood flow conditions , such as stretching force over the endothelial cells . the morphological alignment of endothelial cells along the direction of blood flow and the organization of cytoskeletal components were shown to be independently regulated . further studies are required to gain insight into the organization of cytoskeletal components and associated changes in cell shape in response to mechanical stimuli applied to endothelial cells in both in situ and in vitro .

the cytoskeletal components of endothelial cells in the renal artery were examined by analysis of en face preparations under confocal laser scanning microscopy . renal arterial endothelial cells were shown to be elongated along the direction of blood flow , while stress fibers ran perpendicular to the flow in the basal portion . focal adhesions were observed along the stress fibers in dot - like configurations . on the other hand , stress fibers in the apical portion of cells ran along the direction of flow . the localizations of stress fibers and focal adhesions in endothelial cells in the renal artery differed from those of unperturbed aortic and venous endothelial cells . tyrosine - phosphorylated proteins were mainly detected at the sites of cell - to - cell apposition , but not in focal adhesions . pulsatile pressure and fluid shear stress applied over endothelial cells in the renal artery induce stress fiber organization and localization of focal adhesions . these observations suggest that the morphological alignment of endothelial cells along the direction of blood flow and the organization of cytoskeletal components are independently regulated .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC3312276

drug delivery systems ( ddss ) are useful for reducing drug side effects and maximizing drug action . the design of drug carriers for dds is the most important activity in this area . the advent of molecular biology studies has enabled the identification of many disease - causing proteins . because some of these are effective as drugs , a variety of nanoparticles such as liposomes , micelles , and polymers have been adopted as drug carriers [ 14 ] . because polymers are similar in size to proteins , they are not suitable as protein carriers . whereas liposomes and micelles are larger than polymers and proteins , they can be used as protein carriers . however , because these are self - assembled nanoparticles , some treatments are necessary for their preparation as carriers . since most proteins are sensitive to temperature , ph , and organic solvents , it is possible that such treatments induce protein denaturation . hydroxyapatite ( ha ) , ca10(po4)6(oh)2 , is a major component of hard tissues such as bones and teeth and has been used as a biomaterial [ 5 , 6 ] . because it has been reported that some proteins , such as bovine serum albumin ( bsa ) and lysozyme , can bind to ha just by mixing , it is a good candidate for a protein carrier [ 79 ] . in this study , we investigated the association and dissociation behavior of two bioactive proteins , cytochrome c and insulin , to ha . it is known that the release of cytochrome c from mitochondria to the cytosol induces apoptosis . therefore , the delivery of cytochrome c into the cytosol of cancer cells should induce apoptosis , which may be useful for cancer therapy . insulin , a key protein of diabetes , is commonly injected into diabetic patients to suppress blood sugar levels , and its controlled release can markedly improve their quality of life . because the delivery of these two proteins is important , we attempted to use ha for a delivery system . the absorption and desorption on ha were affected by the surface conditions dependent on the preparation procedure of ha . considering universal use of ha for protein delivery , commercially available ha was used as a carrier in this study . ha nanoparticles were purchased from sigma - aldrich ( mo , usa ) . according to the material data sheet ( no . 677418 ) , the size and surface area were smaller than 200 nm and larger than 9.4 m / g , respectively . cytochrome c is cationic , and insulin is anionic at physiological ph , and the molecular weight of cytochrome c is larger than that of insulin . adsorption experiments with cytochrome c were performed by mixing ha ( 0 , 10 , 20 , and 30 mg ) with an aqueous solution ( 2.5 mg / ml , 200 l ) . after 4 h mixing with a rotator , centrifugation ( 15,000 g , 20c , 60 min ) was performed to collect the supernatants . these were analyzed by reversed phase high performance liquid chromatography ( hplc ) to estimate the residual concentration of cytochrome c. the hplc system was equipped with a cosmosil 5c18-ms - ii column ( nacalai tesque , inc . , kyoto , japan ) and a uv detector ( 220 nm ; uv-2075plus , jasco inc . , tokyo , japan ) . samples ( 5 l ) were injected with an autosampler ( as-2057plus , jasco inc . , tokyo , japan ) and eluted with acetonitrile/0.05% trifluoroacetic acid = 20/80 ( a ) and acetonitrile/0.05% trifluoroacetic acid = 60/40 ( b ) at 1.0 mlmin by pu-2089plus ( jasco inc . , a linear gradient elution was performed over 20 min from an initial state ( a ) 100% to the final state ( b ) 100% . in the case of insulin adsorption , the same experimental procedures were performed except the insulin solution was prepared by dissolving it in 0.01 n hcl and adjusting to ph 3 . association ratio ( % ) was calculated as [ ( c0c)/c0 ] 100 , at which c0 and c are the initial concentration and the supernatant concentration of proteins , respectively . during desorption experiments , ha ( 10 and 20 mg ) absorbing cytochrome c and insulin was transferred into 400 l of phosphate buffer saline ( pbs ; 8 mm na2hpo4 , 2 mm kh2po4 , 137 mm nacl , 3 mm kcl ) , and rotated . after predetermined incubation times , centrifugation ( 15,000 g , 20c , 60 min ) was performed to collect the supernatants . the residual concentrations of cytochrome c and insulin were estimated by hplc . in the case of insulin , dissociation ratio ( % ) was calculated as [ c / c0 ] 100 , at which c0 and c are the total concentration of the associated proteins and the supernatant concentration , respectively . therefore , insulin was dissolved in an acidic solution ( ph 3 ) . after the incubation and subsequent centrifugation , the residual cytochrome c and insulin in the solution were estimated from the hplc analysis . cytochrome c and insulin were eluted after 10 min and 13 min , respectively , under the running conditions ( figure 1(a ) ) , and the peak areas were proportional to the protein concentrations ( figure 1(b ) ) . thus , the protein concentrations in the supernatants were evaluated by hplc analysis and the adsorbed amounts were calculated by subtracting the concentrations in the supernatant from the initial ones . the adsorption efficiency of insulin was higher than that of cytochrome c. as less as 10 mg ha was sufficient to load almost 0.5 mg insulin , but 30 mg ha is necessary to load the same amount of cytochrome c under this condition . this may be because cytochrome c is larger than insulin . from the surface area of the ha ( > 9.4 m / g ) and the protein diameters ( d ) of insulin ( 3 nm ) and cytochrome c ( 4 nm ) , the percent of occupied area can be estimated . in the experiment , 0.5 mg of proteins , that is 9 10mol ( 5 10 molecules ) of insulin and 4 10mol ( 2 10 molecules ) of cytochrome c , were used . the projected area of proteins may be approximated as (d/2 ) , assuming the protein to be spherical . given that all molecules were absorbed , the occupied areas of insulin and cytochrome c were 0.4 m and 0.3 m , respectively . the surface area of ha ( 10 mg , 20 mg , and 30 mg ) can be calculated as > 0.094 m , > 0.19 m , and > 0.28 m , respectively . even though the absorption amount of cytochrome c could be correlated to the total surface area of ha , that of insulin could not . long - term incubation contributed to the efficient loading of cytochrome c even with a small amount of ha . the initial absorption occurred in less than 1 h , and the subsequent absorption occurred more slowly , in the range of an hour . the first absorption phase might be attributed to surface absorption and the latter by penetration into the pores . the release profiles also occurred over two phases , in less than 1 h and over the hour range ( figure 3 ) , similar to the adsorption profile . this result suggests that absorbed proteins at the surface were released very fast and those within the pores were released more slowly . thus , regulation of release can be achieved by the control of protein size and the pore size of ha . it is possible that step - by - step protein release can be performed in an ha - based delivery system . less than 40% of the bound insulin was released from ha , even though 70% of cytochrome c was released after 24 h. insulin is smaller than cytochrome c and readily bound to ha ( table 1 and figure 2 ) , but the release of insulin is slower than that of cytochrome c ( figure 4 ) . it is likely that the slow release may be caused by its poor solubility in pbs , compared to cytochrome c. the release of insulin was next examined at ph 3 , because insulin is easily soluble in acidic solution , which is a condition of the association . however , the release at ph 3 was slower than that at ph 7.4 and was perhaps affected by the charge of insulin . insulin has an isoelectric point ( pi ) of 5.3 so is positively charged at ph 3 and negatively at ph 7.4 . a decrease in insulin release was observed , especially at ph 7.4 after more than 5 h. the readsorption of the released insulin to ha might have occurred , because the desorption conditions differed from the absorption condition . our results suggest that the association and dissociation properties to ha were affected by both the charge and size of proteins . ha has a hexagonal structure , in which the c ( ca - rich ) site is arranged in the a c and b c planes and the p ( ca - deficient ) site is in the a it was reported that anionic molecules bind to the c site and cationic ones to the p site . therefore , ha - based protein delivery is suitable for ph - dependent controlled release . cationic cytochrome c and anionic insulin at the physiological ph were absorbed and desorbed in different manners . because the charge of insulin was changed with decreasing ph , it markedly influenced the adsorption and desorption behaviors . the absorption behavior may be very complex , because large protein molecules bind to ha at multiple points . therefore , the regulation of controlled release of protein is still to be investigated ( for further information , see supplementary material available online at doi:10.1155/2012/932461 . ) . in conclusion , we prepared protein - associated ha and characterized its association and dissociation properties . . however , their association and dissociation behaviors differed , depending on the size and charge of the proteins .

hydroxyapatite ( ha ) is a precursor of bone and has been studied as a biomaterial . we attempted ha to apply to protein delivery systems . in this study , the association and dissociation properties of two types of bioactive proteins , cytochrom c and insulin , to ha were investigated . cytochrom c was less associated with ha than insulin , which was easily released from it . however , the release of insulin from ha was slow . insulin was released from ha at ph 7.4 more rapidly than at ph 3 . the association and dissociation properties might be influenced by the size , solubility and net charge of protein . ha is a potential protein carrier with controlled release .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

PMC4610885

cognitively able adolescents and adults with a diagnosis of autism spectrum disorder ( asd ) , such as those once diagnosed with asperger s syndrome ( as ) or high functioning autism , commonly have restricted social participation as a result of social and communication deficits.14 they frequently report social isolation and negative experiences in social situations , which may influence their motivation for social engagement . whether individuals with asd wish to engage socially ( ie , the term autism is drawn from the greek word , autos , meaning self . the name was first applied to children who were socially isolated , seemingly by choice . thus , the popular belief that individuals with asd lack the motivation to engage socially . recently , chevallier et al proposed a social motivation theory of autism , describing it as an extreme case of diminished social motivation.5 other researchers studying social motivation concluded that children with asd display less desire for interaction than typically developing peers,6 a finding explicitly and directly gleaned from asking participants to view pictures of people taken from the side view and indicate whether they wanted to interact with those individuals . for example , deckers et al asked children with asd to indicate whether they would like to get acquainted with individuals presented in profile pictures of faces.6 if they wanted to know a person , they pulled on a joystick and turned the photo toward them . if they did not want to meet the person , they pushed on the joystick which turned the picture away ( ie , face turn approach - avoidance task).7 unlike asking them explicitly , using this implicit approach , deckers et al found that children with asd were inclined to express a desire to approach others . other researchers8,9 have used electroencephalography with cognitively able children with asd , finding greater left than right anterior cortical activity while at rest , a pattern associated with a disposition to approach socially.10 a third hypothesis is that social motivation in individuals with asd is context dependent . that is , individuals are motivated to engage socially in specific contexts and not in others . for example , using experience sampling methodology ( esm ) , hintzen et al found that cognitively able adults with asd chose to spend more time with familiar people than did adults without asd.11 in addition , we found that cognitively able adolescents and adults with asd reported enjoying everyday social situations when they were conversing with friends compared with other people or being alone ( chen et al , unpublished data , 2015 ) . self - determination theory ( sdt),1214 which focuses on how social contexts affect engagement , may help understand the contextual nature of motivations for social interaction in people with asd . instead of treating motivation as a unitary construct , sdt addresses the orientation / type of motivation which concerns the reasons or goals that give rise to an action.14 according to sdt , motivations can be self - determined in situations that individuals find compelling ( ie , intrinsically motivated ) or that they think are good for them ( ie , identified regulation ) . in contrast , individuals sometimes perform behaviors to satisfy external demands ( ie , external regulation ) , or to avoid guilt or attain recognition ( ie , introjected regulation ) ; both are extrinsically motivated . however , amotivated behaviors occur in situations where an individual lacks a reason for or an intention to act . sdt suggests that orientation of motivation can be predicted by the degree to which an action satisfies basic psychological needs . deci and ryan described three basic psychological needs inherent to all human beings:12 autonomy , competence , and relatedness . human needs play a mediating role between the actions of individuals and the social context.1214 deci and ryan suggested that participation in a supportive social environment is crucial to satisfaction of psychological needs ; which , in turn , facilitates self - determined behaviors and positive experiences . our previous findings that cognitively able adolescents and young adults with asd enjoyed social interactions with certain contexts suggests that those interactions met basic needs . in contrast , whitehouse et al found that adolescents with as have little intrinsic motivation for establishing friendships , probably because their needs are not met.15 the lack of clarity around the motivation for social participation of adolescents and adults with asd may , in part , reflect differences in methodology . while we have used esm16,17 to explore social experiences in everyday contexts , most previous researchers have used retrospective interview or global self - rating scales . for example , whitehouse et al15 used the friendship motivation questionnaire,18 which required participants to indicate how true statements were of them . this task is highly cognitive , requiring aggregation of experiences over time.19 retrospective responses are easily contaminated by memory bias20 or by the emotional states elicited by reflective questioning.19 more importantly , retrospective and one - time reporting are divorced from real life contexts and experiences of social participation . thus , the low motivation for social engagement identified by whitehouse et al15 may not truly reflect their social desires in everyday life . esm is an ecological momentary assessment used to identify dynamic relationships between subjective experiences and everyday contexts.19 esm allows participants to report their actions , momentary thoughts , and feelings in real time , across natural settings and over a period of time . this method enables to identify fluctuations in perceptions about everyday experiences within a person.21 as such , it is suited to capturing the variability inherent to the asd population . unlike single - case studies , data collected with esm can be aggregated and analyzed at within- and between - person levels simultaneously.21 in addition , compared with retrospective approaches , data collected through esm have greater ecological validity and are less subject to recall or social appropriateness bias.16,19 further , esm survey questions are short , straightforward , and address only the immediate context , which involves less cognitive demand than retrospective methods . recent research has established the validity and usability of esm with the asd population.11,2226 the purpose of the present study was to examine motivation for social participation in everyday contexts among cognitively able adolescents and adults with asd . we used sdt as a theoretical framework and esm as a methodology . to examine the extent to which the basic needs of competence and relatedness were met in everyday social experiences , we investigated perceptions of difficulty and social reciprocity . given the heterogeneity of people with asd and our previous findings ( chen et al , unpublished data , 2015 ) , we examined severity of asd symptomology and global social anxiety as moderators of motivation for and perceptions of everyday participation . in addition , because we had found that australian females with asd are more socially active than australian males and taiwanese people ( chen et al , unpublished data , 2015 ) , we also examined whether participant group ( australian females vs other people ) served as a moderator . the study had approval from the university of sydney human research ethics committee , the autism spectrum australia research approval committee , and the research ethics committee of national taiwan university hospital ( ntuh ) . all participants provided written consent , and parents provided additional consent for participants younger than 18 . because the study began prior to release of the diagnostic and statistical manual of mental disorders ( dsm ) 5th edition,27 we used dsm - iv criteria for asd diagnosis . thirty - two individuals with as or high functioning autism agreed to participate : 14 australians ( four males ) were recruited via research flyers circulated around australia and 18 taiwanese ( 14 males ) were referred by psychiatrists employed at ntuh . all individuals met the following inclusion criteria : 1 ) a formal diagnosis from a psychiatrist or psychologist using the dsm - iv criteria;3 2 ) aged between 16 and 45 years , and 3 ) having sufficient reading comprehension to understand the surveys . reading comprehension of australian participants was confirmed by standard scores of 85 on the reading comprehension subtest of woodcock reading mastery test-3rd edition.28 because there are no equivalent reading assessments for taiwanese adults , the reading comprehension of taiwanese participants was confirmed by a verbal intelligence quotient 70 on the wechsler adult intelligence scale - iv.29 no participants had a diagnosis of intellectual disability , neurological or other developmental disorders ( eg , cerebral palsy ) . in order for their data to be included , participants had to have completed at least 17 of 49 surveys ( > 33% ) in accordance with suggestions from previous researchers.30 data from two taiwanese males were excluded from the final sample . the second completed only 15 surveys in 7 days ; the minimum requirement was 17 surveys ( see procedure ) . thus , the final sample included data from 14 australians ( four males ; aged 1643 years [ mean = 24.8 , standard deviation { sd } = 9.0 ] ) and 16 taiwanese ( 12 males ; aged 1945 years [ mean = 27.8 , sd = 6.3 ] ) . in the final sample , the main diagnosis was as ( n=12 [ 86% ] in the australian group and n=13 [ 81% ] in the taiwanese group ; table 1 ) . half of the australian participants and a quarter of the taiwanese participants had concomitant mental health diagnoses ( eg , attention deficit / hyperactivity disorder , depression , anxiety , obsessive compulsive disorder ) . regarding current education and employment status , seven ( 50% ) australians and four ( 25% ) taiwanese were still students . we developed an esm survey to explore what participants were doing ( everyday activities , figure 1 for options ) and who they were communicating with ( social interaction conditions , figure 2 for options ) . each time they were signaled , participants chose the main activity in which they were engaged and as many social partners as were relevant . they indicated why they were doing the activity ( from a list ) and their experiences of : a ) difficulty of activity engagement , and b ) social reciprocity during interactions . social reciprocity included questions about being listened to ( do you think they were really listening to what you had to say ? ) and caring about others ( did you care about what they were doing / saying ? ) . participants responded yes or no to perceived difficulty and from 0 ( not at all ) to 10 ( very much ) for perceived social reciprocity . the esm survey was loaded to the participation in everyday life survey application ( piel app ) , an esm platform designed for ios devices.31 the piel app prompted participants to complete the esm surveys , time stamped the responses , and stored the data . the social responsiveness scale second edition ( srs-2)32 was used to evaluate the severity of asd symptoms . in taiwan , the chinese version of srs33 was used . the srs-2 is a parent / teacher - report or self - report questionnaire for individuals 2.5 years or older . in this study , participants who were aged 19 years and above completed the self - report version . the srs-2 comprises 65 items organized into five subscales : social awareness , social cognition , social communication , social motivation , and restricted interests and repetitive behaviors . items are rated on a 4-point likert scale from 0 ( not true ) , to 3 ( almost always true ) . total standard scores of 60 or higher are clinically significant and indicate deficiencies in social reciprocal behavior that may interfere with everyday social interactions . the srs-2 has evidence of sound psychometric properties when used with adults.32,34 to evaluate the severity of global social anxiety in australian and taiwanese participants , the social interaction anxiety scale ( sias)35 and the chinese version of the sias36 were used . the 20-item self - report scale measures experiences in social situations associated with social anxiety according to dsm - iv criteria.3 participants rated the items using a 5-point scale ( 0= not at all characteristic of me to 4= extremely characteristic of me ) . the sias and chinese version of the sias have good evidence of reliability and validity across clinical and community populations.35,36 we provided participants with an ipod touch or delivered the piel app to their ios device ( ie , ipod touch or iphone ) . we conducted a 30- to 60-minute individual training session with each participant on the use of the piel app and the ios device . the training was held at a university campus , outpatient clinic of the hospital or a quiet public place ( eg , a library ) . during the training parents of participants under 19 received the school - age version of srs-2 by mail . the device prompted participants seven times each day at random intervals to respond to the same esm survey . they were instructed to answer as many surveys as possible but to ignore signals that occurred at inconvenient times ( eg , when bathing ) . the survey became dormant if participants did not respond to a prompt within 2 minutes . we coded activities into five types : non - activity , productivity , solitary / parallel leisure , social activities , and self - care we coded reasons for engaging in activities into five levels based on degree of self - determination drawn from sdt:12 a ) intrinsic motivation ( i like it ) , b ) three levels of extrinsic motivation : identified regulation ( i am doing it for my own good ) ; introjected regulation ( i want people to like me ; i want to look occupied ) , and external regulation ( i have to ) , and c ) amotivation ( it s just a habit ; nothing else to do ) . for each participant , we calculated the proportion of each motivation associated with each activity and interaction with different people . we also calculated the proportion of perceived difficulty and overall means of social reciprocity associated with each activity and interaction , respectively . prior to calculating overall means , we centered each participant s ratings at his or her mean to account for individual differences . esm data have a hierarchical structure with multiple surveys ( level 1 ) nested within data from each participant ( level 2 ) . we performed multilevel analyses to examine how motivation and perceptions were associated with everyday participation . given that multilevel analysis takes into account the dependency of the surveys from the same participant,37,38 the method is preferred over the conventional ordinary least squares approaches for esm data.39,40 in addition , multilevel analysis allows examination of relationships between variables at different levels and also accounts for potential moderating effects of level 2 variables on the relationships of level 1 variables.37,38 previous researchers suggested a minimum level 2 sample size of 30 to achieve sufficient power.41,42 we examined associations between everyday activities and the dependent variables , degree of self - determination and perceived difficulty , in two multilevel logistic analyses . in each analysis , non - activity , productivity , solitary / parallel leisure , and social activities were the level 1 independent variables and self - care was the reference . to examine source of motivation as a dependent variable in multilevel logistic analysis , the five motivation levels were coded as relatively self - determined ( intrinsic motivation , identified regulation ) or relatively externally determined ( introjected regulation , external regulation , amotivation ) ; the relatively externally determined was the reference . to examine perceived social reciprocity , we conducted two multilevel linear analyses with being listened to and caring about others as continuous dependent variables . because participants were able to select multiple social partners in a single interaction , we coded each social partner as a binary ( yes / no ) variable : no one , family members , casual / intimate friends , or people at school / work all the four types of social partners were included as level 1 independent variables in the analysis of motivation . the analyses of perceived social reciprocity included only three independent variables : family members , casual / intimate friends , and people at school / work because social reciprocity requires the presence of a social partner . in each multilevel analysis , we simultaneously analyzed individual characteristics as level 2 independent variables to examine their associations with motivation and perceived social reciprocity . these variables were severity of asd ( ie , total standard scores of srs-2 ) , severity of global social anxiety ( ie , total raw scores of sias ) , and participant group ( australian females vs other participants ) . srs-2 and sias data were grand mean centered for comparisons across participants.37,38 to examine potential moderating effects of individual characteristics ( severity of asd , global social anxiety , and participant group ) , we added the interactions between level 1 and level 2 independent variables to the original multilevel analyses . these additional analyses helped identify whether the level 2 independent variables moderated the relationships between level 1 independent and dependent variables . to reduce model complexity and maximize stability , we removed cross - level interactions which showed no significant relationships to the dependent variable when establishing the final model.37,38 we used hierarchical linear and nonlinear modeling ( hlm 6.08 ) software43 to perform the multilevel analyses . associations between independent and dependent variables were evaluated through the estimation of the odds ratios ( ors ) and corresponding 95% confidence intervals ( cis ) for the multilevel logistic analysis , and a fixed regression coefficient ( ) and standard errors for the multilevel linear analysis . significance in cross - level interaction indicates a significant moderating effect of a level 2 independent variable on the relationship between level 1 independent and dependent variables . thirty - two individuals with as or high functioning autism agreed to participate : 14 australians ( four males ) were recruited via research flyers circulated around australia and 18 taiwanese ( 14 males ) were referred by psychiatrists employed at ntuh . all individuals met the following inclusion criteria : 1 ) a formal diagnosis from a psychiatrist or psychologist using the dsm - iv criteria;3 2 ) aged between 16 and 45 years , and 3 ) having sufficient reading comprehension to understand the surveys . reading comprehension of australian participants was confirmed by standard scores of 85 on the reading comprehension subtest of woodcock reading mastery test-3rd edition.28 because there are no equivalent reading assessments for taiwanese adults , the reading comprehension of taiwanese participants was confirmed by a verbal intelligence quotient 70 on the wechsler adult intelligence scale - iv.29 no participants had a diagnosis of intellectual disability , neurological or other developmental disorders ( eg , cerebral palsy ) . in order for their data to be included , participants had to have completed at least 17 of 49 surveys ( > 33% ) in accordance with suggestions from previous researchers.30 data from two taiwanese males were excluded from the final sample . the second completed only 15 surveys in 7 days ; the minimum requirement was 17 surveys ( see procedure ) . thus , the final sample included data from 14 australians ( four males ; aged 1643 years [ mean = 24.8 , standard deviation { sd } = 9.0 ] ) and 16 taiwanese ( 12 males ; aged 1945 years [ mean = 27.8 , sd = 6.3 ] ) . in the final sample , the main diagnosis was as ( n=12 [ 86% ] in the australian group and n=13 [ 81% ] in the taiwanese group ; table 1 ) . half of the australian participants and a quarter of the taiwanese participants had concomitant mental health diagnoses ( eg , attention deficit / hyperactivity disorder , depression , anxiety , obsessive compulsive disorder ) . regarding current education and employment status , seven ( 50% ) australians and four ( 25% ) taiwanese were still students . we developed an esm survey to explore what participants were doing ( everyday activities , figure 1 for options ) and who they were communicating with ( social interaction conditions , figure 2 for options ) . each time they were signaled , participants chose the main activity in which they were engaged and as many social partners as were relevant . they indicated why they were doing the activity ( from a list ) and their experiences of : a ) difficulty of activity engagement , and b ) social reciprocity during interactions . social reciprocity included questions about being listened to ( do you think they were really listening to what you had to say ? ) and caring about others ( did you care about what they were doing / saying ? ) . participants responded yes or no to perceived difficulty and from 0 ( not at all ) to 10 ( very much ) for perceived social reciprocity . the esm survey was loaded to the participation in everyday life survey application ( piel app ) , an esm platform designed for ios devices.31 the piel app prompted participants to complete the esm surveys , time stamped the responses , and stored the data . the social responsiveness scale second edition ( srs-2)32 was used to evaluate the severity of asd symptoms . in taiwan , the chinese version of srs33 was used . the srs-2 is a parent / teacher - report or self - report questionnaire for individuals 2.5 years or older . in this study , participants who were aged 19 years and above completed the self - report version . the srs-2 comprises 65 items organized into five subscales : social awareness , social cognition , social communication , social motivation , and restricted interests and repetitive behaviors . items are rated on a 4-point likert scale from 0 ( not true ) , to 3 ( almost always true ) . total standard scores of 60 or higher are clinically significant and indicate deficiencies in social reciprocal behavior that may interfere with everyday social interactions . the srs-2 has evidence of sound psychometric properties when used with adults.32,34 to evaluate the severity of global social anxiety in australian and taiwanese participants , the social interaction anxiety scale ( sias)35 and the chinese version of the sias36 were used . the 20-item self - report scale measures experiences in social situations associated with social anxiety according to dsm - iv criteria.3 participants rated the items using a 5-point scale ( 0= not at all characteristic of me to 4= extremely characteristic of me ) . the sias and chinese version of the sias have good evidence of reliability and validity across clinical and community populations.35,36 we provided participants with an ipod touch or delivered the piel app to their ios device ( ie , ipod touch or iphone ) . we conducted a 30- to 60-minute individual training session with each participant on the use of the piel app and the ios device . the training was held at a university campus , outpatient clinic of the hospital or a quiet public place ( eg , a library ) . during the training parents of participants under 19 received the school - age version of srs-2 by mail . australian participants took the woodcock reading mastery test-3rd edition to ensure reading comprehension ability . following training , the device prompted participants seven times each day at random intervals to respond to the same esm survey . they were instructed to answer as many surveys as possible but to ignore signals that occurred at inconvenient times ( eg , when bathing ) . the survey became dormant if participants did not respond to a prompt within 2 minutes . we coded activities into five types : non - activity , productivity , solitary / parallel leisure , social activities , and self - care ( figure 1 for types ) . we coded reasons for engaging in activities into five levels based on degree of self - determination drawn from sdt:12 a ) intrinsic motivation ( i like it ) , b ) three levels of extrinsic motivation : identified regulation ( i am doing it for my own good ) ; introjected regulation ( i want people to like me ; i want to look occupied ) , and external regulation ( i have to ) , and c ) amotivation ( it s just a habit ; nothing else to do ) . for each participant , we calculated the proportion of each motivation associated with each activity and interaction with different people . we also calculated the proportion of perceived difficulty and overall means of social reciprocity associated with each activity and interaction , respectively . prior to calculating overall means , we centered each participant s ratings at his or her mean to account for individual differences . esm data have a hierarchical structure with multiple surveys ( level 1 ) nested within data from each participant ( level 2 ) . we performed multilevel analyses to examine how motivation and perceptions were associated with everyday participation . given that multilevel analysis takes into account the dependency of the surveys from the same participant,37,38 the method is preferred over the conventional ordinary least squares approaches for esm data.39,40 in addition , multilevel analysis allows examination of relationships between variables at different levels and also accounts for potential moderating effects of level 2 variables on the relationships of level 1 variables.37,38 previous researchers suggested a minimum level 2 sample size of 30 to achieve sufficient power.41,42 we examined associations between everyday activities and the dependent variables , degree of self - determination and perceived difficulty non - activity , productivity , solitary / parallel leisure , and social activities were the level 1 independent variables and self - care was the reference . to examine source of motivation as a dependent variable in multilevel logistic analysis , the five motivation levels were coded as relatively self - determined ( intrinsic motivation , identified regulation ) or relatively externally determined ( introjected regulation , external regulation , amotivation ) ; the relatively externally determined was the reference . to examine perceived social reciprocity , we conducted two multilevel linear analyses with being listened to and caring about others as continuous dependent variables . because participants were able to select multiple social partners in a single interaction , we coded each social partner as a binary ( yes / no ) variable : no one , family members , casual / intimate friends , or people at school / work . all the four types of social partners the analyses of perceived social reciprocity included only three independent variables : family members , casual / intimate friends , and people at school / work because social reciprocity requires the presence of a social partner . in each multilevel analysis , we simultaneously analyzed individual characteristics as level 2 independent variables to examine their associations with motivation and perceived social reciprocity . these variables were severity of asd ( ie , total standard scores of srs-2 ) , severity of global social anxiety ( ie , total raw scores of sias ) , and participant group ( australian females vs other participants ) . srs-2 and sias data were grand mean centered for comparisons across participants.37,38 to examine potential moderating effects of individual characteristics ( severity of asd , global social anxiety , and participant group ) , we added the interactions between level 1 and level 2 independent variables to the original multilevel analyses . these additional analyses helped identify whether the level 2 independent variables moderated the relationships between level 1 independent and dependent variables . to reduce model complexity and maximize stability , we removed cross - level interactions which showed no significant relationships to the dependent variable when establishing the final model.37,38 we used hierarchical linear and nonlinear modeling ( hlm 6.08 ) software43 to perform the multilevel analyses . associations between independent and dependent variables were evaluated through the estimation of the odds ratios ( ors ) and corresponding 95% confidence intervals ( cis ) for the multilevel logistic analysis , and a fixed regression coefficient ( ) and standard errors for the multilevel linear analysis . significance in cross - level interaction indicates a significant moderating effect of a level 2 independent variable on the relationship between level 1 independent and dependent variables . participants completed an average of 38 , of a possible 49 ( average response rates : 77.6% ) , esm surveys ( sd = 7 , range = 2349 ) , providing a total of 1,131 surveys . overall , participants spent the majority of time engaging in activities related to solitary / parallel leisure ( 42.4% ) , followed by 25.0% in productivity ( ie , home chores and class , work or meeting ) , and 12.2% in non - activity . they engaged infrequently ( 10.8% ) in social activities and they did not interact with another person most of the time ( 71.6% ) . casual friends / an intimate friend ( 11.4% ) were the ones they mainly interact with when they engaged in conversation . figure 1 shows that participants were most frequently intrinsically motivated to engage in social and solitary / parallel leisure activities , except running / jogging / fitness / sport where the behaviors were identified as regulated . conversely , participants were frequently externally regulated ( the least autonomous response ) when they engaged in self - care and productive activities . they described productive activities as difficult only 16.3% of the time , social activities figure 2 shows that participants were intrinsically motivated to interact with family members and casual / intimate friends . however , they frequently perceived interaction with people at school / work as externally regulated . in terms of perceived social reciprocity , participants perceived relatively high levels of being listened to while interacting with family members ( mean [ m ] = 0.313 , sd = 1.139 ) and casual / intimate friends ( m = 0.281 , sd = 0.753 ) compared with people at school / work ( m = 0.004 , sd = 0.804 ) . further , they cared more about family members ( m = 0.345 , sd = 0.876 ) and casual / intimate friends ( m = 0.035 , sd = 0.985 ) than people at school / work ( m = -0.305 , sd = 0.736 ) . table 2 summarizes the results of the multilevel analyses of motivation and perceived difficulty of activities . participants were more likely to be self - determined while engaging in solitary / parallel leisure and social activities than in other activities . however , australian females displayed a weak relationship between self - determined motivation and solitary / parallel leisure ( or = 0.44 , 95% ci = 0.220.89 ) . regarding perceived difficulty , participants were more likely to report productive activities as difficult than other activities . participants with higher levels of global social anxiety had stronger relationships between perceived difficulty and engagement in productive activities ( or = 1.05 , 95% ci = 1.011.09 ) and social activities ( or = 1.08 , 95% ci = 1.031.14 ) . multilevel analyses in table 3 showed that participants were more likely to be self - determined while interacting with family members and casual / intimate friends than with other social partners . further , they were more likely to perceive higher levels of being listened to during interaction with casual / intimate friends . upon close inspection , global social anxiety moderated the relationship between thought of caring about others and interaction with casual / intimate friends ( =0.10 , standard error = 0.02 , p<0.01 ) . that is , participants with higher levels of global social anxiety were less likely to care about casual / intimate friends while interacting with them . table 2 summarizes the results of the multilevel analyses of motivation and perceived difficulty of activities . participants were more likely to be self - determined while engaging in solitary / parallel leisure and social activities than in other activities . however , australian females displayed a weak relationship between self - determined motivation and solitary / parallel leisure ( or = 0.44 , 95% ci = 0.220.89 ) . regarding perceived difficulty , participants were more likely to report productive activities as difficult than other activities . participants with higher levels of global social anxiety had stronger relationships between perceived difficulty and engagement in productive activities ( or = 1.05 , 95% ci = 1.011.09 ) and social activities ( or = 1.08 , 95% ci = 1.031.14 ) . multilevel analyses in table 3 showed that participants were more likely to be self - determined while interacting with family members and casual / intimate friends than with other social partners . further , they were more likely to perceive higher levels of being listened to during interaction with casual / intimate friends . upon close inspection , global social anxiety moderated the relationship between thought of caring about others and interaction with casual / intimate friends ( =0.10 , standard error = 0.02 , p<0.01 ) . that is , participants with higher levels of global social anxiety were less likely to care about casual / intimate friends while interacting with them . we explored the motivation for social engagement in everyday contexts among cognitively able adolescents and adults with asd through the lens of sdt , using esm as a methodology . we investigated the controversy whether cognitively able adolescents and adults with asd desire to engage socially . three possible perspectives have been proposed : they prefer not to engage socially;5,6 they wish to engage socially but express that desire more implicitly than explicitly;6,8,9 and the desire for social engagement is context specific as suggested by sdt.1214 our findings support the latter view . in line with sdt,12,14 the results suggest that cognitively able adolescents and adults with asd were motivated to interact in situations where they felt competent and experienced social reciprocity . specifically , they were motivated to engage in social activities where they did not experience significant difficulty . further , cognitively able adolescents and adults with asd were motivated to interact with friends and perceived their friends as listening to them during interactions . these findings indicate that social activities and interactions with friends are the everyday contexts that support basic psychological needs and facilitate motivation for engagement . consequently , they reported enjoying these social situations ( chen et al , unpublished data , 2015 ) . thus , our findings highlight the importance of context when exploring motivation for , and perceptions of , everyday social participation . perceiving the social contexts of interaction with friends as being listened to may reflect how cognitively able adolescents and adults with asd characterize friendship . our findings are similar to those of howard et al44 who found that adolescents with asd defined a friend as a person who provides care and responds to needs . these findings suggest that , whereas children consider friendships in terms of companionship,45 adolescents and adults value the affective components of friendship . in addition to the impact of context , our findings suggest that global social anxiety , an individual characteristic , influences perceptions of competence and social reciprocity in social interactions . individuals with higher global social anxiety were more likely to perceive social activities as difficult than those with lower levels of social anxiety . this finding may suggest that cognitively able individuals with asd who concurrently have social anxiety may be more self - aware of their social difficulties than those without social anxiety.46 the association between perception of difficulty in social activities and social anxiety may explain our previous finding of a negative relationship between time spent in social situations and severity of social anxiety ( chen et al , unpublished data , 2015 ) . in contrast to our current findings , other researchers15 have identified that adolescents with as are not intrinsically motivated to develop friendships . in addition to the difference in methodology used , the discrepancy in the findings may be explained by the extent to which basic needs to feel competent and related are met at different stages of a social interaction . when our participants responded to a signal from the ios device and indicated that they were already engaged in a social interaction with a trusted friend or partner other researchers , however , have described difficulty with the initiation of social interactions and establishing friendships,4 which may reflect a gap between social demands and their beliefs about their social skills.47,48 conscious awareness of being different and fear of being excluded may contribute to reduced feelings of relatedness;4 which , in turn , may result in fewer attempts at initiating everyday social interactions.11,49,50 presumably , friendships develop out of a motivation to engage with others ; if motivation is low because of unmet basic needs , then friendships will not follow . however , clearly our participants managed to establish some valued friendships . we did not find a significant relationship between interacting with friends and caring about what they were thinking or doing . this may reflect a core characteristic of asd : a lack of social reciprocity.3 deficits in understanding social or emotional cues of others and considering others perspectives may influence willingness to care about others.51,52 although our participants nominated friends , further research is required to investigate the meaning and true nature of their friendships . first , we did not recruit neurotypical adolescents and adults because the purpose of the study was to explore the motivation for social engagement and identify individual characteristics related to motivation and needs satisfaction among individuals with asd . thus , we do not know how the experiences of our participants compare with those of neurotypical adolescents and adults . further , because of the relatively low incidence of cognitively able adolescents and adults with autism , our participants represented a wide age range . second , due to different recruitment procedures , we were unable to match the number of males and females in australia and taiwan . we had more female participants in australia , probably due to their interest and willingness to volunteer.16 nonetheless , we did not find differences between groups as a function of nationality . finally , half of the participants from australia and a quarter of those from taiwan had comorbid mental health diagnoses . because of the relatively small sample size , we were not able to take into account the effects of additional diagnoses or medications on social experience . further research is needed to examine the effects of comorbidities and compare the findings with neurotypical adolescents and adults . while the application of multilevel analysis may offset the unequal numbers of male and female participants in the two countries,37 a larger sample size is required before the findings can be generalized . our findings highlight several important considerations for research and practice . exploring in - the - moment experiences may provide contextually accurate and detailed information to facilitate intervention to enhance social participation of cognitively able adolescents and adults with asd . although our participants were motivated and did not perceive significant difficulty while being engaged socially , they still spent limited time in social situations . researchers and professionals may need to consider ways to improve social skills and enhance perceived competence for initiating social interactions . perceived challenges with initiating social interaction may be the result of previous experiences ( eg , being excluded ) . given that severity of social anxiety was often associated with perceptions of social engagement , strategies targeting social anxiety management should be incorporated into interventions . because cognitively able adolescents and adults with asd were motivated to interact with friends , service providers and researchers may need to consider incorporating peers in intervention and promoting friendship as an important intervention outcome .

objectivethe purpose of the present study was to examine motivation for the contextual nature of motivations for social participation in cognitively able adolescents and adults with autism spectrum disorder , using self - determination theory as a theoretical framework.methodsfourteen australians and 16 taiwanese ( aged 1645 years ) with asperger s syndrome and high functioning autism were asked to carry a device which prompted them seven times / day for 7 days , to record what they were doing , with whom , perceived difficulty and social reciprocity , and the reasons for engaging in a situation , which were then coded into degree of self-determination.resultsmultilevel analyses showed that participants were more likely to be self - determined while engaging in solitary / parallel leisure and social activities than in other types of activities . interactions with family members and casual / intimate friends were also positively associated with self - determined motivation . further , participants were more likely to perceive higher levels of being listened to during interaction with casual / intimate friends than in interaction with other people . global social anxiety served as a moderator for their perceptions of difficulty and social reciprocity during social engagement.conclusionthe findings highlight the context - dependent motivations for social engagement of cognitively able individuals with autism spectrum disorder .

Introduction Methods Participants ESM Measures of ASD severity and global social anxiety Procedure Data analysis Results Relationships of motivation and perception of difficulty with everyday activities Relationships between motivation and perceived social reciprocity and types of social interaction Discussion Conclusion

PMC4013555

many studies have shown that lyric / aeg-1 has a pivotal role in the development and progression of tumourigenesis ( brown and ruoslahti , 2004 ; chen et al . ; emdad et al . , 2009 ; hu et al . , 2009 ; jiang et al . , 2012 ; , 2011 ; song et al . , 2009 ; wang et al . , 2011 ; xia et al . ; yoo et al . , 2009 ; yu et al . , differential lyric / aeg-1 expression and altered localisation has been demonstrated in a variety of cancers , with increased expression frequently linked to a poor prognosis ( chen et al . ; jiang et al . , 2012 ; lee et al . , 2009 ; li et al . , 2008 ; liao et al . , 2011 ; song et al . , 2009 ; thirkettle et al . , 2011 ; xia et al . ; yoo et al . , 2009 ; yu et al . , 2009 ) . previous studies have shown that lyric / aeg-1 increases chemo - resistance and cell survival in response to hypoxia and glucose starvation , possibly by initiating protective autophagy ( bhutia et al . ; lyric / aeg-1 is regulated by h - ras and pi3k and there is mounting evidence for its role in regulating ubiquitination ( ash et al . , 2008 ; emdad et al . , 2006 ; ubiquitin modifications are known to result in a broad range of responses , dependent upon the number of ubiquitin molecules and their linkage ( denuc and marfany , 2010 ; ikeda and dikic , 2008 ) . the addition of k48-linked ubiquitin moieties is associated with proteasomal dependent degradation of the substrate protein , whereas k63-linked ubiquitin moieties are associated with endocytosis and dna repair { aguilar and wendland , 2003 # 48}. signalling activation and protein trafficking are more closely associated with monoubiqutination . ubiquitin monomers can also elicit a response analogous to phosphorylation ( ikeda and dikic , 2008 ) that regulates signalling or sub - cellular localisation of proteins such as pten , p53 and foxo3a ( salmena and pandolfi , 2007 ) . topors is a unique e3 ligase which can ligate both ubiquitin and small ubiquitin - like modifier ( sumo ) to substrate proteins ( denuc and marfany , 2010 ) . it contains an n - terminal ring - domain which ligates ubiquitin to its target proteins including dna topoisomerase i , p53 and nkx3.1 ( guan et al . , 2008 ; depending on cellular context , topors has been documented as a proto - oncogene or tumour suppressor ( guan et al . lyric / aeg-1 contains an usually large number of lysine residues ( > 12% ) with the majority clustered within extended nuclear localisation signal regions ( exnls ) . these regions have previously been defined as exnls-1 ( aa78130 ) , exnls-2 ( aa415486 ) and exnls-3 ( aa546582 ) and shown to act as nuclear and nucleolar localisation signals ( thirkettle et al . , 2009a ) . however , only exnls-2 , which contains 18 lysine residues , was shown to be modified by mono - ubiquitin ( thirkettle et al . , 2009a ) causing almost all of lyric / aeg-1 to run at a higher mw of 105 kda . here we identify the specific sites of lyric / aeg-1 ubiquitination by topors in the exnls-2 region . mutagenesis of these residues leads to a highly significant reduction in lyric / aeg-1 ubiquitination which results in altered sub - cellular distribution and impairs the interaction between p65 , importin- and lyric / aeg-1 . gfp - tagged lyric / aeg-1 constructs were created using pqbi25-fc3 plasmid ( q - biogene ) as described ( thirkettle et al . , 2009a ) . site directed mutagenesis was performed using quickchange ii site - directed mutagenesis kit ( stratagene ) following the manufacturer 's instructions to create gfp - k486r - lyric / aeg-1 alone , gfp - k491r - lyric / aeg-1 alone or a gfp - k486r / k491r - lyric / aeg-1 double mutant . stefan weger ( the free university of berlin , germany ) ( weger et al . , 2005 ) . cos-7 cells purchased from the cancer research uk cell bank were routinely cultured in dmem ( gibco ) supplemented with 10% fetal bovine serum ( labtech ) . for transfection cells were grown to 40% confluency . dna ( 1 g/6 wells of a 24 well plate or 6 g / per 14 cm dish ) was transfected using fugene 6 ( roche ) following the manufacturer 's protocol . cells were grown for a further 48 h prior to confocal microscopy or western analysis . all western blotting procedures were performed as described ( whitaker et al . , 2007 ) including the addition of 0.5% ( w / v ) sodium deoxycholate . membranes were incubated with primary antibody ; anti - gfp ( 1:5000 ) , anti - actin ( 1:5000 ) , anti - p65 ( 1:1000 ) , anti - importin- ( 1:5000 ) ( all from abcam ) , anti - ubiquitin ( 1:1000 , santa cruz biotechnology ) and anti - topors ( 1:1000 , sigma ) . protein bands were detected with ecl - plus ( ge healthcare ) and where they exceeded the dynamic range of film diaminobenzidine ( vector laboratories ) was used . immunoprecipitation was performed at 4 c using the method previously described ( thirkettle et al . , 2009a ) . for densitometry all available western blots ( n > 3 ) were scanned using a typhoon scanner ( ge healthcare ) and analysed using imagequant tl ( ge healthcare ) . to overcome any variation in transfection / translation efficiency the amount of ubiquitination was normalised using gfp - lyric / aeg-1 values ( ubiquitination / gfp - lyric / aeg-1 ) . proteins were separated using pre - cast 8% gels ( pierce ) and stained using biosafe coomassie ( molecular probes ) . protein bands were excised and incubated overnight with destain ( 1:1 ratio of acetonitrile and 10 mm triethylammonium bicarbonate ) . bands were washed in destain , dehydrated and reduced using 10 mm dtt , alkylated with 50 mm iodoacetamide then washed and dehydrated once more . proteins were digested at 37 c overnight using porcine trypsin ( promega ) ( 10 ng/l ) . digestion was stopped by flash freezing before the addition of 5% formic acid ( fluka , uk ) and sonication for 15 min , and repeated prior to sample dehydration . digested samples were resuspended in 8 l 1% formic acid for 20 min prior to analysis using a nano - reverse phase lc- ms using a qtof 6510 mass spectrometer with chip cube source interface and 1200 series hplc running masshunter b.01.03 ( agilent , usa ) . samples were loaded onto a chip containing integrated 40 nl enrichment and 150 mm analytical columns and electrospray needle using 3 l / min 0.1% formic acid . nl / min using a linear gradient from 10 to 70% elution buffer ( 80% acetonitrile/20% 0.1% formic acid ( v / v ) ) for 10 min . after 1 min the gradient was increased to 100% elution buffer and 100% buffer was continued for a further 6 min . ms data was recorded in the 2902500 m / z range at 6 spectra per second . ms / ms data was acquired in the 573000 m / z range at 4 spectra per second . data was searched against uniprot ( kb15.5j ) and the known lyric / aeg-1 constructs using mascot daemon version 2.2.2 ( matrix science , uk ) using a peptide tolerance of 10 ppm and a fragment tolerance of 0.05 da in addition to statistical scoring , the validity of the data was confirmed by manual assessment of the data using scaffold software 2.1 ( proteome science , usa ) . data were acquired using the agilent 's lc - chip cube- 6510 qtof data - dependent mode with the settings described above with the exception of collecting profile data . data was acquired randomly as determined by list randomizer ( http://www.random.org/lists/ ) . using trapper version 4.2.0 ( natalie tasman , institute for systems biology , usa ) files were converted to mzxml format and imported into progenesis lc - ms version 2.5.3478.16299 ( nonlinear dynamics , uk ) for lc - ms run alignment and ms peak extraction for label - free quantitation after ion peak identification by mascot . peaks of interest were analysed and validated using the statistical software r ( version 2.10.1 ) ( team , 2010 ) . to normalize for variability in protein input the data was analysed using a mixed effects model , where the two fixed effects were gfp - lyric / aeg-1 constructs and ubiquitin . to reflect the pairing of measurements within these fixed effects ( induced by the fact that each replicate provides two measurements ) we also had a single mixed effects term indicating which replicate ion abundance mass spectrometric measurement it came from ( analogous to the paired t - test that would have been carried out for a single fixed effect ) . analysed data was the log 2 median of the unmodified ubiquitin normalized abundances obtained from progenesis lc - ms label - free quantitation software . cells were fixed stained and mounted as previously described ( thirkettle et al . , 2009a ) . a yeast two - hybrid assay was performed by dualsystems biotech ag , zurich , switzerland using plexa - dir - lyric / aeg-1 aa73 - 582 as bait and a human placental cdna library as described ( thirkettle et al . , 2009b ) . previously , we have demonstrated that cytoplasmic lyric / aeg-1 is modified within the exnls-2 region by mono - ubiquitin , leading to a 105 kda mw band and no increase in lyric / aeg-1 degradation ( thirkettle et al . , 2009a ) . using immunoprecipitation of gfp - tagged wtlyric / aeg-1 and exnls constructs ( shown in figure 1a ) we have previously demonstrated that deletion of the entire exnls-2 region ( aa415486 ) , and not exnls-1 ( aa78130 ) or exnls-3 ( aa546582 ) , leads to an almost complete loss of lyric / aeg-1 mono - ubiquitination ( thirkettle et al . , 2009a ) . we identified two potential specific mono - ubiquitination sites by mass spectrometry at lysine residues k486 and k491 . k486 lies within the exnls-2 region while k491 lies 5 residues upstream of exnls-2 ( figure 1b , top panel ) . to determine if k486 and k491 are the primary sites of ubiquitin modification single and double point mutations were made to ablate mono - ubiquitination without disrupting the protein charge or the potential to act as an nls ( k486r , k491r and k486/491r ) . gfp - tagged wild - type and mutant constructs and ubiquitin were over - expressed in mammalian cells and immunoprecipitated using the gfp - tag . mutation of either k486 or k491 resulted in a significant reduction in lyric / aeg-1 mono - ubiquitination . when both k486 and k491 were mutated mono - ubiquitination was almost entirely ablated ( figure 1b ) . to validate these findings we employed a mass spectrometry quantification method . ubiquitination was normalised to the amount of gfp - tagged construct using the number of defined gfp peptides per construct as a control for gfp - lyric / aeg-1 input ( figure 1c ) . due to the lysine - rich nature of lyric / aeg-1 the standard mass spectrometry methods were unsuitable and ubiquitination of each construct was quantified using a label - free mass spectrometry and a mixed effects model . using this method the most consistent and robust ubiquitin and gfp peptides that could be detected were defined and measured for each construct ( shown in supplementary figures s2 and s3 ) . using mass spectrometry we confirmed that deletion of exnls-2 resulted in a significant loss of ubiquitination compared to wtlyric / aeg-1 ( p = 0.0012 ) ( figures 1a and 1c ) . when k486 and k491 were modified a highly significant loss of ubiquitination was seen ; k486r mutant ( p = 0.0028 ) , k491r mutant ( p = 0.0004 ) and the k486/491r double point mutant ( p = 0.0009 ) ( figures 1b and 1c ) . there was also a small , but not significant , loss of ubiquitination when exnls1/3 was deleted ( p = 0.43 ) . we have previously demonstrated that deletion of the entire exnls-2 region had no effect on lyric / aeg-1 function or stability and that treatment with mg132 failed to promote lyric / aeg-1 poly - ubiquitination , both of which are consistent with mono - ubiquitination ( thirkettle et al . , 2009a ) . to establish if ubiquitination of lyric / aeg-1 had any effect on its function we examined the interaction between lyric / aeg-1 and p65 , a known interacting protein proposed to shuttle into the nucleus with lyric / aeg-1 ( emdad et al . wtlyric / aeg-1 interacts with p65 but this interaction was lost with the non - ubiquitinated k486r / k491r mutant ( figure 2a ) . mass spectrometry data identified a number of peptides corresponding to importin- , a nuclear import chaperone , suggesting that it may interact with lyric / aeg-1 ( supplementary figure s4 ) ( harel and forbes , 2004 ) . this interaction was confirmed by immunoprecipitating gfp - lyric / aeg-1 or the gfp - lyric / aeg-1 k486/491r mutant from cos-7 cells overexpressing the constructs . importin- was shown to interact with the wtlyric / aeg-1 but only at very low levels in the k486/491r mutant suggesting the ubiquitination of lyric / aeg-1 might regulate its interaction with importin-. using confocal microscopy we examined the localisation of the gfp - tagged lyric / aeg-1 constructs . previously we have reported that wtlyric / aeg-1 localises throughout the cytoplasm , nucleolus and to a lesser extent the nucleoplasm , as shown by co - localisation with fibrillarin ( figure 3 ) ( thirkettle et al . , 2009a ) . all of the lyric / aeg-1 ubiquitin mutants are expressed at lower levels than wtlyric / aeg-1 despite being transfected with identical amounts of dna . all of these mutants exhibit loss of the wild - type diffuse cytoplasmic and nucleoplasmic localisation . k491r and all nuclear lyric / aeg-1 in the k486r , k491r and k486r / k491r double mutants resides within the nucleolus and nuclear bodies and is lost from the nucleoplasm . to identify potential e3 ligases for the ubiquitination of lyric / aeg-1 we used a yeast two - hybrid assay with c - terminal lyric / aeg-1 , ( aa 73582 ) , as bait ( thirkettle et al . the screen identified only one e3 ligase , the n - terminal fragment of topors ( aa 45250 ) , containing the ring finger domain , as an lyric / aeg-1 interacting partner ( figure 4a ) ( rajendra et al . , 2004 ) . the interaction between wtlyric / aeg-1 was supported using immunoprecipitation of gfp - tagged wtlyric / aeg-1 from cos-7 cells co - transfected with wtgfp - lyric / aeg-1 and topors ( figure 4b ) . to determine if lyric / aeg-1 is a potential substrate for the topors ubiquitin e3 ligase activity we immunoprecipitated wtlyric / aeg-1 from cos-7 cells transfected with an increasing concentration of topors ( figure 4c ) . western blot analysis showed that the ubiquitination of lyric / aeg-1 increased with increasing topors expression . we have shown in previous studies using mg132 that ubiquitination of lyric / aeg-1 does not regulate its degradation by the proteasome ( thirkettle et al . , 2009a ) . mono - biquitination is known to regulate the localisation and function of a number of proteins including pten , p53 and foxo3a ( denuc and marfany , 2010 ; salmena and pandolfi , 2007 ) . the large number of functions attributed to lyric / aeg-1 suggests it plays a pivotal role in tumourigenesis ( ash et al . , 2008 ; bhutia et al . ; brown and ruoslahti , 2004 ; emdad et al . , 2009 ; hu et al . , 2009 ; li et al . , 2012 ; yoo et al . ) . previously we have shown that the exnls-2 region of lyric / aeg-1 is modified by mono - ubiquitin , rather than poly - ubiquitin and that this modification does not alter lyric protein stability ( thirkettle et al . , 2009a ) . we also saw modification by sumo-1 , but at much lower levels than mono - ubiquitin suggesting that mono - ubiquitin may play a more important role in post - translational modification of lyric / aeg-1 . deletion of exnls-2 , but not exnls-1 or exnls-3 , results in an abrogation of lyric / aeg-1 modification by mono - ubiquitin ( figure 1a ) . we have previously reported that lyric / aeg-1 localisation and expression is up - regulated in prostate tumourigenesis supported by reports of overexpression multiple other cancers ( brown and ruoslahti , 2004 ; chen et al . ; , 2011 , 2009 , 2008 ; liao et al . , 2011 ; song et al . ; ; yoo et al . , 2009 ; yu et al . , 2009 ) . we and others have previously shown that lyric / aeg-1 can move between the cytoplasm and nucleus ( figure 3 ) ( emdad et al . , 2006 ; sarkar et al . , 2008 ; thirkettle et al . , 2009a ) . we have demonstrated that shuttling into the nucleus and interaction with proteins known to shuttle into the nucleus is attenuated by mono - ubiquitination of two key lysine regions at k486 and k491 ( figure 3 ) . mutating k486 and k491 of lyric / aeg-1 resulted in a dramatic effect on its localisation to peri- and sub - nuclear compartments and complete loss of cytoplasmic and nucleoplasmic lyric / aeg-1 ( figure 3 ) . this suggests the retention of non - ubiquitinated lyric / aeg-1 within inactive sub - cellular compartments where it is unable to bind with known interacting protein p65 and the nuclear import protein importin- ( figure 2 and supplementary figure s4 ) ( emdad et al . , 2006 ; we have shown that mutation of k486 and k491 results in a dramatic loss of lyric / aeg-1 mono - ubiquitination ( figure 1b and c ) . this suggests that k486 and k491 are the major sites of mono - ubiquitination and that multiple other neighbouring lysine residues do not act co - operatively to compensate for the loss of a single lysine at either k486 or k491 . although k486 lies with the reported exnls-2 region , k491 lies just outside the exnls-2 region previously identified as the mono - ubiquitinated region of lyric - aeg-1 . this is consistent either with co - operativity between the k486 and k491 mono - ubiquitination sites or binding of the e3 ligase needed to modify k491 to a site within exnls-2 . lyric / aeg-1 has previously been demonstrated to interact with brca2 and cdkn1a - interacting protein and indirectly alter its stability ( ash et al . , we identified topors , an oncogene and e3 ligase known to ubiquitinate proteins including p53 , topoisomerase i and ib kinase epsilon ( renner et al . ) , as an e3 ligase that potentially interacts with lyric / aeg-1 . topors was shown to interact with lyric / aeg-1 by yeast two - hybrid assay and co - immunoprecipitation figure 4a and b. in addition , increasing amounts of topors alters lyric / aeg-1 ubiquitination in a dose - dependent manner ( figure 4c ) . lyric / aeg-1 has been implicated in tumour development , progression and treatment resistance ( bhutia et al . ; chen et al . ; hu et al . , 2009 ; jiang et al . , 2012 ; lee et al . , 2009 ; li et al . , 2012 , 2008 ; thirkettle et al . , 2009a ; wang et al . , 2011 ; xia et al . understanding the mechanism of lyric / aeg-1 modification by ubiquitin and its effects on lyric / aeg-1 sub - cellular trafficking provides new information on the how lyric / aeg-1 function is regulated in cells and offers potential new avenues for therapeutic intervention .

the pivotal role of lyric / aeg-1 in malignant transformation , tumourigenesis and chemo - resistance has previously been demonstrated in different cell types and sub - cellular compartments . the localisation of lyric / aeg-1 appears crucial to its function and is regulated by three lysine - rich nuclear localisation signal regions , one of which was previously demonstrated to be modified by ubiquitin . here we show that mutation of lyric / aeg-1 at k486 and k491 results in a loss of ubiquitination . a k486/491r double mutant that is incapable of ubiquitination shows reduced binding to the nfb subunit p65 or importin- resulting in a distinctive peri - nuclear localisation of lyric / aeg-1 . we also provide evidence to suggest that topors , an e3 ligase that also regulates p53 modification may be responsible for lyric / aeg-1 ubiquitin modification . overall we demonstrate that specific sites of lyric / aeg-1 ubiquitination are essential for regulating lyric / aeg-1 localisation and functionally interacting proteins .

Introduction Materials and methods Results Discussion

PMC3351132

in humans , intracerebral hemorrhage ( ich ) causes secondary damage in the brain through the induction of cerebral edema and perihematomal injury [ 1 , 2 ] . perihematomal edema with mass effect is an almost universal complication of ich , elaborated over several days after the initial insult . glutamate release , bioenergetic failure , inflammation , and apoptosis play a key role in the pathogenesis of secondary injury [ 3 , 4 ] . in animal models of ich , molecular and cellular studies also indicate that hemorrhage induces inflammation , apoptotic cell death , and progressive tissue destruction in perihematomal tissue [ 57 ] . various knockout mice have been bred to study the gene expression of the pro - oxidants heme and ferrous iron , proteolytic enzymes such as metalloproteinases , thrombin , and inducible nitric oxide synthase , as well as leucocyte adhesion molecules ( cd18 ) in the perihematomal area . their genetic variability has turned the mouse into a key study animal for intracerebral hemorrhage despite the small size of its brain . the hematoma is either induced by stereotactic injection of collagenase or autologous blood into the basal ganglia . longitudinal studies of hematoma kinetics in mice are sparse and generally use one modality only . the purpose of our pilot study was ( i ) to develop t1-and t2-weighted mri protocols for high - resolution studies of mouse brain in vivo which allow for the identification of hematoma and edema , and ( ii ) to perform a direct comparison of hematoma and edema volumes on mri with morphometric studies on cryosections . serial mr imaging would allow longitudinal studies of the temporal and spatial evolution of intracerebral hemorrhages and perihematomal injury . that would not only substantially reduce the amount of animals needed , but also better depict the time - dependent action mechanism of various pathogenetic factors . c57bl/6 mice ( jackson laboratories , bar harbor , me , usa ) were maintained in the department of laboratory animal research at harvard medical school with access to food and water ad libitum . all experiments were performed in accordance to the national institutes of health guide for the care and use of laboratory animals as well as the institutional guidelines established by the institutional animal care and use committee ( iacuc ) at harvard medical school . a total of 11 male adult mice , 1012 weeks of age , with a body weight of 2030 g , were used . mice were anesthetized by intraperitoneal injection of 0.5 mg / g body weight of avertin ( tribromoethanol , sigma - aldrich , st . , mice were injected with 0.075u collagenase type vii - s ( sigma - aldrich , st . louis , mo , usa ) in 500 nl saline into the left caudate putamen using the following stereotactic coordinates : 0.5 mm posterior , 3.0 mm lateral to the bregma , and 4.0 mm in depth . collagenase was infused over 2 minutes using a stereotaxic injector ( stoelting , wood dale , il , usa ) and the 1 l - syringe ( hamilton , reno , nv ) stayed in place for 10 minutes to prevent reflux . , two mice underwent mr imaging 2 , 8 , and 24 hours after collagenase injection to define the best early time points for mr imaging . in part 2 , nine mice were subjected to an imaging schedule from days 1 ( n = 9 ) , 3 ( n = 7 ) , 10 ( n = 5 ) , and 21 ( n = 2 ) based on the different phases of edema ( hyperacute , acute , subacute , chronic ) that bear their own pathophysiology . animals were evaluated using a modified 28-point neurological scoring system before isoflurane anesthesia for the mr . the test included body symmetry , gait , climbing , circling behaviours , front limb asymmetry , and compulsory circling . each point was graded from 0 to 4 , establishing a maximum deficit score of 24 . mr imaging was performed with a high - field 4.7 tesla mr scanner ( biospin , bruker , germany ) at the center for basic mr research at beth israel deaconess medical center . anesthesia was induced with 2% isoflurane and maintained with 1.6% isoflurane in 100% o2 via an mr - compatible nose cone apparatus to minimize motion artifacts . mice were positioned headfirst and prone inside a plexiglass cradle with respiratory monitoring and a constant flow of isoflurane delivered directly to the nose of the mouse . the magnet compatible apparatus allows a precise and reproducible fixation of the head at the center of the bruker linear birdcage radiofrequency coil with an inner diameter of 22 mm . t1- and t2-weighted images were acquired at a 256 256 matrix and a 2.56 2.56 cm field of view . a relaxation enhancement ( rare ) sequence was used for both t1-weighted ( echo time / relaxation time [ te / tr ] = 12/500 , rare factor = 1 ) and t2-weighted ( te / tr = 75/2000 , rare factor = 12 ) imaging . t2 * -weighted imaging parameters were te = 14 ms , tr = 1500 ms , fov = 2.56 2.56 cm , and fa = 30 , matrix 128 128 . in all acquisitions , hematoma size was determined from t1 and t2 * images , and t2 images were used for edema quantification . the measurements were performed by an examiner blinded to the exam date using a computer - assisted image analysis program ( imagej , nih , available at website http://rsb.info.nih.gov/ij/ ) . the values for hematoma and edema volumes were compared with those determined from cryosections . following final mr imaging , brains were removed , placed in 4% paraformaldehyde and cryoprotected in 30% sucrose for 3 days at 4c before they were frozen . coronal sections of 20 m - thickness were taken at 300 m intervals over the rostral - caudal extent of the lesion . sections were stained with luxol fast blue and cresyl violet according to previously published protocols [ 15 , 17 , 18 ] . lesion volumes ( mm ) were computed as running sums of lesion area multiplied by the thickness of each section ( 300 m ) over the extent of the lesion expressed as an orthogonal projection . given the small sample size at different examination dates , the correlation coefficient ( spearman coefficient ) for non - parametric correlations and two - tailed p values at a confidence interval ci = 95% were analyzed using one - way anova . in two animals , mr imaging was performed as early as 2 hours after hematoma induction , and the hematoma was observed as a focal hypointensity on t1- , t2- , and t2 * -weighted images . the hematoma remained stable without any evidence of expansion when scanned again 8 hours and 24 hours following collagenase injection ( figure 1 ) . in mice with serial mr imaging up to 72 hours , 10 and 21 days , the predominantly low - signal hematoma within the left caudate / putamen in t1- , t2- , and t2 * -weighted images were consistent with deoxyhemoglobin and intracellular methemoglobin after 24 hours . the signal converted to predominantly high intensity at 72 hours to 10 days on t2-weighted rare spin - echo images , consistent with extracellular methemoglobin ( figure 2 ) . the initially hypointense hematoma on t1-weighted images became hyperintense on days 3 and 10 . even on t2 * -weighted images , the signal of the hematoma core became hyperintense at 10 days . on day 21 , a low - signal streak was left at the site of the former space - occupying hematoma on t1- , t2- , and t2 * -weighted images ( figures 3 and 4 ) . table 1 illustrates the mean hematoma sizes on t1- and t2 * -weighted images and the mean edema sizes on t2 on day 1 for nine mice , on day 3 for seven mice , on day 10 for four mice , and for the remainder ( n = 2 ) on day 21 . after final imaging , the equivalent hematoma and edema volumes were compared with the hematoma and edema areas on histology as also shown in table 1 . the spearman correlation coefficient between hematoma volume on t2 * -weighted images and histology was r = 0.61 ( p < 0.09 ) over all times points , thus not reaching statistical significance . the spearman correlation coefficient between hematoma volume on t1 and histology , however , was statistically significant r = 0.7 ( p < 0.04 ) . so was the coefficient for edema volume on t2 and histology on all examination dates with r = 0.73 ( p < 0.03 ) . ongoing research in intracerebral hemorrhage and the prospects of therapy have raised the need for a noninvasive method to investigate ich in rodent models . the quantification of hematoma volume is currently based on spectrophotometry assays [ 8 , 20 ] , histomorphometry on stained brain slices , or computer - assisted outlining of brain slices , thus excluding follow - up measurements during the disease progression and for the evaluation of novel therapeutic interventions . the same accounts for edema volumetry that uses brain water content measurements [ 11 , 16 , 17 ] , blood - brain barrier permeability for evans blue dye on histology , or hemispheric enlargement for quantitation . among the biological imaging techniques , magnetic resonance imaging ( mri ) constitutes an excellent tool for neuroimaging in rats and pigs [ 22 , 23 ] . owing to the variety of knockout genotypes in mice , repeated studies of the same animal are important research tools to study hematoma and edema evolution in a longitudinal fashion . previous mri studies of the mouse brain have either been postmortem imaging of excised specimen with a near - microscopic spatial resolution [ 24 , 25 ] or in vivo studies with a high in - plane resolution at the expense of a much poorer section thickness . at high magnetic fields , the ratio between surface and volume in the head is important because of the presence of high susceptibility effects and poor magnetic field homogeneity in the whole head . even with optimization of the shimming procedure and a reduction in tr , it was not possible to suppress this artifact completely . for t1-weighted sequence , the echo time was minimized to remove any t2 weighting and minimize susceptibility artifacts at air / tissue interfaces . in t2-weighted sequence , a rare factor of 12 increased the effective echo time and reduced the scan time . the echo time in t2 * sequence was optimized to provide adequate signal - to - noise ratio while maximizing the contrast for measurement of hematoma . a review of the literature revealed only two reports [ 13 , 27 ] with murine experimental ich and quantitation of hematoma size on t2 * alone or t2-weighted images . given these extremely limited data on the changes in signal intensities in the course of hematoma resorption and the lack of data for edema formation , we undertook this study to describe the signal characteristics of hematoma resorption and edema evolution in mice over time . in our pilot study , we show for the first time that intracerebral hemorrhages appear hypointense on t1 at high field strength as early as 2 hours after the ictus . although less well demarcated compared to t2 * images , the hematoma remained discernable on t1 in the course of hematoma resorption . hematoma volumes on t1 correlated well with those on histology ( p < 0.04 ) . t2 images approximated edema size well in the acute to chronic phase of edema formation in mice when compared with histology ( p < 0.03 ) . van der weerd et al . had found t2-weighted images useful for quantitation of lesion volume 24 hours after stroke in a mcao model . as our results indicate , t2 * changes do not correlate with hematoma volumes on histology . that might be due to blooming artifacts that render the suitability of t2 * images for quantification of hematoma at this field strength questionable . knight et al . assessed the evolution of intracerebral hemorrhage in rats by mri estimates of t2 relaxation time and hematoma - induced changes in cerebral blood flow and blood - brain barrier permeability over 14 days using a 7-tesla , 20-cm bore magnet . the lesion core and adjacent rim were identified by windowing of t2 values and changed in a consistent manner over time . the mri and histological estimates of tissue loss were well correlated . despite the novel approach for following the temporal and spatial resolutions of intracerebral hemorrhages in mice by high field mr imaging , the high costs for experimental mr imaging at high field are a major drawback to large study groups . the reproducibility of the murine ich model used in our study that was pioneered by clark et al . made it one of the principal models of murine ich [ 11 , 12 , 1517 , 30 ] . theoretical concerns that bacterial collagenase might induce an inflammatory response , independent of that elicited by parenchymal blood , have not been confirmed for the activation of microglia in cell cultures after the addition of collagenase at various concentrations . whether the morphological substrate of what we depict as hyperintensity on t2-weighted images exclusively represents edema , remains questionable . alternate techniques imply the injection of autologous blood into the striatum [ 8 , 10 ] , but lack the pathophysiologic event of vessel rupture . repetitive multimodal mri scanning at 4.7 t in mice with collagenase - induced ich allows studying the individual kinetics of signal intensities underlying hematoma resolution and edema formation in a noninvasive manner . that would allow studying a wide range of therapeutic treatments in a longitudinal fashion in mice .

background and purpose . pilot study to examine the use of t1- , t2- , and t2 * -weighted images for evaluating hematoma size and extent of edema in mouse brain at high field . methods . following collagenase - induced intracerebral hemorrhage , nine mice were imaged at 4.7 t using t1- , t2- , and t2 * -weighted images for hematoma and edema quantitation on days 1 , 3 , 10 , and 21 after surgery . values were compared with morphometric analysis of cryosections at the time of final mr imaging . results . for hematoma quantitation , the spearman correlation coefficient ( r ) between t1 signal change and histology was 0.70 ( p < 0.04 ) compared with r = 0.61 ( p < 0.09 ) for t2*. the extent of perihematomal edema formation on cryosections was well reflected on t2 with r = 0.73 ( p < 0.03 ) . conclusions . within the limits of our pilot study , mr imaging on 4.7 t appears to approximate the temporal changes in hematoma and edema sizes in murine ich well , thus laying the groundwork for longitudinal studies on hematoma resorption and edema formation .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC2099161

the concept of minimal invasive dentistry has evolved as a consequence of an increased understanding of caries and the development of adhesive restorative materials . within this concept , prevention and hard tissue preservation are the primary goals , and dentists are encouraged to prefer a more conservative and biological approach rather than a surgical approach , although the latter is sometimes unavoidable . the atraumatic restorative treatment ( art ) technique is part of a minimal invasive approach and , as such , a technique that meets the specific goals mentioned above . in brief , with art , soft demineralized carious tooth tissue is removed using hand instruments only , followed by restoration of the tooth with an adhesive restorative material , often glass ionomer cement [ 4 , 7 ] . because neither electricity nor running water is required for this treatment approach , art can be applied in almost any setting . although initially developed to provide restorative dental treatment in outreach or rural areas , art or modified art techniques are increasingly introduced into dental clinics in industrialized countries [ 1 , 11 , 14 ] . since its introduction in the mid-1980s these studies served mainly to obtain information on technical aspects of the process , handling characteristics of the restorative material , and on the survival of the restorations . they led to improvement of the technique and to the development of new , more appropriate glass ionomer restoration materials , especially for art purposes . studies focussing on the survival of art restorations have shown that the art approach is very successful in restoring single - surface dentine lesions in the permanent dentition : 3-year survival rates of 7192% have been reported [ 5 , 6 , 9 , 10 , 12 , 20 ] . regarding the survival rates of art restorations in the primary dentition , only a few field studies were performed . they showed acceptable survival rates ( 6596.7% ) for single - surface art restorations , but generally low success rates ( 3176.1% ) for multi - surface art restorations , even with the newer glass ionomer materials [ 2 , 3 , 1113 , 19 , 23 , 24 , 28 ] . although its performance under multisurface conditions is disappointing , art is considered a valuable approach towards the treatment of dental caries . the use of art has resulted in the retention of many teeth that would otherwise have been extracted in a later stage . nevertheless , there still remain some controversies towards the technique , presumably based on the inconsistency in survival results . moreover , a recent study , investigating the influence of dental treatment on the oral health of a surinamese child population , concluded that performing art restorations only , did not contribute significantly to an improvement of the oral health , suggesting that art alone is not a sufficient solution in the battle against dental decay ( van gemert - schriks et al . frencken et al . described comprehensively that art should be part of a basic package of oral care in which prevention and urgent care are also represented . however , within this package , these three components should be geared to one another as much as possible and the individual effects of all three components must be sufficient and beneficial under different circumstances . when the success of either component , particularly art , can not be guaranteed , its contribution in the package should be reduced . thus , the evaluation of art in different countries or communities , among different kinds of caries - risk populations and under diverging conditions remains useful . therefore , the aim of this study is to evaluate the survival of both single- and two - surface art restorations in the primary and permanent dentitions of children from a high - caries population in a field setting on a longitudinal base . it was part of a large - scale project investigating the influence of dental treatment on the oral health of children ( van gemert - schriks et al . , submitted , 2007 ) . within the scope of that particular project , 380 6-year - old children were divided randomly among four different treatment groups . material presented in the current article concerns only those children who received restorative treatment , according to the art method , either in their primary or permanent dentitions . the restorative treatments were performed in accordance with the art guidelines [ 4 , 7 ] and took place in empty classrooms where four children were treated at the same time . ketac - molar ( 3m - espe ) was used as the restorative material of choice . the treatments were carried out by four dutch dentists who were trained in art during a 1-week art course and by using art in children from their own practices , for a period of 3 months , before the start of the treatment phase of the study . they were assisted by six surinamese health care assistants from the medical mission who completed an art course supplemented with some basic dental knowledge . the dentists were asked to note any contamination with blood and/or saliva during the restoration of the cavity . furthermore , the presence or absence of adjacent teeth was noted . during the treatment , one of the authors ( mgs ) , who was not involved in the treatment phase , observed and classified the overall behavior of the child , based on a modified venham scale [ 23 , 29 ] . before the study , this observer was trained in using the venham behavior scale by scoring 42 videotapes of children in a dental situation . this comparison resulted in a cohen s kappa of 0.87 , implying an excellent agreement . the children were revisited for evaluation of the art restorations 6 months ( t1 ) , 1 year ( t2 ) , 2 years ( t3 ) , and 3 years ( t4 ) after the initial treatment . the same author and dentist mentioned above ( mgs ) , evaluated the restorations according to the art criteria ( table 1 ) using a cpitn probe , a mouth mirror and a headlamp . before the study , this person was calibrated against a gold standard ( kappa 0.94 ) . this gold standard was achieved by the consensus of two experienced dentists during the assessment of 24 extracted molars with art restorations . restorations scored code 00 or 10 were considered successful , codes 1140 were classified as failures , and codes 5090 were assigned in case the tooth was unavailable for evaluation . if a tooth or restoration showed multiple defects , a marginal defect dominated an over- or underfilled restoration ( 10 , 11 > 12 , 13 ) , secondary caries dominated a marginal defect ( 20 , 21 > 10 , 11 ) , absence of a restoration dominated secondary caries ( 30 > 20 , 21 ) , and an overfilled cavity dominated an underfilled cavity ( 13 > 12 ) . table 1evaluation criteria for the art restorationscodeevaluation characteristics00restoration present , correct10restoration present , slight marginal defect / wear of surface ( < 0.5 mm ) . no repair needed.11restoration present , gross marginal defect / wear of surface ( > 0.5 mm ) . repair needed.20secondary caries , discoloration in depth , surface hard and intact , caries within dentin . repair needed.40inflammation of the pulp ; signs of dentogenic infection ( abscesses , fistulae , pain complaints ) . extraction needed.50tooth not present because of extraction60tooth not present because of shedding70tooth not present because of extraction or shedding90patient not present evaluation criteria for the art restorations statistical analyses were performed using spss for windows , version 12.0.1 ( spss , chicago , usa ) . meier survival analyses were performed on the censored data of both single- and two - surface restorations . statistical analyses were performed using spss for windows , version 12.0.1 ( spss , chicago , usa ) . meier survival analyses were performed on the censored data of both single- and two - surface restorations . as stated in the materials and methods section , the children in this study were derived from a larger study population of children participating in another project . the overall caries prevalence , expressed in terms of decayed , missing and filled surfaces ( dmfs ) among that group of children , was 11.51 ( sd 10.5 ; range 053 ) in the primary dentition and 0.20 ( sd 0.62 ; range 05 ) in the permanent dentition . according to the standards of the world health organisation , this denotes a high - caries child population based on the caries prevalence in the primary dentition . within the larger group , 194 children ( mean age 6.09 0.48 years ) received art restorations in either their primary or permanent teeth , or both . their baseline caries prevalence was 12.75 ( sd 9.88 ; range 053 ) in the primary dentition and 0.23 ( sd 0.67 ; range 05 ) in the permanent dentition . at baseline ( t0 ) , 475 art restorations were placed in the primary dentition ( mainly first and second molars ) and 54 in the first permanent molars ( predominantly mandibular ) . a mann whitney u test showed that children who received two - surface restorations scored higher on the venham behavior scale ( p = 0.005 ) than children that received single - surface restorations , in the primary dentition . furthermore , dentists reported more contamination ( chi - square = 25.02 , df = 1 , p < 0.001 ) when placing two - surface restorations than single - surface restorations . table 2baseline data for the art restorations primary dentitionpermanent dentitionnumber of filled surfaces121number of restorations13334254number of children ( n)6114734mean number of restorations per child ( sd ; range)3.50 ( 1.61 ; 17)3.64 ( 1.73 ; 18)2.07 ( 0.97 ; 14)dentist143 ( 32.3%)74 ( 21.6%)12 ( 22.2%)241 ( 30.8%)84 ( 24.6%)16 ( 29.6%)334 ( 25.6%)89 ( 26.0%)7 ( 13.0%)415 ( 11.3%)95 ( 27.8%)19 ( 35.2%)adjacent tooth presentyes117 ( 88.0%)303 ( 88.6%)45 ( 83.3%)no16 ( 12.0%)39 ( 11.4%)9 ( 16.7%)contamination blood / salivayes13 ( 9.8%)110 ( 32.2%)5 ( 9.3%)no120 ( 90.2%)232 ( 67.8%)49 ( 90.7%)venham behavior score050 ( 37.6%)78 ( 22.8%)8 ( 14.8%)144 ( 33.1%)137 ( 40.1%)27 ( 50.0%)226 ( 19.5%)82 ( 24.0%)13 ( 24.1%)313 ( 9.8%)33 ( 9.6%)6 ( 11.1%)412 ( 3.5%)5sd standard deviationstatistical significant difference at p = 0.005 . baseline data for the art restorations sd standard deviation statistical significant difference at p = 0.005 . the lost - to - follow - up percentage of the restorations originally placed was 4.63% . after 3 years , the cumulative survival of the single - surface art restorations in the primary dentition was 43.4% ( standard error ( se ) 10.9% ) . for the two - surface restorations , a cumulative survival of 12.2% ( se 2.99% ) was observed . 1a and b. the cumulative survival of the single - surface art restorations in the permanent dentition was 29.6% ( se 8.2% ) after 3 years ( fig . 2 ) . 2survival curve single - surface art restorations , permanent dentition a survival curve single surface art restorations , primary dentition . b survival curve multisurface art restorations , primary dentition survival curve single - surface art restorations , permanent dentition table 3 represents the failure characteristics for the restorations in both primary and permanent dentitions at 3 years . the main failure characteristics of both single- and two - surface art restorations in the primary dentition were gross marginal defects ( score 11 ) and total or partial losses ( score 30 ) . for restorations in the permanent dentition , the main failure characteristics were secondary caries ( score 21 ) and gross marginal defects ( score 11 ) . table 3failure characteristics for the art restorations at 3 years primary dentitionpermanent dentition1 surface2 surface1 surfacerestorations baseline ( n)13334254failures ( n)4225132failure scoregross marginal defect ( 11)21 ( 15.8%)86 ( 25.1%)13 ( 24.1%)restoration present , underfilled ( 12)1 ( 0.8%)9 ( 2.6%)restoration present , overfilled ( 13)2 ( 1.5%)11 ( 3.2%)2 ( 3.7%)sec . caries , discoloration ( 20)sec . caries , surface defect ( 21)5 ( 3.8%)1 ( 0.3%)14 ( 25.9%)total or partial loss ( 30)13 ( 9.8%)120 ( 35.1%)3 ( 5.6%)pulpal inflammation ( 40)24 ( 7.0%)restoration missing , extracted ( 50)scores 6090 were not included ( censored data ) failure characteristics for the art restorations at 3 years scores 6090 were not included ( censored data ) a log - rank test indicated that there were no statistically significant differences in survival times between the four dentists regarding single - surface restorations in both primary and permanent teeth . however , regarding the two - surface restorations in the primary dentition , statistically significant differences between the four dentists appeared ( log - rank statistic 11.7 , df = 3 , p = 0.009 ) . 3survival curves per dentist , multi - surface art restorations primary dentition survival curves per dentist , multi - surface art restorations primary dentition to detect any confounding variables on the survival of the art restorations , a cox regression analysis was performed . no significant relation could be found , indicating that neither the presence or absence of an adjacent tooth , nor contamination with blood and/or saliva , nor the behavior of the child during the restorative phase of the treatment had an influence on the 3-year survival of the restorations in the primary dentition . neither of these variables had an effect on the survival rates in the permanent dentition , except for the presence of adjacent teeth . restorations in teeth where no adjacent tooth was present were found to be more likely to fail ( hazard ratio = 6.53 , 95% ci 2.6616.02 , p < 0.001 ) . in contrast with other studies , the results of this study show extremely low survival rates for both single- and two - surface art restorations in the primary and permanent dentitions . an operator effect was observed for two - surface restorations only . neither the behavior of the child during restoration , and the number of restorations per child , nor the contamination of preparations with blood or saliva had a significant influence on the survival of the restorations in this study . this field study was performed correctly and the statistical power was sufficiently high to detect at least medium effects . however , because it was part of a large - scale randomized controlled clinical trial , no comprehensive criteria were formulated beforehand regarding , for example , the number of restorations per patient , and the location and the size of the cavities . this aspect is inherent to many cohort studies and it does not imply an inferior study quality , but it limits a meaningful comparison with other survival studies . although all possible efforts were exercised to trace the participating children over the evaluation period , 22 restorations ( 4.63% , eight children ) , all in primary molars , could not be evaluated at any of the recall visits . either the children did not show up , or the teeth concerned had exfoliated before the first evaluation . these restorations were regarded as missing data and , therefore , excluded from further analysis . twenty - six restorations ( 5.47% , 21 children ) were lost for evaluation because the teeth either exfoliated or the child moved to another district during the course of the study , but after the first evaluation . these restorations ( scores 6090 ) were treated as censored data and not as true failures because they survived up to a certain moment . many causative factors could be suggested that might explain the failure of the art restorations , such as secondary caries , cervical margin gaps , material properties , and field conditions ( outside temperature , atmospheric humidity ) . however , many other art studies face these or comparable problems and , therefore , these factors can not sufficiently explain the extremely low survival rates found in this particular study . the operator difference for the survival rates of the two - surface restorations was not unique , and not a sufficient explanation for the disappointing survival results . operator effects are often found in art studies [ 4 , 9 , 15 , 21 , 26 ] and , as in every profession , there will always be individual differences in technical skills . the finding that the absence of an adjacent tooth was related to a lower 3-year survival of single - surface art restorations in permanent molars could not be explained . one can only speculate about possible reasons for this relationship , such as that these freestanding molars experience larger occlusal forces . a possible influence of the relatively high caries prevalence on the survival of the restorations could be hypothesized , but is very doubtful . a study in indonesia , where the child population exhibited a much higher caries prevalence , also found disappointing survival rates for two - surface art restorations , but these rates were not as extreme as those found in the current study . the survival rates for single - surface art restorations , derived from other earlier cited studies , were all very promising regardless of the caries profile of the study populations . furthermore , no effect on the survival rates of the restorations was found when the number of restorations per child was included in the analysis . the art protocol prescribes not to eat or drink within at least 1 h after the completion of the restorative treatment . the children in the current study were not supervised after they received restorative treatment and consequently , their food intake could not be controlled . other patient - related factors that may influence the survival of the restorations are the behavior and saliva flow of the child . the survival of the art restorations in this study was analyzed at the restoration level . this method requires independency of the restoration data and , with respect to the mentioned patient - related possible bias , this assumption could not be guaranteed . to control for this lack of independency , the survival analyses also were performed at the patient level , including only one randomly selected restoration per child . the predominant failure characteristics for both single- and two - surface art restorations in the primary dentition were gross marginal defects and total or partial losses . this agrees with previous studies concerning the survival of art restorations in the primary dentition [ 6 , 13 , 25 , 26 ] . gross marginal defects could be induced by occlusal forces or insufficient wear resistance of the restorative material . ketac - molar was specifically developed for art purposes , and it has shown excellent results for posterior restorations in the primary dentition [ 16 , 22 ] . glass ionomer restorations can be dislodged for a number of reasons , such as insufficient cleaning and conditioning of the cavity , and improper mixing of the material . however , all dentists and chair - side assistants followed the art guidelines and the manufacturer s instructions as much as possible under the given circumstances . the main reasons for failure of the single - surface restorations in the permanent dentition were gross marginal defects and secondary caries . this latter finding is somewhat surprising and contrasts with earlier art studies [ 5 , 6 , 10 , 26 ] . glass ionomer cement has been the restorative material of choice for the art technique , based mainly on its fluoride releasing and , thus , caries - preventive properties . many studies underline these characteristics of glass ionomer [ 18 , 26 , 30 , 31 ] . circumstances that were not recognized as possible interfering factors at the start of the study might have played an important role , including cultural and seasonal dietary influences . people living in the rainforest of suriname eat seasonal fruits such as mangos and fruits of the fiber palm ( awarra ) . in particular , the latter may influence the survival of the restorations , given the frequency and method in which they are consumed . the authors have seen unusual wear patterns , also in adult dentitions , which might have been caused by excessive consumption of awarras . a possible causality between these dietary habits and the survival of the art restorations can only be disclosed by future controlled studies . although previous studies have suggested that art should not be considered as a routine procedure to restore multisurface cavities [ 13 , 24 ] , based on the results of this study , even the art restoration of single - surface cavities might be reconsidered . the uncertain predictability for the success of art may introduce further discussion about alternative treatment strategies , especially in those situations where choices have to be made with respect to a well - balanced , cost - effective package of basic oral health care . to gain insight into factors determining the cumulative success rate of art restorations , future studies should focus in more detail on variables that could possibly contribute to the failure of restorations .

the aim of this study was to evaluate the survival of single- and two - surface atraumatic restorative treatment ( art ) restorations in the primary and permanent dentitions of children from a high - caries population , in a field setting . the study was conducted in the rainforest of suriname , south america . art restorations , made by four dutch dentists , were evaluated after 6 months , 1 , 2 , and 3 years . four hundred seventy - five art restorations were placed in the primary dentition and 54 in first permanent molars of 194 children ( mean age 6.09 0.48 years ) . three - year cumulative survivals of single- and two - surface art restorations in the primary dentition were 43.4 and 12.2% , respectively . main failure characteristics were gross marginal defects and total or partial losses . three - year cumulative survival for single - surface art restorations in the permanent dentition was 29.6% . main failure characteristics were secondary caries and gross marginal defects . an operator effect was found only for two - surface restorations . the results show extremely low survival rates for single- and two - surface art restorations in the primary and permanent dentitions . the variable success for art may initiate further discussion about alternative treatment strategies , especially in those situations where choices have to be made with respect to a well - balanced , cost - effective package of basic oral health care .

Introduction Materials and methods Statistical analysis Results Discussion Conclusion

PMC4853568

endovascular treatment of large vessel acute ischemic strokes in appropriately selected patients has been endorsed as evidence - based care.1 2 the next steps in advancing this therapy are to develop systems of care that can be divided into prehospital and intra - hospital pathways . other time - dependent treatments , such as trauma and acute myocardial infarction , provide a valuable model for endovascular stroke therapy . the value of an initial golden hour in improving outcomes has been shown for treatment of trauma3 to neonatal resuscitation.4 early reperfusion for large vessel strokes is a critical determinant of endovascular therapy outcomes.5 6 emergency medicine publications provide useful insight into developing checklists and protocols geared towards seamless resuscitation of injured or sick patients.7 these pit - crew-type protocols clearly define the role of each team member , allowing for synchronized , parallel delivery of care . our center has performed endovascular stroke interventions for the past 15 years and participated in several clinical trials . we had noticed a fall in the volume of patients in the past 3 years as an after effect of negative stroke trials8 9 and also because we had decided to offer stroke interventions only in the setting of a randomized clinical trial . , we instituted a quality improvement ( qi ) process to reduce our door - to - needle times and develop a process that might consistently reduce times to treatments at all hours of the day and all days of the week . the qi process was based on six - sigma which , although used by corporations for years , has only recently been adopted in medicine.10 this paper provides the results of those efforts , comparing the treatment times before and after implementation of the qi process . to determine whether our times for different steps in endovascular stroke care improved as a result of the qi process ; the null hypothesis stating that there would be no difference in these times . our setting was a rural , tertiary level , academic medical center with almost 700 beds , which is also the regional level-1 trauma center . prospectively recorded treatment times before and after the qi implementation provided the data for this analysis . the pre - qi treatment times included all endovascular patients from 2011 to 2014 who had presented to our emergency room ( er ) . since the project was specifically targeted at improving efficiency between the er and interventional neuroradiology ( inr ) , the following patients were excluded : in - house patients undergoing an intervention for stroke , patients undergoing another procedure in the hospital with a stroke and patients treated with unknown symptom onset . the post - qi treatment times were those for all patients undergoing endovascular therapy between january 2015 and october 2015 . the following treatment times were compared : er to imaging ( ct ) ( er ct)imaging to inr lab ( ct lab)inr lab to groin puncture ( lab puncture)imaging to needle ( groin puncture ) ( ct puncture)door to needle ( er puncture ) . er to imaging ( ct ) ( er ct ) imaging to inr lab ( ct lab ) inr lab to groin puncture ( lab puncture ) imaging to needle ( groin puncture ) ( ct puncture ) door to needle ( er puncture ) . the qi process was started in the fall of 2014 and incrementally implemented over almost 3 months . the impetus to initiate the process was treatment delays in stroke interventions , inconsistencies in times and care between working hours and on - call hours , ad hoc roles of different members , and suboptimal handover of patients between different services . the qi team was led by two six - sigma trained engineers and comprised physicians , allied staff , and management from interventional neuroradiology , neurology , emergency medicine , and anesthesia . meetings were held to develop a baseline understanding of the existing practice , followed by observance of a final protocol was signed off towards the end of 2014 and in 2015 all cases followed the updated protocol . important features of the protocol ( figure 1 ) included identification of key personnel in the team , definition of a clear role for every member , and emphasis on parallel processing of assigned tasks . the protocol contained explicit details , such as how a nurse enters the hospital , where a technician would stand , and the position of the detectors in the inr laboratory . the protocol focused on the three main time - points along the stroke pathway : er ct , ct lab , and lab puncture . an overview of the protocol is presented along a timeline from patient arrival to arterial puncture . er , emergency room ; geta , general endotracheal anesthesia , off hours treatment : stroke intervention performed before 7:00 or after 17:00 or at the weekend ; icu , intensive care unit ; inr , neurointerventionalist ; lcfa , left common femoral artery ; lsn , last seen normal ; lvo , large vessel occlusion ; nihss , national institutes of health stroke scale ; onset - er , time from symptom onset to er arrival ; rcfa , right common femoral artery ; rt - pa , recombinant tissue plasminogen activator . the emergency medical services evaluate stroke patients using the cincinnati prehospital stroke scale11 and alert the stroke team ( neurology , radiology , emergency medicine , and laboratory staff ) by paging the group . on arrival , the er staff and on - call neurology residents evaluate the patient to confirm the stroke diagnosis . at least one iv line is placed while the patient is in the er for the purpose of drawing laboratories and for contrast administration . after a point - of - care creatinine test ( istat ; abbot laboratories , abbott park , illinois ) , the stroke patient is transferred to the ct scanner by a dedicated stroke nurse and the neurology team . the ct technologists already alerted by the emergency medical services have the scanner ready for the patient . the radiology resident meets the stroke team in the scanner control room for live review of imaging for all patients presenting within 6 h with a national institutes of health stroke scale ( nihss ) score 6 . before implementation of the qi process any ct scanner was used , but after the qi process a designated scanner ( aquilion - one , toshiba america medical systems , tustin , california , usa ) was used for all stroke patients . the scanner is equipped with 3200.5 mm detector rows covering 16 cm of volume per rotation , allowing for a uniform protocol comprising non - contrast ct ( ncct ) , volumetric ct perfusion ( ctp ) imaging , and ct angiography ( cta ) . this switch was crucial in allowing earlier detection of a large vessel occlusion ( lvo ) and alerting the attending neurointerventionalist who could then review the ncct and cta ( onsite or offsite ) , while ctp imaging was being carried out and processed . another critical component was installation of an image router to simultaneously receive and disseminate images while they were being acquired . this reduced the scanner to transfer time of all images from 12 min to just over 5 min . the ct stroke scan is performed with a gantry speed of 3 rotations / s . after the ncct , the cta is initiated from the aortic arch to the cranial vertex with an injection of 4060 ml of optiray 350 ( covidien , hazelwood , missouri , usa ) through an 1820 g antecubital iv line at a rate of 45 ml / s , followed by a similar volume of saline chaser . images acquired are automatically sent via the router for immediate review . a volumetric whole brain , time - resolved ctp sequence follows . the imaging router enables transfer of 6080 ( 19320 ) images to a picture archiving and communication system and the perfusion post - processing software ( vital images , minnetonka , minnesota , usa ) . iv recombinant tissue plasminogen activator ( rt - pa ; activase , genentech inc , san francisco , california , usa ) is administered if indicated after exclusion of hemorrhage on the ncct . a collaborative decision about endovascular treatment is made based on clinical presentation , comorbidities , and imaging . if the decision is to treat , the radiology resident places a single order set . this was designed to simultaneously trigger multiple tasks with one click of the button namely , anesthesia ( in our view , use of anesthesia makes the procedure safer ) , intensive care unit ( icu ) bed request , inr order set , foley placement , peripheral iv line , etc . a single paging system was implemented allowing the resident to contact the neurointerventional team ( one nurse , and two technicians ) with one phone call . medcom provides the relevant patient information and location in the er . if within 5 min the staff has not responded a repeat page is sent . the system also documents all paging times , which can be reviewed for qi purposes . the radiology resident places a preoperative note in the chart documenting the decision to treat . the neurology resident 's duty running in parallel with this is to notify the icu service of the eventual admission of the stroke patient . the inr nurse arrives through the er , checks the patient , and alerts the er staff to initiate transfer to the inr suite . the minimum staffing requirement for all neurovascular procedures is set at two technicians and one nurse . one technician is scrubbed and one floats . during working hours , a third technician is available for complex elective cases . when two emergent cases occur simultaneously after hours , the technicians are split and the backup nurse is called in . improvement in groin puncture times upon patient arrival in the angiography suite required planning to ensure parallel preparation of patient , tray , and anesthesia . the inr technicians prepare the room for patient arrival by opening pre - packaged stroke trays and devices as indicated by the inr attending . a separate stroke cart is stocked with the catheters , wires , thrombectomy devices , and syringes to hold everything in one place for this purpose . the biplane detectors are positioned in such a way as to facilitate patient transfer to the angiography table . once the patient is placed on the table , a coordinated effort is made between the anesthestist working at the head of the patient and left arm ( which is positioned out ) , and one of the inr technicians working at the groins for sterile preparation . the inr nurse assists the anesthesiologist , if required , prepares the flushes , and performs documentation . an 8 fr right common femoral arterial sheath is placed as soon as the groin is prepped . if by that time a radial arterial line has not been established by the anesthesiologist , a second 4 fr sheath is immediately placed in the left common femoral artery for invasive blood pressure monitoring . the 8 fr sheath is removed at the end of the procedure and , if placed , the second 4 fr sheath is sutured in place to be used in the icu . an anesthesia cart and ventilator is permanently stationed in each of two adjacent biplane angiography suites , allowing performance of parallel emergent cases . small coordinated steps in the patient preparation process were designed to emulate defined roles and parallel tasks seen in trauma resuscitation . inr : neurointerventionalist , t1 : technician-1 , t2 : technician-2 , t3 : technician-3 , n1 : nurse-1 , n2 : nurse-2 , a1 : anesthesiologist / certified registered nurse anesthetist ( crna)-1 , a2 : anesthesiologist / crna-2 . during the patient preparation stage ( a ) , t1 sets up the procedure trays and prepares the devices and catheters . t2 prepares the patient and helps the attending technician , who punctures the right femoral artery and typically places an 8 fr sheath . the patient 's left arm is extended out on an arm board for simultaneous access to anesthesia for placement of lines and administration of drugs . if there is no radial arterial access by the time the right femoral sheath is placed , the inr punctures the left femoral artery and places a 4 fr sheath for invasive blood pressure monitoring . even though it is possible to obtain arterial tracing via the 8 fr right femoral sheath , placement of the 4 fr sheath allows removal of the larger right femoral sheath at the end of the procedure . the patient is transferred to the intensive care unit with the 4 fr sheath in place for pressure monitoring . the nurse takes a report , prepares the continuous flush lines , assists the anesthesiologist , and charts all times . the a - plane detector is stationed in such a way as to allow easy positioning over the groin in case fluoroscopy is required . for the interventional stage ( b ) , t2 scrubs up and functions as the float . one anesthesiologist ( a1 ) stays to cover the case , assisted by the nurse . this setup with stocked anesthesia cart is duplicated in an immediately adjacent second interventional biplane room . during working hours an additional technician ( t3 ) and nurse ( n2 ) if two simultaneous emergent cases occur after hours , the technicians split and the backup nurse ( n2 ) is called in . the patient demographics and stroke severity the significance of simple bivariate associations was assessed using the fisher exact test for categorical variables . wilk w test demonstrated a non - normal distribution of the continuous time - point data . non - parametric wilcoxon rank sum tests were thus performed to compare the time - point means all statistical analysis was performed using the jmp pro 12.0.1 software package ( sas institute inc , cary , north carolina , usa ) . to determine whether our times for different steps in endovascular stroke care improved as a result of the qi process ; the null hypothesis stating that there would be no difference in these times . our setting was a rural , tertiary level , academic medical center with almost 700 beds , which is also the regional level-1 trauma center . prospectively recorded treatment times before and after the qi implementation provided the data for this analysis . the pre - qi treatment times included all endovascular patients from 2011 to 2014 who had presented to our emergency room ( er ) . since the project was specifically targeted at improving efficiency between the er and interventional neuroradiology ( inr ) , the following patients were excluded : in - house patients undergoing an intervention for stroke , patients undergoing another procedure in the hospital with a stroke and patients treated with unknown symptom onset . the post - qi treatment times were those for all patients undergoing endovascular therapy between january 2015 and october 2015 . the following treatment times were compared : er to imaging ( ct ) ( er ct)imaging to inr lab ( ct lab)inr lab to groin puncture ( lab puncture)imaging to needle ( groin puncture ) ( ct puncture)door to needle ( er puncture ) . er to imaging ( ct ) ( er ct ) imaging to inr lab ( ct lab ) inr lab to groin puncture ( lab puncture ) imaging to needle ( groin puncture ) ( ct puncture ) door to needle ( er puncture ) . the qi process was started in the fall of 2014 and incrementally implemented over almost 3 months . the impetus to initiate the process was treatment delays in stroke interventions , inconsistencies in times and care between working hours and on - call hours , ad hoc roles of different members , and suboptimal handover of patients between different services . the qi team was led by two six - sigma trained engineers and comprised physicians , allied staff , and management from interventional neuroradiology , neurology , emergency medicine , and anesthesia . meetings were held to develop a baseline understanding of the existing practice , followed by observance of a final protocol was signed off towards the end of 2014 and in 2015 all cases followed the updated protocol . important features of the protocol ( figure 1 ) included identification of key personnel in the team , definition of a clear role for every member , and emphasis on parallel processing of assigned tasks . the protocol contained explicit details , such as how a nurse enters the hospital , where a technician would stand , and the position of the detectors in the inr laboratory . the protocol focused on the three main time - points along the stroke pathway : er ct , ct lab , and lab puncture . an overview of the protocol is presented along a timeline from patient arrival to arterial puncture . er , emergency room ; geta , general endotracheal anesthesia , off hours treatment : stroke intervention performed before 7:00 or after 17:00 or at the weekend ; icu , intensive care unit ; inr , neurointerventionalist ; lcfa , left common femoral artery ; lsn , last seen normal ; lvo , large vessel occlusion ; nihss , national institutes of health stroke scale ; onset - er , time from symptom onset to er arrival ; rcfa , right common femoral artery ; rt - pa , recombinant tissue plasminogen activator . the emergency medical services evaluate stroke patients using the cincinnati prehospital stroke scale11 and alert the stroke team ( neurology , radiology , emergency medicine , and laboratory staff ) by paging the group . on arrival , the er staff and on - call neurology residents evaluate the patient to confirm the stroke diagnosis . at least one iv line is placed while the patient is in the er for the purpose of drawing laboratories and for contrast administration . after a point - of - care creatinine test ( istat ; abbot laboratories , abbott park , illinois ) , the stroke patient is transferred to the ct scanner by a dedicated stroke nurse and the neurology team . the ct technologists already alerted by the emergency medical services have the scanner ready for the patient . the radiology resident meets the stroke team in the scanner control room for live review of imaging for all patients presenting within 6 h with a national institutes of health stroke scale ( nihss ) score 6 . before implementation of the qi process any ct scanner was used , but after the qi process a designated scanner ( aquilion - one , toshiba america medical systems , tustin , california , usa ) was used for all stroke patients . the scanner is equipped with 3200.5 mm detector rows covering 16 cm of volume per rotation , allowing for a uniform protocol comprising non - contrast ct ( ncct ) , volumetric ct perfusion ( ctp ) imaging , and ct angiography ( cta ) . . this switch was crucial in allowing earlier detection of a large vessel occlusion ( lvo ) and alerting the attending neurointerventionalist who could then review the ncct and cta ( onsite or offsite ) , while ctp imaging was being carried out and processed . another critical component was installation of an image router to simultaneously receive and disseminate images while they were being acquired . this reduced the scanner to transfer time of all images from 12 min to just over 5 min . the ct stroke scan is performed with a gantry speed of 3 rotations / s . after the ncct , the cta is initiated from the aortic arch to the cranial vertex with an injection of 4060 ml of optiray 350 ( covidien , hazelwood , missouri , usa ) through an 1820 g antecubital iv line at a rate of 45 ml / s , followed by a similar volume of saline chaser . images acquired are automatically sent via the router for immediate review . a volumetric whole brain , time - resolved ctp sequence follows . the imaging router enables transfer of 6080 ( 19320 ) images to a picture archiving and communication system and the perfusion post - processing software ( vital images , minnetonka , minnesota , usa ) . iv recombinant tissue plasminogen activator ( rt - pa ; activase , genentech inc , san francisco , california , usa ) is administered if indicated after exclusion of hemorrhage on the ncct . a collaborative decision about endovascular treatment is made based on clinical presentation , comorbidities , and imaging . if the decision is to treat , the radiology resident places a single order set . this was designed to simultaneously trigger multiple tasks with one click of the button namely , anesthesia ( in our view , use of anesthesia makes the procedure safer ) , intensive care unit ( icu ) bed request , inr order set , foley placement , peripheral iv line , etc . a single paging system was implemented allowing the resident to contact the neurointerventional team ( one nurse , and two technicians ) with one phone call . medcom provides the relevant patient information and location in the er . if within 5 min the staff has not responded a repeat page is sent . the system also documents all paging times , which can be reviewed for qi purposes . the radiology resident places a preoperative note in the chart documenting the decision to treat . the neurology resident 's duty running in parallel with this is to notify the icu service of the eventual admission of the stroke patient . the inr nurse arrives through the er , checks the patient , and alerts the er staff to initiate transfer to the inr suite . the minimum staffing requirement for all neurovascular procedures is set at two technicians and one nurse . one technician is scrubbed and one floats . during working hours , a third technician is available for complex elective cases . when two emergent cases occur simultaneously after hours , the technicians are split and the backup nurse is called in . improvement in groin puncture times upon patient arrival in the angiography suite required planning to ensure parallel preparation of patient , tray , and anesthesia . the inr technicians prepare the room for patient arrival by opening pre - packaged stroke trays and devices as indicated by the inr attending . a separate stroke cart is stocked with the catheters , wires , thrombectomy devices , and syringes to hold everything in one place for this purpose . the biplane detectors are positioned in such a way as to facilitate patient transfer to the angiography table . once the patient is placed on the table , a coordinated effort is made between the anesthestist working at the head of the patient and left arm ( which is positioned out ) , and one of the inr technicians working at the groins for sterile preparation . the inr nurse assists the anesthesiologist , if required , prepares the flushes , and performs documentation . an 8 fr right common femoral arterial sheath is placed as soon as the groin is prepped . if by that time a radial arterial line has not been established by the anesthesiologist , a second 4 fr sheath is immediately placed in the left common femoral artery for invasive blood pressure monitoring . the 8 fr sheath is removed at the end of the procedure and , if placed , the second 4 fr sheath is sutured in place to be used in the icu . an anesthesia cart and ventilator is permanently stationed in each of two adjacent biplane angiography suites , allowing performance of parallel emergent cases . small coordinated steps in the patient preparation process were designed to emulate defined roles and parallel tasks seen in trauma resuscitation . inr : neurointerventionalist , t1 : technician-1 , t2 : technician-2 , t3 : technician-3 , n1 : nurse-1 , n2 : nurse-2 , a1 : anesthesiologist / certified registered nurse anesthetist ( crna)-1 , a2 : anesthesiologist / crna-2 . during the patient preparation stage ( a ) t2 prepares the patient and helps the attending technician , who punctures the right femoral artery and typically places an 8 fr sheath . the patient 's left arm is extended out on an arm board for simultaneous access to anesthesia for placement of lines and administration of drugs . if there is no radial arterial access by the time the right femoral sheath is placed , the inr punctures the left femoral artery and places a 4 fr sheath for invasive blood pressure monitoring . even though it is possible to obtain arterial tracing via the 8 fr right femoral sheath , placement of the 4 fr sheath allows removal of the larger right femoral sheath at the end of the procedure . the patient is transferred to the intensive care unit with the 4 fr sheath in place for pressure monitoring . the nurse takes a report , prepares the continuous flush lines , assists the anesthesiologist , and charts all times . the a - plane detector is stationed in such a way as to allow easy positioning over the groin in case fluoroscopy is required . for the interventional stage ( b ) , one anesthesiologist ( a1 ) stays to cover the case , assisted by the nurse . this setup with stocked anesthesia cart is duplicated in an immediately adjacent second interventional biplane room . during working hours an additional technician ( t3 ) and nurse ( n2 ) if two simultaneous emergent cases occur after hours , the technicians split and the backup nurse ( n2 ) is called in . the significance of simple bivariate associations was assessed using the fisher exact test for categorical variables . a shapiro wilk w test demonstrated a non - normal distribution of the continuous time - point data . non - parametric wilcoxon rank sum tests were thus performed to compare the time - point means all statistical analysis was performed using the jmp pro 12.0.1 software package ( sas institute inc , cary , north carolina , usa ) . before ( n=64 ) and after ( n=30 ) group based on the implementation of the qi process . there were no differences in baseline demographics , stroke severity , symptom onset , comorbidities , and the use of iv rt - pa or general endotracheal anesthesia ( geta ) between the two groups ( table 1 ) . a comparison of the time intervals showed a significant reduction in times in the after qi implementation group across all parameters ( figure 3 ) . the time intervals were separately compared for interventions performed during working hours that is , 7:00 to 17:00 on weekdays ( table 2 ) , and for off - hours and at weekends ( table 3 ) . there was significant reduction in times for both working hours and off - hours interventions . we found no correlation between age , nihss , onset to er or any of the comorbidities and the door - to - needle time . we also looked at the impact of geta on the patient preparation and puncture time after the implementation of the qi process . the mean time from inr room arrival to groin puncture was 15 ( 4 ) min in patients who received geta versus 15 ( 3 ) min in patients treated without geta ( p=0.87 ) , indicating that geta caused no delays . comparison of baseline demographics , comorbidities and treatment variables afib , atrial fibrillation ; dm , diabetes mellitus ; er , emergency room ; geta , general endotracheal anesthesia , off hours treatment : stroke intervention performed before 7:00 or after 17:00 or at the weekend ; hpl , hyperlipidemia ; htn , hypertension ; nihss , national institutes of health stroke scale ; onset - er , time from symptom onset to er arrival ; rt - pa , recombinant tissue plasminogen activator ; smk , smoking . comparison of time parameters during working hours on weekdays er , emergency room ; qi , quality improvement . comparison of time parameters after hours or on weekends er , emergency room ; qi , quality improvement . the six - sigma process , developed at motorola in the 1980s because quality was lagging behind that of japanese companies , was adopted by many corporations as a qi practice . however , healthcare is neither of those and preventable errors cause 44 00098 000 deaths a year.12 six - sigma has been applied in medicine to improve many processes from catheter - related10 and surgical - site infections13 to er,14 surgery,15 and behavioral health systems.16 six - sigma achieves higher levels of quality by understanding and improving a process and decreasing , if not eliminating , variability . control.17 each step in the dmaic chain is fundamental to the success of the qi process.18 in our setting , the process of endovascular stroke therapy required improvement of treatment times . the first step was to define specific goals for example , door - to - needle time of no more than 120 min , split into key intervals such as er ct , ct lab , and lab puncture . different members of the team were clearly identified for each stage and unambiguous roles assigned to each of them . the process was refined over a period of almost 3 months at weekly meetings , where all stroke interventions performed during that week were dissected , with iterations based on feedback from all involved . the performance of the different subprocesses and of the overall process was measured and incrementally improved . our protocols mandated that processes should be performed simultaneously and not sequentially . for example , once it is decided to proceed with an intervention , the radiology and neurology residents perform their tasks independently and concurrently . likewise , once the patient is in the inr suite , the nurse , the technicians , the attending physician , and the anesthesia team move at the same time . furthermore , this reduction in time is consistent regardless of the type of anesthesia used . our overall door - to - needle times are now at just over 90 min and the reduction in times is significant for both working hours ( 7:0017:00 on weekdays ) and off - hours interventions ( tables 2 and 3 ) . our goal is to reduce this to 60 min . this may require the presence of an in - house interventional team , which could bring down the technicians / nurses response time from 30 min to < 5 min . that alone will allow treatment to start within an hour of patient arrival . we have significantly reduced our times for image acquisition , processing , and dissemination through a separate informatics project and it now takes about 10 min from placing the patient on the table to display of images ; this includes ncct , cta , and ctp . by eliminating cta and ctp we will gain , at most , 58 min . we do not think it is worth losing the large amount of information obtained from cta / ctp merely to reduce the time by an additional 58 min . information about clot location and burden , vascular anatomy , tandem lesions , access , and state of collaterals can make the subsequent intervention safer and faster . endovascular stroke therapy is entering its next phase of growth , which will require streamlining of both prehospital and intra - hospital care processes . delivery of endovascular stroke therapy is resource intensive and costly,19 requiring round - the - clock readiness . similar processes have been developed for acute coronary syndromes , specifically st segment elevation myocardial infarctions ( stemis).20 however , the number of current and projected future endovascular stroke interventions is much lower than stemi interventions,21 owing to a higher prevalence of ischemic heart disease than stroke.22 this means that while both stemi and lvo interventions require similar resources and investments to operate a round - the - clock service , the cost of lvo interventions will be lower owing to the smaller number of procedures performed . it may be beyond the capability of smaller hospitals ( defined as 300 beds ) to invest in high - quality round - the - clock stroke therapy but the temptation could be there from a marketing perspective and a perceived financial benefit . add to that an oversupply of physicians21 and we might find small regional hospitals with one to two physicians offering endovascular stroke care , not realizing that the postoperative care by dedicated neurospecialists and allied staff is as important as the procedure itself . the consequences of such a model could be dire with patients scattered among competing hospitals with variable standards of care and inconsistent quality metrics . additionally , the comparatively lower prevalence of ischemic stroke will result in multiple centers carrying out a small volume of procedures instead of a regional high - volume center to which all such patients could be efficiently transferred and treated . the process at our institution was developed based on the manpower and resources within our system a tertiary level academic medical center with in - house resident teams . we realize that our specific methodology may not be applicable to other systems with different resources . the process at our institution was developed based on the manpower and resources within our system a tertiary level academic medical center with in - house resident teams . we realize that our specific methodology may not be applicable to other systems with different resources . time - critical interventions such as endovascular stroke therapy can benefit from a systemically implemented protocol - driven approach . while the methodology can be different in different places based on available resources , as the field grows it is important to regionalize systems of care based on population densities , facility capabilities , and health system networks .

backgrounddelays in delivering endovascular stroke therapy adversely affect outcomes . time - sensitive treatments such as stroke interventions benefit from methodically developed protocols . clearly defined roles in these protocols allow for parallel processing of tasks , resulting in consistent delivery of care.objectiveto present the outcomes of a quality - improvement ( qi ) process directed at reducing stroke treatment times in a tertiary level academic medical center.methodsa six - sigma - based qi process was developed over a 3-month period . after an initial analysis , procedures were implemented and fine - tuned to identify and address rate - limiting steps in the endovascular care pathway . prospectively recorded treatment times were then compared in two groups of patients who were treated before ( n=64 ) or after ( n=30 ) the qi process . three time intervals were measured : emergency room ( er ) to arrival for ct scan ( er ct ) , ct scan to interventional laboratory arrival ( ct lab ) , and interventional laboratory arrival to groin puncture ( lab puncture).resultsthe er ct time was 40 ( 29 ) min in the before and 26 ( 15 ) min in the after group ( p=0.008 ) . the ct lab time was 87 ( 47 ) min in the before and 51 ( 33 ) min in the after group ( p=0.0002 ) . the lab puncture time was 24 ( 11 ) min in the before and 15 ( 4 ) min in the after group ( p<0.0001 ) . the overall er arrival to groin - puncture time was reduced from 2 h , 31 min ( 51 ) min in the before to 1 h , 33 min ( 37 ) min in the after group , ( p<0.0001 ) . the improved times were seen for both working hours and off - hours interventions.conclusionsa protocol - driven process can significantly improve efficiency of care in time - sensitive stroke interventions .

Introduction Methods Objective Patient population The QI process ERCT CTLab Labpuncture Data analysis Results Discussion Limitations Conclusion

PMC2771145

metabolic syndrome describes a combination of metabolic abnormalities that include central obesity , dyslipidemia , hypertension , insulin resistance , and a pro - inflammatory and pro - thrombotic state . an important group of pharmacological targets for the treatment of metabolic syndrome are the peroxisome proliferator activated receptors ( ppars ) . ppars are ligand - activated transcription factors belonging to the superfamily of nuclear receptors , which include numerous cellular receptors for nutrients and steroids . so far , three ppar isotypes ( , / , ) have been identified in a wide range of species , each displaying a different tissue distribution and ligand specificity . ppars share a similar structure and a common molecular mechanism of action by forming an obligate heterodimer with the 9-cis retinoic acid receptor rxr . ppar - rxr heterodimers selectively bind genomic sequences consisting of a direct repeat of the hexameric nucleotide sequence aggtca separated by 1 nucleotide ( direct repeat-1 ) . these so - called peroxisome proliferator response elements ( ppre ) are located in the promoter of ppar target genes or in intronic regions [ 25 ] . the ppar isotype ( nr1c1 ) is highly expressed in liver , and governs the adaptive response to fasting [ 68 ] . ppar is an extremely important regulator of hepatic nutrient metabolism including fatty acid oxidation ( peroxisomal and mitochondrial ) , fatty acid uptake , amino acid metabolism , glycerol metabolism , and lipoprotein assembly and transport [ 911 ] . in addition , ppar potently suppresses the hepatic inflammatory response [ 12 , 13 ] , an effect that is also observed in extra - hepatic tissues such as the vascular wall . much less is known about the role of ppar in other tissues , although evidence is accumulating that ppar induces cardiac and skeletal muscle fatty acid oxidation [ 15 , 16 ] . importantly , ppar mediates the effects of hypolipidemic fibrate drugs , which decrease plasma triglycerides and increase plasma hdl concentrations . in contrast to ppar , ppar ( nr1c3 ) is highly expressed in white adipose tissue ( wat ) , where it promotes lipid storage . ppar is a key transcription factor in the adipogenesis program and is essential for adipocyte survival [ 17 , 18 ] . it also serves as the molecular target for the thiazolidine - dione ( tzd ) class of insulin - sensitizing drugs that are widely used in the treatment of type 2 diabetes . ppar promotes whole body glucose utilization ; however , it has been difficult to identify the molecular mechanisms behind this effect . much of the attention has been focused on possible cross - talk between adipose tissue and skeletal muscle , as muscle is responsible for the major share of whole body glucose utilization . however , adipose tissue is a large organ , especially in the obese , and accordingly it can also be envisioned that the insulin - sensitizing effect of tzds on glucose uptake is partially exerted at the adipose tissue level . while ppar and ppar have been extensively studied over many years , much less is known about the function of the ppar/ isotype ( nr1c2 ) . studies with genetically modified ppar/ mice have illustrated the importance of this nuclear receptor in wat and skeletal muscle , two organs that have a key role in glucose homeostasis [ 1922 ] . it was shown that activation of ppar/ in adipose tissue protects against adiposity and hyperlipidemia by inducing fatty acid catabolism . moreover , pharmacological activation as well as specific constitutive over - expression of ppar/ leads to a shift in muscle fiber composition towards type i muscle fibers , resulting in increased muscle oxidative capacity [ 20 , 22 ] . ppar/ has also been shown to stimulate hepatic vldl production , influence wound healing , and affect colon carcinogenesis [ 2325 ] . however , whether ppar/ has a functional role in glucose homeostasis , in analogy with other ppar isotypes , remains to be firmly established . here we show that glycogen synthase 2 ( gys-2 ) , the rate - limiting enzyme for glycogen synthesis in liver and adipose tissue , is a target gene of ppar , ppar/ and ppar. transcriptional regulation is achieved via a ppar response element present in the first intron . we show that an additional direct repeat response element identified in the gys-2 promoter mediates transactivation by hepatic nuclear factor 4 ( hnf4 ) . chemicals . wy14643 was obtained from eagle picher technologies laboratories ( lenexa , ks , usa ) . dulbecco s modified eagles medium ( dmem ) , fetal calf serum ( fcs ) , calf serum and penicillin / streptomycin / fungizone were from cambrex bioscience ( seraing ) . otherwise , chemicals were from sigma ( zwijndrecht , the netherlands ) . animal experiments . ppar/ mutant null mice ( ppar/-/- ) and ppar heterozygous mice ( ppar+/ ) were on a mixed background ( svl29/c57bl/6 ) and have been described previously [ 26 , 27 ] . ppar-/- mice and corresponding wild - type mice on an svl29 background were purchased at jackson laboratories ( bar harbor , me , usa ) . liver - specific hepatocyte nuclear factor 4 ( hnf4)-null mice were generated as described previously . livers were collected from 45-day - old hnf4 x albumin - cre ( ko ) and hnf4 x albumin - cre ( flox ) mice . for the fasting experiment , 3-month - old male mice were fasted for different periods of time starting at the onset of the light cycle . for the refeeding experiment , mice were fasted for 24 h after which they were put back on chow for 7 h before sacrifice . after sacrificing the animals , the animal experiments were approved by the animal experimentation committee of wageningen university or the etat de vaud ( switzerland ) . total rna was prepared from epididymal wat of wild - type and ppar/-/- mice ( five animals of each genotype ) using trizol reagent ( invitrogen , breda , the netherlands ) and subsequently pooled per group . pooled rna was further purified using qiagen rneasy columns , and the quality was verified using bioanalyzer 2100 ( agilent , amsterdam ) . for one cycle crna synthesis ( affymetrix , santa clara , usa ) 10 g of rna was used . hybridization , washing and scanning of affymetrix genechip mouse genome 430 2.0 arrays was according to standard affymetrix protocols . fluorimetric data were processed by affymetrix genechip operating software and the gene chips were globally scaled to all the probe sets with an identical target intensity value . 3t3-l1 fibroblasts were grown in dmem plus 10 % calf serum and plated for final differentiation in dmem plus 10 % fcs . at 2 days after reaching confluence , the medium was changed and the following compounds were added : isobutyl methylxanthine ( 0.5 mm ) , dexamethasone ( 1 m ) , and insulin ( 5 g / ml ) . after 3 days , the medium was changed to dmem plus 10% fcs and insulin ( 5 g / ml ) . after 6 days the medium briefly , after cannulation of the portal vein , the liver was perfused with calcium - free hbss , which was pre - gassed with 95 % o2 /5 % co2 . next , the liver was perfused with a collagenase solution until swelling and degradation of the internal liver structure was observed . cells were cultured in william s medium e supplemented with 10% fcs , penicillin / streptomycin / fungizone , insulin and dexamethasone . cells were plated in collagen ( serva feinbiochemica , heidelberg , germany ) -coated wells with a density of 0.510 cells / ml . after 4 h of incubation , the medium was removed and replaced with fresh medium . total rna was extracted from tissues with trizol reagent ( invitrogen ) ; 1 g total rna was then reverse - transcribed with iscript ( bio - rad , veenendaal , the netherlands ) . cdna was pcr - amplified with platinum taq dna polymerase ( invitrogen ) on a bio - rad icycler or mylq pcr machine . primers were designed to generate a pcr amplification product of 100200 bp and were taken from primerbank ( http://pga.mgh.harvard.edu/primerbank/ ) . specificity of the amplification was verified by melt curve analysis and evaluation of efficiency of pcr amplification . expression was related to the control gene 36b4 , which did not change under any of the experimental conditions studied . the following primer pairs were used : mgys-2 ( forward ) : ccagcttgacaagttcgaca , mgys-2 ( reverse ) : at - caggcttcctcttcagca , m36b4 ( forward ) : agcgcgtcctggcattgtgtgg , m36b4 ( reverse ) : gggcagcagtggtggcagcagc , mppar ( forward ) : tattcggctgaagctggtgtac , mppar ( reverse ) : ctggcatttgttccggttct , mppar ( forward ) : ttgagcccaagttcgagtttg , mppar ( reverse ) : cggtctccacacagaatgatg , mppar ( forward ) : ca - caatgccatcaggtttgg , mppar ( reverse ) : gctggtcgatatcactggagatc . the proximal part of the mouse gys-2 promoter was pcr amplified from mouse genomic dna ( strain c57bl/6 ) using the forward primer : 5 cttgctgcctttcag - gagagggcag 3 and reverse primer : 5 ttctctttagc - catta ag atag 3. the resulting 553-bp fragment was used for a second pcr amplification step introducing hindlll and kpnl sites , which were used for subcloning into the pgl-3 basic vector ( invitrogen ) . a 156-bp nucleotide fragment surrounding the putative ppre within the mgys-2 promoter was pcr amplified from mouse genomic dna ( strain c57bl/6 ) and subcloned into kpnl / bg / ii sites of the pgl3 sv40 promoter vector ( pgl3-tk - luc , promega , leiden , the netherlands ) using the forward primer : 5aaatcg - cagctgaaacct 3 , and reverse primer : 5 ctcctgcttgtgcttctgc 3. a 314-nucleotide fragment surrounding the putative ppre within intron 1 of the mouse gys-2 gene was pcr amplified from mouse genomic dna ( strain c57bl/6 ) and subcloned into the kpnl and bglll sites of the pgl - tk - luc reporter gene . reporter vectors were transfected into human hepatoma hepg2 cells , together with an expression vector ( psg5 ) for mppar , mppar , or mpparyl , in the presence or absence of wy14643 ( 50 m ) , l-165041 ( 5 m ) , or rosiglitazone ( 5 m ) , respectively . a -galactosidase reporter vector was co - transfected to normalize for differences in transfection efficiency . luciferase activity was measured 24 h post transfection using the promega luciferase assay kit ( promega ) on a fluoroskan ascent fl apparatus ( thermo labsystems , breda , the netherlands ) . -galactosidase activity was measured in the cell lysate by a standard assay using 2-nitrophenyl-d - galactopyrano - side as a substrate . to disable the mouse gys-2 ppre within the mgys-2 promoter , two separate ( a and b ) partially overlapping pcr fragments were generated using the wild - type mgys-2 promoter as a template . primers sets used to generate part a of the mutated mgys-2 promoter fragment were : 5-tttggtctaaaggcctttggccaaagg-3 and 5-cttgctgcctttcaggagagggc ag-3. primers sets used to generate part b of the mutated mgys-2 promoter fragment were : 5-cctttggccaaaggccttta - gaccaaa-3 and 5-ttctctttagccattaagatagg - gattg-3. pcr was carried - out using the two upper dna fragments and the following primers : 5- cccaagcttcttgctgcctttcaggag-3 and 5- ggggtaccttctctttagccattaagatag-3. the pcr fragment was subsequently cloned into the pgl3 basic reporter vector ( hindiii / kpni cloning site ) and verified by automated sequencing . the hhnf4 expression plasmid was constructed by amplifying human hepatoma hepg2 cdna using the following primers , forward primer 5-gaatgcgactctccaaaacc-3 and reverse primer 5-atccttcccattcctgctct-3 , followed by subcloning of the resulting pcr product into an pgem - teasy vector ( promega ) . the insert was excised by noti digestion and further subcloned into pcdna3.1/v5-hisa ( invitrogen ) . hrxr and mppar proteins were generated from psg5 expression vectors , using the tnt coupled in vitro transcription / translation system ( promega ) . the following oligos were annealed to generate the double - stranded dna probe ; for dr - lint : 5-caggactttggtgacctctggcctatat-3 and 5-acacatataggccagaggtcaccaaagtc-3. for nonspecific competition , the following primers were used : etsf 5-tggaatgtaccggaaataacacca-3 , etsr 5- tggtgttatttccggtacattcca-3. oligonucleotides were annealed and labeled by klenow filling ( new england biolabs , leusden , the netherlands ) using redivue [ -p]dctp ( 3000 ci / mmol ; amersham , roosendaal , the netherlands ) . in vitro - translated proteins ( 0.50.8 l / reaction ) were pre - incubated for 15 min on ice in 1 binding buffer ( 80 mm kcl , 1 mm dtt , 10 mm tris - hcl ph 7.4 , 10 % glycerol , plus protease inhibitors ) in presence of 2 g poly[di.dc ] , 5 g sonicated salmon sperm dna and competitor oligonucleotides in a final volume of 20 l . then 1 ng ( 1 ng/l ) of radiolabeled oligo was added and incubation was continued for another 10 min at room temperature . complexes were separated on a 4 % polyacrylamide gel ( acrylamide / bisacrylamide 37.5:1 ) equilibrated in 0.5 tbe at 25 ma . chromatin immunoprecipitation ( chip ) on 3t3-l1 cells and mouse liver was carried out as described previously . sequences of primers used for pcr were 5-tctggcaggcataaggacccgagtt-3 and 5- ggaagccaggacagagtgcaaatacaat-3 for dr - lint ( intron ) . for dr - lprom , the following primers were used : 5-aaaactgcttgtgtctgagggaaac-3 and 5-agag - gacagactgagcatgacaagag-3. control primers used were 5-gctgcgagatccatcacccactaaac-3 and 5-agccatctcaccagccccaactt-3. antibodies against ppars were from santa cruz biotechnology ( santa cruz , ca , usa ) . chip on rat hepatocytes was done using a commercially available kit ( active motif , rixensart , belgium ) . the primers used to amplify the sequence surrounding the dr - lprom were 5-gaatgccgctgtgcctgagggaaac-3 and 5-agaggacagaagaagagtgacaagag-3. for dr - lint : 5-tctgtcaggcataaggacctgggtt-3 and 5-attgtatttgaactctgtcctggtctct-3. histology . liver tissue from wild - type and ppar-/- mice was embedded in tissue - tek o.c.t compound from sakura finetek ( zoeterwoude , the netherlands ) and frozen . cryosections of 5 m from frozen liver were made and analyzed for glycogen accumulation using the periodic acid - schiff ( pas ) reaction . a mouse anti - glycogen synthase monoclonal antibody was used ( clone gs-7h5 mab3106 ) ( chemicon international , hampshire , uk ) . the primary antibody was used at a dilution of 1:1000 and the secondary antibody ( anti - mouse igg , dako , glostrup , denmark ) was used at a dilution of 1:8000 . accordingly , we compared gene expression in wat of wild - type versus ppar/-/- mice using affymetrix micro - array analysis . the expression of several genes involved in glucose and lipid metabolism was down - regulated in ppar/-/-mice , including ppar , pgc - l , and glut4 , which was confirmed for several genes by real - time quantitative pcr ( qpcr ) ( table 1 ) . expression of gys-2 was most significantly down - regulated in ppar/-/-mice , and therefore gys-2 was selected for more detailed investigation . q - pcr confirmed the marked down - regulation of gys-2 mrna in wat of ppar/-/- mice ( fig . furthermore , expression of gys-2 also appeared to be down - regulated in wat of ppar+/ and ppar-/- mice , although the former result did not achieve statistical significance . in ppar/-/- mice , the decrease in gys-2 mrna was paralleled by a significant down - regulation of ppar and ppar expression , while in ppar mice expression of ppar/ was significantly down - regulated ( fig . these data show that ppars are crucial for maintaining gys-2 expression in fat , although it is difficult to ascertain which ppar isotype is the main regulator of gys-2 expression in wat . figure 1expression of glycogen synthase 2 ( gys-2 ) in white adipose tissue ( wat ) is regulated by peroxisome proliferator - activated receptors ( ppars ) . expression of gys-2 ( a ) and ppars ( b ) in wat of ppar/ mice , ppar mice , and pparmice , as determined by qpcr . the effects of ppar deletion were evaluated by student s mest ( * p<0.05 ; * * p<0.01 ) . ( c ) differentiated 3t3-l1 adipocytes were treated with the synthetic ppar agonists rosiglitazone or ciglitazone , or the ppar/ agonist l165041 for 24 h. expression of gys-2 was determined by qpcr . data shown are representative results from three independent experiments , ( d ) gys protein expression was analyzed in lysates from 3t3-l1 adipocytes treated with either ppar/ ( l165041 , 2.5 m ) or ppar ( rosiglitazone , 1 m ) agonist.table 1genes involved in glucose and lipid metabolism that were differentially expressed between white adipose tissue of wild - type and ppar/ mice.geneproductfold decrease micro - arrayq - pcrglucose metabolismgys2glycogen synthase 213.279.28pik3r1phosphatidylinositol 3-kinase , regulatory subunit , polypeptide 1 ( p85 alpha)2.99pik3r1phosphatidylinositol 3-kinase , regulatory subunit , polypeptide 1 ( p85 alpha)2.64gys3glycogen synthase 3 , brain2.25ppp1r3cprotein phosphatase 1 , regulatory subunit 3c , protein targeting to glycogen2.19slc2a4solute carrier family 2 ( facilitated glucose transporter ) , member 42.161.56slc2a4solute carrier family 2 ( facilitated glucose transporter ) , member 42.041.56pfk-26-phosphofructo-2-kinase / fructose-2,6-bisphosphatase2.01gdc1glycerol phosphate dehydrogenase 1 , cytoplasmic adult1.93ppp1r3cprotein phosphatase 1 , regulatory subunit 3c , protein targeting to glycogen1.91gdc1glycerolphosphate dehydrogenase 1 , cytoplasmicadult1.84pyglliver glycogen phosphorylase1.71lipid metabolismppargperoxisome proliferator activator receptor gamma2.993.63c5dsterol - c5-desaturase2.85lrp1low density lipoprotein receptor - related protein 12.22cd36cd36 antigen2.203.09]fads3fatty acid desaturase 32.06lipelipase , hormone sensitive1.961.66lrp2low density lipoprotein receptor - related protein 21.91fabp5fatty acid binding protein 5 , epidermal1.87dgat1diacylglycerol acyltransferase1.87decr12,4-dienoyl coa reductase 1 , mitochondrial1.85slc27a1solute carrier family 27 ( fatty acid transporter ) , member 11.85phyhphytanol - coa hydroxylase1.77dgat2/1diacylglycerol o - acyltransferase 2-like 11.75ppargc1peroxisome proliferative activated receptor , gamma , coactivator 11.741.97 expression of glycogen synthase 2 ( gys-2 ) in white adipose tissue ( wat ) is regulated by peroxisome proliferator - activated receptors ( ppars ) . expression of gys-2 ( a ) and ppars ( b ) in wat of ppar/ mice , ppar mice , and pparmice , as determined by qpcr . the effects of ppar deletion were evaluated by student s mest ( * p<0.05 ; * * p<0.01 ) . ( c ) differentiated 3t3-l1 adipocytes were treated with the synthetic ppar agonists rosiglitazone or ciglitazone , or the ppar/ agonist l165041 for 24 h. expression of gys-2 was determined by qpcr . data shown are representative results from three independent experiments , ( d ) gys protein expression was analyzed in lysates from 3t3-l1 adipocytes treated with either ppar/ ( l165041 , 2.5 m ) or ppar ( rosiglitazone , 1 m ) agonist . genes involved in glucose and lipid metabolism that were differentially expressed between white adipose tissue of wild - type and ppar/ mice . to investigate whether expression of gys-2 in adipocytes is under direct control of ppars , the effect of ppar ligands on gys-2 mrna was studied in differentiated mouse 3t3-l1 adipocytes . it was observed that the ppar/ agonist l165041 , and the ppar agonists ciglitazone and rosiglitazone significantly induced gys-2 mrna levels ( fig . thus , gys-2 may represent a direct target gene of ppar and ppar/ in adipocytes . it was observed that liver glycogen levels were reduced in refed ppar-/- mice compared to refed wild - type mice [ 30 , 31 ] , which we confirmed using histochemical staining ( fig . expression of gys-2 is highest in liver , followed by wat ( our unpublished data ) . we confirm that hepatic gys-2 mrna is markedly reduced in ppar-/- mice ; however , only in the 24-h fasted and refed state ( fig . 3a ) . to further examine the role of ppar in gys-2 expression , primary hepatocytes from wild - type and ppar-/- mice were treated with the synthetic ppar agonist wy14643 , allowing for a direct evaluation of the effect of ppar activation on gys-2 expression . basal expression of gys-2 was about fourfold reduced in ppar-/- hepatocytes , indicating a requirement for ppar ( fig . furthermore , wy14643 stimulated gys-2 expression in wild - type but not in ppar-/- hepatocytes ( fig . 3b ) . induction of gys-2 expression by synthetic ppar agonists was also observed in rat primary hepatocytes ( fig . 3c ) . together , these data suggest a direct role of ppar in governing hepatic gys-2 expression . figure 2staining for hepatic glycogen is higher in refed wild - type mice compared to refed ppar-/- mice , ( a ) representative hematoxylin and eosin staining of liver from two wild - type and two ppar-/- mice , ( b ) representative pas staining of liver from two wild - type and two ppar-/- mice . mice were fasted for 24 h followed by refeeding for 7 h before sacrifice.figure 3ppar governs hepatic expression of gys-2 . ( a ) relative expression of gys-2 in fed , fasted and refed wild - type and ppar-/- mice , as determined by qpcr . mice were fasted for 0,6 , 12 or 24 h , with refeeding for 7 h following 24 h fasting . significant differences between wild - type and ppar-/- mice were observed in the 24-h fasted and the refed state ( b ) relative expression of gys-2 in freshly isolated wild - type and ppar-/- hepatocytes treated for 24 h with vehicle ( dmso ) or wy14643 ( 10 m ) , as determined by qpcr . significant effects were observed by two - way anova for genotype ( p<0.001 ) , and for the interaction between the genotype and wy14643 ( p<0.05 ) . ( c ) relative expression of gys-2 in freshly isolated rat hepatocytes which were treated for 24 h with either vehicle ( dmso ) , wy14643 ( 50 um ) or fenofibrate ( 50 m ) . the effects of wy14643 and fenofibrate were statistically significant ( student s mest : p<0.01 ) . glycogen is higher in refed wild - type mice compared to refed ppar-/- mice , ( a ) representative hematoxylin and eosin staining of liver from two wild - type and two ppar-/- mice , ( b ) representative pas staining of liver from two wild - type and two ppar-/- mice . ( a ) relative expression of gys-2 in fed , fasted and refed wild - type and ppar-/- mice , as determined by qpcr . mice were fasted for 0,6 , 12 or 24 h , with refeeding for 7 h following 24 h fasting . significant differences between wild - type and ppar-/- mice were observed in the 24-h fasted and the refed state ( b ) relative expression of gys-2 in freshly isolated wild - type and ppar-/- hepatocytes treated for 24 h with vehicle ( dmso ) or wy14643 ( 10 m ) , as determined by qpcr . significant effects were observed by two - way anova for genotype ( p<0.001 ) , and for the interaction between the genotype and wy14643 ( p<0.05 ) . ( c ) relative expression of gys-2 in freshly isolated rat hepatocytes which were treated for 24 h with either vehicle ( dmso ) , wy14643 ( 50 um ) or fenofibrate ( 50 m ) . the effects of wy14643 and fenofibrate were statistically significant ( student s mest : p<0.01 ) . identification of a putative ppre in the proximal promoter of the mouse gys-2 gene . to determine what genomic region could be responsible for the ppar - induced up - regulation of gys-2 mrna , the mouse gys-2 gene was scanned for potential ppres ( nubiscan algorithm and hidden markov model framework ) [ 33 , 34 ] . a direct repeat-1 motif ( dr - lprom ) was localized to the proximal gys-2 gene promoter , about 169 bp upstream from the transcription start site . with the exception of two nucleotides , dr - lprom is identical to the consensus sequence , suggesting that this sequence could serve as a functional ppre ( fig . figure 4the dr-1 present in the gys-2 promoter does not mediate ppar - dependent transactivation . ( a ) alignment of the consensus ppre sequence with the sequence of rat and mouse gys-2 dr - lprom and gys-2 dr - lint . ( b ) hepg2 cells were transfected with a reporter vector containing a 553-nucleotide fragment of the proximal mouse gys-2 promoter gene and ppar expression vectors , ( c ) hepg2 cells were transfected with a sv40 reporter vector containing an isolated 156-nucleotide fragment surrounding dr - lprom of the proximal mouse gys-2 promoter gene and ppar expression vectors . luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m of rosiglitazone . chromatin immunoprecipitation of dr - lprom using antibodies against mppar ( d ) or mppar/ ( e ) . the gene sequence spanning dr - lprom and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes . ( f ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . ( a ) alignment of the consensus ppre sequence with the sequence of rat and mouse gys-2 dr - lprom and gys-2 dr - lint . ( b ) hepg2 cells were transfected with a reporter vector containing a 553-nucleotide fragment of the proximal mouse gys-2 promoter gene and ppar expression vectors , ( c ) hepg2 cells were transfected with a sv40 reporter vector containing an isolated 156-nucleotide fragment surrounding dr - lprom of the proximal mouse gys-2 promoter gene and ppar expression vectors . luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m of rosiglitazone . chromatin immunoprecipitation of dr - lprom using antibodies against mppar ( d ) or mppar/ ( e ) . the gene sequence spanning dr - lprom and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes . ( f ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . to examine whether the promoter region containing the putative ppre is responsible for ppar - dependent up - regulation of gys-2 expression , a 553-nucleotide fragment of the mouse gys-2 promoter gene was cloned in front of a luciferase reporter gene and transactivation studies were carried out in hepg2 cells . surprisingly , co - transfection of ppar or pparl expression vectors in combination with ppar agonists slightly decreased luciferase activity , while ppar/ activation had little effect ( fig . transactivation assays performed with a small genomic fragment surrounding dr - lprom cloned in front of sv40-luciferase led to a similar overall ppar - mediated repression for ppar and pparl , while ppar/ had little effect ( fig . co - transfection of rxr or of different co - activators such as cbp and pgcl did not change this pattern ( data not shown ) . thus , the ppre identified in the gys-2 promoter probably does not mediate the effect of ppars on gys-2 expression . nevertheless , chip experiments carried out in 3t3-l1 cells indicated that ( 1 ) ppar was bound to dr - lprom in mature adipocytes , but not in pre - adipocytes ( fig . 4d ) , and ( 2 ) ppar/ was bound to dr - lprom in pre - adipocytes and , more strongly , in mature adipocytes ( fig . 4e ) . thus , despite dr - lprom behaving poorly as a ppre in classical transactivation assay , it binds both ppar and ppar/ in adipocytes . this suggests that in vivo binding of ppar and ppar/ to dr - lprom does not translate into transcriptional activation of the gys-2 gene , and accordingly that activation of gys-2 expression by ppars may be mediated by another genomic region . it should be mentioned that chip did not reveal any binding of ppar to dr - lprom in hepatocytes ( data not shown ) . interestingly , using the same strategy as described above , a putative ppre that is homologous to the consensus dr-1 sequence was identified in intron 1 of the mouse gys-2 gene ( fig . 4a ) . to assess whether dr - lint was able to mediate ppar - dependent transactivation , a 314-nucleotide genomic fragment surrounding dr - lint was cloned in front of the sv40 promoter followed by a luciferase reporter gene . in hepg2 cells treatment with the synthetic ppar agonist wy14643 induced reporter activity and this activation was further enhanced upon co - transfection of mppar ( fig . similar inductions of reporter activity were observed for ppar/ and ppar and their respective agonists ( fig . thus , dr - lint is able to mediate ppar - dependent transactivation , irrespective of the ppar isotype , suggesting that it may at least be partially responsible for ppar - dependent regulation of gys-2 expression . figure 5gys-2 up - regulation by ppars is mediated by a ppre present in intron 1 of the gys-2 gene ( dr - lint ) . ( a ) hepg2 cells were transfected with a 314-nucleotide fragment of intron 1 of the mouse gys-2 gene and ppar expression vectors . luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m rosiglitazone . ( b ) binding of the ppar / rxr heterodimers to dr - lint as determined by gel shift assays . a double - stranded response element containing gys-2 dr - lint was incubated with in vitro transcribed / translated mppar protein ( left panel ) , mppar/ protein ( middle panel ) and mpparl protein ( right panel ) together with in vitro transcribed / translated hrxr protein . fold excess of specific ( malic enzyme ppre ) or nonspecific ( ets oligonucleotide ) cold probe is indicated . gys-2 up - regulation by ppars is mediated by a ppre present in intron 1 of the gys-2 gene ( dr - lint ) . ( a ) hepg2 cells were transfected with a 314-nucleotide fragment of intron 1 of the mouse gys-2 gene and ppar expression vectors . luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m rosiglitazone . ( b ) binding of the ppar / rxr heterodimers to dr - lint as determined by gel shift assays . a double - stranded response element containing gys-2 dr - lint was incubated with in vitro transcribed / translated mppar protein ( left panel ) , mppar/ protein ( middle panel ) and mpparl protein ( right panel ) together with in vitro transcribed / translated hrxr protein . fold excess of specific ( malic enzyme ppre ) or nonspecific ( ets oligonucleotide ) cold probe is indicated . in agreement with the transactivation data , ppar , ppar/ and ppar proteins a retarded heterodimeric complex was observed only in the presence of both ppar and obligate binding partner rxr ( fig . the complex disappeared in the presence of an excess of cold specific oligonucleotide , but not nonspecific oligonucleotide . examination of in vivo ppar binding to dr - lint by chip yielded very similar results as for dr - lprom : ppar was bound to dr - lint in mature 3t3-l1 adipocytes , but not in pre - adipocytes ( fig . 6a ) , whereas ppar/ was bound to dr - lint in both pre- and mature adipocytes ( fig . chip analysis demonstrated binding of ppar to dr - lint in wild - type but not ppar mice , and binding was enhanced by fasting and wy14643 ( fig . together , these data indicate that mouse gys-2 is a direct ppar target gene and that regulation by ppars is at least partially mediated by a ppre present in intron 1 . figure 6ppar , ppar/ , and ppar bind to the gys-2 dr - lint in vivo . chromatin immunoprecipitation of gys-2 dr - lint using antibodies against mppar ( a ) , mppar/ ( b ) or mppar ( c ) . the gene sequence spanning dr - lint and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes ( a , b ) or mouse liver ( c ) . pi , preimmune serum , cntl , random control sequence , ( d ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . ppar , ppar/ , and ppar bind to the gys-2 dr - lint in vivo . chromatin immunoprecipitation of gys-2 dr - lint using antibodies against mppar ( a ) , mppar/ ( b ) or mppar ( c ) . the gene sequence spanning dr - lint and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes ( a , b ) or mouse liver ( c ) . pi , preimmune serum , cntl , random control sequence , ( d ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . gys-2 is a novel direct target of the liver enriched factor hnf4. as explained above , ppar caused a reduction in gys-2 promoter activity via dr - lprom ( fig . a similar decrease of promoter activity in response to ppar despite the presence of a putative ppre has been reported for other genes . indeed , it was found that ppar decreases expression of the apociii and transferrin genes via competition with the hnf4. since hnf4 is known to recognize dr-1 sequences as well , we examined whether hnf4 might control the expression of gys-2 in liver , possibly via dr - lprom . gys-2 mrna levels were markedly decreased in liver - specific hnf4null mice , thus supporting a role for hnf4 in regulating gys-2 expression ( fig . 7a ) . in transactivation assays using the gys-2 promoter , hnf4 markedly activated reporter activity , suggesting the presence of a hnf4 response element within the 0.55-kb promoter fragment ( fig . 7b ) . mutating dr - lprom resulted in an approximately 50 % reduction in hnf4-dependent activation of the gys-2 promoter ( fig . 7c ) , which suggests that ( a ) hnf4 responsiveness is partially mediated by dr - lprom , or ( b ) the mutations with dr - lprom only partially disabled hnf4 responsiveness . regardless of these explanations , these data suggest that hnf4 directly regulates the hepatic expression of gys-2 at least partially via dr - lprom . finally , chip clearly showed hnf4 binding to dr - lprom but not dr - lint in rat primary hepatocytes ( fig . figure 7gys-2 dr - lprom is a binding site for the nuclear receptor hnf4. ( a ) gys-2 mrna levels in liver of liver - specific hnf4-null ( hnf4-/- ) and wild - type ( flox ) mice were analyzed by qpcr ( n=3 per group ) . ( b ) hepg2 cells were transfected with a reporter vector containing 553-bp of the mouse gys-2 proximal promoter and increasing amounts of hhnf4 expression vector , ( c ) hepg2 cells were transfected with a reporter vector containing 553 bp of the wild - type and mutated mgys-2 proximal promoter and hhnf4 expression vector , ( d ) chromatin immu - noprecipitation of gys-2 dr - lprom using antibodies against hnf4. the gene sequence spanning dr - lprom and a control sequence were analyzed by pcr in the immunoprecipitated chromatin of rat primary hepatocytes . ( e ) hepg2 cells were transfected with a reporter vector containing 553 bp of the mouse gys-2 proximal promoter , an expression vector for hhnf4 , and increasing amounts of mppar expression vector . normalized luciferase activity of the mgys-2 reporter vector in the absence of mppar , hhnf4 and wy 14643 was set at 1 . dr - lprom is a binding site for the nuclear receptor hnf4. ( a ) gys-2 mrna levels in liver of liver - specific hnf4-null ( hnf4-/- ) and wild - type ( flox ) mice were analyzed by qpcr ( n=3 per group ) . ( b ) hepg2 cells were transfected with a reporter vector containing 553-bp of the mouse gys-2 proximal promoter and increasing amounts of hhnf4 expression vector , ( c ) hepg2 cells were transfected with a reporter vector containing 553 bp of the wild - type and mutated mgys-2 proximal promoter and hhnf4 expression vector , ( d ) chromatin immu - noprecipitation of gys-2 dr - lprom using antibodies against hnf4. the gene sequence spanning dr - lprom and a control sequence were analyzed by pcr in the immunoprecipitated chromatin of rat primary hepatocytes . ( e ) hepg2 cells were transfected with a reporter vector containing 553 bp of the mouse gys-2 proximal promoter , an expression vector for hhnf4 , and increasing amounts of mppar expression vector . normalized luciferase activity of the mgys-2 reporter vector in the absence of mppar , hhnf4 and wy 14643 was set at 1 . whereas hnf4 activates the gys-2 promoter via dr - lprom , ppar does the opposite , suggesting that ppar may interfere with gys-2 promoter activation by hnf4. to examine whether this is the case , the effect of ppar on hnf4-mediated transactivation of the 0.55-kb gys-2 promoter was studied . ppar activation significantly reduced hnf4-dependent transactivation , indicating competition between hnf4 and ppar in the regulation of the gys-2 promoter ( fig . as already mentioned above , we failed to find any evidence for binding of ppar to dr - lprom in hepatocytes . thus , the inhibitory effect of ppar on transcriptional activation of gys-2 by hnf4 likely does not occur via competition with hnf4 for actual binding to dr - lprom . accordingly , we compared gene expression in wat of wild - type versus ppar/-/- mice using affymetrix micro - array analysis . the expression of several genes involved in glucose and lipid metabolism was down - regulated in ppar/-/-mice , including ppar , pgc - l , and glut4 , which was confirmed for several genes by real - time quantitative pcr ( qpcr ) ( table 1 ) . expression of gys-2 was most significantly down - regulated in ppar/-/-mice , and therefore gys-2 was selected for more detailed investigation . q - pcr confirmed the marked down - regulation of gys-2 mrna in wat of ppar/-/- mice ( fig . furthermore , expression of gys-2 also appeared to be down - regulated in wat of ppar+/ and ppar-/- mice , although the former result did not achieve statistical significance . in ppar/-/- mice , the decrease in gys-2 mrna was paralleled by a significant down - regulation of ppar and ppar expression , while in ppar mice expression of ppar/ was significantly down - regulated ( fig . these data show that ppars are crucial for maintaining gys-2 expression in fat , although it is difficult to ascertain which ppar isotype is the main regulator of gys-2 expression in wat . figure 1expression of glycogen synthase 2 ( gys-2 ) in white adipose tissue ( wat ) is regulated by peroxisome proliferator - activated receptors ( ppars ) . expression of gys-2 ( a ) and ppars ( b ) in wat of ppar/ mice , ppar mice , and pparmice , as determined by qpcr . the effects of ppar deletion were evaluated by student s mest ( * p<0.05 ; * * p<0.01 ) . ( c ) differentiated 3t3-l1 adipocytes were treated with the synthetic ppar agonists rosiglitazone or ciglitazone , or the ppar/ agonist l165041 for 24 h. expression of gys-2 was determined by qpcr . data shown are representative results from three independent experiments , ( d ) gys protein expression was analyzed in lysates from 3t3-l1 adipocytes treated with either ppar/ ( l165041 , 2.5 m ) or ppar ( rosiglitazone , 1 m ) agonist.table 1genes involved in glucose and lipid metabolism that were differentially expressed between white adipose tissue of wild - type and ppar/ mice.geneproductfold decrease micro - arrayq - pcrglucose metabolismgys2glycogen synthase 213.279.28pik3r1phosphatidylinositol 3-kinase , regulatory subunit , polypeptide 1 ( p85 alpha)2.99pik3r1phosphatidylinositol 3-kinase , regulatory subunit , polypeptide 1 ( p85 alpha)2.64gys3glycogen synthase 3 , brain2.25ppp1r3cprotein phosphatase 1 , regulatory subunit 3c , protein targeting to glycogen2.19slc2a4solute carrier family 2 ( facilitated glucose transporter ) , member 42.161.56slc2a4solute carrier family 2 ( facilitated glucose transporter ) , member 42.041.56pfk-26-phosphofructo-2-kinase / fructose-2,6-bisphosphatase2.01gdc1glycerol phosphate dehydrogenase 1 , cytoplasmic adult1.93ppp1r3cprotein phosphatase 1 , regulatory subunit 3c , protein targeting to glycogen1.91gdc1glycerolphosphate dehydrogenase 1 , cytoplasmicadult1.84pyglliver glycogen phosphorylase1.71lipid metabolismppargperoxisome proliferator activator receptor gamma2.993.63c5dsterol - c5-desaturase2.85lrp1low density lipoprotein receptor - related protein 12.22cd36cd36 antigen2.203.09]fads3fatty acid desaturase 32.06lipelipase , hormone sensitive1.961.66lrp2low density lipoprotein receptor - related protein 21.91fabp5fatty acid binding protein 5 , epidermal1.87dgat1diacylglycerol acyltransferase1.87decr12,4-dienoyl coa reductase 1 , mitochondrial1.85slc27a1solute carrier family 27 ( fatty acid transporter ) , member 11.85phyhphytanol - coa hydroxylase1.77dgat2/1diacylglycerol o - acyltransferase 2-like 11.75ppargc1peroxisome proliferative activated receptor , gamma , coactivator 11.741.97 expression of glycogen synthase 2 ( gys-2 ) in white adipose tissue ( wat ) is regulated by peroxisome proliferator - activated receptors ( ppars ) . expression of gys-2 ( a ) and ppars ( b ) in wat of ppar/ mice , ppar mice , and pparmice , as determined by qpcr . the effects of ppar deletion were evaluated by student s mest ( * p<0.05 ; * * p<0.01 ) . ( c ) differentiated 3t3-l1 adipocytes were treated with the synthetic ppar agonists rosiglitazone or ciglitazone , or the ppar/ agonist l165041 for 24 h. expression of gys-2 was determined by qpcr . data shown are representative results from three independent experiments , ( d ) gys protein expression was analyzed in lysates from 3t3-l1 adipocytes treated with either ppar/ ( l165041 , 2.5 m ) or ppar ( rosiglitazone , 1 m ) agonist . genes involved in glucose and lipid metabolism that were differentially expressed between white adipose tissue of wild - type and ppar/ mice . to investigate whether expression of gys-2 in adipocytes is under direct control of ppars , the effect of ppar ligands on gys-2 mrna was studied in differentiated mouse 3t3-l1 adipocytes . it was observed that the ppar/ agonist l165041 , and the ppar agonists ciglitazone and rosiglitazone significantly induced gys-2 mrna levels ( fig . thus , gys-2 may represent a direct target gene of ppar and ppar/ in adipocytes . it was observed that liver glycogen levels were reduced in refed ppar-/- mice compared to refed wild - type mice [ 30 , 31 ] , which we confirmed using histochemical staining ( fig . expression of gys-2 is highest in liver , followed by wat ( our unpublished data ) . we confirm that hepatic gys-2 mrna is markedly reduced in ppar-/- mice ; however , only in the 24-h fasted and refed state ( fig . 3a ) . to further examine the role of ppar in gys-2 expression , primary hepatocytes from wild - type and ppar-/- mice were treated with the synthetic ppar agonist wy14643 , allowing for a direct evaluation of the effect of ppar activation on gys-2 expression . basal expression of gys-2 was about fourfold reduced in ppar-/- hepatocytes , indicating a requirement for ppar ( fig . furthermore , wy14643 stimulated gys-2 expression in wild - type but not in ppar-/- hepatocytes ( fig . 3b ) . induction of gys-2 expression by synthetic ppar agonists was also observed in rat primary hepatocytes ( fig . 3c ) . together , these data suggest a direct role of ppar in governing hepatic gys-2 expression . figure 2staining for hepatic glycogen is higher in refed wild - type mice compared to refed ppar-/- mice , ( a ) representative hematoxylin and eosin staining of liver from two wild - type and two ppar-/- mice , ( b ) representative pas staining of liver from two wild - type and two ppar-/- mice . mice were fasted for 24 h followed by refeeding for 7 h before sacrifice.figure 3ppar governs hepatic expression of gys-2 . ( a ) relative expression of gys-2 in fed , fasted and refed wild - type and ppar-/- mice , as determined by qpcr . mice were fasted for 0,6 , 12 or 24 h , with refeeding for 7 h following 24 h fasting . significant differences between wild - type and ppar-/- mice were observed in the 24-h fasted and the refed state ( b ) relative expression of gys-2 in freshly isolated wild - type and ppar-/- hepatocytes treated for 24 h with vehicle ( dmso ) or wy14643 ( 10 m ) , as determined by qpcr . significant effects were observed by two - way anova for genotype ( p<0.001 ) , and for the interaction between the genotype and wy14643 ( p<0.05 ) . ( c ) relative expression of gys-2 in freshly isolated rat hepatocytes which were treated for 24 h with either vehicle ( dmso ) , wy14643 ( 50 um ) or fenofibrate ( 50 m ) . the effects of wy14643 and fenofibrate were statistically significant ( student s mest : p<0.01 ) . glycogen is higher in refed wild - type mice compared to refed ppar-/- mice , ( a ) representative hematoxylin and eosin staining of liver from two wild - type and two ppar-/- mice , ( b ) representative pas staining of liver from two wild - type and two ppar-/- mice . ( a ) relative expression of gys-2 in fed , fasted and refed wild - type and ppar-/- mice , as determined by qpcr . mice were fasted for 0,6 , 12 or 24 h , with refeeding for 7 h following 24 h fasting . significant differences between wild - type and ppar-/- mice were observed in the 24-h fasted and the refed state ( b ) relative expression of gys-2 in freshly isolated wild - type and ppar-/- hepatocytes treated for 24 h with vehicle ( dmso ) or wy14643 ( 10 m ) , as determined by qpcr . significant effects were observed by two - way anova for genotype ( p<0.001 ) , and for the interaction between the genotype and wy14643 ( p<0.05 ) . ( c ) relative expression of gys-2 in freshly isolated rat hepatocytes which were treated for 24 h with either vehicle ( dmso ) , wy14643 ( 50 um ) or fenofibrate ( 50 m ) . the effects of wy14643 and fenofibrate were statistically significant ( student s mest : p<0.01 ) . identification of a putative ppre in the proximal promoter of the mouse gys-2 gene . to determine what genomic region could be responsible for the ppar - induced up - regulation of gys-2 mrna , the mouse gys-2 gene was scanned for potential ppres ( nubiscan algorithm and hidden markov model framework ) [ 33 , 34 ] . a direct repeat-1 motif ( dr - lprom ) was localized to the proximal gys-2 gene promoter , about 169 bp upstream from the transcription start site . with the exception of two nucleotides , dr - lprom is identical to the consensus sequence , suggesting that this sequence could serve as a functional ppre ( fig . figure 4the dr-1 present in the gys-2 promoter does not mediate ppar - dependent transactivation . ( a ) alignment of the consensus ppre sequence with the sequence of rat and mouse gys-2 dr - lprom and gys-2 dr - lint . ( b ) hepg2 cells were transfected with a reporter vector containing a 553-nucleotide fragment of the proximal mouse gys-2 promoter gene and ppar expression vectors , ( c ) hepg2 cells were transfected with a sv40 reporter vector containing an isolated 156-nucleotide fragment surrounding dr - lprom of the proximal mouse gys-2 promoter gene and ppar expression vectors . luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m of rosiglitazone . chromatin immunoprecipitation of dr - lprom using antibodies against mppar ( d ) or mppar/ ( e ) . the gene sequence spanning dr - lprom and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes . ( f ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . ( a ) alignment of the consensus ppre sequence with the sequence of rat and mouse gys-2 dr - lprom and gys-2 dr - lint . ( b ) hepg2 cells were transfected with a reporter vector containing a 553-nucleotide fragment of the proximal mouse gys-2 promoter gene and ppar expression vectors , ( c ) hepg2 cells were transfected with a sv40 reporter vector containing an isolated 156-nucleotide fragment surrounding dr - lprom of the proximal mouse gys-2 promoter gene and ppar expression vectors . luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m of rosiglitazone . chromatin immunoprecipitation of dr - lprom using antibodies against mppar ( d ) or mppar/ ( e ) . the gene sequence spanning dr - lprom and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes . ( f ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . to examine whether the promoter region containing the putative ppre is responsible for ppar - dependent up - regulation of gys-2 expression , a 553-nucleotide fragment of the mouse gys-2 promoter gene was cloned in front of a luciferase reporter gene and transactivation studies were carried out in hepg2 cells . surprisingly , co - transfection of ppar or pparl expression vectors in combination with ppar agonists slightly decreased luciferase activity , while ppar/ activation had little effect ( fig . transactivation assays performed with a small genomic fragment surrounding dr - lprom cloned in front of sv40-luciferase led to a similar overall ppar - mediated repression for ppar and pparl , while ppar/ had little effect ( fig . co - transfection of rxr or of different co - activators such as cbp and pgcl did not change this pattern ( data not shown ) . thus , the ppre identified in the gys-2 promoter probably does not mediate the effect of ppars on gys-2 expression . nevertheless , chip experiments carried out in 3t3-l1 cells indicated that ( 1 ) ppar was bound to dr - lprom in mature adipocytes , but not in pre - adipocytes ( fig . 4d ) , and ( 2 ) ppar/ was bound to dr - lprom in pre - adipocytes and , more strongly , in mature adipocytes ( fig . 4e ) . thus , despite dr - lprom behaving poorly as a ppre in classical transactivation assay , it binds both ppar and ppar/ in adipocytes . this suggests that in vivo binding of ppar and ppar/ to dr - lprom does not translate into transcriptional activation of the gys-2 gene , and accordingly that activation of gys-2 expression by ppars may be mediated by another genomic region . it should be mentioned that chip did not reveal any binding of ppar to dr - lprom in hepatocytes ( data not shown ) . interestingly , using the same strategy as described above , a putative ppre that is homologous to the consensus dr-1 sequence was identified in intron 1 of the mouse gys-2 gene ( fig . 4a ) . to assess whether dr - lint was able to mediate ppar - dependent transactivation , a 314-nucleotide genomic fragment surrounding dr - lint was cloned in front of the sv40 promoter followed by a luciferase reporter gene . in hepg2 cells treatment with the synthetic ppar agonist wy14643 induced reporter activity and this activation was further enhanced upon co - transfection of mppar ( fig . similar inductions of reporter activity were observed for ppar/ and ppar and their respective agonists ( fig . thus , dr - lint is able to mediate ppar - dependent transactivation , irrespective of the ppar isotype , suggesting that it may at least be partially responsible for ppar - dependent regulation of gys-2 expression . figure 5gys-2 up - regulation by ppars is mediated by a ppre present in intron 1 of the gys-2 gene ( dr - lint ) . ( a ) hepg2 cells were transfected with a 314-nucleotide fragment of intron 1 of the mouse gys-2 gene and ppar expression vectors . luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m rosiglitazone . ( b ) binding of the ppar / rxr heterodimers to dr - lint as determined by gel shift assays . a double - stranded response element containing gys-2 dr - lint was incubated with in vitro transcribed / translated mppar protein ( left panel ) , mppar/ protein ( middle panel ) and mpparl protein ( right panel ) together with in vitro transcribed / translated hrxr protein . fold excess of specific ( malic enzyme ppre ) or nonspecific ( ets oligonucleotide ) cold probe is indicated . gys-2 up - regulation by ppars is mediated by a ppre present in intron 1 of the gys-2 gene ( dr - lint ) . ( a ) hepg2 cells were transfected with a 314-nucleotide fragment of intron 1 of the mouse gys-2 gene and ppar expression vectors . luciferase and -galactosidase activities were determined 24 h after exposure of the cells to different ppar agonists : 50 m wy14643 , 5 m l-165041 and 10 m rosiglitazone . ( b ) binding of the ppar / rxr heterodimers to dr - lint as determined by gel shift assays . a double - stranded response element containing gys-2 dr - lint was incubated with in vitro transcribed / translated mppar protein ( left panel ) , mppar/ protein ( middle panel ) and mpparl protein ( right panel ) together with in vitro transcribed / translated hrxr protein . fold excess of specific ( malic enzyme ppre ) or nonspecific ( ets oligonucleotide ) cold probe is indicated . in agreement with the transactivation data , ppar , ppar/ and ppar proteins a retarded heterodimeric complex was observed only in the presence of both ppar and obligate binding partner rxr ( fig . the complex disappeared in the presence of an excess of cold specific oligonucleotide , but not nonspecific oligonucleotide . examination of in vivo ppar binding to dr - lint by chip yielded very similar results as for dr - lprom : ppar was bound to dr - lint in mature 3t3-l1 adipocytes , but not in pre - adipocytes ( fig . 6a ) , whereas ppar/ was bound to dr - lint in both pre- and mature adipocytes ( fig . chip analysis demonstrated binding of ppar to dr - lint in wild - type but not ppar mice , and binding was enhanced by fasting and wy14643 ( fig . together , these data indicate that mouse gys-2 is a direct ppar target gene and that regulation by ppars is at least partially mediated by a ppre present in intron 1 . figure 6ppar , ppar/ , and ppar bind to the gys-2 dr - lint in vivo . chromatin immunoprecipitation of gys-2 dr - lint using antibodies against mppar ( a ) , mppar/ ( b ) or mppar ( c ) . the gene sequence spanning dr - lint and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes ( a , b ) or mouse liver ( c ) . pi , preimmune serum , cntl , random control sequence , ( d ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . ppar , ppar/ , and ppar bind to the gys-2 dr - lint in vivo . chromatin immunoprecipitation of gys-2 dr - lint using antibodies against mppar ( a ) , mppar/ ( b ) or mppar ( c ) . the gene sequence spanning dr - lint and a random control sequence were analyzed by pcr in the immunoprecipitated chromatin of 3t3-l1 preadipocytes and mature adipocytes ( a , b ) or mouse liver ( c ) . pi , preimmune serum , cntl , random control sequence , ( d ) schematic overview of localization of primers used for amplification of immunoprecipitated dna . gys-2 is a novel direct target of the liver enriched factor hnf4. as explained above , ppar caused a reduction in gys-2 promoter activity via dr - lprom ( fig . a similar decrease of promoter activity in response to ppar despite the presence of a putative ppre has been reported for other genes . indeed , it was found that ppar decreases expression of the apociii and transferrin genes via competition with the hnf4. since hnf4 is known to recognize dr-1 sequences as well , we examined whether hnf4 might control the expression of gys-2 in liver , possibly via dr - lprom . gys-2 mrna levels were markedly decreased in liver - specific hnf4null mice , thus supporting a role for hnf4 in regulating gys-2 expression ( fig . 7a ) . in transactivation assays using the gys-2 promoter , hnf4 markedly activated reporter activity , suggesting the presence of a hnf4 response element within the 0.55-kb promoter fragment ( fig . 7b ) . mutating dr - lprom resulted in an approximately 50 % reduction in hnf4-dependent activation of the gys-2 promoter ( fig . 7c ) , which suggests that ( a ) hnf4 responsiveness is partially mediated by dr - lprom , or ( b ) the mutations with dr - lprom only partially disabled hnf4 responsiveness . regardless of these explanations , these data suggest that hnf4 directly regulates the hepatic expression of gys-2 at least partially via dr - lprom . finally , chip clearly showed hnf4 binding to dr - lprom but not dr - lint in rat primary hepatocytes ( fig . figure 7gys-2 dr - lprom is a binding site for the nuclear receptor hnf4. ( a ) gys-2 mrna levels in liver of liver - specific hnf4-null ( hnf4-/- ) and wild - type ( flox ) mice were analyzed by qpcr ( n=3 per group ) . ( b ) hepg2 cells were transfected with a reporter vector containing 553-bp of the mouse gys-2 proximal promoter and increasing amounts of hhnf4 expression vector , ( c ) hepg2 cells were transfected with a reporter vector containing 553 bp of the wild - type and mutated mgys-2 proximal promoter and hhnf4 expression vector , ( d ) chromatin immu - noprecipitation of gys-2 dr - lprom using antibodies against hnf4. the gene sequence spanning dr - lprom and a control sequence were analyzed by pcr in the immunoprecipitated chromatin of rat primary hepatocytes . ( e ) hepg2 cells were transfected with a reporter vector containing 553 bp of the mouse gys-2 proximal promoter , an expression vector for hhnf4 , and increasing amounts of mppar expression vector . normalized luciferase activity of the mgys-2 reporter vector in the absence of mppar , hhnf4 and wy 14643 was set at 1 . dr - lprom is a binding site for the nuclear receptor hnf4. ( a ) gys-2 mrna levels in liver of liver - specific hnf4-null ( hnf4-/- ) and wild - type ( flox ) mice were analyzed by qpcr ( n=3 per group ) . ( b ) hepg2 cells were transfected with a reporter vector containing 553-bp of the mouse gys-2 proximal promoter and increasing amounts of hhnf4 expression vector , ( c ) hepg2 cells were transfected with a reporter vector containing 553 bp of the wild - type and mutated mgys-2 proximal promoter and hhnf4 expression vector , ( d ) chromatin immu - noprecipitation of gys-2 dr - lprom using antibodies against hnf4. the gene sequence spanning dr - lprom and a control sequence were analyzed by pcr in the immunoprecipitated chromatin of rat primary hepatocytes . ( e ) hepg2 cells were transfected with a reporter vector containing 553 bp of the mouse gys-2 proximal promoter , an expression vector for hhnf4 , and increasing amounts of mppar expression vector . normalized luciferase activity of the mgys-2 reporter vector in the absence of mppar , hhnf4 and wy 14643 was set at 1 . whereas hnf4 activates the gys-2 promoter via dr - lprom , ppar does the opposite , suggesting that ppar may interfere with gys-2 promoter activation by hnf4. to examine whether this is the case , the effect of ppar on hnf4-mediated transactivation of the 0.55-kb gys-2 promoter was studied . ppar activation significantly reduced hnf4-dependent transactivation , indicating competition between hnf4 and ppar in the regulation of the gys-2 promoter ( fig . as already mentioned above , we failed to find any evidence for binding of ppar to dr - lprom in hepatocytes . thus , the inhibitory effect of ppar on transcriptional activation of gys-2 by hnf4 likely does not occur via competition with hnf4 for actual binding to dr - lprom . in the present study , we have identified the mouse gys-2 gene as a direct ppar and hnf4 target gene . we have shown that the effects of ppars and hnf4 on gys-2 expression occur via two distinct response elements . indeed , while transcriptional activation of the gys-2 gene by ppars was found to be mediated by a ppre present in intron 1 of the mgys-2 gene ( dr - lint ) , the stimulatory effect of hnf4 was mediated by a response element in the immediate upstream promoter ( dr - lprom ) . our data are suggestive of the following scenario . in liver , which expresses high amounts of hnf4 , dr - lprom is occupied by hnf4 but not ppar , while dr - lint is bound by ppar but not hnf4. hence , hnf4 and ppar activate gys-2 expression via different response elements . nevertheless , important negative cross - talk between the two nuclear receptors was observed . in the absence of any mutual binding to the response elements , it can be hypothesized that competition may take place at the level of binding to common co - activator proteins in a mechanism that is often referred to as squelching . in adipose tissue , which does not express hnf4 , dr - lprom is occupied by ppar/ and ppar , but this does not result in transcriptional activation . rather , transactivation occurs via binding of ppar/ and ppar to dr - lint . our data indicate that hnf4 is an extremely powerful activator of mouse gys2 transcription , explaining the marked reduction in hepatic gys-2 expression in liver - specific hnf4-null mice . as mentioned above , regulation of mgys-2 expression by hnf4 at least partially occurs via dr - lprom . a recent study that combined chip with promoter micro - arrays showed that the gys-2 promoter is bound by hnf4 in human liver , thus establishing gys-2 as a direct target of hnf4 in human as well . it is not very clear why disabling the dr - lprom reduced hnf4-dependent trans - activation by only 50 % . it is possible that the 0.55-kb gys-2 promoter fragment contains an additional hnf4 response element , although in silico analysis failed to reveal such an element . alternatively , it is possible that mutating the wild - type dr - lprom ( aggccaaaggcca ) into a mutated dr - lprom ( aggcctttggcca ) only partially disabled the response element . while functional ppres are commonly located within regulatory sequences , i.e. , proximal promoters , ppres have also been identified in intronic sequences . examples are ppres within intron 3 of human / mouse angptl4 and rat peroxisomal thiolase b genes , and within intron 1 of the rat acyl - coa binding protein gene and human carnitine palmitoyltransferase 1a [ 25 ] . our data demonstrate that regulation of gys-2 expression by ppars is also mediated by an intronic ppre . in the past few years , our understanding of the function of ppar/ in vivo has improved greatly thanks to studies using various transgenic mouse models [ 19 , 37 ] . at the level of metabolism , ppar/ over - expression promotes skeletal muscle fatty acid oxidation and type i fiber content in mice , resulting in improved endurance exercise performance . conversely , deletion of ppar/ in cardiomyocytes is associated with impaired fatty acid oxidation and expression of fatty acid oxidative genes , whereas glucose uptake is increased . in wat , ppar/ stimulates fatty acid oxidation and uncoupling , thereby diminishing adiposity . it is thus clear that ppar/ plays a pivotal role in governing fatty acid oxidation in a variety of tissues . in contrast , data linking ppar/ to regulation of glucose homeostasis remain scarce [ 20 , 39 , 40 ] . our data reveal that ppar/ is a critical regulator of the adipose expression of the gys-2 gene . furthermore , micro - array and qpcr analysis indicated that expression of numerous other genes involved in lipid and glucose metabolism was markedly down - regulated in ppar/-null mice , including glut4 , p85 , and cd36 . since ppar and pgc - l were significantly down - regulated as well , it is possible that many of the observed changes are not linked to the absence of ppar/ per se but rather reflect indirect effects mediated via decreased ppar and pgc - l mrna . although such an effect may contribute to some extent to the down - regulation of gys-2 in ppar/-null mice , the in vitro studies leave no doubt that gys-2 is a direct target gene of ppar/ , as well as of ppar. glycogen is stored in many tissues , yet it is particularly abundant in liver , muscle , and adipose tissue . in liver , glycogen serves to maintain blood glucose levels between meals , while skeletal muscle glycogen is used to fuel muscle contractions . in contrast , adipose tissue glycogen serves as a source of glycerol 3-phosphate , which is required for ( re)-esterification of fatty acids into triglycerides . several alternative pathways exist to produce glycerol 3-phosphate , including synthesis from glucose , and conversion of gluconeogenic precursors ( glyceroneogenesis ) . since expression and activity of glycerol kinase are very low in adipose tissue , direct phosphorylation of glycerol is not considered as a major pathway to generate glycerol 3-phosphate . however , recent studies suggest that this may change after treatment with synthetic ppar agonists , which markedly up - regulate glycerol kinase expression in human and mouse adipocytes [ 11 , 42 ] . in fact , it has been hypothesized that stimulation of glycerol kinase expression by tzds , resulting in increased fatty acid re - esterification , may at least partially account for the suppressive effect of tzds on plasma free fatty acid levels . stimulation of fatty acid esterification is part of a general lipogenic and adipogenic effect of ppar in the adipocyte . since adipose glycogen stores yield glycerol 3-phosphate as a precursor for fatty acid ( re-)esterification , up - regulation of gys-2 expression by ppar can be placed in the context of the lipogenic role of ppar in the adipocyte , which is aimed at promoting energy storage . besides contributing to lipogenesis , synthesis of glycogen permits continued uptake of glucose into cells . accordingly , it can be speculated that up - regulation of adipose gys-2 by ppar might partially account for the stimulation of glucose uptake into adipocytes by ppar agonists . it is currently still ambiguous whether ppar/ serves a general anabolic or catabolic function in the adipocyte . on the one hand , it has been reported that ppar/ promotes fatty acid oxidation in adipocytes [ 20 , 21 ] . on the other hand , ppar/ also seems to have a facilitative , yet important role in lipo- and adipogenesis . as discussed above for ppar , up - regulation of gys-2 expression by ppar/ may indicate a role for ppar/ in fatty acid ( re-)esterification , thus contributing to a lipogenic role for ppar/. the highest levels of glycogen are found in liver and fluctuate with nutritional status . remarkably , expression of gys-2 in liver increases during fasting , at the same time when glycogen stores are actively broken down . the reason behind this seemingly counterintuitive regulation is not very clear , but it may serve to prime the glucose synthesizing system for when dietary glucose becomes available again . in the absence of ppar , we observed that the expression of gys-2 drops markedly during prolonged fasting and refeeding . the reduced gys-2 expression is likely responsible for the diminished rate of glycogen formation upon refeeding , as observed by us and previously by others . indeed , the effect of ppar deletion on liver glycogen is minor except under conditions of refeeding [ 3032 ] . it has been reported that after a short - term fast the gluconeogenic flux in ppar-null mice is directed more towards glycogen , leading to a decrease in hepatic glucose output . however , it is unclear what happens to the gluconeogenic flux toward glycogen in the fasted - refed state , although our and other data clearly indicate that total glycogen synthesis is decreased in ppar-/- mice . mutation of the gys2 gene in humans leads to lower hepatic glycogen levels and fasting hypoglycemia , biochemical features that are also observed in ppar-/- animals . opposite to that observed in patients with a dysfunctional gys2 gene , ppar-/- mice show low plasma ketones , which is explained by the stimulatory effect of ppar on fatty acid oxidation and ketogenesis . overall , our data suggest that the decreased hepatic glycogen levels in ppar-/- and liver - specific hnf4-null mice [ 28 , 35 ] may be due to decreased activation of gys-2 expression via dr - lint and dr - lprom , respectively . although ppar and hnf4 stimulate gys-2 expression via different response elements , important interplay exist between signaling of the two nuclear receptors . in conclusion , we show that gys-2 is a direct target gene of ppars .

abstract.glycogen synthase 2 ( gys-2 ) is the ratelimiting enzyme in the storage of glycogen in liver and adipose tissue , yet little is known about regulation of gys-2 transcription . the peroxisome proliferator - activated receptors ( ppars ) are transcription factors involved in the regulation of lipid and glucose metabolism and might be hypothesized to govern glycogen synthesis as well . here , we show that gys-2 is a direct target gene of ppar , ppar/ and ppar. expression of gys-2 is significantly reduced in adipose tissue of ppar-/- , ppar/-/- and ppar+/- mice . furthermore , synthetic ppar/ , and agonists markedly up - regulate gys-2 mrna and protein expression in mouse 3t3-l1 adipocytes . in liver , ppar deletion leads to decreased glycogen levels in the refed state , which is paralleled by decreased expression of gys-2 in fasted and refed state . two putative ppar response elements ( ppres ) were identified in the mouse gys-2 gene : one in the upstream promoter ( dr-1prom ) and one in intron 1 ( dr-1int ) . it is shown that dr-1int is the response element for ppars , while dr-1prom is the response element for hepatic nuclear factor 4 alpha ( hnf4 ) . in adipose tissue , which does not express hnf4 , dr-1prom is occupied by ppar/ and ppar , yet binding does not translate into transcriptional activation of gys-2 . overall , we conclude that mouse gys-2 is a novel ppar target gene and that transactivation by ppars and hnf4 is mediated by two distinct response elements .

Introduction Materials and methods Results Expression of Gys-2 in WAT is regulated by PPARs. Discussion

PMC3982805

animals and treatment : the experimental design was approved by the laboratory animal care and use committee of iwate university , iwate , japan . eight clinically healthy holstein bull calves , aged 10 3 weeks , were purchased from a government farm ( national livestock breeding center , morioka , japan ) and were housed in a 4 5 m , open - sided straw bed and naturally ventilated barn at the cattle research center , iwate university . the calves had been weaned within 4 weeks after birth and fed starter pellets containing ground corn ( standard diet ) and had access to fresh water ad libitum . calves were assigned to a 4 2 experimental design for the probiotic and control groups . a probiotic ( miyarisan pharmaceutical co. , ltd . , tokyo , japan ) , which included lactobacillus plantarum strain 220 [ 9 10 colony forming units ( cfu)/g ] , enterococcus faecium strain 26 ( 9 10 cfu / g ) and clostridium butyricum strain miyari ( 9 10 cfu / g ) , was administered daily at 3.0 g/100 kg body weight ( bw ) to the calves once daily for 5 consecutive days . the probiotic was mixed with 50 ml tap water in a beaker , and the probiotic suspension was administered orally to each calf in a 50 ml drencher in the morning , prior to feeding . calves given tap water alone , with no probiotic , served as the control . no abnormal changes in the health of any calf were observed during the experimental period . blood collection and pbmc isolation : blood was drawn by jugular venipuncture from each calf at predose and on days 7 and 14 . the first administration day was considered day 1 . a blood specimen ( 6 ml ) was collected in a heparinized tube ( bd vacutainer , belliver industrial estate , plymouth , u.k . ) and used for pbmc isolation for flow cytometry analysis . blood ( 4 ml ) was also collected in two edta tubes ( bd vacutainer , franklin lakes , nj , u.s.a . ) and used for total rna extraction from peripheral leukocytes and white blood cell ( wbc ) counts . wbc count was determined using an automatic hematology analyzer device equipped with software for bovine samples ( poch-100iv diff , sysmex , kobe , japan ) . the percentage of pbmcs was determined using a hemogram method with the dif - quick staining protocol . briefly , to purify pbmcs and to lyse red blood cells , 2 ml heparinized blood was mixed with 8 ml 0.83% ammonium chloride solution . after 5 min at room temperature , samples were centrifuged ( 2,000 rpm , 10 min , 4c ) . leukocytes were purified by washing twice in phosphate - buffered saline ( pbs , ph 7.4 ) followed by centrifugation ( 2,000 rpm , 5 min , 4c ) . after the final wash , the cell pellet was diluted in 500 l pbs , and cells were counted . then , the cell concentration was adjusted to 1 10/ml in pbs , and the cells were kept at 4c until used for the flow cytometry assay . flow cytometry assay : the primary monoclonal antibodies used in the present study are shown in table 1table 1.monoclonal antibodies used for flow cytometry assayitems target molecule specificity mab clone isotype supplier cd3 tcr complex pan - t cells mm1a igg1vmrd cd4 cd4 moleculet - helper cells cact183aigg1vmrd cd8 cd8cytotoxic t cells bat82aigg1vmrd cd45r ptpr pan - leukocytes gc6a igmvmrd wc1 unknown t cellsil - a29igg1vmrd cd14 lps receptor monocytes , macrophages cam36a igg1vmrd cd21 cr2 b cells baq15aigmvmrd cd282 tlr2 monocytes hca151f igg abd - serotec tcr , t cell receptor ; ptpr , protein tyrosine phosphatase receptor ; lps , lipopolysaccharide ; cr2 , complement component receptor 2 ; tlr2 , toll - like receptor 2 .. flow cytometry analysis was performed as described previously . briefly , pbmcs were labeled with primary antibody and incubated for 60 min at 4c . cd3 t cells and each of cd4 and cd8 t cell subsets were additionally labeled with cd45r monoclonal antibody to mark pan - leukocytes . labeled cells were stained with specific purified igg conjugated to phycoerythrin ( pe ) polyclonal secondary antibody ( goat anti - mouse igg1 rpe , abd serotec , kidlington , oxford , u.k . ) or fluorescein isothiocyanate ( fitc)-conjugated antibody ( goat anti - mouse igg or igm fitc , southern biotech , birmingham , al , u.s.a . ) and incubated for 30 min at 4c . flow cytometry was performed using a facscan analyzer ( becton dickinson , franklin lakes , nj , u.s.a . ) , equipped with a computer running the cell quest software ( becton dickinson , milan , italy ) . tcr , t cell receptor ; ptpr , protein tyrosine phosphatase receptor ; lps , lipopolysaccharide ; cr2 , complement component receptor 2 ; tlr2 , toll - like receptor 2 . total rna extraction from leukocytes and cdna synthesis : total rna was extracted from 1 ml whole blood leukocytes using the sv total rna isolation system ( promega , tokyo , japan ) . trace genomic dna in the crude total rna samples was removed by incubation with 46 units dnase i per 100 mg total rna ( ambion , austin , tx , u.s.a . ) for 30 min at 37c . the concentration of total rna was verified on a 1% agarose gel , and purity was determined using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , rockland , de , u.s.a . ) . cdna was prepared using an oligo(dt ) primer and a thermal cycler ( mycycler ; bio - rad , tokyo , japan ) . real - time pcr assay : real - time pcr analysis was carried out using the iq sybr green supermix kit ( bio - lad ) as described previously . specific individual cytokine primer sequences and the internal standard gene -actin used are shown in table 2table 2.primers used for real - time pcrgenegenbank access . no.sequence ( 53 ) annealing temp ( c ) concentration ( nm ) -actin ay141970f : ggccgagcggaaatcg r : gccatctcctgctcgaagtc65750 il-6 nm173923f : gtcttcaaacgagtgggtaaag r : tgaccagaggagggaatgc65250 il-8 nm173925f : cactgtgaaaattcagaaatcattgtta r : cttcacaaatacctgcacaaccttc59250 tnf- z48808 f : cggtggtgggactcgtatg r : gctggttgtcttccagcttca65 750 ifn- nm174086 f : taggcaagtctatgggatttc r : gcattcattacatcatcaagtg59 250 . amplification was carried out using a mini opticon ( bio - rad ) thermal cycler with the following profile : one cycle of 95c for 2 min , followed by 45 cycles of 95c for 30 sec , 65 or 59c for 30 sec and 74c for 30 sec , followed by 74c for 7 min . melting curve analysis was performed from 50 to 95c , read every 0.5c holding for 5 sec . relative gene expression was calculated using the comparative threshold cycle number ( 2 ) method , as described by livak and schmitge . results are summarized as ct values , where ct is the difference in the threshold cycle for the target and -actin , as an internal control . statistical analysis : the number of cd - positive or cd - negative cells was calculated from the percentages of leukocytes gated by flow cytometry analysis and total pbmcs . 5.01 ) was used for statistical calculations , and one - way repeated - measures analysis of variance followed by the tukey - kramer multiple comparison method was used to evaluate differences in values among the groups . subpopulations of peripheral leukocytes : the number of cd - positive cells is shown in figs . 1 and 2fig.1.number of cd3 , cd4 , cd8 , cd14 , cd21 and wc1 cells in the peripheral blood of calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black columns ) . * values in the treatment group compared to the predose day in the same group ( p<0.05 ) . 2.number of tlr2 ( cd282 ) cells in the peripheral blood of calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black columns ) . numbers were counted at predose ( pre ) and on days 7 and 14 . * values in the treatment group compared to the predose day in the same group ( p<0.05 ) . the first administration day of the probiotic was deemed day 1 .. compared to the value on the predose day and the control value on the same day , increased ( p<0.05 ) numbers of cd3 t cells ( cd3 cd45r / cd45r ) , wc1 t cells and cd282 monocytes were noted in the treatment group on day 7 . the values remained higher on day 14 ; however , the difference was not statistically significant . compared to the value on the predose day , cd8 t cells were increased ( p<0.05 ) in the treatment group on day 7 . compared to the control value on the same day , the cd4 t cell subset was also increased ( p<0.05 ) in the treatment group on day 7 . the values of cd3 , cd4 and cd8 cell subsets decreased slightly from day 7 to day 14 , and the differences were not statistically significant on day 14 . discrete analysis of data indicated that the numbers of cd45r and cd45r t cells were increased ( p<0.05 ) in the cd3 cells in the treatment group . however , the values were increased ( p<0.05 ) only in the number of cd4cd45r and cd8cd45r t cells on day 7 compared to the values on the predose day and/or the control ; they were not significantly different among the groups on day 14 ( table 3table 3.the numbers of peripheral blood mononuclear cell ( pbmc ) and t cell subpopulations ( cell/l ) in probiotic - treated and control calvestreatment controlpre day 7day 14pre day 7day 14pbmc 5,283 193 6,323 3185,650 260 4,836 346 4,467 415 5,303 513 cd3cd45r1,425 117 2,199 123 * 1,940 136 1,321 157 1,431 206 1,777 202 cd3cd45r727 60 1142 158 * 998 82 668 92 729 76 857 106 cd4cd45r191 47 315 44244 52 201 35 171 37 228 42 cd4cd45r326 62 625 79 * 394 39 328 47 305 59 333 49 cd8cd45r53 80 77 1065 12 68 13 65 28 69 13 cd8cd45r209 41 526 89 * 340 41 207 28 304 84 280 43 a ) calves received 3.0 g/100 kg of probiotic for 5 consecutive days , and additional calves given the vehicle alone served as the control . compared to the predose ( pre ) values in the same group ( p<0.05 ) . # compared to the values in the control on the same day ( p<0.05 ) . ) . the numbers of cd14 and cd21 cells changed slightly in the treatment group on days 7 and 14 , but there was no statistically significant difference between the treatment and control groups . number of cd3 , cd4 , cd8 , cd14 , cd21 and wc1 cells in the peripheral blood of calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black columns ) . * values in the treatment group compared to the predose day in the same group ( p<0.05 ) . number of tlr2 ( cd282 ) cells in the peripheral blood of calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black columns ) . numbers were counted at predose ( pre ) and on days 7 and 14 . values in the treatment group compared to the predose day in the same group ( p<0.05 ) . a ) calves received 3.0 g/100 kg of probiotic for 5 consecutive days , and additional calves given the vehicle alone served as the control . compared to the predose ( pre ) values in the same group ( p<0.05 ) . # compared to the values in the control on the same day ( p<0.05 ) . mrna expression of cytokines : expression of il-6 and ifn- mrna was increased slightly in the treatment group on day 7 compared to the control , and the values were increased ( p<0.05 ) on day 14 compared to the predose day ( fig . 3fig . 3.relative mrna expression of il-6 , il-8 , ifn- and tnf- in peripheral leukocytes of calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black columns ) . * values in the treatment group compared to the predose day in the same group ( p<0.05 ) . expression of il-8 mrna was similar between the treatment and control groups on days 7 and 14 . expression of tumor necrosis factor - alpha ( tnf- ) was slightly higher on days 7 and 14 in the treatment group , but the differences were not statistically significant ( p=0.07 ) between the treatment and control groups . relative mrna expression of il-6 , il-8 , ifn- and tnf- in peripheral leukocytes of calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black columns ) . * values in the treatment group compared to the predose day in the same group ( p<0.05 ) . certain probiotics have been suggested to have immune - stimulating properties in pre - weaned calves and adult cattle [ 14 , 15 , 24 ] . in the present study , toll - like receptor 2 ( tlr2 ; cd282 ) cell surface molecule antibody was used as a marker to distinguish monocytes in peripheral blood of healthy weaned calves receiving probiotic and a control group . thus , in addition to cd282 cells , the numbers of cd3 t cells , cd4 , cd8 and wc1 t cell subsets and cd14 and cd21 cells were evaluated . likewise , the expression of cytokine mrna , such as il-6 , il-8 , inf- and tnf- , was examined to assess the possible immune - stimulating effects of the probiotic on the activation of peripheral leukocytes . in a previous study , the number of cd4 cells was higher after repeated administration of a probiotic consisting of clostridium butyricum strain miyairi in dairy cows . in another study , cd8 cells were stimulated in the peripheral blood of pre - weaned calves by administration of a bacteria - based probiotic . furthermore , a number of in vitro and in vivo studies on human pbmcs have suggested that a probiotic containing e. faecium , c. butyricum or lactobacillus subspecies shows enhancing effects on t cell subsets and monocytes [ 12 , 35 ] . in young mammals , most cd3 , cd4 and cd8 cells express the high - molecular - weight isoform of cd45r , and an increase in the number of t lymphocytes expressing cd45r facilitates regular activation of t cells . in the present study , the increase in the number of cd3cd45r t cells in the treatment group , compared to the predose day or control , may indicate a nonspecific , but enhanced immune state with possibly multiple causes after the 5-day repeated administration of the probiotic . similar to cd3 cells , increased numbers of cd4cd45r and cd8cd45r t cells were observed . they may be involved in immune system responses , along with direct or indirect immune - stimulatory effects of the probiotic in the calves , as suggested by previous studies in cattle [ 6 , 15 , 24 ] . the wc1 molecule is a member of the scavenger receptor cysteine - rich ( srcr ) family found on t cells , which are negative for cd4 and cd8 . in cattle , the regulatory potential of wc1 t cell subsets is comparable to that of cd14 cells , and the wc1 t cell subpopulation is larger in calves . bovine lymphocyte subsets are activated indirectly through cytokines secreted by t cells , antigen - presenting cells ( apcs ) and other accessory cells . as our results show , the higher number of wc1 t cells in the calves receiving the probiotic was associated with an increased number of cd4 cells , which might suggest stimulation of cellular immunity in the calves . although wc1 t cells and cd14 cells are generated comparably in bovines , we found that there were more wc1 t cells than cd14 cells . on the other hand , wc1 cells are immune suppressors , secreting ifn- in bovines [ 11 , 27 ] , consistent with our results . the expression of tlrs has been correlated with the population of total bacteria and lab in the gastrointestinal tract , including the rumen of calves , during the first 6 months of life . moreover , the expression of tlr2 was evident on bovine antigen presenting cells with high level expression on peripheral blood monocytes and monocyte - derived macrophages . the tlr2 cell surface molecule on bovine monocyte - derived dendritic cells ( dcs ) and gastrointestinal macrophages is most responsible for recognition of gram - positive bacteria [ 4 , 9 , 10 ] . mechanisms indicating a direct role of tlr2 include bacterial reorganization by epithelial macrophages in calves . the transcytosis processing of bacterial particles via ileal peyer s patch m cells and the later appearance of tlrs on dcs in the lamina propria play a key role in the mechanism of probiotic immunomodulation in cattle [ 7 , 20 , 21 ] . thus , dcs are the principal stimulators of t cells [ 7 , 12 ] . the tlr signaling pathway is involved with the actions of a probiotic containing c. butyricum , and the absence of tlr2 expression might indicate the need for tlr2 in the recognition of clostridium bacteria [ 9 , 10 ] . in the present study , we provide evidence for an increased number of tlr2 monocytes in the peripheral blood of calves receiving a probiotic consisting of three species of gram - positive bacteria , including c. butyricum . the increased number of peripheral monocytes expressing tlr2 might be associated with the formal reaction of innate immunity in response to increasing and changing bacterial diversity in the gastrointestinal tract of the calves receiving the probiotic . furthermore , the cd14 cell surface molecule is essential for the response to most gram - negative bacteria , including lipopolysaccharides , via the tlr4 and md2 complex [ 22 , 28 ] . the tlr2 molecule recognizes microbial - associated molecule patterns independently [ 4 , 7 ] . although some differences were noted between the different antibodies assessed the expression of tlr2 and cd14 monocytes in bovine . moreover , tlr2 is expressed on peripheral blood b lymphocytes ; however , a b cell receptor complex including the cd21 molecule is present on mature b cells and also ileal peyer s patch - derived b lymphocytes in healthy calves . similar to these reports , the numbers of cd14 and cd21 b cells in the present study were slightly increased in the calves on days 7 and 14 . probiotics including lactobacillus and c. butyricum induce the release of proinflammatory cytokines , such as il-6 and tnf- , which are involved in nonspecific immune responses [ 9 , 10 ] . a probiotic consisting of e. faecium and c. butyricum upregulates the level of ifn- and downregulates tnf- in the supernatant of human pbmcs . in our study , increased expression of il-6 , inf- and tnf- mrna was observed in the treatment group on days 7 and 14 . increased expression of il-6 , ifn- and tnf- in peripheral leukocytes may be dependent on the period after administration and numbers of activated t lymphocytes or monocytes in the treatment group . , in which increased expression of ifn- was observed only 2 weeks after trickle stimulation of cultured pbmcs from calves . on the other hand , expression of il-8 is related to neutrophil function , especially inflammatory stimulation , in bovine . in the present study , the expression of il-8 was unchanged compared to the predose day and the control value on the same day , likely because the calves were healthy . in conclusion , repeated 5-day administration of a bacteria - based probiotic resulted in increased numbers of cd3 t cells , cd4 , cd8 and wc1 t cell subsets and cd282 monocytes in healthy holstein calves . moreover , higher mrna expression of pro - inflammatory cytokines , such as il-6 , inf- and tnf- , in peripheral leukocytes was noted . these results suggest that the peripheral blood leukocytes and cellular immune function in the healthy calves may have been stimulated via effects on the gastrointestinal microbiota , which generally increased , with overall stability of the rumen bacterial flora , by the oral probiotic treatment .

abstracteight holstein calves ( 10 3 weeks ) were used to examine the interaction between a bacteria - based probiotic agent ( probiotic ) and the function of peripheral blood mononuclear cells ( pbmcs ) . the probiotic , consisting of lactobacillus plantarum , enterococcus faecium and clostridium butyricum , was administered orally at 3.0 g/100 kg body weight to calves once daily for 5 consecutive days . calves given the vehicle alone with no probiotic served as the control . in the treatment group , increases in numbers of cd282 + ( tlr2 ) monocytes , cd3 + t cells and cd4 + , cd8 + and wc1 + t cell subsets were noted on day 7 post - placement compared to predose day and the control group . expression of interleukin ( il)-6 , interferon - gamma ( inf- ) and tumor necrosis factor - alpha ( tnf- ) was elevated in peripheral leukocytes on days 7 and 14 . these results suggest that peripheral blood leukocytes in healthy calves may be stimulated via the gastrointestinal microbiota , which was increased by the oral probiotic treatment , with overall stability of the rumen bacterial flora . the 5-day repeated administration of a bacteria - based probiotic may enhance cellular immune function in weaned calves .

