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PMC4530252

insulin resistance in muscle and liver and -cell failure represent the core pathophysiologic defects in type 2 diabetes mellitus ( t2 dm ) . most patients with t2 dm also develop serious complications because of chronic hyperglycemia , such as nephropathy , retinopathy , neuropathy , and cardiovascular diseases , like coronary artery disease , cerebrovascular , and peripheral vascular disease . t2 dm involves a complex interaction between genetic variants and the environment , with obesity being a primary risk factor . the correlations between body fat and insulin resistance have been very well established , and the associations between genetic polymorphisms and t2 dm were demonstrated to be reliant on obesity status . evidence from various studies has shown that genetic susceptibility to t2 dm is polygenic [ 5 , 6 ] , and genome wide association studies ( gwas ) have reported more than 20 genetic loci associated with the risk of t2 dm [ 7 , 8 ] . the single nucleotide polymorphisms ( snps ) in some of these genes have only a small effect on the disease status , but few , like those at the transcription factor 7-like 2 gene ( tcf7l2 ) and proliferator - activated receptors-2 ( ppar-2 ) have a profound effect on the t2 dm prevalence , as has previously been reported in large scale studies or meta - analyses conducted in various populations and ethnic groups [ 911 ] . tcf7l2 variant rs7903146 a c - to - t ( genomic position : 114748339 ) substitution in intron 3 and ppar-2 pro12ala have been most extensively studied in all major ethnic groups and were found to be more consistently associated with the risk of developing t2 dm in most of populations , such as asians , africans , and caucasians [ 12 , 13 ] . these associations were also observed in most of the arab populations of mena region like tunisians , lebanese , [ 1418 ] , iranian [ 19 , 20 ] , and north africans . although these polymorphisms were established as risk factor for t2 dm in most of the populations studied so far , however , some of the studied polymorphisms seem not to be a major contributor to t2 dm susceptibility in the saudi and emirati arab populations [ 2224 ] . there is considerable inter - population variation in the frequency of the risk allele ( pro12 ) which ranges from a high of 0.960.98 in populations including the japanese , chinese , and african americans to 0.91 in pima indians and a low of 0.81 in the finnish population . tcf7l2 is an important regulator of the proglucagon gene , which encodes glp-1 and is believed to play a role in glucose metabolism . evidence also suggests that this transcription factor itself is critical for beta cell proliferation , antiapoptotic activity , and insulin secretion [ 26 , 27 ] . moreover , in some studies the associations of snps in ppar-2 and tcf7l2 have previously been shown to be modulated by obesity status . ppar-2 pro12ala polymorphism was found more associated with t2 dm susceptibility in obese populations while tcf7l2 rs7903146 t allele was shown to be more prevalent in t2 dm nonobese population compared to t2 dm obese individuals , suggesting a pivotal role of obesity status in genetic association with t2 dm . therefore , inconsistence regarding the genetic association studies among the arab populations might be partly explained by the obesity status . based on these findings , we decided to study the effect of obesity status on association of ppar-2 pro12ala variant with t2 dm risk in emirati population . in addition , we have also chosen to evaluate genetic variant rs10885409 in tcf7l2 which is c114798062 t substitution and was reportedly associated with t2 dm risk in north indians but has not been examined in earlier genetic epidemiologic studies in any other previously studied populations , including arabs . we also studied the effect of these variants on diabetic complications , which had not been previously reported in any of the arab populations studied to date . this study was conducted on four hundred and eighty - six ( n = 486 ) unrelated emiratis who were identified during their routine visit to a diabetes clinic in dubai and al ain , united arab emirates , between the period of june 2012 and december 2013 . the case group of 272 type 2 diabetes patients had mean age of 58 12 years and consisted of 60% females . the control group consisted of 216 healthy individuals who had mean age 45 16 years and was 68.37% female . the individuals selected for control group were nondiabetic , normotensive individuals with no indication of any complications . this study was approved by the ethics committees of the dubai health authority and al ain medical district human research ethics committee in the united arab emirates . after obtaining an informed consent from the participant , one ml of saliva was collected by a registered nurse using oragene kit ogr-500 ( dna genotek , ottwa , canada ) . the genomic dna was extracted from saliva using prepittml2p ( dna genotek , ottwa , canada ) in accordance with the manufacturer 's instructions . all the saliva samples were processed in the molecular cell biology laboratory at khalifa university , abu dhabi , for dna extraction and genotyping . in addition , a clinical assessments and lifestyle questionnaire was completed at the clinic to study any correlation between lifestyle variables and genetic variation . an individual was classified as t2 dm if the subject was diagnosed with t2 dm by a qualified physician , was on a prescribed drug treatment regimen for t2 dm , and returned biochemical test results of a fasting plasma glucose level of at least 126 mg / dl based on the criteria outlined by the world health organization ( who ) consultation group report . the individuals with bmi more than 30 were considered obese and those with a bmi less than 30 were grouped into the nonobese population . according to jnc 8 classification , all the individuals with blood pressure more than 140/90 mmhg were considered hypertensive . taqman assays were used for snp genotyping ( applied biosystems , foster city , ca ) . these assays use fluorogenic probes in a 5 nuclease assay to identify differences in dna sequences . all pcr reactions took place in optical 96-well reaction plates ( applied biosystems ) with a final reaction volume of 10 l that contained 10 ng of genomic dna , 5 l of taqman genotyping master mix ( applied biosystems ) , and 0.5 l of assay mix ( 20x ) . the pcr thermal cycling conditions were set as follows : 95c for 10 min to activate dna polymerase , followed by 40 cycles of 92c for 15 s and 60c for 1 min . all pcrs were performed using a 9700 fast thermal cycler ( applied biosystems ) , and the end point fluorescence readings were obtained on a viia 7 real - time pcr system ( applied biosystems ) . upon running 5% samples in duplicate , the genotyping success rates were found to be 99.9% for both snps . student 's t - test was used to compare the clinical parameters to different groups . the genotype frequencies were tested for the hardy - weinberg equilibrium using a test . fisher exact test was used to analyze the statistical significance of the difference in allelic distribution of various polymorphisms in patients and controls , with p values , odds ratios ( ors ) , and confidence intervals reported . the following gene transmission models were considered for tcf7l2 to study the effect of t allele : ( 1 ) recessive ( ttvs ct + cc ) and ( 2 ) dominant ( cc vs ct + tt ) effect of t allele for association analysis . a p value of < 0.05 was considered statistically significant . the demographic characteristics and clinical and biochemical parameters of t2 dm patients and nondiabetic control patients are described in table 1 . the mean age and male / female ratios significantly differed in the affected individuals and controls ( table 1 ) . the mean glucose level , mean arterial blood pressure , triglycerides , ldl , and total cholesterol by tcf7l2 rs10885409 genotype did not significantly differ ( cc vs ct + tt ) ( table 2 ) . the allele and genotype frequencies of the rs10885409 snp at tcf7l2 and ppar-2 pro12ala variant in control and t2 dm groups without consideration of obesity status are summarized in table 3 . the distribution of the tcf7l2 polymorphism ( rs10885409 ) was consistent with the hardy - weinberg equilibrium in t2 dm patients ( = 2.513 , p = 0.2846 ) as well as in nondiabetic controls ( = 1.296 , p = 0.5230 ) . the frequencies of tt , ct , and cc genotypes for rs10885409 snp at tcf7l2 were 22.5 , 44.6 , and 32.9% , respectively , in t2 dm cases and 23.3 , 46.5 , and 30.2% , respectively , in nondiabetic subjects . t allele was found with minor allele frequencies of 0.45 and 0.48 in t2 dm cases and control groups , respectively . the genotype and allele frequencies were not statistically different between the groups . the genotyping results for ppar-2 demonstrated g allele ( 12ala ) as a minor allele with a frequency of 0.04 and which was found only in heterozygote form . deviations from the hardy - weinberg equilibrium were not observed in any of the groups ( = 0.669 , p = 0.7156 for control group ) . association analyses demonstrated no significant association between the rs10885409 snp of tcf7l2 and t2 dm susceptibility when the obesity status was not considered ( tables 3 and 4 ) . moreover , the genotype and allele frequency distributions among the t2 dm case groups with or without hypertension or any of the complications related with t2 dm did not significantly differ ( table 6 ) . when the data were analyzed considering the obesity status , the frequency of the cc genotype was found to be significantly higher ( 26.1 vs 41.1 , p = 0.0176 ) in nonobese ( bmi < 30 ) t2 dm patients , but not in the obese affected patients ( bmi > 30 ) ( table 5 ) . the nonobese subjects with cc genotypes were at an approximately two - fold higher risk of t2 dm than those with ct or tt genotypes ( or 1.975 ( 95% ci 1.1273.461 ) , p = 0.017 ) ( table 5 ) . in this investigation , we carried out genetic association study of the snps in ppar-2 and tcf7l2 with t2 dm susceptibility and its interaction with the obesity status for the first time in the emirati population . while many variants have been identified in ppar-2 gene , the most prevalent and best studied is the pro12ala polymorphism which has been shown to impair the function of the ppar-2 isoform of the receptor and to be associated with obesity and/or diabetes and insulin sensitivity related phenotypes in different populations [ 9 , 12 , 13 ] . grant et al . found that common genetic variants of the tcf7l2 gene were associated with t2 dm risk , and these findings were consistently reproduced in several populations . most of these studies focused on the genetic variant rs7903146 , which was consistently found to be associated with a risk for t2 dm in most populations studied to date . studies of two major arab populations , saudi arabs and emirati arabs [ 22 , 23 ] , were not consistent with these findings . rather , we investigated another snp rs10885409 that was found to be associated with t2 dm in a previous study conducted in indians sikhs residing in the usa . the genotyping results for ppar-2 showed 12ala allele as minor allele and we established the risk allele ( pro12 ) frequency in emirati population among the highest observed so far and was comparable to saudi , japanese , chinese , and african but failed to observe any association with t2 dm risk . these findings are consistent with saudi and some caucasian studies [ 30 , 31 ] . moreover , given the very high incidence of the pro12 allele in this population , the study size was extremely underpowered and the data could not be analysed for the association with incidence of t2 dm related complications and interaction with obesity status . the results of the genotyping for rs10885409 demonstrated that neither the t nor the c allele was associated with t2 dm . similarly , rs10885409 did not correlate with t2 dm when the data for males and females were analyzed separately , ruling out the possibility of any gender - based association . we also observed that the genotype did not affect any of the clinical or biochemical parameters , such as the bmi , fasting glucose level , or lipid profile ( table 2 ) , which are considered to be related to t2 dm and were found significantly altered in the cases and controls ( table 1 ) . the allele and genotype distribution were not in concordance with the only available study conducted in an indian sikh population on the same allele . the c allele was established to be minor , with an allele frequency of 0.46 in nondiabetics and significantly higher frequency of 0.53 in t2 dm cases , and the strongest association was suggested ( 1.64 ; 95% ci [ 1.202.24 ] ; p = 0.001 ) in dominant models . the data presented here and in the two previous studies in arabs [ 16 , 17 ] suggested that tcf7l2 polymorphism is not related to t2 dm risk in the arabian peninsula . a coding variant ( pro477thr ) in exon 14 of tcf7l2 and the recently identified rs290487 variant of tcf7l2 were also not associated with t2 dm in japanese and chinese populations . however , rs7903146 was associated with t2 dm in both of these populations [ 9 , 10 , 30 ] , suggesting the possibility of allele - specific associations only . a literature exploration revealed inconsistencies in the reports in even the most thoroughly studied and widely accepted t2 dm risk variant rs7903146 , especially in arab populations . in arabian peninsula , the life style is more westernized with reduced physical activity and excessive calorie intake . these divergent results of the association could be explained by the interaction between the bmi and environmental factors in modulating t2 dm risk , as previous reports suggested that different genetic architectures could increase the t2 dm susceptibility according to the obesity status . in fact , the tcf7l2 rs7903146 t allele was associated with t2 dm in nonobese tunisian arabs , whereas no effect was detected in overweight and obese individuals . we divided our t2 dm cases and controls into two groups each of bmi < 30 and bmi > 30 . the obese group included 147 t2 dm and 88 healthy subjects , and the nonobese group included 112 and 115 t2 dm and control individuals , respectively . we observed the frequency of the cc genotype to be significantly higher ( 41.1 versus 26.1 , p = 0.0176 ) in nonobese t2 dm cases compared to matching nondiabetic subjects ( 0.61 versus 0.51 , p = 0.0376 ) , and the same was also true for the c allele ( table 4 ) . the nonobese cc genotype carriers were found to be at high risk for t2 dm ( or 1.975 , 95% ci 1.1273.461 , p = 0.017 ) ( table 5 ) ; however , rs10885409 was not associated with t2 dm risk in the obese group . these results were in complete concordance with those of the tunisian study and were sufficient to explain the inconsistencies that were observed regarding the effect of the tcf7l2 variant on t2 dm risk . most of the studies were conducted without considering the obesity status , and our results strongly suggested that the genetic associations might be modulated by the presence and absence of obesity , especially in arab populations . the allelic and genotypic associations were also analyzed in diabetic patients with at least one complication and without any complications . we did not observe any significant differences in the allele and genotype frequencies between the two groups ( table 6 ) , indicating that rs10885409 did not affect the presence of diabetes - related complications . studies regarding the effect of variant rs10885409 on diabetes - related complications were not available , and the studies on rs7903146 were largely inconsistent [ 34 , 35 ] . unfortunately , we could not analyze this association for each complication separately or based on obesity status because of the very small sample size of the groups formed for this purpose . however , we observed significant differences in the biochemical parameters in different genotypes in nonobese subjects . the mean levels of cholesterol , ldl , and tgs were significantly higher in t2 dm cases with either ct or tt genotypes compared to matching controls with the same genotype ( table 7 ) . interestingly , the ldl levels were significantly higher in t2 dm subjects with the ct + tt genotype compared to t2 dm cases with the cc genotypes ( p = 0.0001 ) ( table 7 ) . in conclusion , our results show that the pro12ala mutation in the ppar-2 gene is unlikely to serve as clinically useful predictor of t2 dm and/or obesity in emiratis . tcf7l2 variation may be a risk factor for the occurrence of t2 dm in arab populations , but this association relies on the obesity status . our case control study on rs10885409 established cc genotype as a risk factor in nonobese emiratis . however , the association was absent when the obesity status was not considered and in the obese population with t2 dm risk and related complications .

background . transcription factor 7-like 2 gene ( tcf7l2 ) and peroxisome proliferator - activated receptors-2 ( ppar-2 ) have a profound effect on the incidence of type 2 diabetes mellitus ( t2 dm ) and had previously been found to be associated with t2 dm risk in various ppopulations . however , studies in the arab population are inconsistent . we conducted a case control study to confirm the association of variants rs10885409 of tcf7l2 and pro12ala ( rs1801282 ) of ppar-2 with risk of t2 dm and related complications in emirati population of arab origin . we also investigated the interaction of these associations with obesity status . methods . dna was extracted from the saliva samples of 272 t2 dm patients and 216 nondiabetic emiratis . genotyping for rs10885409 ( tcf7l2 ) and rs1801282 ( ppar-2 p12a ) variants was accomplished with a taqman assay . the subgroups were constituted according to obesity status . results . in the nonobese group , the rs10885409 c allele in the recessive model was significantly associated with the incidence of t2 dm ( or 1.975 [ 95% ci 1.1273.461 ] , p = 0.017 ) , but this association was not observed in the obese group or when bmi was not considered . ppar-2 risk allele pro12 frequency ( 0.96 ) was similar in the groups tested and more than 90% population was homozygous for this allele . conclusions . our case - control study is the first of its kind in emiratis which establishes tcf7l2 rs10885409 c allele as a t2 dm risk factor in emiratis and this association is modulated by obesity status . we also confirmed that pro12ala mutation in ppar-2 is not associated with t2 dm risk in this population .

1. Introduction 2. Methodology 3. Results 4. Discussion

PMC4329833