MATERIALS AND METHODS RESULTS DISCUSSION

PMC3136081

hibernation is a behavioural and physiological adaptation of endothermic animals which enhances survival during extended seasonal periods of reduced food supply and low ambient temperature when core body temperatures can be close to 0c . the ability to hibernate is found throughout the class mammalia and involves differential expression of genes common to all mammals , rather than induction of novel gene products unique to hibernation . most hibernators periodically interrupt the state of hibernation ( torpor ) by euthermic episodes or arousal , a process responsible for up to 90% of the energy consumed during hibernation . previously , using golgi and electron microscope studies in hippocampal tissue of hibernating ground squirrels we have shown marked structural alterations in the components of neural circuitry , with the reversible retraction of dendritic spines and synapses in ca1 and ca3 [ 1 , 2 ] . with the exception of hibernators , mammals as a whole are much more sensitive to hypothermia , than are their individual constituent cells . even though the optimum growth of mammalian cells occurs at 3537c , in culture many continue to grow and divide at 2533c , although substantially more slowly than at optimal body temperatures . prolongation of the cell cycle at suboptimal ( 25c33c ) temperatures is attributed to a temporal expansion of the g1 and s phases , with g2 being the least sensitive . however , research into the process by which hypothermia affects the cell cycle has been largely neglected . this is despite the fact that work on the response of mammalian cells to lower than optimal temperatures , first published in the mid 20th century , suggested several intriguing effects on cell cycle progression . although the mechanisms involved remain largely unexplored , a sudden but severe cold shock can be used to generate a high degree of cell cycle synchrony in some mammalian cultures . other hypothermic regimes can also be used to enrich cultures for g2 cells and to significantly slow otherwise rapid cellular processes , including mitosis . in this respect hibernating mammals provide a rich resource to study the process of cell division and neurogenesis . neurogenesis in the mammalians in hippocampus persists through adulthood mainly within the two neurogenic structures , the dentate gyrus of the hippocampus and the subventricular zone of the forebrain [ 58 ] . in these areas neural progenitor cells continuously divide and give birth to new neurons [ 5 , 6 ] . previous studies have demonstrated that behavioural and physiological stimuli , such as learning , voluntary wheel running exercise , kindling , and environmental enrichment , enhance hippocampal neurogenesis . however , although the veracity of hippocampal neurogenesis is now generally accepted , there is uncertainty surrounding the extent of adult neurogenesis . moreover , whether neurogenesis involves only progenitor cells or also more mature hippocampal cells is unclear and the absence of definitive phenotypic markers at ultrastructural level has limited the development of the field . investigations initiated in the 1970s by kaplan using h - thymidine light microscopic autoradiography and ultrathin sections at electron microscope level reexamined the initial observations and provided evidence not only that could neurogenesis occur in the adult brain , but also that the cells appear ultrastructurally , similar to sister cells in the dentate gyrus of the hippocampus , one of the structures shown to be neurogenic [ 1214 ] . also , studies performed by gould and mcewen demonstrated that mitosis and apoptosis in hippocampus are regulated by adrenal steroids , possibly through excitatory amino acids which may also participate in the development and maintenance of the dentate gyrus . the siberian ground squirrel ( spermophilus undulatus ) which has adapted to survive under very low environmental temperatures and a short summer season is a unique animal for investigation of phenotypic metabolism changes [ 1618 ] this is because there are 2 euthermic states : ( i ) a winter interbout state and ( ii ) summer animals , with typical features for nonhibernating rodents . in the present study we have utilised this natural hibernation model in the adult arctic ground squirrel to study mitosis at different stages of cell cycle . our data suggest that immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus . adult ground squirrels , spermophilus undulatus , of both sexes and 600700 g in weight , estimated as between 2 and 3 years of age were caught in yakutiya ( siberia ) and kept in individual cages in a cold vivarium under natural photoperiodicity . food was supplemented with sunflower seeds and carrots , and nesting material was provided ad libitum . in november , the animals were individually placed in wooden hibernation boxes ( 20 20 25 cm ) and transferred to a dark room having a temperature of 13c . functional states were initially characterized by telemetry of heart rate ( hr ) and body temperature ( tb ) , by using a special transducer . later , continuous monitoring of functional states was performed simply by recording the nest temperature . at the bottom of the wooden animal box , a thermistor ( sensitivity 0.2c ) the temperature of the bedding was in the range 14c , whereas in interbout events it increased to 1420c . the course of torpor - activity cycles could thus be monitored in individual animals and allowed us to predict the length of a subsequent bout . samples were taken from animals killed in the predicted middle of a hibernation bout . in december , hibernation bouts had a 1-week duration , but this became 1.52 weeks in january - february . hr was determined by using electrodes fixed onto the skin of the shoulder blade and the left foreleg . directly after the start of perfusion procedure , the heart temperature ( th ) was determined with an electrical point thermometer ( sensitivity 0.2c ) . the functional states were defined as follows : ( i ) deep torpor , in the middle of an extended bout of deep hibernation ( usually 3 to 4 days of hibernation bout ; brain temperature 24c ; hr 68 beats per min ) ; ( ii ) interbout ( usually at the second day after natural arousal ) , quasiactive animals , which had increased their brain temperature spontaneously to about 36c between two hibernation bouts ; s. undulatus relies on its fat depots and does not need food in these brief intervals of normothermic arousal ( hr 150300 beats per min ) ; ( iii ) 2.5 h provoked arousal : 22.5 h awakening from the middle of hibernation bout , provoked by transfer to a warm room . in the last case the provoked awakened ground squirrels had a tb of 34c ( hr 300400 beats per min ) . three animals from each group received an intraperitoneal injection of brdu ( 5-bromo-2-deoxyuridine ; sigma ) in dose 150 ug / g body weight in sterile 0.9% nacl solution . the animals were injected according to their group ( 3 - 4 animals per group ) as follows . using nest temperature monitoring ( as above ) 4 animals were chosen with interbout normothermic interval 2 days . 1 day before entrance in hibernation brdu injection was done in interbout / normothermic animals ( tb 35 - 36c ) . after 3 days of hibernation the animals were perfused with solution temperature similar to tb 46c . animals were sacrificed with an overdose of ketamine and perfused transcardially with 100 ml physiological saline in 0.01 m phosphate buffer ( ph 7.4 ) ( pbs ) , followed by 300 ml of 0.5% glutaraldehyde and 3% paraformaldehyde in 0.05 m cacodylate buffer ( ph 7.4 ) . the brains were stored in the fixative overnight . all antibodies were diluted in 0.1 m phosphate buffer ( ph 7.4 ) containing 0.05% triton x-100 and 2% fish gelatin ( sigma ) ( incubation buffer ) . the primary antibodies used in this study were monoclonal rat anti - brdu ( cat . ab6326 , abcam , uk ) , 1 : 300 , and polyclonal rabbit anti - dcx ( cat . ab18723 , abcam , uk ) , 1 : 700 . for immunohistochemistry with the peroxidase technique , biotinylated donkey anti - rat igg ( cat . 712 - 065 - 150 , jackson , usa ) , 1 : 200 and biotinylated donkey antirabbit igg ( cat . 711 - 065 - 152 , jackson , usa ) , 1 : 100 , were used as secondary antibodies and detected with avidinbiotin - peroxidase complex ( abc , vectastain elite , vector laboratories , uk ) ( 9 l / ml ) . after quenching excess of aldehyde groups with 1% sodium borohydride in pb for 30 minutes the 50 um sections were incubated in 2 m hcl for 30 min at 37c and washed in borate for 5 minutes . the sections were blocked in incubation buffer ( ib ) for 2 hours , followed by incubation in primary antibody in ib overnight at 4c . after rinses in pb , the sections were incubated in the secondary antibody in ib for 4 hours at room temperature . after another set of rinses , abc elite reagent ( vector laboratories , uk ) was applied for 1 hour . as substrate for the peroxidase reaction , diaminobenzidine ( dab , sigma , usa ) was applied for 5 minutes at a concentration of 0.22 mg / ml in tris buffer ( ph 7.4 ) with 0.01% hydrogen peroxide . sections were thoroughly washed , mounted , air - dried , dehydrated , and cover - slipped . to control for nonspecific labelling , adjacent sections were incubated without primary or secondary antibody ; no labelling was detected following this procedure . statistica ( statsoft , tulsa , okla , usa ) was used to obtain means sds to perform statistical analyses , and the kolmogorov - smirnov and shapiro - wilk normality tests to compare distributions ( criterion p < .05 ) . one - way anova test followed by bonferroni 's or tukey 's unequal n honest significant differences tests was performed with the originpro 7.5 . figures 1(a ) , 1(b ) , 1(c ) and 1(d ) show the distribution of immature neurons with dcx - immunoperoxidase staining in the dorsal portion of dentate gyrus in the 4 different functional states as previously described in , which corresponds to the distribution of newborn neurons in this brain region observed using other markers for newly generated neurons [ 1923 ] . the sgz appears discontinuous because dcx - labelled granule cells formed clusters [ 3 , 19 ] and the appearance of the labelled cells was similar to mature granule cells . dcx - positive cells were mainly found in the sgz at the hilar border . labelled cells with branched apical dendrites were found only in summer animals , and many had bifurcating and trifurcating processes ( figure 1(a ) ) . comparative analysis of dcx - labelled dentate gyrus ( dg ) cells in summer ( figure 1(a ) ) , and hibernating animals ( figure 1(d ) ) , shows retraction of dendritic branches during hibernation supporting previous data on reversible retraction of dendritic branches . the appearance of these dcx - labelled neurons was similar to that of dg cells . it is notable that in summer , normothermic animals showed intensive labelling of neurons in layer ii of entorhinal cortex ( figure 1(e ) ) . axons of neurons from layer ii of the perforant path input directly to the dg and both entorhinal cortex and dg participate in spatial memory . these data suggest that immature neurons may persist into adulthood rodents and that these cells appear to undergo development and differentiation . it is possible that these dcx immunoreactive neurons enable the high degree of plasticity of the neuronal network . our study was designed only to show that mitotically active granule cells were localized in the dg . theoretically brdu insertion occurs during dna repair and may not detect a distinct proliferative state . however ; we did not observe brdu labelling of either pyramidal neurons or interneurons in different brain regions where there could be intensive dna repair . hypothermia can stop the cell in any phase of the cell cycle , though mainly the g2 phase . for mitotically active intestinal epithelial cells during hibernation the mitotic index in the intestinal crypts of ground squirrels entrance into hibernation stimulated suspension of the cells in different phases of the cell cycle . figure 2 shows brdu immunoreactive cells in 3 different phases of mitosis : metaphase , anaphase , and telophase including ultrastructural patterns of metaphase immature granule neuron . figure 3 shows that after telophase many brdu - labelled cells in the dg formed cellular doublets . brdu - labelled cells such as those shown in figures 2 and 3 originate apparently from the dg layer supporting the idea that mitosis occurs within dcx clusters . previously using serial ultrathin sections we found that , within dcx - labelled clusters of immature granule cells , neurons are present during mitosis . show interphase and metaphase granule cells where definitive markers are somatic synapses that are absent in mature granule cells . uncovering such an event is exceptional because the cell cycle time is approximately 24.7 hours [ 25 , 27 ] whilst the duration of mitosis has been assessed as only 10% of total time of the cell cycle . by determining the length of the cell cycle for dividing cells and the total number of dividing cells , it may be estimated that approximately 9,000 new cells are generated in the adult rat dentate gyrus each day . the daily regulation of the hippocampal neurons , an apparent daily change in the number of s - phase cells , has been reported in the hilus . thus , proliferation in the hilus seems to depend on the time of the day . cell divisions in other mammalian tissues ( e.g. , tongue epithelium , intestinal epithelium , and skin ) are associated with specific periods in the day [ 2931 ] . in the hippocampus , it has been observed that m - phase cells show a clear day / night variation , with a significant increase during the night but the number of s - phase progenitors remains unchanged across the day . it is therefore very difficult to demonstrate mitotic cells in dentate gyrus of adult rodents amongst more than 2 millions granule cells . however , in this connection hibernation of arctic ground squirrel presents unique model for accumulation and the study of different stages of cell cycle including mitosis . our immunohistochemical studies of doublecortin- ( dcx ) and brdu - labelled cells in the dentate gyrus of the adult arctic ground squirrel hippocampus have demonstrated a population of immature cells which appear to exist in a mitotic state and may potentially represent a renewable pool for generation of new neurons within the dentate gyrus .

neurogenesis occurs in the adult mammalian hippocampus , a region of the brain important for learning and memory . hibernation in siberian ground squirrels provides a natural model to study mitosis as the rapid fall in body temperature in 24 h ( from 35 - 36c to + 46c ) permits accumulation of mitotic cells at different stages of the cell cycle . histological methods used to study adult neurogenesis are limited largely to fixed tissue , and the mitotic state elucidated depends on the specific phase of mitosis at the time of day . however , using an immunohistochemical study of doublecortin ( dcx ) and brdu - labelled neurons , we demonstrate that the dentate gyrus of the ground squirrel hippocampus contains a population of immature cells which appear to possess mitotic activity . our data suggest that doublecortin - labelled immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

PMC5056538

limited hospital financial and personnel resources coupled with the need for established evidence - based clinical practice have forced surgeons and anaesthetists to consider appropriate triage for the immediate postoperative management of patients who have been subjected to major head and neck surgery . poor nutritional status , diabetes mellitus , and chronic diseases of the respiratory and cardiovascular systems as a result of long standing alcohol and tobacco use are factors frequently encountered in patients with head and neck cancers . neoadjuvant radiotherapy , extensive surgery , and flap reconstruction further influence the postoperative recovery and risk of complications . it is common practice in centres worldwide to admit patients to critical care unit ( ccu ) for 24 hours following major head and neck surgery in order to allow for airway oedema to stabilise , facilitating safe extubation prior to stepping down to the ward . in the case of patients undergoing tl , a cuffed tracheostomy tube is placed into the newly formed stoma to protect the airway ; however , admission to ccu remains routine practice in many hospitals including ours . the drawbacks of such practice may include cancellation of operations due to lack of ccu beds , the intrinsic expense of critical care , and lack of continuation of care for patients . multiple studies [ 36 ] have provided evidence on the safety of nursing head and neck cancer patients on a specialised ward with adequately trained nursing staff with no evidence of increase in the risk of morbidity or mortality . patients undergoing a total laryngectomy ( tl ) at the royal glamorgan hospital ( rgh ) , a district general hospital ( dgh ) in south wales , uk , are routinely admitted to level 2 ( high dependency unit , hdu ) or level 3 ( intensive therapy unit , itu ) postoperatively depending on patient comorbidity and bed availability . following 24 hours of close monitoring this case series aims to quantify ccu care received by patients following tl at a dgh and compare morbidity and mortality rates , in an attempt to inform current practice . classifications of levels of care ( adapted from ) are as follows : level 0 : patients that can be managed on a normal ward care in an acute hospital level 1 : patients at risk of their condition deteriorating , who can be managed on an acute ward with additional advice and support from the ccu 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 level 3 : patients requiring respiratory support and/or support of at least two organ systems . anonymised data were collected retrospectively for those undergoing tl , coded e2910 at rgh between 1st march 2010 and 31st january 2015 . as this was extracted from routinely collected data , formal ethical approval was not required . head and neck centres across wales were invited to complete an online ( see appendix ) and telephone survey to obtain data regarding postoperative level of care for tl patients . the patients ' operative risk as assessed prior to the procedure was characterized by the american society of anesthesiology ( asa ) grade . intraoperative data collected were the procedure type , operating time , estimated blood loss , and documented complications . the patient 's vital signs and blood results were obtained from the computerised ccu records and patient notes to allow calculation of possum and p - possum . all patients were graded with an operative severity of major as per griffiths et al . ( table 1(a ) ) . each patient was allocated a score depending on severity ( 1 , 2 , 4 , or 8) for each of the 12 physiological parameters ( ps ) and 6 operative parameters ( os ) as shown in table 1(b ) , reproduced from copelands work on possum ( physiological and operative severity score for the enumeration of mortality and morbidity ) . from these values predicted morbidity and mortality were calculated using formulae below possum morbidity % risk ( r ) is(1)lnr1r=5.91 + 0.16ps+0.19os . possum mortality % risk ( r ) is(2)lnr1r=7.04 + 0.13ps+0.16os.the p - possum ( portsmouth possum ) , a refinement of the original scoring system , utilises the same 18 physiological and operative parameters , but a different formula is applied to determine predicted mortality . details regarding complications encountered by patients were classified into surgical site complication , superficial infection , wound dehiscence , bleeding , myocardial infarction , pneumonia , fluid overload , renal failure , return to theatre , mechanical ventilation after surgery , ards , cardiac arrest , and death . each complication , for the purpose of statistical analysis , was treated as a single episode ( table 3 ) . statistical analyses were carried out using the pearson correlation . a p - value of < 0.05 was considered statistically significant . from 2010 to 2015 , 22 patients electively underwent tl with or without neck dissection and hemithyroidectomy . they ranged in age from 43 to 89 years ( median 65 years ) , including 17 males and 5 females . no patients died during surgery and all patients were admitted to hdu postoperatively for close monitoring . the average length of the procedure was 7 hours and 56 minutes ( sd 1 hour 21 mins ) . table 2 summarises the mean age , procedure length , asa class , and possum scores in relation to the operative procedure . patient 1 required invasive mechanical ventilation for a period of 6 hours on admission to hdu postoperatively following total laryngectomy + partial pharyngectomy + hemithyroidectomy + bilateral neck dissection ( tlppht ) . the length of the surgery was 8 hrs and 4 mins and the patient was transferred back to the ward after a total hdu stay of 22 hours and 15 mins ( asa 3 , p - possum 1.7 ) . patient 2 returned to theatre to arrest a haemorrhage encountered 6 hours after tlppht requiring invasive mechanical ventilation on return to itu for 9 hours ( length of ccu stay 46 hours , p - possum 2.5 , and asa 3 ) . patient 3 returned to theatre 10 days following tlppht due to intermittent chyle discharge from a wound dehiscence . the 72-year - old underwent a washout and repair of chyle leak and primary closure before being discharged to the ward . two days later , the patient suffered from haematemesis , returning to theatre for a second time for repair of a defect in the internal jugular vein ( ijv ) and pharyngeal fistula . three weeks postoperatively this patient underwent a debridement and pectoralis major flap reconstruction due to wound dehiscence ( length of hospital stay 85 days , p - possum 3.9 , and asa 3 ) . patient 4 suffered from a cardiac arrest thirteen days following tlppht after a myocardial infarction as a result of an estimated 3-litre haemorrhage . this patient was resuscitated with blood products and transferred to emergency theatres to control the haemorrhage originating in the right ijv and later moved to itu for mechanical ventilation and inotropic support . the patient developed acute respiratory distress syndrome and died 3 days later ( asa 4 , p - possum 7.1 ) . patient 5 was fluid overloaded in the immediate postoperative period and was treated effectively with intravenous diuretic and biochemical monitoring of renal function ( length of hdu stay 20 hours 10 mins , p - possum 2.8 , and asa 3 ) . two within the first 24 hours : both patients 6 and 7 had undergone tlppht and were found to have increased oxygen and suction requirements as well as radiological evidence of consolidation ( mean length of hdu stay 23 hours 45 mins , p - possum 9.2 , and asa 3 ) . the third , patient 8 , developed pneumonia 6 days following tlppht and recovered following treatment with intravenous antibiotics ( length of hdu stay 16 hours 15 mins , p - possum 3.1 , and asa 3 ) . patient 9 was admitted to itu postoperatively for level 2 care ( p - possum 2 , asa 2 , and length of stay in ccu 23 hrs and 50 mins ) and patient 10 cancelled due to lack of hdu bed . patient 11 , a 64-year - old with previous neck irradiation , had a pharyngeal leak 9 days following tl . he was successfully managed conservatively and began oral feed at postoperative day 14 ( asa 3 , p - possum 0.8 , and length of hdu stay 22 hours ) . the decision to transfer this patient to the ward was made at 16 hours ; however , due to a lack of bed - space on the ent ward , the transfer was delayed . the mean delay in discharge amongst the average length of ccu stay for the 22 patients was 25 hours and 27 minutes . head and neck centres across wales were invited to complete a telephone and online questionnaire survey ( see appendix ) regarding postoperative practice for patients following tl . pearson correlation analysis between the number of complications per patient and individual p - possum mortality and possum morbidity risk scores did not show a significant relationship ( p = 0.648 and p = 0.362 , resp . ) . this case series investigates the morbidity and mortality amongst a cohort of patients undergoing tl with or without additional procedures . it focuses on the incidence and timescale in the development of postoperative complications whilst comparing the outcomes to p - possum mortality risk . the linear average for p - possum predicted mortality was 6.68% which on a cohort of 22 patients is 1.5 predicted deaths . the observed mortality of 1 echoes previous studies [ 8 , 10 ] suggesting that p - possum overpredicts mortality rates . 's possum and its refined p - possum version by prytherch et al . following its publication , large cohorts of patients have been used to validate variations of possum for specific surgical groups including cr - possum for colorectal surgery and v - possum for vascular surgery , deemed more sensitive and specific for predicting patient outcomes following these operations . there was no significant correlation between the number of complications with possum ( p = 0.362 ) and that with p - possum ( p = 0.648 ) within our patient cohort . griffiths et al . concluded , based on a sample of 301 head and neck patients , that possum and p - possum scoring did not appear to have relevance in predicting mortality . variables such as peritoneal soiling and the glasgow coma scale designed for a general surgical population would not be relevant for head and neck patients . in an attempt to make possum applicable to the head and neck population , griffiths et al . suggested inclusion of radiotherapy and previous surgery as variables . the royal college of surgeons in england working group recommended that patients with a predicted p - possum mortality of 10% should be admitted to a critical care location postoperatively . amongst the 22 patients , 3 patients would have fallen within this category and therefore would have required admission to critical care based on these recommendations . cardiopulmonary exercise testing ( cpet ) has become increasingly popular for preoperative assessment as it represents a noninvasive simulation of the requirements of major surgery . cpet has been deemed a powerful diagnostic and prognostic tool for a variety of medical disorders , including coronary artery disease , cardiac failure , and restrictive and obstructive pulmonary disease . it can be used as a valuable resource for providing detailed evaluation of patients ' functional status before major surgery [ 15 , 16 ] . cost control and efficient use of resources are becoming increasingly vital in modern medicine , but it remains essential that cost saving measures should not adversely affect the quality of patient care . some of the factors influencing this challenge are the changing patient demographics and the high cost of medical equipment and technology in addition to rising patient and public expectation of higher standards of surgical care . inappropriate utilisation of critical care facilities has been discussed most notably by knaus et al . . the authors found that , during 49% of admissions and 65% of the nursing shifts , the emphasis was on close nursing care and observations and not intensive treatment . they also reported that 86% of patients admitted for close monitoring did not require active treatment prior to discharge . in our case series , one patient required invasive mechanical ventilation immediately postoperatively and was therefore appropriately placed to be managed in ccu . 21 patients ( 95% ) admitted to hdu for close monitoring did not require any intervention that could not be delivered on ent ward . in 1999 , godden et al . demonstrated that it was safe to care for head and neck patients outside the itu , provided it was done in an appropriate environment with adequately trained nursing staff , close consultant support , and sufficient throughput to provide experiential training . they found little difference in the general complications between the group of patients nursed on the ward with special care for 48 hours and those admitted directly to itu for 24 hours before being transferred to the ward for a further 24 hours of special nursing . hanna et al . developed a clinical pathway for patients undergoing tl and assessed its impact on their outcomes . patients were transferred to itu only for specific indications requiring an increased level of care such as respiratory or haemodynamic instability . this supported the findings from husbands et al . who introduced care pathways to the management of head and neck surgical patients and found significant reductions in hospital costs as well as shorter lengths of hospital stay . prospective study involving 304 patients following radical cystectomy assessed the difference in outcomes between patients who were managed on the ward and those requiring intensive care monitoring . 93.7% of their patients were managed safely on a surgical ward and did not require ccu . they stress the importance of preoperative optimisation , early consultation with specialists , and working closely with intensivists during the perioperative period . close monitoring on an ent specialist ward within the immediate postoperative period would involve 1 : 1 care provided by a trained ent nurse for 24 hours . the aim of specialist nursing care would include hourly monitoring of urine output , respiratory rate , blood pressure , and pulse ; care of the laryngectomy stoma with humidified air , oxygen , and suction with continuous monitoring of oxygen saturations ; and care of the wound with 2 hourly checks for bleeding or haematoma , administering intravenous fluids , and standard care of pressure areas which would involve turning the patient and palpating the skin every 8 to 24 hours . it is impossible to avoid all postoperative complications ; however , in order for postoperative sequelae to be minimised , close monitoring is essential , enabling rapid recognition and treatment of ensuing complications . by avoiding the use of ccu beds and instead ensuring that the nurses in the head and neck ward are adequately trained to manage these patients , it is likely that their recovery and discharge are expedited other than the four patients three with a p - possum > 10% and one requiring active intervention in ccu18 patients from the case series may have been nursed on ent specialist ward with 1 : 1 nursing for the first 24 postoperative hours . the cost per night of a bed in the ent ward is 290 , and the additional cost of a specialist ent nurse for the 24-hour period is an estimated 290 within the trust . based on the throughput for the period of the study this would cost a total of 10,440 which represents a minimum saving of 7,848 . this is because ccu is an expensive commodity costing 1016 in hdu and 2,661 in itu per day . in wales there was an average of 3.2 critical care beds per 100,000 people in 2014 , lower than the number available for the population in the rest of the uk . the welsh government has recommended that units run at an average occupancy of 6570% ; however , all units in wales report occupancy rates greater than 80% , with many often operating at over 100% occupancy at times . it is suggested that occupancy of 80% is likely to result in nonclinical transfers and failure of admittance in a timely manner with associated morbidity and mortality . in our case series , one patient was admitted to itu postoperatively for level 2 care and another was cancelled due to lack of a hdu bed . not using ccu for these patients would mean that operations are not dependent on the availability of a bed and therefore not cancelled on the day of operation , ultimately benefiting the patients and their families who will be spared the anxiety that this causes . benefits to the hospital are harder to estimate but would include loss of revenue if operating lists were cancelled . the institute for innovation and improvement estimates running costs for an average operating theatre at 1,200 per hour ; therefore , greater efficiency and careful use of resources are paramount . critical care beds may become blocked when patients are unable to be discharged due to a lack of ward beds . if a shortage of critical care beds were to cause an increase in cancellation rates of major elective surgery , hospitals may fail to meet targets . the limitations of our study include its retrospective methodology and possible selection bias inevitable in an operative series . it is difficult to predict whether the outcome of the patients within our case series would have differed had they not been admitted to hdu for the initial 24 hours due to the lack of a control group . further studies should aim to compare the outcomes of patients nursed on different levels of care within wales and their cost implications . the inconsistency of the levels of care for post - tl patients across wales may be due to several factors : funding issues , critical care capacity and workload , expertise of nursing staff available , adequate throughput , and dedicated medical teams to care for the patient once on the ward . several studies [ 3 , 24 ] have shown that head and neck cancer surgery does not necessitate admission to critical care and therefore patients can be safely managed in a ward environment . there is no substitute for sound clinical judgement and decisions must be tailored to each individual . however , the evidence suggests that admission to ccu can safely be the exception and not the norm . based on a small patient sample , the observed 30-day mortality was encountered on the fifteenth postoperative day , well beyond the first 24 hours following discharge from ccu to the ward . close monitoring and nursing care necessary within this period could be provided on a specialist ent ward with appropriately trained one - to - one nurses . the authors would advocate a change towards ward - based care for postlaryngectomy patients and meticulous preoperative assessment to identify patients requiring ccu admission in order to reduce unplanned admissions . keeping appropriately selected patients out of ccu allows nursing staff more familiar with head and neck cancer patients to care for them and helps maintain an esprit de corps amongst ent nurses and surgeons , whilst freeing up critical care beds for those in most clinical need .

critical care unit ( ccu ) beds are a limited resource and in increasing demand . studies have shown that complex head and neck patients can be safely managed on a ward setting given the appropriate staffing and support . this retrospective case series aims to quantify the ccu care received by patients following total laryngectomy ( tl ) at a district general hospital ( dgh ) and compare patient outcomes in an attempt to inform current practice . data relating to tl were collected over a 5-year period from 1st january 2010 to 31st december 2015 . a total of 22 patients were included . all patients were admitted to ccu postoperatively for an average length of stay of 25.5 hours . 95% of these patients were admitted to ccu for the purpose of close monitoring only , not requiring any active treatment prior to discharge to the ward . 73% of total complications were encountered after the first 24 hours postoperatively at which point patients had been stepped down to ward care . avoiding the use of ccu beds and instead providing the appropriate level of care on the ward would result in a potential cost saving of approximately 8,000 with no influence on patient morbidity and mortality .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

PMC5021387

the self - organization of supramolecular structures driven by weak intermolecular interactions is one of the most fascinating thermodynamic phenomena in biology , chemistry , and physics . because the interactions are weak , many different structures can be formed with the same type of building blocks . in biology , 20 different amino acids serve as building blocks for many unique three - dimensional structures . each of these structures has been optimized by evolution in order to perform a specific function , e.g. , to catalyze a chemical reaction or to create an efficient solar energy converting photosystem . an artificial chemical system with the same goal is an organic solar cell . in some bulk - heterojunction solar cells an interpenetrating network of a polymer phase and a fullerene phase is formed spontaneously . again , weak interactions between the side groups of the polymer and the fullerene determine the morphology of the solar cell that is key to its function . another example is self - assembled dye aggregates , called chlorosomes , that function as light - harvesting complexes in green sulfur bacteria . the present work deals with j - aggregates of self - assembled cyanine dyes . while the optical properties of cyanine dyes are mainly determined by the polymethine chromophore , their solution behavior and aggregate morphology can decisively be influenced by the substituents . cyanines with long alkyl substituents ( amphiphilic cyanines ) form bilayers in aqueous solution with the side chains facing inward and the polar / charged part of the molecule facing outward , thus stabilizing the aggregate by increasing the entropy of the surrounding water molecules . besides this so - called hydrophobic interaction , dispersive and electrostatic couplings are important for the formation of the aggregate . they are significant for cyanine dyes due to the large polarizability of their delocalized -electron systems . since the dye molecules are charged , electrostatic interactions between dye molecules among each other and also between dye molecules and solvated counterions are important as well . the interplay of all of the above interactions determines the three - dimensional structure of the aggregate . their relative weight can be changed by varying the character of the substituents , the solvent properties , or even the preparation conditions . it is a challenging thermodynamical problem to predict the structure of a j - aggregate . most likely , the free energy surface is highly frustrated , that is , it consists of many local minima , and slight changes in the above - discussed interactions can shift the global minimum and thereby the equilibrium structure of the aggregate . in recent years v. berlepsch and co - workers were able to elucidate the morphologies of aggregates formed by many different cyanine dyes by using cryogenic transmission electron microscopy ( cryo - tem ) . cryo - tem provides highly resolved direct images of supramolecular structures in their native environment that are free of the drying artifacts due to the specific sample preparation technique . detected architectures of dye aggregates range from thread - like fibers across ribbons to fascinating single- and double - walled tubes . however , due to the smallness of dye molecules the resolution of cryo - tem is generally not sufficient to directly resolve their mutual orientation within the aggregates . probably the simplest way to distinguish different structures is to measure their optical spectra . the coulomb interaction between the delocalized -electrons in one cyanine monomer and those in another monomer gives rise to excitonic interaction between the local optical excitations . for a pair of monomers subject to excitonic interaction , if one monomer is excited and another one in the ground state , the first one gets de - excited and simultaneously the second one gets excited . if the excitonic coupling is strong compared to static and dynamic disorder , the excited states of the aggregate are delocalized , the excitation energies of the delocalized states of the aggregate are shifted , and the oscillator strength is redistributed with respect to those of the isolated monomers . the redistribution of oscillator strength depends on the mutual geometry of optical transition dipole moments of the monomers . in the classical aggregates of jelley and scheibe the transition dipoles are oriented in line , and the oscillator strength in this case is shifted to the lowest exciton state . the corresponding spectral band is very narrow and red shifted with respect to that of the isolated monomers . because all of the oscillator strength is redistributed to this narrow band it is also said to possess scheibe the red - shifted band is called a j - band and self - aggregated cyanine dyes possessing a dominant j - band are called j- or scheibe aggregates . if the mutual geometry of neighboring dye molecules is such that the line connecting molecular centers is perpendicular to the transition dipole moments of both dyes , the oscillator strength is shifted to the exciton state with the highest energy , which is blue shifted with respect to the excitation energy of the isolated monomer . due to this hypsochromic shift this band is called a h - band , in contrast to the red - shifted j - band discussed before . in any case , there is a critical dependence of the optical spectra of the aggregate on the mutual orientation of the transition dipole moments of the monomers that allows one to use the optical spectra for structure prediction . a subtlety in the interpretation of spectra of dye aggregates concerns the coupling of excitons to intramolecular vibrations of the chromophores . the vibrational progression that is observed in optical spectra of monomers in solution is distinctly changed by the excitonic coupling in the aggregate whereas j - bands do show weaker vibrational side lines , in the case of h - bands these side lines get stronger than those of the isolated chromophores . this remarkable redistribution of oscillator strength was explained by spano with a model that included one effective vibrational mode per chromophore in the exciton hamiltonian , which was numerically diagonalized . an alternative description of this effect was given by briggs and co - workers using a greens - function approach . here , we will use the same approach as spano and in addition investigate a simplified model ( assuming the same local electronic transition energies of the chromophores ) that can explain the observed redistribution of oscillator strength semianalytically . of course , the optical excitation energy of the monomers is not only shifted by excitonic coupling but also by dispersive and electrostatic couplings between monomers in the aggregate . however , the effect of those couplings can be taken into account by shifting the energies of the localized ( one - exciton ) states of the aggregate . in the simplest approximation these localized energies are assumed to be equal to some reference value that describes at which energy a monomer in the aggregate would absorb if it was not coupled excitonically to the other monomers . in this case can be obtained by comparison of calculated and measured spectra , since variation of just shifts the calculated spectrum along the energy ( wavelength ) axis . pioneering work in the relation of optical spectra to the structure of chromophore aggregates was provided by kuhn and later continued by knoester and co - workers . in the models for simple sheet - like aggregates the dye molecule is represented by a brick containing the transition dipole that is described by two atomic partial charges at a certain distance . the discovery of tubular dye aggregates has led to increasingly complex theoretical models . created tubular aggregates by wrapping a brick - layer lattice in certain directions , where the optimal direction was determined by comparing the resulting absorbance and linear dichroism spectra with experimental data , and the radius was estimated by cryo - tem . of course , the final geometry depends also on the organization of the layer used to create the tube , and it has to be checked whether the proposed organization is also consistent with the minimum free energy of the molecular aggregate . again , pioneering work was provided by knoester and co - workers , who studied the spontaneous self - aggregation of an alkyl - substituted pseudoisocyanine ( amphi - pic ) in aqueous solution by molecular dynamics simulations . cylindrical and ribbon - like conformations were suggested as possible candidates for the equilibrium structure , but due to the finite simulation times ( 200700 ns ) it could not be decided which of the two structures is more likely . in contrast , cryo - tem images suggest that the investigated amphi - pic dye forms double - walled tubes and multilamellar spherical aggregates , at least at high dye concentrations . it seems that there is a need for either longer simulation times or some type of preselection of possible structures that are closer to the equilibrium structure . knoester and co - workers suggested to take for this purpose the phenomenological dipole models that describe the optical spectra . in the case of the amphiphilic cyanine dye c8s3 , which forms double - walled tubular aggregates , megow et al . successfully modified the phenomenological dipole model of knoester et al . by incorporating information from high - resolution cryo - tem data and running md simulations , arriving at a model of a stable structure that still describes the optical spectra . a crucial step in the description of optical spectra was to take into account the dispersive shift in site energies that was found to be different for molecules in the outer and the inner cylinder of the aggregate . in the present work dye molecular crystals as a basis for generating starting structures for md simulations . the rationale behind this approach is the expectation that the three - dimensional arrangement of dye molecules in the respective single crystals reflects their intermolecular interactions . the question to be addressed is whether and if so to what degree these interactions are varied if soft colloidal structures , tubes , or sheets are formed in solution . nevertheless , we believe that the packing in a dye aggregate is closer to the packing in a molecular crystal than to a random geometry . as an example , we study the cyanine derivative 5,5,6,6-tetrachloro-1,1,3,3- tetraethyl - benzimidacarbocyanine ( ttbc ) ( figure 1 ) . single - walled tubes have been observed in ref ( 30 ) in aqueous solutions for this dye after aggregation with cl or i counterions under different solvent conditions . a cryo - tem image of fibrous aggregates of ttbc - cl is reproduced here ( figure 2 ) together with an averaged density profile across a single aggregate , which proves its tubular architecture . ttbc - cl forms aggregates with an absorption spectrum ( type i ) that contains a strong broad h - band and a weaker and more narrow j - band , independent of the preparation conditions . in the recent literature this type of aggregate is referred to as hybrid hj - aggregate . in the case of ttbc - i , tubular aggregates with the same diameter as for ttbc - cl are seen in cryo - tem , but the measured absorption spectrum depends strongly on the solvent conditions . besides the type i spectrum observed at high ph values , an absorption spectrum with a single j - band ( in the following referred to as type ii ) was reported at low ph values . structure of the ttbc molecule with color - coded atoms ( light blue , c ; dark blue , n ; green , cl ; white , h ) . ( a ) network of single fibrous aggregates and bundles of fibers at low magnification . interestingly , for ttbc there exist also two distinct single - crystal structures that were obtained for two different solvents . in the case of a 2:1 methanol : ttbc - i solvate , ttbc organizes in separated layers where the molecules in each layer form a herringbone - like arrangement . the structure can be described as a crystal lattice with two molecules per unit cell . referring to its solvent , it was termed dyem . in the alternative dyea structure ( an 1:1 acetonitrile : ttbc - i solvate ) just a single molecule per unit cell is found and the molecules inside crystal sheets are arranged in a brick - layered fashion . compared to dyem the distances between layers of ttbc molecules in dyea crystals are much smaller . details about these crystal structures are given in the supporting information ( si ) . a simple explanation of the two types of optical spectra found for the j - aggregates of ttbc discussed above has been suggested in the experimental work . the explanation has been that the two peaks found in the type i spectrum just represent a davydow split spectrum , as expected for crystals with two molecules per unit cell as found in dyem . in fact , the present type i spectrum of ttbc aggregates has been explained by assuming a 2d flat herringbone structure of transition dipole moments in a phenomenological model by glen and co - workers . at that time , no information from tem was available , which later revealed a tubular structure of the aggregate , as discussed above . here , based on our assumption of a crystal - like arrangement , we want to find out whether a tubular herringbone structure can also explain the optical spectra and if such a structure is stable . an alternative brick - layered structure with one molecule per unit cell as found in dyea crystals could then be responsible for the spectrum with the single j - band ( type ii ) . from analytical work of knoester and co - workers it is known that tubes with rotational symmetry and one chromophore per unit cell in general exhibit two bands , where one is polarized in parallel to the symmetry axis of the tube and the other is polarized perpendicular to this axis . thus far only tubes have been found , where both transitions are red shifted with respect to the transition energy of the isolated monomer . in the present work we show that the analytical model also contains a solution with one red - shifted and one blue - shifted exciton band that is a hybrid hj - aggregate , which seems to be realized in monowalled ttbc tubes with a type i spectrum . during the rest of our considerations we will focus on the latter type i spectrum , since it contains a rich substructure that we want to understand on a molecular basis . for this purpose we will use the crystal structures of dyem and dyea to generate tubes , the resulting optical spectra of which will be compared to experimental data . interestingly , in both cases tubes can be created which explain the absorption and linear dichroism spectra . we will use those structures as starting geometries for molecular dynamics simulations in order to determine their relative stability and , in an iterative procedure , provide a proposal for the molecular structure of the ttbc - cl single - walled hybrid hj - aggregate that can explain all available experimental spectroscopic data and is stable within the time range of our md simulations ( 100 ns ) . we treat the intramolecular vibrational degrees of freedom of the monomers quantum mechanically by introducing one effective vibrational mode per monomer with frequency vib and huang rhys factor s. the coupling of electronic excitations to these modes is treated within the one - particle approximation , that is , vibrational excitations are taken into account only in the electronic excited state of the chromophores . the rationale behind this approximation is that the energy of the quanta of this intramolecular mode is large compared to the thermal energy and , therefore , an electronic transition induced by an external field will always start from the vibrational ground state of the electronic ground state . however , the coulomb coupling between the vibronic transitions of different monomers mixes the excited intramolecular states of the electronic ground state of the monomers into the excited eigenstates of the aggregate . all intermolecular degrees of freedom are treated classically by molecular dynamics simulations . neglecting electron exchange between the monomers results in the following frenkel exciton hamiltonian1the are the local excitation energies of the monomers between the electronic and intramolecular vibrational ground state and the electronic excited state with intramolecular vibrational quanta . the off - diagonal element describes the coupling of the transition between the electronic and the intramolecular ground state and the electronic and the th intramolecular excited vibrational state of monomer m with the transition between the electronic excited state with vibrational excitations of monomer n and its electronic and vibrational ground state . in the condon approximation this coupling , termed excitonic coupling , condon factors of the monomers ff , that is2where the franck condon factor f is given as3for the present system we estimated s = 0.83 and vib = 800 cm for the effective intermolecular vibrational mode from optical spectra of isolated monomers , as described in detail later . please note that the explicit time dependence of the above hamiltonian results from the classical treatment of the intermolecular vibrational degrees of freedom . the interaction of the aggregate with a monochromatic external field e(t ) = e0e cos t is described in rotating wave approximation by the hamiltonian4where m0 is the local electronic transition dipole moment of monomer m , f is the franck condon factor introduced in eq 3 , and h.c . denotes the hermitian conjugate of the first expression on the righthand side . the optical spectra are obtained after diagonalization of hexc(t ) ( eq 1 ) , resulting in exciton states5with excitation energies m ( t ) . the influence of intermolecular degrees of freedom determines the time - dependence of the above eigenstates and energies . in the calculation of the absorbance ( ) this time dependence is taken into account in the static limit , where it leads to inhomogeneous broadening6with the transition dipole moment7the average over inhomogeneous conformations is denoted as and is performed taking into account the time - dependent trajectories resulting from the classical propagation of intermolecular degrees of freedom . in the case of linear dichroism , the difference in absorbance of light polarized parallel and perpendicular to the symmetry axis of the tube is measured reading8where m(t ) is the angle between the exciton transition dipole moment dm(t ) and the symmetry axis of the tube . in the following , we consider a simplified hamiltonian for which it is possible to disentangle the diagonalization of hexc(t ) ( eq 1 ) into a diagonalization of the bare exciton hamiltonian and a subsequent diagonalization of the exciton vibrational hamiltonian , separately for every eigenstate of the bare exciton hamiltonian . in this way a simple explanation of the different redistribution of oscillator strength by the exciton vibrational coupling for j- and h - bands will be obtained . we consider a simplified hamiltonian without static disorder , that is , the hamiltonian in eq 1 is assumed to be time independent . in addition , we assume that all local electronic transition energies of the chromophores are equal , i.e. , we have , where is the transition energy between the vibrational ground states of the electronic excited and ground state . the coefficients cm(m ) of the eigenstates |m ( eq 5 ) then fulfill the following equation9where em is the eigenenergy of |m. in addition , we investigate the bare exciton hamiltonian hexc that does not include the intramolecular vibrational mode of the chromophores10where is the local vertical transition energy of the chromophore in the tube taken at the equilibrium position of nuclei in the electronic ground state11with the huang rhys factor s introduced in eq 3 . the eigenstates |k of hexc read12the coefficients cm(k ) are obtained from the solution of the eigenvalue problem13with the eigenenergy k of hexc . we rewrite the second expression in eq 9 as14where we used the orthogonality of wave functions , that is , kcm(k)cm(k ) = m , m and ncn(k)cn(l ) = k , we proceed by introducing coefficients15hence , it holds that16using the above expressions , eq 9 can be rewritten as17multiplying this equation on both sides by cm(l ) and summation over l then results in the final eigenvalue problem18the optical transition dipole moment dm then follows from the eigenvalue coefficients cm(m ) ( eq 9 ) using eq 16 as19where dk = mcm(k)dm is the transition dipole moment of the bare exciton state |k ( eq 12 ) , f is the franck condon factor defined in eq 3 , and ck(m ) is obtained from the solution of the eigenvalue problem in eq 18 . since eq 18 is diagonal with respect to k , no mixing of different exciton states k and l occurs due to the intramolecular exciton vibrational hamiltonian hex obtained for n chromophores taking into account nvib vibrational levels per chromophore can be arranged into n subgroups |mk for k = 1 ... n . the eigenstates in each subgroup are obtained by solving eq 18 , that is , diagonalizing a nvib times nvib matrix that describes the vibrational progression of a given exciton state k. therefore , the quantum number m in eqs 18 and 19 can be replaced by mk , and the respective transition dipole moments of eigenstate subgroup mk are then obtained as20the resulting optical spectrum reads21where mk = emk/ is the optical transition energy that is obtained from the eigenenergy em = emk in eq 18 and the respective eigenvectors contain the coefficients ck(mk ) which determine whether there is constructive or destructive overlay of different vibronic transition dipole moments fdk of a given bare exciton state k. the above equations also show that only those bare exciton states k contribute to the absorbance spectrum , which have a nonvanishing bare exciton transition dipole moment dk , that is , there is no mixing with dark exciton states induced by the coupling to intramolecular vibrational modes . therefore , in the present homogeneous case we can analyze the optical spectrum in two steps , first considering the bare exciton spectrum only and in the second step including the influence of the intramolecular vibrational mode . the latter effect can be qualitatively understood by a perturbative treatment of the off - diagonal couplings in eq 18 that couple different vibrational states and . for this purpose we can identify the eigenstate |mk in the vibronic manifold of the kth exciton state with a state |k that for absent off - diagonal coupling equals |k|. taking into account the off - diagonal coupling in eq 18 up to first order , the coefficient ck(k ) is obtained as22the resulting transition dipole moment dmk = dk then reads using eq 2023where the coefficient c( ) mixing different transitions is given as24the franck rhys factor s < 1 , decreases with increasing . therefore , in the case of the low - energy exciton states ( ) we have c( ) > 0 for > . in particular , it holds that c(0 ) > 0 for all = 1 ... nvib in eq 23 , where nvib denotes the number of vibrational levels included per chromophore . hence , there is constructive superposition of vibronic transition dipole moments dkf and dkc()f in the optical transition to the vibrational ground state ( = 0 ) of the low - energy exciton states ( with nonvanishing bare exciton transition dipole moment dk ) . in the case of high - energy exciton states we have and obtain constructive superposition , that is , c( ) > 0 , if it holds that . the magnitude of c( ) goes rapidly to zero for large or , because the respective franck condon factors are small . hence , in general , the mixing of vibronic transitions goes to zero in this case . therefore , the redistribution toward higher vibronic transitions , as may occur in the case of high - energy exciton states , will never be as strong as the redistribution toward low - energy vibronic transitons in the case of low - energy exciton states . this effect in essence explains the difference in the vibrational progression of j- and h - bands . concerning the shift in energies due to the off - diagonal coupling in eq 18 we note that the first nonvanishing contribution is obtained in second order in this coupling and the resulting energy e reads25with26for large , the respective franck condon factors f are practically zero and , that is , there is no influence of the excitonic interactions and the aggregate absorbs light as the monomers . for small , however , there is a -dependent change of the vibronic transition energies , which become nonequidistant and experience a further shift e( ) by coupling with other vibronic transitions . along the same line of discussion used above , we see that for low - energy exciton states ( ) and huang rhys factors s < 1 it holds that e( ) < 0 for < , that is , there is always a red shift due to the coupling with higher energy vibronic transitions . in the case of high - energy exciton states ( ) it depends on the relative magnitude of vib and whether the shift goes to the red or to the blue . in the following it will be discussed how the aggregate hamiltonian in eq 1 is parametrized by combining quantum chemical , electrostatic , and molecular mechanics calculations . the diagonal elements of the exciton hamiltonian ( eq 1 ) describe the energies at which the chromophores would absorb light if they were not coupled excitonically . these diagonal elements contain the vertical excitation energy of a ttbc molecule taken at the equilibrium position of nuclei in the electronic ground state and a shift that is described by taking into account intermolecular electrostatic coupling and intermolecular london dispersive interactions , respectively . the electrostatic transition energy shift of chromophore m is obtained as27where jmk(eg , ge ) denotes the coulomb coupling between the charge densities of the electronic excited state of chromophore m and that of the electronic ground state of the chromophore k , which may represent another chromophore or a solvent molecule including the counterions , and jmk(gg , gg ) is the same type of coulomb coupling but between the ground state charge densities of chromophore m and its environment . the charge density couplings are evaluated by performing quantum chemical calculations on the isolated chromophore and fitting the electrostatic potentials by that of atomic partial charges , which are then used to calculate the couplings28here qi(m)(a , a ) is the partial charge placed at atom i of chromophore m for the ground ( a = g ) and excited state ( a = e ) and qj(k)(g , g ) is the partial charge at atom j of the environmental molecule k in its electronic ground state . we used ( time - dependent ) density functional theory and the b3lyp exchange correlation functional for the calculation of the electrostatic potential of the ground ( excited ) state of the isolated chromophores . the numerical values of the atomic partial charges that fit the respective potentials are given in the si . in inductive shifts arise due to the polarization of the wave function of a molecule by the charge density of its environment and vice versa . we follow our previous work on j - aggregates and neglect the inductive shifts , noting , however , that for some systems these shifts were reported to be significant . in order to allow for a microscopic description of static disorder we will include besides the in eq 27 a description of the dispersive transition energy shifts , introduced in our previous work as29where vmk(t ) is the coupling of two effective extended dipoles carrying unit charges and being located at molecules m and k. the distance between the two charges on a given chromophore should reflect the extent of the -electron system of the chromophore . for the present ttbc molecule the factor q in the above equation is a constant that was derived using second - order perturbation theory in the intermolecular interaction and contains information about energies and oscillator strengths of higher excited states . this factor can be obtained from experiments on transition energy shifts of the isolated chromophores in different solvents . describing the latter by a homogeneous dielectric of refractive index nr2 and the solute as a sphere of radius r results in a dispersive shift of30with the prefactor that contains the difference in the variance of the permanent dipole moment between the excited and the ground state of the solute . from the absorption peaks of ttbc in methanol and water we obtain and extraplolate a vacuum transition energy of the chromophore . by taking many different tube geometries and averaging the dispersive shift ( eq 29 ) of a molecule with its neighbors one should obtain a similar result as from the continuum approximation in eq 30 . hence , q follows as31where < >orient denotes an average over many tube geometries and over time and is taken from the experimental shift measured in different solvents . with this q the dispersive shifts in eq 29 can be calculated for every monomer m if the refractive index nr is known . for a monolayer of a related compound nr = 1.64 was measured in ref ( 45 ) . as discussed before , the main effect of the diagonal elements of the exciton hamiltonian is to shift the center of the spectrum along the energy ( wavelength ) axis . in the present work , we find that for nr 1.5 we can explain the shift of the absorbance spectrum of our final suggestion for the ttbc tube with respect to that of the isolated monomer in methanol ( nr = 1.33 ) . the deviations between the above nr = 1.5 and nr = 1.64 measured in ref ( 45 ) could be due to the difference in morphology ( tube versus plane monolayer ) , but it could also reflect the neglect of inductive transition energy shifts , discussed above . using nr 1.5 we obtain q = 2.28 ev from eq 31 , which is used to obtain dispersive shifts from eq 29 along the md trajectories , that will be used for a microscopic description of static disorder . the excitonic coupling between the transition densities of the monomers is obtained from the poisson - tresp method , which allows us to take into account polarization effects of the environment . the environment in the present case comprises the transitions between the ground state and higher ( than first ) excited states of the other monomers and the excitations of solvent molecules . the excitonic coupling jmn between monomers m and n is obtained in the following way . first , the transition density of the isolated ttbc monomer is calculated with time - dependent density functional theory using the b3lyp exchange - correlation functional . the electrostatic potential of this transition density in vacuum is fitted by atomic transition charges qi(g , e ) . a poisson equation is solved in order to obtain the electrostatic potential m(r ) of the transition charges of monomer m32where (r ) equals 1 if r points into the cavities of monomers m or n and (r ) = nr2 otherwise . please note that in the original poisson - tresp method all chromophore cavities ( and not just those of chromophores m and n ) have = 1 and the protein solvent environment has = 2 . in the present case there is no protein environment forming binding pockets for the chromophore , but the chromophores self - aggregate . since the excitonic coupling between the low - energy excitations is taken into account explicitly in the hamiltonian a certain amount of polarization is included in this way . it is this residual polarization that contributes to the screening in the present possion - tresp calculations . we take nr = 1.5 as has been estimated from the dispersive shift in monomer transition energies discussed above . the excitonic coupling jmn then follows as33 in order to simplify the above procedure , the above coupling is compared with the coupling in vacuum34and a screening / local field correction factor fmn = jmn / jmnvac is determined . as will be shown below fmn varies between 0.50 and 0.60 and depends on the mutual geometries of monomers in the tube aggregate . the variation in fmn by the nuclear dynamics is found to be small in the md simulations ( see below ) . therefore , time - independent factors fmn can be assumed . in rough estimations of optical spectra , we take an average screening / local field correction factor f 0.55 , determined as described in detail later . since different quantum chemical methods give a similar shape of the transition density but magnitudes that may differ by a factor of up to 1.5 , it is necessary to rescale the quantum chemical transition density by a constant factor that is chosen such as to reveal the experimental transition dipole moment . as shown by knox and spring the dipole strength d ( in units of d ) of a chromophore in a certain spectral region is given as 9.186 10nra( ) , where nr is the refractive index of the medium and a( ) = d()/ , with the absorption coefficient ( ) . we measured the absorption coefficient of ttbc in dmso , which has a refractive index nr = 1.48 that is practically identical to the one estimated for the tube and obtained a dipole strength of 14.4 d. taking into account an average screening / local field correction factor of f 0.55 results in an effective transiton dipole moment of deff = = 10.7 d , which is close to the deff = 11.4 d that has been used before for this type of chromophore . please note that the present estimate of deff is an upper bound , since the d in poisson - tresp refers to the vacuum rather than the medium transition dipole moment of the chromophore . smaller deff however did not lead to any reasonable agreement between calculated and measured spectra . the franck condon factor s of the effective vibrational mode has been estimated from optical spectra of ttbc monomers in methanol . the monomer absorption is given as35with the franck condon factor f that is related to the huang for comparison with experimental data we have dressed the -functions in eq 35 with gaussians of width = ( + 1 ) 0 in order to take into account inhomogeneous broadening . the larger broadening of the higher vibrational states corrects for the limitations of the effective mode model . the absorption spectrum calculated for s = 0.83 , vib = 800 cm and a 00 transition energy corresponding to a wavelength of 515 nm , is compared in figure 3 to the experimental data , revealing good agreement . in addition , we also show a homogeneous spectrum , calculated by dressing the sticks in eq 35 by lorentzians of width 30 cm , in order to visualize the vibrational progression that will be important later when we will discuss how it is changed by the excitonic coupling in the aggregate . experimental absorption spectrum of ttbc monomers in methanol ( black dashed line ) compared to an effective mode model that is inhomogeneously ( red line ) or homogeneously ( blue line ) broadened . for a direct consistency check of our final structural model , we also simulated the tem images . to this end , we estimated the actual degree of noise in the raw data and determined the contrast transfer function ( ctf ) of the used microscope . the latter data allow one to calculate projection images of the theoretical molecular model structures with a resolution and degree of noise as found for the raw data . as raw cryo - tem image data are of poor contrast we used the single - particle analysis approach to construct a noise - reduced sum image of some tube segments . to get an impression of the reached consistency this sum image and the simulated images , the class sum image of tubes or only their density profiles technical details of the image processing and simulation procedures are given in the si . cryo - tem images show thin elongated thread - like structures which are interpreted as a projection of single - walled dye tubes . this interpretation becomes clear at high magnification , where a pattern of fine lines with a spacing of about 19 is visible for isolated individual fibers . a line scan perpendicular to the long axis of a single fiber reveals a cylindrical structure , where the parallel lines with high contrast correspond to the walls of a tubular aggregate . the tem data provide one constraint for our structure model ; the compatibility with the crystal lattice of dyea or dyem provides another one . these constraints will be fulfilled simultaneously by using the following lattice - wrapping procedure . first , a crystal layer is defined . because of its dense packing the crystal plane ( 100 ) is taken for both dyea and dyem . then a wrapping vector c is chosen , the length of which becomes the circumference of the tube after wrapping . to be compatible with the crystal lattice , c has to be a lattice vector itself c = n1c + n2b . from the range of likely tube diameters each of them can be described by lattice indices ( n1 , n2 ) . the two half circles in the ( 100 ) plane define the range of possible tube circumferences , and the different vectors c define the wrapping directions considered . the inner circle corresponds to dmin = 16 and the outer one to dmax = 26 . choice of wrapping vectors c = n1c + n2b in the ( 100 ) plane of dyea ( left ) and dyem ( right ) . each wrapping vector is depicted as a solid line ; its label corresponds to its indices ( n1 , n2 ) . minimum and maximum circumference as estimated from cryo - tem data are drawn as half circles . examples of tubes generated from the ( 100 ) plane of dyea crystals ( left ) and dyem crystals ( right ) applying the wrapping vector c = 6c + 0b . this procedure may fail to provide a reasonable structure , because it creates steric overlaps between dye molecules in some cases . some of the overlapping structures can be repaired by increasing the tube diameter or increasing the spacing between dye molecules in the direction along the tube axis . this change corresponds to a distortion of the primitive unit cell of the wrapped lattice which increases the unit cell area and decreases the packing density . for the wrapping directions of figure 4 the resulting lattice parameters ( after removing steric overlaps ) are given in the si . after wrapping a representative subset of tubes for dyea and dyem , linear optical spectra were calculated according to eq 6 . the following simplifications , which turned out to be uncritical for a qualitative interpretation of the spectra , were applied in order to save computational time . ( i ) static disorder was described by applying a gaussian distribution function for the local transition energies of the monomers . a width of 118 mev ( fwhm ) was estimated for this distribution function based on a comparison of the calculated and measured low - energy absorbance peak . ( ii ) the mean local electronic transition energy of all monomers was set equal to , corresponding to a wavelength of 557 nm , based on a comparison of calculated and experimental spectra . ( iii ) an average screening local field correction factor f = 0.55 was used for the calculation of excitonic couplings with the tresp method resulting in an effective transition dipole moment of 10.7 d , as discussed before . these approximations allow us to obtain the optical spectra from a static structure instead of a whole set of structures generated by md simulations . as will be shown later the inhomogeneous spectra obtained by evaluating the variations of site energies from the md trajectories are very similar to the spectra obtained with the present simple method . here , the average over static disorder in local optical transition energies is performed by generating random combinations of transition energies and averaging the resulting optical spectra . we find that sufficient convergence of the spectra is obtained if 10 000 configurations of disorder are taken into account in the monte carlo average . we varied the length of the tube and found that taking into account around 100 chromophores leads to convergence of the absorption spectra . the calculated linear optical spectra resulting from the different wrapping directions are compared to experimental data in figures 6 and 7 for dyea and dyem tubes , respectively . we see that for structures generated from dyea only the wrappings in ( 6,0 ) and ( 5,5 ) directions qualitatively match the experimentally determined linear dichroism and absorbance spectra . the intensity ratio between the main peaks is somewhat closer to the experimental value for the ( 6,0 ) tube . for dyem the wrapping direction ( 6,0 ) fits best , although the red - shifted peak carries somewhat too little intensity . we take into account the ( 6,0)-dyea and ( 6,0)-dyem structures for further optimization . absorbance ( left ) and linear dichroism ( right ) spectra of dyea tubes that were generated by wrapping the ( 100 ) plane of the dyea crystal in directions as described by the wrapping vector c = n1c + n2b denoted by ( n1 , n2 ) in comparison to experimental data ( bottom ) . vertical dashed lines are placed at the experimental maxima for easy comparison with the calculations . absorbance ( left ) and linear dichroism ( right ) spectra of dyem tubes that were generated by wrapping the ( 100 ) plane of the dyem crystal in directions as described by the wrapping vector c = n1c + n2b denoted by ( n1 , n2 ) in comparison to experimental data ( bottom ) . vertical dashed lines are placed at the experimental maxima for easy comparison with the calculations . to test the stability of the ( 6,0)-dyea and ( 6,0)-dyem candidate structures , md simulations were carried out using the namd program package , the amber force field , and the gaff parameter set . the particle mesh ewald method was utilized in order to establish periodic boundary conditions . two snapshots for ( 6,0)-dyea taken at 60 ps and 2.7 ns are shown in figure 8a and 8b , respectively . the instability can be attributed to intermolecular distances being too large in the initial structure . the large intermolecular distances stem from removing steric clashes as described before . at 2.7 ns a well - ordered tube is recovered in the central region of the simulation box . snapshots of structures during md simulation with initial structures obtained from wrapped crystal planes : ( a ) ( 6,0)-dyea after 60 ps , ( b ) ( 6,0)-dyea after 2.7 ns , and ( c ) ( 6,0)-dyem after 0.5 ns . in the case of the alternative ( 6,0)-dyem starting geometry we find that the structure breaks apart in the course of the md simulations . at 0.5 ns the molecules partially realign and form a structure which is similar to the ( 6,0)-dyea tube discussed above ( figure 8c ) . hence , we conclude that the ( 6,0)-dyem structure is further away from the stable tube structure than the one of ( 6,0)-dyea . in a next step , the partially ordered central segment of the md - simulated dyea tube at 2.7 ns ( figure 8b ) was used to determine new structure parameters . a least - squares fit between the disordered tube and an ideal helical tube revealed the primitive lattice vectors of the latter , given in the si , which deviate from the lattice vectors of the original crystal plane of dyea . next , these new lattice vectors were used to create a new ( 6,0 ) tube . this geometry was found to be stable within a new md run which took 100 ns . ( a ) md snapshot of the final tube structure viewed along the symmetry axis of the tube ( top ) and perpendicular to this direction ( bottom ) . ( b ) space - filling model of the final ideal tube structure with lattice parameters c = 13.582 , b = 8.832 , = 140.344 , and = 74.000 extracted from the final md structure by a least - squares fit . the structure obtained after 100 ns md simulation was used again to determine primitive lattice vectors used to create an ideal tube , which represents our suggestion for the structure of the single - walled tube of ttbc molecules with type i spectrum . the lattice parameters of this final ( 6,0 ) tube are c = 13.582 , b = 8.832 , = 140.344 , and = 74.000 , where c and b are the magnitudes of unit cell vectors c and b ( figure 4 ) , is the angle between these vectors , and the angle denotes the angle between the transition dipole moment of the dye molecule and the symmetry axis of the tube . the optical spectra resulting for this structure are compared in figure 10 with the experimental data revealing good agreement for both linear absorbance and linear dichroism . close inspection reveals a somewhat too large intensity of the calculated spectra in the intermediate wavelength region ( 515560 nm ) . linear absorbance ( solid lines ) and linear dichroism ( dashed lines ) spectra calculated for the final structural model for the ttbc - cl tube ( green lines ) compared to experimental data ( black lines ) . for a qualitative understanding of the structure of the spectrum , we neglected static disorder and dressed the exciton stick spectra with lorentzians of width 10 mev ( fwhm ) . as before , a mean local transition energy , corresponding to a wavelength of 557 nm , has been assumed for the monomers in the tube . the resulting absorbance spectra are shown in figure 11 ( green and red solid lines ) together with the experimental data ( black solid line ) . for comparison , we also included the homogeneous spectra resulting from the bare exciton hamiltonian , that is , without including the effective vibrational mode ( green and red dashed lines ) . homogeneous absorption spectra obtained by diagonalizing the bare exciton hamiltonian ( dashed lines ) and the full hamiltonian that includes the coupling to one effective intramolecular vibrational mode per chromophore ( solid green and red lines ) . for illustrative purposes , stick spectra were broadened by loretzians of width 10 mev ( fwhm ) . red lines indicate a polarization parallel to the symmetry axis of the tube , and green lines mark those transitions which are polarized perpendicular to this axis . for comparison , we also show the experimental absorption spectrum ( black solid line ) . in the homogeneous spectrum we find a single low - energy peak at around 590 nm ( the j - band ) , the transition dipole moment of which is polarized along the symmetry axis of the tube , and a number of peaks between 450 and 550 nm ( the h - band ) with transition dipole moments that are polarized perpendicular to this axis . in the bare exciton spectra it is seen that part of the broadening of the h - band is due to the presence of multiple exciton transitions with nonzero transition dipole moments . in the case of the j - band switching on the coupling of excitons to the effective intramolecular vibrational mode changes the h - band qualitatively but leaves the j - band unchanged , except for a 10 nm red shift and a slight reduction in intensity . interestingly , practically no vibrational progression is visible for the j - band . this remarkable behavior is explained by our analytical treatment and the perturbation theory . as shown by the analytical treatment of the homogeneous spectrum , the coupling to the effective vibrational mode does not lead to a mixing between different eigenstates of the bare exciton hamiltonian . the perturbation theory explains how for every eigenstate of the bare exciton hamiltonian the vibrational progression , which one would naively expect , is changed by the off - diagonal elements of the exciton vibrational coupling . whereas in the case of the j - band the majority of the oscillator strength is redistributed to the lowest energy transition , that is , to the = 0 state of the lowest exciton state , in the case of the transitions in the h - band , there is redistribution of oscillator strengths toward transitions with larger . for the present system we have s < 1 , and therefore , all of the mixing coefficients c(0 ) ( eq 24 ) for = 0 in the case of the j - band are positive . in addition , we have for all h - band exciton states with strong transition dipole moments , and therefore , it holds that c( ) > 0 for < . hence , there is redistribution of oscillator strength toward the states with larger . this effect is illustrated in figure 12 , where for the bare exciton transition with the largest oscillator strength of the h - band and that of the j - band the redistribution of oscillator strength by the exciton as seen in figure 12 the exciton vibrational coupling ( the first part in eq 18 ) leads to a vibrational progression in the exciton spectrum ( figure 12b ) , which is , however , qualitatively changed by the off - diagonal elements ( the second part in eq 18 ) of the exciton whereas for the exciton transitions in the j - band practically all oscillator strength is redistributed to the = 0 transition , for the transitions in the h - band it is redistributed toward several vibronic transitions with larger , as expected . the perturbative analysis already explains the effect qualitatively as figure 12c ( perturbation theory ) in comparison to figure 12b ( no redistribution of oscillator strength between different vibronic transitions of the same excitonic transition ) and figure 12d ( exact numerical diagonalization ) demonstrates . the most intense transitions of the h - band and the j - band are marked by a green and red triangle , respectively . those two transitions are investigated further in c and d. ( b ) including the coupling to the effective vibrational mode but neglecting the off - diagonal parts of this coupling ( the sum over in eq 18 ) . vibrational coupling in first - order perturbation theory ( eqs 23 and 25 ) . we note that the present finding of a different redistribution of oscillator strength for h- and j - bands is in line with earlier numerical results by spano . we are , however , not aware of such a simple analytical explanation of the effect , as presented in this work . experimentally this effect is known since the work of jelley and scheibe on the classical j - aggregates . we also have to note that spano went beyond the single - particle approximation , used here for the inclusion of vibrational states , and found that the redistribution can be somewhat less if a two - particle approximation is used . an alternative way to relate the vibronic line shape of the isolated monomer to that of the aggregate was offered by briggs and co - workers using a greens - function approach termed coherent exciton scattering ( ces ) . this approach was recently shown to be equivalent to a direct diagonalization of the exciton we want to exploit the rotational symmetry of our structural model to explain the general structure of its linear absorption and dichroism spectra by applying analytical solutions of the bare exciton problem worked out by knoester and co - workers before in different approximations . we keep in mind that the inclusion of exciton vibrational coupling will lead to additional redistribution of oscillator strength and splitting of exciton vibrational eigenstates but independently for every eigenstate of the bare exciton hamiltonian , as has been shown above . our best - fitting structure model possesses 6-fold rotational symmetry with respect to its symmetry axis . the exciton states |k in such a structure can be written as36where |n1 , n2 denotes an excited state that is localized at the n2th chromophore in the n1th ring of the tube . the rotational symmetry demands that the wave function k(n1 , n2 ) possesses bloch form in ring direction . using 2 = 2/n2 , where n2 denotes the number of chromophores within a ring , and counting the eigenstate of the ring by k2 , we get for k2 > 0 ( see eq 12 in ref ( 23))37hence , the eigenstate |k = |k1k2 of the tube in eq 36 can be written as38where39denotes the contribution of the excited state that is localized at the k2th eigenstate of the n1th ring to the overall exciton state |k1k2. in particular , it holds that the transition dipole moment40is only nonzero for those k2 values for which the transition - dipole moment 0 , n1 k2 of the isolated rings is nonzero . transforming the bare - exciton hamiltonian from the localized basis |n1n2 to that of delocalized states of the rings |n1k2 gives rise to off - diagonal elements , that is , coupling j(n1 , k2 ) between the eigenstates of different rings in the same k2 state , that are n1 rings apart . this coupling reads41here j(n1 , n2 ) is the excitonic coupling between a pair of chromophores in the two rings , where the position inside the rings differs by n2 . the prime in the sum indicates that for n1 = 0 we do not sum over n2 = 0 . for n1 = 0 both chromophores are in the same ring and the excitation energy of the k2 state of the isolated ring follows as42where denotes the local vertical exciton energy of the chromophore in the tube ( eq 11 ) . the transition dipole moments 0 , n1 k2 of the delocalized states in the rings are obtained as43thereby it is seen that only k2 = 0 and k2 = 1 lead to a nonzero transition dipole moment 0,n1k2 of the ring . if the transition dipole moment of the chromophores in the ring 0 , n1n2 have an angle with respect to the symmetry axis of the tube , the parallel component 0 , n1n2 = 0 , n1n2 cos adds up constructively only for k2 = 0 in eq 43 , whereas the perpendicular components 0 , n1n2 = 0 , n1n2 sin of the single chromophores give a nonzero transition dipole moment of the ring only for k2 = 1 because of the rotational symmetry of arrangement of transition dipole moments . the linear absorption spectrum is obtained from the transition dipole moments of exciton states 0 , k1k2 in eq 40 . taking into account the rotational symmetry in the location of chromophore transition dipole moments 0,n1n2 and that of the eigenstates in the rings , where eigenstates with k2 = 1 and those with k2 = 1 are degenerate , and performing an average over random orientations of tubes with respect to the polarization of the external light field gives for the absorption spectrum44where the oscillator strength reads45here , the angle describes the relative rotation of neighboring rings around the symmetry axis of the tube . using the parameters n1 , n2 , c , b , and , which define the unit cell and the circumference vector , this can be written as46for the present system we find = 30. contrary to the rest of this section , in the above equation n1 and n2 stand for the components of the circumference vector as described in the si . in the case of linear dichroism , the tubes are oriented in a flow cell and the absorption is measured with light polarized parallel and perpendicular to the symmetry axis of the tube . linear dichroism is defined as the difference between these two absorbances ( parallel minus perpendicular ) . taking into account an average over random orientations of the complexes with respect to rotation around the symmetry axis yields the linear dichroism spectrum47 as in the case of isotropic absorption , bands with nonzero oscillator strength are created for the three values of k2 = 0 , 1 . the internal states of the bands are counted by k1 . as discussed above , due to the rotational symmetry of the tube , the k2 = 0 band is polarized parallel to the symmetry axis of the tube and the k2 = 1 bands are polarized perpendicular to this axis . the oscillator strengths m ( k1 , k2 ) ( eq 45 ) of the different transitions will be calculated below in different approximations . before a general conclusion can be drawn about the angle between the chromophore s transition dipole moments and the symmetry axis of the tube . as seen from the expression for ld( ) in eq 47 , equals the ratio between the negative and the positive part of the ld spectrum . hence , within the present approximations of neglecting static disorder and homogeneous broadening we can get an estimate for the angle directly from the positive and negative parts of the experimental ld spectrum . analyzing this spectrum in this way results in 70 , which is close to the = 74 obtained indirectly by optimizing the structure and calculating optical spectra with a theory that includes static disorder . due to the presence of static disorder the rotational symmetry in the rings is broken and a mixing between transitions that are polarized in parallel and perpendicular directions occurs , which changes the relatives oscillator strengths in the ld spectrum . integrating the oscillator strength calculated in the presence of disorder and using the same analysis as applied to the experimental ld spectrum results in an angle 71 , which is practically identical to the 70 inferred above . the oscillator strength m ( k1 , k2 ) in eq 45 can be obtained analytically using either a nearest neighbor approximation for the inter - ring couplings or periodic boundary conditions for the ends of the tube . inspection of the inter - ring couplings for the present system ( table 1 ) suggests that in particular for the k2 = 1 bands the coupling between rings falls off too weakly with increasing n1 to justify a nearest neighbor approximation . indeed , we find large deviations between the spectrum obtained in this approximation and the one resulting from an exact numerical diagonalization , as discussed in the si . for periodic boundary conditions the eigenenergies of the tube are obtained as48 the oscillator strengths of the transitions in the k2 = 0 band read49and that of the k2 = 1 bands are50hence , in the case of k2 = 0 all oscillator strength is concentrated in the k1 = 0 transition , which occurs at a transition energy51by applying the values for j(n1 , k2 = 0 ) in table 1 it is seen that e0 = 2.145 ev ( 578 nm ) is strongly red shifted with respect to the monomer excitation energy since all j(n1 , k2 = 0 ) are negative . in the case of the k2 = 1 bands , the oscillator strength is distributed over several transitions and the one with the largest oscillator strength occurs at a transition energy that is close to52 as shown in ref ( 23 ) , for very long tubes and the periodic boundary conditions considered , this transition is the only one carrying oscillator strength for k2 = 1 . for the present system ( table 1 ) we obtain eh = 2.460 ev ( 504 nm ) , which is blue shifted with respect to the monomer transition energy . as seen in tables 1 and 2 , the inter - ring couplings are responsible for the blue shift . in figure 13 the absorption spectrum resulting from numerical diagonalization of the bare exciton hamiltonian is compared to the one obtained analytically by using periodic boundary conditions , described above . the qualitative behavior of the numerical exact spectrum is well reproduced , in particular , the position and intensity of the k2 = 0 transition that is polarized parallel to the symmetry axis of the tube and the splitting between this transition and the strongest transition of the k2 = 1 bands . there are however quantitative deviations between the intensities of the different transitions in the k2 = 1 bands obtained in the two different calculations . the quantity re[j*(n1 ; k2)e ] in the last column is used in eq 52 to estimate the energy of the most intense transition of the h - band . homogeneous absorption spectrum resulting from the bare exciton hamiltonian by numerical diagonalization ( black ) or analytical theory ( eqs 48 , 49 , and 50 ) using periodic boundary conditions ( green ) . stick spectra ( vertical lines ) were dressed with lorentians of fwhm 10 mev . dashed lines represent transitions that are polarized perpendicular to the symmetry axis of the tube and solid lines those which are polarized parallel to this axis . vertical dashed finally , we use the md trajectories for a microscopic description of static disorder in transition energies and excitonic couplings . for this purpose 500 snapshots between 22 and 72 ns of the md trajectories were taken , and for each of the nuclear configurations the fluctuation in transition energies ( eq 27 ) was calculated . in addition , we used this parametrization to explain the redshift between the vacuum transition energy estimated for the monomer in methanol and the ( 557 nm ) estimated above from the calculations of the optical spectra of the tube . for this purpose , we take the and explicitly calculate the electrostatic ( eq 27 ) and dispersive ( eq 29 ) transition energy shift induced by the interchromophore coulomb coupling in the tube . concerning the excitonic couplings , an analysis of the screening factors fmn of selected snapshots revealed that the fluctuations of fmn along the md trajectories are small ( figure 14 ) . the screening factors fmn , however , depend on the mutual orientation of chromophores , and 6 major groups of screening factors are identified . for these groups we determined averaged screening factors that range between 0.58 for group 1 down to 0.47 for group 2 , the overall average being 0.55 used before . in the calculation of static disorder along the md trajectories we distinguish between the different groups and apply these average screening values to the calculation of excitonic couplings with the tresp method53where the fluctuation of vmn(t ) is given solely by the variation of ri(m)(t ) and rj(n)(t ) and not by that of fmn , which is taken from the respective group of screening factors the chromophore pair ( m , n ) belongs to . the resulting optical spectra are compared in figure 15 to the experimental data and show only slightly less agreement than the spectra obtained by the phenomenological model of static disorder , discussed above ( figure 11 ) . the calculated spectrum was shifted by 80 mev to the red in order to implicitly take into account the dispersive shift by the solvent molecules . this shift is within the range of 50100 mev estimated before for this type of interaction . screening factors of excitonic couplings , obtained by the poisson - tresp method , as a function of the vacuum coupling , taking into account nuclear coordinates from 5 different snapshots along the md trajectories ( at 37 , 42 , 47 , 52 , and 57 ns ) . screening factors are grouped by the vertical lines , and dashed horizontal lines denote the average screening factor for a given group . measured absorption ( black solid line ) and linear dichroism ( black dashed line ) spectra are compared to calculated spectra ( red and green lines , respectively ) . in the calculations static disorder in site energies and excitonic couplings was described microscopically taking into account md trajectories . for comparison of theoretical model structures with experimental cryo - tem data , projection images of the model structures were simulated , filtered , and normalized in the same way like the experimental data . at the available resolution the visual comparison demonstrates a high level of consistency ( figure s11 in the si ) . however , due to the small size of dye monomers and the high degree of statistical noise details of the molecular packing within the tubes can not be resolved by cryo - tem and are also invisible in the mimicked model tem images . to provide a quantitative assessment we generated cross - sectional density profiles of both the simulated images and the sum image of the tem data . as shown in figure 16 the curve progression of the density profile derived from the experimental data ( red line ) corresponds very well to the progression of the density plots of the simulated micrographs derived from tomographic projection views of the ( 6,0 ) dyea tube model ( black lines ) . in other words , within the achievable resolution the deviations within the model projections due to different orientations are greater than the differences between the experimental structure and the theoretical model . quite similar results were obtained for other tube models with suitable diameter and suggest that a clear proof of one particular structure model against others is not possible in this way . calculated cryo - tem 2d - density profiles of the ( 6,0 ) dyea tube model ( black lines ) for 10 tomographic projections . experimental density profile from the sum image of cryo - tem of the tube ( red line ) . horizontal straight line corresponds to the mean gray value of the background ( 121.6 11.0 ( std . therefore , in the present study , the optical spectra and the molecular stability were analyzed in addition to the cryo - tem data . the structure proposed on these grounds is fully consistent with the experimental cryo - tem data , as figure 16 demonstrates . a systematic procedure for the structure prediction of self - organized single - walled tubes of cyanine dyes has been developed and successfully applied to ttbc . the method combines information from cryo - tem with that from molecular crystals of ttbc from molecular mechanics and optical spectra of the monomer and the aggregate . the frenkel exciton hamiltonian of the aggregate , used to calculate optical spectra , is parametrized by using quantum chemical / electrostatic methods , developed before . the arrangement of molecules in dyea crystals , containing one molecule per unit cell , is found to be closer to the final structure than that of the alternative dyem containing two molecules per unit cell . thus far we have not found any dyem tube that can explain the optical spectra and is also stable during the md simulations . in contrast , our iterative refinement of the ( 6,0 ) dyea tube , which effectively adjusted the unit cell , finally resulted in a stable tube . although this result at first glance is somewhat counterintuitive , it sheds some light on our strategy of choosing the starting structure for the md simulations . obviously the interaction in a plane in a 3d crystal does not always contain the major interaction of a solvent - exposed single - walled tube . the proposed structure of the ( 6,0 ) dyea tube consists of rings of 6 chromophores with rotational symmetry . the transition dipole of each chromophore forms an angle of = 74 with respect to the symmetry axis of the tube . neighboring rings are rotated by an angle = 30 with respect to each other . this structure explains all experimental data available so far and allows for a detailed investigation of the effect of interchromophore couplings on the optical properties of the aggregate that after all allowed for the prediction of the structure . from the monomer spectrum in methanol a mean 00 transition energy corresponding to a wavelength of 515 nm , an effective vibrational frequency of 800 cm , and a huang the interchromophore coupling in the tube leads to a striking change in the optical properties . two main absorption bands , the transition - dipole of which are orthogonally polarized , appear : a sharp low - energy j - band that is polarized along the symmetry axis of the tube and a much broader high - energy h - band . such hybrid h- and j - aggregate - like properties have been reported before for semiconducting polymers and terrylene dye crystals and have led to the classification as hj - aggregates . the difference in widths of the two bands is shown to result from a redistribution of oscillator strengths among vibronic transitions that is induced by exciton delocalization and by coupling of excitons to the effective vibrational mode . an analytical model in combination with perturbation theory how unique the present proposal is , additional optical experiments like pump probe and 2d electronic spectra would be helpful . it will be interesting to study exciton transfer / relaxation between the h- and the j - bands in this system . we expect that the inclusion of exciton relaxation - induced lifetime broadening will further improve the agreement between calculated and measured spectra .

cryogenic transmission electron microscopy ( cryo - tem ) studies suggest that ttbc molecules self - assemble in aqueous solution to form single - walled tubes with a diameter of about 35 . in order to reveal the arrangement and mutual orientations of the individual molecules in the tube , we combine information from crystal structure data of this dye with a calculation of linear absorbance and linear dichroism spectra and molecular dynamics simulations . we start with wrapping crystal planes in different directions to obtain tubes of suitable diameter . this set of tube models is evaluated by comparing the resulting optical spectra with experimental data . the tubes that can explain the spectra are investigated further by molecular dynamics simulations , including explicit solvent molecules . from the trajectories of the most stable tube models , the short - range ordering of the dye molecules is extracted and the optimization of the structure is iteratively completed . the final structural model is a tube of rings with 6-fold rotational symmetry , where neighboring rings are rotated by 30 and the transition dipole moments of the chromophores form an angle of 74 with respect to the symmetry axis of the tube . this model is in agreement with cryo - tem images and can explain the optical spectra , consisting of a sharp red - shifted j - band that is polarized parallel to to the symmetry axis of the tube and a broad blue - shifted h - band polarized perpendicular to this axis . the general structure of the homogeneous spectrum of this hybrid hj - aggregate is described by an analytical model that explains the difference in redistribution of oscillator strength inside the vibrational manifolds of the j- and h - bands and the relative intensities and excitation energies of those bands . in addition to the particular system investigated here , the present methodology can be expected to aid the structure prediction for a wide range of self - assembled dye aggregates .

Introduction Theory Results and Discussion Conclusions

PMC3136207

metabolic syndrome refers to a collection of issues including visceral obesity , elevated blood glucose levels , dyslipidemia ( elevated fasting triglycerides and low high - density lipoprotein ( hdl ) cholesterol levels ) , and hypertension . it leads to an increase in the risk of developing of cardiovascular disease ( cvd ) , type 2 diabetes , and cancer . thus , effective strategies preventing metabolic syndrome are required to decrease the incidence of diseases and promote healthy aging . the development of metabolic syndrome is primary caused by a sedentary lifestyle and overnutrition ; however , genetic characteristics are also involved to some extent . daily physical activity directly influences obesity and metabolic syndrome associated with the metabolic function of skeletal muscle ( figure 1 ) . thus , dietary exercise , along with adequate diet , is well known to be one of the major preventive therapies against metabolic syndrome . in the last few decades , it has been shown , in epidemiological and experimental studies , that exercise reduces obesity , improves glucose tolerance , and decreases the risk of diabetes and cvd . the effects of exercise are brought about by elevated energy consumption , improvement of insulin sensitivity , and a reduction in inflammation . a single bout of exercise drastically changes various physiological parameters such as hormone production , blood flow , and the activity of the nervous system , in addition to altering the expression / activity of certain genes and proteins in the skeletal muscle . this paper reviews evidence regarding the influence of exercise on the progress of metabolic syndrome along with its underlying molecular mechanisms . it particularly focuses on the skeletal muscle , a major metabolic organ , and describes the benefits of functional food factors combined with exercise therapy . sedentary behavior and persistent low levels of physical activity are known to induce progression toward metabolic syndrome , type 2 diabetes , and cvd [ 15 ] . energy consumption depends on the intensity and amount of physical activity ; therefore , a sedentary life tends to result in a positive energy balance and leads to an accumulation of body fat . adipose tissue secretes bioactive factors , adipocytokines , such as tumor necrosis factor alpha ( tnf- ) , plasminogen activator inhibitor , and resistin , into circulation . it has been considered that there is aclose relationship between these adipocytokines and health problems , such as obesity and metabolic and cardiovascular disorders , as they cause insulin resistance , injury to the endothelium , and inflammation . this decrease may be due to atrophy of skeletal muscle , a major energy - consuming tissue in the body . it has been reported that the loss of fat - free mass with inactivity and age explains a reduction in the resting metabolic rate ( rmr ) . muscle atrophy may be due to both muscle fiber atrophy and loss of complete muscle fibers [ 8 , 9 ] due to several factors including the apoptosis of muscle cells , decreased differentiation of satellite cells , and reduced protein levels as a result of decreased protein synthesis and increased protein degradation . the activity of enzymes involved in aerobic metabolism and glucose uptake in muscle is also decreased by inactivity and aging . laboratory studies have shown that significant protein degradation is seen within 2 days of muscle immobilization leading to loss of muscle mass within 1 week . insulin - induced glucose uptake into the muscle is also reduced along with a reduction in its signaling pathway , within 2 days of immobilization . at the same time , the activity of lipoprotein lipase ( lpl ) , a protein important for controlling plasma triglyceride catabolism hdl cholesterol and other metabolic risk factors was lost . consistent with the decrease in lpl function , the clearance of plasma triglyceride by skeletal muscle was significantly decreased and plasma hdl cholesterol concentration declined . recently , it has been established that low - grade continuous inflammation and oxidative stress are associated with metabolic disorders and cvd [ 1618 ] . low levels of physical inactivity lead to chronic inflammation and oxidative stress in the skeletal muscle , the circulatory system , and other tissues . some adipocytokines , such as tnf- and interleukin-6 ( il-6 ) , which are secreted from accumulated visceral adipose tissue can cause this inflammation . insulin - induced activation of the insulin receptor ( ir ) , phosphatidylinositol 3-kinase ( pi3k ) , and akt is prevented along with ib kinase ( ikk ) activation and degradation of ib in the muscle tissue [ 1921 ] . furthermore , ikk- silencing prevents tnf--induced impairments in insulin action on akt phosphorylation and glucose uptake . growing evidence suggests that additional adipocytokines including resistin , fatty - acid - binding protein ( fabp ) , and visfatin have also induced insulin resistance associated with inflammation [ 2325 ] . in addition , a reduction of circulating adiponectin , an adipocytokine with anti - inflammatory properties , occurs with obesity and leads to insulin resistance in skeletal muscle and liver [ 2628 ] . a recent study clearly showed that adiponectin directly improves glucose and lipid metabolism along with mitochondria biogenesis and activation of the key metabolic modulators via adiponectin receptor 1 ( adipor-1 ) in skeletal muscle . this inflammation of skeletal muscle is caused by muscle inactivity even when body fat is low . indeed , expression of tnf- in skeletal muscle is elevated with insulin resistance in human . this indicates that tnf- generated from not only other cells but also myocytes disturbs insulin signaling . an increased level of oxidation of lipids , dna , and proteins is also observed in muscles of sedentary subjects compared to that of active subjects [ 3134 ] . furthermore , continuous activation of intracellular oxidative - stress - sensitive factors such as the nuclear factor - kappa b ( nf-b ) and mitogen - activated protein kinase is seen in the muscle of sedentary men [ 35 , 36 ] . oxidative - stress is also strongly associated with development of insulin resistance in the skeletal muscle . thus , oxidative - stress - induced insulin resistance in muscle leads to the initiation of diabetes and potentially late diabetic complications . it is without doubt that insulin sensitivity is inversely correlated with the plasma levels of free radicals in diabetic patients [ 17 , 18 ] . several studies have demonstrated that reactive oxygen species ( ros ) impair insulin - mediated glucose uptake and storage by disrupting signaling control points such as glycogen synthase kinase-3 , akt phosphorylation , and actin remodeling [ 3739 ] . in addition , we have recently found that 3-nitrotyrosine modification of adenylate kinase 1 ( ak1 ) , a key enzyme in synthesis ; equilibration ; regulation of adenine nucleotides is elevated in older muscle and that the modification of ak1 is involved in the impairment of glucose uptake via inhibition of amp - activated protein kinase ( ampk ) . furthermore , it has been suggested that metabolic regulation of adiponectin is associated with reduction of oxidative - stress in skeletal muscle . inflammatory cytokines and ros are also associated with protein degradation via activation of the ubiquitin - proteasome pathway . have revealed that the addition of oxidants and tnf- to myotubes increases protein degradation rates , ubiquitination of proteins such as myosin , and expression of the main components of the ubiquitin - proteasome pathway [ 4042 ] . muscle ring finger 1 ( murf1 ) and atrogin-1 have been identified as the ubiquitin ligases whose activities increase during atrophy [ 43 , 44 ] . nf-b can regulate the ubiquitin - proteasome proteolytic pathway through the induction of murf1 and proteasome expression [ 4547 ] . furthermore , it has been shown that the 20s proteasome can selectively degrade oxidatively modified proteins without ubiquitination [ 48 , 49 ] . these observations suggest that protein degradation could be the link between oxidative stress , inflammatory cascade , and muscle atrophy . in fact , hyperactivity of nf-b and the ubiquitin - proteasome pathway has been identified as a major cause of aged - related muscle atrophy [ 50 , 51 ] . many large cohort studies have found that higher level of physical activity is associated with reduced risk of developing diabetes and cvd [ 5258 ] . one of the first major trials to examine the effect of physical activity was the university of pennsylvania alumni health study . in this study , the level of physical activity was found to be inversely related to the development of type 2 diabetes in 202 male subjects . the incidence declined by 6% for each 500 kcal increment in energy expenditure from less than 500 to 3500 kcal . in addition , the osaka health survey showed , in 444 men , that regular exercise , at least once a week , reduced the relative risk of type 2 diabetes to 0.75 compared with in those engaging in exercise less often . subjects who engaged in intense exercise at least once a week , at weekends , exhibited further reduction of the multiple - adjusted relative risk of type 2 diabetes to 0.55 compared with sedentary subjects . cardiorespiratory fitness is also protective against diabetes and metabolic syndrome [ 60 , 61 ] . studies lasting for 312 months involving exercise sessions of 3060 min in a week have shown that regular exercise reduces fat mass and improves insulin sensitivity without dietary caloric restriction in overweight men and women [ 6266 ] . moreover , regular exercise decreases plasma levels of triglyceride and hdl cholesterol and lowers blood pressure [ 6769 ] . consistent with the improvement , regular exercise reduces circulating adipocytokines , such as resistin , visfatin , and fabp , involving development in metabolic disorder and inflammation [ 7072 ] . on the other hand , several studies have suggested that the improvement in insulin sensitivity induced by regular exercise is not mediated by changes in plasma adiponectin [ 7375 ] . however , the ratio of high - molecular - weight form to total adiponectin was increased by regular exercise and there was a positive correlation between the increase of the adiponectin ratio and the improvement of insulin sensitivity in older insulin - resistant adults . in addition , it has been shown that muscle adipor-1 is elevated in response to physical exercise , which elevates metabolic signal transduction of adiponectin and then improves oxidative metabolism . therefore , the regulation of these adipocytokines including adiponectin likely contributes to the prevention of metabolic syndrome by daily exercise . aerobic training has traditionally been adopted as the main form of exercise therapy in epidemiological and laboratory studies . however , recently , the inclusion of resistance training as an integral part of an exercise therapy program has recently been endorsed by the american heart association , the american college of sports medicine , and the american diabetes association . cross - sectional studies have shown that muscle mass is inversely associated with mortality and the prevalence of metabolic syndrome , independent of cardiorespiratory fitness levels . even in the elderly , resistance training increases muscle mass from , 7.4% to 10.0% , along with muscle strength after 1016 weeks [ 83 , 84 ] . one study demonstrated that twice - weekly resistance training could prevent age - associated loss of lean body mass ( lbm ) and rmr , which is closely correlated to losses in lbm . resistance training contributes to an elevation in rmr as a result of a greater muscle protein anabolism . theoretically , a gain of 1 kg in muscle mass should result in an increase of approximately 21 kcal in rmr . thus , resistance training , when sustained over years or decades , translates into clinically important differences in daily energy expenditure and can prevent age - associated fat gains . however , resistance training can rather inhibit lbm loss when combined with dietary restriction in antimetabolic syndrome therapy . in several randomized control trials , where obese men were randomly assigned to either a diet - only group or a diet with resistance training group , lbm was preserved by exercise training [ 8795 ] . furthermore , there is a strong support for the notion that resistance training is at least as effective as aerobic training in reducing some major cvd risk factors . findings from several studies demonstrate that resistance training significantly decreases glycosylated hemoglobin levels in people with an abnormal glucose metabolism and has a tendency to improve lipoprotein - lipid profiles [ 90 , 96 , 97 ] , independent of changes in body weight or composition . in addition , several factors such as hormones , growth factors , oxidative stress , and heat stress also affect signaling . numerous human and animal studies have shown that metabolic improvement due to exercise occurs along with changes in the expression / activity of muscle proteins and alterations in their mrna transcription ( figure 2 ) . a single exercise bout improves glucose uptake in skeletal muscles via insulin - dependent and insulin - independent signal transduction mechanisms [ 98101 ] . this effect is observed for several hours after exercise , often persisting until the next day . the increase in glucose uptake is caused by the translocation of the glucose transporter 4 ( glut4 ) to the plasma membrane after activation of the ir / pi3k / akt signaling pathway [ 102 , 103 ] . elevated ampk activity and intracellular calcium levels can also induce glut4 translocation independent of the insulin signaling pathway [ 104 , 105 ] . in addition , a considerable amount of attention has been given to the peroxisome proliferator - activated receptor gamma coactivator-1 alpha ( pgc-1 ) as a target for the prevention or treatment of metabolic syndrome . pgc-1 has been shown to have the central role in a family of transcriptional coactivators involved in aerobic metabolism and is activated by exercise . activation of pgc-1 alters the metabolic phenotype through interaction with nuclear respiratory factor and the peroxisome proliferator - activated receptor [ 107 , 108 ] . improved understanding of the activation of the pgc-1 protein by exercise has implications beyond improving athletic performance [ 109 , 110 ] . it may be target for the treatment of various diseases such as the mitochondrial myopathies and diabetes [ 111113 ] . the activity / expression of lpl , a protein important for controlling triglyceride catabolism and cholesterol levels in plasma , is also elevated for 3 to 22 hours after exercise [ 114 , 115 ] . these beneficial adaptations persist in humans and animals who perform regular exercise , independent of the acute effects of exercise . initiation of protein synthesis appears to be regulated by the akt / the mammalian target of rapamycin ( mtor ) signaling proteins . akt phosphorylation regulates the catabolic pathway by preventing the induction of muscle - specific ubiquitin ligases such as atrogin-1 and murf1 and activates the anabolic pathway by phosphorylating mtor . mtor then initiates translation via the activation of translation regulators p70 and the eukaryotic initiation factor-4e ( eif-4e ) complex , following phosphorylation of eif-4e - binding protein-1 , one of the main translational inhibitors . in addition , regular exercise can inhibit the apoptotic signaling pathway ; this is associated with reducing oxidative stress and inflammation , and it results in the preservation of skeletal muscle fibers . this inhibitory effect results from the upregulation of antiapoptotic mediators , such as b - cell lymphoma / leukemia , x - linked inhibitors of apoptosis , and heat shock protein 70 and from the downregulation of proapoptotic mediators such as caspase-3 and bax [ 119 , 120 ] . a decrease in tnf- , a factor that accelerates the caspase cascade , may be involved in antiapoptotic signaling , as regular exercise blunts tnf- expression in aged muscle [ 121 , 122 ] . furthermore , the inhibition of tnf- and oxidative stress would lead to a reduction in age - related muscle dysfunction , including prevention of protein degradation and impaired glucose uptake . the study of the mechanisms underlying the effects of exercise has been enhanced by the analysis of the function of micrornas ( mirnas ) in recent years . the mirnas are small noncoding sections of rna that regulate gene expression by degrading mrna molecules or , more frequently in mammalian cells , inhibiting their translation [ 123 , 124 ] . it has been suggested that mirna - mediated gene regulation is a part of the fundamental mechanism of posttranscriptional regulation and may have diverse functional effects . several of these mirnas have been suggested to have a role in a wide range of biological processes , including development , cell death , carcinogenesis , and response to stress [ 126129 ] . some mirnas , including mir-1 , mir-133 , and mir-206 , which are referred to as myomirs , have also been suggested to act as modulators of skeletal muscle function [ 130 , 131 ] . we along with other researchers have shown that physical activity elevates muscle metabolism associated with pgc-1 , via regulation of some mirnas [ 132 , 133 ] . furthermore , there is growing evidence that secreted proteins derived from muscle , also known as myokines , are elevated in plasma in response to exercise and regulate various functions of other organs . muscle - derived il-6 is well known as a representative myokine that is markedly elevated in muscle and secreted into plasma following muscle contraction [ 134 , 135 ] . this myokine could mediate some of the exercise - induced metabolic changes and anti - inflammatory effects in other organs such as the liver , adipose tissue , and blood vessels . subsequently , other muscle - derived proteins , il-15 , and fibroblast growth factor-21 have been reported to regulate nutrient metabolism in other organs . furthermore , it has been shown that brain - derived neurotrophic factor is produced in skeletal muscle in response to contraction and has been suggested to increase fat oxidation in skeletal muscle in both an autocrine and paracrine fashion . the potential effects of several food factors on muscle lipid metabolism in exercise have been investigated . some of them have been found to accelerate lipid utilization ; however , their efficacy is still controversial . a rate - limiting step in lipid metabolism in myocytes is the entry of long - chain fatty acids into the mitochondria . carnitine palmitoyltransferase i ( cpt i ) , located on the outer mitochondrial membrane , plays an important role in the entry of fatty acids into mitochondria . we found that a novel antioxidant astaxanthin limits the oxidative modification of cpt i by hexanoyl lysine . this causes , along with elevated cpt i activity during exercise , acceleration of the reduction in body fat due to exercise training . ikeuchi et al . also showed that astaxanthin supplementation accelerates a catabolism in body fat along with a reduction of blood lactate . catechin , one of the polyphenols contained in japanese green tea , accelerates the utilization of fatty acids as an energy source for skeletal muscle contraction during exercise . it has been suggested that the effect of catechin is related to the enhancement of -oxidation activity and the level of fatty acid translocase / cd36 mrna in muscle . intake of -lipoic acid , combined with endurance exercise training , further accelerates glucose uptake and activity of the insulin signaling pathway compared with training alone . this leads to an increase in circulating free fatty acids and a further improvement in endurance . capsaicin , obtained from hot red peppers , is likely to enhance fat metabolism by increasing lipolytic hormones and promoting fat oxidation in the skeletal muscle . however , growing evidence indicates that a large dose of dietary antioxidants prevents the adaptation generally seen as a result of regular exercise . vitamin c supplementation ( 1 g / day , for 6 weeks ) decreases the improvement of vo2 max associated with training . antioxidant vitamins and n - acetylcysteine reduced mitochondria biogenesis associated with the expression of pgc-1 , a key modulator of aerobic metabolism in skeletal muscle cells [ 143 , 144 ] . more recently , in a prospective randomized intervention study , a combination of vitamin c ( 1000 mg / day ) and vitamin e ( 400 iu / day ) has been shown to inhibit the improvement of insulin sensitivity and elevation of plasma adiponectin along with the cancellation of pgc-1 induction in response to exercise training in healthy men . these observations suggest that the intake of antioxidants is not always beneficial in counteracting muscle dysfunction related with inactivity and that oxidative stress is involved in signal transduction of exercise adaptation . it has been shown that the protein requirement for subjects performing resistance training is higher than that for sedentary individuals . the daily recommended protein intake is estimated to be 1.41.8 g / kg for those performing resistance exercise when the intake of calories and carbohydrates is adequate . however , it is not only the amount of protein , but also the timing of intake that is important for the efficient building of muscle . eating protein immediately after exercise is more effective , in terms of increasing protein synthesis , compared with several hours later . the cross - sectional area of the quadriceps muscle after a 12-week resistance training program is greater . additionally , protein synthesis in muscle can be promoted by intake of proteins combined with carbohydrates via the actions of insulin as this accelerates the increase in muscle mass and strength . in addition , it has been reported that the intake of amino acids and peptides is beneficial . amino acids are not only utilized in the synthesis of muscle protein but can also exert a variety of physiological effects . attention has been focused on the effects of branched - chain amino acids ( bcaas ) , including valine , leucine , and isoleucine , which are known to be found in relatively high concentrations in both muscle proteins and food proteins . therefore , when bcaas are not supplied in the diet , muscle protein is catabolized to obtain them . furthermore , dietary bcaas modulate muscle protein metabolism to promote the synthesis and inhibit the degradation of proteins . it is the most abundant free amino acid in muscle tissue , and its intake leads to an increase in myocyte volume and results in the stimulation of muscle growth . glutamine is also found at relatively high concentrations in many other human tissues and has an important homeostatic role . therefore , during catabolic states such as exercise , glutamine is released from skeletal muscle into the plasma to be utilized for maintenance of the glutamine level in other tissues . -hydroxy--methylbutyrate ( hmb ) is a metabolite of the branched - chain amino acid leucine . it increases muscle mass by inhibiting the degradation of protein via its influence on the metabolism of branched - chain amino acids . a meta - analysis supported the use of hmb while performing resistance exercise to augment lbm and strength . several studies have demonstrated that an intake of hmb for at least 4 weeks achieved a greater increase in lbm or muscle power output . previously , numerous studies on prevention and development of metabolic syndrome have focused on effects of adipose tissue . on the other hand , because skeletal muscle plays an important role as a metabolic organ in the development of metabolic syndrome , recently these relationships have been established , as described in this paper . daily exercise habit and physical fitness level are associated with a reduction in risk of metabolic syndrome , independently of body fat level . therefore , although adequate diet is important for prevention and treatment of metabolic syndrome , regular exercise it needed for the therapies .

a sedentary lifestyle can cause metabolic syndrome to develop . metabolic syndrome is associated with metabolic function in the skeletal muscle , a major consumer of nutrients . dietary exercise , along with an adequate diet , is reported to be one of the major preventive therapies for metabolic syndrome ; exercise improves the metabolic capacity of muscles and prevents the loss of muscle mass . epidemiological studies have shown that physical activity reduces the risk of various common diseases such as cardiovascular disease , diabetes , and cancer ; it also helps in reducing visceral adipose tissue . in addition , laboratory studies have demonstrated the mechanisms underlying the benefits of single - bout and regular exercise . exercise regulates the expression / activity of proteins associated with metabolic and anabolic signaling in muscle , leading to a change in phenotype . the extent of these changes depends on the intensity , the duration , and the frequency of the exercise . the effect of exercise is also partly due to a decrease in inflammation , which has been shown to be closely related to the development of various diseases . furthermore , it has been suggested that several phytochemicals contained in natural foods can improve nutrient metabolism and prevent protein degradation in the muscle .

1. Introduction 2. Progression of Metabolic Syndrome due to a Sedentary Lifestyle 3. Evidence for the Beneficial Effects of Exercise 4. Molecular Mechanisms Underlying the Benefits of Exercise 5. Effects of Combining Food Factors with Exercise Therapy 6. Conclusion

PMC4943148

extensive global evidence highlights a lack of communication skills in doctors that leads to dysfunctional consultations . it has also been seen that doctors lack skills required for patient - centered consultations . literature confirms that communication skills can be learned and taught and indicates that patient centeredness decreases as medical students progress into the clinical years . effective communication is a key competency required by international regulatory and accreditation bodies for both under and postgraduates . various communication models and teaching methodologies for teaching and learning have been reported in international literature . pakistan medical and dental council , the national regulatory authority , stresses the development of effective communication skills . however , among the over ninety registered medical colleges , communication skills are taught in a handful only . in 2002 - 2003 , a private medical college in pakistan instituted problem - based learning with an integrated longitudinal communication skills program . the cambridge - calgary model of communication skills is used as the foundational framework for this program . in the first 2 years , teaching involves the development of basic to complex communication skills including disclosing bad news , communicating with angry patients , and dealing with myths and misconceptions . the skills are reinforced in the subsequent 3 years within the clinical rotations of the medical students . learning strategies include plenary lectures , small group experiential sessions , video discussion , and reflective assignments . formative and summative assessment of communication skills takes place annually through specific objective structured clinical examination ( osce ) stations . the objective of this study was to evaluate the effectiveness of the communication skills program by comparing communication skills of its medical students ( medical college 1 [ college with integrated longitudinal communication skills program ] ) with medical students of a matching medical college without formal communication skills teaching ( medical college 2 [ comparable college without communication skills program ] ) . a private medical college ( medical college 2 ) without formal communications skills teaching was chosen as the comparison group . it matched regarding socioeconomic background , curriculum , and academic schedule with the college teaching communication skills ( medical college 1 ) . communication skills of the students of the two medical colleges were assessed longitudinally through an osce conducted in years three and five on the same cohort . for each osce , we invited all students available at the time according to the curricular schedules to participate in the osce . the osce 's consisted of four communication skills stations as this number has been found to measure communication skills with a good level of reliability . each station had a specific patient perspective built into the scenario and required an empathetic , nonjudgmental , and honest approach to elicit patients ideas , fears , and expectations [ table 1 ] . patient scenarios in the 4-station objective structured clinical examination validity was ensured through multidisciplinary development of the stations on commonly encountered clinical presentations and was reviewed by faculty involved in teaching communication skills . to ensure inter - rater reliability , equal numbers of examiners were drawn from both the institutions . examiner training and standardization was conducted through specially made teaching videos demonstrating desirable communication skills on the selected scenarios . standardized patients were trained by examiners before each osce to ensure uniformity , and the angoff method was used for setting the standard of each station . ethical approval was granted by the ethics review committees of both participating universities ( 1182-fm / erc - aku and 0769-zu ) . we used a rating scale based on the cambridge - calgary guide 1 for each station to assess the communication skills . this contains six communication skills constructs in the form of subscales with individual items in each . the constructs included initiating the session ( its ) , gathering information ( gi ) , understanding patient perspective ( upp ) , providing structure to the consultation ( psc ) , building the relationship ( btr ) , and closing the session ( cts ) . for the disclosing bad news station , breaking the news ( btn ) we analyzed the mean construct scores and the mean total scores with 95% confidence interval for each station of both osces . results of each osce station were converted into percentage scores derived from the sum of the construct scores divided by the total score and multiplied by 100 . smirnov and shapiro wilk tests were used to observe normality of the scores . as the scores were not distributed normally , the mann whitney u - test was used to compare differences in the distribution of construct scores and percentage scores by osce stations , universities , and year . cronbach 's alpha was calculated for each osce and for each station at the end of both years to test reliability . spss statistics for windows , version 21.0 , ibm , armonk , new york , was used for data entry and statistical calculations . we used a rating scale based on the cambridge - calgary guide 1 for each station to assess the communication skills . this contains six communication skills constructs in the form of subscales with individual items in each . the constructs included initiating the session ( its ) , gathering information ( gi ) , understanding patient perspective ( upp ) , providing structure to the consultation ( psc ) , building the relationship ( btr ) , and closing the session ( cts ) . for the disclosing bad news station , breaking the news ( btn ) construct was used instead of gi . we analyzed the mean construct scores and the mean total scores with 95% confidence interval for each station of both osces . results of each osce station were converted into percentage scores derived from the sum of the construct scores divided by the total score and multiplied by 100 . smirnov and shapiro wilk tests were used to observe normality of the scores . as the scores were not distributed normally , the mann whitney u - test was used to compare differences in the distribution of construct scores and percentage scores by osce stations , universities , and year . cronbach 's alpha was calculated for each osce and for each station at the end of both years to test reliability . spss statistics for windows , version 21.0 , ibm , armonk , new york , was used for data entry and statistical calculations . students of medical college 1 had higher mean percentage scores on all stations in both years . there was an increase in the scores from third to fifth year on stations focusing on history taking and patient 's perspective in both medical colleges . the scores on stations focusing on the exploration of the patient 's perspective decreased from third to fifth year in both medical colleges . the difference in scores between the two groups reduced in the fifth year . at the end of the third year , 21 ( 31.34% ) ( 10 male and 11 female ) from the medical college with the longitudinally integrated communication skills program ( medical college 1 ) and 31 ( 46.26% ) ( 19 female and 12 male ) students from ( medical college 2 ) consented out of the 67 available students on each site . similarly , at the end of the fifth year , out of the available 65 students , 19 students ( 29.3% ) ( 8 male and 11 female ) from medical college 1 and 22 ( 34% ) ( 11 male and 11 female ) students from medical college 2 participated after giving consent . at the end of the third year , medical college 1 achieved a significantly higher overall mean total station score of 68.0% ( standard deviation [ sd ] = 13.5 ) versus 57.2% ( sd = 15.4 ) ( p < 0.001 ) . at the end of the fifth year , the overall mean total station score of medical college 1 remained significantly higher , although the difference had reduced from 9.2% to 7.1% ( 70.2% ) ( sd = 13.7 ) versus 63.1% ( sd = 15.2 ) ( p = 0.004 ) [ table 2 ] . mean percentage scores and mean p values of the constructs and total objective structured clinical examination scores of both medical colleges at the end of the third year , the mean overall percentage score of each construct was significantly higher in medical college 1 . at the end of the fifth year , medical college 1 received higher scores in the btr construct on worried patient presenting with fever ( wppf ) and patient presenting with positive hepatitis c report ( phcr ) stations with p = 0.026 and p = 0.017 , respectively . in the upp construct , marginally significantly higher scores were obtained on two stations again , anxious patient presenting with headache ( apwh ) and wppf with ( p = 0.052 ) and ( p = 0.083 ) , respectively . in the breaking the news(btn ) construct on the phcr station , the difference in scores between the two medical colleges was insignificant . reliability was checked by calculating cronbach 's alpha for each osce at the end of each osce and was found to be 0.751 and 0.815 , respectively [ table 3 ] . longitudinal comparison of the mean percentage construct and total scores for each station between and within universities the mean station scores in apwh station of medical college 1 and medical college 2 increased in the fifth year with a significant increase in medical college 2 scores with p = 0.048 and p = 0.004 , respectively . the mean score of wppf station increased significantly in both medical colleges with p 0.001 and p < 0.001 . however , the mean station score of medical college 1 decreased significantly in the disclosing bad news in phcr station ( p = 0.004 ) , whereas it increased insignificantly in medical college 2 ( p = 0.775 ) . the mean station scores of patient request for faith healing therapy for diabetes mellitus station decreased in the fifth year with p = 0.0046 and p = 0.036 , respectively , for both medical colleges 1 and 2 [ table 3 ] . as this was a pilot study , the results can not be extrapolated widely . a pretest was not realistic in such a situation where students are entering the program for the first time . increasing the number of stations to more than four responder bias is a possibility as students with better communication skills may have been more likely to have accepted the invitation to participate in the osce . at the end of the third year , medical college 1 achieved a significantly higher overall mean total station score of 68.0% ( standard deviation [ sd ] = 13.5 ) versus 57.2% ( sd = 15.4 ) ( p < 0.001 ) . at the end of the fifth year , the overall mean total station score of medical college 1 remained significantly higher , although the difference had reduced from 9.2% to 7.1% ( 70.2% ) ( sd = 13.7 ) versus 63.1% ( sd = 15.2 ) ( p = 0.004 ) [ table 2 ] . mean percentage scores and mean p values of the constructs and total objective structured clinical examination scores of both medical colleges at the end of the third year , the mean overall percentage score of each construct was significantly higher in medical college 1 . at the end of the fifth year , medical college 1 received higher scores in the btr construct on worried patient presenting with fever ( wppf ) and patient presenting with positive hepatitis c report ( phcr ) stations with p = 0.026 and p = 0.017 , respectively . in the upp construct , marginally significantly higher scores were obtained on two stations again , anxious patient presenting with headache ( apwh ) and wppf with ( p = 0.052 ) and ( p = 0.083 ) , respectively . in the breaking the news(btn ) construct on the phcr station , the difference in scores between the two medical colleges was insignificant . reliability was checked by calculating cronbach 's alpha for each osce at the end of each osce and was found to be 0.751 and 0.815 , respectively [ table 3 ] . longitudinal comparison of the mean percentage construct and total scores for each station between and within universities the mean station scores in apwh station of medical college 1 and medical college 2 increased in the fifth year with a significant increase in medical college 2 scores with p = 0.048 and p = 0.004 , respectively . the mean score of wppf station increased significantly in both medical colleges with p 0.001 and p < 0.001 . however , the mean station score of medical college 1 decreased significantly in the disclosing bad news in phcr station ( p = 0.004 ) , whereas it increased insignificantly in medical college 2 ( p = 0.775 ) . the mean station scores of patient request for faith healing therapy for diabetes mellitus station decreased in the fifth year with p = 0.0046 and p = 0.036 , respectively , for both medical colleges 1 and 2 [ table 3 ] . a pretest was not realistic in such a situation where students are entering the program for the first time . increasing the number of stations to more than four responder bias is a possibility as students with better communication skills may have been more likely to have accepted the invitation to participate in the osce . this is the first pilot study exploring the outcome of communication skills training in the undergraduate curriculum in pakistan . improvement in communication skills as a result of formal teaching using a variety of approaches has been demonstrated repeatedly in various studies . based on this principle , the longitudinal integrated communication skills program of medical college 1 utilizes a range of evidence - based teaching and learning strategies in delivering the content . the cambridge - calgary guide assessment instrument has been rated very highly on the measures of psychometric properties , practicality , and overall value in a review of assessment tools . improvement in communication skills was sustained in the medical college with the longitudinally integrated communication skills program ( medical college 1 ) , even though the difference between the two colleges decreased over time . this can be attributed to a longitudinal program with earlier initiation of communication skills teaching in this college . research has shown that communication skill programs introduced earlier initially lead to a greater improvement which declines over time , although it never reaches preintervention level . this decrease was demonstrable in both colleges and can be attributed to an overall decline in patient - centeredness , increased cynicism , increased doctor centeredness , and matches previous literature findings . in our case , this decline may also be due to the concentrated communication skills teaching sessions in the first and second year , resulting in much better scores in the first osce , followed by a reduced rate of improvement in the clinical years commensurate with the reduced curricular time for communication skills teaching . one of the main challenges was deficiency of faculty training in teaching communication skills in clinical years in addition to lack of time in busy clinics . limited time for communication skills teaching has often been reported as a challenge for teaching programs . other reasons included a deficit of desirable role modeling compounded by a lack of concurrent faculty development . the majority of the undergraduate training is based in a private , highly specialized tertiary care setting with a focus on volumes and specific organ systems in specialty clinics causing the consultation to become disease oriented . it is well recognized that private hospitals volume and revenue - oriented goals are frequently in conflict with an ideal environment for the reinforcement of skills that are oriented to elicit the patient 's perspective that includes concerns , fears , and expectations of the patients . the vast majority of patients complaints stem from a real or perceived lack of patient - centered communication skills and supports this position . the consultation skills of the comparison medical college also improved longitudinally over time , and can be attributed to maturation of the students commensurate with experience . we postulate that training in wards and clinics through role modeling and observation on real patients improves consultation skills . to counteract the drift toward the disease - oriented model , contextual and repetitive teaching that is patient - oriented and is supported by appropriate role modeling by trained faculty in all clinics and wards will allow greater integration . in addition , at all stages of teaching , special focus and attention need to be given to specific communication skills required for a patient - centered consultation that can build a therapeutic relationship and elicit the patient 's perspective . running long - term and sustainable longitudinal programs for specific communication skills teaching / learning in appropriate contexts , with the support of trained and motivated faculty , is a well - recognized challenge . longitudinally integrated communication skills teaching in an undergraduate curriculum positively impacted communication skills of the students . undergraduate curriculum positively impacted communication skills . community - based undergraduate training in the clinical years , supported by a longitudinal communication skills program with on - going faculty development , should ensure appropriate role modeling to reinforce a patient - centered approach . a large - scale study evaluating the effectiveness of undergraduate communication skills training in community - based settings is required to assess the success of the above - mentioned interventions in preventing the drift toward the disease - oriented approach .

background : evidence highlights a lack of communication skills in doctors leading to dysfunctional consultations . to address this deficit , a private medical college instituted curricular reforms with inclusion of a longitudinal communication skills program . a pilot study was undertaken to evaluate the effectiveness of this program by comparing the consultation skills of medical students of this college with a medical college without a communication skills program.methods:a 4-station objective structured clinical examination ( osce ) was conducted in the third and final year . mann whitney u - test was used to compare the difference in the distribution between osce stations total and construct scores.results:at the end of the third year , 21 ( 31.34% ) , students of the study site ( medical college 1 [ college with integrated longitudinal communication skills program ] ) and 31 ( 46.26% ) students from the comparison site ( medical college 2 [ comparable college without communication skills program ] ) consented . medical college 1 achieved a significantly higher overall mean total station score of 68.0% ( standard deviation [ sd ] = 13.5 ) versus 57.2% ( sd = 15.4 ) ( p < 0.001 ) . significantly higher mean scores were achieved on three stations . at the end of the final year , 19 students ( 29.3% ) from medical college 1 and 22 ( 34% ) students from medical college 2 consented . the difference in overall mean total station score reduced from 9.2% to 7.1% ( 70.2 ) ( sd = 13.7 ) versus 63.1 ( sd = 15.2 ) ( p = 0.004 ) . the mean scores of both colleges decreased in patient presenting with hepatitis c report station ( p values 0.004 and 0.775 ) and in patient request for faith healing therapy in diabetes mellitus station ( p values 0.0046 and 0.036 ) , respectively.conclusion:longitudinal communication skills in an undergraduate curriculum positively impacted consultation skills . community - based training and faculty development are required to develop effective patient - centered consultation skills .

Introduction Methods Instrument Statistical analysis Results Comparison of mean percentage Objective Structured Clinical Examination station scores between the two medical colleges Comparison of mean percentage construct scores between the two medical colleges Comparison of mean station and construct scores within medical colleges over time Limitations Discussion Conclusion Financial support and sponsorship Conflicts of interest

PMC4157479

a 13-year - old girl had a history of partial atrioventricular septal defect ( pavsd ) total correction at the age of 4 years . however , a right ventricular assist device was applied at the operation because of the akinetic right ventricle ( rv ) . two years later , based on our echocardiography results , the patient was transferred due to severe tricuspid regurgitation and rv chamber enlargement with rv dysfunction . she underwent tricuspid septal commissuroplasty , de vega - type tricuspid annuloplasty , right atrial reduction plasty , and isthmus ablation . further , we performed one - and - a - half ventricle repair . in spite of the operation , six years after the one - and - a - half ventricle repair , cardiac magnetic resonance imaging was performed to evaluate her cardiac function and measure the left ventricle ( lv ) volume . the patient s rv end diastolic volume index and rv ejection fraction were 500.4 ml / m , and 13.2% , respectively . the lv stroke volume index and ejection fraction were 38.2 ml / m and 28% , respectively . echocardiographic evaluation showed global rv akinesia and lv ejection fraction of 25% with paradoxical interventricular septal motion . we suspected that the rv enlargement affected both the rv and the lv function and it resulted in decreased lv contractility . she underwent extracardiac conduit fontan operation with polytetrafluoroethylene ( ptfe , gore - tex ; wl gore & associates , flagstaff , az , usa ) 24-mm tube graft , rv exclusion , atrial septectomy , and permanent pacemaker implantation . rv exclusion procedures include tricuspid valve obliteration ( from the rv side ; 5 - 0 prolene double layer , reinforcement suture from the ra side ; 4 - 0 polyester ptfe pledget - supported interrupted mattress suture ) and pulmonary valve obliteration ( 6 - 0 prolene running suture ) to reduce the rv volume with no flow connection , thrombin soaked gel - foam packing to the rv , and rv free - wall wide resection , and it was performed under the condition of cardiac arrest ( fig . permanent pacemaker bipolar leads were implanted at the lv apex , rv apex , left atrial roof , and ra free wall owing to a history of frequent atrial flutter and junctional rhythm . we did not perform arrhythmia surgery because the patient underwent an electrophysiology study and radiofrequency catheter ablation for supraventricular arrhythmia before the operation . the cardiopulmonary bypass time was 308 minutes , and the aortic cross clamp time was 146 minutes . we performed the computed tomographic angiography not cardiac magnetic resonance imaging to evaluate the patient s postoperative cardiac function and chamber size , because she was implanted with a permanent pacemaker . the rv volume was markedly reduced , and the rv was occluded with thrombosis ( fig . further , the stroke volume index and the ejection fraction of lv were increased to 48.03 ml / m and 33% , respectively ( table 1 ) . the patient s vital signs were stable with a central venous pressure of 18 to 21 mmhg . she was extubated in 14 hours , and she stayed in the intensive care unit for 90 hours . we removed the chest tubes 5 days later , and she was discharged on postoperative day 21 . she has been followed up for 21 months without any complaint of dyspnea and palpitation . 3 ) , and in the latest echocardiography , the ejection fraction of the lv had increased to 54% . recently , the importance of rv failure has been noted , in light of the incomplete understanding of the rv failure mechanism and a poorer prognosis than lv failure has . further , it has been reported that an increased rv volume and decreased ejection fraction are associated with lower survival rates in patients with congestive heart failure . the rv volume overload is one of the causes of right ventricle failure ( rvf ) . further , it is known that the rv volume over load leads to the leftward displacement of the interventricular septum and changes in the lv geometry , thus resulting in decreased lv contractility . , several reports have shown the successful outcome of the original and the modified rv exclusion procedures in the case of ebstein s anomaly . . she could have lived in a biventricular state if she had undergone the total correction of pavsd at the appropriate time . however , this case is meaningful in determining how to manage patients with rvf . we performed an rv exclusion to normalize the motion of the interventricular septum by reducing the rv volume . further , we demonstrated that both the end diastolic volume index and the stroke volume index increased after the operation . another beneficial effect of rv exclusion is lung expansion , because an enlarged heart can compress the lungs . in addition , adequate lung expansion helps to reduce pulmonary vascular resistance , and decreased pulmonary vascular resistance is a good prognostic factor among functional univentricular patients . in conclusion , the rv exclusion procedure in selected patients with severe rvf might be a safe and beneficial option to improve lv function .

a 13-year - old girl , who had undergone the total correction of partial atrioventricular septal defect at the age of 4 years , was admitted with severe tricuspid regurgitation in echocardiography . she had received one - and - a - half ventricle repair during follow - up . her right ventricle showed global akinesia , and the ejection fraction of the left ventricle was 25% with paradoxical interventricular septal motion . we performed right ventricular exclusion adjunct to the fontan procedure . she is doing well two years after the operation without complications .

CASE REPORT DISCUSSION

PMC3391667

patellofemoral pain ( pfp ) accounts for approximately 25% of knee injuries in athletes ( taunton et al . , 2002 ) . pfp patients are often not able to perform exercises like running , as increased patellofemoral joint compression forces ( pfjcf ) may aggravate pfp pathology . overloading of the patellofemoral joint in pfp patients could eventually lead to severe chronic injuries , such as osteoarthritis ( buckwalter and brown , 2004 ) . conservative treatment ( such as rehabilitation ) is important to manage pfp ( dixit et al . , 2007 ) . although it has been reported that br has lower pfjcf compared to fr , this may be due to methodological issues ; as running speeds were lower for br than fr trials ( flynn and soutaslittle , 1995 ) . another study found no difference in pfjcf between fr and br at similar , but unnaturally slow speed ( sussman et al . , 2000 ) . further research is therefore required to establish differences between pfjcf in br compared to fr at the same speed . pfjcf is influenced by multiple inter - related factors : knee extensor moment , patellar moment arm , quadriceps muscle force and patellar tendon force . the quadriceps muscle force is related to the knee extensor moment and the patellar tendon moment arm and the patellar tendon force is dependent on the knee angle and the quadriceps force ( gill and o'connor , 1996 ) . the within subject differences in maximum pfjcf between fr and br will therefore depend on the peak knee extensor moment and the knee angle at this peak moment . the main factors that influence the knee moment are the magnitude of the ground reaction force ( grf ) , position of the knee relative to the grf vector , and angular accelerations of the lower limb segments . besides these individual biomechanical factors , propulsive mechanisms of br and fr might also explain differences in pfjcf . the telescopic inverted pendulum ( tip ) approach can be used to explore the predominant propulsive mechanisms in fr and br ( jacobs and van ingen schenau , 1992 ; papa and cappozzo , 1999 ) . pendular movement , such as observed in walking , can be simulated by a simple inverted pendulum model where the stance limb is modeled as a rigid segment that rotates around the ankle ( mcgeer , 1990 ; garcia et al . , 1998 ) . such movement would have relatively low knee extensor and high hip flexor moments . running on the other hand involves a large compression and passive recoil of the stance limb ( telescopic motion ) and can therefore be better modeled by a spring mass model ( seyfarth et al . , 2002 ) . the aims of this study were to ( 1 ) investigate if br had a reduced peak pfjcf compared to fr at the same speed , and ( 2 ) if this force was reduced in br , to investigate how changes in relevant biomechanical parameters resulted in this reduced pfjcf . it was hypothesized that ( 1 ) pfjcf would be lower in br compared to fr , and ( 2 ) that this would coincide with a reduced peak knee moment in br as a result of grf alterations in br . heel strike running has been associated with increased grf ( lieberman et al . , 2010 ) and foot ground contact has been found to be reversed in br relative to fr ; toe heel contact in br versus heel twenty moderately active healthy subjects , without any recent knee injury or pain , were recruited for this study . ethical approval was obtained from the human research ethics committee of the school of healthcare studies at cardiff university , and written consent was obtained from all subjects . the subjects were asked to run along a 7-m walkway in forward ( fr ) and backward ( br ) directions at a speed between 2.8 and 3.4 m / s . kinematic data were collected at 200 hz using an eight camera vicon mx motion analysis system ( oxford metrics group ltd . , uk ) . 16 reflective markers were placed using the lower limb plug - in - gait marker set . ground reaction force data were collected at 1000 hz using two kistler force plates ( kistler instruments ltd . , switzerland ) . data of three subjects were excluded from analysis , due to missing pelvis markers during part of the data collection . the data of 17 subjects ( 7 males and 10 females , age : 286 years , height : 1.710.07 m and mass : 70.720.3 inverse dynamics calculations were performed within vicon nexus software ( version 1.6.1 , oxford metrics group ltd . , uk ) . , usa ) , combining kinematic and kinetic data with values for the patellar tendon moment arm ( dpt ) from literature ( tsaopoulos et al . , 2006 ) . a polynomial was fitted to these extrapolated data , resulting in an equation for dpt based on knee angle ( ) , and body height . tsaopoulos et al . ( 2006 ) demonstrated that dpt was expected to be scaled by body height , but not by body mass:(1)dpt = a(1.092 + 0.020.0000012)witha=0.04ifheight1.65ma=0.045if1.65m < the pfjcf was calculated as follows:(2)pfjcf = rfqfplfqwith(3)fq = mk(max)dptand(4)rfqfpl=0.633 + 0.010.000052where rfq - fpl is the the ratio of the quadriceps to patellar tendon force , fq is the quadriceps tendon force and mk(max ) is the peak knee moment . ( 4 ) ) . to investigate the kinematics and kinetics of br and fr , a telescopic inverted pendulum ( tip ) model approach was used , as described in the introduction ( fig . the magnitude of the grf was calculated at the time of peak knee moment ( mk(max)):(5)|grf|=fy2+fz2with fy as the horizontal and fz as the vertical component of the grf . the orientation of the grf relative to the ground ( grf ) at the time of mk(max ) was calculated in the sagittal plane , with 0 being perpendicular to the ground , grf>0 pointing in anterior and grf<0 in posterior direction . the location of the grf relative to the foot ( coploc ) was calculated at the time of mk(max ) by dividing the distance between the projection of the center of pressure ( cop ) on the foot and the metatarsal marker by the length of the foot . the position of the cop relative to the foot was also calculated at foot strike ( coplocfs ) , with foot the foot , shank and thigh segment angular accelerations ( foot , shank and thigh ) were calculated using the line between the calcaneus and metatarsal marker , the calcaneus and knee marker , and the asi and knee marker respectively . statistical differences for the output variables between fr and br were determined in spss ( version 18.0.2 ) with an independent t - test . stepwise linear regression analysis was used to investigate which variables most influenced pfjcf and subsequently mk(max ) . the pfjcf and mk(max ) were significantly higher and the knee was slightly more flexed in fr ( table 1 ) . peak hip flexor moments ( mh(max ) ) were significantly higher in br ( table 1 ) . 2 ) showed that the stance leg is shortened during the deceleration phase and extended during the push - off phase in both fr and br . in fr the stance leg flexed slightly more at mk(max ) ( table 1 ) and extended more during the push - off phase than in br ( fig . 2 ) . in both fr and br , mk(max ) occurred at similar though significantly different approach angles of the contact leg ( l ; fig . therefore in both situations the body was upright and leaning forward slightly at mk(max ) ( as l was close to , but smaller than 90 ) . there was no significant difference between fr and br for the magnitude ( |grf| ) and orientation of the grf ( grf ) at mk(max ) ( table 2 ) . the cop location on the foot ( coploc ) at mk(max ) was further backward and moving slower forward along the foot in fr ( table 2 ) . the angular acceleration ( foot ) of the foot at mk(max ) was significantly different and in opposite directions between fr and br ( table 3 ) . the acceleration of the shank segment ( shank ) at mk(max ) was in the same direction , but significantly smaller in fr ( table 3 ) . there was no significant difference between the angular accelerations of the thigh segment ( thigh ) at mk(max ) ( table 3 ) . stepwise regression analysis with the pfjcf as dependent variable and mk(max ) , mh(max ) , k ( knee flexion angle ) , l , and |grf| at mk(max ) as predictors confirmed that mk(max ) predicted the majority of variance in pfjcf ( 93.0% , adjusted r=0.930 ) . another stepwise regression analysis with mk(max ) as the dependent variable and k , l , |grf| , grf , coploc , copdt , foot , shank , thigh at mk(max ) as potential predictors showed that 54.8% ( adjusted r=0.548 ) of the variance in mk(max ) was predicted by k , coploc , and |grf| . the cop location on the foot at foot strike ( coplocfs ) was closer to the forefoot in br compared to fr ( table 2 ) . when investigating fr and br separately pfjcf was not correlated to coplocfs ; however when data were pooled there was a significant but weak correlation ( r=0.260 and p=0.008 , as shown in fig . pfjcf was significantly lower in br than in fr and this difference was not due to running speed , confirming our first hypothesis . the reduction in pfjcf was lower than that found by flynn and soutaslittle ( 1995 ) ( 27% versus 46% decrease ) . the higher decrease in pfjcf observed by flynn and soutaslittle ( 1995 ) was most likely due to the difference in running speed between their fr and br trials . kinematics at the peak knee moment ( mk(max ) ) were similar between br and fr which seems consistent with an earlier study ( van deursen et al . , 1998 ) . however , kinetics differed with higher mk(max ) in fr and higher mh(max ) in br . this predominantly agreed with flynn and soutaslittle ( 1995 ) , as knee flexion angles were similar ( fr : 44 and 38 , br : 41 and 38 ) and peak knee moments were similar for br ( 124 and 123 nm ) but lower in fr ( 157 and 246 nm ) . tip ( telescopic inverted pendulum ) analysis demonstrated that in both br and fr mk(max ) occurred during the loading response , with the body upright and leaning slightly forward ; mk(max ) thereby positively contributed to the support moment . the forward lean indicated that mk(max ) contributed to push - off in fr only , as in br a backward lean would be required for mk(max ) to contribute to push - off . this was demonstrated by the reduced mk(max ) and increased mh(max ) in br , which would be expected in pendular movement . also , in fr the stance leg extended more during the push - off phase ( and flexed slightly more during the deceleration phase ) than in br , consistent with our interpretation that fr involves a more telescopic movement . regression analysis showed that mk(max ) could predict the majority of the differences in pfjcf , confirming the first part of the second hypothesis that reduced pfjcf in br was caused by a reduced mk(max ) . although the magnitude ( |grf| ) and orientation ( grf ) of the grf at mk(max ) did not differ between br and fr , the location of the grf was further back on the foot in fr ( larger coploc ) . this would result in a larger moment arm between the grf vector and the knee joint , as the knee flexion angle ( k ) at mk(max ) was similar between br and fr . stepwise regression analysis showed that the variance in mk(max ) was best predicted by k , coploc and |grf| . k and coploc both influence the magnitude of the moment arm of the grf vector relative to the knee joint . mk(max ) therefore relied most on the position and magnitude of the grf , partly confirming the second part of our second hypothesis . the main factor influencing the peak knee moment was therefore coploc , indicating that foot strike has a large impact on pfjcf . although angular accelerations of the lower limb segments and joint angles were different between br and fr trials , these were not significant predictors of mk(max ) , and therefore are considered to have minimal influence on pfjcf . the differences in pfjcf observed between br and fr were not consistent in all subjects ( fig . investigation of the cop location at foot strike ( coplocfs ) showed that this was closer to the heel in fr . there was a weak correlation between coplocfs and pfjcf when fr and br data were pooled . the relatively low pfjcf observed in some of the subjects during fr may therefore be due to running style ( such as heel versus forefoot strike ) . we would expect lower knee moments resulting in lower pfjcf in forefoot strike runners , as they have lower loading rates of the foot ( oakley and pratt , 1988 ) and a lower grf ( lieberman et al . , 2010 ) . this agrees with our findings that pfjcf was reduced if the cop was closer to the forefoot . however , this conclusion did not apply to all subjects during fr ( see fig . 4 , right lower corner ) . clearly , further research is required to investigate whether it is the br style that resulted in a reduced pfjcf or whether an adapted fr style could also be advised to pfp patients . this study had several limitations ; as pfjcf can not be measured in vivo it was estimated with simplified models . this study focused on compressive forces only and did not include the direction and location of the forces acting on the patellofemoral joint . the use of more complex models of the knee and the additional calculation of patellofemoral joint stresses ( ratio of pfjcf to the contact area ( mcginty et al . , 2000 ) ) would have provided insight into the distribution and direction of the forces acting on the joint surface . there is however a strong relationship between the patellofemoral contact area and knee flexion angles ( salsich et al . , 2003 ; besier et al . , 2005 ; escamilla et al . , 2008 ) . as mk(max ) occurred at similar knee flexion angles in br and fr , it can be assumed that the patellofemoral contact area would be comparable , and patellofemoral joint stresses would be directly related to pfjcf . estimation of joint stresses requires complex and computationally intense methods ( farrokhi et al . , 2011 ) ; as similar trends could be expected in patellofemoral joint stresses and compression forces between br and fr , this study included compression forces only . future research may involve more detailed analysis of the forces acting on the patellofemoral joint during backward and forward running . the patellar tendon moment arm was important in the calculations of the pfjcf , as it defined the magnitude of the pfjcf relative to the knee moment . there is controversy in literature on how this moment arm should be estimated ( tsaopoulos et al . , 2006 ) . we assumed the patellar tendon moment arm depended on knee angle , as the majority of studies demonstrated that the patellar tendon moment arm changes significantly during the first 45 of knee flexion ( smidt , 1973 ; herzog and read , 1993 ; baltzopoulos , 1995 ; kellis and baltzopoulos , 1999 ; tsaopoulos et al . , 2006 , 2007 ) , and only limited studies found the moment arm to change little with knee angle ( gill and o'connor , 1996 ) . this study demonstrated that pfjcf was reduced in br compared to fr , and that this was not due to a difference in running speed . it can be concluded that br can be used as part of rehabilitation of pfp patients , to continue to exercise without increased pfjcf . although br can be suggested for rehabilitation , only a limited number of studies investigated br as part of rehabilitation of knee injured patients . a case study showed that br allowed exercising with decreased pfp ; however if implemented incorrectly it can lead to overuse injury ( satterfield et al . , obviously , rehabilitation programs need to include other components , such as muscle strengthening ( dixit et al . , 2007 ; dixit et al . , 2007 ) and/or taping ( dixit et al . , 2007 ) . the reduced pfjcf in br compared to fr may also prevent overloading and thereby the development of chronic conditions such as osteoarthritis . however pfjcf was not decreased in br compared to fr in all subjects , and pfjcf was lower when the cop was closer to the forefoot . the cop location , that was closer to the heel at peak knee moment in fr than in br , was the main predictor of the increased knee extensor moments . certain fr styles may therefore also be able to reduce pfjcf , and could be useful in injury prevention or rehabilitation .

patellofemoral pain ( pfp ) is a common injury and increased patellofemoral joint compression forces ( pfjcf ) may aggravate symptoms . backward running ( br ) has been suggested for exercise with reduced pfjcf.the aims of this study were to ( 1 ) investigate if br had reduced peak pfjcf compared to forward running ( fr ) at the same speed , and ( 2 ) if pfjcf was reduced in br , to investigate which biomechanical parameters explained this . it was hypothesized that ( 1 ) pfjcf would be lower in br , and ( 2 ) that this would coincide with a reduced peak knee moment caused by altered ground reaction forces ( grfs).twenty healthy subjects ran in forward and backward directions at consistent speed . kinematic and ground reaction force data were collected ; inverse dynamic and pfjcf analyses were performed.pfjcf were higher in fr than br ( 4.51.5 ; 3.41.4bw ; p<0.01 ) . the majority of this difference ( 93.1% ) was predicted by increased knee moments in fr compared to br ( 15754 ; 12451 nm ; p<0.01 ) . 54.8% of differences in knee moments could be predicted by the magnitude of the grf ( 2.30.3 ; 2.40.2bw ) , knee flexion angle ( 446 ; 417 ) and center of pressure location on the foot ( 2511 ; 126% ) at time of peak knee moment . results were not consistent in all subjects.it was concluded that br had reduced pfjcf compared to fr . this was caused by an increased knee moment , due to differences in magnitude and location of the grf vector relative to the knee . br can therefore be used to exercise with decreased pfjcf .

Introduction Materials and methods Results Discussion Conflict of interest statement

PMC3238806

peptides are small ( low molecular weight , lmw ) proteins , built up of amino acids connected by peptide bonds . the shortest peptide is two amino acids long , and with increasing length of the amino acid chain , the name changes from peptide over polypeptide to protein , with a fuzzy border between them . also the international union of pure and applied chemistry ( iupac ) has no clear weight or amino acid chain length limit . a somewhat arbitrary but quite generally acknowledged definition puts the boundary between a peptide and a protein at a chain length of 50 amino acids ( wikipedia ) . in literature , often pragmatic definitions are used , such as the small proteins typically running off a typical 2d polyacrylamide gel or the small proteins with zero or maximally one tryptic cleavage site , and , therefore , different upper molecular weight limits for peptides can be found from 10 kda and even beyond [ 16 ] . as proteomics is for proteins , peptidomics is the comprehensive study of all ( native ) peptides in a biological sample . in the last decade , proteomics has gained increasing recognition as a reliable and reproducible approach to study molecular processes in high throughput at a global level . hot topic as it is recognized that peptides play complex regulatory roles in many if not all biological processes , for example , intercellular signaling [ 7 , 8 ] . as such , peptide signals secreted into the extracellular medium , reflect the state of a cell in a certain condition , and , by definition , are potential biomarkers indicative for specific physiological / pathological processes [ 911 ] . currently , with a general proteomic approach , it is possible to detect and identify several hundreds to a few thousands of proteins in a single experiment [ 1214 ] . with modern mass spectrometers excelling in sensitivity and dynamic range , also the cell 's peptidomes become much more comprehensively accessible for analysis , and the discovery of novel biomarkers becomes possible , such as in innovative cancer research . for example , ueda et al . used size - exclusion - based enrichment of the peptidome in combination with label - free quantitation by nano - lc - ms / ms for peptidome profiling in lung carcinomas . body fluids , especially blood serum or plasma , and , in particular cases , ( primary ) cell culture media , serve as typical and readily available sources for a however , the detection of peptide biomarkers typically present at low concentrations is hampered by the masking effect caused by a number of highly abundant proteins [ 11 , 1619 ] . the large dynamic concentration range , in which peptides and proteins are present in a biological system presents a major bottleneck for peptidomic discovery of new biomarkers . figure 1 reflecting the large dynamic range of proteins and peptides in human blood plasma is very illustrative in this respect . especially - the presence of albumin in a sample has been shown to prevent the successful identification of low - abundance biomarkers in many peptidomic studies [ 17 , 20 , 21 ] . hence , different methods for capturing , partitioning , fractionating , depleting , or enriching a sample have been developed [ 22 , 23 ] . liquid chromatography ( lc ) coupled with tandem mass spectrometry ( ms / ms ) is the analytical method of choice in today 's proteomics and peptidomics research . its major benefits include enhanced specificity ( particularly over the gc - ms technologies of 25 years ago , which had very limited applicability for peptide separations ) , its potential for high - throughput analyses , no requirement for expensive analyte - specific reagents , high speed of assay development , and a relatively low cost per assay ( the instrument itself , however , not being that cheap ) . in an ideal world , no sample preparation would be required for the analysis of a sample as every manipulation can lead to problems such as loss of sample and , even worse , loss of quantifiability . in particular , for the proteomic / peptidomic analysis of native peptides , which typically do not require protease ( trypsin ) digestion prior to lc - ms / ms analysis , a so - called top - down however , as the complexity of samples still far exceeds the capacity of currently available analytical systems , specific sample preparation remains a crucial part of the analysis in a whole . peptidomics sample preparation is often time consuming and laborious , involving multiple steps [ 25 , 26 ] . in this paper , we present an overview of different techniques ( see figure 2 ) used for the simplification of complex biological samples and review advantages and possible problems related to them ( see table 1 ) . our focus will be on biofluids such as blood , urine , or cerebrospinal fluid ( csf ) , but some issues related to the extracellular peptidome ( part of the so - called secretome ) conditioned media are cell culture media whose cells have grown in for a certain period of time . the cells condition the media by releasing / secreting proteins , cytokines , and other biomolecules . as such , culture supernatants or conditioned media ( cm ) can be considered yet another ( body ) fluid that can serve as a source for the identification of novel biomarkers , for example , in cancer research . it is important to note that in order to promote a healthy growth of cells in culture , the culture medium has to be supplemented with a standard cocktail of nutrients and growth factors . in mammalian cell cultures , this is typically achieved by the addition of a substantial volume of fetal calf serum ( fcs , up to 10% solution ) . when studying the intracellular proteome ( or peptidome ) , cells are washed several times with fcs - free cell culture medium and/or phosphate - buffered saline ( pbs ) prior to lysis to reduce contamination of the sample with ( bovine ) serum proteins . however , this is not possible when the extracellular peptidome is under investigation . as a compromise , in most studies , the medium containing fcs is replaced by fcs - free medium or medium containing reduced amounts of fcs just before starting the secretome experiment ( e.g. , giving a biological / physiological stimulus ) . for adherent cells however , when studying cells in suspension , several centrifugation steps are required before the medium can be replaced , which arguably is a source of unnatural stress to the cells , possibly even causing cell lysis . a main disadvantage of working with reduced amounts of fcs ( or no fcs at all ) is that also this is known to cause metabolic stress to the cells , not seldom inducing cell death and consequently altering the cell culture 's secretome [ 11 , 2834 ] . a traditional well - established technique in proteomics is one- or two - dimensional ( 1d or 2d ) polyacrylamide gel electrophoresis ( page ) . although more a protein than a typical peptide separation technique , 1d and 2d page are used in the latter can then be analyzed by ms after extraction from the gel as proteolytic fragments , after in - gel digestion . denaturing agents , such as sds , are used to unfold the macromolecules and disrupt noncovalent intra- and intermolecular protein / protein interactions . its poor resolving power , however , often poses a problem in the analysis of complex mixtures . 1d and particularly 2d page are employed to increase the depth of proteome / peptidome analysis , that is , through fractionation of the sample components and removal of lmw impurities , particularly salts , which interfere with subsequent ms analyses [ 3541 ] . 2d page is very sensitive predominantly to molecular charges of a protein ( by the isoelectric focusing step ) , making it a very effective method to reveal / separate certain posttranslational modifications like phosphorylations , sulfations , or glycosilations . limitations are that proteins / peptides with extreme pi values can not be separated and that the smaller peptides are typically not retained in the second ( mw separation ) dimension . the dynamic range for detection in 2d page is 1010 , which is less than the protein expression range observed in biological systems . in order to achieve the detection of low abundant proteins , such higher loads , however , often further compromise the technique 's resolution due to spot fusion and comigration [ 19 , 21 , 39 , 4246 ] . important in page is the visualization of the separated proteins , although selected areas of a gel can be processed for ms from unstained preparative gels after comparison / alligning with an analytical reference or master gel . the most commonly used visible stains are coomassie brilliant blue ( cbb ) and silver nitrate staining . cbb staining is easy , ms compatible , and linear over , at least , one order of magnitude , so it is usable for quantification to a limited extent . silver nitrate is a more sensitive staining method0.5 ng versus 50 ng for cbb but the staining procedure is more labor intensive and has a more limited linear range ( due to its polychrome results ) . although widely regarded as the standard of rigor by which all other ultrasensitive staining methods are judged , silver staining remains quite a complex and variable protein - gel - staining methodology , with many dozens of published protocols , all of them requiring several steps . silver staining quantitation is never simple , due to the complex polychromatic nature of the color development and to considerable differences in response factors between different proteins . fluorescent dyes are also popular to visualize proteins in gels ; however , they are not cheap reagents and require expensive scanners / image analyzers . they exhibit detection sensitivity rivaling that of silver staining , with workflow advantages similar to cbb staining . fluorescence offers linear quantitation ranges 10100-fold greater than other colorimetric methods [ 4750 ] . because of the limitations associated with gel - based techniques , recently with respect to detection of the smaller proteins and peptides ( see above ) , attention has gone to off - gel methods for peptide / protein separations , in particular in solution - pi - based peptide separations without the need for carrier ampholytes . it focuses proteins and peptides on an immobilized ph gradient ( ipg ) gel , which is sealed against a multichamber frame that contains both sample and focusing solutions . the sample is separated by migration through the gel , followed by diffusion into the well adjacent to the section of the ipg strip . it allows for multiple samples to run simultaneously and requires only small sample volume and no prior sample cleanup . disadvantages are that it has a rather long separation time and requires an insulated cooling system [ 5153 ] . recently described a variation of the mostly used offgel system ( agilent technologies , usa ) . off - gel separation has not only shown applicability for proteins but also for peptides . hubner et al . compared the technique to in - gel digestion and found that using peptide off - gel separation led to a third more protein / peptide identifications comparing off - gel separation to reversed phase liquid chromatography ( rplc ) at high ph , manadas et al . conclude that rplc leads to the identification of more peptides and also more unique peptides . in general , off - gel separation has clear advantages over a gel - based approach with respect to focusing and concentrating peptides , but it still requires further optimization to reach the same level of identifications as an rplc - based separation . many different approaches exist to separate proteins based on their biochemical and biophysical properties such as molecular weight , mass , and hydrophobicity . another way to reduce a sample 's complexity is to specifically remove the most abundant protein(s ) , by doing ( immuno- ) affinity capturing [ 17 , 20 , 32 , 39 , 5661 ] . an example is the removal of albumin from serum , plasma , or cell culture samples . the most used dye for removal of albumin is cibacron blue ( often in combination with protein g for the removal of igg ) . this dye however does not only show affinity for albumin but also for nad , fad , and atp binding sites of proteins , which often results in the unwanted removal of proteins of interest [ 6264 ] . several comparisons have been made on cibacron - blue - based depletion of highly abundant proteins from a complex sample versus immunodepletion of the same sample . the overall conclusion is that the dye method is less performant than the immuno - based affinity removal : it does not only incompletely bind all albumin from the sample ( lower efficacy ) , but it also appears to remove a substantial portion of proteins other than the targeted albumin ( lower specificity ) [ 62 , 65 , 66 ] . immunodepletion based on monoclonal antibodies ( mabs ) is generally not preferred as , besides being very expensive , these antibodies typically remove only proteins or protein fragments with the specific targeted epitope , whereas other fragments of the protein remain untouched . therefore , immunodepletion systems are generally based on polyclonal igg and/or igy antibodies , targeting multiple epitopes on the same proteins . moreover , a mixture of polyclonal antibodies to distinct proteins are nowadays commonly used for removing multiple highly abundant proteins at once . antibody - based depletion of a sample can be performed in ( low - pressure ) spin cartridges or ( high - pressure ) liquid chromatography ( lc ) columns . when using antibodies , one has to consider the number of proteins that has to be depleted from the sample . depending on the system used , it is possible to remove between 1 and 20 abundant proteins . they observed that increasing the number of depleted proteins from 12 to 20 had only little beneficial effect and could in fact even increase the removal of peptides and proteins of interest which are associated with the abundant proteins . also tu et al . did a comparison of 2 types of multiple affinity removal system ( mars ; agilent technologies , inc . ) depleting , respectively , 7 ( mars-7 ) or 14 ( mars-14 ) abundant proteins . the mars-14 column removed 7 extra proteins but showed no substantial advantage over the mars-7 in improving peptide analysis / global protein identifications from plasma . recently , a creative way of depleting a sample was developed , the so - called hexapeptide library of combinatorial peptide ligands . high abundant proteins are expected to quickly saturate their specific affinity ligands leaving nonbound high abundant proteins to be washed away . in contrast , low and medium abundant proteins and peptides do not saturate their ligands and hence are concentrated on the beads . this technique has the advantage that peptides and proteins are adsorbed under native conditions and thus allow monitoring of their biological activity [ 68 , 69 ] , although its efficacy is still debated . limited comparative studies are published on the different depletion and enrichment methods . in these few comparisons , typically , the methods compared all lead to identification of a number of peptides and proteins , a part of which is generally identified by all methods under investigation and another part which has been identified uniquely in a sample that was treated with one of the methods [ 58 , 68 ] . the only exception to this is the comparison of dye - based depletion to immunodepletion of albumin ( see above ) . in this case , immunodepletion invariably resulted in the most efficient enrichment of low abundant proteins and peptides [ 62 , 65 , 66 ] . they , however , did not compare based on identification of proteins but on number of spots found by 2d page and also took into account the costs of all methods . when only looking at protein spots , they concluded that one column ( seppro mixed12-lc20 , genway biotech ) had the best overall performance leading to the largest number of new proteins spots . a second column ( multiple affinity removal column human 6 , agilent technologies ) in the same study led to almost the same quality results , while being cheaper and thence representing a more economical option which could become the preferred method when budgets are limited . in those cases where larger members of the peptidome need to be addressed , another crucial part of sample pretreatment is alkylation and digestion of the peptides / small proteins . this can be performed prior to or after the previously described techniques , that is , just before lc - ms / ms analysis . to break the tertiary structure of peptides , disulfide bridges have to be disrupted ( reduced ) and blocked to prevent reoxidation . breaking of disulfide bonds is traditionally achieved using reducing agents such as dithiothreitol ( dtt ) or tris(2-carboxyethyl)phophine hydrochloride ( tcep ) [ 72 , 73 ] . after reduction , the thiol groups are blocked , typically by alkylation , with iodoacetamide ( iam ) being the mostly used alkylating agent . another alkylating agent is 2-bromoethylamine ( brea ) , which transforms cysteine into s - aminoethyl cysteine , a pseudo - lysine , hereby creating an extra cleavage site for proteolytic enzymes such as trypsin [ 74 , 75 ] , which can be an advantage in certain cases . when larger ( poly)peptide members of the peptidome are envisaged , a bottom - up approach , requiring proteolytic digestion prior to mass spectrometric analysis , is sometimes to be considered . trypsin by far is the most used proteolytic enzyme for the degradation of proteins or peptides . this protease has the advantages of having a high cleavage specificity and being stable in a wide range of conditions . it cleaves c - terminal to arginine or lysine residues ( except where the subsequent amino acid in the parent sequence is a proline ) . thanks to the biological distribution of these amino acids among all proteins , the resulting peptide masses typically fall within the range required for analysis by mass spectrometry . some larger peptides or even proteins have been described in literature that contain only 1 tryptic cleavage site , producing a peptide still too large to be readily detected by mass spectrometry . in those cases , a combination of 2 or more proteases and/or alkylating with brea i.s.o . trypsin is very similar to chymotrypsin in primary structure , however chymotrypsin prefers cleaving c - terminal to amino acids with bulky aromatic residues such as phenylalanine , tyrosine , and tryptophan [ 48 , 7679 ] . also other sequence - specific proteases have their advantages in view of subsequent ms analysis of the resulting peptides . a good example is metalloendopeptidase lys - n , which guarantees a prominent positive charge at the aminoterminus of the cleaved product . ultrafiltration is a rather easy and widely used technique to fractionate a proteomics sample into almw fraction ( the peptidome ) and ahmw fraction ( the rest of the proteome ) by centrifugation . for example , in a recent study on csf , zougman et al . demonstrated the power of ultrafiltration to separate neuropeptides from the set of larger proteins present in csf . the ultrafiltrate low molecular weight fraction of the csf was directly separated by nano - lc - ms / ms . ultimately , this led to the identification of 563 peptides derived from 91 protein precursors . for example , they exhibited evidence of being produced by proconvertase cleavage , having a high cysteine content , bearing an aminoterminal pyroglutamate and/or carboxyterminal amidation . interestingly , in the same study , a parallel proteome profiling by 1d page was performed , and this yielded 798 proteins . for many of the identified neuropeptides in the ultrafiltrate , the corresponding precursor protein was not identified in the proteome screen , which evidently demonstrates the necessity of separating peptides from larger protein species . many different devices are available , each with specific molecular weight cut - off ( mwco ) and type of membrane . ultrafiltration is not seldom reported as a poorly reproducible technique , with possible removal of proteins and peptides below and above the stated mwco [ 8284 ] . to disrupt potential protein - protein / peptide interactions , acetonitrile ( acn ) . acn this way improves the recovery of lmw peptides [ 20 , 85 ] . harper et al . investigated several factors which might influence the ultrafiltration such as centrifugation time and speed . in their study , slower speed and longer time than advised by the manufacturer lead to improved recovery of lmw proteins and peptides . however , at the same time , also more hmw proteins appeared to pass through the filters . they concluded that devices with vertical or angular membrane configuration reduce membrane fouling , allowing continuous flow rates , even with high protein concentrations . this approach is known as filter - aided sample preparation ( fasp ) and it can be used to combine the advantages of in - gel and in - solution digestion [ 88 , 89 ] . the addition of organic solvents to serum causes high molecular weight ( hmw ) proteins to precipitate , leaving the lmw protein fraction including the peptides in solution . by also adding ion - pairing reagents such as trifluoroacetic acid , peptides , and smaller proteins can be dissociated from high abundant proteins , thereby facilitating their extraction [ 9092 ] . in general , proteins are precipitated by adding ice - cold acetone or acn . tucholska et al . compared several solvents for organic precipitation and concluded that acetone precipitated the serum proteins most completely . they found that methanol was actually the least favorable solvent for precipitation as it still contained the largest amount of albumin in solution . report that the use of methanol also leads to a much larger and less dense precipitate which was more difficult to separate from the supernatant . polson et al . likewise compared several precipitation methods , concluding that acn is the most efficient precipitant for protein removal . as an alternative to organic solvents , although as is a very efficient precipitant , it can cause interface contamination when combining with lc - ms . this method consists of 2 steps , the first of which denatures proteins and peptides and by dropping the denatured sample in ice - cold acetone causes all proteins and peptides to precipitate . in the second step , the lmw proteins and peptides which dissolve in 70% acn containing 12 mm hcl are separated from most of the other proteins . when compared to normal organic precipitation ( with acn ) and ultrafiltration , they conclude this method gives a much better yield for low molecular weight peptide extraction from human serum ( see figure 3 ) . another analytical tool to separate lmw compounds from hmw compounds is size exclusion chromatography ( sec ) . this is a widely used technique for the purification and analysis of synthetic and biological polymers based on their size , which is not by definition the same as their molecular weight . the material used for sec consists of porous beads , which either exclude the peptide / protein analytes from the internal space or allow them to enter based on their size . peptides and smaller proteins , which can enter the beads , will move at a slower rate through the column than bigger proteins which can not penetrate the beads , thus migrating faster . a disadvantage of this technique is its low resolving power , which can be improved by using it in combination with other separations , such as in multidimensional separation approaches ( see below ) . other drawbacks are the high elution volumes which cause dilution of the sample , increased costs when having to use multiple columns , and the need for high sample loads . in literature , some reports have appeared on the use of a one - step peptidome enrichment method based on sec [ 15 , 95 , 96 ] . mention the separation of over 12,000 molecules by using sec followed by c18 nano - lc - ms . they show that their procedure allows a precise separation of serum proteins and peptides based on their molecular weights . tucholska et al . showed that sec can be used to remove substantial amounts of albumin from serum ( although a small amount could still be detected in the depleted fraction ) . performed a comprehensive peptidome analysis of mouse liver by combining sec prefractionation with nano - lc - msms . from the low molecular weight fractions ( <3 kda ) that eluted from the sec column , reversed phase liquid chromatography ( rplc ) separates molecules based on differences in their hydrophobicity . the mobile phase is a water and nonpolar organic solvent mixture , whereas the stationary phase is hydrophobic . factors influencing the selectivity and resolution of separation include the hydrophobic ligand , the particle size , sample volume , column length , and ph . the most commonly used hydrophobic ligand for peptide analysis is c18 , but c4 and c8 are preferred for the larger peptides and proteins . in terms of peptides extraction , the solid phase material can be packed in syringe - shaped cartridges , or even in 96-well plate formats , which allows for high throughput extractions . another advantage of this technique is its capability to desalt samples , which is desirable prior to mass spectrometric analysis [ 43 , 44 , 98 , 99 ] . in proteomics / peptidomics analysis , nowadays , capillary reversed phase columns with an internal diameter of 75 to 100 m are commonly used , mainly because of the increased sensitivity this offers . because of these small columns , low flow rates have to be used , without decreasing the sensitivity , and ( often environmentally unfriendly ) solvents are saved . reducing the particle size to 2 m or less allows the use of a wider variety of linear velocities while maintaining acceptable chromatographic resolution in substantially reduced analysis times . this , however , requires new hardware since typical hplc systems generally are not able to handle the high pressures required to pump the mobile phase through a column packed with such minute particles . ultrahigh - pressure lc ( uplc ) systems have been developed for this purpose , allowing pressures of 400 bar and higher . thanks to the greater analytical power of uplc , downscaling of sample volumes and increasing the throughput of sample handling have become possible [ 98 , 100103 ] . ion exchange chromatography ( iex ) separates peptides ( and proteins ) based on their charge in a specific salt environment and ph of the mobile phase . two types of iex exist , namely , cation or anion exchange ( respectively cx and ax ) . iex has a disadvantage , being the use of salts which makes the eluate incompatible with ms . the use of salts can also lead to irreversible peptide or protein absorption to the resin , resulting in sample loss . the principles of iex are well understood , and other advantages include the high resolution that can be achieved , high capacity , selectivity , and robust operation . usually , simple salt buffers are sufficient and concentrations are used in a defined range , in which the so - called salting - in effect on proteins is observed . cation exchangers are negatively charged , and anion exchangers ; are positively charged . above its pi , a protein is negatively charged and binds to an anion exchanger ; below its pi , it is positively charged and binds to cation exchangers . the ion exchanger itself behaves like an acid or base , and the disproportionation of the charges depends on the ph . strong ion exchangers behave like a strong acid or base and do not change the charge within a wide range of ph , whereas weak ion exchangers do . this property can also be exploited to gain selectivity or by applying ph gradients for elution [ 44 , 105 , 106 ] . restricted access material ( ram ) has been described for use in the separation of large biomolecules and the extraction of lmw analytes . ram could be considered to be an upgrade of normal size exclusion material . the outer surface of the ram particles is coated with a protective , nonadsorptive hydrophilic packing , while the surface of the pores can be coated with a variety of different affinity matrices . in principle , it prevents access of macromolecules ( e.g. , proteins ) to the bonded phase in the pores , which can be compared to the size exclusion process . simultaneously , lmw target compounds ( such as peptides ) are selectively retained on the affinity matrix in the pores [ 108 , 109 ] . the most used type of internal selection material is strong cation exchange ( scx ) as this type of material is highly suitable for the online extraction of target peptides from complex biological samples such as plasma [ 110 , 111 ] . the method allows repetitive , direct injections of untreated sample matrices and can also handle higher injection volumes in comparison to sec [ 95 , 98 ] . a few setups have been described , in which ram is even used in an online lc - ms / ms setup [ 108 , 110113 ] . even described the use of a macroscopic ram cartridge online with nano - lc - ms for the quantification of a peptide analyte in samples containing high amounts of bovine serum albumin ( bsa ) . although the separation process can be automated , important parameters in ram chromatography , such as loading flow rate , ph , and amount of dilution and injected volume should be optimized as they have a large effect on the amount of adsorbed peptides and proteins as well as on their recovery from the column . the use of ram has been demonstrated for the analysis of peptides in complex biological matrices . isolated and detected more than 1500 peptides and protein fragments from only 3 ml of urine using an online ram - scx system . because of the high complexity of biological samples , often a single fractionation or separation step is insufficient therefore , several techniques can be combined to what is referred to as a multidimensional separation . combining 2 ( different ) separation techniques leads to an increased number of peptides measurable , an enlarged overall dynamic range and thus an improved peptidome coverage . a multidimensional approach can be achieved both offline and online . for the offline separation , fractions are collected after the first dimension , which are later reinjected into the second dimension separation . in between both steps a disadvantage of this method is the potential sample loss when transferring between both dimensions . when doing online multidimensional separation , the samples are automatically transferred from the first to the second dimension . also the solvents should not cause salt precipitation or immediate elution of the compounds in the second dimension . most often , rplc is the second dimension for its high speed , desalting capability , and compatibility with mass spectrometry [ 19 , 43 , 115118 ] . it is clear that , besides selecting the best methodology to comprehensively separate peptides from a biological mixture , an essential part of peptidomics sample preparation is the preservation of the integrity of the in vivo peptidome . various protease inhibitor cocktails can be introduced in the sample as early as viable , to try and inhibit ubiquitous peptide degrading enzymes [ 118 , 119 ] . in addition , peptidomics samples are traditionally snap - frozen , at a very low temperature as soon as possible , and subsequently freeze dried . yet , such strategies donot always appear adequate ( rapid enough ) to prevent bioactive peptide degradation ( several peptidases survive repeated freeze - thaw cycles ) . this has clearly been demonstrated for the ( neuro)peptidome , which appears particularly sensitive to rapid ex vivo ( postsampling ) degradation ( figure 4 ) . microwaves were found to be effective to rapidly and permanently inactivate enzymes [ 104 , 120 ] , although poorly controllable . an elegant automated instant thermal denaturation system , based on conductive heating , at controlled pressure under vacuum , was developed for successful and reproducible blocking of ex vivo sample degradation , thereby stabilizing the peptidome ( denator , gothenburg , sweden ) . depending on the sample - origin - specific indicator peptides can be used to monitor / assess the sample quality , prior to analysis . one example is the post mortem appearance in mammalian neuronal tissue of the peptide stathmin 220 , which indicates proteolytic degradation . in general , it can be concluded that sample preparation for the purpose of capturing the peptidome from biofluids still has room for improvement , and a single generic methodology is not ( yet ) available . many methods exist for the analysis of proteins which can , sometimes with minor adjustments , be used for peptides as well . ideally , sample preparation / handling should be minimal , but current mass spectrometers are not quite able to handle complex biological matrices . for example , over the last decades , the sensitivity of mass spectrometers has tremendously increased , where at the moment they have reached the low attomole level . also , improvements in mass accuracy and advances in peptide fragmentation techniques permit us now to obtain highly confident identification and even fully de novo sequence novel peptides . despite these ongoing improvements , it should be noted that at the moment , a peptidomics analysis ms system that can operate without sample preparation is still far way , which is mainly due to the high complexity of the biological matrices . the biggest bottleneck in biofluid research is the high dynamic range at which the proteins / peptides occur . for example , albumin concentration is ~40 mg / ml in serum , whereas the concentration of biologically active compounds such as cytokines is ~1 pg / ml . the removal of abundant plasma / serum proteins by dye- or immuno - based depletion is at moment the most popular strategy to mine however , one should also realize that albumin and other major proteins in blood plasma carry some number of other adsorbed peptides and even small proteins on their surface , and , therefore , these will risk to get lost during this depletion step . the precipitation of the abundant protein fraction in human biofluids by organic solvents has probably the longest history among all the methods used for abundant protein removal . this procedure is still one of the most effective methods as it permits fast and , although not always highly reproducible , cheap obtainment of the peptide fraction for their analysis by mass spectrometry . several other analytical strategies are at hand that aid in the reduction of the complexity of samples , and some specifically focus on capturing the lmw fraction , that is , the smaller proteins and peptides from the bulk of larger proteins , for example , ultrafiltration as method to split a sample into a proteome and a peptidome fraction . however , one should be aware that several studies have shown dramatic differences in the performance ( both in terms of reproducibility , recovery , and separation ) of commercially available ultrafiltration devices . a more preferred analytical tool to separate biomolecules on basis of size is liquid chromatography . size exclusion chromatography and more specifically restricted access material have high potential in peptidome research , due to their high resolution and selectivity . these last two chromatography techniques also can be miniaturized , making them even more favorable in term of sensitivity . in general , as sample preparation is often time consuming and laborious , high - throughput and automated approaches are highly desirable . whereas up to now , when doing multidimensional lc , the first dimension led to the collection of fractions in an offline setup , online multidimensional lc setups gain popularity . also the column material used in liquid chromatography is continually being improved , and the possibility of combining separation based on different physicochemical properties , such as ram , is a big step forward . even though it usually takes some effort to completely set up and optimize an lc - based method , once done , it usually results in a very robust and reliable technique , which can be fully automated . also miniaturization is an important aspect of the evolution , as a miniature system ( e.g. , lab - on - a - chip ) will allow for lower amounts of solvents and less complicated equipment to be used , decreasing the cost of an analysis . with the development of new sample pretreatment methods and lower detection limits on mass spectrometers , future peptidome analysis will aid substantially in biological and medical research , for example , by discovering new biomarkers and uncovering novel signaling pathways .

although big progress has been made in sample pretreatment over the last years , there are still considerable limitations when it comes to overcoming complexity and dynamic range problems associated with peptide analyses from biological matrices . being the little brother of proteomics , peptidomics is a relatively new field of research aiming at the direct analysis of the small proteins , called peptides , many of which are not amenable for typical trypsin - based analytics . in this paper , we present an overview of different techniques and methods currently used for reducing a sample 's complexity and for concentrating low abundant compounds to enable successful peptidome analysis . we focus on techniques which can be employed prior to liquid chromatography coupled to mass spectrometry for peptide detection and identification and indicate their advantages as well as their shortcomings when it comes to the untargeted analysis of native peptides from complex biological matrices .

1. Introduction 2. Sample Preparation Techniques Used for the Analysis of LMW Secretome/Signaling Proteins 3. General Sample Preparation in Peptidomics 4. Sample Quality: Preserving the Integrity of the Peptidome 5. Conclusion and Future Perspective

PMC2745458

precise preoperative planning is a vital principle of success in orthopaedic surgery . arguably , no field in medicine is as dependent for its success on accurate planning and implementation of alignment correction and implant placement . traditionally , preoperative planning has been performed on standard radiographs with various techniques , including the use of clear plastic templates [ 79 , 11 ] . recently , digital templating was proposed as a method to electronically overlay templates from a digital library on clinical radiographs for arthroplasties . the advocates of this technique cite the wide variety of available templates , the speed and precision of the technique , and elimination of hard - copy printouts of radiographs with their associated cost . the disadvantages of digital templating are the dependence on the digital library , cost of the software , and limitations in software design for each application . the purposes of this study are to describe ( 1 ) the techniques used in deformity analysis and preoperative surgical planning using standard radiographs for joint arthroplasty and corrective osteotomies of the extremities , ( 2 ) the use of ct scans to analyze rotational deformities in the presence and absence of joint prostheses and in planning corrective rotational osteotomies or revision joint replacement , and ( 3 ) the techniques for analyzing angular deformities of the spine . for all these applications , the specific use of a widely available image analysis software is discussed . for this study i proposed using commercially available software ( adobe photoshop 6.0 ; adobe systems inc , san jose , ca ) for a wide variety of orthopaedic surgical applications . however , any software program that allows calibration of measurements for lines and angles can be used . they can be used with a step - by - step technique for orthopaedic applications with lower cost and increased flexibility than commercial orthopaedic software ( tables 13 ; figs . 1 , 2).table 1general techniques for adobe photoshoptechniques / steps1 . multiple layers can be created by going to the layer menu at the top and selecting new and then layerb . 1 , large white arrow ) on the toolbarc . draw the path for the lines by pressing the left mouse button and dragging the mouse to the end of the line and then releasing the left mouse buttond . finalize the line on the new layer by placing the cursor over the line , pressing the right mouse button and selecting fill path . the line will now be placed definitively on the new layer established as shown in 1a2 . 1 , small black arrows)b . to measure an angle abc , press the left mouse button and drag from points a to b c. at this point , a linear measurement is given at the top of the screend . keep the cursor over point b and press the alt button ; the ruler is then converted to an angle icon on the imagee . press the left mouse button again and drag the mouse to point cf . at this point , an angular measurement is given for the angle abc in the " info " window ( under " window " menu at top of screen)3 . placement of texta . creating a new layer and then selecting the text tool ( fig . 1 , letter t ) b. press the left click button and drag the mouse to create a text box ; text can be typed into the text box c. if text is not visible , be sure the font is large enough to match the size of the image and change the color to improve contrast table 2technique for radiographic templating using adobe photoshopprocedures / steps1 . image and template software entrya . convert images to black and white by selecting the image toolbar followed by mode ; select grayscalec . save template image files in separate folders as jpeg ( .jpg ) files ( select file toolbar followed by save as and select a file name with a format as jpeg)d . enter desired radiographs , ct images , or mr images into software by directly downloading them or by scanning theme . in cases of standard radiographs , some form of calibration template must be placed on the skin at the level of the jointf . in cases of ct or mri , menu on the photoshop menu window at the top of the screen containing the items : file , edit , image , layer , etch . collect and save all images used for that specific case on the computer desktop or in a separate folderj . the template image is inverted to make it more visible by selecting the image menu , followed by from the photoshop toolbar ( fig . 1 ) , select the top left marquee tool ( black arrow ) in the shape of a rectangle d. on the template image , the marquee tool is used to outline the template along with a 10-cm segment of the calibration ruler found on most implant templatese . select the preoperative radiograph image and press ctrl v to paste the desired portion of the template image onto the preoperative radiograph imageh . at this point , under the layers palette 2 ) , one will see a new layer ( that of the template image)i . toggle the visibility of this layer by pressing the visibility toggle , which appears as an eye on the layers palette ( fig . 2 , black arrow)j . change the opacity of the new template image by selecting the template layer on the layers palette ( fig . 2 ) and using the opacity tool on the upper right of the layers palette ( fig . 2 , large black arrowhead ) ; slide it to 50% ; at this point , the template image becomes partially transparentk . change the brightness / contrast of the new template image by selecting the template layer as described previously and selecting the image toolbar followed by adjust ; select brightness / contrast3 . transforming the template imagea . next , the size of the selected portion of the template image is adjusted to the radiographb the template layer c. once this has been selected , a rectangle encloses the entire template image layerd . the rectangle can be resized either vertically or horizontally by passing the cursor over any of its sides , pressing the left mouse button , and dragging the mousee . the rectangle can be rotated by passing the cursor over its corners , pressing the left mouse button , and dragging the mousef . ensure the magnification change seen at the top of the page is equal for both the height and width to avoid template image distortiong . select the layer of the template image in the layers palette ( fig . 2 , template image not shown)b . use the move tool in the top right of the photoshop toolbar ( fig . 1 , large black arrowhead)c . left click on the template image and drag it to the desired position with the mouse5 . calibrating the template imagea . rotate the template image , resize , and reposition it as directed in 3a e so that its ruler portion superimposes the calibration of the preoperative radiograph ; for example , the 100-mm ruler should be exactly matched to the 100-mm radiographic marker on the skinb . once the sizing has been selected , the template image can again be rotated and repositioned ( but not resized ) and placed in the desired position over the bonec . the same technique can be performed for multiple templates ( ie , femoral and acetabular for tha , femoral and tibial for tka , calibrated rulers , etc)d . while working on a new template , make the previous template layers invisible by toggling the layer visibility icon on the layers palette ( fig . 2 , black arrow)table 3technique for rotational analysis of ct / mr images using adobe photoshopstepsa . copy and paste desired cuts as separate layers into new image document ( fig . make all images invisible by toggling the layer visibility icon on the layers palette ( fig . 2 , black arrow)c . alter the opacity of the image listed higher on the layer list to 50% ( fig . 2 , black arrowhead)d . draw lines along the axes that are to be measured ( eg , femoral neck axis , transmalleolar axis , etc ) as directed in general techniques ( table 1)f . 1the toolbar from the software package is shown with common tools needed for digital templating : line tool ( white arrow ) ; measure tool ( small black arrows ) ; text tool ( letter t ) ; marquee tool ( black arrow ) ; and move tool ( large black arrowhead).fig . 2the software layers palette is shown with visibility toggle ( black arrow ) and layer opacity adjustment ( large black arrowhead ) . general techniques for adobe photoshop technique for radiographic templating using adobe photoshop technique for rotational analysis of ct / mr images using adobe photoshop the toolbar from the software package is shown with common tools needed for digital templating : line tool ( white arrow ) ; measure tool ( small black arrows ) ; text tool ( letter t ) ; marquee tool ( black arrow ) ; and move tool ( large black arrowhead ) . the software layers palette is shown with visibility toggle ( black arrow ) and layer opacity adjustment ( large black arrowhead ) . the alignment objective in tka is to restore a projected anteroposterior weightbearing axis of the lower extremity to pass through the center of the knee . traditionally , in the coronal plane , the goal is to perform the distal femoral cut exactly perpendicular to a line from the femoral head to the apex of the femoral notch distally . the anatomic axis is defined as the line of best fit in the femoral diaphysis that passes through the center of the distal femur . the position of this axis is obtained intraoperatively by placement of an intramedullary rod in the femur starting at a point just anterior to the origin of the posterior cruciate ligament on the inferior trochlea . on the femur , the angle between the mechanical axis and the anatomic axis ( fig . this angle is measured in the software package and usually is between 4 and 7 ( fig . 3b , black arrow ) . this angle is equivalent to the valgus angle set on the distal femoral cutting guide , thus achieving a distal femoral cut perpendicular to the mechanical axis of the femur . on the tibia , the goal is to cut the tibial surface exactly perpendicular to the line connecting the midpoint of the medial and lateral tibial spines and the center of the ankle . a perpendicular line is drawn to this mechanical axis line in the software package , which then can be translated proximally and distally depending on the desired degree of bone resection . by placing the distal femoral and proximal tibial cut lines on their respective bones , the profile and thickness of each cut can be predicted preoperatively ( fig . 3b ) and both bone cuts will be perpendicular to their respective mechanical axes in the coronal plane . the same strategy can be applied in the sagittal plane with the measurement of the native proximal tibial slope and distal femoral flexion / extension as well as the analysis of any post - traumatic or congenital deformities involving the femur or tibia.fig . a preoperative radiograph shows the mechanical axis of the femur connecting the central distal femur and femoral head and the anatomic axis of the femur ( the line running from the distal femur center up the femoral shaft ) . the tibial mechanical axis is shown as the line connecting the midpoint between the medial and lateral tibial eminence and the center of the ankle . ( b ) a higher - magnification view of lower extremity alignment is shown at the level of the knee . the black arrow represents the angle between the mechanical and anatomic axes of the femur , which can be used to set the distal femoral valgus cut angle used intraoperatively during tka . ( c ) the radiographic templates are imported into the software and are pasted onto the preoperative radiograph ( these templates do not contain a calibration ruler ) . postoperative ( d ) anteroposterior and ( e ) lateral radiographs after the tka are shown . ( a ) a preoperative radiograph shows the mechanical axis of the femur connecting the central distal femur and femoral head and the anatomic axis of the femur ( the line running from the distal femur center up the femoral shaft ) . the tibial mechanical axis is shown as the line connecting the midpoint between the medial and lateral tibial eminence and the center of the ankle . ( b ) a higher - magnification view of lower extremity alignment is shown at the level of the knee . the black arrow represents the angle between the mechanical and anatomic axes of the femur , which can be used to set the distal femoral valgus cut angle used intraoperatively during tka . ( c ) the radiographic templates are imported into the software and are pasted onto the preoperative radiograph ( these templates do not contain a calibration ruler ) . postoperative ( d ) anteroposterior and ( e ) lateral radiographs after the tka are shown . a second objective in planning tka using digital templating is to select the size of the implants preoperatively . this can be performed by scanning each of the templates into a separate image file and calibrating the templates to the calibration markers placed on the radiograph ( table 2 ) . the preoperative plan can be performed by overlaying the template images digitally on the radiographs ( fig . this technique allows a close approximation of the ultimate implant sizes preoperatively ( fig . the goal of tha is to replace the painful hip . for long - term success , many believe that the restoration of hip biomechanics may lead to improved outcomes . with appropriate preoperative planning , the hip center of rotation and the offset between the proximal femur and the acetabulum can be improved . the offset is defined as the lateral displacement of the femur relative to the pelvis . the surgeon can adjust these factors based on the depth of reaming and subsequent cup placement , vertical placement of the femoral implant , type of implant used ( standard versus high offset ) , use of offset polyethylene liners , and variations in femoral head sizes . digital templating allows the surgeon to precisely preplan these factors to restore hip biomechanics and to minimize leg length inequality ( fig . ( a ) a preoperative radiograph with acetabular and femoral components is shown with the implants in the desired positions and with the templates resized to match the image calibration . ( a ) a preoperative radiograph with acetabular and femoral components is shown with the implants in the desired positions and with the templates resized to match the image calibration . resurfacing tha has gained worldwide interest in recent years as a method to address younger patients with advanced hip arthritis while preserving bone for future surgical procedures . varus positioning of the femoral component in the coronal plane is one risk factor for such failure resulting from the tensile stresses applied to the lateral neck bone . this can be the result of an excessively small femoral component diameter or a femoral preparation that is excessively in the valgus position in which the cylindrical reamer penetrates the cortical bone of the lateral neck . on the lateral projection , the femoral head sometimes can be translated posteriorly on the neck forcing the starting point for the guide pin into a more anterior position on the head to remain central in the neck . additionally , excessive anteversion , retroversion , anterior translation , or posterior translation of the femoral component could lead to notching or impingement against the acetabular component . my preferred technique is to minimize the size of the femoral component while avoiding notching and thus to decrease bone resection on the acetabular side ( fig . 5 ) . based on these observations , it is clear there is little room for error in placement of the femoral component in either varus or valgus directions . some guidance systems use a pin in the lateral femoral cortex to determine the trajectory of the guide pin , measuring the vertical distance from the top of the greater trochanter to the entry site on the femoral shaft ( 65 mm ; fig . 5b , white arrows ) . others reference the intertrochanteric crest and measure the distance between the projection of the guide pin and the lesser trochanter ( 45 mm ; fig . 5b , black arrow ) . both of these measurements can be preplanned by placing calibrated rulers in the desired position in the software.fig . ( a ) a preoperative radiograph shows the size template ( 10 cm ) on skin . ( b ) a preoperative radiograph with the calibrated template is shown in the desired position on the femoral component to avoid notching and to achieve the desired stem shaft angle ( acetabular sizing requires different positioning of the template ) . calibrated rulers have been imported to determine the distance for guide pin placement along the lateral femoral cortex or along the intertrochanteric crest ( based on preferred guidance technique ) . ( a ) a preoperative radiograph shows the size template ( 10 cm ) on skin . ( b ) a preoperative radiograph with the calibrated template is shown in the desired position on the femoral component to avoid notching and to achieve the desired stem shaft angle ( acetabular sizing requires different positioning of the template ) . calibrated rulers have been imported to determine the distance for guide pin placement along the lateral femoral cortex or along the intertrochanteric crest ( based on preferred guidance technique ) . the indications for osteotomy of the proximal tibia include localized osteoarthritis and deformities of the lower extremity from congenital or traumatic etiologies . i have used the technique of digital templating to preplan a proximal tibial osteotomy for a posttraumatic valgus deformity ( fig . 6 ) and to perform a virtual correction in the coronal plane with the software . using this technique , the size and shape of an opening wedge and closing wedge can be predicted and planned before surgery . additionally , plate templates can be used in the same way as for joint arthroplasty to determine optimal plate length and the potential need for plate bending . the virtually osteotomized tibia can then be freely transformed and rotated until the desired correction is achieved on the image ( fig . the final mechanical axis of the extremity also can be confirmed on this image to run in the desired position . in addition to the coronal plane planning , the same strategies can be used in correcting congenital or posttraumatic deformities in the sagittal plane and in avoiding an iatrogenic sagittal deformity during correction of a pure coronal plane deformity.fig . 6a d digital preoperative planning of opening wedge varus high tibial osteotomy in a posttraumatic deformity is shown . in this case , a lateral opening wedge osteotomy is selected . ( a ) osteotomy level selection : the level of the initial transverse osteotomy is drawn on the long , standing radiograph . next , the selection tool is used to select a rectangle to include the entire tibia , ankle , and foot ( not shown ) . ( b ) osteotomy fragment reorientation : this image selection is copied and pasted into a new layer in the software . the virtually osteotomized tibia then can be rotated using the free transform function ( white box ) . this view allows assessment of desired correction by drawing a line from the center of the femoral head to the center of the ankle ( not shown ) . the final mechanical axis of the extremity also can be confirmed on this image to run in the desired position and the lateral cortical opening can be measured on the preoperative plan . ( d ) postoperative radiograph : a postoperative radiograph was obtained after stabilization with a locking plate ( tomofix ; synthes , paoli , pa ) and after placement of a tricalcium phosphate wedge in the osteotomy site . digital preoperative planning of opening wedge varus high tibial osteotomy in a posttraumatic deformity is shown . in this case , a lateral opening wedge osteotomy is selected . ( a ) osteotomy level selection : the level of the initial transverse osteotomy is drawn on the long , standing radiograph . next , the selection tool is used to select a rectangle to include the entire tibia , ankle , and foot ( not shown ) . ( b ) osteotomy fragment reorientation : this image selection is copied and pasted into a new layer in the software . the virtually osteotomized tibia then can be rotated using the free transform function ( white box ) . this view allows assessment of desired correction by drawing a line from the center of the femoral head to the center of the ankle ( not shown ) . the final mechanical axis of the extremity also can be confirmed on this image to run in the desired position and the lateral cortical opening can be measured on the preoperative plan . ( d ) postoperative radiograph : a postoperative radiograph was obtained after stabilization with a locking plate ( tomofix ; synthes , paoli , pa ) and after placement of a tricalcium phosphate wedge in the osteotomy site . rotational malalignment of the lower extremity is an important source of disability and pain . in many cases physical examination methods such as the quadriceps angle ( q angle ) , j sign , femoral anteversion , and tibial torsion are examiner - dependent and do not provide quantitative information on the degree and location of deformities ( ie , femoral or tibial ) . thus , axial ct scans of the lower extremity provide a valuable role in measurement of these deformities . by superimposition of these images using the software package and by changing the opacity and visible layers , these relationships can be measured precisely and the optimal corrective procedures can be selected ( table 3 ) . the technique requires a ct scan of the pelvis , which includes the femoral heads down to the lesser trochanters , the knee from the distal femur to the level of the tibial tubercle , and the ankle to include both malleoli . to determine the femoral neck axis , i have used the method described by teitge and torga - spak ( fig . 7a ) by superimposing the axial image with the femoral head center on a second axial image at the base of the neck with its center . using the layering functions of the software , a line is drawn connecting these two points ( tables 1 , 2 ) . a second line is drawn along the posterior femoral condyles ( fig . the angle between the femoral neck axis and the posterior femoral condylar line is measured and defined as the femoral anteversion . this can be performed by superimposition of the layers of the distal femur and those of the femoral neck and head ( fig . an identical technique can be used to determine external tibial torsion ( fig . 7d , white arrow ) using a coronal line bisecting the tibial plateau . measurements of these values are important in the determination of congenital and posttraumatic deformities and in planning corrective osteotomies.fig . ( a ) superimposed axial images of the femoral head centers and bases of femoral necks are shown . ( b ) an axial ct scan of the distal femur is shown with posterior condylar lines . the angle between the posterior condylar lines and femoral neck axis is defined as the femoral anteversion . ( d ) tibial torsion is measured by bisecting the proximal tibiae ( white arrow ) and defining the transmalleolar axis ( black arrowhead ) . ( a ) superimposed axial images of the femoral head centers and bases of femoral necks are shown . ( b ) an axial ct scan of the distal femur is shown with posterior condylar lines . the angle between the posterior condylar lines and femoral neck axis is defined as the femoral anteversion . ( d ) tibial torsion is measured by bisecting the proximal tibiae ( white arrow ) and defining the transmalleolar axis ( black arrowhead ) . berger et al . used rotational analysis for assessment of patellar subluxation and dislocation in tka . they compared 30 patients with patellofemoral complications after tka with 20 patients with successful outcomes . they developed a method to measure the component rotation of the femoral and tibial components independently by superimposing the axial ct images . femoral component rotation was measured relative to the transepicondylar axis and tibial component rotation was measured relative to the tibial tubercle ( fig . they reported a combined internal rotation of 1 to 4 was associated with lateral tracking and patellar tilt . larger degrees of combined internal rotation ( 717 ) were associated with early patellar dislocation or late patellar failure . in the case of tka , the ct images need only include the knee down to the level of the tibial tubercle unless there is an issue regarding proximal femoral or distal tibial deformity . using this technique , the relationship between the transepicondylar axis ( fig . 8a , black arrow ) and 8c ) can be evaluated to determine whether tibial malrotation is an underlying factor leading to patellar subluxation or dislocation ( fig . 8a drotational malalignment in tka can be determined according to the method of berger et al . . ( a ) the transepicondylar axis ( black arrow ) and posterior condylar axis of the prosthesis ( white arrow ) are marked on this axial ct section of the distal femur . ( b ) an axial ct scan of the proximal tibia is shown at the level of the polyethylene liner with the center point of the liner shown ( white dot ) . ( c ) an axial ct of the proximal tibia is shown at the level of the tibial tubercle . is calculated as the angle between a line bisecting the insert sagittally from anterior to posterior running through the center point and a line bisecting the tibial tubercle and running through the center point . rotational malalignment in tka can be determined according to the method of berger et al . . ( a ) the transepicondylar axis ( black arrow ) and posterior condylar axis of the prosthesis ( white arrow ) are marked on this axial ct section of the distal femur . ( b ) an axial ct scan of the proximal tibia is shown at the level of the polyethylene liner with the center point of the liner shown ( white dot ) . ( c ) an axial ct of the proximal tibia is shown at the level of the tibial tubercle . is calculated as the angle between a line bisecting the insert sagittally from anterior to posterior running through the center point and a line bisecting the tibial tubercle and running through the center point . numerous methods have been used to measure kyphotic , scoliotic , and pelvic deformities in the axial skeleton . they can include measurements of the cobb angle in scoliosis , kyphotic angles after vertebral fractures , and pelvic obliquity . one such example would be measurement of the kyphotic angle in guiding operative versus nonoperative treatment of burst fractures of the spine ( fig . 9 ) . in the method described by knight et al . , the angle subtended by a line overlying the inferior end plate of the vertebra above the fracture and the line overlying the inferior end plate of the fractured vertebra is measured using the techniques described here ( fig . 9a c the kyphotic angle for a thoracic burst fracture is calculated according to the method of knight et al . . ( a ) a lateral radiograph of the thoracic spine is shown from a patient who has sustained a burst fracture . ( c ) the kyphotic angle is measured according to the method of knight et al . . the kyphotic angle for a thoracic burst fracture is calculated according to the method of knight et al . . ( a ) a lateral radiograph of the thoracic spine is shown from a patient who has sustained a burst fracture . ( c ) the kyphotic angle is measured according to the method of knight et al . . the purpose of this article is to provide technical guidelines for use of a widely digital imaging software program to achieve three objectives : the use of deformity analysis and preoperative surgical planning of standard radiographs in joint arthroplasty and corrective osteotomies , the use of ct scans to analyze rotational deformities , and the analysis of angular deformities of the spine . using these techniques , preoperative planning can be performed from anywhere and at any time as long as the images can be scanned or downloaded directly from the radiographic archive into the software program . the critical requirements of any software program used for this application include the ability to select and resize certain segments of any image , the use of multiple layers , and the ability to alter the opacity of various layers . the same approaches can be used for essentially any orthopaedic procedure requiring linear or angular correction and/or implantation of size - specific implants . the techniques are limited to two - dimensional imaging and each view needs to be templated independently . this limitation makes management of complex three - dimensional deformities difficult , if not impossible . another limitation of this technique is that the accuracy and precision have not been validated . the techniques require a baseline investment of time from each operator with a variable learning curve . this can lead to insufficient interobserver and intraobserver reliability . finally and most importantly , when used in preoperative planning , the techniques presented here provide a tool to place the implants and perform osteotomies based on a preoperative goal . however , the ideal position of implants and extremity alignment in arthroplasty and corrective osteotomies are points of ongoing debate . detailed a method to optimize images of radiographs obtained with a digital camera using photoshop software . however , the emphasis of their study was not on preoperative planning and/or the use of the overlay template technique discussed in this article . the segmentation of mr images has been a difficult challenge often requiring either semiautomated or fully manual segmentation of images to separate bone from the surrounding soft tissues . park et al . described the use of special functions of photoshop software to perform semiautomatic segmentation of mr images . they then used these segmented images to make three - dimensional reconstructions of various anatomic structures , including the skin , bones , digestive tract , respiratory tract , urinary tract , cardiovascular system , and nervous system . they were able to fulfill their objective of substantially accelerating the segmentation process over manual segmentation . in yet another medical application of the same type of software , carvalho et al . described a digital subtraction radiography technique to evaluate chronic periapical dental lesions . however , by using the digital subtraction technique , the authors were able to determine if the lesions were healing or expanding and also to quantify the size of the osseous lesions at each time point . the techniques for image rotation , sizing , and the use of layers are similar to those discussed in the current article . despite the limitations , the techniques presented here recapitulate the same techniques that have been used for decades using planning tools such as tracing paper , plastic templates , and printed radiographs . the ultimate objective of this study was to show that a universally available digital imaging software package can be used for a wide variety of orthopaedic applications , including analysis of deformities , size - specific templating of implant - related procedures , and planning of corrective surgical procedures . with progression of the digital age and gradual elimination of hard - copy radiographs , digital deformity analysis and surgical planning may soon become the standard technique .

analysis of deformity and subsequent correction are the basis for many orthopaedic surgical procedures . in advanced cases of joint degeneration , arthroplasty may be the only available treatment option . until recently , these analyses and preoperative surgical plans have been performed using standard radiographs , tracing paper , and/or plastic overlays . numerous customized , commercially available , computer - based preoperative planning software programs have been introduced . the purposes of this study were to describe ( 1 ) the techniques used in deformity analysis and preoperative surgical planning using standard radiographs for joint arthroplasty and corrective osteotomies of the extremities , ( 2 ) the use of computed tomography ( ct ) scans to analyze rotational deformities in the presence and absence of joint prostheses and in planning corrective rotational osteotomies or revision joint replacement , and ( 3 ) the techniques for analyzing angular deformities of the spine . all these applications were performed with a widely available image analysis software .