nitric oxide ( no ) is an important biological second messenger in humans , which plays a critical role in cell and neuronal signaling , blood pressure regulation , and the immune response . no is produced from oxidation of l - arginine ( l - arg ) in the presence of nadph by a class of heme - dependent enzymes , nitric oxide synthases ( nos ) . mammals have three dominant isoforms of nos : constitutively expressed neuronal nos ( nnos ) , present throughout the nervous system and skeletal muscles , endothelial nos ( enos ) , also a constitutive enzyme located in the endothelium and functioning in regulation of blood pressure and blood flow , and inducible nos ( inos ) , which is associated with the immune response . in the brain , low nanomolar concentrations of no produced by nnos are neuroprotective , and downstream no , along with cyclic guanosine 5-monophosphate ( cgmp ) in the protein kinase g ( pkg ) signaling pathway , plays an important role in neurotransmission and other metabolic processes . however , overexpression and overactivation of nnos following neuronal damage causes no levels to jump several orders of magnitude , which is neurotoxic . such no - mediated neurotoxicity leads to protein degradation , misfolding , and aggregation through tyrosine - nitration , s - nitrosylation , and oxidative stress damage through formation of reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) . this neurotoxicity has been implicated in several neurodegenerative disorders that include alzheimer s , parkinson s , and huntington s diseases and amyotrophic lateral sclerosis ( als ) . furthermore , progressive neuronal damage and loss of neural tissue associated with no overproduction are seen in cerebral palsy , stroke , ischemic brain damage , and migraine headaches . therefore , with the increasing human and economic costs associated with neurodegenerative diseases , and the lack of existent treatments , there is an urgent need for the development of new therapeutics that would prevent , cure , or attenuate neurodegeneration . with high levels of no implicated in these neurodegenerative conditions , and target validation linking nnos to these pathological conditions all nos isoforms are active only as homodimeric enzymes , where each monomer contains an n - terminal oxygenase domain and a c - terminal reductase domain . the reductase domain contains the fmn , fad , and nadph binding - sites , while the oxygenase domain binds the cofactor ( 6r)-5,6,7,8-tetrahydrobiopterin ( h4b ) and the substrate l - arg at the heme catalytic site . upon dimerization and activation of nos by ca - mediated calmodulin binding , electrons flow from nadph to fad and fmn in the reductase domain to the heme in the oxygenase domain , where conversion of l - arg to l - citrulline and no takes place in the presence of oxygen . therefore , quite predictably , the major approach in the development of nos inhibitors involves the utilization of arginine mimetics as competitive nnos inhibitors . however , in addition to potent inhibition of nnos , there are many challenges associated in designing nnos inhibitors . first , selectivity of nnos inhibitors over enos and inos ( nos isoforms share nearly identical active site ) is essential to minimize undesired side effects . second , as arginine isosteres , these inhibitors are highly polar ; therefore , they suffer from poor bioavailabilty . hence , the design of potent nnos inhibitors with improved pharmacokinetic properties that address high isoform selectivity , blood brain barrier permeability , and minimal off - target efficacy is crucial . in this respect , continuing efforts from our laboratories , guided by structure - based drug design and fragment hopping , have resulted in a series of highly potent and selective small molecule nnos inhibitors based on pyrrolidinomethyl-2-aminopyridine scaffolds ( figure 1 , compounds 1 and 2 ) . compounds 1 and 2 showed high potency and excellent selectivity over inos and enos , while 2 , when administered intravenously to a pregnant rabbit dam with induced uterine hypoxia , showed a complete reversal of hypoxia ischemia induced death in the newborn kits . however , further development of these compounds stalled because of their inability to cross the blood brain barrier . this was presumably the result of the hydrophilic nature of the molecule ( too many basic amines and hydrogen - bond donors , high polar surface area , and a large number of rotatable bonds ) . modifications of these pyrrolidinomethyl-2-aminopyridine scaffolds , such as reducing the number of polar charges and basicity by alkylation , fluorination , and intramolecular hydrogen bonding , met with either diminished potency or selectivity or without notable improvement in cellular permeability . furthermore , synthesis of these pyrrolidinomethyl-2-aminopyridine scaffolds involved more than 12 steps , difficult chiral resolutions , and diastereomer separations . later modifications of simplified double - headed 2-aminopyridine scaffolds displayed good potency and selectivity ; however , they still suffered from poor permeability in a caco-2 assay , which is used to estimate intestinal cellular permeability and also reflects potential brain permeation . therefore , as bioavailability was a major challenge in these nnos inhibitors , one avenue we explored to improve the cellular permeability of the nos inhibitors , while maintaining a good potency and selectivity , was replacement of the more basic arginine isosteres with fewer basic groups that engage in heme - coordination in the active site of nos to arrest l - arginine turnover ( for example , the pka of conjugate acid of 2-aminopyridine is 7.1 ) . compared to many inhibitors designed as arginine mimetics , only a few reports exist that explore the heme - coordination of nos . however , imidazole ( pka of its conjugate acid is 6.9 ) is known to weakly bind to nnos ( ic50 200 m ) , while the less - donating 1-phenylimidazole shows nearly 10-fold improved potency ( ic50 25 m ) . furthermore , 2-(1-imidazolyl)pyrimidine scaffolds , such as 3 , have shown very good potency as inos dimerization inhibitors . in addition , 3 demonstrated good cellular permeability and in vivo efficacy against inos in rats . toward this end , we designed simplified compounds 4 and 5 with improved predicted physicochemical properties by hybridizing selective molecular fragments from inhibitors 1 and 3 . the key hydrophobic tail of 1 was incorporated into structures 4 and 5 based on previous precedence , where contacts between 1 and residues lining a hydrophobic pocket adjacent to the substrate access channel were implicated in improving isoform selectivity . on the basis of inhibitory assay results , docking studies , and crystallographic analysis , further modifications on 5 , such as changing the linker length between the two aromatic heads , the number of secondary amines , and utilizing various substitutions on the aromatic rings , were employed to maximize rat and human nnos potency , improve isoform selectivity , enable cellular permeability , and minimize off - target effects . figure 2 summarizes the different nnos inhibitors designed and studied in this series . through these structure these compounds showed improved cellular permeability , high selectivity against a panel of central nervous system ( cns ) receptors , and attenuated cyp inhibition . synthesis of the 2,4-disubstituted pyrimidine scaffolds in this study was designed to utilize common intermediates and proceed in relatively few steps . thus , the synthesis of the 2,4-disubstituted pyrimidine cores in 4 and 5 was obtained by following a modified procedure reported by davey et al . alkylation of n - boc piperazine ( 21 ) by 3-fluorophenethyl bromide gave 22 , which underwent a sequential one - pot substitution on 4-chloro-2-methanesulfonyl pyrimidine by itself and imidazole , respectively , to provide target compound 4 ( scheme 1 ) . similar substitutions on 4-chloro-2-methanesulfonyl pyrimidine by 26 and imidazole , respectively , gave the primary framework as a boc - protected precursor of 5 , which was subsequently boc - deprotected with trifluoroacetic acid and obtained as a trihydrochloride salt upon precipitation from methanolic hcl . compound 25 was synthesized following a reported procedure , from where a mitsunobu reaction with diphenylphosphoryl azide gave the corresponding azide , which was subsequently reduced to give the primary amine 26 . reagents and conditions : ( a ) ( i ) nah , thf , 0 c , 1 h , then 3-fluorophenethyl bromide , tbai , 60 c , 48 h , 56% , ( ii ) tfa , ch2cl2 , 0 c rt , 3 h , 85% ; ( b ) 4-chloro-2-methanesulfonyl pyrimidine , k2co3 , mecn , 40 c , 19 h , then imidazole , 65 c , 30 h , 82% ; ( c ) 3-fluorophenyl acetaldehyde , nabh(oac)3 , rt , 14 h , 91% ; ( d ) ( i ) h2 , pd / c , meoh , 12 h , 86% , ( ii ) boc2o , ch2cl2 , 3 h , 76% ; ( e ) ( i ) diad , pph3 , dppa , thf , 12 h , ( ii ) pph3 , thf / h2o , 41% ; ( f ) ( i ) same as ( b ) , 60% , ( ii ) tfa , ch2cl2 , 2 h , ( iii ) hcl in meoh , 10 min , 83% . in 6 and 7 , where the secondary amine was benzylic to the pyrimidine , a reductive amination to form the c n bond with an aromatic aldehyde was conceived as a favorable disconnection ( scheme 2 ) . therefore , 2-methylthio-4-pyrimidine carboxaldehyde ( 30 ) was first obtained by condensation between pyruvaldehyde dimethylacetal ( 27 ) and thiourea ( 28 ) and then acetal deprotection . this intermediate underwent efficient reductive amination with 3-fluorophenylethylamine and 3-fluorophenylpropylamine to form secondary amines 33 and 34 , respectively . then boc protection of the secondary amine and oxidation of the thioether group to methanesulfonyl by mcpba enabled the successive displacement by imidazole to get the main structural frameworks of 6 and 7 . finally , deprotection of the boc group under acidic conditions and subsequent precipitation from methanolic hcl gave 6 and 7 as dihydrochloride salts . reagents and conditions : ( a ) ( i ) n , n - dimethylformamide dimethyl acetal , 110 c , 8 h , ( ii ) naome , meoh , rt , 16 h , ( iii ) mei , 60 c , 12 h , 87% ; ( b ) hcl , 85 c , 6 h , 58% ; ( c ) nabh(oac)3 , cat . acoh , 3 sieves , ch2cl2 , 14 h , 8589% ; ( d ) ( i ) boc2o , thf , 12 h , 9799% , ( ii ) mcpba , ch2cl2 , 12 h , 7589% ; ( e ) imidazole , k2co3 , mecn , 60 c , 4 h , 8493% ; ( f ) ( i ) tfa , ch2cl2 , 3 h , ( ii ) hcl in meoh , 10 min , 7993% . 2,4-disubstituted pyrimidine scaffolds possessing two methylene units between the secondary amine and the pyrimidine ring ( 814 ) were synthesized via a michael addition on the electron - deficient vinyl pyrimidine ring ( scheme 3 ) . therefore , the 2-methylthio-4-vinyl pyrimidine ( 40 ) was synthesized by a stille coupling between commercially available 4-chloro-2-methylthio pyrimidine ( 39 ) and tributylvinyltin in the presence of tetrakistriphenylphosphine pd(0 ) . a michael addition between 40 and the particular primary amines gave the corresponding homobenzylic amines ( 4147 ) . the primary amines ( 3132 , s3s12 ( see supporting information ) , 62 ) were synthesized either from commercially available bromides or carboxylic acids in 23 steps , as elaborated in the supporting information . note that in the case of the primary amines containing a cyclopropyl group , the cis and trans isomers were completely separable by silica gel column chromatography ( see supporting information for further details ) , and the trans isomer was carried forward for the michael addition reaction . reagents and conditions : ( a ) tributylvinyl tin , pd(pph3)4 , dce , 70 c , 48 h , 92% ; ( b ) r - nh2 , cat . acoh , etoh , 848 h , 6297% ; ( c ) ( i ) boc2o , thf , 3 h , 8095% , ( ii ) mcpba , ch2cl2 , 3 h , 6591% ( note in case of 46 , oxone was used instead of mcpba in a 1:1 thf / h2o mixture for 4 h ) ; ( d ) imidazole , k2co3 , mecn , 65 c , 510 h , 7692% ; ( e ) ( i ) tfa , ch2cl2 , 3 h , ( ii ) hcl in meoh , 10 min , 8099% . following a similar route as illustrated for the synthesis of 6 and 7 , final compounds 814 were obtained ( scheme 3 ) . however , in the case of 46 , after boc protection , mcpba oxidation ( regardless of conditions ) always led to undesired oxidation of the pyridine ring to the n - oxide . finally , oxidation of 46 in a 1:1 mixture of thf and h2o by oxone at room temperature for 4 h gave desired sulfone 53 . similar imidazole substitution , boc deprotection , and acidification gave final compound 13 as a pure trihydrochloride salt . when separation of the two enantiomers of 12 became crucial , the trans - isomer of 2-(3-fluorophenyl)-1-cyclopropyl amine ( 62 ) was subjected to dcc - mediated amidation with ( s)-(+)--methoxyphenylacetic acid ( scheme 4 ) . the diastereomers , 63a and 63b , were obtained pure by silica gel column chromatographic separation . finally , hydrolysis of the auxiliary under refluxing ethanolic hcl gave the enantioenriched amines 62a and 62b in > 95% enantiopurity . these amines were then independently converted to the final enantiomerically enriched isomers ( r , r)-12 and ( s , s)-12 by following the exact same route as used to synthesize 12 from 62 and 2-methylthio-4-vinylpyrimidine ( 40 ) . reagents and conditions : ( a ) ( i ) ( s)-(+)--methoxyphenylacetic acid , dcc , ch2cl2 , 20 c to rt , 12 h , ( ii ) chiral resolution of diastereomers on silica gel column ; ( b ) hcl in etoh , reflux , 12 h. when the 2,4-disubstituted pyrimidine was replaced by a 3,5-disubstituted pyridine ring , as in 15 , synthesis was initiated from commercially available 5-bromo nicotinic acid ( 64 ) by reduction to alcohol 65 ( scheme 5 ) . homologation by one methylene unit was obtained by conversion of the alcohol to a nitrile ( 66 ) and then to its aldehyde by dibal . as the intermediate aldehyde was photosensitive and unstable , it was synthesized in the dark , and the crude reaction , after work up , was immediately subjected to reductive amination with 3-fluorophenethyl amine ( 31 ) to provide 67 in a 41% yield ( over two steps ) . boc protection of the secondary amine , followed by a cu - catalyzed amination of the 3-bromopyridine group , gave 68 , which was boc deprotected and treated with methanolic hcl to give final compound 15 as a trihydrochloride salt . reagents and conditions : ( a ) ( i ) isobutyl chloroformate , et3n , thf , rt , 1 h , ( ii ) nabh4 , h2o , 12 h , 43% ; ( b ) ( i ) socl2 , ch2cl2 , 2 h , ( ii ) kcn , dmf , rt , 12 h , 70% ; ( c ) ( i ) dibal , ch2cl2 , 78 c , 1 h , ( ii ) 3-fluorophenethylamine , cat . acoh , mgso4 , nabh(oac)3 , 12 h , 41% ; ( d ) ( i ) boc2o , thf , 12 h , 92% , ( ii ) cubr , 8-acetyl-5,6,7,8-tetrahydroquinoline , imidazole , cs2co3 , dmso , 100 c , 12 h , 67% ; ( e ) ( i ) tfa , ch2cl2 , 3 h , ( ii ) hcl in meoh , 10 min , 88% . the different aromatic substitutions at the 2-position of the pyrimidine in 1620 were synthesized from advanced intermediate 49 by substituting the sulfone with the different methyl imidazoles or triazoles ( scheme 6 ) . thereafter , a similar tfa - mediated boc deprotection and salt formation gave the compounds 1620 . however , when 4-methylimidazole was used , both 4- and 5-methyl-2-imidazolyl pyrimidines 72 and 73 were obtained , which were inseparable by chromatographic conditions . therefore , a subsequent boc deprotection of the mixture of 72 and 73 , followed by a chromatographic separation , provided the free bases of 19 and 20 . these were subsequently treated with methanolic hcl to obtain pure dihydrochloride salts 19 and 20 . reagents and conditions : ( a ) imidazole / triazole , k2co3 , mecn , 65 c , 524 h , 8288% ; ( b ) ( i ) tfa , ch2cl2 , 1 h , ( ii ) hcl in meoh , 10 min , 8799% . table 1 summarizes the binding affinity of 420 in the in vitro enzyme inhibitory assays against several isoforms of nos . an oxyhemoglobin no capture assay was used to determine the ic50 value of inhibitors against the purified rat and human nnos , murine macrophage inos , and bovine enos . the apparent ki values of the inhibitors were determined from ic50 and substrate km values using the cheng prusoff equation , and the corresponding isoform selectivities as ratios of their respective ki values . compounds 420 were assayed in vitro against four purified nos isoforms : rat nnos ( rnnos ) , bovine enos ( enos ) , murine inos ( inos ) , and human nnos ( hnnos ) using known literature methods . prusoff equation directly from ic50 values ( see experimental section for details ) . ic50 values are the average of at least two replicates with nine data points ; all experimental standard error values are less than 15% , and all correlation coefficients are > 0.9 . selectivity values are the ratios of respective ki values . nt = not tested . between the two initial inhibitors designed , 4 and 5 , compound 4 only weakly inhibited nnos ( ki 5 m ) while 5 displayed a stronger inhibition of nnos ( ki 0.368 m ) . this nearly 10-fold difference in potency can be attributed to the key structural difference in the linker between the pyrimidine and 3-fluorophenyl ring in 4 and 5 . while a piperazine ring may be sterically favorable in a more open active site , in case of 3 binding to an inos monomer ( and hence preventing dimerization ) , the same may cause unfavorable steric clashes with the peptide backbone in the nnos dimer . furthermore , the open and flexible linker in 5 can also engage in favorable interactions via the secondary amines , thus orienting the hydrophobic end of the molecule properly . to gain more insight into the structural basis for potencies and selectivity , we determined the crystal structure of 5 bound to nnos and enos . indeed , the crystal structure of 5 bound to nnos ( figure 3a ) shows that while the 2-imidazolyl pyrimidine head in the molecule ligates to the heme fe , the void left between the imidazole ring and glu592 is filled with a water molecule . the secondary amine next to the pyrimidine ring is engaged in a salt bridge with heme propionate a , while the other secondary amine makes a hydrogen bond with a water bridging in between h4b and the heme . this also orients the rest of the linker toward the hydrophobic pocket lined by tyr706 , met336 , leu337 , and trp306 ( from the other monomer ) in rat nnos . the aromatic ring of 5 engages in quite a few van der waals interactions with these four residues at distances from 3.5 to 4.0 , except for the closer distance ( 3.3 ) from the fluorine atom of 5 to the carbonyl oxygen of met336 . this also represents the first structure of nnos with a type ii imidazole - based heme - bound inhibitor . the crystal structure of 5 bound to enos ( figure 3b ) reveals two major differences compared to its binding conformation seen in nnos . first , similar to nnos , the tail fluorophenyl ring can still make van der waals contacts with tyr477 , leu107 , and trp76 ( the other monomer ) , but the fluorine atom is no longer inserted into the pocket as in the nnos case . instead , the aromatic ring simply caps the pocket at its edge with the f atom pointing sideways . the reason for this binding orientation is very likely an amino acid variation , val106 ( enos ) vs met336 ( nnos ) , which is also part of this hydrophobic pocket . the more degrees of freedom of met336 in nnos can more readily adapt to inhibitor binding than val106 in enos . as a result , the cg1 atom of val106 in enos would clash with the aromatic ring of 5 if it were in its position seen in nnos . consequently , in enos the secondary amine in the linker of 5 makes a hydrogen bond with heme propionate d , rather than with a water molecule as in nnos . the val / met variation is , therefore , the structural basis for the observed 100-fold selectivity for 5 ( table 1 ) . active site structure of 5 bound to rat nnos ( ( a ) pdb 4d3b ) and bovine enos ( ( b ) pdb 4d33 ) . key hydrogen bonds are shown by dashed lines , and distances are in . fc map for the ligand is contoured at 2.5 . the heme pyrrole rings are labeled in order to identify the propionate positions . all structural figures were prepared with pymol ( www.pymol.org ) . because the part of inhibitor 5 positioned directly over the heme appeared optimal , we sought to improve the enzyme inhibitor interactions beyond the active site ( compounds 69 ) , utilizing different linker lengths between the pyrimidine and 3-fluorophenyl rings . the rationale behind the design of the shorter 4-atom - linker , as in 6 , was to avoid the twisting of methylene units on top of each other , as seen in the conformation of 5 , but still enable the aromatic end to reach the hydrophobic pocket . also , we reasoned that moving the secondary amine away from the pyrimidine by one or two methylene units might enable it to hydrogen