Introduction Materials and Methods Discussion

PMC4262734

the cell cycle is a fundamental cellular process that governs the ability of cells to divide . control of the cell cycle is crucial for the generation of tissues from dividing stem cells , such as the development of the nervous system . exit from the cell cycle is associated with the control of differentiation because differentiated cells tend to be postmitotic . re - entry of differentiated neurons into the cell cycle leads to apoptosis ( folch et al . , 2012 ) , although there are some examples where the cells have been shown to differentiate even if cell - cycle exit is prevented ( lobjois et al . , 2008 ) . recently , not only cell - cycle exit , but also the length of the cell cycle , in particular the g1 phase length , has been associated with the decision of cells to differentiate . during mouse cortical development , elongation of the g1 phase by inhibiting the g1 cyclin - dependent kinases ( cdk4/6 ) promotes neurogenesis , whereas shortening of g1 by overexpressing g1 kinase cdk4/cyd1 promotes proliferative divisions ( lange and calegari , 2010 ; lange et al . , 2009 ) . a correlation between cell cycle / g1 length and the propensity for differentiation has also been documented in embryonic stem cells ( roccio et al . , 2013 ; coronado et al . , 2013 ) and neural progenitors in the chicken spinal cord ( wilcock et al . , 2007 ) . apart from g1 , the other two phases of interphase , g2 and s , have also been linked to neuronal differentiation . expanding progenitors in the mouse cortex have been shown to have a longer s phase ( arai et al . , 2011 ) and elongation of the g2 phase has also been shown to promote progenitor proliferation ( peco et al . , 2012 ) , although in the latter case , the effect has been linked back to the shortening of the g1 phase . this is not surprising because g1 is the key cell - cycle phase where both intrinsic and extrinsic signaling pathways impinge to instruct the cell whether to go for another round of division or differentiate ( hindley and philpott , 2012 ) . why do some progenitors have a longer cell - cycle / g1 phase ? an intriguing observation is that in the cortex , progenitors with a longer g1 phase are nonpolar ( basal progenitors ) whereas progenitors with shorter g1 are apicobasally polarized ( apical progenitors ; arai et al . , 2011 ) . it is also known that in many systems such as drosophila neuroblasts , and mouse , xenopus , and chicken neuroepithelium , polarized progenitors have a lower propensity to differentiate than their nonpolar daughter cells ( sabherwal and papalopulu , 2012 ) . these results point to a link between polarity and the cell cycle , however , a direct mechanistic link between these two biological processes is not known . any such mechanism is likely to involve key regulators of cell - cycle progression , such as the cyclin / cyclin - dependent kinase ( cy / cdk ) complexes and their inhibitors ( cyclin - dependent kinase inhibitors / cdkis ) . inhibition of cdk activity by stronger association with cdkis has been shown to trigger neuronal differentiation ( kranenburg et al . , 1995 ) . conversely , loss of cdkis has been shown to inhibit differentiation ( carruthers et al . , 2003 ; mairet - coello et al . , 2012 ; vernon et al . , , we have used the neuroectoderm of the xenopus embryo to investigate how the apicobasal polarization of neural progenitor cells affects cell - cycle kinetics and consequently , neuronal differentiation . xenopus neuroectoderm exhibits a clean segregation of polarized and nonpolar progenitors into two distinct layers and provides a simple and accessible model for answering such fundamental questions . in xenopus , deep ( inner ) nonpolar cells have a high propensity to differentiate , whereas superficial ( outer ) apicobasally polarized neuroepithelial cells are intrinsically resistant to primary neuronal differentiation ( chalmers et al . , 2002 ) . using this system , we have previously shown that the key regulator of polarity , the atypical ( serine - threonine ) protein kinase c ( apkc ) has an instructive role in promoting proliferation and suppressing differentiation in polarized progenitors ( sabherwal et al . , 2009 ) . in this paper , we show that , first , the cell - cycle kinetics of polarized and nonpolar neuroectodermal cells differ significantly , with polarized cells having shorter total cell - cycle length and shorter s and g1 phases than nonpolar cells . overexpression of p27xic1 , a member of the cip / kip cdki family , elongates the g1 phase of the cell cycle and promotes terminal differentiation during xenopus primary neurogenesis , a phenotype opposite to that of overexpression of an activated membrane - targeted form of apkc , apkc - caax . apkc - caax overexpression rescues the increased neuronal differentiation phenotype of p27xic1 overexpression , as would be expected if apkc counteracted the activity of p27xic1 . then , we show that apkc directly phosphorylates p27xic1 in the n terminus of the protein . phosphomimetic p27xic1 shows reduced binding to the g1-s related cyclin - dependent kinase 2 ( cdk2 ) , resulting in reduced inhibition and higher kinase activity , which in turn causes a faster cell cycle . this study thus identifies a direct mechanistic link between apicobasal cell polarity and the cell cycle . dual - pulse s phase labeling ( dpsl ) analysis on outer layer apicobasally polarized progenitors and inner layer nonpolar neural progenitors ( both sox3 + ) , showed that polarized progenitors have a significantly shorter cell cycle length and a shorter s phase length than nonpolar progenitors at open neural plate stage , nf13 ( tc , polarized versus nonpolar , mean sem , 282 14 min versus 411 21 min , and ts , polarized versus nonpolar , 40 4 min versus 141 14 min , figure 1a ) . we also analyzed these progenitors for percentage of labeled mitoses ( plm ) to estimate g2 + 1/2 m phase length and compared them for their mitotic indices ( figure 1b ) . putting the numbers together from these experiments ( see supplemental experimental procedures available online for details ) showed that polarized progenitors have a shorter g1 phase ( tg1 , 94 min versus 160 min for nonpolar cells ) but a longer g2 phase than the nonpolar progenitors ( tg2 , 132 min versus 89 min ) . the lengths of mitoses were marginally different between the two layers ( tm , 16 min versus 21 min ) ( figure 1c ) . thus , establishing the cell - cycle kinetic parameters for polarized and nonpolar progenitors showed that they differ significantly and that polarized progenitors cycle faster . overexpression of constitutively active , membrane - targeted apkc ( apkc - caax ) suppressed neuronal differentiation , as judged by the reduction in the expression of a terminal differentiation marker n - tubulin . conversely , nuclear dominant - negative apkc ( nls - apkc - nt ) promoted neuronal differentiation as n - tubulin was enhanced ( figure s1 available online ) . extending these observations further , we found that on apkc - caax overexpression , elrc , a marker of committed neural progenitors ( carruthers et al . , 2003 ) , was also suppressed , whereas cells expressing the neural progenitor marker sox3 showed expansion ( sox3 + area was increased on the injected side as shown by in situ hybridization ) ( figure s1 ) . this suggested that apkc - caax suppressed neuronal differentiation by promoting neural progenitor expansion . in apkc - caax - overexpressing embryos , the number of sox3 + progenitors on the injected side was significantly higher ( shown by immunostaining , figure 2a ) and cells on the injected side had significantly shorter tc and ts , than on the noninjected side , calculated by the dpsl technique . control embryos overexpressing gfp - caax exhibited no such differences ( figure 2a ) . to further see the effects of apkc on different phases of the cell cycle , we treated and imaged hela fucci cells ( sakaue - sawano et al . , 2008 ) for 4860 hr in the presence of a myristoylated , cell - permeable inhibitor specific against apkc ( sajan et al . , 1999 ) . cells inhibited for apkc showed significant lengthening of the total cell - cycle time ( tc , 25.32 0.58 this was mainly attributed to the lengthening of the g1 phase ( tg1 , 17.04 0.47 hr versus 11.79 0.51 hr for controls ) , with small effects observed in the early s phase ( ts , 5.68 0.78 hr than 3.38 0.40 hr for controls ) . the effect on the length of s , g2 , and m together ( tsg2 m , 14.15 0.67 hr versus 10.92 0.43 hr for controls ) indicated that the g2 phase was also largely unaffected by the inhibitor treatment ( figure 2b and movies s1a and s1b ) . the pseudosubstrate myristoylated inhibitor used here is highly specific and has no effect on classical and novel pkcs ( standaert et al . , 1999 ) . nevertheless , these data were further substantiated by the experiment with a chemical inhibitor against apkc , bisindolylmaleimide g6983 . this inhibitor has been used previously to show that apkc is involved in te formation ( eckert et al . , 2004 ) and can maintain es cells in an undifferentiated state in the absence of lif , through the inhibition of pkc ( dutta et al . , 2011 ) . g6983 showed similar effects on cell - cycle kinetics as shown by myr inhibitor against apkc ( figure 2b and movies s1a and s1c ) . the fucci data along with dpsl data suggested that both gain and loss of apkc signaling activity affects cell cycle length via g1 and s phases , with g2 and m phases remaining largely unaffected . these data suggested that the effects of apkc on progenitors proliferation might be mediated via its effects on cell - cycle kinetics . to test this further , we investigated the interaction of apkc with cell - cycle regulators . g1 kinases ( cdk4/6 ) and g1/s kinase ( cdk2 ) are positively regulated by cyd and cye / a , respectively , and negatively regulated by a cip / kip family cyclin - dependent kinase inhibitor ( cdki ) p27xic1 ( ohnuma and harris , 2003 ) . because cyclins d , e , a , and p27xic1 are regulated by posttranslational modifications , mainly phosphorylations ( alberts et al . , in vitro kinase assays showed that only bacterially expressed p27xic1 is a direct phosphorylation target of gst - apkc ( commercially supplied ) ( figure 3a and data not shown ) . the specificity of this in vitro phosphorylation was confirmed by performing the kinase assay in the presence of a pseudosubstrate inhibitor specific against apkc ( sajan et al . , 1999 ) . this reduced the kinase signal in a dose - dependent manner ( figure 3b ) . to see if apkc can phosphorylate p27xic1 in a complex embryonic milieu as well , we performed in vitro kinase assay using embryonic lysate , instead of kinase buffer , as the medium for interaction between exogenous gst - p27xic1 ( bacterially expressed ) and his - apkc ( commercially supplied ) . the result demonstrated that phosphorylated gst - p27xic1 significantly increases in the presence of apkc , whereas it drops down to basal level in the presence of pseudosubstrate inhibitor against apkc ( figure 3c ) . to further substantiate these observations in vivo , we performed in vivo kinase assay measuring the incorporation of -p32-labeled orthophosphate by p27xic1 , overexpressed in hela cells along with apkc - caax or in the presence of myristoylated pseudosubstrate apkc inhibitor . the experiment showed that p27xic1 incorporated more orthophosphate when expressed along with apkc - caax ; the incorporation was reduced when the same cells were cultured in the presence of the inhibitor ( figure 3d ) . p27xic1 and apkc - caax also showed direct physical interaction in coimmunoprecipitation ( coip ) assays . first , flag - p27xic1 and ha - apkc - caax constructs were co - overexpressed in hela cells and ha - apkc - caax was detected after ip of flag - p27xic1 using anti - flag beads ( figure 3e ) . more importantly , the endogenous p27xic1 and apkc from embryonic lysate also showed direct physical interaction in coip experiments where either p27xic1 or apkc is immunoprecipitated with antibodies and apkc or p27xic1 is detected by western blot in the pull - down , respectively ( figures 3f and s2 ) . the effect of apkc inhibitors on hela fucci cell - cycle length ( figure 2b ) suggested that human p27kip1 might also be negatively regulated by apkc and may be a phosphorylation target of apkc . however , our in vitro kinase assay using immunoprecipitated p27kip1 from hela cells showed that human p27kip1 is not a direct phosphorylation target of apkc ( data not shown ) . this suggests that apkc negatively regulates p27kip1 in an indirect manner . to gain some insight into where the interaction of apkc and p27xic1 may be taking place , we performed immunostaining on neurula stage embryos overexpressing flag - p27xic1 and ha - apkc - caax . these showed that p27xic1 was largely in the nucleus of the cells , and a portion of apkc - caax , which was mostly localized to the cell cortex , was also found in the nucleus ( figure s3 ) . these findings are consistent with previous reports showing nuclear enrichment of p27xic1 ( chuang and yew , 2001 ; chuang et al . , 2005 ) and some nuclear localization of apkc - caax ( sabherwal et al . , 2009 ) and lend support to the idea that apkc interacts with p27xic1 in the nucleus . in our previous publication ( sabherwal et al . , 2009 ) , we showed that apkc - caax is more active and nuclear than apkc and hypothesized that after getting activated in the membrane , a small proportion of apkc - caax is translocated to the nucleus . this suggests that although apkc - caax is predominantly membrane localized and weakly nuclear , it is highly active and sufficient to influence nuclear events like interacting with p27xic1 and modulating its activity . to identify domains of p27xic1 phosphorylated by apkc as shown in figure 4a , the n - terminal and middle parts of the protein showed positive kinase signal whereas the c - terminal fragment showed no sign of phosphorylation during in vitro kinase assays . phosphosite identification using liquid chromatography / tandem mass spectrometry ( lc / ms / ms ) analysis on flag - p27xic1 phosphorylated by apkc in vitro ( i.e. , in vitro kinase reaction on immunoprecipitated / iped flag - p27xic1 from hela cells ) identified multiple phosphorylation sites with significant ascore values ( 13 , ascore is a measurement of the confidence of phosphorylation ; beausoleil et al . , 2006 ) within the n - terminal , cdk - binding domain ( figure 4a ) . to see if the sites identified correspond to the phosphosites in vivo , similar lc / ms / ms analysis was carried out on flag - p27xic1 iped from hela cells co - overexpressing it with ha - apkc - caax . this analysis identified a phosphosite ( t68 ) with a significantly high ascore ( ascore = 27 ; figure 4a ) , located within the cdk interaction domain of p27xic1 ; another site ( t99 , located immediately after the cdk interaction domain ) was picked with a low ascore value ( ascore = 13 ; figure 4a ) . none of these sites was identified on flag - p27xic1 iped from hela cells overexpressing it alone . xenopus p27xic1 is 44% identical to human p27kip1 and 40% identical to human p21cip1 in the conserved n terminus . it also possesses a pcna - like binding site characteristic of p21cip1 in the c terminus ( su et al . , 1995 ) . the apkc phosphosite in p27xic1 ( t68a ) is not conserved in mammalian p27kip1 but is conserved in mammalian p21cip1 ( although it has not been identified as an apkc phosphosite by others ) and p27xic1 from zebrafish ( figure 4b ) , suggesting some degree of functional conservation linking apkc activity and cell - cycle regulation ; the other phosphosite identified ( t99 ) shows no conservation with other members of cip / kip cdkis . identification of apkc phosphorylation sites prompted us to check if these phosphorylation events are functionally important by generating phospho ( s / t to a ) or phosphomimetic ( s / t to e ) mutants . initial protein abundance experiments showed that the level of flag - p27xic1 protein is decreased in embryos injected with ha - apkc - caax and increased in embryos injected with the dominant - negative ha - apkc - nt ( figure s4a ) . protein stability experiments using cycloheximide showed that the half - life of overexpressed flag - p27xic1 was reduced when it was co - overexpressed in hela cells along with ha - apkc - caax ( figure s4b ) . however , nonphosphorylatable mutants ( like t68a ) were also destabilized in comparison to the wild - type protein , whereas phosphomimetic mutants ( like t68e ) of p27xic1 appeared more stable ( see figures s4c and s4d for examples ) , suggesting that the effect of apkc phosphorylation on p27xic1 stability is complex and can not be reproduced by single amino acid changes . not only the stability of cdkis , but also the inhibition of cdk kinase activity by cdkis has been shown to promote differentiation ( hasan et al . , 2013 ) . we tested whether the binding of p27xic1 to cdks is affected by apkc phosphorylation , which could explain the shortening of g1 and s phases of cell cycle . p27xic1 interacts with cdk2 and cdk4 and inhibits their kinase activities ( finkielstein et al . , 2001 ) . coip assays using hela cells co - overexpressing flag - p27xic1 along with either ha - cdk2 or ha - cdk4 showed that p27xic1 binds to cdk2 much stronger than to cdk4 ( data not shown ) . similar coip assays showed that phosphomimetic mutant t68e showed almost 50% reduction in its binding to cdk2 in comparison to the wild - type p27xic1 ( figures 4c , 4e , and s5 ) , whereas the phosphomutant t68a showed enhanced binding ( increased by almost 140% ) to cdk2 ( figures 4d , 4e , and s5 ) . other mutants showed binding similar to the wild - type p27xic1 ( figures 4c , 4d , and s5 ) . these assays suggested that a single phosphorylation event at amino acid t68 of p27xic1 by apkc is enough to affect its interaction with cdk2 . using histone h1 as a substrate for active cdk2/cyclina , we found that in comparison to the wild - type p27xic1 , phosphomutant t68a abolished the kinase activity of cdk2 almost completely , while the phosphomimetic mutant t68e had a negligible effect on cdk2 activity in the same assay ( figure 4f ) . taken together , these results mean that phosphorylation of p27xic1 by apkc essentially inhibits the activity of p27xic1 by reducing its binding to cdk2 , which in turn results in failure to negatively regulate the cdk2 kinase activity . p27xic1 overexpression by injecting mrna has been shown to promote neurogenesis ( vernon et al . , 2003 ) , while its morpholino - mediated knockdown blocked neurogenesis and promoted progenitor proliferation ( carruthers et al . , 2003 ) . here , we have used dna injections , because in xenopus the g1 phase appears in post - mid - blastula transition cell cycles . we found that such p27xic1 dna injections promoted neurogenesis ( figure 5a ) in a cell autonomous fashion ( figure s6 ) and suppressed neural progenitor proliferation , consistent with results obtained previously with rna overexpression . a subset of p27xic1-overexpressing embryos was used for fluorescence - activated cell sorting ( facs ) cell - cycle profiling on isolated nuclei . experimental embryos showed significantly higher percentage of nuclei in g1 phase than the control embryos ( 35.4% versus 27.4% ) , with a concomitant decrease of nuclei in s phase ( 40.7% versus 49.7% for control ) and negligible effect on the proportion of nuclei in g2 phase ( 23.9% versus 22.9% for control , figure 5b ) , indicating that g1 phase elongates and proliferation decreases upon p27xic1 overexpression . whereas 89% of p27xic1-injected embryos showed enhanced staining for elrc , the percentage dropped to 33% with a milder phenotype when p27xic1 was coinjected with apkc - caax ( figure 5c ) . thus , p27xic1 s overexpression phenotype of promoting neuronal differentiation was rescued by unilateral injection of apkc - caax . the corresponding numbers for n - tubulin staining were 88% and 20% after overexpression of p27xic1 alone or with apkc - caax ( data not shown ) . this supported the idea that apkc promotes progenitor proliferation , at the expense of neuronal differentiation upstream of p27xic1 , by inhibiting its activity . the idea was further supported by the observation that the phosphomutant of p27xic1 ( t68a ) could promote neuronal differentiation similar to p27xic1 , but apkc - caax could not effectively rescue this effect ( figure 5c ) . to understand if the effects of apkc and p27xic1 activities on neuronal differentiation were mediated via their effects on the cell - cycle length , we manipulated the cell cycle of embryos by nongenetic , chemical means using olomoucine ( olo ) , an inhibitor that lengthens the g1 phase by inhibiting the activity of g1 kinases ( calegari and huttner , 2003 ) . facs analysis of nuclei from embryos treated with olo showed that experimental embryos had more nuclei in g1 and g2 phases than control embryos , with a corresponding decrease in the proportion of nuclei in s phase , suggesting a decrease in proliferation and an elongation of growth phases ( figure 6a ) . experimental embryos also showed a significantly higher number of myt1-positive cells ( a marker of differentiated neurons , 12.53 2.44 per section ) than the dmso controls ( 8.75 0.38 ) ( figures 6b and 6c ) , supporting the idea that elongating g1 phase promotes differentiation . cell polarization and cell division are two fundamental biological processes that have been independently linked to cellular differentiation . in this work , we showed that apicobasal polarity and cell - cycle control are directly linked in neural progenitor cells in a way that leads to distinct differentiation potential of polarized versus nonpolar progenitors . our work has uncovered a remarkable similarity in the endogenous cell - cycle kinetics of apicobasally polarized and nonpolar progenitors between xenopus neuroectoderm and the mouse embryonic cortex ( arai et al . , 2011 ) . in both cases , the total length of the cell cycle and the length of g1 in apicobasally polar progenitors ( apical progenitors in the mouse , superficial progenitors in xenopus ) are shorter than those found in nonpolar ones ( basal progenitors in the mouse , deep progenitors in xenopus ) . in addition , in both cases , the basal nonpolar progenitors have a higher propensity to differentiate than the apicobasally polarized ones ( chalmers et al . , 2002 ; chenn and mcconnell , 1995 ) . thus , although the xenopus neuroectoderm shows a much simpler structure , some of the basic principles relating to the cell - cycle control during neurogenesis are highly conserved . we have used this system to specifically address the role of the apicobasal polarity in controlling the cell cycle via the apically localized key kinase apkc . apkc is a ubiquitous kinase that gets activated in cell cortex in a pi-3,4,5-trisphosphate ( pip3)-dependent manner ( reviewed by hirai and chida , 2003 ) . polarized cells are thought to contain a highly active pool of apkc because it is recruited to the apical cortex and/or the junctional complexes ( joberty et al . , 2000 ; lin et al . , 2000 ) where it should get activated . therefore membrane targeting of apkc by attaching it to a caax motif makes it constitutively active and mimics the effects of apicobasal polarization on apkc activity ( lee et al . , 2006 ; sabherwal et al . , apkc was the first molecule shown to promote the proliferation of polarized neuroblasts in drosophila and neural progenitors in xenopus both by gain- and loss - of - function experiments ( rolls et al . , 2003 ; chabu and doe , 2008 , 2009 ; sabherwal et al . , , the interaction of apicobasal polarity kinase apkc with the cell - cycle machinery in polarized cells was thought to be indirect , via the hippo pathway ( reviewed in genevet and tapon , 2011 ) . for example , apkc phosphorylates and negatively regulates kibra , an upstream positive regulator of the hippo pathway ( bther et al . , 2004 ; overexpression of apkc mislocalizes apical hippo to the cytoplasm with its negative regulator rassf , leading to the dampening of the hippo pathway and resulting in enhanced cell proliferation ( grzeschik et al . , 2010 ) . our current results establish a direct link between apkc and the cell cycle in context of cell polarity . in this study , we show that apkc - caax directly phosphorylates the nuclear cell cycle / cyclin - dependent kinase inhibitor p27xic1 . phosphorylation primarily takes place in t68 , located in the n - terminal domain , although we can not exclude contribution from other sites . apkc - caax overexpression leads to a faster cell cycle in the neuroectoderm with shorter g1 and s phases , decreased neuronal differentiation , and enhanced neural proliferation ( this work and sabherwal et al . , 2009 ) . apkc has also been shown to have a role in cell proliferation of nonpolarized cells . for example , apkc has been shown to directly phosphorylate and degrade p21cip1 in human colorectal ( hct116 ) cells ( scott et al . , 2002 ) , to upregulate cyd1 transcription , and to reduce p27kip1 s nuclear translocation in a ras - dependent manner in mcf7 cells ( castoria et al . , 2004 ) . we assume that these nonpolar cells have a basal activity of apkc , which is further enhanced by recruitment to the apical membrane in polarized cells . how does phosphorylation of p27xic1 by polarity kinase apkc lead to a faster cell cycle with shorter g1 and s phases ? p27xic1 physically interacts with cdks and cyclins through its cdk / cy interaction domains and reduces the activities of g1 kinase ( cdk4/cyd ) , g1-s transition kinase ( cdk2/cye ) and s progression kinase ( cdk2/cya ) with different half - maximal inhibitory concentration values ( su et al . , 1995 ; finkielstein et al . , our data show that phosphomimetic p27xic1 has a reduced ability to bind and inhibit cdk2 . enhanced cdk2 activity through cye overexpression causes the cells to cycle faster through g1 and enter s phase prematurely ( ohtsubo and roberts , 1993 ) , whereas inhibition of cye / cdk2 complex delays entry into s phase ( van den heuvel and harlow , 1993 ) . thus , inhibition of cdk2 kinase activity by cdkis , like p27xic1 , is key in inhibiting the cycling of progenitors and promoting their differentiation . we suggest that in polarized progenitors , unrestricted kinase activities of g1/s - specific cdk / cy complexes due to lower p27xic1 activity would shorten their g1 phase and concomitantly promote s phase entry , thereby promoting their cycling and interfering with the ability to differentiate . the prolongation of total cell - cycle length , and g1 in particular , as cells approach differentiation seems to be a widespread phenomenon ( takahashi et al . mechanistically , it is thought that elongation of the g1 phase promotes neuronal differentiation by providing sufficient time for the accumulation and posttranscriptional modification of differentiation - promoting factors , such as neurogenin and neurod ( reviewed in lange and calegari , 2010 ; hardwick and philpott , 2014 ) . in support of this idea , it was recently shown that the activity of neurogenin is controlled by time - dependent , rheostat - like sequential ( de)phosphorylation events in a cell - cycle - dependent manner ( ali et al . , 2011 ) . in conclusion , our findings provide mechanistic evidence for a direct link between apicobasal polarity and the cell cycle through apkc and p27xic1 , which is used during development to endow polarized neural progenitors with lower propensity for differentiation than their nonpolar counterparts . p27xic1 has a complex mode of regulation , which includes phosphorylation , ubiquitination , and degradation in the nucleus , and changes in the activity through the cell cycle ( chuang and yew , 2001 ; chuang et al . , 2005 ; lin et al . , regulation of apkc is likely to be equally complex , including shuttling between the membrane and the nucleus ( sabherwal et al . , 2009 ) . one challenge for the future is to understand the dynamic interaction between apkc and p27xic1 during the cell cycle . this method enables the estimation of the total cell - cycle length ( tc ) and s phase length ( ts ) by injecting two different thymidine analogs ( martynoga et al . , 2005 ) . embryos collected at open neural plate stage ( nf13 ) were injected with ethynyl deoxyuridine ( edu , 2 10 nl , 10 mm in dmso [ life technologies ] ) in the neural plate midline at two different positions . after 2 hr incubation , bromodeoxyuridine ( brdu , 2 10 nl , 10 mm in dmso [ roche ] ) was injected in the same place ( figure 1a ) . embryos were fixed in memfa 30 min later and processed for edu , brdu , and sox3 ( as a marker of both polar and nonpolar neural progenitors ) staining as described elsewhere ( auger et al . , 2012 ) . this method involves labeling the mitotic ( phosphohistone h3/ph3 + ) cells with a thymidine analog ( edu or brdu ) and gives an estimate of the lengths of total cell cycle , s and g2 + 1/2 m phases , depending on the duration of the experiment ( shackney and ritch , 1987 ; peco et al . , 2012 ) . embryos at nf13 were injected with edu as described above and collected every 30 min until 240 min as the last point of our experiment ( figure 1b ) . embryos were fixed , sectioned , and stained for edu , ph3 , and sox3 as described elsewhere ( auger et al . , 2012 ) . nuclei from nf15 embryos ( 20 per set ) for facs analysis were prepared as described elsewhere ( frederick and andrews , 1994 ) , followed by cell - cycle profiling using a cyan adp flow cytometer from beckman coulter running summit v4.3 software . the modfit analysis on the data was carried out using flojo software . hela fucci cells were maintained and propagated as described elsewhere ( sakaue - sawano et al . , 2008 ) . control and experimental cells with inhibitors were imaged on a heated stage at 37c and 5% co2 supply using nikona1 confocal microscope for 23 days . for dual - pulse s phase labeling and percentage of labeled mitoses analyses , five to ten sections per embryo from the mid - anterior neural plate were imaged on a nikona1 confocal microscope . still and time - lapse ( for fucci ) images were processed and analyzed using imagej software . depending on the outcome of the normality test , appropriate tests for significance ( mentioned in the figure legends ) were chosen from the software and applied to the data . animal experiments were approved by the university s ethical review panel and were undertaken under uk home office project license ppl 70/7648 . culturing and overexpression in xenopus laevis embryos were carried out as described elsewhere ( sabherwal et al . , 2009 ) . staging was made according to the nieuwkoop and faber table of development ( nf stages ; nieuwkoop , 1967 ) . the following amounts of mrna or dna were injected : apkc - caax 0.250.5 ng , nls - apkc - nt 0.250.5 ng , and p27xic1 0.15 ng ( 0.075ng 2 ) ; 0.5 ng gfp or -galactosidase ( lacz ) mrna was coinjected as lineage n - tubulin , elrc , and sox3 have been described ( chalmers et al . , 2002 fixation , x - gal staining , in situ hybridization , and sectioning were carried out as described elsewhere ( bourguignon et al . , 1998 ) . gst - p27xic1 was expressed in bl21 escherichia coli cells and purified as described by the plasmid supplier ( ge healthcare ) . for in vitro kinase assay , 20 l of eluted gst - p27xic1 was mixed with kinase buffer ( 20 mm tris - hcl [ ph 7.5 ] and 5 mm mgcl2 ) , gst - apkc ( 1 l/0.1 g , calbiochem)/his - apkc ( 1 l/0.1 g , millipore ) , and p32-radiolabelled atp ( 1 l , 0.37 mbq [ perkin elmer ] ) . the final 30 l reaction was incubated at 30c for 30 min , boiled with sample buffer , and loaded on 10%12% polyacrylamide gel . post - run , dried gel on whatman paper was used to expose x - ray film to get an autoradiogram impression of the kinase reaction . to perform in vitro kinase assays in an embryonic environment , the reaction was performed exactly as described above , except that instead of kinase buffer , nf13 - 14 embryonic lysate ( as described below but without edta and egta ) was used as the incubation medium with 10 l ( 3.7 mbq ) of p32-radiolabelled atp . for in vivo kinase assays , hela cells were transfected with flag - p27xic1 alone or with ha - apkc - caax . for inhibitor treatment , flag - p27xic1 transfected cells were incubated with the media containing the inhibitor for 24 hr . p32-radiolabelled orthophosphate ( perkin elmer ) incorporation was performed as described elsewhere ( baril et al . , 2000 ) , followed by immunoprecipitation of flag - p27xic1 and autoradiography as described above . for the immunocomplex kinase assay , 10 l of protein a / g sepharose beads ( santa cruz biotechnology ) was mixed with 100 l of cleared lysate from hela cells ( overexpressing the protein of interest ) and 25 g of mouse anti - flag antibody ( m2 clone [ sigma ] ) . for histone h1 in vitro kinase assay , 0.5 g of histone h1 ( sigma ) , 100 ng of active cdk2/cyclina protein ( millipore ) , and 1 l of p32-radiolabelled atp ( 0.37 mbq ) were mixed with immunoprecipitated wild - type or mutant p27xic1 in 1 kinase buffer and incubated at 30c for 30 min . the reaction was stopped and autoradiography was performed as described above . hela cells were maintained and transfected as described elsewhere ( sabherwal et al . , 2009 ) . coip assays from hela cells overexpressing proteins of interest were performed as described elsewhere ( roth et al . , 2010 ) . proteins were immunoprecipitated using 510 g of the following antibodies : mouse anti - flag ( m2 clone [ sigma ] ) , mouse anti - ha ( sigma ) , and mouse anti - myc ( santa cruz biotechnology ) . coip from embryos was performed using the pierce crosslink ip kit , following the manufacturer s protocol using rabbit anti - apkc ( c20 clone , santa cruz biotechnology ) and rabbit anti - p27xic1 antibodies ( custom made against the antigen as described by shou and dunphy , 1996 ) . for making embryo lysates , embryos were dissociated in lysis buffer ( 50 mm tris ph7.5 + 150 mm nacl + 0.5% np40 + 5 mm edta + 5 mm egta ) . cleared lysates were used for western blot analysis . the following antibodies were used for detection : rat ha - hrp ( roche ) , mouse flag - hrp ( sigma ) , mouse myc - hrp ( santa cruz biotechnology ) , rabbit apkc ( c20 clone , santa cruz biotechnology ) , rabbit anti - p27xic1 , and mouse anti--tubulin ( dm1a clone [ sigma ] ) . myristoylated pseudosubstrate inhibitor against apkc ( invitrogen life technologies ; sajan et al . , 1999 ) was used at a working concentration of 12.5 m . chemical inhibitor against apkc ( g6983 [ calbiochem ] saiz et al . , 2013 ) was used at a working concentration of 5 m . olomoucine ( calbiochem ; calegari and huttner , 2003 ) was used at a working concentration of 120 m . antibody staining on sections and whole mounts was performed as described elsewhere ( sabherwal et al . , 2009 ) . the following primary and secondary antibodies were used : rat anti - ha ( roche ) , rabbit anti - flag ( sigma ) , mouse anti - brdu ( mobu clone [ life technologies ] ) , mouse anti - phosphohistoneh3 ( abcam ) and rabbit anti - sox3 ( custom made ; zhang et al . , 2003 ) , rabbit anti - myt1 ( custom made ; sabherwal et al . , 2009 ) , alexa488-coupled anti - mouse , and alexa647-coupled anti - rabbit ( life technologies ) . for phosphopeptide mapping , lc / ms / ms analysis of phosphosites was performed at the taplin biological mass spectrometry facility ( harvard medical school ) as described ( roig et al . , 2005 ) . please refer to the supplemental experimental procedures for information regarding sample preparation for ms analysis .

summaryduring the development of the nervous system , apicobasally polarized stem cells are characterized by a shorter cell cycle than nonpolar progenitors , leading to a lower differentiation potential of these cells . however , how polarization might be directly linked to the kinetics of the cell cycle is not understood . here , we report that apicobasally polarized neuroepithelial cells in xenopus laevis have a shorter cell cycle than nonpolar progenitors , consistent with mammalian systems . we show that the apically localized serine / threonine kinase apkc directly phosphorylates an n - terminal site of the cell - cycle inhibitor p27xic1 and reduces its ability to inhibit the cyclin - dependent kinase 2 ( cdk2 ) , leading to shortening of g1 and s phases . overexpression of activated apkc blocks the neuronal differentiation - promoting activity of p27xic1 . these findings provide a direct mechanistic link between apicobasal polarity and the cell cycle , which may explain how proliferation is favored over differentiation in polarized neural stem cells .

Introduction Results Discussion Experimental Procedures

PMC5002936

there is an urgent need to better understand the molecular factors that govern influenza a virus ( iav ) virulence . seasonal ( human - adapted ) iav is a major cause of morbidity and mortality each year , but the pandemic potential of iav strains that originate in animal hosts poses an even more serious threat to public health . there have been four influenza pandemics in the past century , in 1918 , 1957 , 1968 , and 2009 . the precise origin of the 1918 pandemic strain , which killed roughly 50 million people , is unclear . however , the three subsequent pandemic strains resulted from the process of genetic reassortment , in which gene segments from different viruses mix during host coinfection to produce novel viral strains . there is much concern that a new pandemic strain might result from reassortment involving the highly pathogenic h5n1 and h7n9 avian influenza viruses currently circulating in asia . the majority of these viruses undergo a deletion in the stalk of the neuraminidase surface protein , a characteristic that is associated with virulence when causing outbreaks in domestic poultry . it is thus vitally important to understand the effect of stalk deletion on viral function . influenza a strains are named according to the alleles coding for their two surface antigens , the hemagglutinin ( ha ) and neuraminidase ( na ) glycoproteins . hemagglutinin ( ha , 18 antigenically distinct alleles identified to date ) facilitates viral adhesion to human host cells by binding to cellular sialylated - oligosaccharide receptors , and neuraminidase ( na , 11 antigenically distinct alleles ) facilitates viral release by cleaving sialic acid linkages . for example , hyperglycosylation and active - site mutation tend to reduce ha / sialic acid binding . na activity is generally proportional to the length of the highly variable na stalk , and recent neuraminidase stalk deletions often produce highly pathogenic avian influenza viruses such as h5n1 . influenza virulence is determined in large part by the balance between na and ha activities at the virion surface . recent viral strains with stalk - deletion na glycoproteins ( nadel ) are more virulent because the reduced nadel activity alters this balance . for example , the virulence of both a pandemic 2009 h1n1 virus and a more recent 2013 h7n9 strain was enhanced when deletion mutations containing 20 and 5 amino acids , respectively , were introduced into the corresponding na stalks . in these strains , reduced na activity due to a stalk deletion may enhance viral infectivity by reducing oligosaccharide - receptor cleavage and thus increasing the number of receptors available for ha binding . this strategy is particularly advantageous when ha activity is itself compromised ( e.g. , due to hyperglycosylation , active - site mutations , etc . ) , leading many to express concerns that a current h5n1 strain might obtain a long - stalk na through reassortment with a human - adapted virus . optimal viral replication is achieved only when the activities of na and ha are ideally complementary . the mechanism by which reduced na stalk height impacts na activity remains uncertain . one prevailing theory suggests that nadel glycoproteins have reduced sialidase activity relative to wild type ( nawt ) because their diminished height hinders access to cellular sialylated - oligosaccharide receptors ( i.e. , the the implication is that a towering canopy of ha effectively buries the short - stalk nadel such that cell - bound oligosaccharides simply can not reach the na enzymatic pockets . while there is merit to the limited - access theory , especially in cases where the stalk length is profoundly reduced , experiment and computational modeling suggest it may not fully explain the reduced nadel activity . we hypothesize that , in addition to any limited - access effects , na stalk length also reduces na enzymatic activity by altering the binding affinity of sialic acid , the native substrate , to the enzymatic pocket . given that the stalk is roughly 40 from the active site , we propose an indirect mechanism by which stalk length impacts binding - pocket dynamics at a distance . other studies have demonstrated that the na enzymatic pocket is highly flexible and so samples many conformations . for example , x - ray crystallography and molecular dynamics simulations of the isolated neuraminidase catalytic domain suggest that key loops surrounding the active site ( i.e. , the 150- and 430-loops ) are highly mobile , permitting large cavities to form immediately adjacent to the main pocket . given that the enzyme is known to adopt a variety of conformational states , it is reasonable to hypothesize that stalk length may have a quasi - allosteric impact on ligand binding affinity by shifting the binding - pocket conformational population toward a subtly different distribution . to properly study the impact of stalk length , we built a full model of 2009 h1n1 neuraminidase as well as a model that included a 20 amino acid stalk - deletion mutation similar to one known to enhance virulence in a recent strains . , who replaced the long - stalk nawt of a 2009 h1n1 pandemic virus with a short - stalk h5n1 nadel . they attributed the resulting decrease in transmissibility at least in part to a reduction in the ability of the short - stalk nadel to cleave tethered substrates and penetrate the host mucus barrier . rather than exactly replicating the work of blumenkrantz et al . , who inserted a foreign na ( from h5n1 ) into a h1n1 virus , we made our deletion directly in the h1n1 nawt stalk itself . our models were constructed using data from both x - ray crystallography ( used to model the enzymatic head ) and also protein protein docking and modeling ( used to model the stalk , transmembrane , and intravirion domains , which have never been crystallized ) . all na models were embedded in complex - mixture lipid bilayers ( figure 1 ) . equilibrated neuraminidase models used in the current study : ( a ) wild - type ( long - stalk ) nawt and ( b ) stalk - deletion nadel . subsequent atomistic molecular dynamics simulations of these systems support the theory that the na stalk length impacts the dynamics of the na binding pocket and therefore the affinity for the endogenous substrate . furthermore , these simulations reveal previously uncharacterized druggable hotspots that may open up additional avenues for structure - based drug design . previous simulations have included only the na head ; our models , which include additional na domains as well as the associated lipid bilayer , allow us to take the next steps toward more realistic atomic simulations of viral components to generate a complete model of long - stalk ( wild - type ) apo nawt , a monomer of the tetrameric na head was built by homology using schrdinger s prime and the na sequence of the 2009 h1n1 pandemic reference strain a / california/04/2009 ( genbank accession fj966084 ) , which codes for a na protein identical to that of the a / england/195/09 virus used by blumenkrantz et al . default prime parameters were used . the monomeric homology model , based on the 3nss crystal structure , was missing a single lysine residue on the c terminus , which was added manually . the appropriate tetramer was formed by aligning copies of the homology - model monomer to chains a d of the 2hu4 crystal structure using multiseq , as implemented in vmd . the resulting tetrameric head was then processed with schrdinger maestro s protein preparation wizard and subjected to repeated minimization in the maestro environment . both sspro 4.5 , a neural - network - based algorithm for predicting secondary structure from primary sequence , and abtmpro , a svm algorithm for determining whether a given transmembrane segment is helical or barrel , suggested that the stalk has an -helical structure . we therefore used the amber xleap module to generate a single helix with the appropriate long - stalk sequence . rosettadock 3.4 was used to assemble four identical copies of this helix into a four - helix bundle , one helix at a time . at each step , the top 200 best - scoring structures were clustered by root - mean - square distance ( rmsd ) using cutoffs ranging from 0.75 to 3.0 , and representative dockings were examined visually . the top representative docked pose that positioned the newly introduced helix in the expected parallel conformation was retained . when no such pose was identified by clustering , all docked poses were examined sequentially , starting with the pose that had the highest score , until an acceptable pose was identified . the resulting four - helix bundle was then processed with schrdinger maestro s protein preparation wizard and subjected to repeated minimization in the maestro environment . we assumed that the cysteine residues located in the extra - virion region of the stalk likely participate in interchain disulfide bonds , but that the cysteine residues located in the transmembrane region likely do not . the tetrameric - head and stalk / transmembrane - region models were next positioned near each other using vmd . the combined models were subsequently loaded into maestro and again subjected to multiple minimizations . finally , maestro was used to build and position the residues of the short intravirion topological domain . based on the assumption that this domain lies at the interface of the inner membrane leaf and bulk intravirion water , all hydrophobic side chains were manually pointed upward toward the bilayer , and all polar side chains were pointed downward . to generate a model of apo na with a virulence - enhancing stalk deletion ( hereafter called nadel ) , the appropriate mutant amino acid sequence was first identified . these residues were manually removed from the nawt model , and the flanking na segments were juxtaposed and fused using vmd and schrdinger s maestro , followed by multiple minimizations as needed . our efforts to construct a complex - mixture lipid - membrane model were guided by a recent paper describing the composition of the influenza viral envelope . we identified and classified all listed membrane components with mol % > 1.5 , taking care to keep the composition of the inner and outer leaflets separate . as our ultimate goal was to perform molecular dynamics simulations of this system , we were limited to the lipids of the charmm 36 force field . consequently , we had to substitute some experimentally identified lipids with available lipid models . 3-palmitoyl-2-oleoyl - d - glycero-1-phosphatidylethanolamine ( pope ) was used for any phosphatidylethanolamine ( pe ) variant , 2,3-distearoyl - d - glycero-1-phosphatidylserine ( dsps ) for phosphatidylserine ( ps ) , 3-palmitoyl-2-oleoyl - d - glycero-1-phosphatidylglycerol ( popg ) for the forssman glycolipid hapten , and 3-palmitoyl-2-oleoyl - d - glycero-1-phosphatidylcholine ( popc ) for sphingomyelin ( sm ) . cholesterol and phosphatidylcholine , also major components of the envelope membrane , are included in the charmm 36 force field . the na models were then positioned within this generated bilayer , and clashing lipid residues were deleted with pymolecule . in preparation for molecular dynamics simulations , we resolved multiple steric clashes in both the long - stalk and stalk - deletion systems using serial iterations of minimization and geometry optimization in schrdinger s maestro , coupled with manual atomic and molecular manipulation in vmd . partial charges were assigned to all atoms according to the specifications of the charmm22 all - hydrogen force field for proteins and the charmm36 all - hydrogen force field for lipids . the vmd plug - in cionize(47 ) was used to position sodium and chloride ions as required to bring the systems to electrical neutrality and to simulate a 20 nm solution . limited manual adjustments to the positions of some ions that had been placed far from any protein or lipid molecule were made . for both the nawt and nadel systems , amber s xleap module was used to generate a water box extending 15 beyond any atom . water molecules were subsequently removed if they were not positioned directly above the bilayer , so that the 15 margin was ultimately retained only along the z axis , perpendicular to the bilayer itself . the nawt and nadel systems had 366 293 and 351 699 atoms , respectively . psfgen was used to fully parametrize each system according to the charmm22 , charmm36 , and tip3p force fields for proteins , lipids , and water molecules , respectively . namd 2.9 running on the stampede supercomputer at the texas advanced computing center was used for all md simulations . periodic boundary conditions were employed with the particle mesh ewald method to account for electrostatic effects ( smoothing cutoff : 14 ) . because of observed instabilities when our standard minimization protocols were used , we subjected the initial nawt system to only 1000 steps of unrestrained conjugate gradient minimization , with the first and max initial steps for the line minimizer reduced to 1.0 10 and 1.0 10 , respectively . the structure was minimized in four distinct steps : hydrogen atoms were first relaxed for 5 000 steps ; hydrogen atoms , water molecules , and ions were next relaxed for 5 000 steps ; hydrogen atoms , water molecules , ions , protein side chains , and lipid tails were then relaxed for 10 000 steps ; and , finally , all atoms were relaxed for 25 000 steps . following minimization , each system was subjected to a constrained equilibration using an npt ensemble at 310 k. the equilibration was realized in four steps , using stepwise harmonic - constraint force constants of 4 , 3 , 2 , and 1 kcal / mol / on the protein backbone . for each force constant ( 1 fs time step ) , 250 000 steps of md simulation were performed . langevin dynamics were applied to maintain the temperature , and a modified langevin piston nos the nawt model underwent an additional 5 million 2 fs steps of unconstrained simulation , followed by another 21 million 1 fs steps . the nadel model underwent an additional 5.5 million 2 fs steps of unconstrained simulation , followed by another 36.5 million 1 fs steps . satisfied that both systems were sufficiently equilibrated , five distinct 100 ns productive runs were performed for each ( 100 million 1 fs steps ) using distinct random seeds in order to sample diverse protein configurations . ( 5 simulations ) ( 100 ns per simulation ) ( 2 systems ) ( 4 monomers per system ) = 4 s of monomeric simulation total . 5000 and 7000 frames were extracted from the last 50% and 70% of the tetrameric nawt and nadel simulations , respectively , for subsequent analysis . for comparison purposes , we also analyzed an archived simulation of the isolated apo na head ( hereafter called nahead ) , described previously . in brief , the na head ( pdb i d : 3nss(26 ) ) was simulated for 100 ns using the amber99sb force field , yielding 400 ns of monomeric trajectory . for the current study , we extracted 5000 frames from this simulation for subsequent analysis . we used root - mean - square - distance ( rmsd ) calculations to judge the geometric similarity between the primary na enzymatic pockets of crystallographic holo and simulated apo models . we first identified active - site residues as those that came within 5.0 of a crystallographic oseltamivir ligand ( pdb i d : 2hu4:a , h5n1 ) . these residues are arg118 , glu119 , asp151 , arg152 , arg156 , trp178 , ser179 , ile222 , arg224 , glu227 , ser246 , glu276 , glu277 , arg292 , asn294 , tyr347 , gly348 , arg371 , and tyr406 . for the purposes of our rmsd analysis , we discarded tyr347 because that tyrosine is an asparagine in h1n1 na . hydrogen atoms were also removed prior to performing all rmsd calculations ; rmsd alignment was performed only on the remaining heavy atoms . we performed principal component analysis ( pca ) on these same active - site heavy atoms . after rmsd aligning the nawt trajectories by these atoms using vmd , we used the python modules scikit - learn and numpy to calculate their first two principal components and to project each trajectory frame onto those components . the frames of the nadel trajectory were similarly projected onto the same two nawt principal components to facilitate comparison . the povme algorithm was used to measure the volume of the primary sialic - acid - binding pocket over the course of the nawt , nadel , and nahead simulations . to calculate the volume of the binding pocket for a given trajectory frame , we monitored the space within a pocket - centered sphere of radius 16.0 . in all cases , the pocket volume was calculated from the portion of the sphere that was unoccupied by protein atoms . only volumetric regions contiguous with the pocket grid spacing and padding parameters were set to 1.0 and 1.09 , respectively . all - atom , residue root - mean - square fluctuations ( rmsf ) were calculated for each monomer of each simulation . for the nawt and nadel simulations , for the nahead simulation , there were 4 associated monomeric simulations ( 1 run 4 monomers ) . in all cases , the monomeric trajectories were first aligned by the carbons of residues ser90 to asp469 ( i.e. , the residues of the na head ) . the residue center - of - mass rmsf values were calculated for each monomeric simulation separately . to identify druggable hotspots , we first identified representative protein conformations from the md simulations . the heads of the aligned monomeric trajectories were subjected to rmsd clustering , as implemented in the gromacs computer package . we adjusted the rmsd cutoff until the trajectory frames were separated into five clusters . the required cutoffs were 1.72 , 1.74 , and 1.74 for the nawt , nadel , and nahead simulations , respectively . the centroid members of each of these clusters were considered representative of the corresponding simulated model . the 15 representative na structures ( 3 models 5 cluster centroids ) were then submitted to the ftmap server to identify druggable hotspots . to simplify the analysis , all the docked molecular probes associated with each simulation ( nawt , nadel , and nahead ) were considered collectively . to identify druggable hotspots that were specific to a given model , the associated docked probes were superimposed , and any probe atom associated with one model that came within 5 of any probe atom associated with the other was deleted . the remaining probe atoms congregated at hotspots that were unique to their respective models . as described in the introduction , the limited - access theory holds that stalk - deletion neuraminidase glyocproteins ( nadel ) have reduced sialidase activity relative to nawt because their diminished height hinders access to cellular sialylated - oligosaccharide receptors . the fact that na enzymatic activity varies depending on ligand bulkiness is perhaps the most compelling evidence in favor of this theory . studies have shown that short - stalk nadel glycoproteins have significantly reduced activity when a bulky substrate ( e.g. , fetuin ) is used , presumably because the large substrate can not physically reach the na enzymatic sites . in contrast , na activity is largely stalk - independent when cleaving the small molecule munana ( essentially a single sialic acid with a 4-methylcoumarin fluorophore attached to the 2-carbon ) , apparently because this smaller substrate is not subject to the same large - scale steric hindrance . however , several lines of evidence suggest that the limited - access theory may not fully explain the reduced nadel activity . binding to these receptors , which are extended , large , and cell bound , is likely diffusion limited ; in contrast , munana is a relatively small molecule with an affinity that is likely little dependent on kon . while munana may be a fine substrate for measuring relative na enzymatic activity in the context of a small - molecule screen , it is arguably an inadequate surrogate for the actual receptor . second , fetuin ( -2-hs - glycoprotein ) is also very different than the endogenous receptor , and the theory that its bulk alone prevents it from accessing the nadel enzymatic sites is hardly certain . we note that all available monoclonal antibodies known to target na antigenic sites bind equally well to virions with nadel and nawt glycoproteins , despite the fact that antibodies are much larger than fetuin ( 160 and 1000 kda for igg and igm , respectively , vs 40 kda for fetuin ) . furthermore , recent cryo - electron microscopy studies of whole - virion particles demonstrate that the na glycoproteins are not evenly interspersed among the more prevalent ha s as the limited - access theory suggests ; rather , the na particles tend to cluster together on the viral surface . even when stalk mutations substantially reduce the na height relative to ha , this clustering effect alone should in theory preserve much access to the na enzymatic head . computational evidence likewise suggests that simple steric considerations are not solely responsible for reduced nadel activity . our models of 2009 h1n1 glycoproteins demonstrate that each stalk amino acid contributes only 1.2 to the total na height ( data not shown ) . the 2013 h7n9 five - amino - acid stalk deletion with enhanced virulence mentioned in the introduction therefore reduced the na height by only 6 ( roughly 4% ) . more substantial stalk deletions do not necessarily force the na enzymatic head to retreat entirely behind a towering canopy of ha glycoproteins either . in our models of h1n1 ha and na , we found the height of nawt was nearly equal to that of ha ( 149 vs 154 , respectively ) . when a virulence - enhancing 20 amino acid na stalk deletion was modeled ( nadel ) , the height of the na fell to only 125 , still 81% of the ha height . given that the na head is itself a boxlike structure 80 square , the resulting 24 height reduction seems unlikely to substantially interfere with access . we suggest that , beyond limited - access effects , actual differences in the affinities of nawt and nadel for the endogenous sialylated - oligosaccharide receptors contribute to the differences in enzymatic activity . as support for our reduced - affinity hypothesis , we analyze atomistic molecular dynamics simulations of three na models : five 100 ns simulations of 2009 h1n1 nawt ; five 100 ns simulations of 2009 h1n1 na with a 20-amino - acid deletion in the stalk ( nadel ) ; and one 100 ns simulation of the isolated h1n1 na head , nahead ( figure 1 ) . these simulations indicate that stalk length does in fact impact the dynamics of the enzymatic sialic acid binding pocket , suggesting a potential mechanism for the proposed stalk - length - dependent differences in affinity . to verify that the simulations of the three na systems ( i.e. , nawt , nadel , and nahead ) had properly equilibrated , we rmsd aligned each tetrameric trajectory by its c atoms using vmd . the rmsd was then calculated between each trajectory frame and a corresponding reference structure ( figure s1 ) . despite extensive pre - equilibration , the rmsd values of the whole - glycoprotein nawt and nadel simulations continued to drift slightly throughout the productive runs . to further investigate the source of this drift , we calculated separate c rmsd values for the residues of the na head ( figure 2 ) , the lower ( membrane - embedded ) stalk , and the upper stalk . the dynamics of the head and lower stalk were in fact stable in both the nawt and nadel simulations . it was the upper stalk that was dynamically unstable . given that the upper stalk serves as a linker connecting two fairly ridged bodies ( the lower stalk and the head ) , it is reasonable to suppose that this region is legitimately hypermobile . rmsds of all trajectory monomers to their respective reference structures . in all cases , only the c of the head domain ( i.e. , the c of all residues homologous to the 388 residues present in the 3nss crystal structure ) were considered . ( a ) nawt ( 5 tetrameric trajectories 4 monomers per tetramer = 20 runs ) ; ( b ) nadel ( 5 tetrameric trajectories 4 monomers per tetramer = 20 runs ) ; and ( c ) nahead ( 1 tetrameric trajectory 4 monomers per tetramer = 4 runs ) . subsequent analyses ( described below ) focused primarily on the residues of the na binding pocket , located on the enzymatic head . given the instability of the upper stalk , however , we chose to discard the initial 50% and 30% of the nawt and nadel trajectories , respectively , to ensure that subsequent analysis focused on the most equilibrated portion of the simulations . in contrast , nahead , based on the 3nss crystal structure , equilibrated far faster . we therefore discarded only the first 20% of the nahead simulation ( figures 2 and s1 ) . we next sought to characterize the effects of the stalk on binding - pocket dynamics by comparing the geometries of our simulated apo binding sites to that of holo ( ligand - bound ) na . we calculated the rmsd values between the simulated binding - pocket conformations and that of a reference crystallographic structure , 2hu4 ( h5n1 ) , which shares in common with h1n1 a total of 18 out of 19 binding - pocket residues . in calculating these rmsd values , the single differing residue was ignored . a histogram of the rmsd values suggests that the long - stalk nawt pocket exists in two conformational states ( figure 3a ) , with rmsds to the apo ( reference ) conformation of roughly 1.8 and 2.7 , respectively . in contrast , the pocket conformations sampled during the nadel and nahead simulations were not binary , suggesting the pocket is less flexible in these systems . ( a ) simulated apo binding - pocket conformations are compared to the crystallographic ( 2hu4 ) holo conformation . geometric similarity was judged by calculating the rmsds between simulated and crystallographic pocket residues . . geometric similarity was judged by calculating the rmsds between simulated and crystallographic triad conformations . ( c ) representative nawt arginine - triad conformations sampled from the 2.4 state ( in yellow ) and the 3.4 state ( in red ) . the crystallographic position of the triad residues is shown in thick , white licorice . an oseltamivir molecule has been positioned within the binding pocket for reference ( in orange ) , though the simulations did not include any ligand . ( d ) the simulated conformations of nawt and nadel active - site heavy atoms , in black and red , respectively , projected onto the first two principal components of the nawt simulation . the nawt site assumes a greater variety of conformations , in harmony with the rmsd analysis . an arginine triad formed by arg118 , arg292 , and arg371 is a key feature of the enzymatic pocket . this triad forms a positively charged arginine nest that surrounds the negatively charged carboxylate groups of the endogenous substrate as well as all fda - approved na inhibitors . as with the whole pocket , the average rmsd between the atom positions of the simulated apo triad configurations and the holo 2hu4 conformation were 2.5 and 2.4 for the nawt and nadel simulations , respectively . a t - test led us to reject the null hypothesis that these two averages are statistically equal ( p = 0.0 ) . a histogram of the arginine - triad rmsd values suggests that , unlike the nadel triad , the nawt triad again assumes two distinct conformations ( figure 3b ) . to further characterize these two nawt states , we visually inspected representative conformations from each ( figure 3c ) . in the state with rmsd = 2.4 , arg371 is near its crystallographic conformation , and arg292 is displaced only slightly in the direction opposite of arg371 . in contrast , in the state with rmsd = 3.4 , arg371 is generally displaced in the direction of arg118 , and arg292 is even further from arg371 . arg118 possesses roughly the same conformation in both states . to further verify that the nawt pocket assumes a greater variety of conformational states than the nadel pocket , we next performed a principal component analysis ( pca ) of the nawt active - site heavy atoms ( figure 3d ) . pocket conformations from both the nawt and nadel simulations were projected onto the first two principal components . the nawt pocket ( in black ) sampled the same region of pca space as the nawt simulation ( in red ) , but the nawt pocket additionally sampled other conformations not seen in the nadel simulation , confirming that the nawt pockets adopted a greater number of conformational states . having studied the impact of stalk length on binding - pocket dynamics generally , we next considered the dynamics of specific binding - pocket residues . we calculated the associated all - atom , residue root - mean - square fluctuations for each of the 20 monomeric trajectories associated with the nawt and nadel simulations . for each residue , we compared the ensembles of nawt and nadel rmsf values using a t - test . a number of residues exhibited statistically significant average rmsf differences when a liberal significance level was used ( t - test , = 0.10 ) . most interestingly , the rmsf values of two of the three arginine residues of the key active - site triad ( arg371 and arg118 ) differed significantly ( table 1 ) . this analysis again implicates the arginine triad and 371 loop as contributing to differential , stalk - length - dependent binding - pocket dynamics . data from the corresponding nahead ( stalk - absent ) simulations are also included . the average rmsf value is shown plus or minus the standard deviation . the residue - specific populations of all nawt and nadel rmsf values were compared using t - tests . to better visualize differences in active - site dynamics , we colored the residues of a nawt conformation by rmsf = rmsfwt rmsfdel ( figure 4 ) . the 371 , 406 , and 430 loops were generally more flexible in the nawt simulation ( shown in red ) . however , a portion of the 150 loop was more flexible in the nadel simulation ( shown in blue ) . na active - site residues colored by rmsf = rmsfwt rmsfdel , from 0.2 to 0.2 . residues in red and blue were more flexible in the nawt and nadel simulations , respectively . a crystallographic oseltamivir molecule is shown for reference , though no ligand was included in the simulations . finally , we also used our molecular dynamics simulations to study na druggability . to get an initial impression of the full extent of pocket dynamics , we first performed a volumetric analysis of the primary sialic - acid - binding pocket using the povme algorithm . the trajectory frames with both the largest and smallest volumes from the nawt , nadel , and nahead simulations were compared separately ( figure 5 ) . in all cases , the pocket fluctuated between a closed conformation ( figure 5a ) and an open conformation ( figure 5b ) , suggesting a highly dynamic pharmacophore . interestingly , the nawt pocket tended to be larger than the nadel pocket ( figure 5c , p = 0.0 per a t - test ) , implying possible differences in the pharmacophore distributions of these two na variants that may have important implications for drug discovery . to further study variant - dependent differences in na - pocket druggability . ftmap seeks to replicate in silico the experimental multiple solvent crystal structures ( mscs ) method developed by mattos et al . in brief , the ftmap algorithm uses computer docking to flood the surface of a protein with small organic molecular probes . these probes are then clustered to identify regions of the protein surface that are most likely to bind small molecules . ( a ) the smallest and ( b ) largest binding - pocket conformations sampled by the nawt simulation . the smallest and largest conformations from the nadel and nahead simulations were similar ( data not shown ) . ( in licorice ) have been placed in each pocket for reference , though the simulations included no ligand . ( ) histograms of the volumes sampled over the course of the nawt and nadel simulations , in black and gray , respectively . we used ftmap to identify druggable hotspots on the surfaces of five representative na structures extracted from each of our molecular dynamics simulations . for the most part , the druggable regions of the enzymatic site did not differ substantially between the nawt and nadel simulations . however , ftmap did position some molecular probes near a 430-loop - adjacent region of the nawt pocket that was not deemed druggable when the nadel conformations were considered , suggesting subtle differences that may be pharmacologically relevant ( figure 6a ) . unique druggable hotspots ( outlined ) . the 371 , 430 , and 150 loops are shown in mauve , pink , and green , respectively . ( a ) a druggable hotspot was identified near the 430 loop of the nawt pocket . ( b ) a second druggable hotspot was identified beneath the 371 loop of both the nawt and nadel pockets . ( c ) a large druggable hotspot was identified near the base of the nawt and nadel heads . the nawt and nadel hotspots were then merged and similarly compared to the nahead hotspots . surprisingly , in both stalk - inclusive na simulations ( nawt and nadel ) , the 371 loop occasionally flipped upward , opening a novel druggable hotspot that was not apparent when the representative structures of the nahead simulation were considered ( figure 6b ) . this pocket has been described previously , but the nawt and nadel simulations confirm its importance . to date , no known na inhibitor exploits this pocket , and it has not been the focus of previous cadd efforts . finally , another large druggable hotspot located at the base of the na head was unique to the stalk - inclusive ( i.e. , nawt and nadel ) simulations ( figure 6c ) . factors that alter the sialic acid binding / association mechanism may profoundly alter na affinity and activity in additional ways not explained by the theory of limited access . the length of the stalk appears to influence the dynamics of the 150 , 430 , 371 , and 406 binding - pocket loops . the 371 loop and the critical arginine catalytic triad are particularly affected . as a consequence , the apo nawt pocket may adopt a greater variety of conformational states than does that of nadel . specifically , the nadel pocket tends to sample a single conformational state that is generally similar to that of the holo conformation . in contrast , nawt additionally samples more distant pocket geometries ( figure 3 ) . while the impact of stalk length on binding - pocket dynamics may well be partially responsible for the reduced nadel affinity , we note also that stalk length likely has a profound influence on the long - range electrostatics of the whole - virion surface , which could also impact kon . future efforts will focus on elucidating stalk - length - dependent differences in this effect as well . finally , the predicted druggable hotspots evident in the stalk - inclusive simulations also suggest novel and urgently needed opportunities for drug discovery . prior to the 2009 h1n1 pandemic , seasonal h1n1 in the united states was almost entirely oseltamivir resistant . fortunately , that pandemic strain itself remained largely susceptible to oseltamivir , but given the viral proclivity for rapid mutation and adaptation , multidrug combinatorial therapies may be necessary in the future . these novel hotspots also provide means for experimentally testing some of the hypotheses generated in the current study . virtual screening into the na pocket conformations sampled over the course of the nawt and nadel simulations could identify compounds that are uniquely suited to the corresponding pocket geometries . if subsequent experimental validation confirms binding , x - ray crystallography could be used to determine if the novel ligand(s ) stabilize one of the predicted na conformations .

deletions in the stalk of the influenza neuraminidase ( na ) surface protein are associated with increased virulence , but the mechanisms responsible for this enhanced virulence are unclear . here we use microsecond molecular dynamics simulations to explore the effect of stalk deletion on enzymatic activity , contrasting na proteins from the a / swine / shandong / n1/2009 strain both with and without a stalk deletion . by modeling and simulating neuraminidase apo glycoproteins embedded in complex - mixture lipid bilayers , we show that the geometry and dynamics of the neuraminidase enzymatic pocket may differ depending on stalk length , with possible repercussions on the binding of the endogenous sialylated - oligosaccharide receptors . we also use these simulations to predict previously unrecognized druggable hotspots on the neuraminidase surface that may prove useful for future efforts aimed at structure - based drug design .

Introduction Materials and Methods Results and Discussion Conclusion

PMC3649120

the following case report highlights a potentially life threatening complication of undertaking an incision biopsy on a soft tissue tumour , namely surgically uncontrollable haemorrhage from an unsuspected vascular lake . the case report demonstrates the value of interventional radiology for acute bleeding and radiotherapy for more chronic tumour bleeding . a 75 year old male presented with a 3 month history of painful progressive left buttock swelling and non - productive cough . past medical history included carcinoma of the prostate 5 years previously , which had been treated with surgery and radiotherapy . examination revealed a fixed 10 by 10 cm mass in the left buttock extending over the sacro - iliac joint . a pleural aspiration cytology confirmed a malignant effusion , although did not differentiate the cell type . the pelvic mri scan demonstrated a large 10 x 10 x 5 cm heterogeneous lesion in the left buttock , occupying much of the posterior portion of the gluteus maximus and invading the iliac bone and sacrum . the primary lesion was not operable and the presence of the malignant effusion denoted stage 4 disease , ( t2 , n0 , m1 ) . percutaneous needle core biopsy of the lesion was inconclusive and incision biopsy was requested after drainage and talc pleurodesis of the pleural effusion . at the time of incision biopsy the capsule of the lesion this resulted in profuse bleeding from a deep , fresh blood filled , cavity within the cystic tumour . a large vascular lake in the centre of the tumour was evident with a significant vascular flow rate . the cavity was therefore tightly packed with haemostatic material and firm pressure applied to the wound , causing partial cessation of the haemorrhage . full surgical control of the bleeding was not possible due to the fact that the tumour was in - operable and the feeding vessels originated from the internal iliac vessels running through the invaded bone of the ileum . angiography revealed a tumour blush in the gluteal region supplied by the median sacral artery and the posterior division of the internal iliac artery ( iia ) . the iia was catheterised revealing multiple feeding branches with extravasation from one ( figure 1 ) . angiograms pre - embolisation with histo - acryl angiograms post - embolisation with histo - acryl over the ensuing 3 weeks , despite the initial successful embolisation the patient haemorrhaged on a further 2 occasions despite pressure dressings to the wound area . on both of these occasions on each occasion , a different set of feeding vessels was noted to be bleeding and was embolised . histology from the incision biopsy revealed a diagnosis of epithelioid angiosarcoma ( figure 2 ) which is known to be associated with the development of vascular lakes . ( cd31 immunohistochemistry : immunoperoxidase , original magnification x100 ) to prevent the occurrence of further haemorrhage , the patient was given a course of radiotherapy ( 30gy in 10 treatments over 14 days ) . palliative chemotherapy to control respiratory symptoms from the pleural effusion was declined and the patient died 6 months after initial presentation . the epithelioid phenotype of angiosarcoma was first described in 1976 by rosai and colleagues who documented cases of cutaneous angiosarcomas . it was later documented by fletcher and colleagues in 1991 who described deeper lesions found in the soft tissues where they are now most commonly found . ( 1 ) epithelioid angiosarcomas are rare , high - grade , malignant , vascular tumours , which have an aggressive course and tend to recur locally , spread widely , and have a high rate of lymph node and systemic metastases.(2 ) it often presents in middle - aged and elderly males with a peak incidence in the seventh decade of life . prognosis for these types of tumours is very poor with death usually occurring from 6 months to 2 years after presentation . epithelioid angiosarcomas have been described arising in various soft tissues around the body and are most often metastatic in origin . histologically these tumours consist of solid and infiltrative sheets of epithelioid cells characterised by large , oval to round cells with abundant eosinophilic cytoplasm , vesicular nucleus and eosinophilic nucleolus.(3 ) in most cases there are vascular channels or cystically dilated spaces . vascular lakes are therefore a common finding associated with these types of sarcoma . a distinctive starry - sky histological pattern has also been reported in epithelioid angiosarcomas . immunohistochemically the neoplastic cells show positivity for endothelial cell markers such as cd31 , cd34 , factor viii - related antigen , and vimentin.(4 - 5 ) due to cytokeratin expression and epithelioid growth pattern these tumours can occasionally be misdiagnosed as carcinomas . endothelial differentiation can be confirmed ultrastructually by the presence of weibel - palade bodies , intermediate filaments and basal lamina . intracytoplasmic vacuoles containing red blood cells are often present in some cells.(4 ) irradiation of soft tissues for the treatment of various primary cancers is well documented as a cause for several types of sarcoma . as in this case , the patient had undergone a course of radiotherapy for prostate cancer several years prior to presenting with the pelvic sarcoma and this therefore may have been the causative factor . other risk factors for the development of angiosarcomas include carcinogens , chronic lymphoedema and hormonal status . however one such case documented a lesion of the mons pubis 4 years following radiotherapy for vulval carcinoma.(5 ) angiography and tumour embolisation is a well established treatment of tumours both pre- and post - operativley . tumour embolisation may be carried out for acute bleeding from tumour vessels , and to reduce the blood supply to tumours prior to surgery . treatment usually consists of delivering one or two fractions of 5 - 8 gy each on successive days .

epithelioid angiosarcoma is a rare , highly malignant tumour with a poor prognosis . we present the case of a 75 year old man who underwent an incision biopsy to diagnose the soft tissue tumour and suffered from surgically uncontrollable haemorrhage . the case report demonstrates the value of interventional radiology for acute bleeding and radiotherapy for more chronic tumour bleeding .

INTRODUCTION CASE REPORT DISCUSSION

PMC4831861

post - activation potentiation ( pap ) is a neuromuscular phenomenon in which the force and power output of a muscle is immediately enhanced after heavy resistance exercise ( 9 ) . the underlying principle of the ability of pap to increase force and power output of muscle is theorized to be a result of phosphorylation of myosin regulatory light chains ( 21 ) . it has been hypothesized that the phosphorylation of myosin regulatory light chains cause the interaction between actin and myosin to become more sensitive to ca released from the sarcoplasmic reticulum , resulting in an increased level of myosin cross bridge activity in response to submaximal resistance exercise ( 20 , 21 ) . the majority of pap research has investigated a practice referred to as complex training , in which a heavy resistance training exercise is executed prior to performing an explosive bodyweight movement with similar biomechanical characteristics ( 12 ) . the heavy resistance exercise is hypothesized to induce pap , thus increasing the acute performance of the bodyweight exercise , with the prospect of superior chronic neuromuscular adaptations ( 9,12 ) . this increase in central nervous system stimulation causes greater motor unit recruitment and force production that may last from 5 to 30 minutes ( 18 ) . to date , pap research has shown a few general trends . first , trained individuals appear to respond more favorably to eliciting the pap response than do untrained subjects ( 15,18 ) . second , moderate to heavy intensity of resistance exercise elicits the most favorable pap response ( 4,16,18 ) . third , there appears to be a critical window of time for power output due to pap , as an initial decrease in power is observed immediately after heavy resistance exercise , followed by an optimal time of pap where peak power can be harnessed ( 11,16,24 ) , followed by gradual diminishing power output ( 3,4,6,7,11 ) . collectively , a recent meta - analysis by wilson et al . ( 25 ) suggested the optimization of the pap effect would utilize a combination of multiple sets , moderate intensity ( 6084% 1rm ) , and moderate rest periods ( 710 minutes ) . whereas the majority of previous pap research has examined male subjects within athletic or recreationally trained populations , minimal research exists as to how females respond to pap , with even fewer studies in regards to female athletes ( 8) . previous studies on females have found evidence of a pap response , albeit with small effect sizes ( es=.20 ) ( 13,14,25 ) , yet the pap response in female athletes is largely unknown . common limitations in previous pap studies that have examined female responses are small sample sizes leading to underpowered studies , and often no differentiation between male and female responses in statistical analysis ( 5,13,14,18 ) . according to national collegiate athletic association ( ncaa ) data , nearly 204,000 females participated in college and university sponsored athletics in 20122013 ( 17 ) . the paucity of research concerning female athletes should be a top priority for sport scientists to address , providing strength and conditioning coaches who work with female athletes evidence - based recommendations on best practices . therefore , the purpose of this study was to investigate the acute pap response of back squats on static squat jump performance among ncaa division ii female athletes . ncaa division ii female collegiate athletes from a midwestern university were recruited for this study . inclusion criteria sought healthy female athletes in the off - season of their respective training macrocycle . subjects with any musculoskeletal injuries or other health problems that would inhibit the ability to jump and/or squat were excluded from the study . female athletes who volunteered for this study ( n=29 , age 19.9 1.0 years ) were current roster members from the university s basketball ( n=11 ) , softball ( n=10 ) , and volleyball ( n=8 ) teams . all subjects signed informed consent documents and completed a physical activity readiness - questionnaire ( par - q ) before participation . this study was approved by the university s institutional review board , and all participants completed the study with no injuries reported . additionally , all testing of athletes was coordinated with the strength and conditioning staff of each team to minimize any potential disruption in off - season workouts . upon reporting for testing , subjects prepared for 1rm testing by performing a light 3-minute bicycle warm - up at a self selected pace . next , subjects performed a 3-minute dynamic warm - up consisting of a series of dynamic stretches spanning 15 meters . following the warm - up , back squat 1rm subjects were instructed to descend to a depth in which the femur was parallel to the ground . a certified strength and conditioning specialist ( cscs ) and experienced spotters were present at all times to ensure safety of subjects and appropriate exercise technique execution . in addition , the spotters visually inspected and confirmed the depth of the squat and provided verbal feedback when needed . upon determination of 1rm , subjects were familiarized with the squat jump technique . a static squat jump ( ssj ) was used to increase consistency and minimize variability of jumping form ( 22 ) . in accordance with proper ssj technique identified by sayers et al . ( 22 ) , subjects assumed a squat position , feet shoulder width apart , knees bent at approximately a 90-degree angle , with arms back and ready to be swung upward ( figure 1 ) . subjects were instructed to refrain from performing any cardiovascular or strength training 24 hours prior to pap testing . subjects prepared for pap testing by performing the same bike and dynamic stretching warm - ups as performed on day 1 in which 1rm was tested . after the 6-minute warm - up , subjects performed 3 ssj s for maximal height , as measured by a vertec ( sports imports , hilliard , oh ) . each subject performed 3 jumps in which the highest jump height was recorded , with 30 seconds of rest separating each jump attempt . the pre - pap jumps served as a control for examining the influence of the resistive loads on jump performance . after the pre - pap vertical jumps , the subjects rested for 3-minutes before performing the following 4-stage squat warm - up : 1 ) 5 repetitions at 50% 1rm , 2 ) 5 reps at 60% 1rm , 3 ) 3 repetitions at 75% 1rm , and 4 ) a final pap inducing set of 3 repetitions at 90% 1rm . after the final pap set at 90% 1rm , each subject rested for 5 minutes while height and body mass were recorded . height was measured to the nearest 0.1 cm using a stadiometer ( seca 213 , chino , ca ) and body mass was measured to the nearest 0.1 kg using a calibrated scale ( befour , saukville , wi ) . once the 5-minute rest period was complete , the subject performed 3 ssj s utilizing the same procedure as the pre - pap jumps . all 3-jump heights were recorded with the highest number being used for subsequent data analysis . maximal jump height was determined by calculating the difference between the highest jump and the subject s reach height . this calculation was used for both pre - pap jump height and post - pap jump height . peak power of each subject s pre - pap highest jump and post - pap highest jump were calculated using the sayers equation in which peak power ( w ) = 60.7 ( jump height [ cm ] ) + 45.3 ( body mass [ kg ] ) 2055 ( 22 ) . a mixed between - within repeated measures anova was performed to determine main effects for time ( pre vs. post power ) and team ( volleyball vs. basketball vs. softball ) , as well the interaction effect ( time team ) . in the presence of significant interaction effects , follow - up contrasts were used to evaluate the simple - effects of time within levels of team . to maintain global = 0.05 , the level of significance was set at p < 0.0167 for each of the 3 simple effects contrasts ( pre vs. post for each team ) . ncaa division ii female collegiate athletes from a midwestern university were recruited for this study . inclusion criteria sought healthy female athletes in the off - season of their respective training macrocycle . subjects with any musculoskeletal injuries or other health problems that would inhibit the ability to jump and/or squat were excluded from the study . female athletes who volunteered for this study ( n=29 , age 19.9 1.0 years ) were current roster members from the university s basketball ( n=11 ) , softball ( n=10 ) , and volleyball ( n=8 ) teams . all subjects signed informed consent documents and completed a physical activity readiness - questionnaire ( par - q ) before participation . this study was approved by the university s institutional review board , and all participants completed the study with no injuries reported . additionally , all testing of athletes was coordinated with the strength and conditioning staff of each team to minimize any potential disruption in off - season workouts . upon reporting for testing , subjects prepared for 1rm testing by performing a light 3-minute bicycle warm - up at a self selected pace . next , subjects performed a 3-minute dynamic warm - up consisting of a series of dynamic stretches spanning 15 meters . following the warm - up , back squat 1rm subjects were instructed to descend to a depth in which the femur was parallel to the ground . a certified strength and conditioning specialist ( cscs ) and experienced spotters were present at all times to ensure safety of subjects and appropriate exercise technique execution . in addition , the spotters visually inspected and confirmed the depth of the squat and provided verbal feedback when needed . upon determination of 1rm , subjects were familiarized with the squat jump technique . a static squat jump ( ssj ) was used to increase consistency and minimize variability of jumping form ( 22 ) . in accordance with proper ssj technique identified by sayers et al . ( 22 ) , subjects assumed a squat position , feet shoulder width apart , knees bent at approximately a 90-degree angle , with arms back and ready to be swung upward ( figure 1 ) . subjects were instructed to refrain from performing any cardiovascular or strength training 24 hours prior to pap testing . subjects prepared for pap testing by performing the same bike and dynamic stretching warm - ups as performed on day 1 in which 1rm was tested . after the 6-minute warm - up , subjects performed 3 ssj s for maximal height , as measured by a vertec ( sports imports , hilliard , oh ) . each subject performed 3 jumps in which the highest jump height was recorded , with 30 seconds of rest separating each jump attempt . the pre - pap jumps served as a control for examining the influence of the resistive loads on jump performance . after the pre - pap vertical jumps , the subjects rested for 3-minutes before performing the following 4-stage squat warm - up : 1 ) 5 repetitions at 50% 1rm , 2 ) 5 reps at 60% 1rm , 3 ) 3 repetitions at 75% 1rm , and 4 ) a final pap inducing set of 3 repetitions at 90% 1rm . after the final pap set at 90% 1rm , each subject rested for 5 minutes while height and body mass were recorded . height was measured to the nearest 0.1 cm using a stadiometer ( seca 213 , chino , ca ) and body mass was measured to the nearest 0.1 kg using a calibrated scale ( befour , saukville , wi ) . once the 5-minute rest period was complete , the subject performed 3 ssj s utilizing the same procedure as the pre - pap jumps . all 3-jump heights were recorded with the highest number being used for subsequent data analysis . maximal jump height was determined by calculating the difference between the highest jump and the subject s reach height . this calculation was used for both pre - pap jump height and post - pap jump height . peak power of each subject s pre - pap highest jump and post - pap highest jump were calculated using the sayers equation in which peak power ( w ) = 60.7 ( jump height [ cm ] ) + 45.3 ( body mass [ kg ] ) 2055 ( 22 ) . a mixed between - within repeated measures anova was performed to determine main effects for time ( pre vs. post power ) and team ( volleyball vs. basketball vs. softball ) , as well the interaction effect ( time team ) . in the presence of significant interaction effects , follow - up contrasts were used to evaluate the simple - effects of time within levels of team . to maintain global = 0.05 , the level of significance was set at p < 0.0167 for each of the 3 simple effects contrasts ( pre vs. post for each team ) . post - activation potentiation results by athletic team are presented in table 2 with all data as mean sd . there was a significant interaction effect observed between time and team , f(2,26)=4.5 , p=.022 . post - hoc tests indicated that the volleyball team had a significant decrease in power output from pre to post , 96.454.5 w , p=.008 . the main effect for time ( pre vs. post power ) was not significant , f(1,26)=1.2 , p=.279 , indicating for the group as a whole , there was no evidence of a pap effect . the main effect for team was also non - significant , f(2,26)=1.8 , p=.173 , indicating no difference in power outputs by team . the purpose of this study was to determine the acute effect of back squats on static squat jump performance in ncaa division ii female athletes . the results of this study found that a pap set of 3 reps at 90% of 1rm followed by a 5-minute rest period did not potentiate subsequent jump height in this cohort of female athletes . whereas a small subset of athletes did successfully potentiate , these differences were not observed for the cohort as a whole , or when examined by each of the three athletic teams . additionally , this study provided descriptive data on absolute strength ( 1rm ~ 92 kg ) and relative strength ( 1rm / body mass ~1.3 ) back squat levels in female athletes , which are noticeably lacking within the strength and conditioning literature . previous studies have found the pap response to be highly individualized ( 5,10,14 ) thus the results of our study clearly mirror this trend . the challenge to consistently elicit the pap response stems from a careful balance of the training status of the athlete , the level of strength attained by the athlete , the intensity of the conditioning activity , and the period of time following the potentiating activity ( 20,23,25 ) . whereas our study consisted of trained female athletes , the absolute and relative strength levels of our subjects may provide clues as to why the majority failed to potentiate . within our study , of the athletes above the 80th percentile for 1rm / body mass ( > 1.44 , n=5 ) , 4 of the 5 did successfully potentiate . previous research provides evidence that stronger athletes have a greater potential to potentiate ( 2,26 ) , which is hypothesized to be due to an ability to become more fatigue resistant as strength levels increase ( 25 ) . additionally , emerging research suggests that individuals who have the strength to squat twice their body mass are able to display both earlier and greater pap responses than weaker individuals ( 19,23 ) . given that our subjects average 1rm / body mass ratio was 1.3 , it is thus very plausible that the combination of low levels of absolute and relative strength , in conjunction with 90% 1rm pap load , and 5-minute rest period , was too fatiguing to successfully potentiate with a protocol commonly used within several previous pap research studies within an athletic population ( 5,23 ) . as previously stated , minimal research exists on female athletes as a whole ( 8) , let alone the pap response , therefore comparisons and potential conclusions are somewhat limited . nonetheless , our results are similar to those of jensen and ebben , in which ncaa division i anaerobic female athletes utilized a pap protocol consisting of 5rm back squats followed by countermovement vertical jumps at 10 sec , 1 min , 2 min , 3 min , and 4 min post - squat jumps , respectively , which resulted in no significant effect on subsequent jump heights or ground reaction forces ( 13 ) . however , our results contrast those of mccann and flanagan , who in a study of ncaa division i female volleyball athletes , found that a pap protocol of 5rm back squats followed by 4 min rest , resulted in significant increases in vertical jump height ( 14 ) . likewise , in a study of national and international hockey and softball female athletes , duthie et al . found a pap protocol of 3rm half squats followed by 5 min rest , resulted in significant increases in both peak power and maximal force for athletes with a 1rm > 139 kg ( 10 ) . are in stark contrast to the female athletes in our study ( 91.8 kg ) , providing additional evidence on the impact of maximum strength on the ability to successfully potentiate . additionally , when comparing ncaa division ii , ncaa division i , and international caliber female athletes , the inherent difference in athleticism , genetic potential , and skill at each level of competition , does warrant serious consideration when contrasting previous findings . first , the subjects in this study were from a select population of healthy , exercise ready , off - season ncaa division ii female athletes . thus , care should be taken if attempting to extrapolate our findings to other populations of athletes . therefore , the results of this study are best generalized to female athletes with a similar strength profile as observed in our subjects . second , this study used a single pap protocol of 90% 1rm followed by a 5 min rest period . in order to create minimum disruption to the athlete s training cycle , the above protocol was deemed acceptable by the university s strength staff when consulting with our research team as per how and when to best gain access to the athletes limited availability . additional manipulation of pap load , rest period , or combination of each , could have certainly resulted in a different temporal pattern of pap responses among our subjects , and is duly noted as a limitation . however , we feel the inclusion of trained female athletes , albeit with limited access , provides a much more valuable understanding to the pap response than had we utilized recreationally trained females . finally , whereas jump height was objectively measured via the use of a vertec , peak power measurements were determined by utilizing the sayers equation , and are thus noted as estimates . when the use of a force plate is impractical , the sayers equation has been cross - validated to be an accurate estimate of peak power for both r and see ( 22 ) . in conclusion , the results of this study found that a pap set consisting of 90% 1rm followed by 5 min rest , failed to potentiate static squat jump performance in a cohort of ncaa division ii female athletes . strength and conditioning coaches who work with female athletes should be aware that the successful implementation of pap complexes within this population appears to be highly individualized . therefore , the use of pap complexes in female athletes should consider both the relative strength of each athlete and the length of the rest period when attempting to optimize the pap response . based on the findings of our study , we recommend that future research that investigates the pap response in female athletes to consider the following : 1 ) the absolute strength of the athlete , 2 ) the relative strength of the athlete , 3 ) the level of competition that the athlete is at , and 4 ) for athletes who are unable to squat twice their body mass , the utility of pursuing such pap complexes .

post - activation potentiation ( pap ) is a phenomenon in which the power output of a muscle is immediately enhanced after heavy resistance exercise . whereas the majority of pap research has examined males , minimal research exists as to how female athletes respond . therefore , the purpose of this study was to investigate the acute pap response of back squats on static squat jump ( ssj ) performance among ncaa division ii female athletes . female athletes ( n=29 ) who were current roster members from basketball , softball , and volleyball , performed 3 ssj prior to 3 repetitions of the back squat exercise at 90% 1rm . after a 5-min rest , athletes once again performed 3 ssj for maximal height , with peak power calculated using the sayers equation . there was a significant interaction effect observed between time and team , p=0.022 ; post - hoc tests indicated that the volleyball team had a significant decrease in power , p=0.008 . the main effect for time was not significant , p=0.279 , indicating for the group as a whole , there was no evidence of a pap response . the main effect for team was also non - significant , p=0.173 , indicating no difference in power outputs by team . strength and conditioning coaches who work with female athletes should be aware that the successful implementation of pap complexes within this population appears to be highly individualized . therefore , the use of pap complexes in female athletes should consider both the absolute and relative strength of each athlete in conjunction with the length of the rest period when attempting to optimize the pap response .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC4490316

5-amp - activated protein kinase ( ampk ) , a key regulator of cellular energy homeostasis , is activated in response to various metabolic stresses including starvation , hypoxia , and ischemia . ampk is a heterotrimeric enzyme , which is activated by phosphorylation of thr172 on its subunit . several upstream kinases can modify thr172 , including liver kinase b1 ( lkb1 ) , calcium / calmodulin - dependent protein kinase kinase 2 ( camkk2 ) , and transforming growth factor activated kinase-1 ( fig . master switch that regulates intracellular processes such as the cellular uptake of glucose , -oxidation of fatty acids , biogenesis of glucose transporter 4 , and mitochondrial biogenesis [ 1 - 4 ] . upon activation , ampk increases cellular energy levels by inhibiting anabolic energy - consuming pathways ( including fatty acid synthesis and protein synthesis ) and stimulating energy - producing catabolic pathways ( including fatty acid oxidation and glucose transport ) ( fig . 2 ) . because it is a major regulator of both lipid and glucose metabolism , ampk has been considered as a potential therapeutic target for the treatment of diabetes , obesity , and cancer . recently , however , many reports show that ampk actually plays a critical role in tumor survival and growth [ 5 - 8 ] . the metabolism reprogramming triggered by the activation of ampk is important for the survival of cells in the tumor microenvironment . thus , it would not be surprising if , in some cases , ampk acts as a tumor promoter . in contrast to normal cells , which produce energy through a relatively low rate of glycolysis and oxidation of pyruvate in the mitochondria , the energy production of most cancer cells relies on a high rate of glycolysis followed by lactic acid fermentation in the cytosol ; this phenomenon is known as the warburg effect [ 9 - 11 ] . rapidly growing cancer cells achieve glycolytic rates that are up to 200 folds higher than those of normal cells , even when oxygen is plentiful . the warburg effect may reflect adaptation to low - oxygen environments within tumors , mitochondrial damage , or oncogene - mediated shutdown of mitochondria , which would otherwise induce apoptosis in cancerous cells . on the other hand , the effect might also be associated with rapid cell proliferation . because glycolysis provides most of the building blocks required for cell proliferation , cancer cells may need to activate glycolysis despite the availability of oxygen ( fig . 3 ) . in several types of cancer , genetic defects in the enzymes of the tricarboxylic acid cycle are involved in tumorigenesis , whereas oncogenic h - ras transformation damages mitochondrial function . upon initiation of the warburg effect , cells depend on aerobic glycolysis with lactate production , rather than oxidative phosphorylation , to maintain adenosine triphosphate ( atp ) levels . pfkfb3 is overexpressed and its phosphorylation is elevated in several types of cancer ( including colon and breast cancers ) as a consequence of ampk activation . various stress signals , such as hypoxia and nutrient depletion , can drive pfkfb3 expression , and ampk contributes to the elevation of pfkfb3 levels and glycolytic flux in response to reduced ph . in addition , expression of pfkfb2 , a phosphorylation target of both akt and ampk , is elevated in human cancers . ampk can act as a negative regulator of the warburg effect under nutrient- and growth factor - rich conditions . ampk depletion further increases mammalian target of rapamycin complex 1 ( mtorc1 ) activation , hypoxia - inducible factor 1 ( hif1 ) levels , and aerobic glycolysis in myc over - expressing cells . depletion of ampk leads to increased lactate production , promoting the use of nonglucose carbon sources for anabolism , maintenance of bioenergetics , and lactate production . the role of lactate production in proliferating tumor cells , the primary characteristic of the warburg effect , remains poorly understood . the key glycolysis enzyme phosphofructokinase-1 is inhibited by o - glcnacylation , which is necessary for optimum tumor growth , suggesting that suppression of lactate - producing glycolysis can also confer survival and growth advantages on cancer cells , depending on the genetic background and cellular context . although the ampk signaling pathway was originally characterized as a tumor - suppressive pathway , recent studies have demonstrated that many factors in the tumor microenvironment , as well as some oncogenic signals , are ampk activators . the oncogene src ( proto - oncogene tyrosine - protein kinase ) , which is overexpressed and activated in many cancers , activates ampk through the activation of the protein kinase c -lkb1 pathway in ovcar3 and a431 cells . similarly , the expression of myc and h - ras activates ampk in some cancers . moreover , androgen receptor ( ar ) signaling activates ampk through the transactivation of camkk2 in prostate cancer [ 22 - 24 ] . in addition to the activation of oncogenes , the loss of tumor suppressor gene folliculin ( flcn ) , which is associated with birt - hogg - dub syndrome , also activates ampk . initially , two groups reported that lkb1 and ampk play protumorigenic roles in the context of oncogene - induced transformation of mouse embryonic fibroblasts ( mefs ) . they showed that both lkb1-null mefs and ampk1/2-null ( ampk knockout ) mefs are resistant to oncogenic effects such as h - ras / sv40t - induced anchorage - independent growth and solid tumor growth in vivo . remarkably , several studies demonstrated that ampk is also essential for other types of oncogene - induced tumorigenesis , such as mycinduced development of hepatocellular carcinoma ( hcc ) , h - raspten deletioninduced astrocytic tumor cell proliferation , and kinase suppressor of ras 2-induced anoikis resistance in mefs . depletion of ampk activity in breast and pancreatic cancer cells hampers anchorage - independent growth and tumor formation in vivo [ 30 - 32 ] . the lkb1-ampk signaling pathway is also essential for glioma cell survival under low - glucose conditions . two recent approaches have identified the ampk1 and ampk1 subunits as essential protumorigenic genes in melanoma and prostate cancer , respectively . activation of ampk following loss of the tumor suppressor flcn increases mitochondrial biogenesis by inducing peroxisome proliferator - activated receptor ( ppar ) coactivator 1 ( pgc1 ) expression , which in turn results in the production of reactive oxygen species ( ros ) . . also demonstrated that high levels of ros activate hif1 transcriptional activity , resulting in elevated expression of glycolytic enzymes , which induce atp production . the ar - camkk2-ampk axis in prostate cancer increases aerobic glycolysis and anabolic metabolism partly through pgc1-mediated mitochondrial biogenesis , which can provide both the energy and the building blocks required for rapid cell growth . recently , mechanisms associated with the metabolic functions of ampk , other than those involved in the production of energy or building blocks , have attracted increasing attention . jeon et al . showed that in the absence of ampk activity , oxidative stress is the major cause of cancer cell death during glucose deprivation and matrix detachment . under such stressful metabolic conditions , maintenance of nadph levels rather than atp levels is crucial for ampk - induced solid tumor formation in vivo , tumor cell survival , and anchorage - independent growth . acetyl - coa carboxylase ( acc ) 1 and acc2 decrease nadph consumption by inhibiting fatty acid synthesis and increase nadph production by activation of fatty acid oxidation , respectively . they both need to be inhibited to maintain the glutathione / oxidized glutathione ratio and to reduce h2o2 levels , thereby protecting against oxidative stress - induced cell death . in osteosarcoma loss of flcn results in the constitutive activation of ampk , which in turn induces autophagy , inhibits apoptosis , improves cellular bioenergetics , and confers resistance to energy - depleting stresses such as oxidative stress , heat , anoxia , and serum deprivation . despite accumulating evidence highlighting the oncogenic role of ampk , this enzyme was originally identified as a tumor suppressor following the discovery of lkb1 and mtorc1 [ 38 - 41 ] . germline mutations in the gene encoding ampk were detected in peutz - jeghers syndrome . additionally , somatic mutations in the tumor suppressor lkb1 were observed in lung and cervical cancers . mtorc1 , which is inactivated by the lkb1-ampk axis , is an important regulator of growth factor and nutrient signaling that controls protein synthesis and cell growth , and is over - activated in most human cancers . since lkb1 activates ampk and ampk inhibits mtorc1 , it is reasonable to classify ampk as a tumor suppressor . although lkb1 deletion alone is not sufficient to cause lung cancer , it elevates mtorc1 and src activity , which enhances lung cancer induced by k - ras activation or pten deletion [ 44 - 46 ] . conversely , in the endometrium , lkb1 deletion alone is sufficient for the development of invasive endometrial cancer , which can be effectively treated by rapamycin , an mtorc1 inhibitor , suggesting that mtorc1 activation is the major downstream target of lkb1 inhibition [ 48 - 50 ] . notably , lkb1 also plays an ampk / mtorc1-independent role in tumor suppression that depends on microtubule affinity - regulating kinase - mediated regulation of snail or hippo signaling . deletion of prkaa1 , the gene that encodes ampk1 , accelerates development of myc - induced lymphoma . this observation contradicts the findings of liu et al . , who showed that ampk plays an important role in the development of myc - induced hcc . . also demonstrated that deletion of either lkb1 or prkaa1 induces mtorc1/hif1-dependent metabolic reprogramming that helps cancer cells meet their energetic and anabolic demands . this observation also conflicts with the findings of yan et al . , who suggested that ampk activates aerobic glycolysis through the pgc1-ros - hif1 axis in the absence of flcn . although this discrepancy could be due to tissue - specific and context - dependent functions of ampk , further research is needed to obtain an unequivocal interpretation of these seemingly contradictory findings . several additional studies have reported conflicting pharmacological evidence regarding the role of ampk in cancer . the antidiabetic drug metformin , which indirectly activates ampk by inhibiting mitochondrial metabolism , decreases the incidence and mortality rate of breast cancer . moreover , several lines of evidence support the idea that ampk activators , such as 5-aminoimidazole-4-carboxamide-1-b - d - ribofuranoside ( aicar ) , metformin , and phenformin , suppress cancer cell growth and proliferation [ 56 - 59 ] . however , in contradiction to these findings , liu et al . showed that aicar and metformin reduce glioblastoma proliferation and viability . furthermore , the direct ampk activator a769662 has no effect on proliferation , suggesting that the antitumor effects of aicar and metformin are ampk - independent . shackelford et al . also demonstrated that phenformin selectively kills lkb1-deficient lung tumor cells in vivo , suggesting that the antitumor effect of phenformin is not dependent on activation of ampk . notably , vincent et al . showed that all these nonspecific and indirect ampk activators strongly inhibit cell proliferation and viability in the absence of ampk , whereas such cytotoxic effects are reduced in the presence of ampk , suggesting that the activation of ampk actually opposes the cytotoxic effect of the indirect ampk activators . along these lines , vincent et al . demonstrated that the direct ampk agonist a769662 promoted cell proliferation under metabolically stressful conditions , supporting the idea that ampk plays an oncogenic role in the tumor microenvironment . therefore , we must consider the effects of organ , cancer type , and acute vs. long - term ampk activation and inactivation when investigating the mechanistic contribution of ampk to cancer [ 63 - 65 ] . non muscle - invasive bladder cancer ( nmibc ) and muscle - invasive bladder cancer ( mibc ) are associated with distinct molecular pathways . nmibc is characterized by activation of the ras pathway through mutations in h - ras , fgfr-3 , and pi3k , while mibc is associated with a loss of tumor suppressor genes , including p53 , rb , and pten [ 66 - 72 ] . activation of the ras pathway occurs in approximately 80% of all nmibcs , whereas more than 50% of mibcs exhibit loss of p53 function [ 70 - 72 ] . importantly , there is crosstalk between these pathways and the mtor pathway , which means that alterations in either pathway are predicted to influence mtor activity . reported that intravesical rapamycin instillation exerts striking inhibitory effects on tumor progression in a bladder - specific pten and p53 double knockout transgenic mouse model , which develops carcinoma in situ lesions that progress to mibc . in addition to the h - ras , p53 , and pten pathways , diverse upstream signals such as ampk and insulin can affect the mtor pathway . although no direct evidence about the role of the ampk pathway in bladder cancer is currently available , several studies support the idea that the ampk pathway might influence both bladder cancer development and progression . liu et al . reported that rhodiola rosea extract and salidroside inhibit the mtor pathway and translational initiation via activation of ampk in umuc-3 bladder cancer cells . metformin inhibits the growth of bladder cancer cells via indirect activation of ampk , which in turn suppresses the mtor / p70 s6 kinase-1 ( s6k1 ) pathway in 253j and rt4 bladder cancer cell lines . . demonstrated that troglitazone ( a synthetic ligand of ppar ) activates autophagy concurrent with the activation of ampk and suppression of the mtor signaling pathway in t24 cells . activation of mtor occurs via a multistep process including upstream pi3k and ras activation , or inactivation of ampk , leading to phosphorylation and inactivation of the tuberous sclerosis complex 1 and 2 ( tsc1/tsc2 ) heterodimer . inactivation of tsc1/tsc2 results in release of rheb inhibition and subsequent mtor activation through rheb - gtpase activity . finally , mtor activity regulates the effects of a number of downstream molecules , including s6k and elongation - initiation factor 4e binding protein-1 . in mibc , pten loss appears to correlate with an increased mtor activity , suggesting that it exerts a direct effect on downstream signaling components in bladder cancer . thirty - nine percent of mibc cases exhibit either loss - of - heterozygosity ( loh ) or homozygous deletion of the pten locus , and occasionally , mutations in the pten coding region occur . additionally , loss of p53 function appears to synergize with pten loss to promote activation of the mtor signaling pathway . finally , loh of tsc1 has been reported in approximately 50% of bladder cancer cases . taken together , the deregulation of components upstream of the mtor pathway may occur in bladder tumors , suggesting that mtor signaling may be elevated in this type of cancer . the results of our analysis revealed that patients with bladder cancer have elevated levels of urinary acetyl - coa and carnitine . because carnitine is crucial for the entry of fatty acids into the mitochondria for oxidation , and acetyl - coa is the final product of this oxidation event , we speculate that -oxidation of fatty acids might be an important factor in the development of bladder cancer . when we examined the gene expression levels of the enzymes involved in fatty acid oxidation using our published microarray data , we found that bladder cancer specimens expressed significantly higher levels of carnitine palmitoyl transferase 1a ( cpt1a ) than normal bladder mucosae . cpt is a key enzyme that uses carnitine to transfer fatty acids to the mitochondria for oxidation . in particular , our microarray data also showed that bladder cancers express carnitine acylcarnitine translocase - like protein ( cacl ) , another enzyme involved in fatty acid transport to mitochondria , at higher levels than normal bladder mucosae . although cacl has not been studied as extensively as cpt1a , strategies that target it may also have therapeutic potential in bladder cancer . thus , in line with other studies implicating fatty acid oxidation in various types of tumorigenesis , our metabolomics study and microarray analysis indicated that -oxidation of fatty acid plays an important role in bladder tumorigenesis and aggressiveness . the level of acetyl - coa , another molecule associated with -oxidation , can be influenced by input from pyruvate through the pyruvate dehydrogenase complex . we also found that the level of a third component of the complex , dihydrolipoyl dehydrogenase , is significantly reduced in bladder cancer . this observation suggests that the higher acetyl - coa levels in bladder cancer are largely due to elevated -oxidation , rather than conversion from pyruvate . our speculation is concordant with the warburg effect in cancer cells , in which pyruvate is converted to lactate rather than acetyl - coa . although the ampk - fatty acid -oxidation pathway has not been completely characterized in the context of bladder cancer , fatty acid -oxidation is mainly controlled by the ampk pathway , suggesting that ampk acts as an oncogenic regulator of bladder cancer . the pathways related to ampk - fatty acid -oxidation that are putatively affected by bladder cancer , based on data from our two studies , are summarized in fig . 4 . several lines of evidence suggest that activation of the mtor pathway and stimulation of the fatty acid -oxidation pathway might be important in the development of bladder cancer . , no study to date has described the connection between the ampk , mtor , and -oxidation pathways . additional research into the missing link between mtor and -oxidation of fatty acids following ampk activation may reveal novel mechanisms underlying the roles of ampk in bladder cancer . although ampk activation ( resulting in torc1 inhibition ) is considered to be a reasonable anticancer tactic , ampk may in fact exert very complex effects in tumor cells . the antidiabetic drug metformin , which inhibits mitochondrial metabolism , inhibits mtorc1 in both an ampk - dependent and ampk - independent manner . compound c , a selective ampk inhibitor , binds to the ampk subunit and acts as an atp - competitive inhibitor . however , several studies have demonstrated that compound c can also inhibit many other kinases as well as the bone morphogenetic protein receptor , and this promiscuity raises doubts about whether its effects are mediated by ampk inhibition . sunitinib , a multiple tyrosine kinase inhibitor that is used clinically against advanced clear - cell renal cell carcinoma , can directly inhibit ampk by binding the ampk subunit . table 1 summarizes how ampk activity can be affected not only in vitro , but also in growing cells , organs , or whole organisms . these data were collected from several publications describing the molecular mechanisms and tissue - specific effects of ampk activity . currently , several lines of evidence suggest that ampk could act as a tumor promoter rather than tumor suppressor . notably , secondary activation of ampk by many metabolic inhibitors actually induces resistance to apoptosis , suggesting that inhibition of ampk with concurrent induction of metabolic stress represents a more reasonable anticancer therapeutic strategy than activating ampk . such an approach might provide an effective way to investigate the synergistic anticancer effects of combination therapy using current treatment modalities in conjunction with the relevant ampk inhibitors . although ampk was originally considered a tumor suppressor that acted via mtorc1 inhibition , today the consensus is that ampk might in fact be a tumor promoter . this suggests that inhibition of ampk is a more reasonable strategy for treating cancers than activating the enzyme . although activation of the mtor pathway and elevated fatty acid -oxidation are closely associated with the ampk pathway , no publication has described the connection between the ampk , mtor , and -oxidation pathways . therefore , further research aimed at identifying the missing link between mtor and -oxidation of fatty acid following ampk activation will provide better insight into the role of ampk in bladder cancer .

the 5-amp - activated protein kinase ( ampk ) is a key regulator of cellular metabolism and energy homeostasis in mammalian tissues . metabolic adaptation is a critical step in ensuring cell survival during metabolic stress . because of its critical role in the regulation of glucose homeostasis and carbohydrate , lipid , and protein metabolism , ampk is involved in many human diseases , including cancers . although ampk signaling was originally characterized as a tumor - suppressive signaling pathway , several lines of evidence suggest that ampk plays a much broader role and can not simply be defined as either an oncogenic regulator or tumor suppressor . notably , several recent studies demonstrated that the antitumorigenic effects of many indirect ampk activators , such as metformin , do not depend on ampk . conversely , activation of ampk induces the progression of cancers , emphasizing its oncogenic effect . bladder cancer can be divided into two groups : non muscle - invasive bladder cancer ( nmibc ) and muscle - invasive bladder cancer ( mibc ) . the molecular mechanisms underlying these two types of cancer are distinct : nmibc is associated with activation of the ras pathway , whereas mibc is characterized by loss of major tumor suppressors . importantly , both pathways are connected to the mammalian target of rapamycin ( mtor ) pathway . in addition , our recent metabolomic findings suggest that -oxidation of fatty acids is an important factor in the development of bladder cancer . both mtor and -oxidation are tightly associated with the ampk pathway . here , i summarize and discuss the recent findings on the two distinct roles of ampk in cancer , as well as the relationship between bladder cancer and ampk .

INTRODUCTION AMPK AND THE WARBURG EFFECT AMPK: AN ONCOGENIC REGULATOR? AMPK: A TUMOR SUPPRESSOR? REGULATION OF AMPK IN BLADDER CANCER AMPK AS A THERAPEUTIC TARGET FOR CANCER CONCLUSIONS

PMC3977414

hiv / aids has been recognized as one of the most important public health problems in the recent past , especially in asian countries such as malaysia , china , thailand , and iran . according to the world health organization 's ( who 's ) report of hiv , iran is categorized as one among the countries with gradually accumulating levels of infection . preventing hiv infection is one of the top priorities in 2010 to lead a healthy life . three transmission ways have been identified for the disease : sexual activity with an infected person , contact with infected blood through sharing injection needles , and mother - to - infant infection . in order to prevent hiv transmission through sexual activities , which accounts for 85% of the disease , the three following options have been recommended : abstinence , faithfulness , and condom use , among which the first one is the safest . due to the lack of global access to aids treatment and also based on the fact that hiv / aids transmission requires a behavioral process , behavioral change interventions are the best approach to prevent the spread of the disease . since most health problems are closely related to human behavior , behavioral theories and models can provide insights into finding ways to prevent health problems such as hiv / aids . most of the studies conducted have been based on cognitive - behavioral , health belief models , and theories such as social cognitive theory ( sct ) and theory of reasoned action ( tra ) . considering the results , and in order to increase the efficiency of the models , a selective mixture of aforementioned theories has been employed in the present study . in his survey of aids - behavior change theories , noar ( 2007 ) mentioned 13 theories used in the studies , including belief- and intervention - based , message - driven , and aids - related theories , and other common hygienic behavior changes . some researchers used a questionnaire based on behavior change theories or models such as motivation - behavioral skills model ( imb ) , social cognitive theory ( sct ) , and the theory of reasoned action ( tra ) . furthermore , the constructs such as knowledge , attitude , self - efficacy ( se ) , behavioral intention ( bi ) , and high - risk sexual behavior were used in some studies . therefore , given the cultural differences between the western countries and iran and the sensitive nature of the disease , the hiv / aids behavior change questionnaires derived from the previous studies conducted in non - islamic countries can not be applied here . although the validity and reliability of the questionnaire has been confirmed in a previous study , this study was conducted only with male students . however , to our knowledge , no study has been carried out with female students so far . as females , in comparison with males , are physiologically more vulnerable to hiv / aids , female students were selected as the target group . considering the lack of appropriate tools and questionnaires , the study focused on designing and validating a culturally suitable sexual abstinence questionnaire among unmarried iranian female students . the study was cross - sectional in design and was conducted from february to june 2010 . the criteria based on which the participants were selected were as follows : age range of 15 - 25 years , single status , living alone , and residing in tehran for at least 1 year at the time of the study . those with physical disability and those reluctant to participate in the study were excluded . for achieving an appropriate score maximum fitness and predicting the number of students , the participants were randomly selected from the three areas of humanities , engineering , and science at the university of tehran . then , the participants were selected through cluster systematic sampling ( with each class as one cluster ) . for each question , a minimum of 10 samples were used according to munro 's method , and 348 female students were randomly selected to reach construct validity . descriptive ( mean , standard deviation , and cronbach 's alpha ) and inductive statistics ( exploratory and confirmatory factor analysis ) functions of spss and amos ver . 16 were used to analyze the data . a self - administered questionnaire was used which comprised the following four parts : socio - demographic characteristics , se regarding sexual abstinence , perceived benefits ( pb ) toward sexual abstinence , and behavioral intention ( bi ) . the demographic information included age , residency region , family income , parents education , and previous knowledge about hiv . there were four se questions about individual restraint , avoidance of risky situations , and the behavioral skills of saying no . for unifying the results and facilitating analysis , the questions were designed with five options ranging from strongly agree to strongly disagree . for each item , a typical 5-point likert scale ranging from strongly disagree ( 1 ) to strongly agree ( 5 ) was used . the scale of pb consisted of six items measuring individual and social benefits of abstinence , risky behavior avoidance , negative answer to risky offers , and high - risk situation avoidance . furthermore , one question with regard to the features of female students in the studied society was added to the aforementioned questions . bi questions consisted of four items based on the dual behavior theory of gibbons and gerrard and a domestic questionnaire designed by ghaffari et al . these questions were about the bi of abstinence , a negative answer to risky advances , and avoidance of highly risky situations . also , given the fact that the participants were young and prone to highly risky situations , one of the questions required them to give their own opinions about their willingness to avoid high - risk situations . descriptive statistics , cronbach 's alpha , and exploratory and confirmatory factor analysis functions of spss and amos ver . the study was approved by the ethics committee of tehran university and ethical principles were adhered to throughout the study . after the researchers explained the purpose and procedures of the study to the participants , they consented to participate in the study . the study was cross - sectional in design and was conducted from february to june 2010 . the criteria based on which the participants were selected were as follows : age range of 15 - 25 years , single status , living alone , and residing in tehran for at least 1 year at the time of the study . those with physical disability and those reluctant to participate in the study were excluded . for achieving an appropriate score maximum fitness and predicting the number of students , the participants were randomly selected from the three areas of humanities , engineering , and science at the university of tehran . then , the participants were selected through cluster systematic sampling ( with each class as one cluster ) . for each question , a minimum of 10 samples were used according to munro 's method , and 348 female students were randomly selected to reach construct validity . descriptive ( mean , standard deviation , and cronbach 's alpha ) and inductive statistics ( exploratory and confirmatory factor analysis ) functions of spss and amos ver . 16 were used to analyze the data . a self - administered questionnaire was used which comprised the following four parts : socio - demographic characteristics , se regarding sexual abstinence , perceived benefits ( pb ) toward sexual abstinence , and behavioral intention ( bi ) . the demographic information included age , residency region , family income , parents education , and previous knowledge about hiv . there were four se questions about individual restraint , avoidance of risky situations , and the behavioral skills of saying no . these questions were also designed according to the iranian standardized general se questionnaire . for unifying the results and facilitating analysis , the questions were designed with five options ranging from strongly agree to strongly disagree . for each item , a typical 5-point likert scale ranging from strongly disagree ( 1 ) to strongly agree ( 5 ) was used . the scale of pb consisted of six items measuring individual and social benefits of abstinence , risky behavior avoidance , negative answer to risky offers , and high - risk situation avoidance . furthermore , one question with regard to the features of female students in the studied society was added to the aforementioned questions . bi questions consisted of four items based on the dual behavior theory of gibbons and gerrard and a domestic questionnaire designed by ghaffari et al . these questions were about the bi of abstinence , a negative answer to risky advances , and avoidance of highly risky situations . also , given the fact that the participants were young and prone to highly risky situations , one of the questions required them to give their own opinions about their willingness to avoid high - risk situations . descriptive statistics , cronbach 's alpha , and exploratory and confirmatory factor analysis functions of spss and amos ver . the study was approved by the ethics committee of tehran university and ethical principles were adhered to throughout the study . after the researchers explained the purpose and procedures of the study to the participants , they consented to participate in the study . in order to determine the face validity , the questionnaire was distributed to 30 students with similar characteristics . they were asked to pinpoint the weaknesses and ambiguities of the questionnaire , comment on its clarity , rationality , brevity , and appropriateness , and finally , improve it . content validity ratio ( cvr ) was applied to assess the extent of experts comment upon the clarity , rationality , brevity and appropriateness of itthen , the questionnaire was given to 20 specialists in the fields of health education , nursing , medicine , and psychology to take the experts ideas into account . content validity was assessed by each panel member with a 3-item likert scale ( the items were necessary , useful but not necessary , and unnecessary ) . in case the number of questions was reduced to 50 after measuring the cvr . in order to verify the reliability of the questionnaire , cronbach 's alpha or the internal consistency for pb , bi , and se variables was found to be 0.87 , 0.77 , and 0.85 , respectively . all these values indicate the desirable level of the scales . in order to determine the construct validity of the questionnaire , one way of investigating construct validity is by running a factor analysis for identifying clusters of questions related to the instruments . this method helps the researcher to determine whether the available tool measures one construct or several ones . the exploratory factor analysis using principal components analysis with varimax rotation was used to determine compliance and naming of the extracted factors . using all observations ( n = 348 ) , the factor analysis helped in identifying three decisive factors with a variance greater than 61% and 88% of kaiser meyer olkin ( kmo ) , both of which are appropriate indicators of factor analysis . after inserting three retention factors in the orthogonal varimax rotation , each of the factors was given a name and estimated using the probability method . based on the loadings , and also the content of questions , the three factors were identified as se , bi , and pb , respectively . as presented in the chart , only the first three values have eigen values more than one . six pb questions were omitted , and four se and four bi questions were used in the model ; at the final stage and after determining factor analysis , one se question was deleted . the results of the exploratory factor analysis with varimax rotation after running exploratory factor analysis , in order to confirm the supposed factor structure of the measure as well as the contribution of each question in measuring the components of self - efficacy , bi and pb were analyzed using amos software . the insignificance of the statistics indicated the model 's fitness with the data , but the pitfall of the statistics is that it is sensitive to the sample size , which means that with larger sample sizes , the possibility of the statistics being insignificant t decreases . values less than 0.05 for the index root mean square error of approximation ( rmsea ) , values above 0.9 for goodness of fitness index ( gfi ) , and adjusted goodness of fitness index ( agfi ) were used as the criteria for model compliance with the observed data [ table 3 ] . as it was stated , the model 's of the goodness of fitness indexes are all acceptable . therefore , the confirmatory factor analysis also approved the construct validity of the questionnaire [ figure 1 ] . structural equation fitness to model on the whole , 348 female students participated in the study the characteristics of the study sample goodness fitness indexes of the sexual abstinence behavior of hiv / aids questionnaire the average of cvr for the entire questionnaire was 0.85 . the cvr for the subscales of pb , bi , and se was calculated to be 0.83 , 0.84 , and 0.89 , respectively . the time needed for completing the questionnaire was about 6 min . in order to verify the reliability of the questionnaire , cronbach 's alpha score was calculated . cronbach 's alpha or the internal consistency for pb , bi , and se variables was found to be 0.87 , 0.77 , and 0.85 , respectively . all these values indicate the desirable level of the scales . in order to determine the construct validity of the questionnaire , one way of investigating construct validity is by running a factor analysis for identifying clusters of questions related to the instruments . this method helps the researcher to determine whether the available tool measures one construct or several ones . the exploratory factor analysis using principal components analysis with varimax rotation was used to determine compliance and naming of the extracted factors . using all observations ( n = 348 ) , the factor analysis helped in identifying three decisive factors with a variance greater than 61% and 88% of kaiser meyer olkin ( kmo ) , both of which are appropriate indicators of factor analysis . after inserting three retention factors in the orthogonal varimax rotation , each of the factors was given a name and estimated using the probability method . based on the loadings , and also the content of questions , the three factors were identified as se , bi , and pb , respectively . as presented in the chart , only the first three values have eigen values more than one . six pb questions were omitted , and four se and four bi questions were used in the model ; at the final stage and after determining factor analysis , one se question was deleted . the results of the exploratory factor analysis with varimax rotation after running exploratory factor analysis , in order to confirm the supposed factor structure of the measure as well as the contribution of each question in measuring the components of self - efficacy , bi and pb were analyzed using amos software . the insignificance of the statistics indicated the model 's fitness with the data , but the pitfall of the statistics is that it is sensitive to the sample size , which means that with larger sample sizes , the possibility of the statistics being insignificant t decreases . values less than 0.05 for the index root mean square error of approximation ( rmsea ) , values above 0.9 for goodness of fitness index ( gfi ) , and adjusted goodness of fitness index ( agfi ) were used as the criteria for model compliance with the observed data [ table 3 ] . as it was stated , the model 's of the goodness of fitness indexes are all acceptable . therefore , the confirmatory factor analysis also approved the construct validity of the questionnaire [ figure 1 ] . the characteristics of the study sample goodness fitness indexes of the sexual abstinence behavior of hiv / aids questionnaire the average of cvr for the entire questionnaire was 0.85 . the cvr for the subscales of pb , bi , and se was calculated to be 0.83 , 0.84 , and 0.89 , respectively . the time needed for completing the questionnaire was about 6 min . considering that there is lack of valid and reliable questionnaire on the behavioral changes of hiv / aids among iranian females , we aimed to design an appropriate abstinence questionnaire and determine its validity and reliability . this point has been taken into account to a lesser extent in the questionnaires developed in the western countries . for example , some iranians believe that avoiding unsafe sexual encounters would give them better chances of getting married in the future . other studies on the validity and reliability of domestic questionnaires have mainly investigated the knowledge of people and their behaviors and attitudes toward hiv , and reported the reliability of the scales . for instance , in a study by khatoni et al . , face - to - face web - based training was employed to verify the content validity of nurses knowledge questionnaire by the experts , and also , the reliability of the questionnaire was confirmed through test retest ( 0.9 ) . furthermore , the cronbach 's alpha for the hiv / aids knowledge and attitude questionnaire developed by diclemente et al . was reported to be 0.72 . in the study conducted by nije - carr on hiv / aids knowledge , attitudes , and beliefs patient questionnaire ( hakabpq ) , also , the internal consistency of hiv - related knowledge , attitudes , and sexual risk - taking behaviors was reported to be 0.073 in the study by ugarte et al . in the study of koopman et al . ( 1990 ) , both the knowledge and belief instruments were indicated to have a high internal consistency , test retest reliability , and successfully avoided ceiling effects . the highest reported figure was 0.96 for the validity of st andrews sexual knowledge and attitudes instrument ( saskai ) , which was conducted among female employees by kappa test . of course , the high internal consistency is a result of high level of similarity among the questions . in the study of hughes and admiraal entitled systematic review of hiv / aids knowledge measures , the results showed low reliability and a validity rate of hiv knowledge questionnaire . in general , the measuring instruments of knowledge , attitudes , and beliefs about hiv do not have a high level of internal consistency and have rarely been tested by factor analysis . lux and petoza designed a questionnaire based on which the cronbach 's alpha was estimated to be 0.64 and 0.79 for healthy sexual bi and se of sexual discussion , respectively . in another study conducted by mahoney thombs and ford , the cronbach 's alpha se subscale for using condoms the scale of mukoma et al . , which was applied to evaluate school - based hiv / aids intervention in south african courtiers and tanzania , reported a cronbach 's alpha of higher than 0.5 . regarding sexual behaviors , the cronbach 's alpha was lower than 0.5 , based on which the author verified the utility of the scales . in a similar study conducted by ghafari et al . , the alpha values for a 10-scale instrument about hiv / aids prevention were 0.69 , 0.78 , and 0.74 for se , pb , and bi , respectively , which were lower than those of the present instrument . it is worth noting that the alpha values between 0.8 and 0.9 show a high internal consistency and the alpha values of 0.7 and higher present a good internal stability . in the present study , the cronbach 's alpha was between 0.77 and 0.87 , indicating a good internal correlation of the instrument . also , the reliability coefficient of the scale was approximately 0.85 , which is satisfactory when seen in the light of the studies listed . there were three main measures of sexual behaviors : abstinence , number of sexual partners , and condom use , which were measured in some articles , the psychological measures of sexual abstinence instrument have not been mentioned . one of the strengths of the study was using different methods of testing reliability and validity and exploratory and confirmatory factor analysis . it should be noted that the questionnaire was optimal in terms of structure validity and scale clustering and the goodness of fitness indexes . other researchers are suggested to work on the construction of theories of behavior change scale in islamic contexts as well as to research into different groups of adolescents , youth , and occupations with the risk of hiv / aids infection . this study have showed that the sexual behavioral abstinence , and avoidance of high - risk situation questionnaire ( sbahaq ) questionnaire can be used as a valid and reliable tool to measure abstinence and avoidance of high - risk situations regarding hiv / aids infection . however , it is recommended to test the validity of the tool in various communities such as students , scholars , etc .

background : this study was designed to assess the validity and reliability of the designed sexual , behavioral abstinence , and avoidance of high - risk situation questionnaire ( sbahaq ) , with an aim to construct an appropriate development tool in the iranian population.materials and methods : a descriptive analytic study was conducted among female undergraduate students of tehran university , who were selected through cluster random sampling . after reviewing the questionnaires and investigating face and content validity , internal consistency of the questionnaire was assessed by cronbach 's alpha . explanatory and confirmatory factor analysis was conducted using spss and amos 16 software , respectively.results:the sample consisted of 348 female university students with a mean age of 20.69 1.63 years . the content validity ratio ( cvr ) coefficient was 0.85 and the reliability of each section of the questionnaire was as follows : perceived benefit ( pb ; 0.87 ) , behavioral intention ( bi ; 0.77 ) , and self - efficacy ( se ; 0.85 ) ( cronbach 's alpha totally was 0.83 ) . explanatory factor analysis showed three factors , including se , pb , and bi , with the total variance of 61% and kaiser meyer olkin ( kmo ) index of 88% . these factors were also confirmed by confirmatory factor analysis [ adjusted goodness of fitness index ( agfi ) = 0.939 , root mean square error of approximation ( rmsea ) = 0.039].conclusion : this study showed the designed questionnaire provided adequate construct validity and reliability , and could be adequately used to measure sexual abstinence and avoidance of high - risk situations among female students .

INTRODUCTION MATERIALS AND METHODS Participants and study design Instruments Statistical analysis Ethical issues RESULTS Reliability Demographic information DISCUSSION CONCLUSION

PMC2694299

the light microscopic study was performed on 40 human horizontal rectus muscles obtained from 3 normal subjects and 34 patients with oculomotor disorders . six control horizontal rectus muscles were obtained from the right globe of 3 multiorgan donors ( 2 muscles from each ) a few hours after postmortem in conformity with legal requirements . patients with oculomotor disorders were composed of 20 patients with acquired strabismus , 11 with infantile strabismus , and 3 with congenital nystagmus . one horizontal rectus muscle from each patient with oculomotor disorder was obtained during resection of a rectus muscle . the electron microscopic examination was carried out on 6 horizontal rectus muscles from the left globe of 2 multiorgan donors ( 8 month infant and 61-year - old female ) and 4 patients , including 10-year - old subject with intermittent exotropia , 3-year - old with infantile exotropia , 49-year - old with acquired paralytic strabismus and 4-year - old with congenital nystagmus . methods for securing human tissues were humane , included proper consent and approval , and complied with the tenets of the declaration of helsinki and austrian federal transplantation law . the 20 patients with acquired strabismus consisted of 6 with paralytic strabismus , 5 with constant exotropia , 5 with intermittent exotropia , 3 with sensory strabismus , and 1 with secondary esotropia after retinal reattachment . the 11 patients with infantile strabismus consisted of 5 with infantile exotropia , 5 with infantile esotropia , and 1 with congenital third cranial nerve paralysis . the mean age in patients was 29.4 ( range , 2 - 64 ) years in patients with acquired strabismus , 3.45 ( range , 1 - 10 ) years in patients with infantile strabismus , and 4.3 ( range , 3 - 6 ) years in patients with congenital nystagmus ( table 1 ) . before surgery , all patients affected by strabismus demonstrated large - angle deviations ( 50 prism diopters ) , and all patients with congenital nystagmus showed large - angle face turn ( 30 degrees ) . all samples from the eoms were obtained by excising a full width strip of rectus muscle during strabismus surgery . eoms were placed on slight stretch and care was taken to include the distal myotendinous junction in the 8 - 12 mm long tissue strip . all specimens were gently stretched with 2 forceps and fixed with pins to the bar during the fixation process to prevent folding and curling . the specimens were fixed for at least 24 hours in 10% buffered formalin and then embedded in paraffin for routine histopathological processing . complete series of paraffin cross sections ( 10 m ) were cut on a microtome and mounted on glass slides . paraffin cross sections were stained with hematoxylin and eosin at approximately 200 m intervals , and we examined samples progressing from muscle to tendon to select the most distal section just before reaching the first evidence of tendon . from the distal part from this selected section , we made 5 serial sections at intervals of 50 m , which totaled to 200 m . series of these 5 sections in the myotendinous junction were stained with hematoxylin and eosin . total imc counts in each eom were evaluated based on an examination of these 5 serial sections with 50 m intervals in the distal myotendinous junction ( 200 m ) . when the imc was found , 2 nearest sections ( 10 m intervals ) were impregnated with silver for light microscopy . for conventional transmission electron microscopy , tissues were immersed in a fixative solution consisting of 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) and allowed to remain in this solution for more than 12 hours . after rinsing in the same buffer , samples were postfixed in 1% osmium tetroxide in phosphate buffer 0.1 m ( ph 7.4 ) , dehydrated in ethanol at increasing concentrations , and embedded in epon . when the imc was found , approximately 10 ultra - thin sections alternating with semi - thin ones were cut at appropriate intervals ( 50 - 60 nm ) , mounted on copper grids , stained with uranyl acetate and lead citrate , and examined under a hitachi h-7100 transmission electron microscope ( hitachi , tokyo , japan ) . all specimens were gently stretched with 2 forceps and fixed with pins to the bar during the fixation process to prevent folding and curling . the specimens were fixed for at least 24 hours in 10% buffered formalin and then embedded in paraffin for routine histopathological processing . complete series of paraffin cross sections ( 10 m ) were cut on a microtome and mounted on glass slides . paraffin cross sections were stained with hematoxylin and eosin at approximately 200 m intervals , and we examined samples progressing from muscle to tendon to select the most distal section just before reaching the first evidence of tendon . from the distal part from this selected section , we made 5 serial sections at intervals of 50 m , which totaled to 200 m . series of these 5 sections in the myotendinous junction were stained with hematoxylin and eosin . total imc counts in each eom were evaluated based on an examination of these 5 serial sections with 50 m intervals in the distal myotendinous junction ( 200 m ) . when the imc was found , 2 nearest sections ( 10 m intervals ) were impregnated with silver for light microscopy . for conventional transmission electron microscopy , tissues were immersed in a fixative solution consisting of 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) and allowed to remain in this solution for more than 12 hours . after rinsing in the same buffer , samples were postfixed in 1% osmium tetroxide in phosphate buffer 0.1 m ( ph 7.4 ) , dehydrated in ethanol at increasing concentrations , and embedded in epon . when the imc was found , approximately 10 ultra - thin sections alternating with semi - thin ones were cut at appropriate intervals ( 50 - 60 nm ) , mounted on copper grids , stained with uranyl acetate and lead citrate , and examined under a hitachi h-7100 transmission electron microscope ( hitachi , tokyo , japan ) . small nerves enclosed by a loose capsule of connective tissue cells terminated in the myotendinous region . the imcs were identified with a light microscope by two investigators ( sep , hss ) . golgi tendon organs or muscle spindles were not observed in any stained eom region . in the control eoms , nerve ending and muscle fibers were sheeted by thin capsules , which constituted the imcs ( fig . 1a ) . the presence of nerve fibers within the imcs was revealed by silver staining ( fig . examinations of the distal myotendinous junctions of eoms of patients with acquired strabismus showed normal features . in the congenital strabismus eoms , the distance of neighboring nerve fibers increased gradually from distal to proximal muscle fibers ( fig . a single myelinated nerve fiber ran without branching , and the axon was completely sheathed by a schwann cell . most imcs possessed normal features , however some schwann cell degenerations were observed in the eoms of patient with intermittent exotropia ( fig . examination also found atrophic muscle fiber and flattened schwann cell process in some of the imcs of patient affected by paralytic strabismus ( fig . most nerve fibers at the distal myotendinous junction in patient with infantile strabismus were observed to be altered . these modifications consisted of schwann cell degeneration and axon alteration with maintenance of a practically normal general architecture ( fig . . examinations of the distal myotendinous junction of patient with congenital nystagmus revealed small , anomalous nerve terminal endings . some schwann cells and axons lacked neurotubules and neurofilaments , and golgi apparatuses and mitochondria were also absent . axonal myelin was anomalous , discontinuous , and unevenly distributed , and unmyelinated nerve fibers demonstrated fragmented neurilemma , swelling , and degeneration . small nerves enclosed by a loose capsule of connective tissue cells terminated in the myotendinous region . the imcs were identified with a light microscope by two investigators ( sep , hss ) . golgi tendon organs or muscle spindles were not observed in any stained eom region . in the control eoms , nerve ending and muscle fibers were sheeted by thin capsules , which constituted the imcs ( fig . 1a ) . the presence of nerve fibers within the imcs was revealed by silver staining ( fig . examinations of the distal myotendinous junctions of eoms of patients with acquired strabismus showed normal features . in the congenital strabismus eoms , the distance of neighboring nerve fibers increased gradually from distal to proximal muscle fibers ( fig . a single myelinated nerve fiber ran without branching , and the axon was completely sheathed by a schwann cell . most imcs possessed normal features , however some schwann cell degenerations were observed in the eoms of patient with intermittent exotropia ( fig . examination also found atrophic muscle fiber and flattened schwann cell process in some of the imcs of patient affected by paralytic strabismus ( fig . most nerve fibers at the distal myotendinous junction in patient with infantile strabismus were observed to be altered . these modifications consisted of schwann cell degeneration and axon alteration with maintenance of a practically normal general architecture ( fig . . examinations of the distal myotendinous junction of patient with congenital nystagmus revealed small , anomalous nerve terminal endings . some schwann cells and axons lacked neurotubules and neurofilaments , and golgi apparatuses and mitochondria were also absent . axonal myelin was anomalous , discontinuous , and unevenly distributed , and unmyelinated nerve fibers demonstrated fragmented neurilemma , swelling , and degeneration . small nerves enclosed by a loose capsule of connective tissue cells terminated in the myotendinous region . the imcs were identified with a light microscope by two investigators ( sep , hss ) . golgi tendon organs or muscle spindles were not observed in any stained eom region . in the control eoms , nerve ending and muscle fibers were sheeted by thin capsules , which constituted the imcs ( fig . 1a ) . the presence of nerve fibers within the imcs was revealed by silver staining ( fig . examinations of the distal myotendinous junctions of eoms of patients with acquired strabismus showed normal features . in the congenital strabismus eoms , the distance of neighboring nerve fibers increased gradually from distal to proximal muscle fibers ( fig . at the distal myotendinous junction of control eoms , several imcs were observed near the muscle fibers . within the imc capsule , a single myelinated nerve fiber ran without branching , and the axon was completely sheathed by a schwann cell . most imcs possessed normal features , however some schwann cell degenerations were observed in the eoms of patient with intermittent exotropia ( fig . 4 ) . examination also found atrophic muscle fiber and flattened schwann cell process in some of the imcs of patient affected by paralytic strabismus ( fig . most nerve fibers at the distal myotendinous junction in patient with infantile strabismus were observed to be altered . these modifications consisted of schwann cell degeneration and axon alteration with maintenance of a practically normal general architecture ( fig . . examinations of the distal myotendinous junction of patient with congenital nystagmus revealed small , anomalous nerve terminal endings . some schwann cells and axons lacked neurotubules and neurofilaments , and golgi apparatuses and mitochondria were also absent . axonal myelin was anomalous , discontinuous , and unevenly distributed , and unmyelinated nerve fibers demonstrated fragmented neurilemma , swelling , and degeneration . although still under debate , proprioceptive input from the eoms probably plays a role in the control of ocular alignment . steinbach and smith5 have given supports for a proprioceptive contribution to spatial localization from the study of strabismus patients . they have demonstrated patients undergoing strabismus surgery for a second time showed more errors in perceiving shifts of eye position when compared with those undergoing their first surgery , and suggested that some nervous organs may be important for eye position proprioception . palisading nerve endings in imcs ruskell denied not only the existence of imcs at birth in human,21 but also their role in proprioception in his review article.22 however , lukas et al.23 have given important arguments against ruskell 's study , and suggested that imcs may serve as proprioceptors as well as effectors . despite many studies on the subject , uncertainty persists of the exact roles of imcs in visual spatial perception and ocular motor behavior . several groups have suggested that imcs are sensory,16,21,22,24 - 29 but others have considered them as motor30 - 32 structures , or both.23 billig et al.26 identified sensory nerve endings in cat extraocular muscles by injecting neuronal tracers into the trigeminal ganglion , which contains the sensory cells that innervate the eye muscles . after the application , three types of nerve endings were labeled within eoms , one type confirmed palisading endings of imcs at the myotendinous junction of each eom , whilst the other types were sensory terminals within the muscle belly . they reported imcs establish contacts with tendon fibrils and attach muscle fibers , and reveal sensorylike nerve terminals . however , they also presented the morphological features of motor terminals , which were confirmed by -bungarotoxin staining . based on these features , they proposed that imcs might function as " propriocept - effectors " , by combining sensory and motor qualities . another view have highlighted imcs as motor structures and suggested that visual spatial perception might be served by efference copy.14,30 - 32 sas and schb30 observed degenerated palisade endings in eoms after removing small stereotactic lesions in oculomotor nuclei . blumer et al.31 studied imcs in rabbit eoms , and based on fine structure and -bungarotoxin binding suggested that myoneural contacts in imcs are exclusively motor . a recent study on cat eoms also provided evidence that palisade endings are exclusively motor . specifically , it was found that palisade endings arise from nerve fibers that establish motor contacts on muscle fibers , and also that neuromuscular contacts in palisade endings exhibit -bungarotoxin staining.32 specific research attention has also been focused on the possible correlation between oculomotor disorders and structural modifications of proprioceptors at the myotendinous junction . however , data on the possible morphological alterations of imcs in the various forms of strabismus is still lacking . corsi et al.7 observed alterations in proprioceptors located at the scleral myotendinous junction of the eoms of patients suffering from congenital strabismus . another study20 on congenital esotropia also reported the presence of altered sensory nerve endings at the myotendinous junction , and of normal motor nerve endings in the muscle body . this damage to nerve endings consisted of alterations to both contractile structures and mitochondria , and resulted in severer lesions at the myotendinous junction than in the muscle body.20 in patients with congenital nystagmus , the myotendinous and tendinoscleral area of eoms showed not only anomalous imcs but also anomalous vascular endothelial cells.33 all of these alterations support the hypothesis that the most important eom functional alteration in congenital oculomotor disorders concerns the distal myotendinous junction , and that imcs , as proprioceptors , play a prominent role in disease pathogenesis . in this study , we observed that almost normal features in the nerve endings at the myotendinous junction are demonstrated in acquired strabismus , including constant exotropia , intermittent exotropia , paralytic strabismus , sensory strabismus , and secondary esotropia after retinal reattachment . however it is important to note that some imcs in noninfantile strabismus exhibited the morphologic alterations , which should be interpreted as rather acquired abnormalities from strabismus than the causes of oculomotor disorders . , it appears that congenital and early - onset oculomotor disorder may be related to a degeneration and/or dysgenesis of imcs . furthermore , when considering the normal features of most imcs in acquired strabismus our study supports the hypothesis that some disturbance of imcs may play an important role in the pathogenesis of oculomotor disorder , and that this is not the consequence of strabismus due to other causes . we speculate that imcs are supposed to provide proprioceptive information , and a proprioceptive feedback system should be stimulated and calibrated early in life for the development of binocular vision . our study provides evidence that some disturbances in the proprioceptive feedback network , such as degeneration and/or dysgenesis of imcs , could result in oculomotor disorders . this concept is supported by the findings of previous studies,34,35 in which neonatal de - efferentation in the rat resulted in a reduced size of proprioceptors , whereas de - afferentation in young rats caused the formation of receptors that maintained their general architecture , but completely lacked the nerve component . in conclusion , imcs are supposed to function as proprioceptors , although they may have also effector properties . it should be emphasized in strabismus surgery that imcs , as ocular proprioceptors , are located in the myotendinous region .

purposeto analyze innervated myotendinous cylinders ( imcs ) in the extraocular muscles ( eoms ) of normal subjects and strabismic patients.methodsthe rectus muscles of 37 subjects were analyzed . distal myotendinous specimens were obtained from 3 normal subjects , 20 patients with acquired strabismus , 11 with infantile strabismus , and from 3 with congenital nystagmus , and were studied by using light microscopy . some specimens ( 6 rectus muscles ) were also examined by transmission electron microscopy.resultsimcs were found in the distal myotendinous regions of eoms . the imcs of patients with acquired strabismus showed no significant morphological alterations . however , significant imcs alterations were observed at the distal myotendinous junction of patients with congenital strabismus and congenital nystagmus.conclusionsthis study supports the notion that imcs in human eoms function mainly as proprioceptors , along with effector properties , and a disturbance of ocular proprioceptors plays an important role in the pathogenesis of oculomotor disorder . we suggest that a proprioceptive feedback system should be stimulated and calibrated early in life for the development of binocular vision .

Materials and Methods Light Microscopy Electron Microscopy Results Morphology of Innervated Myotendinous Cylinders Light Microscopy Electron Microscopy Discussion

PMC3022164

osteoporosis , a disorder characterized by the progressive loss of bone tissue and microarchitectural deterioration , remains a public health problem and reduces the quality of life for the aging population . it has been estimated that with increasing age , bone mineral density ( bmd ) in women decreases 1 - 2% per year at the femoral neck and spine ( excluding the influence of corticosteroid use ) . to date , the predominant medical strategies to prevent and/or treat postmenopausal bone loss have focused on antiresorptive medications ( i.e. , bisphosphonates ) . however , these treatments might be limited due to adverse side effects , questionable compliance , and long - term safety concerns . simple aerobic exercises ( aex ) like walking , jogging , and running could provide an important role in maintaining and/or increasing bone density in women . therefore , implementing nonpharmacological treatments that have little or no inherent side effects ( like exercise ) , either alone or in combination with pharmaceutical agents , is critical . although regular aex may improve bone status and/or maintain bone mass preventing fractures , relatively vigorous aerobic , weight - bearing , or strength training regimens are even more effective . however , in some cases vigorous exercise may increase the risk of injury , particularly in the elderly . also , compliance to vigorous exercise is likely to be low in the older population . the published studies examining the positive role for aex in relation to bone mineral density ( bmd ) are inconclusive . several controlled intervention studies have shown positive effects on bmd , while others have yielded either mixed or negative results . these differences could be attributed to different study populations ( young or old adults ) , the level of intensity tested ( low or high ) , and/or kind of activities ( running , jogging , etc . ) employed . to narrow down the discussing realm for this review , we focus on the impact of organized aerobic activities performed on treadmill and other gym equipment , as well as running and/or walking , on bmd in older adults . whole - body vibration ( wbv ) or vibration training is a relatively new type of exercise in the wellness industry . previously used for astronauts , wbv has been reported to have the positive effect on bmd , similar to that of aex in both animal [ 10 , 11 ] and human [ 12 , 13 ] studies . some studies also compared the effect of wbv and resistance training on muscle strength with positive results [ 14 , 15 ] . the resonance frequencies of the spine occur between 5 and 15 hz which are also the frequencies considered to be a causative factor in low back pain and circulatory disorders like raynaud 's phenomenon . however , wbv - related injuries ( back pain , muscular discomfort , etc . ) can be prevented by limiting the duration to a maximum of 10 minutes and maintaining the posture of the participant in a semisquat stance which involves the leg muscles reducing the transmission of vibrations to the head . therefore , wbv may be safer to apply and easier to comply with than aex , particularly in a community setting . however , there is no consensus regarding the benefits , safety , and long - term application of wbv . to our knowledge , there is also no comparison of the effects of wbv and aex on bmd in elderly . therefore , the purpose of this paper was to evaluate and compare the effect of aex and wbv on bmd at various skeletal sites in older population and to explain the possible mechanisms of their action on bone . papers on aex and whole - body vibration published in english were searched using pubmed and medline ( firstsearch ) databases . combinations of the following phrases as keywords were used in the search aerobic exercise / whole - body vibration , bone mineral density / content , older women / female , older men / male , and running / walking / jumping and would have to be present in the title , abstract , or keywords . studies with strength and resistance exercise were not included in this context . after excluding studies with vitamin d and calcium supplementation , or those involving subjects on medications and different chronic conditions , 17 studies were selected and are discussed in this paper for the effects of aex . after excluding studies that examined wbv but focused on bone implants and those in which medications and other special treatment were used , 4 studies were selected and discussed in this paper . a snapshot of the relevant studies and their overall description is shown in tables 1 and 2 . women entering menopause face many challenges regarding their bone health , especially those with a history of or current inactivity . therefore , engaging the elderly in exercise programs is necessary to maintain bmd and increase the quality of life . most of resistance exercises have shown a positive effect on increasing or maintaining bmd in postmenopausal women [ 38 , 39 ] . whether aex has the same effect is questionable . results from a meta - analysis revealed that aex maintained spine and significantly increased femoral neck bmd , suggesting that the femoral neck might be more responsive to high - impact aex than the lumbar spine . welsh and rutherford found that 1-year aex including high - load step and jumping significantly increased femoral neck and trochanter bmd in a group of previously sedentary men and postmenopausal women . in addition , chien et al . found that a 6-month aex intervention , including graded treadmill - walking and stepping , significantly increased femoral neck , but not spine bmd in chinese postmenopausal women . even in the chronic stroke population , aex maintained the femoral neck bmd , while a significant reduction in bmd was observed in controls . these results suggest that aex has positive impact on femoral neck bmd . confounding results still exist as some studies have shown that aex did not enhance bmd in postmenopausal women [ 28 , 30 , 41 ] . these conflicting results were most likely due to insufficient intensity and frequency of exercise , small sample sizes , and the choice of measurement site employed in each study . the most frequently used duration and frequency of aex were 3060 minutes per session , 2 - 3 times a week . in a community exercise program , it was found that 1 year of aex combined with strength training did not show an improvement in bmd at several skeletal sites in women aged 60 years and over . martin and notelovitz showed that 1 year of moderate aex did not improve spine or forearm bmd due to inadequate sample sizes to detect small changes in bone . other limitations were the low intensity of exercise and the lack of the involvement of upper extremities activities . results from the meta - analysis showed that exercise did not improve femoral neck bmd in postmenopausal women thought resistance training was included in their data analysis . the conflicting conclusion may be due to the combination of aerobic and resistance exercise in their data analysis and the different choice of measurement such as forearm or spine . if we only consider the effect of aex on bmd , not including other kinds of exercises , aex seems to increase femoral bmd in older adults . . found that 54 male runners aged 40 to 80 years maintained their hip and spine bmd over a 4- to 5-year running period by self - reporting training hours compared to their baseline measurements , the age range used may be too large to draw meaningful conclusions . a similar study revealed that runners exhibited a lower age - related bone loss compared to controls although both runners and controls had a significant decrease in spine bmd after a 5-year followup . a 9-year longitudinal study following the running subjects showed that those who maintained their training volume lost less bone in the spine than those who did not maintain their running regimens . in contrast , kirk et al . found that postmenopausal runners tended to have lower spine bmd than age- and height - matched controls , indicating that running prevents hip bmd loss , but it may not maintain age - related reduction of spine bmd in older men and women . while these results did not demonstrate positive effect on spine , they are consistent with the mechanostat theory that the bmd of a lower limb , a primary weight - bearing site , benefits the most from running . ideally , running and walking could be an effective measure to prevent the loss of bone mass for the older population ; however , compliance with moderate to intensive exercise is questionable . low - intensity aex such as walking has a lower impact force upon the skeleton compared to running ; therefore , it might offer an inferior osteogenic stimulus . walking as a physical activity may be beneficial for postmenopausal women as well as elderly population , but it may depend on walking speed , with brisk and fast pace being more advantageous . krall and dawson - hughes examined the impact of current and past walking on bmd in 237 healthy caucasian women ( 4372 years ) and found that women who walked more than 7.5 miles / week had higher whole body , leg , and trunk bmd compared to those who walked less than 1 mile / week . furthermore , the number of miles walked per week during a 1-year period was positively correlated with the rate of bmd increase in the lower limbs . a recent meta - analysis reported that walking had favorable effects on hip , but not spine . however , an earlier meta - analysis results showed that walking had a positive significant effect on spine but not on femoral bmd . this contradiction is probably due to including the same groups of population twice in their analysis and the combination of other exercise modes with walking . based on the observational and intervention studies investigating the influence of different walking regimens on bmd [ 20 , 23 , 24 ] , it was suggested that brisk and fast walking pace is more beneficial in specific skeletal sites , for example , foot and calcaneal bones . evaluated the effect of self - reported past and current walking routines ( including normal , fast , and brisk pace ) on bmd in older caucasian women and found that both forearm and hip bmd were higher in the subjects that were able to walk at a brisk or fast pace . similarly , in a 3-year evaluation of the effects of walking and other habitual physical activities ( not necessarily aerobic ) on bmd in postmenopausal women , ilich and brownbill reported that walking at a faster pace , involvement in sports / recreational activities , and even participation in low - impact physical activities were essential in augmenting bone mass . in a randomized study , it was shown that one year of brisk walking significantly increased calcaneal bmd and slightly increased spine bmd , although not significantly . similarly , a 2-year brisk walking regimen significantly reduced loss of bmd at femoral neck , but there was no difference at the spine bmd . these results support walking as an effective method of increasing bmd and confirm that a brisk and fast walking pace may , in addition to femoral bmd , also benefit calcaneus and forearm bmd . however , most of the studies have been done in women while older men have been less investigated regarding the osteogenic response to walking and running . there is a consensus that a combination of aerobic and anaerobic exercise is more effective in improving bone mass than either one alone . one year of brisk walking combined with gymnastic training either increased or maintained the spine bmd in postmenopausal women . results from an earlier meta - analysis showed that walking with other aexs significantly affected the bmd at spine , but not hip . bone mass increases or decreases in response to mechanical loading depending on whether the thresholds controlling bone formation and resorption have been reached . a few studies have demonstrated the importance of walking intensity on bmd preservation in postmenopausal women . hatori et al . reported that 7 months of walking with an intensity above 110% of the heart rate at its anaerobic threshold attenuated bone loss in the spine of postmenopausal women , whereas walking at an intensity below 90% of the heart rate at its anaerobic threshold had no influence on bmd . similarly , borer et al . confirmed that fast walking pace increased leg and total bmd in early postmenopausal women who were engaged in 15 weeks of walking . the greater response to the higher intensity walking may be due to the elevated ground reaction forces that occur at a faster walking pace . a failure to show increase in spine bmd during a walking study by cavanaugh and cann in 55-year - old postmenopausal women may be due to the employment of lower walking intensities . therefore , a combination of different kinds of aexs may be the most efficient approach to reach desired exercise intensity to enhance or maintain bone mass at different skeletal sites in postmenopausal women . it is generally accepted that mechanical loading on bones is probably one of the ways to induce skeleton 's structural changes and increase bone mass . physical activity induces a mechanical load on bone tissues due to external forces and muscle contractions , the latter exerting the greater force on bones than any other weight - associated gravitational forces . to withstand the rigor of various functional activities , bone tissue rapidly accommodates changes in its microenvironment . although the mechanism of mechanical loading effects on bone is not completely understood , it is postulated that it is due to the mechanotransduction of a load . this mechanotransduction is carried out through fluid flow near and between osteocytes ( mature cells within the mineralized bone matrix ) . in vivo and in vitro studies indicate that mechanical stimuli increase strain , loading frequency , and fluid flow , all of which have an osteogenic effect . however , several conditions must be met to affect bone positively : ( 1 ) the strain produced by loading must be of high enough magnitude to exceed the minimum effective strain ( or threshold ) ; ( 2 ) the strain should be applied in an intermittent fashion ; ( 3 ) loading should produce a different from normal strain distribution within the bone . in addition , recent research implies that frequent loading on bone without rest may not allow sufficient time for osteocyte fluid flow to recover from inertial dumping between each load cycle . therefore , fluid flow and subsequent osteocyte stimulation might be reduced or completely inhibited after the first loading cycle . if so , inserting short time periods between loadings will allow for recovery from the inertial dumping effect and facilitate osteocyte stimulation . this effect was recently shown in vivo , in both young and older animals and in two different species ( avian and murine ) ; by inserting 10 sec , rest period between each loading cycle greatly enhanced the osteogenic potential of the low - magnitude regimen . while much emphasis is given to the resident bone cells ( primarily osteocytes ) and their response to the local load imposed on bone , new research implies that response to the mechanical load ( exercise ) may also be neuronally regulated and therefore systemic , with a resultant effect on multiple bones . . showed that in young rats , intense mechanical stimulation of one limb can illicit a response in other limbs and even in the entire skeleton . this newest discovery may explain why increased bmd after localized mechanical stimulation could be recorded in skeletal sites that were not directly stimulated ( e.g. , increased forearm bmd with walking ) . wbv is a new approach that is currently being tested for its effect on bmd and bone strength [ 51 , 55 ] . there are two main types of vibration devices / techniques : ( a ) up - and - down oscillating vibration plates and ( b ) reciprocating vertical displacements on the left and right sides of a fulcrum , providing the lateral oscillations . [ 11 , 51 ] observed an increase in bone formation in weight - bearing sites and a substantial increase in the quality and quantity of trabecular bone in sheep exposed to a low - intensity , high - frequency ( 2050 hz ) mechanical stimuli . it is well established that decreased estrogen due to ovariectomy in animal models decreases the bone formation rate resulting in a decrease in bone mass . in rats , flieger et al . found that low - level , high - frequency mechanical loading ( 50 hz ) was effective in preventing bone loss shortly after ovariectomy . even in aging mice , low- and high - intensity wbv significantly increased mineralized bone surfaces . bone is known to adapt to different loading conditions and the loading - induced strains are believed to be based on the adaptation of the bone tissue . for that reason , researchers usually administered frequencies at 1535 hz to obtain the maximum transmissibility of the mechanical loading produced by the vibrating plate . while the adaptation to the mechanical loading is most likely the limiting factor , elderly subjects have been shown to benefit from simulated mechanical loading . early - postmenopausal women who stood on a vertical plate of low magnitude ( 0.3 g ) and high frequency ( 30 hz ) , twice / day ( 10 minutes each ) for a year , showed no difference in bmd compared with controls . however , evaluating those with 80% compliance , controls lost 2.13% of bone mass in the femoral neck , whereas treatment group gained 0.04% over one year . when the analysis focused on the lower weight ( < 65 kg women , a known risk factor for osteoporosis ) and those who were compliant , the benefit of treatment became significant and demonstrated a 3% and > 2% positive difference at the spine and femoral neck bmd , respectively . any potential benefit of wbv strongly depends on compliance and vibration stimulus that can be varied in multiple ways ( including type , magnitude , frequency , and duration ) , and different types of vibration loading are likely to result in different effects on bmd . low - level mechanical stimuli may be more effective in lighter than in heavier women , particularly for hip bmd . a recent meta - analysis ( although published only as an abstract ) also showed that low - intensity wbv effectively attenuated postmenopausal bmd decline in hip but not in spine . another study in postmenopausal women where intervention was employed on a vertical plate , higher magnitude ( 2.285.09 g ) , and high - frequency ( 3540 hz ) for 6 months showed a significant 1.51% net increase in total hip bmd but not in total body or spine bmd . a 5-month study examining an older man ( 79 years ) employing multiple vibration intensities and frequencies to evaluate bmd showed detectable increase in spine , femoral neck , trochanter , and forearm bmd . though this study demonstrated positive results on bmd , only one participant was examined , therefore , the results can not be representative of the larger group . gusi et al . investigated whether wbv is more effective than walking for maintaining or increasing bmd . they compared the effect of walking and wbv using a reciprocating plate , low amplitude ( 3 mm ) , and medium frequency ( 12.6 hz ) , for 8 months on bmd in postmenopausal women . after 8 months , femoral neck bmd was significantly ( 4.3% ) higher in the wbv group than in the walking group . the difference in bmd at spine and other sites of the hip was not significant between two groups . the results suggest that the vibration could be an easier approach to increase bmd at the femoral neck than walking and could be applied to provide a surrogate for suppressed bone loss of hip after menopause . however , at the present time , it is too early to make a conclusion due to insufficient research in elderly adults and varied vibration protocols . possible mechanism by which wbv affects bone may be based on the same principle as aexs that activate the osteoblasts while reducing the activity of the osteoclasts . the strain , magnitude , and frequency are essential factors for the effect of wbv . controlled loading study has shown that high - strain magnitude and high - strain rate are the most osteogenic . it has been hypothesized that mechanical stimulation recruits additional osteoblasts and increases the percentage of mineralizing surfaces therefore , increasing the rate of bone formation and decreasing the rate of bone resorption . it has been suggested that the high - frequency vibration may have played an important role in the osteogenic effect . there is a general perception that signals must be large enough to elicit a positive influence on bone mass and morphology . however , the high - frequency stimulation may be capable of influencing skeletal architecture by distributing uniform stresses on bones . [ 62 , 63 ] hypothesized that this influence may be achieved directly by mechanical strain , or indirectly through amplification of the signal by intramedullary pressure or fluid flow in bone tissue . the mechanism behind the frequency - dependent adaptive response of bone to stimuli might be the stochastic resonance . stochastic resonance is a phenomenon in which mechanical noise ( broad - band frequency of vibration ) enhances the response of a nonlinear system to a weak signal by boosting it over a threshold . the stochastic threshold may be modified through a system such as neuromuscular feedback amplified by the low - level signal or by stimulating skeletal muscle pump activity , resulting in significant effects on circulatory flows and fluid flow through the bone tissue . in addition , previous study has shown that stochastic resonance can enhance mechanosensitivity of different mechanoreceptors in the body , for example , muscle spindles . these findings indicate that vibration stimulation employs multiple ways to influence bone mass and structure . in summary , evaluation of the published literature provides evidence of the effectiveness of aex and wbv in increasing or at least maintaining bone mass in the elderly . the mechanism could be due to increasing the circulation of fluid and activating the osteoblasts while reducing the activity of the osteoclasts via mechanical stimulation . the osteogenic effects of both aex and wbv could be site specific ( to the spine or hip ) , depending on the exercise load and the type of exercises . the beneficial effects on bone can be maintained for a longer time if the exercise continues although the exercise may not maintain the age - related reduction of bmd in elderly . in addition , the risk of injuries or falls could be high and the compliance to aex particularly in elderly low . walking is an inexpensive , practical exercise associated with low injury rates and demonstrates high acceptability by elderly . for these reasons however , there is evidence that the osteogenic effect of load bearing may decline with aging , suggesting either a decrease in osteoblast activity or a desensitizing of osteocytes to mechanical stimuli . therefore , alternative , more acceptable strategies with a lower risk of injury need to be explored . vibration , with increased stresses on the bone , stimulates remodeling but may also decrease bone resorption . studies show that it may increase femoral neck bmd in postmenopausal women and in lower - weight women , in addition to inhibiting bone loss after menopause . however , just a few studies have investigated the effect of wbv on bmd in older population and different protocols were employed in the studies . in addition , it is still unknown if these short - term effects of low - intensity wbv will persist or whether body will adapt ( although the parameters can be constantly changed to account for adaptation ) . it is not known yet whether the benefits of wbv will disappear after the intervention is terminated , as it has been shown previously with other types of exercise . the on - going vibes trial investigated the effects of wbv on various bone and muscle parameters in postmenopausal women over a 2-year period , and results , when available , should provide more insight into the issue . this may shed further light on the mechanism by which wbv operates and may yield future areas of study . overall , future studies are required to confirm these short - term findings and to investigate whether the long - term wbv and aex still have positive effect on bone .

osteoporosis and its associated fractures are common complications of aging and most strategies to prevent and/or treat bone loss focused on antiresorptive medications . however , aerobic exercise ( aex ) and/or whole - body vibration ( wbv ) might have beneficial effect on bone mass and provide an alternative approach to increase or maintain bone mineral density ( bmd ) and reduce the risk of fractures . the purpose of this paper was to investigate the potential benefits of aex and wbv on bmd in older population and discuss the possible mechanisms of action . several online databases were utilized and based on the available literature the consensus is that both aex and wbv may increase spine and femoral bmd in older adults . therefore , aex and wbv could serve as nonpharmacological and complementary approaches to increasing / maintaining bmd . however , it is uncertain if noted effects could be permanent and further studies are needed to investigate sustainability of either type of the exercise .

1. Introduction 2. Methods 3. Effect of Aerobic Exercise on Bone in Older Population 4. Possible Mechanism by Which Aerobic Exercise Affects Bone 5. The Effect of Whole-Body Vibration on Bone 6. Possible Mechanism by Which Low-Intensity Whole-Body Vibration Affects Bone 7. Conclusions

PMC4765253

as stated by who , each year over two million people die from diarrheal diseases , many of which are acquired from eating contaminated food from food service . food borne diseases remain responsible for high levels of morbidity and mortality in the general population . the most frequently reported food worker 's errors were handling of food by a person either actively infected carrying a pathogen , bare - hand contact with food , failure to wash hands properly . whenever necessary and insufficient cleaning of processing or preparation equipment orkitchen tools . such unhygienic practise would cause contamination of the food and cross - contamination of ready - to - eat ( rte ) foods . food businesses have become widespread in recent times , in response to the changing lifestyle and food consumption of people . they offer convenience and ease to access food to busy individuals , who are unable to pre- pare their own meals regularly at home . in large scale cooking , food passes through many hands , thereby increasing the chances of food contamination due to improper handling . deliberate or accidental contamination of food during large production might endanger the health of consumers , and have very expensive repercussions on a country , as such outbreaks feature prominently in national statistics . there was general agreement revealed from several authors as good levels of knowledge towards food safety among food handlers and the effective practices of such knowledge in food handling were imperative in ensuring the safe production of food in any catering operations . recently , many studies pinpoint the need for training and education of food handlers in public hygiene measures due to their lack of knowledge on microbiological food hazards , temperature ranges of refrigerators , cross contamination and personal hygiene . education on food safety should be given to all staff in food processing businesses so as to bring behavioral changes besides adoption of positive attitudes . assessment of impact of food safety measures require reliable epidemiological estimates while no precise and consistent data exists regarding burden of food borne illnesses . a descriptive type of cross - sectional study was carried out during one year i.e. from august 2013 to july 2014 by purposefully selecting all the food handlers / mess - workers / canteens - workers worked any period of time during above said one year duration in all the 18 food establishments ( messes and canteens ) run / managed by the same management running / managing all the five important teaching hospitals in the bareilly district of uttar pradesh . on an average these 18 foods establishments have been serving eatables to approximately 2399 medical students in the campuses of five teaching hospitals in the bareilly city of uttar pradesh . the survey was carried out by of four researchers conducted several visits till all handlers worked during study period covered . the tool of the study was a research schedule which was used to evaluate the knowledge and practices of food handlers about their personal hygiene and sanitary measures adopted in food establishments . it also included the questions assessing the health status of food handlers and preventive measures used by them in last 3 months . the schedule has been designed to obtain information about the demographic characteristics e.g. , age , sex , education , caste / category , below poverty line or above poverty line . 999 per capita per month respectively as given by planning commission of india ( pci ) for twelfth five year plan ( 2012 - 17 ) . by using schedule we also got information about occupational characteristics of food handlers e.g. job type , job duration and training taken etc . during survey the health status of food handlers was assessed for communicable diseases especially food borne infections e.g. acute diarrheal disease and systemic disorders e.g. , hypertension , coma and diabetes mellitus . we also assessed whether the food handlers were vaccinated or given chemoprophylaxis against important communicable diseases like typhoid , tetanus and worm infestations during last three months . chi - square test was used as test of significance and regression analysis was done thereafter to know the independent association of the predictors of health status of food handlers . the health status of mess workers with respect to caste did not show significant association [ table 1 ] . the educational and occupational status also did not show significant association while there was significant association of the rural and urban background with the health status [ tables 24 ] . it has been clearly depicted in table 4 that 78.3% of the mess workers who were diseased , belonged to urban background while only 21.7% of those from rural background found to be diseased . also , there was no significant relationship of the health status with socio - economic status i.e. apl or bpl [ table 4 ] . besides this , the duration of job and the training done had no significant effect on health status [ figure 1 and table 5 ] . association of health status of mess workers with educational status association of health status of mess workers with occupational status association of health status of mess workers with residence association of health status of mess workers with poverty line association of health status of mess workers with duration of job . chi - square value = 1.049 , df = 3,p value = 0.789 association of health status of mess workers with training done as far as the immunization and deworming of health workers is concerned , there was no significant effect of it on their health status [ tables 68 ] . though the personal hygiene had no significant relationship with the health status of mess workers but the use of glove had been found to be a useful measure in the prevention of diseases [ tables 9 and 10 ] . similarly , the practice of nail cutting and hand washing after toilet had no significant association with the health status of the mess workers but the habit of hand washing before cooking or serving food had significant association with the health status of mess workers [ tables 1113 ] . religion and caste also had no significant association with health status of mess workers [ figures 2 and 3 ] . association of health status of mess workers with t.t.immunisation association of health status of mess workers with anti - typhoid immunisation association of health status of mess workers with use of de - worming tablets association of health status of mess workers with personal hygeine association of health status of mess workers with use of gloves association of health status of mess workers with nail cutting habit association of health status of mess workers with hand washing after toilet association of health status of mess workers with hand washing before cooking association of health status of mess workers with caste . chi - square value = 2.63 , df = 2 , p value = 0.268 association of health status of mess workers with religion . chi - square value = 0.146 , df = 1 , p value = 0.702 on applying logistic regression , the health status of the mess workers was found to be significantly associated with use of glove , hand washing after toilet and hand washing before cooking and serving food . though there was a significant association with rural and urban background on application of chi - square but on nullifying the effect of other confounders by regression analysis , the result was not found to be significant . conversely , the habit of washing hands after toilet showed no significant association on application of chi - square but on applying logistic regression , there was a significant association present . there is an inverse association observed between caste , occupational status , anti - typhoid immunization , deworming , personal hygiene , use of gloves and hand - washing before cooking / serving . there is 0.035 times less chances of developing disease in health workers with the use of gloves . also , there are less chances of disease among the health workers who wash their hand before cooking as compared to those who do not [ table 14 ] in the present study , 87.29% food handlers were apparently healthy and 12.71% were unhealthy . dental caries ( 6.6% ) , anaemia ( 2.2% ) , hypertension ( 1.1% ) , cough / cold ( 1.1% ) , scabies ( 0.6% ) and fungal infection ( 0.6% ) were the diseases that are attributed to their unhealthy conditions while working in the food establishment of various teaching hospitals included in this study . in this study with the help of chi - square test and multinomial logistic regression analysis we had analysed any association of certain demographic characteristics and personal hygiene practices for their unhealthy status during being in work infood establishment . after analysis it was observed that the only background characteristic i.e. residence of food handlers was significantly associated with the unhealthy conditions of food handlers in this study as it was observed that food handlers who were belonged to the urban background found to be unhealthier as compared with the food handlers of rural background when examined clinically during survey period in this study . though there was a significant association with rural and urban background on application of chi - square but independently on applying logistic regression , there was no such association found . other background characteristics i.e. age , gender , castes , educational status , occupational status were also not significantly found to be associated with their health status when analysed using same tests in this study . on analysis of health status and preventive measures the health status of the mess workers was found to be significantly associated with use of glove , hand washing after toilet and hand washing before cooking and serving food . though the habit of washing hands after toilet showed no significant association on application of chi - square but on applying logistic regression , there was a significant association present . in one of the similar study , the authors had revealed that majority ( 62.74% ) of food handlers were apparently healthy in their study but 23.52% had injuries , 19.60% had fever and hypertension , 10% had anaemia , diabetes and poor nutritional status . in their study , again majority ( 68.09% ) of food handlers reported some illness in the previous 3 months . another study observed that only 25.33% of food handlers suffered from one or more disease in the past 6 months . this observation was more close to our study as only small proportion of food handlers were had disease attributing to their unhealthy status . to compare the findings of our study , we had reviewed several studies , carried out among food handlers in food establishments in various parts of the world . these studies have been conducted on the health status of the food handlers with their background characteristics but none has shown the association of these factors with it unlike the present therefore this study is an attempt towards the analysis of such predictors of food handlers using regression analysis to nullify the effect of confounders . on the basis of observations , analysis and discussion in this study it can be concluded here that only belonging or holding good socio - economic background characteristics can not help food handlers to stay healthy . good personal hygiene practices adopted by food handlers while working in food establishments of teaching hospitals were the important measures to stay healthy . in this study it was also revealed that preventive measures were of no much useful in retaining good health by food handlers as preventive measures i.e. , anti - worm , anti - typhoid , tetanus vaccines can only prevent worm infestations , typhoid fever , tetanus only . it can be well concluded that use of gloves , washing of hand after toilet and before cooking and serving has significant role in maintaining good health status of food handlers and subsequently maintaining good health status of students i.e. , medical / dental / nursing students served by them in these teaching hospitals .

introduction : the us centers for disease control and prevention ( usdhhs - cdc 1996 ) revealed that the outbreaks of food borne diseases include inadequate cooking , heating , or re - heating of foods consumption of food from unsafe sources , cooling food inappropriately and allowing too much of a time lapse . as we all know that the food handlers have been working in various types of community kitchen and their health status can affect the status of food hygiene which can lead to contamination of foods attributing to acute gastroenteritis and food poisoning in various subgroups of the population e.g. , medical / dental / nursing students . the background characteristics of these food handlers may have important role to affect health status of these handlers.methods:the indexed study was carried out among the food handlers working in the food establishments the 5 teaching hospitals of bareilly city in u.p . india during one year i.e. , from august 2013 to july 2014 . the survey method using schedule was conducted to get information about the background characteristics and food handlers and each food handler was examined clinically for assessing health status . chi - square test was used as test of significance and regression analysis was also done to nullifying the effect of confounders.results:the health status of the mess workers was found to be significantly associated with use of gloves , hand washing after toilet and hand washing before cooking and serving food.conclusion:the rationale of this study was that though many studies have been carried out to show the health status of the food handlers and their background characteristics , no study has highlighted the association of these background characteristics and personal hygiene practices with the health status of food handlers .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

PMC3241943

the goal of the transition strategy is to ensure a sustained hiv response through an effective transition. this goal thus emphasizes both the need for an effective transition and also the broader purpose which is to sustain impact . the bmgf program team has been actively planning and refining the transition strategy since 2007 , and the strategy has been adapted over time as the challenges and implementation issues have become clearer . the strategy identifies three main stakeholders to whom hiv / aids prevention activities will be transferred . the most significant of these is the goi . a 2009 memorandum of cooperation between the national aids control organization and the bmgf that builds on an earlier 2006 memorandum of understanding sets out clear agreements regarding the transition process , with 10% of tis to be transferred to government by april 2009 , a further 20% by april 2011 , and the remaining 70% by april 2012 . the bmgf program team has worked intensively to promote and ensure continuation of government commitment to prevention among high - risk groups . by sharing its own experience of prevention among most - at - risk groups , and through technical support programs , avahan has also sought to enhance government capacity to manage the hiv prevention program at multiple levels . finally , avahan has sought to prepare the tis that it supports for the financial and managerial handover through , for example , aligning interventions with government guidelines and ensuring that necessary management skills are transferred to the ngos responsible for administering the programs . community groups or community - based organizations ( cbo ) , made up of the affected communities , are also seen as natural owners of the interventions . they play a key role not only in delivering services but also in sustaining the demand for services and holding the government accountable to its commitment to provide such services . transition activities related to community groups include both organizational development activities to build the capacity of such groups and develop networks between them and structural interventions that aim to shape the environment through , for example , strengthening links with and promoting understanding among the police force . finally , it is acknowledged that other potential stakeholders , such as other donors or other local institutions , may also play a role in the transition process . for example , nacp iii includes a stronger focus on scaled prevention , in comparison to previous phases of the nacp . the hiv / aids prevention strategies such as tis , prevention of mother - to - child transmission , voluntary counseling and testing , condom promotion , sexually transmitted infection ( sti ) management , and blood safety account for 67.2% of the nacp iii estimated resource needs , and the government is clearly committed to saturated coverage of marps . in addition , bmgf along with other development partners,2 is currently making substantial investments in capacity enhancement at national , state , ngo , and community levels . for example , bmgf is currently supporting the training of trainers , the development of training tools and materials , and support to the national aids control organization 's information education and communications program to ease the transition . the first 10% of avahan - supported tis were transferred to government during 2009 , a further 20% transitioned this year 2011 , and the remaining tis will transition in 2012 ( fig . much of the research in the development field that has addressed the phasing out of donor support to a project or a whole country ( donor exit ) takes the form of small - scale , retrospective case studies based on short - term consultancy work , which draw the bulk of their data from discussions with stakeholders ( 69 ) . the studies focus in particular on how communication and transition planning affect the overall success of the transition . in high - income country contexts , and particularly within the health promotion field , there have been a number of studies with stronger theoretical and conceptual foundations that have sought to identify what factors have promoted sustainability , once external funding for a program has been withdrawn . these studies have typically identified three core dimensions of sustainability , all of which are relevant to the avahan transition ( 1012 ) : community continued capacity of a community to develop and deliver services , particularly relevant when the initial program worked via a community structure.continuation of health programs continuation of program activities within an organization ( or by another organization).maintenance of health benefits continued health benefits for individuals after the initial program - funding ends community continued capacity of a community to develop and deliver services , particularly relevant when the initial program worked via a community structure . continuation of health programs continuation of program activities within an organization ( or by another organization ) . maintenance of health benefits continued health benefits for individuals after the initial program - funding ends literature on sustainability planning identifies processes that help lead to such sustainability . these include routinization that is typically viewed as the extent to which the program has been integrated into existing organizational systems and practices , and institutionalization that considers the role of institutional standards , and the extent to which innovation and learning not only gets adopted and sustained , but is also reflected in institutional standards and norms that govern multiple organizations within the broader health system ( 11 , 13 , 14 ) . it may also be important to assess whether and how the transfer of the stand - alone program into the broader health system triggers further adaptation and change throughout the system ( 15 ) . if the health system is viewed as a complex adaptive system ( 16 , 17 ) , then the transfer of a stand - alone program into the health system will stimulate interactions between the program and the broader system that it is embedded within . this dynamic interaction is argued to be an important dimension of sustainability that can potentially trigger system - wide changes over time ( 12 ) . for example , a review of sustainability studies identified 19 studies that sought to assess the sustainability of health programs after their completion ( 11 ) . the studies were typically implemented 15 years after the cessation of program funding or the formal completion of the program and sought to asssess which if any program components or effects continued . for example , sridharan et al analyzed strategic plans to identify strategies that aimed to promote sustainability ( 18 ) . in one of the few developing country papers , hanh et al . developed a framework to assess and predict the likely sustainability of different dengue control projects in vietnam ( 19 ) . one longitudinal study sought to predict sustainability and then assess actual sustainability against predictions ( 20 ) . however , none of the studies identified so far have prospectively sought to analyze sustainability and to guide processes so as to promote program sustainability . building on case studies and conceptual work by yin ( 21 ) , scales to assess the degree of routinization and institutionalization have been developed ( 13 , 22 ) ; however , these are quite context specific and require careful adaptation to different contexts . in general , there appears to be a dearth of empirical work on routinization and institutionalization ( 23 ) . 2 ) was developed through an iterative process involving review of avahan documentation , relevant literature , and interactions with government and avahan staff involved in the design and implementation of the transition . the headings at the top of the figure represent a logical progression , from the activities conducted under the second phase of avahan , to the immediate proximal impacts of these activities ( defined as a state of transition preparedness ) , to the institutionalization of avahan activities within the government system and finally the achievement of the transition goal ( impact is sustained through an effective transition ) . achievement of the goal in turn contributes to india 's national aids control goals and also to the overall purpose of avahan in terms of maintaining or improving trends in reduction of new hiv infections among marps and the general population in india . the first column of the figure draws on discussion of the avahan transition strategy above to identify five main clusters of activities in preparation for transition . three of these sets of activities relate to supporting and adding to the capacities of various entities , namely : 1.supporting government capacity activities include ( 1 ) enhancing the technical and managerial skills of government staff members through training and mentoring ; ( 2 ) supporting quasi - government hiv / aids prevention structures and the systems necessary for those structures to operate effectively , and ( 3 ) supporting the development and production of training materials as well as government norms and guidelines.2.supporting ngo capacity including the provision of capacity development support to implementing partners ( ngos / cbos ) to prepare them for transition and to enable them to take over some of the analytical and management functions previously conducted by avahan contractors , in the post - transition period.3.supporting community capacity including support to community organizations both through strengthening management and governance structures and through building networks of cbos . supporting government capacity activities include ( 1 ) enhancing the technical and managerial skills of government staff members through training and mentoring ; ( 2 ) supporting quasi - government hiv / aids prevention structures and the systems necessary for those structures to operate effectively , and ( 3 ) supporting the development and production of training materials as well as government norms and guidelines . supporting ngo capacity including the provision of capacity development support to implementing partners ( ngos / cbos ) to prepare them for transition and to enable them to take over some of the analytical and management functions previously conducted by avahan contractors , in the post - transition period . supporting community capacity including support to community organizations both through strengthening management and governance structures and through building networks of cbos . the final two activities under the first column concern : 4.alignment of interventions alignment of the technical , managerial , and cost elements of avahan programs with government norms so as to facilitate transition . for example , avahan programs have often operated their own sti clinics , and these services have sometimes provided a broader range of primary - care services ( 24 ) . under government guidelines , ngos are encouraged to establish public private partnerships with private health care providers for sti services and/or develop linkages with government sti services.5.sustaining and monitoring commitment government commitment to high levels of service coverage for tis for high - risk groups is critical to the successful transition of avahan programs . securing and maintaining such commitment is inherently political and thus may be difficult to plan for . while government commitments are already documented in the current nacp and in the 2009 memorandum of cooperation with the bmgf , naco and the development partners , including bmgf , alignment of the technical , managerial , and cost elements of avahan programs with government norms so as to facilitate transition . for example , avahan programs have often operated their own sti clinics , and these services have sometimes provided a broader range of primary - care services ( 24 ) . under government guidelines , ngos are encouraged to establish public private partnerships with private health care providers for sti services and/or develop linkages with government sti services . sustaining and monitoring commitment government commitment to high levels of service coverage for tis for high - risk groups is critical to the successful transition of avahan programs . securing and maintaining such commitment is inherently political and thus may be difficult to plan for . while government commitments are already documented in the current nacp and in the 2009 memorandum of cooperation with the bmgf , naco and the development partners , including bmgf , jointly monitor these commitments , so as to help ensure that they are sustained . these packages of activities are intended to ensure that all organizations involved in program planning , management , delivery , and oversight , both among avahan partners and their counterparts within the government who will take over these functions , reach a state of transition preparedness, so that avahan programs can be transitioned into government systems with minimal disruption . transition preparedness column relate very directly to the five clusters of activities in the first column and indicate that transition preparedness has been achieved with respect to each of these activities . the third column of the transition logic model reflects the processes that need to occur during and after the transition for the final goal ( sustained impact ) to occur . we have called this set of processes institutionalization : by institutionalization , we mean that the key elements of the avahan program are integrated into the organizational procedures and behaviors of government agencies and other key implementing partners . drawing on the dimensions of institutionalization discovered during the literature review , three different levels of institutionalization are identified . at the most basic level , routinization needs to occur , that is , key selected practices associated with avahan - supported tis need to be adapted to better fit government systems , adopted , and implemented on a routine basis . second , representing a higher degree of institutionalization , select avahan practices need to be reflected in government norms , standard operating procedures , guidelines , and policies . to some degree , this process of institutionalization has already occurred through the development of the current nacp and the norms and guidelines associated with this phase of the program . however , this process of institutionalization needs to continue and be consolidated , particularly at the state level . the final dimension of institutionalization concerns the extent to which the transitioning of avahan into the broader government health system has provoked dynamic changes within that system . for example , the transition process should lead to greater use of government sti services by marps and increased uptake of government counseling , and testing services . to the extent that these high - risk populations have been empowered by community mobilization and are accustomed to services that treat them with respect , they may provide a strong voice within the government health system that calls for improved standards of care for marps . furthermore , there has been substantial concern in the literature about how stand - alone programs may drain government of scarce expertise ( 25 , 26 ) . although these concerns are particularly acute in sub - saharan african countries facing severe and generalized epidemics , the integration of avahan programs into government may still release such scarce technical skills , previously employed by avahan , and make them available to provide broad support across government programs . integration of the avahan program into the government health system may thus give rise to a variety of possible feedback loops and unanticipated consequences of dynamic interactions between actors . the final column reflects both the avahan transition goal ( sustained hiv response through an effective transition ) and the broader outcomes that the achievement of this goal is meant to lead to , including improved coverage of hiv / aids prevention services and a sustained impact on the hiv / aids epidemic in india . finally , the arrow at the bottom of the figure illustrates that learnings from early transition waves will inform the strategies and practices for later phases of the avahan transition . the research protocol was submitted to the johns hopkins school of public health institutional review board ( irb ) , where it was exempted and also to the yrg care irb in chennai , india where it was approved . the protocol was also reviewed by the who - led evaluation advisory group of avahan . the research seeks to address both shorter term questions regarding the implementation of different activities and their proximal effects as well as higher level , and longer term questions regarding the overall achievement of avahan goals . our overarching research question is : 1.has avahan successfully transferred its program to government and other stakeholders in a manner that sustains its effects ? has avahan successfully transferred its program to government and other stakeholders in a manner that sustains its effects ? lower level research questions include : 2.did avahan implement all the elements of its transition strategy , as set out in plans originally agreed with naco and the states ? if not , why not?3.how effective are the different transition elements in achieving transition preparedness?4.what are the elements necessary for effective transition and are any not addressed?5.how effectively have the elements of the avahan transition strategy translated into institutionalization of the program ? did avahan implement all the elements of its transition strategy , as set out in plans originally agreed with naco and the states ? if not , why not ? how effective are the different transition elements in achieving transition preparedness ? how effectively have the elements of the avahan transition strategy translated into institutionalization of the program ? in developing the overall study design , the researchers sought to not only address the research questions identified above but also to craft the study so that it produced early findings that could inform later rounds of transition programming as well as broader overarching conclusions . furthermore , a research study to assess the readiness of cbos to transition has already been initiated separately , and a multidonor process to track the implementation of nacp iii and government 's commitment to the plan is in place . this study will not replicate the other work , but rather will draw on relevant findings from the other assessments to inform the overall evaluation of the transition process . the assessment will focus only on capacity with respect to hiv / aids prevention functions where avahan has provided additional support to naco and select state aids control societies ( sacs ) , and as it relates to the transition . it will assess the extent to which avahan support has contributed to ensuring the availability of staff with appropriate skills and training , in light of their job responsibilities , and with access to relevant norms , guidelines , and job support tools at national and state levels . wp1 plans to employ a structured survey of all staff in relevant units , semi - structured interviews with selected managers , and an administrative record review . this wp assesses the extent to which avahan tis are aligned with government norms prior to transition and how well prepared avahan funded ngos and cbos are for the transition . based on reviews of government norms and standards , a standardized checklist was developed that identifies the key issues in transition alignment ( such as the composition of the ti team , adjustments to budgets and reporting formats , and adherence to guidelines on sti syndromic management ) . structured surveys employing the checklist are planned to be administered at all of the tis just prior to their transitions in 2011 and in 2012 . simple indicators of transition readiness will then be developed so as to measure transition readiness across various dimensions ( such as ngo capacity and alignment of program elements such as costs , sti services ) . it is anticipated that given the phasing of the transition process , evidence from this assessment can be used to inform subsequent rounds of the transition . using a longitudinal case study design , a series of case studies of select tis will be studied to explore in detail how transition preparedness affects transition and institutionalization . the case studies employ both qualitative and quantitative data to describe and analyze these processes and will provide both the details of transitions as well as a picture of the transition process over time . the case studies will also look for any unanticipated consequences of the transition , including any dynamic changes in the system provoked by the transition . it is anticipated that this small - scale qualitative work may also help inform the development of wp4 . this wp seeks to examine the routinization of avahan processes among tis post - transition and more broadly the adoption and institutionalization of avahan learnings within the government . in a preliminary step , the delphi method ( 27 ) was used with avahan staff and partners to identify key features of the avahan approach that should be institutionalized . the study will then assess if key practices associated with avahan tis are adopted and implemented on a routine basis , and whether avahan practices are reflected in government norms , standard operating procedures , guidelines , and policies post - transition . structured questionnaires will be implemented to assess the uptake of avahan learnings and their routinization in ngo and cbo practices . institutionalization is planned to be examined using semi - structured interviews among naco and sacs staff and an administrative record review . the summative evaluation will seek to synthesize findings from different elements of the study , and from relevant studies and assessments conducted by others ( notably on community preparedness for transition , and government commitment to implementation of nacp iii ) so as to consider the entire set of links illustrated in the logic model for transition . drawing on existing data on service coverage and health impacts , this analysis will assess how differing degrees of transition preparedness , government capacity and commitment , and program institutionalization have contributed to sustaining effective services and program outcomes . the evaluation design described here is being implemented by a team of independent evaluators ; however , the team clearly needs to work closely with those implementing avahan , and for many dimensions of the evaluation , the team is reliant on existing sources of secondary data . early experience with implementation of the research has highlighted a number of challenges : secondary data is not always available in consistent formats , there are differences across states , but also before transition ( collected through the avahan monitoring system ) and after transition ( collected by the government system ) . these differences make it very difficult to detect real trends in service delivery for example.although the research is intended to serve the needs of program implementers and inform their actions , program implementers have heavy burdens , particularly at the time of transition , and the evaluation can sometimes be seen as intrusive and yet another burden in an already busy schedule.the research can be viewed as politically sensitive . although the evaluation is primarily concerned with the transition process and the effects of the transition , if not carefully framed , it can appear as an assessment of government performance ( post - transition ) . secondary data is not always available in consistent formats , there are differences across states , but also before transition ( collected through the avahan monitoring system ) and after transition ( collected by the government system ) . these differences make it very difficult to detect real trends in service delivery for example . although the research is intended to serve the needs of program implementers and inform their actions , program implementers have heavy burdens , particularly at the time of transition , and the evaluation can sometimes be seen as intrusive and yet another burden in an already busy schedule . although the evaluation is primarily concerned with the transition process and the effects of the transition , if not carefully framed , it can appear as an assessment of government performance ( post - transition ) . 2 ) was developed through an iterative process involving review of avahan documentation , relevant literature , and interactions with government and avahan staff involved in the design and implementation of the transition . the headings at the top of the figure represent a logical progression , from the activities conducted under the second phase of avahan , to the immediate proximal impacts of these activities ( defined as a state of transition preparedness ) , to the institutionalization of avahan activities within the government system and finally the achievement of the transition goal ( impact is sustained through an effective transition ) . achievement of the goal in turn contributes to india 's national aids control goals and also to the overall purpose of avahan in terms of maintaining or improving trends in reduction of new hiv infections among marps and the general population in india . the first column of the figure draws on discussion of the avahan transition strategy above to identify five main clusters of activities in preparation for transition . three of these sets of activities relate to supporting and adding to the capacities of various entities , namely : 1.supporting government capacity activities include ( 1 ) enhancing the technical and managerial skills of government staff members through training and mentoring ; ( 2 ) supporting quasi - government hiv / aids prevention structures and the systems necessary for those structures to operate effectively , and ( 3 ) supporting the development and production of training materials as well as government norms and guidelines.2.supporting ngo capacity including the provision of capacity development support to implementing partners ( ngos / cbos ) to prepare them for transition and to enable them to take over some of the analytical and management functions previously conducted by avahan contractors , in the post - transition period.3.supporting community capacity including support to community organizations both through strengthening management and governance structures and through building networks of cbos . supporting government capacity activities include ( 1 ) enhancing the technical and managerial skills of government staff members through training and mentoring ; ( 2 ) supporting quasi - government hiv / aids prevention structures and the systems necessary for those structures to operate effectively , and ( 3 ) supporting the development and production of training materials as well as government norms and guidelines . supporting ngo capacity including the provision of capacity development support to implementing partners ( ngos / cbos ) to prepare them for transition and to enable them to take over some of the analytical and management functions previously conducted by avahan contractors , in the post - transition period . supporting community capacity including support to community organizations both through strengthening management and governance structures and through building networks of cbos . the final two activities under the first column concern : 4.alignment of interventions alignment of the technical , managerial , and cost elements of avahan programs with government norms so as to facilitate transition . for example , avahan programs have often operated their own sti clinics , and these services have sometimes provided a broader range of primary - care services ( 24 ) . under government guidelines , ngos are encouraged to establish public private partnerships with private health care providers for sti services and/or develop linkages with government sti services.5.sustaining and monitoring commitment government commitment to high levels of service coverage for tis for high - risk groups is critical to the successful transition of avahan programs . securing and maintaining such commitment is inherently political and thus may be difficult to plan for . while government commitments are already documented in the current nacp and in the 2009 memorandum of cooperation with the bmgf , naco and the development partners , including bmgf , alignment of the technical , managerial , and cost elements of avahan programs with government norms so as to facilitate transition . for example , avahan programs have often operated their own sti clinics , and these services have sometimes provided a broader range of primary - care services ( 24 ) . under government guidelines , ngos are encouraged to establish public private partnerships with private health care providers for sti services and/or develop linkages with government sti services . sustaining and monitoring commitment government commitment to high levels of service coverage for tis for high - risk groups is critical to the successful transition of avahan programs . securing and maintaining such commitment is inherently political and thus may be difficult to plan for . while government commitments are already documented in the current nacp and in the 2009 memorandum of cooperation with the bmgf , naco and the development partners , including bmgf , jointly monitor these commitments , so as to help ensure that they are sustained . these packages of activities are intended to ensure that all organizations involved in program planning , management , delivery , and oversight , both among avahan partners and their counterparts within the government who will take over these functions , reach a state of transition preparedness, so that avahan programs can be transitioned into government systems with minimal disruption . transition preparedness column relate very directly to the five clusters of activities in the first column and indicate that transition preparedness has been achieved with respect to each of these activities . the third column of the transition logic model reflects the processes that need to occur during and after the transition for the final goal ( sustained impact ) to occur . we have called this set of processes institutionalization : by institutionalization , we mean that the key elements of the avahan program are integrated into the organizational procedures and behaviors of government agencies and other key implementing partners . drawing on the dimensions of institutionalization discovered during the literature review , three different levels of institutionalization are identified . at the most basic level , routinization needs to occur , that is , key selected practices associated with avahan - supported tis need to be adapted to better fit government systems , adopted , and implemented on a routine basis . second , representing a higher degree of institutionalization , select avahan practices need to be reflected in government norms , standard operating procedures , guidelines , and policies . to some degree , this process of institutionalization has already occurred through the development of the current nacp and the norms and guidelines associated with this phase of the program . however , this process of institutionalization needs to continue and be consolidated , particularly at the state level . the final dimension of institutionalization concerns the extent to which the transitioning of avahan into the broader government health system has provoked dynamic changes within that system . for example , the transition process should lead to greater use of government sti services by marps and increased uptake of government counseling , and testing services . to the extent that these high - risk populations have been empowered by community mobilization and are accustomed to services that treat them with respect , they may provide a strong voice within the government health system that calls for improved standards of care for marps . furthermore , there has been substantial concern in the literature about how stand - alone programs may drain government of scarce expertise ( 25 , 26 ) . although these concerns are particularly acute in sub - saharan african countries facing severe and generalized epidemics , the integration of avahan programs into government may still release such scarce technical skills , previously employed by avahan , and make them available to provide broad support across government programs . integration of the avahan program into the government health system may thus give rise to a variety of possible feedback loops and unanticipated consequences of dynamic interactions between actors . the final column reflects both the avahan transition goal ( sustained hiv response through an effective transition ) and the broader outcomes that the achievement of this goal is meant to lead to , including improved coverage of hiv / aids prevention services and a sustained impact on the hiv / aids epidemic in india . finally , the arrow at the bottom of the figure illustrates that learnings from early transition waves will inform the strategies and practices for later phases of the avahan transition . the research protocol was submitted to the johns hopkins school of public health institutional review board ( irb ) , where it was exempted and also to the yrg care irb in chennai , india where it was approved . the protocol was also reviewed by the who - led evaluation advisory group of avahan . the research seeks to address both shorter term questions regarding the implementation of different activities and their proximal effects as well as higher level , and longer term questions regarding the overall achievement of avahan goals . our overarching research question is : 1.has avahan successfully transferred its program to government and other stakeholders in a manner that sustains its effects ? has avahan successfully transferred its program to government and other stakeholders in a manner that sustains its effects ? lower level research questions include : 2.did avahan implement all the elements of its transition strategy , as set out in plans originally agreed with naco and the states ? if not , why not?3.how effective are the different transition elements in achieving transition preparedness?4.what are the elements necessary for effective transition and are any not addressed?5.how effectively have the elements of the avahan transition strategy translated into institutionalization of the program ? did avahan implement all the elements of its transition strategy , as set out in plans originally agreed with naco and the states ? if not , why not ? how effective are the different transition elements in achieving transition preparedness ? how effectively have the elements of the avahan transition strategy translated into institutionalization of the program ? in developing the overall study design , the researchers sought to not only address the research questions identified above but also to craft the study so that it produced early findings that could inform later rounds of transition programming as well as broader overarching conclusions . furthermore , a research study to assess the readiness of cbos to transition has already been initiated separately , and a multidonor process to track the implementation of nacp iii and government 's commitment to the plan is in place . this study will not replicate the other work , but rather will draw on relevant findings from the other assessments to inform the overall evaluation of the transition process . the assessment will focus only on capacity with respect to hiv / aids prevention functions where avahan has provided additional support to naco and select state aids control societies ( sacs ) , and as it relates to the transition . it will assess the extent to which avahan support has contributed to ensuring the availability of staff with appropriate skills and training , in light of their job responsibilities , and with access to relevant norms , guidelines , and job support tools at national and state levels . wp1 plans to employ a structured survey of all staff in relevant units , semi - structured interviews with selected managers , and an administrative record review . this wp assesses the extent to which avahan tis are aligned with government norms prior to transition and how well prepared avahan funded ngos and cbos are for the transition . based on reviews of government norms and standards , a standardized checklist was developed that identifies the key issues in transition alignment ( such as the composition of the ti team , adjustments to budgets and reporting formats , and adherence to guidelines on sti syndromic management ) . structured surveys employing the checklist are planned to be administered at all of the tis just prior to their transitions in 2011 and in 2012 . simple indicators of transition readiness will then be developed so as to measure transition readiness across various dimensions ( such as ngo capacity and alignment of program elements such as costs , sti services ) . it is anticipated that given the phasing of the transition process , evidence from this assessment can be used to inform subsequent rounds of the transition . using a longitudinal case study design , a series of case studies of select tis will be studied to explore in detail how transition preparedness affects transition and institutionalization . the case studies employ both qualitative and quantitative data to describe and analyze these processes and will provide both the details of transitions as well as a picture of the transition process over time . the case studies will also look for any unanticipated consequences of the transition , including any dynamic changes in the system provoked by the transition . it is anticipated that this small - scale qualitative work may also help inform the development of wp4 . this wp seeks to examine the routinization of avahan processes among tis post - transition and more broadly the adoption and institutionalization of avahan learnings within the government . in a preliminary step , the delphi method ( 27 ) was used with avahan staff and partners to identify key features of the avahan approach that should be institutionalized . the study will then assess if key practices associated with avahan tis are adopted and implemented on a routine basis , and whether avahan practices are reflected in government norms , standard operating procedures , guidelines , and policies post - transition . structured questionnaires will be implemented to assess the uptake of avahan learnings and their routinization in ngo and cbo practices . institutionalization is planned to be examined using semi - structured interviews among naco and sacs staff and an administrative record review . the summative evaluation will seek to synthesize findings from different elements of the study , and from relevant studies and assessments conducted by others ( notably on community preparedness for transition , and government commitment to implementation of nacp iii ) so as to consider the entire set of links illustrated in the logic model for transition . drawing on existing data on service coverage and health impacts , this analysis will assess how differing degrees of transition preparedness , government capacity and commitment , and program institutionalization have contributed to sustaining effective services and program outcomes . the assessment will focus only on capacity with respect to hiv / aids prevention functions where avahan has provided additional support to naco and select state aids control societies ( sacs ) , and as it relates to the transition . it will assess the extent to which avahan support has contributed to ensuring the availability of staff with appropriate skills and training , in light of their job responsibilities , and with access to relevant norms , guidelines , and job support tools at national and state levels . wp1 plans to employ a structured survey of all staff in relevant units , semi - structured interviews with selected managers , and an administrative record review . this wp assesses the extent to which avahan tis are aligned with government norms prior to transition and how well prepared avahan funded ngos and cbos are for the transition . based on reviews of government norms and standards , a standardized checklist was developed that identifies the key issues in transition alignment ( such as the composition of the ti team , adjustments to budgets and reporting formats , and adherence to guidelines on sti syndromic management ) . structured surveys employing the checklist are planned to be administered at all of the tis just prior to their transitions in 2011 and in 2012 . simple indicators of transition readiness will then be developed so as to measure transition readiness across various dimensions ( such as ngo capacity and alignment of program elements such as costs , sti services ) . it is anticipated that given the phasing of the transition process , evidence from this assessment can be used to inform subsequent rounds of the transition . using a longitudinal case study design , a series of case studies of select tis will be studied to explore in detail how transition preparedness affects transition and institutionalization . the case studies employ both qualitative and quantitative data to describe and analyze these processes and will provide both the details of transitions as well as a picture of the transition process over time . the case studies will also look for any unanticipated consequences of the transition , including any dynamic changes in the system provoked by the transition . it is anticipated that this small - scale qualitative work may also help inform the development of wp4 . this wp seeks to examine the routinization of avahan processes among tis post - transition and more broadly the adoption and institutionalization of avahan learnings within the government . in a preliminary step , the delphi method ( 27 ) was used with avahan staff and partners to identify key features of the avahan approach that should be institutionalized . the study will then assess if key practices associated with avahan tis are adopted and implemented on a routine basis , and whether avahan practices are reflected in government norms , standard operating procedures , guidelines , and policies post - transition . structured questionnaires will be implemented to assess the uptake of avahan learnings and their routinization in ngo and cbo practices . institutionalization is planned to be examined using semi - structured interviews among naco and sacs staff and an administrative record review . the summative evaluation will seek to synthesize findings from different elements of the study , and from relevant studies and assessments conducted by others ( notably on community preparedness for transition , and government commitment to implementation of nacp iii ) so as to consider the entire set of links illustrated in the logic model for transition . drawing on existing data on service coverage and health impacts , this analysis will assess how differing degrees of transition preparedness , government capacity and commitment , and program institutionalization have contributed to sustaining effective services and program outcomes . the evaluation design described here is being implemented by a team of independent evaluators ; however , the team clearly needs to work closely with those implementing avahan , and for many dimensions of the evaluation , the team is reliant on existing sources of secondary data . early experience with implementation of the research has highlighted a number of challenges : secondary data is not always available in consistent formats , there are differences across states , but also before transition ( collected through the avahan monitoring system ) and after transition ( collected by the government system ) . these differences make it very difficult to detect real trends in service delivery for example.although the research is intended to serve the needs of program implementers and inform their actions , program implementers have heavy burdens , particularly at the time of transition , and the evaluation can sometimes be seen as intrusive and yet another burden in an already busy schedule.the research can be viewed as politically sensitive . although the evaluation is primarily concerned with the transition process and the effects of the transition , if not carefully framed , it can appear as an assessment of government performance ( post - transition ) . secondary data is not always available in consistent formats , there are differences across states , but also before transition ( collected through the avahan monitoring system ) and after transition ( collected by the government system ) . these differences make it very difficult to detect real trends in service delivery for example . although the research is intended to serve the needs of program implementers and inform their actions , program implementers have heavy burdens , particularly at the time of transition , and the evaluation can sometimes be seen as intrusive and yet another burden in an already busy schedule . although the evaluation is primarily concerned with the transition process and the effects of the transition , if not carefully framed , it can appear as an assessment of government performance ( post - transition ) . there are a number of aspects of the current study design , which we believe are quite innovative . first , to the best of our knowledge , this is the first prospective analysis of a transition and institutionalization process that seeks to determine the effects of these processes on sustainability . second , the fact that this is a mixed - method study enables us to triangulate between different data sources and use the rich detail available in the qualitative research to inform the design and analysis of quantitative research components . third , the study has been designed in a phased manner , so that early practical lessons from the research can be used to inform later rounds of the transition process . it is difficult at this point in time to know whether the right balance has been struck . there are clearly elements of the avahan transition process that our logic model and research design do not play close attention to ( for example , efforts to change structural issues such as attitudes toward fsws , high - risk msm , and transgenders ) and yet could be critical for transition success . conversely , although we are conceptually clear as to how different components of the overall study design relate , we believe that there will be substantial challenges in synthesizing data collected through different wps and understanding the broader chain of connections between transition preparedness , program institutionalization , and sustainability outcomes . although this study is tailored to one particular , albeit very large program , we believe that both its methods and findings are likely to be relevant to other programs and contexts . although substantial effort is frequently invested in the design of a new program , there is rarely a comparable investment in ensuring the sustainability of program benefits . in the current financially constrained context , carefully planned program transitions may be the key to ensuring allocative efficiency and the sustainability of benefits . there are a number of aspects of the current study design , which we believe are quite innovative . first , to the best of our knowledge , this is the first prospective analysis of a transition and institutionalization process that seeks to determine the effects of these processes on sustainability . second , the fact that this is a mixed - method study enables us to triangulate between different data sources and use the rich detail available in the qualitative research to inform the design and analysis of quantitative research components . third , the study has been designed in a phased manner , so that early practical lessons from the research can be used to inform later rounds of the transition process . it is difficult at this point in time to know whether the right balance has been struck . there are clearly elements of the avahan transition process that our logic model and research design do not play close attention to ( for example , efforts to change structural issues such as attitudes toward fsws , high - risk msm , and transgenders ) and yet could be critical for transition success . conversely , although we are conceptually clear as to how different components of the overall study design relate , we believe that there will be substantial challenges in synthesizing data collected through different wps and understanding the broader chain of connections between transition preparedness , program institutionalization , and sustainability outcomes . although this study is tailored to one particular , albeit very large program , we believe that both its methods and findings are likely to be relevant to other programs and contexts . although substantial effort is frequently invested in the design of a new program , there is rarely a comparable investment in ensuring the sustainability of program benefits . in the current financially constrained context , carefully planned program transitions may be the key to ensuring allocative efficiency and the sustainability of benefits . work on this assessment has been funded by the bill and melinda gates foundation that also supports the implementation of the avahan program .

backgroundsustainability is the holy grail of many development projects , yet there is limited evidence about strategies that effectively support transition of programs from donor funding to national governments . the first phase of avahan , the india aids initiative supported by the bill and melinda gates foundation ( 20032009 ) , aimed to demonstrate an hiv / aids prevention program at scale , primarily targeted at high - risk groups . during the second phase ( 20092013 ) , this large - scale program will be transitioned to its natural owners : the government of india and local communities . this paper describes the evaluation design for the avahan transition strategy.methods/designa detailed logic model for the transition was developed . the avahan transition strategy focuses on three activities : ( 1 ) enhancing capacities among communities , non - governmental organizations ( ngos ) , and government entities , in line with india 's national aids control strategy ; ( 2 ) aligning technical and managerial aspects of avahan programs with government norms and standards ; and ( 3 ) promoting and sustaining commitment to services for most - at - risk populations . it is anticipated that programs will then transfer smoothly to government and community ownership , become institutionalized within the government system , and support a sustained hiv / aids response.the research design evaluates the implementation and effectiveness of ( 1 ) activities undertaken by the program ; ( 2 ) intermediate effects including the process of institutionalization and the extent to which key avahan organizational procedures and behaviors are integrated into government systems ; and ( 3 ) overarching effects namely the impact of the transition process on the sustained delivery of hiv / aids prevention services to high - risk groups . both qualitative and quantitative research approaches are employed so that the evaluation will both assess outcomes and explain why they have occurred.conclusionsit is unusual for donor - supported projects in low- and middle - income countries to carefully plan transition processes , and prospectively evaluate these . this evaluation is designed so as to both inform decision making throughout the transition process and answer larger questions about the transition and sustainability of donor programs .

Avahan transition strategy Previous evaluations of transition Methods and design A transition logic model Management and governance Research objectives and questions Research design WP1 government capacity assessment (addressing research questions 2 and 3) WP2 NGO and TI transition preparedness (addressing research questions 2, 3, and 4) WP3 longitudinal case studies of TIs (addressing research questions 4 and 5) WP4 institutionalization assessment (addressing research questions 1 and 5) WP5 summative evaluation (research question 1) Research implementation Discussion Strengths and limitations Contribution Conflict of interest and funding

PMC3005839

histone proteins package eukaryotic dna into chromatin , a multilevel array in which nucleosomes comprise the basic unit . the nucleosome consists of a core , ~147 base pairs ( bp ) wrapped around a histone octamer in 1.67 left - handed turns , in addition to a variable length of linker dna . by influencing dna site exposure and factor association , nucleosome positioning provides a platform for regulation , such as gene - specific control of transcription . in conjunction with a variety of histone variant substitutions , posttranslational modifications , and chromatin remodeling activities , nucleosome organization and dynamics allow for site selectivity in genomic transactions well beyond dna primary structure . many nuclear activities require open chromatin states and at least transient alteration of nucleosomes [ 4 , 5 ] . as the most abundant nonhistone proteins in the nucleus , high mobility group factors assist in these processes by modifying chromatin architecture . within this protein family , hmgns are unique in having specific affinity for the nucleosome core , wherein they act to facilitate processes including dna repair , replication , and transcription by unfolding chromatin [ 7 , 8 ] . hmgn variants modulate distinct histone modifications , and they display cell cycle - dependent binding to chromatin and differential expression during development . under physiological conditions , hmgn1 and hmgn2 bind cooperatively to the nucleosome core to form species containing two molecules of either one or the other variant ( not mixed pairs ) [ 10 , 12 ] . specificity and affinity are conferred by an ~30 amino acid n - terminal motif the nucleosome - binding domain ( nbd ) [ 13 , 14 ] . in contrast to the highly positive - charged nbd , the c - terminus contains a very acidic region , encompassing the regulatory domain ( rd ) , which is required for high - affinity binding to chromatin , transcription stimulation , and disruption of linker histone h1-mediated chromatin compaction [ 15 , 16 ] . in spite of extensive biochemical and functional analysis of the hmgns , their chromatin - modifying activities are intricate , and mechanisms of action have remained elusive . in this work , we have composed a reconstituted xenopus laevis system to investigate the nature of structural and thermodynamic alterations arising from hmgn - nucleosome interactions . our findings hold significance for understanding the architectural changes in chromatin elicited by this family of nuclear factors . xenopus laevis hmgn1 and hmgn2 expression constructs were generated by inserting codon - optimized genes into the ndei and bamhi sites of pet-3a vector ( ezbiolab inc . , proteins were overexpressed in e. coli and purified using a similar approach as applied previously for recombinant hmgns . the supernatant of the cell lysate , obtained by homogenization in a buffer of 50 mm tris - hcl ( ph 7.5 ) , 0.5 m nacl , 1 mm edta , 1 mm -mercaptoethanol , and 1 mm phenylmethylsulphonyl fluoride ( pmsf ) , was subjected to size - exclusion chromatography using a 26/60 sephacryl s-200 column ( ge healthcare , uppsala , sweden ) pre - equilibrated with a buffer of 20 mm tris - hcl ( ph 7.5 ) , 0.2 m nacl , 1 mm edta , and 1 mm pmsf . further chromatographic purification was carried out using a mono s or resource s cation - exchange column ( ge healthcare , uppsala , sweden ) equilibrated with a buffer of 20 mm tris - hcl ( ph 7.5 ) , 0.1 m nacl , and 1 mm edta . hmgn eluted over a gradient of 0.2 to 0.4 m nacl ( 20 mm tris - hcl ( ph 7.5 ) , 1 mm edta ) . purified hmgn1 ( 11.4 kd ) , hmgn2 ( 9.4 kd ) , and hmgn1 t ( 8.9 kd ) were subjected to n - terminal sequencing and mass spectrometry analysis to establish composition . nucleosome core particle ( ncp ) was assembled with xenopus laevis histones as described previously . ncp147 and ncp146b are composed , respectively , of 147 bp and 146 bp derivatives of human -satellite dna . ncp-601 was produced from a 145 bp fragment , atcagaatcccggtgccgaggccgctcaattggtcgtagacagctctagcaccgcttaaacgcacgtacgcgctgtcccccgcgttttaaccgccaaggggattactccctagtctccaggcacgtgtcagatatatacatcgat , corresponding to the strong positioning array and 12-nucleosome array ( gifts from t. richmond , eth - zurich ) , composed , respectively , of 167 bp and 177 bp repeats with the 601 core element , were produced using a modified version of the original protocol [ 22 , 23 ] . trace amounts of residual plasmid vector fragments arising from the ecorv digest for 12-nucleosome array or exogenously added 145 bp dna for 4-nucleosome array served as an excess histone octamer sink during reconstitution to prevent oversaturation of the array dna . contaminating ncp and small nucleosomal assemblies were eliminated by differentially precipitating array with the addition of 2.5 to 4 mm mgcl2 . histone octamer saturation of array was confirmed via scai restriction digestion analysis , whereby cleavage occurs at the linker dna midpoint between nucleosome sites ( see supplementary figure 1 in supplementary material available online at doi : 10.4061/2010/143890 , which includes 5 figures ) . electrophoretic mobility shift assays ( emsas ) were carried out with three different running buffers ; approximate physiological ionic strength buffer corresponded to 1x tbe ( 89 mm tris - hcl , 89 mm boric acid , 1.0 mm edta , and ph 8.3 ) , and low ionic strength buffer was , with two exceptions , 0.25x tbe . for the 12-array samples in the upper panels of figure 2(a ) and figure 3(c ) , the buffer was 0.25x tae ( 10 mm tris - hcl , 10 mm acetic acid , 0.25 mm edta , and ph 8.3 ) . electrophoresis under divalent metal conditions was conducted using a 1x tb buffer ( 89 mm tris - hcl , 89 mm boric acid , and ph 8.3 ) with the addition of 1 mm mgcl2 or cacl2 . gels were loaded with aliquots having nucleosome core site concentrations of 0.5 m for both ncp and 12-array samples . preassembled ncp or array was incubated with hmgn or nbd peptide in respective 0.25x or 1x running buffer for approximately 30 min at room temperature prior to electrophoresis at 4c . for coassembly trials , hmgn was introduced either at the onset of salt dialysis - based histone - dna reconstitution or at a kcl concentration of 0.4 m , corresponding to the midpoint . ncp was analyzed with 6% polyacrylamide gels , whereas analysis of array was conducted using 1% agarose/1.3% polyacrylamide composite gels . hmgn was added at desired stoichiometry to 0.75 m dna or ncp in 1x tb or 1x tb + 1 mm mgcl2 buffer , and samples were allowed to incubate at room temperature for at least 10 minutes before taking measurements using a varian cary 300 bio uv / vis spectrophotometer equipped with a temperature controller . data collection entailed monitoring uv absorbance at 260 nm wavelength from ~20 to ~95c over 1 intervals . first derivative of absorbance versus temperature values ( h/t ) were calculated with the instrument software using standard settings . initial h/t versus temperature profiles were normalized by adjusting with respect to a h/t maximum setting of 1.0 ( yielding h/t * values in figure 3 ) . in the production of recombinant xenopus hmgn1 and hmgn2 , we also obtained a c - terminal truncation product , hmgn1 t , lacking 25 amino acids from the rd ( figure 1 ) . the three proteins bind cooperatively to form 2 : 1 hmgn : nucleosome core particle ( ncp ) species ( s1 ) at near physiological ionic strength ( see figure 1(a ) ) . the relative electrophoretic mobility of s1 for hmgn1 compared to hmgn2 changes with buffer conditions , whereby hmgn1-s1 migrates slowest at low ionic strength . at near physiological ionic strength , the relative migration becomes roughly equivalent , and with the addition of 1 mm mg , hmgn2-s1 is slowest . therefore , binding of two molecules of hmgn1 or hmgn2 to the ncp results in different ion - dependent conformations . in the presence of mg and at two and above protein to ncp molar ratio , a distinct species ( s2 ) is observed for hmgn2 or hmgn1 t binding , which displays dramatically reduced electrophoretic mobility ( see figures 1(a ) and 1(b ) ) . this s1-s2 transition has no apparent dna sequence dependence since both ncp constructs ( ncp147 and ncp-601 ) , composed of unrelated sequences , display nearly identical behavior . furthermore , mg - dependent s2 formation is also observed for hmgn1 t or hmgn2 binding to a 12-nucleosome array ( 12-array ; figure 2 ) . similar to ncp , hmgn : nucleosome ratios of 2 : 1 and above can trigger the s2 transition for the 12-array . in order to determine the basic components required for s2 formation , we investigated whether the phenomenon is mg specific . however , ca is equally effective at eliciting the transition , which therefore displays a divalent metal dependency ( figure 3(a ) ) . also , considering that the truncated form of hmgn1 is capable of causing s1-s2 transition , we tested whether the nbd alone has such activity . yet , 32 amino acid peptides corresponding to the nbd of either hmgn1 or hmgn2 ( see figure 1(d ) ) are unable to evoke s2 , even at very high stoichiometry ( figures 3(b ) and 3(c ) ) . therefore , the residues necessary for producing the transition reside outside the nbd of hmgn1 and hmgn2 . moreover , although the hmgn1 nbd peptide is able to bind to the 12-array at near physiological ionic strength in the presence of mg , association of the hmgn2 nbd peptide under these chromatin compacting conditions is not observed ( see figure 3(c ) ) . it is important to note that the hmgn2 nbd peptide does bind readily to ncp under identical conditions ( see figure 3(b ) ) . the electrophoretic mobility shift assays ( emsas ) reveal analogous behavior for both ncps and the 12-array , and the extreme reduction in migration rate of s2 relative to s1 is consistent with a pronounced conformational change of the nucleosome core to a less compact state . the nature of this transition suggests that it could involve irreversible nucleosome disassembly or modification . however , we find that the s1-s2 conversion can be completely reversed by either removing mg or reducing the hmgn : nucleosome stoichiometry ( supplementary figure 2 ) . although the s1-s2 transition is readily reversible , structural rearrangements in the nucleosome may be influenced by the presence of cofactors during chromatin assembly . we therefore tested whether introduction of hmgn at the beginning or midpoint of the nucleosome reconstitution process , as opposed to protein addition after assembly , affects the outcome ( supplementary figures 3 and 4 ) . differences could arise , for instance , through competition between hmgn and h2a - h2b dimer for the same dna - binding sites . however , ncp or 12-array coassembled with hmgn behaves in an apparently identical fashion as preassembled material , with the former also capable of s1 and s2 formation . in order to delineate the influence of buffer condition and hmgn binding , we conducted thermal denaturation experiments to assess nucleosome stability ( figure 4 ) [ 2528 ] . this assay is based on the hyperchromic effect of increased dna uv absorbance arising from base unstacking . in this way , the unwinding of the double helix can be monitored , yielding a sigmoidal melting profile , for which the first derivative is equivalent to change in enthalpy with respect to temperature ( h/t ) . the sigmoidal inflexion points , or maximal h/t values , correspond to melting temperatures ( tm ) associated with one or more transitions . moreover , the relative areas in h/t plots associated with multiple transitions are proportional to the relative extents of double helix unwinding . thermal denaturation of the nucleosome is generally associated with two transitions [ 2528 ] . this is followed by the major transition at elevated temperature , in which the remaining double helix melts . as observed previously for samples in monovalent cation buffer , the magnitude of the minor transition is much smaller relative to the major transition for ncp thermally denatured in near physiological ionic strength buffer ( see figures 4(b ) and 4(c ) ) . however , with the addition of 1 mm mg or ca , the minor transition becomes much more pronounced , indicative of a substantially greater extent of dna unwinding arising from divalent metal - mediated destabilization of the nucleosome core termini . moreover , the tm of the major transition can be lowered from the presence of divalent metal in particular for ca . the overall destabilization of the ncp from the presence of divalent metal is particularly striking in comparison with the naked dna fragments , which display the opposite behavior as a consequence of double helix stabilization by mg and ca ( see figure 4(a ) ) . as a result , under the present divalent metal conditions , the tm for the initial minor ncp transition is similar to the tm for the respective naked dna fragment . this indicates that , from the modulating influence of divalent cations , histone octamer association may provide only weak stabilization of the terminal dna arms in the nucleosome . in conjunction with the divalent metal conditions , the presence of saturating stoichiometry of either hmgn2 or hmgn1 t shows a profound further reduction in stability of the nucleosome termini relative to ncp alone ( see figures 4(e)4(h ) ) . moreover , what is initially the minor transition at lower temperature under hmgn - free conditions becomes the main transition , encompassing the majority of the double helix . in addition , the tm for this early transition is significantly reduced relative to ncp alone . thus , both the ease and extent of unwinding the nucleosome core termini is increased dramatically with hmgn2 or hmgn1 t association . although a slight destabilization of the termini is apparent from saturating amounts of hmgn2 or hmgn1 t in near physiological ionic strength monovalent cation buffer , the major effect is clearly contingent on the presence of divalent metal ( see figure 4(d ) ) . in contrast to the divalent metal - dependent influence of hmgn2 or hmgn1 t association , for hmgn1 binding , one sees a much diminished effect on reducing nucleosome termini stability ( see figures 4(d)4(h ) ) . reconstitution of ncp typically yields off - centered and centered forms , which differ in positioning of the histone octamer on the dna by 10 bp ( figure 5 ) . compared to the thermodynamically favored centered ncp , the off - centered form lacks a histone h3 n - terminal tail - dna interaction at the recessed end and contains an additional section of linker dna on the opposing half . in order to illuminate potential mechanistic features for hmgn - induced decompaction of the nucleosome , we investigated protein binding to off - centered ncp . under low ionic strength conditions , a distinct specific species for hmgn binding to off - centered ncp is observed , which has electrophoretic mobility intermittent between the 1 : 1 and 2 : 1 species seen for purely centered ncp ( see figure 5(b ) ) . moreover , under near physiological ionic strength conditions , where cooperative binding to centered ncp occurs , two additional specific species are observed for off - centered ncp . the migration rates of the new off - centered species relative to those for centered ncp and their prevalence at either only low or high hmgn stoichiometry suggests that they coincide with 1 : 1 and 2 : 1 assemblies . the existence of a 1 : 1 off - centered complex at physiological ionic strength would in turn imply that cooperative hmgn binding to the nucleosome core requires intact elements at the termini . in the presence of mg , off - centered ncp displays a dramatic reduction in electrophoretic mobility relative to the centered form , consistent with a substantial decrease in compactness of the nucleosome core ( see figure 5(b ) and supplementary figure 5 ) . furthermore , in addition to hmgn2 and hmgn1 t , hmgn1 can associate with this off - centered species to generate an assembly with migration properties very similar to s2 . this suggests that disruption of histone - dna interactions at one terminus may render the nucleosome core susceptible to transition to a decompacted conformation , to which hmgns can stably associate . hmgn1 and hmgn2 have both been shown to enhance transcription from and promote nuclease digestion of minichromosomes assembled in nuclear / ovum extracts , but the effect has largely been observed only if hmgn is present during , as opposed to being introduced after , chromatin assembly [ 7 , 16 , 29 , 30 ] . here , we have constructed a bottom - up system in order to uncover fundamental distinctions between hmgn variants . we do not observe any significant differences arising from whether hmgn is present from the start or midpoint of nucleosome assembly , as compared to protein addition subsequent to reconstitution . in conjunction with the observation that a significant fraction of hmgn in the nucleus is associated with metastable multiprotein complexes , this indicates that there are likely multiple factors , such as histone tail modifications , which can regulate or influence hmgn activity in vivo . although hmgn1 and hmgn2 are known to modulate distinct activities [ 9 , 11 ] and localize to separate regions in the nucleus , differences in chromatin structural attributes associated with the two variant types are not known . with the trials of divalent metal - containing conditions notwithstanding , our results do not reveal any pronounced conformational or stability differences of hmgn - nucleosome assemblies between the two variants . on the other hand , association of hmgn2 in the presence of mg or ca the ubiquitous divalent cations in the nucleus leads to a substantial reduction in stability of the nucleosome core dna termini and appears to trigger a conformational switch in the nucleosome to a less compact state . however , in contrast to hmgn2 , we do not observe a pronounced stability or structural transition elicited through hmgn1 association , which is nonetheless consistent with a previous hmgn1-nucleosome array investigation . although residues n - terminal to the nbd may play a role , the activity we observe is likely conferred by elements within or in the vicinity of the rd , since it is also displayed by our c - terminal truncation species , hmgn1 t , but not by the nbd peptides . in fact , hmgn1 t is similar to the hmgn3b variant , which also lacks a substantial portion of the rd compared to hmgn1 . in this regard , the acidic 10 amino acid region at the c - terminus of hmgn1 t has similar character compared to an element within the hmgn2 rd situated at roughly identical distance in primary structure from the nbd ( see figure 1(d ) ) . thus , variations in the hmgn c - terminus arising from differences in expression or posttranslational modification could allow fine regulation of nucleosome stability and structural transitions . hmgn1 and hmgn2 have been found to localize to separate clusters of nucleosomes in vivo , and this differential clustering in the nucleus is promoted a priori by the exclusive nucleosome binding behavior of these two variants . the distinct conformational and thermodynamic properties we observe for hmgn1 versus hmgn2 assemblies support a previously proposed allosteric mechanism underlying the absence of mixed variant pairs bound to an individual nucleosome . in low ionic strength buffer , the electrophoretic migration rate for s1 displays a typical proportionality to the molecular weight of the hmgn - ncp assembly . under more physiological ionic conditions , however , the hmgn2-s1 migrates equally or more slowly with respect to hmgn1-s1 , in spite of the greater mass of the latter . this suggests a conformational change to a somewhat less compact state upon hmgn2 association with the nucleosome under cooperative binding conditions . in addition , under approximate physiological ionic conditions with mg , the hmgn1 nbd peptide can associate with ncp or 12-array , whereas binding of the hmgn2 nbd peptide to ncp , but not 12-array , is observed . considering that the isolated hmgn2 nbd also displays homopaired association suggests that the binding of this nbd alone elicits a conformational change as well , which is suppressed by nucleosome compaction . thus , overall it appears that binding of one hmgn2 induces a conformational change in the nucleosome , which facilitates association of a second hmgn2 while preventing that of hmgn1 . although previous work has shown that association of hmgn1 and hmgn2 increases the stability of the nucleosome core , these thermal denaturation studies were conducted at low ionic strength with monovalent cation buffers . in contrast to the inhibitory effect of mg and ca on dissociation of the double helix , we find a pronounced stability reduction for the nucleosome core in the presence of only low concentrations of these divalent cations . although there is a general decrease in tm for the final high - temperature transition , the primary influence relates to destabilization of the dna ends at the nucleosome core termini . we recently completed a crystallographic study characterizing in detail counterion binding in the nucleosome core and found that divalent metal hydrates and the histone n - terminal tails can compete with each other for association with the same sites in the dna minor groove . therefore , considering that the terminal histone - dna interactions are the weakest and most dynamic [ 19 , 3840 ] , the main divalent metal - mediated stability reduction apparently arises through binding competition , such that the nucleosomal dna arms are more frequently in a disassociated state . this is consistent with the apparent divalent metal - mediated decompaction we observe for off - centered ncp , which is missing an h3 n - terminal tail - dna interaction at one terminus , and previous work has indicated that the h3 tail is critical for maintaining nucleosome stability in any case . accordingly , hmgn2 and hmgn1 t apparently act in a synergistic capacity with divalent metal to further decrease stability of the nucleosome termini . although the combined nucleosome destabilization arises , at least in part , from a direct effect of mg or ca , divalent metal binding to acidic residues abundant in the hmgn c - termini potentially altering protein conformation may also be involved in the activity . we observe divalent metal - dependent stability of the nucleosome core and differences in the nucleosome modulation activities of hmgn1 and hmgn2 . this emphasizes the potential importance of testing the influence of mg and ca in chromatin studies . within our in vitro system established here , hmgn2 appears capable of destabilizing the nucleosome core , while hmgn1 may require additional factors for similar activity . further investigations , which include extra nuclear components or probe the influence of posttranslational modifications , could help elucidate the mechanism of chromatin unfolding by the hmgns .

high mobility group n proteins ( hmgns ) bind specifically to the nucleosome core and act as chromatin unfolding and activating factors . using an all - xenopus system , we found that hmgn1 and hmgn2 binding to nucleosomes results in distinct ion - dependent conformation and stability . hmgn2 association with nucleosome core particle or nucleosomal array in the presence of divalent metal triggers a reversible transition to a species with much reduced electrophoretic mobility , consistent with a less compact state of the nucleosome . residues outside of the nucleosome binding domain are required for the activity , which is also displayed by an hmgn1 truncation product lacking part of the regulatory domain . in addition , thermal denaturation assays show that the presence of 1 mm mg2 + > or ca2 + gives a reduction in nucleosome core terminus stability , which is further substantially diminished by the binding of hmgn2 or truncated hmgn1 . our findings emphasize the importance of divalent metals in nucleosome dynamics and suggest that the differential biological activities of hmgns in chromatin activation may involve different conformational alterations and modulation of nucleosome core stability .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC5371393

heat - shock proteins are a highly conserved family of molecular chaperones that facilitate the folding , stability , and cellular localization of their substrate proteins . up - regulation of the pathways associated with the heat - shock response has been implicated in a number of disease areas , including cancer . recent focus has been on the inhibition of the molecular chaperone heat - shock protein 90 ( hsp90 ) using adenosine triphosphate ( atp ) competitive inhibitors , an approach that has resulted in considerable success as several compounds have now entered clinical trials . the heat - shock protein 70 ( hsp70 ) family of molecular chaperones represents another potential target for small - molecule mediated antagonism of the heat - shock response pathway . the hsp70 isoform , heat - shock cognate 70 ( hsc70 ) , is ubiquitously expressed in tissues , while the inducible isoform , heat - shock protein 72 ( hsp72 ) , is largely expressed in response to stress , including treatment with hsp90 inhibitors , and aids cell survival through inhibition of several apoptotic pathways . we have previously shown that dual knockdown of these two hsp70 isoforms in human colon and ovarian tumor cell lines results in apoptosis , which was in contrast with nontumorigenic cell lines where apoptosis was not observed , indicating a potential therapeutic window for hsp70 inhibitors . to execute their refolding activity , the hsp70 proteins utilize the hydrolysis of atp to adenosine diphosphate ( adp ) and inorganic phosphate ( adp / pi ) in a complex catalytic cycle involving a number of protein conformational changes and through a process which is tightly regulated by various cochaperones such as the heat - shock protein 40 ( hsp40 ) proteins and the nucleotide exchange factor bag family molecular chaperone regulator 1 ( bag1 ) protein . while this complexity presents numerous opportunities to antagonize the refolding activity of hsp70 , the clearest strategy remains atp - competitive binding of inhibitors to the conserved nucleotide - binding domain of the protein . there remains only one published chemotype which displays atp - competitive submicromolar inhibition of hsp70 and has been shown to be effective in cellular assays , a chemotype derived from adenosine ( figure 1 ) . the affinity of three known hsp70 inhibitors derived from adenosine and measured by spr , see ref ( 7 ) for details . the atpase domain of hsp70 is a member of the actin atpase family of proteins , a target class which has delivered very little success in the discovery of high affinity ligands . a recent study to assess the potential of the hsp70-atp binding site for antagonism with small molecules using sitemap described the target as difficult , while a separate analysis using a fragment - based screening approach returned a very low hit rate ( 0.4% ) , a result generally associated with low ligandability . several studies into the biochemical mechanism of hsp70 refolding activity and atp hydrolysis have demonstrated that the atp binding site of hsp70 in solution is highly flexible in nature , undergoing numerous conformational changes . with the challenge of finding atp - competitive hit matter against hsp70 hindering the potential development of inhibitors for this important target , we sought to investigate the binding mechanism of adenosine - derived ligands to the atp site of hsp70 . the aim was to improve our understanding of how high affinity ligands bind to this region of the protein so that this knowledge could be applied to future inhibitor design . the slow turnover of atp by hsp70 , and the potent product inhibition by adp / pi , means that using functional assays is a challenge for the characterization of hsp70 ligand binding . therefore , we focused on surface plasmon resonance ( spr ) as a biophysical method to assess the affinity of ligands . unfortunately , full - length human hsp72 gave poor spr data in our hands , displaying erratic and difficult to interpret sensorgrams . therefore , the nucleotide - binding domain ( nbd ) of human hsc70 ( hsc70-nbd residues 1381 ) was used in all spr experiments . adenosine 1 is a relatively weak ligand for hsc70-nbd , displaying a pkd= 3.95 0.01 ( kd = 110 m , n = 3 ) , when measured by spr , but we decided to use this compound as a starting point for our investigations into the binding mechanisms of this chemotype to the hsp70 proteins . we began by analyzing the importance of the ribose motif to the binding affinity of adenosine 1 . removing either the 2- or 3-hydroxyl groups from the sugar motif or changing their relative and absolute stereochemistry resulted in no measurable removal of either the 6-amino group or the 3-nitrogen of the adenine ring also resulted in the loss of all measurable affinity . these results demonstrate the importance of the ribose motif and the adenine aminopyrimidine motif to binding of adenosine - derived ligands to the hydrophilic region of the protein . in contrast , removal of the 5-hydroxyl was well tolerated , as compound 2 retained its affinity in the spr assay with a pkd = 3.88 0.02 ( kd = 130 m , n = 3 ) ( figure 2 and supporting information table 1 ) . the heteroatoms and stereochemistry highlighted in red are important for affinity to hsp70 and could not be replaced by hydrogen or ch or the stereochemistry changed . it has previously been shown by massey et al . that addition of a primary or secondary amine to the 8-position of adenosine resulted in a significant increase in affinity for these ligands . in our hands , 8-aminoadenosine 3 gave a pkd = 5.16 0.01 ( kd = 7.0 m , n = 3 ) , a 16-fold increase in affinity when compared to adenosine 1 . we decided to investigate this substituent in order to better understand its role in the improved affinity of these compounds ( table 1 ) . all results are quoted as the geometric mean standard error of the mean ( sem ) of three independent experiments unless otherwise stated , pkd = log10(kd(m ) 10 ) . methyl substitution on the 8-amino group ( entry 2 ) gave ligand 4 and resulted in a 2.5-fold drop in affinity , while dimethyl analogue 5 ( entry 3 ) displayed a kd greater than 1 mm . replacement of the 8-amino substituent with a methoxy group to give 6 ( entry 4 ) also resulted in a complete loss of activity . this dramatic effect on binding suggested that one of the hydrogens of the 8-amino group was involved in hydrogen bonding . to assess whether an intramolecular hydrogen bond to the 5-hydroxyl was important for affinity , we removed this group to give 7 ( entry 5 ) . however , only a 1.6-fold drop in affinity was observed with analogue 7 when compared with 8-n - methylaminoadenosine 4 ( entry 2 ) . these results suggest that an intramolecular hydrogen bond between the 8-amino substituent and the 5-hydroxyl group can not rationalize the increased affinity observed with the 8-aminoadenosine series . we speculated that the role of the 8-amino substituent as a hydrogen bond donor is through interactions with the cyclic ribose oxygen to stabilize a gauche conformation of adenosine , which facilitates binding by reducing the energy barrier to the initial binding event . finally , we sought to investigate the role of the imidazole ring of adenosine to the binding of these ligands to hsp70 ( table 2 ) . all compounds were purchased from the relevant commercial suppliers and used without further purification . all results are quoted as the geometric mean sem of three independent experiments unless otherwise stated , pkd = log10(kd(m ) 10 ) . we screened three commercially available bacterial natural products , all based on the replacement of the purine of adenosine with a pyrrolopyrimidine scaffold , exchanging the nitrogen at the 7-position with carbon . comparing this change in scaffold to adenosine 1 ( entry 1 ) : the more lipophilic derivative tubercidin 8 ( entry 2 ) displayed a 4-fold improvement in affinity , substitution at the 7-postion was well tolerated , with the nitrile derivative toyocamycin 9 ( entry 3 ) displaying comparable affinity to adenosine 1 , and the primary amide derivative sangivamycin 10 ( entry 4 ) gave a 35-fold increase in affinity at 3.3 m . with knowledge of the increased affinity observed with the pyrrolopyrimidine scaffold in hand , we planned to combine the scaffold hop with the improved affinity previously described for 8-amino substitution of the adenosine scaffold . 8-amino substitution of tubercidin proved synthetically intractable due to the absence of an electron - withdrawing group at the 7-position , making aromatic substitution at the 8-position challenging . therefore , we focused our efforts on the synthesis of 8-aminotoyocamycin 12 and 8-aminosangivamycin 13 ( scheme 1 ) . tribenzoyl intermediate 11 was prepared in four steps and 20% yield using a previously described procedure . despite repeated attempts , however , we were successful using methylamine as a nucleophile to give 8-n - methylaminotoyocamycin 12 , which underwent in situ deprotection of the benzoyl groups under the reaction conditions . treatment of intermediate 11 with basic hydrogen peroxide resulted in hydrolysis of the nitrile group , and subsequent addition of methylamine gave 8-n - methylaminosangivamycin 13 in low yield and moderate purity . 8-n - methylaminotoyocamycin 12 gave a pkd = 5.47 0.02 ( kd = 3.3 m , n = 3 ) against hsc70-nbd . this result represented a 27-fold improvement in activity compared to toyocamycin 9 ( table 2 , entry 1 ) and a 5-fold increase in affinity when compared to the corresponding 8-n - methyladenosine 12 ( table 1 , entry 2 ) . kd = 30 m , n = 1 ) , which is a 7-fold drop in affinity compared to sangivamycin 10 ( table 2 , entry 3 ) . we rationalized this structure activity relationship ( sar ) through the potential effect of the 7-substituent on the hydrogen bonding ability of the key 8-n - methylamino group . the primary amide substituent of 13 can form an intramolecular hydrogen bond with the 8-n - methylamino group to give a resonance stabilized six - membered intramolecular hydrogen bond , which would mask the hydrogen bond donor effect of the 8-n - methylamino substituent and block the binding of this ligand . to develop the toyocamycin scaffold further , we sought to introduce a benzylic substituent to the 8-position amine . previously , massey et al . have shown that n - benzyl substitution at the 8-amino position could improve the affinity of adenosine derived inhibitors of hsp70 ( figure 1 ) . because our toyocamycin derived scaffold 12 had displayed a 5-fold improvement in affinity compared to the corresponding adenosine scaffold 4 , we hypothesized that addition of an 8-n - benzyl substituent to toyocamycin would result in an improved chemotype for hsp70 inhibition . 8-n - benzyltoyocamycin 14 was prepared via a similar method to 12 using benzylamine as a nucleophile . for comparison , 8-n - benzyladenosine 15 was prepared in one step via a literature procedure from commercially available 8-bromoadenosine ( scheme 2 ) . 8-n - benzyladenosine derivative 15 gave a pkd = 5.84 0.02 ( kd = 1.5 m , n = 3 ) , representing a 12-fold improvement in binding affinity compared to the 8-n - methyladenosine analogue 4 ( table 1 , entry 2 ) ; however , the 8-n - benzyltoyocamycin analogue 14 gave a pkd = 5.56 0.02 ( kd = 2.8 m , n = 3 ) , representing little change in affinity compared to 8-n - methyltoyocamycin derivative 12 despite the increase in molecular weight and lipophilicity . even though toyocamycin 9 was apparently an improved scaffold , when compared to adenosine , for the inhibition of hsp70 , we had been unable to develop the compounds beyond a micromolar affinity ligand . we believed that improving our understanding of the complex sar surrounding the nucleoside core and lipophilic 8-n - benzyl substituent through analysis of ligand / hsp70 co - crystal structures would be crucial to the discovery and development of small - molecule inhibitors of hsp70 . in 1995 , kinetic studies by mckay and ha using changes in tryptophan fluorescence indicated that the hsp70 isoform , hsc70 , undergoes a number of conformational changes during its catalytic cycle . the energy released from atp hydrolysis drives subsequent conformational changes in the substrate binding domain , allowing hsp70 to carry out its refolding function on client proteins . however , mckay and ha discovered that atp and adp have two distinct binding mechanisms . while adp binds and then dissociates in an apparent single - step mechanism with slow - off kinetics , the binding of atp is a two - step process , requiring an additional conformational change of the nucleotide - binding domain prior to atp hydrolysis . because it had been demonstrated that the two nucleotide substrates of hsp70 can jump between binding mechanisms , we hypothesized that the complexity surrounding the apparent sar of the nucleoside derived hsp70 inhibitors was due to a change in mechanism between a one - step and an induced conformational change two - step mechanism , even if kinetically this would be difficult to observe . to investigate whether a two - step binding model is consistent with the observed nucleoside sar , we decided to probe hsp70 ligand binding by x - ray crystallography . there is currently no crystal structure of full - length human hsp70 ; therefore , we focused our crystallography efforts on the nbd of the human hsp70 isoform hsp72 ( hsp72-nbd ) . in our hands , adp 16 gave an affinity of pkd = 6.49 ( kd = 0.32 m , n = 1 ) when measured by spr against the hsc70-nbd . the co - crystal structure of adp / pi bound to the hsp72-nbd has previously been solved and analysis of this structure clearly revealed a closed conformation of the protein . the two -helices flanking the sides of the atp binding site , closed around the adenosine motif of adp , forming multiple hydrogen bonds with the ribose and adenine moieties of adp ( figure 3 ) . pdb 3atu , important hydrogen bonding interactions , with their distances in , and residues are indicated . the key salt - bridge interaction between lysine-56 and glutamic acid-268 was measured at 2.8 ( 2 sf ) . only selected residues are shown and solvent has been omitted for clarity . from analysis of this structure , the two phosphate groups of adp form multiple hydrogen bonds within the phosphate binding region of the hsp72-nbd . it is also clear that the hsp72-nbd would need to undergo a conformational change in order for adp / pi to dissociate , resulting in the previously described slow - off kinetics . it has been suggested previously that the closed conformation is apparently stabilized by the formation of a solvent exposed salt - bridge between glutamic acid-268 and lysine-56 . intrigued by the formation of this intramolecular interaction , we then sought to generate a co - crystal structure with the bacterial natural product sangivamycin 10 ( kd = 3.3 m ) ( figure 4 ) . pdb 5aqz , key hydrogen bonding interactions and their distances in are indicated . the crystal structure of sangivamycin 10 revealed a similar hydrogen bonding framework to the adenosine motif of the adp / pi structure ( figure 3 ) . the pyrrolopyrimidine ring interacts with serine-275 and the 2- and 3-hydroxyls of the ribose interact with lysine-271 . however , in contrast to the adp / pi hsp72-nbd structure , sangivamycin 10 co - crystallized in a more open conformation . the two -helices of the nucleotide binding domain are no longer in close proximity , and the solvent exposed salt - bridge between glutamic acid-268 and lysine-56 is absent . it is this difference in the hsp70-nbd conformation which we hypothesize is an important factor in the complexity of sar observed for nucleoside - derived inhibitors . the binding of both adp / pi and sangivamycin 10 are dominated by the formation of multiple hydrogen bonds to key residues in the nucleoside binding cleft but the crucial difference between the two ligands is their ability to induce and stabilize the closed conformation of the hsp72-nbd . it is this ligand - driven induced conformational change that leads to high affinity for hsp70 . the hsp72-nbd closed conformation observed in the adp / pi bound structure is presumably brought about by the interactions of the -phosphate group with the two glycine - rich loops of the phosphate binding region . we speculated that this conformational change could force water molecules from the nucleotide binding domain cleft , strengthening the many hydrogen bond contacts surrounding the adenosine due to the more hydrophobic environment . the absence of the phosphate groups , and in particular the -phosphate group , in sangivamycin 10 means there is no induced conformational change so the affinity is only dependent on the multiple hydrogen bonds ( figure 5 ) . induced open and induced closed conformations of hsp72 . pdb 3atu and 5aqz , the copper - colored structure represents the co - crystal structure of adp / pi bound to the hsp72-nbd in the induced closed conformation due to interactions of the phosphate groups through hydrogen bonds with the glycine rich loops and stabilized by the salt - bridge . the gray - colored structure represents the co - crystal structure of sangivamycin 10 bound to the hsp72-nbd . the overlay shows sangivamycin 10 bound to a more open nbd conformation , in which the formation of the glu - lys salt - bridge is not possible . only selected residues are shown and solvent has been omitted for clarity . mimicking phosphate groups with druglike ligands represents a significant challenge in medicinal chemistry . however , if it were possible to induce the closed conformation of the hsp70 nucleotide binding domain via an alternative mechanism , then mimicking the -phosphate would be unnecessary . because the solvent exposed salt - bridge between glutamic acid-268 and lysine-56 was the only additional enthalpic intradomain interaction that we observed in the hsp70-nbd closed conformation structure , when compared with the more open sangivamycin 10 structure , we hypothesized that a ligand stabilizing this interaction would stabilize the closed conformation of hsp70 , leading to increased affinity . we therefore proposed that the previously described potent 8-n - benzyladenosine derived ligands were able induce a conformational change in hsp70 but via a lipophilic mechanism , which would be more amenable to drug discovery . to investigate this hypothesis , we synthesized the known quinoline derived hsp70 inhibitor 17 in one step and 68% yield using our previously described method ( scheme 3 ) . although several 8-n - substituted hsp70 inhibitors have been described in the literature , the 8-n - quinoline adenosine derived ligand 17 was chosen because it was reported to be the highest affinity ligand which was only substituted at the 8-position . in our hands , 17 gave a pkd = 6.14 0.01 ( kd = 0.72 m , n = 3 ) against hsc70-nbd when measured by spr , which was consistent with the data reported in the literature . 8-n - quinoline aminoadenosine derived ligand 17 was then submitted to our co - crystallization protocol with hsp72-nbd ( figure 6 ) . co - crystallization of 8-n - quinoline aminoadenosine derived ligand 17 bound to hsp72 . pdb 5ar0 , the hsp72 structure clearly demonstrates the formation the key salt - bridge when co - crystallized with 8-n - quinolineadenosine 17 . co - crystallization of 17 with the hsp72-nbd showed the nbd in a closed conformation ( figure 5 ) , nearly identical to the conformation of the hsp72-nbd in the adp / pi 16-bound structures ( figure 6 ) . to our knowledge , this is the first example of a co - crystal structure demonstrating a non - nucleotide ligand binding to the closed conformation of hsp70 . the similarity of the respective hsp72 conformations is consistent with a potential role of the induced conformational change in enhancing the affinity of the 8-n - benzyl nucleoside derived hsp70 ligands . however , the mechanism by which the inhibitor induced conformational change occurs must be distinctly different from the mechanism of nbd closure upon nucleotide binding because the quinoline moiety of 17 forms no interactions within the phosphate binding region . to rationalize the induced conformational change observed with 17 , we further analyzed the closed structure of hsp72-nbd to identify the key binding interactions . 8-n - quinoline adenosine 17 displays a similar hydrogen bonding network with the same key residues observed in both adp / pi and sangivamycin 10 , with an additional solvent exposed water molecule bound to the quinoline nitrogen . however , in contrast with the more open conformation observed in the hsp72-nbd sangivamycin 10 complex , the key salt - bridge between glutamic acid-268 and lysine-56 , which is present in the nucleotide - bound hsp70-nbd structures , is clearly formed in the 17-bound hsp72-nbd structure ( figure 5 ) . in the adp / pi/ hsc70-nbd co - crystal structure , the salt - bridge is solvent exposed , weakening its effect ( figure 7 ) . by contrast , in the 17-bound hsp72-nbd co - crystal structure , the quinoline moiety is able to form a -stack with arginine-272 , although this interaction is solvent exposed so is only likely to be weak ; the result is to place the quinoline group directly in front of the salt - bridge . because the quinoline is highly lipophilic , this creates a more lipophilic environment surrounding the salt - bridge , protecting it from water and strengthening the interaction , which leads to increased affinity for 8-n - benzyl aminonucleoside derived ligands . we propose the binding mechanism of these ligands to hsp70 is analogous to a door and latch . the initial binding event is similar for all nucleoside derived ligands of hsp70 and is dominated by hydrogen bonds to serine-275 and lysine-271 . the nucleotide binding domain is then able to close around the ligand but for this process to be favorable , when balanced by the entropy cost of restricting the conformational freedom of the protein , it must be stabilized by the ligand . adp can achieve this through interactions with the phosphate binding region , while quinoline ligand 17 stabilizes the key salt - bridge through hydrophobic desolvation . sangivamycin 10 has neither of these substituents so predominately binds to the open conformation . overlay of adp / pi and 8-n - quinoline adenosine 17 co - crystal structures with hsp72 . pdb 3atu and 5ar0 , overlay of the adp / pi - hsp72 co - crystal structure ( copper ) and the 8-n - quinoline adenosine 17-hsp72 co - crystal structure ( gray ) . only selected residues are shown and solvent has been omitted for clarity . to test this hypothesis , and to generate more active ligands of hsp70 for further development , we synthesized a series of 8-n - benzylaminoadenosine analogues using our previously described method ( table 3 ) . all results are quoted as the geometric mean sem of three independent experiments unless otherwise stated , pkd = log10(kd(m ) 10 ) . the 8-n - benzyl derivative 15 , as shown previously , gave a pkd = 5.84 0.02 ( kd = 1.4 m , n = 3 ) when measured by spr against hsc70-nbd . the weaker activity observed with the benzyl group 15 compared to quinoline 17 we rationalized was due to its less efficient desolvation of the glutamic acid / lysine salt bridge by the smaller lipophilic group . therefore , we decided to add a number of lipophilic substituents to assess whether we could improve the desolvation effect , promote the induced closed conformation , and improve the affinity of the ligands . para - chloro - substitution gave adenosine derivative 18 ( entry 3 ) with a pkd = 6.55 0.01 ( kd = 0.28 m , n = 9 ) , a 5-fold improvement in affinity compared to compound 15 and a 60-fold improvement compared to 8-n - methylaminoadenosine 4 ( table 1 , entry 2 ) . although the kinetics of the these ligands binding to hsp70 were at the limit of what could be accurately measured by spr , analysis of the off - rate did reveal that the quinoline ligand 17 and the para - chlorobenzyl derivative 18 possessed measurably slow off - rates compared to sangivamycin 10 , whose half - life was too short to be observed with this technique ( see supporting information for details ) . similar improvements were also observed for the para - fluoro derivative 19 ( entry 4 , pkd = 6.34 0.01 , kd = 0.46 m , n = 3 ) and para - methyl derivative 20 ( entry 5 , pkd = 6.53 0.01 , kd = 0.30 m , n = 3 ) . because the crystal structure of this ligand class shows that the benzylic moiety resides in the cleft formed by the two -helices in the nucleotide binding domain of hsp70 , it is unlikely that the para - lipophilic substituents interact with hsp70 via a lipophilic pocket , as the group is essentially solvent exposed . this effect could not be explained by an increase in the overall lipophilicity of the ligand to exploit the nonspecific hydrophobic effect because dichlorobenzyl derivative 21 ( entry 6 , pkd = 5.45 0.01 , kd = 3.5 m , n = 3 ) displayed a significant drop in affinity . the atp binding site of hsp70 is a challenging region of the protein to target with small molecules due to its hydrophilic nature and high flexibility . to target the nucleotide binding domain it is important to understand the conformational changes that this region of the protein undergoes . using protein / ligand x - ray crystallography , we have demonstrated that non - nucleotide ligands of hsp70 can induce conformational changes in the protein and that these changes can play an important role in the binding of hsp70 inhibitors . in solution , kinetic studies suggest that this protein undergoes a number of conformational changes of not just the nucleotide binding domain but also the substrate - binding domain . also , interactions between these two domains and the role of cochaperones in these conformational changes have yet to be addressed . better understanding of the flexibility of hsp70 and its effect on the affinity of ligands will contribute to better assay design and more efficient inhibitor optimization . unless otherwise stated , reactions were conducted in oven - dried glassware under an atmosphere of nitrogen using anhydrous solvents . thin layer chromatography ( tlc ) was performed on precoated aluminum sheets of silica ( 60 f254 nm , merck ) and visualized using short - wave uv light . flash column chromatography was carried out on merck silica gel 60 ( partial size 4065 m ) . h nmr spectra were recorded on bruker amx500 ( 500 mhz ) spectrometers using an internal deuterium lock . chemical shifts are quoted in parts per million ( ppm ) using the following internal references : cdcl3 ( h 7.26 ) , meod ( h 3.31 ) , and dmso - d6 ( h 2.50 ) . signal multiplicities are recorded as singlet ( s ) , doublet ( d ) , triplet ( t ) , quartet ( q ) , multiplet ( m ) , doublet of doublets ( dd ) , doublet of doublet of doublets ( ddd ) , apparent triplet ( app t ) broad ( br ) , or obscured multiplet ( obs m ) . c nmr spectra was recorded on bruker amx500 spectrometers at 126 mhz using an internal deuterium lock . chemical shifts are quoted to 0.01 ppm , unless greater accuracy was required , using the following internal references : cdcl3 ( c 77.0 ) , meod ( c 49.0 ) , and dmso - d6 ( c 39.5 ) . high resolution mass spectra were recorded an agilent 1200 series hplc and diode array detector coupled to a 6210 time - of - flight mass spectrometer with dual multimode apci / esi source . analytical separation was carried out on a merck purospher star rp-18 , 30 mm 4 mm column using a flow rate of 1.5 ml / min in a 4 min gradient elution , uv detection was at 254 nm . all compounds were > 95% purity by hplc analysis unless otherwise stated . to a solution of 8-bromoadenosine ( 0.051 g , 0.15 mmol ) in etoh ( 1.5 ml ) was added methylamine solution ( 33% solution in etoh , 0.25 ml , 3.0 mmol ) , and the mixture was heated to 80 c for 12 h. after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was purified by silica gel chromatography eluting with 2 m meoh / nh3:etoac ( 8:2 ) to give the desired compound as a white solid ( 0.031 g 71% ) ; h ( 500 mhz , dmso - d6 ) 7.90 ( s , 1h ) , 6.95 ( d , j = 4.8 hz , 1h ) , 6.57 ( s , 2h ) , 5.92 ( dd , j = 6.0 , 4.0 hz , 1h ) , 5.86 ( d , j = 7.3 hz , 1h ) , 5.26 ( d , j = 6.7 hz , 1h ) , 5.16 ( d , j = 4.0 hz , 1h ) , 4.68 ( td , j = 7.0 , 5.3 hz , 1h ) , 4.12 ( ddd , j = 5.6 , 4.1 , 2.1 hz , 1h ) , 3.97 ( d , j = 2.3 hz , 1h ) , 3.703.58 ( m , 2h ) , 2.89 ( d , j = 4.5 hz , 3h ) ; c ( 126 mhz , dmso - d6 ) 152.86 , 152.52 , 150.28 , 148.89 , 117.64 , 86.99 , 86.13 , 71.42 , 71.20 , 62.15 , 29.59 . ( m + h ) requires 297.1306 , found 297.1299 . to a solution of 8-bromoadenosine ( 0.064 g , 0.19 mmol ) in etoh ( 1.9 ml ) was added dimethylamine ( 40% solution in water , 0.42 ml , 3.70 mmol ) , and the mixture was heated to 80 c for 12 h. after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was purified by silica gel chromatography eluting with 2 m meoh / nh3:etoac ( 9:1 ) to give the desired product as a white solid ( 0.027 g , 47% ) ; h ( 500 mhz , meoh ) 8.05 ( d , j = 1.1 hz , 1h ) , 5.91 ( d , j = 7.5 hz , 1h ) , 5.16 ( dd , j = 7.5 , 5.3 hz , 1h ) , 4.36 ( dd , j = 5.2 , 1.4 hz , 1h ) , 4.14 ( d , j = 1.9 hz , 1h ) , 3.86 ( dd , j = 12.6 , 2.3 hz , 1h ) , 3.73 ( dd , j = 12.6 , 2.7 hz , 1h ) , 3.03 ( s , 6h ) ; c ( 126 mhz , meod ) 157.19 , 153.92 , 149.92 , 149.22 , 116.86 , 89.06 , 86.97 , 71.96 , 71.79 , 62.86 , 41.89 . hrms ( esi ) c12h19n6o4 ( m + h ) requires 311.1462 , found 311.1464 . to a solution of 8-bromoadenosine ( 0.11 g , 0.33 mmol ) in meoh ( 4 ml ) was added naome ( 0.18 g , 3.3 mmol ) , and the mixture was stirred at room temperature for 12 h. after this time , the solvent was removed under reduced pressure and the resulting residue was purified by silica gel chromatography eluting with 2 m meoh / nh3:etoac ( 9:1 ) to give the desired product as a yellow oil ( 0.025 g , 26% ) . h ( 500 mhz , meod ) , 8.06 ( s , 1h ) , 5.91 ( d , j = 7.1 hz , 1h ) , 4.93 ( dd , j = 7.0 , 5.2 hz , 1h ) , 4.33 ( dd , j = 5.2 , 2.0 hz , 1h ) , 4.21 ( s , 3h ) , 4.164.09 ( m , 1h ) , 3.86 ( dd , j = 12.6 , 2.5 hz , 1h ) , 3.71 ( dd , j = 12.6 , 2.9 hz , 1h ) ; c ( 126 mhz , meod ) 155.19 , 154.01 , 150.10 , 148.34 , 115.35 , 87.65 , 86.82 , 72.43 , 71.55 , 62.62 , 56.69 . hrms ( esi ) c11h16n5o5 ( m + h ) requires 298.1146 , found 298.1152 . to a solution of 5-deoxyadenosine ( 0.024 g , 0.096 mmol ) in dioxane ( 0.48 ml ) and water ( 0.48 ml ) was added khpo4 ( 0.065 g , 0.29 mmol ) and bromine ( 0.023 g , 0.14 mmol ) as a solution in water ( 0.50 ml ) , and the mixture was stirred for 15 min . the mixture was then quenched satd sodium thiosulfate solution ( 5.0 ml ) , extracted with etoac , dried ( mgso4 ) , and the solvent removed under reduced pressure . the product from the previous step ( 0.032 g , 0.097 mmol ) was dissolved in 2 m menh2 in etoh ( 1.9 ml ) , and the mixture was heated to 70 c for 12 h. after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was purified by silica gel chromatography eluting with 2 m meoh / nh3:etoac ( 1:9 ) to give the desired product as a white solid ( 0.009 g , 33% over two steps ) ; h ( 500 mhz , dmso - d6 ) 7.90 ( s , 1h ) , 6.75 ( q , j = 4.5 hz , 1h ) , 6.45 ( s , 2h ) , 5.58 ( d , j = 4.2 hz , 1h ) , 5.23 ( d , j = 5.0 hz , 1h ) , 5.09 ( q , j = 5.0 hz , 1h ) , 4.99 ( d , j = 5.7 hz , 1h ) , 4.144.04 ( m , 1h ) , 3.83 ( app p , j = 6.2 hz , 1h ) , 2.88 ( d , j = 4.5 hz , 3h ) , 1.24 ( d , j = 6.3 hz , 3h ) ; c ( 126 mhz , dmso - d6 ) 152.97 , 152.91 , 150.19 , 149.12 , 117.95 , 88.07 , 79.10 , 74.87 , 71.05 , 29.73 , 18.96 . hrms ( esi ) c11h17n6o3 ( m + h ) requires 281.1357 , found 281.1354 . ( 2r,3r,4r,5r)-2-(4-amino-6-bromo-5-cyano-7h - pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl dibenzoate 11 ( 120 mg , 0.17 mmol ) was dissolved in 2 m methylamine in ethanol ( 5.0 ml ) , and the reaction was heated in a microwave reactor at 140 c for 1 h. after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was purified by silica gel chromatography with the biotage sp1 purification system ( column , 10+s ; flow rate , 15 ml / min ; gradient starting with 100% dcm from 0 to 1 cv then from 100% dcm to 2% nh4oh/18% meoh/80% dcm from 1 cv to 21 cv ) to give the desired compound as a white solid ( 0.045 g , 80% yield ) ; h ( 500 mhz , meod ) 3.22 ( s , 3h ) , 3.833.84 ( m , 2h ) , 4.14 ( q , j = 1.9 hz , 1h ) , 4.27 ( dd , j = 5.6 , 2.0 hz , 1h ) , 4.65 ( dd , j = 7.6 , 5.6 hz , 1h ) , 6.29 ( d , j = 7.9 hz , 1h ) , 8.01 ( s , 1h ) ; c ( 126 mhz , meod ) 29.55 , 56.92 , 61.31 , 70.91 , 71.19 , 86.12 , 87.35 , 101.19 , 118.75 , 148.27 , 149.87 , 151.26 , 154.16 . hrms ( esi ) c13h17n6o4 ( m + h ) requires 321.1306 , found 321.1303 . to a solution of ( 2r,3r,4r,5r)-2-(4-amino-6-bromo-5-cyano-7h - pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl dibenzoate 11 ( 87 mg , 0.13 mmol ) in thf ( 1.0 ml ) was added hydrogen peroxide ( 33% , 1.0 ml ) and 1 m naoh ( 1.0 ml ) . the reaction was stirred at room temperature for 18 h before 1 m hcl was added until the mixture reached ph 6 , when the solvent was removed under reduced pressure . the resulting residue was dissolved in 2 m methylamine in ethanol ( 5.0 ml ) , and the reaction was heated to 120 c in a microwave reactor for 50 min . after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was purified by silica gel chromatography using the biotage sp1 purification system ( column , 10+s ; flow rate , 15 ml / min ; gradient starting with 100% dcm from 0 to 1 cv then from 100% dcm to 2% nh4oh/18% meoh/80% dcm from 1 cv to 21 cv ) to give the desired product as a yellow solid ( 0.009 g , 10% yield ) ; h ( 500 mhz , meod ) 2.89 ( s , 3h ) , 3.78 ( dd , j = 12.2 , 1.9 hz , 1h ) , 3.86 ( dd , j = 12.2 , 2.2 hz , 1h ) , 4.18 ( q , j = 1.7 hz , 1h ) , 4.33 ( dd , j = 5.5 , 1.3 hz , 1h ) , 4.85 ( m , 1h ) , 6.12 ( d , j = 7.9 hz , 1h ) , 8.02 ( s , 1h ) ; c ( 126 mhz , meod ) 35.35 , 62.23 , 71.62 , 72.28 , 86.70 , 87.49 , 98.51 , 101.02 , 146.72 , 147.31 , 150.61 , 157.30 , 167.61 . ( 2r,3r,4r,5r)-2-(4-amino-6-bromo-5-cyano-7h - pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl dibenzoate 11 ( 31 mg , 0.045 mmol ) was dissolved in etoh ( 2.0 ml ) , and benzylamine ( 29 mg , 0.27 mmol ) was added to the solution . the reaction was heated in a microwave reactor at 160 c for 1 h. after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was dissolved in methanol ( 0.5 ml ) , and 0.5 m sodium methoxide ( 1.0 ml ) was added . the reaction was stirred at room temperature for 1 h , then the solvent was removed under reduced pressure . the resulting residue was purified by silica gel chromatography with the biotage sp1 purification system ( column , 10+s ; flow rate , 15 ml / min ; gradient starting with 100% dcm from 0 to 1 cv then from 100% dcm to 2% nh4oh/18% meoh/80% dcm from 1 cv to 21 cv ) to give the desired compound as a white solid ( 0.007 g , 39% yield ) ; h ( 500 mhz , meod ) 3.803.86 ( m , 2h ) , 4.18 ( q , j = 1.8 hz , 1h ) , 4.28 ( dd , j = 5.6 , 1.7 hz , 1h ) , 4.73 ( dd , j = 7.8 , 5.7 hz , 1h ) , 4.78 ( d , j = 10.3 hz , 2h ) , 6.37 ( d , j = 7.9 hz , 1h ) , 7.257.27 ( m , 1h ) , 7.337.36 ( m , 2h ) , 7.417.43 ( m , 2h ) , 8.01 ( s , 1h ) ; c ( 126 mhz , meod ) 46.28 , 61.40 , 71.14 , 71.29 , 86.27 , 86.81 , 87.38 , 101.06 , 118.30 , 126.72 , 126.98 , 128.25 , 138.41 , 148.25 , 149.94 , 150.10 , 154.22 . hrms ( esi ) c19h21n6o4 ( m + h ) requires 397.1619 , found 397.1614 . to a solution of 8-bromoadenosine ( 0.061 g , 0.18 mmol ) in n - butanol ( 1.76 ml ) was added pr2net ( 0.092 ml , 0.53 mmol ) and benzylamine ( 0.038 ml , 0.35 mmol ) , and the mixture was heated to 120 c for 16 h. after this time , the mixture was cooled to room temperature and the resulting residue was purified by silica gel chromatography eluting with etoac : meoh ( 95:5 ) to give the desired product as a white solid ( 0.017 g , 26% ) ; h ( 500 mhz , meod ) 8.00 ( s , 1h ) , 7.457.38 ( m , 2h ) , 7.377.32 ( m , 2h ) , 7.287.23 ( m , 1h ) , 6.11 ( d , j = 7.7 hz , 1h ) , 4.81 ( dd , j = 7.6 , 5.4 hz , 1h ) , 4.69 ( d , j = 15.8 hz , 1h ) , 4.64 ( d , j = 15.8 hz , 1h ) , 4.30 ( dd , j = 5.6 , 1.6 hz , 1h ) , 4.18 ( q , j = 1.9 hz , 1h ) , 3.85 ( dd , j = 12.0 , 2.3 hz , 1h ) , 3.79 ( dd , j = 11.9 , 1.7 hz , 1h ) ; c ( 126 mhz , meod ) 152.12 , 151.99 , 149.65 , 148.63 , 138.75 , 128.11 , 126.68 , 126.52 , 116.56 , 87.29 , 86.41 , 71.64 , 71.52 , 61.75 , 45.42 . hrms ( esi ) c17h21n6o4 ( m + h ) requires 373.1619 , found 373.1623 . to a solution of 8-bromoadenosine ( 0.099 g , 0.29 mmol ) in n - butanol ( 2.9 ml ) was added quinolin-6-ylmethanamine ( 0.045 g , 0.29 mmol ) and pr2net ( 0.15 ml , 0.85 mmol ) , and the mixture was heated to 120 c under n2 for 24 h. after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was purified by chromatography eluting with etoac:2 m meoh / nh3 ( 80:20 ) to give the desired product as a white solid ( 0.008 g , 7% ) ; h ( 500 mhz , meod ) 8.83 ( dd , j = 4.4 , 1.7 hz , 1h ) , 8.37 ( dt , j = 8.4 , 1.2 hz , 1h ) , 8.04 ( d , j = 8.7 hz , 1h ) , 8.02 ( s , 1h ) , 7.95 ( d , j = 1.8 hz , 1h ) , 7.86 ( dd , j = 8.8 , 2.0 hz , 1h ) , 7.54 ( dd , j = 8.3 , 4.3 hz , 1h ) , 6.16 ( d , j = 7.6 hz , 1h ) , 4.924.83 ( obs m , 3h ) , 4.32 ( dd , j = 5.4 , 1.7 hz , 1h ) , 4.21 ( q , j = 1.9 hz , 1h ) , 3.87 ( dd , j = 12.0 , 2.3 hz , 1h ) , 3.80 ( dd , j = 11.9 , 1.7 hz , 1h ) ; c ( 126 mhz , meod ) 151.98 , 151.94 , 149.72 , 149.52 , 148.44 , 146.69 , 137.89 , 136.94 , 129.30 , 128.46 , 127.90 , 125.07 , 121.35 , 116.59 , 87.38 , 86.47 , 71.70 , 71.53 , 61.75 , 45.29 . hrms ( esi ) c20h22n7o4 ( m + h ) requires 424.1728 , found 424.1725 . to a solution of 8-bromoadenosine ( 0.078 g , 0.23 mmol ) in n - butanol ( 2.3 ml ) was added pr2net ( 0.12 ml , 0.68 mmol ) and ( 4-chlorophenyl)methanamine ( 0.055 ml , 0.45 mmol ) , and the mixture was heated to 120 c for 12 h. after this time , the mixture was cooled to room temperature and the resulting residue was purified by silica gel chromatography eluting with etoac : meoh ( 95:5 ) to give the desired product as a white solid ( 0.033 g , 36% ) ; h ( 500 mhz , meod ) 8.00 ( s , 1h ) , 7.40 ( d , j = 8.3 hz , 2h ) , 7.35 ( d , j = 8.6 hz , 2h ) , 6.10 ( d , j = 7.5 hz , 1h ) , 4.79 ( dd , j = 7.6 , 5.4 hz , 1h ) , 4.66 ( d , j = 15.9 hz , 1h ) , 4.62 ( d , j = 16.0 hz , 1h ) , 4.30 ( dd , j = 5.4 , 1.7 hz , 1h ) , 4.18 ( q , j = 1.9 hz , 1h ) , 3.85 ( dd , j = 11.9 , 2.3 hz , 1h ) , 3.79 ( dd , j = 12.0 , 1.7 hz , 1h ) ; c ( 126 mhz , meod ) 152.19 , 151.83 , 149.68 , 148.72 , 137.67 , 132.42 , 128.22 , 128.16 , 116.54 , 87.30 , 86.40 , 71.65 , 71.50 , 61.73 , 44.80 . hrms ( esi ) c17h20cln6o4 ( m+h ) requires 407.1229 , found 407.1237 . to a solution of 8-bromoadenosine ( 0.048 g , 0.14 mmol ) in n - butanol ( 1.4 ml ) was added ( 4-fluorophenyl)methanamine ( 0.032 ml , 0.28 mmol ) and pr2net ( 0.073 ml , 0.42 mmol ) , and the mixture was heated to 120 c for 12 h. after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was purified by silica gel chromatography eluting with etoac : meoh ( 9:1 ) to give the desired product as a white solid ( 0.043 g , 79% ) ; h ( 500 mhz , meod ) 7.99 ( s , 1h ) , 7.50 ( dd , j = 8.3 , 5.3 hz , 1h ) , 7.447.38 ( m , 2h ) , 7.237.16 ( m , 1h ) , 7.097.04 ( m , 2h ) , 6.09 ( d , j = 7.6 hz , 1h ) , 4.78 ( dd , j = 7.6 , 5.4 hz , 1h ) , 4.65 ( d , j = 15.7 hz , 1h ) , 4.60 ( d , j = 15.7 hz , 1h ) , 4.29 ( dd , j = 5.4 , 1.6 hz , 1h ) , 4.17 ( q , j = 1.9 hz , 1h ) , 3.83 ( dd , j = 12.0 , 2.2 hz , 1h ) , 3.78 ( dd , j = 11.9 , 1.7 hz , 1h ) ; c ( 126 mhz , meod ) 164.14 , 163.00 , 161.06 , 152.14 , 151.85 , 149.66 , 148.65 , 134.75 , 134.72 , 130.99 , 130.92 , 128.53 , 128.46 , 116.55 , 115.67 , 115.50 , 114.79 , 114.62 , 87.26 , 86.41 , 71.60 , 71.50 , 61.72 , 44.79 . hrms ( esi ) c17h20fn6o4 ( m + h ) requires 391.1525 , found 391.1517 . to a solution of 8-bromoadenosine ( 0.062 g , 0.18 mmol ) in n - butanol ( 1.79 ml ) was added p - tolylmethanamine ( 0.045 ml , 0.36 mmol ) and pr2net ( 0.093 ml , 0.54 mmol ) , and the mixture was heated to 120 c for 48 h. after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was purified by silica gel chromatography eluting with etoac : meoh ( 9:1 ) to give the desired product as a white solid ( 0.042 g , 61% ) ; h ( 500 mhz , meod ) 7.99 ( s , 1h ) , 7.28 ( d , j = 7.8 hz , 2h ) , 7.16 ( d , j = 7.7 hz , 2h ) , 6.09 ( d , j = 7.6 hz , 1h ) , 4.80 ( dd , j = 7.6 , 5.4 hz , 1h ) , 4.64 ( d , j = 15.6 hz , 1h ) , 4.58 ( d , j = 15.6 hz , 1h ) , 4.29 ( dd , j = 5.4 , 1.7 hz , 1h ) , 4.18 ( d , j = 1.9 hz , 1h ) , 3.84 ( dd , j = 12.0 , 2.3 hz , 1h ) , 3.78 ( dd , j = 11.9 , 1.7 hz , 1h ) , 2.32 ( s , 3h ) ; c ( 126 mhz , meod ) 152.08 , 152.01 , 149.64 , 148.59 , 136.39 , 135.64 , 128.70 , 126.57 , 116.57 , 87.27 , 86.40 , 71.61 , 71.50 , 61.74 , 45.26 , 19.70 . hrms ( esi ) c18h23n6o4 ( m + h ) requires 387.1775 , found 387.1779 . to a microwave vial was added 8-bromoadenosine ( 0.30 g , 0.87 mmol ) and 3,4-dichlorobenzylamine ( 0.29 ml , 2.2 mmol ) in ethanol ( 1.7 ml ) . the reaction mixture was concentrated , and the crude residue was purified by silica gel column chromatography eluting with etoh : dcm ( 3:7 ) to give the desired product as a white solid ( 0.28 g , 73% ) ; h ( 500 mhz , dmso - d6 ) 7.90 ( s , 1h ) , 7.707.53 ( m , 3h ) , 7.37 ( dd , j = 8.3 , 2.0 hz , 1h ) , 6.55 ( s , 2h ) , 5.995.85 ( m , 2h ) , 5.32 ( d , j = 6.6 hz , 1h ) , 5.18 ( d , j = 4.0 hz , 1h ) , 4.72 ( td , j = 7.0 , 5.3 hz , 1h ) , 4.56 ( d , j = 6.0 hz , 2h ) , 4.12 ( ddd , j = 5.5 , 3.9 , 1.9 hz , 1h ) , 3.99 ( q , j = 2.3 hz , 1h ) , 3.62 ( dt , j = 7.0 , 3.2 hz , 2h ) 3.453.35 ( obs . m , 1h ) ; c ( 126 mhz , dmso - d6 ) 153.01 , 151.48 , 150.25 , 149.17 , 141.70 , 131.27 , 130.86 , 129.66 , 129.58 , 128.05 , 117.38 , 87.00 , 86.30 , 71.53 , 71.38 , 62.23 , 44.76 . hrms ( esi ) c17h19cl2n6o4 ( m + h ) requires 442.0867 , found 442.0859 . to a solution of 8-bromoadenosine ( 0.073 g , 0.21 mmol ) in n - butanol ( 4.2 ml ) was added ( 4-methoxyphenyl)methanamine ( 0.083 ml , 0.63 mmol ) and pr2net ( 0.22 ml , 1.3 mmol ) , and the mixture was heated to 120 c for 12 h. after this time , the mixture was cooled to room temperature and the solvent removed under reduced pressure . the resulting residue was purified by silica gel chromatography eluting with etoac : meoh ( 95:5 ) to give the desired product as a white solid ( 0.049 g , 58% ) ; h ( 500 mhz , meod ) 7.98 ( s , 1h ) , 7.32 ( d , j = 8.8 hz , 2h ) , 6.89 ( d , j = 8.8 hz , 2h ) , 6.07 ( d , j = 7.6 hz , 1h ) , 4.78 ( dd , j = 7.6 , 5.4 hz , 1h ) , 4.60 ( d , j = 15.3 hz , 2h ) , 4.55 ( d , j = 15.3 hz , 1h ) , 4.28 ( dd , j = 5.6 , 1.7 hz , 1h ) , 4.16 ( q , j = 1.9 hz , 1h ) , 3.83 ( dd , j = 12.0 , 2.3 hz , 1h ) , 3.78 ( s , 3h ) , 3.77 ( obs . dd , j = 11.9 , 1.7 hz , 1h ) ; c ( 126 mhz , meod ) 158.96 , 152.09 , 151.98 , 149.63 , 148.59 , 130.63 , 127.94 , 116.59 , 113.49 , 87.27 , 86.38 , 71.61 , 71.48 , 61.73 , 54.26 , 45.05 . hrms ( esi ) c18h22n6nao5 ( m + na ) requires 425.1544 , found 425.1540 . all surface plasmon resonance ( spr ) experiments were carried out on a biacore t100 enhanced to t200 sensitivity ( ge life sciences , amersham place , uk ) . amine coupling chemistry was used to immobilize the truncated hsc70-nbd domain ( residues 1381 ) on a research grade cm5 sensor chip . the running buffer used in the immobilization step consisted of 1 phosphate buffered saline ( 10 mm nahpo4/nah2po4 ph 7.4 , 2.7 mm kcl , 137 mm nacl ) , and the chip surface was activated for 10 min using a 1:1 mixture of 100 mm n - hydroxysuccinimide and 400 mm 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide . hsc70-nbd was injected at a concentration of 70 g / ml in a 10 mm acetate buffer ph 5.0 with 750 m adp as protection for the active site lysines . the reaction was monitored in real time and stopped when a target immobilization level of 5000ru was obtained . finally , the surface was blocked via an injection of 1 m ethanolamine at ph 8.5 for 7 min . the flow rate was maintained at 10 l / min for all the above procedures . flow cell one was left unmodified and used as the reference surface . following protein immobilization , the running buffer was changed to 1 phosphate buffered saline containing 0.05% ( v / v ) tween20 and 5% ( v / v ) dmso in order to reduce the nonspecific binding and increase solubility of the compounds . all liquid handling was carried out using an echo 550 acoustic liquid dispenser ( labcyte , dublin , ireland ) , and compounds were added to 384-well polypropylene v - bottomed plates ( greiner , stonehouse , uk ) , which were used as sample plates for the spr experiments . for each compound , an eight - point dose response experiment was carried out using a concentration range of 502000 m , 5200 m or 0.06252.5 m , depending on the potency of the compound . the experiments were performed at a flow rate of 30 l / min , a sample injection time of 60 s , and a dissociation time of 120 s. the cm5 surface was not regenerated between sample injections . kd values were calculated from the background normalized binding curve generated from the sensorgrams under equilibrium conditions , using the 1:1 binding model in the biacore software version 2 ( ge life sciences , amersham place , uk ) . all results are reported as the geometric mean standard error of the mean ( sem ) of at least three independent measurements unless otherwise stated . the human hspa1a ( hsp72 ) gene was amplified from the image clone 3345864 ( accession no . bc002453 ) by pcr using the respective forward and reverse primers 5-gatcgaccatatggccaaagccgcggcga-3 and 5-acagaattcctaatctacctcctcaatgg-3. the hspa1a gene was cloned into a ptwoe vector , which is a modified version of pet-17b ( merck chemicals ltd . , nottingham , uk ) encoding a n - terminal 6xhis - tag followed by a human rhinovirus 3c protease cleavage site . bl21-ai cells ( invitrogen , paisley , uk ) transformed with the vector containing the hspa1a gene were grown in luria bertani medium to an optical density at 600 nm of 0.6 and induced with 0.5 mm isopropyl--d-1-thiogalactopyranoside and 0.2% ( w / v ) arabinose at for 16 h at 20 c . cells were harvested by centrifugation at 6000 rpm for 40 min at 4 c using an avanti centrifuge j-26xp ( beckman coulter , high wycombe , uk ) with a jla 8.100 rotor . cell pellets were resuspended in 3 volumes of lysis buffer consisting of 25 mm tris , 50 mm nacl , 5% ( v / v ) glycerol , 1 complete edta - free protease inhibitors ( roche , basel , switzerland ) , 25 u / ml benzonase nuclease ( merck chemicals ltd . ) at ph 7.5 . cell lysis was performed by sonication using a vibra - cell vcx500 ( sonics & materials inc . , newtown , ct , usa ) with a 13 mm solid probe for 24 cycles of 5 s on , 55 s off with amplitude set at 50% . the lysate was clarified by centrifugation at 20000 rpm for 30 min at 4 c using an avanti centrifuge j-26xp ( beckman coulter ) with a ja 25.50 rotor . the supernatant was passed through a 1.2 m syringe filter ( sartorius stedim , germany ) and loaded onto a 5 ml histrap ff column ( ge healthcare , chalfront st . giles , uk ) equilibrated in buffer a , comprising 25 mm tris , 50 mm nacl , ph 7.5 , and eluted with a gradient of 0100% buffer b ( buffer a + 250 mm imidazole ) over 10 column volumes ( cv ) . fractions containing hsp72 were pooled , concentrated , and loaded onto a superdex 200 ( 16/60 ) size exclusion column ( ge healthcare ) equilibrated in 25 mm tris , 400 mm nacl , 2 mm edta 5% ( v / v ) glycerol , ph 7.5 , for further purification . fractions containing hsp72 were pooled and further purified to remove contaminating nucleotides using a 6 ml resource q column ( ge healthcare ) equilibrated in 20 mm tris , 2 mm edta , 5% ( v / v ) glycerol , ph 7.5 . following a 10 cv wash with the same buffer , hsp72 was eluted using a gradient from 0 to 500 mm nacl over 6 cv and loaded onto a superdex 200 16/60 column ( ge healthcare ) equilibrated in a buffer containing 25 mm tris , 400 mm nacl , 15 mm edta , 5% ( v / v ) glycerol , ph 7.5 . the removal of contaminating nucleotides was followed by measuring the ratio of absorbance at 260 and 280 nm ( a260/a280 ) using a nanodrop nd-1000 uv spectrophotometer ( thermofisher , wilmington , de , usa ) . samples with a260/a280 below 0.6 were regarded as nucleotide free . purified hsp72-nbd protein was thawed ; buffer exchanged into fresh 100 mm hepes ph 7.5 and incubated with 5 mm of the inhibitor for 30 min on ice prior to crystallization . / inhibitor co - crystals were grown at 18 c in sitting drops by mixing equal volumes of protein solution ( 512 mg / ml ) and precipitant solution containing 1728% ( v / v ) peg3350 , 0.1 m hepes ph 7.5 , 2 mm mgcl2 , and 2 mm nah2po4 . co - crystals of approximate dimensions 100 100 300 m typically formed overnight . crystals were in liquid nitrogen , using 22.5% ( v / v ) ethylene glycol for the co - crystals with sangivamycin 10 and 25% ( v / v ) glycerol for compound 17 . x - ray diffraction data were collected at 100 k at diamond light source ( oxfordshire , uk ; beamlines i041 and i24 ) . all data were imported to mtz format with pointless , then scaled and merged with aimless and the ccp4 suite . the structures were solved by molecular replacement with phaser , with the public domain hsp72-nbd structure 1s3x as the search model after removal of all nonprotein atoms . structures were refined in iterative cycles of model building with coot and refinement with buster . the data collection and refinement statistics are presented in supporting information table s15 , and for fo fc electron density figures for all structural data , see supporting information figure s14 .

hsp70 is a molecular chaperone and a key component of the heat - shock response . because of its proposed importance in oncology , this protein has become a popular target for drug discovery , efforts which have as yet brought little success . this study demonstrates that adenosine - derived hsp70 inhibitors potentially bind to the protein with a novel mechanism of action , the stabilization by desolvation of an intramolecular salt - bridge which induces a conformational change in the protein , leading to high affinity ligands . we also demonstrate that through the application of this mechanism , adenosine - derived hsp70 inhibitors can be optimized in a rational manner .

Introduction Results and Discussion Conclusions Experimental Section

PMC5276769

postural anomalies are widespread and are exhibited by people of all demographics . while commonly attributed to lack of physical activity , poor posture is especially prominent in individuals who spend long periods of time sitting at a desk or computer , as is necessary with many jobs in developed countries1,2,3 . poor posture is a major causative factor of many musculoskeletal problems , including joint stress , pain , and various diseases4,5,6,7,8,9,10,11 . detecting irregularities in posture is an important component in understanding sources of pain and limited movement , and for subsequently developing mitigation strategies such as corrective exercises . due to its dynamic nature , objective analysis often utilizes elaborate technology , such as x - ray imaging and 3d motion capture , which provide a valid and reliable assessment ; however , these capabilities are found only in specialized clinics12 . in an effort to address this limitation , portable device technology has been developed hand - in - hand with medical technology to provide non - invasive , user - friendly , and affordable posture - assessing software designed for use by physical therapists , chiropractors , and other health and fitness professionals . , trinity , fl , usa ) is a novel , commercially sold photographic mobile application that enables the identification of deviations from the ideal standing posture . the posturescreen mobile ( psm ) application facilitates the assessment of posture in a variety of settings . while the convenience of this tool is evident , the information provided would only be useful if the measurements were repeatable between multiple visits and reproducible between multiple examiners . therefore , the purpose of this study was to investigate the inter- and intra - rater agreement when using psm , as well as understanding whether standard clothing can obscure the reliability of such measurements . one experienced licensed physical therapist ( a doctor of physical therapy ) and two inexperienced examiners ( undergraduate students ) performed repeated postural assessments of 10 subjects wearing both full and minimal clothing on two nonconsecutive days using psm installed on a tablet ( ipad ; apple inc . , cupertino , ca , usa ) . an initial set of posture images was captured with subjects dressed in standard street clothing . a second set of images was captured on the same visit with subjects wearing minimal clothing ( shorts for men , and shorts and a sports bra for women ) . a third set of images was captured 48 hours later in the minimally clothed condition . subjects were recruited randomly from the ucla community , based on availability , not on postural qualifications . this study was approved by the ucla institutional review board and all participants gave their written informed consent . participants completed standard health , medical , and exercise history forms following enrollment and prior to data acquisition . before each assessment , the room was set up in a standardized way to ensure uniform conditions throughout the test . tape was placed on the floor to indicate where the subject should stand for the anterior picture . a second piece of tape was laid perpendicular to the first piece to indicate the positioning for the lateral picture . the tablet was placed on a stand exactly 10 feet away from the subject markers at a height of 4.5 feet to standardize the image angle . height was measured to the nearest 0.5 cm using a wall - mounted stadiometer , and weight was measured to the nearest 0.1 kg with an octopolar bioelectrical impedance scale ( biospace inbody r20 ; inbody co. ltd . , subjects were instructed to stand at the tape marks in a comfortable position with weight evenly distributed on both legs and to look at a marker placed on the wall in front of them at eye level . to help eliminate potential deviations from their routine posture , subjects were asked to stand on their right foot for a few seconds and then shift their balance to the left foot for a few seconds . subjects then placed both feet on the ground and an image was captured using psm and the camera function of the ipad . this process was repeated to obtain both an anterior and right lateral view of each subject . an initial set of images was captured with subjects dressed in standard street clothing with their shoes removed . a second set of images was captured on the same visit with subjects wearing minimal clothing ( shorts for men , and shorts and a sports bra for women ) also with their shoes removed . an identical third examination was conducted 48 hours later in the minimally clothed condition . subjects were asked to refrain from strenuous physical activity between visits in order to reduce external factors influencing postural variation . three examiners analyzed each set of data by following the psm prompts to select 17 specific anatomical landmarks . twelve landmarks were placed on the anterior view image at the following locations : the right pupil , left pupil , midpoint between the nose and upper lip , top of the right acromioclavicular ( ac ) joint , episternal notch , top of the left ac joint , right lateral rib t8 level , left lateral rib t8 level , right anterior superior iliac spine ( asis ) , left asis , center of the right talus , and center of the left talus . five landmarks were placed on the lateral view image at the following locations : the external auditory meatus , center of the shoulder at the cervicothoracic junction , greater trochanter , center of the tibiofemoral joint , and center of the malleolus . psm then calculated the following 13 quantitative data points using proprietary algorithms : head shift ( lateral ) , head shift ( longitudinal ) , head tilt , shoulder shift ( lateral ) , shoulder shift ( longitudinal ) , shoulder tilt , ribcage shift , hip shift ( lateral ) , hip shift ( longitudinal ) , hip tilt , head weight , effective head weight , defined as how heavy the head feels to its supporting structures based on any postural deviations , and knee shift . intraclass correlation coefficients ( iccs ) were derived by calculating between - subject variance as a fraction of the total variance of each of the 13 postural measurements made by all three examiners to determine inter - rater agreement . inter - rater agreement was calculated separately for the fully clothed and minimally clothed conditions . additionally , iccs were calculated for the measurements of the two minimally clothed examinations conducted by the experienced examiner on separate occasions to determine the intra - rater agreement of the measurements . iccs greater than 0.60 but less than or equal to 0.80 ( 0.60<icc0.80 ) were considered to indicate substantial agreement , while iccs equal to or greater than 0.81 ( icc0.81 ) were considered to indicate the postural measures of head weight and effective head weight were omitted from the analysis because these were considered to be affected more by body weight than by the placement of landmarks by the raters . ten subjects ( five females ) completed the study ( age 21 1 years ; height 172 11 cm ; weight 70 14 kg ; mean sd ) . inter - rater agreement for both the fully and minimally clothed conditions and the intra - rater agreement for the minimally clothed condition are reported in table 1table 1.levels of agreement of repeated measurementsposturalmeasurementsintraclass correlation coefficientsfully clothed(3 examiners)minimally clothed(3 examiners)minimally clothed(2 visits)head shift lateral0.87 * 0.83 * 0.46head shift ap0.550.720.73head tilt0.91 * 0.86 * 0.78shoulder shift lateral0.670.640.33shoulder shift ap0.750.720.68shoulder tilt0.580.88 * 0.78hip shift lateral0.95 * 0.93 * 0.76hip shift ap0.640.550.84*hip tilt0.100.260.59head weight1.001.000.90effective head weight0.980.960.84rib cage shift0.190.580.59knee shift0.88 * 0.750.86*data are intraclass correlation coefficients ( iccs ) where 0.40<icc0.60 indicate moderate agreement , 0.60<icc0.80 indicate substantial agreement and icc0.81 indicate almost perfect agreement . head weight and effective head weight were omitted from the analysis because they were considered to be affected more by body weight than by the placement of landmarks by the raters . inter - rater agreement of the fully clothed exam was at least substantial ( icc>0.60 ) for seven measures and specifically almost perfect ( icc0.81 ) for : head shift lateral , head tilt , hip shift lateral , and knee shift . inter - rater agreement of the minimally clothed exam was almost perfect for the previously identified measures minus knee shift but plus shoulder tilt . in the minimally clothed condition , only hip tilt failed to reach at least moderate agreement ( 0.40<icc0.60 ) . intra - rater agreement ( between visits ) for the minimally clothed exams was at least substantial ( icc>0.60 ) for seven measures and specifically almost perfect for : hip shift anterior / posterior ( hip shift ap ) and knee shift . there were large differences between inter- and intra - rater agreement for head shift lateral ( 0.826 vs. 0.457 ) and shoulder shift lateral ( 0.642 vs. 0.332 ) , with both failing to have substantial intra - rater agreement despite having met or exceeded this level of agreement in the inter - rater analysis . in contrast , hip tilt possessed a low inter - rater agreement ( icc=0.256 ) but registered a much higher intra - rater agreement ( icc=0.588 ) . data are intraclass correlation coefficients ( iccs ) where 0.40<icc0.60 indicate moderate agreement , 0.60<icc0.80 indicate substantial agreement and icc0.81 indicate almost perfect agreement . head weight and effective head weight were omitted from the analysis because they were considered to be affected more by body weight than by the placement of landmarks by the raters . to the best of our knowledge , this is the first published study that has examined both the inter- and intra - rater agreement of the mobile application , posturescreen mobile , for screening static posture . acceptable levels of agreement were found among the measurements of three different examiners of varying experience . overall inter - rater agreement was substantial whether the examination was conducted on a fully clothed or minimally clothed subject , with 7 of the 11 measures exceeding an icc of 0.60 in the fully clothed condition . in the fully clothed condition , the examiners evaluation of head position in the frontal plane ( head shift lateral , head tilt ) , weight shift in the frontal plane ( hip shift lateral ) , and one of three measures relating to weight shift in the sagittal plane ( knee shift ) using psm were especially reproducible ( icc0.81 ) . unsurprisingly , removing the shirt ( i.e. , creating a minimally clothed condition ) improved the agreement of measures relying on upper body landmarks on the acromioclavicular joints and rib cage . for example , the rib cage shift measure improved from slight agreement ( 0.01<icc0.20 ) to moderate agreement . importantly , the head shift anterior / posterior ( head shift ap ) measure showed substantial agreement ( icc=0.724 ) in the minimally clothed condition . head shift ap may be the most clinically relevant measure provided by psm as it enables a quantitative diagnosis of forward head posture . forward head posture is a common postural deviation in developed countries and it is believed to be a causative factor of many musculoskeletal diseases , including migraine and chronic tension headaches10 , 11 . traditional means of diagnosing this postural condition involve manually determining the angle between the horizontal and a line running from the c7 vertebra to the tragus of the ear from a sagittal photograph14 . a quick , yet reliable , means of determining this angle could greatly simplify management of cervical - spine - related disorders . posture measures that failed to reach substantial agreement in the minimally clothed condition tended to rely on anatomical landmarks that are either difficult to observe ( e.g. , anterior superior iliac spine , greater trochanter ) or non - specific ( e.g. , rib cage ) . hip tilt stood out as having notably poor inter - rater agreement . even among highly experienced examiners , identification of pelvic deviation via hands - on palpation can prove challenging and inexact15,16,17 . therefore , it is unsurprising that attempts to identify this phenomenon via photographic observation were less reliable . however , the strong intra - rater agreement of hip tilt could indicate that this measure , unlike the other 10 , requires an experienced examiner , meaning the undergraduate examiners were not able to reliably estimate the location of the landmarks . however , since we did not assess intra - rater agreement for the inexperienced examiners , this can not be stated conclusively . alternatively , it is possible that each examiner has their own consistent , even if inaccurate , idea of where the asis is located when analyzing the photographs . in addition to evaluating the performance of the examiner , intra - rater agreement could be considered a partial measure of postural consistency within participants , at least among measures with strong inter - rater agreement . between - visit variations suggest certain aspects of posture may be highly inconsistent within individuals on a day - to - day basis . all lower body weight shift measures ( hip shift ap , hip shift lateral , and knee shift ) appear to have remained fairly consistent across visits , possibly because we asked subjects to unweight each foot prior to each exam , thus standardizing the lower extremity weight shift preference for each visit . in contrast , none of the weight shift measures above the waist reached almost perfect agreement , indicating the variable nature of weight shift preference from moment - to - moment even with a stable lower body . a previous study by hopkins18 , tested the validity and intra - rater agreement of psm by comparing it with the vicon 3d motion analysis system ( vicon motion systems , oxford , united kingdom ) as the gold - standard measure19 . this investigator found that the shift and tilt measurements using psm were repeatedly higher than those measured by the vicon system in the frontal view . in the lateral view , the results reversed these significant differences in variation persisted with and without the use of anatomical markers . unlike this study , hopkins found that the trial - to - trial variance with psm exhibited low intra - rater agreement . one such posture assessment tool with 29 measurements was found to be highly reliable in a study by ferreira et al.20 , with just 14.0% of measurements for inter - rater agreement and 19.3% for intra - rater agreement failing to reach an icc of at least 0.70 . while these findings were better than those of our present study of psm ( with 30.7% of inter - rater and 38.5% of intra - rater measures with iccs lower than 0.700 ) , there is a trade - off between ease - of - use and time spent performing analysis . the study of ferreira et al.20 involved five fully trained physical therapists and the software required identification of 32 anatomic points . it is noteworthy that psm performed reliably while requiring identification of only 17 points and when employed by inexperienced examiners . one important finding of this study is the inconsistency of posture between subject visits . there are several factors that may influence day - to - day posture including energy level , recent exertion , and involuntary compensation to alleviate temporary discomfort . in order to minimize these confounding factors , subjects were instructed not to perform any heavy lifting between visits . nevertheless , any of these factors may have led to lower iccs that underestimate the true inter - rater agreement . future studies should build on the work of raine et al.14 to identify acceptable ranges for each postural measure to allow categorization ( e.g. , normal , at risk , abnormal ) , which would likely improve the clinical utility of this device . psm appears to be a reliable and user - friendly tool for characterizing static standing posture . to maximize agreement , assessments should be conducted with the subject wearing minimal clothing so as to better identify anatomical landmarks . our results indicate that usage of psm requires minimal formal training since two inexperienced examiners reliably matched the trained physical therapist in the determination of most measures . moreover , psm may provide a cheaper , more feasible alternative to more advanced methods for initial patient screenings or for situations that do not require high precision . for assessments where a very high degree of accuracy is required , this study did not receive any funding , and authors have no conflicts of interest to declare . this study did not receive any funding , and authors have no conflicts of interest to declare .

[ purpose ] to determine the intra- and inter - rater agreement of a mobile application , posturescreen mobile ( psm ) , that assesses static standing posture . [ subjects and methods ] three examiners with different levels of experience of assessing posture , one licensed physical therapist and two untrained undergraduate students , performed repeated postural assessments of 10 subjects , fully clothed or minimally clothed , using psm on two nonconsecutive days . anterior and right lateral images were captured and seventeen landmarks were identified on them . intraclass correlation coefficients ( iccs ) were calculated for each of 13 postural measures to evaluate inter - rater agreement on the first visit ( fully or minimally clothed ) , as well as intra - rater agreement between the first and second visits ( minimally clothed ) . [ results ] eleven postural measures were ultimately analyzed for inter- and intra - rater agreement . inter - rater agreement was almost perfect ( icc0.81 ) for four measures and substantial ( 0.60<icc0.80 ) for three measures during the fully clothed exam . during the minimally clothed exam , inter - rater agreement was almost perfect for four measures and substantial for four measures . intra - rater agreement between two minimally clothed exams was almost perfect for two measures and substantial for five measures . [ conclusion ] psm is a widely available , inexpensive postural screening tool that requires little formal training . to maximize inter- and intra - rater agreement , postural screening using this mobile application should be conducted with subjects wearing minimal clothing . assessing static standing posture via psm gives repeatable measures for anatomical landmarks that were found to have substantial or almost perfect agreement . our data also suggest that this technology may also be useful for diagnosing forward head posture .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION Conflicts of interest

PMC3987469

as the power of medical technology advances , more and more difficult questions are raised about what sorts of rights to such technologies people might have , especially in resource - poor countries . people surely have a right to basic medical care , therefore global access to infertility care should be seen as a fundamental human right , with respect to socio - cultural , ethical and political differences worldwide . although ivf and related procedures get all the public attention , infertility care can not be reduced to assisted reproductive techniques alone . other options are equally important such as a listening ear and psychological support for infertile couples , the availability of basic diagnostic procedures , easy methods of ovarian stimulation and timed coitus , intrauterine insemination , reproductive surgery etc . the level of infertility care we are aiming for will differ from country to country . many variables can be important such as the economical and political situation of the country , the level of education and reproductive health care , actual facilities concerning medical care including the quality of the hospitals , the available equipment , facilities to perform surgery in case of complications , the level of mother care and many others . it is our aim to describe the facts and our views and vision on the issue of childlessness and infertility in developing countries . the large majority of childless couples are residents of developing countries . according to the who it is a silent population of more than 180 million couples facing the consequences of infertility day by day ( rutstein and iqbal , 2004 ) . infertility care is probably the most neglected and underestimated health care issue in developing countries . although the negative consequences of childlessness are much more pronounced in developing countries when compared to western societies , interest of the international community and local health care providers is still lacking ( ombelet et al . , 2008 ) . in less - developed countries the 12-month infertility prevalence rate ranges from 6.9 to 9.3% ( boivin et al . , 2007 ) . substantial geographical differences are noted and these differences can be explained by different environmental , cultural and socioeconomic influences . in sub - saharan africa infertility is caused by infections in over 85% of women compared to 33% worldwide ( cates et al . approximately 70% of pelvic infections are caused by stds while the other 30% are attributable to pregnancy - related sepsis ( ericksen and brunette , 1996 ) . similarly , many cases of male factor infertility are caused by previous infections of the male genitourinary tract ( kuku and osegbe , 1989 ) . both conditions are preferably treated by assisted reproductive technologies but most infertile couples in developing countries ca nt afford art because the techniques are too expensive and mostly limited to private centres ( nachtigall , 2006 ; murage et al . the consequences of involuntary childlessness are much more dramatic in developing countries and can create more wide ranging societal problems compared to western societies , particularly for women . negative psychosocial consequences are often severe and childless women are frequently stigmatised , isolated , ostracized , disinherited and neglected by the entire family and even the local community . this may result in physical and psychological violence and polygamy ( daar and merali , 2002 ; dyer 2004 , 2005 ; umezulike and efetie , 2004 ; ombelet et al . , 2008 ) . women are usually blamed for infertility and can be ostracized and assaulted by their families , even driven to suicide or killed ( anonymous , 2006 ) . because many families in developing countries completely depend on children for economic survival , childlessness has to be regarded as a social and public health issue and not only as an individual medical problem ( gerrits and shaw , 2010 ; papreen 2000 ; van balen and gerrits 2001 , 2009 ) . reduced fecundity in hiv - infected individuals has been described and marital instability and polygamy secondary to infertility may in turn increase the spread of hiv-1 infection . hiv is 3 times more prevalent in infertile couples when compared to fertile controls in the same population ( nabaitu et al . , 1994 ; both conditions are more common in resource - poor countries , may lead to stigmatisation and isolation and are strongly influenced by socio - cultural and economic conditions . treatment options are expensive and in both cases the final result is a diminished population . on the other hand , hiv treatment is becoming more effective and available at lower prices which are not the case for infertility treatment . awareness , attention , documentation and research of the hiv problem are much more pronounced compared to the infertility problem which remains mainly hidden . public solutions are being applied for hiv , for infertility the solution is mainly found in the private sector . it is striking , almost fascinating , that budgets for hiv research are huge and the information on hiv is easily available while the contrary is true for infertility . do we care ? despite the well documented observations of the social and economic consequences a surprisingly low interest is shown on the issue of infertility and childlessness on a national and international level . the two key arguments against treatment of infertility in developing countries are overpopulation and limited resources . the argument of overpopulation suggests that in countries where overpopulation poses a demographic problem , infertility management should not be supported by the government . it is well known that the world population is expected to increase from 6.7 billion inhabitants in 2005 to 9.2 billion in 2050 ( united nations , 2007 ) . by 2050 , the population of the developing world would be adding 35 million annually , 22 million of whom would be absorbed by the least developed countries . therefore , national and international health strategies have always focussed on reducing total fertility rates ( number of children per woman ) while infertility care has received little or no attention ( hamberger and janson , 1997 ) . but even if infertility treatment could be made more accessible in developing countries it would probably account for less than 1% of all deliveries . increasing efforts on family planning and health education can readily overcome this small contribution to the fertility rate . united nations data not only show that the majority of developing countries already succeeded to drop their global fertility rate below 2.5 , the data also show that the expected population growth in developing countries is mainly due to an improved life expectancy and not to high fertility rates ( united nations , 2007 , fig . 1 , fig . : it is well known that the fertility rate of a specific country is positively related to infant mortality , which is understandable because aged couples will depend economically from their children in many developing countries ( palloni and rafalimanana , 1997 ) . according to the limited resources argument it is hard to justify expensive fertility treatment in settings with few resources and more important challenges to deal with . can expensive techniques be justified in countries where poverty is still an important issue and where health care systems still struggle with the huge problem of infectious diseases such as malaria , tuberculosis and hiv ? in most developing countries the reduction of maternal mortality and the promotion of contraception are considered to be the reproductive health priorities ( aboulghar , 2005 ) . improved reproductive health education programmes have proven to be an excellent preventive tool against overpopulation , sexually transmitted diseases ( stds ) and pregnancy - related infections . but even with better education and preventative care programmes , involuntary childlessness will remain an important problem for millions of couples . reproductive autonomy is the main argument in favor of the provision of infertility treatment in developed countries . people have the right to decide when , how many and how to have children . why would citizens of developing countries not have the right to have at least one child , especially if we succeed to simplify the methods of infertility care and make them affordable for a much larger part of the population ? we recently carried out an internet - search for possible donors . a questionnaire dealing with the scope of their actions and the interest in infertility care in developing care was sent to the most important foundations , ngos and international societies linked to reproductive health ( fig . they all showed interest in the issue of childlessness in developing countries , but in none of these organisations infertility care has been funded before and no future projects were planned . considering local governments : it is not only the resource constraint which prevents the providing of infertility services in many developing countries . with the dominant discourse focusing on controlling overpopulation it is no wonder that infertile women are marginalised and consequently excluded from health sector interventions . infertile women are victims of the systematic process of cultural exclusion , but in some countries they are also international statements : promises and promises : men and woman of full age , without any limitation due to race , nationality or religion , have the right to marry and to raise a family . this statement was adopted 60 years ago at the 1948 un universal declaration of human rights and ca nt be misunderstood : it implies the right to access to fertility treatments when couples are unable to have children . at the united nations international conference on population and development in cairo in 1994 the following statement reproductive health therefore implies that people have the capability to reproduce and the freedom to decide if , when and how often to do so and to have the information and the means to do so . in 2004 the world health assembly proposed five core statements , including the provision of high - quality services for family - planning , including infertility services ( world health assembly , 2004 ) . the international federation of obstetricians and gynaecologists ( figo ) stated that women and men have the right to the highest available standard of health care for all aspects of their sexual and reproductive health political statements and commitments need to result in appropriate actions but progress towards the attainment of these goals on the subject of infertility in developing countries remains however slow . brain drain , lack of collaboration , budgetary constraints and lack of political commitment ( fathalla et al . , 2006 ) . on the other hand , the most important non - profit international organisations including family health international , who , international planned parenthood federation ( ippf ) and the population council still focus on safe motherhood , the reduction of unsafe abortions , prevention of stds and hiv / aids . the implementation of infertility treatment in developing countries is not a priority for these organizations . the level of reproductive health care education is very low in most developing countries although it is the most cost - effective strategy in the prevention of unwanted pregnancies and sexually transmitted diseases ( lutz and goujon , 2001 ) . a better education is clearly associated with lower fertility rates and an increasing access to education for women is probably the best strategy for an optimal population control ( fig . 4 ) . incorporation of reproductive health education in general health education should be supported by the governments . data from social researchers have shown that an important barrier to use contraception is the fear for consequent infertility . family - planning and infertility are clearly linked and should be handled in the same centres . public education on prevention of infertility includes not only prevention of stds and pregnancy - related infections , but also life style factors , iatrogenic infertility , environmental pollution and contamination . prevention of infertility remains the most cost - effective treatment strategy particularly in countries with a high prevalence of pregnancy - related infections and stds . advantages are numerous : prevention programmes are more cost - effective and benefit a greater number of people , they are more effective in eliminating the social consequences of infertility and will improve the health status of women in other ways . last but not least , prevention programmes can easily be integrated within existing mother - care and family - planning services . on the other hand , when resources are exclusively allocated to prevention programmes due to limited resources , millions of childless couples are ignored because prevention will fail in a substantial number of couples . although it is our belief that the governments should give priority to education and prevention programmes , this does not mean that no money should go to infertility treatment at all , especially if we could make infertility care less expensive and affordable . from an ethical and socio - cultural point of view we have to ask ourselves whether we can justify withholding infertility treatment , including art , in those cases where prevention has failed . believing that the arguments of overpopulation and limited resources are strong enough for not helping infertile couples in developing countries does nt take into account the human rights in general and the reproductive rights in particular ( vayena 2002 ) . efforts should be made to reduce the excessive social reactions to infertility inspired by pronatalism . therefore infertility treatment should be part of an integrated reproductive care programme including family planning and contraception , mother care , and reproductive health . education , empowerment of women and economic prosperity are the most effective solutions to most problems related to both population growth and infertility . simultaneously , investments in low - cost interventions are justified ( pennings et al . , 2009 ) . reproductive autonomy , justice and equity support our efforts to make art available and accessible worldwide subject to political stability and a basic level of medical infrastructure . most important is to focus on changing the existing moral and socio - cultural beliefs in so far that childless couples are no longer isolated and discriminated . we will need the media , patient organisations and interested politicians to reach this goal . obstacles will be numerous and will depend on local socio - cultural , political and religious influences . providing infertility care in developing countries can only be successful if we are able to diminish the socio - cultural , psychological and economic consequences of unwanted childlessness . patient support networks already claimed the right to equitable access to infertility treatment all over the world ( dill , 2007 ) . patients voices will be crucial when the issue of infertility has to be discussed with policy makers and health care providers . if we want to implement accessible fertility services in developing countries , the first objective is to simplify the diagnostic procedures ( malpani and malpani , 2002 ) . moreover , all of these procedures can be performed by a small team of health care providers within a short period of time in an inexpensive setting ( ombelet and campo , 2007 ) . implementing low cost art is only possible if we succeed to simplify the methods of treatment in such a way that they still are effective and safe , but affordable . for the recruitment of oocytes in an ivf programme we should avoid the use of high doses of expensive ovarian stimulation medication such as gonadotrophins , gnrh agonists and gnrh antagonists , if possible . the use of clomiphene citrate , a very cheap oral drug , has been proven in many studies to be an optimal alternative with acceptable results , minimal side effects and a very low complication rate ( ingerslev et al . , 2001 ; verberg et al . , 2009 ; aleyamma et al . , 2011 ) . lowering the costs associated with laboratory procedures , namely fertilization and culture of eggs and embryos , represents another challenge . humidicrib , a plastic box which is commonly used for keeping newborns snug , instead of an expensive laminar flow hood ( hovatta and cooke , 2006 ; pilcher , 2006 ) . for a tenth of the price this box can be modified to be used as a portable , near sterile environment for the handling of gametes and embryos . expensive cylinders of carbon dioxides required to incubate the embryos may be abandoned in favour of exhaling across the culture medium before sealing it in a plastic bag . this bag , containing the petri dish with the embryos , can be dropped into a warm bath without the need for expensive incubators . this technique has been successfully used for more than ten years in veterinary ivf ( vajta et al . , 1997 , 2004 ) . other strategies using very simple incubation systems are presently tested and the preliminary results are very promising ( van blerkom , personal communication ) . intravaginal fertilization and culturing is another inexpensive method for low cost ivf ( frydman and ranoux , 2008 ) . a tube filled with culture medium containing the oocytes and washed spermatozoa is hermetically closed and placed in the vagina . over 800 cycles cycles have been published worldwide with a very reasonable clinical pregnancy rate of almost 20% ( frydman and ranoux , 2008 ) . presently we do nt know if ivf , even minimal stimulation ivf , is feasible in less than ideal conditions when compared to centres with high standards of laboratory equipment and using standard stimulation protocols . therefore , studies on simplified , low - cost diagnostic procedures and art techniques are urgently required in a low - cost setting . we have to avoid that accessible art does nt mean poorer quality , otherwise we will create an unacceptable double standard in therapy . risks of implementing art in developing countries include the inability to deal with complications following infertility treatment . ovarian hyperstimulation syndrome , multiple pregnancies , premature babies , ectopic pregnancies are not uncommon in an art programme . these complications should be avoided at all price and the facilities and knowledge of the staff should be adequate to handle these problems . reproductive tourism by attracting international clients from western countries because treatment can be offered at lower prices is a real danger and is already reported in india ( vayena , 2009 ) . we also have to realize that even if universal access to infertility care will be available , barriers will always remain considerable . not only in many islamic countries ( serour , 2006 ) but also in latin america the use of art is severely restricted because of religious doctrines ( inhorn , 2003 ) . in india a conflict with the normative value of hindu because of the involvement of a third party which compromises the process of conception has been described ( bharadwaj , 2003 ) . patients in many non - western countries upholding traditional belief systems are mostly unfamiliar with the technology of modern medicine and therefore might be not willing to accept art as such ( qiu 2003 ) . consequently , traditional healers will play an important role and have to be educated about the new developments in infertility practice . because they speak the language of the local people and infertility care has to be an essential part of a more comprehensive reproductive health care program including infertility and hiv prevention , family - planning and safe mother - care , a go - together of prevention and treatment programs ( sharma et al . , 2009 ) . december 2007 the special task force developing countries and infertility of eshre ( european society of human reproduction and embryology ) organised an expert meeting on in arusha , tanzania : the arusha - project was born ! global access to infertility care is the key message but can only be implemented and sustained if they are supported by local policy makers as well as the international community . accessible infertility care should only be provided in developing countries if there is a political will to support actions for gender equality and women s empowerment through education . the implementation of new reproductive technologies will require well - organised education and training programmes . regular audits and systems of accreditation and registration should be implemented in order to maintain appropriate standards of care in all centres involved . the need for funding is crucial and is likely to require input and collaboration from various role players . funding is needed for the fixed costs of new fertility centres ( building , equipment , ... ) , the running services ( consumables , medication , medical interventions , staff salaries ) , for training the medical , paramedical and administrative staff and for education the public which implies contacts with schools , politicians , traditional healers and the media ( sallam , 2008 ) . i hope that the medical and pharmaceutical industry will also make relevant contributions such as providing cheap medication , manufacturing of basic ultrasound and laboratory equipment at low price etc . foundations have to be convinced about the value of this project , taking into account the growing demand from the developing countries itself and the case of equity and social justice . many international organizations have already expressed their will to participate in this initiative such as the who , eshre , iffs , icmart ( international society for monitoring assisted reproductive technology ) and ismaar ( international society for mild approaches to assisted reproduction ) . bilateral tubal occlusion due to sexually transmitted diseases ( stds ) and pregnancy - related infections is the most common cause of infertility in developing countries . consequently most cases of infertility are only treatable by using assisted reproductive technologies ( art ) which are either unavailable or very costly and only within reach of the happy few who can afford it . prevention remains the number one priority , not only the prevention of stds but also the prevention of infertility due to unsafe abortions and deliveries . we urgently need a better public education on reproductive health and raising awareness of health care providers and politicians on the importance of childlessness . most striking is the total lack of interest of the international society including foundations and non - governmental organizations working in the field of reproductive health . time has come to change policies and to realize that access to infertility care is one of the largest emerging fields in global medicine . the immense problem of childlessness in developing countries requires greater attention at national and international levels for reasons of social justice and equity . keystones in the successful implementation of infertility care in low - resource settings include simplification of art procedures in order to establish accessible good quality infertility services at low cost . to conclude , i believe that global access to infertility care in developing countries can only be achieved when good quality but affordable infertility care is linked to more effective family planning and safe motherhood programmes . only a global project with respect to socio - cultural , ethical , economical and political differences can be successful and convince those who believe that the issue of overpopulation is still an absolute argument to deny the forbidden desire of many childless women in developing countries . in a world that needs vigorous control of population growth , concerns about infertility may seem odd , but the adoption of a small family norm makes the issue of involuntary infertility more pressing . if couples are urged to postpone or widely space pregnancies , it is imperative that they should be helped to achieve pregnancy when they so decide , in the more limited time they will have available . mother or nothing the agony of infertility - m. fathalla , who bulletin , 2010

according to who data more than 180 million couples in developing countries suffer from primary or secondary infertility . the social stigma of childlessness still leads to isolation and abandonment in many developing countries . differences between the developed and developing world are emerging because of the different availability in infertility care and different socio - cultural value surrounding procreation and childlessness.although reproductive health education and prevention of infertility are number one priorities , the need for accessible diagnostic procedures and new reproductive technologies ( art ) is very high . the success and sustainability of art in resource - poor settings will depend to a large extend on our ability to optimise these techniques in terms of availability , affordability and effectiveness.accessible infertility treatment can only be successfully introduced in developing countries if socio - cultural and economic prerequisites are fulfilled and governments can be persuaded to support their introduction . we have to liaise with the relevant authorities to discuss the strengthening of infertility services , at the core of which lies the integration of infertility , contraceptive and maternal health services within public health care structures.after a fascinating period of more than 30 years of ivf , only a small part of the world population benefits from these new technologies . time has come to give equitable access to effective and safe infertility care in resource - poor countries as well .

Introduction The Facts Views Vision Conclusion

PMC4045514

globalization is not a new trend in auxology . for decades , researchers have compared growth and maturation in populations across the world , exploring racial and regional differences and studying the effects of changes in lifestyle . in particular , there has been a longstanding interest in the differences in tempo of maturation among populations and their secular trend . such studies have preferentially been performed using bone age ( ba ) determinations . unlike secondary sexual characteristics , ba can reveal the entire course of maturation from birth to adulthood . however , bone age rating is associated with considerable operator variability , and it is particularly difficult to ensure consistency between raters from widely separated parts of the world . the emergence of a fully automated and therefore rater - independent ba rating method has the potential to overcome this problem and , thereby , revitalize comparative studies of maturation across the world . we present a reanalysis of a large study of hand radiographs involving modern , normal chinese children using this automated bone age system . due to the one - child policy , parents have a strong focus on growth and development of their child , and currently , there are more than 500.000 ba determinations in china per year . this need is growing , but there is a shortage of persons qualified to do the rating . adult height prediction in asian children based on automated ba will be reported in a separate paper . our study had the following objectives : to validate the automated method by assessing its ability to analyse these images and its agreement with manual tanner - whitehouse ( tw ) ratings ; to derive reference curves for greulich - pyle ( gp ) ba of chinese children , to study their regional variation in china , and to compare the curves to american children studied with the same bone age method ; to construct a chinese ba scale ( called bx - china05 ) as an adaptation of the gp ba for the chinese population in 2005;to construct a chinese tw ba scale called tw - china05 . to validate the automated method by assessing its ability to analyse these images and its agreement with manual tanner - whitehouse ( tw ) ratings ; to derive reference curves for greulich - pyle ( gp ) ba of chinese children , to study their regional variation in china , and to compare the curves to american children studied with the same bone age method ; to construct a chinese ba scale ( called bx - china05 ) as an adaptation of the gp ba for the chinese population in 2005 ; to construct a chinese tw ba scale called tw - china05 . left hand radiographs of 2883 males aged 220 years and 3143 females aged 219 years were extracted from a previous study of children of the han ethnicity from five different cities in china . the full age range was covered in the children from dalian , wenzhou , and shijiazhuang , while the age range started at 5 years in guangzhou and at 7 years in shanghai . the images were taken close to the children 's anniversaries ; in addition , images were taken at 2.5 and 3.5 years of age ( the age distribution can be appreciated from figure 2 ) . the original study was approved by the ethical committee of the medical department , hebei sports science institute , in 2002 . before the study started , a questionnaire was given to children 's parents for surveying background such as parent 's occupation , education level , income , and the medical history of the children and their family , and a written informed consent was obtained from the parents and/or the children . the films were scanned using a vidar diagnostic pro advantage scanner ( hemdon , usa ) in 150 dpi and 12 bits per pixel . the radiographs formed a representative sample of the previous study which comprised 17,000 radiographs recorded on films . these radiographs had previously been ba rated by the first author using the tw method . the images were analysed using the bonexpert version 2.1 automated method for ba determination ( visiana , denmark , http://www.bonexpert.com ) , which determines gp and tw3 ba [ 57 ] . the method was developed on caucasian children and has been validated on asian children from japan and california [ 8 , 9 ] . the tw ba rating , more specifically , the assignment of stages to each bone , was recently recalibrated in order to coincide with ratings from the first zurich longitudinal study . this rating , called standard tw rating , has been shown to be consistent with tanner 's ratings of the so - called gold series of reference images . in order to compare bone maturation in different regions , reference curves of ba ca versus chronological age ( ca ) were derived for each city separately and for all cites combined , using gp ba . it is fast and reliable , because the image is rated by direct comparison to reference images . however , the gp ba scale refers to mid- to upper - class american caucasian children from the 1930s , a standard group remote from present - day chinese children . we therefore developed a variant of gp ba called bx - china05 as follows . the gp ba is considered to be not an estimate of ca , but rather an abstract maturity measure , and for each age , we determined the median gp ba of chinese children . the relationship between ca and median gp ba was tabulated and used to transform an observed gp ba value into an age value , defined as bx - china05 . for example , if the median gp ba is 13.5 at a ca of 13 years , a child with a gp ba of 13.5 has a bx - china05 of 13 years . this method of mapping a maturity measure to a population - specific bone age is not new ; it was originally conceived in the context of the tw method , and we also carried it through for the tw method in this study . therefore , for each age , we derived the median sum maturity score ( sms ) of the automated tw rating in order to derive a table for transforming an observed sms value into a bone age , which we call tw - china05 ( such a table had been estimated before using the manual tw ratings of these image data . by reestimating the table using automated tw ratings , we ensured that the table was consistent with bonexpert 's standard tw rating of bone stages ) . thirty images were rejected by bonexpert before arriving at the 6026 images of this study . most of these rejections were due to poor image quality or a bone age below the lower limit of the intended range of bonexpert ( 2.5 years for boys and 2.0 years for girls ) , but 12 of the rejected images were of good quality , corresponding to 0.2% , so the observed efficiency of the method was 99.8% . figure 1 shows the agreement between the manual tw3 rating and bonexpert 's tw3 rating , expressed as a bland - altman plot , that is , the difference is plotted versus tw3mid , the average of the two tw3 bone ages . focusing on the tw3mid range of 2.516 years for boys and 2.514.5 years for girls , the average difference between automated and manual tw3 ratings was 0.00 years for boys and 0.37 years for girls . the root mean square ( rms ) deviations were 0.64 for boys and 0.68 years for girls in the same tw3mid range . to study the difference in tempo of maturation across various populations and ethnicities , we used the gp ba minus the age ( ca ) . figure 2 displays the observed ba ca data , and the mean and 2 sd curves are indicated . the sd for boys within the age range of 817 years was 1.26 years , and the sd for girls aged 7 to 15 years was 1.12 years ( the sd curve was computed in 1-year intervals , and the quoted sd numbers are the averages over the entire age interval ) . figure 3 shows the average curves for each of the five cities , and figure 4 compares the ba ca curves with data on four ethnicities from los angeles , usa . the new bone age , bx - china05 , is reported in table 1 . to calculate bx - china05 for a new image , one should first determine the bonexpert gp ba and then apply the correction from table 1 . it is seen that the average difference was close to zero for all ages as expected . the sd for boys over the age range of 814 years was 1.08 years , and for girls aged 712 years , the sd was 1.01 years . table 2 shows the relationship between bonexpert 's sms and tw - china05 , the new tw ba adapted to this population , and figure 6 compares bonexpert 's tw - china05 ba and the manual tw - china05 ba . the sd of tw - china05 ca for boys aged 814 years was 1.09 , and for girls aged 712 years , it was 1.01 years . we found that bonexpert was able to determine bone age for 99.8% of the good - quality images of chinese children . this result compares well with previous results in caucasian children , despite that this is a new ethnic group , and the similar performance is probably due to fact that the variation between the average caucasian and asian child is much smaller than the variation within the caucasian population itself . there was good agreement between automated standard tw3 rating and manual tw3 as shown in figure 1 . firstly , there was only a small average difference ( bias ) of 0.00 years in the boys and 0.37 years in the girls , which indicates that the chinese tw rater was largely consistent with the european raters underlying the calibration of bonexpert 's standard tw rating . this agreement was in contrast to that reported in where a japanese rater had a bias of up to 1.19 years . secondly , the rms deviation ( 0.64 years for boys and 0.68 years for girls ) was smaller than that observed for the zurich raters . one reason for this slightly better result may lie in the fact that the chinese children were rated by a single rater while the zurich images were rated by one of two raters . the chinese rater was never trained by authoritative raters on the tw gold series radiographs in london or on other series . he learned the rating from the book but still achieved good agreement with the automated rating . the most striking aspect is that , at the end of puberty , the average ba ca has reached approximately 1 year . in other words , chinese children reach maturity around one year earlier than the children that made up the gp ba reference , in agreement with previous studies . this dramatic deviation was the main motivation for setting up the bx - china05 ba scale . figure 2 also shows that the spread in maturity is rather large in chinese children . this is partly due to the fact that the children were from several cities , separated by large distances . to illustrate this effect , figure 2 shows the average ba ca for the two cities at either end of the ba ca spectrum , dalian , a middle - size city in the north , and shanghai , a large city in the south . our pooling of all five cities contributed to a larger sd of ba ca . even within each city , there was a relatively large spread , presumably due to differences between social classes . figure 3 reveals the significant differences between the five cities , with shanghai being the most advanced in ba and dalian the most delayed , and the other cities lie in between these two extremes . in principle , these data could be used to set up city - specific reference curves for ba ca . however , we believe that this would be impractical , and one would be faced with ambiguities when a child moves from one city to another , or if the child is from a city not among the five investigated here . we prefer to pool all children and treat city differences as a statistical uncertainty . a limitation of this sample is that all data come from urban regions and middle and upper social classes , so the sample might not be representative for children from a rural community or children living under poor social conditions . figure 4 compares the chinese ba ca curves with data on four ethnicities from los angeles , usa . this comparison exploits the strength of the automated method . for the first time , one is able to compare maturity of chinese children with children in the west using a method guaranteed to have no rater bias . the upper part of the figure compares los angeles children of different ethnicities , showing that asians and hispanics in los angeles have similar maturation tempo , while the lower plot compares asian children from los angeles and china . we see that asians , averaged over the two sexes , reach maturity 0.6 years before american caucasians . this comparison of the populations with each other is more interesting than a comparison to the outdated gp standard . in , it was reported that american caucasians reach maturity 0.4 years before europeans , so we infer that asians reach maturity a full year ahead of european caucasians . the new bone age bx - china05 is defined by table 1 , which can conveniently be built into the automated method . the table can also be used to transform a manually determined gp ba to bx - china05 . the table can even be used to adjust the ages of each plate of the gp atlas , thereby translating it to chinese . for instance , the female plate with gp ba 12.0 years can be labelled 11.5 years for chinese females . the new bx - china scales end 1.2 years earlier than the original gp ba scale . the new tw - china05 scale ends at ages 16.1 years for boys and 15.1 years for girls , similar to the endings of the corresponding manual tw - china05 scale of 16 and 15 years , respectively , as also illustrated in figure 6 . both are adapted to agree , on average , with the age in the chinese population . however , there are important differences , because bx - china05 is based on the gp method whereas tw - china05 is based on the tw method . gp and tw ba rating represent two rather different methods . when used for manual rating , the tw method has the advantage that it employs an explicit procedure that ensures that the same bones are used , with the same weights , by all raters . the gp method , on the other hand , has the advantage that it easier to learn and can be performed in a fast and intuitive manner . furthermore , its reliance on a direct comparison of the image with the plates in the atlas ensures that raters are constantly calibrated to the correct level . the tw method based on manual rating has been used extensively in china , rather than the gp method , because the latter refers to pre - ww ii ohio children which are far removed from modern chinese children . this paper has introduced bx - china05 for chinese children , based on the gp method . the gp atlas is both a method and a ba standard . with bx - china05 , one uses the gp method but escapes its ba standard by employing the corrections in table 1 . with the introduction of automated methods , and the recalibration of gp ba in the shape of bx - china05 , the balance between the tw and the gp methods changes in favour of the gp method , which has several advantages relative to the tw method . in the automated method used in this paper , the tw and gp methods are both based on the 13 rus bones ( radius , ulna , and 11 short bones ) chosen by tanner for the tw method . the gp method assigns a uniform weight of 7.7% to all 13 bones , while the tw method places a larger weight of 20% on both radius and ulna . any uncertainty in the interpretation of radius and ulna will , therefore , have a disproportionately large effect on the tw ba , and , indeed , the precision of the automated gp ba is considerably better ( 0.18 years sd ) than that of the automated tw ba ( 0.30 years ) .adult height prediction has been found to be more accurate using gp compared to tw ba , primarily due to the large weight of the radius and ulna in the tw method . the bonexpert method for adult height prediction is , therefore , based on gp ba ( this is also true for the method for chinese children , presented in a separate paper).the tw3 ba terminates at 16.5 and 15 years for boys and girls , respectively , while the gp ba terminates at 19 and 18 years . in other words , this also holds for the automated versions where tw3 and gp agree well up to the ba where tw3 saturates . the same findings are noted in the chinese version where tw - china05 stops 1.7 years before bx - china05 . when rating the maturity of adolescents , the gp method is the more powerful method . notice , however , that the automated method has the general limitation that it becomes more uncertain above a certain maturity level , which is 17 and 15 years of gp ba for boys and girls , respectively . this translates to the limits of 15.8 and 13.8 years , respectively , on the bx - china05 scale . above these limits , there are only small maturity - related changes in the appearance of the short bones . in the automated method used in this paper , the tw and gp methods are both based on the 13 rus bones ( radius , ulna , and 11 short bones ) chosen by tanner for the tw method . the gp method assigns a uniform weight of 7.7% to all 13 bones , while the tw method places a larger weight of 20% on both radius and ulna . any uncertainty in the interpretation of radius and ulna will , therefore , have a disproportionately large effect on the tw ba , and , indeed , the precision of the automated gp ba is considerably better ( 0.18 years sd ) than that of the automated tw ba ( 0.30 years ) . adult height prediction has been found to be more accurate using gp compared to tw ba , primarily due to the large weight of the radius and ulna in the tw method . the bonexpert method for adult height prediction is , therefore , based on gp ba ( this is also true for the method for chinese children , presented in a separate paper ) . the tw3 ba terminates at 16.5 and 15 years for boys and girls , respectively , while the gp ba terminates at 19 and 18 years . in other words , this also holds for the automated versions where tw3 and gp agree well up to the ba where tw3 saturates . the same findings are noted in the chinese version where tw - china05 stops 1.7 years before bx - china05 . when rating the maturity of adolescents , the gp method is the more powerful method . notice , however , that the automated method has the general limitation that it becomes more uncertain above a certain maturity level , which is 17 and 15 years of gp ba for boys and girls , respectively . this translates to the limits of 15.8 and 13.8 years , respectively , on the bx - china05 scale . above these limits , there are only small maturity - related changes in the appearance of the short bones . in rare cases where the automated method fails , it is recommended to record a new x - ray , unless the child has abnormal bone structure . one can also perform a manual rating using the gp atlas and then use table 1 to correct the gp ba to the bx - china05 . it would even be acceptable to apply the manual tw - china05 method , in case the rater is more familiar with this method . we have presented an important step toward the application of automated ba assessment to asian populations around the world . we have found that asian children in los angeles , and in mid- to large - sized cities in china , have approximately the same maturation rates . asians reach full maturity 1.2 years before the age indicated by the gp ba reference , and , thus , the use of this standard in china is considered misleading by some . on the other hand , as we have pointed out , there are many advantages of the gp atlas methodology , and we have presented an adaptation of the gp ba to chinese children , called bx - china05 .

rationale and objective . large studies have previously been performed to set up a chinese bone age reference , but it has been difficult to compare the maturation of chinese children with populations elsewhere due to the potential variability between raters in different parts of the world . we re - analysed the radiographs from a large study of normal chinese children using an automated bone age rating method to establish a chinese bone age reference , and to compare the tempo of maturation in the chinese with other populations . materials and methods . x - rays from 2883 boys and 3143 girls aged 220 years from five chinese cities , taken in 2005 , were evaluated using the bonexpert automated method . results . chinese children reached full maturity at the same age as previously studied asian children from los angeles , but 0.6 years earlier than caucasian children in los angeles . the greulich - pyle bone age method was adapted to the chinese population creating a new bone age scale bx - china05 . the standard deviation between bx - china05 and chronologic age was 1.01 years in boys aged 814 , and 1.08 years in girls aged 712 . conclusion . by eliminating rater variability , the automated method provides a reliable and efficient standard for bone age determination in china .

1. Introduction 2. Subjects and Methods 3. Results 4. Discussion 5. Conclusion

PMC4868243

in the development of nutritional and physical activity interventions , understanding resting metabolic rate ( rmr ) is important to identify the absolute daily energy requirement and achieve desirable energy consumption1 . rmr provides energy necessary to maintain body functions at rest and accounts for 6080% of total energy expenditure2 . an imbalance of energy intake and expenditure leads to weight gain or loss in the long term3 . physical exercise improves health by improving cardiorespiratory fitness , body composition , and psychosocial well - being . in addition , physical exercise is an important tool in the prevention and treatment of obesity4,5,6 . physical exercise improves body composition and metabolic activity , thereby reducing excess weight and related comorbidities7,8,9,10,11 . vo2max measures the maximal oxygen consumption during exercise to the point of exhaustion of physical strength and is one of the best methods of predicting cardiorespiratory endurance and aerobic preparation . the amount of energy needed by individuals is associated with body mass , and vo2max is associated with body weight . regular aerobic physical activities increase vo2max and indirectly reduce the effects of many diseases12 . the respiratory system plays an important role in providing the energy required by different body systems for metabolism . therefore , the respiratory system is greatly affected by short- and long - term exercise . owing to its important role in physical activities , the respiratory system is studied by many researchers13 . the level of cardiorespiratory fitness varies with the condition of the respiratory , cardiovascular , and musculoskeletal systems , and its evaluation is important because of its relationship to health and wellness . poor cardiorespiratory fitness increases the rate of all causes of premature death , and especially that due to cardiovascular diseases . improvement in cardiorespiratory fitness is associated with reduced premature death rates due to all causes14 , 15 . many previous studies examined changes in rmr and vo2max after intervention with defined exercise for a certain period of time16,17,18,19 . in addition , studies have compared rmr and vo2max for habitual physical activities , regularly performed types of exercise , and different fitness levels . gilliat - wimbery et al.20 studied differences in rmr and vo2max between the two groups with different habitual physical activities , and toth et al.21 studied differences in rmr and vo2max among three groups with different types of regularly performed exercise . in addition , mccargar et al . compared the levels of vo2max for different fitness levels22 . however , studies that subdivide individuals by amount of exercise at normal times are lacking . therefore , the present study intended to examine the effects of individual weekly exercise time on rmr and vo2max , which are important components of individual health indexes . this study can be helpful in preventing and treating health problems using subdivided exercise programs customized for individuals . thirty healthy adult male and female subjects between the ages of 20 and 60 who satisfied the following selection criteria were studied : without cardiopulmonary , metabolic , or musculoskeletal disorders ; not pregnant ; not taking any mood - altering medications or other drugs , including anti - inflammatory drugs , antihistamines , pain medications , antidepressants , or beta - blockers23 ; not athletes ; had not participated in similar studies ; understood the purpose of the study , and agreed to participate . all participants were informed about the potential risks and experimental design and provided informed consent for participation , with the knowledge that they could withdraw at any time . the general characteristics of the study subjects are shown in table 1table 1.general characteristics of the study subjectsgroup a(n=25)meansdgroup b(n=15)meansdgroup c(n=17)meansdgender ( male / female)12/136/97/10age ( years)41.213.140.212.442.312.7height ( cm)164.17.2162.98.0165.76.9weight ( kg)64.112.162.711.763.414.2values are mean sd . the frequency of walking at moderate or high intensity24 of exercise awareness ( 12 or more on the borg scale ) at normal times , and the duration of exercise per session were surveyed . the subjects were divided into group a , who did not perform any walking exercise at all per week , group b , who performed walking for less than 200 minutes per week , and group c , who performed walking for 200 minutes or more per week . a body composition analyzer ( inbody720 , biospace , south korea ) was used , and general characteristics including age , height , weight , body mass index ( bmi ) , fat free mass ( ffm ) , muscle mass , skeletal muscle mass , and obesity indexes such as visceral fat area ( vfa ) and waist - hip ratio ( whr ) were recorded . rmr was measured using a respiratory gas analyzer ( quark b2 , cosmed , italy ) . all subjects were prohibited from performing exercise for four hours before measurement of rmr between 10:00 am and 4:00 pm . rmr was measured in supine subjects for 31 minutes after a five - minute rest25 . graded exercise tests were conducted using a 12-channel electrocardiogram scanner ( case 6.0 , ge , usa ) , respiratory gas analyzer ( quark b2 , cosmed , italy ) , and automatic blood pressure ( bp ) and pulse measuring instrument ( tango , suntech medical , usa ) on a treadmill ( t2100 , ge , usa ) under supervision by a researcher and safety guard . subjects were instructed to stop exercise in one or more of the following situations : heart rate ( hr ) did not increase with exercise intensity ; respiratory exchange ratio was 1.15 or higher ; exercise awareness ( borg scale ) was 17 or higher ; hr was 90% or higher than age - predicted maximum ( 220 minusage ) ; or o2 uptake did not increase with exercise intensity27 . along with vo2max , exercise ability indexes were measured , including hemodynamic indexes such as bp and hr at the time of maximum exercise , the ventilatory equivalent for o2 ( ve / vo2 ) , the ventilatory equivalent for carbon dioxide ( ve / vco2 ) , and the physiological dead space / tidal volume ratio ( vd / vt ) . in addition , respiratory indexes , such as breathing reserve ( br ) , and metabolic indexes , such as anaerobic threshold ( at ) and maximum o2 pulse , were measured . to analyze the data in this study , statistics program spss 18.0 for windows was used . for general characteristics of this study , frequency analysis , means , and standard deviations were calculated . to compare differences between the groups in headache and stiffness changes , one - way analysis of variance in addition , to examine differences in headache and stiffness before and after each intervention , a paired t - test was conducted . the results of graded exercise tests of the three groups are shown in table 2table 2.graded exercise testvariablesgroup agroup bgroup cmax . bp ( mmhg)83.021.987.014.191.216.2ve / vo2 ( l / min)46.87.545.77.045.66.4ve / vco2 ( l / min)37.65.139.05.436.15.0vd ( ml)323.5107.7376.7107.7390.0118.0vd / vt0.2140.0290.2250.0430.2100.037br ( max . ve / mvv , % ) 88.03.587.82.688.63.5vo2max ( ml / min)1,735.4467.52,113.0443.62,445.1616.6at ( % ) 61.921.082.629.992.438.6o2pulse ( ml / beat)13.04.415.97.322.220.9values are meansd , * p<0.05 , group a did not perform any walking exercise at all per week , group b performed walking for less than 200 minutes per week , group c performed walking for 200 minutes or more per week , max . bp : maximum diastolic blood pressure , ve / vo2 : ve for o2 , ve / vco2 : ve for co2 , vd : physiological dead space , vd / vt : physiological dead space / tidal volume ratio , br : breathing reverse ( maximum exercise ventilation / maximal voluntary ventilation ) , vo2max : maximal oxygen uptake , at : anaerobic threshold ( % , o2 uptake at at / peak o2 uptake ) , o2 pulse : peak o2 pulse ( vo2/hr ) . significant differences in vo2max and at were found among the three groups ( p<0.05 ) . however , the remaining indexes did not show any statistically significant differences ( p>0.05 ) . the results for the rmr and obesity indexes of the three groups are shown in table 3table 3.results for rmr and obesity indexesvariablesgroup agroup bgroup crmr ( l / min)1,083.3267.81,314394.01,420.2244.0bmi ( kg / m)22.32.123.182.924.62.9ffm ( kg)45.99.349.88.853.69.7muscle mass ( kg)43.38.947.08.550.69.3skeletal muscle mass ( kg)25.35.827.65.430.06.0vfa ( cm)75.436.793.044.489.626.8whr0.80.00.80.00.90.0values are meansd , * p<0.05 , group a did not perform any walking exercise at all per week , group b performed walking for less than 200 minutes per week , group c performed walking for 200 minutes or more per week , rmr : resting metabolic rate , bmi : body mass index , ffm : fat free mass , vfa : visceral fat area , whr : waist - hip ratio . values are meansd , * p<0.05 , group a did not perform any walking exercise at all per week , group b performed walking for less than 200 minutes per week , group c performed walking for 200 minutes or more per week , max . bp : maximum diastolic blood pressure , ve / vo2 : ve for o2 , ve / vco2 : ve for co2 , vd : physiological dead space , vd / vt : physiological dead space / tidal volume ratio , br : breathing reverse ( maximum exercise ventilation / maximal voluntary ventilation ) , vo2max : maximal oxygen uptake , at : anaerobic threshold ( % , o2 uptake at at / peak o2 uptake ) , o2 pulse : peak o2 pulse ( vo2/hr ) values are meansd , * p<0.05 , group a did not perform any walking exercise at all per week , group b performed walking for less than 200 minutes per week , group c performed walking for 200 minutes or more per week , rmr : resting metabolic rate , bmi : body mass index , ffm : fat free mass , vfa : visceral fat area , whr : waist - hip ratio the present study examined the effects of normal weekly exercise time on vo2max and rmr in adults . vo2max and rmr were significantly different among the three groups divided by weekly exercise at normal times . gilliat - wimberly et al . studied healthy female subjects aged 3550 years who were divided into groups performing physical activity for at least nine hours per week or for approximately one hour per week20 . the rmr of the group performing activity for at least nine hours was significantly higher than that of the group performing physical activity for approximately one hour . in a study by toth et al . , individuals aged 3659 years were divided into three groups based on types of exercise : a resistance - trained group , an aerobic - trained group , and an untrained group . the aerobic - trained group showed significantly higher rmr than the untrained group and higher rmr values than the resistance - trained group , although the differences were not significant . in addition , the aerobic - trained group showed significantly higher vo2max than the untrained and resistance - trained groups . in a study by mccargar et al . , overweight women aged 2549 years were divided into groups with above or below average fitness levels by using a 1-mile timed walking test22 . the vo2max of the group with below average fitness levels was statistically significantly lower than the group with above average levels . therefore , exercise duration and type at normal times and fitness levels have positive effects on vo2max and rmr . shim et al . reported that when individuals aged 55 years or older underwent muscle strength training once per week for eight weeks , rmr significantly increased from weeks 4516 . reported that when male subjects aged 3545 were divided into a fat group and a normal group and performed aerobic exercise three times per week for eight weeks , both groups showed statistically significant increases in vo2max17 . reported that when university student athletes were divided into an aerobic swimming group and an anaerobic swimming group and swam intermittently at least three times per week for six weeks , both groups showed statistically significant increases in vo2max18 . in a study by osei - tutu et al . , individuals were divided into two groups and walked five times per week for eight weeks ; both the group that walked for 30 minutes continuously per day and the group that walked for 10 minutes three times per day showed statistically significant increases in vo2max19 . the subjects in the present study showed differences in vo2max and rmr for different amounts of exercise at normal times . the above studies show that diverse exercises that are performed consistently will positively affect vo2max and rmr . in the present study , not only vo2max and rmr but at is a direct indicator of aerobic capacity28 and is widely used in setting the intensity of aerobic training of healthy subjects29 and individuals with chronic diseases ( e.g. , coronary heart disease30 , hypertension31 , or obesity32 ) . a study by farrel et al.33 reported that long - distance running was associated with both vo2max and at . group c in the present study is thought to have shown higher at values compared with the other groups because group c performed larger amounts of exercise at normal times . in the present study , weekly exercise at normal times affected vo2max , rmr , and at , all of which are indicators of individual physical abilities and health , and these three values increased with the amount of exercise . in addition , vo2max , rmr , and at are considered to be closely related , consistent with the results of previous studies . the results of the present study should be helpful for the development of exercise programs to enhance individual physical abilities .

[ purpose ] the present study examined the effect of individual weekly exercise time on resting metabolic rate and vo2max ( maximal oxygen uptake ) , which are important components of individual health indexes . [ subjects and methods ] thirty healthy adults participated in this study . questionnaires were used to divide the participants into groups based on average weekly walking . resting metabolic rate was measured using a respiratory gas analyzer . graded exercise tests were conducted using a treadmill , and the modified bruce protocol was used as an exercise test method . [ results ] vo2max , anaerobic threshold , and resting metabolic rate were significantly different among the groups . [ conclusion ] average weekly exercise time affected vo2max , resting metabolic rate , and anaerobic threshold , all of which are indicators of individual physical ability and health . these values increased as the individual amount of exercise increased . in addition , vo2max , resting metabolic rate , and anaerobic threshold were found to be closely correlated . these findings were consistent with the results of similar previous studies .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4053279

k. pneumoniae belonging to the family of enterobacteriaceae is an important opportunist human pathogen associated with hospital - acquired infections such as pneumonia , urinary tract infections , or bacteraemias [ 13 ] . this species naturally produces a broad - spectrum -lactamase of the shv-1 type or penicillinase len that confers resistance to penicillins and to first - generation cephalosporins . the widespread use of expanded - spectrum antibiotics for treatment of serious infections due to gram negative bacteria has led to the appearance of extended - spectrum -lactamases ( esbls ) which are able to hydrolyze these compounds [ 48 ] . since their first identification in germany and the united states of america , extended - spectrum -lactamases ( esbls ) have spread worldwide [ 6 , 7 ] . these enzymes are mostly plasmid - encoded variants of tem-1 , tem-2 , and shv-1 by one or more amino acids or are from a rapidly evolving class called ctx - m . they confer resistance to broad - spectrum penicillins , monobactams , and narrow - spectrum and third - generation cephalosporins , respectively [ 4 , 8 ] . the prevalence of esbl producing k. pneumoniae isolates in hospitals ranges from 5 to 25% in several parts of the world . in september 2012 , more than 100 shv variants have been identified ( http://www.lahey.org/ ; j. a. jacoby and k. bush ) . in tunisia , shv-2a and shv-12 were the predominant shv - type esbl produced by k. pneumoniae . in this work , we searched for the presence of potential new shv 's variants produced by k. pneumoniae isolates in tunisia clinical settings . from this survey , we isolated a k. pneumoniae strain ( ml2011 ) resistant to third - generation cephalosporin and aztreonam . our goal was to study the contribution of shv-104 in the multiresistant phenotype of the strain . k. pneumoniae ml2011 was collected in july 2004 , from intensive care unit of the military hospital of tunis ( tunisia ) . identification of strains was performed by using both the vitek automated system ( biomrieux , marcy l'etoile , france ) and api 20 e system ( biomrieux , marcy l'etoile , france ) . biochemical and serological confirmation of the identity of this strain was done at the laboratory of bacteriology , military hospital , tunisia . e. coli dh5 and e. coli hb101 were used , respectively , for the transformation and conjugation experiments . e. coli bl21 and bl21 ( de3 ) ( plyss ) were used for the resistance profile of the different strains and for the overexpression of shv-104 , respectively . disc susceptibility testing was performed according to clinical and laboratory standards institute ( clsi ) guidelines on mueller - hinton agar ( bio rad , france ) . detection of esbl activity was achieved with a double - disk synergy test , by screening isolate for synergism between amoxicillin - clavulanate and cefotaxime , ceftriaxone , ceftazidime , and aztreonam , respectively . minimal inhibitory concentrations ( mics ) of ampicillin , ticarcillin , ceftriaxone , cefotaxime , ceftazidime , cefoxitin , imipenem , streptomycin , and chloramphenicol were determined by etest strips , according to the manufacturer 's instructions ( ab biodisk , solna , sweden ) , and interpreted according to clinical breakpoints from the clsi and eucast . pi of the -lactamases produced by k. pneumoniae ml2011 , the hb101 transconjugants , and the bl21 transformants were determined as described previously . plasmid dna of the clinical isolate was extracted with a plasmid extraction kit gfx micro plasmid prep ( amersham biosciences , uk ) , according to the manufacturer 's instructions . plasmid dna electrophoresis was performed in 1% agarose gel and visualized with ethidium bromide under uv light . the plasmid dna of e. coli rp4 ( 50 kb ) was used as a molecular size marker [ 13 , 14 ] . transformants were selected on luria bertani ( lb ) medium agar plates supplemented with ampicillin ( 100 g / ml ) . transfer of resistance phenotypes was performed by a liquid mating method on lb broth medium . culture mixtures were incubated overnight at 37c with transformants e. coli dh5/pml2011 as donors and e. coli hb101 as recipient strain . after conjugation , bacterial suspensions were plated onto agar containing ampicillin ( 100 g / ml ) and streptomycin ( 100 g / ml ) . amplifications of the blatem , blashv , and blactx - m genes by pcr were carried out using the plasmid pml2011 and the chromosomal dna of k. pneumoniae ml2011 as the template . the pcr products ( corresponding to shv and ctx - m genes ) were visualized by agarose gel electrophoresis , purified with a gfx pcr dna and gel band purification kit ( amersham biosciences , uk ) . prior to their cloning into ptz57r / t vector ( fermentas , usa ) , the sequence of the pcr fragment was verified by giga genomics facility ( ulg , belgium ) . the ligation mix was transformed into e. coli dh5 competent cells . positive colonies were selected on lb broth medium supplemented with ampicillin ( 100 g / ml ) . the insert sequences were performed by giga genomics facility in belgium , using forward and reverse m13 universal primers : puc / m13f ( 5-cgc cag ggt ttt ccc agt cac gac-3 ) and puc / m13 r ( 5-tca cac agg aaa cag cta tga c-3 ) . ndei and bamhi restriction sites were inserted at the 5 and 3 extremities of blashv , respectively , by pcr . the nucleotides sequences of the pcr - generated fragments were firstly verified by sequencing and secondly cloned into the pet26b ( + ) vector ( novagen inc . , / shv-104 , was used to transform e. coli bl21 ( de3 ) ( plyss ) competent cells ( novagen inc . , the shv-104 enzyme was produced in terrific broth ( tb ) medium containing kanamycin ( 50 g / ml ) and chloramphenicol ( 30 g / ml ) as selecting agents during growth of the bacteria at 37c under orbital shaking . 40 ml of an overnight preculture in tb was inoculated to 1 l of fresh tb supplemented with kanamycin and chloramphenicol . at an absorbance value of 0.7 at 600 nm , iptg ( final concentration 0.5 mm ) was added and the culture was incubated for 5 additional hours . cells were harvested by centrifugation ( 5,000 g for 10 min at 4c ) ; the pellet was resuspended in 50 ml of 20 mm tris / hcl buffer ph 7.5 ( buffer a ) . bacteria were disrupted with the help of cell disrupter equipment ( emulsiflex c3 , germany ) . cell debris were removed by centrifugation ( 30 000 g for 45 min at 4c ) and the supernatant was dialysed overnight against buffer a at 4c . the crude extract was loaded onto a q - sepharose ff column ( 2.6 by 34 cm , pharmacia , sweden ) equilibrated in buffer a. the -lactamase shv-104 was eluted in the flow through . the solution was dialysed overnight against 20 mm tris / hcl buffer ph 8.5 and loaded in a hitrap q - sepharose hp ( 5 ml ; ge healtcare ) . the enzyme was eluted by a linear nacl gradient ( 00.7 m ) in ten column volumes . the active fractions were collected and concentrated on ym-10 membrane ( amicon , beverly , mass . ) to a final volume of 3 ml . the sample was loaded in a molecular sieve sephacryl-100 ( 1.5 56 cm ) column equilibrated in 25 mm sodium phosphate buffer ph 7.0 containing 0.2 m nacl . the fractions exhibiting -lactamase activity were collected and their specific activity was followed by measuring the rate of nitrocefin hydrolysis . the fractions exhibiting a constant specific activity were pooled and concentrated to a final concentration of 0.5 mg / ml . the enzyme preparation was stored at 20c in 25 mm sodium phosphate buffer ph 7.0 . the n - terminal sequence was determined with the help of a gas - phase sequencer ( prosite 492 ; applied biosystems , calif ) . all the measurements were done on a spectrophotometer specord 50 analytik jena ( analis , belgium ) connected to a personal computer via an rs232c interface . bovine serum albumin ( 20 g / ml ) was added to diluted solutions of -lactamase in order to prevent enzyme denaturation . the steady state kinetic parameters ( km and kcat ) were determined using the hanes ' linearization of the michaelis - menten equation . the kcat and km values were determined for a representative set of -lactam antibiotics . a model of the shv-104 structure mics for k. pneumoniae ml2011 showed that this strain was resistant to all -lactams tested except imipenem ( table 2 ) . k. pneumoniae ml20011 exhibited a high level of resistance to oxyimino - cephalosporins ( cefotaxime , ceftriaxone , and cefpirome : mic 512 mg / l ; ceftazidime : mic 256 mg / l ) . the strain was also resistant to chloramphenicol , ciprofloxacin , nalidixic acid , and tetracycline . the disk diffusion method performed according to the clsi guidelines showed synergistic effect between ceftazidime , cefotaxime , aztreonam , ceftriaxone , and amoxicillin - clavulanic acid suggesting the presence of an esbl enzyme . plasmid analysis revealed the presence of a 50 kb transferable plasmid by transformation and conjugation experiments . the frequency of conjugational transfer performed with k. pneumoniae ml2011 as donors and e. coli hb101 as the recipient was 10/donor . the e. coli transformants and transconjugants were resistant to penicillins and expanded - spectrum cephalosporins but were susceptible to cefoxitin , aminoglycosides , quinolones , chloramphenicol , and imipenem ( table 2 ) . the examination of the crude extract of k. pneumoniae ml2011 by isoelectric focusing showed the presence of four -lactamase bands with apparent pis of 5.5 , 7.3 , 7.6 , and 8.6 . only the -lactamases with pi 8.6 and pcr analyses confirmed the presence of blatem , blactx - m-1 group , and blashv in k. pneumoniae ml2011 . blashv can be present on both the chromosomal dna of k. pneumoniae ml2011 and the plasmid pml2011 . the complete nucleotide sequences were performed and compared for shv gene with strain k. pneumoniae kp297 ( ddbj / embl / genebank accession number ef035567 ) and ctx - m gene with ctx - m-1 genebank accession number x92506 . these comparisons revealed the presence of ctx - m-28 previously described in and 2 shv : blashv-1 on the chromosomal dna and an open reading frame that was similar to blashv-1 with one mutation on the plasmid pml2011 . the mutation consisted of the replacement of the ( r ) residue at position 202 ( codon cgt ) to s ( codon agt ) . as this substitution was not present in all known shv -lactamases , the sequence of blashv was named blashv-104 and was deposited in genbank ( accession number eu274581 ) . the analysis of the shv sequences coding mutation at amino acid 202 suggests that shv-104 has evolved directly from shv-1 . moreover , the modification r202s may result in a change of the pi to 7.3 . the best results , in terms of production of shv-104 , were found in growing e. coli bl21(de3 ) ( plyss ) in tb , added 0.5 mm iptg after 5-hour incubation . the shv-104 was extracted from the periplasmic space and purified by three chromatographic steps ( q - sepharose ff , hitrap q - sepharose hp , and molecular sieve sephacryl-100 ) to obtain at the end ~10 mg ( 90% protein pure ) for liter . kinetic parameters values , kcat and km , of shv-104 -lactamase for representative -lactam antibiotics were shown in table 3 . kcat values of shv-104 against ticarcillin , nitrocefin , cephalothin , and cefuroxime were higher than those of shv-1 , and km values were higher ( 43 to 540 m versus 21 to 100 m ) except for ticarcillin . overall , the catalytic efficiency against penicillins was higher for shv-104 ( kcat / km , 0.001 to 19 m s ) than for shv-1 ( kcat / km , 0.3 to 14 m s ) , except for cefuroxime ( kcat / km , 0.001 m s versus 0.005 m s ) . in contrast , shv-104 had a low catalytic efficiency against benzylpenicillin ( 30-fold ) than shv-1 and exhibited catalytic activity against cefotaxime ( an oxyimino - cephalosporin ) . kinetic analysis showed that shv-104 was more active against nitrocefin and cephalothin , but hydrolysis remained inefficient , owing to a high km . , only cefotaxime was slowly hydrolysed by shv-104 . on the basis of the fact that a group 2be enzyme should hydrolyze one or more oxyimino--lactams such as cefotaxime , ceftazidime , or aztreonam at 10% the rate at which benzylpenicillin is hydrolyzed [ 17 , 18 ] , the activity of the novel enzyme was insufficient for the enzyme to count as an extended - spectrum -lactamase ( esbl ) . finally , a model of the shv-104 structure was built with the help of easypred program using shv-1 as template . the r202s mutation may affect a salt bridge present in shv-1 between r202 and e92 via a water molecule ( figure 1 ) . analysis of shv-104 structure model showed that it is reasonable to think that this mutation can not have a direct effect on the broadening of the activity profile of the shv enzyme . a plausible hypothesis relies on the fact that the absence of a salt bridge in shv-104 compared to shv-1 could increase the overall flexibility of the protein and indirectly the flexibility of the -lactamase active site . the conversion of a non - esbl to an esbl for the shv family can be achieved by different mutations . unlike the earliest described esbls ( shv-4 , -5 , -7 , -9 , -10 , -12 , -15 , -22 , -45 , -46 , -55 , -64 , and -66 ) , shv-104 does not possess these mutations , g238s and e240k , thought initially to be necessary for the hydrolysis of cefotaxime . mutation at position 179 was responsible for the esbl phenotype in shv-6 , -8 , and -24 . random mutagenesis of 179 in tem-1 produced three e. coli transformants ( 179n , g , and y ) with increased levels of resistance to ceftazidime . shv-7 variant was found with a serine at position 43 and is resistant to cefotaxime , ceftazidime , and aztreonam . the hydrogen bonding from r43 to the segment at positions 64 to 69 running behind the -sheet may be important in maintaining the position of s70 . in conclusion , we could demonstrate that the production of shv-104 is not the major factor for the resistance pattern of k. pneumonia ml2011 . our study indicates that shv-104 can be an intermediate in the process of selection of a new shv boras spectrum -lactamase . we could also conclude that the coexpression of ctxm-28 by this strain will increase the resistance profile of the strain toward beta - lactam antibiotics .

klebsiella pneumoniae ml2011 , a multiresistant isolate , was isolated from the military hospital of tunis ( tunisia ) . the determination of the minimal inhibitory concentrations exhibited by k. pneumoniae ml2011 was performed by etest . the crude extract of the isolates contains four different -lactamases with pi 5.5 , 7.3 , 7.6 , and 8.6 . only the -lactamases with pi 7.3 and pi 8.6 were transferred by transformation and conjugation experiment . molecular characterization of these genes was performed by pcr and sequencing . the chromosomal -lactamases are tem ( pi 5.5 ) and shv-1 ( 7.6 ) . ctx - m-28 ( pi 8.6 ) and the novel variant of shv named shv-104 ( pi 7.3 ) were encoded by bla gene located on a 50 kb highly conjugative plasmid . the shv-104 -lactamase was produced in e. coli and purified . its profile of activity was determined . compared to shv-1 , shv-104 contains one mutation , r202s . their kinetic parameters were similar except for cefotaxime . the analysis of the predicted structure of shv-104 indicated that the r202s mutation suppresses a salt bridge present in shv-1 . therefore , the overall flexibility of the protein increased and might improve the hydrolysis of cefotaxime . we can conclude that the multiresistant phenotype of k. pneumoniae ml2011 strain is mainly linked to the production of ctx - m-28 since shv-104 possesses a narrow spectrum of activity .

1. Introduction 2. Materials and Methods 3. Results and Discussion

PMC4438175

rupture of the anterior cruciate ligament ( acl ) of the knee is a common injury with a growing incidence in professional and recreational sportspersons [ 1 , 2 ] , reflected in the growing number of surgical acl reconstructions performed each year . knee stability is generally restored by arthroscopic ligament reconstruction based on the transplantation of free autologous tendon grafts . despite being a well - established and highly standardized surgical procedure , approximately 10% of patients require operative revision because of graft failure and persistent joint instability [ 5 , 6 ] . the interface between tendon and bone is of critical importance for the successful osseous integration of the transplant into the femoral and tibial tunnels . however , the clinical issue of bone - tendon integration is not limited to acl reconstruction but is a rather prominent challenge to orthopaedic surgeons treating ligament and tendon injuries at many other anatomical structures , including rotator cuff lesions , rupture of the distal tendon of the biceps brachii and scapholunar ligament , and the reconstruction of the medial patellofemoral ligament and lateral ankle joint stabilizators . conventional nonselective nonsteroidal anti - inflammatory drugs ( nsaids ) and selective cyclooxygenase- ( cox- ) 2 inhibitors are widely used following musculoskeletal trauma and as postoperative analgesics . while the cox-1 isoform has been identified as a housekeeper enzyme that is constitutively expressed in almost all tissues , cox-2 is the product of an immediate - early gene that is rapidly inducible and tightly regulated . the expression of cox-2 is highly restricted but is sharply upregulated during inflammatory processes . both cox-1 and cox-2 are key enzymes in the synthesis of prostaglandins ( pgs ) , which are important factors in bone metabolism and fracture healing . whereas cox-1 and cox-2 inhibitors reportedly impair bone formation , they both appear to enhance tendon - bone integration . we examined the effects of nonselective cox inhibition and selective cox-2 inhibition on the interaction between osteoblasts and fibroblasts at the tendon - bone interface in vitro . if not otherwise specified , reagents were sourced from sigma - aldrich ( taufkirchen , germany ) and consumables from sarstedt ( nmbrecht , germany ) . cells from the murine mc3t3-e1 ( preosteoblast ) and 3t3 ( fibroblast ) lines ( dsmz , braunschweig , germany ) were cultivated at 37c in a humidified atmosphere of air and 5% co2 in eagle 's minimum essential medium , modification ( mem ) , and dulbecco 's modified essential medium ( dmem ) , respectively . both media were supplemented with 10% fetal calf serum , 100 u / i penicillin , and 100 g / ml streptomycin . mc3t3-e1 cells were adapted to dmem 10 days before the initiation of the coculture . a murine coculture model providing osteoblast , interface , and fibroblast regions as described by wang and colleagues was used to study the effects of cox inhibition on bone - tendon healing in vitro . a sterile agarose divider ( 1.2 cm width ) was fixed to the base of a plastic cell culture dish , and 1 10 mc3t3-e1 cells were seeded on the left and 1 10 3t3 cells on the right of the divider ( figure 1 ) . cells allowed become adherent over 10 min ; then the cocultures were covered with fully supplemented dmem with 10 g / ml ascorbic acid and 1 mm glycerol-2-phosphate . after 3 days under standard culture conditions , the agarose divider was removed . cultures were stimulated with lipopolysaccharide ( lps ) at a concentration of 1 ng/l over 4 hours . next , the supernatant was decanted and the nonselective cox inhibitor ibuprofen ( sigma - aldrich , steinheim , germany ) or the specific cox-2 inhibitor parecoxib ( pfizer , new york city , ny , usa ) was added . at days 2 and 3 , medium was carefully renewed . medium was removed and cocultures were rinsed with ice - cold phosphate - buffered saline ( pbs ) . a region - specific cell harvest was performed by applying 600 l buffer rlt ( lysis buffer in the rneasy kit , qiagen , hilden , germany ) to the upper border of the respective region , while the plate was held in a tilted position . for each area , lysed cells were removed from the plate using a cell scraper and collected in three separate tubes ( osteoblast , interface , and fibroblast regions ) . cultures treated with dimethyl sulfoxide ( dmso , 520 m ) and nacl ( 36.02 m ) served as negative experimental controls . as a positive control , the stimulatory effects of recombinant bone morphogenetic protein- ( bmp- ) 2 at 500 ng / ml ( inductos , dibotermin alfa , pfizer , berlin , germany ) were measured at day 3 . the application of bmp-2 resulted in a significant regulation of the target genes at the osteoblast region ( figure 2 ) . a rneasy mini extraction kit ( complementary dna ( cdna ) synthesis was performed in a flexcycler thermal cycler ( analytik - jena , jena , germany ) using the iscript cdna synthesis kit ( bio - rad , munich , germany ) according to the manufacturer 's instructions . , the ssofast evagreen supermix ( bio - rad ) and 1 g of cdna were processed in a bio - rad c1000 thermocycler running the cfx96 real - time system . cycling conditions were 30 sec at 95c , 40 cycles of 5 sec at 95c , and 10 sec at 60c . finally , a melt - curve analysis with 0.5c increments every 5 sec from 65c to 95c was performed . the target genes were alpl ( alkaline phosphatase ) , bglap ( bone gamma - carboxyglutamate ( gla ) protein or osteocalcin ) , fmod ( fibromodulin ) , and runx2 ( runt - related transcription factor 2 ) ; reference genes were actb ( actin beta ) and hprt ( hypoxanthine guanine phosphoribosyl transferase ) . the primer covered at least one exon - intron junction and the negative first - deviation plots of the melting curve revealed specificity . target gene expression was assessed using cfx manager 3.1 software ( bio - rad ) and normalized to the reference genes . to measure the influence of ibuprofen and parecoxib on the viability of mc3t3-e1 and 3t3 cells , the xcelligence real - time cell analyzer ( roche applied science , mannheim , germany ) and rtca 1.2.5 software were employed according to the manufacturer 's instructions . briefly , 5,000 cells were seeded per well and incubated for 24 h ( 3t3 ) or 48 h ( mc3t3 ) under standard conditions . next , the medium was changed and cells were exposed to final concentrations of ibuprofen or parecoxib of 0 m , 5 m , 10 m , 25 m , 50 m , or 100 m . controls were treated with either nacl ( 36.02 m ) or dmso ( 520 m ) . the experiment was performed over 100 h at 37c in a humidified atmosphere of air and 5% co2 . the cell index was acquired automatically every 15 min . to measure proliferation and migration at the interface region , the established coculture model was performed on sterile object slides ( gerhard menzel , braunschweig , germany ) as outlined above . the divider was removed 24 h after seeding and cultures were stimulated with lps ( 1 ng/l ) over 4 h. next , cells were exposed to ibuprofen ( 100 m ) or parecoxib ( 12.63 m ) ; dmso ( 520 m ) and nacl ( 36.02 m ) treated cultures served as controls . object slides were rinsed with pbs and cells were fixed for 15 min in 4% formalin . next , slides were carefully washed with pbs , and a hematoxylin and eosin stain was performed . slides were observed and photographed with a leica dm lb microscope ( leica mikrosysteme vertrieb , wetzlar , germany ) . quantification of combined migration and proliferation was performed by counting all visible cells in one - quarter of a visual field ( at 20x magnification ) of the osteoblast , interface , and fibroblast regions . for each experimental condition , statistical analysis was undertaken using spss for mac , version 22 ( spss , chicago , il , usa ) . groups were compared by employing one - way analysis of variance with dunnett 's multiple comparison . statistical significance was set at p < 0.05 . graphs were plotted using microsoft excel for mac , version 14.1.0 ( microsoft , redmond , wa , usa ) . the dose - dependent effects of ibuprofen and parecoxib on the viability of cultured mc3t3-e1 and 3t3 cells are shown in figure 2 . ibuprofen provoked a dose - dependent reduction in the viability of mc3t3 cells from 48 h onwards when compared with untreated controls ( figure 3(a ) ) , but there was no apparent effect on 3t3 cells ( figure 3(b ) ) . in contrast , parecoxib led to a significantly reduced cell viability of 3t3 cultures ( figure 3(d ) ) , but no dose - dependent impairment of cell viability in mc3t3 cells ( figure 3(c ) ) . the region - specific effects of ibuprofen and parecoxib treatment on the expression of alpl , bglap , fmod , and runx2 in lps - stimulated and unstimulated cultures were analyzed by qpcr at 24 , 48 , and 72 h. unstimulated cocultures served as controls . isolated lps - exposure led to significantly decreased rates of alpl expression at all regions ( figures 4(a ) and 4(e ) ) , while bglap , fmod , and runx2 showed no definite regulation by lps . ibuprofen treatment led to reduced alpl expression at the osteoblast and interface regions of lps - treated and -untreated cultures , while no distinct regulation was noted at the fibroblast region ( figure 4(a ) ) . bglap expression was significantly downregulated by ibuprofen in lps - untreated cultures at the interface and fibroblast regions , while expression in lps - stimulated cells was increased significantly by ibuprofen at the interface and fibroblast regions ( figure 4(b ) ) . although there were significant increases in bglap expression in the lps - stimulated cultures following parecoxib treatment , a nonsignificant trend towards a decrease was also noted in unstimulated cultures ( figure 4(f ) ) . there was no consistent regulation of fmod by ibuprofen ( figure 4(c ) ) , while parecoxib resulted in decreased fmod expression ( figure 4(g ) ) . runx2 expression was downregulated by ibuprofen and parecoxib at all three regions ( figures 4(d ) and 4(h ) ) . histological growth patterns of mc3t3-e1 and 3t3 cells at the osteoblast interface and fibroblast regions at day 7 are depicted in figure 5 . the quantitative analysis of cell outgrowth ( combined proliferation and migration ) revealed no inhibitory effects by ibuprofen in unstimulated cultures at the interface or fibroblast regions ( figure 6(a ) ) , whereas the cell count was significantly increased in the osteoblast region following exposure to ibuprofen ( p < 0.001 , figure 6(a ) ) . however , in lps - stimulated cocultures , ibuprofen treatment resulted in a significant decrease in the number of detectable cells at the osteoblast region ( p < 0.05 ) and a trend towards reductions at the interface and fibroblast regions . these results were mirrored following exposure to parecoxib ( figure 6(b ) ) , where unstimulated cultures showed increased cellular outgrowth at the osteoblast region , and inhibitory effects were noted at all three regions following lps - stimulation . ibuprofen , parecoxib , and other nsaids are a key part of many postoperative analgesic regimes following musculoskeletal surgery . while the development of cox-2 specific drugs aimed to improve the efficacy and safety of nsiads , their superiority over conventional nonselective cox inhibitors remains a matter of substantial controversy . induction of cox-2 and its biochemical product prostaglandin h2 is an important part of the healing process in musculoskeletal tissues , including bone and tendon . in musculoskeletal tissue , cox-2 is involved in a multitude of complex biological processes , including the recruitment and activity of proinflammatory cells and the formation and maturation of restored tissue . we used a highly controlled in vitro model to investigate the influence of cox inhibition on bone - tendon integration . while we found marked suppression of markers of bone and noncalcified extracellular matrix formation following ibuprofen and parecoxib exposure , the published literature on the role of cox inhibition on bone - tendon healing remains controversial . oak and colleagues showed that inhibition of 5-lipoxygenase , cox-1 , and cox-2 led to improved tendon healing , and krivic and colleagues reported that tendon healing was enhanced by an immunosuppressive protocol involving the peptide bpc 157 and systemic methylprednisolone in vivo . further evidence indicating the advantageous effects of limiting inflammation in injured tendons was provided by the study of mccarrel and colleagues , who reported that leukocyte - reduced platelet - rich plasma was superior to standard platelet - rich plasma in an in vitro model . in contrast , kocaoglu and colleagues reported that immunosuppression with mitomycin - c had no effect on the tensile load required to rupture repaired tendons , even though microscopic signs of inflammation were significantly decreased . although another group found that specific selective cox-2 inhibition with celecoxib did not influence tendon healing , it has been reported that systemic ibuprofen administered in the first postoperative week has detrimental effects on tendon healing in a rat supraspinatus tendon repair model ( although ibuprofen administered only in the second postoperative week did not ) . the role of cox inhibition on bone formation and fracture healing has been studied extensively , but there is still significant controversy about the specific effects of selective cox-2 and nonselective cox inhibition . the majority of published studies reported reduced rates of callus formation and delayed fracture healing following treatment with selective and nonselective cox inhibitors [ 2431 ] . interestingly , utvg 's group found that both selective cox-2 inhibition and nonselective cox inhibition administered for only the first 7 days after the fracture did not significantly influence bone regeneration and concluded that short - term treatment with an nsaid might not impair fracture healing . while o'connor and colleagues reported that treatment with the selective cox-2 inhibitor rofecoxib resulted in less effective fracture healing compared with ibuprofen , gerstenfeld et al . found that parecoxib had only a minimal effect on healing , even at doses that are known to fully inhibit prostaglandin production the latter authors ' conclusion that a selective cox-2 inhibitor has less effect on fracture repair compared with nonselective nsaids has been challenged , as the pharmacokinetic characteristics of the drugs used in the study can be unpredictable . while we found some drug - specific differences , both selective and nonselective cox inhibition led to significant impairment of in vitro bone - tendon healing . a relevant limitation of the majority of the previously published in vitro studies is the lack of an adequate proinflammatory stimulus in the experimental setup . for the present study lps was administered to induce inflammatory signaling in both cell lines , thus simulating the early postoperative situation following acl reconstruction . lps has been reported to be a proinflammatory stimulus in mc3t3-e1 cells by guo et al . . the authors noted an lps triggered activation of the jnk pathway , eventually leading to an inhibition of osteoblast differentiation and programmed cell death . in their classic paper , arakawa et al . reported on the effects of lps on the expression of the prostaglandin receptor gene ep4 in 3t3 cells , underlining the proinflammatory effect of lps in this murine mesenchymal cell line . while using an established in vitro coculture model enabled highly standardized and reproducible experiments to be performed , the complexity of bone - tendon healing , including the involvement of other cell types in a complex three - dimensional extracellular matrix , could not be reproduced . another potential limitation could be the use of established murine cell lines ; however , the rationale for using murine cell lines instead of human cell cultures was primarily the excellent characterization of their molecular background by previous studies . next , a wide range of molecular tools have been constructed for murine cells , enabling complex experimental approaches for future studies . furthermore , mc3t3-e1 and 3t3 cells are globally available , thus improving the reproducibility of our data . furthermore , the expression analysis was limited to four genes serving as surrogate parameters of osteointegration ( alpl , bglap , and runx2 ) and extracellular matrix formation and maturation ( fmod ) . the role of alpl , bglap , and runx2 in osteoblast differentiation has been studied extensively , and their importance for bone - tendon integration has been underlined previously [ 37 , 38 ] . fibromodulin ( fmod ) has been identified to be a key participant in the organization of extracellular matrix by interacting with collagen fibrils and has been described as an important structural component of tendon and cartilage tissue . however , the authors are aware that the analysis of the effects of the cox inhibition on noncalcified extracellular matrix formation and maturation is limited and should be further dissected by future studies employing long - term cultured tendon specimens and animal models . another drawback of the study is the limitation on two cox inhibitors , that is , ibuprofen and parecoxib . however , both ibuprofen and parecoxib are widely used as anti - inflammatory drugs and have become the postoperative therapeutic standard in orthopedic practice . thus , the present study aimed to transfer the clinical issue of a potential interference between ibuprofen or parecoxib and bone - tendon healing to the highly controlled experimental setup of a cell based in vitro study . finally , the induction or inhibition of cox-1 and cox-2 was not specifically measured . nonselective cox inhibition and the specific inhibition of cox-2 resulted in region - specific reductions in expression of markers of calcification and extracellular matrix synthesis in vitro . furthermore , parameters indicative of cell viability , proliferation , and migration were suppressed by cox inhibition . as nsaids are in widespread clinical use to manage pain after acl surgery , further in vitro and in vivo studies examining the biologic and biomechanical effects of cox inhibition are needed .

the effects of cyclooxygenase ( cox ) inhibition following the reconstruction of the anterior cruciate ligament remain unclear . we examined the effects of selective cox-2 and nonselective cox inhibition on bone - tendon integration in an in vitro model . we measured the dose - dependent effects of ibuprofen and parecoxib on the viability of lipopolysaccharide- ( lps- ) stimulated and unstimulated mouse mc3t3-e1 and 3t3 cells , the influence on gene expression at the osteoblast , interface , and fibroblast regions measured by quantitative pcr , and cellular outgrowth assessed on histological sections . ibuprofen led to a dose - dependent suppression of mc3t3 cell viability , while parecoxib reduced the viability of 3t3 cultures . exposure to ibuprofen significantly suppressed expression of alpl ( p < 0.01 ) , bglap ( p < 0.001 ) , and runx2 ( p < 0.01 ) , and although parecoxib reduced expression of alpl ( p < 0.001 ) , fmod ( p < 0.001 ) , and runx2 ( p < 0.01 ) , the expression of bglap was increased ( p < 0.01 ) . microscopic analysis showed a reduction in cellular outgrowth in lps - stimulated cultures following exposure to ibuprofen and parecoxib . nonselective cox inhibition and the specific inhibition of cox-2 led to region - specific reductions in markers of calcification and cell viability . we suggest further in vitro and in vivo studies examining the biologic and biomechanical effects of selective and nonselective cox inhibition .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

PMC4798823

the practice of science was once the realm of amateur practitioners , and those without formal scientific training , such as antonie van leeuwenhoek , joseph priestly , and benjamin franklin , have made major contributions . however , the scientific research process has become increasingly professionalized over the last century , allowing few opportunities for people outside the establishment to participate . the specialized knowledge , sophisticated equipment , and rigorous training that scientists undergo have resulted in tremendous scientific advances but have also built barriers between scientists and the public , who indirectly funds their research . although the proliferation of films and popular science publications bear witness to the public s continued interest in science , few venture into one of the laboratories where modern biology research occurs . however , the diy - bio ( do - it - yourself biology ) movement seeks to remove obstacles that prevent participation by hobbyists , small entrepreneurs , and curious individuals . diy - bio encompasses a diverse set of activities ( reviewed in 5 and 9 ) , all involving lab research outside of traditional settings . diy - bio has established a parallel infrastructure , including a google group and listserv ( http://diybio.org ) , journal ( www.oreilly.com/biocoder/ ) , and code of ethics ( http://diybio.org/codes ) . although some diy - biologists practice at home in makeshift laboratories , community labs ( biohacker spaces ) are increasingly common . community labs are communal spaces that provide shared instrumentation ( typically second - hand equipment ( 10 , 12 ) ) , reagents , management , and communal projects . although the physical space and resources are important , equally important is the community itself , which provides the collective expertise to engage in meaningful projects . for example , on a typical night , you may find an artist , an engineer , and a computer programmer huddled over a 3d printer that they have programmed to extrude agar and plant cells into a form that will grow and evolve over time . the collaborations that develop from bringing individuals with varied expertise and backgrounds together is something that many established scientific institutions strive for , but these often happen spontaneously in the community lab environment . while some community projects are meant to test hypotheses , projects such as the plant bioprinting may be initiated for their artistic merit and their ability to develop new biological technology . community labs generally do not seek to compete directly with academic researchers in inquiry - driven research and instead often focus on the design and manipulation of novel biological systems , including those with practical applications . based loosely on technology hackerspaces and drawing inspiration from the synthetic biology community , community labs vary greatly in the types and extent of activities . some , such as genspace in new york ( www.genspace.org ( 7 ) ) and biocurious in california ( www.biocurious.org ( 13 ) ) , have robust participation and programming while others fluctuate in the extent of their activities . we are both founding members of the board of directors and one of us ( tb ) is executive director of baltimore underground science space ( bugss , www.bugssonline.org ) , a community laboratory in baltimore , md , established in 2012 . because the community lab movement is growing and there is a dearth of resources for those establishing community labs , our goal is to share our experience setting up and maintaining a community lab . we discuss our successes , problems , approach to overcoming those problems , and continuing challenges . we respect the great diversity of community lab spaces , and we therefore seek to use our particular experience only as a case study rather than attempting to speak for the movement as a whole . many labs hold the diy ethos paramount and therefore concentrate on research projects ; for example , the glowing plant project introduced the luciferase gene into arabidopsis thaliana ( 2 ) , and real vegan cheese seeks to make dairy products from the cloned casein genes of diverse organisms ( 3 ) . some groups retain their strong connection to technology hackerspaces and focus on development of low cost technology such as open pcr ( polymerase chain reaction ) ( 12 ) or providing broader access to cutting - edge technologies such as crispr ( clustered regularly - interspaced short palindromic repeats ) ( 11 ) . the options are not mutually exclusive , and most spaces support a variety of educational , artistic , or even commercial activities , which may change as different opportunities present themselves . at bugss , we focus on both member projects and classes for the public . the educational aspects support the development of member projects by equipping novice diy - biologists with the skills necessary to function safely and effectively in the lab ; this allows us to engage both novices who want highly structured courses and members who seek to pursue independent investigations . while having more than one focus can draw in the greatest number of participants , the institution must retain a level of focus to maintain a coherent identity , enable like - minded people to find one another , and not draw its leadership in conflicting directions . traditionally , hackerspaces develop from a core group of people with a shared interest who organize themselves in a democratic , bottom - up manner . the formation of bugss , legally organized as a maryland non - profit corporation , was no different . the board oversees long - term strategic planning and the executive committee oversees day - to - day operations . this top - down approach for organizing bugss was chosen both to protect the initial financial investment of the founders and to address concerns regarding biological and chemical safety , biosecurity , and the public s perception of biohacking . public perception of diy - biology was foremost in our minds during the formation of bugss . questions are often raised about the risks of bioterrorism or the biological safety of members and the community at large ( 1,11 ) . in order to allay those fears as well as demonstrate a level of responsibility and seriousness , we generated a number of foundational documents , including bylaws to establish organizational responsibility and governance , a membership agreement to define the rights and responsibilities of all participants , a safety policy to set rules for the types of experiments permitted in the space , and lists to delineate what chemicals and biological agents were permissible . as new situations arise , it has been important to have a revision mechanism in place that respects the needs and desires of both members and the broader community . having foundational governance documents has also been instrumental in allowing bugss to adapt to changes in leadership without the potentially chaotic disruption that could ensue . initially , the burden of organizing and running the lab was borne by the founders who formed the executive committee . however , as the demands on the organization grew , members of the board of directors have increasingly stepped in by teaching courses , fundraising , and leading community projects . currently , bugss is undergoing another transition , as funding has been obtained from a local philanthropic organization to hire a staff person . as the needs and demands of the organization have grown , operating on an all - volunteer basis became increasingly problematic , and it was necessary to have a full - time paid staff position to manage the day - to - day activities as well as to develop and implement new programming as directed by the board . questions often arise as to what regulations apply to community laboratories , and the answer is that there are surprisingly few in the united states . in the absence of federal funding , the presence of employees , or the generation of hazardous waste , most regulation occurs at the state and local level ( fire department inspections , zoning restrictions , waste water discharge permits , and business licenses ) . however , bugss adheres to the spirit , if not the letter , of federal agencies such as the occupational safety and health administration ( osha ) and environmental protection agency ( epa ) . critical to this effort was the inclusion on our board of a professional safety consultant with intimate knowledge of the safety and environmental health regulations and practices of the biotechnology industry . under his guidance , bugss developed a chemical hygiene plan , chemical inventory plan , and member safety training protocol that meet the regulatory requirements of state and federal agencies . recognizing the need for qualified safety professionals , the diy - bio movement has instituted the ask a biosafety expert service ( http://ask.diybio.org/ ) , which allows members of the community to get answers to questions that may arise . for example , if a person has a question on how to properly dispose of a live culture or the proper biosafety level for a particular experiment , those questions are answered by a team of professional experts . this is just one example of how the larger diy - bio community is proactively addressing safety concerns . finding a reliable funding model that will cover start - up costs , monthly rent , utilities , and supplies is probably the most significant issue that community labs face . start - up funding is the first hurdle ; several labs , including biocurious and counter culture labs ( https://counterculturelabs.org/ ) have successfully used crowdsourcing venues such as kickstarter . others , such as bugss , have relied on funds and resources provided by the founders . regardless of the source of the start - up funds , one essential feature is that there be sufficient resources to enable the organization to survive from inception to the point where income can cover the organization s operational expenses , which may be months or even years . although usually organized as non - profit entities , community labs are businesses . before we made any financial commitments to starting bugss , we held several meetings to gauge local community interest and developed a business plan with cash - flow projections and revenue - generating strategies . although the reality was a bit different , having a plan allowed us to determine whether we were on the path to sustainability . in retrospect , it would have been useful to have a board member with business experience to guard against some costly mistakes made early in bugss s development . for example , two areas that we did not pay sufficient attention to were gaining exempt status from the internal revenue service under section 501(c)3 of the tax code and developing timely methods for billing members and collecting payments . one mechanism that was crucial to our initial success was sharing space ; for our first 2.5 years , we substantially reduced our rent by subleasing space from a for - profit startup company , chesapeake bioworks , formed by one of us ( tb ) . sharing space with other hackerspaces , schools , or businesses is a cost - effective way to establish a community lab . however , these arrangements require negotiation of a comprehensive joint use agreement to govern the use of space and equipment and are only likely to be successful if the other entity has an interest in seeing the community lab succeed . bugss generates income through member fees ( monthly fees that entitle members to use of the space and a defined amount of supplies and reagents ) , course fees , donations , and grants . our most significant sources of operating income are course and membership fees , with substantially reduced rates for students and teachers . while grants are a potentially large source of funding , most philanthropic organizations are reluctant to fund operational expenses and instead restrict their funds to specific programming or activities . in addition , the majority of grant - making organizations can only give money to nonprofits that are tax exempt under section 501(c)3 of the internal revenue code . obtaining that tax exempt status is an involved task that took us almost a year ( although a simpler online application has recently been implemented ) . community labs encompass myriad activities , allowing them to serve diverse audiences . at bugss , we focus on talks , courses , and member projects . talks are the most popular and well - attended activity , and for many individuals , they are the first exposure to the community lab movement . the topics of these public lectures range widely and have encompassed immunology , synthetic genomics , bioinformatics , and bioethics . given the ideal location of bugss within the research - intensive maryland and washington dc region , we have been able to host leading scientists from academic , government , and private research institutes ; these public lectures offer citizens exposure to a diverse array of topics while simultaneously enabling researchers to explain the broader impacts of their work to the voting public . courses allow novices to learn both basic and advanced lab techniques , and most of our class participants are professionals in diverse nonbiology fields , including engineering , law , and art . we intentionally created two scaffolded series of courses ( one in molecular and synthetic biology and the other in 3d printing ) , with the intent of enabling members to build proficiency in lab techniques . yet , what we find instead is that most individuals take courses sporadically rather than as a way to become fluent with lab skills . we have yet to surmount this problem , which impacts our ability to support independent research projects . at bugss , we feel that the heart of the community lab movement lies in enabling citizens to pursue their own creative projects in biology . indeed , we have consistent interest from members who seek to develop their project ideas . membership at bugss entitles individuals to use of the space and a limited number of standard molecular biology reagents and chemicals , but members are individually responsible for incurring the costs of their experiments . we seek to overcome this by teaching skills in our courses and have found that the courses can serve a secondary function : training student members who can subsequently serve as teachers themselves . second , project ideas come to us at varying stages of development . some members have extensively researched their project and have a well - formulated series of experiments . others are kernels of ideas that require guidance to narrow their scope into feasible projects and to determine what resources are available and what has already been accomplished ( or what has previously failed to work ) . this can be even more challenging when members are interested in fast - moving fields with extensive literature . to balance the flow of ideas coming in with the time required to convert those ideas into realistic lab projects , we focused on cultivating two projects : using synthetic biology to create a sensor for environmental contamination and bioprinting of plant cells . because these two projects are consonant with the two focuses of our lab courses , we are able to build expertise within the community to understand and support these two projects . for example , in our synthetic biology courses , we teach members how to assemble biobricks , standardized vectors that are used to create composite parts ( 14 ) ; in our member project to create a sensor for contamination , we use biobricks to assemble a lead - sensitive promoter with a reporter gene , ribosome binding site , and terminator to create the synthetic genetic system . we have also just initiated a series of monthly lab meetings that allow the community to come together , receive project updates , and offer feedback on these member projects . most importantly , we have built small teams that work on each of these projects , allowing us to funnel new members with limited expertise into a community of citizen researchers who are thereby able to make meaningful progress on a finite number of projects . contrary to the initial perception of diy - biology as comprised largely of secretive garage hackers , most participants in the diy - bio enterprise accomplish their work within community lab spaces ( 4 ) . the presence of laboratory equipment and the odd working hours can raise questions from the community about what is occurring in the lab space . are potentially dangerous experiments being conducted ? are illegal drugs being manufactured ? are pathogens being used ? however , due to their limited space and communal nature , community labs can actually guard against clandestine activities and enable safety and regulatory oversight of amateur scientists ( 6 , 1 , 8) . many community labs also have reached out to the law enforcement community , and early in its development , bugss invited representatives from the fbi to present a talk on the diy - bio movement and bioterrorism concerns . both transparency and proactive supervision of experiments will add legitimacy to the community lab movement as well as assuage concerns concerning these unorthodox establishments . indeed , the bigger impediment to citizen - science spaces may be that they become overly cautious in an attempt to alleviate fears . community labs will be an important connection between citizen scientists and security and regulatory agencies as they attempt to ensure public safety without overburdening the creativity that flourishes in these spaces . community labs also have an important role to play in communication between institutional researchers and the public . many lessons have been learned from the public backlash to genetically modified foods , which is not always grounded in accurate understanding of the underlying science . as exciting new fields develop on the cutting edge of biology , direct engagement between researchers and citizens at community labs can help to alleviate concerns before they fully develop . this interaction can ensure that there is public support for the application of these new technologies to healthcare and agriculture . the impetus behind the formation of community labs is not to keep pace with the leading edge of scientific research ; instead , for many , the motivation is to do science within a community of diverse expertise . given the need for top - down leadership and the movement toward having professional staff , the challenge remains to preserve the community lab as a bottom - up , member - driven organization . this demands that the members themselves retain ownership of the organization by equitably sharing the work of mentoring research , maintaining communications and social media presence , and recruiting new members . while this broad participation is challenging to establish and maintain , it is the only path forward for ensuring sustainable growth . it remains to be seen whether community labs will develop into alternative educational institutions , develop innovative products , or contribute to the scientific knowledge base , but they have already opened an avenue back into the scientific research experience for the engaged layperson .

the highly specialized nature of scientific research has erected substantial barriers between professional scientists and the laity , who have become distanced from the process of discovery . the do - it - yourself biology movement seeks to remove these impediments , with community laboratories serving as vehicles for public engagement and participation in scientific inquiry . we describe our experience establishing and maintaining the bugss community lab in baltimore . while each community lab is distinct in its structure , culture , and programming , we hope that this review of our experience will serve as a resource to inform those who seek to understand this growing movement and those who plan to establish their own community labs .

INTRODUCTION SETTING UP A COMMUNITY LAB REGULATIONS FUNDING ACTIVITIES AND PROGRAMMING CONCLUSION

PMC3800383

in the long intervening period between initiation of carcinogenic tobacco habits and the development of invasive oral cancers , well - defined oral potentially malignant lesions may occur , of which leukoplakia is the most common . leukoplakia is a white plaque of questionable premalignant risk , having excluded other known lesions that carry no increased risk for cancer , with histological presentation ranging from mild hyperkeratosis to squamous cell carcinoma . more than 75% or oral cancers are reported to occur in a preexisting leukoplakia and the prevalence of leukoplakia in the indian population is reported to range from 0.7 to 5.0% in various regions in india and the global prevalence of leukoplakia is 2.6% . because of their varied histological presentations , it is important to distinguish between these lesions and assess the associated risk in order to determine the clinical management and predict prognosis . currently the severity of these lesions is usually assessed by histological demonstration of epithelial dysplasia in biopsy samples . to date , no clear markers for grading of epithelial dysplasia have evolved , and histological criteria for diagnosing a it is known that hypertrophy of the nucleolus is one of the most distinctive cytological features of cancer cells . dysplastic cells more frequently display a larger nucleolus than benign cells and nucleolar size might represent morphological parameters of the cell proliferation rate in cancer tissue . nucleolar organizer regions ( nors ) are loops of deoxyribonucleic acid ( dna ) that transcribe to rrna , and the nucleolus is a structure containing this chromosomal part and in addition the material which accumulate around the nor , most notably the ribosomal ribonucleic acids ( rrnas ) and their precursors as well as specific ribosomal proteins . the qualitative or quantitative changes in interphase nors may be visible in relation to proliferative activity or transformation , and hence could aid diagnosis or prognostication of malignancy . nors and associated proteins show affinity for metallic silver and can be visualized by a one - stage argyrophil ( agnor ) method , by staining nor associated proteins . high agnor counts have been found to reflect proliferative status of cell and correlate with poor prognosis in malignant conditions . agnor quantification by image cytometry has proven useful which indicates marked changes from the basal to the upper malpighian layers in tissue with functional polarity ( both normal and pathological ) would support an association between differentiation linked cellular activities and nor patterns . the present study used morphometry to evaluate nor related parameters including agnor count , agnor area , agnor perimeter , and agnor proportion ( mean agnor area / mean nuclear area ) in normal oral epithelium , dysplastic and nondysplastic leukoplakia and significance of these morphometric parameters in distinguishing nondysplastic leukoplakias ( ndlks ) from dysplastic ones . the study sample included biopsy specimens from 50 cases of oral leukoplakia ( 22 nondysplastic , 28 dysplastic ) and 10 specimens of normal oral epithelium . all specimens were collected from the archieves of the department of oral pathology and microbiology , saraswati dental college and hospital , lucknow . the diagnoses were reviewed using routine hematoxylin and eosin stained sections and were considered as the histopathological grading was made using world health organization ( who ) criteria for potentially malignant lesions . the first group , named as nondysplastic dysplasia ( ndlk ) consisted of lesions diagnosed as hyperkeratosis , epithelial hyperplasia and mild epithelial dysplasia . the second group , named as dysplastic leukoplakia ( dlk ) consisted of moderate epithelial dysplasia , severe dysplasia and carcinoma in situ . following this , the tissue sections were stained for agnor using ploton technique : for each case , 3 m thick sections of routinely processed specimens from formalin fixed , paraffin - embedded blocks were dewaxed in xylene and dehydrated through alcohols to deionized water . the sections were then incubated in dark for 30 min at room temperature ( 25c ) in a fresh solution made by mixing two parts of 2% gelatin in 1% formic acid with one part of 50% aqueous silver nitrate solution . the sections were then washed in running deionized water , dehydrated in ascending alcohol concentrations , cleared in xylene , and mounted with dpx . the agnors were visualized as intranuclear brown to black dots of different sizes under light microscopy . microscopic fields , representative of the lesion , were identified and photographs were taken using olympus live view digital slr camera e-330 in 46 different fields moving from left to right to avoid any overlapping of cells . the photographs were analyzed using image pro express 6.0 for windows , ( media cybernetics ) after calibrating the software with photomicrograph of stage micrometer . agnors from 100 randomly selected nuclei of epithelial cells in basal and parabasal layers were assessed in four different fields at 100x magnification for their numbers , area , and perimeter using magic wand and trace wand tool of image analysis . mean agnor count / nuclei = total agnor count/100 mean agnor area ( m)/nuclei = total agnor area/100 agnor proportion = mean agnor area / mean nuclear area . the results were statistically analyzed for relationship between agnor count and other agnor related parameters in normal oral epithelium , ndlk and dlk . one way analysis of variance ( anova ) was performed as to compare the mean agnor parameters among normal epithelium , ndlk , and dlk to determine the differences among the three groups . for statistical evaluation , the statistical analysis was done using spss ( statistical package for social sciences ) version 15.0 statistical analysis software . mean agnor count / nuclei = total agnor count/100 mean agnor area ( m)/nuclei = total agnor area/100 agnor proportion = mean agnor area / mean nuclear area . the results were statistically analyzed for relationship between agnor count and other agnor related parameters in normal oral epithelium , ndlk and dlk . one way analysis of variance ( anova ) was performed as to compare the mean agnor parameters among normal epithelium , ndlk , and dlk to determine the differences among the three groups . for statistical evaluation , the statistical analysis was done using spss ( statistical package for social sciences ) version 15.0 statistical analysis software . the results of the present study [ table 1 ] show mean agnor / nucleus of different groups in various agnor parameters like count , area , perimeter , and proportion reveals increase in these parameters of agnor in dlk indicating an order of normal < non - dysplastic < dysplastic . calculation of mean agnor count / nucleus in various parameters in all the groups including control and leukoplakia ( ndlk and dlk ) tables 2 demonstrates the results of the anova f test which were used to compare the values in between and within groups , as well as it also shows the p value . from this analysis of variance revealed statistically significant difference ( p ) for agnor count ( < 0.001 ) , agnor area ( 0.004 ) , agnor perimeter ( 0.015 ) and agnor proportion ( 0.027 ) indicating increase in values from control to dlk . calculation of analysis of variance of agnor in various parameters in between the groups table 3 shows intergroup differences for agnor parameter in control and leukoplakia groups using pairwise student the results revealed that there was a statistically significant difference in mean agnor count agnor area , agnor perimeter while the proportions were not statistically significant . intergroup differences for various agnor parameters in between two groups i.e. , control versus leukoplakia and within leukoplakia group i.e. , ndlk versus dlk the presence of dysplastic areas in the epithelium is believed to be associated with a likely progression to cancer . when architectural disturbance is accompanied by cytological atypia ( variation in size and shape of keratinocytes ) the term dysplasia applies . it should be emphasized that dysplasia is a diagnosis defined by the presence of certain histological and cytological features . ideally , the diagnosis corresponds to the nature of the lesion meaning that a mucosa with epithelial dysplasia has an increased risk of developing into carcinoma when compared to normal mucosa . leukoplakia as a clinical diagnosis may have varied histological presentations ranging from mildly hyperkeratotic lesions to those exhibiting severe dysplasia . many oral leukoplakias regress or stay quiescent , but many progress and about 3 - 6% turn into squamous cell carcinoma in future . it is , therefore , important to distinguish between these lesions as their management may differ . mildly , hyperkeratotic / hyperplastic lesions may be kept under observation and may resolve spontaneously , whereas dysplastic lesions need to be excised . image analysis provides a unique advantage of reducing some of the difficulties by giving the operator opportunity to control the magnification and resolution of the images : hence reducing the errors in counting of nonspecific silver stains . it also provides a permanent record of the stained tissue and helps in assessment of various morphological characteristics of nors which is not possible by manual counting . the literature search revealed a very few studies on oral dysplasia using morphometry which made the present study a pioneer attempt in this field . as seen from the results of this study , the mean agnor count per nucleus was found to be higher in patients with dlk [ figure 1 ] as compared to ndlk [ figure 2 ] and controls [ table 1 ] . the agnor count showed statistically significant difference on comparison between group and within group [ table 2 , p < 0.001 ] . these findings are in concordance with previous studies done by warnakulasuriya and johnson , gomez et al . , and chattopadhyay et al . , who stated that mean agnor count increased gradually from normal epithelium to nondysplastic to dlk to squamous cell carcinoma . these higher counts found in many carcinomas were due to dispersion of agnors within the nucleoplasm . the results of this study support the view that in dysplastic tissue , chromosomal disarray with multiple nucleoli appears to result in an increase in agnors , and higher agnor counts suggesting a poor prognosis in oral cancer . the increase in agnor number and area could be the expression of an alteration of the mechanism controlling cellular proliferation and perhaps cellular differentiation . this is in accordance with the high values of agnor in severe and even moderate dysplasia and could represent a marker of proliferative cell hyperactivity , therefore may be a possible indication for a strict clinical management and/or a more incisive treatment of preneoplastic lesions . neoplastic cells generally exhibit a rise in the synthesis of normal and abnormal products ; hence there is a significant rise in agnor material . agnor counts rise with increased cell ploidy , with increased transcriptional activity and in stages of active cell proliferation . agnor staining in dysplastic leukoplakia ( arrow showing agnor dots ) agnor staining in nondysplastic leukoplakia ( arrow showing agnor dots ) apart from the agnor counts , various other morphometric parameters of agnor were also analyzed in this study . these included mean agnor area / nucleus , mean agnor perimeter / nucleus , and agnor proportion . from the results of this study , it was observed that the mean agnor area / nucleus [ figure 3 ] was higher in patients with dlk as compared to ndlk and controls [ table 1 ] , [ figure 4 ] . when intergroup comparison [ table 3 ] was made , it is seen that mean agnor area / nucleus was able to differentiate control group from leukoplakia group ( p = 0.006 ) but was not able to differentiate ndlk from dlk ( p = 0.053 ) . increased agnor areas with increasing grades of malignancy have been reported in non - hodgkins lymphoma , in colonic tumors , and melanocytic neoplasm of skin . similar findings have also been reported in oral cavity by muzio et al . , who found increased mean agnor area / nucleus in patient with moderate and severe dysplasia as compared to mild dysplasia . cabrini et al . , also reported increasing agnor area from normal oral epithelial to benign lesions and to scc . derenzini et al . , evaluated nucleolar size , using silver staining , in 10 tumor cell lines and found that nucleolar size can reliably indicate the rapidity of cell proliferation . they postulated that the higher agnor protein value corresponds to a worse clinical outcome , and since nucleolar size is a representation of agnor protein value ; thus it could be used as a parameter to assess rate of proliferation and prognosis in individual tumors . the results of our study are consistent with findings of muzio et al . , who found that the high value of agnor area in oral dysplasia could be a risk marker which could help in identifying the subgroup of lesions with worse prognosis . photomicrograph showing analysis of agnor area through image pro express mean agnor count in various parameters another variable analyzed in our study was mean agnor perimeter and was found to be higher for dysplastic cases as compare to nondysplastic cases [ table 1 ] , [ figure 4 ] . when the various groups were analyzed with each other [ table 3 ] a statistically significant difference was found between the control and leukoplakia group ( p = 0.008 ) , though agnor perimeter failed to differentiate dysplastic from nondysplastic lesions ( p = 0.204 ) . perimeter of agnors will also be higher in cases of numerous , small , fragmented , and scattered nors as compared to lesser and larger nors in a nucleus . hence , increase in agnor perimeter per nucleus is indicative of fragmented and more irregular nors . cabrini et al . suggested that increase in number and irregularity of nors and a decrease in their size would be an expression of altered proliferation and differentiation mechanism in conjunction with synthesis of new oncogenic proteins present in carcinomas . hence , smaller and more irregular nors are expected in dysplastic epithelium as compared to normal epithelium . when the volume fraction of agnor per nuclei was assessed , it gave an indication of proportion of nuclear volume occupied by agnor . significantly , higher values were obtained for leukoplakia ( dlk and ndlk ) patients as compared to controls [ table 1 ] , [ figure 4 ] , though this difference was statistically significant [ table 2 ] , ( p = 0.027 ) but in intergroup comparison , no significance was seen when differentiating nondysplastic from dysplastic group [ table 3 ] , ( p = 0.360 ) . these findings are in agreement to those in some of the previous studies by weeks et al . , and nyska et al . , who found that the ratio of agnor area / nuclear area increases from control to benign and benign to malignant tissue . other authors hypothesized that the ratio obtained more accurately reflected the proliferative status of a cell . on the other hand , cabini et al . thus , the results of the present study show a linear relationship between the various agnor parameters and increasing grades of dysplasia as assessed by image analysis . their role in diagnosis of epithelial dysplasia is still subject to further investigation . in summary , results indicate a change in the nor pattern in oral mucosal lesions ( dysplastic and nondysplastic leukoplakia ) when compared with normal oral epithelium . the present study reveals that mean agnor parameters including count , area , perimeter , and proportion were found to be increasing in dysplastic lesions when compared with nondysplastic and control group . to conclude , the computerized morphometry of agnor related parameters could be valuable tool for defining objective criteria for diagnosis / determination of dysplasia in oral leukoplakia . we suggest larger studies of this nature to determine accurate , effective , and meaningful cutpoint to distinguish between ndlk and dlk .

background : silver stainable nucleolar organizer regions ( agnors ) are replicatory markers which may have a place in objectively characterizing dysplasia.materials and methods : a study of various morphometric parameters related to agnors was performed in basal and parabasal layers of normal human oral epithelium , nondysplastic leukoplakia , and dysplastic leukoplakia employing photomicrographs of silver stained paraffin embedded sections using image analysis , to assess the usefulness of these parameters in distinguishing dysplastic leukoplakia from nondysplastic oral leukoplakia.results:out of various mean agnor related parameters , agnor count , area , perimeter , and proportion were found to be higher in dysplastic leukoplakia as compared to nondysplastic leukoplakia . on statistical analysis , agnor count showed statistically significant differentiation between dysplastic and nondysplastic leukoplakia . while other parameters can distinguish normal oral epithelium from dysplastic and nondysplastic leukoplakia.conclusion:to conclude , the agnor count is the most appropriate marker to differentiate between dysplastic and nondysplastic leukoplakia .

I M Calculation of morphometric parameters Statistical analysis R D

PMC4393047

there are more than 100,000 colours of synthetic dyes produced commercially , and over 7 10 tons of dyes are produced annually worldwide . dyes are used throughout the world in textile , paper , cosmetic , pharmaceutical , and food industries and also used as additives in petroleum products . synthetic dyes are extensively used particularly in the textile and dyeing industries . in the process of dyeing , about 1520% of the dyes used for dyeing do not bind to the fibres and are lost in the effluent . it has been estimated that about 280,000 tons of textile dyes are discharged in such industrial effluents every year worldwide . the discharge of textile dyes into rivers or lakes is the most visible sign of water pollution because several dyes are visible even at a low concentration of less than 1 ppm . in addition to changing the colour of water , the presence of dye in the receiving water bodies also impedes sunlight penetration that in turn decreases photosynthetic activity , dissolved oxygen concentration , and water quality and causes acute toxic effects on aquatic flora and fauna . many synthetic dyes are also toxic and carcinogenic to aquatic and human lives because they are made from compounds such as benzidine and other aromatic compounds . reductive cleavage of azo dyes , which comprises about 70% of all dyes used , also resulted in the production of amines that are mutagenic to human and are also retained in the anaerobic compartment of the lower intestine by intestinal microflora after ingestion of azo dyes [ 79 ] . therefore , industrial effluents containing dyes must be treated prior to their discharge into the environment . among industrial effluents , wastewater from textile and dyestuff industries is one of the most difficult to be treated since the dyes used are usually synthetic and contain complex aromatic molecular structures making them more stable and difficult to degrade . conventional wastewater treatment plants using activated sludge treatment are unable to treat dye - containing wastewater . it has been estimated that up to 90% of reactive textile dyes still persist even after the treatment . several physical and chemical methods including membrane filtration , adsorption , ion exchange , ozonation , flocculation - coagulation , and oxidation have been used to treat dye containing wastewater . however , due to high cost involved , disposal problems , and less adaptable to a wide range of dye wastewaters , most of these methods have not been widely applied [ 6 , 12 ] . bioremediation of dye containing effluents using effective dye degrading microorganisms is still seen as an attractive alternative solution due to its low - cost , environmentally friendly , and publicly acceptable treatment technology . white rot fungi have been shown to be able to degrade a wide range of organic pollutants including synthetic dyes [ 1316 ] . in recent years , many studies have focussed on the use of white rot fungi to decolourise synthetic dyes due to its ability to produce nonspecific , ligninolytic enzymes [ 1618 ] . dye decolourisation , in particular by the white rot fungus phanerochaete chrysosporium , has been intensively studied , and the degradation pathway for sulfonated azo dye by this isolate has also been elucidated [ 6 , 9 ] . however , there is no report on the use of endophytic fungi for the decolourisation of dye . most studies on endophytic fungi have focussed on studying its relationship with its host and its diversity and also to isolate bioactive compounds of medical value from these fungi [ 1921 ] . in this view , the present investigation is an attempt to find out and examine the possible application of very little studied endophytic fungi isolated from melastoma malabathricum to decolourise several synthetic dyes belonging to two major dye groups widely applied in the dyeing industries . here we report the screening and isolation of dye decolourising endophytes on agar medium and dye decolourisation analysis to select the best isolate that decolourises synthetic dyes . the endophytic fungi used in this study were isolated from the stem of a healthy flowering plant , m. malabathricum . plant samples were collected from the surrounding areas of universiti malaysia sarawak ( unimas ) and kota samarahan campus and processed immediately after collection . the plant samples were washed under running tap water to remove debris and air - dried before being cut into 5 cm in diameters . in order to eliminate epiphytic microorganisms , the samples were surface - sterilized [ 21 , 22 ] by immersion in 5% ( v / v ) clorox for 5 minutes , followed by 70% ( v / v ) ethanol , and rinsed twice with sterilized distilled water . the surface sterilized samples were blot - dried using sterile filter paper and then aseptically cut into 2 cm in diameters . the pieces of stems were then transferred aseptically onto potato dextrose agar ( pda ) ( merck , germany ) plates ( 3 pieces per petri plate ) and incubated at room temperature for a period of 2 weeks . the plates were observed daily , and hyphal tips of developing fungal colonies were subcultured individually . the isolated endophytes were grown onto glucose minimal ( gm ) agar plates and initially screened for their ability to decolourise anthraquinone dye ( remazol brilliant blue r , rbbr ) and three azo dyes ( orange g , congo red , and methyl red ) . the gm agar medium contained ( g / l ) : k2hpo4 , 1 ; znso47h2o , 0.01 ; cuso45h2o , 0.05 ; mgso47h2o , 0.5 ; feso47h2o , 0.01 ; kcl , 0.5 ; glucose , 10 ; nano3 , 3 ; and agar , 20 . the ph of the agar medium was adjusted to 5.5 before being autoclaved at 121c for 15 minutes . dyes were added into the agar from a stock solution to a final concentration of 200 mg l. the agar plates were inoculated with a 5 mm agar plug from a 7-day old fungal culture and incubated in the dark at room temperature . plates were regularly monitored and observed for visual disappearance of colour for a period of 16 days . the best dye decolourising isolate was selected for further dye decolourisation in liquid gm medium . dyes were added to the 20 ml gm liquid medium in 100 ml erlenmeyer flask to a final concentration of 200 mg l. each flask was inoculated with 2 pieces of 5 mm agar plugs from a 7-day old fungal culture and incubated in the dark at room temperature under static condition . each culture condition was prepared in triplicate , incubated for a period of 16 days , and sampled at 4-day interval . during the sampling , each culture was harvested and centrifuged at 6000 rpm for 10 minutes to separate the fungal mycelium from the culture medium . fungal biomass was determined by drying the fungal mycelium to a constant weight at 70c . dye decolourisation by the isolated fungus was measured by monitoring the absorbance of each dye in the culture medium at its respective maximum absorption wavelength ( 595 nm for rbbr , 475 nm for orange g , 497 nm for congo red , and 520 nm for methyl red ) using a uv - vis spectrophotometer ( libra s12 , biochrom ) . percentage of decolourisation was calculated according to the following formula : ( 1)percentage of decolourisation ( % ) = acasac100 , where ac is the absorbance at the maximum absorption wavelength of dye in the control flask at time , t , and as is the absorbance at the maximum absorption wavelength of dye in the sample flask at time , t . the selected fungal isolate was identified by morphological characteristic as well as comparison of internal transcribed spacer ( its ) sequences . the morphological appearances of the selected fungal isolate were characterized based on mycelium colours , growth patterns , and structure of fruiting bodies . genomic dna of the selected fungus was extracted according to the method of cubero et al . . extracted fungal dna was then pcr - amplified using universal primer pair of its1 and its4 under the following condition : initial denaturation at 95c for 5 minutes , 30 cycles of denaturation at 95c for 1 minute , annealing at 55c for 1 min , extension at 72c for 1 min , and a final extension at 72c for 7 minutes . a neighbour - joining tree was constructed , and the distances between sequences were calculated . bootstrap analysis was performed with 1000 replications to assess the confidence limits of the branches . a total number of twenty endophytic fungi were successfully isolated from the stem of m. malabathricum . these fungi were named according to the source from which they were isolated and followed by a number ( ms120 with m = melastoma and s = senduduk ) . dye decolourisation activity of the isolated endophytic fungi was screened using an agar plate method with decolourisation observed by the production of clear halo . all of the tested fungi were able to grow on the dye containing minimal agar medium , and 14 of the fungal isolates were able to decolourise at least one of the dyes ( table 1 ) . among the 20 tested fungi , 13 isolates were able to decolourise rbbr , 2 isolates decolourised orange g , 13 isolates decolourised congo red , and 9 isolates decolourised methyl red . only one endophyte , isolate ms8 , was able to decolourise all four different dyes tested ( figure 1 ) . dye decolourisation by isolate ms8 was also the most rapid among all of the 20 fungal isolates tested . both rbbr and orange g dye in the agar medium started to decolourise within 2 to 3 days after fungal inoculation and were completely decolourised within a period of 8 days with no visible traces of dye adsorption on the fungal mycelium . congo red and methyl red were also partially decolourised by isolate ms8 with the production of halo , and no observable dye was absorbed by the fungal mycelium . isolates ms4 and ms17 were also able to completely decolourise rbbr by day 14 and orange g by day 12 , respectively , with no dye adsorption to the fungal mycelium . isolate ms8 was selected for further dye decolourisation in glucose minimal liquid medium based on the ability to decolourise all the dyes on the agar plate . the results obtained showed that isolate ms8 was able to decolourise all of the four different dyes tested to a different extent ( figure 2 ) . among the four different dyes tested , rbbr was decolorized the fastest and to the greatest extent by isolate ms8 , up to 97.4% decolourisation in 4 days . the three azo dyes were also decolourised by isolate ms8 with methyl red being the highest , reaching 56% decolourisation after 16 days followed by congo red ( 48.1% ) and orange g ( 32.6% ) . among the two different dye groups tested , the anthraquinonic dye rbbr was decolourised to a greater extent as compared to the three azo group dyes . decolourisation of the monoazo dye methyl red was also greater as compared to the decolourisation of both diazo dyes congo red and orange g. this shows that dyes belonging to chemically different groups are not decolourised to the same extent and the structural differences in the dye molecule strongly affect the decolourisation process . similar results have also been reported on a white rot fungi irpex lacteus which showed a lower efficiency in removal of monoazo and diazo dyes in liquid medium as compared to anthraquinone , phthalocyanine , and triphenylmethane dyes . decolourisation of a monoazo dye , xylidine ponceau , by a basidiomycetous fungal isolate rck-3 was also reported to be more efficient than the diazo dye , congo red . as decolourisation of a dye requires the destruction of the chromophore , the complexity and amount of chromophore a dye carries in its chemical structure therefore determine the resistance of the dye towards decolourisation . decolourisation of the monoazo dye methyl red was also greater as compared to the decolourisation of the diazo dye congo red and monoazo dye orange g even though all three are of the same azo type dye . decolourisation of orange g by isolate ms8 in liquid medium however was surprisingly lower than the extent of orange g decolourisation achieved by the isolate on agar plate . a possible reason for this would be that the presence of dye in liquid medium was much more toxic towards the isolate as compared to the toxicity of dye on agar medium . further quantification of the biomass produced by isolate ms8 with the presence of the tested dye in liquid medium shows that the biomass produced was greatly reduced as compared to the control biomass of the isolate produced in the absence of the dye ( table 2 ) . the presence of azo type dye in liquid medium has previously been reported to reduce gas solubility , and therefore high azo dye concentration can decrease fungal growth . a white rot fungus ischnoderma resinosum , capable of degrading a wide spectrum of chemically and structurally different synthetic dyes , has also been reported to be more sensitive to dyes in liquid culture than in agar plate . the biomass produced by i. resinosum in media containing malachite green and crystal violet also reached only about 10% when compared to the control biomass produced in the media without dyes . besides that , the production of some other dye oxidizing activities by the isolate might also be better on agar as compared to liquid medium . isolate ms8 was selected for further identification based on its morphological characteristic and also by comparison of the its sequences . isolate ms8 is a fast growing fungus with mycelium that covers the whole 90 mm petri dish in 7 days ( figure 3(a ) ) . the fungal isolates , when observed microscopically , showed the presence of clamp connection ( figure 3(b ) ) , the characteristic feature of basidiomycetous fungi . the isolate however did not produce any spore like structures , which normally provided the basis for fungal identification . isolation of a mycelial sterilia , fungi that do not sporulate in culture , such as isolate ms8 however is not uncommon during the isolation of endophytic fungi . for a given host , mycelial sterilia can take up to an average of 20% of the population of fungal endophytes . pcr amplification of the its region of isolate ms8 using a universal primer pair , its1 , and its4 , resulted in a pcr product with an approximate size of 620 bp in molecular weight . comparison of the its sequence with the established fungal its sequences in the genbank through a standard nucleotide - nucleotide blast homology search shows that isolate ms8 shared 99% sequence similarity with a basidiomycetes , marasmius cladophyllus . the sequence had been successfully deposited in genbank database ( accession number : kf241549 ) . a phylogenetic tree was constructed to determine the phylogenetic relationship of isolate ms8 with other marasmius species using m. epiphyllus as the outgroup ( figure 4 ) . the short branches and clustering of isolate ms8 together with m. cladophyllus show that isolate ms8 was closely related to m. cladophyllus . isolate ms8 was also distantly related with m. rotula . on a whole , endophytic fungus ms8 identified to be m. cladophyllus was able to decolourise the 4 different dyes tested especially anthraquinonic dye which was known to resist degradation due to their fused aromatic structures . the dye decolourization ability of this isolate was also comparable to that of white rot fungi such as i. lacteus and thelephora sp [ 27 , 34 ] . novel peroxidases capable of degrading beta - carotene were recently found to be produced by marasmius scorodonius . these novel peroxidases may be assigned to the group of dyp - type peroxidases ( dye decolourising peroxidases ) that is able to degrade synthetic anthraquinone dyes . these novel peroxidases may also be produced by isolate ms8 which was shown to effectively decolourise the anthraquinonic dye , rbbr . further enzymatic studies therefore are necessary in order to elucidate the possible enzymatic activities involved in dye decolourisation by our m. cladophyllus isolate ms8 . endophytic fungi isolated from the medicinal plant m. malabathricum in particular isolate ms8 was able to decolourise both the anthraquinone and azo type of synthetic dye to a different extent . this therefore suggests , that besides white rot fungi , endophytic fungi were also capable of decolourising the synesthetic dyes and of interesting potential to be used in the decolourisation of dyestuff effluents .

a total of twenty endophytic fungi successfully isolated from melastoma malabathricum ( senduduk ) were examined for their ability to decolourise azo dyes : congo red , orange g , and methyl red and an anthraquinone dye , remazol brilliant blue r. initial screening on the glucose minimal media agar plates amended with 200 mg l1 of each respective dye showed that only isolate ms8 was able to decolourise all of the four dyes . the isolate decolourised completely both the rbbr and orange g in the agar medium within 8 days . further quantitative analysis of the dye decolourisation by isolate ms8 in aqueous minimal medium showed that isolate ms8 was able to decolourise all the tested dyes at varying levels . dye decolourisation by the isolate ms8 was determined to be 97% for rbbr , 33% for orange g , 48% for congo red , and 56% for methyl red , respectively , within a period of 16 days . molecular identification of the fungal isolate ms8 using primer its1 and its4 showed that isolate ms8 shared 99% sequence similarity with marasmius cladophyllus , a basidiomycete . the ability to decolourise different types of dyes by isolate ms8 thus suggested a possible application of this fungus in the decolourisation of dyestuff effluents .

1. Introduction 2. Materials and Methods 3. Results and Discussion

PMC4622555

copd is one of the main causes of morbidity and mortality worldwide , and is expected to become the third - leading cause of death and the fifth - leading cause of disability - adjusted life years lost in 2020.1 patients with copd suffer recurrent exacerbations that require hospitalization . reducing admissions to the hospital is one of the primary aims in the management of the disease,2 due to the high burden to the patient and also the economic and social cost.3 furthermore , gold ( global initiative for chronic obstructive lung disease ) recently proposed a way to categorize risk in patients with copd using history of exacerbations for identifying patients that have a higher risk of being hospitalized . there is a need for simple ways to identify patients who are at the highest risk of being hospitalized within the clinic , in order to design preventive interventions and proper care , including palliative care . quality of life ( qol ) , measured with disease - specific questionnaires , has been shown to offer additional information to predict copd patients risk of hospitalization.4 however , disease - specific questionnaires are currently done in the context of clinical trials , and are rarely used in daily practice . not using the predictive information from such questionnaires as the chronic respiratory questionnaire self - administered survey ( crq - sas)57 or st george s respiratory questionnaire ( sgrq)8 may be a missing opportunity for better forecasting the risk of hospitalization . while the entire questionnaire might not be feasible for routine use , some questions might be significantly associated with the likelihood of hospitalization and merit further investigation . for example , while inflammatory and physiologic changes associated with copd have been implicated in the onset of depression and anxiety,9 the perception of qol is a better predictor of difficult emotions in copd than biological or physiological markers.10 the objective of the present study was to investigate the predictive value of individual questions from a disease - specific qol questionnaire , the crq - sas , on the risk of hospitalization in a well - characterized cohort of copd patients . the ultimate goal of this work is to develop a clinically relevant and easy algorithm that clinicians can use in routine practice to identify patients with an increased risk of hospitalization . patients with copd ( n=493 ) were prospectively recruited from an outpatient pulmonary clinic at mayo clinic in rochester , mn , usa . the following inclusion criteria were used in an attempt to obtain a uniform patient population : diagnosis of copd based on the gold 2011 guidelines,11 age > 40 years , history of smoking more than 10 pack - years , and the ability to complete questionnaires . this study received mayo clinic institutional review board approval , and all patients signed research consent . at the time of recruitment , investigators recorded demographics , clinical information , and health care utilization ( hospitalization and er visits ) during the previous year and self - reported by participants . pulmonary function tests were performed according to the current guidelines and established reference values.12 dyspnea status was assessed using the modified medical research council ( mmrc ) dyspnea scale ( range : 04 ) , as has been previously described.13 perceived qol was assessed using the crq - sas , a 20-item validated questionnaire that measures the health status of patients with copd . the inventory is divided into four dimensions : dyspnea , fatigue , emotional function , and mastery . lower scores in each dimension indicate a greater degree of dysfunction on a 7-point scale.6,7 finally , patients were classified subsequently according to the gold severity and risk criteria ( gold 2013 ) into gold a , gold b , gold c , and gold d groups.14 the composite index of copd severity ( age , dyspnea , obstruction)15 was calculated for all patients . the goal of this study was to define subsets of items ( individual questions from the crq - sas ) that could predict the risk of hospitalization after adjusting for age , lung function , history of hospitalizations , and mmrc dyspnea that can be used in daily clinical practice to make decisions on individuals at highest risk of hospitalization . methods used to define the subsets included variable cluster analysis , factor analysis using scree plots and varimax rotation , recursive partitioning ( classification and regression tree [ cart ] models ) , univariate and stepwise logistic regression models , and model validation using bootstrapping . recursive partitioning was done using rpart version 4.18.16 all other analyses were done using sas 9.2 ( sas institute inc , cary , nc , usa ) . development of the algorithm to identify patients at risk of hospitalization began with a confirmatory analysis of the internal psychometric structure of the existing measures . factor- and cluster - analysis routines were followed to demonstrate that indeed the internal structure of the original measures was demonstrated in our application setting . univariate logistic models were fitted to see how well each of the individual questions and subscales could predict hospitalizations . collinearity diagnostics were then run to determine whether any combinations of variables were too highly correlated to be included together in multivariate modeling . these diagnostic measures showed that the four crq subscales ( mastery , dyspnea , fatigue , and emotional ) were highly related to the individual crq questions , so subsequent multivariate models only used individual crq items . the logistic regression models then identified a subset of the individual items that were related to the incidence of hospitalization , indicating that a small number of items might be useful in identifying patient subgroups at higher risk of hospitalization . the akaike information criterion was used as the primary guide for item selection , along with the c - statistic . cart models were used to find combinations of items that could predict hospitalization using the variables identified in the previous factor analysis , cluster analysis , and logistic regression work . cart models were created once using only the individual crq questions and once using the crq variables plus sex , age , forced expiratory volume in 1 second ( fev1 ) percentage predicted , and mmrc scale . stepwise logistic regression models were then used to confirm and refine the predictive power of the cart models . finally , the stepwise logistic models were confirmed by applying stepwise logistic modeling to 1,000 bootstrapped samples . the goal of this study was to define subsets of items ( individual questions from the crq - sas ) that could predict the risk of hospitalization after adjusting for age , lung function , history of hospitalizations , and mmrc dyspnea that can be used in daily clinical practice to make decisions on individuals at highest risk of hospitalization . methods used to define the subsets included variable cluster analysis , factor analysis using scree plots and varimax rotation , recursive partitioning ( classification and regression tree [ cart ] models ) , univariate and stepwise logistic regression models , and model validation using bootstrapping . recursive partitioning was done using rpart version 4.18.16 all other analyses were done using sas 9.2 ( sas institute inc , cary , nc , usa ) . development of the algorithm to identify patients at risk of hospitalization began with a confirmatory analysis of the internal psychometric structure of the existing measures . factor- and cluster - analysis routines were followed to demonstrate that indeed the internal structure of the original measures was demonstrated in our application setting . univariate logistic models were fitted to see how well each of the individual questions and subscales could predict hospitalizations . collinearity diagnostics were then run to determine whether any combinations of variables were too highly correlated to be included together in multivariate modeling . these diagnostic measures showed that the four crq subscales ( mastery , dyspnea , fatigue , and emotional ) were highly related to the individual crq questions , so subsequent multivariate models only used individual crq items . the logistic regression models then identified a subset of the individual items that were related to the incidence of hospitalization , indicating that a small number of items might be useful in identifying patient subgroups at higher risk of hospitalization . the akaike information criterion was used as the primary guide for item selection , along with the c - statistic . cart models were used to find combinations of items that could predict hospitalization using the variables identified in the previous factor analysis , cluster analysis , and logistic regression work . cart models were created once using only the individual crq questions and once using the crq variables plus sex , age , forced expiratory volume in 1 second ( fev1 ) percentage predicted , and mmrc scale . stepwise logistic regression models were then used to confirm and refine the predictive power of the cart models . finally , the stepwise logistic models were confirmed by applying stepwise logistic modeling to 1,000 bootstrapped samples . cluster and factor analysis arrived at the same conclusions and verified the original crq - sas domains . these two multivariate methods resulted in the following four groups of the crq - sas variables : group 1 , questions 15 ; group 2 , questions 8 , 11 , 15 , and 17 ; group 3 , questions 6 , 12 , 14 , 16 , 18 , and 20 ; and group 4 , questions 7 , 9 , 10 , 13 , and 19 . these four factors are the same as the four factors defined in the development of the crq : dyspnea , fatigue , emotional , and mastery.5 the only exception was that question 9 clustered with the mastery variables in this analysis , even though it was originally part of the emotional subscale . this is not a surprising result , since question 9 contains both emotional issues and concerns about how well the subjects can overcome copd barriers . logistic regression models were used to determine variables associated with hospitalization . before running these models , the subscales based on factor analysis were strongly related to the individual crq - sas variables , so those variables were not used together in the logistic models . all of the other variables had condition numbers less than 100 , indicating there were no other collinearity concerns . recursive partitioning was done using just the crq - sas variables and again using the crq - sas variables along with the mmrc dyspnea scale , age , sex , and fev1% predicted . the resulting cart model using the crq - sas variables demonstrated an algorithm to predict hospitalization ( figure 1 ) . univariate logistic modeling ( table 2 ) showed that fev1% predicted , mmrc dyspnea scale , each of the individual crq - sas questions , the subscales created from factor analysis , and the cart groups were all significantly related to hospitalization . the two different cart models ( crq - sas variables alone versus crq - sas variables plus other variables : mmrc dyspnea scale , fev1% predicted , age , and sex ) performed almost the same in predicting the risk of hospitalizations . in fact , the cart model using just the crq - sas variables had a better c - statistic ( 0.70 ) than the other cart model using the mmrc dyspnea scale , fev1% predicted , age , and sex ( c=0.684 ) . stepwise logistic modeling found that the cart model using the crq - sas variables was the best predictor of the risk of hospitalization ( table 3 ) . the algorithm obtained through the cart modeling ( figure 1 ) identified two subsets of patients that accounted for 40% of the sample with a combined hospitalization risk of 50% ( double the risk of hospitalization compared to the rest of the patients ) . the first subset , approximately 20% of the sample ( 103 of 493 ) , indicated that they felt fear or panic when they were short of breath most of the time or all the time . these participants carried a 57% risk of hospitalization compared to only 27% for the rest of the sample ( all other groups combined ) . the second subset , approximately a further 20% of the sample ( 101 of 493 ) , included individuals who indicated they felt at least mildly short of breath while caring for their basic needs and also felt discouraged or down in the dumps , at least a little of the time , and had a 43% risk of hospitalization . we built an algorithm using three questions from the crq - sas following a rigorous statistical method that can help clinicians to forecast risk of hospitalization and increase our portfolio of methods to determine hospitalization risk , an event associated with poor prognosis and mortality.17 we identified important outcomes in copd fear of breathlessness , dyspnea with activities of daily living , and depressive symptoms ( figure 1 ) that have been previously reported with an increased risk of hospitalization.14,1820 importantly , we defined specific cut points ( a defined score for each question ) that made them simple for interpretation and definition of risk . we identified a simple way to test the construct of fear of breathlessness in copd that is associated with hospitalizations . the construct of fear has been shown to have an impact on disease disability in copd.21 specific measures of the construct of fear to use in daily care are lacking , but currently existing measures have been reported to be predictive of copd - treatment outcomes after controlling for general anxiety.22 disease - specific fear is defined as anxiety in the face of severe physical symptoms and their consequences.23 keil et al showed in 2011 the importance of specific fears in the context of copd and their association with disability.21 our study supports the hypothesis of keil et al feeling fear or panic due to shortness of breath was the most predictive question in our algorithm : a 57% risk of hospitalization . our results , which further emphasize the importance of symptoms of depression and anxiety ( fear ) , have profound consequences for the health of patients with copd . anxiety ( fear ) is associated with increased risk of exacerbations , poorer health - related qol , worse physical activity19 relapse within 1 month of receiving emergency treatment,24 and hospital readmission.25 depression is associated with increased mortality , impaired health - related qol , and excessive health - care - utilization rates and costs , including longer hospital stay after acute exacerbation,20 increased risk of exacerbation and hospital admission,26 hospital readmission,27 and lower physical activity.28 we support and extend previous reports about the impact emotions ( anxiety and depression ) have on health care utilization ( in addition to physical functioning and qol).19,29,30 gold has recently highlighted the importance of effective screening and management of anxiety and depression.14 furthermore , gathering patient - reported outcome data regarding sensitive issues , such as fear and depression , from a questionnaire rather than through a personal and potentially difficult conversation has been seen to elicit information that a patient may not be willing to verbalize to a health care provider.31,32 our findings also extend previous reports suggesting that individual items within questionnaires can be as sensitive , if not more sensitive , than the overall scores and content of larger questionnaires to forecast specific outcomes.33,34 furthermore , items drawn from general questionnaires have been demonstrated to be useful indicators for more refined clinical interventions.35 the overall measures and longer questionnaires remain useful tools , but by looking at the psychometrics of individual items we can capitalize on the most clinically relevant information . we followed a strict psychometric approach with multiple sensitivity analyses via alternative analytical approaches and bootstrapping resampling sets of 1,000 patients . the algorithm is intended to be a supplementary source of information that the clinician can incorporate into their personal assessment of the patient that can impact the monitoring and care plan for the patient . importantly , the algorithm incorporates patient - reported information that has not been used routinely or systematically in clinical practice . current algorithms in the area of copd care have prognostic characteristics that are similar to what we have produced . the body mass , obstruction , dyspnea , and exercise capacity index that has universal application in clinical practice have an area under the curve of 0.74 , which is comparable to the area under the curve of 0.70 obtained for our algorithm.36 our group previously showed that a single item measuring qol was actually more informative and sensitive to change than longer scales purported to measure the same thing.37 specifically , in copd we initially reported that a single general question on health status is independently associated with the risk of hospitalization.18 the retrospective health - care - utilization measure ( history of a hospitalization in the last year ) can be viewed as a limitation . however , a recent report from the eclipse group38 has clearly shown a very strong association between a hospitalization in the previous year and hospitalization in the subsequent year , supporting the validity of the measure used in this study . there are ( undoubtedly ) more variables involved to accurately predict individualized risk for hospitalization ( a previous hospitalization and gold risk assessment ) ; our results provide a simple screening tool to identify a subset of the population that might benefit from closer monitoring , specific therapies , or even palliative care . in particular , the data indicate that if a patient perceives fear related to breathlessness , their risk of hospitalization doubles from a mean value of 30% in the copd - severe population to approximately 50%.39 this allows the limited resources available to the clinical team to be directed to patients who are more likely to be hospitalized . this algorithm to identify patients at higher risk of hospitalization will have false positives and negatives ; however , it is not intended as a diagnostic laboratory test , but a supplementary source of information for the clinician . the retrospective health - care - utilization measure ( history of a hospitalization in the last year ) can be viewed as a limitation . however , a recent report from the eclipse group38 has clearly shown a very strong association between a hospitalization in the previous year and hospitalization in the subsequent year , supporting the validity of the measure used in this study . there are ( undoubtedly ) more variables involved to accurately predict individualized risk for hospitalization ( a previous hospitalization and gold risk assessment ) ; our results provide a simple screening tool to identify a subset of the population that might benefit from closer monitoring , specific therapies , or even palliative care . in particular , the data indicate that if a patient perceives fear related to breathlessness , their risk of hospitalization doubles from a mean value of 30% in the copd - severe population to approximately 50%.39 this allows the limited resources available to the clinical team to be directed to patients who are more likely to be hospitalized . this algorithm to identify patients at higher risk of hospitalization will have false positives and negatives ; however , it is not intended as a diagnostic laboratory test , but a supplementary source of information for the clinician . we created a simple algorithm following a very robust statistical method that may become informative to the practicing clinician to forecast the risk of hospitalization in copd patients . we identified clinically relevant constructs ( fear of breathlessness , significant dyspnea with activities of daily living , and depressive symptoms ) embedded in the crq questionnaire that are independently associated with hospitalization risk . our work is not implying that we have a better way to predict hospitalization , but an alternative way to augment our accuracy in forecasting the risk of hospitalization and the opportunity to provide our patients the most individualized and precise care possible .

purposeforecasting hospitalization in patients with copd has gained significant interest in the field of copd care . there is a need to find simple tools that can help clinicians to stratify the risk of hospitalization in these patients at the time of care . the perception of quality of life has been reported to be independently associated with hospitalizations , but questionnaires are impractical for daily clinical use . individual questions from valid questionnaires can have robust predictive abilities , as has been suggested in previous reports , as a way to use patient - reported outcomes to forecast important events like hospitalizations in copd . our primary aim was to assess the predictive value of individual questions from the chronic respiratory questionnaire self - assessment survey ( crq - sas ) on the risk of hospitalization and to develop a clinically relevant and simple algorithm that clinicians can use in routine practice to identify patients with an increased risk of hospitalization.patients and methodsa total of 493 patients with copd prospectively recruited from an outpatient pulmonary clinic completed the crq - sas , demographic information , pulmonary function testing , and clinical outcomes . the cohort had a mean age of 70 years , was 54% male , with forced expiratory volume in 1 second percentage predicted 42.816.7 , and modified medical research council dyspnea scale score of 21.13.resultsour analysis validated the original crq - sas domains . importantly , recursive partitioning analysis identified three crq - sas items regarding fear or panic of breathlessness , dyspnea with basic activities of daily living , and depressive symptoms that were highly predictive of hospitalization . we propose a robust ( area under the curve = 0.70 ) but short and easy algorithm for daily clinical care to forecast hospitalizations in patients with copd.conclusionwe identified three themes fear of breathlessness , dyspnea with basic activities of daily living , and depressive symptoms as important patient - reported outcomes to predict hospitalizations , and propose a short and easy algorithm to forecast hospitalizations in patients with copd .