bond with both the heme propionates and improve the potency of the molecule . with this amine moving up along the linker , the other secondary amine in 5 becomes obsolete and thus can be removed to reduce the basicity of the inhibitors . from evaluation of the binding affinity of these inhibitors against nnos ( table 1 ) , we found two crucial factors that contribute to their potency and selectivity : the position of the secondary amine and the length of the linker . moving the secondary amine by one more methylene unit from the pyrimidine ring ( 8 and 9 ) dramatically decreased the ki values relative to 6 and 7 ( 8.7 and 2.7 m , respectively ) . the structure of 7 bound to nnos ( figure 4a ) reveals that the secondary amine can no longer make a hydrogen bond with either of the heme propionates , thus suggesting that this interaction is critical for imparting potency to these pharmacophores . this polar interaction was regained in 8 and 9 , where the secondary amine is one carbon farther away from the pyrimidine than in 6 and 7 . the structure of 8 bound to nnos ( figure 4b ) indicates that this position of the amine enables it to engage in dual salt bridges with both the heme propionates , with the secondary amine positioned equidistant between the two . however , the five - atom linker of 8 is not long enough to bring the fluorophenyl ring into tighter van der waals contacts with the hydrophobic pocket . this , in fact was attained with the six - atom linker between the fluorophenyl and the pyrimidine to provide maximum potency . thus , 9 is one of the most potent nnos inhibitors in this series of 2-imidazolylpyrimidine scaffolds ( ki = 0.019 m ) . indeed , the structure of nnos9 ( figure 5a ) demonstrates the structural basis for this good potency . like 8 , the secondary amine of 9 can establish dual salt bridges with both heme propionates . in addition , owing to the longer linker of 9 , the fluorine atom of the aromatic ring can be inserted into the hydrophobic pocket , as in the case of 5 . extensive van der waals contacts provided by the inhibitor s aromatic ring should be responsible for the 7-fold higher potency when comparing 9 with 8 ( figure 5a vs figure 4b ) . furthermore , 9 displayed 260-fold selectivity over enos , with an improved 41-fold selectivity over inos . the differences in interactions with residues lining the hydrophobic pocket also contribute to isoform selectivity . as shown in figure 5b , even though 9 in enos can retain the dual salt bridges from its secondary amine in the linker , its fluorophenyl ring can not insert into the hydrophobic pocket as it does in nnos . this is the result of an amino acid variation ( val106 in enos vs met336 in nnos ) , as postulated for 5 . the lack of these van der waals contacts reflects in the poorer potency of 9 to enos , which is supported by the fact that both 8 and 9 share similar potency to enos ( see figure s1 in the supporting information for structure of 8 bound to enos ) . active site structures of 7 ( ( a ) pdb 4v3v ) and 8 ( ( b ) pdb 4v3w ) bound to rat nnos . key hydrogen bonds are shown by dashed lines and distances are in . the omit fo fc map for the ligand is contoured at 2.5 . active site structures of 9 bound to rat nnos ( ( a ) pdb 4v3x ) and bovine enos ( ( b ) pdb 4d35 ) . fc map for the ligand is contoured at 2.5 . encouraged by the good potency and selectivity of 9 , we sought to replace the fluorophenyl with a chlorophenyl ring at the hydrophobic end of 10 . prior studies have shown that halogen replacements could result in enhanced inhibitory potency and selectivity from contacts between the inhibitor and residues in the hydrophobic pocket . however , in our case , this change results in slightly diminished inhibitor potency in nnos ( 32 nm for 10 vs 19 nm for 9 ) . the structure of nnos10 ( figure s2 in supporting information ) shows that because of the bulkiness of the chlorine atom , the aromatic ring actually retreats from the hydrophobic pocket , leading to less extensive van der waals contacts with the protein compared to 9 . as expected , the chlorophenyl ring of 10 in the enos10 structure ( figure s3 in supporting information ) is entirely outside of the pocket , similar to the situation in enos9 ( figure 5b ) , and therefore 10 still maintains good n / e selectivity ( 253-fold ) . while 9 showed a high level of selectivity against enos , we looked deeper into further improving its inos selectivity . the three nos isoforms have a 5060% overall sequence identity and highly conserved , almost identical active site sequences . therefore , specificity among these nos isoforms have relied on key differences in residues and their conformational flexibility along the substrate access channel that connects the active site to the hydrophobic pocket ( vide supra ) lying at the farthest end of this channel . previous designs of nnos inhibitors have shown that contacts between the inhibitor and residues of this pocket could be in part responsible for imparting isoform selectivity . in murine inos , for example , a polar asn115 replaces leu337 of rat nos in this pocket , which would strongly disfavor hydrophobic interactions . in addition , superimposition of the nnos9 and inos crystal structures reveals subtle differences in spatial orientations of residues along the substrate access channel . we reasoned that conformational restrictions and an increase of steric bulk in 9 , by introduction of cyclopropane rings along the methylene linker , might impose steric clashes and thereby lower binding affinity to inos . furthermore , installing a cyclopropane ring as in 11 increases the metabolic stability of a more labile benzylic methylene group in 9 , while the electron - withdrawing nature of the ring can reduce the basicity of the secondary amine as in 12 , thereby increasing the probability of blood brain barrier permeation . although both 11 and 12 displayed a slightly lower binding affinity for nnos , they displayed a weaker inhibitory effect on inos than parent compound 9 . in fact , 12 demonstrated an improved 73-fold selectivity over inos , more than the previous lead , and the highest n / e selectivity ( 363-fold ) obtained so far in this series of scaffolds . to gain more concrete evidence , we determined the crystal structures of both 11 and 12 in nnos ( parts a and b of figure 6 , respectively ) and enos ( supporting information , figures s4 and s5 , respectively ) . because of the rigidity of the linker s cyclopropyl ring , the fluorophenyl ring of both inhibitors in nnos retreats from the deeper position seen for 9 ( figure 5a ) but is oriented similarly to 10 ( supporting information , figure s2 ) . again , less extensive van der waals contacts for 11 and 12 relative to those by 9 are the reason for the slightly weaker potency of these two inhibitors ( table 1 ) . interestingly , although racemic samples of 11 and 12 were used for the crystal preparations , the electron density indicated that only one enantiomer preferably binds in each case : ( r , r)-11 in nnos11 and ( r , r)-12 in nnos12 . similarly , the same enantiomer also dominates the binding of 11 or 12 to enos ( supporting information , figures s4 and s5 , respectively ) ; however , in both cases , the fluorophenyl ring caps at the edge of the hydrophobic pocket . inhibitor contacts are the origin of poorer binding affinity for 11 and 12 toward enos . owing to a different location of the cyclopropane in the linker , the kink caused by the cyclopropyl ring in 12 pulls the fluorophenyl ring away from the protein more severely than that observed in 11 . this explains the even poorer affinity of 12 to enos , which results in better n / e selectivity ( table 1 ) . active site structures of 11 ( ( a ) pdb 4v3z ) and 12 ( ( b ) pdb 4d2y ) bound to nnos . there is preferential binding of one enantiomer : ( r , r)-11 and ( r , r)-12 for 11 and 12 , respectively . key hydrogen bonds are shown by dashed lines , and distances are in . fc map for the ligand is contoured at 2.5 . the observation that one enantiomer of 12 dominates the binding prompted us to synthesize and test the two enantiomers of 12 separately to identify the more potent enantiomer in the pair and determine the effect of chirality on the binding of the inhibitor to the nos active site . table 2 summarizes the binding affinity to noss by the two enantiomers of 12 . while the ring up ( r , r)-12 shows a much tighter binding to nnos , comparable to the affinity of 9 , this particular enantiomer also preferably binds to enos and inos . however , the very low ki for nnos ( 18 nm ) compared to that for enos and inos makes ( r , r)-12 the best inhibitor in this series with 573- and 119-fold selectivities against enos and inos , respectively . ic50 values are the average of at least two replicates with nine data points ; all experimental standard error values are less than 10% , and all correlation coefficients are > 0.95 . these results are also in agreement with the structural studies . the nnos structure with ( r , r)-12 bound confirms that this enantiomer is indeed the one that dominates the binding to nnos when racemic 12 was used for crystal preparation ( figure 6b ) . it is surprising that the structure of ( s , s)-12 enantiomer bound to nnos ( figure s6 in supporting information ) also shows a binding mode that is not that much different from that of ( r , r)-12 ; both the positions of the secondary amine and the fluorine of the aromatic ring more or less overlap between the two enantiomers , even though different chiralities at the cyclopropane lead to differences in the linker conformation and phenyl ring orientation ( figure s6 in supporting information ) . therefore , the differences in linker conformation and position of the cyclopropane ring may result in the 8-fold variation in the nnos affinities between these two enantiomers ( table 2 ) . alongside determining the potencies of these inhibitors in the three lower animal isoforms of nos , we assayed these compounds with the human nnos enzyme to see if the potency and selectivity ratios are also reflected similarly in the human isoform . prior work on isosteric arginine mimetics developed from our lab , such as the highly potent and selective inhibitor 1 , showed a human nnos potency of 0.070 m , with a 5-fold selectivity difference between the human and rat isoform . this is one of the most potent human nnos inhibitors developed in our lab to date . when compounds 812 were assayed against human nnos , we were pleased to see that inhibitors 912 displayed good potency in human nnos , and a similar 410-fold selectivity difference between the human and rat isoform of nnos , as seen with previously reported inhibitors from our lab . the human nnos exhibits an exact sequence identity with the rat isoform , except for the hydrophobic pocket , where a hydrophilic his342 replaces the leu337 present in rat nnos . thus , the pocket in human nnos is smaller and more hydrophilic on one side . therefore , the modifications that we made in our chemical structures to improve nnos potency , although generally well tolerated in human nnos , can be modified to improve their human nnos potency and lower the rat / human selectivity . for this target , we designed and tested compounds 1314 , with smaller and polar aromatic ends that may interact effectively with his342 residue in the pocket in human nnos . indeed , we found that 13 is a potent inhibitor of human nnos ( 0.125 m ) and rat nnos ( 0.054 m ) , where the selectivity between the two has dropped down to 2.3-fold ( table 1 ) . the structure of the human nnos13 complex ( figure 7a ) shows , as expected , that the nitrogen of the pyridine ring indeed makes a hydrogen bond with the side chain of his342 . although the bond distances vary from 2.8 to 3.2 in the four independent copies of 13 , all are well ordered in structure . in contrast , the polar pyridine ring of 13 does not behave as well in the structure of rat nnos13 ( figure 7b ) , where the ring shows the sign of disordering with weak electron density . the more hydrophobic pocket in rat nnos prefers a phenyl ring over the pyridine ring for interactions . active site structures of 13 bound to human nnos ( ( a ) pdb 4v3u ) or rat nnos ( ( b ) pdb 4d30 ) . key hydrogen bonds are shown by dashed lines and distances are in . the omit fo fc map for the ligand is contoured at 2.5 . compound 10 ( with a 3-chlorophenyl tail ) also displayed potent human nnos inhibition similar to 13 . with a 3-chloro substitution being bigger than a 3-fluoro , it is difficult to explain why 10 is a more potent human nnos inhibitor than 9 . with the known behavior of 10 in rat nnos ( figure s2 in supporting information ) , the bulkier chlorophenyl ring in human nnos is likely pulled out a bit from the depth of the hydrophobic pocket compared to the position of a fluorophenyl ring in 9 . this movement may allow the 3-chlorophenyl ring to fit better in the pocket , which might contribute to the slightly better affinity of 10 than 9 to human nnos . on the other hand , it is easier to explain the preference for more polar aromatic heads being tolerated better in human nnos with 14 , where a polar 3-cyanophenyl with a shorter 4-atom linker between the arene and the pyrimidine head , demonstrated a 138 nm binding affinity for human nnos , while a 183 nm binding affinity for rat nnos . thus , this is the first case of a potent human nnos inhibitor from our lab , where the selectivity is reversed in preference toward its human isoform potency over the rat isoform . with a shorter linker , the cyanophenyl ring of 14 locates right outside the hydrophobic pocket in rat nnos ( figure s7 in supporting information ) . however , in human nnos , the larger side chain of his342 is at the right distance for a hydrogen bond , as shown in figure s8 in supporting information , which makes 14 a better inhibitor for human nnos than rat nnos . therefore , these inhibitors clearly demonstrate that we can utilize the same pocket in the substrate access channel for both potency and selectivity determining factors in designing the nos inhibitors . finally , with compounds 1520 , we investigated variations in the 2-imidazolylpyrimidine part of the scaffold that may result in improving potency or minimizing off - target affinity . to this end , preliminary docking studies indicated that a 3,5-disubstituted pyridine could replace the 2,4-disubstituted pyrimidine in 8 and engage in a salt bridge formation with the glu592 in the active site of nnos , the same residue that forms crucial hydrogen bonds with the guanidine group of l - arginine or other arginine mimetics . however , when 15 was assayed against nnos , it showed a poor 5.5 m inhibition against nnos . therefore , further variations on the imidazole ring fragment were made based on 9 , keeping the pyrimidine ring intact . we were interested from the literature examples of using of heme - nitrogenous ligands as inhibitors of aromatase enzymes for the treatment of breast cancer , and inhibitors of lanosterol demethylase enzymes as antifungal drugs . among the triazoles and methyl - substituted imidazoles incorporated as 2-pyrimidyl substituents in compounds 1620 , both the 1,2,4- and 1,2,3-triazole - substituted scaffolds were poor inhibitors of nnos , presumably because of a major depletion of the heme - fe binding affinity by the electron - deficient triazoles . among the substituted imidazoles , only ( 5-methyl)-2-imidazolylpyrimidine ( 20 ) retained very high potency against nnos , and along with high selectivity against enos , it demonstrated a remarkably high 400-fold selectivity against inos as well , in sharp contrast to all the imidazole - substituted inhibitors mentioned previously . this presumably arises from the residues lining the active site over the heme - porphyrin , which makes a smaller and less flexible pocket in inos compared to nnos . this pocket is surrounded by pro565 , val567 , and phe584 from two -strands in rat nnos ( figure 8a ) , but by pro344 , val346 , and phe363 in murine inos . while residues facing the heme are conserved , the residue that stabilizes the second -strand is ser585 in nnos and asn364 in inos . asn364 is involved in more hydrogen bonds than ser585 , which should help rigidify this part of the structure ; however , the asn364 polypeptide chain in inos also shrinks the active site pocket to accommodate its larger side chain compared to ser585 in nnos . the crystal structure of 20 bound to nnos ( figure 8a ) shows that the methyl group sticks in to this pocket just described , and the imidazole ring is bent away from the pyrimidine ring plane at a larger angle than usual because of steric hindrance between the pyrimidine ring and the methyl group . therefore , the methyl group might create some clashes in the active site pocket of inos , thereby making it a very poor inos inhibitor . on the other hand , the 257-fold selectivity of 20 against enos stems from different structural features at the other end of inhibitor . similar to parent compound 9 , the fluorophenyl ring of 20 in enos ( figure 8b ) can not insert into the hydrophobic pocket on the far end of the substrate access channel . thus , lack of good van der waals contacts in the pocket in enos compared to those encountered by 20 in nnos leads to poorer affinity for enos . active site structures of 20 bound to rat nnos ( ( a ) pdb 4d32 ) and bovine enos ( ( b ) pdb 4d3a ) . key hydrogen bonds are shown by dashed lines and distances are in . the omit fo fc map for ligand is contoured at 2.5 . encouraged by the high nnos affinity and good isoform selectivities of several compounds in this series , as one of the lead nnos inhibitors , 9 was subjected to a caco-2 monolayer permeability assay ( table 3 ) . the caco-2 cell line is a human intestinal epithelial line used to approximate the compound s permeabililty in the gastrointestinal tract as well as the blood compound 9 showed good cellular permeability , thus reflecting a better potential for oral bioavailability . in addition , the efflux ratio of 9 was below 2 , which indicates that it is likely not a favorable substrate for p - gp or any active transport system that might shuttle it out of the cells . we also sought to determine the off - target effects of 9 against a panel of 50 cns receptors , which included g - protein coupled receptors such as the serotonin , adrenergic , dopamine , and histamine receptors , as well as muscarinic and receptors . compound 9 showed significant inhibition ( > 75% ) at a high 10 m concentration in a primary binding assay only at the following targets : human serotonin 5-ht1a and 5-ht2a ( 83 and 88% ) , adrenergic -2c ( 95% ) , -1 and -2 receptors ( 94 and 88% ) , and dopamine d3 receptor ( 87% ) . therefore , a consecutive secondary binding assay on these targets were evaluated , which revealed a binding affinity ( ki ) of 0.2 m only for -2c and receptors , while the rest were 0.5 m or higher . so , even a 0.2 m off - target affinity holds 10-fold selectivity ( relative to the nnos ki ) , which nonetheless is about 7% of the total receptors assayed . thus , overall 9 displays a good safety profile , which is very promising for further development of this class of scaffolds . finally , the presence of a heme - coordinating group increases the likelihood of these compound s ability to inhibit cytochromes p450 ( cyps ) , the xenobiotic - metabolizing enzymes in humans . therefore , 9 was evaluated against five major human liver microsomal p450s and , at 10 m concentration , was found to decrease the activity of cyp2c19 , cyp2d6 , and cyp3a4 by more than 70% . because cyp3a4 is the major liver and intestinal p450 that metabolizes the majority of drugs , we determined ic50 values for 9 , ( r , r)-12 , 13 , and 20 using an in vitro cyp3a4 activity assay and 7-benzyloxy-4-trifluoromethylcoumarin as a substrate ( table 4 ) . compound 9 displayed moderate inhibitory potency for cyp3a4 ( ic50 of 2.5 m ) and maintained > 130-fold selectivity toward nnos . when hydrophobicity in the molecules is increased through the cyclopropane ring insertion or methyl substitution of the imidazole in ( r , r)-12 and 20 , respectively , the cyp3a4 inhibitory potency is substantially increased ( ic50 < 1 m ) . this is understandable given the bigger and more hydrophobic active site of cyp3a4 that preferably binds large , nonpolar molecules . the more polar and potent human nnos inhibitor 13 acts as a weak cyp3a4 inactivator ( ic50 of 70 m ) , which suggests that modulation of hydrophobicity and bulkiness of compounds containing the heme - coordinating group can attenuate cyp inhibition , even with the presence of heme - ligating groups like imidazole . ic50 values are the average of at least two replicates with nine data points ; all experimental standard error values are less than 10% , and all correlation coefficients are > 0.95 . we have designed and synthesized a new series of 2,4-disubstituted pyrimidine scaffolds by exploiting the much less - explored heme coordinating ability of inhibitors in the active site of nos . this design was based on the rationale that 2-imidazolylpyrimidines might bind and inhibit nnos with groups that are less polar and less basic than the 2-aminopyridines and therefore more bioavailable . we also speculated that by modulating other parts of the inhibitor molecule , we would be able to incorporate selectivity into the inhibitor against other nos isozymes and heme - containing cyp enzymes . indeed , we were able to obtain 2,4-disusbtituted pyrimidines that are highly potent inhibitors of nnos , as shown by its low nanomolar binding affinity to both rat and human nnos , and > 200-fold and > 100-fold selectivity over enos and inos , respectively . crystal structures of the compounds bound to both rat and human nnos indicate heme - fe coordination by the 2-imidazolyl fragment , and the noncoordinating aryl rings are stabilized in a hydrophobic pocket at the far end of the substrate access channel . access to this pocket is important for gaining inhibitory potency in both rat and human nnos isoforms ( via extensive van der waals contacts and polar interactions ) and isoform selectivity ( owing to the sequence diversity in the pocket among nos isoforms ) . very promising compounds that came from this study were 9 , ( r , r)-12 , and 13 , which showed good caco-2 permeability , minimal off - target binding efficacy , and good selectivity . even with the presence of imidazole rings , cyp3a4 inhibition could be attenuated by modifications to other parts of the molecule . therefore , these results exhibit high potential among these compounds to be orally bioavailable and brain permeable . hence , further developments of these compounds are currently in progress . anhydrous solvents were purified by passage through a solvent column composed of activated alumina and a supported copper redox catalyst . all remaining solvents and reagents were purchased from commercial vendors and used without further purification . moisture or oxygen - sensitive reactions were performed under an atmosphere of dry n2 or argon . analytical thin - layer chromatography was performed on silicycle precoated silica gel 60 f254 plates . an agilent 971-fp flash purification system with various siliasep ( silicycle , 4063 m , 60 ) prepacked silica gel cartridges was used for flash column chromatography . h nmr and c nmr spectra were recorded at 500 and 126 mhz respectively on a bruker avance - iii instrument . low - resolution esims was performed on a thermo finnigan lcq or bruker amazon sl mass spectrometer consisting of an electrospray ionization ( esi ) source . high - resolution mass spectral data were obtained at the integrated molecular structure education and research facility ( northwestern university ) on an agilent 6210a tof mass spectrometer in positive ion mode using electrospray ionization , with an agilent g1312a hplc pump and an agilent g1367b autoinjector . the purity of the compounds was evaluated on a beckman gold reverse phase analytical hplc system using a phenonemex gemini c-18 ( 4.6 mm 250 mm , 5 m ) reverse phase column with uv absorbance and evaporative light scattering detection . purities of all final compounds that were subjected to enzymatic assays were found to be > 95% . preparative hplc was performed at the northwestern university center for molecular innovation and drug discovery chemcore lab , using an agilent 1200 series hplc and agilent 6120 quadrupole mass spectrometer ( api - ms mode ) , and a phenomenex gemini - nx 5 m c18 column ( 150 mm 21.2 mm ) . chiral hplc to determine enantiopurity of precursors of racemic and enantiomers of 11 was performed on an agilent 1260 series hplc using a 0.46 cm 25 cm chiralpak ad - h column , with hexanes and 2-propanol ( isocratic 10% 2-propanol in hexanes ) as the mobile phases , and the flow rate of 0.5 ml / min with uv detection . 4-chloro-2-methanesulfonyl pyrimidine,24 , 25 , 26,40,65 , and 66(43 ) were synthesized following previously reported procedures . syntheses of the remaining primary amines are detailed in supporting information . to a solution of 22 ( 0.152 g , 0.729 mmol ) in mecn ( 4 ml ) , 4-chloro-2-methanesulfonyl pyrimidine ( 0.14 g , 0.729 mmol ) and k2co3 ( 0.21 g , 1.522 mmol ) were added . the mixture was heated at 40 c for 19 h and monitored by tlc for complete consumption of 22 . then , imidazole ( 0.248 g , 3.645 mmol ) was added and the resulting mixture heated at 65 c for an additional 30 h. the mixture was cooled , diluted in ch2cl2 ( 40 ml ) , washed with h2o ( 2 30 ml ) , and the organic layer dried over anhydrous sodium sulfate . the solution was concentrated , and the resulting crude oil was purified by flash chromatography ( ch2cl2/meoh ) to yield 4 as clear sticky oil ( 0.289 g , 82% ) . h nmr ( 500 mhz ; cdcl3 ) : 8.51 ( s , 1 h ) , 8.13 ( d , j = 6.2 hz , 1 h ) , 7.80 ( s , 1 h ) , 7.267.21 ( m , 1 h ) , 7.10 ( s , 1 h ) , 6.98 ( d , j = 7.8 hz , 1 h ) , 6.946.86 ( m , 2 h ) , 6.36 ( d , j = 6.2 hz , 1 h ) , 3.71 ( s , 4 h ) , 2.82 ( t , j = 10.5 , 8.6 hz , 2 h ) , 2.64 ( dd , j = 10.1 , 8.3 hz , 2 h ) , 2.59 ( t , j = 5.2 hz , 4 h ) . c nmr ( 126 mhz ; cdcl3 ) : ( 163.74 , 161.79 , d , j = 245.9 hz , 1 c ) , 162.1 , 156.7 , 154.1 , ( 142.43 , 142.37 , d , j = 7.3 hz , 1 c ) , 136.0 , 129.9 , ( 129.81 , 129.74 , d , j = 8.4 hz , 1 c ) , ( 124.27 , 124.25 , d , j = 2.5 hz , 1 c ) , 116.5 , ( 115.53 , 115.36 , d , j = 21.0 hz , 1 c ) , ( 113.08 , 112.92 , d , j = 21.0 hz , 1 c ) , 100.5 , 59.7 , 53.4 , 43.8 , 33.2 . hrms ( esi ) : calcd for c19h22fn6 [ m + h ] , 353.1884 ; found , 353.1887 . compound 26 ( 0.146 g , 0.516 mmol ) was diluted with mecn ( 3 ml ) , followed by addition of 4-chloro-2-methanesulfonyl pyrimidine ( 0.1 g , 0.516 mmol ) and k2co3 ( 0.143 g , 1.033 mmol ) and heated at 40 c for 19 h. imidazole ( 0.176 g , 2.583 mmol ) was added to the resulting mixture , and heating continued at 65 c for another 30 h. the mixture was cooled , diluted in ch2cl2 ( 30 ml ) , washed with h2o ( 2 20 ml ) , and the organic layer dried over anhydrous sodium sulfate . the solution was concentrated , and the resulting crude oil was purified by flash chromatography ( etoac / meoh ) to yield the boc - protected precursor of 5 as a clear sticky oil ( 0.132 g , 60% ) . this intermediate boc - protected carbamate was dissolved in ch2cl2 ( 3 ml ) , cooled to 0 c , and trifluoroacetic acid was added ( 1.5 ml ) to it . the resulting solution was stirred at room temperature for 2 h , when it was diluted with more ch2cl2 ( 15 ml ) , and treated slowly with saturated k2co3 ( 10 ml ) . the layers were separated , and the aqueous layer was extracted with ch2cl2 ( 2 10 ml ) . the combined organic layers were dried with anhydrous sodium sulfate , concentrated , and chromatographed with etoac / meoh to give an oily residue . methanolic hcl ( 2 m , 2 ml ) was added to the residue , stirred for 10 min , concentrated to 1 ml , and treated with excess et2o , when a white crystalline precipitate formed . the solid was collected by filtration and dried to obtain 5 as a white crystalline solid ( 0.11 g , 83% ) ; mp = 208210 c . h nmr ( 500 mhz ; dmso - d6 ) : 10.32 ( s , 1 h ) , 9.49 ( s , 2 h ) , 8.66 ( t , j = 6.0 hz , 1 h ) , 8.52 ( t , j = 1.8 hz , 1 h ) , 8.22 ( d , j = 6.0 hz , 1 h ) , 7.89 ( t , j = 1.7 hz , 1 h ) , 7.39 ( td , j = 7.9 , 6.3 hz , 1 h ) , 7.197.07 ( m , 3 h ) , 6.69 ( d , j = 6.0 hz , 1 h ) , 3.903.86 ( m , 2 h ) , 3.23 ( dq , j = 11.4 , 6.3 hz , 2 h ) , 3.183.14 ( m , 2 h ) , 3.06 ( dd , j = 9.7 , 6.5 hz , 2 h ) , 1.30 ( dd , j = 15.1 , 6.8 hz , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : ( 164.16 , 162.23 , d , j = 243.9 hz , 1 c ) , 164.1 , 155.8 , 153.1 , ( 141.09 , 141.00 , d , j = 11.3 hz , 1 c ) , 136.2 , ( 131.52 , 131.46 , d , j = 7.6 hz , 1 c ) , ( 125.79 , 125.78 , d , j = 1.3 hz , 1 c ) , 122.1 , 119.7 , ( 116.48 , 116.31 , d , j = 21.4 hz , 1 c ) , ( 114.69 , 114.48 , j = 26.4 hz , 1 c ) , 107.7 , 48.4 , 46.7 , 37.2 , 32.0 . hrms ( esi ) : calcd for c17h20fn6 [ m + h ] , 327.1728 ; found , 327.1731 . general method a for trifluoroacetic acid mediated boc - group deprotection : compound 37 ( 0.257 g , 0.647 mmol ) was diluted in ch2cl2 ( 5.5 ml ) and cooled to 0 c . trifluoroacetic acid ( 2.75 ml ) was added to the resulting solution and stirred at room temperature for 3 h. the reaction mixture was diluted with more ch2cl2 ( 25 ml ) and treated with saturated k2co3 ( 15 ml ) . the layers were separated , and the aqueous layer was extracted again with ch2cl2 ( 2 15 ml ) . the resulting oily residue was purified by flash column chromatography ( ch2cl2/meoh ) to give the free base of 6 as a yellow oil . the oil was treated with methanolic hcl ( 2 m , 2 ml ) for 10 min , when a white precipitate started forming . this suspension was concentrated to 0.5 ml and treated with excess et2o and sonicated . the white solid was filtered , washed twice with et2o , and dried to give a white amorphous solid of 6 as a dihydrochloride salt ( 0.223 g , 93% ) ; mp = 220222 c . h nmr ( 500 mhz , dmso - d6 ) : 10.07 ( s , 2 h ) , 10.02 ( s , 1 h ) 9.05 ( d , j = 5.1 hz , 1 h ) , 8.57 ( s , 1 h ) , 7.78 ( d , j = 5.0 hz , 2 h ) , 7.447.37 ( m , 1 h ) , 7.217.09 ( m , 3 h ) , 4.61 ( t , j = 5.2 hz , 2 h ) , 4.01 ( br s , 1 h ) , 3.393.28 ( m , 2 h ) , 3.243.15 ( m , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : 164.8 , ( 164.17 , 162.23 , d , j = 244.4 hz , 1 c ) , 161.2 , 153.4 , ( 141.05 , 140.99 , j = 7.6 hz , 1 c ) , 136.9 , ( 131.51 , 131.44 , d , j = 8.8 hz , 1 c ) , ( 125.80 , 125.78 , d , j = 2.5 hz , 1 c ) , 125.1 , 120.7 , 119.3 , ( 116.48 , 116.31 , d , j = 21.4 hz , 1 c ) , ( 114.68 , 114.45 , d , j = 29 hz , 1 c ) , 49.7 , 48.2 , 31.9 . hrms ( esi ) : calcd for c16h17fn5 [ m + h ] , 298.1463 ; found , 298.1462 . compound 7 was obtained as a white solid in its dihydrochloride salt form ( 0.37 g , 79% ) from 38 ( 0.502 g , 1.22 mmol ) following general method a ; mp = 153155 c . h nmr ( 500 mhz , dmso - d6 ) : 9.61 ( br s , 3 h ) , 9.01 ( d , j = 4.9 hz , 1 h ) , 8.40 ( s , 1 h ) , 7.69 ( d , j = 4.6 hz , 1 h ) , 7.60 ( s , 1 h ) , 7.407.34 ( m , 1 h ) , 7.147.05 ( m , 3 h ) , 4.57 ( t , j = 5.5 hz , 2 h ) , 3.69 ( br s , 1 h ) , 3.093.03 ( m , 2 h ) , 2.74 ( t , j = 7.6 hz , 2 h ) , 2.162.00 ( m , 2 h ) . c nmr ( 126 mhz ; dmso - d6 ) : 168.0 , 164.9 , ( 164.26 , 162.32 , d , j = 244.44 , 1 c ) , 161.3 , 153.7 , ( 144.75 , 144.69 , d , j = 7.56 hz , 1 c ) , 137.1 , ( 131.38 , 131.31 , d , j = 8.8 hz , 1 c ) , ( 125.52 , 125.5 , d , j = 2.5 hz , 1 c ) , 120.3 , 119.0 , ( 116.14 , 115.97 , d , j = 21.42 hz , 1 c ) , ( 114.03 , 113.86 , d , j = 21.42 hz , 1 c ) , 49.7 , 47.3 , 32.5 , 27.7 . hrms ( esi ) : calcd for c17h19fn5 [ m + h ] , 312.1619 ; found , 312.1625 . compound 8 was obtained as a pale cream - colored amorphous solid ( 0.182 g , 80% ) from 55 ( 0.244 g , 0.594 mmol ) following general method a ; mp = 200201 c . h nmr ( 500 mhz , dmso - d6 ) : 9.88 ( s , 1 h ) , 9.39 ( s , 2 h ) , 8.94 ( d , j = 5.1 hz , 1 h ) , 8.44 ( s , 1 h ) , 7.82 ( s , 1 h ) , 7.70 ( d , j = 5.1 hz , 1 h ) , 7.42 ( q , j = 7.8 hz , 1 h ) , 7.287.05 ( m , 3 h ) , 4.08 ( br s , 1 h ) , 3.563.53 ( m , 2 h ) , 3.39 ( t , j = 6.9 hz , 2 h ) , 3.313.25 ( m , 2 h ) , 3.133.03 ( m , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : 170.1 , ( 164.26 , 162.32 , d , j = 244.44 hz , 1 c ) , 160.5 , 153.5 , ( 141.21 , 141.15 , d , j = 7.56 hz , 1 c ) , 136.6 , ( 131.63 , 131.56 , d , j = 8.82 hz , 1 c ) , 125.9 , 125.8 , ( 122.23 , 122.19 , d , j = 5.0 hz , 1 c ) , 119.4 , ( 116.57 , 116.40 , d , j = 21.42 hz , 1 c ) , ( 114.74 , 114.57 , d , j = 21.42 hz , 1 c ) , 48.5 , 44.9 , 33.4 , 32.0 . hrms ( esi ) : calcd for c17h19fn5 [ m + h ] , 312.1619 ; found , 312.1622 . title compound 9 was obtained as a white amorphous solid ( 0.385 g , 89% ) from 56 ( 0.462 g , 1.086 mmol ) following general method a ; mp = 196198 c . h nmr ( 500 mhz , dmso - d6 ) : 9.94 ( s , 1 h ) , 9.39 ( s , 2 h ) , 8.94 ( d , j = 5.1 hz , 1 h ) , 8.45 ( s , 1 h ) , 7.85 ( s , 1 h ) , 7.70 ( d , j = 5.1 hz , 1 h ) , 7.37 ( q , j = 7.9 hz , 1 h ) , 7.12 ( t , j = 8.4 hz , 2 h ) , 7.06 ( td , j = 8.9 , 2.3 hz , 1 h ) , 4.25 ( br s , 1 h ) , 3.533.48 ( m , 2 h ) , 3.433.37 ( m , 2 h ) , 3.02.94 ( m , 2 h ) , 2.74 ( t , j = 7.6 hz , 2 h ) , 2.03 ( p , j = 8.2 hz , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : 170.2 , ( 164.17 , 162.24 , d , j = 243.18 hz , 1 c ) , 160.4 , 153.3 , ( 144.72 , 144.66 , d , j = 7.56 hz , 1 c ) , 136.5 , ( 131.29 , 131.22 , d , j = 8.82 hz , 1 c ) , ( 125.44 , 125.42 , d , j = 2.52 hz , 1 c ) , 123.9 , 122.3 , 119.5 , ( 116.07 , 115.91 , d , j = 20.16 hz , 1 c ) , ( 113.92 , 113.75 , d , j = 21.42 hz , 1 c ) , 47.3 , 44.7 , 33.4 , 32.6 , 27.7 . hrms ( esi ) : calcd for c18h21fn5 [ m + h ] , 326.1776 ; found , 326.1779 . compound 10 was synthesized as a white amorphous solid ( 0.5 g , 99% ) from 57 ( 0.541 g , 1.224 mmol ) following general method a ; mp = 184186 c . h nmr ( 500 mhz , dmso - d6 ) : 10.03 ( s , 1 h ) , 9.59 ( s , 2 h ) , 8.94 ( d , j = 5.1 hz , 1 h ) , 8.47 ( s , 1 h ) , 7.88 ( s , 1 h ) , 7.71 ( d , j = 5.1 hz , 1 h ) , 7.397.32 ( m , 2 h ) , 7.29 ( d , j = 8.2 hz , 1 h ) , 7.24 ( d , j = 7.6 hz , 1 h ) , 4.01 ( s , 1 h ) , 3.533.47 ( m , 2 h ) , 3.453.36 ( m , 2 h ) , 2.992.93 ( m , 2 h ) , 2.73 ( t , j = 7.6 hz , 2 h ) , 2.04 ( p , j = 7.8 hz , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : 170.2 , 160.4 , 153.2 , 144.4 , 136.3 , 133.9 , 131.2 , 129.1 , 128.0 , 127.0 , 123.2 , 122.4 , 119.6 , 47.3 , 44.7 , 33.4 , 32.5 , 27.6 . hrms ( esi ) : calcd for c18h21cln5 [ m + h ] , 342.1480 ; found , 342.1483 . compound 11 was prepared from 58 ( 0.25 g , 0.571 mmol ) as an off - white amorphous dihydrochloride salt ( 0.206 g , 88% ) following general method a ; mp = 198200 c . h nmr ( 500 mhz , dmso - d6 ) : 10.00 ( s , 1 h ) , 9.58 ( s , 1 h ) , 9.49 ( s , 1 h ) , 8.94 ( d , j = 5.1 hz , 1 h ) , 8.43 ( s , 1 h ) , 7.88 ( s , 1 h ) , 7.71 ( d , j = 5.1 hz , 1 h ) , 7.31 ( q , j = 7.9 hz , 1 h ) , 7.076.97 ( m , 3 h ) , 4.48 ( br s , 1 h ) , 3.593.50 ( m , 2 h ) , 3.443.39 ( m , 2 h ) , 3.183.15 ( m , 1 h ) , 3.012.96 ( m , 1 h ) , 2.21 ( dt , j = 9.2 , 5.2 hz , 1 h ) , 1.49 ( dq , j = 12.5 , 7.9 hz , 1 h ) , 1.221.09 ( m , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : 170.2 , ( 164.21 , 162.28 , d , j = 243.18 hz , 1 c ) , 160.4 , 153.2 , ( 145.83 , 145.77 , d , j = 7.56 hz , 1 c ) , 136.3 , ( 131.0 , 130.93 , d , j = 8.82 hz , 1 c ) , 123.4 , ( 123.03 , 123.01 , d , j = 2.52 hz , 1 c ) , 122.4 , 119.5 , ( 113.63 , 113.46 , d , j = 21.42 hz , 1 c ) , ( 113.40 , 113.24 , d , j = 20.16 hz , 1 c ) , 51.2 , 44.2 , 33.3 , 22.6 , 20.0 , 15.4 . hrms ( esi ) : calcd for c19h21fn5 [ m + h ] , 338.1776 ; found , 338.1778 . compound 12 was synthesized as a white amorphous solid ( 0.256 g , 93% ) from 59 ( 0.294 g , 0.672 mmol ) following general method a ; mp = 210212 c . h nmr ( 500 mhz , dmso - d6 ) : 9.85 ( s , 1 h ) , 9.69 ( s , 1 h ) , 9.60 ( s , 1 h ) , 8.93 ( d , j = 5.1 hz , 1 h ) , 8.39 ( s , 1 h ) , 7.82 ( s , 1 h ) , 7.65 ( d , j = 5.1 hz , 1 h ) , 7.37 ( td , j = 8.0 , 6.4 hz , 1 h ) , 7.227.14 ( m , 2 h ) , 7.05 ( td , j = 8.4 , 2.3 hz , 1 h ) , 4.2 ( br s , 1 h ) , 3.553.42 ( m , 2 h ) , 3.33 ( t , j = 7.2 hz , 2 h ) , 2.77 ( m , 1 h ) , 2.682.59 ( m , 2 h ) , 1.731.66 ( m , 1 h ) , 1.23 ( ddd , j = 9.7 , 5.7 , 4.0 hz , 1 h ) , 0.84 ( q , j = 6.1 hz , 1 h ) . c nmr ( 126 mhz , dmso - d6 ) : 170.0 , ( 164.11 , 162.17 , d , j = 244.44 , 1 c ) , 160.4 , 153.4 , ( 144.24 , 144.18 , d , j = 7.56 hz , 1 c ) , 136.4 , ( 131.21 , 131.14 , d , j = 8.82 hz , 1 c ) , 125.5 , 124.2 , 122.1 , 119.3 , ( 116.16 , 115.99 , d , j = 21.42 hz , 1 c ) , ( 113.97 , 113.80 , d , j = 21.42 hz , 1 c ) , 45.3 , 36.7 , 35.8 , 33.0 , 18.6 , 10.7 . hrms ( esi ) : calcd for c19h21fn5 [ m + h ] , 338.1776 ; found , 338.1776 . title compound 13 was obtained as a trihydrochloride salt , and as a pale - green amorphous solid ( 0.26 g , 91% ) from 60 ( 0.28 g , 0.684 mmol ) following general method a ; mp = 212214 c . h nmr ( 500 mhz , dmso - d6 ) : 10.12 ( s , 1 h ) , 9.77 ( s , 2 h ) , 8.97 ( s , 1 h ) , 8.95 ( d , j = 5.1 hz , 1 h ) , 8.85 ( d , j = 5.4 hz , 1 h ) , 8.61 ( d , j = 7.8 hz , 1 h ) , 8.50 ( s , 1 h ) , 8.07 ( dd , j = 6 , 7.5 hz , 1 h ) , 7.92 ( s , 1 h ) , 7.73 ( d , j = 5.1 hz , 1 h ) , 3.563.39 ( m , 4 h ) , 3.022.99 ( m , 4 h ) , 2.15 ( p , j = 7.4 hz , 2 h ) . note that the pyridinium and the imidazolium protons appear very broad on the baseline due to exchange with solvent . c nmr ( 126 mhz , dmso - d6 ) : 170.3 , 160.4 , 153.1 , 147.0 , 141.9 , 141.8 , 140.4 , 136.3 , 127.9 , 122.7 , 122.5 , 119.7 , 46.8 , 44.7 , 33.3 , 29.5 , 26.9 . hrms ( esi ) : calcd for c17h21n6 [ m + h ] , 309.1822 ; found , 309.1824 . compound 14 was synthesized as a white amorphous solid ( 0.277 g , 95% ) from 61 ( 0.313 g , 0.774 mmol ) following general method a ; mp = 220222 c . h nmr ( 500 mhz , dmso - d6 ) : 9.93 ( br s , 3 h ) , 8.93 ( d , j = 5.1 hz , 1 h ) , 8.41 ( s , 1 h ) , 8.17 ( s , 1 h ) , 8.02 ( d , j = 7.9 hz , 1 h ) , 7.92 ( d , j = 7.8 hz , 1 h ) , 7.85 ( s , 1 h ) , 7.727.66 ( m , 2 h ) , 4.33 ( t , j = 5.3 hz , 2 h ) , 3.583.49 ( m , 2 h ) , 3.44 ( t , j = 7.0 hz , 2 h ) . note that the imidazolium proton appears very broad on the baseline due to exchange with solvent . c nmr ( 126 mhz , dmso - d6 ) : 170.0 , 160.4 , 153.3 , 136.4 , 136.1 , 134.9 , 134.5 , 133.5 , 130.7 , 123.8 , 122.3 , 119.4 , 119.3 , 112.3 , 50.0 , 44.5 , 33.2 . hrms ( esi ) : calcd for c17h17n6 [ m + h ] , 305.1509 ; found , 305.1512 . compound 15 was obtained as a white amorphous solid ( 0.260 g , 88% ) from 68 ( 0.288 g , 0.704 mmol ) following general method a ; mp = 230232 c . h nmr ( 500 mhz , dmso - d6 ) : 9.96 ( s , 1 h ) , 9.61 ( s , 2 h ) , 9.02 ( d , j = 2.4 hz , 1 h ) , 8.73 ( d , j = 1.4 hz , 1 h ) , 8.47 ( t , j = 1.6 hz , 1 h ) , 8.42 ( t , j = 2.0 hz , 1 h ) , 8.02 ( s , 1 h ) , 7.42 ( td , j = 7.9 , 6.5 hz , 1 h ) , 7.19 ( dd , j = 10.0 , 1.9 hz , 1 h ) , 7.16 ( d , j = 7.7 hz , 1 h ) , 7.13 ( td , j = 8.7 , 2.5 hz , 1 h ) , 5.57 ( s , 2 h ) , 3.36 ( dt , j = 11.9 , 6.8 hz , 2 h ) , 3.273.19 ( m , 4 h ) , 3.153.03 ( m , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : ( 164.19 , 162.25 , d , j = 244.44 hz , 1 c ) , 151.3 , 142.2 , ( 141.18 , 141.12 , d , j = 7.56 hz , 1 c ) , 136.0 , 135.2 , 132.6 , 131.6 , ( 131.56 , 131.49 , d , j = 8.82 hz , 1 c ) , 125.8 , 122.1 , 121.9 , ( 116.50 , 116.34 , d , j = 20.16 hz , 1 c ) , ( 114.65 , 114.49 , d , j = 20.16 hz , 1 c ) , 48.3 , 47.4 , 31.9 , 29.1 . hrms ( esi ) : calcd for c18h20fn4 [ m + h ] , 311.1667 ; found , 311.1673 . title compound 16 was obtained as a pale - yellow hygroscopic solid ( 0.186 g , 89% ) from 69 ( 0.224 g , 0.525 mmol ) following general method a. h nmr ( 500 mhz , dmso - d6 ) : 8.94 ( d , j = 5.0 hz , 1 h ) , 8.69 ( br s , 2 h ) , 8.25 ( s , 2 h ) , 7.64 ( d , j = 5.0 hz , 1 h ) , 7.38 ( q , j = 7.9 hz , 1 h ) , 7.167.00 ( m , 3 h ) , 3.62 ( br s , 1 h ) , 3.543.47 ( m , 2 h ) , 3.303.27 ( m , 2 h ) , 3.053.02 ( m , 2 h ) , 2.742.70 ( m , 2 h ) , 1.991.96 ( m , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : 169.6 , ( 164.16 , 162.22 , d , j = 244.44 hz , 1 c ) , 160.5 , 155.7 , ( 144.56 , 144.52 , d , j = 5.04 hz , 1 c ) , 138.9 , ( 131.27 , 131.21 , d , j = 7.56 hz , 1 c ) , ( 125.37 , 125.35 , d , j = 2.52 hz , 1 c ) , 121.5 , ( 116.00 , 115.83 , d , j = 21.42 hz , 1 c ) , ( 113.92 , 113.75 , d , j = 21.42 hz , 1 c ) , 47.3 , 45.5 , 33.6 , 32.4 , 27.8 . hrms ( esi ) : calcd for c17h20fn6 [ m + h ] , 327.1728 ; found , 327.1735 . compound 17 was obtained as a white amorphous solid in its dihydrochloride form ( 0.125 g , 99% ) from 70 ( 0.135 g , 0.316 mmol ) following general method a ; mp = 177179 c . h nmr ( 500 mhz , dmso - d6 ) : 9.56 ( s , 1 h ) , 9.20 ( s , 2 h ) , 8.89 ( d , j = 5.1 hz , 1 h ) , 8.35 ( s , 1 h ) , 7.60 ( d , j = 5.1 hz , 1 h ) , 7.37 ( td , j = 8.0 , 6.4 hz , 1 h ) , 7.157.08 ( m , 2 h ) , 7.06 ( td , j = 8.8 , 2.5 hz , 1 h ) , 6.29 ( br s , 1 h ) , 3.46 ( p , j = 7.0 hz , 2 h ) , 3.33 ( t , j = 7.2 hz , 2 h ) , 2.99 ( dt , j = 12.3 , 6.3 hz , 2 h ) , 2.74 ( t , j = 7.6 hz , 2 h ) , 2.01 ( p , j = 7.8 hz , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : 169.7 , ( 164.15 , 162.22 , d , j = 243.18 , 1 c ) , 160.4 , 154.6 , 154.3 , 145.6 , ( 144.67 , 144.61 , d , j = 7.56 hz , 1 c ) , ( 131.24 , 131.18 , d , j = 7.56 hz , 1 c ) , ( 125.38 , 125.36 , d , j = 2.52 hz , 1 c ) , 121.3 , ( 116.02 , 115.85 , d , j = 21.42 hz , 1 c ) , ( 113.88 , 113.71 , d , j = 21.42 hz , 1 c0 , 47.2 , 45.0 , 33.4 , 32.5 , 27.7 . hrms ( esi ) : calcd for c17h20fn6 [ m + h ] , 327.1728 ; found , 327.1728 . compound 18 was prepared from 71 ( 0.141 g , 0.32 mmol ) following general method a to give the dihydrochloride salt , which was obtained as an off - white hygroscopic solid ( 0.115 g , 87% ) . h nmr ( 500 mhz , dmso - d6 ) : 9.33 ( s , 2 h ) , 8.98 ( d , j = 5.1 hz , 1 h ) , 8.36 ( d , j = 2.2 hz , 1 h ) , 7.79 ( d , j = 2.1 hz , 1 h ) , 7.73 ( d , j = 5.1 hz , 1 h ) , 7.38 ( td , j = 8.0 , 6.5 hz , 1 h ) , 7.167.09 ( m , 2 h ) , 7.07 ( td , j = 8.8 , 2.3 hz , 1 h ) , 3.51 ( br s , 1 h ) , 3.403.37 ( m , 4 h ) , 3.02 ( s , 3 h ) , 2.992.94 ( m , 2 h ) , 2.74 ( t , j = 7.6 hz , 2 h ) , 2.01 ( p , j = 7.8 hz , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : 169.9 , ( 164.14 , 162.21 , d , j = 243.18 , 1 c ) , 160.3 , 154.5 , 147.4 , ( 144.65 , 144.59 , d , j = 7.56 hz , 1 c ) , ( 131.25 , 131.18 , d , j = 8.82 hz , 1 c ) , 125.4 , 122.0 , 121.3 , 119.7 , ( 116.01 , 115.84 , d , j = 21.42 hz , 1 c ) , ( 113.89 , 113.72 , d , j = 21.42 , 1 c ) , 47.2 , 45.0 , 33.5 , 32.5 , 27.6 , 15.8 . hrms ( esi ) : calcd for c19h23fn5 [ m + h ] , 340.1932 ; found , 340.1933 . compounds 19 and 3-(3-fluorophenyl)-n-2-[2-(5-methyl-1h - imidazol-1-yl)pyrimidin-4-yl]ethylpropan-1-amine dihydrochloride ( 20 ) were obtained in an inseparable 15:1 ratio ( by h nmr ) from the reaction of 15:1 mixture of compounds 72 and 73 ( 0.668 g , 1.52 mmol ) following general method a , as a white amorphous solid of the corresponding dihydrochloride salts ( 0.62 g , 99% total ) . this mixture was separated by preparative hplc on an agilent 1200 series instrument using a phenomenex gemini - nx 5 m c18 column ( 150 mm 21.2 mm ) , using a gradient of 115% mecn in h2o with 0.1% formic acid , to isolate 19 and 20 separately in > 95% purity . following this , 19 and 20 were converted to their corresponding dihydrochloride salts by similar precipitation with methanolic hcl and excess et2o . white amorphous solid ; mp = 2 03205 c . h nmr ( 500 mhz , dmso - d6 ) : 9.53 ( s , 1 h ) , 8.95 ( s , 2 h ) , 8.90 ( d , j = 5.1 hz , 1 h ) , 8.07 ( s , 1 h ) , 7.62 ( d , j = 5.1 hz , 1 h ) , 7.38 ( td , j = 8.0 , 6.4 hz , 1 h ) , 7.157.09 ( m , 2 h ) , 7.07 ( td , j = 8.9 , 2.2 hz , 1 h ) , 3.63 ( br s , 1 h ) , 3.50 ( dt , j = 12.8 , 7.1 hz , 2 h ) , 3.31 ( t , j = 7.2 hz , 2 h ) , 3.052.95 ( m , 2 h ) , 2.73 ( t , j = 7.6 hz , 2 h ) , 2.34 ( s , 3 h ) , 1.99 ( p , j = 7.7 hz , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : 170.0 , ( 164.14 , 162.21 , d , j = 243.18 hz , 1 c ) , 160.4 , 153.2 , ( 144.65 , 144.60 , d , j = 6.3 hz , 1 c ) , 135.6 , 133.4 , ( 131.25 , 131.18 , d , j = 8.82 hz , 1 c0 , 125.4 , 122.0 , ( 116.01 , 115.85 , d , j = 20.16 hz , 1 c ) , 115.8 , ( 113.88 , 113.71 , d , j = 21.42 hz , 1 c ) , 47.3 , 44.7 , 33.4 , 32.5 , 27.6 , 11.3 . hrms ( esi ) : calcd for c19h23fn5 [ m + h ] , 340.1932 ; found , 340.1937 . h nmr ( 500 mhz , dmso - d6 ) : 9.74 ( s , 1 h ) , 9.25 ( s , 2 h ) , 8.96 ( d , j = 5.1 hz , 1 h ) , 7.72 ( d , j = 5.1 hz , 1 h ) , 7.59 ( s , 1 h ) , 7.437.32 ( m , 1 h ) , 7.11 ( t , j = 7.4 hz , 2 h ) , 7.097.03 ( m , 1 h ) , 3.68 ( br s , 1 h ) , 3.483.43 ( m , 2 h ) , 3.37 ( q , j = 6.7 hz , 2 h ) , 2.97 ( dt , j = 14.9 , 9.5 hz , 2 h ) , 2.73 ( t , j = 7.6 hz , 2 h ) , 2.65 ( s , 3 h ) , 2.051.97 ( m , 2 h ) . c nmr ( 126 mhz , dmso - d6 ) : 169.9 , ( 164.14 , 162.21 , d , j = 243.18 hz , 1 c ) , 160.3 , 154.7 , ( 144.63 , 144.57 , d , j = 7.56 hz , 1 c ) , 137.5 , ( 131.25 , 131.18 , d , j = 8.82 hz , 1 c ) , 131.1 , ( 125.37 , 125.35 , d , j = 2.52 hz , 1 c ) , 122.1 , 121.1 , ( 116.00 , 115.84 , d , j = 20.16 hz , 1 c ) , ( 113.89 , 113.72 , d , j = 21.42 hz , 1 c ) , 47.2 , 45.0 , 33.5 , 32.5 , 27.6 , 13.13 . hrms ( esi ) : calcd for c19h23fn5 [ m + h ] , 340.1932 ; found , 340.1937 . to a suspension of nah ( 0.107 g , 2.686 mmol ) in thf ( 4 ml ) cooled to 0 c , a solution of 21 ( 0.5 g , 2.686 mmol ) in thf ( 2.5 ml ) was added dropwise . the reaction was stirred at room temperature for 1 h followed by the addition of 2-(3-fluorophenyl)ethyl bromide ( 0.66 g , 3.242 mmol ) in thf ( 2 ml ) and a pinch of tetrabutylammonium iodide ( 0.05 g , 0.13 mmol ) . the resulting solution was heated at 60 c for 48 h , when it was cooled to room temperature and diluted with ethyl acetate ( 20 ml ) and water ( 20 ml ) . the layers were separated , and the aqueous layer was washed with etoac ( 2 10 ml ) . the combined organic layers were washed with brine ( 20 ml ) , dried over sodium sulfate , and concentrated . the residue was purified by flash column chromatography ( hexanes / etoac ) , and the resulting oil ( 0.464 g , 1.504 mmol , 56% ) was diluted with ch2cl2 ( 12 ml ) . this solution was cooled to 0 c and treated with trifluoroacetic acid ( 6 ml ) . the reaction was stirred for 3 h at room temperature , after which it was concentrated . the residue was diluted with etoac ( 20 ml ) and treated with saturated k2co3 ( 15 ml ) . the organic layer was extracted , and the aqueous layer was re - extracted with more etoac ( 2 10 ml ) . the combined organic layers were dried and concentrated to yield a colorless oil ( 0.266 g , 85% ) , clean by nmr . h nmr ( 500 mhz , cdcl3 ) : 7.22 ( q , j = 7.2 hz , 1h ) , 6.96 ( d , j = 7.7 hz , 1h ) , 6.926.85 ( m , 2h ) , 3.98 ( s , 1h ) , 2.96 ( t , j = 5.0 hz , 4h ) , 2.78 ( dd , j = 11.2 , 8.3 hz , 2h ) , 2.58 ( dd , j = 11.0 , 8.5 hz , 2h ) , 2.53 ( s , 4h ) . c nmr ( 126 mhz , cdcl3 ) : ( 163.75 , 161.80 , d , j = 245.7 hz , 1 c ) , ( 142.71 , 142.65 , d , j = 7.56 hz , 1 c ) , ( 129.75 , 129.69 , d , j = 7.56 hz , 1 c ) , ( 124.29 , 124.27 , d , j = 2.52 hz , 1 c ) , ( 115.55 , 115.38 , d , j = 21.42 hz , 1 c ) , ( 112.98 , 112.81 , d , j = 21.42 hz , 1 c ) , 60.4 , 53.5 , 45.5 , 33.0 . compound 30 ( 0.2 g , 1.3 mmol ) was added in ch2cl2 ( 20 ml ) to a flask containing oven - dried 3 molecular sieves . compound 31 ( 0.17 ml , 0.18 g , 1.3 mmol ) was added as a solution in ch2cl2 ( 6 ml ) , followed by glacial acetic acid ( 20 l ) . the mixture was stirred for 1.5 h and cooled to 0 c , and sodium triacetoxyborohydride ( 0.413 g , 1.95 mmol ) was added in one portion . the mixture was stirred at room temperature for 14 h , after which it was diluted with ch2cl2 ( 30 ml ) and filtered . the filtrate was washed with saturated aqueous nahco3 ( 30 ml ) , and the aqueous layer was extracted with ch2cl2 ( 2 20 ml ) . the organic phase was washed with brine ( 40 ml ) , dried over anhydrous sodium sulfate , and concentrated . the resulting residue was purified by flash column chromatography , eluting with a gradient of ch2cl2/meoh to yield the intermediate 33 ( 0.306 g , 85% ) as an off - white sticky oil . h nmr ( 500 mhz , cdcl3 ) : 8.42 ( d , j = 5.0 hz , 1 h ) , 7.25 ( q , j = 7.5 , hz , 1 h ) , 6.98 ( d , j = 7.6 hz , 1 h ) , 6.946.88 ( m , 3 h ) , 3.83 ( s , 2 h ) , 2.90 ( t , j = 6.6 hz , 2 h ) , 2.83 ( t , j = 6.7 hz , 2 h ) , 2.50 ( s , 3 h ) , 2.13 ( s , 1 h ) . c nmr ( 126 mhz , cdcl3 ) : 172.4 , 168.6 , ( 163.84 , 161.88 , d , j = 246.96 hz , 1 c ) , 157.1 , ( 142.29 , 142.23 , d , j = 7.56 hz , 1 c ) , ( 129.92 , 129.85 , d , j = 8.82 hz , 1 c ) , ( 124.36 , 124.34 , d , j = 2.52 hz , 1 c ) , ( 115.60 , 115.43 , d , j = 21.42 hz , 1 c ) , 114.2 , ( 113.21 , 113.04 , d , j = 21.42 hz , 1 c ) , 53.9 , 50.2 , 36.1 , 14.0 . ms ( esi ) m / z [ m + h ] : calcd , 278.1 ; found , 278.06 . compound 34 ( 0.594 g , 89% ) was prepared from 30 ( 0.352 g , 2.285 mmol ) and 32 ( 0.35 g , 2.285 mmol ) by the exact procedure used to prepare 33 . h nmr ( 500 mhz , cdcl3 ) : 8.42 ( d , j = 5.0 hz , 1 h ) , 7.21 ( q , j = 7.5 hz , 1 h ) , 6.96 ( d , j = 5.0 hz , 1 h ) , 6.93 ( d , j = 7.6 hz , 1 h ) , 6.886.83 ( m , 2 h ) , 3.80 ( s , 2 h ) , 2.682.63 ( m , 4 h ) , 2.54 ( s , 3 h ) , 2.22 ( s , 1 h ) , 1.83 ( p , j = 7.3 hz , 2 h ) . c nmr ( 126 mhz , cdcl3 ) : 172.3 , 168.7 , ( 163.77 , 161.82 , d , j = 245.7 hz , 1 c ) , 157.1 , ( 144.47 , 144.41 , d , j = 7.56 hz , 1 c ) , ( 129.69 , 129.63 , d , j = 7.56 hz , 1 c ) , ( 123.94 , 123.92 , d , j = 2.52 hz , 1 c ) , ( 115.16 , 115.0 , d , j = 20.16 hz , 1 c ) , 114.3 , ( 112.70 , 112.54 , d , j = 20.16 hz , 1 c ) , 54.0 , 48.7 , 33.1 , 31.3 , 14.1 . ms ( esi ) m / z [ m + na ] : calcd , 314.11 ; found 314.22 . compound 33 ( 0.306 g , 1.103 mmol ) was dissolved in thf ( 10 ml ) , and a solution of di - tert - butyl dicarbonate ( 0.265 g , 1.213 mmol ) in thf ( 6 ml ) was added . the resulting solution was stirred overnight , and thereafter , it was diluted with etoac / h2o ( 30 ml , 1:1 ) . the layers were separated and the aqueous layer extracted with etoac ( 2 10 ml ) . the combined organic layers were dried , concentrated , and purified by column chromatography ( hexanes / etoac ) . the resulting oil ( 0.412 g , 1.091 mmol , 99% ) was dissolved in ch2cl2 ( 2 ml ) and added to a solution of m - chloroperbenzoic acid ( 0.69 g , 4.0 mmol ) in ch2cl2 ( 4 ml ) at 0 c . the reaction was gradually warmed to room temperature while it was stirred overnight . the resulting suspension was then filtered and the white solid washed with cold ch2cl2 ( 5 ml ) . the collected filtrate was washed with 10% aqueous k2co3 ( 5 ml ) and the organic fraction dried , concentrated , and purified by flash column chromatography to give a sticky white solid ( 0.335 g , 75% ) . h nmr ( 500 mhz , cdcl3 ) : ( 8.81 ( d , j = 5.0 hz ) , 8.79 ( d , j = 4.9 hz ) , 2:3 , 1 h ) , ( 7.39 ( d , j = 4.8 hz ) , 7.32 ( d , j = 4.0 hz ) , 3:2 , 1 h ) , 7.23 ( q , j = 7.8 hz , 1 h ) , 6.966.83 ( m , 3 h ) , ( 4.53 ( s ) , 4.46 ( s ) , 3:2 , 2 h ) , 3.573.52 ( m , 2 h ) , 3.33 ( s , 3 h ) , ( 2.89 ( t , j = 6.7 hz ) , 2.84 ( t , j = 7 hz ) , 2:3 , 2 h ) , ( 1.45 ( s ) , 1.35 ( s ) , 3:2 , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 170.85 , 170.17 , 1 c ) , ( 165.82 , 165.61 , 1 c ) , ( 163.78 , 161.83 , d , j = 245.7 hz , 1 c ) , 158.51 , ( 155.53 , 154.77 , 1 c ) , ( 140.92 , 140.87 , d , j = 6.3 hz , 1 c ) , ( 130.09 , 130.03 , d , j = 7.56 hz , 1 c ) , 124.48 , ( 121.18 , 120.17 , 1 c ) , ( 115.72 , 115.55 , d , j = 21.42 hz , 1 c ) , ( 113.54 , 113.37 , d , j = 21.42 hz , 1 c ) , ( 81.03 , 80.93 , 1 c ) , ( 52.99 , 52.17 , 1 c ) , ( 49.90 , 49.83 , 1 c ) , ( 39.12 , 39.03 , 1 c ) , ( 34.70 , 34.20 , 1 c ) , 28.20 . ms ( esi ) m / z [ m + na ] : calcd , 432.14 ; found , 432.10 . compound 36 ( 0.745 g , 89% ) was synthesized from 34 in two steps , following the exact same procedure as used to synthesize 35 . h nmr ( 500 mhz , cdcl3 ) : ( 8.85 ( d , j = 6.0 hz ) , 8.83 ( d , j = 4.5 hz , 4:5 , 1 h ) , ( 7.45 ( d , j = 4.5 hz ) , 7.40 ( d , j = 4.5 hz , 5:4 , 1 h ) , 7.23 ( q , j = 6.9 hz , 1 h ) , 6.936.84 ( m , 3 h ) , ( 4.60 ( s ) , 4.56 ( s ) , 5:4 , 2 h ) , ( 3.37 ( t , j = 7 hz ) , 3.28 ( t , j = 7 hz ) , 4:5 , 2 h ) , 3.33 ( s , 3 h ) , 2.652.55 ( m , 2 h ) , 1.87 ( p , j = 7.4 hz , 2 h ) , ( 1.45 ( s ) , 1.35 ( s ) , 5:4 , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 171.07 , 170.43 , 1 c ) , ( 165.86 , 165.66 , 1 c ) , ( 163.81 , 161.86 , d , j = 245.7 hz , 1 c ) , 158.59 , ( 155.71 , 154.90 , 1 c ) , ( 143.69 , 143.58 , d , j = 13.8 hz , 1 c ) , ( 129.92 , 129.85 , d , j = 8.82 hz , 1 c ) , 123.85 , ( 121.16 , 120.17 , 1 c ) , ( 115.11 , 114.95 , d , j = 20.16 hz , 1 c ) , ( 113.01 , 112.84 , d , j = 21.42 hz , 1 c ) , ( 80.92 , 80.82 , 1 c ) , ( 52.30 , 51.98 , 1 c ) , ( 47.98 , 47.81 , 1 c ) , ( 39.11 , 39.03 , 1 c ) , ( 32.78 , 32.64 , 1 c ) , ( 29.66 , 29.59 , 1 c ) , 28.25 . ms ( esi ) m / z [ 2 m + na ] : calcd , 869.31 ; found , 869.23 . compound 35 ( 0.292 g , 0.713 mmol ) was dissolved in mecn ( 4 ml ) , and imidazole ( 0.243 g , 3.567 mmol ) and k2co3 ( 0.197 g , 1.427 mmol ) added to the solution . the resulting mixture was heated at 65 c for 4 h , after which it was cooled and diluted with ch2cl2 ( 20 ml ) . water ( 20 ml ) was added to the organic layer and the layers separated . the aqueous layer was washed once with ch2cl2 ( 10 ml ) , and the combined organic layers were dried over anhydrous sodium sulfate , concentrated , and purified by flash column chromatography using hexanes / etoac . compound 37 was obtained as a faint - yellow sticky oil ( 0.263 g , 93% ) . h nmr ( 500 mhz , cdcl3 ) : 8.59 ( s , 1 h ) , 8.57 ( d , j = 5.2 hz , 1 h ) , 7.86 ( s , 1 h ) , 7.23 ( q , j = 7.8 hz , 1 h ) , 7.15 ( s , 1 h ) , ( 7.07 ( d , j = 4.9 hz ) , 7.01 ( d , j = 4.7 hz ) , 3:2 , 1 h ) , 6.996.83 ( m , 3 h ) , ( 4.44 ( s ) , 4.36 ( s ) , 3:2 , 2 h ) , ( 3.58 ( t , j = 7.5 hz ) , 3.54 ( t , j = 7.2 hz ) , 2:3 , 2 h ) , ( 2.91 ( t , j = 7.2 hz ) , 2.86 ( t , j = 7.1 hz ) , 2:3 , 2 h ) , ( 1.46 ( s ) , 1.36 ( s ) , 3:2 , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 170.13 , 169.55 , 1 c ) , ( 163.80 , 161.85 , d , j = 245.7 hz , 1 c ) , 158.85 , ( 155.58 , 155.00 , 1 c ) , 154.43 , ( 141.15 , 141.10 , d , j = 6.3 hz , 1 c ) , 136.13 , ( 130.63 , 130.52 , d , j = 13.86 hz , 1 c ) , 130.05 , 124.45 , ( 116.49 , 116.09 , 1 c ) , ( 115.71 , 115.55 , d , j = 20.91 hz , 1 c ) , 115.29 , ( 113.49 , 113.32 , d , j = 21.42 hz , 1 c ) , 80.63 , ( 52.81 , 51.91 , 1 c ) , 49.89 , ( 34.83 , 34.35 , 1 c ) , 28.26 . ms ( esi ) m / z [ m + h ] : calcd , 398.21 ; found , 398.42 . compound 38 was synthesized from 36 ( 0.648 g , 1.53 mmol ) following the same procedure as used above to synthesize 37 from 35 . compound 38 was obtained as an off - white viscous oil after purification ( 0.531 g , 84% ) . h nmr ( 500 mhz , cdcl3 ) : 8.61 ( d , j = 4.3 hz , 1 h ) , 8.58 ( s , 1 h ) , 7.85 ( s , 1 h ) , 7.21 ( q , j = 6.5 hz , 1 h ) , 7.14 ( s , 1 h ) , ( 7.10 ( d , j = 4.5 hz ) , 7.05 ( d , j = 3.9 hz ) , 5:4 , 1 h ) , 6.956.83 ( m , 3 h ) , ( 4.50 ( s ) , 4.44 ( s ) , 5:4 , 2 h ) , ( 3.40 ( t , j = 6.9 hz ) , 3.31 ( t , j = 7.1 hz ) , 4:5 , 2 h ) , 2.652.59 ( m , 2 h ) , 1.961.82 ( m , 2 h ) , ( 1.46 ( s ) , 1.35 ( s ) , 5:4 , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 170.28 , 169.76 , 1 c ) , ( 163.81 , 161.86 , d , j = 245.7 hz , 1 c ) , 158.84 , ( 155.77 , 155.14 , 1 c ) , 154.42 , ( 143.91 , 143.86 , d , j = 6.3 hz , 1 c ) , 136.12 , ( 130.63 , 130.52 , d , j = 13.86 hz , 1 c ) , 129.81 , 123.84 , 116.47 , ( 116.07 , 115.27 , 1 c ) , ( 115.10 , 114.93 , d , j = 21.42 hz , 1 c ) , ( 112.98 , 112.81 , d , j = 21.42 hz , 1 c ) , 80.50 , ( 52.18 , 51.76 , 1 c ) , 47.84 , ( 32.83 , 32.69 , 1 c ) , 29.66 , 28.29 . ms ( esi ) m / z [ m + h ] : calcd , 412.20 ; found , 412.00 . to a solution of 40 ( 0.3 g , 1.971 mmol ) in ethanol ( 12 ml ) , 31 ( 1.3 ml , 1.371 g , 9.85 mmol ) and acetic acid ( 0.06 ml , 0.06 g , 0.985 mmol ) were added . the reaction was stirred at room temperature for 8 h , after which it was diluted with ch2cl2 ( 25 ml ) . the resulting solution was treated with saturated nahco3 ( 20 ml ) , and the layers were separated . the aqueous layer was washed with ch2cl2 ( 3 10 ml ) , and the combined organic layers were dried over anhydrous sodium sulfate and concentrated . the obtained residue was purified by flash column chromatography ( ch2cl2/meoh ) to obtain 41 as a clear oil ( 0.356 g , 62% ) . h nmr ( 500 mhz , cdcl3 ) : 8.37 ( d , j = 5.0 hz , 1 h ) , 7.267.20 ( m , 1 h ) , 6.95 ( d , j = 7.6 hz , 1 h ) , 6.936.87 ( m , 2 h ) , 6.79 ( d , j = 5.1 hz , 1 h ) , 3.03 ( t , j = 6.8 hz , 2 h ) , 2.91 ( t , j = 7.0 hz , 2 h ) , 2.86 ( t , j = 6.8 hz , 2 h ) , 2.79 ( t , j = 7.1 hz , 2 h ) , 2.52 ( s , 3 h ) , 1.61 ( br s , 1 h ) . c nmr ( 126 mhz , cdcl3 ) : 172.4 , 168.8 , ( 163.86 , 161.91 , d , j = 245.7 hz , 1 c ) , 156.8 , ( 142.30 , 142.25 , d , j = 6.3 hz , 1 c ) , ( 129.92 , 129.85 , d , j = 8.82 hz , 1 c ) , ( 124.29 , 124.27 , d , j = 2.52 hz , 1 c ) , 115.8 , ( 115.52 , 115.36 , d , j = 20.16 hz , 1 c ) , ( 113.21 , 113.04 , d , j = 21.42 hz , 1 c ) , 50.5 , 47.7 , 37.4 , 35.9 , 14.0 . ms ( esi ) m / z [ m + h ] : calcd , 292.1 ; found , 291.84 . compounds 4247 were synthesized from 40 and the corresponding primary amines ( 32 , 62 , s3s12 ; see supporting information ) , following the same procedure used to synthesize 41 . h nmr ( 500 mhz , cdcl3 ) : 8.36 ( d , j = 5.0 hz , 1 h ) , 7.237.18 ( m , 1 h ) , 6.93 ( t , j = 7.7 hz , 1 h ) , 6.886.83 ( m , 2 h ) , 6.81 ( d , j = 5.0 hz , 1 h ) , 2.98 ( t , j = 6.6 hz , 2 h ) , 2.85 ( t , j = 6.7 hz , 2 h ) , 2.752.68 ( m , 2 h ) , 2.672.59 ( m , 2 h ) , 2.52 ( s , 3 h ) , 1.76 ( p , j = 7.3 hz , 2 h ) , 1.57 ( br s , 1 h ) . c nmr ( 126 mhz , cdcl3 ) : 172.3 , 169.1 , ( 163.75 , 161.80 , d , j = 245.7 hz , 1 c ) , 156.8 , ( 144.60 , 144.54 , d , j = 7.56 hz , 1 c ) , ( 129.65 , 129.58 , d , j = 8.82 hz , 1 c ) , ( 123.93 , 123.91 , d , j = 2.52 hz , 1 c ) , 115.8 , ( 115.13 , 114.97 , d , j = 20.16 hz , 1 c ) , ( 112.62 , 112.45 , d , j = 21.42 hz , 1 c ) , 48.9 , 47.9 , 37.6 , 33.2 , 31.2 , 14.0 . ms ( esi ) m / z [ m + h ] : calcd , 306.1 ; found , 305.85 . pale - yellow oil ( 0.591 g , 75% ) . h nmr ( 500 mhz , cdcl3 ) : 8.39 ( d , j = 5.0 hz , 1 h ) , 7.217.14 ( m , 3 h ) , 7.04 ( d , j = 7.2 hz , 1 h ) , 6.82 ( d , j = 5.0 hz , 1 h ) , 3.02 ( t , j = 6.6 hz , 2 h ) , 2.88 ( t , j = 6.6 hz , 2 h ) , 2.66 ( d , j = 7.5 hz , 2 h ) , 2.63 ( d , j = 8.0 hz , 2 h ) , 2.55 ( s , 3 h ) , 1.90 ( br s , 1 h ) , 1.81 ( p , j = 7.5 hz , 3 h ) . c nmr ( 126 mhz , cdcl3 ) : 172.4 , 169.0 , 156.9 , 144.0 , 134.0 , 129.6 , 128.5 , 126.5 , 126.0 , 115.8 , 48.9 , 47.9 , 37.5 , 33.2 , 31.2 , 14.1 . ms ( esi ) m / z [ m + h ] : calcd , 322.1 ; found , 321.91 . h nmr ( 500 mhz , cdcl3 ) : 8.38 ( d , j = 5.0 hz , 1 h ) , 7.18 ( q , j = 7.8 hz , 1 h ) , 6.876.78 ( m , 3 h ) , 6.766.68 ( m , 1 h ) , 3.093.02 ( m , 2 h ) , 2.89 ( t , j = 6.7 hz , 2 