Introduction Patients and methods Statistical analysis Results Discussion Limitations Conclusion

PMC4383429

for the last years both competent authorities , the us food and drug administration ( fda ) and the european medicines agency ( ema ) , force the development of high quality child - appropriate medications and intend to improve the availability of information on the pediatric use . due to the current lack of sufficient evidence - based pharmacotherapy in children , sophisticated clinical investigations in all pediatric age groups ( particularly in neonates and infants ) unfortunately , most bioanalytical assays are not yet tailored to meet current ethical and analytical burdens for research in children . although the blood sample volume for determination of drug concentration is limited to microliters , it is essential for a valuable determination in pediatric patients to keep the calibration range as broad as or even broader than in assays applied in adult studies . hplc - ms / ms is a predestinated analytical technique that appears to be the most suitable to deal with small sample volumes obtained from children and is linked with high selectivity for the quantification of the analytes of interest in diverse biological matrices . however , both the chromatographic equipment and the mass spectrometer ( ms ) encounter problems caused by the matrix . for example , the lifetime of the hplc column is reduced if the sample purification is insufficient . additionally , the detection by ms is susceptible to matrix effects leading to ionization suppression or enhancement [ 1 , 2 ] . the matrix has a profound impact and restrains a precise quantification especially at the lower concentration levels . this ends up in nonrobust methods that do not encompass the broadest calibration range possible . therefore , the role played by proper sample preparation and extraction is important to overcome and control the interference caused through the biological matrices . a sophisticated sample clean - up removes material that chromatographically interferes with the analyte , enables appropriate recoveries , and erases matrix compounds that shorten column lifetime and affect the detection by ms . attempts to simplify sample preparation and to reduce the preparation time ended in the awareness that this process accounts for less accuracy and precision in quantification . biological fluids like plasma , serum , urine , and saliva present a varying composition of , for example , lipids , proteins , electrolytes , cells , coeluting metabolites , impurities , and degradation products . all of these components might interfere with the analyte of interest . to reduce this interference several approaches had been developed in the past . commonly used extraction techniques are protein precipitation ( ppt ) , liquid / liquid extraction ( lle ) , and solid - phase extraction ( spe ) . the purpose of the present work was to illustrate the importance of sample preparation exemplified by solid - phase extraction for the bioanalytical method development of low - volume assays for pediatric studies according to international agency guidelines . using the method validation of enalapril , enalaprilat , and benazepril ( internal standard ) , the encountered challenges and advances in sample preparation and solid - phase extraction as well as their effects on bioanalytical method validation of small volume samples were emphasized . the drug substances enalapril maleate crs and enalaprilat dihydrate crs ( both european pharmacopoeia reference standards ) were purchased at the european directorate for the quality of medicine & healthcare ( strasbourg , france ) . benazepril hydrochloride ( 98% , hplc ) and ethyl acetate ( 100% p.a . ) were obtained from sigma - aldrich ( seelze , germany ) . methanol ( hipersolv chromanorm hplc grade ) , water ( super gradient grade ) , and acetone ( analar normapur ) were purchased from vwr ( germany ) . alternative supplier of methanol ( hplc grade ) was fisher scientific ( loughborough , united kingdom ) . formic acid ( 98100% p.a . ) was delivered by applichem ( gatersleben , germany ) . ammonium formate ( 99% , hplc grade ) was obtained from fluka ( seelze , germany ) . blank human serum was provided by employees of the institute of clinical pharmacy and pharmacotherapy ( dsseldorf , germany ) . oasis 96-well plates ( 30 and 10 mg ) and xbridge beh c18 3.5 m columns ( 3.0 mm 150 mm ) were obtained from waters ( eschborn , germany ) . stock solutions of enalapril , enalaprilat , and benazepril ( internal standard ) were prepared at 0.10 mg / ml in methanol . these stock solutions were diluted with water to obtain working solutions with 10 g / ml enalapril and enalaprilat as well as 166 ng / ml benazepril . for the calibration curve , blank serum was spiked with the analytes of interest and serially diluted . the final calibration range of the mass spectrometry was 0.2200 ng / ml enalapril and 0.18180 ng / ml enalaprilat . quality control ( qc ) samples were independently prepared at four concentration levels over the whole calibration range ( lloq , low , medium , and uloq ) . based on preliminary investigations on the degree of sample dilution prior to solid - phase extraction a dilution ratio of 1 : 23 using water led to a robust method with high recovery rates for all analytes of interest . solid - phase extraction was chosen for the extraction process of the biological fluid owing to the superior purification performance if compared to protein precipitation or liquid - liquid extraction . based on the compound properties of enalapril and enalaprilat , strong mixed - mode ion exchangers were chosen as sorbent material for the purification . both a cation exchanger that interacts with the carboxylic acid groups and an anion exchanger that binds with the amino group of all above - mentioned substances were evaluated on their applicability ( oasis mcx and max material ) . the spe protocol included a conditioning step utilizing methanol to enable optimal wetting of the cavernous sorbent material . for the subsequent equilibration step , different ph values and acids in aqueous solutions were tested to warrant for best interaction conditions prior the aqueous sample was loaded into the cavity . namely , 2% formic acid ( v / v ) , 4% phosphoric acid ( v / v ) , 0.2 n hydrochloric acid , and pure water were evaluated . to purify the sample as much as possible , washing steps from hydrophilic to lipophilic properties were evaluated ( water , 2% formic acid , hydrochloric acid , methanol , isopropanol , acetone , ethyl acetate , and mixtures of the aforementioned ) . elution was assessed by acidified methanol for max sorbent material and ammonium in methanol for mcx sorbent material . the kind of acid ( or base ) , its concentration , and the corresponding elution volumes were investigated to obtain all analytes within one fraction by reducing the coeluted residual matrix to a minimum . to meet current demands in sample throughput within a clinical study , the switch to 96-well format came along with a changeover from vacuum extraction to positive pressure extraction . spe - formats with higher amounts of spe cavities per plate were commercially not available . for the critical steps of sample load , washing , and sample elution , the positive pressure was kept between 1 and 2 psi to warrant an intensive interaction between analyte and sorbent material as well as reproducible flow rate . figure 1 illustrates the corresponding scale - up . the utilized modular hplc system ( shimadzu deutschland gmbh , duisburg , germany ) consisted of a controller scl-10avp , two separate pumps lc-10advp , three - channel online degasser dgu-20a prominence , autosampler sil-10advp , and a column oven ( l-2300 , vwr / hitachi ) . for the separation of enalapril and enalaprilat an xbridge beh c18 3.5 m column ( 3.0 mm 150 mm ) was used . after injection of 10 l sample solution ( methanol / water 40 : 60 , v / v ) the samples were separated under gradient conditions within 6-minute run time utilizing a methanol / water mixture ( 40 : 60 , v / v ) buffered with formic acid ( 1% , v / v ) and ammonium formate ( 2 mm ) . the applied gradient started with 40% of methanol and increased stepwise after 0.5 minute to 60% and after 1 minute to 80% . between 1 and 4 minutes the amount of methanol was continuously increased to 95% and reduced at 4.5 minutes to 40% of methanol again . the flow rate was 0.4 ml / min and the column temperature was maintained at 50c which resulted in a moderate back pressure of 125 bar . triple - quadrupole tandem mass spectrometric detection was performed on an applied biosystems sciex api 2000 ( applied biosystems / mds sciex , concord , canada ) with an electrospray ionization ( esi ) interface running in positive ionization mode . the device screened the transitions channels 377.2 to 234.2 m / z ( enalapril ) , 349.1 to 206.1 m / z ( enalaprilat ) , and 425.3 to 351.2 m / z ( benazepril ) in multiple reaction monitoring ( mrm ) mode . the bioanalytical method was fully validated according to current fda and ema bioanalytical guidelines as a quantitative confirmatory method in terms of linearity , specificity , accuracy , precision , recovery , matrix effect , and stability [ 4 , 5 ] . the latter was in particular validated on recovery of the extraction process and absolute plus relative matrix effect . additionally , extraction process efficiency and interference caused by hyperlipidemic and hemolyzed samples were evaluated . the ratio of peak area of serum spiked with analyte prior to solid - phase extraction ( areaa ) with the peak area of blank serum spiked with analyte after the extraction ( areab ) yielded the recovery of the assay . calculation was performed as follows:(1)re%=areaaareab100 , where re = recovery ; areaa = peak area of serum spiked with analyte prior to extraction ; areab = peak area of blank serum spiked with analyte after the extraction . although the blood composition and ph value are strongly controlled and vary only slightly within a healthy subject , the overall consequence of all compounds in a biological sample matrix contributes to the matrix effect that alters the accurate and precise determination of the analyte of interest . therefore , a suitable sample preparation and purification by spe contributes extremely to a robust method being less - sensitive to the effect . apart from broadly investigated absolute matrix effect , the investigation of the relative matrix effect appears in this contect more relevant for precise and robust methods for the analysis of biological samples . calculation of the absolute matrix effect was conducted by calculating the ratio of the peak area of extracted human serum postspiked with analyte ( areax ) to the peak area of the analyte in the same concentration dissolved in mobile phase ( areay ) . the absolute matrix effect was determined at four concentration levels with five replicates per level . the absolute matrix effect was calculated according to the equation by matuszewski et al . : ( 2)me%=areax areay100 , where me = matrix effect ; areax = detected peak area of extracted human serum postspiked with analyte ; areay = detected peak area of dissolved analyte in mobile phase . to distinguish between ion suppression and ion enhancement caused by the matrix , a value < 0 indicated ion suppression caused by the matrix while ion enhancement was present if the calculated value was > 0 . according to international guidelines , the absolute matrix effect should be evaluated but is not limited to a certain range . however , the applicant needs to be able to estimate the effect of the matrix on the assay 's performance . by contrast the european medicines agency provides a guidance mentioning fixed acceptance criteria for the relative matrix effect . the intersubject variability of the internal standard normalized relative matrix effect ( is - me ) of processed samples should be maximum 15% and is expressed as coefficient of variation ( cv ) . the calculation of the latter was done by evaluation of the individual is - normalized matrix effect being defined as the matrix factor of the analyte divided by the matrix factor of the is . the matrix factor represents the ratio of the peak area in the presence and the absence of the matrix of the corresponding substance . the cv of the is - normalized matrix effects of seven subjects was used to assess the relative me . the relative matrix effect was evaluated at 0.39 ng / ml enalapril and 0.35 ng / ml enalaprilat ( low concentration level ) as well as at 200 ng / ml enalapril and 180 ng / ml enalaprilat ( uloq ) . per concentration level three replicates per subject were analysed:(3)is - me% = peak area of analytepresence of matrixpeak area of analyteabsence of matrix peak area of ispresence of matrixpeak area of isabsence of matrix1100 , where is - me = internal standard normalized matrix effect . to calculate the process efficiency of the solid - phase extraction the following equation by taylor was used:(4)pe%=re%100me%100 , where pe = process efficiency ; re = recovery ; me = absolute matrix effect . further validation parameters , as listed in the international bioanalytical guidelines , were investigated as follows : linearity was evaluated by measuring freshly spiked human serum with enalapril and enalaprilat at 11 concentration levels . the bioanalytical method was evaluated on four different days by four different runs on accuracy and precision . therefore , five independently prepared quality control samples were assessed on four concentration levels ( enalapril : 0.2 , 3.13 , 25 , and 200 ng / ml ; enalaprilat : 0.18 , 2.81 , 22.5 , and 180 ng / ml ) per run . the precision was determined by anova while the accuracy was described by percentage deviation of the mean value to the nominal values of each concentration level . a maximum deviation of 15% ( 20% at the lloq ) was regarded as acceptable . the selectivity was assessed by check for interaction caused by 7 human serum samples spiked with 11 common comedications . the long - term stability of the drugs was evaluated at 80c for at least 60 days , short - term stability conducted at 20c for 24 h , and autosampler stability for 24 h also at 20c . additionally , the stability of the dried eluate after sample extraction was investigated at 20c for 24 h. the high - throughput approach was applied to a phase i study in 24 healthy volunteers . both urine and serum samples were withdrawn and analyzed after administration of 10 mg enalapril maleate . with focus on sample extraction , the applicability was evaluated on the time required to run the extraction , the occurrence of any clotting of the cavity during extraction , and the goodness of extraction by checking for any shift in retention time in samples of different volunteers and different sample conditions ( e.g. , hemolyzed samples ) . preliminary investigations on the degree of sample dilution had been shown to influence the extraction performance and accounted highly for a robust method with high recovery . investigations on suitable sample dilution solvents ( formic acid , phosphoric acid , hydrochloric acid , and water ) and their mixing ratio with the sample itself were conducted . to determine the best suitable mixing ratio of acids or pure water , the ratio was varied between 1 : 1 and 1 : 23 . the conducted investigations on the most appropriate dilution solvent showed that water is sufficient if high dilution factors were applied . by increasing the mixing ratio , the detected peak areas of enalapril and enalaprilat increased in parallel ( figure 2 ) . a mixing ratio of 1 : 10 and 1 : 23 worked best with regard to sample recovery . the highest dilution ratio resulted in a total sample volume of about 1.2 ml . owing to the maximum capacity of a cavity ( ~1.4 ml ) , a higher degree of dilution is not recommended for routine . it increases the risk of carryover and rises the likelihood of sample mix - up as the sample solution needs to be pipetted at least in two parts into the cavity . the final composition of the diluted sample solution consisted of 50 l serum being mixed with 5 l benazepril working solution ( is ) and 1100 l water . specifically , if small biological sample volumes like in pediatric research require purification , the sample extraction plays a particular role . commonly , protein precipitation ( ppt ) , liquid / liquid extraction ( lle ) , and solid - phase extraction ( spe ) are applied as extraction techniques . ppt is a fast and simple approach but works best only in protein - rich matrices such as whole blood , plasma , or serum . nevertheless , the ppt is nonselective and does not remove matrix interferences other than proteins . in the investigations by dams et al . , ppt in combination with lc - ms / ms had the greatest matrix effect amongst the investigated purification approaches . it is a useful and fast technique to optimize lifetime of the equipment but does not increase the analytical sensitivity which is important for low - volume lc - ms / ms assays . lle allows separation of analytes of interest from proteins and other hydrophilic components , but if emulsions are formed , the separation of the organic solvent becomes difficult and might result in incomplete and various - analyte diffusion . jessome and volmer emphasized the cumbersome sample preparation by lle and lc - ms / ms . especially the complex adjustment of the ph value for the transfer in the organic phase , extraction of highly polar substances and necessary multiple extraction steps are some challenges faced . in particular for high - throughput analytics as it is useful in clinical study approaches the lle is not the first choice . consequently , spe was selected owing to its superior purification properties and flexibilities in extraction protocols to cope with diversity of analytes , purifications solvents , and biological fluids . the selected drug combination ( calculated logp values of enalapril : 2.5 ; enalaprilat : 0.9 ; benazepril : 3.5 ) carried the risk of improper binding to the sorbent due to coulomb repulsion , losing hydrophilic analytes during too extensive and not well - balanced washing steps or by incomplete recovery of the more lipophilic compounds from the sorbent material during elution . all this may be attributed to low recovery or bad reproducibility which in turn narrows the calibration range , because especially lower concentrations might not conform to international guideline requirements . available sorbent phases characterized by different interaction possibilities ( van der waals forces , ionic interaction , etc . ) and different amounts of sorbent per cavity and the high flexibility in the spe protocol on how intensive the purification of sample needs to be conducted represent some of many useful tools to overcome those drawbacks . this polymeric material is characterized by a strong cation exchanger ( on sulfonic acid base ) binding the carboxylic acids groups of the analytes of interest . however , purified samples showed a split peak for the selected transition of enalaprilat if determined by hplc - ms / ms . the corresponding chromatograms revealed a peak occurring at an earlier retention time plus a second peak at the expected retention time of the compound ( figure 3 ) . the peak area and intensity of the first peak did not alter with different enalaprilat concentrations per sample and accounted therefore most likely for a residual serum matrix component . it was not possible to erase the peak neither by thought - out spe nor by any number of different lc gradients or by different hplc columns featured with opposed chromatographic properties ( atlantis t3 , xbridge , and xselect ) . the checks for contamination in mobile phase , autosampler , or solutions for spe were additionally negative . at the lowest concentration level of the calibration curve , the first peak and the one of enalaprilat were comparable in shape , intensity , and peak area . this phenomenon carried the risk of less robustness if automated integration of the chromatogram is preferred . after also scanning for the second most intense transition of enalaprilat ( 349.1 303.1 m / z ) , clarity was brought to question as the first peak did not belong to enalaprilat . however , quantification with two transitions would have resulted in a higher lloq which was undesired as very low concentration levels are expected in the scheduled pediatric studies . therefore , the sorbent material of the spe was changed from cation exchanger to strong anion exchanger ( max ) and a new extraction protocol was developed . this brought success to the method , as the compound with the same transition as enalaprilat ( 349.1 206.1 m / z ) could be detached by spe and consequently the split peak was removed . at this stage an excuse to an already established and fully validated extraction method for the same drug entities in urine ( amongst others ) is made to introduce another useful approach to purify the sample matrix , to reduce the relative matrix effects , and to meet the current ema bioanalytical guideline . a two - step solid - phase extraction by a weak anion exchanger followed by a strong cation exchanger significantly reduced the internal standard normalized matrix effect compared to the purification by mcx only ( figure 4 ) . the more hydrophilic the compound was the merrier the improvement of the matrix effect was pronounced . however , it expounds how diverse the several biological fluids are and emphasizes the high required effort in method development to reduce the relative matrix effect . by applying the final two - step extraction the following results for the relative matrix effect were obtained : at the lloq the cv was 4.04% and 6.62% for enalapril and enalaprilat , respectively . a cv of 1.26% for enalapril and 1.25% for enalaprilat was calculated at the uloq and was therefore well within the ema requirements of 15% . this bioanalytical urinary method was fully validated according to the strictest validation parameters of ema and fda bioanalytical guidance . at an early development stage of the extraction protocol in serum at which the extraction was not methodologically sound , we encountered some deviation in retention time when comparing the chromatograms of analyte solved in mobile phase to extracted analyte in serum ( figure 5 ) . by increasing the amount of organic washing steps as well as the elution force of the used organic solvents ( methanol , acetone , and ethyl acetate ) , this timely shift even the intensities and peak areas of both , the analyte in mobile phase and the analyte in purified serum , were finally comparable . this pointed out the gained high degree of sample purification and accounted for a very limited signal suppression by the residual matrix compound in the final extract . however , it illustrates that matrix effects not only affect the signal intensities of the mass spectrometer but can also alter the chromatographic performance . finally , the conducted investigations on the most appropriate elution solvent and its volume yielded 0.4 ml acidified methanol ( 2% formic acid , v / v ) . this elution step did not only served to elute the analytes of interest but also acted as a final step to fraction the analytes of interest and other interfering residual compounds . hereby the choice and elution force of the solvent as well as the amount of solvent were investigated on their effect to attribute to a rugged , reliable , and selective protocol . as illustrated in figure 6 , there was a reciprocal relationship between the elution volume of acidified methanol and the detected peak areas of the analytes of interest . the obtained smaller peak areas after the sorbent material was eluted with higher volumes of acidified methanol might be explained by the fact that more interfering matrix was coeluted . the final protocol for the serum method was destined to the following approach : the samples were extracted with oasis max solid - phase extraction cartridges ( 10 mg , 1 ml)a mixed mode , reverse - phase , and strong anion exchanger ( on quarternary amine base ) . the max 96-well plates chosen allowed for high sample throughput and were primed with 1 ml of formic acid in methanol ( 2% , v / v ) followed by an equilibration step with 1 ml water . on the one hand , this aqueous step prior to sample load ensured that , for example , no denaturation and therefore clotting of sample matrix on the sorbent material happened . on the other hand , it offered the best interaction conditions with the sorbents . after the sample mixture was loaded into the cartridges and passed , the sorbent of the cartridges was washed by 1.0 ml of water , 1.0 ml of methanol - acetone mixture ( 60 : 40 , v / v ) , 1.0 ml of ethyl acetate , and 500 l of methanol . the increasing elution force of the investigated organic solvents was used mainly to wash out phospholipids that are known to be responsible for a large part of matrix effect in blood , serum , and plasma . finally the analytes were eluted from the cartridges once with 0.4 ml of formic acid in methanol ( 2% , v / v ) . the eluate was evaporated to dryness under a gentle stream of compressed air while shaking at 550 rpm at 40c . the residue was reconstituted with 100 l of methanol and water ( 40 : 60 , v / v ) . for reproducible and high - quality solid - phase extraction ( spe ) with a high run - to - run consistency as desired for clinical studies , the switch from single cartridges by vacuum extraction to 96-well positive pressure extraction was conducted . spe - formats with higher amounts of spe cavities per plate were commercially not available . the conventional vacuum manifold had the disadvantage of irreproducible analyte recoveries due to variable processing times in the columns . for the highest possible reproducibility during extraction , the controlled and appropriate flow rate is much more essential than applying either vacuum or positive pressure . however , the positive pressure manifold had the advantage of being equipped with a monitor to check for the flow rate of the liquid . specifically the sample load , washing , and elution step are known to be the most sensitive and critical steps regarding the flow rate . the exact adjustment of the flow rate of the liquids was important to generate a well - balanced setting of high reproducibility , best extraction speed and duration of sample extraction . the transfer from vacuum extraction to positive pressure not only enabled a semiautomated extraction but also highly increased the sample amount up to about thousand samples which can be purified per week by one laboratory technician . since appropriate equipment for rapid and continuous drying was not commercially available , required equipment for the drying process was self - developed to suit best the laboratory preconditions and needs . figure 1 is enclosed illustrating the scale - up from single cartridges using vacuum technique to the positive pressure extraction in 96-well formate . as indicated in figure 7 , the scale - up is a mandatory step in method development if the assay will be applied to analyze hundreds or thousands of samples . in our positive pressure approach , it took about 2 hours from raw sample to sample preparation and extraction to the final sample solution ready to be determined by hplc - ms / ms . in such a run , 96 samples could be prepared which required much more than one full working day ( 8 h ) of one laboratory technician to prepare the same amount by the previously used vacuum manifold approach . the applied pressures of 13 psi were fully sufficient to ensure a continuous and appropriate flow rate of solvent / sample solution through the sorbent material . the chosen calibration range of 0.2200 ng / ml enalapril and 0.18180 ng / ml enalaprilat , respectively , showed guideline - conforming linearity over all eleven concentration levels . best fit of the linear regression was gained by 1/x weighting for both analytes . no additional peaks above the guideline limits within 0.3 minutes of the retention time of enalapril , enalaprilat , and benazepril were observed in the spiked serum samples with coadministered drugs ( acetylsalicylic acid , aliskiren , ramipril , ramiprilat , candesartan , atenolol , bisoprolol , metoprolol , pantoprazole , and pravastatin ) . no interferences in blank samples of the different sources were detected and the signal - to - noise ratios of spiked to blank samples of all sources were above 5 : 1 . obtained results by one - way anova for the intrarun precision ranged from 2.2 to 5.0% for enalapril and from 4.9 to 18.0% for enalapril . mean accuracy results of the quality control samples were likewise within the guideline requirements of 15% at all concentration levels ( at the lloq 20% ) . hemolyzed blood as well as hyperlipidemic blood samples had no detectable influence on the specific ms - channels of enalapril , enalaprilat , and is , respectively . obtained stability results of enalapril and enalaprilat for long - term storage ( 80c , 60 days ) , for short - term storage ( 20c , 24 h ) , and in the autosampler ( 20c , 24 h ) proved the drug stability . additionally , both drug substances showed no significant degradation if they were stored as dried elution extract at 20c for 24 h. the effect of the matrix on the determination of enalapril and enalaprilat was evaluated at the lloq ( 0.2 ng / ml ; 0.18 ng / ml ) , one low concentration ( 3.13 ng / ml ; 2.81 ng / ml ) , one middle concentration ( 25 ng / ml ; 22.5 ng / ml ) , and at the uloq ( 200 ng / ml ; 180 ng / ml ) . by combining spe and chromatographic separation the matrix effect was observably reduced in this setting , leading to an ion suppression of 8.9 to 19.8% for enalapril and of 7.2 2.8% for the internal standard benazepril . the sample matrix had no or a slight ion enhancing effect on detection of enalaprilat . all analytes were almost fully recovered from the sorbent of the mixed - mode anion exchanger , resulting in a process efficiency of 67 to 94% for enalapril , 95119% for enalaprilat , and 71% for benazepril . the relative matrix effects of the extracted serum samples at a low concentration level ( 0.39 ng / ml enalapril and 0.35 ng / ml enalaprilat , resp . ) , coefficients of variation of 1.87% for enalapril and 8.96% for enalaprilat were evaluated for all seven different human sources . details are arranged in table 2 . for the phase i study , in total approximately 1600 serum and 600 urinary samples were analyzed . a shift in the retention times of enalapril , enalaprilat , and benazepril in purified serum samples of 24 different sources was not detected during the analysis by hplc - ms / ms and allowed for an automated intergation . furthermore , hemolyzed samples did not affect the extraction run negatively and showed no significant interference during analysis . the determined pharmacokinetic results of the phase i study will be used to apply a new marketing authorization and remain therefore confidential . however , during sample determination 22 calibration curves in serum and 7 in urine were required to quantify the drug concentration in the corresponding samples . obtained results of the calibration curves on intra- and interrun accuracy proved the applicability of the established bioanalytical method comprising the good sample extraction and their suitable preparation . furthermore , these tailored low - volume assays will be applied to pediatric phase ii and iii studies . the available pediatric study investigating the pharmacokinetics of enalapril and its active metabolite enalaprilat in hypertensive children was published by wells et al . using a radioimmunoassay . lloyd et al . reported enalaprilat concentration values between 0.9 and 12.7 ng / ml in children with heart failure . both reported ranges are covered by the linear range of the assay presented and confirm its applicability for pediatric research . the required sample volume of 50 l serum for reliable determination appears additionally well suitable for clinical trials in all age groups , especially for neonates and newborns . the required sample volumes of published lc - tandem mass spectrometry assays on enalapril and enalaprilat range between 200 and 1000 l blood [ 1217 ] . using the example validation of the low - volume bioanalytical method of enalapril and enalaprilat , pitfalls as well as improvements of the extraction protocol were shown . the aim of an accurate and precise low - volume method encompassing a broad calibration range with very low limits of quantification was only gained by complex extraction protocols . the calibration range of the established assay covers reported enalapril and enalaprilat concentrations in pediatric patients and proves its applicability for pediatric research . it was shown that the undertaken efforts for a sophisticated extraction protocol utilizing solid - phase extraction resulted in a high recovery and high reduction of matrix effect . controlling the latter amongst others warranted for a valuable and reliable bioanalytical method in serum . it allowed successful validation of a low - volume bioanalytical hplc - ms / ms method according the fda and ema bioanalytical guidelines . the applicability of the high - throughput approach was proven by a clinical study in 24 volunteers .

in usa and europe , medicines agencies force the development of child - appropriate medications and intend to increase the availability of information on the pediatric use . this asks for bioanalytical methods which are able to deal with small sample volumes as the trial - related blood lost is very restricted in children . broadly used hplc - ms / ms , being able to cope with small volumes , is susceptible to matrix effects . the latter restrains the precise drug quantification through , for example , causing signal suppression . sophisticated sample preparation and purification utilizing solid - phase extraction was applied to reduce and control matrix effects . a scale - up from vacuum manifold to positive pressure manifold was conducted to meet the demands of high - throughput within a clinical setting . faced challenges , advances , and experiences in solid - phase extraction are exemplarily presented on the basis of the bioanalytical method development and validation of low - volume samples ( 50 l serum ) . enalapril , enalaprilat , and benazepril served as sample drugs . the applied sample preparation and extraction successfully reduced the absolute and relative matrix effect to comply with international guidelines . recoveries ranged from 77 to 104% for enalapril and from 93 to 118% for enalaprilat . the bioanalytical method comprising sample extraction by solid - phase extraction was fully validated according to fda and ema bioanalytical guidelines and was used in a phase i study in 24 volunteers .

1. Introduction 2. Methods 3. Results and Discussion 4. Conclusion

PMC3015972

laparoscopic colon resection is superior to open surgery in regards to postoperative pain , recovery , and hospital stay . however , there are no standardized techniques , and data on technique - specific outcomes are lacking . various terms are used for laparoscopic colon surgery in the literature , for example , laparoscopic - assisted colectomy ( lac , usually with extracorporeal anastomosis ) , hand - assisted colectomy ( hac or hals ) , and laparoscopic colectomy with intracorporeal anastomosis ( lcia ) . additionally , there are various techniques for mobilization of the mesentery ( medial - to - lateral vs. lateral - to - medial ) and ligation of the vasculature ( extra- vs. intracorporeally ) . laparoscopic - assisted colectomy ( lac ) with creation of an extracorporeal ileocolonic anastomosis ( ea ) for right - or extended right colectomies remains the preferred approach in most centers . however , this technique limits the ability to choose an extraction site , which is usually a small midline incision . in addition , problems with intestinal alignment after extraction are known to occur . a completely intracorporeal anastomosis ( ia ) may reduce the likelihood or intestinal twists and offers the possibility of using any abdominal location for specimen extraction . the question of whether there is any advantage or disadvantage between these 2 techniques remains unanswered . the goal of this retrospective study was to evaluate the safety and feasibility of an intracorporeal anastomotic technique for laparoscopic right hemicolectomies . all patients requiring a right hemicolectomy for neoplasm who presented to city of hope from september 2004 to april 2008 were analyzed . data such as sex , age , body mass index ( bmi ) , pathology , operative technique , blood loss , operative times , intra- and postoperative complications , and length of stay were entered into a prospective database approved by the institutional review board . during the study period , 80 laparoscopic right hemicolectomies with either an intracorporeal ( ia , n=23 ) or extracorporeal ( ea , n=57 ) anastomosis were performed . a retrospective analysis of all data collected in regards to these 80 laparoscopic right hemicolectomies was performed . to ensure accuracy of the collected data , the 3 right hemicolectomies that were converted from a laparoscopic to an open approach were not included , as conversion rate was not an outcome parameter in this study . due to the retrospective nature of this study , an intention to treat analysis could not be performed . all cases with ia were performed by a single surgeon ( a. pigazzi ) starting in january 2006 with all his cases performed with an ia by june 2006 . all patients had preoperative colonoscopies with biopsy and tattooing of lesions located in areas other than the cecum . postoperative ileus was defined as abdominal distension requiring either conversion to an npo - status after a diet was started , placement of a nasogastric tube for decompression , or radiological imaging . pneumoperitoneum was created either via the percutaneous insertion of a veress needle or with the open hassan technique as per surgeon 's preference . four to 5 ports were used : a 10-mm to 12-mm umbilical camera port for a 30-degree laparoscope , one 10-mm working port for stapling devices in the left lower abdomen , and 2 to 3 five - mm working ports located in the left upper abdomen and suprapubic region . the mobilization of the right colon and mesentery was carried out in a medial - to - lateral fashion in most cases as previously described . in the ea group , the ileocolic pedicle and the right branch of the middle colic artery were divided close to their origin intracorporeally with a vascular endo - gia stapler or hemoclips in 26 patients . in 15 cases , the ileocolic pedicle was divided intracorporeally , whereas branches of the middle colic artery and the remaining mesentery were ligated after the colon was exteriorized . in the remaining 16 cases , the exteriorization of the colon and creation of the external anastomosis was carried out in most cases through extension of the umbilical port to a 4-cm to 8 cm midline incision after sufficient mobilization of the right colon and hepatic flexure . a side - to - side , stapled ileocolonic anastomosis was created in all cases with a stapled closure of the enterotomy in 91% of cases and a double - layer , hand - sewn closure in the remaining 9% . the anastomosis was re - evaluated in situ after closure of the incision and reinsertion of the laparo - scope in all cases . in the ia group , the ileocolic pedicle and after completion of the medial - to - lateral mobilization , the terminal ileum and transverse colon were divided intracorporeally with a 60-mm endo - gia stapler , blue load . in 21 patients , we used a pfannenstiel incision covered with a wound protector to retrieve the specimen . the specimen was always opened on the side table to ensure that the tumor or inked lesion was included in the resection . after closure of the incision and re - insufflation of the pneumoperitoneum , the intracorporeal ileocolonic anastomosis was created in a side - to - side , isoperistaltic fashion by using the 60-mm endo - gia . the enterotomy was then closed laparoscopically with a 2-layer , running suture with 3.0 vicryl . wound protectors were used in all 80 cases ; bags for specimen extraction were not used in the ia group . quantitative and categorical variables were analyzed with a 2-tailed , unpaired student t test and the chi - square or fisher exact probability test , respectively . all patients requiring a right hemicolectomy for neoplasm who presented to city of hope from september 2004 to april 2008 were analyzed . data such as sex , age , body mass index ( bmi ) , pathology , operative technique , blood loss , operative times , intra- and postoperative complications , and length of stay were entered into a prospective database approved by the institutional review board . during the study period , 80 laparoscopic right hemicolectomies with either an intracorporeal ( ia , n=23 ) or extracorporeal ( ea , n=57 ) anastomosis were performed . a retrospective analysis of all data collected in regards to these 80 laparoscopic right hemicolectomies was performed . to ensure accuracy of the collected data , the 3 right hemicolectomies that were converted from a laparoscopic to an open approach were not included , as conversion rate was not an outcome parameter in this study . due to the retrospective nature of this study , an intention to treat analysis could not be performed . all cases with ia were performed by a single surgeon ( a. pigazzi ) starting in january 2006 with all his cases performed with an ia by june 2006 . all patients had preoperative colonoscopies with biopsy and tattooing of lesions located in areas other than the cecum . postoperative ileus was defined as abdominal distension requiring either conversion to an npo - status after a diet was started , placement of a nasogastric tube for decompression , or radiological imaging . pneumoperitoneum was created either via the percutaneous insertion of a veress needle or with the open hassan technique as per surgeon 's preference . four to 5 ports were used : a 10-mm to 12-mm umbilical camera port for a 30-degree laparoscope , one 10-mm working port for stapling devices in the left lower abdomen , and 2 to 3 five - mm working ports located in the left upper abdomen and suprapubic region . the mobilization of the right colon and mesentery was carried out in a medial - to - lateral fashion in most cases as previously described . in the ea group , the ileocolic pedicle and the right branch of the middle colic artery were divided close to their origin intracorporeally with a vascular endo - gia stapler or hemoclips in 26 patients . in 15 cases , the ileocolic pedicle was divided intracorporeally , whereas branches of the middle colic artery and the remaining mesentery were ligated after the colon was exteriorized . in the remaining 16 cases , the exteriorization of the colon and creation of the external anastomosis was carried out in most cases through extension of the umbilical port to a 4-cm to 8 cm midline incision after sufficient mobilization of the right colon and hepatic flexure . a side - to - side , stapled ileocolonic anastomosis was created in all cases with a stapled closure of the enterotomy in 91% of cases and a double - layer , hand - sewn closure in the remaining 9% . the anastomosis was re - evaluated in situ after closure of the incision and reinsertion of the laparo - scope in all cases . in the ia group , the ileocolic pedicle and after completion of the medial - to - lateral mobilization , the terminal ileum and transverse colon were divided intracorporeally with a 60-mm endo - gia stapler , blue load . in 21 patients , we used a pfannenstiel incision covered with a wound protector to retrieve the specimen . the specimen was always opened on the side table to ensure that the tumor or inked lesion was included in the resection . after closure of the incision and re - insufflation of the pneumoperitoneum , the intracorporeal ileocolonic anastomosis was created in a side - to - side , isoperistaltic fashion by using the 60-mm endo - gia . the enterotomy was then closed laparoscopically with a 2-layer , running suture with 3.0 vicryl . wound protectors were used in all 80 cases ; bags for specimen extraction were not used in the ia group . quantitative and categorical variables were analyzed with a 2-tailed , unpaired student t test and the chi - square or fisher exact probability test , respectively . demographic and pathologic data for the study cohort are listed in table 1 . in 57 patients , there was no statistically significant difference in operative time , estimated blood loss , number of nodes harvested , or length of hospital stay between the 2 groups . the length of the incision was significantly shorter in the ia group ( 4 cm vs. 5 cm , p=0.004 ) . overall , 17.5% ( 14/80 ) of patients had postoperative ileus ( table 3 ) . however , there was no difference in the incidence of ileus between the 2 groups ( 22% ia vs. 16% ea , p=0.75 ) . patient demographic data ia : intracorporeal anastomosis ; ea : extracorporeal anastomosis ; asa : american society of anesthesiology score . ia = intracorporeal anastomosis ; ea = extracorporeal anastomosis ; ns = not significant . in the ea group , 3 ( 5.3% ) patients had intraoperative complications that included 2 cases of twisted mesentery requiring intraoperative revision of the anastomosis , and one case of bleeding from the extracorporeally divided mesentery . one volvulus of the anastomosis caused a complete bowel obstruction requiring reoperation on postoperative day # 8 . one patient in the ea group experienced an anastomotic leak , which was treated with an end - ileostomy . overall , 4 ( 7% ) complications were directly related to the anastomosis in the ea group compared with one ( 4.3% ) in the ia group ( p=1.0 ) . the one anastomotic - related complication in the ia group included a leak from the anastomosis in a patient with a bmi ( body mass index ) of 37kg / m . overall , the major complication rate for all patients ( 2 anastomotic leaks , 1 reoperation for volvulus , and 1 death ) was 6.25% ( 5/80 ) with no statistically significant difference between groups ( table 3 ) . the only death in our series occurred in a patient from the ea group ( overall 30-day mortality 1.3% ) . this patient was initially discharged on postoperative day # 7 , but readmitted on postoperative day # 12 for acute abdomen . a significant amount of ischemic small bowel proximal to the anastomosis due to vascular compromise was found , and the patient developed multisystem organ failure . injury to the sma / smv during the dissection or forceful stretching of the mesentery may have caused this fatal bowel ischemia ; however , we can not conclude whether this was related to the anastomotic technique . one patient in the ia group was converted from an extracorporeal approach due to a short mesentery and a high bmi of 32 kg / m , but was counted in the ia group . late complications consisted of 4 ( 7% ) incisional hernias in the ea group , 2 ( 8.7% ) in the ia group , and 1 internal hernia requiring reoperation 6 months after surgery in the ea group . the 2 incisional hernias in the ia group occurred in the only 2 patients in whom the specimen was not removed through a pfannenstiel incision . the literature is limited comparing outcomes between the different surgical techniques in laparoscopic colon resections . bernstein at al compared laparoscopic - assisted versus completely laparoscopic colectomies and found no difference in the length of hospital stay or the duration of postoperative ileus . the series by franklin et al is the largest series comparing intracorporeal anastomosis for right colon resections ( n=82 ) with 10 cases with extracorporeal anastomosis . their intracorporeal approach was found to be safe and feasible with similar operative times and complication rates . these findings are confirmed by bergamaschi et al who recently described the short - term outcomes of 111 intracorporeal right colectomies . however , the most commonly applied technique for creation of an anastomosis after laparoscopic right colectomy remains an extracorporeal , stapled ileocolonic anastomosis . in this laparoscopic - assisted technique , some authors argue that , once mobilized , the right colon is a midline structure and can be easily exteriorized through a 4-cm to 6-cm midline incision that directly overlies the base of the ileocolic pedicle , allowing for easy proximal ligation . the limitations of this approach include poor exposure of the ileocolic pedicle in obese patients through a small incision as well as limitations in regards to the location of the incision . difficult exposure of the base of the mesentery could lead to compromise of a high mesenteric ligation necessary for optimal oncologic outcome . therefore , many series describe the technique of intracorporeal high - vessel ligation combined with an extracorporeal anastomosis . we did not see a difference in the number of lymph nodes in either group ; however , our numbers may be too low to detect any significant difference . the creation of the anastomosis in an obese patient may be facilitated by an internal approach , because this technique eliminates the need to exteriorize heavy mesentery and large specimens through a small incision in a thick abdominal wall . raftopoulos et al compared laparoscopic right hemicolectomies with intracorporeal anastomosis in the obese and nonobese patients and reported the same incision length , conversion rate , morbidity , and length of stay for thin and obese patients . this compares very favorably with other reports in the literature with conversion rates up to 39% and morbidity rates up to 52% for laparoscopic - assisted colectomies with extracorporeal anastomosis in obese patients . in our series , one patient with a bmi of 32 kg / m scheduled for an ea was found to have shortened , thick small bowel mesentery so that the terminal ileum could not be exteriorized adequately . he was therefore converted to an ia , thus avoiding conversion to an open procedure . this may suggest an advantage of the intracorporeal anastomosis in patients with short or very heavy mesentery . an additional benefit of the intracorporeal technique is the ability to remove the specimen through any type of incision . incisional hernia rate in laparoscopic colon surgery is described as high as 17% to 24% with a higher rate for midline versus off - midline incisions . in comparison , the pfannenstiel incision is known for excellent cosmetic results and rare incisional hernia rates of 0% to 2% . interestingly , the 2 incisional hernias in the ia group occurred in the only 2 patients in which the specimen was removed though a small midline incision . overall , 6 hernias occurred in 59 patients with midline incisions , which equals a hernia rate of 10.2% with the midline incision compared with 0% ( 0/21 ) with the pfannenstiel incision . therefore , our practice has been modified to only perform pfannenstiel incisions for specimen extraction after intracorporeal anastomosis . in the ea group , it appears that alignment of the mesentery or volvulus of the anastomosis itself can be a problem when an extracorporeal anastomosis is performed . in 2 patients in the ea group , the anastomosis had to be redone due to twisting of the mesentery , and in one patient a volvulus of the anastomosis was missed leading to reoperation for bowel obstruction . references in the literature in regards to this problem are scarce . senagore et al reported a 1.6% incidence of operative small bowel obstruction in their series of 70 laparoscopic - assisted right hemicolectomies . some opponents of laparoscopic colectomy with ia argue that the operative time is longer , especially because the ia approach requires laparoscopic suturing skills . in our series , the median operative time of 190 minutes for the ia group was not significantly different from the operative time of 180 minutes for the ea group . the 4 studies on intracorporeal anastomosis with right colectomies reported operative times from 120 minutes to 218 minutes comparing favorably to operative times of 85 minutes to 190 minutes recorded for laparoscopic - assisted colectomies with extracorporeal anastomosis . these early results show that an intracorporeal anastomosis with transabdominal extraction has similar outcomes compared with extracorporeal anastomosis for laparoscopic right colectomies . however , there appears to be a trend towards smaller incision length in the ia group compared with a trend of more anastomosis - related complications in the ea group . because our study is a retrospective analysis of a small number of cases , a larger prospective trial will be necessary to confirm these findings .

background : during laparoscopic right hemicolectomy , the anastomosis can be created intra- or extracorporeally . this study aimed to determine whether a difference exists in short - term outcomes between these techniques.methods:prospectively collected data of 80 consecutive patients who underwent laparoscopic right hemicolectomies since 2004 were reviewed retrospectively . an intracorporeal anastomosis was performed in 23 patients , an extracorporeal anastomosis in 57.results:there were no significant differences in median length of stay ( 4 days ) , number of removed lymph nodes , estimated blood loss , operative time ( 190 minutes intracorporeal vs. 180 minutes ) and postoperative ileus ( 22% intracorporeal vs. 16% ) . the incision length was significantly shorter in the intracorporeal group ( 4 cm vs. 5 cm ; p=0.004 ) . complications related to the anastomosis including twisting of the mesentery ( n=2 ) , anastomotic volvulus ( n=1 ) , or leak ( n=1 ) occurred in 4 patients in the extracorporeal group compared with one minor anastomotic leak in the intracorporeal group . major complication rates were similar between the 2 groups ( 4.3% intracorporeal vs. 5.3% extracorporeal).conclusion : the type of anastomosis does not influence short - term outcomes after laparoscopic right hemicolectomy . an intracorporeal anastomosis results in shorter incision length and may decrease wound - related complications .

INTRODUCTION MATERIALS AND METHODS Patients Surgical Technique Statistical Analysis RESULTS DISCUSSION CONCLUSION

PMC3773588

prostate - specific antigen ( psa ) is one of the most important tools for the early detection of prostate cancer . an elevated serum psa level is an indication to pursue additional diagnostic evaluation , including ultrasound - guided prostate biopsies , to rule out prostate cancer . however , the major drawback of serum psa testing is that psa is prostate - specific but not prostate - cancer - specific . various physiological and benign pathologic processes , including prostatitis , urinary retention , and ejaculation , can affect the serum psa level [ 3 - 8 ] . also , there are limited data on the effect of hospitalization on the serum psa level . serum psa levels measured at least 24 hours after admission in hospitalized patients have been shown to be decreased compared with those measured in the outpatient department before admission , which suggests that the lack of activity associated with bed rest during hospitalization might decrease serum psa values . however , there have been no reports comparing serum psa levels measured just after admission with those measured in the outpatient department before admission . if serum psa levels measured just after admission are decreased compared with those measured in the outpatient department , only the serum psa levels measured in the outpatient department should be considered for the serial monitoring of psa levels in patients with equivocal serum psa values . therefore , in this study , inpatient serum psa ( ipspsa ) values measured just after admission were compared with the preadmission outpatient serum psa ( opspsa ) values to investigate whether hospitalization influences serum psa values . transrectal ultrasound - guided needle biopsies of the prostate were performed for detecting prostate cancer in 2,017 patients between february 2001 and april 2011 at our institution . the indications for prostate biopsy were serum psa elevation or abnormal findings on a digital rectal examination or transrectal ultrasonography . young patients with serum psa elevation usually underwent prostatitis workup and were given oral antibiotic treatment if needed . the decision to biopsy was based on an elevated repeat psa level after a period of time and the patient 's preference despite being informed of the low risk of prostate cancer . among the total 2,017 patients , the patients who underwent prostate biopsies on an outpatient department basis , in whom preadmission opspsa was not measured at ajou university hospital , who had a pathologic diagnosis of prostate cancer , who had recent clinical evidence of acute urinary retention , or who had an interval of more than 1 month between opspsa and ipspsa were excluded . patients who had been taking 5-reductase inhibitors or phytotherapeutic agents within 6 months of biopsy were also excluded from the analysis . all other medications including -blockers were allowed to be taken until the day of biopsy , except for antiplatelet drugs and anticoagulants , which were instructed to be stopped or switched to short - acting drugs a few days before . finally , we retrospectively reviewed the data of 416 patients who were hospitalized for prostate biopsies ; whose opspsa and ipspsa values were measured in our hospital within 1 month of admission and just after admission , respectively ; and whose prostate biopsy results revealed no evidence of cancer . blood sampling for psa was always instructed to be taken before any procedures that could influence the serum psa level , such as digital rectal examination , prostatic massages , transrectal ultrasonography , and cystoscopy . five milliliters of blood was collected from an antecubital vein and was immediately sent to our hospital 's central laboratory . psa was measured in serum samples by using a microparticle enzyme immunoassay in an asym immunoassay analyzer ( abbott diagnostics , abbott park , il , usa ) until 2006 and afterwards by using an electrochemiluminescence immunoassay ( roche diagnostics gmbh , mannheim , germany ) in a modular e170 analyzer ( hitachi , ibaraki , japan ) . the patients were usually hospitalized at 17:00 to 18:00 in the afternoon for prostate biopsies . samples were obtained for blood tests , including psa , immediately after admission , and prostate biopsies were performed the next day after admission , irrespective of the serum psa level during hospitalization . the opspsa and ipspsa measurements were compared according to age , the presence or absence of chronic prostatitis in biopsy pathology , serum psa levels , and prostate size . statistical analysis was performed by using paired sample t - tests with spss ver . 16.0 ( spss inc . , the mean age of the 416 patients included in this study was 61.6 years ( range , 29 to 85 years ) . among all 416 patients , the mean interval between opspsa and ipspsa measurements was 22.2 days ( range , 3 to 30 days ) . the mean prostate size measured by transrectal ultrasonography was 53.63 ml ( range , 12.8 to 197.9 ml ) . among all 416 patients , the mean ipspsa levels ( 6.69 ng / ml ) were significantly lower than the opspsa levels ( 8.01 ng / ml , p<0.001 ) ( table 1 ) . when the patients were stratified according to age , mean ipspsa levels were significantly lower than opspsa levels in patients aged 40 to 59 years ( p<0.001 ) and 60 years ( p<0.001 ) . the mean ipspsa levels were lower than opspsa levels in patients aged 20 to 39 years , but not significantly so ( p=0.141 ) . when the patients were stratified according to the presence or absence of chronic prostatitis in biopsy pathology , mean ipspsa levels were significantly lower than opspsa levels , irrespective of the presence or absence of chronic prostatitis ( p<0.001 and p=0.001 , respectively ) . when the patients were stratified according to the serum psa level , mean ipspsa levels were significantly lower than opspsa levels in patients with psa of 4 to 9.9 ng / ml ( p<0.001 ) and 10 ng / ml ( p<0.001 ) . the mean ipspsa levels were lower than the opspsa levels in patients with psa < 4 ng / ml , but not significantly so ( p=0.116 ) . when the patients were stratified according to prostate size , mean ipspsa levels were significantly lower than opspsa levels , irrespective of prostate size ( table 1 ) . various physiologic and benign pathologic processes can lead to elevations of the serum psa level . these factors include nonmalignant diseases of the prostate , such as benign prostatic hyperplasia , acute and subacute prostatitis , prostatic ischemia , and infarction of the prostate . urinary retention and ejaculation can also lead to increased psa levels in the absence of prostatic diseases . in addition , physical manipulation of the prostate , such as prostatic massages , cystoscopy , or in rare cases , digital rectal examination , can also increase serum psa levels [ 3 - 8 ] . there have also been many studies of biological variations in serum psa levels [ 11 - 13 ] . a more recent survey of 27 published studies suggested that a single measurement of the serum psa level may not be sufficiently precise for screening and diagnosis of prostate cancer , because the mean biological variation of psa is 20% in the range of 0.1 to 20 ng / ml for men over 50 years . with regard to the daily variability in the serum psa level , mermall et al . reported a circadian variation of serum psa with a nadir value in the early morning and a peak value at midafternoon . by contrast , tekin et al . demonstrated a diurnal rhythm in psa with a peak value at 08:00 and a gradual decline throughout the day until midnight . however , many other studies have reported that the variations in serum psa values during a 24-hour period are random without any diurnal or circadian pattern [ 17 - 20 ] . therefore , it is unlikely that there is an optimal time point during a 24-hour period at which to measure psa , and blood samples for serum psa values can be drawn from patients at any time of day . there have also been a few reports about discrepancies between outpatient and inpatient serum psa values in the same man . stamey et al . reported that serum psa levels measured 24 hours after admission decreased by a mean of 18% with a maximum of 50% when compared with preadmission levels . this may be due to the lack of activity associated with bed rest during hospitalization , and physical exertion owing to ambulatory status could cause an increase in serum psa values . confirmed the findings of stamey et al . by showing a significant difference between outpatient values and inpatient values measured after a minimum of 24 hours of bed rest . however , they found that the stressful exercise of cardiac stress testing during hospitalization had no effect on serum psa values . . also found that the serum psa levels measured 72 hours after hospitalization significantly decreased by a median of 12% compared with the values obtained immediately before admission . although the reasons for this decrease in the serum psa level are uncertain , it is probable that the lack of activity compared with the preadmission ambulatory status could contribute to the decrease in serum psa levels , because blood samples were obtained 24 to 72 hours after admission in the above 3 studies . the effect of exercise on the serum psa level is controversial . whereas exercises such as bicycle riding increased the serum psa levels in some reports , exercises such as bicycle riding or high - altitude trekking did not affect the serum psa levels in others . in the most recent study by loprinzi and kohli , participants were 16% more likely to have an elevated serum psa level for every 1-hour increase in sedentary behavior , and were 18% less likely to have an elevated serum psa level for every 1-hour increase in light physical activity . therefore , those authors suggested that individuals who engage in more sedentary behavior and lower levels of light physical activity have higher serum psa values . in the current study , we obtained the blood samples for ipspsa testing just after admission and found that the ipspsa values were decreased compared with the preadmission levels . therefore , the effects of resting and sedentary behavior did not seem to influence the results . the reasons for the decrease in serum psa levels after hospitalization are still not certain . the decrease in ipspsa levels compared with opspsa levels , although not significant , in patients aged 20.39 years and with psa < 4 ng / ml might have been due to the small sample size of these subgroups compared with the other subgroups . psa variability may be related to analytical variation according to the methods of psa measurement , sample handling , and laboratory processing . therefore , we excluded patients in whom opspsa was not measured in our hospital to eliminate the effects of analytical variation . serum psa was measured by using two different immunoassay systems according to the period of psa testing in our hospital . however , because our focus of analysis was the comparison of opspsa and ipspsa in the same man with an interval between the two psa measurements of within 1 month , and opspsa and ipspsa in the same man were measured by using the same immunoassay system , there should be no analytical variation according to the laboratory processing . we also excluded patients with an interval of more than 1 month between opspsa and ipspsa measurements to minimize the possible effect of biological variation in serum psa levels . we also excluded patients who had a pathologic diagnosis of prostate cancer , because the serum psa level might be influenced during the time interval between the opspsa and ipspsa . in our previous report about short - term ( median interval , 29 days ; range , 7 to 90 days ) psa velocity before prostate biopsy in 670 patients with serum psa levels of 2.5 to 20 ng / ml , median short - term psa velocity ( ng / ml / mo ) , which was defined as ( [ ipspsa - opspsa]/interval [ days])30 , was significantly different between patients with benign histology and those with prostate cancer ( -0.70 and -0.20 , respectively ; p=0.021 ) . the factor of recent ejaculation , which can affect the serum psa level , was not analyzed in this study . another limitation of our study is that the time of blood sampling was different for opspsa and ipspsa . the blood sampling for the former was mostly done in the morning or early afternoon , whereas that for the latter was done in the late afternoon . as discussed earlier , the influence of the timing of psa measurement within a day is controversial . moreover , the steps following the blood sampling , i.e. , from the handling of samples until the analysis , were identical for both , excluding any possible influence from preanalytical differences . nevertheless , we believe that the ipspsa levels were decreased compared with opspsa levels ; thus , the hospitalization itself decreased the serum psa levels . therefore , serum psa values should be measured on an outpatient basis for the serial monitoring of psa . a larger prospective hospitalization decreases serum psa values compared with those measured on an outpatient basis in patients with benign prostatic diseases . therefore , serum psa values should be measured on an outpatient basis for the serial monitoring of psa levels .

purposeto investigate whether hospitalization influences serum prostate - specific antigen ( psa ) values.materials and methodstransrectal ultrasound - guided prostate biopsies were performed for detecting prostate cancer in 2,017 patients between february 2001 and april 2011 at ajou university hospital . of those patients , 416 patients who were hospitalized for prostate biopsies , whose serum psa values were measured at the outpatient department within 1 month of admission and also just after admission , and who had negative prostate biopsy results were included in the present study . we retrospectively reviewed the data of the 416 patients and compared the serum psa values measured in the outpatient department with those measured during hospitalization.resultsamong all 416 patients , the interval between the two psa measurements was 22.2 days ( range , 3 to 30 days ) and the prostate size measured by transrectal ultrasonography was 53.63 ml ( range , 12.8 to 197.9 ml ) . among all patients , mean serum psa levels measured during hospitalization were significantly lower than those measured in the outpatient department ( 6.69 ng / ml vs. 8.01 ng / ml , p<0.001 ) . when stratified according to age , the presence or absence of chronic prostatitis in the biopsy pathology , serum psa levels , and prostate size , the serum psa levels measured during hospitalization were significantly lower than those measured in the outpatient department in all subgroups , except in cases aged 20 to 39 years and those with psa < 4 ng / ml , in whom no significant differences were shown.conclusionshospitalization decreases serum psa values compared with those measured on an outpatient basis in patients with benign prostatic diseases . therefore , serum psa values should be checked on an outpatient basis for serial monitoring .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

PMC4306396

cardiovascular disease is a major and growing cause of morbidity , mortality , and disability worldwide . atherosclerosis , a chronic low - grade inflammatory condition , is the underlying cause of most heart attacks and strokes . the atherosclerotic process , which begins early and progresses over the life course , can be influenced by a number of traditional proinflammatory risk factors ( e.g. , obesity , physical inactivity , high fat diet , dyslipidemia , hypertension , diabetes , and smoking ) and their interactions with genetic susceptibility [ 2 , 3 ] . exposure to urban air pollution also has been strongly implicated in promoting atherosclerosis and adverse cardiovascular events . it has been estimated that urban air pollution may be responsible for as much as one - tenth of all global cvd - related deaths and total disability - adjusted life years [ 1 , 3 , 4 ] . traffic - related air pollution is a major contributor to poor air quality especially in urban centers with high vehicular traffic volume where it can be responsible for as much as 90% of outdoor pollutants present in the local airshed [ 3 , 5 ] . vehicular emissions are composed of a complex mixture of co , co2 , nox , particulate matter ( pm ) , volatile organic compounds ( e.g. , benzene , formaldehyde , and acetaldehyde ) , ozone , and other secondary byproducts ( e.g. , nitrates and inorganic and organic acids ) and other substances . the specific mixture of pollutants associated with mobile source emissions is influenced by fuel type ( gasoline , diesel ) and quality ( e.g. , high versus lower sulfur content ) , vehicle type ( heavy- or light - duty ) , vehicle age , operating and maintenance conditions , exhaust treatment , engine lubricants , wearing of mechanical gears , brake pads , and tires , and road dust present , among other factors [ 3 , 5 ] . steep spatial gradients exist for traffic - related pollutants such as ultrafine pm ( ufp ) , fine pm ( pm2.5 ) , coarse pm ( pm10 ) , co , and nox . the highest exposures occur 50100 meters from roadways but diminish to approximate background levels by 100400 meters depending on the type of pollutant and other factors ( e.g. , meteorological conditions , local topography , and other environmental characteristics ) [ 58 ] . the evidence from diverse toxicological and epidemiologic studies has linked pm , especially pm2.5 , with many of the adverse cardiovascular effects reported as associated with exposure to traffic emissions [ 5 , 6 ] . emerging evidence also suggests that ufp may also be important for reasons of its small particle size which allows for deep penetration into lung tissue , a large surface area which facilitates toxicant absorption , and its inflammatory chemical properties [ 5 , 6 ] . the evidence from toxicological and epidemiological studies suggests that exposure to pm and other urban air pollutants appears to promote the release of proinflammatory cytokines , reactive oxygen species , and/or endothelial injury and vascular remodeling indicators in adults [ 5 , 9 ] . it has been hypothesized that repeated inflammatory responses caused by long - term exposure to traffic - related pollutants may promote arterial remodeling and accelerate the atherosclerotic process [ 5 , 10 , 11 ] . some recent studies have linked long - term exposure to urban traffic pollution with increased arterial calcification , stiffness , and/or carotid intima - media thickness ( cimt ) in adults with or without preexisting cardiovascular disease or other chronic conditions [ 10 , 1215 ] but others did not [ 1618 ] . little is known about the potential atherosclerotic effects of traffic - related exposures in children . the special behavioral , anatomical , and physiological characteristics of children increase their potential vulnerability to the negative cardiovascular effects of traffic - related pollutants . this , along with their smaller lung volumes , higher baseline ventilation rates , tendency to mouth breathe , and other attributes , can expose children to greater pollutant loads compared to older individuals [ 19 , 20 ] . several authors have reported that higher exposure to urban air pollutants is associated with increased blood markers of oxidative stress , systemic inflammation , and endothelial dysfunction in children [ 5 , 2124 ] . however , only one published study has investigated whether long - term residence in close proximity to urban traffic promotes ultrasound - detectable endothelial remodeling in children . iannuzzi and associates reported that healthy schoolchildren aged 614 years ( n = 52 ) who resided 30300 meters from a major road in a small town on the amalfi coast of italy exhibited significantly increased carotid arterial stiffness but not thickness ( i.e. , cimt ) compared to those living further away , that is , 330730 and 7801450 meters . it is possible that long - term exposure of the children who lived closest to road traffic was sufficient for arterial stiffness to be detected but not enough for cimt . in addition , it is also possible that the small sample size ( n = 52 ) may have reduced the power of the study to detect potential between - group differences in cimt . the study of the potential effects of exposure to traffic - generated air pollutants in children is important line of inquiry for the reason that small but progressive increases in fatty streak deposition and arterial thickening over time can lead to earlier progression to clinical disease and premature mortality . the identification of proatherogenic environmental factors such as traffic - related pollution exposure can help to identify underlying mechanisms and target specific strategies to slow atherosclerosis progression and future adverse cardiovascular outcomes . evidence from such studies can also be used in support of changes in environmental health policy and regulations . the major objective of the present study was to investigate the hypothesis that living close to heavy urban traffic , a proxy measure for exposure to traffic pollutants , promotes arterial remodeling in healthy children that is detectable as increased cimt . in addition , we investigated whether close residential proximity to heavy urban traffic is associated with increased systemic inflammation as indicated by c - reactive protein and interleukin-6 ( il-6 ) serum levels . the study was conducted in the quito metropolitan district ( qmd ) , the ecuadorian capital city . this major urban center of 2.2 million residents is situated in a narrow valley between the eastern and western andes mountain chains and has an average elevation of 2850 meters ( range : 5004790 m ) above sea level . in the last 15 years annual concentrations of all major air pollutants are reported to exceed world health organization guidelines excepting ozone . motor vehicles are a major and growing source of air pollution in the qmd airshed . private vehicle ownership increased by 29% from 145 vehicles/1000 persons in 2002 to 187/1000 persons in 2008 with an estimated 30,000 additional vehicles added annually . approximately 8% of circulating buses , trucks , cars , and vans in the qmd are powered by diesel but , by volume , diesel accounts for approximately 35% of the fuel sold in quito for use in vehicles . the diesel premium ( 500 ppm ) and gasoline ( 2000 ppm ) sold in the qmd for motor vehicle fuel contain the highest sulfur content in latin america . mobile sources account for an estimated 98% of co , 76% of no2 , 67% of co2 , 53% of nox , and 46% of pm annual emissions in the qmd airshed . nearly one - fourth of qmd residents live within 100 meters of heavily traveled roads and streets . the present study was conducted in three low - income qmd urban neighborhood areas ( figure 1 ) . the three sites were selected based on expected differences concentrations of pm and other ambient air pollutants collected from qmd central air monitors during the 12 months prior to the start of the study [ dr . these were el camal ( most heavily polluted ) , cotocollao ( medium polluted ) , and los chillos ( least polluted ) . one public elementary school in the neighborhood areas was identified based on its close proximity to a qmd central air monitor ( 5 km ) and estimated air pollutant concentrations . the investigators first held meetings with school officials , teachers , students , and their parents to explain the purpose of the study and answer questions . a computerized random numbers list was generated to select ~100 child participants from each school site . prospective participants from the three elementary schools were eligible for study inclusion if they were aged 712 years , were healthy , and resided within five miles of a qmd central air monitor in the same neighborhood . they were excluded from participation if they had showed clinical evidence of or had a positive history for cardiovascular disease , dyslipidemia , diabetes , or other serious chronic or infectious conditions ( e.g. , tb , hiv / aids ) or lived in a home with indoor smokers or if another child from their household was already enrolled in the study . thirteen prospective child participants were excluded from the study because they showed evidence of a chronic disease ( n = 1 ) , had indoor smokers present in their household ( n = 6 ) , or had siblings already enrolled in the study ( n = 4 ) or if their parents declined to complete the informed consent process ( n = 2 ) . the study protocol was approved by the university of texas at el paso and the university of central ecuador biomedical ethics institutional review boards . verbal and written informed assent was obtained from the child participants and informed consent for their child to participate in the study was obtained from parents / guardians . trained interviewers who were native ( ecuadorian ) spanish speakers collected detailed data on child participants , household , and neighborhood characteristics during face - to - face interviews with participants parents / guardians . the sociodemographic characteristics data included child age , sex , ethnicity , parental occupation , marital status , residential history , monthly household and per capita income , and family size and composition . household and neighborhood environmental characteristics data included child residential history , home construction , household operation behaviors involving cooking , heating , trash burning , and other pollutant sources ( e.g. , candles , incense ) , home ventilation practices , household hobbies / recreational activities ( e.g. , woodworking , metal work , and plastic models ) , and any outdoor smoking behavior by household members . detailed data also were collected on the source and location of local neighborhood pollutant sources ( e.g. , street food stands , lpg depositories , gasoline stations , car repair shops , and factories ) . these focused on cardiovascular and other serious chronic ( e.g. , diabetes ) and infectious conditions ( e.g. , tb ) . data collected on child activity patterns included the timing , physical location , and type of indoor and outdoor activities . these were obtained by physical activity recalls administered to the child participants and their parents / guardians . weight , height , and midupper arm circumference measurements were obtained for each child participant . weight was measured to the nearest kilogram on a calibrated electronic balance ( detecto , in ) . a portable stadiometer ( seca , chino , ca ) midupper arm circumference was measured to the nearest centimeter with a semiflexible anthropometric measuring tape ( seca , chino , ca ) . a 12-hour fasting blood sample ( 7 ml ) was donated by the child participants for the purpose of measuring total cholesterol , hdl , triglyceride , and glucose with the modular p-800 chemistry analyzer ( roche diagnostics , in ) . a portion of the blood sample was also used to analyze high sensitivity c - reactive protein and il-6 ( roche diagnostics , in ) . all samples were analyzed in the biomedical research institute at the central university of ecuador medical campus . the cimt studies were performed using the micromaxx 3.4.1 high - resolution m - mode digital ultrasound portable system linked to a 510 mhz multifrequency high - resolution linear transducer ( sonosite , bothwell , wa ) . participants were placed in a supine position with the head extended and slightly rotated to the opposite side using a 45-degree head block . semiautomatic measurements of mean and maximum cimt were made using sonocalc version 4.1 edge detection software ( sonosite , bothwell , wa ) . thickness was assessed as both the mean and maximum of three predefined angles ( anterior , lateral , and posterior ) capturing the media - adventitia interface of the near and far arterial walls , 1 cm proximal to the bulb from both right and left carotid arteries . the pediatric cardiologist also performed the blood pressure measurements using a calibrated manual sphygmomanometer with adjustable pediatric cuff following the american heart association recommendations . three readings were taken ( 2-minute intervals ) and the average was recorded as mean systolic and diastolic blood pressure . these measurements were used to calculate mean arterial pressure ( map = [ ( 2 diastolic blood pressure ) + systolic blood pressure]/3 . we obtained data on the current address and residential history of each child participant from their parents . the unique residential addresses of participants were physically verified by the study team and subsequently geocoded into latitude and longitude coordinates . the major traffic arteries for each quito neighborhood zone were identified based on traffic volume studies collected by the qmd department of transportation and public works . google earth ( mountain view , ca ) was used to obtain x and y coordinates for longitude / latitude orientation and straight line distance ( in meters ) from each unique residence to the nearest major traffic artery in each zone . the first was residential proximity to the nearest heavily trafficked road , defined as having a daily volume of 10,000 vehicles / day . we performed sensitivity analyses for both cimt mean and cimt maximum to determine the optimum roadway distance cut - off point . based on the sensitivity analysis results , residential proximity in this study was defined as < 100 , 100199 , and 200 meters from the nearest heavily trafficked road ( 10,000 vehicles / day ) . we also calculated a distance - weighted traffic density ( dwtd ) value for each child participant . ( 230 m ) radius buffer around the residence of each participant and used a basic model to estimate motor vehicle exhaust dispersion from roadways . the dwtd model is based on the assumption that vehicular exhaust emissions follow a gaussian distribution in which 96% of pollutants emitted undergo dispersion in vertical and horizontal directions at a distance of approximately 500 feet ( 152.4 m ) from the center of the road . published studies have confirmed that dispersal of vehicle exhaust pollutants approximate the 500 ft , distance from roadways although exact dispersion distances varied by study site and pollutant type . in the dwtd model used for the study , d represents the shortest distance from the child participant 's residence to each major roadway within the buffer we constructed . y represents the value that was used in the model to weigh the vehicle flow obtained for each road within this area : ( 1)y=10.42exp0.5d/50020.42 . data on vehicle flow for the roadways of interest in the study were obtained from traffic volume studies conducted by the qmd department of transport and public works . historical data from qmd central air monitoring stations were used to obtain estimates on one - year and five - year annual mean pm2.5 and ozone background concentrations for each of the three study neighborhoods for the respective 12- and 60-month periods immediately prior to cimt measurement in the children . we were not able to calculate lifetime pm2.5 and ozone exposure due to the lack of reliable qmd central air monitor data prior to 2005 . since all child residences ( and schools ) were located within close distance ( i.e. , 5 kilometers ) from one of the three qmd central monitors , we used these data from these to estimate annual residential exposure . we estimated childhood background exposures corresponding to the prior five - year period ( 20052010 ) by averaging exposures across the relevant residential histories for those time periods . we used published qmd central air monitor station data to estimate annual mean pm2.5 and ozone background concentrations for the study children . in the event that they had lived in multiple residences during the exposure periods of interest , time was split by weighting the location - specific exposure estimates according to duration of residence spent at that location . historical data on background annual pm2.5 for 20052010 were available from qmd central air monitors for the el camal and cotocollao but not the los chillos neighborhoods . however , annual pm10 data were available for los chillos and cotocollao but not el camal . to address this challenge , it was decided to estimate annual background pm2.5 for the los chillos site using the pm2.5/pm10 ratio method in which we used a factor of 0.57 ( mean annual pm2.5/pm10 ratio reported for qmd for 20052010 ) . data were not available on co , nox , or so4 from all three central air monitoring sites and there was no way to estimate these so we did not include these background pollutants in the analyses . initial bivariate analyses were conducted to compare differences in proportions using or fisher 's exact test , as appropriate . these initial analyses analyzed mean differences using independent t - tests or one - way analysis of variance to examine between - group differences in continuous variables , as appropriate . generalized linear models were constructed to examine the association of residential distance to traffic and dwtd with child cimt mean and cimt maximum . potential confounders selected for a priori inclusion in the statistical models were those previously reported to be associated with cimt in healthy children ( i.e. , age , sex , bmi , and blood pressure ) or documented risk factors for atherosclerosis such as positive family cvd history , blood cholesterol , systemic inflammation ( hscrp , il-6 ) , neighborhood background air pollutants ( pm2.5 , o3 ) , and presence of an outside tobacco smoker living in the household . in addition , the analyses also adjusted for other exposure variables identified in the initial analyses which altered effect estimates by > 10% , that is , exterior residential window ventilation practices and time spent outdoors . of the 302 children who were initially enrolled in the study , 15 were excluded from the present analyses because they failed to show up for their first scheduled visit ( n = 3 ) or subsequent cimt ultrasound exams ( n = 12 ) . however , their sociodemographic characteristics were comparable to those of the 287 children who completed the study including age ( 8.6 1.4 yrs versus 8.9 1.4 yrs , p = 0.42 ) , male gender ( 66.7% versus 55.1% , p = 0.43 ) , ethnic minority ( 0% versus 3.8% , p = 0.49 ) , average time of residence in the current neighborhood ( 8.1 1.7 yrs versus 8.4 2.1 yrs , p = 0.6 ) , and monthly household per capita income ( $ 73 55 versus $ 83 49 , p = 0.5 ) . table 1 displays the selected sociodemographic , nutrition , and health characteristics of the child participants and their households stratified by residential distance to traffic . all 287 of the study children were from low - income , working class households and attended one of three local public elementary schools located in cotocollao ( 100 ; 34% ) , el camal ( 94 ; 32.8% ) , and los chillos ( 93 ; 32.4% ) . three - fourths of the child participants had a normal weight for age and sex ; one - fifth were overweight or obese . one - quarter had a positive family history for hypertension and 1520% had a positive family history for diabetes , hypercholesterolemia , or heart attack / stroke . as table 1 also shows , although the proportion of overweight / obesity was marginally higher among children who resided the furthest away from traffic ( > 200 meters ) , no other statistically significant between - group differences were identified regarding participant sociodemographic and health - related characteristics including crp and il-6 . the same general associations shown in table 1 for residential proximity to traffic were also identified for the characteristics stratified by dwtd tertile ( data not shown ) . all children had lived in their current qmd neighborhood during the past 12 months and most ( 87.8% ; 252 ) were lifetime residents of the same . another 8.4% ( 24 ) had previously resided in another qmd neighborhood and 3.8% ( 11 ) had lived in another city . forty - four percent of the children lived less than 100 meters from the nearest road with a daily traffic volume of 10,000 or more vehicles / day . as table 2 indicates , no statistically significant differences were identified among the three groups regarding the reported residential and individual exposure characteristics with two exceptions . the mean altitude of participant homes was 141 and 95 meters lower in los chillos compared to el camal and cotocollao , respectively . in addition , the daily vehicular volume was increased among children who resided 100199 meters from the nearest heavily trafficked road with > 10,000 vehicles / day compared to those who lived further away . table 2 also shows that the three residential distance groups were similar regarding the presence of air pollutant - emitting industries in their neighborhood and background concentrations of ozone . annual 1-year and 5-year pm2.5 background concentrations were higher among children who lived less than 200 meters from the nearest heavily trafficked road . table 3 shows the results of both the unadjusted and adjusted statistical models examining the association of the two traffic exposure indicators , residential distance to traffic and distance - weighted traffic density , with the cimt mean and maximum ultrasound measurements . the unadjusted analysis results revealed that close residential proximity to the nearest heavily trafficked road was positively associated with higher cimt mean and maximum measurements . specifically , children who lived less than 100 meters from traffic had respective average cimt mean and maximum measurements that were 1115% greater compared to those who resided 200 meters away from traffic . children living 100199 meters away also had greater mean and maximum cimt measurements compared to those residing 200 meters away although this difference was not statistically significant . children exposed to the highest dwtd tertile had respective cimt mean and maximum measurements that were increased by 12% and 10% compared to those living in the lowest tertile . those in the middle tertile also had greater average cimt mean and maximum values compared to those residing 200 meters away but the difference was not statistically significant ( table 3 ) . adjustment for covariates reported to influence cimt in healthy children , that is , age , sex , bmi , and blood pressure , cardiovascular risk factors , that is , total cholesterol and family cvd history , or systemic inflammation markers such as hscrp and il-6 ( model 1 ) resulted in only minimal changes in model estimates for both residential proximity to traffic and dwtd analyses . the final cimt mean and maximum models were adjusted for exposure variables ( table 3 ) . the exposure variables included 5-year neighborhood background annual pm2.5 and ozone , altitude of home residence , presence of an outside smoker in the home , time spent outdoors , and time that exterior windows were left open ( model 2 ) . the latter two variables were added to the model as preliminary analyses indicated that child participants who spent an average of 240 minutes / day outdoors ( e.g. , engaged in play , walking to and from school , and running errands ) had significantly increased average cimt mean ( 3.9 m 0.07 versus 3.7 m 0.07 ; f = 5.7 ; p = 0.017 ) and maximum measurements ( 5.4 m 0.06 versus 5.2 m 0.07 ; f = 5.8 ; p = 0.016 ) . likewise , children who lived in households where outside windows were open for an average of 60 minutes or more per day also exhibited significantly increased cimt mean ( 3.8 m 0.07 versus 3.4 m 0.08 ; f = 7.5 ; p = 0.007 ) and maximum measurements ( 5.3 m 0.07 versus 4.8 m 0.09 ; f = 8.7 ; p = 0.003 ) . however , the model estimates changed a little even with the addition of the exposure covariates ( table 3 ) . this study is the first to demonstrate that long - term residential proximity ( < 100 meters ) to highly trafficked roadways , a proxy measure for traffic - related pollution , promotes ultrasound - detectable arterial remodeling in children . our study findings are strengthened by factors such as the apparent dose - response effect of increasing traffic - related exposure and cimt , control for covariates reported to specifically influence cimt in healthy children and other documented atherosclerosis risk factors , and other individual , residential , and neighborhood exposures . the study adds to the growing body of evidence documenting the proatherogenic effects of human exposure to traffic - related air pollution . this is important because even small but progressive increases in arterial thickening that begins early in childhood due to air pollutant exposures could potentially lead to earlier progression to clinical atherosclerosis and its associated sequelae . an estimated 600 million urban inhabitants worldwide are currently exposed to high levels of traffic - related air pollution . a substantial proportion of these , including children , live within 100 meters of heavily trafficked major roadways in cities . the proportion of children and other exposed persons is expected to rise over the next several decades as the proportion of humans living in cities increases . only one other published study has reported on the relationship between long - term residential proximity to traffic and ultrasound - detectable vascular remodeling markers in children . different from our results , iannuzzi and colleagues reported that close residential proximity to traffic was not associated with significantly increased cimt thickness in similarly aged , healthy italian children . however , they did report that it was associated with another early , preclinical atherosclerosis marker which we did measure in our study , that is , increased stiffness of the carotid artery . increased arterial stiffness is reported to be detectable earlier than arterial thickness in children with cardiovascular risk factors . we did not measure arterial stiffness in the present study so we can not compare this result . although methodological differences ( i.e. , sample size , model covariates , distance proxy categories , and traffic distance categories ) may partially explain the discrepancy in findings , it also seems likely that differences in the level of exposure to traffic pollutants may also be responsible . our study was conducted in the quito metropolitan district ( qmd ) , a large major urban center with 2.2 million residents , whereas that of iannuzzi et al . took place in a small italian coastal town , vietri sul mare ( 8,600 residents ) . at the time of the study , the qmd had 414,788 circulating vehicles and the volume of traffic measured on major roadways near the residences of the study participants averaged 27,354 16,060 vehicles / day ( range : 10,00066,484 vehicles / day ) . we were unable to locate data on vehicular traffic for vietri sul mare but it seems likely that , due to its small size and location , the amount of traffic - related air pollution produced by traffic on the relatively small coastal roadway was several orders of magnitude lower compared to our major urban study site . it is also possible that the higher altitude of the ecuadorian study site ( 2820 m ) could have increased the exposure of children to traffic - related air pollutants compared to that of the italian location ( 240 m ) . at this altitude , combustion engines work less efficiently and emit more pollution due to reduced atmospheric oxygen content . in addition , children living at high altitude have higher baseline respiratory rates due to the lower oxygen content of the air which may have also increased their exposure to any traffic - related pollutants present . systemic inflammation is one of several complementary biological mechanisms hypothesized to explain the reported association between exposure to traffic - generated and other urban air pollutants , especially pm , and cardiovascular health indicators including atherosclerosis [ 4 , 5 , 11 ] . however , the findings from epidemiological studies are inconsistent regarding whether exposure to specific pm fractions and other common urban air pollutants is associated with increased systemic inflammation as measured by blood crp or il-6 [ 9 , 10 , 13 , 3946 ] . relatively few studies have investigated the association of specific residential indicators of exposure to traffic emissions with these two systemic inflammation indicators . in our study , close residential proximity to traffic or distance - weighted traffic density was not associated with either inflammation indicator . our results are consistent with several studies conducted in adults [ 45 , 47 ] and children [ 25 , 48 ] which reported a lack of association with crp or il-6 . however , they differ from findings from two other studies indicating that crp was positively associated with residential traffic proximity and density indicators in adults [ 49 , 50 ] , especially long - term residential proximity . the reason for the reported discrepancies among studies could be due to differences in the amount and length of exposure to traffic - related air pollution or other methodological or population differences related to age , bmi , and other attributes . it is also possible that since both crp and il-6 are nonspecific indicators of systemic inflammation , infections , injury , obesity or proinflammatory chronic conditions , or other sources of inflammation may have accentuated or masked the effects of traffic pollutant exposure . this is especially likely in our ecuadorian population where clinical and subclinical respiratory and gastrointestinal and other infections are common . the potential limitations of our study should be considered when interpreting its results . one of these is that the cross - sectional design of the study limits the ability to prove causation between traffic - related exposure and cimt . as in any epidemiological study however , since the study was specifically designed to investigate atherosclerosis risk factors , we were able to control for multiple potential health , exposure , and sociodemographic confounders although we did not control for residential exposure traffic noise . we used proxy indicators of exposures at child residences instead of individual measures of pm and other traffic - related pollutants reported to promote atherogenesis . one of the two proxies used in our study for exposure to traffic - related air pollution was straight line distance from participant residence to the nearest major traffic artery . however , residential distance to a major roadway is reported to be a useful proxy measure for long - term exposure to traffic - related pollutants when assessed on a very small scale such as in our study . in addition , we used a second proxy measure , distance - weighted traffic density ( dwtd ) , which combines data on residential proximity to traffic with vehicular traffic volume to estimate exposure to air pollutants . it is also possible that other unmeasured factors also could have affected residential exposure to traffic - related pollution levels including wind flow , side of street , precipitation , urban canyons , and other physical structures . moreover , assessment of residential exposure may not be an accurate reflection of individual exposure levels . however , we also controlled for child outdoor activity and outdoor air filtration into residential indoor spaces . in conclusion , the results of our study indicated that childhood exposure to traffic - generated air pollutants , as indicated by residential proximity and traffic density proxy markers , promotes atherogenesis in healthy elementary school age children . the present work provides support for the hypothesis that living close to heavily trafficked roadways is detrimental to cardiovascular health . prospective cohort studies are needed to confirm our findings and identify the major traffic pollutants responsible since traffic - related pollution is potentially modifiable through changes in urban planning policy , strengthening and enforcement of clean air laws , use of cleaner technology , and other methods .

chronic exposure to urban traffic pollution is documented to promote atherosclerosis in adults but little is known about its potential effects in children . our study examined the association of long - term exposure to traffic with carotid intima - media thickness ( cimt ) in 287 healthy children . residential proximity and distance - weighted traffic density ( dwtd ) were used as proximity markers for traffic - related air pollution exposure . the multivariable analyses revealed that children residing < 100 meters from the nearest heavily trafficked road had cimt mean and maximum measurements that were increased by 15% and 11% compared to those living 200 meters away ( p = 0.0001 ) . similar increases in cimt were identified for children in the highest versus lowest dwtd tertile . children who resided 100199 meters from traffic or in the middle dwtd tertile also exhibited increased cimt but these differences were not statistically significant . no statistically significant differences were identified between residential distance to traffic or dwtd and systemic inflammation indicators ( crp , il-6 ) . the study results suggest that exposure to urban traffic promotes arterial remodeling in children . this finding is important since even small increases in cimt over time can potentially lead to earlier progression to atherosclerosis . it is also important because traffic - related pollution is potentially modifiable .

1. Background 2. Methods 3. Results 4. Discussion

PMC2447722

signature genes are genes that are unique to a certain taxonomic clade , and are common throughout all its major subclades . the plausible evolutionary explanation for such a phylogenetic distribution is that the gene was invented in the ancestral lineage , and constituted an important innovation that possibly allowed the clade to radiate . the significance of signature genes in biological research is both functional and taxonomic ( 1 ) . signature genes contain a wealth of functional signals that are related to clade - specific processes , many of which remain to be described . furthermore , they hold a strong evolutionary signal that can be used to phylogenetically characterize a set of sequences , such as a metagenomics sample . the recent breakthroughs in nucleotide sequencing ( 2 ) disclose a wealth of environmental niches , each harboring an abundance of undiscovered ( microbial ) life . as the metagenomic sequencing of such niches accelerates ( 35 ) , one of the first challenges lies in the taxonomic characterization of the contributing organisms . traditionally , sequence analyses are the method of choice in taxonomy , and the first approaches to characterize metagenomic data on the basis of sequence have recently been seeing the light ( 69 ) . signature genes are based on gene content , an intermediate between genotype and phenotype ( 10,11 ) that gives a view on phylogeny that is complementary to sequence - based analyses ( 12 ) . for example , using a prototype of our new signature gene method ( 13 ) , we showed that the anaerobic ammonium oxidizing bacterium kuenenia stuttgartiensis is closely related to the chlamydiae , a finding that independently supported the traditional phylogenomic analysis based on a superalignment of 49 proteins . in the current article , we introduce signature , an interactive web server that allows a user to identify signature genes in a set of query sequences . the server first assigns each sequence to one of the orthologous groups from the string 7.0 database ( 14 ) using a sequence similarity search . the phylogenetic distribution of this orthologous group throughout the tree of life is then assessed to determine if the sequence is a signature gene . the server produces a list of taxonomic clades that share signature genes with the set of query sequences , a coverage score for each signature in that clade , and an insightful image of the tree of life , in which the clades are color coded based on the number of signature genes present . this allows the user to quickly see from which part(s ) of the taxonomy the query sequences likely originate . using 1-fold and k - fold cross - validation analyses we confirmed that 93% of all signature genes were re - assigned to their correct clade . the tool uses mrs ( 15 ) , a fast data retrieval system that also allows for blast searches . signature currently assigns 2000 unknown protein sequences to orthologous groups in about 10 h , and in 10 s if the sequences occur in the database . the first is a protein sequence database containing the proteomes of 373 completely sequenced organisms , in which every protein may or may not be assigned to an orthologous group of proteins ( og ) . the database presently incorporated in signature is string version 7.0 that contains 43 577 cogs , kogs and nogs ( 14 ) . the second type of information used by signature is a reference tree of life that defines the evolutionary relationships between the species . , signature provides either the tree of life used in string 7.0 ( consistent with the sequence database ) , or a recently published phylogenomic tree based on a superalignment of 31 universal protein families ( 16 ) . if the selected tree of life includes bootstrap values , the user can choose to set a bootstrap threshold , so that only the reliable nodes will be considered and the nodes with lower bootstrap value will be collapsed to form a multifurcating branch . typically , a user will have a set of sequences obtained from an unknown organism or environment and wants to know to which taxonomic clade(s ) the sample belongs . alternatively , the user will want to know whether a protein sequence has a signature status in any clade , and may thus represent a process particular for that clade . with either of these questions , the user will go to www.cmbi.ru.nl/signature , enter the amino acid sequences in the input field ( in fasta format ) and click go. signature stores the input sequences and the selected options for the blast ( 17 ) search and the og assignment , and then starts by assigning each input sequence to an orthologous group . if , by md5 checksum and a subsequent sequence identity check , the query sequence is identical to one of the proteins already in the database , it is directly assigned to the corresponding og(s ) . otherwise , the sequence is assigned according to a cognitor - like rule ( 18 ) if the majority of the top n blast hits belong to the same og ( this option can be adjusted ) . note that a query sequence will be assigned to multiple ogs if they occur as non - overlapping regions ( the maximum overlap between two regions can be adjusted , see figure 1 ) . the blast searches can be time - limiting , depending on the number and length of the query sequences . while running , signature displays an estimate of the duration until the search is finished , based on the running times of searches with a comparable size that were carried out previously . it is always safe to refresh the page for the most up - to - date results . signature currently identifies the orthologous groups in 2000 unknown protein sequences within 10 h ( depending on the length of the sequences ) , and in 10 s if the sequences occur in the database ( by checksum ) . as the sequence similarity searches are the time - limiting step , we are presently extending the tool with a much faster og assignment algorithm , making it feasible to analyse metagenomics scale data sets and dna data sets in six translated frames in a limited time . figure 1.assignment of a query sequence to ogs using the default og assignment rule ( 3 of first 5 ) . disparate regions are first parsed from all the significant blast hits ( max 30 ) if they overlap less than 35 amino acids ( all these parameters can be adjusted ) . then , each region is assigned to an og : region_1 is not assigned as only two of the first five blast hits belong to the same og , while region_2 is assigned , as at least three of the first five blast hits ( in that region ) belong to og_b . assignment of a query sequence to ogs using the default og assignment rule ( 3 of first 5 ) . disparate regions are first parsed from all the significant blast hits ( max 30 ) if they overlap less than 35 amino acids ( all these parameters can be adjusted ) . then , each region is assigned to an og : region_1 is not assigned as only two of the first five blast hits belong to the same og , while region_2 is assigned , as at least three of the first five blast hits ( in that region ) belong to og_b . once the query sequences have been automatically assigned to the appropriate ogs , signature permits the user to review these assignments and manually change them if desired . it is also possible to include additional ogs for the signature search in the second step . if the user prefers to rely on a personal og assignment tool , the first step may be bypassed by leaving the initial input field empty ( see previous paragraph ) , and entering a list of cogs , kogs or nogs here . upon clicking go , signature will assess for each og whether it is a signature for any clade in the selected tree of life . a signature og for a certain clade is defined as an og that occurs in every daughter lineage of that clade , but nowhere outside it [ figure 2 ( 1 ) ] . to minimize the number of false negatives , the basic signature definition does not require a signature og to be present in every species in the clade . we have developed a nested coverage score that indicates the level of species coverage of a signature og , taking into account potential asymmetrical taxon sampling [ see the figure 2 caption for an example ( 1 ) ] . using the coverage score cutoff , the user can direct signature to report only high - scoring signature ogs . figure 2.definition of signature ogs in a ( partially unresolved ) tree of life . for every species , presence ( 1 ) or absence ( 0 ) of three ogs is indicated . in this example , only og1 is a signature for clade a , as it is present in the daughter lineages a1 , a2 and a3 , but not in clade b. although og2 and og3 are present in more species within clade a , they are not a signature for clade a because og2 is not present in the daughter clade a1 and og3 is present outside of clade a. the coverage score of og1 as a signature for clade a is recursively calculated as the average of the scores in the daughter clades : [ ( 1/1 ) + ( 2/3 ) + ( 1/2)]/3 = 0.72 . definition of signature ogs in a ( partially unresolved ) tree of life . for every species , presence ( 1 ) or absence ( 0 ) of three ogs is indicated . in this example , only og1 is a signature for clade a , as it is present in the daughter lineages a1 , a2 and a3 , but not in clade b. although og2 and og3 are present in more species within clade a , they are not a signature for clade a because og2 is not present in the daughter clade a1 and og3 is present outside of clade a. the coverage score of og1 as a signature for clade a is recursively calculated as the average of the scores in the daughter clades : [ ( 1/1 ) + ( 2/3 ) + ( 1/2)]/3 = 0.72 . the first result of a signature search is a list of all the signature ogs contained in the set of query sequences , along with their coverage score . for each taxonomic clade with signature ogs , signature calculates a log - likelihood significance that is based on the expected number of signature ogs for that clade ( see significance score section below ) . furthermore , signature generates an insightful image of the selected tree of life , where the internal clade branches are color coded based on their number of signature ogs ( shades of green ) , and the species , the terminal leaves in the tree , are color coded based on the cumulative number of signature ogs contained in all the ancestor clades they belong to ( shades of red ) . thus , the user can easily assess which species are most closely related to the source of the query sequences ( figure 3 ) . it should be noted that signature can also handle situations where the sequences are derived from several species . figure 3.image of the tree of life that results from a signature search ( cropped to the archaeal clade ) . the internal clade branches are color coded based on the number of signature ogs they share with the query ( shades of green ) , the terminal species are color coded based on the sum of the signature ogs of all their parent clades ( shades of red ) . note that all species in one clade are colored in the same shade of red . this means that an individual species does not necessarily contain all , or even any , of the signature genes assigned to its parental clades , especially if many signatures were found with a low coverage score . sequences analysed are the ferroplasma acidarmanus type ii scaffolds obtained from dna sequencing of acid mine drainage [ see text ( 4 ) ] . image of the tree of life that results from a signature search ( cropped to the archaeal clade ) . the internal clade branches are color coded based on the number of signature ogs they share with the query ( shades of green ) , the terminal species are color coded based on the sum of the signature ogs of all their parent clades ( shades of red ) . note that all species in one clade are colored in the same shade of red . this means that an individual species does not necessarily contain all , or even any , of the signature genes assigned to its parental clades , especially if many signatures were found with a low coverage score . sequences analysed are the ferroplasma acidarmanus type ii scaffolds obtained from dna sequencing of acid mine drainage [ see text ( 4 ) ] . typically , a user will have a set of sequences obtained from an unknown organism or environment and wants to know to which taxonomic clade(s ) the sample belongs . alternatively , the user will want to know whether a protein sequence has a signature status in any clade , and may thus represent a process particular for that clade . with either of these questions , the user will go to www.cmbi.ru.nl/signature , enter the amino acid sequences in the input field ( in fasta format ) and click go. signature stores the input sequences and the selected options for the blast ( 17 ) search and the og assignment , and then starts by assigning each input sequence to an orthologous group . if , by md5 checksum and a subsequent sequence identity check , the query sequence is identical to one of the proteins already in the database , it is directly assigned to the corresponding og(s ) . otherwise , the sequence is assigned according to a cognitor - like rule ( 18 ) if the majority of the top n blast hits belong to the same og ( this option can be adjusted ) . note that a query sequence will be assigned to multiple ogs if they occur as non - overlapping regions ( the maximum overlap between two regions can be adjusted , see figure 1 ) . the blast searches can be time - limiting , depending on the number and length of the query sequences . while running , signature displays an estimate of the duration until the search is finished , based on the running times of searches with a comparable size that were carried out previously . it is always safe to refresh the page for the most up - to - date results . signature currently identifies the orthologous groups in 2000 unknown protein sequences within 10 h ( depending on the length of the sequences ) , and in 10 s if the sequences occur in the database ( by checksum ) . as the sequence similarity searches are the time - limiting step , we are presently extending the tool with a much faster og assignment algorithm , making it feasible to analyse metagenomics scale data sets and dna data sets in six translated frames in a limited time . figure 1.assignment of a query sequence to ogs using the default og assignment rule ( 3 of first 5 ) . disparate regions are first parsed from all the significant blast hits ( max 30 ) if they overlap less than 35 amino acids ( all these parameters can be adjusted ) . then , each region is assigned to an og : region_1 is not assigned as only two of the first five blast hits belong to the same og , while region_2 is assigned , as at least three of the first five blast hits ( in that region ) belong to og_b . assignment of a query sequence to ogs using the default og assignment rule ( 3 of first 5 ) . disparate regions are first parsed from all the significant blast hits ( max 30 ) if they overlap less than 35 amino acids ( all these parameters can be adjusted ) . then , each region is assigned to an og : region_1 is not assigned as only two of the first five blast hits belong to the same og , while region_2 is assigned , as at least three of the first five blast hits ( in that region ) belong to og_b . once the query sequences have been automatically assigned to the appropriate ogs , signature permits the user to review these assignments and manually change them if desired . it is also possible to include additional ogs for the signature search in the second step . if the user prefers to rely on a personal og assignment tool , the first step may be bypassed by leaving the initial input field empty ( see previous paragraph ) , and entering a list of cogs , kogs or nogs here . upon clicking go , signature will assess for each og whether it is a signature for any clade in the selected tree of life . a signature og for a certain clade is defined as an og that occurs in every daughter lineage of that clade , but nowhere outside it [ figure 2 ( 1 ) ] . to minimize the number of false negatives , the basic signature definition does not require a signature og to be present in every species in the clade . we have developed a nested coverage score that indicates the level of species coverage of a signature og , taking into account potential asymmetrical taxon sampling [ see the figure 2 caption for an example ( 1 ) ] . using the coverage score cutoff , the user can direct signature to report only high - scoring signature ogs . figure 2.definition of signature ogs in a ( partially unresolved ) tree of life . for every species , presence ( 1 ) or absence ( 0 ) of three ogs is indicated . in this example , only og1 is a signature for clade a , as it is present in the daughter lineages a1 , a2 and a3 , but not in clade b. although og2 and og3 are present in more species within clade a , they are not a signature for clade a because og2 is not present in the daughter clade a1 and og3 is present outside of clade a. the coverage score of og1 as a signature for clade a is recursively calculated as the average of the scores in the daughter clades : [ ( 1/1 ) + ( 2/3 ) + ( 1/2)]/3 = 0.72 . definition of signature ogs in a ( partially unresolved ) tree of life . for every species , presence ( 1 ) or absence ( 0 ) of three ogs is indicated . in this example , only og1 is a signature for clade a , as it is present in the daughter lineages a1 , a2 and a3 , but not in clade b. although og2 and og3 are present in more species within clade a , they are not a signature for clade a because og2 is not present in the daughter clade a1 and og3 is present outside of clade a. the coverage score of og1 as a signature for clade a is recursively calculated as the average of the scores in the daughter clades : [ ( 1/1 ) + ( 2/3 ) + ( 1/2)]/3 = 0.72 . the first result of a signature search is a list of all the signature ogs contained in the set of query sequences , along with their coverage score . for each taxonomic clade with signature ogs , signature calculates a log - likelihood significance that is based on the expected number of signature ogs for that clade ( see significance score section below ) . furthermore , signature generates an insightful image of the selected tree of life , where the internal clade branches are color coded based on their number of signature ogs ( shades of green ) , and the species , the terminal leaves in the tree , are color coded based on the cumulative number of signature ogs contained in all the ancestor clades they belong to ( shades of red ) . thus , the user can easily assess which species are most closely related to the source of the query sequences ( figure 3 ) . it should be noted that signature can also handle situations where the sequences are derived from several species . figure 3.image of the tree of life that results from a signature search ( cropped to the archaeal clade ) . the internal clade branches are color coded based on the number of signature ogs they share with the query ( shades of green ) , the terminal species are color coded based on the sum of the signature ogs of all their parent clades ( shades of red ) . note that all species in one clade are colored in the same shade of red . this means that an individual species does not necessarily contain all , or even any , of the signature genes assigned to its parental clades , especially if many signatures were found with a low coverage score . sequences analysed are the ferroplasma acidarmanus type ii scaffolds obtained from dna sequencing of acid mine drainage [ see text ( 4 ) ] . image of the tree of life that results from a signature search ( cropped to the archaeal clade ) . the internal clade branches are color coded based on the number of signature ogs they share with the query ( shades of green ) , the terminal species are color coded based on the sum of the signature ogs of all their parent clades ( shades of red ) . note that all species in one clade are colored in the same shade of red . this means that an individual species does not necessarily contain all , or even any , of the signature genes assigned to its parental clades , especially if many signatures were found with a low coverage score . sequences analysed are the ferroplasma acidarmanus type ii scaffolds obtained from dna sequencing of acid mine drainage [ see text ( 4 ) ] . to assess the significance of finding a set of signature genes for a certain clade within a sample , signature provides a significance score for each clade on the output page . this score is based on the number of expected signature ogs if all ogs were randomly distributed across the genomes . to do this , we composed 1000 randomized sets of genomes , taking care not to place the same og twice in one genome ( 1 ) , and calculated the expected number of signatures for each clade . in these randomizations , the number of signature ogs that we find for a clade depends on the number of daughter clades , the genome sizes of the species therein and on the genome sizes of the species outside the clade . to avoid having to compute the expected number of signature ogs by such randomizations at every signature search we start by calculating the expected the number of ogs present at least once in every daughter lineage , but not in any species outside the clade ( equation 1 ) . ( 1 ) the product runs over all n daughter lineages d of the clade . note that n = 2 in a completely resolved phylogeny , n 2 in a tree where nodes with low bootstrap support have been collapsed to a multifurcating branch . ogsd is the total number of different ogs in all species in daughter d ( union ) , ogsout is the number of different ogs outside the clade , and ogstotal is the number of different ogs in the whole tree . indeed , there is a good correlation between the analytically predicted e and the number of expected signature genes based on randomization ( figure 4 ) . as can be observed in the regression equation , the randomized expected value ( x ) increases faster than the analytical e ( y ) , due to other factors such as variations in genome sizes . thus , taking this correction factor of 0.8146 into account , we can calculate the expected number of signatures from the numbers of ogs present in the daughter lineages as 10 . this value is based on the total number of different ogs in the whole tree , and can be scaled to the number of query sequences by multiplying it with queries / ogstotal . from this expected number of signature ogs , we calculate the observed / expected ratio and present the significance in the signature search output page as the log odds of drawing the observed number of signature ogs ( observed ) from the total set of ogs in the tree , given the number of query sequences ( queries , equation 2 ) . ( 2 ) figure 4.correlation between analytically calculated e ( equation 1 ) and the expected number of signature ogs based on 1000 randomized sets of genomes . equation y = 0.8146x + 0.0526 is the formula for the linear regression line plotted in the figure . ( 16 ) tree of life as provided by signature , where clades with bootstrap values < 80% were collapsed ( 1 ) . the outlier at ( 2.70 ; 7.06 ) are the bacteria , a clade with very low bootstrap support for its earliest branches , leading to 13 daughter lineages and an underestimation of e. for the rest of the clades in this plot , we did not find a correlation between the number of daughter lineages and e ( data not shown ) . correlation between analytically calculated e ( equation 1 ) and the expected number of signature ogs based on 1000 randomized sets of genomes . equation y = 0.8146x + 0.0526 is the formula for the linear regression line plotted in the figure . ( 16 ) tree of life as provided by signature , where clades with bootstrap values < 80% were collapsed ( 1 ) . the outlier at ( 2.70 ; 7.06 ) are the bacteria , a clade with very low bootstrap support for its earliest branches , leading to 13 daughter lineages and an underestimation of e. for the rest of the clades in this plot , we did not find a correlation between the number of daughter lineages and e ( data not shown ) . we assessed the performance of signature by entering a set of sequences from the first metagenomic sample to be published , obtained from random shotgun sequencing of dna from a natural acidophilic biofilm ( 4 ) . from this sample this data set contained 1956 sequences with an average length of 267 amino acids . the sequence - similarity search and og assignment for these proteins took 9 h and 43 min with default parameters , detecting 974 unique ogs ( 1531 total ) in the data set . within 30 s , 196 of these 974 ogs were identified to be a signature for a clade in the default tree of life , highlighting thermoplasmatales as the closest relatives of the species in the metagenomic sample ( figure 3 ) . signature is a web server that identifies signature ogs within a set of query sequences for any clade in a selected or uploaded tree of life . a signature og is an orthologous group that is unique for a clade and is retained in all its daughter lineages ( figure 2 ) . several public databases allow users to download strain- , species- or genus - specific genes , or sets of genes or proteins shared by a chosen set of species ( 1921 ) . signature is unique in that it identifies the signatures in a set of query sequences , for any clade in an adjustable tree of life and places the query in a taxonomic context . signature ogs can provide important new functional insights , as they carry out functions that may characterize the clade . for example , if the sequences in a sample contain many signature genes for the thermoplasmatales and its subclades , then the sample is likely taken from a species in that clade . based on this idea , signature places the signature ogs it discovers in a taxonomic context and produces an insightful image of the tree of life where the branches and leaves are color coded based on the number of signature genes associated to them ( figure 3 ) . thus , the user can easily assess from which clade the sample was likely taken . to test the how well the signature method assigns species to the correct clade , we did cross - validation analyses ( signature provides the option of leaving species out of the analysis ) , in which we removed every species from the data set one by one , and identified the signatures among the ogs in the removed genome in a phylogeny that did not contain that genome ( 1 ) . each of the removed proteins was either a signature og for one of the ancestral nodes of the removed species ( true positive , tp ) , a signature for another node ( false positive , fp ) or not a signature . in that case , the og could have been a signature in the situation where no species were excluded ( false negative , fn ) or not ( true negative , tn ) . using these values , we computed sensitivity [ tp/(tp + fn ) ; 77.6% ] , specificity [ tn/(tn + fp ) ; 98.8% ] , precision [ tp/(tp + fp ) ; 93.1% ] and accuracy ( true / all ; 95.6% ) of the method . removing 10% , 20% or 30% of the species ( k - fold cross - validation ) when interpreting the results of signature , it is important to realize the issue of horizontal gene transfers [ hgts ( 22 ) ] . if a signature gene for a certain clade gets transferred to a distantly related organism , it loses its initial signature status , because it will be found outside its taxon in the new situation . an example of such a case might be og3 in figure 2 , which could have been transferred from a species in clade a to the rightmost species in clade b. in this case , after the hgt , og3 became a signature for the clade ab , as it is present in both its daughter lineages . a solution for this is provided by the coverage score threshold . whereas og3 would have had a coverage score of [ ( 1/1 ) + ( 3/3 ) + ( 2/2)]/3 = 1.00 in clade a before the hgt event , it now has a coverage score of { [ ( 1/1 ) + + ( 3/3 ) + ( 2/2)]/3 } + { [ ( 0/2 ) + ( 1/2 ) ] /2 } thus , by using a ( high ) coverage score threshold , which is provided as an option in the signature search input form , the user can filter out signature ogs with a low coverage score that might be the result of hgt . nevertheless , even without using a coverage score threshold , we find only 1% false positives and a precision of 93% in the k - fold cross validation analyses ( above ) . newly sequenced genomes might of course affect which genes are regarded as hgts , potentially leading to the loss or change of signatures . if hgt would be widespread , genome sequencing could reach a point where all genes have been transferred outside of the taxon for which they were previously a signature . to assess the severity of this problem , we tested the robustness of the set of signature genes against the addition of new genomes by randomly leaving out 10% , 20% or 30% of the species in the tree , and identifying the overlap between the signature ogs in the original set of species and in the subsampled tree . this mimics the situation where up to 30% of the species are newly added to the current tree . new signature genes : 2.0% , 5.1% and 5.9% , respectively [ average of 100 samples ( 1 ) ] . based on the results from these cross - validation experiments , we do not expect to lose many of the current signature genes in an extended species set , and the taxonomic characterization of a query will be consistent . we will continue to add new genomes to the database as they become available . summarizing , signature is a user - friendly web server that facilitates the identification of signature ogs within a set of query sequences . many parameters of the underlying method can be adjusted , providing a comprehensive platform for analyses related to clade - specific genes and processes , both in a functional and in a taxonomic context .

signature genes are genes that are unique to a taxonomic clade and are common within it . they contain a wealth of information about clade - specific processes and hold a strong evolutionary signal that can be used to phylogenetically characterize a set of sequences , such as a metagenomics sample . as signature genes are based on gene content , they provide a means to assess the taxonomic origin of a sequence sample that is complementary to sequence - based analyses . here , we introduce signature ( http://www.cmbi.ru.nl/signature ) , a web server that identifies the signature genes in a set of query sequences , and therewith phylogenetically characterizes it . the server produces a list of taxonomic clades that share signature genes with the set of query sequences , along with an insightful image of the tree of life , in which the clades are color coded based on the number of signature genes present . this allows the user to quickly see from which part(s ) of the taxonomy the query sequences likely originate .

INTRODUCTION DATA A TYPICAL SIGNATURE SEARCH Assigning query sequences to OGs Identifying signature OGs in the tree of life SIGNIFICANCE SCORE PERFORMANCE DISCUSSION

PMC4227742

lysine acetylation plays a critical role in the regulation of transcription , metabolism , and other central biological functions . acetylation of lysine residues can impact protein and genome function through multiple mechanisms , including physical relaxation of histone dna interactions , recruitment of bromodomain - containing effector proteins , covalent active - site modification , and regulation of protein stability . lysine acetylation is a dynamic ptm that represents an equilibrium between the activity of two opposing enzyme classes : lysine acetyltransferase ( kat ) enzymes , which impose the mark , and lysine deacetylases ( kdacs ) , which remove it . while kdacs have been extensively investigated as epigenetic drug targets , several analyses indicate kats can also drive cellular transformation and cancer progression in a tissue - specific manner . for example , fusion of the kat enzyme moz to tif2 is the primary genetic lesion associated with a subset of leukemias and imbues differentiated red blood cells with cancer stem cell - like properties . nonmutant kat activities can also support oncogenic gene expression programs , functioning as essential coactivators for transcription factors such as c - myc and e2a - pbx gene fusions in cancer . proteome - wide studies have revealed lysine acetylation is a prevalent ptm , rivaling phosphorylation in terms of substrate diversity with 4700 human acetylation sites identified to date . however , in contrast to the hundreds of known protein kinases , a recent phylogenetic analysis identified only 18 kats in the human genome . the majority of these canonical kats fall into four families : gcn5/pcaf , p300/cbp , myst , and ncoa , with the rest consisting of transcription factor - related and orphan ( sequence disparate ) kat activities ( supplementary figure s1 ) . kat family members demonstrate significant intrafamily but little interfamily sequence homology , hindering bioinformatics approaches to kat discovery and classification . the functional characterization of kats is also limited by their regulation by protein partners and ptms , factors that are difficult to recapitulate in vitro . furthermore , while individual kats have been shown to be susceptible to inhibition by small molecules and cellular acetyl - coa / coa ratio , methods for comparing the selectivity of these perturbations among multiple kats in parallel do not exist . thus , our ability to discover and characterize acetylation - mediated signaling would be greatly advanced by the development of new methods for the global analysis of kat activity in cellular contexts . chemoproteomic profiling provides a powerful alternative to traditional biochemical assays for measuring enzyme activity in complex biological settings . in this approach , also commonly referred to as activity - based protein profiling ( abpp ) , active - site probes for an enzyme class of interest are modified with chemical handles enabling detection or affinity enrichment . covalent labeling or enrichment of an enzyme by the affinity probe is then used as a proxy for enzyme activity . chemoproteomic probes for kdac enzymes have been used to discover novel kdac complexes and characterize inhibitor selectivity in cell lysates . similar approaches have also been pursued to study kat enzymes , utilizing electrophile - containing analogues of the coa cofactor . however , these probes have not been widely applied to profile kat activity , owing to the fact that most kats do not utilize mechanisms involving active - site nucleophiles . here we report a general strategy for chemoproteomic profiling of kat activity ( figure 1a ) . bisubstrate inhibitors targeting three phylogenetically distinct kat families were converted to clickable photoaffinity probes to enable kat labeling and detection . cofactor - based affinity probes quantitatively report on kat - inhibitor interactions , are applied to identify a previously unknown acyltransferase activity possessed by the canonical kat enzyme gcn5 , and report on kat activity in cell lysates . ms / ms profiling of probe targets led to the identification of two noncanonical kat enzymes , highlighting the existence of several orphan lysine acetyltransferases present in the human genome . in addition to providing insight into the global selectivity and sensitivity of coa - based chemical proteomic probes that will guide future applications , these studies demonstrate the ability of chemical tools for profiling kat activity to provide new insights into kats and their molecular interactions in complex biological contexts . we envisioned a general strategy for chemical profiling of kat activity based on combining molecular recognition elements from kat bisubstrate inhibitors with a clickable photoaffinity tag for covalent cross - linking and detection ( figure 1a ) . pioneered by cole and co - workers , kat bisubstrate inhibitors link coa with the -amino group of a lysine - containing peptide to form high affinity interactions with both the substrate and cofactor binding sites of kat enzymes . these molecules inhibit kat activity with nanomolar potencies , with selectivity for specific kats encoded by the sequence of the bisubstrate peptide . on the basis of literature precedent , we hypothesized that modification of the n - termini of kat bisubstrate inhibitors might be tolerated without a large loss in inhibitory potency . this provides a potential site for incorporation of the clickable photoaffinity element benzophenone - l - propargylglycine ( bpyne ; figure 1 ) , necessary for covalent labeling and detection . to test the scope of this approach , we synthesized a suite of kat probes based on bisubstrate scaffolds that have been shown to target three major families of kats : p300/cbp ( lys - coa - bpyne ; 1 ) , gcn5/pcaf ( h3k14-coa - bpyne ; 2 ) , and myst ( h4k16-coa - bpyne ; 3 ) ( figure 1 ; supplementary scheme s1 ) . kat probes 13 were constructed from bpyne - peptide - bromoacetamide precursors , synthesized on rink amide resin utilizing an orthogonal lys - dde protecting group and postcleavage hplc purification . nucleophilic displacement of bpyne - peptidyl - bromoacetamides , with commerical coa , followed by final hplc purification provided probes 13 on scales ( 1100 mol ) sufficient for biological evaluation ( supplementary scheme s1 ) . to test the affect of our structural modifications on molecular recognition of kat enzymes , we assayed probes 13 against recombinant p300 , pcaf , and mof ( myst1 ) and compared their inhibitory activity to that of non - bpyne - containing parent inhibitor scaffolds ( 46 ; supplementary figure s2 ) . assayed at a single concentration ( 1 m ) , probes 13 demonstrate inhibitory potencies and selectivities that closely mimic those of parent inhibitor scaffolds 46 ( supplementary figures s3 and s4 ) . dose - response analysis of lys - coa - bpyne ( 1 ) demonstrated an ic50 of 26.7 nm toward p300 ( 95% confidence interval [ ci95 ] = 11.7560.64 ) , within error of the inhibition by parent compound 4 ( ic50 = 34.5 nm , ci95 = 17.567.8 nm ; supplementary figure s5 ) . similar results are seen upon comparison of h3k14-coa - bpyne 2 and parent bisubstrate 5 . together , these results suggest the bpyne subunit has minimal effects on kat active - site recognition . next , we evaluated the utility of 13 as kat labeling reagents in vitro . kat probes 13 were incubated with purified recombinant kats , photoirradiated at 365 nm , and subjected to cu - catalyzed [ 3 + 2 ] cycloaddition ( click chemistry ) with a fluorescent azide tag . sds - page analysis of labeling reactions demonstrated dose - dependent fluorescent labeling of kats at probe concentrations between 1 and 10 m . notably , each probe showed sensitive detection of the kat enzyme family it was designed to target , i.e. , kat probe 1 reacted effectively with p300 ( p300/cbp ) , probe 2 with pcaf ( gcn5/pcaf ) , and probe 3 with mof ( myst ) ( figure 2a ) . fluorescent labeling required probe , photo - cross - linking , and cu ; omission of any single component abolished labeling ( supplementary figure s6 ) . kat probes 2 and 3 , which are 3 kda , caused a significant gel shift upon photoirradiation that was visible upon coomassie staining ( figure 2a , supplementary figure s6 ) . this property allowed us to quantify and optimize our photo - cross - linking conditions using gel densitometry . in our optimized photo - cross - linking protocol , we found that 3340% of pcaf was covalently labeled by 2 after 60 min of irradiation at 365 nm on ice . these conditions are consistent with the literature , and cross - linking yields were not found to increase with extended photoirradiation times . ( c ) relative labeling of pcaf by clickable ( left ) and fluorescent ( right ) photoaffinity probes at low ( 0.1 m ) and high ( 1 m ) probe concentrations . the alkyne handle of probes 13 was chosen for its minimal footprint and versatility toward conjugation of diverse azide reporters . however , it is not strictly necessary for ex vivo affinity profiling applications . in order to evaluate the utility of our bioorthogonal detection strategy , we compared the labeling of pcaf by clickable probe 2 with a fluorescent kat photoaffinity probe , tamra - h3k14-coa 7 ( figure 2b ) . both probes facilitate fluorescent detection of pcaf at high probe concentrations ( 1 m ) , suggesting fluorescent bisubstrates may be useful profiling agents for kat enzymes . however , clickable probe 2 exhibits visibly greater labeling than fluorescent probe 7 at low probe concentrations ( 0.1 m ; figure 2b ) . this suggests click chemistry detection strategies improve probe sensitivity , possibly by abrogating negative interactions of the fluorescent tamra reporter on probe - protein recognition that reduce photo - cross - linking . moving toward more complex settings , we assessed the specificity and selectivity of each kat probe ( 13 ) using a cocktail composed of p300 , pcaf , and the myst family acetyltransferase mof . specific probe labeling events were defined as those susceptible to competition by 50100 equiv of parent kat bisubstrate inhibitors 46 ( supplementary figure s7 ) , while selectivity refers to the subset of kat enzymes labeled by each probe . interestingly , each probe showed specific labeling of a unique subset of kat enzymes at 1 m , even in the presence of other kat superfamily members . for example , lys - coa - bpyne 1 showed strong labeling of p300 and pcaf but did not significantly label the myst family member mof ( figure 3 ) . the p300 signal was specifically competed in the presence of excess parent inhibitor , while a small portion of the pcaf signal remained , suggesting a combination of specific and nonspecific interactions between the 1-pcaf pair . h3k14-coa - bpyne 2 demonstrated a clear preference for specific labeling of pcaf and weaker , but detectable , labeling of mof ( figure 3/supplementary figure s8 ) . h4k16-coa - bpyne 3 most strongly labeled the myst family member mof and exhibited fainter labeling of p300 and pcaf ( figure 3 ) . in addition to its parent bisubstrate , the mof-3 interaction was competed by excess lys - coa ( 4 ) , which had no similar effect on the interaction of pcaf with 2 ( supplementary figure s8 ) . for all three probes , selectivities were found to be reduced at higher probe concentrations ( 10 m ) , where 13 exhibit considerable cross - reactivity ( supplementary figure s8 ) . given its combination of broad - spectrum reactivity and straightforward synthesis of probe and competitor , these findings suggest lys - coa - bpyne 1 may be the most well - suited of the probes for applications requiring a general chemoproteomic reporter of kat activity . in contrast , when applied at suitably low concentrations , peptidyl - kat probes 2 and 3 may be better suited for applications that require more selective labeling of specific kat families or as components of kat chemoproteomic probe cocktails designed to achieve broad superfamily coverage . selectivity of kat labeling by probes 13 ( 1 m ) assayed in a mixture of proteins from the p300/cbp , gcn5/pcaf , and myst family . specific labeling events show sensitivity to competition by parent bisubstrate inhibitors ( 46 ; 200 equiv ) . having demonstrated the ability of our probes to label three classes of kats , we next sought to investigate their ability to report on changes in kat activity resulting from exposure to diverse molecular stimuli . first we investigated their ability to report on the affinity and selectivity of small molecule inhibitors . co - incubation of pcaf with the known kat inhibitor garcinol decreased labeling by kat probe 2 in a dose - dependent manner ( figure 4 ) . quantification of fluorescent pcaf labeling by gel densitometry yielded an ic50 of 4.5 1.2 m for garcinol , consistent with the literature ic50 value of 5 m . similarly , labeling of p300 by lys - coa bpyne 1 was sensitive to inhibition by the small molecule inhibitor c646 ( supplementary figure s9 ) . ( a ) fluorescent and coomassie gels from pcaf - garcinol competition experiment . conditions : 2 ( 1 m ) , garcinol ( 0 , 0.08 , 0.16 , 0.33 , 0.63 , 1.26 , 2.5 , 3.75 , 5 , 10 , 20 , 40 m ) . ( b ) dose - response analysis of competitive labeling generated via gel densitometry analysis of fluorescent labeling by kat probe 2 . in addition to small molecule inhibition , recent studies have suggested kat activity may be sensitive to changes in cellular acyl - coa pools , providing a potential mechanism to link changes in the metabolic state of the cell to differential histone acylations and epigenetic control of gene expression . to test whether chemoproteomic probes could provide insight into these mechanisms , we investigated the ability of four different acyl - coas ( acetyl , propionyl , butyryl , and crotonyl - coa ) to compete with 2 for the active site occupancy of gcn5 , a kat whose activity has been proposed to be metabolically regulated ( figure 5 ) . we found that high concentrations of acetyl - coa efficiently competed labeling by probe 2 , consistent with its role as a universal kat cofactor . propionyl - coa also antagonized labeling , while butyryl - coa was a partial antagonist , and crotonyl - coa did not impede labeling ( figure 5b ) . while the ability of gcn5 to utilize propionyl- and butyrl - coa as cofactors has not been previously explored , our results are consistent with a previous biochemical analysis of human pcaf ( which has a highly homologous kat domain ) . these studies indicated a kinetic preference for acyl group donors of acetyl - coa ( kcat / km 92 s m ) butyrl - coa and that malonyl - coa was not utilized as a substrate . indeed , we confirmed gcn5 utilized propionyl- and butyrl - coa as substrates via lc conditions : 2 ( 10 m ) , acyl - coas ( 1000 m ) , garcinol ( 40 m ) . these data suggest competitive affinity profiling provides a useful approach to rapidly gain new insights into kat inhibitor and substrate selectivity . the activity of kat enzymes such as gcn5 and pcaf is ideally studied in cellular settings . commentary has suggested the failure of conventional high - throughput screening campaigns to yield selective kat inhibitors may be due to the inability of these screens to interrogate the ability of small molecules to disrupt native kats , which can exist as multiprotein complexes . therefore , methods to monitor kat activity directly from cell extracts are potentially valuable for next - generation inhibitor discovery efforts . overexpression extracts of epitope - labeled atac ( ada two - a containing ) complex constitute an advanced model system applied for the study of gcn5 family kats in their endogenous setting . we thus asked whether we could use chemoproteomic probe 2 to directly detect kat activity in these systems , without the need for prefractionation , affinity purification , or antibodies . accordingly , pcaf overexpression extracts from hek-293 cells were treated with 2 , followed by photo - cross - linking and click chemistry . weak but detectable labeling of a protein corresponding to the molecular weight of pcaf was observed by fluorescence ( figure 6a ) . notably , labeling was sensitive to competition by h3k14-coa , and immunoblotting confirmed comigration with pcaf . to further verify labeling , we subjected proteins labeled by 2 in pcaf overexpression extracts to click chemistry with biotin azide , followed by affinity purification , tryptic digest , and lc ms / ms protein identification . notably , pcaf peptides were specifically identified in overexpression extracts treated with 2 ( figure 6b ) , but not in extracts pretreated with excess competitor 5 or lysates derived from hek-293 cells transfected with an empty vector control ( figure 6a , bottom , supplementary table s1 ) . in addition to overexpression extracts , clickable photoaffinity probes 1 and 2 are capable of detecting p300 and pcaf , respectively , when spiked into hela cell proteomes ( supplementary figure s11 ) . we used this characteristic to assess the sensitivity of probe 2 and estimate a lower limit at which gcn5/pcaf family enzymes may be detected . this is especially relevant since our inability to enrich pcaf from empty vector transfected hek-293 lysates indicates expression level may be a limiting factor for kat identification . we found that probe 2 could detect as low as 2.5 pmols of pcaf in a standard gel - based experiment against a proteomic background ( figure 6b ) . these findings calibrate the ability of chemoproteomic probes to monitor kat activity in model systems and cell lysates and may be useful for the development of chemoproteomic approaches to screen for selective inhibitors of kats and kat - containing multiprotein complexes . ( b ) proteins identified in lc ms / ms experiments as targets of h3k14-coa - bpyne 2 in pcaf transfected hek-293 extracts . control = competitor treated lane , ev = extract derived from hek-293 cells transfected with empty vector . ( c ) limit of detection of recombinant pcaf spiked into hela cell proteome . conditions : 2 ( 1 m ) , proteome ( 7 g ) , recombinant pcaf ( 0 , 6.25 , 5 , 3.75 , 2.5 , 1.25 pmol ) . having demonstrated the utility of chemoproteomic probes for the targeted study of kat activity in cellular contexts , we last sought to explore their utility for the discovery of potentially novel kat activities directly from cancer cell proteomes . labeling of whole cell extracts by 13 demonstrated a distinct pattern of specific protein labeling events for each probe , with kat probe 1 targeting the largest number of proteins ( figure 7 ) . to identify the proteomic targets of broad - spectrum kat probe 1 , hela cells were lysed , photo - cross - linked in the presence of 1 , and subjected to click chemistry with biotin - azide followed by streptavidin enrichment , tryptic digest , and lc ms / ms analysis . we identified 16 proteins that were abundant ( > 5 spectral counts ) and showed > 5-fold preferential enrichment in the absence of competitor ( figure 7b , supplementary table s2 ) . peptides matching canonical kat p300 and cbp were not observed in our ms / ms data set , likely due to their low abundance in whole cell lysates ( confirmed by western blot ; supplementary figure s12 ) . of the 16 proteins , 2 were acetyltransferases ( green ) , 5 were coa - binding proteins related to primary metabolism ( red ) , with the remaining hits composed of highly abundant proteins and members of the proteasome regulatory complex . to validate lc ms / ms identifications , we performed probe labeling experiments of two overexpressed and purified targets of 1 : atp - citrate lyase ( acly ) , the highest abundance pulldown target of 1 , and n--acetyltransferase 50 ( naa50 ) , an acetyltransferase . both enzymes exhibited competitor - sensitive labeling , indicative of specific molecular recognition by kat probe 1 ( figure 7c ) . conserved domain analysis indicated that each protein specifically labeled by 1 either contains or is closely associated with a protein containing a binding site for an adenine nucleotide - containing cofactor ( coa , atp , or nad(p ) ; figure 7c ) . the off - target engagement of adenosine - binding proteins by kat probe 1 parallels the widespread reactivity that has been observed for atp- and gtp - containing chemical proteomic probes in biological settings . this is likely to be a general limitation of chemical proteomic probes incorporating adenine cofactor - based chemical scaffolds and highlights opportunities for design improvements as well as the requirement for orthogonal validation strategies to support chemical proteomic kat discovery efforts . cofactor - based affinity profiling of endogenous kat activity in a cancer cell proteome . ( a ) labeling of hela cell proteomes by kat probes 13 ( 10 m ) . specific labeling events show sensitivity to competition by parent bisubstrates ( 100 equiv ) . ( c ) affinity labeling of recombinant protein verifies acly and naa50 as targets of 1 . in our data nat10 is a noncanonical kat ( referred to here as orphan kats ) with a gnat - related fold that displays histone and microtubule acetyltransferase activity in cells . biologically , nat10 has been observed to play a role in the regulation of telomerase function and nuclear shape and was recently identified as a druggable target for the treatment of hutchinson gilford progeria syndrome . naa50 ( nat13 ) is the catalytic component of the nate acetyltransferase complex that is required for proper sister chromatid adhesion and chromatin condensation in vivo . the identification of naa50 as a target of lys - coa - bpyne 1 initially struck us as paradoxical , as this enzyme belongs to the n - terminal acetyltransferase ( nat ) family and has been shown to favor protein acetylation of n - terminal met residues . however , literature investigation revealed that , of the 7 nat catalytic subunits encoded in the human genome , naa50 is the only member to have biochemically characterized -lysine acetyltransferase activity , providing a molecular rationale for its targeting by 1 . the selective identification of naa50 by 1 suggests that chemoproteomic profiling may have applications in identifying new kat activities present in acetyltransferase families that are distinct in sequence from canonical kats . the proteomic identification of two orphan kat activities by affinity probe 1 led us to re - evaluate the lysine acetyltransferase literature and consider whether other kat activities were also missing from the list of 18 canonical human kats ( supplementary figure s1 ) . this analysis identified 14 proteins , including nat10 and naa50 , not in the list of 18 canonical kats for which evidence of lysine acetyltransferase activity has been observed . sequence alignment and similarity analyses were used to construct an expanded phylogenetic tree of acetyltransferase proteins , divided into canonical ( p300/cbp , gcn5/pcaf , myst , ncoa ) and orphan kats ( figure 8) . notably , many kats from the initial list of 18 ( atat1 , tf3c4 , elp3 ; supplementary figure s1 ) cluster more closely with orphan kats , indicative of greater sequence similarity . together , these proteins encompass the most comprehensive list of human kats assembled to date . however , we take care to point out that the experimental evidence supporting the activity of all 32 kats , canonical and orphan , ranges widely . in particular , this list contains two proteins ( oga / ncoat , an orphan kat , and src1 , a canonical kat ) for which conflicting observations of kat activity have been made . these discrepancies support the need for universal methodologies capable of directly assaying kat activity in endogenous cells , toward which our current study provides an initial step . in the meantime , our chemoproteomics - inspired census of kat activity provides fertile ground for functional investigation of orphan kat enzymes using traditional structural and biochemical approaches , studies that may facilitate a more complete understanding of lysine acetylation in living systems . expanded phylogenetic tree of kat enzymes , including canonical kats and orphan kat activities observed in this study or annotated in the literature . uniprot accession numbers and literature references for kat activity are provided in the supporting information ( table s3 ) . in summary , here we have described a suite of probes for cofactor - based affinity profiling of kat activity . we have defined key structural features necessary for the covalent labeling and detection of three families of kat enzymes and demonstrated the utility of these probes to monitor kat activity in settings ranging from purified enzymes to kat overexpression extracts to native proteomes . chemoproteomic probes of kat activity provided new insights into kat inhibitor and cofactor selectivity and highlighted the existence of several noncanonical orphan kat activities that may contribute to cellular acetylation signaling pathways . in addition to these advances , it is also important to call attention to the limitations of our initial study . for example , while we demonstrated the ability of kat affinity probes to report on pcaf activity in hek-293 overexpression extracts , we were unable to detect canonical kat enzymes such as cbp and gcn5 directly from hela proteomes . this may be due to the low abundance of these kats in whole cell lysates ( supplementary figure s13 ) or the low cross - linking yields of our clickable photoaffinity probes , which covalently label only 3340% of recombinant pcaf in vitro . this challenge may be addressed in future studies through scale - up , nuclear prefractionation , or utilization of multidimensional protein identification technology ( mudpit ) to increase lc ms / ms detection sensitivity . alternatively , chemical proteomic studies of kinase and kdac activity have shown that noncovalent affinity probe resins can enable enrichment of specific enzyme classes without the need for photo - cross - linking , providing another potential route for the analysis of low abundance kats . furthermore , while the activity of kat complexes have been shown to be preserved in cell extracts , these activities would be ideally studied in living cells and probes 13 are not cell - permeable . cell - penetrating peptides have been used to promote uptake of kat bisubstrate inhibitors , and similar approaches may facilitate live cell profiling of kat activity . these improvements will be important to expand the scope of chemical proteomic analyses of kat activity . regardless , the ability of our current suite of chemoproteomic probes to highlight an expanded landscape of catalytic lysine acetylation provides an example of the power of this approach as currently constituted and sets the stage for the development of chemoproteomic strategies to identify kat inhibitors and the functional characterization of canonical and orphan kat activities in cellular settings . recombinant pcaf , catalytic domain ( aa 492658 ) was obtained from cayman chemical . recombinant p300 , catalytic domain ( aa 12841673 ) and recombinant gcn5 , catalytic domain ( 497663 ) , were obtained from enzo . the plasmid encoding recombinant mof , catalytic domain ( aa 147449 ) was obtained from addgene . his - tagged recombinant mof was expressed in e. coli bl21 and purified via immobilized nickel affinity chromatography using standard conditions . sds - page was performed using bis - tris nupage gels ( 412% ) and mes running buffer in xcell surelock minicells ( invitrogen ) according to the manufacturer s instructions . sds - page fluorescence was visualized using an imagequant las4010 digitial imaging system ( ge healthcare ) . total protein content on sds - page gels was visualized by blue - silver coomassie stain , made according to the published procedure . separation - based assays for kat activity were performed on a labchip ez reader instrument ( perkinelmer ) kindly provided by dr . fluorescence assays for the kat enzyme mof were analyzed on a biotek synergy 2 ( biotek ) . recombinant kat activity was measured by electrophoretic mobility shift assay ( emsa ) as previously reported . this assay measures the separation of fitc - labeled kat substrate peptides ( histone h3 5 - 23 qtarkstggkaprkqlatk - ahx - fitc ; histone h4 1 - 19 sgrgkggkglgkggakrhr - ahx - fitc ) from their acetylated products following incubation with recombinant kat and acetyl - coa . p300 and pcaf assays were performed in 30 l of reaction buffer ( 50 mm hepes , ph 7.5 , 50 mm nacl , 2 mm edta , 0.05% tween 20 , 10 g / ml bsa ) with kat ( p300 [ 50 nm ] or pcaf [ 10 nm ] ) and fitc - peptide ( fitc - h4 for p300 ; fitc - h3 for pcaf ; 1 m ) . acetylation of fitc - h3/h4 peptide by p300 and pcaf was confirmed by lc reactions were plated in 384-well plates , allowed to equilibrate at room temperature for 10 min , and initiated by addition of acetyl - coa ( final concentration = 5 m ) . plates were then transferred to a lab - chip ez - reader at ambient temperature and analyzed by microfluidic electrophoresis . optimized separation conditions were downstream voltage of 400 v , upstream voltage of 2900 v , and a pressure of 2.0 psi for fitc - h3 and downstream voltage of 500 v , upstream voltage of 1500 v and a pressure of 2.0 psi for fitc - h4 . percent conversion is calculated by ratiometric measurement of substrate / product peak heights . percent activity represents the percent conversion of kat reactions treated with inhibitors 16 relative to untreated control kat reactions , measured in triplicate , and corrected for nonenzymatic acetylation . mof showed low activity toward fitc - h3/h4 peptide substrates and was monitored by fluorogenic kat assay using an unlabeled h4 substrate peptide as previously reported . dose - response analysis of p300 and pcaf inhibition by kat probes 1 and 2 and parent inhibitors 4 and 5 were performed in triplicate and analyzed by nonlinear least - squares regression fit to y = 100/(1 + 10(log ic50 purified kat enzymes ( 0.55 g ) or whole cell proteomes ( 20 g ) were incubated with kat probes 13 ( 1 m probe for recombinant labelings ; 10 m probe for proteomic labelings ) in pbs ( ph 7.0 ) for 1 h. control experiments to correct for nonspecific cross - linking were treated with 13 in the presence of 100 equiv of competitors 46 . following equilibration , samples were photo - cross - linked on ice for 1 h using a 365 nm uv light in a fb - uvxl-1000 uv cross - linker . probe labeling was detected by cu(i)-catalyzed [ 3 + 2 ] cycloaddition ( click chemistry ) . click reactions were initiated by sequential addition of tamra - azide 8 ( 100 m ; 5 mm stock solution in dmso , structure given in supplementary figure s15 ) , tcep ( 1 mm ; 100 mm stock in h2o ) , tris(benzyltriazolylmethyl)amine ligand ( tbta ; 100 m ; 1.7 mm stock in dmso / tert - butanol 1:4 ) , and cuso4 ( 1 mm ; 50 mm stock in h2o ) . samples were vortexed and incubated at room temperature for 1 h. cycloaddition reactions were quenched by addition of 5x sds - loading buffer ( strongly reducing ) and subjected to sds - page ( 22 l per well ) . excess probe fluorescence was removed by destaining in a solution of 50% meoh/40% h2o/10% acoh overnight . gels were then washed with water and fluorescently visualized using a imagequant las4010 ( ge healthcare ) with green led excitation ( max 520550 nm ) and a 575df20 filter . for kat probes 2 and 3 , a characteristic intense low molecular weight fluorescence signal 3 kda was observed ( corresponding to the fluorescently labeled kat probe [ 2 , 3 ] ) , indicative of a high - yielding click chemistry reaction . hela s3 cells ( atcc ; manassas va ) were cultured at 37 c under 5% co2 atmosphere in a culture medium of dmem supplemented with 10% fbs and glutamine . hek-293 cells were obtained from the nci tumor cell repository . for isolation of whole cell proteomes , hela cells were grown to 8090% confluency , washed 3 with ice - cold pbs , scraped , and pelleted by centrifugation ( 1400 g 3 min , 4 c ) . after removal of pbs cell pellets were stored at 76 c or immediately processed . cell pellets were resuspended in 12 ml of ice - cold pbs ( 1020 10 cells / ml ) and lysed by sonication ( qsonica xl2000 100 w sonicator , 3 10 s pulse , 50% power , 60 s between pulses ) . lysates were pelleted by centrifugation ( 14,000 g 30 min , 4 c ) and quantified on a qubit 2.0 fluorometer using a qubit protein assay kit . proteomes were diluted to 2 mg / ml and stored in 1 mg aliquots at 76 c until further processing . sds - page gels were transferred to nitrocellulose membranes ( novex , life technologies ) by electroblotting at 30 v for 1 h using a xcell ii blot module ( novex ) . membranes were blocked using startingblock ( pbs ) blocking buffer ( thermo scientific ) for 20 min and then incubated overnight at 4 c in a solution containing the primary antibody of interest ( anti - gcn5 , anti - cbp , cell signaling , 1:1000 dilution ) in the above blocking buffer with 0.05% tween 20 . the membranes were next washed with tbst buffer and incubated with a secondary hrp - conjugated antibody ( anti - rabbit igg , hrp - linked , cell signaling , 1:1000 dilution ) for 1.5 h at room temperature . the membranes were again washed with tbst , treated with chemiluminescence reagents ( western blot detection system , cell signaling ) for 1 min , and imaged for chemiluminescent signal using an imagequant las4010 digitial imaging system ( ge healthcare ) . whole cell proteomes were adjusted to a final protein concentration of 1 mg / ml and incubated with the indicated probe ( 1 or 2 ; 10 m ) for 1 h. hek-293 enrichments utilized 0.5 mg of proteome as starting material , while hela enrichments utilized 1 mg of proteome . control samples to correct for nonspecific cross - linking were preincubated with each probe s cognate competitor ( 4 or 5 ; 100 equiv ) . following equilibration , samples were split into 5 200 l aliquots and photo - cross - linked on ice for 1 h using a 365 nm uv light in a fb - uvxl-1000 uv cross - linker . cross - linked samples were then recombined and subjected to cu(i)-catalyzed [ 3 + 2 ] cycloaddition with tamra biotin - azide 9 ( supplementary figure s15 ) as previously described . final concentrations for click reactions were as follows : hela proteome ( 1 mg / ml in pbs ) , probe 1 ( 10 m ) , tamra biotin - azide ( 40 m ) , tcep ( 1 mm ) , tbta ( 100 m ) , tert - butanol ( 4.8% ) , and cuso4 ( 1 mm ) . samples were vortexed and incubated at room temperature for 1 h. ice - cold 4:1 meoh / chcl3 ( 2.5 ml ) was then added directly to the reaction mixture and mixed vigorously by vortexing . the biphasic solution was centrifuged ( 4000 g 20 min , 4 c ) , and protein precipitated at the interface as a solid disk . liquid layers were carefully discarded , and the resulting precipitate was resuspended in ice - cold 1:1 meoh / chcl3 ( 1 ml ) , sonicated on ice to resuspend , and repelleted by centrifugation ( 14,000 g 10 min , 4 c ) . the resulting cell pellet was air - dried to remove excess methanol and redissolved in 1.2% sds ( 1 ml ) using iterative cycles of heating ( 95 c ) and sonication . redissolved protein was allowed to cool to room temperature and added to 5 ml of pbs to give a final sds concentration of 0.2% . samples were then treated with 100 l of streptavidin - agarose resin ( prewashed 3 with 1 ml of pbs ) and rotated for 1 h at room temperature . streptavidin - agarose bound samples were then washed sequentially with 0.2% sds in pbs ( 3 10 ml ) and pbs ( 3 10 ml ) . samples were then prepared for on - bead digest by reduction with 10 mm tris(2-carboxyethyl)phosphine ( tcep ) and alkylation with 12 mm iodoacetamide . samples were diluted to 2 m urea with 50 mm tris - cl ph 8.0 ( 400 l total volume ) , followed by addition of trypsin and 2 mm cacl2 . after extraction , tryptic peptide samples were acidified to a final concentration of 5% formic acid and frozen at 80 c for lc tryptic peptides enriched by probe 1 were loaded onto a reverse phase capillary column and analyzed by lc separation in combination with tandem ms . peptides were eluted using a gradient of 542% over 40 min with the flow rate through the column set at 0.20 l / min . data was collected in a dual - pressure linear ion trap mass spectrometer ( thermofisher ltq velospro ) set in a data - dependent acquisition mode . the 15 most intense molecular ions in the ms scan were sequentially and dynamically selected for subsequent collision - induced dissociation ( cid ) using a normalized collision energy of 35% . tandem mass spectra were searched against uniprot h. sapiens protein database ( 01 - 13 release ) using sequest ( thermofisher ) . search parameters were fixed as follows : ( i ) enzyme specificity : trypsin ; ( ii ) variable modification : methionine oxidation and cysteine carbamidomethylation ; ( iii ) precursor mass tolerance 1.40 amu ; and ( iv ) fragment ion mass tolerance 0.5 amu . only those tryptic peptides with up to two missed cleavage sites meeting a specific sequest scoring criteria ( delta correlation ( cn ) 0.08 and charge state dependent cross correlation ( xcorr ) 1.9 for [ m + h ] , 2.2 for [ m + 2h ] , and 3.1 for [ m + 3h ] ) were considered as legitimate identifications . spectral count values depicted in figure 7 represent an average of two biological replicates . raw spectral counts for biological duplicates of probe - enriched and control experiments are provided in supplementary table s2 . a pairwise alignment was generated using clustal omega , and a phylogenic tree was constructed using the neighbor - joining method . all phylogenetic trees were displayed in hyperbolic space using hypertree , with branches of the tree designated by different colors and labeled by name where appropriate .

lysine acetyltransferases ( kats ) play a critical role in the regulation of gene expression , metabolism , and other key cellular functions . one shortcoming of traditional kat assays is their inability to study kat activity in complex settings , a limitation that hinders efforts at kat discovery , characterization , and inhibitor development . to address this challenge , here we describe a suite of cofactor - based affinity probes capable of profiling kat activity in biological contexts . conversion of kat bisubstrate inhibitors to clickable photoaffinity probes enables the selective covalent labeling of three phylogenetically distinct families of kat enzymes . cofactor - based affinity probes report on kat activity in cell lysates , where kats exist as multiprotein complexes . chemical affinity purification and unbiased lc ms / ms profiling highlights an expanded landscape of orphan lysine acetyltransferases present in the human genome and provides insight into the global selectivity and sensitivity of coa - based proteomic probes that will guide future applications . chemoproteomic profiling provides a powerful method to study the molecular interactions of kats in native contexts and will aid investigations into the role of kats in cell state and disease .

Introduction Results and Discussion Conclusion Experimental Details

PMC4155811

the role of the adipokine leptin in the regulation of energy balance , has been firmly established since its discovery almost twenty years ago . animal models lacking either the leptin gene ( ob / ob mice ) or the gene encoding for its membrane receptor ( db / db mice ) are extremely obese , hyperinsulinemic and insulin resistant . more importantly , it has been demonstrated that reestablishing leptin signaling in these mice normalizes body weight as well as all metabolic and endocrine alterations . soon after the discovery of this adipokine , it was therefore hypothesized that a reduction in leptin levels and/or an impairment in its secretion would be responsible also for human obesity . however , while leptin deficiency has indeed been shown to cause rare forms of severe human obesity , the majority of obese individuals show markedly increased plasma leptin concentrations , reflecting the greater amount of adipose tissue . this excess of circulating leptin results in a state of resistance , characterized by a reduced response to the hormone action . in fact , hyperleptinemia could be considered a marker of leptin resistance and is not only commonly observed in obese subjects , but also independently associated with insulin resistance and cardiovascular disease ( cvd ) in humans . interestingly , several studies have shown an independent relationship between high leptin and atherosclerosis , myocardial infarction , stroke , and coronary artery intima - media thickness , suggesting that high leptin levels are associated with increased cardiovascular risk . mechanicistically , it has been demonstrated that leptin has a role in several processes relevant to cardiovascular disease , including the regulation of arterial pressure and vascular function and that there is a significant cross- talk between leptin and insulin signaling pathways . these important mechanisms have been reviewed elsewhere , whereas in this review we will focus on the multi - faceted cross talk between leptin and the pro - inflammatory molecule c reactive protein ( crp ) . crp is an acute - phase protein produced mainly by hepatocytes , which belongs to the family of pentraxins and as such consists of five identical non - covalently linked subunits . crp concentration increases 4 to 6 hours after acute tissue injury or inflammation and declines rapidly with the resolution of the inflammatory process . conversely , low - grade chronic inflammation , a condition underlying insulin resistance and associated with cardiovascular disease and type 2 diabetes , produces minor elevations of crp in the 3- to 10-mg / l range . epidemiological evidence indicates that elevated crp levels predict the development of type 2 diabetes and glucose disorders [ 11 - 13 ] . in addition , several cross - sectional studies in nondiabetic subjects , in the general population or in individuals with impaired glucose tolerance ( igt)/impaired fasting glucose ( ifg ) have confirmed that acute - phase reactants , such as crp , are positively correlated with measures of insulin resistance , adiposity , and circulating triglyceride and negatively correlated with hdl cholesterol concentrations [ 14 - 18 ] . furthermore in vitro studies have shown that , in addition to being a sensitive marker of inflammation , crp has direct proinflammatory effects . in endothelial cells , crp decreases nitric oxide and prostacyclin release and increases the expression levels of monocyte chemoattractant protein-1 , interleukin-8 , and plasminogen activator inhibitor-1 . in monocyte - macrophages , crp induces tissue factor secretion , increases reactive oxygen species and proinflammatory cytokine release , promotes monocyte chemotaxis and adhesion , and increases oxidized low - density lipoprotein uptake . also , crp has been shown in vascular smooth muscle cells to increase inducible nitric oxide production , increase nf - kb and mitogen - activated protein kinase activities , and , most importantly , up - regulate angiotensin type-1 receptor resulting in increased reactive oxygen species and vascular smooth muscle cell proliferation [ 19 - 21 ] . more recently , it has also been reported that crp has a direct inhibitory effect on insulin signaling and action in a skeletal muscle cell model . increased plasma levels of both leptin and crp have been reported in a number of conditions , including obesity and inflammation , and have been linked to cardiovascular pathophysiological processes and increased cardiovascular risk [ 9,11 - 18,23 - 26 ] . several studies have demonstrated that a direct correlation exists between the concentrations of the two biomarkers ( table 1 ) . a first , cross - sectional study on 179 apparently healthy japanese male college students aged 18 to 22 reported that crp serum levels had a positive correlation with leptin levels ( r=0.28 , p<0.0002 ) , which was independent from body mass index ( bmi ) . this finding was confirmed by a subsequent study , carried out in a cohort of 100 healthy volunteers ( 48 men , and 52 women ) . in this study , it has been observed that leptin was independently associated with crp after adjustment for age , gender , bmi , waist - to - hip ratio , smoking , and alcohol consumption ( p<0.0007 ) . furthermore , a significant relationship between leptin and crp was reported in both sexes , when men ( r=0.55 , p<0.0001 ) and women ( r=0.61 , p<0.0001 ) were analyzed separately . importantly , the association between leptin and crp remained significant when the analysis was restricted to lean individuals ( bmi < 25 an independent association between leptin and crp levels ( coefficient=0.20 , p<0.0001 ) was found in a study carried out in 946 community - dwelling older subjects ( 398 men , 548 women ; age range 65 to 102 years ) . the independent association between leptin and crp in healthy individuals was also confirmed by a larger population - based study conducted at five health centers in finland comprising 1862 young adults ( 971 women ; 891 men ) aged 24 - 39 yr . this study reported that crp and leptin levels were significantly correlated ( r=0.47 , p<0.0001 for women ; r=0.46 , p<0.0001 for men ) . in multiple regression analyses including age , bmi , waist circumference , insulin , lipids , systolic and diastolic blood pressure , smoking status , and use of oral contraceptives in women , leptin proved to be the main determinant of crp in men ( p<0.0001 ) and the second most important determinant in women ( p<0.0001 ) . similar findings have been reported in obese individuals , even if the results from these studies indicate that , in this group of subjects , the relationship between leptin and crp may be a reflection of fat mass , since the correlation between the two variables was abolished when the data were adjusted for bmi . the independent correlation between leptin and crp levels has also been investigated in subjects with type 2 diabetes . in a study comprising 148 japanese subjects with type 2 diabetes , serum crp levels were positively correlated with leptin ( r=0.330 , p<0.001 ) , and leptin was shown , by multiple regression analysis , to be an independent predictor of crp concentration ( r=0.330 , p<0.001 ) , along with interleukin-6 ( il-6 ) and triglycerides plasma levels . similar results were reported by a study carried out in 150 recently ( 3 years ) diagnosed type 2 diabetic patients showing a significant association between serum crp and leptin ( =0.326 , p=0.01 ) , after adjustment for age and gender . notably , a study that analyzed data from the third national health and nutrition examination survey ( nhanes iii ) with a total of 6,251 participants , including 598 patients with type 2 diabetes , not only confirmed a significant correlation between leptin and crp levels ( spearman correlation =0.22 in men and =0.32 in women , both p<0.0001 ) , but demonstrated also that individuals with high levels of both leptin and crp were at higher risk of cvd than those with high levels of leptin or crp alone . furthermore , while leptin concentration remained independently associated with cvd after adjustment for crp , the reverse was not true , as elevated crp levels were no longer associated with cvd after adjustment for leptin . this observation might explain the suboptimal performance of crp as a biomarker of cardiovascular risk in some studies and suggest that the two markers should be considered in tandem when estimating cvd risk . this suggestion is supported by the results of a more recent study aimed to test the hypothesis that crp levels may modify the relationship of leptin concentration with coronary artery calcium ( cac ) , a measure of coronary atherosclerosis in individuals with excess adiposity . the authors examined 1,460 asymptomatic individuals from two community - based cross - sectional studies , the study of inherited risk of coronary atherosclerosis ( sirca ) and the penn diabetes heart study ( pdhs ) , coordinated at a single , university - based research center at the university of pennsylvania , and analyzed the interaction of log - transformed plasma leptin levels with higher crp levels . the association of plasma leptin with cac was modified by higher crp regardless of the cut - point used ( interaction term p values all < 0.01 in fully adjusted models ) , while no interaction with crp was observed in control analyses with adiponectin , bmi or waist circumference . the authors concluded that crp itself , or the inflammatory pathways that it captures , affects the relationship of circulating leptin concentration to the burden of underlying atherosclerosis in overweight and obese individuals , clinical conditions where chronic inflammation and leptin resistance coexist . leptin is produced by the adipose tissue , and adipocytes are also an important source of circulating inflammatory cytokines , such as il-6 , which in turn promote crp synthesis . however , leptin itself may be able to stimulate crp synthesis from the liver , as suggested by experiments performed in primary human hepatocytes . in fact when human hepatocytes were incubated with human leptin at physiological concentrations ( 1 - 16 nm ) for 24 h a dose - dependent accumulation of secreted crp in the culture medium was observed ; interestingly preincubation with a specific phosphatidylinositol 3-kinases ( pi3k ) inhibitor completely blocked this leptin action , suggesting that leptin - induced hepatic production of crp is a pi3k - dependent process . to exclude the possibility that they were observing a il-6 mediated effect , the authors incubated primary hepatocytes in the presence of il-6 at doses comparable to those observed in vivo in obese individuals and showed that , at these concentrations , il-6 was unable to promote crp synthesis subsequently , following the accumulating evidence indicating that crp can be produced not only from hepatocytes , but also from additional cell types , similar results have been obtained by two independent studies in endothelial cells . a first study showed that in human artery coronary endothelial cells ( haecs ) a dose dependent increase of crp levels was observed with increasing concentration of leptin ( 0 to 400 ng / ml ) . interestingly , the increase of crp expression was attenuated in the presence of anti - leptin receptor antibodies , indicating that a classical leptin signaling was mediating this leptin effect . a subsequent study confirmed these findings , showing that lower , and more physiological , leptin concentrations ( 5 - 10 ng / ml ) were able not only to induce crp synthesis , but also to promote its release in the culture medium . these in vitro data are supported by in vivo experiments showing that exogenous leptin administration is able to increase plasma crp concentration . in a first single - blind , 22-day , placebo / drug/ placebo study , six subjects received recombinant methionyl human leptin ( r - methuleptin ) at the dose of 0.3 mg / kilogram subcutaneously for 6 days . no demonstrable effect of leptin administration on energy metabolism was evident in this small group of never - obese individuals ; leptin treatment induced an elevation in crp , il-6 , and haptoglobin , even if the small number of patients studied made impossible to determine whether these changes were related to leptin itself , to the interaction of leptin with a preexisting inflammatory process ( e.g. , dental abscess reported in one subjects ) , or to mild subcutaneous inflammation at the site of injection ( e.g. , observed in two subjects ) . in a proof - of - concept study aimed to sought evidence for a proinflammatory role of leptin , pegylated human recombinant leptin ( peg - ob 80 mg ) was administered weekly by subcutaneous injections to 12 obese subjects undergoing diet - induced weight loss . the only proinflammatory molecule whose circulatory levels increased significantly upon peg - ob administration was plasma crp ( p<0.05 ) . in a subsequent randomized , placebo - controlled study , twenty healthy , young ( 18 - 35 yr old ) , normal - weight ( bmi=20 - 26 kg / m ) female volunteers underwent a 4-d fasting during which they received r - methuleptin at a dose sufficient to prevent the fasting - induced decline in leptin , thus maintaining physiological concentrations of the hormone . leptin administration increased crp levels ( 6.32.4 vs. 0.70.3 mg / liter ; p<0.04 in leptin - treated vs placebo - treated , respectively ) , and stimulated other inflammatory markers , including circulating p - selectin levels and platelet aggregation , as compared to placebo - treated fasting women . while the study was not specifically designed to discriminate between the direct and indirect effects of leptin , it is worth noting that changes in proinflammatory markers levels at the end of the study significantly correlated with the leptin levels achieved , but not with leptin - induced changes in endocrine and metabolic function . these results were however not replicated from other research groups [ 47 - 49 ] ; notably when r - methuleptin ( 20 mg / daily in two doses ) was administered for 16-weeks to 117 obese subjects with type 2 diabetes , no significant changes in crp levels nor in the levels of other cytokines ( soluble tumor necrosis factor- receptors , interleukin-10 , monocyte chemoattractant protein-1 , il-6 ) were observed as compared to placebo - treated matched obese diabetic subjects . taken together the results from these studies suggest that the ability of leptin to directly induce crp in vivo is highly dependent from the leptin sensitivity state and indeed the more profound changes were observed in a state of enhanced leptin sensitivity , while no effect was reported in obese leptin resistant individuals . it is also worth underlying that exogeneous administration of leptin may not provide an accurate picture of the actions of the adipokine in more physiological settings . the data reviewed above clearly demonstrate that circulating leptin and crp levels are linked by a regulatory loop in which leptin stimulates hepatic and vascular crp expression ( fig . interestingly , there are experimental data suggesting that a feedback inhibitory mechanism may exist with crp directly binding leptin and inhibiting its action ( fig . 2 ) . chen and colleagues reported the presence of five major serum leptin - interacting proteins ( slips ) in human blood , isolated by leptin - affinity chromatography and identified by mass spectrometry and immunochemical analysis . these five proteins were termed slip-1 , slip-2 , slip-3 , slip-4 and slip-5 , and had apparent molecular weights on silver - stained sds - page gels of 30 , 42 , 65 , 70 and 85 kda , respectively . all five human slips had rat counterparts , as passage of rat serum through the mouse leptin affinity column yielded proteins of similar molecular weights . slip-1 has been identified as human crp by excision from the sds - page gel and mass spectrometry analysis . the identity of both human and rat slip-1 was further confirmed by running the correspondent column eluates on sds - page , transferring the gels on nitrocellulose membrane and analyzing them with the appropriate human or rat anti - crp antibodies . direct interaction between crp and leptin was further demonstrated by immunoprecipitation assays , where purified human and rat crp proteins were premixed with recombinant human and mouse leptin , respectively , before addition of species - specific antibodies to leptin and immunoprecipitation . protein precipitates were then subjected to western blot assays using specific antibodies to crp and it was revealed that immunoprecipitation with antibodies to leptin pulled down crp from the leptin - crp mixtures . were unfortunately not confirmed by two other groups , despite the complementary well - designed experimental attempts to reproduce them , and were therefore judged to be experimental artifacts due to the known calcium - dependent binding of crp to the agarose matrix used . by contrast , in a sub - sequent study we were able to demonstrate a direct interaction of crp and leptin and to show that crp inhibits leptin action in both a cellular and an animal model . in this study , leptin was preincubated for 30 min with 0.9 g / ml human recombinant ( hr)crp followed by further incubation with either anti - crp or anti - leptin antibody ; the incubation mixture was then immuno - precipitated and immunoblotted with either anti - crp or leptin antibody . it was observed that the anti - leptin antibody was able to precipitate hrcrp from the complex , and that the anti - crp antibody coimmunoprecipitated leptin from the incubation mixture . next , to address whether the direct interaction between leptin and hrcrp might attenuate physiological functions of leptin , we preincubated leptin with increasing concen - trations of hrcrp and assessed the ability of leptin to stimulate amp - activated protein kinase ( ampk ) and its downstream target acetyl - coenzyme a carboxylase ( acc ) phosphorylation in haecs . pre - incubation of hrcrp with leptin impaired in a dose - dependent manner both ampk thr and acc ser phosphorylation induced by leptin alone . in addition , preincubation of hrcrp with leptin blocked both akt ser and endothelial no synthase ( enos ) ser phosphorylation induced by leptin alone ( fig . pre - incubation of hrcrp with leptin resulted also in a marked reduction in both no production and intracellular cgmp accumulation in response to leptin alone . similar results were obtained in vivo . when c57bl6j mice were treated with leptin alone , we observed a significant increase in ampk thr , acc ser , akt ser , and enos ser phospho - rylation in aorta lysates from these animals ; by contrast , prein - cubation with hrcrp significantly reduced this in vivo physio - logical function of leptin . notably , when a calcium chelant ( egta 10 mm ) was added to the mixture , the inhibitory effect of hrcrp on leptin signaling was blocked , indicating that leptin and crp interaction requires calcium . taken together , these data suggest that during the preincubation of hrcrp with leptin , the two molecules bind each other and are thus unavailable to further bind their respective membrane receptors . the concentration of hrcrp ( 0.9 g / ml ) required to inhibit leptin effect on haecs was within the ranges observed in individuals with cardiovascular disease or obesity , thus suggesting that these findings may be clinically meaningful . an additional level to the complexity of the interactions between crp and leptin is represented by the observation that genetic variants at the leptin receptor locus ( lepr ) are independently related to circulating crp levels . a linkage disequilibrium analysis of 71 single - nucleotide polymorphisms ( snps ) spanning the lepr locus in a cohort of 630 healthy caucasian individuals , revealed four haplotype blocks ; of these , the fourth block was significantly associated with crp levels ( r=0.022 , p=0.049 ) . the strongest effect was observed for the snp rs1805096 , with homozygous carriers of the major allele showing 32% higher crp levels than carriers of the minor allele ( p=0.011 ) . the association of the lepr locus with crp circulating levels was confirmed by a genome - wide study in which 336,108 snps have been evaluated among 6,345 apparently healthy women as potential determinants of plasma crp concentration . overall , seven loci associated with plasma crp at levels achieving genome - wide statistical significance have been identified , including loci in or near the crp gene itself , and the leptin receptor protein gene . notably , virtually identical results were observed when the analysis was restricted to the study participants with crp levels < 10 mg / l , characteristic of chronic inflammation . the data reviewed here demonstrate that a complex interplay links plasma leptin and plasma crp levels . clinical data underlie the importance of both markers in estimating cvd risk and strongly suggest that an additional value will be attained by assessing them in tandem , especially in clinical states , such as obesity , where chronically elevated crp levels and leptin resistance coexist [ 27 - 38 ] . molecular studies indicate that leptin is able to modulate crp expression levels , both indirectly , throughout its action on other proinflammatory molecules , such as il-6 , and directly promoting its hepatic and vascular production . in turn , crp may be able to regulate circulatory leptin bioavailability , as it has been demonstrated that in extracellular settings the two molecules coprecipitate [ 40 , 52 ] and that this interaction impairs leptin ability to bind its receptor and activate intracellular signaling . if confirmed by future in vivo studies in humans , these findings may highlight a potential link between conditions , such as leptin resistance and endothelial dysfunction , that may be amenable of pharmacological treatment targeted to the disruption of leptin - crp interaction . notably , as reported above , leptin induced - vascular no production is impaired in obesity / metabolic syndrome , conditions characterized by hyperleptinemia , higher plasma crp levels and leptin resistance [ 6 - 8 ] . therapies aimed to improve the beneficial effects of leptin by modulating its intracellular signal transduction might thus be helpful in prevention and treatment of leptin resistance - related cardiovascular pathologies . on the other hand , crp , in addition to being a marker of inflammation , has been shown to exert proatherogenic effects on endothelial and vascular smooth muscle cells [ 19 - 21 ] . pharmacological agents able to reduce crp production may therefore not only be useful to prevent the direct negative effects of crp , but also to increase leptin bioavailability . finally , several studies have demonstrated that weight loss and physical aerobic activity are associated with a decrease in crp and leptin levels as well as with improved endothelial function and decreased cvd risk [ 55 - 58 ] . these findings , in addition to confirming the link between leptin , crp levels and cvd , point to weight loss and physical activity as therapeutic approaches able to break the vicious circle feeding cvd pathogenesis .

increased plasma levels of both leptin and c reactive protein ( crp ) have been reported in a number of conditions , including obesity , and have been linked to cardiovascular pathophysiological processes and increased cardiovascular risk ; interestingly these two biomarkers appear to be able to reciprocally regulate their bioavailability , through complex mechanisms that have not been completely clarified yet . here we first review clinical evidence suggesting not only that the circulatory levels of crp and leptin show an independent correlation , but also that assessing them in tandem may result in an increased ability to predict cardiovascular disease . we summarize also molecular studies showing that leptin is able to promote crp production from hepatocytes and endothelial cells in vitro and discuss the studies addressing the possibility that in vivo leptin administration may be able to modulate plasma crp levels . furthermore , we describe two studies demonstrating that crp directly binds leptin in extra - cellular settings , thus impairing its biological actions . finally we report genetic evidence that common variations at the leptin receptor locus are associated with crp blood levels . overall , the data reviewed here show that the chronic elevation of crp observed in obese subjects may worsen leptin resistance , contributing to the pathogenesis of cardiovascular disease , and highlight a potential link between conditions , such as leptin resistance and endothelial dysfunction , that may be amenable of pharmacological treatment targeted to the disruption of leptin - crp interaction .

INTRODUCTION PLASMA LEPTIN AND CRP LEVELS SHOW A DIRECT CORRELATION MOLECULAR MECHANISMS LINKING LEPTIN AND CRP: LEPTIN IS ABLE TO REGULATE CRP EXPRESSION MOLECULAR MECHANISMS LINKING LEPTIN AND CRP: CRP MAY ABLE TO MODULATE LEPTIN ACTION MOLECULAR MECHANISMS LINKING LEPTIN AND CRP: GENETIC VARIANTS AT THE LEPTIN RECEPTOR LOCUS ARE ASSOCIATED WITH CRP PLASMA LEVELS CONCLUSIONS CONFLICT OF INTEREST

PMC4831892

since the formation of the fick equation , physiologists have been trying to further enhance the knowledge base of heart rate , stroke volume , a - vo2 , and their relationship to vo2 . when considering maximal cardiorespiratory values , maximal vo2 ( vo2max ) is reached when maximal heart rate ( hrmax ) , maximal a - vo2 ( a - vo2max ) , and maximal q ( qmax ) are reached ( 21 ) . since a plateau - effect of sv occurs at a level > 50% vo2max ( 30 ) , hr is what drives the value of q , given that maximal sv ( svmax ) remains constant . age is the primary factor related to a decrease in vo2max ( 30 , 31 , 38 , 42 ) . moreover , hrmax decreases with increasing age ( 33 , 34 , 38 , 42 ) . however , we may not always be able to measure hrmax or vo2max values directly , and rely upon hrmax regression equations ( mhres ) to estimate our hrmax . since the early work of robinson on the effects of age on maximal heart rate ( hrmax ) ( 33 ) , researchers have fashioned numerous linear mhres based on age ( 7 , 10 , 11 , 16 , 23 , 27 , 29 , 32 ) . in 1971 , fox et al . published the 220-age mhre ( 13 , 32 ) yet no statistical analysis backed the equation . in 2002 , robergs et al . exposed the precise mhre from a line of best fit , from which 220-age was derived by fox et al . ( 13 ) : 215.4 - 0.9147 age ( 32 ) . today , it is a common practice of athletes and scientists alike to incorporate apocryphal mhres in a generic manner that lacks scientific merit such as 220-age and 226-age ( 4 , 32 , 40 ) . another common problem is the failure to utilize mhres in accordance with the specifications from which they were derived . for example , generalizability of 220-age is lacking as it has been shown to over or under predict based on age ( 15 , 39 , 41 ) , smoking ( 41 ) , bodyweight ( 26 , 41 ) , and conditions such as mental retardation ( 12 ) . furthermore , empirical hrmax values may ( 19 ) or may not ( 10 , 15 , 39 ) vary between sexes , may ( 19 , 22 , 27 ) or may not ( 10 , 30 , 38 , 39 ) vary based on physical activity status , and may ( 24 ) or may not ( 39 ) vary based on testing protocol ( i.e. , treadmill stress test vs. cycle ergometer stress test ) , which may not always be taken into account when applying or creating mhres to predicted hrmax . in 2001 , tanaka et al . ( 39 ) reported a neutral mhre with respect to sex , physical activity status , and testing protocol for which no differences could be seen : hrmax = 208 - 0.7 age . other mhres published by londeree and moeschberger ( 24 ) ( hrmax = 206 0.7 age ) and gellish et al . ( 9 ) ( hrmax = 207 0.7 age ) resemble the mhre reported by tanaka ( 39 ) . furthermore , robergs and landwehr ( 32 ) , through regression analysis of 30 different mhres , reported the mhre of 208.754 0.734 age , which is also similar to that of tanaka et al . therefore , the research supporting 208 0.7 age has been well established despite the many mhres that exist within the scientific community . the current study focused on the ability of scientifically merited and unmerited mhres to predict hrmax based on sex and physical activity specifications . the purpose of this study was twofold : 1 ) to determine the effects of sex and training status on measured hrmax and 2 ) to determine the accuracy of three commonly used mhres ( e.g. 220 age , 226 age , and 208 0.7 age ) to predict hrmax for females and males , aerobically active and sedentary . we hypothesized that sex would have no effect on measured hrmax nor on comparisons made between measured and predicted values between each of the three commonly used mhres , i.e. hrmax = 220 - age , hrmax = 226 - age , and hrmax = 208 ( 0.7 age ) , when compared to their opposite sex counterparts . furthermore , we also hypothesized that there would not be a significant training effect on measured and estimated hrmax . all potential participants were screened for inclusion prior to testing . specifically , the screening included questions from part 4 of the international physical activity questionnaire ( ipaq ) : long last 7 days telephone format ( 8) as well as the physical activity readiness questionnaire ( par - q & you ) ( 1 ) . inclusion criteria for the sedentary and active participants included the following : body mass index ( bmi ) between 18.524.9 ( kg m ) , age of 1825 years , and demonstration of a sedentary lifestyle through ipaq or active running lifestyle . exclusion criteria for any participants consisted of the following : answering yes to any of the questions on the par - q & you questionnaire , diabetes , cancer , and/or any other disease that may have prevented them from exercising to maximal intensity , an eating disorder , abnormal menstrual cycle , currently pregnant , and the use of any medications that affected cardiac , neurological , musculoskeletal , or cognitive function . a total of 52 participants ( 15 aerobically active males , 9 sedentary males , 15 aerobically active females , and 13 sedentary females ) between the ages of 18 and 25 years participated in the study . week for <3 days week and < 8000 steps day over the course of one week ( 6 ) , for a minimum period of 6 months . aerobically active included participants that were engaged in running > 30 min day for 5 dayweek of moderate intensity , or > 20 min day for 3 day week of vigorous intensity ( 18 ) , for a minimum period of 6 months . moderate and vigorous intensity guidelines were established through the american college of sports medicine ( acsm ) and defined as bouts of physical activity lasting longer than ten minutes . those that fell between the two classifications were considered recreationally active and were not included in the study . data were collected in the center for exercise and health fitness research at the university of pittsburgh . following the participant s arrival in the laboratory , experimental procedures were explained and the subject signed an informed consent approved by the institutional review board of the university of pittsburgh . all subjects abstained from alcohol consumption , caffeine , and vigorous exercise for 24 hours and from food intake 3 hours prior to testing . the subjects were then fitted with a strap - on heart rate monitor ( polar electro . , kenpele , finland ) and instructed to be seated for 5 minutes to establish resting hr ( hrrest ) . the mouthpiece , attached to a rudolph model 2700 two - way non - rebreathing respiratory valve ( rudolph , model 2700 , kansas city , mo ) , was fitted comfortably within the subject s mouth to measure respiratory values through the parvo medics truemax 2400 respiratory metabolic analyzer ( truemax 2400 , parvo medics inc . , the subjects were then familiarized to the treadmill during a 5 minute warm - up period at a pace with which they were comfortable and did not allow their hr to be greater than 100 beats min . during this time , they were also given proper instruction on how to prevent injury . subjects performed a standard bruce maximal stress test ( 5 ) on a trackmaster motor driven treadmill ( fullvision inc . , model tmx425c , newton , ks ) . the test was volitionally terminated by the subject due to exhaustion . beginning at the third stage until completion , all subjects were given verbal statements of encouragement every 2060 seconds ( 2 ) . hrmax was defined as the highest hr value attained . during that period of time , vo2 and rer measured by the parvo medic s computer software approximately every 15 seconds was averaged to 30 second values . the vo2max and rer values at the end of the test were recorded . to determine that the subjects achieved a maximal cardiorespiratory effort , the following was required : vo2 < 2.1 ml kg min between stages indicative of a plateau and rer 1.1 . the authors realize such liberally set values to determine a plateau in vo2 and an rer may underestimate true maximal effects , but were deemed necessary for the sedentary group ( 20 ) . data analysis was performed in three stages : 1 ) descriptive statistics , 2 ) effect of sex and aerobic training status on measured hrmax , and 3 ) effect of sex , aerobic training status , and prediction equation on the prediction equation accuracy . prior to performing the statistical analysis , an exploratory data analysis was conducted to determine whether the statistical assumptions were fulfilled for the planned anovas . measures of central tendency , such as means , and measures of dispersion ( i.e. standard deviations and ranges ) were calculated for the measured heart rate and predicted heart rate variables . to screen for marked departures from normality , histograms of the dependent variables were examined along with skewness and kurtosis values . the statistical analyses were performed using spss 16.0 statistical software ( spss , inc . , first , a series of single factor anovas were performed to determine group differences between the following variables : age ( yrs ) , height ( m ) , mass ( kg ) , bmi ( kg m ) , total leisure walking time ( min week ) , total moderate running time ( min week ) , total vigorous running time ( min week ) , hrrest ( beats min ) , hrmax ( beats min ) , vo2max [ ( ml kg ) min ] , and rer . the four groups included active males , sedentary males , active females , and sedentary females . secondly , a two factor anova ( sex aerobic training status ) for measured hrmax was performed . for our third aim , a three factor ( sex aerobic training status prediction equation ) anova with repeated measures on the third factor was performed on the predicted hrmax data . prediction equation had three levels ( 220 - age , 226 - age , 208 - 0.7 age ) . the two dependent variables for this anova were signed residuals ( observed hrmax - predicted hrmax ) and unsigned residuals [ the absolute value of ( observed hrmax - predicted hrmax ) ] . the residual for each participant would be divided by the standard error of prediction for each participant , yielding a signed or unsigned t - score , depending on whether the signed or unsigned residual was used . the sex aerobic training status interaction was included in the model , as was the effects of the prediction equation , prediction equation sex , prediction equation aerobic training status , and prediction equation sex aerobic training status . if any interactions were significant , this indicated that the relative accuracy of the three prediction equations varied according to sex , aerobic training status , or the combination of sex and aerobic training status . all potential participants were screened for inclusion prior to testing . specifically , the screening included questions from part 4 of the international physical activity questionnaire ( ipaq ) : long last 7 days telephone format ( 8) as well as the physical activity readiness questionnaire ( par - q & you ) ( 1 ) . inclusion criteria for the sedentary and active participants included the following : body mass index ( bmi ) between 18.524.9 ( kg m ) , age of 1825 years , and demonstration of a sedentary lifestyle through ipaq or active running lifestyle . exclusion criteria for any participants consisted of the following : answering yes to any of the questions on the par - q & you questionnaire , diabetes , cancer , and/or any other disease that may have prevented them from exercising to maximal intensity , an eating disorder , abnormal menstrual cycle , currently pregnant , and the use of any medications that affected cardiac , neurological , musculoskeletal , or cognitive function . a total of 52 participants ( 15 aerobically active males , 9 sedentary males , 15 aerobically active females , and 13 sedentary females ) between the ages of 18 and 25 years participated in the study . week for <3 days week and < 8000 steps day over the course of one week ( 6 ) , for a minimum period of 6 months . aerobically active included participants that were engaged in running > 30 min day for 5 dayweek of moderate intensity , or > 20 min day for 3 day week of vigorous intensity ( 18 ) , for a minimum period of 6 months . moderate and vigorous intensity guidelines were established through the american college of sports medicine ( acsm ) and defined as bouts of physical activity lasting longer than ten minutes . those that fell between the two classifications were considered recreationally active and were not included in the study . data were collected in the center for exercise and health fitness research at the university of pittsburgh . following the participant s arrival in the laboratory , experimental procedures were explained and the subject signed an informed consent approved by the institutional review board of the university of pittsburgh . all subjects abstained from alcohol consumption , caffeine , and vigorous exercise for 24 hours and from food intake 3 hours prior to testing . the subjects were then fitted with a strap - on heart rate monitor ( polar electro . , kenpele , finland ) and instructed to be seated for 5 minutes to establish resting hr ( hrrest ) . the mouthpiece , attached to a rudolph model 2700 two - way non - rebreathing respiratory valve ( rudolph , model 2700 , kansas city , mo ) , was fitted comfortably within the subject s mouth to measure respiratory values through the parvo medics truemax 2400 respiratory metabolic analyzer ( truemax 2400 , parvo medics inc . , the subjects were then familiarized to the treadmill during a 5 minute warm - up period at a pace with which they were comfortable and did not allow their hr to be greater than 100 beats min . during this time , they were also given proper instruction on how to prevent injury . subjects performed a standard bruce maximal stress test ( 5 ) on a trackmaster motor driven treadmill ( fullvision inc . , model tmx425c , newton , ks ) . the test was volitionally terminated by the subject due to exhaustion . beginning at the third stage until completion , all subjects were given verbal statements of encouragement every 2060 seconds ( 2 ) . hrmax was defined as the highest hr value attained . during that period of time , hr continued to be recorded until a decline was seen . vo2 and rer measured by the parvo medic s computer software approximately every 15 seconds was averaged to 30 second values . the vo2max and rer values at the end of the test were recorded . to determine that the subjects achieved a maximal cardiorespiratory effort , the following was required : vo2 < 2.1 ml kg min between stages indicative of a plateau and rer 1.1 . the authors realize such liberally set values to determine a plateau in vo2 and an rer may underestimate true maximal effects , but were deemed necessary for the sedentary group ( 20 ) . data analysis was performed in three stages : 1 ) descriptive statistics , 2 ) effect of sex and aerobic training status on measured hrmax , and 3 ) effect of sex , aerobic training status , and prediction equation on the prediction equation accuracy . prior to performing the statistical analysis , an exploratory data analysis was conducted to determine whether the statistical assumptions were fulfilled for the planned anovas . measures of central tendency , such as means , and measures of dispersion ( i.e. standard deviations and ranges ) were calculated for the measured heart rate and predicted heart rate variables . to screen for marked departures from normality , histograms of the dependent variables were examined along with skewness and kurtosis values . the statistical analyses were performed using spss 16.0 statistical software ( spss , inc . , first , a series of single factor anovas were performed to determine group differences between the following variables : age ( yrs ) , height ( m ) , mass ( kg ) , bmi ( kg m ) , total leisure walking time ( min week ) , total moderate running time ( min week ) , total vigorous running time ( min week ) , hrrest ( beats min ) , hrmax ( beats min ) , vo2max [ ( ml kg ) min ] , and rer . the four groups included active males , sedentary males , active females , and sedentary females . secondly , a two factor anova ( sex aerobic training status ) for measured hrmax was performed . for our third aim , a three factor ( sex aerobic training status prediction equation ) anova with repeated measures on the third factor prediction equation had three levels ( 220 - age , 226 - age , 208 - 0.7 age ) . the two dependent variables for this anova were signed residuals ( observed hrmax - predicted hrmax ) and unsigned residuals [ the absolute value of ( observed hrmax - predicted hrmax ) ] . the residual for each participant would be divided by the standard error of prediction for each participant , yielding a signed or unsigned t - score , depending on whether the signed or unsigned residual was used . the sex aerobic training status interaction was included in the model , as was the effects of the prediction equation , prediction equation sex , prediction equation aerobic training status , and prediction equation sex aerobic training status . if any interactions were significant , this indicated that the relative accuracy of the three prediction equations varied according to sex , aerobic training status , or the combination of sex and aerobic training status . post hoc tests were done to follow significant interactions . to better describe the active and sedentary groups , the amount of walking , moderate running , and vigorous running performed by each subject was assessed with one - way anova ( see table 2 ) . examination of the distributions indicated that the assumption of normality was not met for the physical activity variables : total walking , moderate running , and vigorous running ( absolute value of skewness 1.5 ) . the square root transformation was applied to the total walking variable and the transformed data were approximately normal . one - way anova was applied to the transformed data which yielded significant results ( p = .002 ) ( see table 2 ) . two nonparametric tests ( mann - whitney u test ) for each variable assessed the differences between active males and females due to the extreme departure from normality for the variables moderate and vigorous running . the results were not significant in either the moderate ( p=.267 ) or vigorous activity levels ( p=.512 ) . one - way anova showed no statistical differences in age between the groups ( see table 1 ) but did demonstrate significant differences between the following variables : total walking ( after square root transformation ) , vigorous running , hrrest , hrmax , vo2max , and rer ( table 2 ) . comparisons also revealed that active and sedentary males had a significantly higher vo2max than the females . both active males and females demonstrated a larger vo2max than their sedentary counterparts signifying a difference between activity levels ( see table 2 ) . two - way anova found significance for sex and activity but not the sex by activity interaction ( see tables 3 ) . for the signed residuals , the males and sedentary participants for sex and activity level respectively demonstrated the least amount of variability with predictions when averaged over all three mhres ( see table 4 ) . equation hrmax = 208 ( 0.7 age ) ( equation 3 ) under predicted by 1.09 beats min whereas the other two equations over predicted by a greater margin ( table 4 ) when averaging all subjects data thereby disregarding sex and activity level . for sex males had the least amount of total error when averaged across the three mhres . when averaging all subjects data and disregarding sex and activity level , equation 3 had the least total error ( table 4 ) . however , once sex was taken into account ( see table 4 equation sex interaction ) , equation 1 and 3 had the least total error for the males and females respectively . when activity level was taken into account ( see table 4 equation activity level interaction ) , equation 3 was the most accurate for the active subjects . interestingly , for the sedentary group , both equation 1 and 3 seemed to have the same amount of accuracy in predicting observed hrmax . tukey post hoc tests indicated significance between equations 1 and 2 for males and between all pairs of equations for females . likewise , a significant difference was found between equations 1 and 2 for sedentary and between all pairs of equations for active . the specific aims of the study were to determine whether there was an effect of sex and/or training status with observed hrmax and if there was a significant difference between three popular mhres versus observed hrmax when sex and training status were taken into account . for the first purpose of the study , activity level and sex affected hrmax independently from one another . though the physiological responses of the heart were not directly measured in the current study , lower hrmax values were demonstrated in active participants suggesting a training effect in our sample . however , such data are controversial as spina et al . demonstrated a decrease in hrmax as a direct result of training ( 37 ) , others noted lower hrmax values with active participants ( 22 , 25 ) , and some have shown no effect in hrmax between either active or sedentary participant ( 10 , 30 , 38 , 39 ) . likewise , a significantly higher hrmax is seen in males , indicating a sex effect , which is also conflicting . hermansen and andersen ( 19 ) , suggest sedentary females have highest hrmax , based on averages , not significance , while more studies claim no significance ( 10 , 15 , 39 ) . lester et al . ( 22 ) utilized cross - sectional data to show an indirect relationship with age for both aerobically active and sedentary males having identical slopes but different intercepts . such results demonstrate the sedentary to be at a greater disadvantage in regard to a greater blunted hrmax with increasing age in opposition to the aerobically active . the results of the current study have been recorded while controlling for age . within our study , only speculation could account for the hrmax response that led to significance within the aerobically active subjects such as increased parasympathetic response ( 9 ) , reduced baroreflex sensitivity due to decreased baroreceptor density ( 36 ) , increase in left ventricular wall thickness ( 16 ) , increase in peak filling rates of blood into the heart ( 23 ) , increased stroke volume ( 28 ) , among other parameters not measured . a decrease in hrmax as a result of training is inconsistent among athletes ( 9 ) and , therefore , such significance may be the result of randomly aerobically active subjects fitting such a profile . literature may suggest a carry - over effect from hrrest to hrmax , thereby establishing cause for lower hrmax seen among physically active . however , whaley et al . ( 41 ) implied a lower hrrest relating to a lower hrmax from three studies whose data never supports such a conclusion ( 3 , 17 , 35 ) . though hrrest may have been measured among such studies the resting values were never reported among sedentary and active females ( 3 ) or active males ( 17 ) , nor emphasized , though measured , among healthy sedentary females ( 35 ) . grimby and saltin ( 17 ) did note a stark contrast in hrmax ( 203 and 148 beats / min ) between two males of near similar vo2max ( l / min ) and blood volume ( l / min ) and corresponding submaximal hr values at the same workload ( 155 and 120 beats / min respectively ) . a training effect can not explain such results as all the males that took part in the study were considered to be aerobically active . nevertheless the concept of a carry - over effect from hrrest to hrmax is a concept not to be ruled out even though not significantly validated in the present study . the significance observed in the active vs. sedentary and male vs. female groups independently , does not remain significant when broken down into dependent groups ( i.e. active females , inactive females , active males and inactive males)(table 3 ) . in other words , the lowest mean hrmax was demonstrated in the active female sample , but the hrrest was lowest in the active male sample . therefore , a lower hrrest does not correspond to a lower hrmax in this study ( table 3 ) . the second purpose of the study was to determine the accuracy of three commonly employed mhres to determine sex and/or training effects . overall , the equation hrmax = 208 - ( 0.7 age ) ( equation 3 ) rendered the most accuracy utilizing these two separate measures for college - age participants . when considering signed residuals a negative mean value specifies the ability of the equation , on average , to over predict , a positive value under predicts , and a value of zero represents perfect accuracy of the mhre to predict the observed . therefore , when sex and activity level groups were combined under the equation effect , equation 3 produced the slightest amount of error representing the more accurate equation over the sample as a whole ( see table 4 ) . a value of zero with the signed residuals indicates the same degree of over and under predictions . because signed residuals do not measure the severity by which the over and under predicting of mhres occurs , the analysis of unsigned residuals is necessary . therefore , the closer to zero , the more accurate the mhre . under the equation , equation activity level effects , equation 3 remains closest to zero . despite hrmax = 220 age ( equation 1 ) being lower under the equation sex ( for males ) and equation activity level ( for sedentary ) effects , the mean differences demonstrated between equation 1 and 3 is 1.02 and .08 beats min respectively . it may be argued that small differences seen between the two equations are unlikely to make for significant differences . combining the effects we would suspect that equation 1 is of better use for the sedentary males , but when examining the non - significant trends of equation sex activity level effect , we see hrmax = 226 age ( equation 2 ) is associated with the least error which is highly inconsistent . yet , if we combined the equation sex and equation activity level active females , sedentary females , and active males the non - significant trends of equation sex activity level follow through as equation 3 having the least error for signed and unsigned residuals alike ( see table 4 ) . in conclusion , we may derive from the data that equation 3 is the better equation to use with the possible exception of sedentary males for college - age participants . in comparison to other studies , the foundation for equation 3 is strong ( 15 , 24 , 32 , 39 ) . ( 39 ) from which equation 3 was derived , demonstrated the equation to be unbiased toward sex and physical activity level with regard to meta - analysis and a laboratory - based portion . in a longitudinal study , gellish et al . ( 15 ) also concluded that sex was not significant factor in predicting hrmax while finding similar mhre to that of equation 3 . since both sexes and physical activity levels of the current investigation favor equation 3 , our data validate the results of other studies concluding that 220 - age and 226 age lacks scientific merit for general use with our sample . the link between age and hrmax has been demonstrated ( 33 , 38 ) , and the small age range of the subjects in this study ( see table 1 ) allowed for greater emphasis to be placed on sex and training effects . differences in treadmill protocol alone ( i.e. balke vs bruce ) may elicit differing mhres ( 14 ) , but would have no bearing on the current study as only one protocol was utilized . we could not assess the effect of bmi on hrmax and the accuracy of the mhre . the current study was also limited in the number of tests performed on each subject . gellish and colleagues ( 15 ) excluded initial tests due to lower hrmax associated with a learning curve , but remains unknown as to how it has affected the current study . while it is understood that a young healthy cohort may not be conducive toward examining cardiorespiratory health , our research allows a greater emphasis to be placed on gender and activity level with regard to the use of certain mhres . hopefully , such research would allow others to contemplate the use of proper mhres in any given setting . future research may focus on experiments involving the impact of exercise protocol to predict hrmax for active and sedentary men and women . the small sample size is also a limiting factor as it gives us smaller statistical power . in addition , understanding the impact of a learning curve and how this may affect observational scores of hrmax when compared to predicted values is of great value . finally , future research should incorporate higher standards in determining vo2max and rer and also utilize additional measurements such as ratings of perceived exertion , blood lactate , and/or estimated hrmax ( 20 , 39 ) . in conclusion , we found the males and sedentary groups to have higher observed maximal heart rates . hrmax = 208 ( 0.7 age ) equation overall had the most accuracy when measuring observed hrmax , with the possible exception of males and sedentary groups . such findings validate the use of the equation in the healthy young college - aged population regardless of sex or training status .

the purpose of the study was to determine if measured maximal heart rate ( hrmax ) was affected by sex or aerobic training status , and to determine the accuracy of three common clinical age - prediction maximal heart rate regression equations used to predict hrmax : hrmax = 220 age , hrmax = 226 age , and hrmax = 208 ( 0.7 age ) . fifty - two participants in total , 30 of which were in the active group ( 15 m , 15 f ) and 22 subjects in the sedentary group ( 9 m , 13 f ) , within the age range of 1825 years and with a normal bmi ( 18.524.9 kgm2 ) underwent a bruce maximal treadmill exercise protocol . the effect of sex and training status on hrmax was analyzed through a two - way anova , and the effect of sex , aerobic training status , and regression equation on accuracy of the hrmax prediction was assessed with a three - way anova ( =0.05 ) . overall , males had a higher hrmax than females ( 198.3 v. 190.4 beats min1 , p<.001 ) and sedentary individuals had higher measured hrmax than active individuals ( 197.3 v. 191.4 beats min1 , p=.002 ) . furthermore , hrmax = 208 ( 0.7 age)(equation 3 ) calculated the smallest signed and unsigned residuals from the difference between observed hrmax and predicted hrmax values for the significant main effects of equation ( 3 ) , equation sex ( females 3 ) , and equation activity level ( active 3 ) . therefore , based on our results , we conclude that hrmax = 208 ( 0.7 age ) has greater accuracy than the other two equations studied for predicting observed values of hrmax in 1825 year olds .

INTRODUCTION Methods Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC3944297

poststroke depression ( psd ) is regarded as one the most common mental sequela of stroke patients1 . psd patients have low physical and cognitive function , and low self - worth compared to those with no depression2 , and they have a decreased quality of life3 . all these factors can have adverse effects on the functional and mental recovery of stroke patients4 . for this reason , various drugs are used to treat psd , such as tricyclic antidepressant , monoamine oxidase inhibitors , and selective serotonin reuptake inhibitors ( ssris ) . however , various adverse reactions to these drugs have been reported , including insomnia , hyposexuality , nausea , and weight gain5 , 6 . exercise can relieve the symptoms of depression without the adverse reactions associated with drugs , by increasing the levels of chemicals related to depression , serotonin , dopamine , and norepinephrine , in the hippocampus7,8,9 . in particular , it has been shown that group exercise is more effective than exercising alone10 . in this respect , the popularity of circuit class training ( cct ) , which involves task - oriented exercise in groups , is growing . cct has been reported to have many benefits , such as improving the mobility of stroke patients and shows better cost efficiency than other therapies11 , 12 . most studies of cct , however , have focused on the improvement of balance , walking ability , or upper extremity functions , and few studies have investigated the potential effects of group cct on depression . accordingly , this study was conducted to investigate the potential effects of cct on depression through changes in branched - chain amino acids ( bcaas ) ( isoleucine , leucine , and valine ) and free - tryptophan ( f - trp ) . the study subjects were stroke patients who were diagnosed with major depressive disorder and dysthymic disorder according to dsm - iv13 guidelines following hospitalization and treatment at d hospital in busan . the degree of depression symptoms was measured with the beck depression inventory ( bdi ) , which is the most widely used self - report measure of depression . forty subjects who had moderate depression , defined as a bdi score between 19 and 29 , were selected for inclusion in this study14 . additional selection criteria were : absence of cognitive problems such as dementia , aphasia , or dysarthria ; a score of 23 or higher on the mini - mental state examination ; absence of other mental problems except depression ; absense of acute musculoskeletal problems ; and an ability to walk 10 m with no physical help . furthermore , those who had taken or were taking antidepressants such as ssris prior to the onset of stroke were excluded . the subjects were group - matched into an experimental group and a control group according to sex , age , height , and weight . the experimental cct group performed gradual task - oriented cct ( 80 min per session ) and received 30 min of general physical therapy . the control group performed stretching exercises and weight - bearing exercises ( 80 min per session)15 . the cct was performed three times per week for eight weeks ( 24 sessions ) . the total cct consisted of four steps : warming up ( 5 min ) , circuit training ( 60 min ) , evaluation and a short break ( 10 min ) , and a group game ( 15 min ) . the control group also performed warming up for 5 min before exercise and cooling down for 10 min after the exercise . two physical therapists received one day of training before this program , and data on the participants attendance and adverse events ( such as falls and heart problems ) were collected during the experiment16 . preliminary training for the whole program was performed one day before the start of the intervention . bdi scores were measured before the start of the experiment and again after eight weeks , at the end of the experiment . blood sampling was performed as follows : a catheter was installed in the forearm vein of the subjects , and 5 ml was collected immediately before the exercise ( 9:10 am ) and immediately after the exercise ( 10:30 am ) at d1 ( the start ) , d6 ( two weeks ) , d12 ( four weeks ) , d18 ( six weeks ) , and d24 ( eight weeks ) . the blood samples were centrifuged ( 3,000 rpm 15 min ) and stored in a refrigerator ( 82c ) until they were analyzed . the f - trp , ( i.e. , not combined with albumin ) was separated from the plasma using a modified version of the method of bloxam and hutson et al17 . to analyze the bcaas and f - trp , the amino acids were sampled with an acetonitrile / methanol solution and diluted to another concentration by an internal standard ( norleucine and d5-trp ; cambridge isotope laboratories , andover , ma ) . the plasma concentration was then analyzed by high performance liquid chromatography ( pharmacia , usa ) . changing trends over time in each group were examined using a two - way repeated measures anova . the paired t - test was used to examine intragroup variations over time , and the independent t - test was used to examine intergroup variations . the statistical significance level was used 5% ( significance was accepted for values of p < 0.05 ) . all the patients gave their written consent after receiving an explanation about the experiment and had been cautioned about potential adverse effects of this experiment . the experiment was approved by the human subject s research ethics committee of the catholic university of pusan ( cupirb-2013 - 019 ) . the independent t - test for the intergroup comparison of general characteristics did not show any significant differences ( table 1table 1 . general characteristics of the subjects ( n=40)groupcct ( n=20)con ( n=20)sexmen 7 ( 35% ) : women 13 ( 65%)men 7 ( 35% ) : women 13 ( 65%)age57.210.858.79.7height ( cm)163.097.1165.45.2weight ( kg)66.4011.968.15.9bmi ( kg / m)24.025.725.04.2paretic sidert 11 ( 55% ) : lt 9 ( 45%)rt 12 ( 60% ) : lt 8 ( 40%)typeinfarction 14 ( 70% ) : hemorrhage 6 ( 30%)infarction 11 ( 55% ) : hemorrhage 9 ( 45%)duration ( mon)8.41.97.92.6 ) . the changes in f - trp , bcaas , and f - trp / bcaas ratios in the blood after the performance of the cct or the extension / weight movement exercise for 80 min by each group are shown in table 2table 2 . responses of blood chemicals to circuit exercise test over timed1d6d12d18d24f - trp ( mol / l)precct54.52.4654.832.6254.212.2254.672.8954.061.89con53.992.8254.622.5554.992.3755.092.0755.892.03postcct60.162.2860.792.6762.663.0764.503.1965.503.29con54.461.9554.242.2754.511.7454.761.8254.371.55bcaas ( mol.l-1)precct540.8028.22538.6025.43533.3718.14534.4811.89535.3013.37con535.8119.45539.0112.49540.9812.96539.6912.26540.6811.84postcct496.5313.71488.7511.88478.1811.39471.7812.37470.5114.00con533.818.71533.8610.97538.2112.41536.3714.48539.4311.78trp / bcaas ( 102)precct1.000.061.020.071.010.051.020.061.010.04con1.000.061.010.051.010.051.020.051.010.05postcct1.210.061.240.061.310.071.370.081.390.09con1.020.031.010.041.010.041.020.041.000.03(mean sd ) different superscripts within the same rows represent significant differences . ( p<0.05 ) , d1 : the first day of circuit exercise ) , d6 ( two weeks of circuit exercise ) , d12 ( four weeks of circuit exercise ) , d18 ( six weeks of circuit exercise ) , and d24 ( eight weeks of circuit exercise ) . ( p<0.05 ) , d1 : the first day of circuit exercise ) , d6 ( two weeks of circuit exercise ) , d12 ( four weeks of circuit exercise ) , d18 ( six weeks of circuit exercise ) , and d24 ( eight weeks of circuit exercise ) before the intervention , the concentrations of f - trp , bcaas , and the f - trp / bcaas ratio in the blood showed no differences between the groups . however , intergroup differences of f - trp ( f = 10.457 , p = 0.00 ) , bcaas ( f= 10.847 , p = 0.00 ) , and f - trp / bcaas ( f = 3.157 , p = 0.00 ) after the intervention were significant . in the cct group , after cct , f - trp significantly increased from the first day of the cct ( d1 ) , and it remained increased at two weeks ( d6 ) , six weeks ( d18 ) , and eight weeks ( d24 ) ( f = 17.635 , p = 0.00 ) . there were no significant differences in f - trp between week one ( d1 ) and two weeks ( d6 ) and between six weeks ( d18 ) and eight weeks ( d24 ) . the values of the bcaas in the cct group showed a significant decrease over time ( f = 10.237 , p = 0.00 ) immediately after the start of the exercise , but no significant difference between six weeks ( d18 ) and eight weeks ( d24)was observed . the f - trp / bcaas ratios also showed a significant increase only in the cct group ( f = 27.277 , p = 0.00 ) . in addition , in contrast to the control group , the bdi score of the cct group exhibited a significant decrease in the final week ( d24 ) compared to the first day of the exercise ( d1 ) from 21.352.28 to 17.651.57 ( t=5.07 , p=0.00 ) ( table 3table 3 . comparison of bdi scores between cct group and control groupgrouppre - test ( d1)post - test ( d24)bdi ( score)cct ( n=20)21.42.317.71.6con ( n=20)22.12.420.12.1(mean sd ) different superscripts represent significant differences . this study investigated the effects of cct on the amelioration of depression in chronic stroke patients who had moderate depression symptoms . in particular , changes in the f - trp / bcaas ratio , which can indirectly determine the level of brain serotonin , an indicator of depression , was measured over eight weeks . bcaas ( isoleucine , leucine , and valine ) are essential amino acids , which can not be synthesized by the body . theoretically , they increase in response to the decomposition of proteins in the body , and they are decomposed again and oxidized to energy sources through the same metabolic process used for carbohydrates and fatty acids18 . in contrast to most general amino acids , which are oxidized in the liver , the amino acids derived from bcaas are oxidized mainly in the skeletal muscles19 . therefore , the use of bcaas increases in the muscles during recovery after exercise . this decreases the concentration of bcaas in plasma and thereby increases the f - trp / bcaas ratio . for the cct group in this study , the plasma concentration of bcaas after cct was significantly lower than prior to the exercise , and the concentration of bcaas after exercise decreased over time . this finding is likely due to increased use of bcaas to improve the recovery rate of fatigued muscles in the recovery process over time . however , as the percentage of f - trp under stable conditions is only 10% of the total trp , it is difficult to convert it into brain serotonin . it has been reported , however , that long - term exercise could help to increase the level of serotonin in the brain by increasing the concentration of f - trp20 . the results of the cct group in the present study provide evidence in support of this hypothesis . long - term cct is believed to increase the concentration of f - trp , which then increases the catecholamine level in the blood while decreasing insulin and glucose levels . this causes an increase in lipocyte - induced free fatty acids in plasma . as a result , trp , which strongly combines with albumin , the latter then combines loosely with albumin , and the level of f - trp in plasma increases . due to increase in f - trp and decrease in bcaas over time , the concentration of f - trp in plasma is expected to rise and aid the competitive passage of f - trp through the cerebrovascular barrier with the help of the amino acid carrier ( system l)21 . consequently , we believed that the ratio of f - trp / bcaas increased over time in the cct group in this study , and that this increase enhanced the concentrations of trp and serotonin in the brains of the subjects in the cct group , likely contributing to the improvement in depression as shown by their bdi scores . limitations of this study were that the subjects were limited to those with moderate depression , and dietary adjustments that could have affected the plasma amino acids were not taken into account . furthermore , although it has been reported that the f - trp / bcaas ratio affects central fatigue , this was not considered . future studies on central fatigue and psd could contribute to the clarification of the effects of the f - trp / bcaas ratio on psd . in conclusion , this study found that cct exercise for eight weeks increased the f - trp / bcaas ratio and might help improve psd .