h ) , 2.72 ( t , j = 6.0 hz , 1 h ) , 2.68 ( d , j = 6.5 hz , 1 h ) , 2.53 ( s , 3 h ) , 1.80 ( br s , 1 h ) , 1.741.69 ( m , 1 h ) , 1.331.28 ( m , 1 h ) , 0.930.86 ( m , 2 h ) . c nmr ( 126 mhz , cdcl3 ) : 172.4 , 169.0 , ( 163.94 , 161.99 , d , j = 245.7 hz , 1 c ) , 156.8 , ( 145.73 , 145.67 , d , j = 7.56 hz , 1 c ) , ( 129.64 , 129.58 , d , j = 7.56 hz , 1 c ) , 121.4 , 115.8 , ( 112.48 , 112.30 , d , j = 22.68 hz , 1 c ) , ( 112.33 , 112.16 , d , j = 21.42 hz , 1 c ) , 53.8 , 47.7 , 37.6 , 23.6 , 21.9 , 15.1 , 14.0 . ms ( esi ) m / z [ 2 m + h ] : calcd , 635.2 ; found , 635.08 . h nmr ( 500 mhz , cdcl3 ) : 8.37 ( d , j = 5.0 hz , 1 h ) , 7.277.20 ( m , 1 h ) , 6.99 ( d , j = 7.5 hz , 1 h ) , 6.966.91 ( m , 1 h ) , 6.89 ( td , j = 8.6 , 2.2 hz , 1 h ) , 6.77 ( d , j = 5.0 hz , 1 h ) , 3.022.97 ( m , 2 h ) , 2.80 ( t , j = 6.6 hz , 2 h ) , 2.59 ( dd , j = 14.8 , 6.8 hz , 1 h ) , 2.54 ( s , 3 h ) , 2.47 ( dd , j = 14.8 , 7.6 hz , 1 h ) , 2.112.05 ( m , 1 h ) , 1.90 ( br s , 1 h ) , 1.070.99 ( m , 1 h ) , 0.690.63 ( m , 1 h ) , 0.470.43 ( m , 1 h ) . c nmr ( 126 mhz , cdcl3 ) : 172.4 , 169.1 , ( 163.82 , 161.87 , d , j = 245.7 hz , 1 c ) , 156.8 , ( 144.17 , 144.12 , d , j = 6.3 hz , 1 c ) , ( 129.73 , 129.67 , d , j = 7.56 hz , 1 c ) , ( 123.92 , 123.89 , d , j = 3.78 hz , 1 c ) , 115.7 , ( 115.15 , 114.99 , d , j = 20.16 hz , 1 c ) , ( 112.85 , 112.68 , d , j = 21.42 hz , 1 c ) , 47.5 , 38.1 , 37.4 , 37.2 , 21.0 , 14.0 , 13.7 . ms ( esi ) m / z [ m + h ] : calcd , 318.1 ; found , 317.9 . h nmr ( 500 mhz , cdcl3 ) : 8.43 ( s , 1 h ) , 8.41 ( d , j = 6.1 hz , 1 h ) , 8.36 ( d , j = 5.0 hz , 1 h ) , 7.47 ( d , j = 7.7 hz , 1 h ) , 7.17 ( dd , j = 7.6 , 4.9 hz , 1 h ) , 6.80 ( d , j = 5.0 hz , 1 h ) , 3.02 ( t , j = 6.7 hz , 2 h ) , 2.88 ( t , j = 6.7 hz , 2 h ) , 2.67 ( t , j = 7.0 hz , 2 h ) , 2.64 ( t , j = 7.5 hz , 2 h ) , 2.52 ( s , 3 h ) , 2.40 ( br s , 1 h ) , 1.81 ( p , j = 7.3 hz , 2 h ) . c nmr ( 126 mhz , cdcl3 ) : 172.3 , 168.8 , 156.8 , 149.8 , 147.4 , 137.0 , 135.7 , 123.2 , 115.8 , 48.7 , 47.7 , 37.2 , 30.9 , 30.5 , 14.0 . ms ( esi ) m / z [ m + h ] : calcd , 289.1 ; found , 289.01 . cream - colored oil ( 0.426 g , 83% ) . h nmr ( 500 mhz , cdcl3 ) : 8.39 ( d , j = 5.1 hz , 1 h ) , 7.63 ( s , 1 h ) , 7.55 ( d , j = 8.0 hz , 1 h ) , 7.52 ( d , j = 7.5 hz , 1 h ) , 7.41 ( t , j = 7.7 hz , 1 h ) , 6.84 ( d , j = 5.1 hz , 1 h ) , 3.84 ( s , 2 h ) , 3.02 ( t , j = 6.6 hz , 2 h ) , 2.89 ( t , j = 6.6 hz , 2 h ) , 2.52 ( s , 3 h ) , 1.77 ( br s , 1 h ) . c nmr ( 126 mhz , cdcl3 ) : 172.2 , 168.8 , 156.7 , 141.6 , 132.3 , 131.3 , 130.5 , 128.9 , 118.7 , 115.7 , 112.1 , 52.6 , 47.1 , 37.3 , 13.8 . ms ( esi ) m / z [ m + h ] : calcd , 285.1 ; found , 284.91 . to a solution of 41 ( 0.356 g , 1.222 mmol ) in thf ( 10 ml ) , a solution of di - tert - butyl dicarbonate ( 0.293 g , 1.344 mmol ) in thf ( 7 ml ) was added . the resulting solution was stirred at room temperature for 3 h and , thereafter , it was diluted with etoac / h2o ( 30 ml , 1:1 ) . the layers were separated , and the aqueous layer extracted with etoac ( 2 10 ml ) . combined organic layers were dried , concentrated , and purified by column chromatography ( hexanes / etoac ) . the resulting oil ( 0.39 g , 0.996 mmol , 82% ) was dissolved in ch2cl2 ( 2 ml ) and added to a solution of m - chloroperbenzoic acid ( 0.636 g , 3.685 mmol ) in ch2cl2 ( 3 ml ) at 0 c . the resulting solution was stirred at room temperature for 3 h. at that point , the reaction was filtered , and the white residue was washed with cold ch2cl2 ( 5 ml ) . the collected filtrate was washed with 10% aqueous k2co3 ( 5 ml ) , the organic layer was dried , concentrated , and purified by flash column chromatography ( hexanes / etoac ) to give a clear viscous oil ( 0.337 g , 80% ) . h nmr ( 500 mhz , cdcl3 ) : 8.76 ( d , j = 4.1 hz , 1 h ) , ( 7.43 ( s ) , 7.31 ( s ) , 7:3 , 1 h ) , 7.23 ( q , j = 6.5 hz , 1 h ) , 6.976.84 ( m , 3 h ) , ( 3.613.53 ( m ) , 3.493.44 ( m ) , 7:3 , 2 h ) , 3.37 ( t , j = 7.1 hz , 2 h ) , 3.34 ( s , 3 h ) , ( 3.183.10 ( m ) , 3.082.98 ( m ) , 7:3 , 2 h ) , ( ( 2.862.80 ( m ) , 2.782.72 ( m ) , 3:7 , 2 h ) , 1.38 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 170.93 , 170.82 , 1 c ) , ( 165.98 , 165.76 , 1 c ) , ( 163.79 , 161.84 , d , j = 245.7 hz , 1 c ) , ( 158.01 , 157.88 , 1 c ) , ( 155.22 , 154.87 , 1 c ) , 141.38 , ( 130.00 , 129.94 , d , j = 7.56 hz ) , 124.54 , 123.56 , ( 115.75 , 115.59 , d , j = 20.16 hz , 1 c ) , ( 113.37 , 113.21 , d , j = 20.16 hz , 1 c ) , 80.05 , ( 49.37 , 49.18 , 1 c ) , ( 46.80 , 46.14 , 1 c ) , 39.09 , ( 36.91 , 36.29 , 1 c ) , ( 34.90 , 34.19 , 1 c ) , 28.25 . ms ( esi ) m / z [ m + na ] : calcd , 446.15 ; found , 446.06 . compounds 4952 were synthesized from compounds 4245 following the same procedures used to synthesize 48 . h nmr ( 500 mhz , cdcl3 ) : 8.818.72 ( m , 1 h ) , ( 7.477.43 ( m ) , 7.377.32 ( m ) , 3:2 , 1 h ) , 7.23 ( q , j = 7.4 hz , 1 h ) , 6.93 ( d , j = 7.5 hz , 1 h ) , 6.87 ( t , j = 8.0 hz , 2 h ) , 3.663.56 ( m , 2 h ) , 3.34 ( s , 3 h ) , 3.243.08 ( m , 4 h ) , 2.58 ( t , j = 7.7 hz , 2 h ) , 1.81 ( p , j = 7 hz , 2 h ) , 1.39 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 170.97 , 170.87 , 1 c ) , ( 165.98 , 165.76 , 1 c ) , ( 163.81 , 161.86 , d , j = 245.7 hz , 1 c ) , 157.89 , ( 155.39 , 155.02 , 1 c ) , ( 143.99 , 143.90 , d , j = 11.34 hz , 1 c ) , ( 129.85 , 129.79 , d , j = 7.56 hz , 1 c ) , ( 123.92 , 123.90 , d , j = 2.52 hz , 1 c ) , 123.54 , ( 115.11 , 114.95 , d , j = 20.16 hz , 1 c ) , ( 112.89 , 112.72 , d , j = 21.42 hz , 1 c ) , 79.93 , ( 47.38 , 47.04 , 1 c ) , 46.03 , 39.08 , ( 36.94 , 36.37 , 1 c ) , 32.76 , ( 29.85 , 29.63 , 1 c ) , 28.30 . ms ( esi ) m / z [ 2 m + na ] : calcd , 897.19 ; found , 897.26 . colorless oil ( 0.613 g , 89% ) . h nmr ( 500 mhz , cdcl3 ) : 8.76 ( d , j = 4.5 hz , 1 h ) , ( 7.45 ( s ) , 7.35 ( s ) , 3:2 , 1 h ) , 7.20 ( q , j = 7.5 hz , 1 h ) , 7.187.13 ( m , 2 h ) , 7.04 ( d , j = 7.3 hz , 1 h ) , 3.673.56 ( m , 2 h ) , 3.34 ( s , 3 h ) , 3.253.06 ( m , 4 h ) , 2.56 ( t , j = 7.5 hz , 2 h ) , 1.81 ( p , j = 7.6 hz , 2 h ) , 1.39 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.96 , ( 166.02 , 165.84 , 1 c ) , 157.91 , 155.36 , 143.42 , 134.13 , 129.69 , 128.35 , 126.48 , 126.15 , 123.50 , 79.96 , ( 47.40 , 47.19 , 1 c ) , 46.07 , 39.11 , ( 37.01 , 36.46 , 1 c ) , 32.74 , 29.89 , 28.34 . ms ( esi ) m / z [ m + na ] : calcd , 476.09 ; found , 476.01 . h nmr ( 500 mhz , cdcl3 ) : 8.66 ( d , j = 4.9 hz , 1 h ) , ( 7.35 ( s ) , 7.25 ( s ) , 1:1 , 1 h ) , 7.10 ( q , j = 7.7 hz , 1 h ) , 6.796.68 ( m , 2 h ) , 6.61 ( dt , j = 2 , 10.3 hz , 1 h ) , 3.683.60 ( m , 2 h ) , 3.323.26 ( m , 1 h ) , 3.21 ( s , 3 h ) , 3.183.02 ( m , 3 h ) , 1.831.75 ( m , 1 h ) , 1.35 ( s , 9 h ) , 1.201.16 ( m , 1 h ) , 0.920.84 ( m , 2 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.70 , ( 165.68 , 165.55 , 1 c ) , ( 163.69 , 161.74 , d , j = 245.7 hz , 1 c ) , 157.74 , 155.01 , 145.08 , ( 129.56 , 129.49 , d , j = 8.82 hz , 1 c ) , 123.33 , 121.26 , ( 112.23 , 112.07 , d , j = 20.16 hz , 1 c ) , ( 112.15 , 111.97 , d , j = 22.68 hz , 1 c ) , 79.74 , ( 51.04 , 50.41 , 1 c ) , 45.83 , 38.87 , ( 36.67 , 36.06 , 1 c ) , ( 31.29 , 30.58 , 1 c ) , 28.08 , ( 22.66 , 22.35 , 1 c ) , ( 14.37 , 13.82 , 1 c ) . ms ( esi ) m / z [ m + h ] : calcd , 450.17 ; found , 450.07 . h nmr ( 500 mhz , cdcl3 ) : 8.70 ( d , j = 5.1 hz , 1 h ) , 7.31 ( d , j = 4.0 hz , 1 h ) , 7.18 ( q , j = 8 hz , 1 h ) , 6.92 ( d , j = 7.6 hz , 1 h ) , 6.86 ( d , j = 9.9 hz , 1 h ) , 6.81 ( td , j = 8.5 , 2.2 hz , 1 h ) , 3.52 ( s , 1 h ) , 3.49 ( t , j = 7 hz , 1 h ) , 3.28 ( s , 3 h ) , 2.992.92 ( m , 2 h ) , 2.79 ( dd , j = 14.5 , 5.3 hz , 1 h ) , 2.35 ( dd , j = 8 , 13.5 hz , 1 h ) , 2.272.23 ( m , 1 h ) , 1.34 ( s , 9 h ) , 0.840.76 ( m , 1 h ) , 0.66 ( q , j = 6.1 hz , 1 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.87 , 165.71 , ( 163.64 , 161.69 , d , j = 245.7 hz , 1 c ) , 157.84 , 156.00 , ( 143.07 , 143.01 , d , j = 7.56 hz , 1 c ) , ( 129.78 , 129.71 , d , j = 8.82 hz , 1 c ) , ( 124.00 , 123.98 , d , j = 2.52 hz , 1 c ) , 123.25 , ( 115.14 , 114.98 , d , j = 20.16 hz , 1 c ) , ( 112.92 , 112.75 , d , j = 21.42 hz , 1 c ) , 79.82 , 45.94 , 38.96 , 37.46 , 36.06 , 34.84 , 28.20 , ( 22.87 , 22.45 , 1 c ) , 14.84 . ms ( esi ) m / z [ m + h ] : calcd , 450.17 ; found , 450.20 . compound 46 ( 0.443 g , 1.536 mmol ) was dissolved in thf ( 15 ml ) , and a solution of di - tert - butyl dicarbonate ( 0.369 g , 1.689 mmol ) in thf ( 6 ml ) was added . the resulting solution was stirred at room temperature for 3 h , and thereafter , it was diluted with etoac / h2o ( 40 ml , 1:1 ) . the layers were separated , and the aqueous layer was extracted with etoac ( 2 15 ml ) . combined organic layers were dried , concentrated , and purified by column chromatography ( hexanes / etoac ) . the resulting oil ( 0.567 g , 1.459 mmol , 95% ) was dissolved in thf ( 12 ml ) . water ( 12 ml ) and oxone ( 1.345 g , 2.188 mmol ) were sequentially added , and the reaction was stirred at room temperature for 4 h. at that point , the reaction was diluted with etoac / h2o ( 30 ml , 1:1 ) and the layers separated . the aqueous layer was basified to ph 10 with 6 n naoh , saturated by addition of solid k2co3 , and extracted with etoac ( 3 15 ml ) . combined organic fractions were dried over sodium sulfate , and concentrated . the resulting light - yellow oil ( 0.527 g , 86% ) was clean by nmr and was used in the next step without further purification . h nmr ( 500 mhz , cdcl3 ) : 8.74 ( d , j = 3.9 hz , 1 h ) , 8.42 ( s , 2 h ) , 7.48 ( d , j = 6.9 hz , 1 h ) , ( 7.42 ( s ) , 7.34 ( s ) , 3:2 , 1 h ) , 7.20 ( dd , j = 7.3 , 4.9 hz , 1 h ) , 3.663.54 ( m , 2 h ) , 3.31 ( s , 3 h ) , 3.243.03 ( m , 4 h ) , 2.56 ( t , j = 7.8 hz , 2 h ) , 1.80 ( p , j = 7 hz , 2 h ) , 1.36 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.87 , 165.81 , 157.85 , 155.27 , 149.53 , 147.30 , 136.70 , 135.74 , 123.47 , 123.37 , 79.93 , ( 47.30 , 46.94 , 1 c ) , 46.01 , 39.05 , ( 36.87 , 36.33 , 1 c ) , 30.11 , ( 29.84 , 29.58 , 1 c ) , 28.25 . ms ( esi ) m / z [ m + na ] : calcd , 443.16 ; found , 443.03 . compound 54 ( 0.396 g , 91% ) was obtained as a colorless oil from 47 following the same procedure used to synthesize 48 from 41 . h nmr ( 500 mhz , cdcl3 ) : 8.76 ( d , j = 4.8 hz , 1 h ) , 7.53 ( d , j = 7.3 hz , 1 h ) , 7.487.37 ( m , 4 h ) , 4.46 ( s , 2 h ) , 3.743.61 ( m , 2 h ) , 3.33 ( s , 3 h ) , 3.203.07 ( m , 2 h ) , 1.38 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.46 , 165.58 , 157.79 , ( 155.26 , 154.98 , 1 c ) , ( 139.96 , 139.55 , 1 c ) , ( 131.83 , 131.28 , 1 c ) , 130.75 , 130.23 , 129.26 , 123.42 , 118.36 , 112.36 , 80.49 , ( 50.65 , 49.71 , 1 c ) , 45.59 , 38.87 , ( 36.34 , 35.94 , 1 c ) , 28.00 . ms ( esi ) m / z [ m + na ] : calcd , 439.14 ; found , 438.99 . compound 48 ( 0.335 g , 0.791 mmol ) was dissolved in mecn ( 5 ml ) , and imidazole ( 0.269 g , 3.958 mmol ) and k2co3 ( 0.22 g , 1.583 mmol ) were added to the solution . the resulting mixture was heated at 65 c for 5 h , after which it was cooled and diluted with ch2cl2 ( 20 ml ) . water ( 20 ml ) was added to the organic layer and the layers separated . the aqueous layer was washed once with ch2cl2 ( 15 ml ) , and the combined organics were dried over anhydrous sodium sulfate , concentrated , and purified by flash column chromatography using hexanes / etoac . the desired compound ( 55 ) was obtained as an off - white viscous oil ( 0.247 g , 76% ) . h nmr ( 500 mhz , cdcl3 ) : 8.61 ( s , 1 h ) , 8.53 ( d , j = 4.9 hz , 1 h ) , 7.88 ( s , 1 h ) , 7.24 ( q , j = 6.0 hz , 1 h ) , 7.16 ( s , 1 h ) , ( 7.08 ( s ) , 6.97 ( s ) , 3:2 , 1 h ) , 6.946.82 ( m , 3 h ) , ( 3.56 ( t , j = 8.5 hz ) , 3.47 ( s ) , 3:2 , 2 h ) , 3.433.36 ( m , 2 h ) , ( 2.99 ( t , j = 8.5 hz ) , 2.90 ( s ) , 3:2 , 2 h ) , ( 2.84 ( s ) , 2.79 ( t , j = 7 hz ) , 2:3 , 2 h ) , 1.38 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 170.39 , 170.27 , 1 c ) , ( 163.81 , 161.85 , d , j = 246.96 hz , 1 c ) , ( 158.43 , 158.33 , 1 c ) , ( 155.18 , 154.98 , 1 c ) , ( 154.55 , 154.38 , 1 c ) , ( 141.54 , 141.47 , d , j = 8.82 hz , 1 c ) , 136.09 , ( 130.55 , 130.47 , 1 c ) , ( 130.00 , 129.94 , 1 c ) , ( 124.48 , 124.46 , d , j = 2.52 hz , 1 c ) , 118.49 , 116.48 , ( 115.74 , 115.57 , d , j = 21.42 hz , 1 c ) , ( 113.38 , 113.22 , d , j = 20.16 hz , 1 c ) , 79.87 , ( 49.37 , 49.23 , 1 c ) , ( 46.97 , 46.21 , 1 c ) , ( 36.84 , 36.07 , 1 c ) , ( 35.00 , 34.29 , 1 c ) , 28.23 . ms ( esi ) m / z [ m + h ] : calcd , 412.20 ; found , 412.08 . compounds 5661 were synthesized from compounds 4954 following the same procedures used to synthesize 55 . h nmr ( 500 mhz , cdcl3 ) : 8.59 ( s , 1 h ) , 8.52 ( d , j = 4.1 hz , 1 h ) , 7.86 ( s , 1 h ) , 7.21 ( q , j = 7 hz , 1 h ) , 7.15 ( s , 1 h ) , ( 7.07 ( s ) , 7.00 ( s ) , 3:2 , 1 h ) , 6.91 ( d , j = 7.5 hz , 1 h ) , 6.886.81 ( m , 2 h ) , 3.663.54 ( m , 2 h ) , 3.243.14 ( m , 2 h ) , 3.042.94 ( m , 2 h ) , 2.58 ( t , j = 7.7 hz , 2 h ) , 1.83 ( p , j = 7.6 hz , 2 h ) , 1.38 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 170.42 , 170.32 , 1 c ) , ( 163.79 , 161.84 , d , j = 245.7 hz , 1 c ) , ( 158.40 , 158.31 , 1 c ) , ( 155.36 , 155.12 , 1 c ) , ( 154.54 , 154.39 , 1 c ) , ( 144.04 , 143.90 , d , j = 17.6 hz , 1 c ) , 136.09 , 130.52 , ( 129.81 , 129.74 , d , j = 8.82 hz , 1 c ) , ( 123.84 , 123.82 , d , j = 2.52 hz , 1 c ) , 118.43 , 116.45 , ( 115.08 , 114.91 , d , j = 21.42 hz , 1 c ) , ( 112.87 , 112.70 , d , j = 21.42 hz , 1 c ) , 79.73 , ( 47.24 , 46.97 , 1 c ) , ( 46.27 , 46.08 , 1 c ) , ( 36.91 , 36.15 , 1 c ) , 32.76 , ( 29.86 , 29.60 , 1 c ) , 28.27 . ms ( esi ) m / z [ 2 m + na ] : calcd , 873.43 ; found , 873.39 . h nmr ( 500 mhz , cdcl3 ) : 8.63 ( s , 1 h ) , 8.54 ( d , j = 4.9 hz , 1 h ) , 7.88 ( s , 1 h ) , 7.18 ( q , j = 7.5 hz , 1 h ) , 7.177.14 ( m , 3 h ) , ( 7.09 ( s ) , 7.05 ( s ) , 3:2 , 1 h ) , 7.03 ( d , j = 7.1 hz , 1 h ) , 3.653.55 ( m , 2 h ) , 3.253.14 ( m , 2 h ) , 3.052.94 ( m , 2 h ) , 2.57 ( t , j = 7.7 hz , 2 h ) , 1.83 ( p , j = 7.6 hz , 2 h ) , 1.39 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.49 , 158.39 , ( 155.38 , 155.21 , 1 c ) , 154.40 , 143.39 , 136.01 , 134.14 , ( 130.34 , 130.21 , 1 c ) , 129.65 , 128.34 , 126.40 , 126.15 , 118.55 , 116.54 , 79.80 , ( 47.27 , 47.06 , 1 c ) , ( 46.28 , 46.12 , 1 c ) , ( 36.99 , 36.21 , 1 c ) , 32.77 , ( 29.91 , 29.73 , 1 c ) , 28.32 . ms ( esi ) m / z [ m + h ] : calcd , 442.19 ; found , 442.06 . h nmr ( 500 mhz , cdcl3 ) : 8.60 ( s , 1 h ) , 8.50 ( d , j = 5.0 hz , 1 h ) , 7.87 ( s , 1 h ) , 7.17 ( q , j = 8 hz , 1 h ) , 7.14 ( s , 1 h ) , ( 7.05 ( s ) , 6.94 ( d , j = 7.7 h ) , 2:3 , 1 h ) , 6.876.78 ( m , 2 h ) , 6.67 ( dt , j = 2.5 , 10.2 hz , 1 h ) , 3.753.63 ( m , 2 h ) , 3.413.30 ( m , 1 h ) , 3.253.16 ( m , 1 h ) , 3.082.95 ( m , 2 h ) , 1.921.78 ( m , 1 h ) , 1.41 ( s , 9 h ) , 1.291.26 ( m , 1 h ) , 0.980.93 ( m , 2 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.40 , ( 163.93 , 161.98 , d , j = 245.7 hz , 1 c ) , 158.34 , 155.26 , 154.47 , 145.25 , 136.05 , 130.44 , ( 129.75 , 129.68 , d , j = 8.82 hz , 1 c ) , 121.37 , 118.41 , 116.46 , ( 112.53 , 112.40 , d , j = 16.38 hz , 1 c ) , ( 112.40 , 112.23 , d , j = 21.42 hz , 1 c ) , 79.89 , ( 51.24 , 50.73 , 1 c ) , 46.24 , ( 36.90 , 36.07 , 1 c ) , 28.32 , 22.85 , 22.08 , 14.65 . ms ( esi ) m / z [ m + h ] : calcd , 438.2 ; found , 438.03 . h nmr ( 500 mhz , cdcl3 ) : 8.62 ( s , 1 h ) , 8.52 ( d , j = 5.0 hz , 1 h ) , 7.88 ( s , 1 h ) , 7.257.18 ( m , 1 h ) , 7.16 ( s , 1 h ) , 6.99 ( s , 1 h ) , 6.96 ( d , j = 7.6 hz , 1 h ) , 6.91 ( d , j = 9.8 hz , 1 h ) , 6.87 ( td , j = 8.5 , 2.3 hz , 1 h ) , ( 3.53 ( t , j = 7 hz ) , 3.50 ( t , j = 7 hz ) , 2:1 , 2 h ) , 2.88 ( t , j = 6.5 hz , 2 h ) , 2.84 ( dd , j = 10 , 5 hz , 1 h ) , 2.43 ( dd , j = 14.0 , 8.0 hz , 1 h ) , 2.36 ( dt , j = 7.2 , 3.6 hz , 1 h ) , 1.37 ( s , 9 h ) , 1.231.20 ( m , 1 h ) , 0.86 ( dt , j = 9.3 , 5.3 hz , 1 h ) , 0.72 ( q , j = 6.1 hz , 1 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.62 , ( 163.80 , 161.85 , d , j = 245.7 hz , 1 c ) , 158.30 , 156.21 , 154.36 , ( 143.16 , 143.11 , d , j = 6.3 hz , 1 c ) , 136.06 , 130.30 , ( 129.86 , 129.80 , d , j = 7.56 hz , 1 c ) , ( 124.08 , 124.06 , d , j = 2.52 hz , 1 c ) , 118.39 , 116.53 , ( 115.29 , 115.13 , d , j = 20.16 hz , 1 c ) , ( 113.09 , 112.93 , d , j = 20.16 hz , 1 c ) , 79.83 , 46.13 , 37.67 , 35.99 , 34.88 , 28.31 , 23.02 , 15.01 . ms ( esi ) m / z [ 2 m + na ] : calcd , 897.43 ; found , 897.32 . pale - yellow oil ( 0.43 g , 84% ) . h nmr ( 500 mhz , cdcl3 ) : 8.57 ( s , 1 h ) , 8.51 ( d , j = 4.7 hz , 1 h ) , 8.40 ( s , 2 h ) , 7.84 ( s , 1 h ) , 7.44 ( s , 1 h ) , 7.17 ( dd , j = 7.3 , 4.9 hz , 1 h ) , 7.12 ( s , 1 h ) , ( 7.05 ( s ) , 6.99 ( s ) , 3:2 , 1 h ) , 3.653.52 ( m , 2 h ) , 3.253.14 ( m , 2 h ) , 3.052.92 ( m , 2 h ) , 2.56 ( t , j = 7.8 hz , 2 h ) , 1.82 ( p , j = 7.6 hz , 2 h ) , 1.36 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.30 , 158.33 , 155.21 , 154.43 , 149.60 , 147.38 , 136.73 , 136.03 , 135.56 , 130.45 , 123.27 , 118.40 , 116.42 , 79.76 , ( 47.16 , 46.97 , 1 c ) , 46.22 , ( 36.85 , 36.09 , 1 c ) , 30.14 , ( 29.83 , 29.58 , 1 c ) , 28.22 . ms ( esi ) m / z [ m + h ] : calcd , 409.22 ; found , 409.08 . h nmr ( 500 mhz , cdcl3 ) : 8.57 ( s , 1 h ) , 8.54 ( d , j = 5.0 hz , 1 h ) , 7.86 ( s , 1 h ) , 7.52 ( d , j = 7.3 hz , 1 h ) , ( 7.46 ( s ) , 7.42 ( s ) , 1:1 , 2 h ) , 7.40 ( d , j = 7.4 hz , 1 h ) , 7.15 ( s , 1 h ) , ( 7.09 ( s ) , 7.01 ( s ) , 1:1 , 1 h ) , 4.45 ( s , 2 h ) , ( 3.71 ( s ) , 3.64 ( s ) , 1:1 , 2 h ) , 3.04 ( s ) , 3.00 ( s ) , 1:1 , 2 h ) , ( 1.45 ( s ) , 1.38 ( s ) , 1:1 , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : 169.90 , 158.35 , ( 155.42 , 155.02 , 1 c ) , 154.26 , 139.80 , 135.85 , 131.71 , 131.17 , 130.78 , 130.57 , 130.31 , 129.21 , 118.38 , 116.31 , 112.47 , 80.52 , ( 50.73 , 49.94 , 1 c ) , 46.09 , ( 36.33 , 35.69 , 1 c ) , 28.07 . ms ( esi ) m / z [ 2 m + na ] : calcd , 831.37 ; found , 831.33 . 2-(3-fluorobenzyl)cyclopropan-1-amine ( 62 ; 0.851 g , 5.15 mmol ) was diluted in ch2cl2 ( 20 ml ) and cooled to 20 c . ( s)-(+)--methoxylphenylacetic acid ( 1.0 g , 6.18 mmol ) was added to the resulting solution , followed by the addition of dicyclohexylcarbodiimide ( 1.275 g , 6.18 mmol ) . the resulting suspension was filtered , and the white precipitate was washed with cold ch2cl2 ( 15 ml ) . the combined filtrate was concentrated and chromatographed in silica gel using a gradient of hexanes / etoac to obtain the two diastereomers ( r , r , s)-63a and ( s , s , s)-63b . h nmr ( 500 mhz , cdcl3 ) : 7.427.29 ( m , 5 h ) , 7.23 ( q , j = 7.8 hz , 1 h ) , 7.01 ( d , j = 7.5 hz , 1 h ) , 6.95 ( d , j = 10.0 hz , 1 h ) , 6.936.86 ( m , 2 h ) , 4.61 ( s , 1 h ) , 3.33 ( s , 3 h ) , 2.91 ( dd , j = 14.9 , 5.8 hz , 1 h ) , 2.67 ( dq , j = 7.2 , 3.6 hz , 1 h ) , 2.39 ( dd , j = 14.9 , 8.0 hz , 1 h ) , 1.201.13 ( m , 1 h ) , 0.83 ( dt , j = 9.3 , 4.8 hz , 1 h ) , 0.76 ( q , j = 6.1 hz , 1 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.8 , ( 163.61 , 161.66 , d , j = 245.7 hz , 1 c ) , ( 143.01 , 142.95 , d , j = 7.56 hz , 1 c ) , 136.7 , ( 129.54 , 129.47 , d , j = 8.82 hz , 1 c ) , 128.3 , 128.2 , 126.8 , ( 123.89 , 123.87 , d , j = 2.52 hz , 1 c ) , ( 115.06 , 114.89 , d , j = 21.42 hz , 1 c ) , ( 112.76 , 112.59 , d , j = 21.42 hz , 1 c ) , 83.5 , 56.8 , 37.3 , 28.4 , 20.4 , 13.1 . h nmr ( 500 mhz , cdcl3 ) : 7.437.30 ( m , 5 h ) , 7.27 ( td , j = 7.9 , 6.2 hz , 1 h ) , 7.07 ( d , j = 7.6 hz , 1 h ) , 7.036.99 ( m , 1 h ) , 6.91 ( td , j = 8.3 , 2.5 hz , 2 h ) , 4.62 ( s , 1 h ) , 3.34 ( s , 3 h ) , 2.87 ( dd , j = 14.9 , 6.3 hz , 1 h ) , 2.68 ( dq , j = 7.4 , 3.7 hz , 1 h ) , 2.48 ( dd , j = 14.9 , 7.6 hz , 1 h ) , 1.261.18 ( m , 1 h ) , 0.820.69 ( m , 2 h ) . c nmr ( 126 mhz , cdcl3 ) : 170.7 , ( 163.59 , 161.64 , d , j = 245.7 hz , 1 c ) , ( 143.04 , 142.98 , d , j = 7.56 hz , 1 c ) , 136.7 , ( 129.54 , 129.48 , d , j = 7.56 hz , 1 c ) , 128.2 , 128.1 , 126.7 , ( 123.89 , 123.87 , d , j = 2.52 hz , 1 c ) , ( 115.05 , 114.88 , d , j = 21.42 hz , 1 c ) , ( 112.75 , 112.58 , d , j = 21.42 hz , 1 c ) , 83.5 , 56.8 , 37.4 , 28.4 , 20.5 , 13.0 . to a solution of ( r , r , s)-63a ( 0.775 g , 2.472 mmol ) in ethanol ( 12 ml ) , 12 n hcl ( 12 ml ) was added , and the reaction was heated to reflux for 12 h. the resulting solution was cooled to room temperature , concentrated , and treated with et2o/6 n hcl ( 30 ml , 1:1 ) . the layers were separated , and the aqueous layer was washed with et2o ( 10 ml ) , basified to ph 1012 with 6 the combined ch2cl2 extracts were dried over sodium sulfate and concentrated to obtain ( r , r)-62a ( 0.347 g , 85% ) , which was pure by nmr and used directly in the next step without further purification . ( s , s)-62b was obtained from ( s , s , s)-63b ( 0.756 g , 2.42 mmol ) in 88% yield ( 0.352 g ) following the same procedure used to synthesize ( r , r)-62a . compounds ( r , r)-12 and ( s , s)-12 were synthesized from 62a and 62b , respectively , following the same procedures used to synthesize racemic 12 from 2-methylthio-4-vinylpyrimidine ( 40 ) and 62 . enantiopurities of ( r , r)-59 ( enantiomeric ratio : 97:3 ) and ( s , s)-59 ( enantiomeric ratio : 96:4 ) were determined by chiral hplc using a chiralpak ad - h column using an isocratic gradient of 10% 2-propanol / hexanes at flow rate 0.5 ml / min . compound 66 ( 0.55 g , 2.78 mmol ) was dissolved in ch2cl2 ( 50 ml ) and cooled to 78 c . dibal in thf ( 25 wt % in toluene ; 2.8 ml , 2.37 g , 4.17 mmol ) was added dropwise , and the reaction continued at 78 c for 1 h. at this point , sodium sulfate decahydrate was added to the reaction mixture , which was gradually warmed to room temperature over 1 h. the resulting suspension was filtered through celite and washed with ch2cl2 ( 20 ml ) , and the combined organic layers were dried and concentrated . to this crude 2-(5-bromopyridin-3-yl)acetaldehyde , chcl3 ( 50 ml ) was added , followed by the addition of anhydrous mgso4 ( 3.0 g ) . 3-fluorophenethylamine ( 31 ; 0.36 ml , 0.387 g , 2.78 mmol ) and acetic acid ( 60 l ) were sequentially added and stirred at room temperature for 1 h. the reaction mixture was cooled to 0 c , and sodium triacetoxyborohydride ( 0.71 g , 3.336 mmol ) was added in one portion . the reaction was filtered , and the filtrate was washed with saturated aqueous nahco3 ( 20 ml ) , and the aqueous layer was extracted with chcl3 ( 2 10 ml ) . the organic phase was washed with brine ( 20 ml ) , dried over sodium sulfate , concentrated , and purified by flash column chromatography with ch2cl2/meoh to obtain 67 ( 0.37 g , 41% ) as a pale - yellow oil . h nmr ( 500 mhz , cdcl3 ) : 8.51 ( d , j = 2.1 hz , 1 h ) , 8.34 ( d , j = 1.6 hz , 1 h ) , 7.64 ( t , j = 1.8 hz , 1 h ) , 7.257.20 ( m , 1 h ) , 6.93 ( d , j = 7.5 hz , 1 h ) , 6.916.84 ( m , 2 h ) , 2.88 ( td , j = 7.1 , 4.0 hz , 4 h ) , 2.76 ( dt , j = 14.5 , 7.1 hz , 4 h ) , 1.31 ( s , 1 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 163.81 , 161.86 , d , j = 245.7 hz , 1 c ) , 148.7 , 148.2 , ( 142.27 , 142.21 , d , j = 7.56 hz , 1 c ) , 138.6 , 137.2 , ( 129.89 , 129.83 , d , j = 7.56 hz , 1 c ) , ( 124.26 , 124.24 , d , j = 2.52 hz , 1 c ) , 120.6 , ( 115.47 , 115.31 , d , j = 20.16 hz , 1 c ) , ( 113.20 , 113.04 , d , j = 20.16 hz , 1 c ) , 50.5 , 50.2 , 36.0 , 33.1 . ms ( esi ) m / z [ 1:1 ; ( m + h ] ) ] : calcd , 323.0 , 325.0 ; found , 322.77 , 324.76 . compound 67 ( 0.37 g , 1.145 mmol ) was dissolved in thf ( 10 ml ) , and a solution of di - tert - butyl dicarbonate ( 0.275 g , 1.26 mmol ) in thf ( 6 ml ) was added . the resulting solution was stirred overnight , and then it was diluted with etoac / h2o ( 30 ml , 1:1 ) . the layers were separated and the aqueous layer extracted with etoac ( 2 10 ml ) . the combined organic layers were dried , concentrated , and purified by column chromatography ( hexanes / etoac ) . the resulting oil ( 0.445 g , 1.051 mmol , 92% ) was added to a vial along with cubr ( 7.5 mg , 0.052 mmol ) and cs2co3 ( 0.685 g , 2.102 mmol ) . the reaction vial was evacuated and backfilled with argon ; anhydrous dmso ( previously purged for 5 min with argon ) , 8-acetyl-5,6,7,8-tetrahydroquinoline ( 17 l , 0.018 g , 0.105 mmol ) , and imidazole ( 0.107 g , 1.576 mmol ) were sequentially added , the reaction sealed , and heated at 100 c for 12 h. it was cooled to room temperature , treated with etoac / h2o ( 30 ml , 1:1 ) , and the layers separated . the aqueous layer was extracted with etoac ( 2 10 ml ) , and the combined etoac extracts were washed with brine ( 20 ml ) , dried , and concentrated . the residue was purified by flash column chromatography using ch2cl2/meoh to obtain 68 ( 0.289 g , 67% ) as a cream - colored oil . h nmr ( 500 mhz , cdcl3 ) : 8.59 ( s , 1 h ) , 8.43 ( s , 1 h ) , 8.01 ( s , 1 h ) , 7.59 ( s , 1 h ) , 7.42 ( s , 2 h ) , 7.24 ( q , j = 6.5 hz , 1 h ) , 7.026.79 ( m , 3 h ) , 3.423.33 ( m , 4 h ) , 2.962.69 ( m , 4 h ) , 1.38 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : ( 163.81 , 161.86 , d , j = 245.7 hz , 1 c ) , 155.17 , ( 154.88 , 154.85 , 1 c ) , 149.15 , 141.51 , ( 140.86 , 140.71 , 1 c ) , 135.86 , 133.89 , ( 130.02 , 129.97 , d , j = 6.3 hz , 1 c ) , 129.13 , ( 124.49 , 124.47 , d , j = 2.52 hz , 1 c ) , ( 115.74 , 115.57 , d , j = 21.42 hz , 1 c ) , ( 113.44 , 113.28 , d , j = 20.16 hz , 1 c ) , 79.98 , ( 49.33 , 49.04 , 1 c ) , 48.19 , ( 34.95 , 34.27 , 1 c ) , ( 32.01 , 31.88 , 1 c ) , 28.27 . ms ( esi ) m / z [ m + h ] : calcd , 411.21 ; found , 411.33 . compounds 6973 were synthesized from 49 following the same procedure used to synthesize 56 . h nmr ( 500 mhz , cdcl3 ) : 8.69 ( s , 1 h ) , 7.93 ( s , 1 h ) , 7.86 ( s , 1 h ) , 7.68 ( s , 1 h ) , 7.14 ( q , j = 7.8 hz , 1 h ) , 6.886.84 ( m , 2 h ) , 6.79 ( d , j = 9.0 hz , 1 h ) , 3.60 ( t , j = 6.8 hz , 2 h ) , 3.223.04 ( m , 4 h ) , 2.50 ( t , j = 7.7 hz , 2 h ) , 1.76 ( p , j = 7 hz , 2 h ) , 1.33 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : 171.28 , ( 163.71 , 161.76 , d , j = 245.7 hz , 1 c ) , 158.73 , ( 155.23 , 155.08 , 1 c ) , 143.91 , 137.50 , 134.86 , ( 129.66 , 129.59 , d , j = 8.82 hz , 1 c ) , 123.89 , ( 123.76 , 123.74 , d , j = 2.52 hz , 1 c ) , 119.95 , ( 114.98 , 114.82 , d , j = 201.6 hz , 1 c ) , ( 112.69 , 112.52 , d , j = 21.42 hz , 1 c ) , 79.62 , ( 47.31 , 46.89 , 1 c ) , 46.34 , ( 37.23 , 36.62 , 1 c ) , 32.71 , ( 29.78 , 29.53 , 1 c ) , 28.18 . ms ( esi ) m / z [ h nmr ( 500 mhz , cdcl3 ) : 9.22 ( s , 1 h ) , 8.66 ( d , j = 4.9 hz , 1 h ) , 8.16 ( s , 1 h ) , ( 7.22 ( s ) , 7.13 ( s ) , 1:1 , 1 h ) , 7.20 ( q , j = 7.2 hz , 1 h ) , 6.90 ( d , j = 7.5 hz , 1 h ) , 6.886.81 ( m , 2 h ) , 3.663.58 ( m , 2 h ) , 3.233.15 ( m , 2 h ) , 3.113.03 ( m , 2 h ) , 2.57 ( t , j = 7.7 hz , 2 h ) , 1.82 ( p , j = 7.7 hz , 2 h ) , 1.38 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : 171.17 , ( 163.82 , 161.87 , d , j = 245.7 hz , 1 c ) , 158.75 , 155.37 , 154.28 , 153.57 , 143.89 , 143.73 , ( 129.81 , 129.74 , d , j = 8.82 hz , 1 c ) , ( 123.84 , 123.82 , d , j = 2.52 hz , 1 c ) , 119.83 , ( 115.09 , 114.92 , d , j = 21.42 hz , 1 c ) , ( 112.88 , 112.72 , d , j = 20.16 hz , 1 c ) , 79.83 , ( 47.28 , 47.06 , 1 c ) , 46.27 , ( 37.06 , 36.45 , 1 c ) , 32.78 , ( 29.86 , 29.65 , 1 c ) , 28.30 . ms ( esi ) m / z [ 2 m + na ] : calcd , 875.39 ; found , 875.33 . colorless oil ( 0.141 g , 85% ) . h nmr ( 500 mhz , cdcl3 ) : 8.53 ( d , j = 4.5 hz , 1 h ) , 7.82 ( s , 1 h ) , 7.18 ( q , j = 7.1 hz , 1 h ) , ( 7.03 ( s ) , 6.97 ( s ) , 1:1 , 1 h ) , 6.92 ( s , 1 h ) , 6.89 ( d , j = 7.5 hz , 1 h ) , 6.856.79 ( m , 2 h ) , 3.653.52 ( m , 2 h ) , 3.233.09 ( m , 2 h ) , 3.012.89 ( m , 2 h ) , 2.78 ( s , 3 h ) , 2.55 ( t , j = 7.7 hz , 2 h ) , 1.80 ( p , j = 7 hz , 2 h ) , 1.37 ( s , 9 h ) . c nmr ( 126 mhz , cdcl3 ) : 169.92 , ( 163.74 , 161.79 , d , j = 245.7 hz , 1 c ) , 158.03 , 155.90 , 155.09 , 146.41 , 143.84 , ( 129.74 , 129.68 , d , j = 7.56 hz , 1 c ) , 127.32 , ( 123.77 , 123.75 , d , j = 2.52 hz , 1 c ) , 118.35 , 117.63 , ( 115.00 , 114.84 , d , j = 20.16 hz , 1 c ) , ( 112.79 , 112.62 , d , j = 21.42 hz , 1 c ) , 79.66 , ( 47.17 , 46.90 , 1 c ) , 46.12 , ( 36.88 , 36.19 , 1 c ) , 32.72 , ( 29.77 , 29.53 , 1 c ) , 28.23 , 18.12 . ms ( esi ) m / z [ m + h ] : calcd , 440.2 ; found , 440.04 . compounds 72 and 73 were obtained as a 15:1 nonseparable mixture of isomers from reaction of 49 with 4(5)-methylimidazole , which was carried to the next step without further purification . all isozymes of nos , rat and human nnos , murine macrophage inos , and bovine enos , were recombinant enzymes , overexpressed in escherichia coli and purified following previously reported procedures . the enzyme inhibition was determined by measuring the production of nitric oxide from l - arginine using the hemoglobin capture assay in the presence of different concentrations of inhibitors . the assay was performed at 37 c in 100 mm hepes buffer with 10% glycerol ( ph 7.4 ) in the presence of 10 m l - arginine and tetrahydrobiopterin , 100 m nadph , 0.83 mm cacl2 , 320 units / ml of calmodulin , and 3 m human oxyhemoglobin . for inos , cacl2 , and calmodulin all assays were performed in 96-well plates using a synergy h1 hybrid multimode microplate reader with automated dispensing of nos enzyme and hemoglobin after 30 s ( maximum delay ) , which initiated the assay . the initial rates of no production were determined by monitoring the formation of methemoglobin ( no mediated conversion of oxyhemoglobin to methemoglobin ) by monitoring the absorbance at 401 nm . the entire kinetic readout was performed for 5 min with measurements at every 22 s interval . each compound was assayed at least in duplicate , and nine concentrations ( 100 m to 10 nm for nnos ; 500 m to 50 nm for inos and enos ) were used to construct dose response curves with slopes from initial readouts . ic50 values were calculated by nonlinear regression using graphpad prism ( standard error values reported are from the log ic50 calculations ) , and apparent ki values were determined using the cheng prusoff equation [ ki = ic50/(1 + [ s]/km ) ] with the following km values for l - arginine : 1.3 ( rat nnos ) , 1.6 ( human nnos ) , 8.2 ( murine macrophage inos ) , and 1.7 m ( bovine enos ) . the selectivity of an inhibitor was defined as the ratio of their respective ki values . the preparations of rat nnos , bovine enos , and human nnos heme domains used for crystallographic studies were carried out by the procedures described previously . the heme domain samples of nnos ( at 9 mg / ml containing 20 mm histidine ) , bovine enos ( 10 mg / ml containing 2 mm imidazole ) , and human nnos ( 13 mg / ml ) were used for the sitting drop vapor diffusion crystallization setup under conditions reported . a new orthorhombic crystal form of human nnos was obtained when the ph was raised to 6.2 from 5.0 and the protein concentration dropped to 10 mg / ml . the well solution compositions were only slightly shifted from what were reported : 911% peg3350 , 40 mm citric acid , 60 mm bis - tris - propane , 10% glycerol , and 5 mm tcep . from the sitting drop setup , plate - like crystals grew to full size at 4 c in 34 days without seeding . fresh crystals were first passed stepwise through cryoprotectant solutions and then soaked with 10 mm inhibitor for 46 h at 4 c before being flash cooled with liquid nitrogen . the cryogenic ( 100 k ) x - ray diffraction data were collected remotely at the stanford synchrotron radiation light source ( ssrl ) or advanced light source ( als ) through the data collection control software blu - ice and a crystal - mounting robot . when a q315r ccd detector was used , 90100 of data were typically collected with 0.5 per frame . if a pilatus pixel array detector was used , 140160 of fine - sliced data were collected with 0.2 per frame . raw ccd data frames were indexed , integrated , and scaled using hkl2000 or mosflm , but the pixel array data were processed with xds and scaled with scala ( aimless ) . for rat nnos or bovine enos structures , the binding of inhibitors was detected by the initial difference fourier maps calculated with refmac . for human nnos structures , one homodimer in the known human nnos structure ( 4d1n ) was used as the search model . the new human nnos structure closely resembles that of rat nnos to have only one homodimer in the asymmetric unit . the inhibitor molecules were then modeled in coot and refined using refmac or phenix . the tls protocol was implemented in the final stage of refinements with each subunit as one tls group . the omit fo fc density maps were calculated by removing inhibitor coordinates from the input pdb file before running one more round of tls refinement in refmac or in phenix ( simulated annealling protocol with a 2000 k initial temperature ) . the resulting map coefficients delfwt and sigdelwt were used to generate maps that are displayed in figures . the refined structures were validated with the validation service in the rcsb protein data bank . the crystallographic data collection and structure refinement statistics are summarized in table s1 of the supporting information , with the pdb accession codes included . caco-2 permeability assays were performed by cyprotex ( watertown , ma ) using the caco-2 epithelial monolayers . caco-2 cells , grown in tissue culture flasks , were trypsinized , suspended in media , and plated in 96-well plates to be grown for 3 weeks ; the proper formation of monolayer was determined by fluorescent measurement of transport of an impermeable dye , lucifer yellow . all assays were performed with compounds at a concentration of 10 m for 2 h. for apical to basolateral ( ab ) permeability , compounds were added on the apical side ( a ) and permeation determined on the basolateral side ( b ) , where the receiving buffer was removed for analysis by lc / ms / ms using an agilent 6410 mass spectrometer ( esi , mrm mode ) coupled with an agilent 1200 hplc . the buffers used were 100 m lucifer yellow in transport buffer ( 1.98 g / l glucose in 10 mm hepes , 1 hank s balanced salt solution , ph 6.5 ) ( apical side ) and transport buffer , ph 7.4 ( basolateral side ) . the apparent permeability ( papp ) is expressed using the following equation : papp = ( dq / dt)/c0a , where dq / dt is the rate of permeation , c0 is initial concentration , and a is the monolayer area . for bidirectional permeability , the efflux ratio was defined as papp(ba)/papp(ab ) ; high efflux ratio ( > 3 ) indicates that a compound is a potential substrate for p - gp or other active transport systems . screening of compound 9 for off - target receptor activity was performed at the nimh psychoactive drug screening program at unc chapel hill . in the primary radioligand binding assays , the compound was tested at a single concentration ( 10 m ) in quadruplicate in 96-well plates . for receptors with which a compound displayed more than 50% inhibition at 10 m concentration , the compound was subjected to secondary radioligand binding assays to determine equilibrium binding affinity at specific targets . in the secondary binding assays , compound 9 was tested at 11 concentrations ( 100.1 m ) and in triplicate . both primary and secondary radioligand binding assays were carried out in a final volume of 125 l per well in the appropriate binding buffer , and the radioligand concentration was at a concentration close to the kd . in a typical assay , 25 l of radioligand was added to each well of a 96-well plate , followed by addition of 25 l binding buffer with or without compound . the reaction started upon addition of 75 l of fresh membrane protein ( typically 2550 g per well ) , and the reaction was incubated in the dark at room temperature for 90 min . the reaction was stopped by vacuum filtration onto cold 0.3% polyethylenimine ( pei)-soaked 96-well filter mats using a 96-well filtermate harvester , followed by three washes with cold wash buffers . scintillation cocktail was then melted onto the microwave - dried filters on a hot plate , and radioactivity was counted in a microbeta counter . for primary binding assay analysis , nonspecific binding in the presence of 10 m of an appropriate reference compound was set as 100% inhibition ; total binding in the absence of test compound or reference compound was set as 0% inhibition . the radioactivity in the presence of test compound was calculated with the equation : % inhibition = ( sample cpm nonspecific cpm ) 100 , where the radioactivity was measured in counts per minute ( cpm / well ) . for secondary binding results , counts ( cpm / well ) were pooled and fitted to a three - parameter logistic function for competition binding in prism to determine ic50 values : y = bottom + ( top bottom)/1 + 1050 , where y is the total binding in the presence of a corresponding concentration of compound ( x , in this case , concentration of 9 ) , and top and bottom are the total and nonspecific binding in the absence and presence of 10 m reference compound . the cyp inhibition assay of 9 was performed against the five major liver microsomal cyp enzymes : cyp1a2 , cy2c9 , cyp2c19 , cyp2d6 , and cyp3a4 ( by sai life sciences ) . in the assay , a 25 l aliquot of microsomes diluted in kphos buffer ( 0.4 mg / ml ) was added to individual wells of the reaction plate . fluvoxamine , sulfaphenazole , quinidine , ticlopidine , and ketoconazole ( positive control inhibitors for cyp1a2 , cyp2c9 , cyp2d6 , cyp2c19 , and cyp3a4 , respectively ) , diluted in buffer ( 25 l ) , were added separately to the respective wells . compound 9 , diluted in dmso to a concentration of 10 m , was directly spiked into microsomal mix ( 2 ) , and 50 l was aliquoted into individual wells . an aliquot of 25 l of phenacetin , diclofenac , bufuralol , s - mephenytoin , and midazolam ( 4 ) for cyp1a2 , cyp2c9 , cyp2d6 , cyp2c19 , and cyp3a4 , respectively , was added separately to wells and incubated for 5 min at 37 c . the reactions were initiated using 25 l of nadph ( 4 ) and further incubated for 5 min for cyp3a4 ( midazolam ) , 10 min for cyp1a2 , cyp2c9 , and cyp2d6 , and 20 min for cyp2c19 . all reactions were terminated using 100 l of ice - cold acetonitrile containing internal standard ( imipramine and glipizide at 1 m ) . the plates were centrifuged at 4000 rpm for 15 min , 100 l aliquots were subjected to lc - m / ms on a shimadzu api 4000 system ( mrm mode ) , and the metabolites were detected . the respective peak area ratios ( pa ) of metabolites and internal standard was used to determine % inhibition , where % activity = ( pa ratios in the presence of compound / pa ratios in dmso control ) 100 , and % inhibition = 100 % activity . the inhibitory potency of 9 , ( r , r)-12 , 13 , and 20 on the 7-benzyloxy-4-(trifluoromethyl)coumarin ( bfc ) debenzylase activity of human cyp3a4 was evaluated fluorimetrically in a reconstituted system with cytochrome p450 reductase ( cpr ) . the reaction was carried out at room temperature in 100 mm phosphate buffer , ph 7.4 , containing catalase and superoxide dismutase ( 2 u / ml each ) . a mixture of 1 m cyp3a4 and 1 m cpr was preincubated for 1 h at room temperature and diluted by 20-fold before measurements . bfc ( 50 m ) and various concentrations of inhibitors were added 2 min prior to initiation of the reaction with 100 m nadph . formation of 7-hydroxy-4-trifluoromethylcoumarin ( ex = 430 nm ; em = 500 nm ) was followed in a hitachi f100 fluorimeter . ic50 values were derived from the [ % activity ] vs [ inhibitor ] plots .