[ purpose ] the purpose of the present study was to investigate the potential effects of circuit class training ( cct ) on poststroke depression through changes in branched - chain amino acids ( bcaas ) ( isoleucine , leucine , and valine ) and free - tryptophan ( f - trp ) . [ subjects ] the study subjects were 40 stroke patients with major depressive disorder . the subjects were group - matched into an experimental and a control group according to sex , age , height , and weight . [ methods ] the experimental cct group performed gradual task - oriented cct ( 80 min per session ) . the control group performed stretching exercises and weight bearing exercises ( 80 min per session ) . both groups performed the exercises three times per week for eight weeks ( 24 sessions ) . blood samples were collected immediately before the exercise ( 9:10 a.m. ) and after the exercise ( 10:30 a.m. ) , every two weeks for eight weeks . [ results ] the f - trp / bcaas ratio in the cct group showed a significant increase compared to the control group over time . [ conclusion ] the results show that the cct may help to improve depression in people with poststroke depression ( psd ) .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3647547

the vascular endothelium provides a cellular interface between the circulating blood and the vascular smooth muscle of the blood vessel walls . it also has an important role in maintaining the balance of the endovascular environment . many factors , such as peroxide , ox - low density lipoprotein ( ldl ) , angiotensin i , and tumor - necrosis - factor-(tnf- ) can induce the production of reactive oxygen species ( ros ) by nadph oxidase in endothelium and vascular smooth muscle , which results in oxidative stress to the endothelial cells ( ecs ) and , thus , the induction of cell apoptosis . ec dysfunction is a trigger factor for the development of atherosclerosis and other cardiovascular diseases . many studies have shown that ec apoptosis is one of several atherogenic factors [ 4 , 5 ] . additional studies have also shown that hemolymph from the silkworm ( bombyx mori ) can inhibit insect and mammalian cell apoptosis that is induced by viruses and several chemical inducers , such as staurosporine , camptothecin , and actinomycin d [ 68 ] . as one such antiapoptotic component in silkworm hemolymph , the 30 k protein has been studied widely [ 6 , 7 ] . a recent study showed that another protein in silkworm hemolymph , storage protein 2 ( sp2 ) , can also inhibit staurosporine - induced hela cell apoptosis and ros generation . owing to the influence of juvenile hormone and ecdysone , sp2 is synthesized by the fat body of feeding larvae and released into the hemolymph . at the end of the feeding period sp2 is presumed to be used as a store of the amino acids required for the development of adult tissues . in the current study , we cloned and expressed the gene encoding silkworm sp2 ( sp2 ) both in a prokaryotic expression system and a silkworm baculovirus system . the expressed protein in escherichia coli was used to generate polyclonal antibodies . the distribution of sp2 in different developmental stages and different tissues was detected by western blot . in addition , the expressed protein in the silkworm baculovirus system was used to study the anti - apoptotic effects of sp2 on human umbilical vein ecs ( huvecs ) induced by peroxidation . this work will lay a foundation for the development and utilization of protein drugs from economically important insects for treatment of vascular diseases . escherichia coli strains tg1 and bl21 ( de3 ) were grown at 37c in lb medium . the silkworm - derived cell line bmn was maintained at 27c in tc-100 medium ( gibco , usa ) supplemented with 10% fetal bovine serum ( fbs ) . b. mori nuclear polyhedrosis virus ( bacmid , conserved by our lab ) was propagated in bmn cells . huvecs were purchased from the american type culture collection ( manassas , va , usa ) and cultured in dulbecco 's modified eagle medium ( gibco , usa ) supplemented with 10% fbs ( gibco , usa ) in a humidified incubator at 37c in an atmosphere of 5% co2 . fifth - instar silkworm larvae ( jingsong haoyue , showa ) were reared on fresh mulberry leaves at 25c . male new zealand rabbits were purchased from the animal research center of hangzhou normal university . a dna gel purification kit and cy3-labeled goat anti - rabbit igg hrp - labeled goat anti - rabbit igg was purchased from dingguo biotechnology ( beijing , china ) . cellfectin ii reagent and a ni - nta purification system were sourced from invitrogen ( carlsbad , usa ) ; a cell death detection elisa kit was sourced from roche diagnostics ( castle hill , australia ) ; the annexin v - fitc / pi apoptosis detection kit was purchased from invitrogen ( carlsbad , usa ) ; staurosporine was sourced from the beyotime company ( shanghai , china ) . similarity analyses for the nucleotide and protein sequences were carried out using genbank blastn and blastp algorithms . the hydrophobicity analysis and the isoelectric point prediction were conducted on the expasy website ( http://www.expasy.org/ ) . simulation for the protein tertiary structure was generated by using the swiss - model program ( http://swissmodel.expasy.org/ ) . the open reading frame ( orf ) of sp2 was amplified by pcr using the cdna library of silkworm pupa constructed by our laboratory as a template . the forward and reverse primers were as follows : p1 , 5-cgcggatccatgaagtctgtcttaatt-3 ; and p2 , 5-ccgctcgagttaattttttggaacaac-3 , which contained bamhi and xhoi restriction sites , respectively . escherichia coli bl21 ( de3 ) was transformed with the recombinant plasmid and cultured in lb media , that included 50 g / ml kanamycin , at 37c until od600 reached approximately 0.5 . recombinant protein expression was induced with 1 mmol / l iptg for 4 h. the his - tagged fusion protein was extracted from the bacteria and purified by ni - affinity chromatography . the protein content was analyzed following the method of bradford and then used to immunize the male new zealand rabbits to prepare the polyclonal antibodies . an elisa was utilized to determine the polyclonal antibody titer , and the specificity of the polyclonal antibody was detected by western blot analysis . western blot analysis was used to evaluate the distribution of sp2 in different tissues of the fifth - instar larvae and other developmental stages of the silkworm . tissues from fifth - instar larvae ( head , epidermis , hemolymph , fat body , silk gland , trachea , the malpighian tubule , and midgut ) were isolated and pulverized into powder in liquid nitrogen . the powdered tissues were then resuspended in a lysis buffer ( 50 mm tris - cl , ph 8.0 ; 0.15 m nacl ; 5 mm edta ; 0.5% np-40 ; 1 mm dithiothreitol ; 5 mg / ml sodium deoxycholate ; 100 mg / l pmsf ; 5 g / ml aprotinin , sigma ) , and the mixture was incubated on ice for 30 min . the supernatants containing total proteins were equalized by assaying the protein content ( following the method of bradford ) . samples were verified by running on a 12% sds - page and electrophoretically transferred onto polyvinylidene difluoride ( pvdf ) membranes . the membranes were blocked with 3% skim milk in phosphate - buffered saline ( pbs ) at 4c overnight , and rabbit anti - sp2 antibody was added and the solution incubated at room temperature for 2 h. after washing with pbst ( pbs + 0.05% tween-20 ) , the bound antibodies were detected by anti - rabbit igg followed by a dab detection system . bmn cells were cultured overnight on a glass coverslip that could be used specifically for confocal microscopy . after removing the culture medium , the cells were washed three times for 5 min in pbs and fixed in 4% polyformaldehyde in pbs ( ph 7.4 ) at room temperature for 15 min , followed by permeabilization with 0.2% triton x-100/pbs for 10 min . the fixed cells were preblocked in 3% bsa in pbs at 4c for 4 h , followed by incubation with the anti - sp2 polyclonal antibody ( diluted 1 : 1000 in blocking buffer ) at room temperature for 2 h ; cells were incubated simultaneously with control serum , which was obtained from the rabbits before immunization with the antigen . after three 10 min pbst washes , cells were incubated with cy3-labeled goat anti - rabbit igg ( diluted 1 : 1000 , promega ) at 37c for 2 h and washed twice in pbst . the cells were then incubated with 46-diamidino-2-phenylindole ( 1 g / ml in pbs ) at 37c for 30 min . after the cells were washed once with pbst , they were photographed on a nikon eclipse te 2000-e confocal microscope ( nikon , japan ) . the sp2 gene was inserted into the mcs of the transfer plasmid pfastbac - htb between bamh i and xho i sites and transformed into dh10bac cells . the sp2 gene was then transferred into a wild type bacmid ( wtbacmid ) dna by homologous recombination to construct the recombinant baculovirus bacmid - sp2 . after white - blue plaque selection , the positive colonies were selected and analyzed by pcr with m13 universal primers , sp2 forward and reverse primers . the recombinant bacmid was transfected into bmn cells for amplification . the third - generation virus ( moi = 10 pfu / cell ) was further used to infect bmn cells ( 1 10 cells / flask ) for subsequent protein expression . after cultivation for 48 h , the cells were harvested by phosphate buffer solution ( pbs , ph 7.4 ) washes , pulsed sonication and centrifugation at 12,000 rpm for 10 min . the supernatant was subjected to ni - affinity chromatography according to the manufacturer 's instructions ( invitrogen ) . after purification by affinity chromatography , the sp2 protein was concentrated , and imidazole was removed by dialysis against 25 mm hepes [ 4-(2-hydroxyethyl ) piperazine-1-ethanesulfonic acid ) , sigma ] ; ph 7.5 , 100 mm nacl . huvecs were isolated by 0.25% trypsinization and cultured in dulbecco 's modified eagle medium ( gibco ) supplemented with 10% fbs and maintained in a humidified atmosphere with 5% co2 at 37c air until 7080% confluence was reached . cells ( from the experimental group ) were pretreated for 24 h with culture medium containing purified sp2 protein ( at a final concentration of 0.5 , 1 , and 2 g / ml ) . after washing twice with hank 's balanced salt solution ( gibco ) , the experimental and negative groups were both exposed to 2 ml sts ( 1 m ) for 2 h to induce apoptosis , whereas the normal group was treated with 2 ml hank 's balanced salt solution . the experimental group was then washed twice with hank 's balanced salt solution and cultured in culture medium in the presence of purified sp2 protein ( at a final concentration of 0.5 , 1 , and 2 g / ml ) . huvec viability and apoptosis were evaluated after 12 h. cell viability was estimated using mtt ( 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide ; sigma ) assay . cells were seeded into 96-well plates at densities of 1 10cells / well and incubated overnight . following the treatments as indicated above , 10 l mtt ( 5 mg / ml ) was added to each well and incubated for 4 h. the insoluble formazan crystals were dissolved in 200 l / well dimethylsulfoxide , and absorbance was measured at 490 nm to calculate the relative cell viability ratio dna fragmentation was evaluated by histone - associated dna fragments using a photometric enzyme immunoassay ( cell death detection elisa , roche ) , according to the manufacturer 's instructions . cells at a density of 1 10 cells / ml were harvested and washed twice with ice - cold pbs . for the quantitative assessment of apoptosis , staining with annexin v - fitc and pi labeling was undertaken before flow cytometric analysis , according to the manufacturer 's recommendations ( annexin v - fitc / pi apoptosis detection kit , invitrogen ) . facs analysis was accomplished using a facscalibur ( becton dickinson , san jose , ca , usa ) . the data were expressed as mean sem and analyzed by the student 's t - test and the newman - keuls test . differences with a value of p < 0.05 were considered statistically significant . sequence analysis revealed that the orf of sp2 ( accession number dq443358 ) was 2112 bp in length and encoded a protein of 703 amino acids , with a predicted molecular mass of 83.45 kd and a theoretical isoelectric point of 5.7 . through blastp comparison , three conservative structural domains of the hemocyanin superfamily were discovered : hemocyanin_n domain , hemocyanin_m domain , and hemocyanin_c domain . hemocyanin can be any of a group of copper - containing respiratory proteins that serve an oxygen - carrying function in the blood of some arthropods and most mollusks . the tertiary structure of silkworm sp2 was predicted using swiss - model ( figure 1 ) . the orf for sp2 was subcloned into the prokaryotic expression vector pet-28a ( + ) . the his - tagged fusion protein was expressed in e. coli bl21 ( de3 ) ( figure 2(a ) ) and purified by ni - affinity chromatography . the purified his - sp2 fusion protein was successfully detected by sds - page ( figure 2(b ) ) . the predicted molecular weight of the fusion protein , including a 3.56-kd his - tag , was 87 kd , which is concordant with the calculated value . the concentration of purified sp2 protein was determined by use of the bradford assay to be 1.5 mg / ml . with the affinity - purified proteins , the titer of the polyclonal antibody , as determined by elisa , was 1 : 6400 . western blot analysis indicated that the antibody reacted specifically with purified his - sp2 fusion protein ( figure 2(c ) ) . to determine the distribution of sp2 in different tissues of the fifth - instar larva and other development stages of the silkworm the results showed that the level of sp2 was very high in the pupal and larval stages but extremely low in the egg and adult stages ( figure 3(a ) ) . in the fifth instar larvae , the sp2 level was highest in the hemolymph and fat body and lowest in the trachea and midgut ( figure 3(b ) ) . the treated cells were examined under a nikon eclipse te2000-e confocal microscope , and images were analyzed using ez - c1 software . dapi - stained nuclei fluoresce red when stimulated with 353 nm light , and cy3-labeled goat anti - rabbit igg fluoresces red when stimulated with 550 nm light . our results indicated that sp2 is mainly located in the cell membrane and only partly in the cytoplasm ( figure 4 ) . to study the function of sp2 protein , recombinant bacmid - sp2 was used to infect bmn cells for expression of the his - tagged protein , and wtbacmid was used as a control ( figure 5(a ) ) . owing to the low expression level of natural sp2 protein in bmn cells , the results of western blot did not show the specific band in the bmn cells lysates infected by wtbacmid ( figure 5(b ) ) . the concentration of purified sp2 protein was determined by use of the bradford assay to be 0.108 mg / ml . the results of sds - page and western blot analysis indicated that the purified fusion protein sp2 had a molecular mass of approximately 87 kd ( figures 5(c ) and 5(d ) ) . sts triggers within the cell both morphological changes and internucleosomal dna fragmentation , which represents apoptosis . apoptotic cells accompanying dna fragmentation were analyzed at 24 h after sts treatment using elisa . a low concentration ( 0.1 m ) of sts did not affect apoptosis , whereas a high concentration ( 2 m ) induced necrosis , and 1 m triggered dna fragmentation efficiently compared with the positive control ( camptothecin ) provided by the kit . based on these data , the following experiments were examined using 1 m sts . cell viability was ameliorated by addition of the purified recombinant sp2 protein in a dose - dependent manner ( figure 6 ) , indicating that sp2 can enhance apoptotic huvec viability . to investigate the inhibitory effect of sp2 protein on sts - induced huvec apoptosis , an elisa was used to detect the dna fragmentation . as shown in figure 7 , huvec apoptosis as measured by dna fragmentation was significantly attenuated by recombinant sp2 protein in a dose - related manner . to quantify the apoptotic huvecs , an annexin v - fitc apoptotic cells were identified by fluorescein ( fitc ) conjugated to the human anticoagulant - annexin v , which was able to bind to phosphatidyl serine ( ps ) in the outer leaflet of the membrane of apoptotic cells . to distinguish cells that had lost membrane integrity , propidium iodide ( pi ) as shown in figure 8 , the apoptosis rate of untreated huvecs ( normal control ) was 1.9% , whereas the percentage of apoptotic huvecs ( negative control ) that had been treated with 1m sts was 52.9% . compared with the negative control , the apoptosis rate of sts + sp2 ( 1 g / ml)-treated huvecs decreased to 42.1% . these results indicate that sp2 has anti - apoptotic effects on huvec apoptosis induced by sts . as the major regulator of vascular homeostasis , the endothelium exerts several vasoprotective effects , such as vasodilation , suppression of smooth muscle cell growth , and inhibition of inflammatory responses . a large body of evidence has suggested that endothelial dysfunction is caused by superoxide and other ros [ 16 , 17 ] . supplementation with antioxidant vitamins c and e can restore endothelial function and , therefore , retard the progression of atherosclerosis . other factors , such as vascular endothelial growth factor ( vegf ) , low concentration of nitric oxide ( no ) in serum , calcium antagonists , prostacyclin , and estrogen , can also inhibit ec apoptosis . many clinical trials have been done to investigate ways of treating diseases associated with apoptosis with anti - apoptotic genes and proteins [ 20 , 21 ] . silkworm hemolymph and its 30 k protein have been reported to exhibit anti - apoptotic activity in various mammalian and insect cell systems and could have therapeutic potential in diseases related to apoptosis . in the current study , another potential anti - apoptotic protein in silkworm hemolymph ( sp2 ) was studied . as a member of the hemocyanin superfamily hemocyanins are large copper - containing proteins that transport oxygen in the hemolymph of many arthropod and mollusk species . most hexamerins are considered to be storage proteins , providing energy , serving as carrier protein or possibly involved in the humoral immune response . owing to the important role of hexamerins during arthropod development , we speculated that silkworm sp2 protein might also have an important role during cell apoptosis . to explore its functions , the sp2 protein was expressed and purified both in a prokaryotic expression system and silkworm baculovirus system . the purified sp2 prokaryotic expression product was used to generate monoclonal antibodies to study the distribution and location of sp2 in silkworm , whereas the purified expression product was used to study its antiapoptotic function . our analysis of the level of sp2 in different tissues of the fifth - instar larva and other developmental stages of the silkworm showed that sp2 levels were highest in the hemolymph and fat body and lowest in the egg and adult stages . these results suggest that sp2 gene activity is affected by juvenile and molting hormones . from the analysis of the subcellular localization of sp2 , we found that , in the bmn cell line , sp2 was localized to both the cell membrane and cytoplasm but was found primarily in the cell membrane . because sp2 is a secreted protein and has a signal peptide predicted by bioinformation ( data not shown ) , our results suggest that it is translocated across the cell membrane guided by the signal peptide . to study the anti - apoptotic activity of sp2 on vascular ec apoptosis , we constructed an sts - induced huvec apoptosis model . the sts treatment was able to induce generation of ros , which results in oxidative stress to the cells . sts at a concentration of 1 m exerted prominent apoptotic effects in cultured huvec . an mtt assay , dna fragmentation detection , and flow cytometric analysis showed that the purified recombinant sp2 protein could significantly enhance the viability of huvec and inhibit huvec apoptosis induced by sts . these findings provide new insights into the prevention of endothelial dysfunction and could ultimately provide therapies for atherosclerosis .

storage protein 2 ( sp2 ) not only is an important source of energy for the growth and development of silkworm but also has inhibitory effects on cell apoptosis . endothelial cell ( ec ) apoptosis is an important contributing factor in the development of atherosclerosis ; therefore , study of the antiapoptotic activity of sp2 on ecs provides information related to the treatment of atherosclerosis and other cardiovascular diseases . in this study , the sp2 gene was cloned and expressed in escherichia coli to produce a 6xhis - tagged fusion protein , which was then used to generate a polyclonal antibody . western blot results revealed that sp2 levels were higher in the pupal stage and hemolymph of fifth - instar larvae but low in the egg and adult stages . subcellular localization results showed that sp2 is located mainly on the cell membrane . in addition , a bac - to - bac system was used to construct a recombinant baculovirus for sp2 expression . the purified sp2 was then added to a culture medium for human umbilical vein ecs ( huvecs ) , which were exposed to staurosporine . a cell viability assay demonstrated that sp2 could significantly enhance the viability of huvec . furthermore , both elisa and flow cytometry results indicated that sp2 has anti - apoptotic effects on staurosporine - induced huvec apoptosis .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC4827900

multiple chemical sensitivity ( mcs ) , also known as chemical intolerance ( ci ) or chemical hypersensitivity , is an emerging disabling illness characterized by chronic adverse effects from exposure to low levels of chemicals in the modern human environment . symptoms can range from mild to disabling and commonly include fatigue , nausea , dyspepsia , rhinitis , confusion , change in heart rate , irritability , dizziness , and headache.1 the most common chemicals described as causing reactions are pesticides , petro chemicals , household cleaners , exhausts , dry cleaning chemicals , perfumes , smoke , and air fresheners.1 prevalence rates vary depending upon the wording of population surveys , but chemical sensitivity appears to be an international problem . in the us , caress and steinemann2 found that 12.6% of a household population study reported being unusually sensitive to common chemical substances . meggs et al3 found that 33% of persons in a us household population survey reported some chemical sensitivity ( cs ) , with 3.9% reporting becoming ill every day from exposures . katerndahl et al4 found that 20% of 400 people in family medicine waiting rooms met the criteria for ci . in the netherlands , 27% reported multiple symptoms from chemicals,5 while in germany , 32% reported similar complaints.6 johansson et al7 found , in sweden , that 32% reported some odor intolerance , with 19% having some life impacts from the intolerance . in the same country , 15.6% of teenagers reported cs.8 and in korea , the prevalence is estimated at 16.4% , with higher numbers among people with atopic dermatitis and those who have lived in new housing.9 thus , sensitivity / intolerance to chemicals is reported to occur in multiple countries . though found internationally , and crossing the lines of race , income , and age,10 mcs does seem to disproportionately affect women.5,8,11 the japanese researchers imai et al12,13 have made pleas for health professionals to attend to indoor air quality and to learn to counsel those with sensitivities as to how to avoid chemical exposure in their daily lives . researchers have , to date , only discussed mcs in the industrialized countries of japan , korea , sweden , germany , canada , usa , spain , france , and the netherlands . however , johnson14 has described in detail the sensitivity - related illness experienced by persons exposed to the september 11 , 2001 , world trade center bombing in new york , the katrina aftermath in new orleans , and the clean - up following the oil spill by the exxon - valdez in alaska , usa . it is certain that indigenous persons experience heavy chemical exposures such as these as a result of industrial sitings , chemical spills , and lesser environmental standards for industrial ventures . texaco for extreme contamination of their forest home,15 a long - running case only recently resulted in an ecuadorean appeals court upholding a ruling against chevron and requiring the company to pay 18.2 billion dollars in damages.16,17 it is well known that nigeria is heavily contaminated as a result of oil extraction18 and that toxic pesticides no longer legal in the us are exported for use in less developed countries . though some progress has been made in understanding sensitivities in industrialized countries , persons with these contested and emerging illnesses still have difficulties in obtaining help with the challenges of living with poorly understood conditions . some are referred to psychologists when medical providers fail to find obvious bodily damage and assume that the problem is psychosomatic . others go for help with any concomitant mental health issues or are unaware that they have reactions to chemicals . additionally , in the usa some persons are required to have a psychological evaluation when applying for social security disability income . the field of sociology preceded psychology in examining and understanding chronic illness.19,20 charmaz21 has described the alienation from the body and the changes in identity goals that accompanied surrendering to chronic illness in 55 adults with chronic illness . though psychology has been slower to recognize the needs of those with chronic illness , writers within this field have focused on the support needs and ways of responding to individuals with chronic illness and their families.22,23 kennedy et al24 admitted in 2001 that the psychological professions were just beginning to address the chronically ill . more specifically , psychological researchers have discussed coping from an empathic perspective for noncontested conditions , such as renal failure,2527 rheumatoid arthritis,28 multiple sclerosis,26 and others.29 over time , the findings of these studies are becoming integrated into health care provision to the benefit of patients . mcs , in particular , has been the subject of heated debate regarding etiology , with some exploring psychogenic pathways . though a thorough summary of etiology research is beyond the scope or space of this paper , research on causation has been fairly polarized . skovbjerg et al30 found neuroticism to be a small predictor of ci and cite this as support for the role of negative effect in the development of ci . bailer et al31 examined trauma in persons with idiopathic environmental intolerance ( iei ) , somatoform , disorder , and in controls . no differences were found regarding general or sexual trauma , but those with iei and somatoform cited more exposure to unspecified and life - threatening illness . the authors believe that these life events may foster the development of dysfunctional beliefs , direct attention toward bodily symptoms of potential illnesses , and lead to enhanced symptom reports . in a later study , bailer et al32 used the modern health worries scale that examines concerns about radiation , environmental pollution , tainted food , and toxic interventions . persons with iei [ sic ] had higher worries than controls , leading the authors to attribute chemical sensitivities to an attributional style . ( but the relationship between the worries measured and the participants health condition calls this interpretation into question due to circular reasoning ) . researchers who view the condition as physiological have found a strong overlap with allergy and asthma,10,11 vitamin and mineral deficiencies,33 enzymatic deficiencies and genetic alleles,34 immunological factors,35 including proinflammatory indicators,36 genetic variations,34 and mixed results regarding upper airway inflammation.37,38 the olfactory kindling hypothesis has found some support39 with evidence from animal studies.40,41 there is some suggestion of elevated histamine both during and between exposures42,43 and some positive correlation between real - time volatile organic compound exposures as measured by canisters and reports of symptoms44,45 and serious neurological changes ( decrease in brain perfusion ) on single - photon emission computed tomography scans in a number of cortical areas during chemical exposure for persons with mcs when compared with controls.46 orriols et al46 also found changes in brain function before and after exposure in the mcs group and say that their findings suggest a neurologic pathogenesis of this disorder . andersson et al8 found that anxiety , but not depression , correlated with cs and speculated that depression may develop as a result of the sensitivity . the relation with anxiety was only correlational , and bloch and meggs11 found that anxiety was related to allergy and asthma as well as cs . similarly , bloch and meggs11 found that panic also has a nonunique relationship with mcs , suggesting that chemical sensitivity is a member of a larger family of diseases that have a common relationship with anxiety and panic . gundersen et al47 found no relationship between mcs and anxiety or depression . in an older study , bertschler et al48 found that mental health indicators improved following treatment for chemical sensitivities , suggesting that psychological discomfort is secondary to the illness . heuser et al49 suggested in 1992 , after finding considerable evidence of neurological abnormality , that the psychiatric presentation by many of these patients may well have a neurological basis . though practitioners who do not believe that mcs is of physiological origin suggest that patients expose themselves gradually to chemicals , fox and sampalli50 found evidence in an exposure booth study that those with mcs did not accommodate to a series of three exposures to dryer sheets , but continued to manifest changes in skin temperature and conductance , respiratory and heart rate , and surface electromyography ( emg ) . cui et al51 argue for an integrative multifactorial psychobiological process model rather than one that is unidirectionally psychogenic or physiological . the authors found that mcs in japanese factory workers did predict mental health issues on the quick environmental exposure and sensitivity inventory ( qeesi ) , though their study model constructed mental health effects as secondary to mcs . dantoft et al52 have created an integrative summary of the etiological research on mcs and call for researchers to set aside the dichotomy and pursue interdisciplinary work in order to positively impact those who experience the illness . dumit53 has referred to conditions such as fibromyalgia , chronic fatigue syndrome , and mcs as illnesses you have to fight to get , in that without a clear medical label , these persons lack the medical validation that confers legitimacy . thus , invisible / emerging / marginalized / contested conditions leave persons with the task of struggling for validation and access to resources . fox and kim54 observed that the barriers faced by individuals from emerging disability groups often prevent experiencing the benefits of participation in society . the life impacts of having cs / ci may include joblessness , lack of medical care , lack of community access,55 social isolation , and even homelessness.1 workplace harassment is not uncommon , and may possibly , along with lack of job accommodations , be a factor in joblessness.56 not surprisingly , persons with these struggles report personal distress . when the symptoms become severe , persons often apply for disability benefits as a result of the inability to continue in the workplace.57,58 despite the use of mental health providers as gatekeepers in the acquisition of disability support , they appear to be generally unprepared to work with this problem . and given the history of women s health care5961 and the sex biases in psychological care,62,63 it is not surprising that persons with ci would report some difficulty in accessing support in the psychological community . gibson et al1 found that 187 of 268 persons with self - reported mcs had seen a total of 549 mental health providers . only 17% of these providers were seen as being informed regarding mcs and 36% were perceived as helpful . respondents reported that their cs was ignored ( n=119 ) , that they received psychogenic labels ( n=102 ) and psychoactive medications ( n=83 ) , and that providers suggested ( n=33 ) or implemented ( n=28 ) psychiatric hospitalization . more recently , doiron64 found that even providers who had been working with this population for some time showed little understanding of clients difficulties or needs . likewise , in japan , imai et al12 found that lack of knowledge about the disorder and difficulty in establishing a diagnosis comprised two of the three major obstacles that aggravate symptoms of sick house syndrome or cs . similar experiences may be encountered when persons with mcs attempt to access services at centers for independent living . gibson et al65 found that services received by consumers with mcs were uneven and unpredictable and that , although some persons received helpful services , advocates turned others away and were unfamiliar with mcs or the needs of those who experience it . in this article , we summarize the experiences of a sample of persons in the us with mcs who sought help from psychological providers for various reasons . we discuss the experiences of persons with sensitivities who visited psychological providers and their views regarding ways to improve services for this population . to our knowledge , this is the first study of the experiences of persons with ci with psychological providers . fifty - five ( 91.7% ) participants were physician diagnosed with mcs , four ( 6.7% ) were self - diagnosed , and one did not answer this question . the 45-question survey contained demographic questions regarding age , sex , and household income , as well as questions regarding how many and what types of mental health providers they had seen and for what reasons . we then asked the responders to concentrate on one specific experience with their most recent mental health provider and to answer questions about that contact in terms of accommodations asked for and received , the reason for the contact , and how the experience could have been better . we also used the satisfaction with life scale ( swls ) comprising five questions on a 7-point likert - type scale ( total scores can range from 5 to 35).66 the questions are broad and not anchored to specific life realms , as diener67 believes that respondents should be free to anchor life satisfaction on their own criteria . the measure has shown satisfactory internal consistency ( r=0.87)68 and test retest reliability ( r=0.82).66 the scale is not correlated with social desirability , sex , age , or educational level.68 the 44-question survey was developed on qualtrics , an online survey maker . after receiving study approval from the james madison university institutional review board , the link to the survey was placed on our mcs research website for 5 months ( www.mcsresearch.net ) . we also informed multiple support and advocacy groups that we were conducting the study and asked for their help in distributing our call to the participants . paper copies were offered for those who could not access the survey online . when accessing the link to the study , respondents first viewed an informed consent document and were advised that pushing the next button after reading the consent implied agreement to participate ; responses were thus anonymous . fifty - five ( 91.7% ) participants were physician diagnosed with mcs , four ( 6.7% ) were self - diagnosed , and one did not answer this question . the 45-question survey contained demographic questions regarding age , sex , and household income , as well as questions regarding how many and what types of mental health providers they had seen and for what reasons . we then asked the responders to concentrate on one specific experience with their most recent mental health provider and to answer questions about that contact in terms of accommodations asked for and received , the reason for the contact , and how the experience could have been better . we also used the satisfaction with life scale ( swls ) comprising five questions on a 7-point likert - type scale ( total scores can range from 5 to 35).66 the questions are broad and not anchored to specific life realms , as diener67 believes that respondents should be free to anchor life satisfaction on their own criteria . the measure has shown satisfactory internal consistency ( r=0.87)68 and test retest reliability ( r=0.82).66 the scale is not correlated with social desirability , sex , age , or educational level.68 the 44-question survey was developed on qualtrics , an online survey maker . after receiving study approval from the james madison university institutional review board , the link to the survey was placed on our mcs research website for 5 months ( www.mcsresearch.net ) . we also informed multiple support and advocacy groups that we were conducting the study and asked for their help in distributing our call to the participants . paper copies were offered for those who could not access the survey online . when accessing the link to the study , respondents first viewed an informed consent document and were advised that pushing the next button after reading the consent implied agreement to participate ; responses were thus anonymous . sixty persons returned usable complete surveys , 57 of which were caucasian ( three classified themselves as other ) . fifty - four considered themselves to be disabled and four did not ( two did not answer ) . there was a range of educational levels : one person had completed some high school , ten had high school degrees , nine had associate s degrees , 12 had technical degrees , 16 had bachelor s degree , ten had master s degrees , and two had doctoral degrees . marital status varied : 14 participants being single , 22 married , 19 divorced , and five living with partners . over two - thirds ( 66.7% ) were not employed , ten ( 16.7% ) worked from home , three ( 5.0% ) worked part - time , and seven ( 11.7% ) worked full - time . most respondents ( 55 ) had lost or had been forced to quit a job because of their sensitivities . respondents had been aware of their disorder for a mean of 14 years and had seen a mean of 4.5 mental health providers , with 20 being the highest number seen . participants rated their level of disability as mild ( 10% ) , moderate ( 19.0% ) , severe ( 46.6% ) , or totally disabled ( 24.1% ) . as we expected , there were people who were forced to seek services as part of the social security or private disability compensation application . others were looking for stress management , medication reviews , or validation that their condition was physiological . people commonly sought counseling for a variety of reasons , including work or social discrimination , difficulty in physical functioning , and other issues related to mcs . many were grieving over their loss of access ( ie , their inability to have contact with much of the world because of their intolerances ) . when asked what type of mental health providers they had seen , 40 ( 66.7% ) reported visiting psychiatrists , 42 ( 70% ) had seen psychologists , 25 ( 41.7% ) had seen counselors , 13 ( 21.7% ) had visited social workers , and eleven ( 18.3% ) had accessed other mental health providers such as neuropsychologists or neurologists ( some saw more than one type of provider ) . ( the tables are not meant to be summative , as not all participants requested accommodations , not all accommodations requested were given , and some participants requested more than one accommodation ) . the most commonly requested accommodations included a fragrance - free environment , being seen in a different location , a chemical - free room , being able to avoid the waiting room , and others as listed in table 2 . some mental health providers were quite accommodating in that they were willing to forego the use of scent for a day or meet outside of their offices . although providers made a variety of accommodations for their clients , some could not control access to their entire buildings . for example , one participant said , the office has signage up that it is a chemical free environment and staff do not wear fragranced products , however , main entry and elevator to the office in the building is [ sic ] often full of chemical smells . while visiting providers , 45 participants ( 75% ) had asked for special accommodations for their sensitivities and 30 people ( 50% ) said they had been met . many respondents felt there was more their providers could have done regarding accommodations . when asked to evaluate their provider s knowledge of mcs on a scale of not knowledgeable , somewhat knowledgeable , or highly knowledgeable , 25 ( 41.7% ) said their provider was not knowledgeable , 27 ( 45% ) said somewhat knowledgeable , and only seven ( 11.7% ) said highly knowledgeable . when rating the experience of the provider regarding mcs , 39 persons ( 65% ) said not experienced , 17 ( 28.3% ) stated somewhat experienced , and only four ( 6.7% ) said highly experienced . thirty - eight percent of participants had received refusals for help from mental health providers in the past because of a lack of knowledge or an inability / unwillingness to accommodate the respondents . when describing how the experience could have been better , respondents named variables related to both the setting and practitioner behavior . persons wanted a safer physical environment in which to be seen that was smoke- and scent - free , with windows that opened . they wanted to avoid waiting rooms and new construction and to be seen in their own homes . many respondents mentioned wanting knowledgeable providers who would believe them and recognize mcs as a physical condition , and offer understanding , compassion , and support for their condition and its associated lifestyle changes ( table 3 ) . when asked about the worst aspect of their experience with mental health providers , respondents listed variables that fell into three categories : the exposures that occurred as a result of the interaction , the tendency to see mcs as psychogenic , and the lack of responsiveness on the part of the providers . exposure problems included fragrance on furniture and on people , having to wear a mask to attempt to avoid such exposures , and becoming sick from the experience . the tendency of the provider to construct mcs as psychogenic caused some clients to receive psychological labels and be given psychogenic medications . one client was required to take the minnesota multiplasic personality inventory , a test usually reserved for clients assumed to have some mental pathology . respondents mentioned being tested only for psychological problems and not for toxic brain syndromes , being seen as trying to get out of working , and having to be on guard so as not to be perceived as having a psychological disorder . the embarrassment of discussing a condition that could be perceived as psychological was also mentioned as the worst aspect of visiting a mental health provider . behaviors from providers that demonstrated a lack of responsiveness on the part of the provider included getting no accommodations , perceiving that the provider had no understanding of mcs , having symptoms dismissed , feeling humiliated , and feeling as if i were talking to a stump . scores on the swls were generally low , with a mean of 12.2 of a possible 35 , a very low score even for persons with chronic medical issues . scores on the swls were generally low , with a mean of 12.2 of a possible 35 , a very low score even for persons with chronic medical issues . although some participants were satisfied with their interactions with mental health providers , many felt that there was much room for improvement , and we gathered valuable data for designing a training module for mental health providers who work with persons with mcs ( to be presented elsewhere ) . we found that more than half of the providers seen were not considered knowledgeable about mcs . egeli et al69 found that patients with fibromyalgia ( another contested illness ) were more satisfied when their physicians were knowledgeable and supportive and listened to their concerns . many of the recommendations suggested by egeli et al s participants for their physicians resembled those of ours regarding their mental health providers . our participants were looking for providers with more knowledge of mcs and alternative treatment methods and were expecting the provider to learn with them and to take extra time to adequately discuss treatments . in our study , over 83.3% of participants considered their mental health provider only somewhat or not experienced . many wished that the providers had been willing to research the subject before their session so they would have a better understanding of the client s dilemma . yet disability benefits from the government would ease the struggles with money and payments to medical centers for mcs clients . a number of respondents reported very negative experiences in having to undergo psychological assessment in order to acquire the needed disability benefits . one of the major themes that the current study has highlighted is that the clients do not see themselves as mentally ill but as disabled by their experiences with mcs . some of their experiences with providers were helpful because they were able to secure help for their problems resulting from the mcs . gibson70 reported that some persons , out of financial desperation , felt forced to accept psychological diagnoses though they believed them to be incorrect . the danger in misinterpreting women s health conditions as psychogenic was addressed by klonoff and landrine,71 who reviewed health conditions that tend to manifest either more often or solely in women ( eg , multiple sclerosis , hypothyroidism , temporal lobe epilepsy ) . the authors believe that the psychological overlay or mimic of psychological symptoms in many physical illnesses may account for the preponderance of women receiving depression and anxiety diagnoses from both primary care and psychological providers . klonoff and landrine state that the use of antidepressants can worsen the condition and even be fatal . women with mcs may face the same risks , as antidepressants were rated as more harmful than helpful by a large sample of persons when used as a treatment for ci.72 life satisfaction scores were lower than even previously found for persons with mcs . gibson et al73 found a mean swls score of 14.86 , ( standard deviation ( sd ) = 7.64 ) in 209 persons with mcs . this is the second study to find low life satisfaction in persons with mcs compared with other published samples . previous researchers found that medical outpatients and elderly patients scored means of 23.5 and 25.8 , respectively , on the swls.66,68,74 in the gibson et al73 study , lower life satisfaction was significantly correlated with the course of illness ( having gotten worse ) , being fatigued , having low leisure satisfaction , having a lower income , and having no romantic relationship . many participants in the current study had lost spouses , jobs , and the freedom to leave their own houses . these cumulative losses are consistent with findings from other studies ; these losses and the lack of access may explain the low mean swls scores . this underscores the need for counseling and support regarding coping with chemical and electrical intolerance.13 there is a great need for more research on the physical causes of mcs , effective interventions , and the support of this population by mental health providers . our results are consistent with those of bernard et al,75 who found that 270 participants with fibromyalgia reported in open - ended questions that they wanted their health care professionals to be more educated about their particular disease and to offer better services to help manage their disease . similarly , lehman et al76 found when studying respondents with chronic fatigue syndrome , that the most popular response ( 36% ) to the question what is the most unhelpful or upsetting thing that a medical professional has said or done to you interestingly , the most popular response ( 52% ) to the query what is the most helpful thing that a medical professional has said or done to you was legitimizing the illness , either through diagnosis or by acknowledging the presence of symptoms . respondents in the lehman et al study , who reported not feeling legitimized by their physicians , also demonstrated significantly higher depression and anxiety scores than those who felt legitimized . when mcs and other emerging disabilities gain legitimacy , people will be able to seek effective services . though disagreement regarding etiology persists , researchers are beginning to hypothesize and uncover neurological effects of chemical exposure in persons with ci . medical acceptance and understanding of this condition , proper financial benefits , and work accommodations would go a long way toward improving the quality of life for persons with environmental intolerances . we believe it is the responsibility of health care providers to study emerging illnesses and disabilities and to contribute positively to the care of those who experience them . koch et al would like rehabilitation counselors to challenge their own biases toward mcs , revise their understanding of universal design , accommodations , and accessibility , and learn to help clients to communicate with their work supervisors and erode their psychosocial isolation.77 recently , gibson et al78 found , in a grounded theory study , that the core activity of participants , healthquest , was literally a euphemism for resisting annihilation , given the tremendous obstacles to seeking medical treatment for persons with mcs . in this study , respondents reported wanting their counselors to understand in detail the triggers , symptoms , and lifestyle requirements of those with mcs , to learn some toxicology , to understand how some psychological conditions such as depression and anxiety can be either toxin - induced or secondary reactions to the lifestyle restrictions , to apply the principles of doing good therapy to this population of disabled people , and to make it a priority to provide an accessible office . until and unless therapists are able to address these needs , persons with mcs will struggle for even a modicum of help from the helping professions . limitations of this study include the nonrandom method of gathering volunteers and the fact that the participants were self - reported to have mcs .

in this paper , we summarize the results of an online survey of persons in the united states with chemical intolerance / multiple chemical sensitivity who sought help from mental health providers , including counselors , psychologists , psychiatrists , and others . respondents reported on their most recent contact with a provider , describing reasons for the contact , accommodations requested and received , and suggestions for how the experience could be more helpful . overall , though clients were accommodated in small ways , some received no accommodation , and many felt that the providers needed to be more knowledgeable regarding chemical intolerance . results are discussed in terms of the importance of providers becoming more aware of multiple chemical sensitivity and more willing to make their services accessible to these clients .

Introduction Methods Participants Materials Procedure Results Satisfaction with Life Scale Discussion

PMC3214519

the successes of these procedures depend on the effective removal of infected dentin , prior to the placement of the restorative material . any leftover bacterial remnants during and after the cavity preparation pose one of the major problem in restorative dentistry . failure to mechanically remove the infected tooth structure and achieve complete sterilization of the cavity preparation can lead not only to microleakage , increased pulp sensitivity , and pulpal inflammation , but also to secondary caries , necessitating replacement of the restoration . therefore , after removal of the carious dentin , it is important to eliminate any remaining bacteria that may be present on the cavity walls , in the smear layer , at the enamel - dentin junction , or in the dentinal tubules . histological and bacteriologic studies have shown that only a small proportion of the teeth are sterile after cavity preparation and that bacteria left in the cavity preparation could survive for longer than a year . today , the application of disinfectants after cavity preparation and before tooth restoration is gaining acceptance as they are considered to eliminate potential risks due to bacterial activity . for two decades , many chemicals have been tested as cavity disinfectants , including chlorhexidine digluconate ( chx ) , benzalkonium chloride , and iodine potassium iodide / copper sulfate , etc . however , there is concern about the use of cavity disinfectants with dentin bonding agents , since they may have an adverse effect on the bond strength of the composite resins . the objective of this study was to compare the effect of the most commonly used cavity disinfection materials on the shear bond strength of composite with either a two - step self - etching or an etch - and - rinse bonding system . two hundred human permanent mandibular molars , free of cracks , caries , and restorations on visual inspection , were used for the study . the teeth were scraped of any residual tissue tags , kept in a 2.6% sodium hypochlorite solution and rinsed under running water for 15 minutes each . later , they were cleaned with pumice and stored in normal saline at 40c until use . the teeth were sectioned with a low - speed diamond disk saw ( markus inc . , michigan , usa ) under water coolant to expose mid - coronal dentin . the sections of the teeth including the roots were embedded in autopolymerizing acrylic resin to form cylinders 2.5 cm in diameter and 5 cm high . the teeth were randomly divided into five main groups of 40 teeth each . in group 1 , the teeth in experimental groups were treated with one of the following cavity disinfectants : 2 % chx solution ( consepsis , ultradent usa ) , 0.1% benzalkonium chloride - based disinfectant ( tubulicid red , dental therapeutics ab , sweden ) , 1% chx gel ( hibitane dental ) , or an iodine potassium iodide / copper sulfate - based disinfectant ( ora-5 , mchenry labs , texas , usa ) for 20 seconds . each group was then randomly divided into two subgroups of 20 teeth each according to the bonding agent used , either clearfil se bond ( clearfil se bond , kuraray , osaka , japan ) or prime & bond nt ( dentsply caulk , milford , de , usa ) . for the clearfil se bond system ( clearfil se bond , kuraray ) , primer was applied to the dentin surface using a sponge supplied by the manufacturer and rubbed for 20 seconds . after this etching and priming step , the se bonding agent was applied to dentin surfaces and light cured for 20 seconds ( spectrum 800 , confident india pvt . ltd . ) . for prime & bond nt ( pbnt ) ( dentsply caulk ) , the dentin surface was etched with 34% phosphoric acid gel ( dentsply caulk ) for 20 seconds , rinsed for 20 seconds with an air -- water syringe and dried with compressed air . then , prime & bond nt was applied , left undisturbed for 30 seconds , lightly air dried for 2 seconds , and light cured for 20 seconds . after adhesive application , the specimens were clamped in the ultradent bonding jig ( ultradent products ; south jordan , ut , usa ) . a hybrid restorative composite ( clearfil apx shade a2 , kuraray ) was carefully inserted into the surface by packing the material into cylindrical - shaped plastic matrices with an internal diameter of 2.30 mm and a height of 3 mm . the composite was incrementally cured with a quartz halogen curing light ( spectrum 800 , confident india pvt . ltd . ) for 60 seconds . after storing in an incubator at 37c in 100% humidity for 24 hours , the specimens were placed in a universal testing machine and the shear bond strength was measured at a crosshead speed of 1 mm / min . the shear bond strength of composite resin to dentin was recorded in newtons ( n ) and calculated in mpa taking into account the cross - sectional area of the composite buildup . one way analysis of variance and tukey hsd tests was performed to determine significant differences in bond strengths between the groups . after the testing procedure , the fractured surfaces were observed with a dissecting microscope ( sz - tp olympus ; tokyo , japan ) at a magnification of 20 to determine failure modes and classified as adhesive failures , cohesive failures within the composite or cohesive failures within the tooth . one specimen from each group was sputter coated with gold after fracture and prepared for sem examination . coated specimens were then observed under the sem ( jmg-5600 , keol ; north carolina , usa ) with different magnifications . the mean and standard deviations of shear bond strengths for each group are presented in table 1 . effect of cavity disinfectants on the shear bond strength of clearfil se bond and prime & bond nt adhesive systems is summarized in table 2 . composition of cavity disinfectants used in the study effect of cavity disinfectants on shear bond strength of clearfil se bond and prime & bond nt adhesive systems . when the results were analyzed , the mean shear bond strength values in the clearfil se bond group which had been treated with chx solution , benzalkonium chloride - based disinfectant and iodine potassium iodide / copper sulfate-(i2ki / cuso4 ) based disinfectants were significantly lower than the other groups ( p < 0.05 ) . the shear bond strength was not adversely affected by any cavity disinfectants in groups in which prime & bond nt was applied after acid etching . regardless of the bonding agent , the chx gel used demonstrated no adverse effect on the shear bond strength of composite resin . the distribution of fracture modes observed with a dissecting microscope at a magnification 20 is shown for all groups in table 3 . the principle mode of failure in groups 2 , 3 , and 5 was adhesive . figures 1 and 2 show the sem micrographs of dentin surfaces after fracture in groups 2 and 3 , respectively . resin tags on dentin surfaces treated with the self - etching system were not clearly visible . however , in figures 3 and 4 , dentin surfaces which were treated with chx solutions ( group 7 ) or chlorhexidine gel ( group 9 ) , respectively , showed clear resin tags when the etch - and - rinse system were used . failure modes of the all test groups sem micrograph of dentin surface treated with 2% chx solution in group 2 . dt : dentinal tubule , ar : adhesive resin sem micrograph of dentin surface treated with 0.1% benzalkonium chloride - based disinfectant in group 3 . rt : resin tag , ar : adhesive resin sem micrograph of dentinal tubules filled with resin tags in group 7 . dt : dentinal tubule , rt : resin tag , ar : adhesive resin sem micrograph of dentin surface treated with chlorhexidine gel and an etch - and - rinse bonding system ( group 9 ) . in the present study , the benzalkonium - based iodine - potassium iodide / copper sulfate ( i2 ki / cuso4)-based disinfectants and chx solution decreased the bond strength when used with self etching bonding system , whereas no adverse effect was observed when used with the etch - and - rinse adhesive . however , irrespective of the bonding system used , the chx gel had no adverse effect on the shear bond strength . benzalkonium chloride , and i2ki / cuso4 -based solutions have been commonly used as cavity disinfectants in clinical practice because of their disinfecting action and wettability property . it has been stated that after deproteinization dentin may exhibit a very porous structure with many irregularities and anastomoses and hence promote the bond strength , but controversial results still exist.[1114 ] from various laboratory results , it has been observed that depending on the testing methodology and the adhesive system used , dentin surface treatment with these disinfecting solutions can increase , decrease , or have no effect on the bond strength . this result is in line with the study by gwinnet et al . that these cavity disinfectants had no effect on the bond strength when the etch - and - rinse technique was used . chx , due to its excellent antibacterial activity , has also been used as a cavity disinfectant for many years . chx has an excellent rewetting capacity and a strong affinity to tooth structure which is thought to improve the bond strengths of the adhesive to dentin . however in the present study , the chx solution exerted an adverse effect on the shear bond strength when used with the self - etching bonding system . this observation is in accordance with results of gurgan et al . that using chlorhexidine prior to or after etching the dentin without rinsing could adversely affect the shear bond strength of the dentin bonding agent yet , few other studies showed that applying chlorhexidine before acid etching did not significantly affect the bond strength.[2224 ] our study presented a similar result for specimens treated with the etch - and - rinse system and the chx solution . we did an extensive research of the concerned literature but did not discover any previous study using the chx gel as a cavity disinfectant . the use of the chx gel in our study did not affect the bond strength of composite regardless of which bonding system was used ( self - etching or etch - and - rinse ) . this finding can be attributed to the fact that chx gel 's lack of effect on the bond strength may be due to its limited penetration into the dentin structure due to its gel form . on the other hand , the gel form may also limit the affinity of the material to the tooth structure . within the limitations of this laboratory study , it may be stated that routinely used disinfectants , except the chx gel , might have an adverse effect on the bond strength of self - etching adhesive systems . thus , when clinicians decide to use benzalkonium chloride based , i2ki / cuso4 based or chx solutions as cavity disinfectants , they should prefer an etch - and - rinse bonding system . however , the use of the chx gel as a cavity disinfectant may be advisable , as it does not have any adverse effect on the bond strength of self - etching and etch - and - rinse systems . further in vitro and in vivo studies are still needed to evaluate the effect of chx gel for cavity disinfection .

aim : the aim was to evaluate the effect of different cavity disinfectants on dentin bond strengths of composite resin applied with two different adhesive systems.materials and methods : two - hundred mandibular molars were sectioned parallel to the occlusal surface to expose dentin in the midcoronal one - third . the dentinal surfaces were polished with waterproof - polishing papers . the specimens were randomly divided into five groups of 40 teeth each as follows : group 1(control ) -- specimens were not treated with any cavity disinfectants . groups 2 - -5 ( experimental groups ) -- dentin surfaces were treated with the following cavity disinfectants , respectively ; 2% chlorhexidine solution , 0.1% benzalkonium chloride - based disinfectant , 1% chlorhexidine gel , and an iodine potassium iodide / copper sulfate - based disinfectant . the specimens were then randomly divided into two subgroups including 20 teeth each to evaluate the effect of different bonding systems . dentin bonding systems were applied to the dentin surfaces and the composite buildups were done . after the specimens were stored in an incubator for 24 hours , the shear bond strength was measured at a crosshead speed of 1 mm / min . the specimens were then statistically analyzed.statistical analysis used : one way analysis of variance and tukey - hsd tests were used.results:there was no significant difference between chlorhexidine gel and control groups regardless of the type of the bonding agent used ( p>0.05 ) . on the other hand , pretreatment with benzalkonium chloride - based , iodine potassium iodide / copper sulfate - based disinfectants or chlorhexidine solutions had a negative effect on the shear bond strength of self - etching bonding systems.conclusions:the findings of this study suggest that when benzalkonium chloride - based , iodine potassium iodide / copper sulfate - based disinfectants or chlorhexidine solutions are used as a cavity disinfectant , an etch - and - rinse bonding system should be preferred .

Introduction Materials and Methods Results Discussion Conclusion

PMC4597829

the food practices of humans are determined by values , attitudes , beliefs , and environmental and religious circumstances ; all of which are the products of tradition , culture , and contacts ( onuorah and ayo , 2003 ) . knowledge and culture affect the intake of a particular food ( asp , 1999 ) . the success in understanding the culture of other countries or ethnic groups lies in understanding their rituals in food consumption customs ( nam et al . , 2010 ) . in developing countries , culture plays a crucial role in determining food patterns ( lahsaeizadeh , 2001 ) . according to kebede ( 2010 ) , cultural diversity is the unique feature of ethiopia ; the country s population composed of about 80 ethnic groups whose cultures are diverse one another . each ethnic group has its own culture manifested to the widely practiced diet ( national foods ) , way of living , celebrations , dressing and dances at the cities and the cultural fabric intertwining is still continuing ( kebede , 2010 ) . it was indicated by twigg ( 1984 ) that almost all cultures build their principal meal around meat . meat has social significance in family gatherings , making friendships , prestige by offering dinners etc . ( mapp , 1994 ) . in ethiopia , there are occasions in which meat plays pivotal and vital parts and its cultural symbolic weight is markedly greater than that accorded to most other foods . these include holidays , initiation rites and visitations by important guests ( kifleyesus , 2007 ) . on feasting days and social ceremonies of ethiopia , according to bea ( 1993 ) , meat products are eaten and the stews are also made mainly from chicken , beef , lamb and mutton . meat and poultry consumptions in ethiopia have associated peculiar cultural practices , for instance : the peoples use the oldest and cultural preservation of meat and prepare traditional dishes from meat , processing and cooking of poultry is a gender based duty and has socio - cultural roles , meat by - products are utilized for preparation of traditional dishes , and the peoples are dependent on limited types of source of animals for meats due to the taboo culturally associated . it is a fact that the share of meat in the human diet has been closely related with a life style , wealth , habits , religious beliefs and human awareness . cultural and religious considerations have always played , and still play , a significant role in the preparation and consumption of meat products ( borowski , 2007 ) . religious - beliefs shape also the social behaviors where differences in religious affiliations tend to influence the way people live , the choices they make , the food they eat , and with whom they associate ( kim et al . , 2004 ) . the population of ethiopia is diverse in religious beliefs : ethiopian orthodox christianity and islam are the major religions , traditional animist religions being practiced by many ethnic groups , and minority of followers of other christian denominations is existing ( united nations , 2004 ) . varying in their beliefs , ethiopia s religions have a significant role to play on consumption of meat and meat products by setting the feeding habits and customs of the people which in turn influences the pattern of meat consumption in the country . therefore , this review will consider various factors , mainly religious and their beliefs , which have a considerable effects on consumption of food of animal origin in ethiopia , positively or the negatively . the consumption of animal products and more specifically meat and meat products is most strictly regulated in cases where religious considerations prevail ( shatenstein and ghadirian , 1997 ) . it was posited by sheikh and thomas ( 1994 ) that the religious groups to which people belong will determine food practices according to their religion . differences in religious affiliations tend to influence the way people live , the choices they make , what they eat and whom they associate with ; the beliefs play significant parts in sculpting social behavior ( kim et al . , 2004 ) and are inbuilt to dictate what a person can eat and what he can not ( onuorah et al . , 2003 ) . the consumption of meat and meat products in ethiopia has very tidy association with religious beliefs , and are influenced by religions . in ethiopia , according to csa ( 2004 ) , christians generally represent 62.8% of the population : 43.5% orthodox christians , 18.6% protestant and 0.7% catholic . because these main religions of ethiopia have their own peculiar doctrines in detecting the feeding pattern of their followers , it will be critical to understand the situation of animals flesh food consumption in relation to the religious influences . the influences of these ethiopian mainly religions on the peoples meat products consumption can be illustrated into two ways ; the beliefs dictate the source animals that should be used or not be used for food , and schedule the days of the years in periodical permeation and restriction of meat products consumptions . the coptic orthodox church , the dominant religious sect , has been dictating many food customs in ethiopia since the fourth century ( bea , 1993 ) . according to the belief of ethiopian orthodox tewahedo christians , the faithful must abstain from eating meat and dairy products to attain forgiveness of sins committed during the year , and undergo a rigorous schedule of prayers and atonement ( teklehaimanot , 2005 ) . therefore , followers do not eat meat and dairy products ( i.e. egg , butter , milk , and cheese ) on fasting days such as wednesdays and fridays except the 50 days running from easter , the fast of the prophets , the fast of nineveh , lent , the fast of the apostles and the fast of the holy virgin mary ( teklehaimanot , 2005 ) . the ethiopian orthodox christians follow fasts in a way similar to other orthodox christians but with a frequency of approximately 250 days in a year ( rakesh and tafesse , 2010 ) . this religious influence along with the poor national economy results in the low per capita meat consumption ( table 1 ) . the three ethiopia s christian sects such as orthodox , protestant and catholic , 62.8% of the total population , have the same ritual process for animal slaughtering , which is , slaughtering the animals in the name of trinity : the name of father god , son jesus and the holly sprit . whereas , muslim s ritual slaughtering entails that the animal is killed in god s name , based on these differences only existing in ritual animals slaughtering between christians and muslims , the municipals abattoirs and slaughter house facilities in most of the ethiopia s cities and towns are constructed and provide service by targeting of the muslims and the christians . on the fasting days of orthodox christian followers , which accounts for 69.27% of the christian and 43.5% of the total population , most of the butchers do nt slaughter and give service except those few owned by muslims . in most of the cases , the christians slaughtering services cease without considering any of the other christian sects followers , for instance those of the protestant christians , who are the abstainers of the orthodox christian s fasting . this is resulted from the dominancy of orthodox christians in the country , according to bea ( 1993 ) , the coptic church of ethiopia is the dominant religious sect since the fourth century that has been dictating many of the food customs . a case study of avery ( 2004 ) which was conducted in the title , red meat and poultry production and consumption in ethiopia and distribution in addis ababa , indicated that the live animals purchased by butcheries or supermarkets are sent directly to the official addis ababa abattoirs enterprise , and sent through the orthodox , muslim , or european slaughter facilities . the third slaughter service does not refer the local / citizens service as it is not common in most the ethiopian abattoirs . the orthodox christians fasting influences consumption of meat by reducing the supply , for example , in the case of the protestant christian followers , 7% population , who their fasting does nt forbid eating meat , children , people with medical case and others are forced to limit or withdraw from consuming meat . according to avery ( 2004 ) , about 85.3% butcheries in addis ababa were closed during the traditional wednesday and friday orthodox christians fasting to sell an average of 313.5 kg raw beef per week . it was reported by tewodros ( 2008 ) that the periods for low demand of cattle meat was observed in correspond to the fasting period of ethiopian orthodox followers . this is because the slaughter houses cease or minimize their services since the butcheries stop ordering cattle slaughter services ( tewodros , 2008 ) . alike to the consumption of meat ( betru and kawashima , 2009 ) , the periods of low bird sales and consumption coincide with orthodox christians fasting ; the pre - easter fasting period which lasts about two months from february through march . the other low sales and consumption period is during the pre - christmas fasting period ( betru and kawashima , 2009 ) . according to aklilu ( 2007 ) , in northern ethiopia particularly in tigray , most strict orthodox christians households - especially in rural areas - abstain from eating animal products during the easter fasting period , pre - christmas fasting period and on wednesday and fridays . there are fluctuations across the months of the year in sales as well as in consumption of both birds and eggs ( betru and kawashima , 2009 ) . in a nutshell , near 62.8% of ethiopia s population , the total christians , withdraw from meat products on an average for about 250 d of the year due to this religious belief . if some christians consuming meat product could be found , it should be by slaughtering chicken as it is on a regular basis done at home or by illegal slaughtering sheep , goat and cattle ( some hotels may be ) at the backyard which is by itself trouble for meat quality and safety control in the country . therefore , this system calls for the attention of government in planning strategy and managing accordingly . regarding the consumption of fish , its demand and supply increase as the demand and supply of meat and poultry declines , and the vice versa . on those fasting days when meat is prohibited by ethiopian coptic orthodox , fish plays an important role because it is not among the forbidden dishes . during lent , for example , christians who abstain from eating meat , milk and eggs consume fish . as a common dish of fasting days , peoples consume fish and vegetarian meals by making the stews from pulses , lentils , peas , field peas , chick peas , peanuts and varieties of vegetables ( teklehaimanot , 2005 ) . generally , there is a limited interest and habit of eating fish with ethiopia s people . this is because of the accessibility problem in related to the shortage of processing and distribution of fish , and religious influences on the consumption pattern . in fact , ethiopian orthodox tewahedo christians do nt restrict the consumption of fish in all over the year inclusively of the fasting schedules . however , ethiopian people have related eating fish with fasting days . because religion may be regarded as the mainspring of culture setting principles for life , and influencing the attitudes in many instances , according to kinsey ( 1988 ) , most of the restaurants and butchers serve fish as substitute of meat . the fish market will have higher supply following the fasting days demand than any the other days , and this leads people to shift their dish towards fish . for the reason that fish consumption trend is also influenced by cultural / geographical regions ( richard and marcia , 2004 ) , it will not be easy to find fish in other times than those fasting days of ethiopian coptic orthodox followers unless it could be in the biggest cities like addis ababa , adama , bahir dare , awassa , and arba minch that have the supply of fish nearby , and many restaurants , super markets and centers where fish are sold exist . therefore , people whose main dish is fish culturally or / and those who prefer fish in their diet may have better access for fish on the fasting days . the religious influence in the consumption fish is observed , therefore , through periodical shifting of the access and the consumption behavior of the followers on fasting day s base . some conservative coptic orthodox followers are , moreover , exhibiting a tendency of withdrawing from eating fish in fasting days with a premise that fish has blood as any of the prohibited animals in nowadays . in respect to the ethiopian muslim religion , there is no any periodical or seasonal restriction for any of the permitted animal products that influence the consumption behavior of the followers . however , there is a belief influence of the muslim religion which is related with the restriction of the source of animal to which their fleshes are not allowed for food . considering the two ethiopian major religions in broad , the christian sects in general and the islam have influential role by restricting their followers for some of the type of animal s flesh foods . both , ethiopian christians and muslims in common do not eat pork as it is forbidden by their religious beliefs ( teklehaimanot , 2005 ) . in fact , the consumption of animal products , more specifically meat and meat products is most strictly regulated in cases where religious considerations prevail ( shatenstein and ghadirian , 1997 ) . the characteristics of the foods that koran and the sunna exhort muslims to eat and not to eat were reported by johanna ( 2009 ) as muslims are expressly forbidden from consuming carrion , spurting blood , pork , and foods that have been consecrated to any being other than god himself . except those that are explicitly prohibited in islam , according to abdullah ( 2004 ) , muslims are allowed to consume all foods ( e.g. , grains , vegetables , fish and meat ) . following the ritual restriction of eating pork for all muslim religion followers as johanna ( 2009 ) indicated , ethiopian muslim similar to other world muslims food by the followers . on the side of all of the christian sects in ethiopia , they have a common restriction for some animal meats which is based on the old testament of holy bible that characterizes the animals used for consumption . for mammals , so as to be served as a food , they need to be ruminants as well as not to have hoof of two fully splits , and for birds , they need to have fingers of fully splits with no attachment one another . by relating with this stated religious belief , therefore , pig does nt fulfill the stated criteria . therefore ethiopian christians do nt eat pigs and it is among the forbidden called as erkuse alike the term haram used with the muslims context . meat of camel is nt also eaten by ethiopian christians using similar affiliation criteria that are used for animal selection in order to be served as food . there is no restriction for the consumption of camel meat by the ethiopian muslim religion as far as religion restriction concerned . lawful or permitted , for ethiopian muslim , and it is also beloved by the followers unless the production states and the agro - ecology of camel production limits the supply and consumption . consumption of chicken in respect to the ethiopian people has very cultural practices , that is , the preparation process of the national dish , doro wat , has strict traditional guidelines and gendered roles . cuisine may vary from region to region but regardless of their religion , ethiopian women learn the ritualized process of making this traditional dish as a rite of passage ( natasha , 2011 ) . killing animals is a job reserved for men but only women know how to cook it - men are not allowed into the kitchen . a proper lady knows how to cut a chicken into 12 perfect pieces ( janet et al . , 2013 ) . when the blood is drained , according to natasha ( 2011 ) , women begin the laborious task of cleaning the carcass . this is done very precisely so that each wing , leg , chest , thigh , back and breast mirror each other and all veins are done away with . in the west , of course , chicken is processed before being packaged and can be bought ready to cook from the grocery store . ethiopian women , however , buy the whole live chicken and cut it up in the traditional manner ( natasha , 2011 ) . in ethiopians culture , a proper lady needs to know how to cut a chicken into 12 perfect pieces . they all have names like feresenga ( a perfect cut of the breast served customarily to the man of the house ) and no matter how many chickens she uses , they all come out the same shape . there are plenty of jokes about a woman who is not able to cut chicken properly ( janet et al . , 2013 ) culturally , it is considered to be a shame when an ethiopian woman failed in the art of cutting and naming the 12 perfect pieces of a chicken . doro wat is a saucy chicken stew which is served with injera and boiled eggs . must dish in ethiopian culture for holidays and social ceremonies independent of the differences in religions . poultry are used for strengthening marriage partnerships and social relationships . in the local culture , women who can provide men with food like a chicken dish ( doro wat ) are considered to be contributing to a stable marriage . serving doro wat is also a demonstration of respect to guests , thus strengthening a social relationship which is especially important for poor households ( demeke , 2008 ) . commonly , people in ethiopia select the types of chicken for consumption ; most consumers prefer to buy local chickens from village producers , since local chickens are considered to be tasty and better suited for preparation of the traditional chicken sauce ( doro wat ) . eggs from local chicken are often favored because of their deep yellow colored yolks . as a consequence of the cultural taboo associated with chicken , the price of live birds varies depending on sex , color and size ( betru and kawashima , 2009 ) . in general , the chickens most frequently preferred by many consumers are the double combs or unique colors of native birds for sacrificial purposes ( avery , 2004 ) . most farmers , therefore , prefer to keep native birds of plumage colors , specially the white ( netch ) and the red ( key ) colors than others , due to the high price they fetch in the local market . this is mainly attributed to the preference of consumers in the market which is based on the cultural value that favor for rose ( double ) comb type of chicken . despite there is no religious recommendation for the types of chicken needed for the sacrificial purposes on the bases of the religious holidays celebrated , majority of the people correspond the plumage color of the chickens with the types of the religious holidays as a result of the cultural taboo associated . for instance , for ethiopian zemen melewecha ( new year ) and for the orthodox christians meskel ( finding of the true cross ) holidays , chicken with white ( netch ) plumage color and double comb type of indigenous chicken are favored , whereas for ethiopian christians fasika ( easter ) holiday , chicken with red ( key ) plumage color and double comb types are favored . in rare cases , chicken with plumage color of black ( tikur ) are preferred by traditional belief followers for sacrificial purposes due the cultural taboo associated . in ethiopia , a cow or an ox can be butchered for the sole purpose of selling within the community . in special occasions and holidays , people have a cultural ceremony for slaughtering cows or oxen , not any other animals and sharing among the group , called kircha , which is a very common option of the rural areas where access of meat is challenging . commonly , a group of 10 to 20 people buy a live animal , slaughter and divide the meat among them . kircha is a form of people s organization and sharing meat among themselves - butchering live animal and sharing the meat in group . the style is also common in some of ethiopia towns people by which the elders especially enjoy being involved in the activity . on holidays , neighbors join together into kircha to buy a large ox and equally divide it then draw lots to decide which pile they get . it is cheaper than going to the butcher ( janet et al . , 2013 ) . according to kifleyesus ( 2007 ) , the meat is divided on the basis of equal portions comprising a package of every internal organ , muscle meat , and bone of the slaughtered animal . size of the package is determined by the number of divisions ( pankhurst , 1988 ) . the greater the number of divisions , the smaller will be the size of a package of meat . the consumption of animal products and more specifically meat and meat products is most strictly regulated in cases where religious considerations prevail ( shatenstein and ghadirian , 1997 ) . it was posited by sheikh and thomas ( 1994 ) that the religious groups to which people belong will determine food practices according to their religion . differences in religious affiliations tend to influence the way people live , the choices they make , what they eat and whom they associate with ; the beliefs play significant parts in sculpting social behavior ( kim et al . , 2004 ) and are inbuilt to dictate what a person can eat and what he can not ( onuorah et al . , 2003 ) . the consumption of meat and meat products in ethiopia has very tidy association with religious beliefs , and are influenced by religions . in ethiopia , according to csa ( 2004 ) , christians generally represent 62.8% of the population : 43.5% orthodox christians , 18.6% protestant and 0.7% catholic . because these main religions of ethiopia have their own peculiar doctrines in detecting the feeding pattern of their followers , it will be critical to understand the situation of animals flesh food consumption in relation to the religious influences . the influences of these ethiopian mainly religions on the peoples meat products consumption can be illustrated into two ways ; the beliefs dictate the source animals that should be used or not be used for food , and schedule the days of the years in periodical permeation and restriction of meat products consumptions . the coptic orthodox church , the dominant religious sect , has been dictating many food customs in ethiopia since the fourth century ( bea , 1993 ) . according to the belief of ethiopian orthodox tewahedo christians , the faithful must abstain from eating meat and dairy products to attain forgiveness of sins committed during the year , and undergo a rigorous schedule of prayers and atonement ( teklehaimanot , 2005 ) . therefore , followers do not eat meat and dairy products ( i.e. egg , butter , milk , and cheese ) on fasting days such as wednesdays and fridays except the 50 days running from easter , the fast of the prophets , the fast of nineveh , lent , the fast of the apostles and the fast of the holy virgin mary ( teklehaimanot , 2005 ) . the ethiopian orthodox christians follow fasts in a way similar to other orthodox christians but with a frequency of approximately 250 days in a year ( rakesh and tafesse , 2010 ) . this religious influence along with the poor national economy results in the low per capita meat consumption ( table 1 ) . the three ethiopia s christian sects such as orthodox , protestant and catholic , 62.8% of the total population , have the same ritual process for animal slaughtering , which is , slaughtering the animals in the name of trinity : the name of father god , son jesus and the holly sprit . whereas , muslim s ritual slaughtering entails that the animal is killed in god s name , based on these differences only existing in ritual animals slaughtering between christians and muslims , the municipals abattoirs and slaughter house facilities in most of the ethiopia s cities and towns are constructed and provide service by targeting of the muslims and the christians . on the fasting days of orthodox christian followers , which accounts for 69.27% of the christian and 43.5% of the total population , most of the butchers do nt slaughter and give service except those few owned by muslims . in most of the cases , the christians slaughtering services cease without considering any of the other christian sects followers , for instance those of the protestant christians , who are the abstainers of the orthodox christian s fasting . this is resulted from the dominancy of orthodox christians in the country , according to bea ( 1993 ) , the coptic church of ethiopia is the dominant religious sect since the fourth century that has been dictating many of the food customs . a case study of avery ( 2004 ) which was conducted in the title , red meat and poultry production and consumption in ethiopia and distribution in addis ababa , indicated that the live animals purchased by butcheries or supermarkets are sent directly to the official addis ababa abattoirs enterprise , and sent through the orthodox , muslim , or european slaughter facilities . the third slaughter service does not refer the local / citizens service as it is not common in most the ethiopian abattoirs . the orthodox christians fasting influences consumption of meat by reducing the supply , for example , in the case of the protestant christian followers , 7% population , who their fasting does nt forbid eating meat , children , people with medical case and others are forced to limit or withdraw from consuming meat . according to avery ( 2004 ) , about 85.3% butcheries in addis ababa were closed during the traditional wednesday and friday orthodox christians fasting to sell an average of 313.5 kg raw beef per week . it was reported by tewodros ( 2008 ) that the periods for low demand of cattle meat was observed in correspond to the fasting period of ethiopian orthodox followers . this is because the slaughter houses cease or minimize their services since the butcheries stop ordering cattle slaughter services ( tewodros , 2008 ) . alike to the consumption of meat ( betru and kawashima , 2009 ) , the periods of low bird sales and consumption coincide with orthodox christians fasting ; the pre - easter fasting period which lasts about two months from february through march . the other low sales and consumption period is during the pre - christmas fasting period ( betru and kawashima , 2009 ) . according to aklilu ( 2007 ) , in northern ethiopia particularly in tigray , most strict orthodox christians households - especially in rural areas - abstain from eating animal products during the easter fasting period , pre - christmas fasting period and on wednesday and fridays . there are fluctuations across the months of the year in sales as well as in consumption of both birds and eggs ( betru and kawashima , 2009 ) . in a nutshell , near 62.8% of ethiopia s population , the total christians , withdraw from meat products on an average for about 250 d of the year due to this religious belief . if some christians consuming meat product could be found , it should be by slaughtering chicken as it is on a regular basis done at home or by illegal slaughtering sheep , goat and cattle ( some hotels may be ) at the backyard which is by itself trouble for meat quality and safety control in the country . therefore , this system calls for the attention of government in planning strategy and managing accordingly . regarding the consumption of fish , its demand and supply increase as the demand and supply of meat and poultry declines , and the vice versa . on those fasting days when meat is prohibited by ethiopian coptic orthodox , fish plays an important role because it is not among the forbidden dishes . during lent , for example , christians who abstain from eating meat , milk and eggs consume fish . as a common dish of fasting days , peoples consume fish and vegetarian meals by making the stews from pulses , lentils , peas , field peas , chick peas , peanuts and varieties of vegetables ( teklehaimanot , 2005 ) . generally , there is a limited interest and habit of eating fish with ethiopia s people . this is because of the accessibility problem in related to the shortage of processing and distribution of fish , and religious influences on the consumption pattern . in fact , ethiopian orthodox tewahedo christians do nt restrict the consumption of fish in all over the year inclusively of the fasting schedules . however , ethiopian people have related eating fish with fasting days . because religion may be regarded as the mainspring of culture setting principles for life , and influencing the attitudes in many instances , according to kinsey ( 1988 ) , most of the restaurants and butchers serve fish as substitute of meat . the fish market will have higher supply following the fasting days demand than any the other days , and this leads people to shift their dish towards fish . for the reason that fish consumption trend is also influenced by cultural / geographical regions ( richard and marcia , 2004 ) , it will not be easy to find fish in other times than those fasting days of ethiopian coptic orthodox followers unless it could be in the biggest cities like addis ababa , adama , bahir dare , awassa , and arba minch that have the supply of fish nearby , and many restaurants , super markets and centers where fish are sold exist . therefore , people whose main dish is fish culturally or / and those who prefer fish in their diet may have better access for fish on the fasting days . the religious influence in the consumption fish is observed , therefore , through periodical shifting of the access and the consumption behavior of the followers on fasting day s base . some conservative coptic orthodox followers are , moreover , exhibiting a tendency of withdrawing from eating fish in fasting days with a premise that fish has blood as any of the prohibited animals in nowadays . in respect to the ethiopian muslim religion , there is no any periodical or seasonal restriction for any of the permitted animal products that influence the consumption behavior of the followers . however , there is a belief influence of the muslim religion which is related with the restriction of the source of animal to which their fleshes are not allowed for food . considering the two ethiopian major religions in broad , the christian sects in general and the islam have influential role by restricting their followers for some of the type of animal s flesh foods . both , ethiopian christians and muslims in common do not eat pork as it is forbidden by their religious beliefs ( teklehaimanot , 2005 ) . in fact , the consumption of animal products , more specifically meat and meat products is most strictly regulated in cases where religious considerations prevail ( shatenstein and ghadirian , 1997 ) . the characteristics of the foods that koran and the sunna exhort muslims to eat and not to eat were reported by johanna ( 2009 ) as muslims are expressly forbidden from consuming carrion , spurting blood , pork , and foods that have been consecrated to any being other than god himself . except those that are explicitly prohibited in islam , according to abdullah ( 2004 ) , muslims are allowed to consume all foods ( e.g. , grains , vegetables , fish and meat ) . following the ritual restriction of eating pork for all muslim religion followers as johanna ( 2009 ) indicated , ethiopian muslim similar to other world muslims food by the followers . on the side of all of the christian sects in ethiopia , they have a common restriction for some animal meats which is based on the old testament of holy bible that characterizes the animals used for consumption . for mammals , so as to be served as a food , they need to be ruminants as well as not to have hoof of two fully splits , and for birds , they need to have fingers of fully splits with no attachment one another . by relating with this stated religious belief , therefore , pig does nt fulfill the stated criteria . therefore ethiopian christians do nt eat pigs and it is among the forbidden called as erkuse alike the term haram used with the muslims context . meat of camel is nt also eaten by ethiopian christians using similar affiliation criteria that are used for animal selection in order to be served as food . there is no restriction for the consumption of camel meat by the ethiopian muslim religion as far as religion restriction concerned . lawful or permitted , for ethiopian muslim , and it is also beloved by the followers unless the production states and the agro - ecology of camel production limits the supply and consumption . consumption of chicken in respect to the ethiopian people has very cultural practices , that is , the preparation process of the national dish , doro wat , has strict traditional guidelines and gendered roles . cuisine may vary from region to region but regardless of their religion , ethiopian women learn the ritualized process of making this traditional dish as a rite of passage ( natasha , 2011 ) . killing animals is a job reserved for men but only women know how to cook it - men are not allowed into the kitchen . proper lady knows how to cut a chicken into 12 perfect pieces ( janet et al . , 2013 ) . when the blood is drained , according to natasha ( 2011 ) , women begin the laborious task of cleaning the carcass . this is done very precisely so that each wing , leg , chest , thigh , back and breast mirror each other and all veins are done away with . in the west , of course , chicken is processed before being packaged and can be bought ready to cook from the grocery store . ethiopian women , however , buy the whole live chicken and cut it up in the traditional manner ( natasha , 2011 ) . in ethiopians culture , a proper lady needs to know how to cut a chicken into 12 perfect pieces . they all have names like feresenga ( a perfect cut of the breast served customarily to the man of the house ) and no matter how many chickens she uses , they all come out the same shape . there are plenty of jokes about a woman who is not able to cut chicken properly ( janet et al . , 2013 ) . culturally , it is considered to be a shame when an ethiopian woman failed in the art of cutting and naming the 12 perfect pieces of a chicken . doro wat is a saucy chicken stew which is served with injera and boiled eggs . must dish in ethiopian culture for holidays and social ceremonies independent of the differences in religions . poultry are used for strengthening marriage partnerships and social relationships . in the local culture , women who can provide men with food like a chicken dish ( doro wat ) are considered to be contributing to a stable marriage . serving doro wat is also a demonstration of respect to guests , thus strengthening a social relationship which is especially important for poor households ( demeke , 2008 ) . commonly , people in ethiopia select the types of chicken for consumption ; most consumers prefer to buy local chickens from village producers , since local chickens are considered to be tasty and better suited for preparation of the traditional chicken sauce ( doro wat ) . eggs from local chicken are often favored because of their deep yellow colored yolks . as a consequence of the cultural taboo associated with chicken , the price of live birds varies depending on sex , color and size ( betru and kawashima , 2009 ) . in general , the chickens most frequently preferred by many consumers are the double combs or unique colors of native birds for sacrificial purposes ( avery , 2004 ) . most farmers , therefore , prefer to keep native birds of plumage colors , specially the white ( netch ) and the red ( key ) colors than others , due to the high price they fetch in the local market . this is mainly attributed to the preference of consumers in the market which is based on the cultural value that favor for rose ( double ) comb type of chicken . despite there is no religious recommendation for the types of chicken needed for the sacrificial purposes on the bases of the religious holidays celebrated , majority of the people correspond the plumage color of the chickens with the types of the religious holidays as a result of the cultural taboo associated . for instance , for ethiopian zemen melewecha ( new year ) and for the orthodox christians meskel ( finding of the true cross ) holidays , chicken with white ( netch ) plumage color and double comb type of indigenous chicken are favored , whereas for ethiopian christians fasika ( easter ) holiday , chicken with red ( key ) plumage color and double comb types are favored . in rare cases , chicken with plumage color of black ( tikur ) in ethiopia , a cow or an ox can be butchered for the sole purpose of selling within the community . in special occasions and holidays , people have a cultural ceremony for slaughtering cows or oxen , not any other animals and sharing among the group , called kircha , which is a very common option of the rural areas where access of meat is challenging . commonly , a group of 10 to 20 people buy a live animal , slaughter and divide the meat among them . kircha is a form of people s organization and sharing meat among themselves - butchering live animal and sharing the meat in group . the style is also common in some of ethiopia towns people by which the elders especially enjoy being involved in the activity . on holidays , neighbors join together into kircha to buy a large ox and equally divide it then draw lots to decide which pile they get . it is cheaper than going to the butcher ( janet et al . , 2013 ) . according to kifleyesus ( 2007 ) , the meat is divided on the basis of equal portions comprising a package of every internal organ , muscle meat , and bone of the slaughtered animal . size of the package is determined by the number of divisions ( pankhurst , 1988 ) . the greater the number of divisions , the smaller will be the size of a package of meat . the consumption of meat was one of the factors that differentiated the society of antiquity . cultural and religious aspects have played a major role in the processing of meat and their consumption ( kim et al . , 2004 ) . in different cultures , pigs , cows , horses , poultry , dogs , camels , deer , cats or rats can be accepted or not as providers of meat ( borowski , 2007 ) . a wide range of mammalian species are also eaten in different parts of the world according to their availability or local custom . thus , for example , the seal and polar bear are important in the diet of the eskimos , and the giraffe , rhinoceros , hippopotamus and elephant in that of certain tribes of central africa ( frederick , 2000 ) . despite all of these animals are used as sources of meat in other parts of the world , they are not incorporated nor have parts in the diet of ethiopian people . in addition , the predominant bird species that is used as a food source by ethiopians is chicken . other species such as ducks , geese , turkeys , pigeons , quails , etc . are rare in ethiopia , and were not seen to be used as food or tried to include in the ethiopian farming system . there is no data available regarding these species except a case that was reported for the opening of a duck farm at chancho in oromiya region in the early 2000 s ( paolo and wossene , 2008 ) . according to sheikh and thomas ( 1994 ) , there is no specific theory considered as a universal theory of product acceptance which generally governs the behavior of people . therefore , people could have different reasons why they are reserved from consuming meats of some animals . as far as the behavior of ethiopian people is concerned , they are sensitive in respect to their culture and very conservative in food . as a result , they commonly tend to be reserved even from touching meats of animals that are not used culturally as sources of meat due to the taboos associated . for instance , avery ( 2004 ) has indicated the uniqueness of behavior of ethiopians , in which many of the people exhibit apprehension towards the meats sold in supermarkets because they are suspicious about the types of meat sold ( i.e. , pork ) . in fact , there is no a well - regarded intervention in ameliorating the meager meat consumption culture of the ethiopian people or / and the utilization of different species of meat animals available in ethiopia as means of income earning . it is also observed that foreigners , who come to ethiopia and have consumption cultures of meats of those animals not used by ethiopians , withdraw from their culture and consume the locally supplied meats . this is because they are aware of the conservative attitude of local people to their culture and therefore , they tend to assimilate themselves in the local cultures than trying to shift the traditional eating habit of local people whose meat source are dependent on limited types of animals . the attempts of changing the meat consumption culture and the supply of meat sources in ethiopia will be anticipated in order to enhance the utilization of meat from varies spices of animals , which could meet the challenge of improving the balance of nutrients available to the population , the opportunity of decreasing malnutrition , thereby improving food security and deriving economic benefits . a gradual change of the people s behavior in their meat consumption culture can be achieved , for instance , by encouraging the supermarkets to sell meats of different animals , by frequently preparing of experience sharing and exhibitions that avail the cultural practices of other worlds , and by celebrating national days of food which promote meat foods of different source animals . according to aberra ( 2006 ) , once the types of food are selected in a gradual adaptation process , they become established as staple diets of a particular society .

the consumption of animal flesh food in ethiopia has associated with cultural practices . meat plays pivotal and vital parts in special occasions and its cultural symbolic weight is markedly greater than that accorded to most other food . processing and cooking of poultry is a gender based duty and has socio - cultural roles . ethiopians are dependent on limited types of animals for meats due to the taboo associated culturally . moreover , the consumption of meat and meat products has a very tidy association with religious beliefs , and are influenced by religions . the main religions of ethiopia have their own peculiar doctrines of setting the feeding habits and customs of their followers . they influence meat products consumption through dictating the source animals that should be used or not be used for food , and scheduling the days of the years in periodical permeation and restriction of consumptions which in turn influences the pattern of meat consumption in the country . in ethiopia , a cow or an ox is commonly butchered for the sole purpose of selling within the community . in special occasions , people have a cultural ceremony of slaughtering cow or ox and sharing among the group , called kircha , which is a very common option of the people in rural area where access of meat is challenging frequently .

Introduction Factors Affecting the Meat Consumption Culture Religion Gender Community spirit Ethiopian Meat Consumption Behavior

PMC5165230

under general anesthetics , decreased hearing sensitivity is common in both animal research and clinical settings . several studies have demonstrated that different anesthetics increase auditory brainstem response thresholds [ 13 ] and depress neural excitability in the auditory midbrain [ 4 , 5 ] and cortex [ 68 ] . the sensitivity of the auditory system could be changed by anesthetics at two levels : the cochlea and the auditory neurons . because any change in cochlear function may influence the response of central auditory neurons , the effects on the cochlea are essential for the anesthetic - induced reduction in hearing sensitivity . however , the majority of studies have focused on the neural responses , whereas few have examined cochlear function . urethane has been widely used in animal research for more than a century because it exerts minimal effects on the cardiovascular and respiratory systems . although urethane has been reported to depress the sound - evoked activity of the auditory system [ 46 ] , its direct effect on the cochlea , particularly sensory hair cells , remains unknown . sensory hair cells in the cochlea not only translate sound vibration into electrical impulses but also amplify the signals of low - level sound . the latter process , defined as cochlear amplification , confers incredible sensitivity on mammalian hearing in a tremendous intensity range . cochlear amplification in mammals is attributed to outer hair cells ( ohcs ) , which can alter their somatic length on the order of micrometers in response to membrane potential changes . this electromotility is powered by the unique motor protein , prestin , on the ohc lateral membrane . the voltage - dependent structural conformation of prestin drives ohc somatic motility , which regulates cochlear amplification [ 11 , 12 ] . however , the voltage - to - length change conversion function ( l - v ) of ohcs is nonlinear and asymmetric : depolarization produces larger cell length changes than comparable hyperpolarization [ 1315 ] . therefore , the changes in the membrane potential may alter the operating point on the l - v function and influence the overall level of cochlear amplification . because their electromechanical conversion occurs via a feedback mechanism , ohcs ohcs are innervated by efferent fibers that originate in the superior olivary complex [ 16 , 17 ] . these efferent fibers form synapses at the base of the ohcs and use acetylcholine ( ach ) as their primary neurotransmitter [ 18 , 19 ] . nicotinic ach receptors ( achr ) have been identified on ohcs [ 18 , 20 , 21 ] , and ach hyperpolarizes the membrane potential of isolated ohcs . the efferent activity of the olivocochlear nerve bundle during electrical stimulation has been shown to be inhibitory , thereby reducing the gain of the cochlear amplifier and providing protection to the ear against overstimulation . a pharmacological study has indicated that urethane enhances the function of nicotinic achrs while inhibiting the responses of nmda and ampa receptors . we hypothesize that urethane influences the micromechanics of the organ of corti via the ohcs and , in turn , the cochlear amplification process . if so , urethane anesthesia provides an alternative strategy to modulate cochlear amplification . by comparing the cochlear microphonic ( cm ) responses between awake and urethane - anesthetized rats we also measured the membrane potential and current as well as prestin activity in isolated ohcs in the presence of urethane . our results indicate that urethane hyperpolarizes the ohc membrane potential , which is at least partially mediated by the achr . all experimental preparations , surgeries , and protocols used in this study were approved by the animal care and use committee of southern medical university of china . healthy young sprague dawley rats of either sex ( 2128 days old , body weight 4070 g ) exhibiting normal hearing were used for the experiments . the cm measurements and whole - cell patch recordings were performed as previously described [ 26 , 27 ] . these methods are briefly described as follows . the scalp was removed , and a metal screw was mounted on the skull using glass ionomer cement . the animals were subcutaneously injected with 0.1 mg / kg buprenorphine and returned to their home cages to recover . during the recovery period , the animals were trained to become accustomed to being head - fixed in the recording setup . to fix the head , the screw was tightly clamped to a metal post . the rat was able to run freely on a plastic plate rotating around its center as described in our recent study . on the day of recording a small incision was made via a dorsolateral approach to the pinna to expose the acoustic bulla . a silver wire recording electrode ( tip diameter , ~500 m ) was placed near the round window membrane through an opening of 3 mm in diameter on the acoustic bulla ( figure 1(a ) ) . the animal was allowed to recover from isoflurane anesthesia for at least 30 min . to acquire the cm under awake conditions , then , the animal was intraperitoneally injected with 1 g / kg urethane using a pipette to examine the effects of this anesthetic . tone bursts ( 50 ms duration , 5 ms rise / fall time ) of various frequencies ( 2 , 4 , 8 , 16 , or 32 khz ) and intensities ( 070 db spl at 5 db intervals ) were presented using a calibrated tdt es1 speaker located 50 cm away from the recorded ear . the frequency - amplitude scan was computer controlled ( tdt system 3 , tucker - davis technologies ) and was delivered in a randomized sequence . the cm responses to the tone bursts were amplified , filtered , and recorded using an a / d converter ( 1440a/700b system , molecular devices ) . customized matlab software was used for offline data processing , such as response averaging and response amplitude extraction . the animals were anesthetized ( co2 inhalation ) and decapitated , and the inner ears were rapidly removed from the temporal bones and placed in leibovitz 's l-15 media ( invitrogen , carlsbad , ca ) . the organ of corti was isolated from the middle and apical turns of the cochlea . after mild enzymatic digestion for 5 min ( 2 mg / ml collagenase iv , sigma , st . louis , mo ) and gentle pipetting , the cells were transferred to a small plastic chamber filled with enzyme - free culture medium ( ~1.5 ml ) . the standard medium was leibovitz 's l-15 , supplemented with 10 mm hepes ( invitrogen , carlsbad , ca ) and adjusted to ph 7.35 and 300 mosm . then , the chamber containing the cells was placed on the stage of an inverted microscope ( nikon , eclipse fn1 ) equipped with a video camera . healthy - appearing solitary ohcs were selected for the electrophysiological experiments if they displayed no obvious signs of shrinkage , swelling , damage , or deterioration such as granularity or translocation of the nucleus . these experiments were performed at room temperature ( 22 4c ) under video monitoring . the ohcs were bathed in l-15 medium buffered with 10 mm hepes ( ph 7.35 , 300 mosm ) . the patch electrodes were pulled from 1.5 mm glass capillary tubes at resistances between 3 and 6 m using a horizontal micropipette puller ( model p-97 , sutter ) . the electrodes were back - filled with a solution containing ( in mm ) 145 kcl , 2 mgcl2 , and 10 hepes . the access resistance typically ranged from 10 to 17 m when the whole - cell recording configuration was established . in most of our recordings , the whole - cell currents were less than 3 na . under computer control , hyperpolarizing and depolarizing voltage steps ( 250 ms duration and ranging from 140 to + 94 mv in 13 mv increments ) the low - pass - filtered currents ( corner frequency of 5 khz ) were amplified using an axopatch 200b amplifier ( axon instruments ) . the urethane - evoked current responses were recorded in voltage - clamp mode . to obtain large urethane - evoked outward currents , the whole - cell currents and evoked current responses were acquired using pclamp 10 software ( molecular devices ) on a computer connected to an a / d converter ( digidata 1322a , axon instruments ) . the data were analyzed using the pclamp software package . for nonlinear capacitance ( nlc ) measurements , the membrane capacitance was measured using a two - sine - wave voltage stimulus protocol ( 10 mv peak at both 390.6 hz and 781.2 hz ) with subsequent fast fourier transform - based admittance analysis at a holding potential of 0 mv . the data were acquired using jclamp software ( scisoft , new haven , ct ) and were analyzed using originpro software ( originlab corporation , northampton , ma ) . the nlc can be described as the first derivative of a two - state boltzmann function of nonlinear charge movement to voltage . the capacitance function is described as(1)cm = clin+qmaxexpvmv1/21+expvmv1/22.four parameters ( qmax , v1/2 , clin , and z ) from the equation were obtained : qmax is the maximum charge transfer ; v1/2 is the peak of the voltage - dependent capacitance ; clin is the linear capacitance ; and = ze / kt is the slope of the voltage dependence of the charge transfer . furthermore , k is the boltzmann constant , t is absolute temperature , z is the valence of the charge movement , and e is the electron charge . clin is the linear capacitance representing the surface area of the membrane ( i.e. , the cell size ) . to compare the magnitude of the nlc and qmax obtained from different cells of varying size , we normalized the nlc and qmax to clin . the drugs were dissolved in standard medium ( l-15 ) adjusted to ph 7.35 and 300 mosm . urethane was delivered via pressure ejection from a micropipette with a tip diameter of ~5 m positioned 2050 m from the bottom of the cell . care was taken to assure that the application of a drug solution did not alter the position of the cell or influence the measurements . in the strychnine coapplication experiments , the strychnine solution was slowly perfused into the bath ( 1 ml / min ) without disturbing the position of the cells . all of the drugs were applied to achieve a final concentration until a consistent response was observed and a washout was performed after each application . a student 's t - test was used to examine the significance of the difference between the responses obtained before and during the drug applications . excel software and originpro software were used for calculating , data fitting , and plotting . the inner and outer hair cells , which are the sensory receptor cells of the inner ear , function as a transducer by converting the mechanical movement of the basilar membrane into an alternating electrical voltage . this alternating voltage is defined as the cm , which mimics the waveform of a sound stimulus . representative cm recordings are shown in figure 1 , in which the effects of urethane anesthesia are presented for the same rat . we performed cm recordings on head - fixed awake rats to monitor the receptor potential before and after urethane application ( figure 1(a ) ) . to avoid the middle ear reflex , mild tone bursts ( less than 70 db spl ) were used to elicit the cm responses . as shown in figure 1(b ) , an 8 khz tone ( level at 70 db spl ) evoked a cm at an amplitude of ~424 v . the amplitude of the cm responses at saturation levels was reduced by 44% to 238 v after the intraperitoneal injection of urethane . the proximity of the recording electrodes to the hair cells may affect the recorded amplitudes . to ensure that our control recordings were not influenced by the activity of the animal , we averaged at least three evoked cm measurements before the urethane injection . for the cms measured from all five rats , urethane induced a significant decrease in the cm of 39.3% on average ( p < 0.01 , student 's t - test ) . the time course of this urethane effect was examined in five rats that exhibited at least an 80% recovery in the cm amplitude . the representative changes in the cm over time after urethane injection are shown in figure 1(c ) . the initial decrease in the cm was observed ~25 min after the urethane injection , reached its lowest value within ~3 hours , and then recovered gradually . the time of the peak reduction was highly variable between different rats , ranging from 45 min to 3 hours ( 45 min , 75 min , 90 min , 3 hours , and 3.2 hours , resp . ) . the cm responses to different sound levels were also measured . as shown in figure 1(c ) , the changes in the cm amplitudes at different sound levels followed a similar time course . we compared the cm thresholds before and after urethane application ( figure 1(d ) ) . the cm thresholds were defined as the minimum sound level that evoked a detectable cm response . consistent with the cm magnitude measurements , the cm thresholds were increased by urethane application at all frequencies examined . the increase of threshold is similar and shows no significance at different sound frequencies ( p > 0.05 , student 's t - test ) . the cm response is dominated by the ohcs in the organ of corti [ 9 , 31 ] . as such , the cm reduction in our experiments represents a significant reduction in ohc activity after urethane application . to determine how urethane alters the responses of ohcs , whole - cell current- and voltage - clamp recordings were performed from ohcs acutely isolated from the middle and apical turns of the rat cochlea . isolated ohcs can easily be identified based on visual inspection : the ohcs display a cylindrical morphology with a nucleus located near the base , whereas inner hair cells are flask - shaped with an upper nucleus position . another indication of ohcs is a functional characteristic : the visible motile responses elicited by the rapid membrane potential changes generated during our patch - clamp measurements [ 22 , 32 ] . the average zero current membrane potential under whole - cell recording conditions was 54 mv ( sd = 7 mv , n = 19 ) . to determine the influence of urethane , different concentrations of urethane were applied to the recorded ohc via local perfusion for ~15 s ( as shown in figure 2(a ) ) . as a control , recordings were also performed with l-15 medium : no membrane potential change was detected during l-15 perfusion ( 0 mm in figure 2(b ) ) . the stability of this recording indicates that our measurements were not affected by the perfusion flow rate . when 100 mm urethane was delivered to an ohc clamped at 0 na , the steady - state membrane potential was hyperpolarized by 28.6 mv and was repolarized shortly after drug application ( figure 2(b ) ) . this reversible hyperpolarization was detected in all five ohcs measured ( mean sd = 27.0 3.9 ) . to rule out the effects of urethane on the ohc membrane potential urethane at a concentration as low as 0.1 mm , which is ~1/100 of the dose typically used to anesthetize animals , elicited a detectable membrane hyperpolarization . figure the smooth curve represents a fit according to the following form of the hill equation : vuret = 100/[(kd/[uret ] ) + 1 ] . a fifty percent reduction in the membrane potential was detected at 15.4 mm ( kd ) , and the slope ( n ) of the membrane potential change to the urethane concentration was 0.91 . subsequent experiments used 100 mm urethane because this concentration consistently evoked an apparent response . we also examined the effects of urethane on the membrane current by using voltage - clamp recordings . a representative example of the whole - cell current recorded from an isolated ohc is shown in figure 3(a ) . when the membrane potential was clamped from 140 to + 94 mv , the cell currents changed from 445 pa ( inward ) to + 1480 pa ( outward ) , resulting in a dynamic range of 1925 pa . the response measured under control conditions is consistent with results previously published for guinea pig [ 22 , 33 ] and gerbil ohcs . the local perfusion of 100 mm urethane significantly increased the current magnitudes , especially at potentials greater than 50 mv . figure 3(b ) shows the current - voltage ( i - v ) curve derived from the steady - state responses shown in figure 3(a ) . the mean change of i - v curves recorded from 11 ohcs was shown in figure 3(c ) . therefore , it is unlikely that the effects of urethane are mediated by a specific urethane receptor . the apparent changes in the ohc current response in the voltage - clamp experiments are most likely due to the effects of urethane on existent ion channels . acetylcholine is the primary efferent neurotransmitter in the cochlea and is released from the efferent chemical synapses at the base of the ohcs [ 19 , 24 ] . the 9 subunit of the nicotinic achr family , which was identified from a rat genomic library , has been demonstrated to play an important role in the ach - induced responses of ohcs [ 18 , 20 , 21 ] . to determine whether the urethane - induced response occurs via this achr , we examined the effect of strychnine ( a potent antagonist of the 9 achr subunit ) on the urethane - induced responses . because the membrane current is directly related to nicotinic achr activity and is easy to record , we measured the urethane - induced current as an indicator of nicotinic achr activity . to obtain the urethane - induced current , the top trace in figure 4(a ) shows a 280 pa upward change of membrane current that correlated in time to the perfusion of 100 mm urethane onto this cell ( ~20 s ) . as indicated in figure 3(b ) , urethane increased the outward current at a membrane potential of 0 mv . therefore , the magnitude of the observed current change reflects the amplitude of the outward current elicited by urethane . coapplication of 0.01 m strychnine reduced the amplitude of the urethane - induced current to 114 pa at saturation level ( figure 4(a ) , middle trace ) . these data suggest that the urethane - induced response is mediated , at least in part , via achr assembled from 9 subunits . then , a higher concentration of strychnine ( 0.1 m ) was coapplied to examine whether the urethane response was blocked in a dose - dependent manner . to minimize desensitization of the receptor , 3 min washout was set between two concentrations . as shown in the bottom trace ( figure 4(a ) ) , the urethane - induced outward current was further reduced to 48 pa in the presence of 0.1 m strychnine . in the seven ohcs measured ( figure 4(b ) ) , the magnitude of the urethane - induced response was significantly reduced by the coapplication of both 0.01 m ( by 54 12% on average ) and 0.1 m strychnine ( by 79 10% on average ) ( both p < 0.001 , student 's t - test ) . mammalian ohcs contract or elongate at acoustic frequencies depending on the membrane potential of the cell [ 10 , 35 , 36 ] . this process , defined as electromotility , is necessary for cochlear amplification [ 3739 ] . prestin , a unique voltage - dependent motor protein found in the membrane of ohcs , mediates the electromotility of ohcs [ 11 , 40 ] . the voltage - sensing and motor functions of mammalian prestin manifest as two characteristics : the nlc and electromotility . the nlc and electromotility are fully coupled in mammals [ 30 , 41 , 42 ] and can be characterized using a simple two - state boltzmann function . because the nlc can be easily and accurately measured experimentally , we measured the nlc to evaluate the effects of urethane on prestin function . figure 5(a ) shows an example of the nlc obtained from an isolated ohc under the whole - cell patch - clamp configuration . as shown in the control ( open circles and gray line ) treatment before urethane application , the nlc is characterized by a bell - shaped dependence on the membrane potential and a peak at 54.8 mv for this cell . no clear change was detected in response to application of 100 mm urethane ( filled circles and black line ) . we examined the nlc from a total of 10 ohcs in response to urethane treatment . figure 5(b ) presents the mean and sd of the normalized nlc from these cells . four parameters ( qmax , clin , v1/2 , and z ) were obtained from a curve fit of the nlc response using the first derivative of the boltzmann function ( heavy lines in figures 5(a ) and 5(b ) ) . the normalized mean values and sds of the four parameters from the 10 ohcs are plotted in figure 5(c ) . no statistically significant difference was found in response to urethane treatment for all parameters ( p > 0.05 , student 's t - test ) . we have shown for the first time that urethane affects the electrical response properties of isolated ohcs . our voltage - clamp data shows that when ohcs were depolarized , the outward current was significantly increased by urethane ( figure 3 ) . this effect was voltage - dependent : it was more pronounced at membrane potentials higher than 50 mv . in mature ohcs , two currents are primarily involved in this process : ( 1 ) the voltage activated outward k current and ( 2 ) the ca - activated k current . the voltage - dependent k current is activated at membrane potentials from 90 mv to 50 mv , displaying half activation at 80 mv . because the urethane - induced current change was clearly detected at membrane potentials > 50 mv , the ca - activated k channel was first reported by ashmore and meech . at membrane potentials > 35 mv , this k channel is opened by an influx of ca , leading to k efflux . under physiological conditions , we assumed that the effect of urethane on ohcs involves this process based on our strychnine experiment . the 9 subunit of the nicotinic achr family has been demonstrated to be the primary nicotinic achr subunit in ohcs . the 9 subunit displays unique pharmacological properties similar to those detected in cochlear hair cells . we found that strychnine , a potent antagonist of 9-containing achrs , significantly blocks the urethane - induced outward current ( figure 4 ) . then , this influx of ca leads to the opening of ca - activated k channels and subsequent k efflux , resulting in hyperpolarization of the ohcs . consistent with our results , a study in xenopus oocytes indicates that urethane enhances the response of nach receptor . our data is also supported by the urethane effects on cochlear function by measuring dpoae . urethane decreases the efferent influence from medial olivocochlear terminus to ohcs via the 9 receptor . the cylindrically shaped ohcs alter their cell length in response to membrane potential changes , exhibited as either a somatic elongation ( upon hyperpolarization ) or contraction ( upon depolarization ) . this somatic motility of ohcs is responsible for cochlear amplification , which contributes to the exquisite frequency selectivity and sensitivity in mammals [ 10 , 37 ] . however , this change in length is asymmetric : the magnitude of contraction is much larger than that of elongation [ 1315 ] . this potential change moves the operating point of the voltage - to - length change conversion function toward lower slope , thereby reducing the overall augmentation of ohc electromotility . based on the voltage - to - length change conversion function for ohcs in the guinea pig , a 30 mv hyperpolarization reduces the total motility magnitude by approximately 2535% . however , in vitro acetylcholine application evokes an increase of electromotile responses of isolated ohcs that develops on a time scale of several seconds . it would increase the driving force for the mechanotransduction current , causing the increase of cochlear microphonic potential . it seems a mismatch between our in vitro data and in vivo cm results . for in vitro experiment , a high concentration urethane was applied to isolated ohcs directly . in addition to a change in the membrane potential , mechanical properties of ohcs could also influence the cochlear amplifier . it is the consensus that normal cell morphology and somatic stiffness of ohcs are essential for cochlear amplification [ 12 , 47 ] . acetylcholine decreases the axial stiffness of the ohcs and reduces the overall mechanical load of the aggregate ohc , resulting in the increase of motile magnitude . it has been suggested that the changes of the ohcs stiffness are mediated by ca - dependent phosphorylation of the ohc cortical cytoskeleton [ 22 , 48 ] . since urethane would also activate 9-containing achrs and induce a ca influx , a similar decrease of ohcs axial stiffness and a reduction of global cochlear activity may be induced by urethane application in vivo . the electromotility of ohcs is presumably attributed to the voltage - dependent activity of the motor protein prestin [ 11 , 40 ] . a generally accepted model for prestin function is that intracellular anions ( in most cases cl ) move toward the extracellular surface upon hyperpolarization and toward the cytoplasmic side in response to depolarization . this anion translocation produces a nonlinear change in the membrane capacitance and , subsequently , triggers a conformational change in prestin , which ultimately alters the somatic length of the ohc . according to this model , any changes in this voltage - dependent activity of prestin may alter the motility of ohcs and affect the overall level of cochlear amplification . for all measured parameters reflecting the properties of prestin function , we did not observe any significant change in response to urethane in our experiments ( figure 5 ) . therefore , we assumed that the influence of urethane was not the alteration of the motile activity of prestin . nonetheless , anion transfer may be significantly reduced by urethane - induced hyperpolarization . in mammalian ohcs , this charge movement is represented as the nlc , which is characterized by a bell - shaped dependence on the membrane potential that peaks between 70 and 20 mv ( 50 mv in our results ) [ 30 , 41 ] . the urethane - induced hyperpolarization shifts the operating range of the membrane potential away from this peak , thus reducing the charge movement and the activity of prestin . this effect is a likely mechanism by which urethane reduces ohc motility in vivo . because the electromotility of ohcs feeds a cycle - by - cycle force to the organ of corti so that the sound vibration is amplified , it is conceivable that even a modest reduction in the motility of ohcs could reduce the overall level of cochlear amplification . the results from isolated ohcs are consistent with our in vivo experimental evidence ( figure 1 ) . although the time courses of the urethane - induced effects were different , all of the rats examined exhibited a similar reduction in the cm magnitude . despite the expanding research to awake animal models of monkeys [ 49 , 50 ] , bats [ 51 , 52 ] , mice , and rats , the frequency selectivity of auditory neurons is composed of two elements : the frequency tuning of the cochlea and the refinement of the auditory neural system . the detected anesthesia - induced changes in neural responses involve the effects of the anesthetic on not only the neurons themselves but also the peripheral receptors ( hair cells ) . therefore , it is critical to identify the influence over hair cells from the integrity . our data show that urethane elicited a ~10 db cm threshold lifting ( figure 1(d ) ) , which indicates that the depression of ohcs leads to a reduction in hearing sensitivity . furthermore , this depression is identical at all sound frequencies , suggesting that urethane affects ohcs along the entire basilar membrane . these results are similar to those of isoflurane and ketamine , which have been assessed using distortion product otoacoustic emissions . the present study found that urethane hyperpolarizes outer hair cells , resulting in a reduction in hearing sensitivity without affecting frequency selectivity . our findings indicate that anesthetics directly affect cochlear hair cells and provide an alternative strategy to modulate cochlear functions .

the cochlea converts sound vibration into electrical impulses and amplifies the low - level sound signal . urethane , a widely used anesthetic in animal research , has been shown to reduce the neural responses to auditory stimuli . however , the effects of urethane on cochlea , especially on the function of outer hair cells , remain largely unknown . in the present study , we compared the cochlear microphonic responses between awake and urethane - anesthetized rats . the results revealed that the amplitude of the cochlear microphonic was decreased by urethane , resulting in an increase in the threshold at all of the sound frequencies examined . to deduce the possible mechanism underlying the urethane - induced decrease in cochlear sensitivity , we examined the electrical response properties of isolated outer hair cells using whole - cell patch - clamp recording . we found that urethane hyperpolarizes the outer hair cell membrane potential in a dose - dependent manner and elicits larger outward current . this urethane - induced outward current was blocked by strychnine , an antagonist of the 9 subunit of the nicotinic acetylcholine receptor . meanwhile , the function of the outer hair cell motor protein , prestin , was not affected . these results suggest that urethane anesthesia is expected to decrease the responses of outer hair cells , whereas the frequency selectivity of cochlea remains unchanged .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC3804439

diabetes mellitus ( dm ) is a major global health problem affecting , according to the world health organization , more than 346 million people worldwide . it is one of the most severe metabolic disorders in humans characterized by hyperglycemia due to a relative or an absolute deficiency of insulin or the resistance of target tissue to regulatory action of the hormone , or both . type 1 , insulin - dependent and type 2 , non - insulin - dependent dm ( t1dm1 and t2dm2 ) both induce a large number of diseases and dysfunctions of the nervous , cardiovascular , excretory , reproductive , and other systems of the organism [ 29 ] . a new view of the nature and pathogenesis of dm - induced complications shared by many specialists nowadays has been prompted by the study of functional activity of hormonal signaling systems regulated by insulin , insulin - like growth factor-1 ( igf-1 ) , and leptin , the principal players responsible for the development of dm and its central and peripheral complications , and by a wide spectrum of other hormones and neurotransmitters , including biogenic amines , purines , glutamate , peptide , and glycoprotein hormones controlling the fundamental cellular processes . the data were obtained showing that the alterations and abnormalities of functional activity of these systems and the changes of expression of hormones and signal proteins in dm led to the disturbances of growth , differentiation , metabolism , and apoptosis in different types of cells and contributed to triggering and developing the pathological processes in the organs and tissues of diabetic individuals [ 1015 ] . it is a common knowledge that in the regulation of physiological and biochemical processes the most important role belongs to a hormone - sensitive adenylyl cyclase ( ac ) signaling system . activating or , alternatively , inhibiting this system , many hormones , growth factors , and hormone - like substances control the activity of camp - dependent signaling cascades and transcription factors and thus regulate gene expression and the other cell processes . the ac system has three main components : ( i ) g protein - coupled receptor ( gpcr ) of the serpentine type that recognizes and specifically interacts with hormone , ( ii ) the -heterotrimeric g protein of the stimulatory ( gs ) and inhibitory ( gi ) types , and ( iii ) the enzyme adenylyl cyclase ( ac ) catalyzing the formation of camp , a second messenger . the system specifically recognizes a hormonal signal , amplifies it , and transmits to the intracellular camp - dependent effector proteins . the gpcr acts as a molecular switch that provides the transduction of hormonal signals from extracellular ligand - binding site to the g proteins located at the cytoplasmic side of the plasma membrane . ligand - activated gpcr induces the conformational changes in the gdp - bound g subunit , leading to gtp exchange for gdp . g-gtp then dissociates from g-dimer complex , and g-gtp and g both interact with the enzyme ac , stimulating or inhibiting its activity . the present review is devoted to the alterations and abnormalities in the gpcr - g protein - ac signaling system regulated by biogenic amines and polypeptide hormones in the brain , heart , skeletal muscles , liver , and the adipose tissue in experimental and human dm and to the role of the changes in ac signaling cascades in the pathogenesis and etiology of dm and its complications . many neurodegenerative disorders , diabetic encephalopathy and alzheimer 's disease in particular , are associated with dm . in diabetic patients , they are manifested as alterations in neurotransmission , electrophysiological abnormalities , structural changes , and cognitive deficit . since the brain is mainly a glucose - dependent organ , a severe hyperglycemia in t1 dm , mild hyperglycemia typical of t2 dm , and recurrent hypoglycemia induced by inappropriate insulin therapy are the major factors responsible for the development of dm - induced cerebral disorders . in the diabetic brain , a large number of ac signaling cascades regulated by different hormones and neurotransmitters and forming a well - coordinated signaling network undergo significant changes due to disturbances in these cascades and abnormalities in overall neuronal signaling , including both camp - dependent and -independent pathways [ 1214 , 1826 ] . currently available data on the hormonal signaling in the diabetic brain indicate that the most essential changes occur in ac signaling cascades regulated by biogenic amines , such as dopamine ( da ) , norepinephrine ( ne ) , and serotonin ( 5-hydroxytryptamine , 5-ht ) and by peptide hormones and adenosine . da is the predominant catecholamine neurotransmitter in the brain , which controls a variety of cns functions ( locomotor activity , cognition , emotion , food intake , and neuroendocrine regulation ) and plays multiple roles in the periphery as a modulator of cardiovascular function , hormone secretion , and gastrointestinal motility . da activates ac via gs - coupled da receptors of types 1 and 5 ( da1r and da5r ) and inhibits the enzyme via gi - coupled da2r , da3r , and da4r . it was found experimentally that in the brain of diabetic animals the dopaminergic signaling was altered , mainly due to the changes at the initial stages of da - induced signal transduction which involves dars , gi , or gs proteins and their effectors , ac in particular , and these alterations are brain region specific . in the cerebral cortex of rats with t1 dm induced by streptozotocin ( stz ) treatment , the expression of da1r and da2r and the total dar binding were increased ; in the cerebellum , da1r was downregulated and da2r upregulated , a total number of dars being however decreased , and in the hypothalamus and brainstem a significant decrease in the da content and the number of da2rs and an increase in da2r affinity were shown [ 22 , 28 ] . the treatment with insulin partially restored dm - induced changes of da1r and da2r expression and da2r receptor binding parameters in the diabetic brain to near control , thereby improving the cognitive and emotional functions impaired in t1 dm [ 28 , 29 ] . the alteration of da - induced signaling in the diabetic brain is associated with their downstream components , such as camp response element binding protein creb , the important transcription factor , playing a pivotal role in dar - mediated nuclear signaling and neuroplasticity . it was found that stz - induced dm produces a significant attenuation of functional activity of creb in the cerebral cortex and cerebellum of diabetic rats and these alterations are eliminated by the insulin treatment . we showed that , in the brain of rats with one - month t1 dm , induced by stz at a dose 65 mg / kg of body weight , and with prolonged , 7-month , t1 dm , induced by three consecutive injections of stz on the first , 10th , and 80th days of experiments at dropped doses 40 , 35 , and 30 mg / kg , the ac stimulating effect of da realized via gs - coupled da1/5rs did not change significantly [ 31 , 32 ] , and , in the brain of rats with the neonatal model of t2 dm , duration 3 to 6 months , this effect even increased a little . the ac inhibitory effect of da2r agonist bromocriptine and its stimulating effect on 5 guanylylimidodiphosphate ( gppnhp ) binding of gi proteins in the synaptosomal membranes isolated from the brain of diabetic rats were decreased , predominantly in t1 dm [ 32 , 34 , 35 ] . as the binding characteristics of dars and the catalytic activity of ac did not change essentially , a suggestion was made that the impairment of bromocriptine - induced ac inhibition in the diabetic brain was due to the reduced function of gi proteins . , the attenuation of da2r agonist - induced suppression of appetite in stz rats might be the result of reduction of gi protein activity in the diabetic brain . the brain serotonergic signaling is involved in the regulation of feeding , locomotion , reproduction , sleep , pain , aggression , and stress responses , as well as thermogenesis , cardiovascular control , circadian rhythm , and pancreatic function . the abnormalities of 5-ht - regulated signaling cascades in the diabetic brain provoke disturbances in neuronal processing and the alteration of plasticity of neurotransmission and play an important role in dm - induced cns disorders [ 38 , 39 ] . this is caused first of all by the alterations of the brain sensitivity to 5-ht , which depends on the functioning of 5-ht - regulated cascades and the decrease of cerebral concentrations of 5-ht and its metabolites as a result of the impairment of biochemical conversion , reuptake and transport of 5-ht . it was shown that 5-ht - regulated ac signaling pathways including gi - coupled 5-hydroxytryptamine receptor of the type 1 ( 5-ht1r ) were altered significantly . one - week stz - induced dm markedly reduced the flat body posture induced by 5-ht1ar agonist 8-hydroxy-2-(dipropylamino ) tetralin hydrobromide ( 8-oh - dpat ) , which indicated that stz - induced t1 dm profoundly affected the sensitivity to drugs acting via 5-ht1ar . in our experiments , no alterations of the sensitivity of ac system in the brain of stz rats to selective agonists of gs - coupled 5-ht6r were detected , while ac sensitivity to agonists of gi - coupled 5-ht1ar and 5-ht1br was decreased significantly [ 32 , 33 , 35 , 41 ] . in the brain of rats with prolonged t1 dm and neonatal t2 dm , the ac inhibitory effects of 5-ht1br - agonist 5-nonyloxytryptamine were reduced by 79 and 70% , respectively . 5-methoxy - n , n - dimethyltryptamine in the case of the neonatal model of t2 dm was reduced by 57 and 51% , respectively [ 35 , 41 ] . in our view , the weakening of 5-ht1r - mediated signaling may be associated with a decrease of expression and activity of gi proteins , because in the diabetic brain the impairment of other gi - coupled cascades was also detected . due to their ability to regulate the synaptic mechanisms , ne and other ligands of adrenergic receptors ( ars ) are essential modulators of memory . the activation of -ars by ne leads to facilitation of the synaptic transmission through the mechanism involving the increase of the level of intracellular camp and protein synthesis de novo , thus contributing to the memory acquisition and maintenance . it was found that the population of gs - coupled -ars in the regional brain of db / db mice was depressed , which demonstrates a considerable modification of adrenergic signaling . the level of ne and the number of gi / o - coupled 2-ars in the frontal cortex , septal area , amygdala , hypothalamus , and medulla of 416-week - old db / db mice with t2dm - like state were , on the contrary , elevated compared to those of control animals and this increase correlated with the changes in blood glucose level and body weight . the alteration of adrenergic signaling in the brain of db / db mice , the genetic model of t2 dm , may be causally associated with the consequences of the changes in structural integrity and neuronal competence of the medial basal hypothalamus . the alterations in the brain adrenergic signaling were also shown in stz - induced t1 dm . in the brainstem of stz - treated rats , the content of epinephrine and the ne / epinephrine turnover were increased , and the affinity of 2-ars to selective agonists was significantly reduced . insulin treatment induced the decrease of the ne content and the restoration of the binding parameters of 2-ars to normal values . in the hypothalamus , thalamus , and amygdala of stz rats , the density of 1- , but not of 2- , ars was increased , and the rate of metabolism of ne was decreased . the neurodegenerative processes in the diabetic brain are largely associated with the damage in the cerebral microvessels whose functions are controlled via different signaling systems , the adrenergic in particular . the number of -ars in the cerebral microvessels of zucker diabetic fatty ( fa / fa ) rats and stz rats was significantly decreased despite the receptor affinity did not change . the isoproterenol - stimulated ac activity in the cerebral microvessels of diabetic rats was significantly lower compared with control , giving evidence for the weakening of -ar - mediated regulation of camp - dependent signaling in t1 dm . we showed the decrease of the ac stimulating effect of isoproterenol in the brain of rats with prolonged t1 dm induced by three consequent injections of moderate doses of stz . these results suggest that alterations of central adrenergic regulation in small vessels may be involved in microvasculature disturbances in the diabetic brain . note that in the cerebral cortex , striatum , and hippocampus of rats with the neonatal model of t2 dm the ac stimulating effect of isoproterenol had no changes . it follows that the alterations of adrenergic signaling are brain region specific and depend to a great extent on the dm model , glycemic control , and duration of dm . in dm , there are significant changes in the brain ac system regulated by peptide hormones and neurotransmitters , such as melanocortin , glucagon - like peptide-1 ( glp-1 ) , somatostatin , relaxin , and pituitary ac - activating polypeptide ( pacap ) . in human and experimental t2 dm , the gene expression of melanocortin receptor of type 4 ( mc4r ) coupled with ac via gs proteins in the brain was restricted , and mc4r - mediated signaling pathways were significantly reduced . it is generally accepted that mc4r - agonists -melanocyte - stimulating hormone ( -msh ) and melanotan ii promote a negative energy balance by decreasing the food intake and increasing the brain activity and energy expenditure , whereas hypothalamic agouti - related peptide ( agrp ) , mc4r antagonist , on the contrary , increases food intake . along with this central injection of mc4r - agonists reduces insulin secretion , while administration of mc4r - antagonists increases plasma insulin level . indeed , elevated insulin level and impaired insulin sensitivity were detected in the young lean mc4r knockout mice , even before the development of pronounced hyperphagia and obesity [ 50 , 53 ] . the mice with functionally inactive mc4r had obesity strikingly reminiscent of the agouti syndrome , which indicates that the disturbances in mc4r signaling pathways were the primary cause of the agouti obesity . the hypothalamic melanocortin system controlling adiposity levels rapidly and more efficiently than the other central signaling systems is regulated by leptin . the decrease of leptin level due to fasting or genetic leptin deficiency led to the reduced expression of proopiomelanocortin gene and to the increased expression of agrp gene , and leptin infusion , on the contrary , increased the hypothalamic content of proopiomelanocortin and mc4r and inhibited the production of agrp . since mc4rs are involved in neuroprotective action , regeneration and promotion of adaptive plasticity of neuronal and glial cells [ 56 , 57 ] , the alterations and abnormalities of hypothalamic melanocortin - sensitive ac system in the hypothalamus are the prime causes of neurodegenerative processes in the diabetic brain . the positive regulatory effects of -msh and selective mc4r - agonists on neuronal plasticity and survival could be mediated by their influence on neuronal signaling regulated by other neurotransmitters , gaba in particular . the contribution of the central melanocortin system in etiology and pathogenesis of t2 dm and obesity is fully confirmed by the data on the immunization of rats with peptides corresponding to the n - terminal extracellular domain mc4r and to the first and third extracellular loops of mc3r [ 59 , 60 ] . in rats injected with peptide corresponding to the n - terminal domain of mc4r , like in the case of the blockade of hypothalamic mcrs by antagonists , the food intake , body weight , plasma insulin , and triglycerides levels increased significantly . the antibodies against the n - terminal domain of mc4r acted as partial mc4r agonists and decreased camp level in cell cultures . antibodies against peptide derived from the first loop of mc3r amplified the ac stimulating effect of -msh , while antibodies against peptides , the derivatives of the third loop of the same receptor , reduced the effect of hormone , acting as a noncompetitive mc3r antagonist . in rats injected with peptide from the third intracellular loop of mc3r , the body weight and blood pressure were increased , and the motor activity was decreased . the plasma levels of triglycerides , insulin , and leptin were much higher compared with control , the same as in t2 dm . but the rats injected with peptide from the first loop had no changes of physiological and biochemical parameters . this is the evidence that peptides derived from the mcrs and the antibodies against these peptides directly influence melanocortin signaling pathways and cause changes in the brain signaling network . their action being receptor and site specific , they can induce either inhibition or enhancement of signal transduction via the cognate receptor . this is in good agreement with the results obtained with synthetic peptides derived from the extracellular and intracellular regions of different gpcrs [ 61 , 62 ] . thus , peptides corresponding to the extracellular loops of mcrs are a promising tool in the study of etiology of dm and its cerebral complications and give a perspective approach to develop new models of dm and obesity based on antibody - induced deregulation of the brain ac signaling . in the brain , glp-1 via specific receptors activates ac and intracellular camp - dependent pathways , modulates ca channels and other effector systems , and functions as a neurotransmitter . glp-1 has the growth factor - like and neuroprotective properties and controls learning , memory , and synaptic plasticity [ 6365 ] . in the periphery , glp-1 is responsible for modulating blood glucose concentrations by stimulating glucose - dependent insulin secretion and by activating -cell proliferation . glp-1 is effective in restoring first - phase insulin response and lowering hyperglycemia in t2 dm . glp-1 receptor agonists , exendin-4 , and liraglutide , like the inhibitors of glp-1 degradation ( dipeptidyl peptidase iv inhibitors ) , have been approved for the treatment of t2 dm [ 67 , 68 ] . liraglutide , analog of human glp-1 with prolonged half - life having a fatty acid palmitoyl group conjugated to the side - chain of lys and an argser substitution , is now widely used in t2 dm therapy . exendin-4 and liraglutide injected subcutaneously for 4 , 6 , or 10 weeks once daily in ob / ob , db / db , and high - fat - diet - fed mice enhanced proliferation rate of progenitor cells by 100150% and stimulated differentiation into neurons in the dentate gyrus . the glp-1 receptor antagonist exendin ( 936 ) significantly reduced progenitor cell proliferation in these mice . exendin-4 , liraglutide , and glp-1 analog with alaval substitution enhanced long - term potentiation in the brain and reduced the number of amyloid dense - core plaques in mice with insulin resistance and in patients with t2dm - associated obesity and alzheimer 's disease . these results give grounds to say that the glp-1 analogs show promise in the treatment of t2dm - induced neurodegenerative diseases , because they cross the blood - brain barrier and increase neurogenesis . glp-1 analogs elicit the insulinotropic activity and improve the central and peripheral symptoms of t2 dm . the pacap is a bioactive peptide with diverse activities in the nervous system ; it functions as a neuromodulator and neurotrophic factor [ 71 , 72 ] . pacap has neuroprotective effect in vitro and in vivo in ischemia , neurodegenerative diseases and retinal degeneration [ 73 , 74 ] . pacap protects neuronal cells from diabetic lipotoxicity , as is shown in the lipotoxicity model induced by the treatment of adult neuronal stem cells isolated from the adult mouse brain subventricular zone with palmitate promoting lipoapoptosis and in the subventricular zone / striatum isolated from obese ob / ob mice . palmitate treatment led to an increase of expression of pacap receptors , such as pac-1 and vpac-2 , in the neuronal cells . in the subventricular zone of ob / ob mice , the level of expression of pac-1 , vpac-1 , and vpac-2 receptors was also significantly increased . these data speak in favor of the fact that pacap counteracted the lipotoxicity , acting via pac-1 and other types of pacap receptors , and protected neuronal cells from apoptosis in the diabetic brain , which suggests a potential therapeutic role of pacap receptor agonists in the treatment of neurological complications induced by t2 dm and obesity . as was found in our experiments , in the brain tissue of rats with prolonged stz - induced t1 dm , the stimulating effects of pacap-38 , the predominant form of pacap in mammals , on ac activity and gppnhp binding were significantly decreased and could be largely restored by the long - term treatment with intranasal insulin . in prolonged neonatal t2 dm , the ac stimulating effect of pacap-38 in the synaptosomal membranes was changed a little in the course of three and six months of the disease but decreased significantly in the case of 18-month t2 dm . in addition to its important role in the protection of neuronal cells from damage and neurodegenerative changes , the decrease of functional activity of pacap-38-regulated ac system in the brain in t1 dm and t2 dm points to abnormalities in pacap - mediated neuroprotection in dm . in the brain , neuropeptide somatostatin specifically binds to five types of gi - coupled somatostatin receptors ( sstrs ) that are widely expressed in the brain with a distinct distribution pattern . the central signaling pathways of somatostatin and its synthetic analogs are involved in the regulation of secretion of many pituitary hormones , for example , growth hormone , prolactin , and thyroid stimulating and adrenocorticotropic hormones . somatostatin controls endocrine , sympathetic , behavioral , and visceral responses to stress , cognition , thermogenesis , food behavior , and tumourgenesis of nervous and peripheral tissues [ 7780 ] . we showed a significant decrease in the functioning of somatostatin - regulated ac system in the brain of rats with stz t1 dm and the neonatal model of t2 dm [ 31 , 32 , 35 , 41 ] . the inhibitory effect of somatostatin on forskolin - stimulated ac activity in the synaptosomal membranes and its stimulatory effect on gppnhp binding capacity of gi proteins in the diabetic brain were markedly reduced , as compared to control , but the therapy with intranasal insulin partially restored both effects . in prolonged , 18-month neonatal t2 dm , the ac inhibitory effect of somatostatin was decreased by 33% compared with the nondiabetic rats of the same age , while , in the case of three- and six - month dm , this effect decreased only by 11% and 21% . this gives evidence that with elongation of t2 dm the alterations of somatostatin - mediated ac signaling in the diabetic brain are progressively strengthening . we regard the decrease of expression and functional activity of gi proteins as one of the main causes responsible for the reduction of ac regulatory effects of somatostatin . at the same time , the reduced number of sstrs can also contribute to the decrease of somatostatin signaling . the expression of some types of sstrs was found to be decreased in the hypothalamus and pituitary of rats with t1 dm . the mrna expression of genes encoding sst1r , sst2r , sst3r , and sst5r , in the diabetic pituitary , was reduced by 5080% , and the level of sst1r and sst5r , restored by insulin treatment . the level of sst5r was reduced by 30% in the hypothalamus of stz rats and completely restored by insulin . taking into account the importance and complexity of the physiological and biochemical effects of somatostatin and its signaling cascades plus their involvement in the pathogenesis of a variety of neurodegenerative diseases , it would be right to say that the disturbances in somatostatin - sensitive ac system are likely to be responsible for the development of dm - induced cns complications . according to the literature data , there are types of ac whose expression and functional activity in t2 dm undergo changes along with the receptor and transducing components of ac system . using real - time rt - pcr , it was shown that the expression of the type 3 ac ( ac3 ) in the striatum and hypothalamus of goto - kakizaki rats , a spontaneous animal model of t2 dm , was higher compared with wistar rats , and the 15-day treatment of goto - kakizaki rats with insulin led to a decrease of the enzyme expression . it was shown that dynamics of alterations of ac3 expression in the striatum and hypothalamus and in the pancreatic islets of diabetic rats was similar , which indicates a high probability of the involvement of ac3 in the glucose homeostasis regulation mediated both by cns mechanisms and insulin secretory activity of pancreatic islets . dm is well known for its cardiovascular complications including acute myocardial infarction , congestive heart failure , and other manifestations of atherosclerosis and often termed as diabetic cardiomyopathy [ 10 , 82 ] . hyperglycemia and insulin deficit in t1 dm and hyperinsulinemia and insulin resistance in t2 dm induce changes in the contractile function and duration of action potential [ 83 , 84 ] , and these changes are closely linked to the alterations and abnormalities in the adrenergic , purinergic , and other signaling systems and their downstream effectors in the cardiac myocytes [ 10 , 8587 ] . in the heart , many effects of the biogenic amines , purines , polypeptide hormones , and other signal molecules are realized via ac signaling system that is implicated in hormonal regulation of arterial tone , reactivity , and cell proliferation . the adrenergic signaling , which has a key role in the regulation of the cardiovascular system , changes to the greatest extent in dm . in the cardiac tissue , there are three pharmacologically distinct subtypes of -adrenergic receptors ( -ars ) . 1- and 2-ars stimulate ac activity via gs proteins , and as a result , the intracellular camp level increased , and the activity of protein kinase a ( pka ) is enhanced . this leads to phosphorylation of l - type calcium channels , phospholamban , troponin , and ryanodine receptors , all being essential for cardiac function , the control of heart rate , and contractility in particular . 3-ar is involved in the regulation of cardiac contractility through activation of gi proteins and no synthase signaling pathway and the stimulation of 3-ar , unlike that of 1- and 2-ars , which decreases the contractile force in human ventricular muscle [ 88 , 89 ] . in the heart of rats with experimental t1 dm , the mrna and protein levels of -ar subtypes and their functional activity and role in the regulation of heart rate and contractility were changed , and these alterations strongly progressed with increasing duration of dm . in the hearts of rats with stz - induced t1 dm duration of 6 to 14 weeks the expression of 1-ar and the density of -ars were decreased significantly compared with control animals [ 86 , 9093 ] . at the same time , the mrna level encoding 2-ar in the heart of rats with 14-week stz t1 dm was increased , but the density of 2-ar protein was , on the contrary , decreased which can be attributed to the increased rate of 2-ar degradation and posttranslational modifications preventing the translocation of this receptor from the endoplasmic reticulum to the plasma membrane . insulin therapy prevented the reduction of the number of myocardial -ars and the level of 1-ar mrna observed in 6-week stz t1 dm . the increase of the number of myocardial -ars to near or above control level was detected in the case of treatment of diabetic rats both with insulin and thyroid hormones . the mrna and protein level of cardiac 3-ar in rats with 14-week stz dm were twice those in control . the estimated ratio of 1- , 2- and 3-ars in the heart of control and diabetic rats was 62 : 30 : 8 and 40 : 36 : 23 , respectively , and the two - week insulin treatment of diabetic animals increased 1- and 2-ar contents , decreased the number of 3-ars and , as a result , restored the ratio to 57 : 33 : 10 . note that the left ventricular cardiomyocytes samples from 29 failing human hearts showed a 2- to 3-fold increase of 3-ar density and a similar increase in the expression of 3-ar - coupled gi2 subunit mediating 3-ar agonist - induced inhibition of ac activity , as compared to nonfailing human hearts . there are data stating that the molecular variations of 3-ar lead to obesity , insulin resistance , and t2 dm . it was shown also that a mutation of the 3-ar gene , resulting in the replacement of tryptophan by arginine at codon 64 , was associated with early onset of t2 dm [ 97 , 98 ] and with diabetic retinopathy and nephropathy in japanese t2 dm patients [ 99 , 100 ] . the decrease of content and functional activity of 1-ar in the diabetic heart led to a blunting of chronotropic effects of ne and other -ar agonists , induced the impaired 1-mediated cardiac stimulation , and led to a decrease of their stimulating effects on cardiac ac activity [ 85 , 92 , 94 , 101 ] . there are grounds to believe that the decrease of 1-ar function in the diabetic heart , especially in the case of prolonged t1 dm , led to the prevention of 1-ar - stimulated apoptosis , which contributes to progressive myocardial dysfunction . it is well known that selective 1-ar - antagonists block apoptosis in the rat ventricular myocytes , while the blockers of 2-ar , on the contrary , enhance this process . the chronotropic response mediated via 2-ar was preserved in diabetic rats with 14-week stz t1 dm , which was demonstrated by the absence of difference between the diabetic and the control atria in their chronotropic and inotropic responses to 2-ar - selective agonist fenoterol . in this case , the maximum chronotropic response to isoproterenol , nonselective -ar agonist , was decreased with no change in pd2 value , while in the case of ne acting preferably via 1-ar both pd2 value and maximum chronotropic effect were decreased significantly . the obtained data showed that the preservation of the 2-ar signaling in the diabetic rats may be a compensatory mechanism triggered by reducing the functional activity of 1-ar - mediated pathways . another evidence in favor of compensatory redistribution of 1-ar and 2-ar - mediated signaling pathways in the diabetic heart is the fact that in the heart of 14-week - stz diabetic rats the contents of 1-ar protein and 1-ar mrna were decreased to 35% and 45% , respectively , whereas the decrease of the maximum chronotropic response of the right atria to 1-ar agonists exceeded 70% of that in control , indicating an increase of the contribution of 2-ar - signaling to -ar - mediated chronotropic effects in t1 dm [ 86 , 103 ] . in the diabetic heart of stz rats , there was an increase in expression and functional activity of 3-ar , resulting in the enhanced negative inotropic effect and the bradycardia . the activation of 3-ar signaling involving both camp- and no - dependent pathways may be a mechanism of preventing the overstimulation of heart muscle by catecholamines , whose level is increased in diabetic cardiomyopathy . it was shown that patients with t1 dm had a decreased -adrenergic responsiveness of the heart to isoproterenol infusion . in ventricular cardiomyocytes and papillary muscle isolated from stz rats , the stimulation influence of -ar agonists on ac was decreased due to the disturbances of the upstream components of this system [ 101 , 104 ] . in the cardiomyocytes and cardiac membrane preparations from rats with stz - induced t1 dm , the ac stimulating effect of isoproterenol was decreased by 10% to 30% [ 94 , 101 , 105 ] . the abnormalities in the ac system sensitive to -ar agonists were detected either at the level of receptor and its coupling to gs proteins or at the level of downstream camp - dependent effector proteins but not at the level of ac , whose functional activity in the diabetic heart remained unchanged . this is indicated by the fact that the basal ac activity and the enzyme stimulation by naf and forskolin in rats with stz t1 dm did not change [ 101 , 106 , 107 ] . it should be noted at this point that in the mesenteric artery of rats with stz t1 dm the changes in the expression and functional activity were identified not only in the downstream components of camp - dependent cascades , such as pka and camp - dependent phosphodiesterase , but also in some types of ac . the mrna expression and the protein content of ac5 and ac6 were significantly lower in the diabetic mesenteric artery , as compared to control , and the stimulation of the basal ac activity by the water - soluble forskolin analog nkh477 , a putative ac5 activator , was significantly impaired in the mesenteric arteries from rats with stz - induced t1 dm . according to the recent finding , transgenic overexpression of ac5 in cardiomyocytes of mice improved their baseline cardiac function but impaired the ability of the heart to withstand stress , while a knockout of gene encoding ac5 and the pharmacological inhibitors of the enzyme , on the contrary , protected the heart against cardiac stress and cardiomyopathy induced by t2 dm and obesity . it follows that the decrease in activity and the changes in the pattern of ac isoforms are a significant contribution in alterations of the blood flow and of the response of heart to catecholamine stimulation in dm . one of the main causes of alterations and abnormalities of 1-ar signaling in the diabetic heart in t1 dm is the selective downregulation of 1-ar due to a significant increase of the level of ne that has high affinity for 1-ar . the elevation of cardiac ne concentration and ne turnover and uptake may be due to the increased sympathetic nerve activity in t1dm - induced diabetic cardiomyopathy , as in the case of other cardiovascular diseases . it was shown that exercise training reduced the cardiovascular morbidity and mortality during t1 dm due to the normalization of the sympathetic outflow and improvement in the responsiveness of the myocardium to autonomic stimulation , which induced the decrease of circulating level of catecholamines and restored the number of 1-ar . it should be mentioned , however , that exercise training of rats with mild t1 dm induced by moderate doses of stz ( 45 mg / kg ) did not prevent dm - induced changes in -ar expression but improved the sensitivity of cardiac ac to isoproterenol . at the same time , excessive exercise training accentuated t1dm - induced decrease in the myocardial -ar responsiveness to hormonal stimulation due to a significant decrease of 2-ar expression without affecting the reduced level of 1-ar . a majority of investigators showed that , as a rule , the alterations and abnormalities in ac sensitivity to -ar agonists in t1 dm as well as t2 dm were associated with the left ventricle , which is the main causal factor of diabetic cardiomyopathy [ 95 , 111 , 113 , 114 ] . however , there are data on the changes in functional activity of ac system in the right atrium ; they differ significantly from those in the left ventricle . it was shown that in the right atrium of swine with stz - induced dm the density of -ar was 14% lower , and the forskolin- and gppnhp - stimulated ac activities were reduced by 34 and 23% , respectively , whereas the basal and isoproterenol - stimulated ac activities as well as the content of gs and gi proteins did not change . despite these alterations , the stimulation of heart rate with isoproterenol and -ar - dependent signaling pathways remained intact in the right atrium of diabetic swine . a significant difference was found in the changes of -ar - mediated signaling in ventricular cardiomyocytes from the heart of stz rats , depending on the gender of animals . in the isolated cardiomyocytes from diabetic female rats , there was no obvious change of the dose - response curve of ac stimulating effect of isoproterenol , whereas in diabetic male rats , there was a considerable decrease in the maximum response to this hormone . one of the causal factors can be a t1dm - induced significant decrease in androgen levels , which negatively affects the -ar regulation of cardiac function . alongside with the altered -ar signaling , there were changes in -ar - regulated signaling pathways in the diabetic heart . in t1 dm , the hormonal sensitivity of myocardial -ars was increased , but the overall number of -ar binding sites was decreased without significant changes in the affinity constants [ 104 , 118120 ] . it was shown that , compared with control , the administration of selective -ar agonists to acute diabetic animals led to a more pronounced -ar - mediated responses of the myocardium , such as maximal change in pressure over time and developed tension [ 121123 ] . the study of rats with one- , four- , and 10-week t1 dm demonstrated that the changes of -ar - dependent cardiac functions were mainly associated with the alterations in the 1-ar - mediated pathways involving gq proteins and phospholipase c , while the ac signaling cascades regulated by 2-ar agonists via gi / o proteins were changed to a lesser extent . at the same time , prolonged t1 dm induced by the treatment of animals with dropping doses of stz ( 3040 mg / kg ) led to a weakening of the inhibitory effect of ne on forskolin - stimulated ac activity in the myocardial membranes and to a decrease of ne - induced stimulation of gi protein gppnhp - binding capacity . we detected a significant decrease of the ac inhibitory effect of somatostatin acting via gi - coupled sstrs . there are all reasons to suppose that the decrease of regulatory influence of somatostatin on the ac system is associated with the decreased function of gi proteins , as well as with the reduced number of sstrs , like in the diabetic atria with markedly reduced levels of sst2r mrna and protein . in the atria of stz - treated rats , the inhibitory effect of somatostatin on atrial natriuretic peptide secretion was attenuated compared to that in the control atria . it means that somatostatin - regulated camp signaling is likely to be involved in the pathogenesis of cardiovascular complications of t1 dm . the data on the weakening of gi - coupled ac cascades regulated by different hormones indicate that the functions of gi proteins in the heart of animals with t1 dm , the same as in the brain , are highly attenuated . this assumption is supported by the findings of other authors who showed the decrease of gi proteins functions in the diabetic heart and no significant changes of gs proteins expression and activity [ 32 , 92 , 101 ] . as early as 1994 , it was shown that in the cardiomyocytes isolated from stz rats the expression of gi2 and go proteins was reduced by 58% and 27% , respectively , whereas the expression of gs proteins did not change . a significant decrease in the level of gi , but not gs proteins , the decrease of the expression of gi subunit and functional activity of gi proteins was identified in the aorta of rats with short - term t1 dm and in the aorta exposed to high glucose , without any changes in the levels and functions of gs subunit [ 126 , 127 ] . the expression of gi2 and gi3 subunits as determined by immunoblotting techniques was reduced by about 40% three days after stz treatment and decreased by about 70% and 50% , respectively , 5 days after the treatment . in the diabetic aorta , the inhibitory effects of gtps and hormonal agents , such as angiotensin ii , oxotremorine , and atrial natriuretic peptide analogs , on forskolin - stimulated ac activity were attenuated compared with control , which points to the decrease of receptor - independent and -dependent functions of gi proteins . the decrease of the expression of the gi2 and gi3 subunits and gi - mediated ac signaling in the case of hyperglycemia induced by short - term stz dm or high concentration of glucose ( above 20 mm ) may be attributed to the increase of o2 and onoo levels , since the treatment with antioxidants reversed the hyperglycemia - induced abnormalities in ac system to control . it should be mentioned that the oxidative stress enhanced in t1 dm and acute hyperglycemia is one of the key causes of the increase of the expression of gq/11 subunits and strengthening of the phospholipase c signaling . in experimental and human t2 dm , the responsiveness of the myocardium to -ar stimulation and ac stimulating effects of -ar agonists was , as a rule , decreased , and , unlike in t1 dm , the number of -ars did not differ significantly from control [ 41 , 130132 ] . as far as alterations in ac sensitivity to agonists are concerned , they depend on the model of t2 dm , its duration and severity . in the myocardium of eight - month - old rats with the neonatal model of t2 dm , the ac stimulating effect of -agonist isoproterenol was increased , but not very much , and in the same tissue of 18-month - old diabetic rats , it was , on the contrary , reduced significantly compared with the respective control . the ac stimulating effect of relaxin , an insulin - like peptide , in the heart of rats with long - term neonatal t2 dm was reduced to a high extent , and in 18-month - old rats it did not exceed 46% of that in the respective control . the ac inhibitory effect of ne on forskolin - stimulated ac activity in the myocardial membranes of eight- and 18-month - old diabetic rats was , at most , 51% and 46% of that in control . these findings suggest a significant decrease of the sensitivity of cardiac ac to adrenergic agonists and relaxin in very prolonged neonatal t2 dm . there were no significant differences between the basal and forskolin - stimulated ac activities in the heart of control rats , on the one hand , and diabetic animals with prolonged neonatal stz t2 dm and zucker fa / fa rats . on the other hand , gppnhp - stimulated ac activity was reduced in the diabetic heart , especially in the case of the neonatal model [ 33 , 41 , 132 ] . these data indicate a weakening of the gs protein function with no change in the catalytic activity of the enzyme . one of the causes of the decline of gs proteins activity is the t2dm - induced hyperhomocysteinemia that leads to the attenuation of gs proteins activity and the decrease of the number of 2-ars , as shown in db / db mice . a decrease of homocysteine level induced by the physical exercises led to the restoration of 2-ar - gs protein - ac system in the diabetic heart . the first data on the functional state of ac system in the skeletal muscles was obtained by garber in 1980 . it was shown that in the skeletal muscles of rats with stz - induced t1 dm epinephrine - stimulated ac activity was reduced by 60% , while the basal and the naf- and 5-ht - stimulated ac activities did not change . later , it was found that the density of -ars and ac stimulating effect of -ar agonists , reduced in different types of the skeletal muscles , for example , the soleus muscles and the vastus lateralis muscles , of rats with stz dm , were partially restored by physical training that was a progressive 10-week treadmill running program [ 135 , 136 ] . we showed that in the skeletal muscles of rats with 30-day stz t1 dm the stimulating effect of -adrenergic agonists and the inhibitory effects of ne and 5-ht on ac activity were reduced , as compared with control [ 31 , 137 ] . the increase of gppnhp binding capacity of g proteins induced by adrenergic agonists and 5-ht in the sarcolemmal membranes of diabetic animals was lower than in control , and the difference between the diabetic and control rats was most pronounced in the case of gi proteins . a special mention deserves the fact that the acute hyperglycemia caused by very short stz t1 dm also led to a decrease of functional activity of gi - coupled ac signaling cascades , as it follows from a significant weakening of inhibition of forskolin - stimulated ac activity and stimulation of gppnhp binding capacity of gi proteins induced by somatostatin in the skeletal muscles of rats with one - day t1 dm . the deficiency of gi proteins in the skeletal muscles in t1 dm not only induced the abnormalities in the camp signaling , but also it reduced the sensitivity of the muscles to insulin action . it was shown that the expression of constitutively activated mutant form of the gi2-subunit with the replacement of asn by leu in the skeletal muscles , liver , and the adipose tissue of mice with stz - induced t1 dm markedly ameliorated glucose tolerance and fasting glucose levels and , in addition , restored glycogen synthase activity in the tissues of transgenic mice . the above is due to the fact that the gi2-subunit is involved in the regulation of the translocation of insulin - sensitive glucose transporter glut4 , and in the in vivo conditions the expression of ql gi2-subunit led to the increase of glucose transport and glut4 translocation in the skeletal muscles even in the absence of insulin stimulation . the data on the functioning of the ac system in the skeletal muscles in t2 dm are rare . there is a report that in the skeletal muscles of young db / db mice the epinephrine - induced lipolytic activation was reduced , but the stimulating effects of this hormone on ac and glycogen phosphorylase activities were unaltered . this gives evidence for the impairment of epinephrine - induced lipolytic activation in the skeletal muscle of db / db mice at the level of components of camp - dependent signaling cascades , downstream of ac . as early as 1984 , it was found that in the hepatocytes of rats with stz t1 dm glucagon - mediated regulation of camp formation was deranged owing to a combined decrease of glucagon receptors , impairment of their coupling with ac , and altered basal ac activity . the basal and naf- , gppnhp- , mn- , and glucagon - stimulated ac activities in the hepatic plasma membranes isolated from the diabetic liver were reduced by 50% , and the content of gs - coupled glucagon receptors was decreased by 67% , all these changes being partially reversed by insulin treatment . later , it was shown that , in the diabetic liver , gi - coupled signaling cascades , camp dependent in particular , underwent significant changes , and these alterations were mainly associated with reduced activity of gi subunits , preferably gi2 [ 142144 ] . the decrease of gi proteins function was due to decreased expression of gi subunits and their increased phosphorylation , depriving gi proteins of the ability to participate in signal transduction . the levels of gi2 and gi3 subunits in the hepatocyte plasma membranes were decreased significantly , but the level of gs subunits was increased . however , in the whole liver , there were no detectable changes in the levels of gi2 and gi3 subunits , but only the decrease of transcription level of gi2 , but not of gi3 , subunit was found . these differences may be related to the fact that dm - induced changes in parenchymal cells ( hepatocytes ) constituting only 60% of cells in the liver may have been masked by the g protein complement of the other , nonparenchymal , cells . it was shown that stz - induced t1 dm caused a profound increase in the steady - state level of gi2 subunit phosphorylation in hepatocytes . gi2 subunit in hepatocytes from diabetic animals was phosphorylated exclusively at the protein kinase c site but not observed at the pka phosphorylation site . this reflected an augmentation of the protein kinase c signalling cascades in hepatocytes from stz - treated rats due to an increased protein kinase c activity and a reduction in protein phosphatases activity . the weakening of gi - mediated signaling was associated with the increase of the signaling cascades involving gs proteins and the enhancement of catalytic function of ac . the influence of stz t1 dm on the basal and hormone - sensitive ac activity and the binding of gs / gi - coupled receptors in the hepatic membranes directly depended on the duration and severity of the diabetic state . no changes in the basal and gppnhp- , mn- , glucagon- , and isoproterenol - stimulated ac activities and the binding affinity and capacity of -ar were observed within 3 days after stz treatment . fifteen days after the treatment , ac stimulation by hormonal and nonhormonal agents and the binding capacity of -ar were greatly decreased , as compared to control . the amount of -ars was also reduced in the hepatic membranes isolated from rats with 15-day stz t1 dm , while it did not change in 3-day diabetic animals . these results may explain the considerable differences in ac signaling detected in the liver of diabetic animals and emphasize the importance to study the dynamics of changes in the ac system when the disease is still developing . the alterations of ac signaling , hormonal sensitivity of -ars , and g protein pattern were detected in the liver in the genetic models of t2 dm . in the liver membranes isolated from ob / ob mice with hyperglycemia , hyperinsulinemia , and obesity typical of t2 dm , the number of 2-ar binding sites was increased threefold , and the response of ac to catecholamines was significantly enhanced as compared to both control animals and db / db mice , the other animal model of t2 dm . the content of gi and gs proteins , especially their gi2 subunits , was reduced in the liver of ob / ob and db / db mice [ 146148 ] . in the hepatic membranes of db / db mice , the levels of expression of gi2 , gi3 , and g subunits were reduced by 75% , 63% , and 73% , respectively , and the maximal inhibitory effect of gppnhp on forskolin - stimulated ac activity was reduced to 60% , as compared to lean animals , which indicates the abolition of gi proteins activity in the liver in t2 dm . regrettably , the data on the lack of significant changes in the liver of stz rats and genetically diabetic mice is very scanty . it may be probably due to the difference in the dm models , duration , and severity of the disease and to the application of different approaches in the study of gi protein functions . for instance , in the early studies , there were no significant differences in the functional activity of gi proteins in the membranes isolated from hepatocytes and the whole liver of the diabetic animals , such as fat zucker ( fa / fa ) rats and rats with short , one - week , stz t1 dm [ 150 , 151 ] . the functioning of adipocytes is regulated via camp - dependent signaling pathways , some of which are canonical pka - dependent pathways , while the others are carried out through the novel exchange protein activated by camp ( epac ) . the deregulation of these signaling cascades and their cross - talk with insulin signaling in adipocytes leads to the alterations in energy expenditure and/or feed efficiency and results in obesity and t2 dm . regulation of metabolism in the adipose tissue has been shown to be impaired in genetically obese animals with hallmarks of developing t2 dm , and the important role in these pathological changes belongs to adrenergic signaling . as is well known , -ar agonists stimulate ac activity via gs - coupled -ars and enhance lipolysis in the adipose tissue . it was found that adipocytes from genetically obese ob / ob c57bl/6j mouse and + /+ c57bl/6j mouse fed on a high fat diet for 16 weeks and zucker fa / fa rats displayed an impaired response of ac to stimulation by -ar agonists due to a significant decrease of 1-ar and , especially , 3-ar expression [ 153159 ] . the decrease of 3-ar and 1-ar expression was found in the white and brown adipose tissues from 12-week - old ob / ob mice , whereas 2-ar mrna level did not change significantly [ 155 , 158 ] . as a consequence , in obese animals , the stimulation of ac activity by selective 3-ar agonist cl316.243 was decreased by 75% in the epididymal white adipose tissue and by 90% in the brown adipose tissue , and the corresponding ac effects of 1/2-ar agonist epinephrine were also decreased considerably , though to a lesser extent compared with those of cl316.243 . the maximal isoproterenol - stimulated lipolysis in the adipocytes from zdf rats was significantly lower , as compared to control animals , and the ec50 values for isoproterenol to increase lipolysis in the adipocytes from normal and obese rats were 2 and 7 nm , respectively . these data demonstrate the significant decrease of -ar - mediated lipolytic activity in the adipose tissue of animals with t2dm - like metabolic disorders . in this connection , the assumption was put forward concerning alterations of the sensitivity of ac in the adipose tissue in t2 dm to other activators of lipolysis , such as glp-1 , glucagon , and glucose - dependent insulinotropic polypeptide-1 . moreover , recently it was shown that the regulation of lipolysis by glucagon at the physiological concentrations ( below 0.1 nm ) was carried out via camp - independent signaling mechanisms . it was established some time ago that endogenous adenosine in the adipocytes tonically activates gi - coupled adenosine receptor of the type 1 , highly expressed in the adipose tissue , reduces ac activity and camp content , and , the same as insulin , inhibits lipolysis . as a result , the plasma free fatty acid concentration elevated in metabolic syndrome , and t2 dm decreases ; this fact may be of use in the treatment of these disorders . it was found that in the adipose tissue of zdf and normal rats the antilipolytic activity of cvt-3619 , a partial agonist of the type 1 adenosine receptor , and ic50 values of cvt-3619-induced inhibition of free fatty acid release from adipocytes were similar . in addition , cvt-3619 is a stronger inhibitor of lipolysis than insulin , especially in the adipocytes isolated from zdf rats resistant to the antilipolytic action of insulin . it seems reasonable to suppose that the maintenance of the activity of gi - coupled purinergic system responsible for antilipolytic activity can partially compensate for a considerable attenuation of insulin antilipolytic effect in the adipose tissue of rats with insulin resistance . in the adipose tissue of stz diabetic rats , gi proteins functions were decreased , but gi - mediated signaling was , on the contrary , unaltered or even strengthened . the significant weakening of functional activity of gi proteins was manifested as decreased inhibitory effects of low concentrations of gppnhp on forskolin - stimulated ac activity and high concentrations of gtp on isoproterenol - stimulated ac activity . the ac inhibitory effects of n-(phenylisopropyl)adenosine , nonmetabolizable adenosine analogue , prostaglandin e2 , and nicotinate were either unchanged or even apparently more effective in the adipocyte membranes from diabetic animals . since insulin - mediated glucose uptake is highly sensitive to the levels of the facilitative glucose transporter protein glut4 , repression of glut4 expression correlated with insulin resistance in the adipose tissue . the differentiation - dependent glut4 transcription was under control of class ii histone deacetylases and regulated by adrenergic agonists that generally increase intracellular camp level and , as a result , promoted the association of histone deacetylases with the glut4 promoter , which led to the decrease of glut4 transcription . the camp - dependent recruitment of histone deacetylases to the glut4 promoter was dependent on the glut4 liver x receptor ( lxr ) binding site , so that the activation of lxr signaling prevented the loss of glut4 expression in dm and obesity . this finding offered a new understanding of how camp - signaling pathways regulated by adrenergic agonists alter gene expression in adipose tissue in dm . this review presents the data concerning the changes in ac signaling system sensitive to biogenic amines , peptide hormones , and purines in dm and its complications , such as diabetic encephalopathy , diabetic peripheral neuropathy , diabetic cardiomyopathy , and others . the main causes of the changes are hyperglycemia , lipidemia , oxidative stress , and other metabolic abnormalities typical of dm , as well as alterations in the central and peripheral signaling cascades induced by the resistance of the cells and tissues to the regulatory action of insulin / igf-1 and leptin as well as by relative or absolute insulin deficiency . these changes largely depend on the type and duration of dm and on the model of the disease in the case of experimental dm . the degree of alterations and abnormalities in the ac signaling system correlates very well with the severity of the dm and its complications . the changes in ac signaling at the early stages of dm are compensatory and , as a rule , reversible , while at the later stages of the disease , when it is accompanied by many complications , these changes occurring in some particular ac signaling cascades induce the alterations in many signaling pathways , both camp dependent and independent , which leads to the disruption and unbalance of global integrated signaling network and , as a result , these changes become irreversible . the most complex pattern and dynamics of the changes of the ac system are in the brain , where they involve a large number of neurotransmitters and signaling cascades regulated by them and are region and cell specific . to know the origin and reversibility of changes in hormonal signaling in dm is very important for the development of effective strategy in diagnostics and treatment of this disease and its complications . one of the most important questions here is whether the abnormalities in dm occur in the tissue or organ in the ac system sensitive to one or a limited number of the related hormones ; if so , how they cover the other signaling cascades , or whether at the initial stage of the disease they occur independent of each other in multiple ac signaling pathways . the latter seems more likely since a few days after t1 dm induction by stz treatment the alterations are detected in multiple ac cascades regulated by various hormones and involving both gs and gi proteins . , there are pieces evidence that at the initial stages of dm or in prediabetes the disturbances occur only in one or a small number of ac signaling cascades , while the others remain unchanged . then the alterations in one signaling cascade can induce , according to the domino principle , changes in the others . the domino principle is well illustrated by the fact that the disturbances in some particular signaling cascades of the brain cause numerous abnormalities in the neuronal network , which may lead to insulin resistance or insulin deficiency and , as a result , to the development of dm and its central and peripheral complications [ 12 , 50 , 52 , 59 , 60 , 165169 ] . summing up , to identify the initial hot spots in ac signaling , the abnormalities of which lead to an avalanche of hormonal disturbances , we must know the etiology and pathogenesis of dm and its prevention at the stage of prediabetic state , such as obesity and metabolic syndrome in t2 dm and autoimmune damage of pancreatic -cells in t1 dm . the causal relation between dm and the altered hormone - sensitive ac system is not a one - way avenue , from dm - induced alterations of hormonal signaling in cns and peripheral organs ; the alterations in ac signaling pathways may be a causal factor of dm and its complications . this speaks in favor of the use on a wide scale not only in the treatment but also in prevention of dm of hormonal and nonhormonal agents that control functional activity of signal proteins , the components of the ac system , and have an influence on availability , transport , and secretion of hormonal molecules . among them are serotonin , d2-agonist bromocriptine , mc3r and mc4r agonists , the regulators of ac activity , and drugs controlling their uptake and transport , selective serotonin reuptake inhibitors in particular [ 35 , 170173 ] . note that these agents may be useful for t2 dm patients with resistance to typical antidiabetic drugs metformin and sulfonylurea , as it is shown in the case of bromocriptine therapy [ 174 , 175 ] .

diabetes mellitus ( dm ) induces a large number of diseases of the nervous , cardiovascular , and some other systems of the organism . one of the main causes of the diseases is the changes in the functional activity of hormonal signaling systems which lead to the alterations and abnormalities of the cellular processes and contribute to triggering and developing many dm complications . the key role in the control of physiological and biochemical processes belongs to the adenylyl cyclase ( ac ) signaling system , sensitive to biogenic amines and polypeptide hormones . the review is devoted to the changes in the gpcr - g protein - ac system in the brain , heart , skeletal muscles , liver , and the adipose tissue in experimental and human dm of the types 1 and 2 and also to the role of the changes in ac signaling in the pathogenesis and etiology of dm and its complications . it is shown that the changes of the functional state of hormone - sensitive ac system are dependent to a large extent on the type and duration of dm and in experimental dm on the model of the disease . the degree of alterations and abnormalities of ac signaling pathways correlates very well with the severity of dm and its complications .

1. Introduction 2. cAMP Signaling in the Brain 3. cAMP Signaling in the Cardiovascular System 4. cAMP Signaling in the Skeletal Muscles 5. cAMP Signaling in the Liver 6. cAMP Signaling in the Adipose Tissue 7. Conclusion

PMC4071986

oral candidiasis is an infectious condition caused by the fungus of the genus candida , the most common of which is candida albicans . oral candidiasis significantly occurs to the immunocompromised individuals , namely , patients with aids immunosuppression , cancer chemotherapy / radiation therapy , corticosteroid therapy , and chronic antibiotic therapy and individuals with xerostomia and diabetes mellitus [ 1 , 2 ] . chronic systemic administration of antifungal agents is required to treat the oral candidiasis , which may produce potential adverse effects . therefore local administration of the antifungal agent is considered as the first line treatment for the oral candidiasis if the drug concentration remained above the minimum inhibitory concentration throughout the treatment . it is difficult to achieve by the conventional dosage forms like oral gels , pastes , solution , suspensions , ointments , mouthwashes , lozenges , mouth paints , and so forth , because less retention time of the dosage form leads to the fluctuation in the salivary drug concentration [ 1 , 2 ] . therefore an ideal dosage form for the treatment of the oral candidiasis is one which sustains the release of drug and retains in the oral cavity and produce antifungal effect for prolonged period of time . this is possible if the drug delivery system possesses sustained release as well as mucoadhesive properties . imidazole antifungals such as miconazole , clotrimazole , itraconazole , and econazole are the major drugs used for the treatment of oral candidiasis . delivery of the antifungals like miconazole to the oral mucosa has been investigated by various workers exploiting novel drug delivery systems like nanostructured lipid carriers . econazole nitrate ( ecn ) is an imidazole antifungal agent which is effective against candida albicans . the ecn interferes with the ergosterol synthesis and thereby alters the normal functions of the cell membrane and causes death of the fungus . ecn is investigated for the topical administration to the skin [ 79 ] , vaginal [ 1012 ] , and buccal applications [ 13 , 14 ] for the treatment of fungal infections . poly(vinyl alcohol ) ( pva ) hydrogels are extensively studied for controlled drug release for various drugs [ 1519 ] . chemical crosslinking of pva hydrogel may lead to the toxic impurities . when pva is exposed to cycles of freezing and thawing freeze / thaw method has many advantages including nontoxicity in comparison to the chemical method . statistical optimization techniques are frequently employed for the development of pharmaceutical formulations [ 2325 ] . 3 full factorial design is the simple experimental design with two variables studied at three levels . the objective of the present work was to develop mucoadhesive hydrogel film ( mhf ) of ecn using pva hydrogel exploiting freeze / thaw technique as crosslinking method . 3 full factorial design was used to optimize the effect of concentration of pva and number of freeze / thaw cycles on drug release . the optimized batch was prepared and studied for physical parameters as well as antifungal efficacy . navsari , gujarat , india ) ; poly(vinyl alcohol ) ( fisher scientific - qualigens fine chemicals , navi mumbai , india ) ; polyethylene glycol 400 ( merck ltd . , mumbai , india ) ; sabouraud dextrose agar ( sda ) with chloramphenicol ( hi - media , mumbai , india ) ultrapure water ( millipore , usa ) were used throughout and all the other chemicals used were of analytical grade . drug - polymer and polymer - polymer interaction studies were done by differential scanning calorimetry and fourier transform infrared spectroscopy . a differential scanning calorimeter ( model pyris-1 dsc , perkin elmer , usa ) was used to determine drug - polymer interaction studies . approximately 65 mg of the pure drug , polymer alone , drug - polymer physical mixture ( 1 : 1 ) , and the formulated mhf was weighed and then vacuum dried for 24 hours to remove all the water . the dried sample ( 16.5 mg ) was heated in the dsc equipment from 45 to 400c at a scanning rate of 10c / min in the nitrogen atmosphere . the heat required to melt the sample was calculated by integrating the peak due to melting . an ftir spectrophotometer ( model spectrum gx with ir quant software , perkin elmer , usa ) was used for the study . pure drug , polymer alone , drug - polymer physical mixture ( 1 : 1 ) , and formulated mhf were subjected to ftir spectroscopy in the range of 4000 to 400 cm . the peaks of pure drug were compared with the physical mixture and the mhf formulations to interpret any drug - polymer interaction . pva dissolved in double distilled water at 90c with different concentrations for 6 hours by continuous stirring on magnetic stirrer with hot plate . accurately weighed ecn dissolved in the mixture of ethanol and polyethylene glycol 400 ( peg400 ) ( 3 : 5 ) and the resulting solution of ecn was added to the pva solution by continuous stirring for 30 minutes at room temperature . the drug containing aqueous solution of pva was poured in to the glass mould of 10 10 cm . the gel containing drug was cross linked by freezing the mixtures in mould at 12c for 16 hr followed by thawing at room temperature for 8 hrs , that is , by freeze / thaw technique . the freeze / thaw cycle was repeated for four , six , and eight times . the resulting crosslinked and drug entrapped hydrogel was dried at room temperature to get the solid films . the amount of ecn was added in such a manner that the area of 2.25 cm ; that is , one film contains 80 mg drug . the resultant hydrogel films were packed individually in aluminium foil and stored in a desiccator at room temperature . the details of the formulation are given in tables 1 , 2 , and 3 . the mhfs were observed for their colour by the naked eyes in the day light . scanning electron microscopy ( sem ) was used to study the surface morphology of the prepared mhfs . samples ( mhfs ) were attached to metal stubs which had been previously covered with coated tape and then sputtered with a layer of gold . they were examined with a scanning electron microscope ( model esem edax xl-30 , philips , netherlands ) . five randomly selected mhfs containing ecn were dissolved in 100 ml methanol by stirring continuously for 2 hours on magnetic stirrer and filtered by filter paper . the amount of drug in the solution was then measured spectrophotometrically at 225 nm . the microenvironment ph of the prepared mhfs was determined to evaluate the possible irritation effects on the mucosa . the films were left to swell in 5 ml of ph 6.8 phosphate buffer in small beakers and the ph was measured after one hour by placing the electrode in contact with the microenvironment of the swollen films . folding endurance was determined on five mhfs in each batch by repeatedly folding the film at the same place till it broke or folded up to 300 times [ 1 , 26 ] . moisture absorption ( ma ) study was performed by a modified method of singh et al . and kanig and goodman [ 1 , 27 ] . accurately weighed preconditioned films ( w0 ) were placed in a constant humidity chamber ( containing a saturated solution of ammonium chloride which gives a relative humidity of 79.5% ) set at room temperature . films were weighed ( wt ) at an interval of 1 , 3 , and 7 days . [ 1 , 2 , 28 , 29 ] was slightly modified to study the bioadhesive character of the prepared mhfs . the apparatus used for study comprised two - arm balance , one side of which contains two glass plates and other side contains a container . one glass plate was attached with the base of the balance and other with the arm of the balance by a thick strong thread . fresh goat buccal mucosa was glued to the upper side of the lower plate and mhf was glued to the lower side of the upper plate by using cyanoacrylate adhesive ( superwiz ) . then 50 gm preload force ( or contact pressure ) was applied on upper plate for 5 minutes ( preload time ) . after removal of the preload force , the water kept in a bottle at some height was siphoned in the container at the rate of 10 ml per minute till the plates were detached from each other . the rate of dropping of water was controlled with on - off switch same as in infusion bottle . the weight of water required for detachment of glass plates was considered as the mucoadhesive strength in mg / cm of the mhf under study . procedure . the water uptake or swelling index of the mhf was determined by equilibrium weight gain method similar to that reported by efentakis and vlachou and roy and rohera . the study was carried out using usp / nf dissolution apparatus i. the mhfs were accurately weighed , placed in dissolution baskets , and immersed in ph 6.8 phosphate buffer maintained at 37 1c in the dissolution vessels . at regular intervals ( 0.5 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , and 12 hours ) , the preweighed basket with mhf was withdrawn from the dissolution vessel , lightly blotted with a tissue paper to remove excess test liquid , and reweighed . the percent swelling index or water uptake was estimated at each time point using the following equation : ( 2)%s=[xtxoxp]100 , where xt is the weight of the swollen mhf after time t and xo is the original mhf weight at zero time and xp is the amount of polymer in the mhf . the water uptake data was subjected to the vergnaud model to determine the rate of water uptake . the generalized form of the vergnaud model is as follows : ( 3)mt = ktn , where mt represents the amount of liquid transferred at time t and k is swelling constant which depends on the amount of liquid transferred after infinite time , the porosity of matrix , and diffusivity . the exponent , n , indicates the mechanism of water uptake . the values of n and k were determined by converting the equation to the logarithm form as follows : ( 4)log(mt)=log(k)+nlog(t ) and plotting the curve log(mt ) versus log(t ) . the values of slope and intercept so obtained by plots are n and log(k ) , respectively . the in vitro residence time was determined using a locally modified usp tablet disintegration testing apparatus , based on the apparatus applied by nakamura et al . . the medium was composed of 700 ml phosphate buffer ( ph 6.8 ) in 1 l glass beaker and maintained at 37 1c . a segment of goat buccal mucosa , 2.5 2.5 cm , was cut and glued by cyanoacrylate adhesive ( superwiz ) on the inside curved surface of 1 l beaker above the level of 700 ml phosphate buffer ( ph 6.8 ) . a glass cylinder ( 100 ml ) the mhf was hydrated from one surface using phosphate buffer ( ph 6.8 ) and then the hydrated surface was brought into contact with the mucosal membrane . the glass cylinder was vertically fixed to the apparatus and allowed to move up and down so that the film was completely immersed in the buffer solution at the lowest point and was out at the highest point . the time necessary for complete detachment of the mhf from the mucosal surface was recorded ( mean of triplicate determinations ) . the drug release studies were carried out using a us pharmacopoeia xxx ( uspxxx ) type ii dissolution apparatus modified to the uspxxx dissolution apparatus type v , that is , paddle over disc apparatus . the dissolution was done at 37 1c and 50 rpm with ph 6.8 phosphate buffer containing 1% sls as a medium ( 900 ml ) . the mhf was fixed with a cyanoacrylate adhesive ( superwiz ) to a thick glass disk placed at the bottom of the vessel of the dissolution testing apparatus containing deaerated , prewarmed the 3 ml sample was withdrawn in the time interval of 1 , 2 , 3 , 4 , 6 , 8 , 10 , and 12 hours . the collected samples were filtered and the amount of drug was measured spectrophotometrically using uv - visible spectrophotometer at 305 nm . the release profiles were fitted in zero order , korsmeyer - peppas equation , higuchi equation , and first order kinetics to determine the mechanism of drug release . the agar plates used in this study were prepared by dissolving sabouraud dextrose agar ( sda ) 65 g in 1 l of distilled water and sterilized by autoclaving ( at 15 lb pressure and 121c ) for 15 min . the agar plates were then allowed to cool and solidify at room temperature ; then they were inoculated ( cultured ) with c. albicans by using a sterile swab . standard dilutions of 5 to 80 g / ml of ecn were prepared in ph 6.8 phosphate buffer containing 1% sls . a 0.1 ml of each dilution was carefully pipetted into 7 mm diameter well of the agar plate . these plates were allowed to prediffuse for 2 hr at room temperature and then incubated for 24 hr . the diameter ( mm ) of the growth inhibition zone surrounding each agar well inoculated with c. albicans was measured and standard calibration curve of concentration of drug versus diameter of zone of inhibition ( mm ) was plotted . antifungal efficacy of the optimized mhfs was determined by subjecting the aliquots of in vitro drug release studies to disc agar diffusion assay [ 36 , 37 ] . aliquots of in vitro drug release samples were collected at 1 , 2 , 3 , 4 , 6 , 8 , 10 , and 12 hour . a 0.1 ml of each sample was carefully pipetted into uniformly spaced 7 mm diameter wells of the agar plates . these plates were allowed to prediffuse for 2 hours at room temperature and then incubated for 24 hour . the diameter ( mm ) of the growth inhibition zone surrounding each agar well inoculated with c. albicans was measured , and the concentrations of ecn were determined from the standard calibration curve constructed under identical conditions , ranging from 5 to 80 g / ml . the mean of three determinations of each sample was determined . statistical analysis . for comparison among the batches , one way anova was used with p value of less than 0.05 to consider as evidence of a significant difference . 3 full factorial design and the regression analysis were used to optimize the concentration of pva and number of freeze / thaw cycles . by using the independent variables and their degrees as well as the values of dependent variables obtained by the experiments , the following type of regression equation was generated for each dependent variable . a statistical model incorporating interactive and polynomial terms was utilized to evaluate the response : ( 5)y=0+1x1+2x2+12x1x2 + 11x12+22x22 , where y is the dependent variable , 0 is the arithmetic mean response of the nine runs , and 1 and 2 are the estimated coefficient for the factors x1 and x2 . the main effects ( x1 and x2 ) represent the average result of changing one factor at a time from its low to high value . the interaction terms ( x1x2 ) show how the response changes when two factors are simultaneously changed . the thermograms of ecn pure drug , pva pure , 1 : 1 physical mixture of ecn with pva , and mhf are given in figures 1 , 2 , 3 , and 4 . ecn has two peaks in the thermogram , one positive ( endothermic ) peak at 166.003c with the peak area of 709.306 mj and another negative peak at 195.338c with the peak area of 1536.433 mj ( figure 1 ) . the reason behind the endothermic peak at 166.003c is the melting of ecn ( melting range of ecn is 161 to 166c ) . both the peaks were found intact in the thermograms of 1 : 1 physical mixture of ecn with pva as well as in the thermogram of mhf . the ftir spectra of ecn pure drug , pva pure , 1 : 1 physical mixture of ecn with pva , and mhf are given in the figure 6 . the ftir spectra showed the prominent peaks of the various bonds between the groups present in the ecn chemical structure ( figure 5 ) . the prominent peaks for various groups are n h stretching at 3426 cm , c cl stretching at 638 cm , c h stretching for aromatic at 3109 cm , c = c stretching for aromatic at 1585 cm , c n stretching at 1330 cm , c o stretching for ether at 1090 cm , and no2 at 1547 cm . it was observed that all the above stated prominent peaks of the ecn pure were present in the ftir spectra of 1 : 1 physical mixtures of ecn with pva and mhf . this confirms that there was no chemical interaction between the drug and the polymers or confirms that the drug is present in pure and unchanged form in the mhf . the sem images of mhfs are given in figures 7 and 8 at 1000x and 500x , respectively . the drug content of the mhfs was found in the range 98.98 0.91% to 100.45 0.45% . the results of microenvironment ph , thickness , weight , and folding endurance of the mhfs are given in the table 4 . the microenvironment ph of the mhfs was between 7.21 0.14 and 7.41 0.18 . the microenvironment ph was unaffected by concentration of pva and number of freeze / thaw cycles . the ph of all the mhfs was near to the ph range of the oral cavity . so , there is minimum possibility of the irritation to the oral mucosa after application . the mhf thickness was found in between 0.32 0.07 mm ( ef3 ) to 0.61 0.05 mm ( ef7 ) . as the concentration of pva increased the thickness of the mhfs increased . this is because pva is the major part of the mhf and increase in its concentration leads to increase in thickness . the mhf weight was found in between 194 1.41 mg ( ef1 ) and 228 2.05 mg ( ef8 ) . as the concentration of pva increased the weight of the mhfs increased . this is because pva is the major part of the mhf and increase in its concentration leads to increase in weight . . this may be due to increase in the water absorbing pva molecules by increasing the concentration of pva . but there was no effect of number of freeze / thaw cycles on the percent moisture absorption . the difference in the moisture absorption from day 3 to day 7 is very less in all the formulations . this may be because of the saturation of the mhf with the moisture absorption within 3 days . this is done to evaluate the moisture absorption ability of the mhfs so that precautions can be taken to avoid it . the highest percent moisture absorption for the mhfs for day 1 , day 3 , and day 7 was found to be 29.14 1.06 , 32.44 1.85 and 32.65 2.03 percent , respectively , for the formulation ef7 and lowest percent moisture absorption was 19.24 1.36 , 22.05 1.14 and 22.12 1.47 percent , respectively , for the formulation ef1 . the mucoadhesive studies were expressed in gm / cm of the mucoadhesive strength ( ms ) . the results of the in vitro mucoadhesive studies are given in the table 4 and figure 9 . as the concentration of pva increased the ms increased and there was decrease in the ms as the number of freeze / thaw cycle increased within the same concentration of pva . this observation agrees with that of the peppas and mongia and tsutsumi et al . . the reason may be the same as explained by the above workers that the work of fracture decreased with increasing number of freezing / thawing cycles . this observation is attributed to loss of adhesive linear pva chains as crystallization occurs due to the slow incorporation of all the linear pva chains in the crystalline structure formed upon repeated cycles . thus , a hydrogel produced after four cycles contains relatively mobile , noncrystalline chains which exhibit strong adhesive behavior either because of hydrogen bonding due to their hydroxyl groups because of significant chain interpenetration or because of both . contrary to this , after six and eight cycles , very few linear pva chains are available for this interaction with mucin . the highest and lowest ms were found to be 153.33 7.09 gm / cm ( ef7 ) and 93.78 2.12 gm / cm ( ef3 ) , respectively . the highest swelling at twelfth hour was found to be 493.94 10.25 percent ( ef7 ) and lowest swelling at twelfth hour was 193.91 14.78 percent ( ef3 ) . it was found that as the concentration of pva increased the percent swelling increased significantly ( p < 0.05 ) . this is because the increase in the water absorbing pva molecules by increase in concentration of pva . it was observed that within the same concentration of pva , as the number of freeze / thaw cycles increased , the percent swelling was decreased significantly ( p < 0.05 ) . the reason for this behavior may be the increase in the crosslinking or the degree of crystallinity , by increase in the freeze / thaw cycles [ 15 , 17 , 20 , 39 ] . the percent swelling or the water uptake data were subjected to the vergnaud model to determine the rate of water uptake and to study the polymer relaxation and the drug release mechanism . according to ebube et al . , 1997 , a value of 0.5 for n indicates a diffusion controlled mechanism in which the rate of diffusion of the liquid is much less as compared with the rate of relaxation of the polymer segment . a value of one for n ( n = 1 ) suggests that the stress relaxation process is very slow as compared with the rate of diffusion . this means that the liquid diffuses through the polymer matrix at a constant velocity showing an advancing front marking the limit of liquid penetration . a value of n between 0.45 and 1 indicates an anomalous or complex behavior in which the rate of diffusion of the liquid and relaxation of polymeric chains are of the same magnitude . n < 0.95 , which is indicative of an anomalous mechanism of water uptake in which solvent diffusion , as well as polymer relaxation , are of the same magnitude . the mucoadhesive and in vitro residence studies are similar on the generalized view but the major difference is that in mucoadhesive studies the force in mg / cm required to detach the mhf from the mucosa surface was considered whereas the in vitro residence time is the time required to detach the mhf from the mucosa surface with continuous motion in the ph 6.8 phosphate buffer . so , in later case , the swelling , water penetration into the polymer matrix , and the motion are the cause of the detachment and simulates the environment of the oral cavity . it was observed that the residence time was decreased by increase in the number of freeze / thaw cycles within the same concentration of pva . the reason behind this behaviour may be the same as for the mucoadhesive studies but one more factor to be considered for this case is the water uptake . the increase in number of freeze / thaw cycles causes increase in crosslinking and the degree of crystallinity [ 15 , 17 , 20 ] . hydrogels produced after four cycles contain relatively mobile , noncrystalline chains which exhibit strong adhesive behavior either because of hydrogen bonding due to their hydroxyl groups or because of significant chain interpenetration or because of both . contrary to this , after six and eight cycles , very few linear pva chains are available for this interaction with mucin . and percent swelling also decreases with increasing the number of freeze / thaw cycles due to the reason discussed under swelling studies . because of all of the above reasons the in vitro residence time decreased by increasing number of freeze / thaw cycles . the highest and lowest in vitro residence time were found to be 734 47 minutes ( ef7 ) and 298 21 minutes ( ef3 ) , respectively . most of the mhfs , especially containing 15% and 20% pva , shown the higher than the required in vitro residence time , that is , > 12 hours or > 720 minutes . because of its poor aqueous solubility , the ecn was incorporated as solid dispersion ( using ethanol and peg400 3 : 5 ) into the mhfs , to increase its dissolution . but it was found that the maximum dissolution was raised to 66.66 g / ml after incorporating ecn as solid dispersion . the volume of dissolution media ( ph 6.8 phosphate buffer with 1% sls ) used was 900 ml . it was observed that the maximum ecn release from the mhfs after an excessively long time of dissolution was found to be ~75% irrespective of the formulation parameters . as the concentration of pva increased , the ecn release decreased significantly ( p < 0.05 ) . the reason behind this type of release behavior may be the same as described by various workers . the higher the concentration of pva , the higher the degree of crystallinity and the higher the pva network system or the denser the crosslinking [ 18 , 19 ] . the above reasons cause the firm entrapment of the ecn into the pva matrix network at higher pva concentrations which leads to the decrease in the release . the release profiles were studied for zero order kinetics , korsmeyer - peppas equation , higuchi square root of time model and first order kinetics to determine the mechanism of drug release . best fit was observed with korsmeyer - peppas equation with r > 0.99 excluding ef1 , ef2 . it clearly indicates that , at lower concentration , that is , 10% of pva , release profile showed poor fit with korsmeyer - peppas equation model . according to the korsmeyer - peppas equation , n = 0.5 indicates fickian release ( diffusionally controlled release ) and n = 1 indicates a purely relaxation controlled delivery which is referred to as case ii transport . n < 1 ) indicate an anomalous behavior ( non - fickian kinetics corresponding to coupled diffusion / polymer relaxation ) . occasionally , values of n > 1 have been observed , which has been regarded as super case ii kinetics [ 41 , 42 ] . the values 0.58 < n < 0.83 of korsmeyer - peppas equation indicate the anomalous behavior , that is , non - fickian kinetics corresponding to coupled diffusion / polymer relaxation . the release behavior showed poor fit with higuchi square root of time model ( r < 0.99 except ef3 and ef4 ) , so the release mechanism is again concluded to be non - fickian diffusion . the release behavior showed poor fit with first order kinetics ( r < 0.99 except ef3 and ef4 ) and zero order kinetics ( r < 0.99 except ef5 ) . as the number of freeze / thaw cycles increased the ecn release decreased significantly ( p < 0.05 ) , within the same concentration of pva . the reason behind this type of release behavior maybe the densification of the crystalline structure , increasing the crosslinking network or high degree of crystallinity by increasing the number of freezing / thawing cycles , thus leading to a reduction of ecn release . the maximum release of ecn at twelfth hour was found to be 75.12 1.02% for ef1 . the ecn release for ef5 to ef9 was less than 75% at twelfth hour but when the dissolution was done for more than twelve hours , it was observed that the maximum ecn release was ~75% . at 10% pva as the number of freezing / thawing cycles increased , the diffusion coefficient or the n of the korsmeyer - peppas equation increased ( table 7 ) . but at 15 and 20% pva there was no significant increase in the n value noticed . the number of freeze / thaw cycles or the degree of crystallinity has no effect on the mechanism of drug release . the optimization was done by 3 full factorial design using response surface methodology ( rsm ) . the independent variables used were concentration of pva ( % w / v of the initial gel ) ( x1 ) and number of freeze / thaw cycles ( x2 ) . the dependent variables used were time required for 50% drug release ( y1 ) , percent of drug release at 8th hour ( y2 ) , and k of zero order equation ( y3 ) . the time required for 50% ecn release ( t50% ) showed correlation coefficient , r value , of 0.99 , indicating a good fit . the t50% value for the nine batches ( ef1 to ef9 ) showed a wide variation ( 3.44 hours to 14.21 hours ) . the data clearly indicate that the t50% values are strongly dependent on the selected variables . in order to determine the levels of factors which yield optimum dissolution responses , the polynomial equation can be used to draw conclusions after considering the magnitude of coefficient and the mathematical sign it carries , that is , positive or negative : ( 6)y=3.4860.186x10.683x2 + 0.0384x1x2 + 0.0232x12 + 0.0635x22 . a linear plot ( figure 12 ) drawn between the predicted and observed responses demonstrates high values of r ( r > 0.9 ) , indicating excellent fit . thus low residuals as well as the significant value of r designate a high prognostic ability of response surface methodology . figure 13 shows the surface plot of t50% ( y1 ) versus concentration of pva ( x1 ) and number of freeze / thaw cycles ( x2 ) . the data demonstrate that both the factors ( x1 and x2 ) affect the drug release ( t50% ) . it may also be concluded from the graph that the high level of x1 ( concentration of pva ) and high level of x2 ( number of freeze / thaw cycles ) favor the sustained release of the ecn from the mhfs , that is , increase in the t50% . the low value of x1x2 coefficient of ( 6 ) also suggests that the interaction between x1 and x2 is not significant . the percent ecn release at 8th hour ( rel8 hr ) showed correlation coefficient , r value , of 0.997 , indicating a good fit . the rel8 hr value for the nine batches ( ef1 to ef9 ) showed a wide variation ( 30.54% to 72.95% ) . the data clearly indicate that the rel8 hr values are strongly dependent on the selected variables . the responses of the rel8 hr are given in ( 7)y=109.3543.712x1 + 0.639x2 + 0.045x1x2 + 0.0109x120.320x22 . a linear plot ( figure 14 ) drawn between the predicted and observed responses demonstrates high values of r ( r > 0.9 ) , indicating excellent fit . thus low residuals as well as the significant value of r designate a high prognostic ability of response surface methodology . figure 15 shows the surface plot of rel8 hr ( y2 ) versus concentration of pva ( x1 ) and number of freeze / thaw cycles ( x2 ) . the data demonstrate that both the factors ( x1 and x2 ) affect the drug release ( rel8 hr ) . it may also be concluded from the graph that the high level of x1 ( concentration of pva ) and high level of x2 ( number of freeze / thaw cycles ) favor the sustained release of the ecn from the mhfs , that is , decrease in the rel8 hr . the low value of x1x2 coefficient of ( 7 ) also suggests that the interaction between x1 and x2 is not significant . k of zero order equation showed correlation coefficient , r value , of 0.997 , indicating a good fit . the k of zero order equation value for the nine batches ( ef1 to ef9 ) showed a wide variation ( 3.79 to 8.20 ) . the data clearly indicate that the k of zero order equation values are strongly dependent on the selected variables . the responses of the k of zero order equation are given in ( 8) . the 1 coefficient is positive ( 0.0192 ) , indicating that k of zero order equation increases by increasing the concentration of pva , and the value of 2 is also positive ( 0.0973 ) , indicating that the k of zero order equation increases by increasing the number of freeze / thaw cycles : ( 8)y=9.194 + 0.0192x1 + 0.0973x20.00566x1x2 0.0100x120.0245x22 . a linear plot ( figure 16 ) drawn between the predicted and observed responses demonstrate high values of r ( r > 0.9 ) , indicating excellent fit . figure 17 shows the surface plot of k of zero order equation ( y3 ) versus concentration of pva ( x1 ) and number of freeze / thaw cycles ( x2 ) . the mhf optimized by the 3 full factorial design with the required values of independent variables was prepared . the optimized mhf was prepared and studied for various physical and physicochemical properties . the mucoadhesive strength and in vitro residence time of the optimized formulation were 126.23 8.56 gm / cm and 736 28 minutes , respectively . the in vitro residence time is higher than the required , that is , 12 hours . the optimized mhf was found to possess excellent mechanical strength and the firmness , that is , folding endurance > 300 number of folding . the water uptake data exhibited an excellent fit in the model with the value of exponent , n , 0.64 with r = 0.998 . the n value indicates an anomalous mechanism of water uptake in which solvent diffusion , as well as polymer relaxation , is of the same magnitude . the optimized formulation was fitted well in the zero order equation ( r > 0.99 ) . the drug released from the optimized mhf was able to inhibit the growth of c. albicans for 12 hours . the maximum diameter of zone of inhibition by the aliquots of optimized formulation was found to be 16.57 0.59 mm at twelfth hour . from the standard calibration curve of ecn agar diffusion assay , the growth inhibition zone of 12 hr dissolution sample of optimized batch corresponds to 75.65 0.67% of ecn , which correlated well with dissolution studies data . it was concluded that the prepared mhfs possess good mucoadhesion and reasonable in vitro residence time which is important for prolonging the contact time of the drug with the buccal mucosa , thus improving the overall therapy of oral candidiasis . mucoadhesive hydrogel film with 15% polyvinyl alcohol and 7 freeze / thaw cycles was optimized by comparing the required drug release rate using polynomial equation generated by running the nine sets of experiments . the optimized batch exhibited the sustained release of drug up to 12 hours with the good fit with zero order kinetics . the in vitro antifungal studies revealed that the drug released from the optimized mhf could inhibit the growth of candida albicans for prolonged period up to 12 hours suggesting better patient compliance and higher therapeutic efficacy .

the mucoadhesive hydrogel film was prepared and optimized for the purpose of local drug delivery to oral cavity for the treatment of oral candidiasis . the mucoadhesive hydrogel film was prepared with the poly(vinyl alcohol ) by freeze / thaw crosslinking technique . 32 full factorial design was employed to optimize the formulation . number of freeze / thaw cycles ( 4 , 6 , and 8 cycles ) and the concentration of the poly(vinyl alcohol ) ( 10 , 15 , and 20% ) were used as the independent variables whereas time required for 50% drug release , cumulative percent of drug release at 8th hour , and k of zero order equation were used as the dependent variables . the films were evaluated for mucoadhesive strength , in vitro residence time , swelling study , in vitro drug release , and effectiveness against candida albicans . the concentration of poly(vinyl alcohol ) and the number of freeze / thaw cycles both decrease the drug release rate . mucoadhesive hydrogel film with 15% poly(vinyl alcohol ) and 7 freeze / thaw cycles was optimized . the optimized batch exhibited the sustained release of drug and the antifungal studies revealed that the drug released from the film could inhibit the growth of candida albicans for 12 hours .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Evaluation of Optimized MHF 5. Conclusion

PMC2859413

dynamic susceptibility - contrast perfusion magnetic resonance ( mr ) imaging is a commonly used method for the noninvasive assessment of cerebral blood perfusion because of the availability of mr imaging units and lack of exposure to ionizing radiation [ 110 ] . after a bolus injection of intravascular contrast agent , the passage of bolus induces the susceptibility inhomogeneity , which in turn causes a relative decrease of image intensities of brain tissues from the baseline . various tissues manifest distinct blood - supply patterns since the contrast agent arrives consecutively , leading to temporal intensity drops at different time instants . based on the perfusion data , we can calculate the hemodynamic parametric maps , that is , relative cerebral blood volume ( rcbv ) , relative cerebral blood flow ( rcbf ) , and mean transit time ( mtt ) , by employing the indicator dilution theory [ 11 , 12 ] . this has been used in the assessment of many brain disorders such as tumors [ 2 , 7 , 13 ] , stroke , infection , and moyamoya disease [ 16 , 17 ] . the estimation of rcbv and rcbf , however , requires arterial concentration of the contrast agent as an arterial input function ( aif ) . this is a demanding task , and many methods have been proposed to address the issue . additionally , classification of blood - supply patterns for various tissue types in brain based on bolus transit profiles is essential for the assessment of brain perfusion . manually selected single or multiple pixels for the tissue of interest and used the averaged temporal profile as a reference in producing a similarity map for segmenting gray matter ( gm ) and white matter ( wm ) from perfusion images . this method is advantageous for easy implementation but limited to extraction of a single tissue type per similarity map . if one attempts to segment multiple tissue types , associated similarity maps need to be created one by one after cumbersome selections of reference pixels , which may be prone to operator influence . alternatively , martel et al . used the conventional factor analysis , under the hypothesis that the observable signal - time curve for each pixel is a weighted sum of pure physiological factors , to classify the signal - time curves of arterial and venous structures from mr dynamic perfusion images . this method has also been applied in nuclear medicine to separate cardiac components and extract left ventricular input function from dynamic h2o pet images [ 2022 ] . it should be noted that , although the factor - analysis - based methods are attractive , additional assumptions of a priori knowledge are required to obliquely rotate the eigenvectors to yield meaningful physiological factors [ 2325 ] . besides , only one or two physiological factors were resolved in past studies . to our knowledge a recent related work was to use the expectation - maximization algorithm with a mixture of multivariate gaussian models for fitting the perfusion mr images so that each pixel can be labeled by the resulting maximal posterior probability . the aim of this study is to automatically classify the spatiotemporal blood - supply patterns which enables us to extract the arterial compartment for modeling the aif and segment other tissue regions as well . to this end , we employ an independent factor analysis ( ifa ) , a data - driven method , allowing us to blindly separate mixed signals into independent - factor ( or independent - source ) components for multitissue hemodynamic classification . that is , the hemodynamics of each tissue type can be dissected without making a priori spatial and temporal assumptions of physiology . the factors in the ifa , in contrast to the conventional factor analysis , are modeled by a finite mixture of gaussian functions that can be used as a constraint to remove rotational factors . this method has been applied to successfully separate the background factors and noise artifacts from the stimulus - evoked meg and eeg sensor data contaminated by large background brain activity . in this study , the use of ifa is based on two inherent assumptions : ( 1 ) signal intensity of each pixel is a linear mixture contributed from different tissues , referred to as the partial volume mixing , which is a well - known phenomenon due to finite resolution of mr scan ; ( 2 ) the anatomic structures of pure tissue types are spatially independent ( nonoverlapped with each other ) . the first step is to identify a dominant tissue type on each independent - factor ( if ) image resolved from the ifa based on the arrival order of corresponding signal - time curve and a priori knowledge of brain anatomy . the second step is to automatically extract regions of the dominant tissue from each selected if image by means of an optimal threshold . the protocol of mr imaging and data recordings is first described followed by the introduction of ifa method and calculation of the pixel - by - pixel rcbv , rcbf , and mtt maps . computer simulations are presented to validate the application of ifa method on two - dimensional independent factors . resultant five if images in conjunction with corresponding signal - time curves from a data set are exhibited . we then calculate and display the rcbv , rcbf , and mtt maps based on the extracted arterial compartment . the averaged ratios for rcbv , rcbf , and mtt between gm and wm from five normal subjects are also computed . five healthy volunteers ( three males and two females ) aged from 18 to 47 were recruited to participate in this study . a multislice gradient - echo epi pulse sequence on a 1.5-tesla scanner ( signa cv / i ; ge medical systems , milwaukee , wi , usa ) was used and the imaging parameters were transaxial imaging , te / tr = 60/1000 milliseconds , flip angle = 90 degrees , fov = 24 cm 24 cm , matrix = 128 128 , slice thickness / gap = 5/5 mm for 7 slices , one acquisition , and 100 images per slice location . twenty milliliters of gd - dtpa - bma ( omniscan , 0.5 mmol / ml ; nycomed imaging , oslo , norway ) followed by 20 ml of normal saline were delivered administratively by a power injector ( spectris ; medrad , indianola , pa , usa ) at a flow rate of 3 - 4 ml / sec in the antecubital vein . the first thirteen and last thirty - seven images were removed from 100 images and fifty images , which exhibited stable baseline and discernible temporal signal changes , were kept in a slice location for analysis . figure 1 displays part of dynamic perfusion images at an upper slice location encompassing the first circulation from a 39-year - old volunteer . with 16384 ( = 128 128 ) pixels for each image , the observation of 50 temporal images can be represented by a 50 16384 matrix . the signal - to - noise ratios ( snrs ) of these five data sets were 51 , 55 , 77 , 83 , and 88 , respectively . in this study , we assume that there are n pure tissue types presented on perfusion images and that signal intensity of each pixel is a linear combination of contributions from n pure tissues due to the effect of partial volume mixing in mr scanning . let the observed noisy mixtures be denoted by a matrix y of size 50 16384 , the linear mixing by a matrix h of size 50 n , the independent factors by a matrix x of size n 16384 , and the zero - mean gaussian noise by a matrix u of size 50 16384 , where each row in x represents an image for a tissue type , each column of h represents a signal - time curve for an associated tissue type , and covariance matrix of the zero - mean gaussian noise is denoted by . under the assumption that the sources x are mutually statistically independent , the ifa - based blind source separation ( bss ) technique attempts to recover the unknown mixing matrix h and hidden if images x from the observed noisy mixtures : in order to recover the if images , we assume that each row of the matrix x is a realization of a random variable whose probability density p(xj ) is in a form of mixture of gaussians ( mog ) given by ( 2)p(xj j)=qj=1njj , qjg(xjj , qj,j , qj)=qj=1njj , qj12j , qjexp [ 12(xjj , qj)2j , qj],j={j , qj,j , qj,j , qj } , where g stands for the gaussian function , qj is a variable with value running over number of gaussians with means j , qj , variances j , qj used in mog model for the source xj , j = 1 , , n , and j , qj 's are the mixing proportions satisfying qjj , qj = 1 . let q = ( q1 , , qn ) denote all possible combinations of the individual qj . the joint probability density p(x ) in n - dimensional space can be formed by the product of the one - dimensional mog 's in ( 2 ) due to the mutual independence of xj , which is itself an mog ( 3)p(x )=j=1np(xj j)=qqg(xq , vq ) , where ( 4)q=j=1nj , qj=1,q1, ,n , qn,q=(1,q1, ,n , qn),vq = diag ( 1,q1, ,n , qn),g(xq , vq)=jg(xjj , qj,j , qj),q=q1 qn . in our implementation , nj was determined to be 2 for each source xj . to estimate the if model parameters , the kullback - leibler distance function is employed to measure the difference between the probability density of the observed signals given the model parameters w , that is , p(y | w ) , and the observed one , that is , p(y ) , which is given by ( 5)(w)=p(y)log p(y)p(y w)dy=e[log p(y w)]hp , where w = { h , , } denotes the unknown mixing matrix h , noise covariance , and mog parameters . the operator e denotes the average over the observed y. the first term in ( 5 ) is the negative log - likelihood of the observed signals given the model parameters w , and the second term is the entropy of observed signals , which is independent of w and will henceforth be dropped . note that , based on p(q , x , y | w ) = p(q)p(x | q)p(y | x ) , p(x | q ) = g(x q , vq ) , and p(y | x ) = g(y hx , ) , the probability density p(y | w ) can be expressed in a closed form : ( 6)p(y w)=qp(q)p(x q)p(y x)dx=qp(q)p(y q ) where p(y | q ) = g(y hq , hvqh + ) . by minimizing (w ) with respect to w based on the expectation - maximization ( em ) method and denoting w the parameters obtained from the previous iteration , the iterative algorithm can be summarized as follows . initialize all the unknown parameters , that is , w = { h , , }. compute the conditional mean of ( 7)xt y , w= xtp(x y , w)dx , xxt y , w=xxtp(x y , w)dx,xj qj , y , w=xjp(xj qj , y , w)dxj , xj2 qj , y , w=xj2p(xj qj , y , w)dxj , p(qj y , w ) . update the mixing matrix and noise covariance : ( 8)h = e[yxt y , w](e[xxt update the source mog parameters : ( 9)j , qj = e[p(qj y , w')xj y , w ) ] , j , qj = e[p(qj y , w)xj2 qj , y , w]e[p(qj y , w)]j , qj2,j , qj = e[p(qj y , w ) ] . rescale the parameters to cancel the extra freedom of scaling for facilitating convergence : ( 10)j2=qj=1njj , qj(j , qj+j , qj2)(qj=1njj , qjj , qj)2,j , qjj , qjj , j , qjj , qjj2 , hijhijj . finally , the if images can be reconstructed from the estimated model parameters w and measured data y using the least - mean - square estimator : ( 11)x^lms(y)=qp(q y)(aqy+bq ) , where p(q | y ) = p(q)p(y | q)/qp(q)p(y | q ) , aq = ( hh + vq)h , and bq = ( hh + vq)vqq . prior to the calculation of pixel - by - pixel rcbv , rcbf , and mtt maps , we computed the concentration - time curve ct(t ) for each pixel using the formula ( 12)ct(t)=kteln ( s(t)s0 ) , where k is an unknown constant , te is the echo time , and s(t ) and s0 are the signal intensities of each pixel at time t and at the baseline , respectively [ 1 , 4 , 5 , 30 ] . by using the indicator dilution theory , one can determine the rcbv for each pixel as a ratio of the area integrating over the first pass ( e.g. , from 1st to 22nd images in figure 1 ) of the contrast agent under the concentration - time curve , ct(t ) , to that under the aif , ca(t ) [ 11 , 12 ] , ( 13)rcbv=first passct(t)dt first passca(t)dt , where ca(t ) is the concentration - time curve for the arterial region . the rcbf can be computed based on the relationship with concentration - time curve for each pixel : ( 14)ct(t)=rcbfca(t)r(t ) , where denotes convolution , denotes multiplication , and r(t ) is the residue function for the pixel . r(t ) curve for each pixel in ( 14 ) can be resolved using the singular value decomposition ( svd ) method and the value of rcbf at each pixel was determined by the maximum value of rcbf finally , the mtt of contrast - agent particles passing through a pixel was defined to be [ 1 , 4 , 5 ] in order to validate the implementation of ifa algorithm , a computer simulation was conducted before perfusion data were processed . since the purpose of this simulation was to verify applicability of the ifa method on the separation of two - dimensional if images , rather than validate the theory of ifa method ( readers are referred to for detailed computer simulations ) , we simply created four hypothetical if images ( 128 128 ) with zero background and foreground related to four major tissue types , that is , artery ( if1 : 603 pixels ) , gm ( if2 : 1683 pixels ) , wm ( if3 : 1514 pixels ) , and others ( if4 : 924 pixels including vein , sinus , choroid plexus , and cerebral spinal fluid ) ( the left four plots in figure 2(a ) ) . in practice , the four tissue - related areas were segmented from the perfusion data and copied to the corresponding locations . the corresponding averaged temporal profile of each area during the first pass was simplified into five points to generate the hypothetical mixing matrix h ( 5 4 ) ( also see the right most plot in figure 2(a ) ) ( 16)h=[11110.43240.69920.87890.89750.25290.46760.69400.70260.61860.76550.84570.74380.84640.92690.96360.8934 ] . we arranged the four if images into a matrix x ( 4 16384 ) which was multiplied by the h to create a matrix y = hx ( 5 16384 ) . additional multivariate gaussian random noises with zero mean and diagonal covariance matrix ( diagonal terms were 10 ) were added to the noise - free simulated data y ( y is displayed in figure 2(b ) ) . now the task was to estimate four if images and mixing matrix from the matrix y , to compare them with the hypothetical ones . the number nj of gaussian functions in the mog model was chosen to be 2 for modeling the probability density function of each if image and minimizing the computational cost since we have experienced that the use of larger nj did not improve the results in either simulated or perfusion data . the values of cost function ( the first term in ( 5 ) ) reduced quickly and converged after around 100 iterations ( figure 2(c ) ) . the correlation value between each pair of hypothetical and estimated if images and that between each pair of hypothetical and estimated mixing weightings were higher than 0.9999 . we further computed a matrix j=(h^th^)-1h^th , where h^ was the estimated mixing matrix , and the result was ( 17)j=[0.99820.00160.00110.00150.00421.00300.00190.00440.00410.00291.00020.00290.00170.00140.00101.0001 ] , which was very close to the identity matrix if the estimate was correct . we repeated the simulation when the snr was reduced to 40 , which was lower than that in the normal data . results showed that the correlation value between each pair of hypothetical and estimated mixing weightings remained as high as 0.9998 and that between each pair of hypothetical and estimated if image only degraded slight to between 0.9883 and 0.9997 . the high correlation values and good approximation of j matrices not only validated the correctness and convergence of our implementation of the ifa method , but also promised the suitability of the ifa method in segregating various tissue types from perfusion mri data . the number of if images ( n ) , that is , number of tissue types , needs to be determined prior to the ifa process . various numbers , ranging from 4 up to 6 , have been assumed in the calculation and we have found that n = 5 elucidated distinctly discernible tissue types , appearing to agree with our knowledge of brain anatomy and physiology . results from five normal image data sets have been confirmed by a neuroradiologist who is one of the coauthors in this study with expertise in perfusion imaging . one of the results was shown in figure 3 , where the upper panel depicts the resultant five if images , which are artery ( ar ) , gm , wm , vein and sinus ( vs ) , and choroid plexus ( cp ) , respectively . figure 3(b ) displays the corresponding signal - time curves ( columns of h ) , respectively , which were all normalized to unit variance and their baselines were shifted to 1.0 for the comparison . the tissue types of if images were identified based on their anatomical structures and the arrival order of contrast agent , that is , artery follows by gm , wm , vs , or cp . among all if images , the if image with brighter pixels representing artery can be easily recognized because of its signal - time curve presenting the fastest signal drop ( red curve in figure 3(b ) ) . the pixels of each major tissue type can be further segmented out from each if image using an automatically optimal thresholding technique , that is , the otsu 's method in this study . the final segmentation result was depicted by a color - coded composite image ( left panel in figure 3(c ) ) and the averaged signal - time curve within each colored area , that is , each tissue type , was computed ( right panel in figure 3(c ) ) . to create the hemodynamic parametric maps , namely , rcbv , rcbf , and mtt shown in the left , middle , and right panels in figure 3(d ) , respectively , we first converted the averaged arterial signal - time curve and temporal intensity profile at each pixel into the concentration - time curves using ( 12 ) followed by ( 13 ) , ( 14 ) , and ( 15 ) . from the segmentation results of the five normal data sets , we have observed that arterial areas were all reliably segmented and the contrast agent consistently arrived first at the artery , followed by gm , wm , vs , and cp . the averaged ratios for rcbv , rcbf , and mtt between gm and wm were 2.139 0.190 , 2.598 0.184 , and 0.789 0.098 , respectively , which were congruent with those in the literature [ 3 , 4 , 9 , 13 ] . this study describes an ifa - based method to classify pixels of the same tissue type on perfusion images based on bolus transit profiles and the assumptions of spatial independence as well as the partial - volume mixing effect . the ifa method is flexible in learning the source densities from observed data so that sources can be more accurately modeled by the mixture of gaussians for the facilitation of subsequent separation . the ifa technique is related to projection pursuit [ 32 , 33 ] , where interesting projections of multidimensional data are pursued for optimal visualization of data and exploratory data analysis . projection pursuit is usually performed by finding directions in which the data is least gaussian distributed . since the independent factors in the ifa are modeled by mixture of gaussian functions , it is interesting to investigate whether the independent factors present maximal non - gaussian clustering structures in future work . our results indicated that the brighter pixels in a cluster were homogeneous in most tissues and can be segmented out using otsu 's method for automatic determination of optimal thresholds . otsu 's method is simple in terms of implementation and computation and is robust to histogram irregularities caused by noise . the determination of the number n of if images is an important issue to be addressed . as suggested by attias , one can determine this number using a comprehensive method , for example , the model comparison method , or a simpler but less precise method , for example , the number of significant eigenvalues of data covariance matrix . in this study , we decided n based on a priori knowledge of the brain anatomy and arrival order of blood flows for different tissues . when n was chosen to be five , each if image exhibited one dominating tissue cluster with brighter intensities , allowing consistent and reliable segregation of the artery , gm , wm , vs , and cp from the normal subjects . when n was less than five , two major tissue types , for examples , artery with gm or vs with cp , can appear at one of if images . on the other hand , if n was larger than five , either one major tissue type was split into two if images , for example , part of sinus was separated from vs , or repeatedly exhibited at two if images . it is worthy to note that the number of unknown parameters depends on the number of dynamic mr images . the more images were used , the more parameters needed to be estimated in the noise covariance and in the mixing matrix h in which columns corresponded to the signal - time curves . we have found that the data points encompassed the duration of the first and second circulations , for example , 50 in the illustrative example ( figure 3 ) , were adequate to resolve the if images . a simple way to determine data length is to average each perfusion image and plot the averaged images with respect to time . the curve delineating the first and second passes is readily seen and the duration can be easily decided . second , the arterial compartment can be modeled consistently on the same slice location for calculation of rcbv , rcbf , and mtt maps . third , bolus transit profiles of these tissues can be well separated , providing information in addition to rcbv , rcbf , and mtt maps , such as temporal scenarios and recirculation of contrast agent . this method also promises differentiation of pathological and nonpathological blood - supply patterns in future clinical applications .

perfusion magnetic resonance brain imaging induces temporal signal changes on brain tissues , manifesting distinct blood - supply patterns for the profound analysis of cerebral hemodynamics . we employed independent factor analysis to blindly separate such dynamic images into different maps , that is , artery , gray matter , white matter , vein and sinus , and choroid plexus , in conjunction with corresponding signal - time curves . the averaged signal - time curve on the segmented arterial area was further used to calculate the relative cerebral blood volume ( rcbv ) , relative cerebral blood flow ( rcbf ) , and mean transit time ( mtt ) . the averaged ratios for rcbv , rcbf , and mtt between gray and white matters for normal subjects were congruent with those in the literature .

1. Introduction 2. Subjects and Data Recording 3. Independent Factor Analysis 4. Calculation of the rCBV, rCBF, and MTT 5. Results 6. Discussion 7. Conclusions

PMC2742651

the human placenta is unique among species . as a matter of fact , of all mammalian organs the placenta shows the greatest variation in terms of anatomy . however , in all species it has the same fundamental function , to nourish the growing fetus , by establishing contact with the maternal blood circulation . in addition , the placenta acts as a protective barrier ; as an example it prevents stress hormones to pass over to the fetus via active transport . in order to prevent rejection of the fetus , the placenta expresses an intricate pattern of major histocompatibility complex molecules , immunizing the mother against the foreign fetal tissue . during pregnancy the placenta also has several important endocrine functions , producing important hormones such as human chorionic gonadotropin and human placental lactogen [ 4 , 5 ] . hence , due to its vast number of functions the placenta expresses more than 20 000 dna sequences and is perhaps the organ expressing the largest number of genes . in fact , it has been used as a fishing pond for finding and sequencing new genes and has been used to identify and clone transporters and receptors , such as human growth hormone - variants , integrin alpha v , and subtypes of the angiotensin receptors [ 79 ] . it affects 3%7% of all pregnancies and is a major cause of maternal and fetal mortality . treatment for pe is symptomatic , and removal of the placenta is the only curative treatment , suggesting that the placenta is a key in the development of pe . the etiology of pe is still largely unknown ; however , it is generally accepted that pe begins with inadequate placentation . invasion of placental trophoblast cells into the maternal spiral arteries is too shallow , leading to inadequate placental perfusion and hypoxia [ 11 , 12 ] . proteomics is a research field undergoing rapid development , especially in regards to the methods being used . the most common method is two - dimensional polyacrylamide gel electhrophoresis ( 2d - page ) . 2d - page for proteome studies gives a map of proteins which reflect changes in protein expression levels , as well different isoforms and posttranslational modifications . lately , three types of protein microarrays have become available , namely , analytical microarrays , functional microarrays , and reverse phase microarrays ( reviewed in ) . highly sensitive and specific recombinant antibody microarrays allowing screening of crude protein extracts are also being developed . due to the large number of proteins , no protein array platform has yet been designed that covers the entire human placental proteome . thus , when screening the placental proteome 2d - page is still among the most accepted methods giving the highest coverage although this may soon change . however , there are several obstacles when working with placental tissue , since it contains large amounts of lipids and glycogen . in order for the first dimension , isoelectric focusing ( ief ) , to be successful , proteins have to be efficiently extracted , and solubilized , and contaminants , such as lipids , polysaccharides , nucleic acids , and salt ions , must be removed . lipids , polysaccharides and nucleic acids bind to proteins which increases the viscosity of the samples and often results in horizontal streaking on the 2d - page . if the salt concentration is too high in the sample , the conductivity in the ipg strips increases and prolongs the ief , resulting in poor 2d - page resolution . for example , precipitating samples with dichloromethane / methanol has been described to remove lipids whereas precipitation with ethanol will remove nucleic acids . for these reasons , the placenta proteome is not fully investigated although some progress has been made in the use of proteomics technology to study pe placenta [ 17 , 18 ] . the primary aim of this study was to establish a robust sample preparation protocol of human placental tissue , by comparing two different solubilization solutions in combination with six different precipitation methods . sixty women admitted at the department of obstetrics and gynecology , lund university hospital , were included and assigned to two groups : pe ( n = 30 ) and control ( n = 30 ) ( table 1 ) . g / l or rise in blood pressure above 20 mmhg from the first trimester of pregnancy . a 10 mm cube of placenta tissue was collected immediately after removal of the placenta and was frozen on dry ice and stored at 80c . this study was approved by the ethical committee review board for studies in human subjects at lund university . human placental tissues were pulverized , and the frozen placental powder ( 25 mg frozen weight ) was homogenized with a lysis solution containing either 3 ml 8 m urea and 2% chaps or 3 ml ice cold 10 mm hepes , ph 7.5 , 24 mm kcl supplemented with a protease inhibitor cocktail ( complete mini plus edta , roche , basel , switzerland ) . samples were stirred for 1 hour followed by centrifugation at 43 000 g for 2 hours . protein concentrations were determined by the bicinchoninic acid ( bca ) method ( thermo scientific , rockford , usa ) with bovine serum albumin ( bsa ) as a standard . pooled samples were prepared by combining equal amounts of proteins from individual samples . the pooled samples were either used directly ( fresh samples ) or stored at 80c prior to use ( frozen samples ) . 30 g were precipitated , and for small and large 2d - page , 100 g and 500 g were precipitated , respectively . acetoneextracted samples were mixed with ice - cold acetone to final concentration of 80% acetone . samples were incubated overnight at 20c followed by centrifugation at 9000 g for 2 minutes . the supernatant was removed , and the protein pellets were allowed to air dry . extracted samples were mixed with ice - cold acetone to final concentration of 80% acetone . samples were incubated overnight at 20c followed by centrifugation at 9000 g for 2 minutes . the supernatant was removed , and the protein pellets were allowed to air dry . acidified acetonesamples were precipitated as above , but the acetone was acidified with acetic acid to a final concentration of 0.05% . samples were precipitated as above , but the acetone was acidified with acetic acid to a final concentration of 0.05% . samples were centrifuged as above , and the protein pellets were allowed to air - dry . samples were centrifuged as above , and the protein pellets were allowed to air - dry . briefly , one volume of sample was mixed with 4 volumes of methanol , 2 volumes of dichloromethane , and 3 volumes of distilled water . the samples were centrifuged 9000 g for 2 minutes at room temperature ( rt ) . the supernatant was discarded , and 3 volumes of methanol were added followed by mixing . the samples were centrifuged at 9000 g for 2 minutes and the supernatant was again discarded after which pellets were air dried . briefly , one volume of sample was mixed with 4 volumes of methanol , 2 volumes of dichloromethane , and 3 volumes of distilled water . the samples were centrifuged 9000 g for 2 minutes at room temperature ( rt ) . the supernatant was discarded , and 3 volumes of methanol were added followed by mixing . the samples were centrifuged at 9000 g for 2 minutes and the supernatant was again discarded after which pellets were air dried . trichloroacetic acid ( tca)one volume of 40% tca was added to one volume of samples and incubated for 1 hour on ice followed by centrifugation at 9000 g for 2 minutes . the supernatant was removed , and the protein pellets were either directly dried or washed with ethanol and then allowed to air dry . the dried pellets were either dissolved in 30 l sodium dodecyl sulphate ( sds ) sample buffer ph 8.5 ( 247 mm tris , 2% sds , 0.5 mm edetate ( edta ) , 16.5 mm dithiothreitol ( dtt ) , 10% glycerol , and 0.025% ( w / v ) bromophenol blue ) followed by 1d - page or dissolved in rehydration sample buffer containing 8 m urea , 2% chaps , 10 mm dtt , and 2% 310 nl ampholines ( ge healthcare , little chalfont , uk ) . one volume of 40% tca was added to one volume of samples and incubated for 1 hour on ice followed by centrifugation at 9000 g for 2 minutes . the supernatant was removed , and the protein pellets were either directly dried or washed with ethanol and then allowed to air dry . the dried pellets were either dissolved in 30 l sodium dodecyl sulphate ( sds ) sample buffer ph 8.5 ( 247 mm tris , 2% sds , 0.5 mm edetate ( edta ) , 16.5 mm dithiothreitol ( dtt ) , 10% glycerol , and 0.025% ( w / v ) bromophenol blue ) followed by 1d - page or dissolved in rehydration sample buffer containing 8 m urea , 2% chaps , 10 mm dtt , and 2% 310 nl ampholines ( ge healthcare , little chalfont , uk ) . samples ( 30 g ) were loaded onto 10-well 4%12% nupage bis - tris gels ( invitrogen , carlsbad , usa ) and run at 200 v for 45 minutes using 2-(n - morpholino ) ethanesulfonic acid ( mes ) as running buffer . the first dimensional isoelectric focusing ( ief ) was performed on a horizontal multiphor ( amersham biosciences , usa ) . immobilized ph gradient ( ipg ) blue native strips 310 nl ( 0.5 3 70 mm or 0.5 3 240 mm ) ( serva electrophoresis gmbh , heidelberg , germany ) were in - gel rehydrated in rehydration sample buffer over night . for 7 cm strips , 130 l ( containing 100 g of proteins ) and for 24 cm strips , 450 l ( containing 500 g of proteins ) rehydration sample buffer were used . the focusing was performed at the following voltage gradients : 150 v for 30 minutes , 300 v for 30 minutes , 600 v for 30 minutes , 1500 v for 1.5 hours , 3000 v for 2 hours ( 7 cm strips ) and 25 hours ( 24 cm strips ) . the strips were equilibrated for 10 minutes in a solution containing 65 mm dtt , 6 m urea , 30% ( w / v ) glycerol , 2% ( w / v ) sds and 50 mm tris - hcl ph 8.8 . a second equilibration step was performed for 10 minutes in the same solution except for dtt , which was replaced by 259 mm iodoacetamide . the strips were soaked in electrophoresis buffer ( 24 mm tris base , 0.2 m glycine and 0.1% sds ) just before the second dimensional gel electrophoresis . the strips were applied on 12% homogeneous tris - glycine gels ( 0.1 8 8 cm or 0.1 20 24 cm ) and overlaid with a solution of 1% agarose in electrophoresis buffer ( kept at 60c ) . electrophoresis was carried out in an xcell mini - cell apparatus ( invitrogen ) using electrophoresis buffer at 125 v for 1.5 hours or in an hoefer dalt gel apparatus ( ge healthcare ) at 20c and constant 80 v for 19 hours . gel staininggels were either stained with silver or stained with sypro ruby ( thermo scientific ) . gels were either stained with silver or stained with sypro ruby ( thermo scientific ) . image analysissmall gels were scanned using a canoscan 995of ( canon , solna , sweden ) , and large gels were scanned using an amersham typhoon 9400 ( ge healthcare ) . the images of the large gels stained with sypro ruby were submitted to ludesi 2d analysis ( ludesi , malmo , sweden , http://www.ludesi.com ) for spot detection , matching , and analysis . small gels were scanned using a canoscan 995of ( canon , solna , sweden ) , and large gels were scanned using an amersham typhoon 9400 ( ge healthcare ) . the images of the large gels stained with sypro ruby were submitted to ludesi 2d analysis ( ludesi , malmo , sweden , http://www.ludesi.com ) for spot detection , matching , and analysis . the purified peptides were eluted directly onto the sample target ( anchorchip target , bruker daltonik gmbh , bremen , germany ) where 0.7 l of matrix , 2,5-dihydroxybenzoic acid ( 10 mg / ml in 30% acn ) had been added . mass spectra of positively charged ions were recorded on a bruker reflex iii instrument ( bruker daltonik ) operated in the reflector mode . the xmass 5.0 and ms biotools software packages provided by the manufactures were used for data processing . the purified peptides were eluted directly onto the sample target ( anchorchip target , bruker daltonik gmbh , bremen , germany ) where 0.7 l of matrix , 2,5-dihydroxybenzoic acid ( 10 mg / ml in 30% acn ) had been added . mass spectra of positively charged ions were recorded on a bruker reflex iii instrument ( bruker daltonik ) operated in the reflector mode . the xmass 5.0 and ms biotools software packages provided by the manufactures were used for data processing . database searchingfor protein identification , human protein sequences in the swissprot database were searched using the mascot software ( matrix science ltd . parameters specified in the search were taxa , homo sapiens ; missed cleavages , 1 ; peptide mass tolerance + /0.1 da ; fixed modification , carbamidomethyl ( c ) ; variable modification , oxidized methionine . for protein identification , human protein sequences in the swissprot database were searched using the mascot software ( matrix science ltd . , parameters specified in the search were taxa , homo sapiens ; missed cleavages , 1 ; peptide mass tolerance + /0.1 da ; fixed modification , carbamidomethyl ( c ) ; variable modification , oxidized methionine . proteins were separated by 1d - page using 10-well 12% nupage bis - tris gels ( invitrogen ) according to manufacturer 's instruction . the gels were loaded with 2.5 g protein per well and run for 60 minutes at 200 v in mops running buffer under reduced conditions and blotted onto polyvinylidene fluoride ( pvdf ) membranes for 8 minutes at 23 v using an iblot dry blotting system ( invitrogen ) . membranes were incubated with primary antibody for 1 hour ( mouse monoclonal anti - apoa1 , sc-69755 , 1 : 2000 dilution ) , followed by washes and then , incubated with the secondary antibody for 1 hour ( goat polyclonal antimouse igg , sc-2031 , 1 : 5000 dilution ) . antibodies were obtained from santa cruz ( santa cruz biotechnology , santa cruz , usa ) . subsequently , the membrane was exposed to enhanced chemilumeniscence ecl+ ( ge healthcare ) for 3 minutes . autoradiographic film hyperfilm ecl ( ge healthcare ) was applied to the blot for various durations to obtain satisfactory exposure . when developed , films were scanned with ultra violet light using a gene flash scanner ( syngene , cambridge , uk ) and imported into syngene gene tools . one reference sample was added to all gels to serve as an internal calibrator to normalize the quantification between gels . a students t - test was used to determine statistical difference between the two groups . immunohistochemistry was performed using envision+system - hrp ( dakocytomation , carpinteria , usa ) on 4 representative pe samples and 4 controls according to the manufacturer 's instructions . briefly , fresh frozen sections , 12 m thick , of the placenta samples were fixed by immersion in 4% phosphate buffered saline ( pbs ) buffered formaldehyde for 15 minutes at rt . sections were incubated in a blocking solution containing 3% h2o2 for 10 minutes at rt . the apoa1 antibody was diluted ( 0.1 g / ml ) in pbs containing 0.25% triton x100 and 1.5% normal goat serum . peroxidase labeled polymer was applied to cover the specimens and then incubated for 30 minutes . after rinsing the sections , liquiddab substrate chromogen solution was applied and incubated for 8 minutes . the sections were rinsed and stained with mayers hematoxylin after which sections were mounted with mountex ( histolab , gothenburg , sweden ) and cover slipped . sections were viewed under an olympus bx-60 microscope and images captured using an olympus dp50cu digital camera ( olympus america inc . , center valley , usa ) . two solubilization solutions were chosen : hepes buffer and the commonly used urea / chaps . the pellet resulted in smearing on 1d - page ( figure 1(a ) ) and without the centrifugation step ; no proteins entered the ief - strip resulting in a blank 2d - page ( data not shown ) . there were no obvious differences in protein patterns between the supernatant and the starting material ( figure 1(a ) ) . 1d - page separation was used to determine protein expression from the two different solubilization solutions . a major difference in the protein pattern between hepes minor differences at higher molecular mass range are also obtained when comparing the two different solubilization solutions ( figures 1(b ) and 1(c ) ) . a number of the protein bands that visibly differed between the two solubilization solutions were identified using maldi - tof ms . the obtained identities included annexin a1 , annexin a2 , endoplasmin , vimentin , and hemoglobin subunit alpha and gamma ( table 2 ) . some of the bands from the 1d - page contained more than one identity due to the limited power of resolution using 1d - page . six different precipitation procedures were tested : acetone , acidified acetone , ethanol dichloromethane / methanol , tca , and tca followed by ethanol wash . to determine the effects of precipitation procedures on protein patterns , precipitated samples were analyzed on 1d - page gels ( figures 1(b ) and 1(c ) ) . samples precipitated with tca , with or without ethanol wash , showed a different protein expression profile compared to the other four precipitation methods . no significant differences in the 1d - page expression profiles were detected between the other four precipitation procedures ( acetone , acidified acetone , ethanol and dichloromethane / methanol ) . samples solubilized with urea / chaps generally resulted in more protein spots and less horizontal streaking , regardless of precipitation procedure ( figure 2 ) . the investigation of whether freezing of solubilized and centrifuged samples influenced the results , both fresh and frozen samples , was analyzed using 2d - page . no major differences could be detected in the protein expression profiles for any of the different solubilization or precipitation methods ( data not shown ) . an increased number of low molecular weight proteins were obtained with samples solubilized in urea / chaps precipitated with acidified acetone ( figure 2 , a5 ) . these spots were not obtained with other combinations of solubilizations and precipitations ( figure 3 ) . electroendosmotic flow ( eof ) was detected during the ief run with samples precipitated with acetone , acidified acetone , or ethanol . this resulted in swelling of the ipg - strip in the alkaline ph gradient and might cause a poor protein separation at an isoelectric point above 8 . urea / chaps in combination with either acidified acetone or dichloromethane / methanol precipitation was judged to be the most efficient extraction methods for 2d - page . however , we chose dicholoromethane / methanol since it is known to remove lipids of which there is abundance in the placenta . in a first attempt to investigate whether it is possible to detect differences between the pe and normal placenta in crude extracts , we pooled crude placenta extracts from 30 preeclamptic and 30 normotensive women respectively . in order to decrease the variation of the differently expressed proteins on the 2d - page , three replicates of each pooled group the number of spots detected was 604 and 747 for preeclamptic and normotensive , respectively . the number of spots was calculated based on the criteria that the spots were present in all triplicate gels in each group . the analysis of the 2d - page showed that 23 proteins were increased and 28 were decreased in the pe placenta ( figure 4 ) . two proteins have so far been identified with maldi - tof ms : apolipoprotein a1 ( apoa1 ) ( acc number : p02647 ) and tropomyosin-3 ( tpm3 ) ( acc number : p06753 ) . apoa1 was 1.63 times increased in the pe placenta pool as compared to the control pool ( pe : 10871 1340 optical density ( od ) units and controls : 6663 1772 od units values presented as mean sd ) . the increased accumulation of apoa1 in the pe placenta was validated by means of western blot ( figure 5(a ) ) . after measuring the absorption of all the apoa1 bands , apoa1 was found to be 1.18 times increased in pe ( pe : 63120 14350 optical density ( od ) units and controls : 52810 13310 od units values presented as mean sd ) . the accumulation of apoa1 was significantly increased in pe ( p = 0.02 ) ( figure 5(b ) ) . both normotensive and pe placenta sections showed a similar pattern with weaker staining in the control sections ( figures 5(c)5(e ) ) . the tpm3 expression could not be validated by means of western blot since the recommended antibodies did not work in our hands ( sc-32516 and sc-18174 , santa cruz biotechnology , santa cruz , usa ) . due to the vast number of functions and many different cell types besides the trophoblasts , including white and red blood cells and endothelial cells , the placenta is a complex organ to study . in order to optimize the protein sample preparation for 2d - page , we compared two different solubilization solutions in combination with six different precipitation methods in order to obtain maximal separation of the proteins . hepes buffer and urea / chaps showed different protein patterns in the low molecular mass range . although no protease inhibitors were added to the urea / chaps solution , urea denatures and inactivates proteases and thus , it is unlikely that the differences are due proteolytic cleavage . several steps in the sample preparations appear to be important to remove contaminating substances . during pregnancy there is a gradual accumulation of glycogen as well as a large amount of lipids in the placenta . glycogen and lipids are known to disrupt ief - separation by blocking the ipg - strip resulting in no or very few spots on 2d - page . the centrifugation did not cause a selective loss of proteins , indicating that most of the placental proteins remained in the supernatant . the freezing of samples directly after solubilization ( without the centrifugation step ) resulted in insoluble aggregates . this observation may facilitate the work flow in proteomics where many samples are analyzed , and the samples can be stored in the freezer until further use without having a major influence on the results . our optimization procedures showed that solubilizing with urea and chaps and precipitating with dichloromethane / methanol or acidified acetone gave the highest number of spots on a 2d - page . solubilization with hepes buffer in combination with dichloromethane / methanol also gave a high number of spots ( figure 2 , b1 ) , although there was considerably more horizontal streaking . dichloromethane / methanol has the advantage or also removing lipids , and since the placenta is rich in lipids , this precipitation method may be the preferred choice . hence , in a first study urea / chaps in combination with dichloromethane / methanol was chosen to examine the differences between the pe and normotensive placental proteome . the differentiating proteins have proven to be hard to identify due to the low concentration of the proteins in the differing spots . the same problem was highlighted in a recent preeclamptic proteomic study , where , as a consequence of low amount of proteins , none of the differentially expressed proteins could be identified . however , we have so far been able to identify two proteins differing between pe and controls : apoa1 ( increased in pe ) and tpm3 ( decreased in pe figure 4 ) . the increase of apoa1 in pe was validated by western blot and immunohistochemistry ( figure 5 ) . due to the difficulties in identifying low abundance proteins in a crude extracted sample , one might have to do purifications of specific cell types , for example , trophoblast to explore the proteome of the placenta . another limitation with 2d - page is that membrane proteins do not easily enter ief , the first dimension since they often precipitate in the ipg - strip . in our first attempt to study differences between pe and normotensive placenta , we found apolipoprotein a1 , a lipid binding protein , to be accumulated in the pe placenta . of the lipoproteins , apoa1 is mainly synthesized in the liver and intestines and is the major fraction of the high - density lipoproteins . the placenta has also been suggested as a site of synthesis for apoa1 although it is not clear whether this is an actual de novo synthesis or an accumulation from the blood stream . in a recent whole genome microarray analysis , it is unlikely that the accumulation of apoa1 in the pe placenta is due to high levels of apoa1 in the maternal plasma since plasma levels apoa1 are not increased in pe [ 26 , 27 ] . in a recent proteomics study of amniotic fluid , accumulation of pro - apoa1 was shown in the amniotic fluid in pe . hence , amniotic fluid may be a possible source of the accumulated apoa1 seen in the pe placenta . apoa1 may server either as nutrient or as a signaling molecule by affecting biological signaling pathways such as protein kinase c dependent pathway ( pkc and the mitogen - activated protein kinases ( mapk ) [ 29 , 30 ] . in conclusion , two solubilization methods in combination with six different precipitation procedures were tested . removal of glycogen by centrifugation is crucial since the glycogen clogged the ipg - strip . both the solubilization solution and the precipitation procedure affected the placenta proteome maps . in this study , solubilization with urea / chaps in combination with dichloromethane / methanol or acidified acetone proved to be the best precipitation procedures , resulting in more spots and less streaking on 2d - page . importantly , freezing samples following centrifugation of the solubilized samples did not affect the proteome map . however , due to the limitation using crude extracts for 2d - page , low abundance proteins can not readily be detected , and thus subcellular fractioning may be required to fully investigate the placenta proteome .

the human placenta is a difficult tissue to work with using proteomic technology since it contains large amounts of lipids and glycogen . both lipids and glycogen are known to interfere with the first step in the two - dimensional polyacrylamide gel electrophoresis ( 2d - page ) , the isoelectric focusing . in order to gain the best possible protein separation on 2d - page , an optimized sample preparation protocol for placental proteins was developed . two different buffers , urea / chaps and hepes , were used for solubilization in combination with six different precipitation methods . the removal of glycogen from the samples by centrifugation was crucial for the final proteome maps . solubilization with urea / chaps in combination with dichloromethane / methanol or acidified acetone proved to be the best precipitation procedures . when applied to clinical placenta samples apolipoprotein a1 was found to be accumulated in the preeclamptic placenta , where it may either have a nutritional effect or act as a modifier of signal transduction .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

PMC3727095

the hypothalamus has long been known to play an important role in feeding behavior . as far back as 1951 , anand and brobeck reported that bilateral destruction of the lateral hypothalamus ( lh ) in rats resulted in the complete absence of eating , leading them to term this area of the brain the feeding center . shortly thereafter , delgado and anand reported that electrical stimulation of the lh in cats resulted in a 1,000 percent increase in total food intake . interestingly , rats will work to receive electrical stimulation of the lh ( self - stimulation ) , indicating that this nucleus also plays a function in reward , but excessive food intake leads them to decrease their rate of self - stimulation by half . while a number of classical neurotransmitters have been implicated in lh - induced feeding , the discovery of neuropeptides in the brain has led researchers to consider several of these local neuromodulatory neurochemicals as major players in feeding and reward . two neuropeptides transcribed in the lh are now understood to play significant roles in feeding and reward . the peptides , orexin a ( ox - a ) and orexin b ( ox - b ) ( also called hypocretin 1 and hypocretin 2 ) , are cleaved from the 130-amino acid precursor neuropeptide preproorexin ( ppox ) , which was independently isolated by two research groups in 1998 [ 5 , 6 ] . neurons containing orexin ( ox ) mrna ( about 6700 in the rat ) lie exclusively within the hypothalamus , spanning the dorsomedial hypothalamic nucleus through the perifornical area and into the lateral hypothalamic area [ 5 , 6 ] . this peptide was immediately recognized for its ability to stimulate food consumption , leading one research group to name the peptide ox after the greek word for appetite , orexis . the peptide melanin - concentrating hormone ( mch ) , isolated from the salmon pituitary in 1983 as an antagonist of alpha - msh - induced skin darkening , neurons containing mrna for the 165-amino acid precursor prepro - melanin - concentrating hormone ( ppmch , numbering about 12300 in the rat ) are distinct from but adjacent to those containing ox , lying predominantly in the lh but also in the perifornical area and subzona incerta . it is now well - established that ox and mch can act as orexigenic neuropeptides , affecting both food intake and processes of reward that influence food intake . despite their similar relationship with consumption , these peptides appear to act in complementary rather than redundant ways with food intake , and in fact have largely opposite roles in physiological processes and reward - related behavior . here , we review the current knowledge about the relationship of these peptides with feeding , while providing a brief discussion of their other actions that may elucidate the mechanisms through which they promote food intake . as with all neuropeptides , the receptors for ox and mch are g protein - coupled receptors . there are two known receptors for ox , called the orexin 1 receptor ( ox1r ) and orexin 2 receptor ( ox2r ) , or the hypocretin 1 and 2 receptors . while ox1r binds to ox - a with an affinity that is two to three orders of magnitude greater than for ox - b , ox2r binds to ox - a and ox - b with nearly equal affinity . orexin receptor binding largely results in neuronal excitation , a rise in cytoplasmic calcium , with ox1r activating gq subunits and ox2r activating gq but also gi / o subunits [ 6 , 11 ] . depending on the species studied , there are either one or two receptors for mch . rats , mice , hamsters , guinea pigs , and rabbits have only one identified mch receptor , mchr1 , but humans , rhesus monkeys , dogs , and ferrets also have a functional mch receptor 2 . as with ox2r , mchr1 binding appears to activate both gi / o and gq subunits , although the major effect of mchr1 binding is a decrease in cyclic amp levels [ 14 , 15 ] . projections from ox- and mch - containing neurons terminate in many of the same brain areas , which may explain why these neuropeptides affect a number of the same behaviors . these brain areas include the locus coeruleus , hippocampus , thalamus , nucleus accumbens ( nac ) , ventral tegmental area ( vta ) , amygdala , cortex , and various nuclei of the hypothalamus [ 16 , 17 ] . the receptors for ox and mch are also located in these same brain areas [ 18 , 19 ] . interestingly , while ox1r and ox2r are often found in the same nuclei , they tend to predominate in different subregions of those nuclei . for example , in the hypothalamus , ox1r is most dense in the anterior hypothalamic nucleus while sparse in the lh and absent from the arcuate and paraventricular nuclei , and ox2r is sparse in the anterior hypothalamic nucleus while dense in the lh , arcuate , and paraventricular nuclei . in support of the idea that ox and mch work in a complementary or even opposite manner , these two peptides have been shown to interact directly with each other . neurons containing ox - a or mch are found to contact each other , and ox1r has been described on mch neurons of the lh . in slice preparation , the addition of ox - a or ox - b evokes long - lasting membrane depolarization and increases spike frequency of mch cells , and the addition of mch inhibits ox - a - induced spike frequency of ox neurons . therefore , while ox directly excites mch neurons , mch prevents excitation of ox neurons . the ox and mch systems largely play opposing roles in the regulation of the sleep - wake cycle and energy balance . whereas ox promotes wakefulness and energy expenditure and is inhibited by a rise in glucose levels , mch plays a role in sleep and energy conservation while being activated by glucose . the firing of ox neurons is robustly tied to arousal during the sleep - wake cycle . these neurons discharge during wakefulness , cease firing with sleep onset , remain silent during slow - wave sleep , discharge periodically during paradoxical ( or rapid eye movement , rem ) sleep , and begin firing again prior to the transition from rem sleep to waking [ 24 , 25 ] . they can fire during sleep when an arousing sound stimulus is presented [ 25 , 26 ] . in support of a direct role for ox in promoting arousal , injection of ox - a or ox - b into the lateral ventricles increases waking and decreases slow - wave and rem sleep , while peripheral injection of the ox2r antagonist jnj-10397049 but not ox1r antagonist sb-408124 decreases the latency for persistent sleep . transgenic mice lacking the ppox gene display behaviors strongly resembling narcolepsy , exhibiting frequent periods of behavioral arrest during the dark ( active ) but not light phase . in fact , the link between ox and narcolepsy was established soon after the neuropeptide 's discovery , with an autosomal recessive mutation of ox2r identified in canine narcolepsy and human narcoleptics found to have a reduction , as much as 95% , in the number of ox neurons . in contrast to ox , mch has been linked with sleep , particularly with paradoxical sleep . rather than discharging during wakefulness , mch neurons fire maximally during rem sleep and occasionally during slow - wave sleep . in support of a direct role for mch in promoting sleep , injection of mch into the lateral ventricles increases the quantity of paradoxical and slow - wave sleep , while both mch and mchr1 knockout mice show increased wakefulness [ 34 , 35 ] . on the other hand human narcoleptics show normal numbers of mch neurons along with reduced numbers of ox neurons [ 31 , 36 ] , and there is no evidence to date linking mutations of mchr1 to narcolepsy . orexin and mch also largely play opposite roles in energy balance , in parallel with their roles in physiological arousal . for example , intracerebroventricular ( icv ) injection of ox potently increases oxygen consumption , and transgenic mice overexpressing ppox are resistant to high - fat diet - induced obesity due to their increased energy expenditure and reduced fat consumption . a specific role for ox2r in promoting energy expenditure is supported by evidence that this resistance to dietary obesity is found in ox overexpressors lacking ox1r but not in those lacking ox2r and that chronic icv injection of [ ala11 , d - leu15 ] ox - b , which binds to ox2r , prevents the development of fat - induced obesity . in addition to the small decrease in oxygen consumption that occurs with icv injection of mch [ 38 , 39 ] , mice overexpressing the ppmch gene show increased body weight on a standard diet , while those lacking mch show both decreased body weight and food intake [ 41 , 42 ] . interestingly , despite hyperphagia , mice lacking mchr1 also exhibit decreased body weight on standard chow and are less susceptible to high - fat diet - induced obesity , likely as a consequence of their hyperactivity . also , genetically obese ob / ob and db / db mice are reported to have elevated mch mrna and peptide levels , and chronic icv mch increases caloric efficiency and body fat mass while an mchr1 antagonist decreases them . together , these results support the idea that ox promotes energy expenditure , while mch reduces it . the activity of ox and mch neurons is also regulated by energy status as indicated by levels of glucose . a physiological rise in glucose , which would occur after normal meal ingestion , is found to inhibit the electrical excitability of ox neurons in the mouse lh [ 46 , 47 ] . this is in contrast to nearby mch neurons , which are excited by elevated glucose levels [ 46 , 48 ] . whereas these two changes together might reflect a role for these peptides in energy balance , the evidence described below suggests that they may similarly be seen as promoting intake of a currently consumed food despite their discordant roles in behavioral state and energy balance , ox and mch both act as orexigenic neuropeptides . while this effect can be seen with standard laboratory chow a notable feature of palatable food is that it is generally overconsumed , such that homeostatic signals are overridden during the course of a meal , resulting in prolonged and excessive intake . importantly , in addition to driving intake , both ox and mch are themselves stimulated by the consumption of palatable food , further contributing to its overconsumption . while similar in this positive feedback circuit , the stimulation of food intake induced by ox and mch appears to occur through different , complementary mechanisms . as described later , ox may increase the seeking and motivation to consume palatable food , whereas mch appears to increase the reinforcing effects of caloric intake . a large body of evidence linking ox and mch with food intake comes from studies of transgenic mice overexpressing or lacking the genes for these neuropeptides and also from studies of outbred rats using injections of the peptides or their antagonists . as described in the introduction , the orexigenic effect of ox was noted at the same time that this peptide was first described , although the magnitude of its effect is much lower than that of the highly orexigenic neuropeptide y ( npy ) . under certain paradigms , this has been shown for central injection of ox - a into the lateral ventricles , hypothalamic paraventricular nucleus [ 49 , 51 ] , lh or perifornical area [ 51 , 52 ] , as well as the nac shell [ 53 , 54 ] . while the feeding effects of icv injection with ox - b are sometimes as potent as those of ox - a [ 50 , 52 ] , these effects of ox appear to occur primarily through ox1r , as a stimulatory effect on food intake with injections into specific brain sites has yet to be observed with ox - b [ 51 , 52 ] . notably , ppox knockout mice show no difference in chow intake when compared to their wild - type littermates [ 55 , 56 ] , supporting the idea that this peptide may not be necessary for normal food intake . a body of evidence indicates that the orexigenic effects of ox are far more robust with palatable food . in a variety of paradigms , peripheral administration of the ox1r antagonist sb-334867 is found to significantly suppress intake of palatable , high - fat food [ 5759 ] , and injection of ox - a into the third cerebral ventricle selectively increases intake of a high - fat diet more than a high - carbohydrate diet . whereas peripheral sb-334867 administration does not consistently decrease sucrose self - administration in rats [ 6163 ] , ad libitum fed ppox knockout mice consume less of a sucrose solution than their wild - type littermates . this link between ox and palatable food intake , similar to chow intake , appears to be mediated by ox1r , with the ox1r antagonist sb-649868 but not ox2r antagonist jnj-10397049 found to decrease binge eating of a high - fat , high - sucrose food in female rats . the ability of ox to increase food intake may occur in large part through its stimulation of arousal as well as an increase in motivation , particularly when palatable food is involved . while similar to wild - type littermates in their chow intake , ppox knockout mice exhibit deficits in their ability to learn about the availability of food . under mild food restriction , they demonstrate delayed acquisition of operant responding for chow and significantly diminished food - anticipatory activity prior to scheduled feeding [ 55 , 66 ] . interestingly , conditional ox gene knockdown via rnai in normal mice causes decreased responding for chow under both variable and progressive ratio schedules , suggesting that ox normally promotes reinforcement - related aspects of food intake . the particular reinforcement - related aspect may be motivation , as ox1r binding largely appears to affect palatable food intake by increasing the motivation for food . peripheral administration of the ox1r antagonist sb-334867 decreases progressive and fixed ratio responding for a high - fat diet and for sucrose [ 57 , 61 , 62 ] , while third ventricle injection of ox - a increases progressive ratio responding for sucrose . together , these results suggest that ox , acting at ox1r , promotes food intake by increasing the motivation for food reward . the orexigenic effect of mch on standard chow is roughly of the same magnitude as ox . intake of chow is increased in rats and mice after injection of mch into the lateral or third ventricles [ 9 , 49 , 67 ] , hypothalamic paraventricular nucleus , and nac shell , while it is decreased after injection of an mchr1 antagonist in the nac shell or periphery . although adult ppmch and mchr1 knockouts compared to wild - type mice actually demonstrate hyperphagia [ 41 , 43 , 72 ] , there is evidence that ppmch knockouts at a young age consume significantly less chow [ 41 , 42 ] , indicating that mch has some role in controlling intake of standard food . similar to ox , the orexigenic effect of mch is more robust with palatable food , particularly with calorically dense food . icv injection of mch promotes the intake of a high- or medium - fat diet , more than a chow diet [ 45 , 73 , 74 ] , and icv or peripheral injection of an mch antagonist decreases intake of and operant responding for these diets [ 45 , 75 , 76 ] . further , transgenic ppmch overexpressors compared to wild - type mice exhibit increased consumption of a high - fat diet . a similar relationship for mch is seen with sucrose , with icv mch stimulating intake of sucrose solutions [ 7779 ] and systemic administration of the mchr1 antagonist gw803430 decreasing sucrose self - administration . notably , the relationship of mch with palatable food does not extend to sweet , noncaloric saccharin [ 79 , 80 ] , indicating that mch may be related more to energy conservation than to the intake of palatable food per se . the ability of mch to stimulate food intake may be due more to its reinforcement of ongoing intake rather than to an effect on the motivation to eat . this is supported by evidence showing that mice lacking the ppmch gene show decreased responding for a high - fat diet under both fixed and progressive ratio schedules and that wistar rats given systemic injection of the mchr1 antagonist gw803430 show reduced fixed and progressive ratio responding for a sucrose solution . further , although the same injection was found to suppress cue - induced reinstatement of lever pressing for sucrose , mch blockade does not affect reinstatement for fat . neither peripheral injection of the mchr1 antagonist snap 94847 in rats nor chronic loss of ppmch in mice significantly affects either cue- or pellet - induced reinstatement of fat seeking [ 58 , 81 ] . together , this evidence indicates that mch promotes food intake primarily by increasing energy conservation , motivating animals to continue consuming energy - dense foods . another set of studies that tie ox and mch to food intake examines the effects of various feeding conditions on their levels of mrna or peptide . neurons containing ox are activated in anticipation of feeding but also following consumption of food , provided that it is palatable food . food deprivation upregulates gene expression and protein levels of ox ( ox - a and ox - b ) and both of its receptors in the hypothalamus as early as twenty - four hours after onset . after a forty - eight hour fast , similar changes have been observed [ 6 , 83 ] , and in female rats , the activity of ox neurons is also upregulated , as indicated by double - labeling of ox with phosphorylated creb . given the relationship of ox with glucose ( see section 3.3 ) , these changes after food deprivation may reflect lowered glucose levels ; however , they may also reflect changes in arousal , as a single day of food deprivation increases wheel running activity and decreases the total number of sleep episodes , effects that occur under conditions of heightened ox activity and are taken to indicate foraging behavior . in line with the effects of food deprivation , ox neuronal activity and gene expression are also upregulated when animals are expecting to receive valued food . in rats with restricted access , ox mrna and double - labeling of ox with c - fos are elevated prior to access to a daily meal of chow , corn oil , or chocolate [ 57 , 87 ] , while ox levels begin to return to baseline within 30 minutes after the start of meal consumption . in sated rats , ox and c - fos double - labeling is also increased by a tone that signals the availability of palatable food in the form of high - sucrose pellets . siegel and colleagues demonstrated that the expression of fos in ox neurons increases in animals working for chow during the light phase but not when working to avoid shock or when receiving unearned rewards . these results suggest that ox neurons are activated in conditions when animals expect to receive specific , often preferred foods . in further support of this idea , c - fos expression in ox neurons is also increased during extinction of sucrose seeking , when animals are motivated to obtain food rewards . after consuming palatable food , ox levels are similarly elevated . with high - fat compared to low - fat , high - carbohydrate food , ox gene expression , and ox - a peptide levels are elevated after a single meal or up to three weeks on the diet [ 9092 ] , with longer periods of exposure leading to compensatory decreases in ox . interestingly , this fat - induced increase in ox may occur more from saturated than unsaturated fat , as consumption of a lard - based diet leads to higher ox mrna levels than does that of a fish oil - based diet [ 94 , 95 ] . similar to the results with fat , ox gene expression is also elevated following consumption of a high - sugar diet . together , these findings indicate that ox is activated both when animals are seeking food and also after they have consumed palatable food , which may in part explain why these foods are consumed in excess . unlike neurons containing ox , those containing mch are not consistently activated in anticipation of feeding , although they are activated following consumption of a palatable , caloric food . gene expression of mch is upregulated after twenty - four hours of food deprivation [ 9 , 97 ] , although longer periods of fasting either increase peptide levels or leave them unchanged [ 9799 ] . the failure to observe an increase may be due to the sensitivity of mch neurons to the sleep - wake cycle . this is indicated by evidence showing mch peptide levels after forty - eight hours of food deprivation to be increased in rats sacrificed during their resting ( light ) phase , but not their active ( dark ) stage when mch neurons are normally quiescent . alternatively , mch may play a less prominent role than ox in food seeking induced by deprivation , particularly as mch neurons are excited by elevated glucose levels ( see section 3.3 ) . thus , these changes in mch in response to food deprivation , unlike with ox , may be related more to changes in caloric efficiency than in circulating glucose . in contrast to ox , there is little evidence linking mch with the expectation of receiving food , with double - labeling of c - fos and mch in sated rats unchanged by a tone signaling the availability of high - sucrose pellets . levels of mch after consuming calorically rich food are clearly increased , supporting its role in palatable food consumption . with maintenance on a high - fat compared to low - fat diet , mch gene expression and hypothalamic peptide levels as well as hypothalamic mchr1 mrna levels are elevated [ 101 , 102 ] . although the type of fat in the diet may not make a difference in this effect , the caloric content appears to be essential , with drinking of noncaloric saccharin having no effect on mch gene expression . thus , consistent with its proposed role described previously in promoting motivation for intake of caloric food , these results indicate that mch is activated after animals have consumed caloric food , further promoting overconsumption . whereas ox and mch each play a significant role in the consumption of palatable food , it is clear that these neuropeptides do not work in isolation . two classes of neurochemicals with which they directly interact are first - order feeding neuropeptides of the arcuate nucleus and dopamine in the limbic system . the orexigenic actions of ox and mch may be due , in part , to their similar downstream activation of neuropeptides in the hypothalamic arcuate nucleus , npy and agouti - related protein ( agrp ) . in the arcuate , ox - positive axon terminals directly contact neurons containing npy , and ox1r protein is located on both npy and agrp - containing neurons . orexin also directly activates npy neurons , as the addition of ox - a or ox - b to the superfusate of isolated arcuate npy neurons increases their intracellular calcium content . this translates into effects of ox on feeding , as the orexigenic effect of icv ox - a or ox - b is greatly reduced by icv pretreatment with an npy receptor antagonist [ 105 , 106 ] . similarly , chronic icv injection with mch , which stimulates feeding , also upregulates npy gene expression , and the orexigenic effect of icv mch is significantly diminished by icv injection of an npy receptor antagonist . in part then , the similar ability of ox and mch to promote food intake may be due to their similar downstream effects on other orexigenic neuropeptides . the dissimilar motivation and arousal - related actions of ox and mch may be due more to their opposing downstream effects on the neurotransmitter dopamine ( da ) , in limbic nuclei such as the nac and prefrontal cortex ( pfc ) . a rise in ox levels results in da release into the nac shell and pfc , which can occur from ox acting directly at da terminals or at their source in the vta . application of ox - a to pfc slices increases phasically evoked da release , an effect inhibited by the ox1r antagonist sb334867 , and application of ox - a or ox - b to vta slices increases the firing frequency of da neurons . similarly , icv injection of ox - a stimulates c - fos in vta da neurons , primarily in those projecting to the nac shell and pfc rather than the nac core . in line with this , icv ox - a is also found to elevate levels of da in the pfc but not the nac core . these findings with ox contrast with those observed with mch , which through transgenic studies appears to inhibit accumbal da . mice lacking ppmch exhibit increased electrically evoked da release in the nac shell and increased da transporter levels in both the nac shell and core [ 81 , 113 ] . as the application of mch to vta slices fails to affect firing of da neurons , these da changes in knockout mice may be due to presynaptic actions in the nac . these opposite effects of ox and mch on limbic da may help to explain their opposite effects on arousal and reward ( see section 6 ) . it is interesting to consider the similar orexigenic actions of ox and mch in light of their opposite effects on da . this neurotransmitter plays an important role in promoting food - seeking behavior and appetitive motivational processes in general , but animals will also work to enhance da levels when they are low . in fact , animals prone to overeating a high - fat diet exhibit markedly reduced basal levels of da in the nac . seemingly then , palatable food intake can be increased from an ox - induced elevation of da , which increases arousal and seeking of palatable food [ 118 , 119 ] , but it can also be increased by an mch - induced reduction in da , which produces anhedonia ( see section 6.2 ) and the need to restore da levels through the consumption of palatable food . this suggests the possibility that ox may contribute to the rise in accumbal da that normally occurs prior to meal consumption , while mch may contribute to the fall observed during the feeding bout . in line with their opposite effects on limbic da , ox and mch largely have opposite effects on processes of reward . whereas ox increases the reinforcing properties of ingested substances , mch instead appears to promote depression and anxiety . the peptide ox plays a role in reward and reinforcement , acting through ox1r or ox2r . in tests of conditioned place preference ( cpp ) following pairing with drug administration , vta injection of ox - a during conditioning induces morphine cpp in a dose - dependent manner , and peripheral administration of the ox1r antagonist sb-334867 or ox2r antagonist tcs - ox2 - 29 suppresses its acquisition and expression [ 122 , 123 ] . while morphine cpp may be perpetuated by ox binding at ox1r or ox2r , ethanol cpp appears to be mediated more by ox2r . acquisition , expression , and reinstatement of ethanol cpp are attenuated by peripheral treatment with the ox2r antagonist jnj-10397049 but not by the ox1r antagonists sb-408124 or sb-334867 . in male rats , conditioned cues associated with sexual behavior in a cpp paradigm induce c - fos in ox neurons , and lesioning of ox neurons prevents the formation of cpp for a chamber paired with sexual behavior . thus , ox may be important in reward processing of both drugs of abuse and also natural rewards , such as sexual activity or palatable food intake . in contrast to ox , mch may not promote reward processing but instead is linked with anxiety and depression . mice with deletions of mchr1 show no difference from their wild - type counterparts in cocaine- or amphetamine - induced cpp and in fact show hypersensitivity to the locomotor activating effects of the da psychostimulant d - amphetamine , suggesting that these mice have enhanced drug - induced reward processing . instead , mch is strongly linked with anhedonia in the forced swim test ( fst ) . the amount of time spent immobile in the fst correlates positively with ppmch mrna , and mchr1 antagonists produce an antidepressant effect in the fst when administered peripherally or directly in the nac shell [ 70 , 131 ] . the mchr1 antagonist snap-7941 also acts as an anxiolytic in tests of rat social interaction and guinea pig maternal - separation vocalization . thus , in contrast to ox , mch may in some cases act as an antireward neuropeptide . in summary , ox and mch , expressed in the lh and acting through their receptors in many of the same nuclei of the brain , are similar both in promoting the consumption of palatable or caloric food and in being stimulated by the intake of this food , responding in a positive feedback cycle to promote excess consumption . importantly , they appear to have complementary roles in controlling this feeding behavior , likely through their largely opposite roles in reward systems and motivation , as well as a number of physiological processes , including the sleep - wake cycle , energy expenditure , and glucose metabolism . perhaps for these reasons , the evidence suggests that ox is activated in situations of food seeking and promotes the motivation for food reward , possibly activating the feeding process , whereas mch plays a larger role during ongoing food intake and reinforces consumption , acting to prolong the intake of energy - dense foods . thus , for a single meal , ox may be involved in initiating palatable food intake and activating mch neurons , and after meal initiation and the consequent rise in glucose , mch functions to prolong the consumption of calories for the sake of energy conservation . these physiological and behavioral effects of ox and mch can be further understood in light of their downstream effects on other neurochemicals . while these neuropeptides both upregulate npy which may contribute to their similar orexigenic effects , they have opposite effects on limbic da , which may contribute to their complementary effects on reward and motivation that themselves can enhance palatable food intake . these two peptides in the lh provide an interesting example of the complex and sometimes opposing physiological , behavioral , and neurochemical processes that are involved in promoting the ingestion of palatable food .

transcribed within the lateral hypothalamus , the neuropeptides orexin / hypocretin ( ox ) and melanin - concentrating hormone ( mch ) both promote palatable food intake and are stimulated by palatable food . while these two neuropeptides share this similar positive relationship with food , recent evidence suggests that this occurs through different albeit complementary effects on behavior , with ox promoting food seeking and motivation for palatable food and mch functioning during ongoing food intake , reinforcing the consumption of calorically dense foods . further differences are evident in their effects on physiological processes , which are largely opposite in nature . for example , activation of ox receptors , which is neuronally excitatory , promotes waking , increases energy expenditure , and enhances limbic dopamine levels and reward . in contrast , activation of mch receptors , which is neuronally inhibitory , promotes paradoxical sleep , enhances energy conservation , reduces limbic dopamine , and increases depressive behavior . this review describes these different effects of the neuropeptides , developing the hypothesis that they stimulate the consumption of palatable food through excessive seeking in the case of ox and through excessive energy conservation in the case of mch . it proposes that ox initiates food intake and subsequently stimulates mch which then acts to prolong the consumption of palatable , energy - dense food .

1. Introduction 2. Receptor Function 3. Physiological Effects 4. Role in Food Intake 5. Interactions with Other Neurochemicals 6. Role in Reward 7. Conclusions

PMC4793031

cerebral palsy ( cp ) is a disorder of movement and posture that appears during infancy or early childhood . it is caused by non - progressive damage to the developing brain before , during , or shortly after birth . spastic cp is the most common form of cp ( accounting for approximately 7080% of cases of cp ) and 50% of children with spastic cp have diplegia . in spastic diplegic cp , the lower extremities are severely affected but the upper extremities are only mildly impaired , intelligence is usually normal , and epilepsy is not common1 . children with cp have neurodevelopmental disorders , such as spasticity , contracture , reduced coordination , selective voluntary control , and muscle weakness2 . among these muscle weakness is more pronounced distally and the hip extensors , knee extensors , and ankle dorsiflexors are relatively weaker than their antagonists3 , 7 . muscle strength and resistance training programs have been used as therapeutic interventions to increase muscle strength and to improve muscle function3,4,5,6,7,8,9,10,11,12,13 . all clinical trials suggest that muscle strength - training programs can increase muscle strength and may improve motor activity without adverse effects in people with cp10 , 11 . the purpose of this study was to determine whether strength - training programs for hip extensors and knee extensors improve gross motor function of myanmar children with cp . forty children ( 25 boys and 15 girls ) diagnosed with spastic diplegic cp , were recruited for this study . the children were both inpatients & outpatients coming for rehabilitation to the national rehabilitation hospital ( nrh ) , yangon , myanmar . the inclusion criteria were : ages between 4 and 12 years ( mean age : 6.07 2.74 years ) ; gmfcs levels i and ii , with the muscle tone of hip and knee extensors lower than grade + 1 of the modified ashworth scale ( mas ) and the ability to follow verbal commands . children were excluded if they : ( i ) had received a nerve block injection or orthopedic surgery ; ( ii ) had hip and/or knee flexion contracture greater than 10 degrees ; or ( iii ) had unstable seizures and other medical conditions . this study was approved by the human research ethics committee of the university of medical technology , yangon ( umty ) , myanmar . all subjects and parents / caregivers received explanations regarding the purpose and procedures of the study before voluntarily agreeing to participate . all parents / caregivers were required to give written informed consent for participation before the start of the study procedures , and were reminded that they had the right to withdraw from the study at any time . all subjects in the study were able to continue their usual therapy at the nrh . flow diagram of the study the study design was one group pretest - posttest design . all forty children in this study were categorized into gmfcs levels i and ii according to the original version of gmfcs . the gmfcs is based on gross motor development of self - initiated movement , and consists of a five - level classification system ( table 1table 1.description of gmfcs levels in general for all age bandslevel abilitylevel i walks without restrictionslevel ii walks without assistive devices but limitations in communitylevel iii walks with assistive deviceslevel iv transported or uses powered mobilitylevel v severely limited dependent on wheelchair ) with descriptions of four age bands : before the 2nd birthday ; between the 2nd and 4th birthdays ; between the 4th and 6th birthdays ; and between the 6th and 12th birthdays14 . the original version of gmfcs used in the present study was in effect prior to the 2007 expended and revised version ( gmfcs - e & r ) with descriptions on five age bands ( younger than 2 , 24 , 46 , 612 and 1218 years)15 . children at level i and level ii learn to walk without aids , children at level iii walk with aids , children at level iv rely mainly on wheelchair mobility and children at level v have no means of independent mobility14 , 15 . one study also confirmed the reliability and validity of the gmfcs , supporting its use in clinical practice and research16 . muscle strength can be defined as the ability of skeletal muscle to develop force for providing stability and mobility within the musculoskeletal system , so that functional movement can take place17 . several types of resistance can be used for strength training19 . the free weights ( adjustable weight cuffs with sand bags and velcro straps ) were used as resistance in this study . the gmfm is a standardized observational instrument designed and validated to measure change in gross motor function over time in children with cp . gmfm is a criterion - based observational measure with 88 items that assesses motor function in five dimensions . the five dimensions and their respective items and maximum scores are shown in table 2table 2.gmfmdimensionitems maximum scores a. lying and rolling 17 51 b. sitting 20 60 c. crawling and kneeling 14 42 d. standing 13 39 e. walking , running and jumping 24 72 total 88 264 . each item is assessed and scored on four - point ordinal scale : 0 = can not initiate activity , 1 = initiates activity , 2 = partially completes activity and 3 = completes activity20 , 21 . dimension d ( 13 items to measure motor function in standing , scoring from 0 to 39 ) and dimension e ( 24 items to measure motor function in walking , running and jumping , scoring from 0 to 72 ) were used as the outcome measures for this study . the amount of training weight in pounds , which was maximum amount of weight that the child could lift in one time , gmfm dimension d and e were assessed at baseline before the start of training program and after the completion of six weeks training program . all forty subjects were given strength - training program , which included progressive resistance hip and knee extension one session ( 45 minutes ) per day , three times a week for total six weeks . the intervention procedure involved passive stretching of hip flexors , adductors , hamstrings , triceps surae for 15 minutes as warm - up . the main exercise , extension exercise for both hip and knee extensors with weight cuff , was done ten times for each muscle group on each side for 30 minutes . for hip extensors , the subject was lying prone , the weight cuff was placed just above knee joint and the subject was asked to lift the thigh with weight to full extension . the subject was lying supine with knees slightly bent , a foam roll or a pillow was placed under knees , the weight cuff was placed just above ankle joint and the subject was asked to lift the lower leg with weight to full extension for knee extensors . the intervention procedure was finished with passive stretching of hip flexors , adductors , hamstrings , triceps surae for 15 minutes as cool - down . the strength - training program was performed 10 repetitions for each muscle group on each side , one session per day for three days per week up to 6 consecutive weeks . a daily diary of physical activity of each subject was kept by the parents or caregivers during the study period . after 6 consecutive weeks training program , the subject was discharged . the parents or caregivers of each subject were requested to ask the subject to perform the same main exercise program at home by the guidance of a written home exercise program . all subjects were requested to visit the study area at once per every 2 weeks up to 6 weeks ( three times ) as follow up . this study was firstly proposed to assess all outcome measures at the end of 6 weeks training and at the end of 6 weeks follow up ( total 12 weeks ) but there were only 7 subjects who were able to come all three times of follow up . more than 75% of the subjects were not able to completely come all three times of follow up because most of the subjects in this study were from suburbs of yangon and other rural areas of myanmar . the subjects who were absent from regular treatment of 3 days for total 6 consecutive weeks were taken as drop - out . descriptive statistics [ mean , median and standard deviation ( sd ) ] were calculated using independent t - test . the outcome measures only at the end of 6 consecutive weeks were calculated as final data . the general characteristics of the subjects are presented in table 3table 3.general characteristics of subjectscharacteristics mean ( sd)frequency ( % ) age , 412 years years 6.07 ( 2.74 ) 40 ( 100)46 years 23 ( 57.5)612 years 17 ( 42.5)gender boys 25 ( 62.0 ) girls 15 ( 38.0)gmfcs level i 30 ( 75 ) level ii 10 ( 25 ) . the mean values and standard deviations ( sd ) of pre - test and post - test outcome measures and p values of the participants are presented in table 4table 4.results of strength trainingpre - test ( mean sd)post - test ( mean sd)training weight ( lb)hip extensors ( right ) 1.81 1.51 3.50 2.16 * hip extensors ( left ) 1.74 1.20 3.09 1.80 * knee extensors ( right ) 1.86 1.49 3.57 2.1 * knee extensors ( left ) 1.77 1.23 3.23 1.75 * gmfmgmfm - d 28.4 11.1 33.2 11.1 * gmfm - e 42.4 19.3 54.9 22.5 * * p<0.05 . statistically significant changes were seen in the amount of training weight in pounds and gmfm - d and e. the purpose of this study was to determine whether strength - training program on hip extensors and knee extensors improves gross motor function of children with spastic diplegic cp ( gmfcs level i & ii ) in myanmar . the results of this study supported the effectiveness of prescribing strength - training program on hip and knee extensor muscle groups of lower limbs in patients with spastic diplegic cp as a method to enhance their functional abilities and to increase their gross motor function . after 6 consecutive weeks of strength - training program , the subjects were found to achieve increased mean values of maximum weight for training for all muscle groups and gmfm dimension d and e. many studies reported that muscle strength and gross motor function activities significantly improved after strength - training program . reported that children with spastic diplegia increased quadriceps femoris muscle strength through heavy resistance exercise , three times per week for 6 weeks using ankle weights at load of approximately 65% of each child s maximum isotonic force production3 , 4 . macphail & kramer stated that there was a direct relationship between knee extensor strength and gross motor ability , which meant improvement in muscular strength , was associated with improvement in walking efficiency and functional abilities in adolescents with cp5 , 6 . in the study of functional outcomes of strength training in spastic cerebral palsy by domiano and abel , the results of their study reinforced the relationship of strength to motor function in children with cp and further demonstrated the effectiveness of strengthening in this population8 . in the randomized clinical trial of strength training in young people with cerebral palsy by dodd et al . , home - based , 6 weeks strength training program on knee extensors and ankle plantar flexors was effective in increasing muscle strength and gmfm - d and e of young people ( mean age 13 years 1 month , sd 3years 1 month : range 8 to 18 years ) with spastic diplegic cp ( gmfcs level i to iii)9 . dodd et al . also reported that strength training programs could increase strength and may improve motor activity in people with cp without adverse effects in their study of a systematic review of the effectiveness of strength training programs for people with cp10 . a pilot study using repeated measure design of the effects of progressive resistance training for children with cp by morton et al . also found that muscle strength and gmfm dimension d and e increased immediately after 6 weeks training program using free weights11 increased muscle strength ( increased amount of training weight in pounds ) and gmfm dimension d and e from baseline to the certain extent until the end of 6 weeks strength training program . although this study was first proposed to assess the outcomes measures at the end of 6 weeks training and at the end of 6 weeks follow up ( total 12 weeks ) but there were only 7 subjects who were able to come all three times of follow up and the result of those 7 subjects were not calculated in the results . the results of the present study supported the effectiveness of prescribing strength training program on muscles of lower limbs in patients with spastic diplegic cp as a method to enhance their functional abilities and to increase their gross motor function . the current physical therapy program including strength training program was as effective as the previous studies , but with much shorter time and utilized unsophisticated and inexpensive equipment which was available in the most of the physiotherapy departments in our country . the limitations of this study were absence of control group , not using standardized assessment of muscle strength , not using gmfm-66 with gross motor ability estimator ( gmae ) software , not involving other physiologic types of cp and presence of weakness that the subjects were able to take usual therapies from the nrh that might have influenced the outcome measures . in conclusion , the results of this study suggested that a simple method of strength training program might lead to improve muscle strength and gross motor function in children with spastic diplegic cp . this simple methodology could provide powerful motivation to these patients group to encourage more active lifestyle and allow increased activity and participation within family and community settings .

[ purpose ] the purpose of this study was to determine whether strength training programs for hip extensors and knee extensors improve gross motor function of children with cerebral palsy in myanmar . [ subjects and methods ] forty children ( 25 boys and 15 girls , mean age : 6.07 2.74 years ) from national rehabilitation hospital , yangon , myanmar , who had been diagnosed with spastic diplegic cerebral palsy , gross motor classification system i and ii participated in a 6-week strength training program ( 45 minutes per day , 3 days per week ) on hip and knee extensors . assessment was made , before and after intervention , of the amount of training weight in pounds , as well as gross motor function measure ( gmfm ) dimensions d ( standing ) and e ( walking , running , jumping ) . [ results ] all scores had increased significantly after the strength - training program . [ conclusion ] a simple method of strength - training program for hip and knee extensors might lead to improved muscle strength and gross motor function in children with spastic diplegic cerebral palsy .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3603208

the protozoan parasite toxoplasma gondii ( t. gondii ) which causes toxoplasmosis , is primarily hosted not only in cats but also in mice , rabbits , dogs , farmyard and wild animals , and domestic fowl , and is transmissible to man [ 15 ] . it has been implicated in the pathogenesis of many diseases , most notably schizophrenia [ 68 ] , but also with bipolar disorder depression and suicide attempts . there is also evidence from serological antibody studies that the parasite may be implicated in the aetiology of alzheimer 's and parkinson 's disease [ 1113 ] and in certain epilepsies of unknown origin . the parasite has also been implicated in a number of autoimmune disorders including antiphospholipid syndrome , cryoglobulinemia , anca - associated vasculitides , autoimmune thyroid diseases , systemic sclerosis , rheumatoid arthritis , inflammatory bowel disease , and systemic lupus erythematosus , possibly related to host / pathogen antigen homology [ 15 , 16 ] . it has already been noted that several schizophrenia susceptibility genes are related to the t. gondii life cycle , as well as to that of other pathogens implicated in this condition ( cytomegalovirus , influenza , rubella , and herpes viruses ) [ 17 , 18 ] and that in both alzheimer 's disease ( herpes simplex , chlamydia pneumoniae , helicobacter pylori , and cryptococcus neoformans ) [ 19 , 20 ] and multiple sclerosis ( epstein - barr virus ) , susceptibility genes are also related to the life cycles of suspect pathogens . in animal models , and without the aid of any gene variant , such agents can , per se , induce pathological features relevant to the disease process , for example amyloid deposition and tau phosphorylation ( induced by herpes simplex , c. pneumoniae , treponemas , borrelia burgdorferi , and other spirochetes ) [ 2224 ] , demyelination induced by various viruses , or dopaminergic overactivity in the case of t. gondii . the h1n1strain of the influenza virus is also able to destroy neurones in the substantia nigra , provoking parkinsonian symptoms in laboratory models . pathogens can thus be regarded as potential causes , likely acting in a gene dependent manner . many such agents show a seroprevalence far above the incidence of the disease with which they are implicated ; for example , t. gondii may infect 30% of the world 's population in comparison to a schizophrenia prevalence of ~1% , and , as is the case with genetic risk factors , conflicting epidemiological data have often cast doubt upon whether such pathogens can truly cause disease . however , this situation also applies to helicobacter pylori , which indubitably causes stomach ulcers and likely gastric cancer [ 31 , 32 ] , although not all of the many infected with this agent ( ~50% of the world population ) succumb to these conditions . any causative effects of such agents in man must therefore be conditioned by other factors , among which are immunity and resistance to the pathogen ; pathogen strain or the timing and severity of infection ; other confounding environmental and medical factors as well as the susceptibility genes for each disease . the effects of risk promoting gene variants , which are also present in control populations , albeit in lower proportion , must also be conditioned by environmental and epigenetic factors , as well as by gene / gene interactions . during its life cycle any pathogen interacts with hundreds of human proteins whose function can only be compromised by their diversion to the attentions of the invader . in addition , bacteria and parasites scavenge important metabolites from host cells or fluids and donate other compounds to the host which must react accordingly . activation of the immune system and inflammatory defence , involving chemokines , cytokines and numerous other mediators are an evident consequence of any infection , as are the resulting fevers . it has also been noted in many bioinformatics studies that pathogen proteins closely resemble our own , and that immune attack directed towards the pathogen may thus result in antibody cross - reactivity with human proteins . the development of pathogen - derived autoantibodies may also play a key role in this pathological scenario [ 18 , 19 , 21 , 3539 ] . as shown below , the hundreds of human proteins implicated in the t. gondii life cycle are highly enriched in the products of susceptibility genes for the numerous conditions with which this parasite has been associated , as well as for others where a link is not yet suspected . subsets of the extensive t. gondii host / pathogen interactome appear to be relatively specific for distinct diseases suggesting that they relate to the cause of the disease , and that they may be able to direct the attentions of the pathogen towards particular pathways , pathologies , and disease . briefly , lists of several hundred susceptibility genes involved in eleven different diseases were compared with a list of several thousand host genes implicated in the t. gondii host / pathogen interactome . any significant enrichment of interactome genes within susceptibility gene datasets ( and vice versa ) was identified by statistical analysis . the genes and environmental factors implicated in the various diseases ( alzheimer 's disease , attention deficit hyperactivity disorder , autism , bipolar disorder , chronic fatigue syndrome , depression , schizophrenia , multiple sclerosis , parkinson 's disease , anorexia , and childhood obesity ) are listed at polygenicpathways ( http://www.polygenicpathways.co.uk/ ) and at sites therein ( including the autism database at mindspec ( autdb ) , the bipolar database at the university of chicago , alzgene , msgene , pdgene and szgene [ 4245 ] ) . genome - wide association data can be accessed at the national human genome research institute http://www.genome.gov/gwastudies/ . host / pathogen interactions for t. gondii and microarray data ( mrna expression changes in response to t. gondii infection ) were collected by literature survey and are listed at http://www.polygenicpathways.co.uk/tgondii.htm . pathway analysis of the human arm of this interactome was performed using kegg mapper http://www.genome.jp/kegg/tool/map_pathway2.html , and the results are posted at http://www.polygenicpathways.co.uk/keggtgondii.htm . the human genome currently contains 26,846 genes , 2792 of which are contained in the t. gondii host / pathogen interactome . in any other dataset , one would expect 2792/26846 genes to be involved with the pathogen ( 10.4% ) . similarly , for n susceptibility genes in any disorder , one would expect n/26846 to appear in the host / pathogen interactome , providing the expected numbers in each colliding dataset . the significance of differences between the observed and expected values was assessed using the chi - squared test . statistical analysis for the enrichment of particular kegg pathways within datasets was performed using the tools at the consensus path database ( cpdb ) developed by the max planck institute for molecular genetics http://cpdb.molgen.mpg.de/cpdb . 2792 proteins or mrnas are involved in the host / pathogen interactome , approximating to 10% of the human genome . a summary of the kegg pathway analysis of the human arm of this interactome is provided in tables 1 and 2 . as might be expected , a high proportion of genes are involved in the immune system and in pathogen defence pathways . many are also involved in the life cycle pathways of a number of viruses , bacteria , and other parasites ( table 1 ) . these stem in part from the common immune and defence mechanisms not only related to the pathogens ( chemokine and cytokine activation , etc . ) , but also related to common signalling networks . the involvement of dedicated bacterial and viral defence pathways in the interactome ( nod , rig1 , and cytosolic dna - sensing pathways ) is likely to impact upon viral defence , although in which direction is impossible to determine . the host intestinal microbiome also influences t. gondii and is also able to act as an adjuvant in response to t. gondii infection by stimulating dendritic cells that provide the immunostimulation necessary to combat the parasite . such effects and the shared pathways between pathogens highlight an important potential cross talk between elements of the microbiome . diverse pathogens are implicated in all of the diseases in this study , and many of the pathways traced out by the disease susceptibility genes , per se , ( posted on the polygenicpathways website ) also involve multiple viral and pathogen life cycle and immune - related pathways . a number of cancer - related pathways are highly represented in the t. gondii interactome ( table 1 ) . while a recent study has suggested its involvement in brain cancer , based on a correlation between cancer mortality and t. gondii seroprevalence , the parasite is able to arrest the growth of other cancerous cells via stimulation of the immune response and inhibition of angiogenesis . antitumour effects have been observed in relation to spontaneous mammary tumours , leukaemia , lung cancer , and carcinogen - induced tumours following injections of toxoplasma antigen or viable parasites in laboratory animals or cells . a high t. gondii antibody seroprevalence ( as well as to the cytomegalovirus and the epstein - barr virus ) has been observed in systemic lupus erythematosus , and it has been suggested that antibodies raised to the pathogen may contribute to the autoimmunity characteristic of this condition via pathogen / host protein mimicry [ 16 , 54 , 55 ] . conversely , t. gondii infection has been shown to prevent the development of lupus - related nephritis in rabbits , a factor perhaps related to the immunosuppressant properties of parasitic infection . toxoplasmosis has been reported to decrease leukocyte , natural killer cell , and monocyte counts in men , while increasing the same in women , with reduced b - cell counts in both . no references were found for relationships between toxoplasmosis and type 1 diabetes , a pathway also figuring in the interactome . prior t. gondii infection has been associated with poor outcome in heart transplant patients ( allograft rejection ) . toxoplasmosis and other infectious agents have also been linked to cardiac myopathy [ 5962 ] , and diverse pathways of which were concentrated in the t. gondii interactome . in relation to asthma , the hygiene hypothesis , linking a reduced incidence of childhood infections ( in general ) to the worldwide increase in asthma and other allergic conditions , may be related to the concentration of t. gondii interactome genes within the asthma pathway , although a positive correlation of t. gondii infection and asthma has also been noted in sweden [ 6365 ] . the parasite clearly has multiple effects on diverse immune - related networks as noted above , and such effects are likely to be both beneficial and nefarious . for example parasite - related immunosuppression may well be useful ( but perhaps not advisable ) in autoimmune diseases such as multiple sclerosis but might also be expected to favour other infections . many of the more specific signalling networks within the interactome ( table 2 ) can be related to the general processes described above . while the map kinase pathway is involved in a multitude of functions , the jak / stat pathway is involved in cytokine signalling , also bridging cytokine activation to cancer pathways . the calcium signalling pathway is also activated by many processes and more specifically by voltage or receptor - gated ion channels ( and is relevant to the channelopathies implicated in autism , depression , bipolar disorder and schizophrenia , and in neurological disorders [ 67 , 68 ] ) or by processes modulating intracellular stores , while the phosphatidylinositol signalling system is also involved in the actions of multiple messengers . tgf beta regulates proliferation , apoptosis , differentiation , and migration ( definition from kegg ) . calcium channel blockers , calmodulin antagonism , or extracellular calcium depletion diminish cellular invasion by the parasite [ 69 , 70 ] . the p53 and growth factor signalling networks ( erbb , vegf ) can be cancer related , while insulin signalling is evidently related to diabetes . ppar receptors control the transcription of many genes especially those related to fatty acid metabolism , but also those involved in cell proliferation and differentiation . these and other pathways control a host of processes from embryonic differentiation to cellular death and apoptosis , and many metabolic pathways that are too numerous to individually review . in relation to the diseases that are the object of this study , the alzheimer 's and parkinson 's disease pathways were both represented , as were the complement , ppar , and terpenoid ( cholesterol synthesis ) pathways relevant to alzheimer 's disease , and the ubiquitin pathway relevant to parkinson 's disease and other degenerative disorders . erbb signalling is highly relevant to the control of peripheral and central myelination , and thus to multiple sclerosis and alzheimer 's disease , but also to a range of psychiatric disorders including autism , anorexia , adhd , bipolar disorder , depression , and schizophrenia . glutathione pathways ) , and the glutathione precursor n - acetylcysteine has been shown to be of benefit in a number of psychiatric disorders [ 7680 ] . the diverse neurotransmitter pathways and many signalling networks are also relevant to most of these conditions . rather than single out any particular pathway from this extensive dataset ( tables 1 and 2 ) , suffice it to say that parasitic infection has massive effects upon a variety of host signalling networks , metabolic pathways , and processes . these are nevertheless relatively selective , in the sense that certain pathways are more affected than others . in addition , within each disease dataset , the spectrum of pathways within the overlapping datasets is distinct and biologically relevant , as detailed below . t. gondii interactome genes were significantly enriched in the susceptibly gene datasets for all diseases with the exception of anorexia and chronic fatigue and represented from ~13% ( autism ) to 33% ( multiple sclerosis ) of the total number of susceptibility genes analysed , with enrichment values from 1.08 to 2.83 fold the expected number ( table 3 ) . for schizophrenia , the fold enrichment ( interactome genes in susceptibility gene dataset ) of 2.03 compares with a recent meta - analysis of t. gondii seroprevalence studies providing an odds ratio ( or ) of 2.71 . a further meta - analysis showed significant associations of schizophrenia with infections by human herpesvirus 2 ( or = 1.34 ) , borna disease virus ( or = 2.03 ) , human endogenous retrovirus w ( or = 19.31 ) , chlamydophila pneumoniae ( or = 6.34 ) , and chlamydophila psittaci ( or = 29.05 ) , including values far in excess of those for any gene . for schizophrenia at least , these data and ample evidence from epidemiological and animal behaviour studies [ 8385 ] firmly advocate toxoplasmosis as a significant cause of the disease , in those with a particular genetic constitution . the ability of the parasite to manipulate dopaminergic metabolism ( via its own tyrosine hydroxylase ) and the involvement of nmda receptor ( e.g. , glutamatergic signalling and long - term potentiation ) , serotonin , or cannabinoid - related signalling networks within the interactome is relevant to the drug - induced psychosis associated with the amphetamines , lsd , cannabis , or phencyclidine ( see ) . dopamine also increases the number of t. gondii tachyzoites in cultured fibroblasts suggesting that neurotransmitters may also be able to manipulate the parasite . for each disease , and across diseases , this was assessed in two ways : firstly by statistical analysis of the enrichment of kegg pathways in each overlapping t. gondii interactome / disease dataset and secondly by a comparison of individual shared and specific overlapping interactome / disease genes across four diseases ( the maximum possible using the venny tool ) . the diseases analysed in this way were alzheimer 's disease and multiple sclerosis , bipolar disorder , and schizophrenia . the permutations of genes common or specific to the various chosen diseases ( alzheimer 's disease , bipolar disorder , schizophrenia , and multiple sclerosis ) are shown by the venn diagram figure 1 summarised in table 4 . all of these genes are members of the host / pathogen interactome . several immune / cytokine and oxidative stress related genes , with different identities , but similar roles , appear as common risk factors across various permutations of diseases , which are all characterised by immune activation [ 8991 ] and oxidative stress [ 92 , 93 ] . bipolar disorder and schizophrenia share many common genes , risk factors , endophenotypes , and subpathologies , and interactome genes relevant to certain of these are related to circadian rhythm , dopaminergic and glutamatergic neurotransmission , growth factors , and signalling networks as highlighted in previous reviews [ 75 , 94 , 95 ] . after sifting through these common subsets , the overlapping t. gondii interactome / susceptibility genes specific to each disease they include app processing , cholesterol and lipoprotein function , complement and immune related genes , and oxidative stress , apoptosis and ubiquitin genes in alzheimer 's disease [ 96100 ] . in bipolar disorder , monoamine / gaba , signalling , adhesion , and ion transport genes are highlighted ( see above and [ 101103 ] ) while in schizophrenia , monoamine / glutamate / neuregulin neuronal development and associated signalling related genes figure prominently , along with those related to adhesion , oxidative stress , and immune activation ( see above ) . in multiple sclerosis , almost the entire common dataset is related to immune function and associated signalling pathways , that are relevant to the autoimmune aspects of the disease [ 104 , 105 ] , with a limited number of genes related to oxidative stress and apoptosis . while the evidence for an involvement of toxoplasmosis in psychiatric disorders is relatively strong , there is less work either in the human condition or in animal models in the case of neurological disorders , such as alzheimer 's or parkinson 's diseases or multiple sclerosis . toxoplasmosis has , however , been associated with a loss of grey matter density in schizophrenic patients , but not in controls , suggesting an influence on degenerative components . for example , it inhibits the development of arthritis in mice deficient in the interleukin receptor antagonist ( il1rn ) . t. gondii infection is also able to reduce infarct size in focal cerebral ischaemia in mice , an effect attributed to the ability of infection to increase the expression of nerve growth factor , as well as that of anti - inflammatory cytokines and of glutathione and oxidative stress protective genes , while reducing the expression of proinflammatory cytokines . parasites have learnt to live with us for many millennia , and their immunosuppressant effects appear to be a relatively common defence mechanism . indeed , the use of helminths ( parasitic worms ) has been suggested in a number of autoimmune settings including irritable bowel disease and multiple sclerosis . a clinical trial with helminth egg infection ( trichuris suis ova ) in autism is also listed at http://clinicaltrials.gov/ , based on anecdotal reports of effectiveness in relation to certain symptoms . the preponderance of immune related host / pathogen genes in the multiple sclerosis dataset ( and to a lesser extent within other datasets ) may be related to these potentially beneficial effects , although the clinical use of t. gondii would be contraindicated by its malevolence directed elsewhere . the kegg pathways influenced by t. gondii ( restricted to the overlapping interactome genes within each disease dataset ) are posted at http://www.polygenicpathways.co.uk/toxoplasmosis.htm , and the cpdb enrichment analysis depicted in tables 5 and 6 . these tables report only the significantly enriched pathways , but many others figure within these overlapping datasets . in all diseases , except for adhd and anorexia , the significantly enriched subsets involved immune or defence related pathways . for the most part ( autism , childhood obesity , depression , bipolar disorder and schizophrenia , alzheimer 's and parkinson 's disease , and multiple sclerosis ) , the bacterial defence nod signalling network was involved , while the similar toll pathway was more restricted ( alzheimer 's and parkinson 's disease and multiple sclerosis , bipolar disorder , and schizophrenia ) . the rig1 and cytosolic dna - sensing pathways recognise viral nucleic acids . the rig-1 pathway was significantly enriched in multiple sclerosis and schizophrenia , while the cytosolic dna - sensing pathway was enriched in alzheimer 's and parkinson 's disease as well as in multiple sclerosis and schizophrenia . diverse pathogen life cycle pathways were enriched in all but the adhd and anorexia datasets . there are few studies relating either obesity or anorexia to toxoplasmosis in man , although both anorexia or subsequent partial weight gain postinfection , as well as hypermetabolism have been associated with t. gondii infection in laboratory and farm animals [ 109111 ] . the only significantly enriched pathways common to the t. gondii interactome in anorexia all relate to neuronal systems ( dopamine , serotonin , and addiction pathways ) . in childhood obesity , a number of autoimmune related pathways were highlighted , as well as the alzheimer 's disease pathway , pathways related to ppar signalling ( regulating fatty acid metabolism ) , and glycerolipid metabolism . a recent review has highlighted the risk promoting effects of midlife obesity ( and several other preventable risk factors ) in relation to alzheimer 's disease . the childhood obesity epidemic , fuelled largely by dietary and sedentary culture , has been associated with an increased risk of affective disorders in adulthood and has also led to an increased incidence of a number of diseases in young children ( dyslipidemia , carotid artery atherosclerosis , cardiac problems , hypertension , the metabolic syndrome , and diabetes and fatty liver disease ) [ 115118 ] that were previously the reserve of old age . many of these are also risk factors for alzheimer 's disease and are able , per se , to increase cerebral beta - amyloid deposition in laboratory models , perhaps a herald for the unwelcome imminence of dementia in young adults . diet , including saturated fat [ 119 , 120 ] , affects the microbiome , and a recent study has shown that , in infants fed formula or breast milk , changes in the gut microbiome can alter the expression of genes related to the innate immune system . this microbiome / immune link may be important in the development of inflammation and metabolic diseases . there do not appear to have been any microbiome studies in relation to t. gondii . however , the parasite scavenges host cholesterol , while host fatty acids and low - density lipoproteins stimulate a t. gondii acyl - coa , cholesterol acyltransferase , which then provides cholesteryl esters that the parasite needs for its survival . fatty diets would certainly be expected to impact upon the success of this parasite , which in turn must influence the lipid metabolism of the host . indeed , t. gondii infection may even possess beneficial effects in hypercholesterolaemic conditions in mice , reducing the development of atherosclerosis via cholesterol and lipoprotein scavenging effects . pathway correlates such as these of course predict relationships but not directionality , which can only be imputed by prior knowledge and future research . certain of the pathways common to the t. gondii interactome and obesity ( and to alzheimer 's disease , see below ) could well reflect a beneficial component of parasitic infection . no clinical studies have specifically linked adhd or autism to toxoplasmosis , although hyperactivity , modified social interactivity , and sensorimotor effects are features of infection in mice that are of relevance to both conditions [ 124126 ] . in adhd , the primary common emphasis was on the calcium signalling pathway to a number of metabolic pathways : phenylalanine and tyrosine ( ddc and maoa ) , tyrosine , histidine ( ddc , hnmt , and maoa ) tryptophan ( acat1 , ddc , and maoa ) , and unsaturated fatty acid synthesis ( fads1 and fads2 ) and to neurotransmitter pathways ( cocaine addiction and ligand / receptor interactions ) . this is a relatively small dataset , but it highlights an important distinction for bacteria or parasites , which , unlike viruses , participate in substrate and metabolite exchange with the host , enabling a much greater effect on metabolic pathways . this influence may be particularly relevant to the reported risks and benefits of various types of diets in many diseases , and in particular , saturated and unsaturated fats . in autism , various cardiomyopathy pathways autistic components have been observed in a number of cardiomyopathy disorders ( melas and timothy syndromes and danon disease ) [ 128130 ] . cellular adhesion and the extracellular matrix play a key role in brain development and in autism [ 131 , 132 ] , and these pathways were the only significantly enriched processes in the overlapping dataset . vegf signalling , dopamine , serotonin , and addiction pathways , but no metabolic pathways , were also enriched . serum vegf levels have been reported to be reduced in severely affected autism cases . although perhaps less evident than with schizophrenia , toxoplasmosis has nevertheless been associated with prenatal depression , depression , bipolar disorder , and with a history of suicide attempts in recurrent mood disorders [ 9 , 10 , 134 , 135 ] . in depression , as well as immune , defence , and diverse pathogen related pathways , autoimmune diseases , hypertrophic cardiomyopathy , rheumatoid arthritis and osteoclast differentiation , cancer pathways , and alzheimer 's disease were overrepresented in the overlapping dataset . depression and arthritis have been reported as comorbid conditions , and prior depression is a significant risk factor in both cardiac conditions and alzheimer 's disease [ 137 , 138 ] . numerous studies have implicated the vegf pathway is relevant to depression and to the mechanism of action of antidepressants . with regard to transforming growth factor , tbf - beta , an anti - inflammatory cytokine , an imbalance of pro- and anti - inflammatory cytokines has been observed in major depression studies . neuronal pathways primarily concerned reward / addiction , glutamate , dopamine , serotonin , and cannabinoid networks . the circadian clock pathway , which was also over - represented , plays a key role in depression and related disorders . in drosophila , the circadian clock regulates the phagocytosis of bacteria , and within its many functions are the control of the immune system . unsaturated fatty acid metabolism again figured in this group , and the general benefits of modifying saturated / unsaturated fat ratios in diet are increasingly recognised , including in the area of psychiatry . the overlapping dataset in bipolar disorder also concerned immune related pathways , several autoimmune disease networks ( type 1 diabetes , arthritis ( and osteoclast differentiation ) , graft - versus - host disease , and allograft rejection ) , and a number of pathogen life cycle pathways . in relation to cancer pathways , slight increases in overall cancer risk have been reported in both bipolar disorder and schizophrenia , which appear to be gender dependent . in relation to the alzheimer 's disease pathway ( which independently figures in all kegg pathways related to susceptibility genes alone in most of these disorders ) , prior psychiatric illness has been shown to be generally associated with an increased risk of developing dementia . common pathological features across many psychiatric disorders and alzheimer 's disease also include white matter changes related to demyelination [ 147 , 148 ] . many of the stressors involved in these conditions ( starvation , viruses , infections and fever , cytokines , oxidative , and endoplasmic reticulum stress ) converge on a pathway that ultimately inhibits translation initiation and protein synthesis . this network is counterbalanced by growth factors and neurotransmitter influences that affect plasticity and growth and is particularly important in regulating oligodendrocyte viability , myelination , and synaptic plasticity ( c.f . the neurotrophin pathway within this dataset and related glutamatergic and growth factor signalling networks in others ) . neurotransmitter networks within the overlapping bipolar / interactome are predominantly related to dopamine and reward pathways and to tyrosine , phenylalanine , and tryptophan metabolism . the link between schizophrenia and toxoplasmosis is perhaps the strongest in relation to published studies [ 6 , 82 , 150152 ] , and of particular relevance is the parasite 's ability to increase cerebral dopamine levels ( see above ) . in this respect , the overlapping interactome / gene dataset was enriched in dopaminergic pathways , and also in those related to serotonergic and glutamatergic transmission as well as cocaine and amphetamine addiction . as in most cases autoimmune and atopic diseases , which are commonly associated with schizophrenia , the link with schizophrenia was positive and gender specific , while an inverse association between schizophrenia and rheumatoid arthritis was observed [ 153 , 154 ] ( c.f . the concentration of osteoclast differentiation pathways in this dataset ) . gluten sensitivity ( characterised by antibodies to a gluten constituent protein , gliadin ) , other food antibodies , and celiac disease have also been associated with schizophrenia . food antigens in schizophrenic patients have been shown to be correlated to the presence of t. gondii antibodies . interestingly , the antiparasitic agent artemisinin reduces the titre of antibodies to gliadin in a subset of schizophrenic patients , and these observations testify to the ability of the parasite to modulate immune function ( and perhaps the antigenicity of other proteins ) . however , artemisinin did not reduce the titre of antibodies to t. gondii , nor did artemisinin ( as add on therapy ) have significant effects on symptomatology [ 155157 ] . artemisinin and its analogues are known to produce neurotoxic effects in laboratory models , an effect possibly linked to excitotoxicity and oxidative stress [ 158 , 159 ] , and clearly more suitable agents are needed in the research domain . the overlapping dataset also included significant enrichment of adhesion molecule , glutathione , and growth factor and related signalling pathways ( vegf , mapk , and wnt , but not erbb signalling , although this pathway is affected by t. gondii ) . the ppar network was also enriched in this dataset and is relevant in relation to the inflammatory arm of this pathway and to the ability of the pathway to regulate cholinergic and dopaminergic function [ 160 , 161 ] . with regard to the cancer pathways in this dataset , schizophrenia has been associated with a reduced cancer incidence , but with no familial explanation , suggesting a nongenetic reason that may conceivably be related to the abilities of t. gondii , and other relevant pathogens , to favour the promulgation of one disease , but perhaps protect against another . in relation to the overlapping alzheimer 's disease pathway within this dataset there are only limited human seroprevalence studies and no apparent animal studies specifically in relation to the substantia nigra , linking toxoplasmosis to parkinson 's disease [ 11 , 12 ] . nevertheless the overlap between the t. gondii interactome and susceptibility genes figures certain key pathways that may merit further research . the interactome / genetic overlap for significantly enriched pathways in parkinson 's disease in relation to neurotransmission was restricted to dopaminergic systems , and a number of key genes including those of the mitochondrial respiratory chain ( atp6 , cytb , and nd2 ) , the quinone reductase nqo2 , and two key parkinson 's disease genes ( pink1 and uchl1 ) figure within the enriched t. gondii interactome . while an ability of t. gondii to promote dopamine synthesis might be considered beneficial in parkinson 's disease , it has also been shown that dopamine promotes synuclein conformational changes , which may directly contribute to pathology . as with other diseases , autoimmune networks , cancer pathways , and alzheimer 's disease any link between alzheimer 's disease and toxoplasmosis is limited to a seroprevalence study and to scattered case reports [ 164 , 165 ] . in alzheimer 's disease , the significantly enriched pathways included ppar signalling , terpenoid biosynthesis ( cholesterol synthesis ) concerned with fatty acid , lipid , and cholesterol homoeostasis , and the arginine and proline metabolism pathway , primarily concerning nitric oxide , all of which play a key role in alzheimer 's disease physiology [ 166 , 167 ] . several pathogens ( herpes simplex , c. pneumoniae , treponemas , and spirochetes ) [ 24 , 168 , 169 ] increase beta - amyloid deposition . the gamma secretase network and app are localised in immunocompetent dendritic cells , and , as the amyloid peptide possesses antimicrobial and antiviral effects [ 170 , 171 ] , beta - amyloid production may well be a general defensive response to pathogen invasion . in normal conditions , it is not known whether beta - amyloid production is also a response to larger parasites , or whether beta - amyloid has antiparasitic activity . in tg2576 transgenic mice ( the swedish app mutation ) , t. gondii infection in fact reduces cerebral beta - amyloid deposition and increases the levels of anti - inflammatory cytokines , effects attributed to the immunosuppressant effects of infection . in relation to the cholesterol related genes in the alzheimer 's disease t. gondii dataset , the parasite can not synthesis its own sterols and scavenges host cholesterol . its growth in macrophages can be inhibited by statins . while a living cholesterol lowering agent might be considered useful in the periphery , such effects may be deleterious if limited to cerebral areas , as the brain synthesises its own cholesterol . this is mostly present in myelin and is generally indispensable for function . in the alzheimer 's disease tg2576 transgenic model , t. gondii lysate antigen inhibits the production of nitrites in microglial cells , contributing to the protective effects of infection in this model . as with obesity , certain interactome / susceptibility gene pathways involved in parasitic infection might well be considered as beneficial . although by far the most enriched dataset in terms of interactome / susceptibility gene overlaps , there appear to have been no studies either in the clinic or in relation to myelination in laboratory studies linking multiple sclerosis and toxoplasmosis . a study in 3 pairs of identical twins reared apart was generally inconclusive , although t. gondii or other pathogen seropositivity were observed in some cases . further work will be of interest in relation to this close association . in multiple sclerosis , the major overlapping pathways primarily concerned cytokine and tgf - beta signaling , the related jak - stat pathway , and the erbb and p53 signalling pathways that plays a key role in myelination [ 175 , 176 ] . within each disease dataset , the susceptibility genes that overlap with the t. gondii interactome , analysed by either method , appear highly relevant to the pathological processes and physiology of the disease . however , while some may be deleterious , ( e.g. , the promotion of dopaminergic activity in relation to psychosis ) , others may be beneficial ( e.g. , immunosuppression in autoimmune diseases ) . even within any particular disease , the diverse effects of the parasite could be either favour or inhibit the development of particular endophenotypes . as suggested below , the overall direction taken and the resulting pathology are likely to depend upon a combination of factors including the strain of parasite , the timing and localisation of infection , our prior immune status , and the susceptibility genes . in many cases , the signalling networks influenced by susceptibility genes either per se or within the overlapping host / pathogen interactome involve many diseases other than the primary disease concerned . for example degenerative disorders may be inversely associated with cancer [ 163 , 178 ] . this may be related to particular signalling networks , for example growth factor signalling pathways are essential for myelination or involved in long - term potentiation , but excessive stimulation will promote cancerous growth . the ability of t. gondii ( and other pathogens ) to affect so many processes , which may be either deleterious or beneficial to various disease - related networks , suggests that pathogens may also be the pivot around which such relationships revolve . several studies have recently shown that the entire human proteome contains short sequences ( pentapeptides to heptapeptides or longer gapped consensi ) that are identical to those within proteins expressed by numerous viruses , bacteria and other pathogens . for diverse pathogens , these human homologues appear to be concentrated within networks that are relevant to diseases in which the pathogen is implicated [ 35 , 37 , 38 , 179 , 180 ] . for example , there are 18,000 pentapeptide overlaps between the poliovirus and the human proteome while a single immunogenic pentapeptide ( vggvv ) within the beta - amyloid peptide is identical to that within proteins from the herpes simplex virus and from 68 other viral species . the extensive host / pathogen interactomes of numerous viruses , bacteria , and parasites no doubt result from this homology which enables pathogen proteins to mimic particular motifs within their human counterparts and to compete for their usual binding partners . such homology must presumably relate to our evolutionary decent from monocellular organisms and to horizontal gene transfer , a process that applies to all living matter . it is now also appreciated that dna derived from both dna and rna viruses ( and not only from retroviruses ) has been extensively incorporated into the human genome , and it seems likely that this has also played a role in our evolution , and evidently in the generation of this protein homology [ 182185 ] . host parasite interactions have also contributed to this gene transfer , and genes from the chagas disease vector , rhodnius prolixus , have been found within the genomes of its tetrapod hosts . peptide homology is more extensive than genetic homology , due to the fact that a number of amino acids can be coded for by several triplet dna codons ( 6 for arginine leucine and serine , 4 for alanine , glycine , proline , threonine , and valine , 3 for isoleucine , and 2 for asparagine , aspartate , glutamate , glutamine , cysteine , histidine , lysine , and phenylalanine ) ( see http://en.wikipedia.org/wiki/dna_codon ) . these essentially correspond to single nucleotide polymorphisms that do not modify the translated amino acid . for short peptide sequences this extensive homology , and more particularly slightly differing rather than identical peptides ( which are more likely to be recognised as nonself ) [ 36 , 187 ] , may well also contribute to autoimmunity problems that are evident in many diseases . for example , in alzheimer 's disease , multiple sclerosis , schizophrenia , and aids , antigenic regions of several autoantigens particular to each disease are homologous to proteins expressed by the pathogens implicated in the same disease ( including t. gondii and schizophrenia ) [ 1821 , 39 ] . diseases currently classified as autoimmune include celiac disease , multiple sclerosis , myasthenia gravis , lupus , rheumatoid arthritis , and inter alia ( see medline plus article at http://www.nlm.nih.gov/medlineplus/ency/article/000816.htm . however , the autoimmune problem appears to be much more extensive than currently appreciated . for example , using a protein array of 9,486 unique human protein antigens , even control blood samples averaged over 1000 autoantibodies , although with extreme intersample variation . as only ~30% of the human proteome was used in this experiment , we may each eventually accumulate over 3000 autoantibodies , irrespective of any particular disease . however , in both parkinson 's and alzheimer 's disease the target profile of the autoantibodies is distinct and can be reliably used as a diagnostic and predictive tool [ 188 , 189 ] . autoimmune signatures , with diagnostic predictive value have also been reported in multiple sclerosis , breast cancer , and nonsmall cell lung cancer . such data , ( in diseases generally not regarded as autoimmune ) and the recognition that so many diseases are characterised by immune activation and inflammation suggest that further research in this area would be fruitful in relation to the understanding of the pathologies and eventual treatment of many diseases . the immune system is trained , in early life , not to recognize the body 's own proteins as self . these bioinformatics data suggest that the multiple autoantibodies seen in man ( even in the absence of disease ) may stem not from some inherent malfunction of the immune system itself , but from antibodies raised to the numerous pathogens that we randomly encounter during the course of our lifetime . because of this extensive host / pathogen homology , such antibodies are also likely to target human proteins , and even if the pathogen is eliminated , continued encounter of these human homologues would sustain an autoimmune response . in this way , pathogens might be able to influence disease processes , even when no longer present , perhaps accounting for numerous studies that have failed to find pathogen dna or protein within diseased tissue , a finding often cited as evidence against pathogen involvement , as recently applied to the controversial implication of the xmrv virus with chronic fatigue or prostate cancer [ 194197 ] . the prospect that autoimmunity is pathogen related suggests that such agents may be able to punch far above their weight and influence biological processes even after their successful removal . this entails a revision of koch 's postulate as already discussed in a recent review on autoimmunity and the metagenome . this autoimmune scenario might also explain why the antiparasitic agent artemisinin failed to influence psychotic symptoms ( as add - on therapy ) in schizophrenia , as destruction of the parasite needs not to affect the behaviour of antibodies raised to it . antibodies to pathogens are clearly cross - reactive with cerebral tissue , although the precise targets remain to be identified . for example 14/25 antibodies to 17 neurotropic pathogens , including borrelia burgdorferi , t. gondii , and various dna and rna viruses were found to bind to western blots of human nervous tissue . it is impossible to verify cross - reactivity solely from sequence analysis , but the ability of pathogen antibodies to react with human proteins could perhaps be tested in bulk using the protein arrays described above . it is now known that antibodies can enter cells , transported by the pathogens to which they bind , , and are also able to traverse the blood brain barrier . for example , in transgenic mice engineered to express nerve growth factor antibodies only in lymphocytes , the blood brain barrier is soon disrupted , with cerebral antibody entry provoking extensive cortical degeneration , cholinergic neuronal loss , tau hyperphosphorylation , and beta - amyloid deposition ( i.e. , the cardinal pathology of alzheimer 's disease ) . this phenomenon is applicable to human diseases , including sydenham 's chorea , believed to be caused by streptococcus induced antibodies which cross - react with basal ganglia antigens . the same streptococcal pathogens ( and likely a similar mechanism ) have been implicated in paediatric autoimmune neuropsychiatric disorders ( panda 's ) whose diverse symptoms include tics , and dystonias , tourette syndrome , and obsessive - compulsive disorder . if autoantibodies do indeed play a key role in the pathogenesis of many diseases , then it is likely that their removal may be of benefit . however , given the large number of autoantibodies , some of which may well be beneficial and also required for pathogen defence , this may be no easy task . however , the research so far suggests that the number of autoantibodies specific to a particular disease may be more limited , allowing scope for analysis of their pathological or redemptive properties . the mechanisms described above provide a general example of multiple gene / environment interactions in relation to a single pathogen interactome , where several thousand genes ( human and protozoan ) are involved . even for a simple population genetics model , with two genes , two risk factors , and a single cause , varying permutations can dramatically influence the eventual outcome . for example , the light and dark coloured genes of the peppered moth , or the light and dark colours of the clean or polluted trees on which they alight , can all be either risk promoting or protective depending on the varying permutations ( the light gene kills the moth alighting on dark trees but is protective on the lighter trees , etc . ) . neither gene , nor risk factor is relevant if there are no hungry birds or at night time . if one splits a complex disease into its component parts and gives the number of interacting processes involved in the t. gondii interactome , even this single pathogen could act either as a cause , a risk promoter , or as a protective agent , depending upon the pathways that it influences the most . for example , its effects on dopamine could promote psychosis or synuclein polymerisation , and its cholesterol scavenging may have beneficial effects in atherosclerosis , but deleterious effects on myelination , immunosuppressive effects might well protect against autoimmunity , but favour other infections , while the host 's inflammatory reaction or associated fever might contribute to inappropriate collateral damage . these complex interactions are nevertheless based on a relatively simple concept ; that each interaction has an effect on the processes and pathways regulated by the human protein concerned . this suggests a model that may have general application to the many other pathogens and environmental agents implicated in these diseases . if one imagines the t. gondii proteins as a number of spheres , each with particular affinity for certain human genes or proteins , and their human interactome partners as a further series of spheres perched on a genetic ledge whose characteristics and apertures are regulated by polymorphisms , mutations , deletions , translocations , or copy number variations , then the trajectory of each , dropped through this genetic sieve or knocked off the ledge and falling through the apertures , will be influenced both by the strain of pathogen with different host / pathogen affinities , the dropping point , the timing , and localisation of infection , when and where different human genes are expressed and by the polymorphic genes themselves ( for both the host and the pathogen ) . each of these human genes controls a particular element of one or many signalling networks , metabolic pathways , structural elements , developmental processes , and so forth , each represented by reception bins at different positions beneath the sieve . depending upon varying permutations of these factors , the eventual number of spheres in each bin will vary , resulting in a diverse spectrum of pathway disturbance . each pathway may be affected either positively or negatively , and the eventual assembly of this pathway mosaic leads to particular endophenotypes or subpathologies , which together combine to assemble into a particular disease . in this way , the same pathogen can produce diverse effects ranging from cause to prevention depending on a permutation of circumstance . the genes , risk factors , and the immune system thus work together to determine the final outcome , while neither per se are likely to provoke a particular disease . while gene / environment interactions are appreciated in both genetic and epidemiological studies , most , particularly in relation to gwas , are performed without data partitioning in relation to other variables . many other pathogens ( each no doubt with extensive host pathogen interactomes ) and many other risk factors are implicated in these and other diseases , and many are able to influence several relevant aspects of pathology ( see section 1 ) . a clearer understanding of these complex effects could perhaps result in a metamorphosis from multiple genes of small effect in large populations to more restricted numbers of greater effect in particular conditions . it is likely that many disease phenotypes have several causes , that subsets of overlapping genes are relevant to each , and that despite the mass of data collection and processing entailed , a dissection of these relationships could eventually lead to disease prevention and cure in multiple conditions . by their very nature , polygenic diseases are complex , with several underlying pathologies and endophenotypes , hundreds of interacting genes , and dozens of environmental risk factors . the failure to replicate either genetic or epidemiological data is a situation peculiar to these diseases , not seen in many other fields . however , the effects of genes and risk factors are clearly conditional and , as illustrated above , may well depend upon each other . replication inconsistency may well be part of the answer and not part of the problem . this may seem a surprisingly high figure , but a similar interactome for the hiv-1 virus , maintained by ncbi http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=detailssearch&term=hiv1interactions[properties ] , contains 1443 human genes ( 5.4% of the human genome ) . bacteria and larger protozoan parasites , which , unlike viruses , also scavenge for host nutrients , as well as injecting their own metabolites into the host 's environment , thus influence a larger spectrum of biochemical rather than signalling pathways . these data were also collected from experiments using various host ( and species ) tissues , and it is likely that brain or other tissue or time - specific interactomes would be more selective . the relevance of many genes to a particular condition is often tested by gene knockout in transgenic models and comforted by the resulting endophenotypes which mimic those of the particular disease . however , risk promoting variants are , for the most part , single nucleotide polymorphisms rather than deletions and while expression may be altered ( in either direction ) in mrna or protein expression studies , there is little to suggest a similar knockout in the human condition ( see for example the microarray geoprofiles database at ncbi http://www.ncbi.nlm.nih.gov/sites/geo/ ) . however , there are two pathogen - related effects that equate to conditional protein knockout which could be cell and regionally and temporally specific . if a host protein is engaged with that of a pathogen , it is effectively taken out of circulation during this period , and the pathways in which it is implicated can but be compromised . secondly , because of extensive homology between pathogen and human proteins , antibody cross reactivity is likely to target the human counterparts of the pathogen antigen , effectively resulting in immunopharmacological knockout . in addition to these knockout effects , immune activation and the general reaction to infection are also likely to influence cellular function , as are the multitude of genes whose mrna levels are influenced by this and other pathogens . in relation to prenatal effects , laboratory models have shown that maternally administered nonspecific viral dna mimics and inflammatory agents or cytokines can also induce behavioural disturbances and psychopathology in the offspring [ 208 , 209 ] . fever during pregnancy also increases the risk of the offspring later developing autism and schizophrenia [ 210 , 211 ] , and it seems likely that prenatal infection in general is able to markedly affect brain development . the consequences would also depend upon which particular brain process and region is concerned at which period of embryogenesis . many other pathogens have been implicated in several of these conditions . in some of the diseases studied , almost one - third of the susceptibility genes were implicated in the t. gondii interactome ( table 3 ) . other pathogens will also have extensive interactomes , specific to each , but with a degree of overlap , and it would not be implausible if the near totality of susceptibility genes , in certain diseases , were involved in the summated life cycles of these diverse environmental triggers . it would thus seem that many susceptibility genes are related to the causes of disease , rather than ( and as well as ) to the disease itself . it is likely that stratification of gwas and other genetic data in relation to infection status and history and many other environmental variables would be useful in determining the contribution of different genes to different risk factors and to their commonly affected pathways . many psychiatric disorders are associated with a degree of social stigma and blame often apportioned to the genes , parentage and upbringing , and behaviour of the affected individuals . these and other chronic diseases also place a heavy long - term burden on family , friends , and caregivers . this analysis suggests that t. gondii is a likely cause of certain aspects of psychiatric disorders , but perhaps a protective agent in others . hopefully , an appreciation that such diseases may well be caused by pathogens and vectored by family pets will help to dispel such prejudice and more importantly create a new framework for the development of new methods of treatment and prevention . given the massive problem of autoimmunity , however , it may be simplistic to suggest that removing the pathogen will halt the disease , although prevention of its initial access might be expected to affect disease incidence . for example if toxoplasmosis in cats and other pets was registered as a notifiable disease requiring obligatory treatment by veterinarians , perhaps the incidence of several diseases could be reduced .

toxoplasma gondii is not only implicated in schizophrenia and related disorders , but also in alzheimer 's or parkinson 's disease , cancer , cardiac myopathies , and autoimmune disorders . during its life cycle , the pathogen interacts with ~3000 host genes or proteins . susceptibility genes for multiple sclerosis , alzheimer 's disease , schizophrenia , bipolar disorder , depression , childhood obesity , parkinson 's disease , attention deficit hyperactivity disorder ( p from 8.01e 05 ( adhd ) to 1.22e 71 ) ( multiple sclerosis ) , and autism ( p = 0.013 ) , but not anorexia or chronic fatigue are highly enriched in the human arm of this interactome and 18 ( adhd ) to 33% ( ms ) of the susceptibility genes relate to it . the signalling pathways involved in the susceptibility gene / interactome overlaps are relatively specific and relevant to each disease suggesting a means whereby susceptibility genes could orient the attentions of a single pathogen towards disruption of the specific pathways that together contribute ( positively or negatively ) to the endophenotypes of different diseases . conditional protein knockdown , orchestrated by t. gondii proteins or antibodies binding to those of the host ( pathogen derived autoimmunity ) and metabolite exchange , may contribute to this disruption . susceptibility genes may thus be related to the causes and influencers of disease , rather than ( and as well as ) to the disease itself .

1. Introduction 2. Methods 3. Statistics 4. Results 5. Summary 6. Conclusion

PMC3385215

hospital supply systems should ensure adequate stock of all the required items to maintain uninterrupted supply . advances in medical care and drugs have disproportionately increased the expenditure on health care delivery . therefore , about one - third of the hospital budget is spent on purchasing various materials and supplies including medicines . this necessitates the effective and efficient management of medical store by keeping a close supervision on important drugs , prevention of pilferage , and priority setting in purchase and distribution of drugs . a study suggested that review and control measures for expensive drugs can bring about 20% savings in medical store budget . the inventory control can bring about substantial improvement not only in patient care but also in the optimal use of resources . continuous quality management in medical store can provide the value added services to the patients . of all inventory control systems available , the abc ( always better control ) and ved ( vital , essential and desirable ) matrix is the most preferred for medical stores . analysis based on pareto 's principle of vital few and trivial many depending on capital investment of the items . although abc analysis is based on the monetary value and rate of consumption of the item , there are items which have low capital investment and consumption but is life saving . ved analysis is based on the need of the drug in the hospital and classified as vital , essential and desirable. combination of abc and ved matrix allows more meaningful control over the inventory . economic order quantity ( eoq ) is another form of inventory control model which balances the possessing cost with the cost of running out of the items . it is the level of inventory that minimizes total inventory holding costs and ordering costs . eoq only applies when demand for a product is constant over the year and each new order is delivered in total when the inventory reaches zero . a fixed cost is charged for each order placed , regardless of the number of units ordered . eoq along with abc - ved analysis is proposed to be the most effective and efficient model for inventory control . ours is a tertiary care centre and number of patients coming to the hospital is increasing with passage of time . to satisfy the healthcare needs of the increased number of patients , increased capital this budget can be accessed from the actual budget consumed in the previous year using the cost inflation index ( cii ) . the application of cii justifies the claim for increased annual drug expenditure ( ade ) . based on previous economic data , prediction for the future can be done using regression analysis . this helps in framing the policies to ensure uninterrupted supply of drugs in the limited budget . detailed literature search revealed scanty data regarding the relevance of cii and regression analysis in medical store management . we therefore considered it worthwhile to carry out an integrated economic analysis of the medical store . the main objectives of the study were to consider abc - ved analysis along with eoq , to assess the indexed cost of acquisition of ade , comparison of indexed cost and the actual cost and to forecast expenditure for the forthcoming years . the annual drug consumption and expenditure on each item was collected from the medical store of a tertiary care hospital in central india , for the financial year 20102011 . the data were transcribed in a ms - excel spreadsheet and arranged in the descending order based on ade . then cumulative cost , cumulative cost percentage , and percentage of number of items were calculated and based on capital investment ; a , b and c items were separated . group a was constituted by approximately first 10% items which consumed 70% of the budget . ved classification of items was done in consultation with physicians and senior staff of department of pharmacology . vital category included the drugs which should be available in the hospital at all times and are critically required for the survival of the patients . essential items , as per who , are those which satisfy the health care needs of the majority of the population and are intended to be available at all times and in adequate amounts . desirable items are those of lowest criticality , the shortage of which would not be detrimental to the health of the patients . two drug categories ( i and ii ) requiring different types of monitoring based on the abc - ved matrix was identified to direct supervisory monitoring . category i comprised of av , ae , ad , bv and be groups and required high management priority . category ii with lower management priority constituted items belonging to bd , cv , ce and cd groups of the abc - ved matrix . the first alphabet in the category denotes its place in abc analysis while the second alphabet is for its place in ved analysis . the annual hospital expenditure ( ahe ) indexed cost of acquisition of ade for the year 20102011 was calculated using the formula where the year 20092010 was taken as the base . percentage of actual ade and percentage of indexed cost in comparison to ade of the previous year were estimated and finally the difference between the two was calculated . linear regression was used to assess the expenditure for the forth coming years taking into consideration the ade of last 10 years . the drug formulary consisted of 210 items , out of which 165 were available in 20102011 . 1,91,44,253 . the drug expenditure included the expenditure for 28,068 indoor admissions and 4,59,569 outdoor patients who were dispensed with drugs for 3 days routinely except for specialty clinics where the drugs were dispensed for 7 days . the split of ade incurred on abc and ved categories of drugs during the financial year 20102011 is shown in table 1 . the cutoff values were not exactly 70% , 20% , and 10% but differed marginally . split of annual drug expenditure depending on abc - ved analysis for financial year 20102011 the abc graph of the year 20102011 using cumulative cost and % items has different curves representing groups a , b , and c consuming about 70% , 20% , and 10% of the total budget , respectively [ figure 1 ] . however , the individual drug budget can not be visualized from this graph which can be best represented by plotting the percentage cost of each drug with respect to ade [ figure 3 ] . this concave type of plotting showed the individual drug expenditure as a percentage of the total ade at a glance which can be further used for eoq . the indexed cost of acquisition of ade for year 20102011 came out to be rs . the index factor for the year 20102011 as fixed by the government of india was 711 and that of 20092010 was 632 . the percentage of actual ade as compared to that of previous year was found out to be 110.39% whereas the percentage of indexed cost as compared to that of previous year was found out to be 112.5% after inflationary correction . the ade for the forthcoming years applying linear regression from 2011 to 2015 is in table 2 and shows a statistically significant value ( p<0.001 ) which will be helpful for framing the policies for the respective years . 87,80,577 ) . using the linear regression function of graph pad prism , version 3.02 , the forecasted ade for the forthcoming years till 2015 showed that for the financial year 20112012 , budgetary requirement is rs . abc analysis of drugs for the financial year 20102011 abc - ved cumulative curve for the financial year 20102011 abc analysis depicting percentage cost of each drug of group a with respect to annual drug expenditure in the year 20102011 projected annual drug expenditure for the next 5 years using linear regression analysis annual drug expenditure for last 10 and forthcoming years using regression analysis we preferred the integrated economic analysis due to the paucity of data in the literature . in a tertiary care centre , along with proper care , timely availability of drugs is a prime concern . from our study if only abc analysis based on cost factor alone is considered , control of only 24 drugs ( out of 165 ) from group a consuming about 70% of the drug budget is needed . this group requires greater monitoring as it has fewer drugs , consuming most of the budget . , the availability of drugs of vital nature from b and c categories ( 12 + 22 ) will be compromised . similarly , if ved analysis is considered alone as ideal , control is to be exerted on 40 vital and 113 essential drugs accounting for 96.8% of ade . however , the ved classification differs from hospital to hospital depending on the health care needs of the patients . moreover , we had a desirable drug in group a , which consumed a good chunk of the budget . thus , not only criticality analysis , but the cost factor also has to be taken into consideration . therefore , the coupling of abc - ved analysis is used for prioritization . from the abc - ved matrix , it is absolutely clear that all stock is not equally valuable and hence , does not require the same management focus . categorization of drugs by the abc - ved matrix helped us to narrow down our focus on category i drugs which required high management priority and stringent control as they consumed the maximum budget . from our study , we found category i drugs consumed about 89.1% of the budget . rational use of drugs with deletion of nonessential drugs and imposition of fixed budget to this category can bring about substantial savings without harming the patient care . the abc - ved matrix can help us in improving the drug availability and lesser emergency purchase and adequate inventory control , thereby reducing the financial budget . the cumulative curve does not depict the actual expenditure consumed by each drug which is well cited from the concave graph . in our study , it was observed that maximum budget was consumed by injection imipinem cilastin combination whereas in the previous study the same place was secured by injection cefotaxim . we could not predict the exact cause for this observed change in our study , but plausible explanation for this may be a resistance pattern developed by the microorganisms or different antimicrobial drug policy in our institution . moreover , the concave curve has helped us to decide eoq and time of order placement . eoq is the amount of inventory to be ordered at one time for the purpose of minimizing annual inventory cost . it is the size of the order which gives maximum economy in purchasing any material and ultimately contributes toward maintaining the materials at the optimum level and at the minimum cost . the subgroups av and ae of category i comprised of only 23 drugs , holding a valuable budget of 69.5% , should be monitored for eoq and ordering of these items must be rational and justifiable . as category ii drugs consumed limited amount of budget , these drugs can be ordered once or twice in a year using eoq and shelved to save management efforts without blocking substantial capital . eoq along with abc - ved analysis can be effectively applicable in non - government hospitals where there is no limitation of maintaining the stock of 3 months only . being a government hospital , we can keep stock of drugs for 3 months only as per directives of government of maharashtra . to avoid unavailability of drugs , the indexed ade for the financial year 20102011 was just 2.11% higher than actual ade . at present , very few studies are available to compare the indexed ade , but still this figure is less as compared to the previous study where it was 2.84% . this may be attributed to smaller drug formulary of our hospital and to some extent , the continuous efforts made by us to satisfy the healthcare needs of our area within limitation of budget of medical store . the inflation index represents the general trend and is not constant for all commodities . in developing country where the value for money is changing every day , the purchasing power of the currency is also changing . due to this impact , the item which cost low in the current year will cost more in the forthcoming years . hence , the application of cii makes sense for prediction of the drug expenditure with the use of inflation factor . as the inflation factor for the future years can not be predicted in advance , hence the drug expenditure prediction for the forthcoming years can not be done with cii but it can be done better using regression analysis . one of the secondary objectives of inventory management is the financial forecasting for the forthcoming years . in government institutions where the financial resources are already fixed , however , such planning does not include the inclusion and exclusion of any drug in inventory or any unavoidable circumstances such as epidemic which can then cause economic burden . moreover , the forecasting of ade for the future years using the ade of the previous year 's helps us to visualize the increasing trend in the consumption of the budget . this also helps for justifying the demand of increased budget during allocation of the budget in government institutions for next year . we found coefficient of determination ( r ) as 0.92 which suggest that 92% of the variation is shared between financial years and forecasted budget , respectively . the application of cii which includes the inflation factor also gives another justification for the increased claim for the budget . forecasting of the budget for coming years helps in deciding the policies in advance to curb the deficiency of drugs and to satisfy the healthcare needs of society . thus , we recommend that abc - ved analysis along with eoq and integrated economic analysis should be strictly followed in every government medical college to utilize the available limited budget for maximum gains . abc - ved analysis identifies drugs requiring stringent control for the optimal use of resources . due to inflation , total expenditure for the drugs is increased each year which supports the higher budgetary requirement for the forthcoming years . at the same time , forecasting of budget helps for better management of medical store . hence , abc - ved along with eoq and integrated economic analysis optimizes the costs of medicare services besides making materials available to the patients which can increase the quality of healthcare services .

economic analysis plays a pivotal role in the management of medical store . the main objectives of this study were to consider always better control - vital , essential and desirable ( abc - ved ) analysis with economic order quantity ( eoq ) , comparison of indexed cost and the actual cost , and to assess the expenditure for the forthcoming years . based on cost and criticality , a matrix of nine groups by combining abc and ved analysis was formulated . drug categories were narrowed down for prioritization to direct supervisory monitoring . the subgroups ae and av of the categories category i and ii should be ordered based on eoq . the difference between the actual annual drug expenditure ( ade ) and the derived indexed cost using the cost inflation index ( cii ) was calculated . linear regression was used to assess the expenditure for the forth coming years . the total ade for the financial year of 20102011 was rs . 1,91,44,253 which was only 7.68% of annual hospital expenditure . using the inflation index , the indexed cost of acquisition of ade for year 20102011 was rs . 1,95,10,387 . the difference between the two was estimated to be 2.11% . thus , the cii justifies the demand of increased budget for next year and prompts us for cautious use of drugs . by taking into consideration the ade of last 10 years , we have forecasted the budget for forthcoming years which will help significantly for making policies according to the available budget .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC4091274

the catalytic functionalization of aldehyde c h bonds represents a mild and atom - economical approach to prepare esters . while there has been much progress in developing hydroacylation of aldehydes , the hydroacylation of ketones is relatively unexplored and warrants attention due to its potential for constructing chiral esters . our laboratory has demonstrated intramolecular ketone hydroacylations , including the first examples of enantioselective lactonizations . due to the propensity of nonchelating aldehydes to engage in decarbonylation , tishchenko dimerizations , and aldol condensations , intermolecular hydroacylations are considerably more challenging . herein , we report the design and development of the first enantioselective intermolecular ketone hydroacylation . we imagined a novel cross - coupling between a broad range of aldehydes 1 and ketones bearing a directing group ( dg ) 2 ( scheme 1 ) . on the basis of previous studies , we proposed that this functional group would promote preferential ketone binding to the rh(i ) center and favor chemoselective cross - coupling over aldehyde dimerization or decarbonylation . in the presence of a suitable bidentate phosphine ligand , coordination of ketone 2 to rh , followed by oxidative addition of aldehyde 1 , would generate coordinatively saturated rh(iii)-hydride i. octahedral complex i has a lower propensity to undergo carbonyl deinsertion , a process that deactivates the rhodium catalyst . ketone insertion into acyl - rh(iii)-hydride i would lead to rh(iii)-alkoxide ii , followed by reductive elimination to give ester 3 with concomitant regeneration of the rh(i ) catalyst . in comparison to conventional approaches to ester synthesis , this catalytic method obviates the need for prefunctionalization of the acyl component and is amenable to enantioselective synthesis of esters . to test our hypothesis , we chose hydrocinnamaldehyde ( 1a ) and -ketoamide 2a as model substrates ( chart 1 ) . a number of rh - bis(phosphine ) catalysts were examined because previous studies in our lab had demonstrated that these complexes promote intramolecular ketone hydroacylation . after studying various ligands , we found rh(i)-josiphos catalysts most promising . commercially available ligand l1 provides modest yield and 13% ee . changing to ligand l2 , which contains a diphenylphosphine and a dialkylphosphine , results in a small improvement in enantioselectivity ( 21% ee ) . josiphos l3 containing a more -accepting difurylphosphine and a -donating di - tert - butylphosphine affords the desired -acyloxyamide 3a in 46% yield and 73% ee . we reason that this c1-symmetric ligand may facilitate the product - determining hydride delivery via the trans effect ; the hydride trans to the electron - rich dialkylphosphine becomes more hydridic , while the ketone trans to the -acceptor becomes more prone to insertion ( scheme 2 ) . aconditions : 1a ( 1.2 equiv ) , 2a ( 1 equiv ) , [ rh(cod)2]bf4 ( 0.1 equiv ) , ligand ( 0.1 equiv ) , dce , rt ( 23 c ) , 24 h. conditions : 1a ( 1.2 equiv ) , 2a ( 1 equiv ) , [ rh(cod)2]bf4 ( 0.1 equiv ) , ligand ( 0.1 equiv ) , dce , rt ( 23 c ) , 24 h. inspired by l3 , we created new josiphos ligands to study the steric influence of the phosphine substituents . in comparison to commercial l3 in contrast , l5 with bulkier -accepting dibenzofurylphosphines affords better results than l3 ( 65% yield , 87% ee ) . the rh - l5 catalyst shows excellent chemoselectivity with no observable aldehyde dimerization . this mild rh(i)-catalyzed method offers an attractive approach to enantioenriched esters that are structurally related to those obtained via the multicomponent passerini reaction . a conventional route to these motifs would involve enantioselective ketone reduction followed by an additional acylation step ( which generally requires stoichiometric activating agents ) to obtain the -acyloxyamides . moreover , the asymmetric reduction of -ketoamides has not been well - studied and remains limited in scope and/or selectivity . a method featuring rh catalysis with bidentate amidophosphine phosphinite ligands appears most promising and has been applied to reduce -ketoamides with good enantioselectivity for a few aryl - substituted ketones . there is one example of ruthenium - catalyzed -ketoamide hydrogenation where excellent ee was achieved following recrystallization for a bulky tert - butyl - substituted ketone . biocatalytic methods have also been reported for reducing -ketoamides , although with low efficiency . thus , with rh - l5 in hand , we studied the coupling of -ketoamides 2 with nonchelating aliphatic aldehydes 1 ( table 1 ) . aryl ketones containing various substituents and substitution patterns undergo hydroacylation with hydrocinnamaldehyde ( 1a ) ( entries 17 ) and hexanal ( 1b ) ( entries 814 ) in good to high yields ( 5398% ) and high enantioselectivities ( 8496% ) . sterically demanding 2-methylphenyl ( entries 2 and 9 ) , mesityl ( entries 4 and 11 ) , and 1-naphthyl ketones ( entries 5 and 12 ) tend to give higher conversions and selectivities even with lower ( 5 mol % ) catalyst loadings , resulting in 7398% yields of the -acyloxyamides with 9496% ee . -ketoamides bearing smaller aryl groups , such as 3-methylphenyl ( entries 3 and 10 ) , 4-chlorophenyl ( entries 6 and 13 ) , and 3,5-difluorophenyl groups ( entries 7 and 14 ) , require a higher catalyst loading to provide the corresponding -acyloxyamides in 5381% yields and 8490% ee . conditions : 0.12 mmol of 1 , 0.1 mmol of 2 . enantiomeric excess determined by chiral sfc analysis . with 1.2 mmol of 1 , 1.0 mmol of 2 . sterically encumbered primary aldehydes ( r = i - bu and neopentyl , entries 1517 ) are effective coupling partners , providing the desired products in high yields and ee . using aldehyde 1d and ketone 2e , we show that the reaction can also be performed on a larger scale ( 1.0 mmol , 353 mg product isolated , 91% yield , 93% ee ) at 5 mol % catalyst loading ( entry 16 ) . the rhodium loading can be further reduced to 3.5 mol % at elevated temperature conditions ( 60 c ) , providing hydroacylation product 3p in 92% yield and 87% ee ( entry 17 ) . transforming -branched cyclohexanecarboxaldehyde ( entry 18 ) requires elevated temperatures ( 50 c ) to proceed and furnishes the desired -acyloxyamide 3q in 57% yield and 90% ee . aryl aldehydes do not participate in hydroacylation to any appreciable extent under the current reaction conditions ( < 5% conversion ) , perhaps due to a higher barrier to c h activation . compared to the n , n-alkylarylamide , ketones containing n , n-diaryl- or dialkylamides are also less effective ( entries 19 and 20 ) and provide -acyloxyamides 3r and 3s with modest yields and enantioselectivities . a cyclic ketoamide is converted to 3-acyloxyindolinone 3 t ( entry 21 ) in 54% yield and 58% ee . the efficacy of this reaction is improved by employing l3 , which provides heterocycle 3 t in 88% yield and 69% ee . the amide motif impacts both reactivity and enantioselectivity , presumably due to a directing group role . the absolute configuration of the products was assigned based on x - ray analysis of -acyloxyamide 3c ( see supporting information ) . the observed s - enantiomer is consistent with a proposed model that invokes the trans - effect in promoting the product - determining ketone insertion step ( scheme 2 ) . in this model , the c1-symmetric ligand cooperatively renders the hydride more nucleophilic and the ketone more electrophilic ( complex a ) , and thus substantially favors formation of the s - stereoisomer in the case of ( sp , r)-l5 . conversely , the alternative complex b is stabilized by the trans - effect which would result in an increased barrier to migratory insertion . we propose the acyl group to be situated on the bottom apical position , with the less hindering n , n-disubstituted amide carbonyl bound to the top apical position . this ligand arrangement minimizes unfavorable steric interactions between the acyl group and the substituents on the phosphines and is supported by dft calculations . we find that morpholine amides direct hydroacylation effectively to yield cross - coupled products in good yields and enantioselectivities ( table 2 ) . a similar trend is observed in which ketones bearing larger aryl groups perform better , both in terms of isolated yields and enantioselectivities . phenyl ketone 4a is hydroacylated with 3,3-dimethylbutanal ( 1d ) to furnish ester 5a in 67% yield and 80% ee , whereas larger mesityl and 1-naphthyl ketones give 5b and 5d in 94 and 75% yields , respectively ( 95 and 92% ee ) . other aldehydes including cyclohexanecarboxaldehyde , hexanal , and hydrocinnamaldehyde are also good coupling partners . the use of the morpholine amide as a directing group provides a handle for further chemical manipulations . the catalyst was hydrogenated at rt for 45 min prior to adding substrates ( see supporting information ) . the rh - l5 catalyst described in this study , however , is not effective with -keto - weinreb amide 2k ( scheme 3 ) . to this end , we have identified an alternative rhodium catalyst derived from methoxy - biphep l6 , which provides good reactivity and modest enantioselectivity for this transformation to yield ester 6 ( 93% , 51% ee ) . the catalyst was hydrogenated at rt for 45 min prior to adding substrates ( see supporting information ) . we initiated mechanistic studies by examining the kinetics for the cross - coupling of aldehyde 1d and -ketoamide 2e by h nmr . these particular substrates were chosen because their h nmr signals are distinct and no products of decomposition were observed over the course of reaction progress , thus simplifying data analysis . initial rates for the hydroacylation of 2e were then measured by varying the concentrations of 1d , 2e , and rhodium catalyst . these experiments revealed a first - order dependence of the rate on both aldehyde 1d ( figure 1 ) and ketone 2e concentrations ( figure 2 ) . more intriguing is the observed second - order rate dependence on catalyst concentration ( figure 3 ) , which suggests the involvement of two rhodium species in the turnover - limiting step . a study on the correlation between the enantiomeric excess of the catalyst and that of the product yielded a small , positive nonlinear relationship ( figure 4 ) . such a phenomenon is consistent with the added degree of complexity in the reaction mechanism as indicated by the kinetic profile . plot of initial rates ( kobs ) with respect to [ aldehyde 1d ] showing first - order dependence ; [ 2e ] = 0.17 m , [ catalyst ] = 0.0083 m. plot of initial rates ( kobs ) with respect to [ -ketoamide 2e ] showing first - order dependence ; [ 1d ] = 0.20 m , [ catalyst ] = 0.0083 m. plot of initial rates ( kobs ) with respect to [ catalyst ] showing second - order dependence ; [ 1d ] = 0.20 m , [ 2e ] = 0.17 m. relationship between the enantioselectivity of the reaction and the ee of the chiral catalyst . the rate data obtained from the kinetics experiments allowed us to calculate initial turnover frequencies . under standard catalytic conditions using 5 mol % catalyst , 1.0 equiv of ketone , and 1.2 equiv of aldehyde at 25 c , the initial turnover frequency is determined to be 4.8 10 s. to gain further insight into the mechanism , we measured the kinetic isotope effect ( kie ) by running two independent , side - by - side experiments using protio-1a and deuterated 1a - d ( scheme 4 ) . aldehyde 1a - d was chosen ( over 1d and others ) for this study due to ease of isolation . examination of the initial rates resulted in a kie of 2.6 and supports c h bond activation to be turnover - limiting . this value is larger than that previously observed for an intramolecular ketone hydroacylation where insertion was implicated as the turnover - limiting step ( kie = 1.79 0.06 ) . the absence of a directing group on the aldehyde in the present system likely increases the barrier to oxidative addition significantly . on the basis of the kinetic analysis and kie , we propose a mechanism invoking homobimetallic activation of the aldehyde where one rhodium catalyst acts as a lewis acid by coordinating the oxygen atom of the aldehyde and a second rhodium catalyst participates in the oxidative addition of the formyl c h bond to produce acyl - rh(iii)-hydride iv ( scheme 5 ) . a report of cationic rhodium complexes behaving as lewis acid catalysts supports this proposal . in addition , lewis acid additives ( i.e. , zncl2 , znbr2 , zni2 , etc . ) have been found to be beneficial in a study on intermolecular alkene hydroacylation , although their role is unclear . the mechanism proceeds with insertion to generate acyl - rh(iii)-alkoxide v , followed by reductive elimination to furnish the product and turnover the rh(i ) catalyst . it is possible for the reaction to occur through bimetallic intermediates following oxidative addition ; however , reactive intermediates were not observed spectroscopically . we believe that rapid coordination and dissociation of solvent and substrate leads to broadening of the p nmr resonance signals and is thus consistent with [ rh(diphosphine)(solvent)2]iii being a resting state . by developing a new rh - josiphos catalyst , we achieved the first enantioselective intermolecular ketone hydroacylation , which features the rare use of nonchelating aldehydes . kinetic analysis reveals a second - order rate dependence on the rhodium catalyst and , together with a study on nonlinear relationship and the kie , points to a unique mechanism involving homobimetallic oxidative addition as the turnover - limiting step . this work provides a foundation for understanding c h activation and hydroacylation using nonactivated aldehydes which will guide future studies and applications .

under rh(i ) catalysis , -ketoamides undergo intermolecular hydroacylation with aliphatic aldehydes . a newly designed josiphos ligand enables access to -acyloxyamides with high atom - economy and enantioselectivity . on the basis of mechanistic and kinetic studies , we propose a pathway in which rhodium plays a dual role in activating the aldehyde for cross - coupling . a stereochemical model is provided to rationalize the sense of enantioinduction observed .

Introduction Results and Discussion Conclusions

PMC5361021

exercise facilitates metabolic functions in the human body and benefits physical health by preventing disease , controlling weight , and maintaining homeostasis1 . the american heart association reported that a lack of exercise is one of the main factors of heart disease ( along with high blood pressure , hyperlipidemia , and smoking ) and recommended regular exercise to treat and prevent heart - related disease2 . measuring the blood biochemical indicators of myocardial damage is conventionally divided into a traditional method that measures creatine kinase myocardial band isoenzyme ( ck - mb ) and a cardiac enzyme test that measures cardiac troponin ( ctn ) . an increase in these elements indicates a strong possibility of acute cardiac infarction3 . of these elements , ctn is an enzyme specific to the heart that increases in response to very small cardiac damage4 . therefore , ctn is a standard biochemical index used for diagnosing myocardial necrosis in patients with coronary artery disease5 . the n - terminal of prohormone brain natriuretic peptide ( nt - probnp ) is an indicator that is useful for diagnosis , the evaluation of treatment effects , and the prediction of the prognosis of patients with cardiac insufficiency . nt - probnp is also known to be effective for predicting a malfunction of the left ventricle after cardiac infarction6 . the material reflects cardiac stress in clinics and has been reported to be excessively expressed over the normal range during serious exercise , such as an ultra - marathon7 . performing regular exercise before fierce endurance exercise is effective for preventing myocardial and muscular tissue damage during the fierce endurance exercise . however , few studies have examined the physiological indicator in cases of one - off high - intensity exercise after moderate- or high - intensity endurance orientation training . therefore , the purpose of this study was to investigate differences in the concentrations of ctni , nt - probnp , and ck - mb in male sprague - dawley rats during one bout of prolonged treadmill exercise ( lasting 3 h ) following different training and non - training regimens . forty male sprague - dawley rats ( age : six weeks , weight : 150180 g each ) were used in this study . all animals were housed ( three rats per cage ) at 23 2 c and 50 5% humidity with a 12-hour light dark cycle . the rats were given a seven - day period for adapting to the environment before the experiment . the rats were randomly divided into four groups ( n=10 in each group ) : control , high - intensity exercise , moderate - intensity exercise , and nonexercise . this study was approved by the institution of animal care and use committee of daegu university , and the experimental procedure complied with the management guidelines for experimental animals . the exercise intensity levels used in this study were set on the basis of the results of a study by shepherd and gollnick ( 1976 ) , in which the maximal oxygen uptakes of white sprague - dawley rats were measured using a metabolic rate - measuring instrument and a treadmill . as a result , the levels were set at 15 m / min ( i.e. , approximately 65% of the maximal oxygen uptake ) and 28 m / min based on the reference data that defined the relevant velocity as being approximately 82% of the maximal oxygen uptake8 . this study s aerobic exercise groups first performed a preliminary aerobic adjustment exercise for 20 min daily for four days and then performed their main treadmill training for 35 min / day six days per week ( except sunday ) for four weeks . the high- and moderate - intensity exercise groups maintained intensity levels of 28 m / min and 15 m / min , respectively . but , non - exercise group carried out preceding exercise for four days and since then did nt carry out intensive aerobic exercise . after the training , the high- and moderate - intensity exercise groups and the nonexercise group simultaneously performed one bout of prolonged treadmill exercise at 15 m / min for 3 h. following this bout of prolonged treadmill exercise , the animals were weighed . each animal s abdominal cavity was dissected immediately , and 10 ml of blood was collected from the main artery using a syringe . the collected blood was put into an eppendorf tube and centrifuged at 18,000 rpm at 4 c for 18 min . the collected sera were stored in a freezer at 70 c prior to analysis . , usa ) was used as a measurement device , and a dimension mmb flextm reagent cartridge was used as a reagent . the blood threshold applied to each experiment was under 0.6 ng / ml and 125 pg / ml for ctni and np - probnp , respectively . a one - way analysis of variance test was performed using pasw ( version 18.0 for windows ) to investigate intergroup differences . table 1table 1.results of prolonged intensive exercise in rats accustomed to high- and moderate - intensity trainingcg ( n=10)hg ( n=10)mg ( n=10)ng ( n=10)weight ( g)230 g220 g220 g230 gctni ( ng / ml)0.01 0.000.08 0.020.59 0.40*1.54 0.74*nt - probnp ( pg / ml)27.8 8.729.7 10.538.5 12.858.5 15.3**p<0.05 . cg : control group ; hg : high - intensity group ; mg : moderate - intensity group ; ng : non - exercise group . * significantly different compared with cg ( p<0.05 ) ; significantly different compared with ng ( p<0.05 ) presents the weights of the rats measured in each exercise group after one bout of prolonged treadmill exercise . the ctni levels differed significantly between the groups : 0.01 0.00 ng / ml for the control group , 0.08 0.02 ng / ml for the high - intensity exercise group , 0.59 0.40 ng / ml for the moderate - intensity group , and 1.54 0.74 ng / ml for the non - exercise group ( p<0.05 ) . the post hoc analysis showed that the high- and moderate - intensity exercise groups had significantly higher ctni levels than the control group ( p<0.05 ) , and the high- and moderate - intensity exercise groups had significantly lower ctni levels than the non - exercise group ( p<0.05 ) ( table 1 ) . cg : control group ; hg : high - intensity group ; mg : moderate - intensity group ; ng : non - exercise group . * significantly different compared with cg ( p<0.05 ) ; significantly different compared with ng ( p<0.05 ) the np - probnp levels differed significantly between the groups : 27.8 8.7 pg / ml for the control group , 29.7 10.5 pg / ml for the high - intensity exercise group , 38.5 12.8 pg / ml for the moderate - intensity exercise group , and 58.5 15.3 pg / ml for the non - exercise group ( p<0.05 ) . the post hoc analysis showed that the non - exercise group had significantly higher np - probnp levels than the control group ( p<0.05 ) , and the high- and moderate - intensity exercise groups had significantly lower np - probnp levels than the non - exercise group ( p<0 0.05 ) ( table 1 ) . this study examined the impact of four weeks of moderate- and high - intensity treadmill warming - up exercise on the heart s condition during serious aerobic exercise using a sample of sprague - dawley white male rats . in this study results , the blood ctni levels of the non - exercise group and the moderate - intensity training group increased significantly after long - distance running compared to the control group , which did not perform long - distance running . however , the high - intensity training group did not show a significant increase , indicating that high - intensity training in advance of long - distance running is effective at preventing myocardial damage during long - distance running . this result is consistent with the results of a study by chen et al.9 , in which ctnt concentrations in white rats decreased remarkably in an experiment group that performed high - intensity warm - up swimming training with an extra weight compared with an experiment group that swam without a weight . the study argued that performing preliminary exercise before high - intensity training can decrease myocardial damage or negative impacts in the body in case of fierce exercise . in the current study , a significant increase in nt - probnp , which is used as an indicator of cardiac insufficiency , was observed in the nonexercise group . several studies reported that elite athletes , non - elite athletes , and leisure players can show a short - term increase in nt - probnp after intense and severe long - term exercise . moreover , nt - probnp concentration increases after long - term exercise or fierce endurance exercise10 , 11 . however , nt - probnp concentrations at rest do not increase among trained athletes compared to untrained people in the same age group10 , 12 . in the current study , the moderate- and high - intensity exercise groups showed a significant decrease of indicator for myocardial damage compared to the non - exercise group when implementing serious long - distance running . among the exercise groups , the prevention effects were larger the high - intensity training than the moderate - intensity training . this implies that performing high - intensity training before severe aerobic exercise is effective at preventing myocardial damage . wannamethee et al.13 noted that the implementation of regular exercise reduced exercise - induced inflammatory responses ; this was attributed to adaptive responses to the exercise and to anti - inflammatory effects . the current study found that , compared to the nonexercise group , rats that underwent preceding exercise showed less cardiac muscle damage and inflammatory responses during prolonged intense exercise . in particular , the effects were greater after one bout of intense exercise following high - intensity training than moderate - intensity training .

[ purpose ] the purpose of this study was to investigate the effects of exercise on myocardial injury in male sprague - dawley rats . two groups of rats were trained with either moderate- or high - intensity treadmill running for four weeks . subsequently , the concentrations of cardiac troponin and the n - terminal of prohormone brain natriuretic peptide ( nt - probnp ) were examined following a single bout of prolonged intensive exercise ( lasting 3 h ) . [ subjects and methods ] the study included 40 six - week - old male sprague - dawley rats weighing 150180 g each . the aerobic exercise group was divided into high - intensity ( 28 m / min ) and moderate - intensity ( 15 m / min ) subgroups . both subgroups were trained for 35 min daily for six days per week ( excluding sunday ) over a four - week period . following training , the high- and moderate - intensity exercise groups and a nonexercise group performed one bout of prolonged treadmill exercise for 3 h at a speed of 15 m / min . [ results ] the cardiac troponin and nt - probnp levels differed significantly between the groups . [ conclusion ] the exercise groups showed lower levels of cardiac troponin and nt - probnp than the nonexercise group after the bout of prolonged intensive exercise .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3276109

enzyme - catalyzed reactions organized into sequential pathways are responsible for producing the molecules needed to sustain life . while many essential metabolic pathways are present in all known life forms , there are also significant differences between microbial and mammalian metabolism . most microbial species can synthesize all of their important metabolic building block molecules , while mammals must acquire many of these from dietary sources . these metabolic differences provide a substantial number of potential protein targets to be examined for the development of selective antimicrobial agents . there is a growing need to identify effective new antibiotics that function against new targets to combat the expanding threat from pathogenic species that are becoming increasingly resistant to existing antibiotics [ 1 , 2 ] . there are , however , fundamental problems that must be resolved for effective biocides to be developed against metabolic enzyme targets . infectious microorganisms often use their host as a source of essential metabolites , thus bypassing inhibitors designed to block key steps in microbial metabolism . in many instances microorganisms have developed alternative routes for the production of important metabolites that can readily bypass inhibited enzyme reactions . although these microbial pathways may not exist in mammals , related enzymes with similar active site geometries or substrate binding motifs can potentially interact with even the most carefully designed inhibitors . the structure and mechanism of a bacterial metabolic enzyme must be thoroughly characterized before a potential drug target can be fully evaluated . aspartate -semialdehyde dehydrogenase ( asadh ) is a key enzyme in an essential amino acid biosynthetic pathway that is not present in mammals . this enzyme has been thoroughly investigated and is now being examined as a target for the development of new antimicrobial agents . the commitment step to this pathway is the phosphorylation of aspartic acid catalyzed by a family of aspartokinases ( ak ) . the next enzyme in the pathway , asadh , catalyzes the production of aspartate semialdehyde ( asa ) that is located at a critical junction in this pathway . from this point one pathway branch leads to the production of lysine through the metabolite diaminopimelate , while the alternative route leads to the synthesis of methionine , threonine , and isoleucine with homoserine serving as their common intermediate ( figure 1 ) . thus , one quarter of the amino acids required for protein synthesis in all organisms are linked through and synthesized by this pathway . the aspartate pathway is exquisitely regulated to control the total amino acid output as well as the relative levels of each amino acid . this regulatory scheme must also maintain the levels of several essential metabolic intermediates during periods of low protein synthesis . this is accomplished by coordinated regulation by the end product amino acids , both through feedback inhibition and by selective gene repression . this regulatory scheme allows each end product to modulate the flux through the initial pathway steps , with branch point allosteric enzymes providing further control over the product levels . in addition to the essential amino acids produced in the aspartate pathway , several important metabolites are synthesized that play crucial roles in important developmental processes , such as cell wall biosynthesis , protective dormancy , and virulence factor production . for example , dihydrodipicolinate is a precursor of dipicolinate , the major component of bacterial spores , and diaminopimelate ( dap ) is required for cross - linking of the peptidoglycan polymers in bacterial cell wall synthesis ( figure 1 ) . another product of this pathway , s - adenosylmethionine , is an essential methyl group donor that also serves as a precursor for quorum sensing signaling molecules with critical roles in triggering virulence factors in infectious microorganisms [ 8 , 9 ] . homoserine lactone signals , for example , are essential for both virulence and biofilm development in the human pathogen acinetobacter baumannii . because this pathway produces many essential compounds that are involved in a range of critical functions , disruptions to the aspartate pathway are fatal to those microorganisms . in particular , selective perturbations of the asd gene that encodes for asadh are lethal to numerous infectious microorganisms . for example , asd mutants of salmonella typhimurium develop an absolute growth requirement for diaminopimelate ( dap ) , a critical cell wall cross - linking component ( figure 1 ) in gram - negative bacteria . this mutated organism undergoes cell lysis when dap is not supplied , and , since this metabolite is not produced in mammals it can not be supplied by the host organism . a similar loss of viability is observed in asd - deficient e. coli strains . during amino acid starvation microorganisms . however , de novo biosynthesis of lysine is essential for the survival of mycobacterium tuberculosis during infection in mice , despite the presence of lysine in the host . even if an organism could mutate to improve lysine transport capacity in response to aspartate pathway inhibition , reversal of the decarboxylation that produces lysine from dap is neither kinetically nor thermodynamically feasible . both of these end products and several additional intermediates of this pathway are thus critical for microbial cell viability , both in culture and during host infection . therefore the identification of effective inhibitors of key aspartate pathway enzymes should provide lead compounds for the development of new biocides . to achieve this aim we have focused on the functional and structural characterization of the microbial asadh family of enzymes . aspartate--semialdehyde dehydrogenase ( asadh ) catalyzes the second reaction in the aspartate pathway , the reductive dephosphorylation of -aspartyl phosphate to aspartate--semialdehyde ( asa ) ( scheme 1 ) , at a critical branch point in this pathway . the asadhs from a variety of organisms encompass a considerable diversity of sequence homologies , ranging from less than 10% to as high as 95% sequence identity when compared to the escherichia coli enzyme ( ecasadh ) . the asadh enzymes in microorganisms can be divided into three branches consisting of the enzymes from gram - negative bacteria , gram - positive bacteria , and archaea / fungi . these branches were initially identified and partitioned through sequence alignments and now , with representative high resolution structures available from each branch , have been compared by structural alignments . the earliest structures of asadhs are from enzymes that were isolated and purified from gram - negative bacteria . these enzyme forms share significant sequence and structural homology and include structures of the asadhs from e. coli [ 14 , 15 ] , vibrio cholerae , and haemophilus influenzae . the overall structure of these asadhs is a homodimer with an extensive contact surface between the subunits . each monomer is composed of a carboxy - terminal domain primarily involved in hydrophobic intersubunit contacts , and a more hydrophilic amino - terminal domain that forms the active site and nadp binding site ( figure 2 ) . the asadh from streptococcus pneumoniae ( spasadh ) is the first member of the gram - positive bacterial branch that was structurally characterized . spasadh is a good representative of the other gram - positive bacterial asadhs with greater than 40% sequence identity to these enzymes , while having less than 25% identity with any of the gram - negative enzymes . unexpectedly , this spasadh has lower sequence homology to the gram - negative enzymes than to those of the archaeal / fungal branch , with sequence identities from this comparison ranging from 18 to 30% . the structure of the asadh from the archaeal hyperthermophile methanococcus jannaschii ( mjasadh ) has a similar overall fold and domain arrangement as the gram - negative enzyme forms despite being less than 10% sequence identity . but the complete set of functionally important active site amino acids has been conserved in mjasadh , suggesting an identical mechanism in the enzyme from this ancient organism despite its lower catalytic efficiency . the first structure of an asadh from a fungal species , candida albicans ( caasadh ) , was recently determined , and it also possesses a similar overall fold , domain organization , and active site structure as the other members of this enzyme family . this fungal enzyme has less than 30% sequence identity to any of the bacterial asadhs and only 40% identity to its closest homologue . alignment of the structures that have been determined from each branch of the asadh family shows that both spasadh and caasadh are most similar to the archaeal asadh from m. jannaschii and more distantly related to the gram - negative enzymes . despite the overall sequence and structural similarities , there are a number of insertions and deletions which serve to differentiate the three asadh branches . structural variations between these branches include a sequence of residues on the enzyme surface that contribute to the binding pocket for the coenzyme nadp . the e. coli asadh is representative of the gram - negative enzymes , with a highly flexible coenzyme binding loop in the absence of nadp that becomes ordered in response to nadp binding . the archaeal mjasadh has three conserved insertions totalling 30 residues when aligned against the gram - negative bacterial ecasadh , with each insertion located on the surface of the structure . a 13-residue insertion in mjasadh leads to an alternative orientation of the coenzyme binding loop , differing from that in the gram - negative enzymes by about 90 , causing it to drape over and occlude the coenzyme binding pocket . in addition , the helical subdomain that comprises a significant fraction of the dimer interface ( figure 2 ) is absent in the archaeal mjasadh . the gram - positive spasadh has the same number of residues in this region as the gram - negative bacterial enzymes , but two short -strands replace the helix - turn - helix structure observed in the helical subdomain of ecasadh ( figure 2 ) . the fungal enzyme from c. albicans is also missing the helical subdomain and contains most of the insertions and deletions observed in the archaeal enzyme . these structural changes suggest differences in how each branch of this enzyme family can carry out its catalytic role , even though each possesses an identical repertoire of highly conserved active site functional groups . in spite of the overall sequence diversity between the different branches of the asadh family the identity of the core active site functional groups has been preserved throughout evolution ( figure 3 ) . a set of active site mutants of asadh from h. influenzae ( hiasadh ) was examined kinetically and structurally with the goal of more precisely establishing the role for each functional group in substrate recognition and binding . a cysteine had been previously identified from biochemical studies as the likely active site nucleophile . replacement of a single sulfur atom with an oxygen in the c136s mutant virtually eliminates catalysis ( table 1 ) , supporting the essential role of this residue as the catalytic nucleophile . some decrease in catalytic activity would be expected because the hydroxyl group of serine is a weaker nucleophile than a cysteine sulfhydryl group . only minor changes are observed in the active site structure of this mutant despite the nearly complete loss of catalytic activity . but a change in orientation of the introduced hydroxyl group is likely responsible for the very low activity of this mutant . the side chain hydroxyl group of the introduced serine rotates by about 90 degrees , with this new orientation stabilized by a hydrogen - bond to an adjacent backbone carbonyl group . this reorientation not only moves this functional group out of the position needed to act as a nucleophile , but also competes with the involvement of this backbone carbonyl in intermediate stabilization and alters the position of the bound phosphate group . mammalian glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) catalyzes a similar reaction , an oxidative phosphorylation , to the reverse reaction catalyzed by asadh and appears to do so by the same mechanism . before the first structure of asadh was available , sequence alignments to the mechanistically related enzyme ( gapdh ) family were used to identify and then test the roles of possible active site functional groups . both enzymes were proposed to contain a cysteine - histidine catalytic dyad ; however the corresponding position of the catalytic histidine in gapdh is occupied by a conserved glutamine in asadh . replacement of this functional amino acid causes a loss in catalytic activity that is not fully recovered even when a histidine is introduced at this position in asadh . this mystery was solved when the first asadh structure revealed that the essential histidine residue actually comes from a completely different position in the primary sequence , with a loop containing h277 folded into the enzyme active site . the h277n mutant of hiasadh has significantly impaired activity ( table 1 ) , highlighting the key role for this residue . however a ternary complex structure with nadp and the active site - directed inactivator , s - methyl - l - cysteine sulfoxide ( smcs ) , showed continuous density extending from the cys136 nucleophile . this structure is consistent with the covalent attachment of smcs to the active site nucleophile . the position of this bound inactivator is shifted in the h277n mutant relative to its position when bound in the wild - type enzyme . this reorientation moves this compound further from the bound phosphate that must attack the carbonyl carbon of the acyl - enzyme intermediate to generate -aspartyl phosphate in the reverse reaction ( scheme 1 ) . so in summary for these catalytic mutants , the c136s mutant still allows substrate binding but formation of the covalently bound intermediate is slowed by a less efficient and misoriented nucleophile . in contrast , the h277n mutant appears to form the initial tetrahedral intermediate efficiently , but its subsequent breakdown is hindered by a shift away from the bound phosphate nucleophile . in each case , small perturbations in the positioning of essential catalytic groups or reactive intermediates are shown to have dramatic effects on enzyme catalysis . additional mutant structures of hiasadh have been determined , with each substitution replacing a putative substrate binding group in order to assess their function . each of these mutants displayed significantly impaired catalytic activity , ranging from 10% to less than 0.1% that of the wild - type enzyme . however , the structural basis for the diminished activities is different for each mutation ( table 1 ) . a conserved arginine ( arg270 ) in the asadh family aligns with a conserved arg231 in the gapdh family , a residue that had previously been assigned a role in binding the substrate phosphate group . based on kinetic studies this arginine residue was proposed to have a comparable role in hiasadh , namely , binding the carboxyl group of the substrate asa . the structures of substrate complexes of the wild - type enzyme and of the inactivator smcs covalently bound to vibrio cholerae asadh ( vcasadh ) support this assignment by clearly establishing the presence of a bidentate interaction between arg270 and the carboxylic group of the substrate asa . arginine270 was replaced by lysine in hiasadh to assess its relative importance in substrate binding by disrupting the bidentate interaction that is formed with asa . maintaining this interaction between the introduced lysyl amino group and the asa carboxyl group will require a shift in the orientation of both the lysyl side chain and the resulting tetrahedral intermediate relative to their positions in the wild - type enzyme . the dramatic loss of activity in r270k can be explained by a shift of the intermediate away from the position that is necessary to form and maintain a productive interaction between its carboxyl group and the altered substrate binding residue during the catalytic cycle . a neutral side chain introduced at this position in ecasadh ( r267l ) can not form any binding interaction with the substrate carboxyl group , leading to a 30-fold increase in the km for asa while also permitting greater flexibility for positioning of the covalent intermediate . this flexibility increases the likelihood that the intermediate can adopt a catalytically viable conformation relative to that imposed in the hiasadh r270k mutant , and this conformational flexibility is manifest in the 100-fold greater activity seen in the r267l ecasadh enzyme form ( table 1 ) . glutamate243 provides a side chain carboxyl group in the active site of asadh whose role in the catalytic cycle had not been definitively established . this group is highly conserved among asadhs from different organisms , and structural studies have shown that it is in position to potentially interact with the amino group of the tetrahedral intermediate in the hiasadh complex . an e243d mutant was produced to assess the possible role of this residue in substrate and intermediate binding . kinetic studies of e243d failed to show the expected detrimental effect on substrate interactions , with the km for asa unchanged from that of the wild - type enzyme . instead the catalytic efficiency of this mutant is significantly compromised , reducing the kcat value to about 1% of that of wild - type hiasadh ( table 1 ) . in this case the position of the bound intermediate ( and presumably that of the bound substrate ) shifts towards the shorter side chain of the introduced aspartate at this position . this shift allows this mutant to maintain substrate binding affinity , but compromises the positioning between the intermediate and the bound cofactor thereby leading to impaired catalytic efficiency . each of these mutations was prepared with the aim of removing and testing critical substrate binding groups . however the structural rearrangements that occur as a consequence of these replacements manifest themselves not in a loss in substrate binding affinity , but in a loss of catalytic activity . structural characterization of these mutants shows that only subtle changes in key active site residues , such as rotation of a side chain to form a new hydrogen - bond or a shift in position relative to a catalytic intermediate , are sufficient to adversely affect catalysis . the phosphate - binding site of asadh is capable of accommodating different tetrahedral oxyanion analogues , leading to different functional consequences . both oxyanion substrates and oxyanion inhibitors bind in the same position by using the same set of ligands ( figure 4 ) , raising the question of what distinguishes a substrate from an inhibitor ? in the apoenzyme the side chain hydroxyl of thr137 forms a hydrogen - bond with asn135 , and this pairing prevents any interactions with the active site glu243 . upon binding of either oxyanion substrate , phosphate or arsenate , thr137 switches hydrogen - bonding partners through a 60 rotation that disrupts its interaction with asn135 . this new conformation moves the threonine hydroxyl group into position to interact with and orient glu243 , an important substrate binding group . glu243 remains in this substrate binding position in the arsenate structure , and this hydrogen - bond with the thr137 hydroxyl group persists even in the absence of bound asa . so , the presence of an oxyanion substrate in the active site helps to position glu243 to interact with the substrate . however , in the presence of the inhibitor periodate this threonine does not change position and switch partners . in this inhibitor - bound structure thr137 remains hydrogen - bonded to asn135 , which is now oriented away from glu243 and can not stabilize the position of this side chain . this single change in hydrogen - bonding partners is apparently sufficient to interfere with the binding of asa . thus , subtle shifts in the position of side chains , even those not directly involved in substrate binding or catalysis , can lead to a sequence of events that result in loss of function for a finely tuned enzyme catalyst . the active site functional groups of asadh are already poised to accommodate amino acid substrate binding in the apoenzyme . however , the binding of nadp is required to induce a domain closure that sets up the active site for catalysis . nadp binding and the coupled domain closure are driven by numerous interactions between the enzyme and the molecular features that are distributed throughout the coenzyme . in vcasadh , arg9 is repositioned during nadp binding to form an electrostatic interaction with the 2-phosphate , with additional hydrogen - bonds to phosphate from thr36 and ser37 ( figure 5 ) . a consensus sequence , sgxg , present in each branch of the asadh family interacts via backbone carbonyl hydrogen - bonds to the amide nitrogen of the nicotinamide , while a conserved glutamine ( gln350 ) in the bacterial enzymes and a corresponding asparagine in the archaeal enzyme are in position to hydrogen - bond to the amide oxygen ( figure 5 ) . binding interactions to the adenine ring of nadp are less conserved between the gram - negative and gram - positive bacterial enzymes , leading to some drastic differences in coenzyme binding . the adenine base in vcasadh is oriented by a cation- interaction with arg9 ( figure 5 ) in the consensus gxxgxxg sequence which is part of the rossmann nucleotide fold . a surface loop spanning from leu189 to ser195 closes around nadp in these gram - negative enzymes , with the exocyclic n6 of the adenine base forming a hydrogen - bond with the backbone carbonyl of a proline ( pro192 ) located on the helical subdomain from the opposite subunit of the dimer ( figure 2 ) . this interaction plays a critical role in the change from an open to a closed enzyme conformation upon coenzyme binding . the overall domain movements encountered in the transition from the apoenzyme to the nadp complex in gram - positive spasadh are similar to those observed for vcasadh . rotation of the n - terminal domain toward the active site in response to nadp binding appears to be a universal mechanism utilized throughout the asadh family . however , none of the interactions that drive domain closure in both ecasadh and vcasadh are observed in spasadh . thus , facilitation of the commonly observed domain closure in spasadh must be driven by a different set of interactions than those that drive the same closure in the gram - negative asadhs . a completely new binding pocket for the adenine base is found in spasadh , along with an altered 2-phosphate binding site , from that previously observed in the gram - negative enzymes . the adenine binding pocket in spasadh is forged between an -helix and the coenzyme binding loop of the n - terminal domain . interactions between the adenine ring and the side chains of thr76 and ser37 , along with a cation- interaction with arg39 , serve to anchor the adenine ring in this pocket . these new interactions cause the adenine ring to adopt an altered position in spasadh formed by rotation around the bonds linking the nicotinamide and adenine ribose with the bridging diphosphates . as a consequence the center of the adenine ring is shifted by about 8.5 with respect to its position in nadp bound in vcasadh , and the position of the exocyclic amine on c6 that formerly interacted with pro192 from the adjacent subunit has been displaced by nearly 14 ( figure 5 ) . however , the 2-phosphate of nadp bound in the spasadh structure moves less than 5 relative to its position in vcasadh , and the phosphate binding groups move to accommodate this shift . comparison of the coenzyme binding site between archaeal mjasadh and the bacterial enzymes also shows some conserved similarities . in bacterial asadhs the backbone amino groups from a methionine and valine are hydrogen - bond donors to the pyrophosphate of nadp ( figure 5 ) , and a change from methionine to serine in the mjasadh structure does not alter these pyrophosphate interactions . a similar reorientation of the adenine ring to that observed in spasadh is seen in the binding of nadp to the asadh from m. jannaschii . an extended surface loop in mjasadh ( from residues 40 to 77 ) specific contacts between the adenine ring and the enzyme include two hydrophobic interactions to leu91 and leu95 that are within 4.0 of the plane of the adenine ring and a cation- interaction with arg39 . similar interactions and a closely related coenzyme orientation are also found in the binding of the adenine ring in the fungal enzyme structure ( caasadh ) ( figure 5 ) . these changes in coenzyme conformation and in the nature of the interactions with the 2-phosphate of nadp in the mjasadh structure suggest the possibility of altered coenzyme specificity relative to that seen for the well - studied gram - negative bacterial enzymes . preference for nadp versus nad binding in enzymes is mediated through specific interactions with either the 2-phosphate of nadp or the 2-hydroxyl of nad . the asadhs from e. coli , v. cholerae , and h. influenzae are each highly specific for nadp . the structure of the nadp complex in vcasadh shows that the 2-phosphate group is bound by the same arginine that stacks against the adenine base , along with three hydrogen bonds that are formed with two serines and a threonine . this arginine in vcasadh is not conserved in mjasadh but instead is replaced by a threonine that is still in the correct position to form a potential hydrogen - bond with the 2-phosphate of nadp but , unlike in vcasadh , is not capable of binding to the adenine base . the possibility of expanded coenzyme specificity for the archaeal enzyme was examined kinetically by testing both nadp and nad as substrates near the physiological temperatures for this thermophilic organism . mjasadh is found to have comparable catalytic rates with either nad or nadp when examined at 70c . however , this enzyme still shows a preference for nadp over nad as its coenzyme , with a michaelis constant that is 130-fold lower for nadp ( knadp = 13 m ) compared to that for nad ( knad = 1700 m ) . it appears that the relaxed specificity for nadp in mjasadh is primarily due to absence of this critical arginine residue , since the positively charged guanidinium group of this arginine interacts directly with the negatively charged 2-phosphate of nadp . an additional explanation for this altered coenzyme specificity comes from the structure of the nadp complex in which the adenine and ribose end of the nadp is bound in a completely different binding pocket in archaeal mjasadh compared to the gram - negative bacterial enzyme family . the subunit interface in the homodimer for both the bacterial and archaeal asadhs is composed primarily of hydrophobic -sheets . the gram - negative bacterial asadhs have the largest dimer interface , formed by a conserved hydrophobic -sheet and complemented by the helical subdomain to create over 3400 of buried surface area ( figure 2 ) . this subdomain is also present in the gram - positive bacterial spasadh , but a 16-residue deletion results in a single -helix followed by an unstructured loop forming the top portion of the dimer interface with about 2600 of total buried surface area . a 48-amino acid deletion in mjasadh results in the complete removal of the helical subdomain that makes a significant contribution to the dimer interface in ecasadh . as a consequence the thermophilic mjasadh structure has a much smaller dimer interface of about 2000 . the enzyme from c. albicans ( caasadh ) is also missing the helical subdomain , with the 44 amino acids that constitute this helix - turn - helix motif in the gram - negative enzyme forms replaced by an unstructured 3 amino acid loop . as a consequence this fungal enzyme has the smallest dimer interface ( 1800 ) and the lowest percentage of buried surface area among the structurally characterized asadhs . interestingly , there is good correlation between the dimer interface area and enzymatic activity , with the enzyme forms possessing the highest buried dimer surface having the highest catalytic activity . this correlation suggests that intersubunit communication in the asadh dimer is critical to promote highly efficient catalysis . a network of hydrogen bonds has been identified across the dimerization interface that is proposed to allow active site to active site communication in the functional asadh dimer from the bacterial enzyme branch . these structural observations support the earlier kinetic experiments that had suggested an alternating site reactivity model for asadh catalysis . the active site glu240 in subunit a of vcasadh is positioned through a hydrogen - bond to gln161 ( figure 3 ) . gln161 also forms a hydrogen - bond with the backbone amide of thr159 , which in turn is hydrogen - bonded through its side chain hydroxyl group to the hydroxyl group of tyr160 across the dimer interface in subunit b ( figure 6 ) . a complementary series of interactions links the active site glu240 in subunit b to tyr160 in subunit a , such that each active site is linked to an amino acid in the adjacent subunit through a network of three hydrogen - bonds . the orientation of the tyr160 residues at the domain interface is crucial to maintaining this network . the tyrosine side chain position is stabilized by -stacking to the tyrosine from the other subunit and also by a perpendicular -stacking interaction with phe345 in the same subunit ( figure 6 ) . the structure of mjasadh does not allow the intersubunit communication route that is seen in the bacterial enzymes . two methionines are present within the dimerization interface region of mjasadh that contribute to the hydrophobic subunit interactions , but replace the conserved tyrosines in each subunit that forms the heart of the hydrogen - bonding network bridging between the two active sites ( figure 6 ) . the phenylalanines that stabilize these tyrosines through -stacking have also been replaced in the archaeal asadh branch with a conserved threonine ( figure 6 ) . these amino acids that are crucial for intersubunit communication have also been replaced in the caasadh enzyme . the base - stacked tyrosine is substituted by either leucine or methionine at this position in all fungal asadhs , while the stabilizing phenylalanine is now a conserved valine in this branch of the enzyme family . these amino acid replacements disrupt the intersubunit hydrogen - bonding network observed in gram - negative asadhs , leading to the loss of this communication channel . the alternating sites reactivity observed in the bacterial forms of asadh is likely absent in both the archaeal and fungal enzymes and could explain the very low catalytic activity in these enzyme forms despite the presence of an identical constellation of active site functional groups throughout the asadh enzyme family . the proposed catalytic mechanism of asadh in the reverse ( nonphysiological ) direction involves the initial attack of the active site cysteine nucleophile ( cys136 ) on the carbonyl carbon of the substrate asa . the enzyme - bound tetrahedral intermediate produced by this reaction ( figure 7 , structure a ) is set up to transfer a hydride ion to the nadp coenzyme bound at an adjacent site , leading to an acyl - enzyme intermediate ( figure 7 , structure b ) . attack of bound phosphate at the carbonyl carbon of this intermediate produces a second enzyme - bound tetrahedral intermediate ( figure 7 , structure c ) . collapse of this intermediate with expulsion of the enzyme thiolate group yields the -aspartyl phosphate product and leaves the enzyme ready to bind another molecule of asa and repeat the catalytic cycle . the chance to characterize a true intermediate in an enzyme - catalyzed reaction is a rare opportunity to bridge a mechanistic gap that is generally only hypothesized between the substrate and the product complexes , or extrapolated from intermediate - analogue structures . for the asadhs this feat has been accomplished twice , capturing and structurally characterizing two different reactive intermediates in the catalytic cycle of this enzyme and lending structural support to this proposed mechanism . a ternary complex produced by diffusing the substrates asa and phosphate into crystals of hiasadh was examined with the aim of determining the role of different active site groups in the catalytic mechanism . however , what was observed was not the structure of the enzyme - substrate complex , but instead the structure of the actual tetrahedral intermediate in the catalytic cycle of asadh ( structure a in figure 7 ) . the bound aspartyl intermediate was modeled into the continuous density emanating from the side chain of the cysteine nucleophile , and this structure clearly shows the tetrahedral geometry around the carbon that is covalently attached to the active site cysteine thiolate group ( figure 8) . subsequent hydride transfer from this tetrahedral intermediate to nadp would lead to formation of the acyl - enzyme intermediate . however , exclusion of the coenzyme from the crystallization conditions eliminates the hydride transfer step that is required for the reaction to proceed , thus stopping the catalytic cycle at this stage and allowing the determination of this important structure . the structure of the acyl - enzyme intermediate ( structure b in figure 7 ) was also determined by running the reaction in the nonphysiological direction . soaking the spasadh - nadp complex crystals with the substrate asa allows the catalytic cycle to proceed up to formation of the acyl - enzyme intermediate . kinetic studies had previously shown that the acyl - enzyme can be quite stable in the absence of phosphate . the refined structures show the key differences between the tetrahedral intermediate and the acyl - enzyme intermediate . the position of the substrate binding groups that interact with these intermediates shift only slightly between these two structures , with the largest movements caused by the conversion from a tetrahedral carbon geometry in the previous intermediate structure to a planar carbon geometry in the acyl - enzyme ( figure 8) . through a combination of kinetic , mutagenic , and structural studies the detailed catalytic mechanism of the aspartate -semialdehyde dehydrogenases has been defined , and the essential functional groups that play a role in substrate binding , catalysis , and regulation have each been identified . subtle changes in any of these critical functional groups are sufficient to alter the finely tuned catalytic machinery of this enzyme , leading to enzyme forms with significantly impaired activity . differences have been observed between the members of this enzyme family , isolated from bacterial , archaeal , and fungal species , regarding their catalytic efficiency , their coenzyme selectivity , and their mode of catalysis . this extensive body of work on the different members of the asadh family now allows us to evaluate this enzyme as a drug target . compounds are now being screened that can potentially recognize these structural and functional differences to produce initial inhibitors that can be developed into species - specific antimicrobials against this key enzyme in an essential metabolic pathway .

the aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals . characterizing the enzyme - catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action . the enzyme aspartate -semialdehyde dehydrogenase ( asadh ) catalyzes an early branch point reaction in the aspartate pathway . kinetic , mutagenic , and structural studies of asadh from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding , catalysis , and enzyme regulation . important structural and functional differences have been found between asadhs isolated from these bacterial and fungal organisms , opening the possibility for developing species - specific antimicrobial agents that target this family of enzymes .

1. Introduction 2. The Aspartate Biosynthetic Pathway in Microbes 3. Sequence and Structural Comparisons among the Aspartate- 4. Role of Active Site Functional Groups 5. Differences in Coenzyme Binding and Specificity 6. The Presence of an Intersubunit Communication Channel 7. Catalytic Mechanism and Covalent Intermediates 8. Concluding Remarks

PMC3659222

the prevalence of prostate cancer increases with age . fortunately , screening using prostate - specific antigen ( psa ) and transrectal prostate biopsies enables the early diagnosis and treatment of prostate cancer . in addition , surgical treatments for localized prostate cancer are associated with safe and favorable outcomes , and the proactive surgical treatment of prostate cancer is encouraged worldwide . the prevalence of bladder neck contracture ( bnc ) , one of the most common complications of prostate cancer surgery , is reported to be variable . in the 1990s , its prevalence was as high as 30% , but the prevalence has been reduced to 2.0% to 5.0% through advancements in surgical techniques and the introduction of new surgical methods , including robot - assisted laparoscopic radical prostatectomy ( rarp ) and pure laparoscopic radical prostatectomy ( lrp ) [ 4 - 6 ] . however , even though the prevalence of bnc has been reduced dramatically , when it does occur , bnc markedly reduces the quality of life of patients , and endoscopic operative procedures such as transurethral bladder neck incisions must be performed in cases refractory to metal sound dilation . there are few studies of bnc in asian populations , including koreans . in this study , therefore , we aimed to identify the prevalence and risk factors of bnc after radical prostatectomy in koreans . this study was conducted as a retrospective research study in 488 patients with prostate cancer who underwent radical prostatectomy at 7 different hospitals from january 2004 to december 2008 . the patient data were collected for analysis from 7 hospitals with the approval of each hospital 's institutional review board . in all patients undergoing open radical prostatectomy ( orp ) through the retroperitoneal approach , vesicourethral anastomosis ( vua ) was performed with six interrupted sutures . if any leakage was identified during saline injection through the foley catheter , additional sutures were added . in patients undergoing lrp and rarp , a double - armed 3 - 0 monofilament suture was performed in a running fashion for vua , and parachute reconstruction of the bladder neck was done without mucosal eversion . with saline irrigation , the anastomosis was confirmed to be watertight . the retroperitoneal approach was used in all rarp procedures , and lrp was performed through the retroperitoneal or transperitoneal approach . cystography was performed 14 to 21 days after the surgery , and when vua healing was confirmed , the foley catheter was removed . if the amount of fluid discharged through the drain was 30 ml / d , the anastomosis was considered healed and the jackson - pratt drain was removed . during a follow - up visit , uroflowmetry and postvoiding residual urine measurement were performed in patients who complained of obstructive voiding symptoms . if a low maximum urinary flow rate or large amounts of residual urine were identified , diagnostic cystoscopy was performed . if it was challenging to advance the 18-fr cystoscope into the bladder , the patient was diagnosed with bnc . patients who were refractory to sound dilatation were treated by means of a cold knife endoscopic incision . univariate analyses ( student 's t - test , mann - whitney 's u test , fisher 's exact test ) were conducted for variables . multiple logistic regression analyses by dummy variables were used to identify the effects of operative method , neurovascular bundle ( nvb ) preservation , racquet handle repair , mucosal eversion , intraoperative blood loss , and length of time before drain removal on the occurrence of bnc . univariate analyses ( student 's t - test , mann - whitney 's u test , fisher 's exact test ) were conducted for variables . multiple logistic regression analyses by dummy variables were used to identify the effects of operative method , neurovascular bundle ( nvb ) preservation , racquet handle repair , mucosal eversion , intraoperative blood loss , and length of time before drain removal on the occurrence of bnc . no significant differences in baseline characteristics were found between patients treated by orp , lrp , or rarp . the mean follow - up period was 32.4 months ( range , 9 to 62 months ) . bnc occurred in 21 patients ( 4.3% ) in total : 17 patients ( 4.7% ) who underwent orp , 4 patients ( 4.0% ) who underwent lrp , and 0 patients who underwent rarp . when the 21 patients with bnc were compared with the patients without bnc , the length of time before drain removal , which reflects postoperative urine leakage from the vua , was significantly longer in the bnc group ( 12 days ) than in the bnc - negative group ( 6.8 days ) . the multivariate analysis also showed that the length of time before drain removal was the only predictor of bnc ( odds ratio [ or ] , 1.12 ; p=0.001 ) ( table 4 ) . the mean time after rp for bnc occurrence was 8.511.5 months ( range , 1.7 to 48.7 months ) . in the patients who had undergone orp , bnc occurred in 17 of 365 patients ( 4.7% ) . the length of time before drain removal was significantly longer in the bnc group ( 11.9 days ) than in the bnc - negative group ( 6.7 days , p=0.017 ) . the amount of intraoperative blood loss was 2,126 ml in the bnc group and 1,487 ml in the bnc - negative group , but the difference was not statistically significant ( p=0.916 ) . a total of 25 of 348 patients ( 7.2% ) in the bnc - negative group had previously undergone turp , whereas no patients in the bnc group had previously undergone turp ( p=0.618 ) . the operative time in the bnc group was longer than in the bnc - negative group ( 273.6 minutes vs. 250.8 minutes ) , but the difference was not significant ( p=0.308 ) ( table 4 ) . the multivariate analysis indicated that the time before drain removal was the only parameter with effects on bnc occurrence ( or , 1.199 ; p<0.001 ) . in the patients who had undergone lrp or rarp , 4 of 99 patients ( 4.0% ) who underwent lrp and 0 of 24 patients who underwent rarp had bnc . the length of time before drain removal was longer in the bnc group than in the bnc - negative group ( 12.3 days vs. 7.2 days , p=0.027 ) . the operation time of the bnc group was also longer than that in the bnc - negative group ( 462.5 minutes vs. 314.3 minutes , p=0.003 ) . however , according to the results of the multivariate analysis , operation time was the only significant predictor of bnc occurrence ( or , 1.013 ; p=0.017 ) . bnc did not occur in the four patients who had previously undergone turp ( table 5 ) . in the present study , the rate of occurrence of bnc was 4.3% , and there was no significant difference in bnc occurrence between orp and lrp patients . the rate of occurrence of bnc in patients undergoing orp was similar to the results of studies conducted after 2000 , but the rate of occurrence in patients undergoing lrp was higher than in previous studies . the length of time before drain removal , which reflects postoperative urine leakage from the vua site , was a significant risk factor affecting the occurrence of bnc in all patients . urinary leakage through the anastomotic gap delays healing of the suture site , while also causing bnc by inducing the proliferation of myofibroblast cells and wound contracture . several previous studies have also suggested urinary extravasation as a risk factor for bnc occurrence . huang and lepor reported that the degree of urinary extravasation on cystography was not related to bnc , and hanson et al . found that the amount of postoperative drain output is not associated with bnc occurrence . however , we thought that the length of time of urinary leakage may have a greater influence on wound contracture than extravasation degree on cystography or amount of leaked urine . significant intraoperative blood loss results in poor visualization of the surgical field , thus complicating mucosa - to - mucosa anastomosis of vua . the presence of blood is also likely to cause bnc by inducing tissue inflammatory responses at the anastomosis site . . demonstrated that excessive intraoperative blood loss at the vua increased bnc occurrence in a study of 156 patients undergoing rrp . . showed that intraoperative blood loss greater than 1,000 ml was related to increased bnc . in the present study , the amount of intraoperative blood loss was greater than in previous studies , but the rate of bnc occurrence was similar . although patients with bnc seemed to experience greater intraoperative blood loss , the difference was not statistically significant ( p=0.387 ) . we suggest that effort to guarantee direct visualization and additional sutures of the vua , if needed , could improve watertight mucosal to mucosal anastomosis , which could then lessen the effect of excessive intraoperative blood loss . watertight vua is the most important and the most challenging part of radical prostatectomy and is considered to be the most crucial factor predicting postoperative bnc . technical modifications and efforts to ensure proper vua have been introduced . according to a study that analyzed 4,132 cases performed by one surgeon from 1983 to 2007 , the initial bnc occurrence rate was nearly 17% but was dramatically reduced over time as the surgeon became more experienced . the rate was as low as 1% for the final 500 cases . during the initial period , the surgeon had used only four sutures for vua but later began to make five sutures at the 2 , 4 , 6 , 8 , and 10 o'clock positions in order to reduce anastomotic leak . in another study , orvieto et al . showed that the rate of occurrence of bnc was 5.7% before 2000 and was reduced to 0.2% after 2000 among 977 cases when the following modifications were applied : 1 ) reconstruction of the bladder neck to a diameter of 28 french ; 2 ) placement of a posterior ( 6 o'clock ) vesicourethral suture during mild traction before placing this suture into the bladder , allowing inspection and , if necessary , replacement of any of the previously placed sutures ; and 3 ) bladder displacement when tying the vesicourethral sutures , which allows the sutures to be tied under direct visual observation . in those studies , technical modification and efforts to complete proper watertight anastomosis of vua resulted in reduced bnc occurrence . in the present study , an additional suture was added to the routine six sutures at the vua site to reduce urinary leakage under direct visualization . although it was not possible to analyze the effects of accumulation of experience or additional sutures on the reduction of urine leakage or the occurrence of bnc , we did observe that decreased urinary leakage at watertight vua sites was related to reduced bnc occurrence . in previous studies that compared orp with lrp or rarp , the rate of occurrence of bnc from lrp or rarp was reported to be lower than that from orp . the magnification of visualization and excellent movement of the device inside the narrow pelvic cavity in lrp and rarp enables the surgeon to more easily and effectively conduct vua , and the running suture technique for vua could be helpful for decreasing the rate of urinary leakage . reported that postoperative cystography performed 7 days after surgery showed that 90% of patients had a watertight anastomosis , which was significantly higher than the 69.4% of patients who had watertight anastomosis after orp . they also reported that it was possible to remove the catheter at 3 days postoperatively , in contrast with the 14 to 21 days after orp . nadu et al . demonstrated that postoperative cystography at 2 to 4 days after lrp showed that 85% of patients had a watertight anastomosis , and that no anatomic strictures occurred in any patients . the possible explanation for the higher success rate of watertight vua in the rarp and lrp groups is that a running anastomosis under better visualization may result in reduced urine extravasations . in the present study , in the univariate and multivariate analyses of bnc occurrence , the rate of occurrence of bnc was 3.6% in patients who underwent lrp or rarp ( 4.0% in patients who underwent lrp and 0% in patients who underwent rarp ) . bnc occurred in 1 of 18 patients ( 5.6% ) who underwent lrp during the early experience of the surgeon , and the rate of occurrence gradually decreased over time , reaching 2.6% ( 1/37 ) in 2008 ( p=0.572 ) . in the 25 patients who had undergone rarp , despite surgeries performed by inexperienced surgeons , no bnc occurred . according to a study by msezane et al . , among 634 rarp cases , bnc occurred in 7 patients ( 1.1% ) and the operation time was significantly longer in the bnc group than in the non - bnc group ( 283 minutes vs. 225 minutes ) . the authors suggested longer operative time with steep trendelenburg position and pneumoperitoneal pressure of 14 to 20 mmhg could result in local tissue ischemia and subsequent bnc occurrence . in the present study , the patients who underwent lrp and those who underwent rarp showed differences in operation time as well as the length of time before drain removal . the number of lrp and rarp cases in the present study was too small to identify statistical significance for operation time . furthermore , each surgeon performed cystography at different times , so we could not compare the length of time that it took for the anastomosis to become watertight after orp , lrp , and rarp . nvb preservation plays an important role in aiding the recovery of potency and continence after radical prostatectomies and also prevents unnecessary removal of periurethral tissues that carry the blood supply to the vua while dissecting the apex of the prostate for nvb preservation . erickson et al . analyzed 4,132 orp cases performed from 1983 to 2007 and reported that during a mean follow - up period of 44 months , bnc occurred in 110 patients ( 2.5% ) . a lack of nvb preservation or surgery prior to 1992 were significant risk factors for bnc , and the prevalence of erectile dysfunction and incontinence was significantly higher in patients with bnc . in the present study , nvb preservation was achieved in 48.4% of all patients , and bnc occurred in 5.2% of patients without nvb preservation compared with 3% of those who had nvb preservation , but this difference was not statistically significant . first , this study was performed retrospectively ; thus , the rate of occurrence of bnc could be underestimated owing to undetected bnc . second , we used the length of time before drain removal as a marker of urinary leakage , but fluid from the drain also contained blood , lymphatics , and peritoneal fluid , particularly in rarp and lrp . surgeons tried to reduce this inaccuracy by estimating fluid creatinine levels and other blood profiles . in addition , because the number of lrp and rarp operations was small , and the number of patients with bnc was also low , it was difficult to analyze the rates of occurrence of bnc according to operative method . postradical prostatectomy bnc occurred in 4.3% of patients in the present study , and the most significant factor related to bnc occurrence was the length of time before drain removal , which in turn reflects urinary leakage from the anastomosis site . urinary leakage can be prevented by elaborate and meticulous anastomosis of the bladder neck and urethra , thereby reducing the frequency of bnc .

purposeto evaluate the prevalence of bladder neck contracture ( bnc ) and its risk factors in patients undergoing radical prostatectomy in korea.materials and methodswe analyzed data from 488 patients with prostatic cancer who underwent radical prostatectomy performed by seven surgeons in seven hospitals , including 365 open radical prostatectomies ( orps ) , 99 laparoscopic radical prostatectomies ( lrps ) , and 24 robot - assisted laparoscopic radical prostatectomies ( rarps ) . patients with bncs were compared with those without bncs to identify the risk factors for bnc occurrence.resultsoverall , bncs occurred in 21 of 488 patients ( 4.3% ) : 17 patients ( 4.7% ) who underwent orp , 4 patients ( 4% ) who underwent lrp , and no patients who underwent rarp . in the univariate analysis , men with bncs had a longer length of time before drain removal ( 12 days vs. 6.8 days , p<0.001 ) , which reflected urinary leakage through the vesicourethral anastomosis . in the multivariate analysis , the length of time before drain removal was the only predictor of bnc ( odds ratio , 1.12 ; p=0.001 ) . intraoperative blood loss was higher in patients with bnc , but the difference was not statistically significant.conclusionsthe most significant factor related to bnc occurrence after radical prostatectomy in our study was the length of time before drain removal , which reflects urinary leakage from the vesicourethral anastomosis . the proper formation of a watertight anastomosis to decrease urinary leakage may help to reduce the occurrence of bnc .

INTRODUCTION MATERIALS AND METHODS 1. Statistical analysis RESULTS DISCUSSION CONCLUSIONS

PMC5011598

muscle damage and delayed onset muscle soreness ( doms ) may occur at different levels depending on the type of contraction ( concentric or eccentric ) , type of exercise ( plyometric jumps , resistance training , downhill runs , long - term runs , etc . ) , movement speed ( high or low angular velocity ) , time interval between sets , and trainability of the individual ( sedentary , physically active , or trained)1 . for a given muscle power , it is known that there is less recruitment of motor units for eccentric contraction than for concentric contraction2 . this higher strength / activation ratio exerts high stress on the tissues involved , and is considered the main factor in structural damage of muscle fibers . additionally , the connective tissue is stretched , causing greater passive strain on the cytoskeleton . these two factors together are responsible for a higher incidence of muscle damage and consequent reduction in contractile capacity , also known as fatigue3 . it has recently been postulated that muscle damage caused by eccentric or any other physical activity may be minimized by performing the same type of exercise a few weeks after the initial training . the protective mechanism of the repeated bout effect appears to be due to an increase in the recruitment of slow contraction motor units , activation of a large number of motor units ( neural adaptation ) , increased dynamic and passive muscular endurance ( mechanical adaptation ) , longitudinal addition of sarcomeres , adaptation to the inflammatory response , and adaptation to maintain muscle excitation - contraction coupling ( cellular adaptation)5 . however , most studies that investigated the repeated bout effect observed a decrease in the ability of muscle to generate strength and power . few studies have verified the repeated bout effect or the effect of repetition of eccentric exercise on subsequent aerobic performance . previous articles reported that both oxygen consumption kinetics and running economy are negatively affected when a prior eccentric exercise is performed6 , 7 . therefore , the present study aimed to evaluate the influence of repeated bouts of eccentric exercise on subsequent high - intensity aerobic performance . it is believed that severe neuromuscular fatigue develops after eccentric high - intensity exercise . however , an analysis of the literature suggests that the harmful effects may be minimized by performing the same activity several weeks later , which could induce favorable adjustments to offset these effects on subsequent aerobic performance . the subject population was a convenience sample comprised of 7 healthy male volunteers , sedentary for at least 3 months , and with no experience in resistance training . demographic data for the sample were as follows ( mean standard deviation [ sd ] ) : age ( 21.7 3.4 years ) ; body mass ( 67.2 9.9 kg ) ; height ( 171.5 0.0 cm ) ; fat percentage ( 15.4 7.1% ) and maximum oxygen consumption ( 44.1 2.8 mlkgmin ) . each volunteer was informed about the investigational procedures and their implications , and provided signed informed consent to take part in the study . the study was approved by the human research ethics committee of unisalesiano , protocol number : 292/09 . anthropometric data included body mass ( bm ) , height ( h ) , and the percentage of body fat from skinfold measurements8 . subjects performed incremental treadmill testing until volitional exhaustion to determine vo2max and the intensity associated with the achievement of vo2max ( vvo2max ) . this test started at 8 kmh , with increases of 1 kmh at each stage . using a metabolic analyzer ( cortex metalyser 3b , leipzig , germany ) , each subject was encouraged to give maximum effort . the test completion was determined by volitional exhaustion or when one of the following criteria was reached9 : ( 1 ) peak heart rate ( hr ) at least equal to 90% of the age - predicted maximum ; ( 2 ) respiratory exchange ratio ( rer ) greater than 1.1 ; and ( 3 ) increased intensity with stable oxygen consumption . the vo2max was defined by the higher average value of oxygen consumption ( vo2 ) found during the last 30 s of each stage . the subjects returned to the laboratory the following week to determine the time limit to vvo2max ( tlim control ) , which was characterized by treadmill running at vvo2max until voluntary exhaustion ; the hr and lactate concentration ( yellow springs 1500 sport , ohio , usa ) were measured before and after tlim . a plyometric activity was performed in the third week , consisting of 10 sets of 10 depth jumps , with a 1-min interval between each set , in which subjects jumped from a high level ( 0.6 m ) . after landing , the subjects were asked to perform the strongest possible vertical jump , to land on another higher plane placed 1 m in front of the first . after 15 min , they performed another time limit activity ( tlim 1 ) . after 6 weeks of limited physical activity , volunteers returned to the laboratory to perform the same procedures as in the third week , with a plyometric activity followed by treadmill running at vvo2max ( tlim 2 ) . for at least 24 h before the experiments , volunteers were advised to avoid any strenuous physical activity and alcoholic or caffeinated drinks , with a night of proper rest and the last meal at least 2 h prior to the test . to avoid any effects of a circadian cycle , all procedures were performed during the same period and time of day for each subject . statistical analysis was performed using spss version 20.0 ( chicago , il , usa ) prior to any analysis , a shapiro - wilk normality test was applied . before the confirmation of normality , one - way analysis of variance ( anova ) was applied with a post - hoc tukey test to compare data for tlim ( control , 1 , and 2 ) and physiological variables related to performance times ( blood lactate and hr ) . student s t - test was used for paired data for the same physiological variables before and after the plyometric activity . in all analyses , a significance level of p0.05 was adopted . table 1table 1.characteristics of volunteersmeansdage ( years)21.73.4body weight ( kg)67.29.9height ( cm)171.50.0fat percentage ( % ) 15.47.1vo2max ( mlkgmin)44.12.8vvo2max ( kmh)12.71.7vo2max : maximum oxygen uptake ; vvo2max : speed associated with maximum oxygen uptake shows the initial data of the sample , including the cardiorespiratory variables ( vo2max and vvo2max ) related to the initial level of physical fitness , as an inclusion criterion . vo2max : maximum oxygen uptake ; vvo2max : speed associated with maximum oxygen uptake table 2table 2.permanence time at vvo2max in control situations ( tlim control ) and after the first and second session of depth jumps ( tlim 1 and tlim 2)tlim control ( s.)tlim 1 ( s.)tlim 2 ( s.)mean283.4125.2192.7sd47.764.171.9significant difference from tlim control ; p<0.05 shows tlim ( control , 1 , and 2 ) . there was a significant difference between tlim 1 and tlim control , suggesting the deleterious effect of previous eccentric exercise . however , after 6 weeks of limited activity , tlim control and tlim 2 showed no statistically significant differences , demonstrating the repeated bout effect on subsequent aerobic performance . significant difference from tlim control ; p<0.05 tables 3 and 4table 3.mean values and standard deviation ( sd ) of physiological responses ( blood lactate concentrations [lac ] , and maximum heart rate hrmax ) after tlimmeansd[lac ] tlim control ( mmol / l)11.684.43[lac ] tlim 1 ( mmol / l)9.083.75[lac ] tlim 2 ( mmol / l)9.843.38hrmax tlim control ( bpm)198.410.6hrmax tlim 1 ( bpm)192.212.6hrmax tlim 2 ( bpm)198.110.6 show the hr and concentrations of lactate after tlim and following the depth jumps , respectively . table 3 shows no statistically significant differences for the 2 variables after 3 time limits . table 4table 4.mean values and standard deviation ( sd ) of physiological responses ( blood lactate concentrations [lac ] , and maximum heart rate hrmax ) after the depth jump ( dj)meansd[lac ] jd 1 ( mmol / l)7.573.21[lac ] jd 2 ( mmol / l)5.912.36hrmax jd 1 ( bpm)177.87.1hrmax jd 2 ( bpm)178.010.3 shows the same results after depth jumps . the purpose of this research was to investigate the influence of the repeated bout effect of eccentric exercise on subsequent high - intensity aerobic performance . the primary finding was that eccentric exercise decreases aerobic performance after the first time limit , but deleterious effects are minimized after performing the same exercises ( jumps ) a few weeks later . this adaptation is called the repeated bout effect11 , 12 . in the current study , thus , one of the criteria used to determine the cardiorespiratory level of subjects was obtained in an incremental ergometry test . thus , the volunteers were considered sedentary , because of their average values for vo2max ( 44 ml / kg / min ) . according to the present investigation and previous studies that also used depth jump exercises to generate muscle damage , low ( 10 jumps ) and high volumes ( 4550 jumps ) , provided the same protective effect against muscle damage induced by the first exposure . however , a larger volume of eccentric movements induces muscle damage as compared to a lower volume5 , 14 . muscle damage is closely related to decreased muscle contractile capacity ; in physiological terms , the greater the damage , the greater the muscle fatigue15 . the protective mechanism of the repeated bout effect appears to be due to an increase in the recruitment of slow contraction motor units , activation of a large number of motor units ( neural adaptation ) , increased dynamic and passive muscular endurance ( mechanical adjustments ) , longitudinal addition of sarcomeres , adaptation to the inflammatory response , and adaptation to maintain muscle excitation - contraction coupling ( cellular adaptation)5 , 14 . however , there is little information about the repeated bout effect of eccentric exercise on subsequent aerobic performance . the effect of repeated bouts of eccentric exercise on running economy16 , 17 and vo2 kinetics during cycloergometric exercise performance7 was verified by some authors . the effect of eccentric exercise on running economy seems to be intensity dependent ; at moderate intensities ( 5575% of vo2max ) , running economy is not affected , but is only influenced at high intensities ( up to 90% of vo2max ) . this is due to prior fatigue of type ii fibers that are additionally recruited at high aerobic exercise intensities18 . moreover , byrne , twist , and eston19 reported that the performance of eccentric exercise at high intensity prior to aerobic activities can lead to reduction in contractile ability of the quadriceps muscles and reduced muscle force development ratio ( fatigue ) , thus increasing the contact time with the ground during running and decreasing efficiency of the exercise . according to billat et al.20 , the determination of vvo2max is dependent on factors that are not related solely to anaerobic participation in the aerobic component , especially with regard to the slow component of oxygen uptake kinetics . eccentric exercise performed prior to aerobic exercise decreases the recruitment level of type ii fibers , thus decreasing the amplitude of the slow component in determining vo2max , and interfering with the time to exhaustion at vvo2max20 , 21 . thus , the adaptations to the repetition of exercise cited by miyama and nosaka5 can improve the level of muscle fiber recruitment after the performance of the second eccentric exercise , which could explain the statistically similar times between tlim control and tlim 2 . for the limit time exercise at vvo2max ( tlim control , tlim 1 and tlim 2 ) , and the depth jump performed by the volunteers , physiologic stress apparently was no different between the moments before and after six weeks without exercise , since the hr values and lactate concentrations were statistically similar . however , the lactate concentrations after tlim 1 and tlim 2 tended to be lower than in tlim control . this lower concentration can be explained by lower lactate removal capacity after exercises with high - intensity eccentric contractions22 . thus , it can be concluded that the repeated bout effect exerts a positive influence on vvo2max , possibly resulting in less deleterious effects on the permanence time after 6 weeks of limited physical activity . however , methodological limitations of this study suggest the need for further research . due to the great variability of the tlim , a larger sample would increase the statistical power . in addition , the kinetic measurement of oxygen uptake , and even electromyographic evaluation of the muscles involved , could help explain the repeated bout effect on subsequent high - intensity aerobic activity , as well as variables related to muscle damage ; these findings could help explain the damage caused by eccentric high - intensity activities .

[ purpose ] it is believed that eccentric high - intensity exercise can decrease performance in subsequent exercise . however , with repetition , the deleterious effects can be minimized . thus , this study evaluated the influence of repeated bouts of eccentric exercise on subsequent high - intensity aerobic performance . [ subjects and methods ] seven healthy and sedentary male volunteers were recruited . a ) visit 1 : determination of maximum oxygen uptake ( vo2max ) and speed associated with maximum oxygen uptake ( vvo2max ) in incremental treadmill testing ; b ) visit 2 : run to exhaustion at vvo2max ( tlim control ) ; c ) visit 3 : 10 sets of 10 depth jumps , followed by a run to exhaustion at vvo2max ( tlim 1 ) ; d ) visit 4 : after 6 weeks without any physical training , the volunteers carried out the same procedures as on the third visit ( tlim 2 ) . data were analyzed using one - way analysis of variance ( anova ) with the post - hoc tukey test . [ results ] significant differences were found between tlim control and tlim 1 ( 283.4 47.7 s vs. 125.2 64.1 s , respectively ) , these were not different from tlim 2 . [ conclusion ] eccentric exercise showed deleterious effects on subsequent high - intensity aerobic performance . these effects were minimized after the exercise protocol was repeated 6 weeks after the first event .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3347222

the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

recent years have seen the emergence of an important new fundamental theory of brain function . this theory brings information - theoretic , bayesian , neuroscientific , and machine learning approaches into a single framework whose overarching principle is the minimization of surprise ( or , equivalently , the maximization of expectation ) . the most comprehensive such treatment is the free - energy minimization formulation due to karl friston ( see e.g. , friston and stephan , 2007 ; friston , 2010a , b see also fiorillo , 2010 ; thornton , 2010 ) . a recurrent puzzle raised by critics of these models is that biological systems do not seem to avoid surprises . we do not simply seek a dark , unchanging chamber , and stay there . this is the dark - room problem . here , we describe the problem and further unpack the issues to which it speaks . using the same format as the prolog of eddington s space , time , and gravitation ( eddington , 1920 ) we present our discussion as a conversation between : an information theorist ( thornton ) , a physicist ( friston ) , and a philosopher ( clark ) .

Conflict of Interest Statement

PMC4698850

prescription medicines constitute a significant proportion of total healthcare expenditure in many countries of the world.12 report from many european countries shows that prescription medicines account for between 10.4% and 27.6% of the total healthcare expenditure.34 nonrational prescribing by healthcare providers contributes significantly to this relatively high proportion . in many developing countries of the world , a significant proportion of the population pays out of pocket to access healthcare , sometimes leading to catastrophic healthcare expenditure.56 this is at variance with the situation in many developed countries where healthcare insurance covers a large chunk of their population.78 health insurance is a form of healthcare financing where financial resources are pooled together and shared to alleviate the burden of healthcare costs and financial risk of the people . additional objectives of healthcare insurance are judicious use of the resources and ensuring the cost - effectiveness of interventions through the use of affordable drugs . in nigeria , the national health insurance scheme ( nhis ) was introduced in 2005 to cater for the healthcare needs of the populace.910 initially , the coverage of the nhis was limited to workers in the formal sectors but has now been expanded to include people in the informal sector of the economy through community - based schemes.11 the scheme has a uniform coverage for all subscribers who pay only 10% of the total cost of medications as co - payment.12 in many hospitals in nigeria , a dedicated outpatients ' clinic has been created to attend only to patients registered under the nhis . prescription audits have been conducted in different settings in nigeria among patients that paid out of pocket revealing various forms of nonrational use of medicines.1314 a study conducted in the north - eastern part of nigeria looked at the prescribing practice in an outpatients ' clinic attending only to patients registered under the nhis while another nigerian study compared the prescribing practices in a military hospital before and postintroduction of the nhis.1516 to the knowledge of the authors , there is no study comparing the prescribing pattern in the general outpatient department of the hospital ( where out of pocket this type of comparison will enable us assess the impact of healthcare insurance on prescribing practices in this facility and may serve as a template for future interventions . the main objective of this study was to compare concurrently the prescribing practices in the general outpatients ' clinic ( noninsured patients ) and the nhis clinic ( patients with insurance coverage ) . this was a cross - sectional study conducted in the general outpatients ' and the nhis clinics of the ekiti state university teaching hospital , ado - ekiti , south - western nigeria . the medical records of patients , who attended these two clinics between the 1 of march and 30 june 2014 were retrieved and used for the study . the hospital is one of the two tertiary healthcare facilities in ekiti state that provide medical care for its populace and that of the neighboring states . the hospital is well staffed with consultants in various fields of family medicine , internal medicine , surgery , obstetrics and gynecology , psychiatry , community medicine , and pediatrics . the general outpatients ' department ( also known as family medicine ) is well staffed with its components of nursing staff , house officers , medical officers , registrars , and consultants . the general outpatients ' clinic is usually the first point of contact for patients brought directly from home or referred from other healthcare facilities . patients aged 18 years and above are usually attended to at the general outpatients ' clinic while the nhis clinic offer services to both adult and children because of its peculiar nature . the attendance registers of both clinics were retrieved , and the medical records of 50 patients were selected monthly for the period of the study using a regular interval ratio . the information retrieved from the case notes included bio - demographic data , working diagnoses , a list of prescribed drugs , and their routes of administration . the following drug use indicators were assessed using the who guidelines on investigation of drug use in health care facilities:17 average number of drugs per prescription , percentage of encounters with antibiotics , percentage of drugs prescribed by generic name , percentage of encounters with injections , and percentage of drugs prescribed from essential drug list . the national essential drug list of nigeria ( 5 edition ) was used to assess prescription of nationally relevant essential medicines . data collected were analyzed using statistical package for social sciences version 17 software ( ibm corporation , armonk , ny , usa ) . comparison of means and frequencies was done using student 's t - test while chi - square was used to determine the level of significance of groups of categorical variables with p < 0.05 considered as significant . ethical approval was obtained from the hospital research ethics committee before the starting the study . the hospital is one of the two tertiary healthcare facilities in ekiti state that provide medical care for its populace and that of the neighboring states . the hospital is well staffed with consultants in various fields of family medicine , internal medicine , surgery , obstetrics and gynecology , psychiatry , community medicine , and pediatrics . the general outpatients ' department ( also known as family medicine ) is well staffed with its components of nursing staff , house officers , medical officers , registrars , and consultants . the general outpatients ' clinic is usually the first point of contact for patients brought directly from home or referred from other healthcare facilities . patients aged 18 years and above are usually attended to at the general outpatients ' clinic while the nhis clinic offer services to both adult and children because of its peculiar nature . the attendance registers of both clinics were retrieved , and the medical records of 50 patients were selected monthly for the period of the study using a regular interval ratio . the information retrieved from the case notes included bio - demographic data , working diagnoses , a list of prescribed drugs , and their routes of administration . the following drug use indicators were assessed using the who guidelines on investigation of drug use in health care facilities:17 average number of drugs per prescription , percentage of encounters with antibiotics , percentage of drugs prescribed by generic name , percentage of encounters with injections , and percentage of drugs prescribed from essential drug list . the national essential drug list of nigeria ( 5 edition ) was used to assess prescription of nationally relevant essential medicines . data collected were analyzed using statistical package for social sciences version 17 software ( ibm corporation , armonk , ny , usa ) . comparison of means and frequencies was done using student 's t - test while chi - square was used to determine the level of significance of groups of categorical variables with p < 0.05 considered as significant . ethical approval was obtained from the hospital research ethics committee before the starting the study . medical records ( including copies of prescriptions ) of 217 and 237 patients were selected from the general outpatients ' and nhis clinics , respectively . malaria was the most frequently diagnosed disease in the two clinics followed by diseases affecting the respiratory , cardiovascular , and gastrointestinal systems [ figure 2 ] . the mean age of patients attending the general outpatients ' clinic was 45.0 17.1 years while that of the nhis clinic was 32.9 16.1 years , a statistically significant difference ( p < 0.0001 ) . the average number of diagnoses was 1.57 0.6 and 1.59 0.75 in patients attending the nhis and general outpatients ' clinics , respectively ( p = 0.75 ) . the average number of prescribed drugs for patients attending the general outpatients ' clinic was 3.9 2.0 while that from the nhis clinic was 4.1 1.6 ( p = 0.24 ) . distribution according to sex comparison of diagnosed conditions about a third of patients from both clinics had more than four medications prescribed . figure 3 shows the grading of a number of prescribed drugs according to the number of prescribed drugs . the classes of drugs prescribed for patients from the two clinics are shown in figure 4 . prescribing by generic names was done in 48.2 23.8% and 45.8 22.9% of prescriptions from the general outpatients ' and nhis clinic , respectively . a comparison of the difference between the two means did not show any statistically significant difference ( p = 0.27 ) . percentage of encounters with antibiotics was 49.4% and 33.6% of patients who attended the nhis and general outpatients ' clinics , respectively . percentage of encounters with injections was 9.3% and 7.4% of patients from the nhis and general outpatients ' clinics , respectively . classification according to number of prescribed drugs classes of prescribed drugs prescribing from the nigerian national essential drug list was done in 70.4% and 71.4% of all cases from the general outpatients ' and nhis clinics , respectively . the lower mean age of patients from the nhis clinic is due to the fact that both adult and pediatric patients are attended to in the nhis clinic whereas only adult patients are seen in the general outpatients ' clinic . the mean number of prescribed drugs found in the general outpatients ' and nhis clinic 3.9 and 4.1 , respectively . a comparison of drug prescribing practices during a pre- and post - insurance era in a nigerian hospital found the mean prescribed drugs to be 3.0 and 2.6 , respectively.16 okoro and shekariin a study conducted in the nhis clinic of a tertiary healthcare facility in the north - east nigeria recorded a mean number of prescribed drugs to be 3.4.15 results from similar general prescription audits conducted in nigeria showed average number of prescribed drugs between 3.0 and 4.0.131819 the difference between the observed values may be due to the status of healthcare facilities where these studies were carried out ; some of the studies were conducted in secondary level healthcare facilities . in a chinese study comparing drug prescribing practices among two groups of patients ( with and without community health insurance ) , the mean number of prescribed drugs observed was 4.6 and 3.1 , respectively.20 polypharmacy has several definitions , but that which defines it as the prescription of more than four medications is the most commonly used.2122 polypharmacy has serious implications on the health and financial state of patients as it may lead to the occurrence of adverse drug reactions and increased healthcare costs.2324 the proportion of patients who had more than four drugs prescribed in this study was 32.4% and 34.1% in the nhis and gopd clinics , respectively . this is more than 16.7% and 15.6% observed during the pre- and post - insurance era in a nigerian military hospital.16 our hospital evolved from a secondary health care center to tertiary care center less than a decade ago as most doctors are yet to adapt to the new modalities of patients ' care . more so , the same group of doctors from the general outpatients ' department also attends to patients in both clinics . nonrational prescription and use of antibiotics have been associated with increased antimicrobial resistance and healthcare expenditure.2526 the likelihood of having an antibiotic prescribed was higher among patients who were seen at the nhis clinic ( 49.4% vs. 33.6% ) . okoro and shekari in a study conducted at a health insurance clinic in the north - eastern part of nigeria found that 56.2% of all encounters had antibiotics prescribed . the percentage of encounters with antibiotics was comparable to other studies conducted in regular outpatients ' clinics in other parts of nigeria ; 34.4% and 35% in studies conducted in the kano , north - west nigeria and lagos , south - west , respectively.1619 sun et al . also found a higher number of encounters with prescribed antibiotics ( 72.4% ) in patients who had community health insurance when compared to those without ( 59.3%).20 this relatively high rate of antibiotic prescription in patients from the nhis clinic may be due to patients ' demand for the drugs and the fact that only 10% of the medication fees are paid as co - payment . the low rate of injections among the two groups of patients is in keeping with recent trends ; the percentage of encounters with injection use in an earlier cited nigerian study fell from 23.9% ( 2009 ) to 8.9% ( 2012).16 the relatively high prevalence level of diseases such as hepatitis b and hiv infection among the populace has re - enforced the need for safe injection practices among healthcare practitioners in nigeria . prescribing by generic name has been identified as a way of reducing health care costs and makes it more cost - effective.2728 prescribing by generic name was done in 48.2% and 45.8% of prescriptions from the general outpatients ' and nhis clinic respectively ; slightly higher than 42.7% and 43.8% found in earlier nigerian studies.1819 however , our values are lower than the 51.5% found in an nhis clinic in northern nigeria and 96.1% recorded in a study conducted in china.1529 the concept of essential medicines was introduced to help streamline the number of drugs deployed within the healthcare system in order to improve rational prescribing and reducing healthcare costs . in this study , 70.4% and 71.4% of all prescribed medications in the general outpatients ' clinic and this is close to 67.1% reported in a health insurance clinic of a teaching hospital in the north - east nigeria.15 the prescribing practice of physicians is affected to some extent by the health insurance status of their patients as has been shown in this study . there was a trend to having more medicines prescribed and more encounters with antibiotics among patients enrolled with the health insurance scheme . there is a need for additional training on the rational use of medicines for all prescribers and especially those attending to this category of patients . the result of this study has clearly shown a trend toward more polypharmacy in patients registered under the nhis in nigeria . this serves as an early warning indicator of nonrational prescribing which could negate the expected economic benefits of the scheme and expose patients to the adverse effects of polypharmacy . early interventions by the health authorities to correct this trend through training and retraining of prescribers , also , prescribing by generic name for patients under the health insurance scheme should be encouraged as this will help in achieving the economic objectives of the nhis . this study may have issues with the accuracy of data since we used the past medical records of patients . the result of this study has clearly shown a trend toward more polypharmacy in patients registered under the nhis in nigeria . this serves as an early warning indicator of nonrational prescribing which could negate the expected economic benefits of the scheme and expose patients to the adverse effects of polypharmacy . early interventions by the health authorities to correct this trend through training and retraining of prescribers , also , prescribing by generic name for patients under the health insurance scheme should be encouraged as this will help in achieving the economic objectives of the nhis . this study may have issues with the accuracy of data since we used the past medical records of patients .

introduction : prescription medicines constitute a significant proportion of total healthcare expenditure in many countries of the world . nonrational prescribing by healthcare providers contributes significantly to this relatively high proportion . in many developing countries of the world , a significant proportion of the population pays out of pocket to access healthcare , sometimes leading to catastrophic healthcare expenditure . healthcare insurance is a form of healthcare financing that promotes judicious use of the resources and ensuring the cost - effectiveness of interventions through the use of affordable drugs . the main objective of this study was to compare concurrently the prescribing practices in the general outpatients ' clinic ( noninsured patients ) and the national health insurance scheme ( nhis ) clinic ( patients with insurance coverage).materials and methods : a cross - sectional study was conducted in the general outpatients ' and the nhis clinics of the ekiti state university teaching hospital , ado - ekiti , south - western nigeria . the medical records of patients , who attended these two clinics between the 1st march and 30th june 2014 were retrieved and used for the study.results:the average number of prescribed drugs for patients attending the general outpatients ' clinic was 3.9 2.0 while that from the nhis clinic was 4.1 1.6 ( p = 0.24 ) . prescribing by generic names was done in 48.2 23.8% and 45.8 22.9% of prescriptions from the general outpatients ' and nhis clinic , respectively . percentage of encounters with antibiotics was 49.4% and 33.6% of patients who attended the nhis and general outpatients ' clinics , respectively.conclusion:there was a trend to having more medicines prescribed and more encounters with antibiotics among patients enrolled under the health insurance scheme .

INTRODUCTION MATERIALS AND METHODS Study setting Selection of cases Statistical analysis Ethical consideration RESULTS DISCUSSION CONCLUSION Implications for clinical practice and policy Study limitations Financial support and sponsorship Conflicts of interest

PMC4531568

local reaction to allergen - specific immunotherapy ( sit ) usually appears within 30 minutes . however , allergens can induce not only an immediate hypersensitivity reaction , but also allergic inflammation or other long - term sequelae that cause episodic symptoms precipitated by nonspecific stimuli . rotne reported two cases that developed local or systemic reactions in connection with physical exercise , and garcia , et al . showed two cases of localized cold urticaria . here , a case that developed local cholinergic urticaria at the sit site , more than ten years after the completion of five years of sit , is reported . a 28-year - old man had suffered from asthma and allergic rhinitis for more than 20 years . when he was an 11-year - old boy , he sought therapy for his diseases , at chonnam national university hospital , and was treated with house dust mite - sit for five years , without adverse reactions ( bencard , brantford , uk ) . his allergic diseases went into remission for about two years after the last injection of sit . he subsequently used bronchodilators as needed , but in december 2002 , revisited the hospital due to dyspnea [ forced expiratory volume in one second ( fev1 ) : 64% of predicted value ] . at this time , he complained of frequently occurring itching eruptions , which appeared on his left upper arm at the former injection sites when he exercised . one month after a short course of anti - asthma therapy and withholding all medications for more than one week , he underwent a six - minute free - running test . one minute after the test , the fev1 had decreased to 1.83 l ( 44% of predicted value ) from the baseline ( 3.0 l , 72% ) , but urticaria was not observed . the patient said that urticaria occurred only when he exercised for a period much longer than the six - minute test . blood sample analyses and skin tests before and 20 minutes after the exercise , and skin biopsies after the exercise were performed . a methacholine intradermal test erythema , swelling , and pruritus occurred at the sit site 30 minutes after the exercise test started , and lasted for one hour after the completion of the test ( figure 1 ) . the biopsy specimens showed no apparent difference between the lesion and the normal sites in the distribution of inflammatory cells and in mast cell degranulation under light and electron microscopic examinations . however , the morphine , but not the histamine , skin test responses were increased after the exercise ( table 1 ) . the control subjects ( n=5 , 20.60.9 year - old men ) showed no significant difference in the skin test responses before and after exercise . rotne reported cases with exercise - induced urticaria at the sit site two to three weeks after the last allergen injection . these cases developed urticaria during sit . in our case , however , more than ten years had passed since the patient completed his five - year sit schedule . it is well known that cholinergic urticaria appears associated with exercise , hot showers , and sweating . the local form of cholinergic urticaria and its association with histamine release has been demonstrated . even though about one - third of patients with cholinergic urticaria show a positive response to a methacholine skin test ( immediate hypersensitivity to cholinergic mediators ) , the exact pathophysiologic mechanisms have not been elucidated . a possible explanation for the complicated phenomena comprised of local urticaria or generalized allergic reactions to stimuli , such as cold , heat , and exercise , allergens can induce allergic inflammation , or other long - term sequelae , which make lesion sites susceptible to various stimuli responses . it has been shown that atopic subjects have an increased releasability of mast cells in response to nonimmunologic stimulation , such as morphine . although the case in the present study showed no evidence of mast cell degranulation on electron microscopic examinations , he showed a greater skin response to morphine than a normal control . moreover , his skin response to morphine , but not to histamine , was increased after the exercise challenge . it is unclear how exercise primes an increase in the histamine release from mast cells in response to morphine . however , late recall urticaria has been observed after injection of allergen in the arm that had not received allergen administration during the sit . in addition , there have been reports of strong local urticaria at the sites of methacholine testing , but only following an exercise test in some patients with cholinergic urticaria , and a marked increase in the wheal response to compound 48/80 ( a cutaneous mast cell degranulating agent ) , but not to histamine , after a combination of food and exercise challenges in patients with food - associated exercise - induced urticaria - angioedema . these observations corroborate the results of our study in several regards . taken together , nonspecific stimuli , such as cold , heat , exercise , and morphine , may precipitate the occurrence of allergic symptoms , including recall urticaria at the sit site , even after a long time period , and especially during the exacerbation period of atopic diseases . allergen - induced long - term sequelae , including an increased releasability of mast cells , may make the lesion sites more susceptible to various stimuli responses .

local reaction to allergen - specific immunotherapy ( sit ) usually appears within 30 minutes , but cases with exercise - induced urticaria at the sit site 23 weeks after the last allergen injection have been reported . a 28-year - old man was treated with house dust mite - sit for 5 years , due to asthma when he was an 11-year - old boy . on a treadmill exercise test for 50 minutes , erythema , swelling , and pruritus occurred at the sit site , which lasted for one hour . there was no evidence of complement activation , and the skin biopsy specimens showed no apparent difference between the lesion and normal sites in the distribution of inflammatory cells and in mast cell degranulation . however , the morphine , but not the histamine , skin test responses were increased after the exercise . there must be a remaining long - term sequela of the sit , including an increased releasability of mast cells , even after more than 10 years .

INTRODUCTION CASE REPORT DISCUSSION

PMC2991639

most exercise protocols designed to induce fat loss have focused on regular steady state exercise such as walking and jogging at a moderate intensity . disappointingly , these kinds of protocols have led to negligible weight loss [ 1 , 2 ] . thus , exercise protocols that can be carried out by overweight , inactive individuals that more effectively reduce body fat are required . accumulating evidence suggests that high - intensity intermittent exercise ( hiie ) has the potential to be an economical and effective exercise protocol for reducing fat of overweight individuals . hiie protocols have varied considerably but typically involve repeated brief sprinting at an all - out intensity immediately followed by low intensity exercise or rest . the length of both the sprint and recovery periods has varied from 6 s to 4 min . most commonly the sprints are performed on a stationary cycle ergometer at an intensity in excess of 90% of maximal oxygen uptake ( vo2max ) . subjects studied have included adolescents , young men and women , older individuals , and a number of patient groups [ 312 ] . the most utilized protocol in past research has been the wingate test which consists of 30 s of all - out sprint with a hard resistance . subjects typically perform the wingate test 4 to 6 times separated by 4 min of recovery . this protocol amounts to 3 to 4 min of exercise per session with each session being typically performed 3 times a week for 2 to 6 weeks . insight into the skeletal muscle adaptation to hiie has mainly been achieved using this type of exercise ; however , as this protocol is extremely hard , subjects have to be highly motivated to tolerate the accompanying discomfiture . thus , the wingate protocol is likely to be unsuitable for most overweight , sedentary individuals interested in losing fat . for example , we have used an 8-second cycle sprint followed by 12 s of low intensity cycling for a period of 20 min . thus , instead of 4 to 6 sprints per session , as used in wingate protocol studies , subjects using the 8 s/12 s protocol sprint 60 times at a lower exercise intensity . total sprint time is 8 min with 12 min of low intensity cycling . for the hiie wingate protocols , total exercise time is typically between 3 to 4 min of total exercise per session . thus , one of the characteristics of hiie is that it involves markedly lower training volume making it a time - efficient strategy to accrue adaptations and possible health benefits compared to traditional aerobic exercise programs . this review summarises results of research examining the effect of different forms of hiie on fitness , insulin resistance , skeletal muscle , subcutaneous , and abdominal fat loss . acute responses to hiie that have been identified include heart rate , hormones , venous blood glucose , and lactate levels , autonomic , and metabolic reactivity . heart rate response is dependent on the nature of the hiie protocol but typically is significantly elevated during exercise and declines during the period between sprint and recovery . for example , weinstein et al . , using the wingate protocol , recorded peak heart rates of 170 bpm immediately after a 30-second maximal all - out cycle sprint . heart rate response to the 8 s/12 s protocol typically averages around 150 bpm after 5 min of hiie which increases to 170 bpm after 15 min of hiie . in this protocol , there is typically a small heart rate decrease of between 58 bpm during each 12-second recovery period . a similar pattern of heart rate response was found for an hiie protocol consisting of ten 6-second sprints interspersed with 30 s recovery . heart rate increased to 142 bpm after the first sprint and then increased to 173 bpm following sprint ten . hormones that have been shown to increase during hiie include catecholamines , cortisol , and growth hormones . catecholamine response has been shown to be significantly elevated after wingate sprints for both men and women [ 17 , 18 ] . catecholamine response to hiie protocols that are less intensive than the wingate protocol have also been shown to be elevated . measured catecholamine response to long ( 24 s/36 s recovery ) and short ( 6 s/9 s recovery ) bout intermittent treadmill exercise and found that norepinephrine was significantly elevated postexercise . found significantly elevated epinephrine and norepinephrine levels after 20 min of hiie cycle exercise ( 8 s/12 s and 12 s/24 s protocols ) in trained and untrained young women . bracken et al . examined the catecholamine response of 12 males who completed ten 6-second cycle ergometer sprints with a 30-second recovery between each sprint . from baseline , plasma epinephrine increased 6.3-fold , whereas norepinephrine increased 14.5-fold at the end of sprinting ( figure 1 ) . the significant catecholamine response to hiie is in contrast to moderate , steady state aerobic exercise that results in small increases in epinephrine and norepinephrine . the hiie catecholamine response is an important feature of this type of exercise as catecholamines , especially epinephrine , have been shown to drive lipolysis and are largely responsible for fat release from both subcutaneous and intramuscular fat stores . significantly , more -adrenergic receptors have been found in abdominal compared to subcutaneous fat suggesting that hiie may have the potential to lower abdominal fat stores . interestingly , in endurance trained women , -adrenergic sensitivity was enhanced , whereas the sensitivity of the anti - lipolytic 2 receptors was diminished . however , no data are available concerning hiie training effects on or 2 adrenergic receptor sensitivity of human adipocytes . nevill et al . examined the growth hormone ( gh ) response to treadmill sprinting in female and male athletes and showed that there was a marked gh response to only 30 s of maximal exercise and the response was similar for men and women but greater for sprint compared to endurance trained athletes . gh concentration was still ten times higher than baseline levels after 1 hour of recovery . venous blood cortisol levels have also been shown to significantly increase after repeated 100 m run sprints in trained males , after five 15-second wingate tests , and during and after brief , all - out sprint exercise in type 1 diabetic individuals . venous blood lactate response to the wingate test protocols has typically ranged from 6 to 13 mmoll . lactate levels after the wingate test are typically higher in trained anaerobic athletes and have been shown to be similar and lower for trained women compared to trained men . trapp et al . showed that 8 s/12 s hiie for 20 min increased plasma lactate levels between 2 and 4 mmoll after 5 min of hiie for both trained cyclists and untrained females . lactate rose to between 4 and 5 mmollafter 15 min of hiie . during a 12 s/24 s hiie condition lactate levels of the untrained were similar but were significantly higher for the trained female cyclists ( between 7 and 8 mmollafter 15 min ) . despite increasing lactate levels during hiie exercise , it appears that free fatty acid transport is also increased . for example , a 20-minute bout of 8 s/12 s hiie produced increased levels of glycerol indicating increased release of fatty acids which peaked for untrained women after 20 min and after 10 min of hiie for trained women . hiie appears to result in significant increases in blood glucose that are still elevated 5 min and 30 min postexercise . hiie appears to have a more dramatic effect on blood glucose levels of exercising type 1 diabetic individuals . bussau et al . examined the ability of one 10-second maximal sprint to prevent the risk of hypoglycemia typically experienced after moderate aerobic exercise in type i diabetics . twenty minutes of moderate - intensity aerobic exercise resulted in a significant fall in glycemia . however , one 10-second sprint at the end of the 20-minute aerobic exercise bout opposed a further fall in glycemia for 120 minutes , whereas in the absence of a sprint , glycemia decreased further after exercise . the stabilization of glycemia in the sprint trials was associated with elevated levels of catecholamines , growth hormone , and cortisol . in contrast , these hormones remained at near baseline levels after the 20 min of aerobic exercise . thus , one 10-second all out sprint significantly increased glucose , catecholamines , growth hormone , and cortisol of type 1 diabetic individuals for 5 min after hiie . authors suggest that the addition of one 10-second sprint after moderate intensity aerobic exercise can reduce hypoglycemia risk in physically active individuals who possess type 1 diabetes . parasympathetic activation was found to be significantly impaired in a 10-minute recovery period after repeated sprint exercise and a 1-hour recovery period in trained subjects . buchhiet et al . have suggested that parasympathetic or vagal impairment is caused by the heightened sympathetic activity that occurs during hiie exercise and the persistent elevation of adrenergic factors and local metabolites during recovery ( e.g. , epinephrine , norepinephrine , and venous blood lactate ) . with regard to metabolic response , hiie initially results in decreased adenosine triphosphate ( atp ) and phosphocreatine ( pcr ) stores followed by decreased glycogen stores through anaerobic glycolysis . gaitanos et al . have suggested that towards the end of an hiie session , which consists of numerous repeat sprints ( e.g. , ten 6-second bouts of maximal sprinting ) , an inhibition of anaerobic glycogenolysis may occur . these authors have further suggested that at the end of the hiie bout , atp resynthesis may be mainly derived from pcr degradation and intramuscular triacylglycerol stores . however , this pattern of fuel utilization during hiie has not been demonstrated in humans . after hard , all - out hiie exercise , complete phosphagen recovery may take 3 - 4 min but complete restoration of ph and lactate to pre - exercise levels may take hours . the recovery of the exercising muscle after hiie to its pre - exercise state is undetermined . after a hard bout of aerobic exercise , recovery has typically been found to be biphasic with an initial rapid phase of recovery lasting 10 s to a few minutes followed by a slower recovery phase lasting from a few minutes to hours . during recovery , oxygen consumption is elevated to help restore metabolic processes to baseline conditions . the postexercise oxygen uptake in excess of epoc during the slow recovery period has been associated with the removal of lactate and h , increased pulmonary and cardiac function , elevated body temperature , catecholamine effects , and glycogen resynthesis . although epoc does not appear to have been assessed after hiie , it is enhanced after split aerobic exercise sessions . for example , magnitude of epoc was significantly greater when 30-minute and 50-minute aerobic exercise sessions were divided into two parts . also an exponential relationship between aerobic exercise intensity and epoc magnitude has been demonstrated . with regard to hiie , it is feasible that the significant increase in catecholamines ( figure 1 ) and the accompanying glycogen depletion described earlier could induce significant epoc . however , aerobic exercise protocols resulting in prolonged epoc have shown that the epoc comprises only 615% of the net total exercise oxygen cost . laforgia et al . have concluded that the major impact of exercise on body mass occurs via the energy expenditure accrued during actual exercise . whether hiie - induced epoc is one of the mechanisms whereby this unique form of exercise results in fat loss needs to be determined by future research . in summary , acute responses to a bout of hiie include significant increases in heart rate , catecholamines , cortisol , growth hormone , plasma lactate and glucose levels , glycerol , and a significant decrease in parasympathetic reactivation after hiie , and depletion of atp , pcr , and glycogen stores . chronic responses to hiie training include increased aerobic and anaerobic fitness , skeletal muscle adaptations , and decreased fasting insulin and insulin resistance ( table 1 ) . surprisingly , aerobic fitness has been shown to significantly increase following minimal bouts of hiie training . for example , whyte et al . carried out a 2-week hiie intervention with three hiie sessions per week consisting of 4 to 6 wingate tests with 4 min of recovery . increases in vo2max of 13% for an hiie program also lasting 2 weeks have been documented . hiie protocols lasting 6 to 8 weeks have produced increases in vo2max of 4% and 68% . longer wingate - type hiie programs lasting 12 to 24 weeks have recorded large increases in vo2max of 41% and 46% in type 2 diabetic and older cardiac rehabilitation patients . the less intense protocols ( 8 s/12 s ) coupled with longer duration conducted over 15 and 12 weeks resulted in a 24% and 18% increase in vo2max . collectively , these results indicate that participation in differing forms of hiie by healthy young adults and older patients , lasting from 2 to 15 weeks , results in significant increases in vo2max from between 4% to 46% ( table 1 ) . mechanisms underlying the aerobic fitness response to hiie are unclear although a major contributor is phosphocreatine degradation during repeated hiie . using thigh cuff occlusion to prevent pcr resynthesis during recovery , trump et al . showed that pcr contributed approximately 15% of the total atp provision during a third 30-second bout of maximal isokinetic cycling . muscle glycogenolysis made a minor contribution to atp provision during the third 30-second bout indicating that aerobic metabolism was the major source of atp during repeated sprinting . similarly , putman et al . showed that repeated bouts of hiie resulted in a progressive increase in atp generation so that by the third out of five 30-second wingate bouts , the majority of atp was generated oxidatively . other mechanisms underlying the hiie increase in aerobic power are undetermined but may involve increased stroke volume induced by enhanced cardiac contractility , enhanced mitochondrial oxidative capacity , and increased skeletal muscle diffusive capacity . there is also evidence indicating that muscle aerobic capacity is increased following hiie due to increases in pgc-1a mediated transcription occurring via ampk activation . have suggested that these marked oxidative adaptations in the exercising muscle are likely to underlie the significant increases in peak and maximal oxygen uptake documented after regular hiie . anaerobic capacity response to hiie has typically been assessed by measuring blood lactate levels to a standardized exercise load or anaerobic performance on a wingate test . a number of studies have demonstrated that hiie lasting from 2 to 15 weeks results in significant increases in anaerobic capacity from between 5% to 28% . for example , tabata et al . used a 20 s/10 s protocol and found that in previously untrained males , anaerobic capacity , measured by maximal accumulated o2 deficit , was increased by 28% . whyte et al . carried out a 2-week hiie intervention and found that previously untrained males increased their anaerobic capacity by 8% , whereas burgomaster et al . found that wingate test performance was increased by 5.4% after two weeks of hiie . a number of studies have taken muscle biopsies after wingate test performance in order to examine skeletal muscle adaptations . in a series of studies , [ 13 , 52 ] have consistently found increased maximal activity and protein content of mitochondrial enzymes such as citrate synthase and cytochrome oxidase after hiie training . for example , talanian et al . carried out an hiie intervention that consisted of 2 weeks of hiie exercise performed seven times with each session consisting of ten 4-minute bouts at 90% vo2max separated by 2-minute resting intervals . vo2max was increased by 13% and plasma epinephrine and heart rate were lower during the final 30 min of a 60-minute cycling steady state exercise trial at 60% of pretraining vo2max . exercise whole body fat oxidation also increased by 36% , and net glycogen use was reduced during the steady state cycling trial . thus , seven sessions of hiie , over two weeks , induced marked increases in whole body and skeletal muscle capacity for fatty acid oxidation during exercise in moderately active women . other studies have found similar results with studies reporting large increases in citrate synthase maximal activity after 2 weeks and 6 weeks of hiie . similarly , hydroxyacyl coenzyme a dehydrogenase activity , which catalyzes a key rate - limiting enzyme step in fat oxidation , also significantly increased after hiie training . increases in oxidative muscle metabolism ( e.g. , hexokinase and citrate synthase activity ) after 7 weeks of hiie training with type 1 diabetic individuals have also been documented . collectively , markers of muscle oxidative capacity have been shown to significantly increase after six sessions of hiie lasting as little as 2 weeks . glycolytic enzyme content and activity has also been shown to increase after exposure to hiie . have shown that 16 weeks of hiie significantly increased phosphofructokinase levels which is a key rate limiting enzyme in glycolysis , whereas macdougall et al . also showed increases in phosphofructokinase with a wingate - type protocol carried out for 7 weeks . in summary , wingate test hiie protocols of between one and seven weeks have demonstrated marked increases in skeletal muscle capacity for fatty acid oxidation and glycolytic enzyme content and activity . the effect of hiie training on fasting insulin and insulin resistance is shown in table 1 . as can be seen all studies that have assessed insulin response to hiie have recorded significant improvements of between 23% and 58% increase in insulin sensitivity . insulin sensitivity has typically been assessed by measuring fasting insulin , homa - ir , and by glucose tolerance tests . in healthy , nondiabetic individuals , the improvement in fasting insulin and insulin resistance has ranged from 23% to 33% [ 37 , 39 , 42 , 45 ] , whereas in individuals possessing type 2 diabetes , two studies have reported greater insulin sensitivity improvements of 46% and 58% . used a glucose tolerance test to assess insulin sensitivity after an intervention that consisted of 2 weeks of hiie performed three times per week with each session consisting of four to six 30-second all out sprints separated by resting interval of between 2 to 4 min . glucose ( 12% ) and insulin areas under the curve ( 37% ) were significantly attenuated with a sustained improved insulin action until at least three days after the last exercise session . this was achieved without a change in body weight and with a total exercise energy increase of only 500 kcal for the two weeks . authors suggest that the small increase in energy expenditure contrasts to the 20003000 kcal per week experienced during a typical aerobic training program . the mechanism(s ) underlying these large increases in insulin sensitivity reported in these studies is likely due to the skeletal muscle adaptations previously discussed involving marked increases in skeletal muscle capacity for fatty acid oxidation and glycolytic enzyme content . in summary , chronic exposure to hiie results in significant increases in aerobic and anaerobic fitness , increased skeletal muscle capacity for fatty acid oxidation and glycolytic enzyme content , and increased insulin sensitivity . the majority of research examining hiie has focused on short - term ( 2 to 6 weeks ) programs on skeletal muscle adaptation . however , some studies have utilized longer programs to determine the effect of hiie on subcutaneous and abdominal fat loss . compared hiie and steady state aerobic exercise and found that after 24 weeks subjects in the hiie group lost more subcutaneous fat , as measured by skin folds , compared to a steady state exercise group when exercise volume was taken into account ( table 1 ) . more recently , trapp et al . conducted an hiie program for 15 weeks with three weekly 20-minute hiie sessions in young women . hiie consisted of an 8-second sprint followed by 12 s of low intensity cycling . another group of women carried out an aerobic cycling protocol that consisted of steady state cycling at 60% vo2max for 40 min . results showed that women in the hiie group lost significantly more subcutaneous fat ( 2.5 kg ) than those in the steady state aerobic exercise program ( figure 2(a ) ) . dunn used a similar hiie protocol together with a fish oil supplementation and a mediterranean diet for 12 weeks . in 15 overweight young women , the combination of hiie , diet , and fish oil resulted in a 2.6 kg reduction in subcutaneous fat ( 8% ) and a 36% increase in insulin sensitivity ( table 1 ) . the amount of subcutaneous fat lost was similar to that observed in the trapp et al . study suggesting that shorter hiie interventions ( 12 versus 15 weeks ) are also effective for reducing subcutaneous fat . with regard to abdominal fat , trapp et al . found that 15 weeks of hiie led to significantly reduced abdominal fat ( .15 kg ) in untrained young women ( figure 2(b ) ) , whereas dunn found that 12 weeks of hiie led to a.12 kg decrease in abdominal fat . as women in these studies possessed relatively low abdominal fat levels , it is possible that the greater abdominal fat of men may demonstrate greater reductions after hiie . for example , boudou et al . , in a study involving older type 2 diabetic males , found that after 8 weeks of hiie no change in body mass occurred ; however , abdominal adiposity was decreased by 44% ( table 1 ) . found a 48% reduction in visceral fat , measured by mri , compared to an 18% decrease in subcutaneous fat following an exercise regimen consisting of steady state exercise two days per week and hiie one day a week for 8 weeks in type 2 diabetic men and women . tjnna et al . examined 32 middle - aged metabolic syndrome men and women who performed 16 weeks of hiie three times per week . examined ten overweight males aged 32 years after two weeks of hiie consisting of 6 sessions of a 46 repeats of a wingate test . vo2max increased ( 8% ) and significant change in waist circumference was also found ( table 1 ) . although the effects of hiie on fat free mass has not been extensively examined , one study using dexa found that trunk muscle mass was significantly increased after 15 weeks , whereas another study using mri showed a significant 24% increase in thigh muscle cross sectional area after hiie . a summary of the results of studies examining the effects of hiie on subcutaneous and abdominal fat , body mass , and waist circumference is illustrated in table 1 . as can be seen studies that carried out relatively brief hiie interventions ( 2 to 6 weeks ) only resulted in negligible weight loss . however , the majority of subjects in these short - term wingate test studies have been young adults with normal bmi and body mass . studies that used longer duration hiie protocols with individuals possessing moderate elevations in fat mass have resulted in greater weight / fat reduction . interestingly , the greatest hiie - induced fat loss was found in two studies that used overweight type 2 diabetic adults ( bmi > 29 kg / m ) as subjects [ 8 , 40 ] . given that greater fat loss to exercise interventions has been found for those individuals possessing larger initial fat mass , it is feasible that hiie will have a greater fat reduction effect on the overweight or obese . thus , more studies examining the effects of hiie on obese or overweight individuals are needed . possible mechanisms underlying the hiie - induced fat loss effect include increased exercise and postexercise fat oxidation and decreased postexercise appetite . as mentioned , gaitanos et al . have suggested that towards the end of an hiie session that consists of numerous repeat sprints ( e.g. , ten 6-second bouts of maximal sprinting ) an inhibition of anaerobic glycogenolysis occurs and atp resynthesis is mainly derived from pcr degradation and intramuscular triacylglycerol stores . that increased venous glycerol accompanied hiie in both trained female cyclists and untrained women supports the notion that acute hiie progressively results in greater fatty acid transport . have shown that 6 to 7 sessions of hiie had marked increases in whole body and skeletal muscle capacity for fatty acid oxidation . as mentioned previously , the epoc or postexercise response to hiie does not appear to have been examined . it is feasible that the catecholamines generated by hiie ( figure 1 ) could influence postexercise fat metabolism . increased fat oxidation after hiie may also occur as a result of the need to remove lactate and h and to resynthesize glycogen . the elevated gh levels documented after a bout of hiie may also contribute to increased energy expenditure and fat oxidation . it is also feasible that hiie may result in suppressed appetite . in rats , hard exercise has been repeatedly reported to reduce food intake . the mechanisms underlying the anorectic effects of exercise are not known but exercise may reduce food intake by facilitating the release of corticotropin releasing factor ( crf ) a potent anorectic peptid . it has been shown that hard running and swimming exercise results in elevated levels of crf in rats [ 57 , 58 ] and increases in indirect markers of crf in humans . rivest and richard and kawaguchi et al . showed that injecting a corticotropin - releasing factor ( crf ) antagonist into the hypothalamus of rats prevented the effects of exercise on food intake and body weight reduction suggesting that crf plays a major role in the anorexia caused by exercise in rats . also provided evidence to support the importance of crf in mediating the long - term effects of exercise on food intake and body weight in rats . however , this exercise - induced anorexia has been observed only for a short time after hard exercise ( > 60% vo2max ) . mechanisms underlying this effect in humans are undetermined but could include the crf peptide effect previously discussed and an exercise - induced redistribution of splanchnic blood flow . for example , a 60%70% decrease in splanchnic blood flow in humans exercising at 70% vo2max has been documented and at maximal exercise splanchnic blood flow is reduced by approximately 80% . in summary , there is evidence to suggest that regular hiie results in increased fat oxidation during exercise ; however , the effects of hiie on postexercise fat oxidation and appetite suppression have not been examined . research examining the effects of hiie has produced preliminary evidence to suggest that hiie can result in modest reductions in subcutaneous and abdominal body fat in young normal weight and slightly overweight males and females . studies using overweight male and female type 2 diabetic individuals have shown greater reductions in subcutaneous and abdominal fat . the mechanisms underlying the fat reduction induced by hiie , however , are undetermined but may include hiie - induced fat oxidation during and after exercise and suppressed appetite . regular hiie has been shown to significantly increase both aerobic and anaerobic fitness and hiie also significantly lowers insulin resistance and results in increases in skeletal muscle capacity for fatty acid oxidation and glycolytic enzyme content . some important issues for future hiie research include optimization of type and nature of hiie protocols , individual fat loss response to hiie , and suitability of hiie for special populations . the most utilized protocol has been the wingate test ( 30 s of all - out sprint ) . this protocol amounts to 3 to 4 min of cycle exercise per session with each session being typically performed 3 times a week . this protocol , although remarkably short in duration , is extremely hard and subjects have to tolerate significant discomfiture . thus , the wingate protocol is likely to be unsuitable for most overweight , sedentary individuals interested in losing fat . other less demanding hiie protocols have included an 8-second cycle sprint followed by 12 s of low intensity cycling for a period of 20 min , a 15-second cycle sprint followed by 15 s of low intensity cycling for a period of 20 min , and a 2-minute cycle sprint followed by 3 min of low intensity cycling for a period of 20 min . a challenge for future research is to identify the minimal dose of hiie for the maximum health benefit . as discussed earlier , reducing the length of hiie training from 15 to 12 weeks still resulted in significant subcutaneous and abdominal fat loss . thus , more research is needed to identify the optimal length and intensity of the hiie protocol for achieving varying health outcomes . with regard to modality , studies have primarily utilized a stationary cycle ergometer , thus , little is known about the effects of other potential hiie modalities such as rowing , walking , running , stair climbing , and swimming . that insulin resistance has recently been shown to primarily be located in leg muscle suggests that hiie exercise that focuses on the legs is likely to show the greatest insulin sensitivity increases . how leg muscle adaptations to hiie impact on subcutaneous and abdominal fat loss and other health markers compared to other regional adaptations is undetermined . it is unclear if the increase in insulin sensitivity following hiie training is simply a response to the last exercise session or a result of more permanent skeletal muscle adaptations . whyte et al . have provided evidence to suggest that for short - term hiie training of two weeks , the increase in insulin sensitivity was largely a result of the last hiie session . they assessed insulin resistance 24 hours and 72 hours after the sixth hiie session in a two - week training program . insulin sensitivity had increased by 25% at 24 hours after hiie but had returned to preintervention levels after 72 hours . in contrast to these results , babraj et al . used a glucose tolerance test to assess insulin sensitivity after a similar intervention and found that insulin sensitivity was improved until at least three days after the last exercise session . why these similar hiiie protocols produced differing results differ is not clear and also whether hiie programs lasting longer than two weeks display a similar effect has not been established . individual variability in fat loss to hiie and other forms of exercise is an important issue for future research . for example , in the intervention previously described there were significant individual differences in the fat loss response to hiie . fat response ranged from a loss of 8 kg to a gain of .10 kg . if fat loss responders alone were examined in this study ( women who lost rather than gained fat ) , then average fat loss was 3.94 kg . as there are likely to be responders and nonresponders in every exercise , fat loss trial calculating mean fat loss alone hides the significant fat loss achieved by some individuals . thus , it is feasible that hiie fat loss programs are effective for producing a clinical decrease in fat ( greater than 6% of fat mass ) for some but not all participants . boutcher and dunn have highlighted a range of program design factors and individual factors that are behavioral , inherited , and physiological in origin that may affect individual fat loss response to exercise . therefore , research is needed to identify the major individual factors that both enhance and impede fat loss response to hiie - based interventions . a small number of studies have examined the effects of hiie fat loss and health of special populations and patients . these have included overweight adolescents , older adults , type 1 and type 2 diabetic individuals , paraplegics , intermittent claudication , chronic obstructive pulmonary disease , and cardiac rehabilitation patients . encouragingly , these studies have shown that hiie appears to be both safe and beneficial for these patient groups . future research needs to establish the most beneficial hiie protocol that is both optimal and sustainable for different types of patients . in conclusion , regular hiie produces significant increases in aerobic and anaerobic fitness and brings about significant skeletal muscle adaptations that are oxidative and glycolytic in nature . the effects of hiie on subcutaneous and abdominal fat loss are promising but more studies using overweight individuals need to be carried out . given that the major reason given for not exercising is time , it is likely that the brevity of hiie protocols should be appealing to most individuals interested in fat reduction . the optimal intensity and length of the sprint and rest periods together with examination of the benefits of other hiie modalities need to be established .

the effect of regular aerobic exercise on body fat is negligible ; however , other forms of exercise may have a greater impact on body composition . for example , emerging research examining high - intensity intermittent exercise ( hiie ) indicates that it may be more effective at reducing subcutaneous and abdominal body fat than other types of exercise . the mechanisms underlying the fat reduction induced by hiie , however , are undetermined . regular hiie has been shown to significantly increase both aerobic and anaerobic fitness . hiie also significantly lowers insulin resistance and results in a number of skeletal muscle adaptations that result in enhanced skeletal muscle fat oxidation and improved glucose tolerance . this review summarizes the results of hiie studies on fat loss , fitness , insulin resistance , and skeletal muscle . possible mechanisms underlying hiie - induced fat loss and implications for the use of hiie in the treatment and prevention of obesity are also discussed .

1. Introduction 2. Acute Response and Chronic Adaptations to High-Intensity Intermittent Exercise 3. High-Intensity Intermittent Exercise and Fat Loss 4. Conclusions and Clinical Implications

PMC4589905

since the 1990s , there has been a dramatic increase in the number of people affected by morbid obesity . unfortunately , even when supported with conscientious and diligent medically controlled dietary methods , many people are unable to maintain a healthy body weight . nonsurgical therapy leads to modest and transient weight loss at best , and surgery has been advocated as the only effective large - scale treatment for obesity . despite the availability of gastric bypass , adjustable banding , sleeve gastrectomy , and duodenal switch , the number of potential candidates who undergo surgical therapy for obesity in the united states remains approximately 1% . a contributing factor to the low number of obese patients seeking surgery may be the unappealing aspects of gastrointestinal division , anastomoses , foreign bodies , and gastric amputation , with the attendant risks of leak , hemorrhage , obstruction , nutritional complications , and socially embarrassing side effects . laparoscopic greater curve plication is gaining interest because it appears to avoid many of the intrinsic risks of more established procedures . the ability to predictably perform plication as an outpatient surgery we present 30-day outcomes in a series of 141 laparoscopic greater curve plications performed as outpatient procedures as well as the 12-month weight loss trend and reported changes in medication management for diabetes and hypertension . laparoscopic greater curve plications ( n = 141 ) were performed by a single surgeon between june 1 , 2009 and august 31 , 2013 . a chart review protocol was approved by an institutional review board , and patients with a minimum body mass index ( bmi ) of 35 who presented for outpatient laparoscopic greater curve plication as a weight loss procedure with an opportunity for at least a 12-month follow - up are reported . the gastrocolic ligament was opened with a harmonic - energy device starting approximately 6 cm proximal to the pylorus and extending to within 2 cm of the left crus of the diaphragm . the greater curve of the stomach was imbricated in layers with interrupted seromuscular nonresorbable sutures ( figure 1 ) . a calibration tube was advanced from the esophagus above the plication to the antrum below the plication to assess resistance and gastric luminal patency . no leak tests , upper endoscopy , or drains were used . completed greater curve plication . the gastrocolic ligament was opened with a harmonic - energy device starting approximately 6 cm proximal to the pylorus and extending to within 2 cm of the left crus of the diaphragm . the greater curve of the stomach was imbricated in layers with interrupted seromuscular nonresorbable sutures ( figure 1 ) . a calibration tube was advanced from the esophagus above the plication to the antrum below the plication to assess resistance and gastric luminal patency . no leak tests , upper endoscopy , or drains were used . completed greater curve plication . procedure data are presented in table 2 . of the 141 patients scheduled for outpatient laparoscopic greater curve plications , 138 patients were discharged , and 3 were admitted for noncritical reasons : nausea in 2 and observation for sleep apnea in 1 . twelve patients ( 9% ) who were discharged as outpatients had 1 episode of outpatient management : 6 of these patients had symptoms of dehydration treated electively at outpatient infusion centers , whereas the remaining 6 patients were evaluated at an urgent care or emergency department and discharged home . data are the number , with the percentage of the total procedures ( n = 141 ) in parentheses . three admissions were for evaluation and treatment of nausea and dehydration ; 1 patient underwent revision surgery , and all were discharged after 1 day . three patients were admitted for other reasons : 1 for acute clostridium difficile colitis ; 1 for deep vein thrombosis evaluation , which was negative ; and 1 for evaluation of gastrointestinal bleeding related to nonsteroidal anti - inflammatory drug ( nsaid ) use . in the 12-month period after surgery , 15 of the 30 patients ( 50% ) who presented on prescription medications for diabetes and 25 of 57 ( 44% ) who presented on prescription medications for hypertension reported a decrease in or discontinuation of medication . average percentage of body weight loss and excess body weight loss ( ebwl ) over time ( n = number of all 141 patients presenting during the specific time interval ) . surgical management of morbid obesity has been shown to provide effective long - term weight loss , weight maintenance , and reduction of comorbidities . a variety of surgical techniques have evolved over the years , but each includes significant potential risks that diminish the attractiveness of surgery to potential patients and referring physicians . in addition , cost considerations continue to have an impact on access provided by health insurance coverage and to influence public policy . perhaps these are among the many reasons that surgical intervention for patients who qualify under traditional weight loss surgery guidelines remains low . although gastric bypass and vertical sleeve gastrectomy have been shown to be relatively safe , many patients voice apprehension about the perceived complexity of these techniques . although adjustable banding has been reported to lead to an excess body weight loss of approximately 50% , it can require intervention for maintenance and surgery for several device - related complications . patients may also have difficulty in maintaining an optimal adjustment schedule , thus contributing to unsatisfactory weight loss . one advantage of gastric banding is that it is often performed as an outpatient procedure , eliminating hospitalization and associated costs . although vertical sleeve gastrectomy is technically more involved , it is possible to perform it as an outpatient procedure in selected patients . limiting gastric volume is a universal component of weight loss procedures . in 1969 , kirk showed in laboratory studies that inversion of the gastric wall slowed , stopped , or reversed weight gain when compared to controls . tretbar et al was inspired by the observation of weight loss after antireflux procedures and proposed gastric wrapping as an alternative to more aggressive techniques of gastric bypass and jejunoileal bypass . wilkinson advanced this idea clinically and added a polypropylene mesh encasement that unfortunately contributed to a significant number of erosions near the gastroesophageal junction . hoekstra et al compared bypass patients to a group of patients who had fundoplication reinforced with teflon mesh and found the weight loss , weight maintenance , and patient satisfaction rates to be higher in the plication group . plication - based concepts essentially disappeared in the transition of bariatric surgery in the laparoscopic era . however , in 2006 , fusco et al presented data suggesting that plication in rats creates significant weight loss and subsequently showed that infolding of the greater curve produces superior results to anterior wall plication . in 2007 , the results supported the idea that plication may be a safe and effective weight loss surgery alternative . these studies routinely present low incidences of complication and intervention , with excess body weight loss approaching that of bypass and sleeve gastrectomy . the operative experiences presented exhibit similar safety with no conversions , no critical complications , and no mortality . effective and efficient intracorporeal suturing has long been considered one of the essential skills of advanced laparoscopic surgery . there is a great deal of variability in suture technique among published authors . in dog models , tensile strength has been shown to be higher with increased density and number of rows of fixation points . running sutures , at least conceptually , may create a constant line of tension , creating areas more prone to ischemia or areas of tension prone to herniation of the plicated segment . previous reports include readmission and reoperation for herniation of the plicated segment between sutures , leading to pain , obstruction , and perforation . several surgeons have reported use of leak testing , intraoperative endoscopy , peritoneal drains , and postoperative upper gastrointestinal radiography . although these choices should be guided by the surgeon 's discretion , the lack of staple lines or gross anatomic changes are likely to lead to a low incidence of positive findings . no special monitoring , endoscopy , leak test , or drain was necessary in this series . the advantage of significant cost reduction , because of the absence of stapling devices or other hardware , has been promoted . cost was not specifically evaluated in the series presented herein , but cost - related factors were obviously absent , including special monitoring , surgical stapling devices , gastric band systems , use of endoscopy , leak test time and materials , use of drains , upper gastrointestinal radiography , and most notably , the routine need for inpatient hospitalization . use of outpatient services and facilities resulted in minimal hospitalization . despite a low complication rate , greater curve plication has routinely been presented as an inpatient procedure with length - of - stay typically between 1 to 2 days . in six of these patients felt unable to take adequate liquids in the first few days , but were otherwise without complaint . these patients were directed to an infusion center for intravenous crystalloid , and all patients rapidly improved . six patients had nausea or discomfort to a degree that they were directed to an urgent care or emergency department for evaluation . in these cases , the evaluations were unremarkable , and patients improved immediately , possibly due to empiric administration of antiemetics and intravenous crystalloid at the time of assessment . one patient experienced syncope on postoperative day 5 and was subsequently diagnosed with c. difficile colitis , which was treated with intravenous antibiotics , and the patient was discharged after 2 days . urgent endoscopy identified a nonbleeding 2-cm well - circumscribed ulcer with a visible vessel along the distal lesser curvature in an area that was undisturbed during the plication technique and remote from suture placement . it was the judgment of the gastroenterologist and surgeon that the ulcer 's appearance was consistent with side effects of nsaids , which the patient subsequently reported using before and after surgery . laparoscopy revealed no gross abnormalities and a calibration tube passed easily , but it was decided to revise the position of the lowest outer sutures by approximately 5 mm . one patient with persistent nausea was also evaluated for pneumonia and discharged the next day ; 1 patient was admitted to an outlying hospital for evaluation of possible deep vein thrombosis , which was negative ; and 1 patient was admitted overnight to an outlying hospital for rehydration . in the 141 patients , the total hospitalization for all patients admitted and readmitted those who underwent reoperation was 12 days . by comparison , if each patient in this series had been hospitalized for 1.5 days the estimated average for plication , according to several published series it is possible that routine admission would have prevented or captured problems that led to some of the subsequent interventions . all patients presented at or after the 30-day postsurgery date , and their postoperative outcomes were documented . longer range outcomes must be viewed with caution , as the attrition rate was significant , with 25% of all patients undergoing surgery presenting at the 11- to 13-month postoperative interval . although the trend for self - reported changes in the use of diabetic and antihypertensive medications reinforces the established benefits of successful weight management , regardless of the method of weight loss , the actual impact may be muted by the attrition rate noted in this series . the simple concept of plication may belie the meticulous skills needed to achieve optimal , sustainable results . ultimately , the minimally invasive aspect of plication may contribute to expanding the benefits of bariatric surgery to obese patients who do not meet the traditional bmi criterion for surgery and may be open to a less complicated technique . the follow - up observations are limited by the small number of patients , the attrition in postoperative presentation , and the retrospective nature of a review . obesity is a burgeoning medical and social concern because of the associated health and financial costs . the current literature for plication is limited but is increasing and fairly consistent in concluding that the technique is a safe and effective adjunct to the current menu of bariatric procedures . the attraction and practicality of plication as a surgical option may be further enhanced by its availability as an outpatient procedure . surgeon experience and skill , patient selection , longer observation times , and additional prospective studies will continue to contribute to the evolution of the role greater curve plication as a primary weight - loss surgery option .

background and objectives : laparoscopic greater curve plication is emerging as a weight loss procedure that avoids many of the complications of other surgeries that require gastrointestinal division , amputation , or use of a foreign body . cost savings and affordability have also been promoted , as plication does not require the use of stapling devices , adjustable gastric bands , or prolonged hospitalization . the ability to predictably perform plication as an outpatient surgery may further define its role as a therapeutic option for treating morbid obesity . we present the 30-day outcomes and supplementary 12-month data in a series of 141 laparoscopic greater curve plication surgeries performed as outpatient procedures.methods:laparoscopic greater curve plication was performed as outpatient surgery in 141 consecutive patients . outcomes including perioperative complications , incidental 12-month follow - up for weight loss , and change in diabetic and hypertensive medication are reported.results:of the 141 plications performed , 138 patients were discharged from the recovery room and 6 were readmitted . there was no conversion to open surgery and no mortality.conclusions:the ability to reliably perform greater curve plication as an outpatient surgery may further define its role as an additional weight loss surgery technique .

INTRODUCTION METHODS Surgical Technique RESULTS DISCUSSION CONCLUSION

PMC3321298

recently , considerable efforts are being made to design new noncentrosymmetric crystal structures by combining amino acids with various interesting organic and inorganic matrices to produce compounds for nonlinear optical ( nlo ) applications . a number of l - histidine compounds exhibiting the nlo behaviour , namely , l - histidine acetate , l - histidine chloride monohydrate , l - histidine tetrafluoroborate , l - histidine hydrochloride monohydrate , l - histidine hydrofluoride dihydrate , l - histidine bromide , and l - histidinium trichloroacetate , were reported earlier . the crystal growth and characterization of l - histidinium trifluoroacetate and l - histidine nitrate were reported from this laboratory [ 8 , 9 ] , recently . in this paper , another new compound possessing the nlo property , namely , l - histidinium 2-nitrobenzoate [ lh2nb , ( i ) ] is reported . to our knowledge , ( i ) is the first reported compound of an amino acid with 2-nitrobenzoic acid . the details regarding the preparation , crystal structure , hydrogen bonding , and shg efficiency of the title compound are discussed . the starting compounds , namely , l - histidine ( loba chemie , 99% ) and 2-nitrobenzoic acid ( alfa aesar , 95% ) were used without further purification . l - histidine and 2-nitrobenzoic acid were mixed in the stoichiometric ratio , in 1 : 1 proportions and dissolved in distilled water . the resultant mixture was stirred continuously to obtain a homogeneous solution , filtered and kept undisturbed for crystallization to take place . good quality single crystals of the title compound were obtained after about a week 's time . three - dimensional intensity data for a crystal of ( i ) were collected on a bruker smart apex ccd area - detector diffractometer using graphite - monochromated mok radiation ( 0.71073 ) . full - matrix least - squares refinement and subsequent fourier synthesis procedures were performed by using shelxl-97 . the hydrogen atoms attached to the c atoms were positioned with idealized geometry and refined using a riding model [ c h = 0.930.97 , uiso = 1.2 ueq ( parent c atom ) ] . h bonding were located from difference fourier map and restrained to a distance of 0.86 . in the absence of significant anomalous scattering effects , friedel pairs ( 1130 ) were merged . the crystal data , structure solution , and refinement parameters are listed in table 1 . crystallographic data ( excluding structure factors ) for the structure of compound ( i ) reported in this paper have been deposited with the cambridge crystallographic data centre as supplementary publication no . copies of the data can be obtained , free of charge , on application to ccdc , 12 union road , cambridge cb2 1 ez , uk ( fax : + 44-(0)1223 - 336033 or email : deposit@ccdc.cam.ac.uk ) . a preliminary study of the powder shg was made with a laser beam of wavelength 1064 nm , using kurtz and perry technique . the beam from a q switched nd : yag laser had an energy of 3.9 mj / pulse , pulse width of 8 ns , and the repetition rate being 10 hz . the crystals were ground to a uniform particle size of about 125150 m and then packed in capillaries of uniform bore and exposed to the laser radiation . a powder of potassium dihydrogen phosphate ( kdp ) , with the same particle size , was used as a reference . the output from the sample was monochromated to collect only the second harmonic ( = 532 nm ) , eliminating the fundamental , and the intensity was measured using a photomultiplier tube . it was found that the shg conversion efficiency for the compound ( i ) is about two times that of the standard kdp crystals . the molecular structure of the compound ( i ) with atom numbering scheme is shown in figure 2 . the asymmetric part of the unit cell contains an l - histidinium cation and a nitrobenzoate anion . the histidine molecule exists as histidinium ion due to the protonation at the n atom of the imidazole ring . the 2-nitrobenzoic acid exists as nitrobenzoate since the proton gets transferred to the amino acid . selected bond lengths , bond and torsion angles of ( i ) are listed in table 2 . the amino and carboxylic groups of histidine are twisted by an angle ( c1c2c3c4 ) of 71.12 ( 2) ( table 2 ) , while this angle is 179.13(2) in the structure of l - histidine . this type of rotation is a common feature in histidine - carboxylic acid structures [ 1 , 8 , 1315 ] . the bond angles in the amino and carboxyl groups are normal as observed in the structure of l - histidine . the plane of the carboxylate group of histidinium is twisted from its normal position , which may be due to the presence of the intermolecular n ho bonds ( figure 3 ) from the nearby histidinium ions . the nitro and carboxylate groups of the nitrobenzoate are twisted with respect to the planar benzene ring , as evident from the values of the torsion angles . the planar six membered and five membered rings in nitrobenzoate and histidinium , respectively , are nearly perpendicular [ 82.99 ] to each other . excepting o6 , all the other oxygen atoms act as acceptors ( figure 3 ) in the formation of n ho and c ho hydrogen bonds ( table 3 ) the level of shg response of a material is inherently dependent upon its structural attributes . on a molecular scale , the extent of charge transfer ( ct ) across the nlo chromophore determines the level of shg output ; the greater the ct , the larger the shg output . the presence of intermolecular interactions , such as hydrogen bonds , can extend this level of ct into the supramolecular realm , owing to their electrostatic and directed nature , thereby enhancing the shg response [ 17 , 18 ] . in lh2nb , the network of n hydrogen bonds running along the a - axis links the cationic histidinium ions ( figure 4(a ) ) . further , the benzoates are interconnected by n ho hydrogen bond to the histidinium molecule . the interconnected molecules of lh2nb are stacked as arrays along the a - axis ( figures 4(a ) and 4(b ) ) and further stabilized by other n ho bonds and weak interactions . the interplanar distances between the two six membered rings ( in 2-nitrobenzoates ) and the two five membered rings ( in the amino acids ) have the values of 3.480 and 3.307 , respectively , which fall in the category of - stacking . such parallel and close stacking is very favorable for the promotion of ct through the lattice . the large shg efficiency ( about two times that of the standard kdp ) of the compound ( i ) possibly arises due to ( i ) the large number of hydrogen bonds and ( ii ) the close stacking of histidinium cations and 2-nitrobenzozte anions , in the structure . the crystals of a new nlo material from the amino acid family , namely , l - histidinium 2-nitrobenzoate ( lh2nb ) , were grown using slow evaporation technique . the crystal structure of lh2nb was elucidated using single crystal x - ray diffraction methods . the proton from 2-nitrobenzoic acid is transferred to the l - histidine , forming l - histidinium 2-nitrobenzoate . the shg efficiency of this material was measured using kurtz and perry method and found to be about two times that of the standard kdp crystals . the large number of hydrogen bonds , the - stacking of l - histidinium anions and 2-nitrobenzoate cations and the presence of strong electron acceptors and electron donors are , in part , the reasons for the large shg efficiency possessed by this material .

a new nonlinear optical organic compound , namely , l - histidinium 2-nitrobenzoate ( abbreviated as lh2nb ( i ) ; ( [ c6h10n3o2]+ [ c7h4no4] ) ) , was synthesized . the molecular structure of lh2nb ( i ) was elucidated using single crystal x - ray diffraction technique . the second harmonic generation ( shg ) efficiency of this compound is about two times that of the standard potassium dihydrogen phosphate crystals .

1. Introduction 2. Experimental Procedures 3. Results and Discussions 4. Conclusions

PMC3059873

acute exercise has a marked effect on the immune system response . increased neutrophil , monocyte , and lymphocyte concentration have been reported following prolonged exercise,1 moderate intensity walking,2 and shortterm swim exercise.3 two hours of cycling at 55% vo2peak significantly increased neutrophil degranulation and oxidative burst,1 while walking for 30min at 60% vo2max increased lymphocyte proliferation and plasma interleukin6 concentration ( il6).2 significant increases in tumor necrosis factor ( tnf ) have been reported after a 30min highintensity run at 85% vo2max4 and following 15min of moderateintensity swimming.5 therefore , it is apparent that aerobic exercise of moderate to high intensity is capable of inducing significant increases in many immune system factors . while acute exercise significantly increases immune parameters , carbohydrate ( cho ) cho supplementation prior to 2.5 h of cycling at 65% vo2max resulted in reduced cell numbers of neutrophils , monocytes , and lymphocytes compared to placebo,6 as well as a significant decrease in plasma il6 concentration.7 similar reductions in immune cell counts and il6 have been reported in trained individuals who received cho supplementation during a 4h cycle ride at 70% of the individual anaerobic threshold compared to nonsupplemented trials.8 regarding the lymphocyte response to acute exercise , an increase in cell concentration is observed with the performance of high intensity exercise , followed by a reduction in concentration that is significantly lower than resting levels.9,10 a portion of this postexercise decrease is likely due to programmed cell death , or apoptosis . previous research from our laboratory has shown that the apoptotic program in lymphocytes can be induced during exercise,11 and that a significant number of apoptotic cells are in evidence immediately following an exercise bout.12,13 to our knowledge , only one previous study has assessed the effect that cho supplementation has on lymphocyte apoptosis . green et al . reported that cho supplementation at 3.2 grams per kg body weight decreased estimated cell death rates in cd4 and cd8 cells in culture taken from endurancetrained individuals who performed 2.5 hours of cycle exercise at 85% of the anaerobic threshold.14 this finding is consistent with previously cited research that cho supplementation may reduce the immunoendocrine changes associated with exercise.6 - 8 a wealth of literature confirms that the central nervous system has a modulatory effect on the immune system during situations of acute stress ( see review by black).15 neuroendocrine responses to stress are mediated by hypothalamic corticotropinreleasing factor16 which acts on the hypothalamicpituitaryadrenal axis to increase corticosteroids and catecholamines which tend to exert an immunosuppressive effect.17 in addition , there is evidence of direct innervation of the sympathetic nervous system into primary and secondary lymphoid tissues.18,19 therefore , it is possible that the immune system can be modified not only by nutritional supplementation as discussed above , but also independently by neural and cognitive factors . the influence of these neural factors may exert an immunosuppressive effect by reducing circulating leukocyte volume , suggesting potential deletion through a cell death mechanism ( apoptosis ) . while carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo , it is not known whether actual carbohydrate ingestion or simply the cognitive awareness of carbohydrate consumption mediates the aforementioned immune response during exercise . it was hypothesized that knowledge of carbohydrate intake would have no effect on the lymphocyte apoptosis response physiologically induced by aerobic exercise . this would hold true whether participants had a correct or incorrect knowledge of supplementation type . therefore , the purpose of this investigation was to determine whether knowledge of carbohydrate intake altered the exerciseinduced lymphocyte apoptotic response , independent of actual carbohydrate intake . endurance trained males and females ( n = 10 , female n = 6 , male n = 4 ) were recruited from local running , cycling , and triathlon clubs voluntarily participated in this investigation . in order to meet inclusion criteria , participants were required to be classified in the excellent category for his or her age group for peak oxygen consumption ( vo2peak ) , and were not currently taking any medications.20 subjects ' characteristics are presented in table 1 . the committee for the protection of human subjects at the university of houston reviewed and approved the testing procedures . prior to testing , participants reported to the university of houston 's laboratory of integrated physiology , where they were asked to read and sign an informed consent form and complete a medical history questionnaire . peak oxygen consumption ( vo2peak ) was determined using an incremental cycle test : stages 13 were 3min in length with 50 watt increases in intensity ; subsequent stages were 2min duration until volitional fatigue . vo2 was determined through automated analysis of expired respiratory gases ( cardio coach plus ; salt lake city , ut ) . vo2peak was identified as the highest level of vo2 measured and sustained for at least 30sec . exercise testing and experimental trials were completed on a velotron pro electronicallybraked cycle ergometer ( racermate , inc . ; seattle , wa ) at a selfselected pedal rate . subjects were randomly assigned into a group with correct cognitive awareness of supplementation type ( female n = 3 , male n = 2 ) , or an incorrect knowledge group ( female n = 3 , male n = 2 ) . for example in the incorrect knowledge group , subjects were informed they were receiving cho , but actually received the placebo ( pla ) beverage ; whereas during the during the cho trial they actually received pla . the correct cognitive awareness group was informed of receiving cho on the cho supplemented trial , as well as placebo during the pla trial . each participant completed two experimental trials in a counterbalanced manner : cho supplementation trial ( 250 ml every 15min , 6% cho beverage ) and control trial separated by at least seven days of recovery . both the cho and pla drinks were designed and supplied by the gatorade sport science institute ( barrington , il ) . the environmental conditions ( room temperature and 60% humidity ) were recorded daily and it was confirmed that there was no significant difference between testing days . all trials were completed between 06000800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24h prior to testing . participants were counseled not to alter their food intake in the days prior to the experimental trial . food intake was documented by 24h food records that were completed by each participant prior to the experimental trial . no significant difference in total calorie and/or macronutrient intake was evident between subjects . with the exception of the immediate postexercise sample , subjects were asked to rest for 20min prior to blood collection and sampling in a quiet , temperature controlled room . venous blood samples were collected from a peripheral arm vein using a multisample butterfly needle into an evacuated tube treated with sodium heparin ( bdbiosciences ; city , state ) . edta treated whole blood was used to determine complete blood counts ( cbc ) , and to measure plasma glucose concentration using a ysi 2300 glucose analyzer ( yellow springs , mo ) . venous blood samples were collected before ( pre ) and immediately after exercise ( post ) and used to make whole blood films within 10min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 briefly , smears were stained in maygrnwald stain ( sigmaaldrich , inc ; st . louis , mo ) for 35 min , and then washed in phosphate buffered saline ( pbs ) for 3 min . afterward , slides were placed in modified giemsa stain ( sigmaaldrich , inc ) for 2 min , washed in deionized water , and allowed to air dry before evaluation . images of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera ( ma88 microscope digital camera , c&a scientific co. , inc . ; manassas , va ) mounted to a light microscope ( cxl plus , labomed , india ) under the oil objective lens . a single investigator evaluated all images in a blinded fashion . according to our previously described procedures , at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 upon evaluation , lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane , while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 cell death due to apoptosis was recorded as the apoptotic index ( % ) , and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted . slides were counted in duplicate and averaged to give the apoptotic index ( ai ) . if duplicate counts varied by greater than 5% , the third slide was evaluated . the intraclass correlation for a single trained observer using the morphological method to determine exerciseinduced lymphocyte apoptosis in our laboratory is r = 0.96 . based on previous studies from our laboratory,11 - 13 a large effect size was anticipated . in an effort to be conservative , our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise . the a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise . post hoc power analysis indicated a statistical power of 92% for the present sample . for power analysis computations , data were analyzed using a factorial 2 ( cognitive awareness : correct or incorrect ) 2 ( supplement : cho or pla ) 2 ( test : pre or post ) anova with repeated measures on the second and third variable and significance accepted at p0.05 . endurance trained males and females ( n = 10 , female n = 6 , male n = 4 ) were recruited from local running , cycling , and triathlon clubs voluntarily participated in this investigation . in order to meet inclusion criteria , participants were required to be classified in the excellent category for his or her age group for peak oxygen consumption ( vo2peak ) , and were not currently taking any medications.20 subjects ' characteristics are presented in table 1 . the committee for the protection of human subjects at the university of houston reviewed and approved the testing procedures . prior to testing , participants reported to the university of houston 's laboratory of integrated physiology , where they were asked to read and sign an informed consent form and complete a medical history questionnaire . peak oxygen consumption ( vo2peak ) was determined using an incremental cycle test : stages 13 were 3min in length with 50 watt increases in intensity ; subsequent stages were 2min duration until volitional fatigue . vo2 was determined through automated analysis of expired respiratory gases ( cardio coach plus ; salt lake city , ut ) . vo2peak was identified as the highest level of vo2 measured and sustained for at least 30sec . exercise testing and experimental trials were completed on a velotron pro electronicallybraked cycle ergometer ( racermate , inc . ; seattle , wa ) at a selfselected pedal rate . subjects were randomly assigned into a group with correct cognitive awareness of supplementation type ( female n = 3 , male n = 2 ) , or an incorrect knowledge group ( female n = 3 , male n = 2 ) . for example in the incorrect knowledge group , subjects were informed they were receiving cho , but actually received the placebo ( pla ) beverage ; whereas during the during the cho trial they actually received pla . the correct cognitive awareness group was informed of receiving cho on the cho supplemented trial , as well as placebo during the pla trial . each participant completed two experimental trials in a counterbalanced manner : cho supplementation trial ( 250 ml every 15min , 6% cho beverage ) and control trial separated by at least seven days of recovery . both the cho and pla drinks were designed and supplied by the gatorade sport science institute ( barrington , il ) . the environmental conditions ( room temperature and 60% humidity ) were recorded daily and it was confirmed that there was no significant difference between testing days . all trials were completed between 06000800 following an overnight fast and participants were instructed to refrain from exercise for a period of 24h prior to testing . participants were counseled not to alter their food intake in the days prior to the experimental trial . food intake was documented by 24h food records that were completed by each participant prior to the experimental trial . no significant difference in total calorie and/or macronutrient intake was evident between subjects . with the exception of the immediate postexercise sample , subjects were asked to rest for 20min prior to blood collection and sampling in a quiet , temperature controlled room . venous blood samples were collected from a peripheral arm vein using a multisample butterfly needle into an evacuated tube treated with sodium heparin ( bdbiosciences ; city , state ) . edta treated whole blood was used to determine complete blood counts ( cbc ) , and to measure plasma glucose concentration using a ysi 2300 glucose analyzer ( yellow springs , mo ) . venous blood samples were collected before ( pre ) and immediately after exercise ( post ) and used to make whole blood films within 10min of collection for the morphological analysis of lymphocyte apoptosis as described previously.12 briefly , smears were stained in maygrnwald stain ( sigmaaldrich , inc ; st . louis , mo ) for 35 min , and then washed in phosphate buffered saline ( pbs ) for 3 min . afterward , slides were placed in modified giemsa stain ( sigmaaldrich , inc ) for 2 min , washed in deionized water , and allowed to air dry before evaluation . images of both normal and apoptotic lymphocytes were captured sequentially from the blood films using a digital camera ( ma88 microscope digital camera , c&a scientific co. , inc . ; manassas , va ) mounted to a light microscope ( cxl plus , labomed , india ) under the oil objective lens . a single investigator evaluated all images in a blinded fashion . according to our previously described procedures , at least 100 cells per slide were captured for subsequent evaluation of apoptotic characteristics.12 upon evaluation , lymphocytes were considered normal if the cell displayed an approximately circular shape with a smooth cell membrane , while lymphocytes that displayed membrane blebbing or apoptotic bodies were considered apoptotic as has been previously reported in the literature.21 cell death due to apoptosis was recorded as the apoptotic index ( % ) , and calculated as the number of apoptotic lymphocytes divided by the total number of lymphocytes counted . slides were counted in duplicate and averaged to give the apoptotic index ( ai ) . if duplicate counts varied by greater than 5% , the third slide was evaluated . the intraclass correlation for a single trained observer using the morphological method to determine exerciseinduced lymphocyte apoptosis in our laboratory is r = 0.96 . based on previous studies from our laboratory,11 - 13 a large effect size was anticipated . in an effort to be conservative , our sample size was calculated using a medium effect size with a 25% increase in apoptosis following exercise . the a priori sample size calculation revealed a minimum of 5 subjects to detect an increase in apoptosis with exercise . post hoc power analysis indicated a statistical power of 92% for the present sample . for power analysis computations , data were analyzed using a factorial 2 ( cognitive awareness : correct or incorrect ) 2 ( supplement : cho or pla ) 2 ( test : pre or post ) anova with repeated measures on the second and third variable and significance accepted at p0.05 . there was a significant supplement x test interaction for blood glucose concentration ( p = 0.04 , fig 1 ) . glucose values following exercise in the cho supplemented conditions were 52% greater than following the pla trials . the only significant main effect was for test , where after exercise there were 93% more lymphocytes ( p<0.001 , fig 2 ) . cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise ( p = 0.43 ) . there was a significant main effect of exercise on lymphocyte apoptosis , where after exercise there was an 84% increase in lymphocyte apoptotic index ( p<0.01 , fig 3 ) . there was a significant supplement x test interaction for blood glucose concentration ( p = 0.04 , fig 1 ) . glucose values following exercise in the cho supplemented conditions were 52% greater than following the pla trials . no interactions were evident with regard to lymphocyte counts . the only significant main effect was for test , where after exercise there were 93% more lymphocytes ( p<0.001 , fig 2 ) . cognitive awareness of drink type did not affect the lymphocyte cell death response before or after exercise ( p = 0.43 ) . there was a significant main effect of exercise on lymphocyte apoptosis , where after exercise there was an 84% increase in lymphocyte apoptotic index ( p<0.01 , fig 3 ) . the main finding of this investigation was that cognitive awareness of drink type had no physiological effect on selected immune parameters following highintensity aerobic exercise . individuals who had correct cognitive awareness of consumed supplementation ( cho or pla ) had statistically similar measures for plasma glucose , lymphocyte concentration , and lymphocyte apoptosis compared to individuals who had an incorrect cognitive awareness of the ingested supplement . in addition , we observed no effect of cho supplementation on either lymphocyte concentration or apoptotic index following 60min of cycle exercise at 80% vo2peak . carbohydrate supplementation significantly increased plasma glucose following exercise regardless of correct or incorrect cognitive awareness of drink type . the rise in glucose response has been confirmed by a number of other investigations.14,22 - 24 we are aware of only one other deception study in which subjects have received an incorrect cognitive awareness of carbohydrate or placebo supplementation prior to exercise.25 as we have shown , cognitive awareness of drink type ( whether correct or incorrect ) did not have an effect on the blood glucose response . the lack of a difference likely means that stimulating factors facilitating the release of glucose into the bloodstream ( i.e. glucagon , epinephrine , etc . ) an increase in lymphocyte concentration following exercise has been reported in numerous investigations.1 - 3,5 - 6 thirty minutes of walking at 60% vo2max increased lymphocytosis by 28% in human subjects2 and 120min of cycling at 65% vo2max resulted in a 20% increase.6 using an animal model , prestes et al . observed increases in lymphocyte counts of 98% and 165% in male wistar rats who performed unloaded swimming for 5 and 15min , and increases of 252% and 191% in animals who swam for 5 and 15min with a 5% load.5 the magnitude of lymphocytosis in the present study was 93% in endurance trained participants who completed 60min of cycle ergometry exercise at 80% vo2peak . an explanation for the lymphocytosis observed with exercise of increasing intensity is the greater expression of adhesion / activation molecules which can serve to facilitate movement of lymphocytes from the marginal pools into the circulation.26,27 while others have reported a significant decrease in lymphocyte count with cho supplementation compared to pla,6 our results did not show a difference . as part of an investigation to assess the effect of carbohydrate supplementation following caffeine ingestion , the participants of walker et al . consumed either caffeine or placebo prior to exercise , and either carbohydrate or placebo during a 2 h cycle ride at 65% vo2max.6 using only the trials in which placebo was consumed prior to exercise ( and excluding the effect of caffeine ) , it was reported that supplementation with cho during exercise reduced lymphocyte count by 21% compared with the trial in which placebo was administered during exercise.6 the common explanation for immunomodulation associated with cho supplementation are effects through hypothalamicpituitaryadrenal ( hpa ) activation and a muted stress hormone response.28 in the present study , cho was only ingested prior to exercise and did not result in suppressed lymphocyte cell count compared to pla . there are two possibilities to explain the differences between the study by walker et al.6 and our findings . first , our participants completed only a 1h cycle ride compared to 2 h. it is possible that the extended exercise duration differentially affected the immune response through increased hpa activation compared to our subjects who completed only half of the duration . second , the combination of supplemented glucose during the 2 h cycle exercise by the subjects of walker et al . could have resulted in a greater prolonged effect toward reducing immune parameters compared to our participants who received a single bolus of carbohydrate prior to exercise , but did not receive supplementation at any other point during their exercise trial . thus it is possible that blood glucose concentration plays a modulating role on cells of the immune system during exercise . in addition , it is likely that cho has a different effect depending on the immune cell subset . while an overall decrease in leukocyte numbers have been reported in various cho supplementation investigations,8,24 - 25 ingestion appears to affect neutrophils to a greater extent than lymphocytes . nieman et al . and sharhag et al . both found carbohydrate supplementation to reduce neutrophil count , but to have no effect on numbers of lymphocytes.8,24 neither correct , nor incorrect , cognitive awareness of drink type altered immunity in terms of lymphocyte apoptosis following exercise . to our knowledge , only one other study has attempted to evaluate the effect of carbohydrate supplementation on exerciseinduced lymphocyte cell death.14 green et al . used carboxyfluorescein succinimidylester fluorescence to estimate lymphocytes undergoing mitosis or cell death using iterative processes until all cells could be accounted for.14 they reported the estimated cell death rates of tcell subsets to be significantly reduced in cho supplemented trials compared to pla bouts in six welltrained male cyclists . in the present study recently , exerciseinduced lymphocyte apoptosis has been measured using either biochemical markers,4,29 - 30 or a morphological technique.11 - 13 in the current investigation , we employed the aforementioned morphological method which we have proposed is potentially more sensitive to cells displaying a wider range of apoptotic characteristics compared to a single apoptotic biomarker . we acknowledge that this method does have drawbacks ( i.e. highly subjective , low number of cells that are evaluated per sample , etc . ) ; however the methodology was the gold standard to which the current biomarker techniques were validated against.31 using a direct evaluation of cells displaying morphological characteristics of apoptosis , we found that carbohydrate supplementation during exercise did not alter exerciseinduced cell death compared to the placebo supplemented trial . while carbohydrate supplementation may alter certain immune parameters , it appears that ingestion has no effect on the mechanisms governing cell death induction during exercise . while we were unable to ascertain differences in select immune parameters due to cognitive awareness of carbohydrate in the present investigation , our study design could be modified to make detection of such differences more likely . the utilization of a single group crossover intervention in which subjects randomly complete the conditions of exercise alone , exercise with carbohydrate supplementation , and exercise in conjunction with placebo , would represent an acceptable experimental design in this regard . it should also be noted that the findings of the present study can only be applied to young and middleaged individuals who are highly aerobically trained , and should not be extended to other populations . in this our findings indicate that cognitive awareness ( either correct or incorrect , of carbohydrate or placebo ) has no effect on blood glucose concentration , lymphocyte concentration , or exerciseinduced lymphocyte apoptosis . while glucose intake itself may reduce the magnitude of various immune responses , knowledge of supplementation and indeed the carbohydrate supplement itself has no effect on altering immunity in terms of lymphocyte cell death following exercise . the authors would like to recognize western kentucky university 's faculty scholarship council for support through a summer faculty award . in addition , the authors would like to thank michael kueht and nisa dadjoo for their technical assistance with the investigation .

objective : the purpose of this investigation was to determine whether cognitive awareness of carbohydrate beverage consumption affects exerciseinduced lymphocyte apoptosis , independent of actual carbohydrate intake.introduction:carbohydrate supplementation during aerobic exercise generally protects against the immunosuppressive effects of exercise . it is not currently known whether carbohydrate consumption or simply the knowledge of carbohydrate consumption also has that effect.methods:endurance trained male and female ( n = 10 ) athletes were randomly assigned to one of two groups based on either a correct or incorrect cognitive awareness of carbohydrate intake . in the incorrect group , the subjects were informed that they were receiving the carbohydrate beverage but actually received the placebo beverage . participants completed a 60min ride on a cycle ergometer at 80% vo2peak under carbohydrate and placebo supplemented conditions . venous blood samples were collected at rest and immediately after exercise and were used to determine the plasma glucose concentration , lymphocyte count , and extent of lymphocyte apoptosis . cognitive awareness , either correct or incorrect , did not have an effect on any of the measured variables.results:carbohydrate supplementation during exercise did not have an effect on lymphocyte count or apoptotic index . independent of drink type , exercise resulted in significant lymphocytosis and lymphocyte apoptosis ( apoptotic index at rest = 6.33% and apoptotic index following exercise = 11.63% , p<0.01).conclusion : neither carbohydrate nor placebo supplementation altered the typical lymphocyte apoptotic response following exercise . while carbohydrate supplementation generally has an immuneboosting effect during exercise , it appears that this influence does not extend to the mechanisms that govern exerciseinduced lymphocyte cell death .

INTRODUCTION METHODS AND MATERIALS Participants 2.2 Protocol 2.3 Statistical Analysis RESULTS Glucose Lymphocyte Concentration 3.3 Lymphocyte Apoptosis DISCUSSION Acknowledgements

PMC3291729

according to a world health organization ( who ) report , cardiovascular diseases ( cvds ) are the number one cause of death globally . it was estimated that 17.3 million people died from cvds in 2008 , representing 30% of all global deaths . in addition , it is predicted that almost 23.6 million people will die from cvds by 2030 ( who , 2011 ) . the most important risk factors of cvds are unhealthy diet , physical inactivity , smoking , and harmful use of alcohol . the effects of unhealthy diet and physical inactivity may show up in individuals as raised blood pressure , raised blood glucose , raised blood lipids , and overweight and obesity . rapid reperfusion of the culprit vessel for salvaging ischemic myocardium and optimal medications reduce complications and improve survival rate . although many drugs and medical devices have been developed , the incidence of cvds remains high . more efficacious therapeutic modalities need to be explored by medical researchers . since the discovery of stem cells , a significant amount of research and development has emerged to be clinically applied for various incurable diseases including cvds . traditionally , the myocardium has been considered as terminally differentiated ; however , there is growing evidence that cardiomyocytes are able to become proliferative when they face substantial damage such as myocardial infarction or heart failure.1)2 ) in addition to intrinsic repair mechanisms , progenitor / stem cell plasticity has emerged as one of the ways to be regenerated.3 ) stem cells are undifferentiated pluripotent multilineage cells with the ability to renew themselves . the sources of stem cells include the embryo , fetus , and various parts of adult tissues . many clinical trials showed excellent safety and feasibility of adult stem cells , but the efficacy of stem cell therapy is not satisfactory to improve cardiac function substantially . the basic mechanisms of stem cell action on the injured myocardium will not be discussed here . we will focus instead on the current status of preclinical / clinical studies regarding therapeutic opportunities . in this review , we summarize recent results of preclinical / clinical trials that have evaluated the safety , feasibility , and efficacy of cell therapy in heart disease . direct or indirect transplantation of adult stem cells to damaged hearts is emerging as an innovative strategy to ameliorate cardiac remodeling and dysfunction after acute myocardial infarction ( ami ) . potential sources of functional cardiomyocytes has been explored and utilized for cell therapy to replace injured cardiomyocytes . stem cells are characterized by their ability to self - renew , clone , and differentiate into multiple tissues . adult stem cells are isolated and characterized from various sources ; peripheral blood,4 ) bone marrow ( bm),5 ) adipose tissue,6 ) umbilical cord blood,7 ) amniotic membrane,8 ) and dental pulp.9 ) a large number of animal studies have demonstrated that stem cells could be engrafted and differentiated within the heart.3)10 - 12 ) in preclinical studies , several large - animal species , including swine , sheep , and dogs , have been used to investigate the effects of stem cell therapy in cvds models . stem cells can be delivered to the heart by intravenous infusion , direct surgical injection , or catheter - based intracoronary infusion . orlic and colleagues first reported the repair of infarct myocardium through transplantation of bone marrow cells ( bmcs ) in mice.3 ) progenitors or stem cells with cardiomyogenic potential were studied both in vitro and in vivo . experimental data showed the expression of cardiac markers such as cardiac contractile proteins in stem cells in transplanted myocardium . one of the popular strategies to increase the cardiomyogenesis of stem cells is the stimulation of stem cells with an anticancer drug , 5-azacytidine , which is a nonspecific deoxyribonucleic acid methylation inhibitor . transient stimulation with 5-azacyticine for one day substantially increased cardiac protein expression in bm - derived mesenchymal stem cells ( mscs ) and sca-1-positive adult cardiac stem cells with spontaneous beating on culture system.13 ) to prove functional stem cell therapy , a small animal model has widely been used . myocardial infarction was experimentally induced by surgical ligation of the coronary artery such as left anterior descending artery in mice or rats , sometimes with reperfusion . various stem cell types , transplantation route , cell number , and transplantation timing have been studied in these small animal models . many studies are reported that stem cell therapy is safe , feasible , and promising for the cure of mi . histological data has revealed that the injected stem cells can survive in ischemic myocardium that participated in neovascularization and cardiomyogenesis . scar size , cardiac remodeling , fibrosis , and inflammation were much more effectively improved . based on these fantastic results of animal studies , many clinical studies were designed and initiated to transfer stem cell therapy to the bedside all over the world . various stem cells or progenitor cells have been introduced for cardiac repair in the last few years , although many past and ongoing clinical trials use predominantly adult autologous bm - derived cells . the use of bmcs in cvds has the advantage that bm can be easily accessed , and isolated cells can be expanded for autologous application . experimental studies of cell priming revealed that it improved cell survival , retention , integration , and differentiation.12)14)15 ) in addition , genetic modification of stem cells before application with the prosurvival gene akt,16 ) vascular endothelial growth factor,17 ) or fibroblast growth factor 218 ) promoted therapeutic efficacy . the anatomy and physiology of the porcine heart is well known to be similar to a human heart , and it is considered that the porcine mi model is the best model for cvds research . cell number , delivery procedure , and surgical techniques in the porcine model are also similar to the clinical setting , and more realistic implications could be provided compared with a small animal model . after successful percutaneous coronary intervention ( pci ) and coronary artery bypass graft surgery with optimal medications , cardiac function is restored only to a limited degree { 3 - 4% improvement in left ventricular ejection fraction ( lvef ) } which may result in cardiac remodeling in approximately 60% of the patients with myocardial infarction.19)20 ) many clinical studies have proceeded for proving their safety and efficacy to reach a final goal " new therapy " . regarding cell type , most clinical trials have used unfractionated bmcs as the delivery product , postulating that stem / progenitor cells are the biologically applicable therapeutic agents . the most widely applicable technique for stem cell delivery is intracoronary infusion from a clinical standpoint . the first human clinical trial of stem cell trial was intracoronary infusion of autologous bm unfractionated mononuclear cells to ami patient.21 ) subsequent clinical studies of stem cells for ami were then initiated . a variety of studies have demonstrated significant improvement of ventricular performance after stem cell therapy in ami , resulting in an increase in lvef and decrease in infarct size . in most cases , stem cell transplantation was performed in a time frame of 12 hours to several days after mi . although there is large variability of hemodynamic data after cell therapy , there is moderate improvement of cardiac performance by stem cell therapy that is more quantitatively effective than therapeutic interventions and pharmacotherapy.22 ) thus , autologous stem cell therapy represents an innovative and effective procedure for regeneration of impaired hearts in the early phase after the infarct . as seen in table 3 , most recent clinical trials utilized bm - derived mononuclear cells isolated from patients after pci . bm has been considered the safest source for autologous transplantation of stem cells , usually mononuclear cells , in clinical trials . for now the most widely used clinically approved source for stem cell therapy is autologous stem cells from bm ( www.clinicaltrials.gov ) . in addition to bm - mononuclear cells , mscs are now actively under investigation for cardiac repair . bm contains a population of hematopoietic stem cells and a rare population of plastic - adherent stromal cells ( 1 in 10000 nucleated cells in bm).23 ) these plastic adherent cells are mscs capable of forming singlecell colonies , expansion in culture , differentiation into osteoblasts , chondrocytes , and adipocytes.24)25 ) mscs were shown to be differentiated into a myogenic phenotype.13 ) animal studies demonstrated that human mscs could be transdifferentiated into endoderm - derived cells10 ) in injected myocardium , and coculture of msc with ventricular myocytes induced transdifferentiation into a cardiomyocyte phenotype in vitro.26 ) large - animal preclinical studies of mscs administration in post - mi heart demonstrated the ability of mscs to engraft , differentiate , and produce substantial functional recovery.12)16)27)28 ) the therapeutic effect of msc on ami has been reported in four clinical trials . chen et al.29 ) infused autologous msc by intracoronary route and demonstrated regional wall motion and global lvef were improved after six months of cell therapy . at that time , vulliet et al.30 ) reported that a microinfarction occurred after intracoronary infusion of mscs in a dog mi model . a prochymal trial31 ) was designed to evaluate the safety of intravenous application of allogeneic bm - derived mscs to ami patients . according to animal studies , a large proportion of infused cells were trapped in the lungs after administration , raising potential concerns regarding compromised pulmonary function.32 ) the results of the prochymal trial did not show any evidence of a pulmonary safety risk after infusion of allogeneic human ( h)bm - mscs . instead , those data revealed improved pulmonary function in the msc - treated patients , compared with baseline status . the rate of arrhythmia event was 4-fold lower in the hmscs group than in the placebo group ( 8.8% vs. 36.8% , p=0.025 ) . in ischemic cardiomyopathy , transendocardial injection of autologous bm - derived progenitor cells including mononuclear cells or mscs produced functional recovery in scarred myocardium and reversed remodeling of the lv chamber.33 ) unfortunately , however , they did not determine superiority between mononuclear cells and mscs . without the placebo control group , the transendocardial autologous cells in an ischemic heart failure trial ( tac - hft ) and percutaneous stem cell injection delivery effects on neomyogenesis ( poseidon ) trials34 ) are in progress to intensify those limitations ( www.clinicaltrials.gov ) . tac - hft is designed for comparing bm - mscs versus mononuclear cells , and poseidon is comparing the effects of allogeneic versus autologous msc therapy in ischemic cardiomyopathy patients . the delivery of multistem , an allogeneic bm - derived stem cell produced by athersys , inc . ( cleveland , oh , usa ) , to ami patients has proved to be safe and well tolerated.35 ) they showed multistem delivered via transarterial adventitia using a microsyringe catheter was safe with improvement of cardiac function in a dose - dependent manner . although the exact mechanism of msc effects is still unresolved , accumulating results of large animal studies and clinical studies have shown that msc - based therapy for cardiac repair is safe and provides substantial improvement in cardiac structure and function . the therapeutic effect of stem cell therapy on heart disease has been shown by experimental studies using small animal models . results have been reported as extraordinarily promising with experimental results such as cardiomyogenesis , neovascularization , and paracrine effect on injured myocardium . intracoronary transfer of autologous bmcs after optimum reperfusion therapy does not dramatically augment recovery of global lv function in patients , but could favorably affect cardiac remodeling after mi . mixed results have been reported in clinical trials of stem cell administered patients after ami with minimal improvement of ejection fraction or only a transient clinical benefit . the different outcomes were attributed to differences in cell preparations , timing and method of cell administration , choice of endpoints , and characteristics of patients . some studies failed to determine persistent clinical benefits after stem cell application ( table 3 ) . arrhythmias have been reported to be associated with intramyocardial rather than intracoronary injection of stem cells in the early clinical studies ; intramyocardial injection could be responsible for arrhythmogenesis . in addition , local injection induces a highly uneven distribution of cells , at least early after injection , which increases electrophysiological heterogeneity . although recent available results of clinical experience35)36 ) so far suggest that proarrhythmic effects may be transient , cardiac arrhythmia occurs unpredictably , and long - term follow - up studies would be essential to understand the arrhythmogenesis induced by stem cell transplantation . the most effective and safe cell type for myocardial repair and the clinical significance of cell therapy - induced arrhythmias will be determined in future pre - clinical and clinical studies . there was a case report about fatal events after autologous hematopoietic stem cell application in a lupus nephritis patient.37 ) after direct renal injection of stem cells isolated from peripheral blood , masses at the sites of injection were developed with hematuria . pathologic analysis revealed the masses were angiomyeloproliferative lesions and suggested to be a possible complication of stem cell therapy . there was no way to find out the detailed cause of death in those cases , but stem cell transplantation could be a causative event . the ultimate goal of cell therapy is the regeneration of lost cardiac muscle along with the reversal of adverse remodeling . despite growing clinical experience , the absence of standardized clinical end point in human trials has left us with fundamental questions . issues to be addressed in the future include determining the ideal cell type , the cell number to be delivered , optimal cell isolation method , efficient cell storage , and optimal time of administration to improve the efficacy of the therapy . after that , more realistic and optimized conditions of stem cell therapy will be applied to patients suffered from cvds with guaranteed safety . the risk of exposing patients to possible adverse outcomes of cell therapy must be seriously considered before clinical application . the argument that clinical trials should be delayed till mechanisms are perfectly understood will deprive a large number of patients from therapeutic chances that may bring them clinical recovery . stem cell therapy is a novel and innovative approach to cardiac therapeutics which has been achieved by numerous preclinical and early clinical studies showing safety , feasibility , and early efficacy . recent evidence from studies in animals and humans demonstrates the important roles of stem cells in cvds . in this review , reports of recent clinical trials of stem cell therapy for myocardial infarction are summarized and some important considerations are suggested for further application . for now , the challenge is to improveme the scientific concept to clinical setting with current treatment modalities .

the contribution of stem cells to cure damaged hearts has finally been unraveled . a large number of preclinical and clinical studies have showed beneficial outcomes after myocardial infarction . in this review , the current understanding of stem cell therapy in preclinical and clinical experiences is summarized . stem cells from bone marrow have shown a potential to improve cardiac performance after myocardial infarction in animal and early clinical studies . clinical trials from all over the world have provided safety assessments of stem cell therapy with marginal improvement of clinical outcomes . thus , further investigations should be encouraged to resolve the discrepancies between studies , clinical issues , and unclear translational findings . this review provides information and commentary on key trials for stem cell - based treat - ment of cardiovascular disease .

Introduction Walking With Animal Study Running With Clinical Study Looking Back With Consideration Conclusion

PMC3820198

the anterior cruciate ligament ( acl ) plays an extremely important role in resistance to anterior tibia deformation and rotational load on the knee joints1 . the treatment goal in acl rupture is to stabilize the knee joint and to recover the range of motion of the knee joint and the muscle strength to a normal level , to restore smooth functioning of the knee joint2 . to improve the range of motion of patients following acl reconstruction , various exercise programs together with electrical therapy , are conducted with the aim of achieving passive joint movement and pain control3 . in closed - chain exercise simultaneous resistance is exerted on the proximal and distal areas while the distal area of the upper and lower extremities remains in a fixed position4 . closed - chain exercise involves simultaneous contraction of the muscles to improve their dynamic stability . when eccentric contraction is predominant , it reduces the shearing force with a joint compression force , thereby providing stability to the joints . sensitive mechanoreceptors respond to changes in the pressure of the articular capsules and promote proprioception5 . impairment of proprioceptive sense has been reported to cause instability of the knee joint6 . proprioception plays an important role in inducing and promoting voluntary and involuntary movements by transmitting basic information to the motor control areas involved in regulating equilibrium and the vestibular senses7 . according to one study , information pertaining to motor control or location is provided largely by muscle spindles8 , whereas ligaments , the subacromial bursa , and capsules perform the role of mechanoreceptors9 . proprioceptive exercises may recover impaired motor senses . to completely recover the functioning of motor senses , proprioceptive exercises should be initiated as early as possible in the early stages of the rehabilitation process10 . in particular , proprioceptive senses are known to affect the preciseness of the articular angles of the knee joint and proprioceptive rehabilitation exercise improves the proprioceptive senses of the operated side after acl construction11 . in a study of the knee joint stability of patients who had undergone acl reconstruction , closed - chain exercise on an unstable surface increased activation of the vastus medialis muscle12 . in a comparison of closed - chain exercise and open - chain exercise , the closed - chain exercise stimulated the mechanoreceptors and increased the muscle mobilization rate more than the open - chain exercise , effectively increasing the strength of the quadriceps femoris muscle and the hamstring muscle13 . therefore , the present study examined the effects of closed - chain exercise performed on a stable surface and on an unstable surface on proprioceptive functions and a functional index of the knee joints . msd : mean standard deviation , seg : stable exercise group , ueg : unstable exercise group the subjects were patients who had undergone acl reconstruction between september 2012 and march 2013 and consented to participation in this study . the subjects were 28 male patients who had undergone acl reconstruction and received exercise treatment in the manual therapy room of g hospital . the stable exercise group ( seg ) performed exercise on a stable floor , and the unstable exercise group ( ueg ) performed exercise on a balance pad . those who had cruciate ligament damage accompanied by multiple fractures or collateral ligament damage , or who had undergone meniscus repair surgery , were excluded from the experiment . the biodex system iii ( biodex medical systems , shirly , ny , usa ) was used to measure the proprioceptive sensory functions of the knee joints on the operated side prior to the exercise and six weeks after the exercise . the subjects ' thighs and abdomen were firmly fixed with straps to ensure that they were vertical at 90 against the knee joints and the dynamometer tube axis , and the isometric muscle strength was measured . the differences in the proprioceptive sense functions of the knee on the operated side at 15 and 45 were measured . with the subjects ' two eyes blinded , the examiner instructed the subjects to perceive the angles that had been measured and to remember them ; the angles were maintained for 10 seconds . after the 10 seconds had elapsed , the subjects were asked to adopt the same angles of the knee joints . the measurements were made three times at 15 and 45 , and the average values of the three measurements were recorded . the knee joint functional index questionnaire was administered prior to the exercise and after six weeks of exercise . the lysholm knee scale , which evaluates the function of the knee joint after knee joint ligament surgery , was used for the functional score test of the knee joints14 . this scale assesses the following : a normal gait ( 5 points ) , instability during activities like gait , running , and jumping ( 30 points ) , pain ( 30 points ) , the degree of edema ( 10 points ) , and femoral amyotrophy ( 5 points ) . the maximum score that can be obtained is 100 points . scores of , zero to 6l , 62 to 81 , 82 to 90 , and 91 to 100 represent to poor , average , good , and excellent knee function , respectively . the paired t - test was conducted to verify differences in the proprioceptive functions and the functional index within each group , and the independent t - test was conducted to verify differences in the proprioceptive functions and the functional index between the groups . * p<0.05 , msd : mean standard deviation , seg : stable exercise group , ueg : unstable exercise group * p<0.05 , msd : mean standard deviation , seg : stable exercise group , ueg : unstable exercise group the between- and within - group changes in the proprioceptive functions of the knee joints and the functional index are presented in table 2 and 3 , respectively . the stable exercise group ( seg ) showed statistically significant differences in the proprioceptive functions and the lysholm score at 15 ( p<0.05 ) . the unstable exercise group ( ueg ) exhibited statistically significant differences in the proprioceptive functions and the lysholm score at 15 and 45 ( p<0.05 ) . prior to the exercise , there were no statistically significant differences between the two groups in any of the variables ( p>0.05 ) . after 6 weeks of exercise , the two groups showed a statistically significant difference in proprioceptive functions at 45 ( p < 0.05 ) . proprioceptive senses play an important role in correcting motor performance and play an important role in neurological senses related to motor skills15 . the biodex system 3 isokinetic dynamometer ( biodex medical systems , shirley , new york , usa ) is a contemporary isokinetic dynamometer with an electrically controlled servomechanism which is used in both clinical and research settings . the biodex system 3 performs with acceptable mechanical reliability and the validity of its measurements has tested16 . a previous study evaluated proprioceptive functions prior to and after acl surgery following rehabilitation exercise and found that the proprioceptive function of both the operated side and the non - operated side significantly improved17 . another study measured the proprioceptive functions of the knee joint at angles of 15 and 45 after a rehabilitation program comprising four weeks of proprioceptive exercises and reported that the proprioceptive functions of the operated sides were enhanced18 . in the present study , the six - week exercise program following acl reconstruction significantly improved the proprioceptive functions at the knee angles of 15 and 45. in the between - group comparison , the proprioceptive functions were significantly different at 45 , but not at 15. in the ueg , the improvement in the proprioceptive senses and the scores of the functional index showed that the exercise was effective . our results demonstrate that closed kinetic chain exercise on an unstable floor promoted proprioception more than closed kinetic chain exercise on a stable floor . a prior study examined knee joint function after acl reconstruction and reported that a swiss ball exercise group showed greater improvement than a control group19 . similarly , in the present study , there were significant differences in the knee joint functional scores after the exercise in both groups , but no significant differences between the two groups . squat exercise on a balance pad or a balance board after acl reconstruction may enhance proprioceptive functions of the knee joint and the knee joint functional index . such exercise is also effective at minimizing cruciate ligament stress . therefore , closed - chain exercise on an unstable surface early after surgery is a very important part of any rehabilitation program . a limitation of this study is that it failed to control for other physical activities . also , we did not control for physiological or psychological factors that may have affected the results . in addition , the subjects were confined to those who underwent cruciate ligament reconstruction at g hospital located in u city , therefore the generalizability of the findings is limited . to address these issues , further studies are required with greater numbers of participants to develop effective exercises programs for a variety of therapeutic interventions .

[ purpose ] the purpose of this study was to examine the effect of closed kinetic chain exercises performed by an unstable exercise group ( ueg ) and a stable exercise group ( seg ) on the knee joint , proprioception , and functional scores of patients who underwent anterior cruciate ligament ( acl ) reconstruction . [ subjects ] twenty - eight patients participated in this study . the exclusion criteria were fracture or neurological disease . [ methods ] the subjects were randomly assigned to one of two groups , each with 14 people . each group took part in a 60-minute exercise program , three times a week for six weeks . [ results ] the results of the clinical evaluation at 45proprioception showed statistically significant differences between the two groups . the results of the clinical evaluation at 15proprioception showed no statistically significant differences between the two groups . [ conclusion ] the proprioception and functional scores of the patients in the ueg who underwent acl reconstruction were superior to those in the seg group .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3753486

insulin resistance is defined as the decreased peripheral tissue response to insulin - mediated cellular actions and the term " insulin resistance " refers to reduced whole - body glucose uptake in response to physiological levels of insulin . insulin resistance is a common feature of obesity and is strongly associated with the etiology of type 2 diabetes , hypertension and coronary heart disease . currently , the gold standard method to evaluate insulin resistance is the hyperinsulinemic - euglycemic clamp technique . as with the increasing epidemic of childhood obesity in recent years , the prevalence of type 2 diabetes has increased among obese youth , particularly in a racial / ethnic minority group . although transient insulin resistance can occur during puberty , obesity is the most prevalent pathophysiological cause of insulin resistance in children and adolescents . reported that impaired glucose tolerance is present in 25% of prepubertal obese children and 21% of obese adolescents based on a 2-hour oral glucose tolerance test ( ogtt ) . a longitudinal study has shown that transition from normal to impaired glucose tolerance and from impaired glucose tolerance to type 2 diabetes is significantly associated with weight gain in children and adolescents . increased abdominal fat , particularly visceral fat is recognized as a strong predictor of insulin resistance in obese youth independent of total adiposity . in adults , epidemiologic studies suggest that regular physical activity is protective against development of type 2 diabetes [ 9 - 11 ] . intervention studies report that engaging in regular exercise is associated with significant improvements in glycemic control and insulin sensitivity in nondieting men and women . further , evidence from well - controlled randomized studies suggests that a combination of aerobic ( e.g. , treadmill walking , jogging ) and resistance ( e.g. , weight lifting ) exercise is more effective than either exercise modality alone to improve insulin sensitivity and glycemic control in men and women . although cross - sectional studies report the beneficial effects of physical activity or having a high cardiorespiratory fitness on insulin sensitivity in children and adolescents , whether engaging in exercise alone is effective in improving insulin sensitivity is not firmly established . the purpose of this mini review is to examine the effects of exercise alone ( e.g. , no calorie restriction ) on glucose tolerance and insulin sensitivity in obese children and adolescents . given that fasting insulin alone or combined with fasting glucose is a poor surrogate measure of insulin resistance , this review will focus on the exercise intervention studies , in which insulin resistance was evaluated by the ogtt , intravenous glucose tolerance test ( ivgtt ) and hyperinsulinemic euglycemic clamp test in nondieting children and adolescents . skeletal muscle is the major site of insulin - mediated glucose uptake in the postprandial state as the majority ( ~85% ) of glucose uptake by peripheral tissues occurs in muscle . exercise has an " insulin - like effect " to facilitate glucose transport from the circulation into the working muscles . it has been suggested that the mechanism by which exercise increases glucose uptake may be via the translocation of glucose transporters ( e.g. , glut-4 ) from an intracellular pool to the surface of the cell , where glucose uptake takes place . indeed , studies demonstrated increased glut-4 concentrations with aerobic training , which is accompanied by increases in insulin - mediated glucose uptake in adults [ 23 - 25 ] . however , the exercise - induced increase in muscle glut-4 concentration and the corresponding increase in insulin sensitivity decreases rapidly after the cessation of exercise , which suggests that exercise should be performed on a regular basis to maintain enhanced insulin sensitivity . currently , we are aware of six intervention studies , wherein the effects of aerobic exercise without calorie restriction on insulin sensitivity and glucose tolerance were examined in obese children and adolescents . inspection of table 1 reveals that the majority of studies have employed short duration of aerobic exercise ( 8 weeks to 3 months ) and reported no significant weight loss after the training . for example , nassis et al . have shown that in overweight and obese girls ( n=19 ) , 12 weeks of aerobic exercise ( 3 days / wk , 40 min / session , running , stair climbing , and jump rope ) resulted in a significant reduction ( -23.3% ) in insulin area under the curve ( auc ) and increase in cardiovascular fitness ( 18.8% ) in the absence of changes in body weight and % body fat . a significant increase ( 6.2% ) in lower limb fat free mass was observed after the training , which was significantly correlated with a reduction in insulin auc ( r=-0.68 ) . similarly , van der heijden et al . performed a nonrandomized controlled study to examine the effect of 12-week moderate intensity ( 70% vo2peak ) aerobic exercise ( 4 days / wk , 30 min / session , treadmill , elliptical , or a bike ) on insulin action in obese hispanic adolescents . in this study , the stable label ivgtt was employed to distinguish peripheral and hepatic insulin sensitivity . despite the absence of weight loss and calorie restriction , significant improvements in peripheral ( 50% ) and hepatic insulin sensitivity ( 23% ) were observed , which was also accompanied by reductions in total fat ( -1.1% ) and increases in lean body mass ( 1.1 kg ) . conversely , monzavi et al . observed no changes in oral glucose tolerance after a 12-week family - based lifestyle intervention program in overweight youth . in adults , some studies suggest that the improvement in insulin sensitivity in response to aerobic exercise training is mediated by reductions in adiposity . reported that in obese men , aerobic exercise alone is associated with significant improvements in insulin sensitivity as measured by hyperinsulinemic - euglycemic clamp technique , and that the improvement in insulin sensitivity is associated with concomitant reductions in visceral fat and not by skeletal muscle characteristics such as intramyocellular lipid and long - chain acyl coa levels . currently , there has only been one randomized controlled study in obese youth that examined the effects of aerobic exercise on insulin action and secretion , and total and regional fat topography . we have recently demonstrated that in obese adolescent boys , 3 months of aerobic exercise ( 3 times / wk , 60 min / session , 60% to 75% of vo2peak ) without calorie restriction results in significant reductions in total ( -2.6% ) and visceral fat ( -6.7% ) and intrahepatic lipid content ( -1.9% ) by comparison to nonexercising controls . however , despite significant improvement in body composition , there were no improvements in oral glucose tolerance , and insulin sensitivity and secretion as accessed by the 3-hour hyperinsulinemic - euglycemic clamp test and the 2-hour hyperglycemic clamp test , respectively . further , no changes in skeletal muscle mass and intramyocellular lipid content were observed in response to aerobic training . at present , we are aware of one randomized controlled trial that examined the effect of different doses of aerobic training on insulin resistance in the pediatric population . davis et al . examined the effects of low - dose ( 5 days / wk , 20 min / session ) versus high - dose ( 5 day / wk , 40 min / session ) of aerobic exercise program ( without dietary restrictions ) on oral glucose tolerance in a large sample of overweight and obese children ( n=222 , 7 to 11 years ) . after 3 months of aerobic exercise , they observed a dose - response trend , such that the reductions in insulin auc was larger in the high - dose exercise ( adjusted mean difference , -3.5610 u / ml ) and the low - dose exercise groups ( adjusted mean difference , -2.9610 u / ml ) than the control group . dose - response associations were also observed for reductions in % body fat and visceral fat , independent of gender and race . observation that the low- and high - dose of exercise groups showed similar improvements on insulin resistance , and that the increment of benefit between the control and low - dose exercise groups was larger than those observed between the low- and high - dose exercise groups , suggest that greater health benefits can be obtained when obese youth move from being sedentary to becoming moderately active . although aerobic types of physical activities have been traditionally recommended for children and adolescents , a recent guideline suggests that properly designed and supervised resistance training is beneficial to improve musculoskeletal strength , cardiovascular risk profiles , motor skills and psychosocial well - being of youth . currently , we are aware of five randomized controlled studies , in which the effects of resistance exercise without calorie restriction on insulin sensitivity and glucose tolerance were examined in obese youth ( table 2 ) . evidence from randomized controlled trials suggests that progressive resistance training is effective in improving insulin sensitivity in previously sedentary obese adolescent boys . shaibi and colleagues examined the effects of a 16-week resistance training alone ( 2 days / wk , 60 min / session ) on insulin sensitivity in overweight hispanic adolescent boys . the exercise program was progressive with respect to the number of sets ( one to three sets ) , repetitions ( three to 15 repetitions ) , and resistance used ( 62% to 97% of baseline 1 repetition maximum ) and each exercise session consisted of single and multiple - joint exercises using both free weights and weight stack equipment . after 16 weeks , insulin sensitivity measured by ivgtt significantly increased in the resistance exercise group ( 45% ) compared with nonexercising controls ( -0.9% ) and the improvement in insulin sensitivity was independent of changes in total fat and lean body mass . similarly , we conducted a randomized controlled study in obese adolescent boys to examine the effects of resistance versus aerobic exercise training , matched for exercise duration and frequency , on insulin sensitivity by a 3-hour hyperinsulinemic - euglycemic clamp technique . we observed a significant increase in insulin sensitivity ( 28% ) in the resistance exercise group alone , which was accompanied by significant increases in skeletal muscle mass ( 1.4 kg ) . we also found that compared with controls , resistance exercise was as effective as aerobic exercise in reducing total ( resistance , -2.5 kg vs. aerobic , -3.0 kg ) and visceral fat ( resistance , -0.2 kg vs. aerobic , -0.1 kg ) , and liver fat content ( resistance , -2.0% vs. aerobic , -1.9% ) as measured by whole - body magnetic resonance imaging and proton magnetic resonance spectroscopy . however , unlike the study of shaibi et al . , we observed that the changes in insulin sensitivity were significantly associated with reductions in total and visceral fat as quantified by whole - body mri technique , which reinforces the importance of adiposity reduction in improving insulin sensitivity in obese adolescent boys . unlike favorable metabolic benefits observed in obese adolescent boys , others report no beneficial effects of resistance exercise on oral glucose tolerance and peripheral insulin sensitivity in obese girls and a mixed sample of obese boys and girls [ 34 - 36 ] . goran 's group reported no improvements in insulin sensitivity in response to a 16-week resistance exercise ( 2 days / wk , 60 min / session ) combined with healthy diet ( decreasing sugar and increasing fiber intake ) in obese african - american and hispanic adolescent boys and girls . similarly , treuth et al . demonstrated significant increases in total and abdominal subcutaneous fat after a 5-month resistance training , which resulted in no improvements in glucose and insulin auc in obese prepubertal girls ( 7 to 10 years ) . although the disparate findings are unclear in the current literature , inclusion of children of both sexes in the analyses [ 34 - 36 ] , significant weight gain and increased energy compensation in response to exercise training may in part explain no improvements in insulin sensitivity . to date , we are aware of two studies wherein the effect of combined resistance and aerobic exercise on insulin sensitivity was examined in obese youth . in a nonrandomized controlled trial , bell et al . examined the effects of circuit training ( 3 times / wk , 60 min / session ) on insulin sensitivity using the hyperinsulinemic euglycemic clamp technique in a mixed sample of obese boys and girls ( n=14 ) . in this study , each exercise session included 1 minute of cycle ergometry ( 65% to 85% of maximum heart rate ) followed by a 1 minute of resistance training ( 12 repetitions / min , 55% to 65% of maximum voluntary contraction ) and two sets of 10 resistance exercise stations . significant increases in insulin sensitivity ( 22% ) and reductions in submaximal heart rate responses ( indicative of improvement in cardiorespiratory fitness ) were observed independent of changes in body composition ( e.g. , total fat and lean body mass ) . conversely , no significant effects of combination of aerobic and resistance training on insulin sensitivity and glucose tolerance were reported in obese hispanic girls . insulin resistant overweight / obese children and adolescents have a high prevalence of the metabolic syndrome and an atherogenic lipoprotein profile of small dense low density lipoprotein , small high density lipoprotein , and large very low density lipoprotein . our group previously demonstrated that for a given body mass index or total fat , insulin resistance is higher in those with high visceral fat and/or high waist circumference . although lifestyle intervention is the primary intervention strategy to treat obese youth , the role of physical activity alone to reduce the risk of type 2 diabetes remains unclear . currently , few randomized controlled studies are available that examined the independent effect of aerobic exercise , resistance exercise or combination of both exercises on glucose tolerance or insulin sensitivity in overweight and obese youth and their findings are inconclusive to date . although there is evidence of dose - response relationships between aerobic exercise and oral glucose tolerance in children , the effect of aerobic exercise alone on in vivo insulin sensitivity has not been firmly established . within the currently limited evidence , two randomized controlled trials suggest the beneficial effects of resistance exercise on insulin sensitivity and skeletal muscle mass in obese adolescent boys , independent of calorie restriction . given the variations in the study designs , the population studied ( single gender vs. both gender combined ; prepubertal vs. pubertal ) and small sample size , intervention studies have failed to conclusively demonstrate the independent effects of exercise on insulin sensitivity and glucose tolerance in children and adolescents . as insulin resistance occurs with puberty , pubertal stage ( e.g. , tanner stage ) should be examined and considered during data analyses / interpretations . as many of the studies did not concurrently assess abdominal fat , a strong independent factor of insulin resistance in youth , it is unclear whether the changes in insulin sensitivity and glucose tolerance are mediated by the changes in abdominal fat . due to the lack of intervention studies comparing the effects of different exercise modalities , the optimal mode of exercise to improve insulin sensitivity is currently unknown in children and adolescents .

as with the dramatic increases in childhood obesity over the past decades , the incidence of type 2 diabetes has increased among children and adolescents in the united states . insulin resistance is a common feature of childhood obesity and increases the risk of type 2 diabetes , metabolic syndrome , and atherogenic lipoprotein profile in obese youth . although cross - sectional studies report beneficial effects of physical activity or cardiorespiratory fitness on insulin sensitivity , the role of regular exercise alone ( e.g. , no calorie restriction ) as a strategy to reduce the risk of type 2 diabetes is unclear in obese children and adolescents . in this mini review , we examined the independent effects of various exercise on glucose tolerance and insulin sensitivity in obese youth .

INTRODUCTION EFFECT OF AEROBIC EXERCISE ALONE ON INSULIN SENSITIVITY AND GLUCOSE TOLERANCE IN OBESE YOUTH EFFECT OF RESISTANCE EXERCISE ALONE OR IN COMBINATION WITH AEROBIC EXERCISE ON INSULIN SENSITIVITY AND GLUCOSE TOLERANCE IN OBESE YOUTH CONCLUSIONS

PMC2982125

one of the endeavors of science is to have the power to predict phenomena , yet my career trajectory has been anything but predictable . from an early age i knew i wanted to be a scientist , but just what kind of science was less certain . i spent many hours as a child looking through my mail order telescope at the moons of jupiter and the rings of saturn . i was convinced i would become an astronaut and then as i got older , perhaps just an astronomer . however , i was quickly drawn to math and theoretical physics , instead of astronomy . but after a summer research fellowship through the college of creative studies at the university of california , santa barbara where i learned to use the atomic force microscope to image dna , i became fascinated with the kinds of questions that biologists ask . i joined the biochemistry , cellular and molecular biology graduate program at johns hopkins university school of medicine . i knew i had a lot to catch up on because i had only taken one or two courses in biology as an undergraduate and the hopkins program was known for its rigorous first - year curriculum . the core courses were very tough , and i quickly learned that vectors were more than just a representation of direction and magnitude . at hopkins , incoming graduate students were fortunate to have graduate student mentors who had gone through the process . she thought that if i was having so much trouble with molecular biology i would surely not pass genetics . but finally my training in physics and deductive reasoning came to my rescue , and i passed genetics with flying colors . after joining one lab and realizing that it was not the home for me , my life took another unexpected turn . my boyfriend ( now husband ) was in tom pollard 's lab , which partway through our graduate careers relocated to the salk institute . i followed my husband to san diego and joined the pollard lab , which was probably one of the best choices i have made . i took up two projects : searching for an actin homolog in archaea and purifying myosin ii from the yeast schizosaccharomyces pombe , neither of which was known at the time to exist in those respective organisms . luckily for me the s. pombe genome project was underway and within a month or so of joining the lab , i discovered a myosin ii in the database and proceeded to knock it out . this was my first foray into reverse genetics , and having seen how powerful this approach can be , i 've never looked back since then . i immediately felt at home with genetics and had by that time become very comfortable with molecular biology . being at the salk institute was also critical , because susan forsburg , a prior wicb junior awardee , made a home for me in her lab while the pollard lab moved to the salk . i had the best of both worlds : two incredibly supportive and rigorous advisors ( one in genetics and one in biochemistry ) . one week , i happened to choose a paper about the cytoskeleton in tomatoes for journal club . having gained all my biology education at a medical school , i was only vaguely aware of research in plants . i read a few more papers about the plant cytoskeleton and quickly discovered a huge gap in knowledge between the animal and plant cytoskeleton fields . my graduate work focused on myosins , so i was curious to find out what classes of myosins were present in plants and what their functions might be . just about then , some of the first myosin phylogenies were described ( hodge and cope , 2000 ; sellers , 2000 ) , and it was clear that plants had evolved their own unique myosins compared with other eukaryotes . from another journal club paper , i became aware of the moss physcomitrella patens and its unique ability among land plants to integrate foreign pieces of dna via homologous recombination ( strepp et al . i was pleasantly surprised to discover that ralph quatrano , a pioneer in the field of the plant cytoskeleton , was establishing p. patens as a model system in his lab at washington university in st . when i first joined the quatrano lab , genomic resources in moss were scarce , so i used traditional methods to clone out three distinct partial myosin clones . after using homologous recombination to knock out these three myosins without any obvious phenotypic consequences , since then , the ability to perform gene targeting and efficient rnai together with growing genomic resources ( including a completed genome ) has served as the basis for my research program . among other studies , we recently uncovered an essential role for class xi myosins in polarized growth ; myosins are important after all ! given the wide range of questions surrounding the role of actin dynamics in plant cell growth , we have plenty to keep us busy for years to come . i have been lucky to assemble a group of enthusiastic and talented postdocs and students , and we have begun to chip away at these questions using many different approaches . we have developed many tools that have expanded the capabilities of the facile reverse genetics system in p. patens . our rnai methodology is rapid and enables phenotypic discovery within a week of transformation ( figure 1 ) . complementation studies are the cornerstone of our research , enabling us to directly address the in vivo significance of our hypotheses . finally by collaborating with others in the field , we aim to marry in vitro knowledge of protein function with in vivo mechanisms of action . class ii , not class i , formins are essential for polarized growth in moss . top , three plants harbor an rnai construct silencing all class i and iii formins . running an academic research lab has simultaneously been the most challenging and the most rewarding part of my career . the main challenge is balancing my new responsibilities particularly managing the lab and mentoring my postdocs and students with committee work and teaching . i have also learned that the colleagues who surround me are far more important than the physical space my lab occupies . supportive colleagues have helped me to craft a collaborative environment in my lab and among neighboring labs . my graduate training in fission yeast genetics within a biochemistry and biophysics lab was a unique experience that still guides my research today and the philosophy by which i mentor my students and postdocs . from the forsburg lab , i learned a wealth of genetics , but most importantly i learned to be keenly observant . one of the most important take - home lessons from the pollard lab was that you should do whatever it takes to answer the biological question .

although i always knew i wanted to be a scientist , i did n't know i would become a cell biologist . events in life that you would never have predicted can greatly impact your career trajectory . i have learned to let those events take me in new directions . following a desire to investigate an understudied area of cell biology , i have found a niche . in this area , my lab is poised to contribute significantly toward understanding the fundamental molecular mechanisms underlying polarized plant cell growth .

BEGINNINGS IN PHYSICS AND THEN BIOLOGY THE FUNGAL CYTOSKELETON THE PLANT CYTOSKELETON LESSONS FROM THE PRESENT AND PAST

PMC4411276

water and small solutes ( e.g. , urea , glucose , amino acids , mineral ions ) in blood plasma freely traverse the gfb while circulating cells such as erythrocytes and high - molecular - weight plasma components such as albumin are selectively retained in blood . intriguingly , the glomerular permeability of proteins , particularly negatively charged proteins such as albumin , is well exceeded by those of neutral dextrans of comparable or even larger sizes ( chang et al . , 1975 ) . additionally , the gfb strongly restricts passage of anionic macromolecules ( thomson and blantz , 2010 ) . size and charge selectivity thus makes the gfb a formidable barrier for the bulk of plasma proteins and results in a urinary product that is virtually protein - free . while the role of tubular reuptake of proteins leaked into urine is well recognized , recent intravital imaging studies with fluorescent albumin conjugates validate the predominant role of the gfb in ensuring minimal loss of albumin in urine ( peti - peterdi and sipos , 2010 ) . the diagnostic hallmark of a compromised gfb is the incidence of protein in urine , a condition called proteinuria , or more specifically albuminuria if measured in terms of urinary albumin content . proteinuria manifests in a host of ailments ranging from congenital nephropathy , hypertension , and diabetes to chronic kidney diseases . over the last two decades , multidisciplinary studies combining genetics , cell biology , physiology , and signal transduction analysis including extensive studies on proteinuric disease models have provided us with valuable insights regarding the physiological importance of each of the three distinctive layers of the gfb and the intricacies of how they may function as an integral unit . in this review , we summarize our current understanding of the cell and molecular basis of renal filtration , highlighting the development , organization , and properties of each compartment of the gfb , and how they contribute to selective permeability . the glomerular vasculature consists of afferent and efferent arterioles and the glomerular capillary tuft ( fig . blood enters and exits the glomerulus via the afferent and efferent arterioles , respectively . inside the glomerulus , the afferent arteriole immediately branches into the elaborate glomerular capillary tuft , a specialized region where blood filters through . unlike the afferent and efferent arterioles , the glomerular capillaries are heavily perforated with transcellular pores and are not surrounded by smooth muscles . these glomerular capillary pores , known as fenestrae ( the plural of fenestra , which means window in latin ) , are 60100 nm wide and comprise 20% of the endothelial surface , making glomerular capillaries efficient portals for the rapid passage of high volumes of fluid characteristic of renal filtration ( fig . 2 d ; levick and smaje , 1987 ) . the idea that the glomerular capillary is a bona fide filtering compartment was previously contentious due to the size of its fenestrae , which are seemingly wide enough to accommodate albumin with a molecular dimension of 8 8 3 nm ( sugio et al . , 1999 ) . nevertheless , biophysical studies demonstrate that fenestrated and nonfenestrated capillaries have comparable permeabilities to macromolecules ( sarin , 2010 ) . recent studies indicate that the glomerular endothelium plays an active role in renal filtration on the basis of its negatively charged surface . the lumen of the glomerular capillaries and the fenestral surfaces are lined with a fibrous lattice of negatively charged glycoproteins called the glycocalyx ( fig . 2 e ; rostgaard and qvortrup , 2002 ; curry and adamson , 2012 ) . additionally , plasma components are adsorbed within the glycocalyx , forming a broader coat > 200 nm thick called the endothelial surface layer ( esl ; hjalmarsson et al . , 2004 ) . the filamentous structure and strongly negative charge of the esl thus effectively make fenestrae narrower and more restrictive . enzymatic destruction of different esl components resulted in elevated albumin excretion accompanied by diminished esl depth and loss of anionic sites on the endothelial surface ( gelberg et al . , 1996 ; jeansson and haraldsson , 2003 ; jeansson and haraldsson , 2006 ; meuwese et al . , 2010 ; dane et al . , 2013 ) similar loss of esl charge density and increased passage of albumin across the gfb was observed when adsorbed esl components are eluted by salt perfusion ( fridn et al . , 2011 ) . the role of the esl in glomerular filtration has also been examined in proteinuric disease models . renal perfusion of adriamycin , a drug used to induce proteinuria in mice , disrupted synthesis of glomerular proteoglycans and dramatically shriveled the glomerular esl , impairing the size selectivity and charge density of the gfb ( jeansson et al . , 2009 ) . in rats , ageing - related proteinuria correlated with the loss of glomerular esl ( salmon et al . , 2012 ) . in both animal models and in human patients , diabetes - induced proteinuria has also been strongly correlated with damage to the esl ( salmon and satchell , 2012 ) . it has been proposed that the esl could serve as a mechanosensor of fluid flow , an argument consistent with the loss of vasodilation upon removal of the esl ( curry and adamson , 2012 ; fu and tarbell , 2013 ) . altogether these findings align with the notion that the esl is an essential feature of the glomerular endothelium and a crucial determinant of glomerular permeability . the establishment of a functional gfb is contingent on the proper development of the glomerular endothelium . nascent podocytes secrete vegfa , a potent chemoattractant and trophic factor for migratory angioblasts that become that glomerular endothelium . vegfa binds the receptors vegfr1 ( flt1 ) , vegfr2 ( flk1/kdr ) , and neuropilin-1 , which are expressed by these angioblasts ( robert et al . , 2000 ) . homozygous ablation of vegfa from podocytes results in arrest of glomerular development and failure to form a gfb . haploinsufficiency for vegfa , however , causes a latent and progressive hypertrophy of gecs with a concomitant disappearance of fenestrae , a phenomenon called endotheliosis ( eremina et al . this breakdown of the glomerular endothelium is seen when vegfa ablation is induced in adult mouse podocytes or when its receptor is absent in gecs , which indicates that vegfa acts in a paracrine manner via vegfr2 ( eremina et al . , 2008 ; sison et al . , 2010 ) . these corroborate earlier findings showing that inhibition of vegfa function causes rapid onset of endotheliosis and proteinuria ( sugimoto et al . , 2003 ) . additionally , compound loss of the phospholipid - binding atpases ehd3 and ehd4 , which are expressed exclusively by gecs , strikingly resembles vegfa haploinsufficiency and vegfr2 deficiency ( george et al . their importance in vesicle trafficking suggests that ehd3 and ehd4 likely regulate the recycling of vegfr2 based on the altered cell surface distribution of vegfr2 in their absence . interestingly , podocyte - specific overexpression of vegfa164 , the predominant vegfa isoform in the kidney , causes global collapse of the glomerular tuft , rapid depletion of gecs , and massive proteinuria ( eremina et al . , 2003 ) . inducible overexpression of moderate levels of vegfa164 in postnatal and adult podocytes , however , causes a reversible disruption of glomerular structure and function ( veron et al . , 2010a , b ) . as diabetic patients are known to have elevated levels of circulating vegfa , these overexpression studies suggest that excessive vegfa signaling could contribute to the progression of diabetic nephropathy ( chiarelli et al . , 2000 ; hovind et al . , these findings further indicate that a delicately balanced dosage of vegfa is necessary to coordinate the development and maintenance of the glomerular vasculature and the gfb . signaling via secreted glycoproteins called angiopoietins intersects with the vegfa - dependent pathway to balance stabilization and remodeling of renal and systemic vasculature ( augustin et al . , the angiopoietin angpt1 is produced by podocytes and mcs , whereas its cognate receptor tie2 is expressed by gecs ( kolatsi - joannou et al . inducible knockout of angpt1 at mid - gestation ( from mouse embryonic day 10.5 ) results in simplified and enlarged glomerular capillary tufts , and the delamination of gecs ( jeansson et al . late gestation ( embryonic day 16.5 ) deletion of angpt1 does not cause overt glomerular maldevelopment but increased susceptibility to diabetic nephropathy . one factor that could contribute to impairment of the gfb is the loss of glomerular endothelial glycocalyx , which is caused by diabetic nephropathy and likely exacerbated by angpt1 deficiency . in systemic vasculature , angpt1 promotes barrier property and reduced permeability to albumin by stimulating the synthesis of glycocalyx and thickening of the esl ( satchell et al . the gbm derives from the fusion of the respective basement membranes of both podocytes and gecs ( abrahamson , 2012 ; miner , 2012 ) . proteomic analysis identified 144 distinct proteins in purified human glomerular ecm including the gbm ( byron et al . , 2014 ; lennon et al . , 2014 ) , with the most abundant being collagens ( types i , iv , vi , and xviiii subunits ) , laminins ( 5 , 2 , and 1 ) , nidogen-1 , heparan sulfate proteoglycans ( hspgs , agrin , and perlecan ) , and tubulointerstitial nephritis antigen - like ( tinagl1 ) protein . the gbm is an integral component of the gfb acting as an intermediary sieving matrix . the gbm may also function as a sink for pro - angiogenic ligands and secreted factors that mediate cellular communication between podocytes and gecs . lastly , the gbm cements podocytes and gec in place by cell ecm adhesive interactions , thus effectively stabilizing the gfb . among the abundant components of the gbm , type iv collagens and laminins are the most indispensable . alport syndrome is a hereditary disorder that targets the gbm , causing mild proteinuria during adolescence and progressing to end - stage renal failure . this ailment is linked to mutations in the genes col4a3 , col4a4 , and col4a5 , which encode the type iv collagen subunits 3 , 4 , and 5 , respectively . maturation of the gbm involves the substitution of the 112 ( iv ) collagen with the 345 ( iv ) collagen trimers as the predominant collagen complex , a developmental change that has been inferred to strengthen the gbm ( miner and sanes , 1994 ) . mutations in alport syndrome disrupt the assembly of 345 ( iv ) collagen trimers , leading to the persistent prominence of 112 ( iv ) collagen complexes . as 345 ( iv ) collagen trimers represent half the total proteins of a mature gbm ( candiello et al . , 2010 ) , it comes as no surprise that alport gbms are grossly perturbed in composition and are morphologically distorted . the importance of the collagen iv complex in the gbm is further highlighted by goodpasture s disease , an autoimmune disorder whereby self - reactive antibodies target the 3 subunit of collagen iv , resulting in glomerulonephritis ( cui and zhao , 2011 ) . mutations linked to pierson syndrome map to the gene lamb2 , which encodes the laminin 2 , impairing the assembly of the laminin complex lm-521 ( a heterotrimer formed among laminin-5 , -2 , and -1 subunits ; zenker et al . , 2004 ; matejas et al . , mice lacking lamb2 also show the abnormal renal and neuromuscular phenotype of pierson syndrome , and reveal a distinctive splitting of the gbm ( noakes et al . , 1995 ) . in mice , loss of lama5 in podocytes or the expression of a hypomorphic allele of lama5 ( causing attenuated expression of laminin-5 ) results in progressive proteinuria and ultrastructural deformation of the gbm ( kikkawa and miner , 2006 ; shannon et al . , 2006 ; goldberg et al . , loss of lm-521 in pierson syndrome causes other laminin complexes ( lm-111 , -211 , -332 , and -511 ) to become more prevalent , although this apparent compensation is insufficient to restore normal gbm structure and gfb function . scanning electron microscopy reveals that the gbm is not amorphous , but is rather a highly organized labyrinth of interconnected polygonal fibrils of varying thickness ranging from 4 to 10 nm ( kubosawa and kondo , 1985 ; hironaka et al . , 1993 ) . the fibrils are most densely packed within the core and have heterogeneous pores averaging 10 nm in diameter . in a proteinuric nephritis disease model in rodents , it was observed that the gbm fibril network was more loosely packed and had enlarged pores as big as 40 nm ( hironaka et al . imaging analysis combining stochastic optical reconstruction microscopy ( storm ) and correlative electron microscopy has revealed nanometer - resolution details of the highly stratified organization of the gbm , delineating the location and orientation of epitopes of major gbm components relative to the adhesion receptor integrin-1 expressed by gecs and podocytes ( fig . in contrast , laminin-5 , agrin , and integrin-1 bimodally align within two distinct layers . interestingly , the 345 and 112 ( iv ) collagen networks are particularly concentrated at the core , closer to the endothelial side , a distribution that is unexpectedly too distant from the extracellular domains of integrin-1 at the surface of podocytes . this indicates that the physiologically important ligands of podocyte integrin-1 are the agrin and laminin complexes . laminin lm-521 and agrin are bimodally distributed , whereas collagen iv complexes are concentrated at the core of the gbm . both the predominant 345 and the less abundant 112 type iv collagens are normally too distant from 1integrin receptor ( ir ) complexes on the podocyte side . this suggests that lm-521 and agrin but not type iv collagens are the normal physiological ligands of ir complexes expressed by podocytes . ( b ) simplified representation of major adhesion receptors ( nephrin , neph1 , and fat1 ) found in the sd . lipid - raft localization of the sd is dependent on the cholesterol - binding podocin . the sd is coupled to both f - actin regulatory ( nck n - wasp arp2/3 and cd2ap arp2/3 ) and cell polarity ( par6apkc/cdc42 ) complexes . application of correlative storm imaging to kidneys of alport mice ( col4a3 mutant mice ) demonstrates the dramatic redistribution of agrin and 112 ( iv ) collagen into a diffuse pattern throughout the width of the gbm . a likely implication of this is that podocytes might be inappropriately exposed to type iv collagens , thereby inducing a pathological transformation as observed in alport disease . these imaging analyses bolster the argument that the ultrafine pore structure of the gbm is key to normal filtration , and that proteinuria results from perturbing the molecular and structural organization of the gbm . the abundance of hspgs such as agrin , perlecan , and collagen xviii confers a net negative charge to the gbm , which prompted a long - held assumption that the gbm is a critical determinant of the charge selectivity of the gfb ( rennke et al . , 1975 ; rennke and venkatachalam , 1977 ; harvey et al . , 2007 ; van den hoven et al . , 2008 ; goldberg et al . , genetic studies in mice aimed at minimizing the net negative charge of the gbm have disputed this argument and failed to result in overt proteinuria ( rossi et al . , 2003 ; treatment of the gbm with heparanase in order to strip glycosaminoglycan - associated anionic charges did not cause overt changes in glomerular morphology or induce proteinuria ( van den hoven et al . it is tempting to speculate that charge repulsion of circulating macromolecules in the gfb is primarily established within the glomerular compartment instead of the gbm . the defining feature of normal fully differentiated podocytes is their elaborate cytoarchitecture , which resembles the stellate body shape of an octopus , characterized by an arborized cell body with multiple projections subdivided into larger major processes and finer pedicels or fps ( fig . major processes are reinforced by microtubules and intermediate filaments while fps are actin - rich projections anchored to the gbm via focal adhesions ( ichimura et al . , 2003 ) . the podocyte cell bodies and their fps wrap around the glomerular capillaries in a strikingly elaborate interdigitating pattern . neighboring podocytes are physically adjoined through their fps via unique intercellular junctions called the sd ( fig . 3 b ) . unlike tight junctions , the sd lacks e - cadherin and is structurally porous ( tassin et al . , 1994 ) . the sd thereby serves as the exit port for primary urinary filtrate and is now well recognized as essential in the selective retention of high - molecular - weight plasma components . the seminal discoveries of the proteins nephrin and podocin as integral components of the sd are instrumental in proving that podocytes and their structural integrity are of key importance in the establishment and maintenance of the gfb ( kestil et al . , 1998 ; inactivating mutations of nphs1 and nphs2 , the respective genes encoding for nephrin and podocin , lead to congenital nephropathy characterized by the collapse of fps and the absence of sds ( kestil et al . this stereotypical pathological transformation of podocytes called effacement is a distinctive hallmark of podocyte injury and is strongly correlated with the onset of proteinuria . several genes , apart from nphs1 and nphs2 , that encode for podocyte - specific proteins are strongly associated with the onset of proteinuric diseases including cd2ap ( shih et al . , 1999 ; kim et al . , 2003 ) , kirrel / neph1 ( donoviel et al . , 2001 ) , fat1 ( ciani et al . , 2003 ) , trpc6 ( reiser et al . , 2005 ; winn et al . , 2005 ) , actn4 ( kaplan et al . , 2000 ) , myo1e ( krendel et al . , 2009 ; mele et al . , 2011 ) , arhgap24 ( akilesh et al . , 2011 ) , arhgdia ( togawa et al . , 1999 ; gupta et al . , 2013 ) , inf2 ( brown et al . , 2010 ) , coq2 ( diomedi - camassei et al . , 2007 ) , coq6 ( heeringa et al . , 2011 ) , plce1 ( sadl et al . , 2002 ; hinkes et al . , 2006 ) , anln ( gbadegesin et al . , 2014 ) , ptpro ( wharram et al . , 2000 ; ozaltin et al . , 2011 ) , and adck4 ( ashraf et al . , 2013 ) . most of these genes encode for intrinsic sd components or their respective interacting partners while the rest encode for proteins needed for the survival , differentiation , cytoskeletal dynamics , and unique morphology of podocytes . the consequences of mutations of these genes highlight the important relationship between podocyte dysfunction and the disruption of the gfb . while many of the molecular constituents of the sds have been identified , their topological assembly into a functional complex is poorly understood . the ectodomains of several adhesion receptors in the sd likely organize the bridge linking juxtaposed fps via a combination of homophilic and heterophilic receptor receptor interactions . by virtue of their large ectodomains , nephrin and fat1 are excellent candidates to associate in trans to connect opposing fps ( inoue et al . , 2001 ; ciani et al . the smaller adhesion receptors such as neph1 and neph3 , however , may interact in cis with nephrin and fat1 ( gerke et al . consistent with its anatomical appearance as a junction between differentiated podocytes , other components of the sd are key molecules associated with adherens and tight junctions including zo-1 , cask , spectrins , magi-2 , jama - a , occludin , cingulin , and iqgap1 ( lehtonen et al . , 2005 ; fukasawa et al . , 2009 ) . the huge scaffold protein zo-1 is essential for the normal interdigitation of fps and the formation of the sd ( itoh et al . , 2014 ) . lack of zo-1 triggers early onset proteinuria with podocyte effacement and the progressive scarring of the glomerulus ( glomerulosclerosis ) . zo-1 appears to be required to maintain the expression and correct spatial distribution of nephrin and podocin . the distinctive morphology of podocytes underscores the importance of cell polarity signaling in podocyte biology . nephrin and neph1 are known to interact with polarity proteins such as par3 , par6 , and apkc/ ( hartleben et al . , 2008 ) . the deletion of apkc/ and the small gtpase cdc42 , which regulates the activation of the par3-par6-apkc/ polarity complex , causes proteinuria and the formation of aberrant junctions between effaced fps ( hirose et al . , 2009 ; scott et al . , 2012 ; blattner et al . , 2013 ) . loss of apkc/ has been shown to interfere with cell surface localization of nephrin , podocin , and neph1 ( satoh et al . , 2014 ) . furthermore , studies in zebrafish demonstrate that the crumbs ( crb ) protein family member crb2b is required for the differentiation of pronephric podocytes , whereas mutations in the human orthologue crb2 have been linked to proteinuric disease ( ebarasi et al . crb proteins are part of the crb patj pals1 polarity complex , which works alongside the par3par6apkc/ complex in directing the apical localization of particular membrane - bound proteins . crb2b suppression in zebrafish leads to loss of polarized distribution of nephrin and the disruption of sd assembly ( ebarasi et al . , 2009 ) . in contrast , podocyte - specific ablation of scribble , a determinant of basolateral trafficking , did not inhibit proper sd formation ( hartleben et al . , 2012 ) . these studies suggest that apical sorting predominates over basolateral sorting mechanisms to specify the polarized and dynamic assembly of the sd complex . parallel bundles of actin filaments and a network of cortical actin form the backbone of terminal fps , and the perturbation of this cytoskeletal assembly is thought to underlie fp effacement and the dismantling of the sd ( ichimura et al . , 2003 ) . compound ablation of the adaptor molecules nck1 and nck2 in podocytes causes proteinuria , which demonstrates that the sd is intimately and dynamically coupled to the actin cytoskeleton ( jones et al . in vitro , oligomerized nephrin interacts with nck adaptors , leading to the recruitment of n - wasp and arp2/3 complex that mediates localized polymerization of actin filaments ( jones et al . , 2006 ; nck adaptors are not only required during podocyte maturation but are also needed to maintain preformed fps ( jones et al . , 2009 ) . since nck proteins also interact with the pinch integrin complex , these adaptors could also help anchor podocytes to the gbm via actin - linked focal contacts ( tu et al . , 1999 ; dai et al . , 2006 ; el - aouni et al . , 2006 ) . similarly , the rho gtpase cdc42 is required to mediate the linkage between the actin cytoskeleton and nephrin complexes ( scott et al . , 2012 ) . the cytoskeletal tethering of the sd is also dependent on the scaffold protein cd2ap , which promotes the stability of actin microfilament network of podocytes ( shih et al . , 1999 ; kim et al . , 2003 ; yaddanapudi et al . , 2011 ; tang and brieher , 2013 ) . equally important to the assembly of the podocyte actin cytoskeleton slit1-robo2 signaling antagonizes nephrin - dependent actin polymerization yet is required to establish a normal pattern of fp interdigitation ( fan et al . , 2012 ) . additionally , depletion of the actin - severing factor cofilin-1 has been shown to cause late - onset proteinuria and ultrastructural defects in podocytes in a manner akin to specific loss of robo2 in podocytes ( ashworth et al . gupta et al . , 2013 ) , arhgap24 ( akilesh et al . , 2011 ) , inf2 ( brown et al . , 2010 ) , myo1e ( krendel et al . , 2009 ; mele et al . , 2011 ) , and anln ( gbadegesin et al . , 2014 ) , which encode for known regulators of the actin cytoskeleton , have all been implicated in the etiology of proteinuric diseases . the cytoskeletal dynamics and structural plasticity of podocytes are also regulated by calcium signaling , lipid protein interactions at the sd , and endocytosis . in podocytes , the ion channels trpc5 and trpc6 mediate distinctive calcium influx in response to angiotensin ii , eliciting the reorganization of the actin cytoskeleton via modulation of the rho gtpases rac1 and rhoa ( tian et al . , 2010 ) . gain - of - function mutations of trpc6 are known to cause proteinuria in humans , whereas genetic loss of trpc5 or trpc6 prevents podocyte injury ( reiser et al . , 2005 ; winn et al . , 2005 ; schaldecker et al . , 2013 ) . these findings suggest that unbalanced elevation of intracellular calcium mitigates podocyte dysfunction and provide an explanation as to the protective benefits of blockade of angiotensin signaling in the progression of proteinuric renal diseases such as glomerulosclerosis and diabetic nephropathy . interestingly , trpc6 interacts with podocin , which suggests how intimately calcium signaling is coupled to the sd complex ( huber et al . , 2007 ; schurek et al . , 2014 ) . lipid - dependent autocrine signaling in podocytes involving sflt1 is required in the regulation of actin dynamics and the proper formation of fps and the sds ( jin et al . , 2012 ) . specifically , sflt1 secreted by podocytes binds to the glycosphingolipid gm3 at the podocyte surface , promoting cell adhesion , nephrin phosphorylation , and consequent remodeling of the cytoskeleton . fp effacement and proteinuria ensues in the absence of endocytosis - related lipid - binding proteins , specifically dynamins , endophilins , and synaptojanin-1 ( soda et al . , 2012 ) . it has been postulated that clathrin - mediated endocytosis could dynamically sculpt fps by regulating the turnover of sd components ( soda and ishibe , 2013 ) . podocytes also play multiple important functions in maintaining the gfb independent of the formation of the sds . podocytes have a vital role in promoting the proliferation , survival , and development of endothelial cells . the pro - angiogenic factors vegfa , angpt1 , and sdf1 are secreted by podocytes and are essential for the normal development of the glomerular endothelium ( simon et al . , 1998 ; yuan et al . , 1999 podocytes together with the glomerular endothelium also collaborate in building the gbm ( byron et al . , 2014 ) . whereas 112 ( iv ) collagen is produced jointly by endothelial cells and podocytes , the 345 ( iv ) collagen network is derived primarily from podocytes , as seen in vivo ( abrahamson et al . , 2009 ) . macromolecules and proteins that traverse the gbm can be sequestered by podocytes via endocytosis , a mechanism that likely prevents the gfb from clogging ( eyre et al . , 2007 ; akilesh et al . , 2008 ) . megalin and cubilin , which form a multifunctional endocytic receptor complex commonly found in absorptive epithelia , are coexpressed in podocytes and could mediate the retrieval of urinary albumin by podocytes ( yamazaki et al . , 2004 ; prabakaran et al . , 2012 ) overwhelming genetic evidence undeniably underscores the fact that the podocyte is an essential component of the gfb . biophysical studies demonstrate that gbm compression reduces permeability to albumin and the polysaccharide ficoll ( robinson and walton , 1989 ; fissell et al . , 2009 ) . it is therefore tempting to speculate based on this that gecs and podocytes physically constrain and mitigate compression of the gbm . consistent with this supposition are genetic studies showing that loss of itga3 ( kreidberg et al . , 1996 ) , itgb1 ( pozzi et al . , 2008 ) , cd151 ( karamatic crew et al . , 2004 ; sachs et al . , 2006 ) , ddr1 ( gross et al . , 2004 ) , ilk ( dai et al . , 2006 ; el - aouni et al . , 2006 ) , tln1 ( tian et al . , 2014 ) , and rap1a / b ( potla et al . , 2014 ) , genes encoding for proteins implicated in the adhesion of podocytes to gbm components such as collagens and laminins , results in impairment of the gfb . intrinsic structural reorganization of the gbm such as in alport and pierson syndrome may also perturb the normal anchorage of podocytes and endothelial cells , cumulatively altering the compressibility of the gbm . this is not at all far - fetched , as ecm stiffness , by way of mechanotransduction via adhesion molecules , is known to influence a diverse range of cellular behavior including restructuring of the actin cytoskeleton , contractility , motility , gene expression , proliferation , and overall differentiation ( dufort et al . , 2011 ) . effaced podocyte fps could very well be indicative of not just the remodeling of cell cell junctions or sds but also of a maladaptive response to reestablish weakening focal contacts on the gbm . in fact , unfastening of podocytes and denuded gbm are common in the progression of diabetic nephropathy and chronic kidney disease ( toyoda et al . , 2007 ; weil et al . , 2012 ; kriz and lemley , 2015 ) , whereas endothelial detachment has been observed in vegfa - null mutant mice ( eremina et al . , 2003 ) . interestingly , comparison of the elastic properties of purified glomeruli by atomic force microscopy reveals that glomerular rigidity is reduced by as much as 30% in mouse models of alport syndrome ( col4a3 knockout ) and hiv - induced nephropathy ( hivan ) before the onset of overt pathological histology ( wyss et al . , 2011 ) . increased glomerular deformability correlating with increased permeability of the gfb is likely symptomatic of an aberrant interaction between the gbm and the cells attached to it . an attractive hypothesis based on electrokinetic principles has been proposed to account for the charge selectivity in renal filtration . micropuncture measurements on salamander ( necturus maculosus ) glomeruli demonstrate that filtration pressure establishes a distinctive streaming potential or charge difference across the gfb , with the bowman s space being more negative than the endothelial lumen ( fig . the phenomenon of streaming potentials arises when electrolytes are forced by a pressure gradient across porous media or a channel carrying a permanent charge . in essence , the electrokinetic model posits that when small cations traverse the gfb , they bind and counterbalance the negatively charged surfaces within the gfb , reaching a threshold at which net ionic movement of small cations lags behind that of small anions ( hausmann et al . , 2012 ; moeller and tenten , 2013 ) . this differential advance of oppositely charged small ions thereby establishes a net charge separation and a measurable electrical field that polarizes the gfb . the streaming potential hypothesis therefore predicts that larger anions such as native albumin would encounter a retrograde electrophoretic field running opposite to the direction of hydraulic flux ( fig . 4 c ) . given the minor importance of negative charges within the gbm , it can be inferred that the streaming potential is largely initiated from the highly charged esl . the finding that neutral albumin traverses the gfb independent of the glomerular filtration rate whereas native anionic albumin passage becomes increasingly more restricted with increasing glomerular pressure is congruent with an electrokinetic model of the gfb ( lund et al . , similarly , albumin readily diffuses and equilibrates across the gfb once plasma flow is halted ( ryan and karnovsky , 1976 ) . ( a ) experimental setup used to demonstrate the existence of a flow - dependent electrical potential ( streaming potential ) across the gfb in salamander ( n. maculosus ) glomeruli ( hausmann et al . small ions , due to differential interaction with the negatively charged gfb , create a net gradient of charges measurable as a streaming potential ( blue arrows ) , making the endothelial lumen ( el ) more positive than the bowman s space ( bs ) . , macromolecules encounter a dynamic electrophoretic field ( green arrow ) that is opposite to that of diffusive and convective fluxes ( purple arrow ) . albumin , a negatively charged macromolecule , would not only encounter size - dependent exclusion by the gfb but would effectively be electrophoresed away from the gfb during the course of active filtration . the hypothesis also predicts that podocyte fp effacement can be detrimental to the generation of a streaming potential as more rapidly advancing small anions bounce back upon encountering the broadened fps , causing the electrical field across the gfb to be short - circuited ( hausmann et al . , 2010 , 2012 ) . hence , normal podocytes with their elaborate and regular network of fps and sds guarantees that streaming potential and filtration occur uniformly across the gfb . further proof regarding this prediction is needed and should ideally be based on recording glomerular streaming potentials in the context of proteinuric disease models in mice . nevertheless , in salamander glomeruli , streaming potentials are reversibly blocked by protamine , a polycationic protein that neutralizes the negative charge of the gfb and is well - known to induce proteinuria ( hausmann et al . , 2010 ) . the robustness of the gfb depends on the plasticity and the dynamic signaling between its distinctive layers . vigorous investigations on this subject over the years have shown that targeted damage to any one layer can lead to collapse of the gfb , and that in many cases compromising one layer has inevitable deleterious repercussions for the other layers . these highlight an emerging theme that the gfb , despite being multilayered , consists of components with dynamically intertwined roles , no single one of which is more important than the others , that harmonize together into one functionally elegant ensemble . continued efforts to refine our understanding of the mechanism of renal filtration and the biology of the gfb are invaluable for the development of better therapeutic strategies to alleviate the burden of proteinuric diseases .

the function of the kidney , filtering blood and concentrating metabolic waste into urine , takes place in an intricate and functionally elegant structure called the renal glomerulus . normal glomerular function retains circulating cells and valuable macromolecular components of plasma in blood , resulting in urine with just trace amounts of proteins . endothelial cells of glomerular capillaries , the podocytes wrapped around them , and the fused extracellular matrix these cells form altogether comprise the glomerular filtration barrier , a dynamic and highly selective filter that sieves on the basis of molecular size and electrical charge . current understanding of the structural organization and the cellular and molecular basis of renal filtration draws from studies of human glomerular diseases and animal models of glomerular dysfunction .

Selective permeability in renal filtration Fenestrated capillaries as primary portals of renal filtration The GBM: A highly ordered ECM and filtration bed The final gatekeepers: Renal podocytes and their slit diaphragms (SDs) The importance of cell adhesion to the GBM Streaming potential and charge selectivity in renal filtration Conclusions

PMC1160246

the integration of genomic sequences with transcription factor function and gene expression to decipher the gene regulatory networks underlying various developmental processes is a major challenge of the post - genomic era ( 1 ) . although the view that regulatory regions manifest as clusters of transcription factor binding sites ( tfbss ) has been around for some time , it was the review by arnone and davidson ( 2 ) that clearly presented the case for emphasizing cis - clusters in both experimental and computational analyses . in fact , it is this paradigm shift that led to important advances in the detection of combinatorial occurrence of cis - elements and understanding transcriptional regulation ( 3 ) . however , the availability of a number of completely sequenced eukaryotic genomes with an ever expanding volume of gene expression profile data has made computationally based strategies for deciphering genetic regulatory networks more viable . the methods range from sophisticated gibbs sampling - based algorithms to more brute force counting and analysis of fixed - length oligonucleotide words ( kmer or ktuple word searching ) ( 4,5 ) . for a complete list of internet resources and tools available to predict transcription regulatory clusters , computational methods have focused primarily on trying to identify the co - occurrence of a set of tfbss in a group of genes co - expressed or functionally related , and most of them have been restricted to the promoter or upstream regions . however , the basic problem of identifying the true positives from a list of combinatorial patterns remains . the problem becomes even more complicated and the results are difficult to interpret when the entire stretch of non - coding regions comprising introns and upstream and downstream regions is considered . adopting a phylogenetic approach allows substantial reduction in the number of false positives in the identification of regulatory regions of individual orthologous gene pairs ( 712 ) . although the need for experimental validation remains critical , at present , predicted cis - acting signature element searches can greatly focus experimental targets for validation studies . the detection of a particular known cis - acting element in all or many of the genes in a particular expression cluster does not necessarily mean that the genes are regulated via that element . the likelihood of this prediction is greater if each of these shared clusters is also conserved in the corresponding inter - species ortholog . cismols analyzer is built based on these two hypotheses and is designed to identify significant cis - regulatory elements from sets of co - expressed or related groups of genes for elements that are also ortholog - conserved . to do this , ortholog - conserved cis - clusters for each individual gene pair are identified and stored in the database . next , a gene list is compiled based on various criteria such as coordinate regulation and then the ortholog - conserved cis - clusters for each of the genes in the list are compared to identify occurrence of common cis - clusters . since the existence of gene regulatory regions in intronic and downstream regions is well proven , our method to identify these sites is not confined to upstream regions alone , but is extended to intronic and 5 and 3 gene - flanking regions . we have successfully validated our algorithm on several data sets comprising skeletal muscle - specific genes , liver - specific genes , pancreas overexpressed genes , olfactory genes ( 13 ) and immune system genes ( 14 ) . genomic regions of orthologous genes are retrieved from ucsc golden path , along with the exon annotations . putative regulatory regions are identified either by using our earlier developed trafac server ( 12 ) or by searching against the potential regulatory regions stored in the genometrafac database (; jegga et al . the conserved cis - element dense regions for each of the ortholog gene pairs are compared to identify the common binding sites in a group of genes . researchers can automatically ( i ) create gene groups and identify shared ortholog - conserved putative regulatory regions and individual binding sites , ( ii ) search genes for known cis - regulatory modules and ( iii ) identify potential novel gene targets for known cis - regulatory modules or novel clusters of individual binding sites . cismols analyzer is designed to analyze a list of genes typically co - expressed or related genes for cis - element clusters that are shared by genes in the list . in contrast , genometrafac is a whole - genome repository of individual genes with specified gene orthologs that have been analyzed , in batch form over the entire genome , for phylogenetically conserved cis - elements ( jegga et al . , manuscript submitted ) . trafac is similarly single - gene oriented , but it allows for the entry of human - curated ortholog gene pairs ( 12 ) . cismols analyzer operates on genes in either database by allowing lists of these genes to be formed and then subjecting the lists to shared cis - element analysis . it is possible to analyze existing gene groups that have been assembled by the system administrators and by other users . however , to create new groups and perform a clustering analysis to detect modules that contain shared cis - elements , a login account is needed . options are also provided to select the genomic regions for a single gene or a group of genes that need to be searched for the occurrence of common tfbss . by default , cismols analyzer searches for clusters in the genomic region comprising the 5- and 3-flanking 10 kb and also the intronic regions . after submitting the genes for analysis , the user will be notified by email of the availability of the results when the clustering is finished . users can customize the search criteria using boolean logic to restrict the search to known validated cis - regulatory modules and/or a combination of individual binding sites . the minimum number of binding sites that must appear in each cluster , or the minimum number of genes in which each cluster must appear , can also be specified . cismols analyzer is designed to analyze a list of genes typically co - expressed or related genes for cis - element clusters that are shared by genes in the list . in contrast , genometrafac is a whole - genome repository of individual genes with specified gene orthologs that have been analyzed , in batch form over the entire genome , for phylogenetically conserved cis - elements ( jegga et al . , manuscript submitted ) . trafac is similarly single - gene oriented , but it allows for the entry of human - curated ortholog gene pairs ( 12 ) . cismols analyzer operates on genes in either database by allowing lists of these genes to be formed and then subjecting the lists to shared cis - element analysis . it is possible to analyze existing gene groups that have been assembled by the system administrators and by other users . however , to create new groups and perform a clustering analysis to detect modules that contain shared cis - elements , a login account is needed . options are also provided to select the genomic regions for a single gene or a group of genes that need to be searched for the occurrence of common tfbss . by default , cismols analyzer searches for clusters in the genomic region comprising the 5- and 3-flanking 10 kb and also the intronic regions . after submitting the genes for analysis , the user will be notified by email of the availability of the results when the clustering is finished . users can customize the search criteria using boolean logic to restrict the search to known validated cis - regulatory modules and/or a combination of individual binding sites . the minimum number of binding sites that must appear in each cluster , or the minimum number of genes in which each cluster must appear , can also be specified . the output generated is a set of putative regulatory modules occurring within an ortholog conserved region of two or more genes . cismolgram ( figure 1 ) , a graphical representation of ortholog - conserved cis - clusters shared by a group of genes , depicts the location of clusters within gene regions . the shared cis - clusters ( two or more than two binding sites ) are represented as variously colored boxes on the gene . each of these shared clusters is linked to its respective ortholog conserved regions and can be visualized as trafacgrams or regulograms ( described in the legend of figure 1 ) . the trafac and regulogram image pages have the option to download the sequences ( corresponding to that cluster window ) in fasta format . links are also provided to the human and mouse ucsc browser , in which the user can see a cismols - identified cluster in the context of other annotations . users can also download the ucsc browser - compatible gff files ( currently gff3 ) for each sequence from the regulogram image page . these can be uploaded directly to ucsc golden path , enabling visualization of the cismols cluster region in the context of all other features available as aligned annotation tracks ( known genes , predicted genes , ests , mrnas , cpg islands , assembly gaps and coverage , chromosomal bands , other species homologies and more ) . the table view or the legend ( figure 1d ) displays a summary of the results . it indicates the details of each cluster ( the individual constituent cis - elements and their total frequency ) and the frequency of the clusters occurring in each of the genes compared and involved in cluster analysis . the default base sequence is the human gene , and it is mapped to the corresponding mouse ortholog . however , the program can be used for any ortholog gene pair after uploading their alignments and binding sites to the trafac database . options are also provided to modify the viewable regions to focus on cis - clusters of interest . the cismolgram can be saved or exported as images ( svg , tiff , pdf , png or jpg format ) . the time taken to analyze a group of genes depends upon factors such as number of genes in the group , gene lengths , percentage conservation between the orthologous pair of genes , and ortholog - conserved cis - element clusters . cismols analyzer looks for conserved cis - element clusters within conserved regions [ blastz - aligned genomic regions of 70% sequence similarity ( 15 ) ] . matinspector ( 16 ) is used to identify the potential binding sites in each of the genomic sequences . the analysis parameters for identification of tfbss were set to 0.85 for the core similarity and optimal for the matrix similarity . a typical analysis on the cismols server for , say , 50 ortholog gene pairs with default parameter settings of 10 kb upstream and downstream for each gene takes approximately 1 h. the current upper limit of processing capability is about 242 ortholog gene pairs ( a total sequence length of 32 mb , of which about 7 mb are blastz - aligned with 70% sequence similarity ) . currently we are also working on providing the statistical significance and comparison between cis - clusters identified for two discrete gene groups . most of the time , the cis - element clusters responsible for tissue specificity tend to be scored relatively low . for example , searching for ortholog - conserved shared cis - clusters in a group of pancreas overexpressed genes without any cis - element filter resulted in the identification of non - specific clusters . however , when the search was performed again restricting the results to only those clusters that have at least one pdx binding site , the resulting shared clusters coincided with the validated regulatory regions of each of the individual genes ( data not shown ) . the top hits , or the cis - element clusters shared by the most genes , tend to be more general , and , although they are important for gene expression , very little knowledge can be extracted from these about conferring tissue specificity for a group of genes . using a control or a negative control does , however , improve understanding of the importance of the shared clusters for instance , comparing the most shared cis - clusters in a group of genes with overexpression in the liver with genes overexpressed in the cerebellum . clearly , there is a paradox in the phylogenetic footprinting approach . to allow the recognition of conserved ( regulatory ) elements , there should be enough evolutionary distance , but , at the same time , this evolutionary distance makes it difficult to recognize tfbss the short conserved elements . nevertheless , the significance of a predicted shared cis - regulatory module for a group of co - expressed genes or a functionally related group of genes will be higher if the shared clusters are additionally conserved in both gene orthologs . detection and visualization of compositionally similar cis - regulatory modules shared by multiples genes . ( a ) cismolgram provides a depiction of cis - element modules that are shared across two or more genes and contain two or more binding sites . each module is represented by variously colored boxes located relative to the promoter of each gene in the analyzed group . genes gathered from the genometrafac database generally have their first exon locations at position 40 000 . the locations of the clusters can be selected to be shown over either set of gene orthologs . by clicking on a cluster module ( circled ) , a link is given to view either ( b ) the density of conserved elements over the regions ( regulogram ) or ( c ) the conserved cis - element arrangement at that location ( trafac image ) . the regulogram shows the two sequences , mouse and human , represented as horizontal bars . the green - colored bars shown parallel to the genomic sequences represent the repeat regions . the regions of sequence alignment are represented as differently colored quadrilaterals that relate one sequence to another . within each shaded block , the percentage sequence similarity and the number of tfbss are represented as two separate line graphs in the lower half . the frequencies of individual binding sites occurring in each of the sequences separately are shown as two running graphs in the top half of the pane . the percentage similarity is the average sequence conservation as determined by the blastz algorithm and the shared cis - element hits are determined by an algorithm that uses a 200 bp moving window to look through the cis - elements present within the conserved sequence block . the regulogram can be clicked to zoom in or to view the tfbss that are common to the two sequences at the click - point coordinate . in the trafac image , the two gray vertical bars are the two genes ( human and mouse ) compared . the tfbss occurring in both the genes are highlighted as variously colored bars drawn across the two genes . ( d ) the table view or the cismolgram legend displays a summary of the results . it indicates the details of each cluster ( the constituent individual cis - elements and their total frequency ) and the frequency of the clusters occurring in each of the genes compared and involved in cluster analysis .

combinatorial interactions of sequence - specific trans - acting factors with localized genomic cis - element clusters are the principal mechanism for regulating tissue - specific and developmental gene expression . with the emergence of expanding numbers of genome - wide expression analyses , the identification of the cis - elements responsible for specific patterns of transcriptional regulation represents a critical area of investigation . computational methods for the identification of functional cis - regulatory modules are difficult to devise , principally because of the short length and degenerate nature of individual cis - element binding sites and the inherent complexity that is generated by combinatorial interactions within cis - clusters . filtering candidate cis - element clusters based on phylogenetic conservation is helpful for an individual ortholog gene pair , but combining data from cis - conservation and coordinate expression across multiple genes is a more difficult problem . to approach this , we have extended an ortholog gene - pair database with additional analytical architecture to allow for the analysis and identification of maximal numbers of compositionally similar and phylogenetically conserved cis - regulatory element clusters from a list of user - selected genes . the system has been successfully tested with a series of functionally related and microarray profile - based co - expressed ortholog pairs of promoters and genes using known regulatory regions as training sets and co - expressed genes in the olfactory and immunohematologic systems as test sets . cismols analyzer is accessible via a web interface at .

INTRODUCTION INPUT Creating and submitting gene groups for analysis Searching for OUTPUT SOFTWARE AND ACCESS CONCLUSION Figures and Tables

PMC4473852

the clinical high - risk state of psychosis ( at - risk mental state , hereafter arms ) is defined by attenuated positive psychotic symptoms , genetic liability and functional deterioration or brief and self - remitting psychotic symptoms ( fusar - poli et al . , 2013 ; yung et al . , 1998 ) . however , affective symptoms , including depressive and anxiety symptoms , are also highly prevalent in these individuals ( salokangas et al . , 2012 ) . a recent meta - analysis , conducted in 1683 high - risk subjects , confirmed that the baseline prevalence of comorbid depressive and anxiety disorder is 41% and 15% , respectively ( fusar - poli et al . , 2014a ) . depressive and anxiety symptoms can precede the onset of attenuated positive psychotic symptoms ( fusar - poli et al . , 2013 ) . some studies indicate that co - occurrence of depressive disorders can predict subsequent transition to psychosis in arms individuals ( salokangas et al . , 2012 ) . however , other studies have not confirmed this finding ( fusar - poli et al . , 2014a ) . additionally , a large study on 3349 twins suggests an association between depressive and/or anxiety symptoms and psychosis - like traits ( schizotypy ) and emphasizes a major role for genetics , especially as regards positive symptoms ( macare et al . , 2012 ) . the comorbidity of psychotic and depressive disorders in the arms population is associated with specific psychopathological features at the time of the presentation to high risk services and with low functional level ( fusar - poli et al . , 2013 ) . because of these problems , clinical high - risk individuals often receive antidepressant medication ( e.g. 42% of arms individuals in our previous study ( smieskova et al . , 2012a ) ) . negative psychotic symptoms are a major source of disability in the psychosis spectrum and are refractory to any effective treatment ( fusar - poli et al . , 2014b ) . negative symptoms group into two factors , one involving diminished expression of affect and alogia and the second involving avolition , including anhedonia and asociality ( fusar - poli et al . , 2014b ) . antidepressants may have a potential benefit for arms individuals , as they may target their negative attenuated psychotic symptoms ( cornblatt et al . however , it is not clear if these improvements are associated with underlying neurobiological changes ( wood et al . , 2011 ) . neuroimaging studies using magnetic resonance imaging ( mri ) have indicated that arms showed brain alterations in the prefrontal ( borgwardt et al . , 2006 ; borgwardt et al . , 2008 ; koutsouleris et al . , 2009 ; mechelli et al . , 2011 ; wood et al . , 2010 ) , cingulate ( fornito et al . , 2008 ; koutsouleris et al . , 2009 ) , superior ( takahashi et al . , 2009 ; takahashi et al . , 2010 ) and medial temporal ( borgwardt et al . , 2007b ; tognin et al . , 2014 ) , insular ( smieskova et al . , 2010 ) and cerebellar regions when compared to healthy controls . furthermore , arms individuals with subsequent transition to psychosis showed volumetric reductions in the prefrontal , insular and cingulate cortex compared to those without transition ( smieskova et al . , 2010 ) . reductions in gray matter volume ( gmv ) in the anterior cingulate gyrus , hippocampus , amygdala ( koolschijn et al . , 2009 ) and prefrontal cortex ( lorenzetti et al . , 2009 ) were associated with major depression . the only available study directly testing the effect of comorbid depressive disorders on the neurobiology of arms uncovered a significant impact on the anterior cingulate region ( modinos et al . , 2014 ) . on the other hand , long - term antidepressant medication can be neuroprotective and some studies have linked the use of antidepressants to an increase in hippocampal volume in patients with major depressive disorder ( amico et al . , 2011 ; it has been shown that antidepressants increase hippocampal neurogenesis ( anacker et al . , 2011 ) . thus , both affective symptoms ( baynes et al . , 2000 ) and antidepressant medication ( kraus et al . , 2014 ) , we addressed for the first time the effect of antidepressant treatment and attenuated negative psychotic symptoms on the neurobiology of arms . firstly , we hypothesized that arms individuals without current antidepressant treatment ( arms - nonad ) would manifest more severe attenuated negative symptoms than arms subjects currently receiving antidepressants ( arms - ad ) . secondly , we hypothesized that arms - ad individuals would have increased gmv in regions associated with depressive symptoms and/or antidepressant medication ( hippocampus , anterior cingulate gyrus , amygdala and precuneus ) compared to the arms - nonad individuals . thirdly , we hypothesized that the volumetric abnormalities in gray matter between arms - ad and arms - nonad would be associated with attenuated negative symptoms . mri data were collected within the framework of a research program on the early detection of psychosis . the subjects were recruited in our specialized clinic for the early detection of psychosis ( fepsy ) at the psychiatric outpatient department , psychiatric university clinics basel , switzerland ( riecher - rssler et al . , 2006 ) . the entire group of arms individuals ( n = 49 ) conforms to yung 's criteria ( yung et al . , 1998 ) and overlaps with previously published data ( borgwardt et al . all the arms individuals were antipsychotic - free and were assessed prior to the neuroimaging session . arms inclusion required one or more of the following:(a)attenuated psychotic symptoms that do not reach full psychosis threshold(b)brief limited intermittent psychotic symptoms ( lasting less than a week with spontaneous remission)(c)a first degree relative with a psychotic disorder plus at least two indicators of a clinical change , such as a marked decline in social or occupational functioning . attenuated psychotic symptoms that do not reach full psychosis threshold brief limited intermittent psychotic symptoms ( lasting less than a week with spontaneous remission ) a first degree relative with a psychotic disorder plus at least two indicators of a clinical change , such as a marked decline in social or occupational functioning . we assessed the subjects using the basel screening instrument for psychosis ( bsip ) ( riecher - rssler et al . , 2008 ) , the brief psychiatric rating scale ( bprs ) ( lukoff et al . , 1986 ) , the scale for the assessment of negative symptoms ( sans ) ( andreasen , 1989 ) and the global assessment of functioning ( gaf ) ( endicott et al . , 1976 ) . negative symptoms , calculated from the bprs as a sum of blunted affect , emotional withdrawal , and motor retardation ( bprs16 , bprs17 and bprs18 ) ( fusar - poli et al . , 2014b ; velligan et al . , 2005 ) . mood disturbance bprs cluster as a sum of anxiety , depression , suicidality and guilt ( bprs02 , bprs03 , bprs04 and bprs05 ) ( thomas et al . , 2004 ) , as well as depression ( bprs03 ) and motor retardation ( bprs18 ) scores alone . we used these scores for stepwise regression analysis with backward elimination . in a second step , we divided the arms individuals into two subgroups , based on whether they were currently being treated with antidepressants ( arms - ad , n = 18 ) or not ( arms - nonad , n = 31 ) ( table 1 ) . antidepressant medication within the ad subgroup included : fluoxetine ( ssri ; n = 2 ) , escitalopram ( ssri ; n = 5 ) , sertraline ( ssri ; n = 1 ) , mirtazapine [ nassa ( noradrenergic and specific serotonergic antidepressant ) ; n = 4 ] , venlafaxine [ ssnri ( selective serotonin norepinephrine reuptake inhibitor ) , n = 2 ] , duloxetine ( ssnri ; n = 2 ) , fluoxetine plus trazodone [ ssri , sari ( serotonin antagonist and reuptake inhibitor ) ; n = 1 ] and st . antidepressant therapy had a mean duration of 50 47 days ( range 4170 days ) . in order to exclude possible biases through antipsychotic therapy in addition , current and previous psychotropic medication , alcohol , nicotine , cannabis , and other illegal drug consumption were assessed using a semi - structured interview , as adapted from the drug and alcohol assessment schedule of the early psychosis prevention and intervention centre ( eppic ) . participants were excluded from the study if they presented with a history of previous psychotic disorder , psychotic symptomatology secondary to an organic disorder , substance abuse , affective psychosis , borderline personality disorder , age under 18 or over 40 , inadequate knowledge of the german language or iq less than 70 ( assessed by multiple - choice vocabulary intelligence test ) ( lehrl et al . , 1995 ) . healthy controls ( n = 24 ) were from the same geographical area as the other groups ( table 1 ) . all participants provided written informed consent . the study was approved by the local ethics committee . for structural imaging , a whole brain 3d t1-weighted mprage ( magnetization prepared rapid acquisition gradient ) sequence was applied using a 3 t magnetic resonance imaging scanner ( magnetom verio , siemens healthcare , erlangen , germany ) and a 12-channel radio frequency head coil . the acquisition was based on a sagittal matrix of 256 256 176 and 1 1 1 mm isotropic spatial resolution , with an inversion time of 1000 ms , repetition time of 2 s , echo time of 3.4 ms , flip angle of 8 and bandwidth of 200 hz / pixel . structural mri data were analyzed using the voxel - based morphometry toolbox ( vbm8 , http://dbm.neuro.uni-jena.de/vbm8/ ) , implemented within spm8 ( wellcome department of cognitive neurology , london , uk ) and running on matlab 7.11 ( mathworks , usa ) . images were then segmented into gray matter , white matter and cerebrospinal fluid using the adaptive maximum a posteriori technique ( in contrast to the classical use of a priori tissue probability maps ) , where local variations in the parameters are modeled by means of slowly varying spatial functions ( rajapakse et al . , 1997 ) . more accurate segmentation can be achieved with partial volume estimation of additional mixed tissue classes in every voxel . all images were dartel - normalized ( diffeomorphic anatomical registration using exponentiated lie algebra ; ashburner , 2007 ) . this method produces more accurate results for registration and additional registration in mni space was unnecessary ( ashburner , 2007 ) . finally , sample homogeneity was reviewed and all images were smoothed using an isotropic 8 mm full - width - at - half - maximum ( fwhm ) gaussian kernel ( shen and sterr , 2013 ) . group differences were explored using a one - way anova . since our groups differed significantly in gender and age , we introduced these two variables as additional covariates of interest . brain region labeling was achieved using the harvard oxford structural atlas ( http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/atlases ( desikan et al . , 2006 ) ) , incorporated within the fmrib software library ( fsl ) . statistical significance was assessed at a cluster level using a threshold of p < 0.005 uncorrected ( cluster - forming threshold ) ; statistical inference was then made at p < 0.05 , adjusted to provide a family - wise error ( fwe ) correction at the peak and cluster levels . on the basis of the previous evidence , we defined 4 specific regions of interest ( rois ) to test for differences in gmv between our two arms groups : the bilateral hippocampus , ( associated with greater risk of depressive disorder ; amico et al . , 2011 ; vasconcelos et al . , 2011 ) , the anterior cingulate gyrus ( reported to be reduced during ongoing depression ; amico et al . , 2011 ; koolschijn et al . , 2009 ) and in co - morbid depression in arms ( modinos et al . , 2014 ) ; the amygdala ( inconsistent changes in depression ; koolschijn et al . , 2009 ) and the precuneus ( associated with increased gray matter density after short antidepressant application in hc ; kraus et al . , 2014 ) . all the regions were individually defined using the wake forest university pickatlas toolbox ( http://fmri.wfubmc.edu/software/pickatlas ) . for each region , a small volume correction was conducted using a 5 mm radius for the hippocampus ( amico et al . , 2011 ; vasconcelos et al . , 2011 ) and amygdala or a 10 mm radius for the precuneus and anterior cingulate cortex ( acc ) ( abutalebi et al . , 2012 ; amico et al . , 2011 ) . mean gray matter volume indices were extracted from these regions using the rex toolbox ( http://web.mit.edu/swg/software.htm ) implemented in matlab 7.11 . the analysis was performed on region of interest basis , with no conjunction mask , no scaling and extraction of the mean within the predefined roi . the extracted values were used for a stepwise backward regression analysis ( see supplementary table ) . only corrected family - wise error values were taken into consideration , in order to avoid a type i error . mean gray matter volume indices were extracted from these regions using the rex toolbox ( http://web.mit.edu/swg/software.htm ) implemented in matlab 7.11 . the analysis was performed on region of interest basis , with no conjunction mask , no scaling and extraction of the mean within the predefined roi . the extracted values were used for a stepwise backward regression analysis ( see supplementary table ) . only corrected family - wise error values were taken into consideration , in order to avoid a type i error . clinical and socio - demographic differences were assessed using one - way anova and -test . for post hoc analysis , the bonferroni correction was conducted . in addition , a stepwise regression analysis with backward elimination was applied , to restrict correlating variables . we included the bprs total score and sans total score ; as well as the bprs clusters for negative symptoms as a sum of blunted affect , emotional withdrawal , and motor retardation ( bprs16 , bprs17 and bprs18 ) ; and mood disturbance as a sum of anxiety , depression , suicidality and guilt ( bprs02 , bprs03 , bprs04 and bprs05 ) ; and the single scores depression ( bprs 3 ) and motor hyperactivity ( bprs 23 ) ( see supplementary table ) . we applied outlier detection using cook 's distance test and no subject had to be excluded from regression analysis . still , we excluded one arms - nonad individual due to missing data in the bprs 16 , 17 , and 18 necessary for calculating the cluster negative symptoms. we then performed a correlation with our significant clinical parameters from the stepwise regression . from the regions of interest with significant differences between arms - ad and arms - nonad ( hippocampus and precuneus ) the data were normally distributed and we performed a series of two - tailed pearson 's correction analyses with statistical threshold set at p < 0.05 . all analyses were performed using the statistical package for the social science ( spss , version 22 ) . clinical and socio - demographic differences were assessed using one - way anova and -test . for post hoc analysis , the bonferroni correction was conducted . in addition , a stepwise regression analysis with backward elimination was applied , to restrict correlating variables . we included the bprs total score and sans total score ; as well as the bprs clusters for negative symptoms as a sum of blunted affect , emotional withdrawal , and motor retardation ( bprs16 , bprs17 and bprs18 ) ; and mood disturbance as a sum of anxiety , depression , suicidality and guilt ( bprs02 , bprs03 , bprs04 and bprs05 ) ; and the single scores depression ( bprs 3 ) and motor hyperactivity ( bprs 23 ) ( see supplementary table ) . we applied outlier detection using cook 's distance test and no subject had to be excluded from regression analysis . still , we excluded one arms - nonad individual due to missing data in the bprs 16 , 17 , and 18 necessary for calculating the cluster negative symptoms. we then performed a correlation with our significant clinical parameters from the stepwise regression . from the regions of interest with significant differences between arms - ad and arms - nonad ( hippocampus and precuneus ) , we extracted the volumes and used them in our in correlation calculations . the data were normally distributed and we performed a series of two - tailed pearson 's correction analyses with statistical threshold set at p < 0.05 . all analyses were performed using the statistical package for the social science ( spss , version 22 ) . mri data were collected within the framework of a research program on the early detection of psychosis . the subjects were recruited in our specialized clinic for the early detection of psychosis ( fepsy ) at the psychiatric outpatient department , psychiatric university clinics basel , switzerland ( riecher - rssler et al . , 2006 ) . the entire group of arms individuals ( n = 49 ) conforms to yung 's criteria ( yung et al . , 1998 ) and overlaps with previously published data ( borgwardt et al . all the arms individuals were antipsychotic - free and were assessed prior to the neuroimaging session . arms inclusion required one or more of the following:(a)attenuated psychotic symptoms that do not reach full psychosis threshold(b)brief limited intermittent psychotic symptoms ( lasting less than a week with spontaneous remission)(c)a first degree relative with a psychotic disorder plus at least two indicators of a clinical change , such as a marked decline in social or occupational functioning . attenuated psychotic symptoms that do not reach full psychosis threshold brief limited intermittent psychotic symptoms ( lasting less than a week with spontaneous remission ) a first degree relative with a psychotic disorder plus at least two indicators of a clinical change , such as a marked decline in social or occupational functioning . we assessed the subjects using the basel screening instrument for psychosis ( bsip ) ( riecher - rssler et al . , 2008 ) , the brief psychiatric rating scale ( bprs ) ( lukoff et al . , 1986 ) , the scale for the assessment of negative symptoms ( sans ) ( andreasen , 1989 ) and the global assessment of functioning ( gaf ) ( endicott et al . , 1976 ) . negative symptoms , calculated from the bprs as a sum of blunted affect , emotional withdrawal , and motor retardation ( bprs16 , bprs17 and bprs18 ) ( fusar - poli et al . , 2014b ; velligan et al . , 2005 ) . mood disturbance bprs cluster as a sum of anxiety , depression , suicidality and guilt ( bprs02 , bprs03 , bprs04 and bprs05 ) ( thomas et al . , 2004 ) , as well as depression ( bprs03 ) and motor retardation ( bprs18 ) scores alone . we used these scores for stepwise regression analysis with backward elimination . in a second step , we divided the arms individuals into two subgroups , based on whether they were currently being treated with antidepressants ( arms - ad , n = 18 ) or not ( arms - nonad , n = 31 ) ( table 1 ) . antidepressant medication within the ad subgroup included : fluoxetine ( ssri ; n = 2 ) , escitalopram ( ssri ; n = 5 ) , sertraline ( ssri ; n = 1 ) , mirtazapine [ nassa ( noradrenergic and specific serotonergic antidepressant ) ; n = 4 ] , venlafaxine [ ssnri ( selective serotonin norepinephrine reuptake inhibitor ) , n = 2 ] , duloxetine ( ssnri ; n = 2 ) , fluoxetine plus trazodone [ ssri , sari ( serotonin antagonist and reuptake inhibitor ) ; n = 1 ] and st . antidepressant therapy had a mean duration of 50 47 days ( range 4170 days ) . in order to exclude possible biases through antipsychotic therapy in addition , current and previous psychotropic medication , alcohol , nicotine , cannabis , and other illegal drug consumption were assessed using a semi - structured interview , as adapted from the drug and alcohol assessment schedule of the early psychosis prevention and intervention centre ( eppic ) . participants were excluded from the study if they presented with a history of previous psychotic disorder , psychotic symptomatology secondary to an organic disorder , substance abuse , affective psychosis , borderline personality disorder , age under 18 or over 40 , inadequate knowledge of the german language or iq less than 70 ( assessed by multiple - choice vocabulary intelligence test ) ( lehrl et al . , 1995 ) . healthy controls ( n = 24 ) were from the same geographical area as the other groups ( table 1 ) . all participants provided written informed consent . for structural imaging , a whole brain 3d t1-weighted mprage ( magnetization prepared rapid acquisition gradient ) sequence was applied using a 3 t magnetic resonance imaging scanner ( magnetom verio , siemens healthcare , erlangen , germany ) and a 12-channel radio frequency head coil . the acquisition was based on a sagittal matrix of 256 256 176 and 1 1 1 mm isotropic spatial resolution , with an inversion time of 1000 ms , repetition time of 2 s , echo time of 3.4 ms , flip angle of 8 and bandwidth of 200 hz / pixel . structural mri data were analyzed using the voxel - based morphometry toolbox ( vbm8 , http://dbm.neuro.uni-jena.de/vbm8/ ) , implemented within spm8 ( wellcome department of cognitive neurology , london , uk ) and running on matlab 7.11 ( mathworks , usa ) . images were then segmented into gray matter , white matter and cerebrospinal fluid using the adaptive maximum a posteriori technique ( in contrast to the classical use of a priori tissue probability maps ) , where local variations in the parameters are modeled by means of slowly varying spatial functions ( rajapakse et al . , 1997 ) . more accurate segmentation can be achieved with partial volume estimation of additional mixed tissue classes in every voxel . all images were dartel - normalized ( diffeomorphic anatomical registration using exponentiated lie algebra ; ashburner , 2007 ) . this method produces more accurate results for registration and additional registration in mni space was unnecessary ( ashburner , 2007 ) . finally , sample homogeneity was reviewed and all images were smoothed using an isotropic 8 mm full - width - at - half - maximum ( fwhm ) gaussian kernel ( shen and sterr , 2013 ) . since our groups differed significantly in gender and age , we introduced these two variables as additional covariates of interest . brain region labeling was achieved using the harvard oxford structural atlas ( http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/atlases ( desikan et al . , 2006 ) ) , incorporated within the fmrib software library ( fsl ) . statistical significance was assessed at a cluster level using a threshold of p < 0.005 uncorrected ( cluster - forming threshold ) ; statistical inference was then made at p < 0.05 , adjusted to provide a family - wise error ( fwe ) correction at the peak and cluster levels . on the basis of the previous evidence , we defined 4 specific regions of interest ( rois ) to test for differences in gmv between our two arms groups : the bilateral hippocampus , ( associated with greater risk of depressive disorder ; amico et al . , 2011 ; vasconcelos et al . , 2011 ) , the anterior cingulate gyrus ( reported to be reduced during ongoing depression ; amico et al . , 2011 ; koolschijn et al . , 2009 ) and in co - morbid depression in arms ( modinos et al . , 2014 ) ; the amygdala ( inconsistent changes in depression ; koolschijn et al . , 2009 ) and the precuneus ( associated with increased gray matter density after short antidepressant application in hc ; kraus et al . , 2014 ) . all the regions were individually defined using the wake forest university pickatlas toolbox ( http://fmri.wfubmc.edu/software/pickatlas ) . for each region , a small volume correction was conducted using a 5 mm radius for the hippocampus ( amico et al . , 2011 ; vasconcelos et al . , 2011 ) and amygdala or a 10 mm radius for the precuneus and anterior cingulate cortex ( acc ) ( abutalebi et al . , 2012 ; amico et al . , 2011 ) . mean gray matter volume indices were extracted from these regions using the rex toolbox ( http://web.mit.edu/swg/software.htm ) implemented in matlab 7.11 . the analysis was performed on region of interest basis , with no conjunction mask , no scaling and extraction of the mean within the predefined roi . the extracted values were used for a stepwise backward regression analysis ( see supplementary table ) . only corrected family - wise error values were taken into consideration , in order to avoid a type i error . mean gray matter volume indices were extracted from these regions using the rex toolbox ( http://web.mit.edu/swg/software.htm ) implemented in matlab 7.11 . the analysis was performed on region of interest basis , with no conjunction mask , no scaling and extraction of the mean within the predefined roi . the extracted values were used for a stepwise backward regression analysis ( see supplementary table ) . only corrected family - wise error values were taken into consideration , in order to avoid a type i error . clinical and socio - demographic differences were assessed using one - way anova and -test . for post hoc analysis , the bonferroni correction was conducted . in addition , a stepwise regression analysis with backward elimination was applied , to restrict correlating variables . we included the bprs total score and sans total score ; as well as the bprs clusters for negative symptoms as a sum of blunted affect , emotional withdrawal , and motor retardation ( bprs16 , bprs17 and bprs18 ) ; and mood disturbance as a sum of anxiety , depression , suicidality and guilt ( bprs02 , bprs03 , bprs04 and bprs05 ) ; and the single scores depression ( bprs 3 ) and motor hyperactivity ( bprs 23 ) ( see supplementary table ) . we applied outlier detection using cook 's distance test and no subject had to be excluded from regression analysis . still , we excluded one arms - nonad individual due to missing data in the bprs 16 , 17 , and 18 necessary for calculating the cluster negative symptoms. we then performed a correlation with our significant clinical parameters from the stepwise regression . from the regions of interest with significant differences between arms - ad and arms - nonad ( hippocampus and precuneus ) the data were normally distributed and we performed a series of two - tailed pearson 's correction analyses with statistical threshold set at p < 0.05 . all analyses were performed using the statistical package for the social science ( spss , version 22 ) . clinical and socio - demographic differences were assessed using one - way anova and -test . for post hoc analysis , in addition , a stepwise regression analysis with backward elimination was applied , to restrict correlating variables . we included the bprs total score and sans total score ; as well as the bprs clusters for negative symptoms as a sum of blunted affect , emotional withdrawal , and motor retardation ( bprs16 , bprs17 and bprs18 ) ; and mood disturbance as a sum of anxiety , depression , suicidality and guilt ( bprs02 , bprs03 , bprs04 and bprs05 ) ; and the single scores depression ( bprs 3 ) and motor hyperactivity ( bprs 23 ) ( see supplementary table ) . we applied outlier detection using cook 's distance test and no subject had to be excluded from regression analysis . still , we excluded one arms - nonad individual due to missing data in the bprs 16 , 17 , and 18 necessary for calculating the cluster negative symptoms. we then performed a correlation with our significant clinical parameters from the stepwise regression . from the regions of interest with significant differences between arms - ad and arms - nonad ( hippocampus and precuneus ) the data were normally distributed and we performed a series of two - tailed pearson 's correction analyses with statistical threshold set at p < 0.05 . all analyses were performed using the statistical package for the social science ( spss , version 22 ) . the arms - ad , arms - nonad and hc showed significant differences in age at mri scan ( p = 0.02 ) , gender ( p = 0.01 ) , years of education ( p = 0.002 ) , smoking ( p = 0.001 ) , and cannabis ( p = 0.006 ) ; but no differences in alcohol consumption or handedness . post hoc analysis showed that smoking ( p = 0.006 ) and cannabis consumption ( p = 0.014 ) were significantly more common in the arms - ad group than in the arms - nonad group ( table 1 ) . we observed significant clinical differences between arms - ad , arms - nonad and hc in the total bprs score ( p < 0.0001 ) , total sans score ( p < 0.0001 ) , gaf total score ( p < 0.0001 ) , bprs cluster for negative symptoms ( p < 0.0001 ) , bprs mood disturbance ( p < 0.0001 ) , bprs depression score ( p < 0.0001 ) , and bprs motor hyperactivity score ( p = 0.005 ) . depression score ( p = 0.011 ) and less motor hyperactivity ( p = 0.029 ) than the arms - nonad ( table 1 ) . the test of our a priori defined contrast in attenuated negative symptoms ( bprs cluster for negative symptoms ) between arms - nonad and arms - ad found no significant difference ( p = 0.220 ) . compared to the hc , there was significantly less gmv in the middle frontal gyrus in the whole arms cohort , and in the superior frontal gyrus in the arms - ad group ( puncorr . the arms - nonad group showed reduced gmv in the left superior parietal lobe compared with the arms - ad group ( puncorr . the arms - ad group had less gmv in the left hippocampus ( fig . 1 ) and right precuneus than the arms - nonad ( pfwe - corr . < 0.05 after small volume correction , table 2 ) however , these results did not survive correction for multiple comparisons ( p < 0.0125 ) . negative symptoms cluster , both in arms subjects ( r = 0.314 , p = 0.030 , fig . 2 ) and in all our subjects , including the hc ( r = 0.293 , p = 0.013 ) . the arms - ad , arms - nonad and hc showed significant differences in age at mri scan ( p = 0.02 ) , gender ( p = 0.01 ) , years of education ( p = 0.002 ) , smoking ( p = 0.001 ) , and cannabis ( p = 0.006 ) ; but no differences in alcohol consumption or handedness . post hoc analysis showed that smoking ( p = 0.006 ) and cannabis consumption ( p = 0.014 ) were significantly more common in the arms - ad group than in the arms - nonad group ( table 1 ) . we observed significant clinical differences between arms - ad , arms - nonad and hc in the total bprs score ( p < 0.0001 ) , total sans score ( p < 0.0001 ) , gaf total score ( p < 0.0001 ) , bprs cluster for negative symptoms ( p < 0.0001 ) , bprs mood disturbance ( p < 0.0001 ) , bprs depression score ( p < 0.0001 ) , and bprs motor hyperactivity score ( p = 0.005 ) . depression score ( p = 0.011 ) and less motor hyperactivity ( p = 0.029 ) than the arms - nonad ( table 1 ) . the test of our a priori defined contrast in attenuated negative symptoms ( bprs cluster for negative symptoms ) between arms - nonad and arms - ad found no significant difference ( p = 0.220 ) . compared to the hc , there was significantly less gmv in the middle frontal gyrus in the whole arms cohort , and in the superior frontal gyrus in the arms - ad group ( puncorr . the arms - nonad group showed reduced gmv in the left superior parietal lobe compared with the arms - ad group ( puncorr . the arms - ad group had less gmv in the left hippocampus ( fig . 1 ) and right precuneus than the arms - nonad ( pfwe - corr . < 0.05 after small volume correction , table 2 ) however , these results did not survive correction for multiple comparisons ( p < 0.0125 ) . we found a negative correlation between the hippocampal volume and the bprs negative symptoms cluster , both in arms subjects ( r = 0.314 , p = 0.030 , fig . 2 ) and in all our subjects , including the hc ( r = 0.293 , p = 0.013 ) . in the present study , we addressed for the first time the effect on psychosis of antidepressant treatment and attenuated negative psychotic symptoms in arms individuals . firstly , we have found no evidence for our first hypothesis , that arms individuals suffer more pronounced attenuated negative psychotic symptoms if they have not been treated with antidepressants . we found that arms - ad individuals had a higher depression score , lower motor hyperactivity and smoked more cigarettes and marijuana than the arms - nonad individuals . the antidepressants that arms - ad individuals were receiving differed in their mode of action some inhibited the reuptake of serotonin and/or noradrenaline , while others enhanced the release of these monoamines ( andrade and rao , 2010 ) . moreover , the antidepressants may take weeks or longer to take effect after dosage ( penn and tracy , 2012 ) . the duration of antidepressant therapy in the arms - ad group varies from 4 to 170 days and thus the observed effect could be indicative of both the predominant depressive and/or attenuated negative psychotic symptoms or of the already developed antidepressant effect of the medication . hence , we can not clearly distinguish the extent to which each of the two components contributes to the current clinical state . secondly , we did not find increased gmv in the regions associated with depressive symptoms in those arms who were receiving antidepressants , compared to those without this medication . thus we could not confirm our second hypothesis , but found gmv deficits in the hippocampus and precuneus only in the arms individuals currently receiving antidepressant medication . our third hypothesis is related to the volumetric abnormalities in arms and their association with the attenuated negative symptoms ; we confirmed this relationship in the hippocampus . we found a clear negative correlation between the bilateral hippocampal volume and attenuated negative symptoms in all arms individuals . this corresponds to studies linking the hippocampus with psychosis ( jun et al . , 2012 ) . previous studies similarly either found negative correlations between the left hippocampal volume and negative symptoms in schizophrenics ( rajarethinam et al . , 2001 ) , or at least a strong trend in this direction ( brambilla et al . , 2013 ) . these findings underline the role of the hippocampus in the pathophysiology of schizophrenia and suggest specific associations between individual structures and both the positive and negative symptoms of the illness ( khn et al . , 2012 ; rajarethinam et al . , 2001 ) . thus , our findings support the importance of hippocampal structures as a region of interest in the early stage of psychosis ( benetti et al . , 2009 ; fusar - poli et al . , 2012 ; walter et al . , 2012 ) our roi analysis demonstrated smaller gmv in the left hippocampus in the arms - ad group than in the arms - nonad group . the hippocampus is involved in various psychiatric conditions , including major depression and psychosis ( videbech and ravnkilde , 2004 ; walter et al . , three meta - analyses have confirmed significant reductions in the hippocampal volume in depression ( campbell et al . , 2004 ; cole et al . , 2011 ; furthermore , the total number of depressive episodes was significantly correlated to the reduction in the right hippocampal volume ( videbech and ravnkilde , 2004 ) . the left - hemispheric deficits in hippocampal volume may reflect brain degeneration , as a consequence of chronic stress ( schmidt and duman , 2010 ) . recent data on antipsychotic - free arms have confirmed that vulnerability to psychosis may be associated with a significant decrease in hippocampal volume ( fusar - poli et al . , 2012 ; wood et al . , 2010 ) . in the right precuneus , we found less gmv in the arms - ad group than in the arms - nonad group . 2013 ) , who found significant reductions in the precuneus volumes , along with several other structural changes in depression . however , the direction of the effect is controversial . for example , a positive association was described between the volume of the precuneus and the severity of depression ( kroes et al . , 2011 ) . it is well established that intrusive imagery and increased self - focus , common in patients suffering from depression , are regularly associated with higher depression scores ( kroes et al . , 2011 ) . since the precuneus is involved in visuospatial processing , imagery and self - related processing ( kjaer et al . , 2002 ; wenderoth et al . , 2005 ) , depression could in principle enhance its gmv . we acknowledge the limitations of a cross - sectional design , which precludes studying clinical and structural abnormalities within the same group of arms before and after antidepressant medication . in order to decide whether brain volumetric deficits are related to distinct depressive symptoms or to the antidepressant effect secondly , other confounders , such as nicotine and cannabis consumption , may have influenced our findings ( higher consumption in arms - ad individuals ) . furthermore , the arms - nonad group shows more motor hyperactivity than the arms - ad group . this could result from the early effect of the antidepressant medication or from the sedative effect of cannabis or nicotine self - medication , which was higher in the arms - ad group ( warburton , 1985 ) . cigarette use may serve as an instrument to alleviate depressive symptoms , although the role of cannabis consumption is unclear . we can only speculate that the attenuated negative symptoms may drive cannabis consumption , although this abuse may exacerbate the positive symptoms observed ( gill et al . , 2013 ) . in addition , the prescribed antidepressant drugs have different affinities to various synaptic receptors and therefore their effects on macroscopic structures and neurogenesis may vary and also be associated with other brain regions . likewise , differences in the duration of antidepressant therapy may affect their impact on brain structure . finally , relatively small sample groups are included , which reduces the statistical power to detect any significant effects . surprisingly , arms individuals without antidepressant medication did not suffer more pronounced attenuated negative psychotic symptoms . the short - term antidepressant medication in this study is more likely to be an indicator of a more serious depressed state than to have a direct effect on attenuated negative psychotic symptoms . these findings emphasize the importance of comorbidity issues , especially in the context of depressive and attenuated negative psychotic symptoms in clinical high - risk individuals and their functional outcomes . the following are the supplementary data related to this article.supplementstepwise regression analysis with backward elimination .

backgroundindividuals with at - risk mental state for psychosis ( arms ) often suffer from depressive and anxiety symptoms , which are clinically similar to the negative symptomatology described for psychosis . thus , many arms individuals are already being treated with antidepressant medication.objectivesto investigate clinical and structural differences between psychosis high - risk individuals with or without antidepressants.methodswe compared arms individuals currently receiving antidepressants ( arms - ad ; n = 18 ) , arms individuals not receiving antidepressants ( arms - nonad ; n = 31 ) and healthy subjects ( hc ; n = 24 ) , in terms of brain structure abnormalities , using voxel - based morphometry . we also performed region of interest analysis for the hippocampus , anterior cingulate cortex , amygdala and precuneus.resultsthe arms - ad had higher depression and lower motor hyperactivity scores than the arms - nonad . compared to hc , there was significantly less gmv in the middle frontal gyrus in the whole arms cohort and in the superior frontal gyrus in the arms - ad subgroup . compared to arms - nonad , the arms - ad group showed more gray matter volume ( gmv ) in the left superior parietal lobe , but less gmv in the left hippocampus and the right precuneus . we found a significant negative correlation between attenuated negative symptoms and hippocampal volume in the whole arms cohort.conclusionreduced gmv in the hippocampus and precuneus is associated with short - term antidepressant medication and more severe depressive symptoms . hippocampal volume is further negatively correlated with attenuated negative psychotic symptoms . longitudinal studies are needed to distinguish whether hippocampal volume deficits in the arms are related to attenuated negative psychotic symptoms or to antidepressant action .

Introduction Materials and methods Subjects Magnetic resonance imaging acquisition Image analysis ROI analysis Statistical analysis of clinical variables Results Clinical and demographic characteristics Whole brain analysis Region of interest analyses Correlation between ROI volumes and clinical parameters Discussion Conclusion