selective inhibition of neuronal nitric oxide synthase ( nnos ) is an important therapeutic approach to target neurodegenerative disorders . however , the majority of the nnos inhibitors developed are arginine mimetics and , therefore , suffer from poor bioavailability . we designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors . in conjunction with extensive structure activity studies , several highly potent and selective inhibitors of nnos were discovered . x - ray crystallographic analysis reveals that these type ii inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency ( 13 ) , as well as high isoform selectivity . interestingly , select compounds from this series ( 9 ) showed good permeability and low efflux in a caco-2 assay , suggesting potential oral bioavailability , and exhibited minimal off - target binding to 50 central nervous system receptors . furthermore , even with heme - coordinating groups in the molecule , modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome p450s from human liver microsomes .

Introduction Chemistry Results and Discussion Conclusions Experimental Section

PMC4070581

the mitochondrial function impairment in skeletal muscle is one of the physiological limitations of aging . however , there is a considerable variability of aging impact on mitochondrial function [ 26 ] . sarcopenia and muscle fatigability in response to aging are associated with the increase of both the reactive oxygen species ( ros ) production and the mitochondrial apoptotic susceptibility , as well as the decrease of transcriptional drive for mitochondrial biogenesis . however , the precise underlying mechanisms of these processes remain unclear . some common regulatory mechanisms that include silent information regulators like sirtuins the silent information regulator 2 homolog 1 ( sirt-1 ) may influence the aging processes and many age - associated diseases , including metabolic disorders such as diabetes . sirt-1 is downregulated in human senescent cells , suggesting that sirt-1 may be required to extend the replicative life span . for example , the deacetylation of lysine residues of the histone tails by sirt induces closed chromatin configuration and transcriptional silencing . besides histone deacetylation , sirt-1 targets a number of transcription factors such as peroxisome proliferator - activated receptor gamma coactivator-1alpha ( pgc-1 ) , which is involved in mitochondrial biogenesis and skeletal muscle differentiation [ 9 , 11 ] . thus , sirt-1-mediated pgc-1 deacetylation is a key factor of mitochondrial biogenesis activation [ 9 , 11 ] . the pgc-1 family of regulated coactivators plays a central role in a regulatory network governing the transcriptional control of mitochondrial biogenesis and respiratory function . these coactivators target multiple transcription factors , including nuclear respiratory factors ( nrf ) 1 and 2 and the orphan nuclear hormone receptor ( estrogen - related receptor alpha err ) , among others . in addition , they themselves are the targets of coactivator and corepressor complexes that regulate gene expression through chromatin remodeling . the proposed role for pgc-1 as a regulator of mitochondrial biogenesis is supported by experiments that verified functional improvements in both cultured cells and transgenic mice . the pgc-1 expression is modulated by extracellular signals that control metabolism , differentiation , or cell growth . in addition , in some cases , pgc-1 activity is regulated by posttranslational modification by the energy sensors , such as sirt-1 and amp - activated protein kinase ( ampk ) . ampk is emerging as a crucial regulator of whole - body energy balance . in skeletal muscle , after being activated , ampk regulates both the fatty - acid oxidation by the phosphorylation of acetyl - coa carboxylase ( acc ) and the mitochondrial biogenesis by the increase of the expression of vital proteins for proper mitochondrial function , such as citrate synthase and succinate dehydrogenase [ 15 , 16 ] . in addition , ampk promotes mitochondrial biogenesis by the increase of both pgc-1 levels and other associated mitochondrial proteins [ 1720 ] . the activation of ampk ( e.g. , by exercise ) triggers an increase in the nad / nadh ratio , which activates sirt-1 . ampk also induces pgc-1 phosphorylation and primes it for subsequent deacetylation by sirt-1 . the impact of ampk and sirt-1 on the pgc-1 acetylation status and other transcriptional regulators will modulate mitochondrial function and activity . in fact , multiple endogenous and exogenous factors regulate mitochondrial biogenesis by pgc-1 , sirt-1 , and ampk , including physical exercise . physical exercise is known to induce metabolic adaptations in skeletal muscle via activation of these molecules [ 3 , 22 ] . however , in the aging process , the influence of different training intensities on molecular alterations remains unclear . thus , this study was designed to test the hypothesis that high training intensity is more effective than low training intensity in restoring sirt-1 , ampk , pgc-1 , and related metabolic enzymes that decrease with aging . based on the fact that aging is related to increased circulating proinflammatory and lower anti - inflammatory cytokines and knowing that besides the role as an activator of mitochondrial biogenesis , pgc-1 also acts as a suppressor of inflammatory cytokines , we also investigated the responses of the tumor necrosis factor alpha ( tnf- ) , interleukin 1 ( il-1 ) , and nuclear factor kappa - b ( nf-b ) to different training intensities in young and middle - aged wistar rats . male wistar rats at 2 ( young ) and 18 ( middle - aged ) months were used during the experiments . the current investigation followed the university guidelines for the use of animals in experimental studies ( protocol number 92/2009 ) . the procedures used in this study received approval from the research ethics committee of universidade do extremo sul catarinense ( cricima , brazil ) . young and middle - aged animals were divided into the following groups ( n = 6 ) : nonexercised ( ne ) , exercised at 0.8 km / h ( 0.8 km / h ) , and exercised at 1.2 km / h ( 1.2 km / h ) . we selected middle - aged rats in order to study molecular and physiological changes related to mitochondrial function during a life period that still allows preventive actions that can lead to healthy aging . all animals were maintained at temperatures ranging from 20 to 25c , with a 12-hour light / dark cycle and fed on a standard rodent chow ad libitum . before the beginning of the exercise protocols , one set of young and middle - aged rats ( n = 8) was evaluated for the following parameters : body weight ( g ) , blood glucose ( mg / dl ) , epididymal fat ( g/100 g ) , and serum insulin ( ng / ml ) . the rats were anesthetized with an intraperitoneal ( i.p ) injection of ketamine chlorohydrate ( 50 mg / kg ; syntec , cotia , sp , brazil ) and xylazine ( 20 mg / kg ; syntec , cotia , sp , brazil ) , and the blood was collected from the cava vein . serum was separated by centrifugation ( 1,100 g ) for 15 min at 4c and stored at 80c for further analysis . the epididymal fat was surgically removed , weighted , and expressed as g/100 g of rats ' body weight . while rest blood glucose ( mg / dl ) was measured by a glucometer ( advantage , boehringer mannheim , irvine , ca ) , the rest serum insulin was determined using a commercially available enzyme linked immunosorbent assay ( elisa ) kit ( crystal chem inc . , all animals were habituated on a nine - channel motor - drive treadmill ( insight ep 131 , ribeiro preto , brazil ) at a velocity of 0.6 km / h for 10 min / day during 1 week in order to reduce stress during the training period . the rats did not receive any electric stimulus to run , but manual stimulation was applied . the exercise group performed a running program at constant speed of 0.8 km / h or 1.2 km / h without inclination for 50 min , 5 days per week , during 8 weeks . the nontrained rats were placed on the switched - off treadmill for the same 8 weeks . forty - eight hours after the last training sessions , the rats were anesthetized with an intraperitoneal ( i.p ) injection of ketamine chlorohydrate ( 50 mg / kg ; syntec , cotia , sp , brazil ) and xylazine ( 20 mg / kg ; syntec , cotia , sp , brazil ) , and quadriceps was removed for biochemical and immunoblotting analyses . the superficial ( i.e. , rectus femoris composed of type i : 1% , type iia : 25% , and type iib : 74% ) and deep quadriceps ( i.e. , vastus intermedius composed of type i : 59% , type iia : 40% , and type iib : 1% ) were homogenized together in extraction buffer ( 1% triton - x 100 , 100 mmtris , ph 7.4 , containing 100 mm sodium pyrophosphate , 100 mm sodium fluoride , 10 mm edta , 10 mm sodium vanadate , 2 mm pmsf , and 0.1 mg of aprotinin / ml ) at 4c ( polytron mr 2100 , kinematica , switzerland ) . the extracts were centrifuged at 11,000 rpm at 4c ( 5804r , eppendorf ag , hamburg , germany ) for 40 min to remove insoluble material , and the supernatants of this tissue were used for protein quantification , according to the bradford method . proteins were denatured by boiling in laemmli sample buffer containing 100 mm dtt , run on sds - page , and transferred to nitrocellulose membranes . antibodies used for immunoblotting were antiphospho ( thr172 ) ampk , antiphospho ( ser79 ) acc , anti - ampk , and anti - acc antibodies ( cell signaling technology , beverly , ma , usa ) ; anti - sirt-1 , anti - pgc-1 , anti - cpt1 , anti - sdh , citrate synthase , anti - cyt - c , and -actin ( santa cruz biotechnology , santa cruz , ca , usa ) ; and anti - tnf- , anti - il-1 , and anti - nf-b ( abcam biotechnology , eugene , oregon , usa ) . chemiluminescent detection was performed with horseradish peroxidase - conjugate secondary antibodies ( thermo scientific , rockford , il , usa ) . the results of the blots are presented as direct comparisons of the area of the apparent bands in autoradiographs and quantified by densitometry using the scion image software ( scion image software , scioncorp , frederick , md ) . the reaction mixture contained 100 mmtris , ph 8.0 , 100 mm acetyl coa , 100 mm 5,5-di - thiobis-(2-nitrobenzoic acid ) , 0.1% triton x-100 , and 24 g supernatant protein , and it was initiated with 100 m oxaloacetate and monitored at 412 nm for 3 min at 25c . succinate dehydrogenase activity was determined according to the method of fischer et al . , measured by the following decrease in absorbance due to the reduction of 2,6-dichloroindophenol ( 2,6-dcip ) at 600 nm with 700 nm as the reference wave length ( = 19.1 mmcm ) in the presence of phenazine methosulphate ( pms ) . the reaction mixture consisting of 40 mm potassium phosphate , ph 7.4 , 16 mm succinate , and 8 m 2,6-dcip was preincubated with 4080 g homogenate protein at 30c for 20 min . subsequently , 4 mm sodium azide , 7 m rotenone , and 40 m 2,6-dcip were added , and the reaction was initiated by adding 1 mm pms and was monitored for 5 min . nadh dehydrogenase ( complex i ) was evaluated according to cassina and radi by determining the rate of nadh - dependent ferricyanide reduction at = 420 nm . the activity of succinate-2,6-dichloroindophenol- ( dcip- ) oxidoreductase ( complex ii ) was determined using the method described by fischer et al . . complex ii activity was measured by following the decrease in absorbance due to the reduction of 2,6-dcip at = 600 nm . the activity of succinate : cytochrome c oxidoreductase ( complex iii ) was determined using the method described by fischer et al . . complex ii - iii activity was measured by cytochrome c reduction using succinate as substrate at = 550 nm . the activity of cytochrome c oxidase ( complex iv ) was assayed according to the method described by rustin et al . and measured by following the decrease in absorbance due to the oxidation of previously reduced cytochrome c ( prepared by reduction of cytochrome with nabh4 and hcl ) at = 550 nm with 580 nm as the reference wavelength ( = 19.1 mmcm ) . the activities of the mitochondrial respiratory chain complexes were expressed as nmol / min / mg of protein . in the present study , the hypothesis is that molecular biomarkers are affected by the following factors : ( 1 ) age : young versus middle - aged and ( 2 ) training intensity : nontrained versus 0.8 km / h speed versus 1.2 speed km / h . the results about the descriptive characteristics of young and middle - aged rats before exercise protocols were expressed as mean and standard error median ( sem ) and were evaluated using the unpaired student 's t - test . while the results about the western blot analyses were expressed as the mean area of the apparent band sem ( arbitrary units were calculated as area versus density ) , the activity of krebs cycle enzymes and the activity of mitochondrial respiratory chain enzymes were expressed as nmol / min / mg of protein sem . differences between the groups for the mentioned parameters were evaluated using two - way analysis of variance ( anova ) followed by the bonferroni post hoc test . a probability of less than 0.05 was considered to be significant . the software used for the analysis of the data was the statistical package for the social sciences ( spss ) version 16.0 for windows . according to table 1 , the middle - aged rats presented higher values of body weight ( 526 12.31 versus 218.9 10.02 g ) , epididymal fat ( 3.9 0.4 versus 1.2 0.2 g/100 g ) , and serum insulin ( 4.2 0.7 versus 1.3 0.5 ng / ml ) compared to young rats . the protein levels of tnf- , il-1 , and nf-b were higher in the quadriceps of middle - aged ne compared to young ne rats ( figures 1(a)1(c ) ) . both groups ( i.e. , young and middle - aged rats ) were trained at 0.8 km / h which decreased the protein levels of tnf- , il-1 , and nf-b compared to their respective ne groups ; however , the middle - aged rats presented higher protein levels of tnf- , il-1 , and nf-b compared to young rats at the 0.8 km / h training intensity ( figures 1(a)1(c ) ) . in addition , both groups were trained at 1.2 km / h which decreased the protein levels of tnf- , il-1 , and nf-b compared to their respective 0.8 km / h groups , but no differences were observed between young and middle - aged rats at the 1.2 km / h training intensity ( figures 1(a)1(c ) ) . we evaluated pivotal molecules involved in mitochondrial function and oxidative metabolism . while ampk phosphorylation decreased in the quadriceps of middle - aged ne compared to young ne rats ( figure 2(a ) ) , no significant differences were observed between these groups ( i.e. , young and middle aged rats ) at ne situation for acc phosphorylation ( figure 2(b ) ) . both groups ( i.e. , young and middle - aged rats ) were trained at 0.8 km / h which increased ampk and acc phosphorylations compared to their respective ne groups ; however , the middle - aged rats presented lower ampk and acc phosphorylations compared to the young rats at the 0.8 km / h training intensity . in addition , both groups were trained at 1.2 km / h which increased the ampk and acc phosphorylation compared to their respective 0.8 km / h groups , but no differences were observed between young and middle - aged rats at the 1.2 km / h training intensity ( figures 2(a ) and 2(b ) ) . total ampk and acc protein levels were similar between the groups ( figures 2(a ) and 2(b)lower panels ) . sirt-1 and pgc-1 protein levels decreased in the quadriceps of middle - aged ne rats compared to young ne rats ( figures 2(c ) and 2(d ) ) . both groups ( i.e. , young and middle - aged rats ) were trained at 0.8 km / h which increased the protein levels of sirt-1 and pgc-1 compared to their respective ne groups ; however , the middle - aged rats presented lower protein levels of sirt-1 and pgc-1 compared to young rats at the 0.8 km / h training intensity . in addition , both groups were trained at 1.2 km / h which increased the protein levels of sirt-1 and pgc-1 compared to their respective 0.8 km / h groups , but no differences were observed between young and middle - aged rats at the 1.2 km / h training intensity ( figures 2(c ) and 2(d ) ) . lower protein levels of cpt1 and cyt - c were observed in the quadriceps of middle - aged ne rats compared to young ne rats . both groups ( i.e. , young and middle - aged rats ) were trained at 0.8 km / h which increased the protein levels of cpt1 and cyt - c compared to their respective ne groups ; however , the middle - aged rats presented lower protein levels of cpt1 and cyt - c compared to young rats at the 0.8 km / h . in addition , both groups were trained at 1.2 km / h which increased the protein levels of cpt1 and cyt - c compared to their respective 0.8 km / h groups , but no differences were observed between young and middle - aged rats at the 1.2 km / h training intensity ( figures 3(a ) and 3(b ) ) . the sdh protein levels did not present significant differences between young and middle - aged rats for the studied experimental situations ( i.e. , ne , 0.8 and 12 km / h ; figure 3(c ) ) . the sdh activity was not different between young and middle - aged rats for the ne situation . the young rats were trained at 0.8 km / h which increased the sdh activity compared to their respective ne group . on the other hand , sdh activity was lower in middle - aged rats compared to young rats at the 0.8 km / h training intensity . in addition , middle - aged rats were trained at 1.2 km / h which increased the sdh activity compared to their respective 0.8 km / h group , but no significant differences were observed between young and middle - aged rats at the 1.2 km / h training intensity ( figure 3(d ) ) . the protein levels and activity of citrate synthase were lower in the quadriceps of middle - aged ne rats compared to young ne rats . both groups ( i.e. , young and middle - aged rats ) were trained at 0.8 km / h which increased the protein levels of citrate synthase compared to their respective ne groups ; however , the middle - aged rats presented lower protein levels and activity of citrate synthase compared to young rats at the 0.8 km / h . in addition , both groups were trained at 1.2 km / h which increased the protein levels and activity of citrate synthase compared to their respective 0.8 km / h groups , but no differences were observed between young and middle - aged rats at the 1.2 km / h training intensity ( figures 3(e ) and 3(f ) ) . the young and middle - aged rats did not present significant alterations for the complexes i , ii , and ii - iii activities at the ne and 0.8 km / h situations . however , the young and middle - aged rats were trained at 1.2 km / h which increased the complexes i , ii , and ii - iii activities compared to their respective 0.8 km / h groups . in addition , for these complexes , we did not observe significant differences between young and middle - aged rats at the 1.2 km / h training intensity ( figures 4(a)4(c ) ) . the complex ii activity was higher in young rats compared to middle - aged rats at the 0.8 km / h training intensity ( figure 4(b ) ) . the complex iv activity decreased in the quadriceps of middle - aged ne rats compared to young ne rats . the young rats were trained at 0.8 km / h which increased the complex iv activity compared to their respective ne group . the complex iv activity of the middle - aged rats was lower compared to young rats at the 0.8 km / h training intensity . in addition , both groups ( i.e. , young and middle - aged rats ) were trained at 1.2 km / h which increased the complex iv activity compared to their respective 0.8 km / h groups , but no differences were observed between young and middle - aged rats at the 1.2 km / h training intensity ( figure 4(d ) ) . the main findings of the present investigation are ( a ) the aging process is associated with the increase of tnf- , il-1 , and nf-b protein levels and with the decrease of the ampk phosphorylation and sirt-1 and pgc-1 protein levels ; ( b ) the young and middle - aged rats were trained at 0.8 km / h which decreased the tnf- , il-1 , and nf-b protein levels and increased the ampk phosphorylation and sirt-1 and pgc-1 protein levels compared to their respective ne groups , although the young rats presented lower values ( for tnf- , il-1 , and nf-b ) and higher values ( for sirt-1 , pgc-1 , and ampk ) compared to the middle - aged rats inside this specific training intensity ; ( c ) the young and middle - aged rats were trained at 1.2 km / h which decreased the tnf- , il-1 , and nf-b protein levels and increased the ampk phosphorylation and sirt-1 and pgc-1 protein levels compared to their respective 0.8 km / h groups ; ( d ) interestingly , at this training intensity ( i.e. , 1.2 km / h ) , the behavior of the analyzed molecules was not different between young and middle - aged rats . the inflamm - aging process is associated with high levels of tnf- , il-1 , and nf-b in skeletal muscle of elderly individuals and rodents [ 23 , 30 ] . the present results are in accordance with della gata et al . showing that exercise training is able to revert partially the high levels of cytokines induced by the inflamm - aging process . on the other hand , up to today , this is the first investigation showing that high training intensities lead to better responses of tnf- , il-1 , and nf-b in skeletal muscle of middle - aged rats . these responses may be linked to the increases observed in the protein levels of pgc-1 , once this molecule acts as a suppressor of inflammatory cytokines . aging process is associated with the reduction of the muscle functionality that , at least in part , is linked to mitochondrial dysfunction [ 1 , 3134 ] . some studies have shown that key molecules are involved in this process , such as sirt-1 , ampk , and pgc-1 [ 912 , 22 , 32 ] . the role of sirt-1 in skeletal muscle is mainly attributed to its ability to deacetylate and activate pgc-1 [ 9 , 12 , 22 ] . pgc-1 orchestrates the genetic program that allows skeletal muscle cell adaptation to meet the energy demands . ectopic expression of pgc-1 in myotubes increases the respiratory chain gene expression ( i.e. , cytochrome c ) and promotes mitochondrial biogenesis . furthermore , it is well known that ampk upregulates sirt-1 activity in skeletal muscles [ 21 , 35 ] . the ampk activity inhibition prevents the deacetylation of pgc-1 by sirt-1 in skeletal muscle cells in response to glucose deprivation , decreasing the mitochondrial complex activity and damaging the fatty acid oxidation metabolism [ 21 , 35 ] . in addition , the study of ampk deficient mice evidenced the impairment of pgc-1 deacetylation in correlation with a failure of muscle metabolism adaptation in response to exercise [ 21 , 35 ] . thus , in the present investigation , we tested the hypothesis that high training intensity is more effective than low training intensity in restoring sirt-1 , ampk , pgc-1 , and related metabolic enzymes that decrease with aging . in summary , these molecules increased when the experimental groups were trained at 0.8 km / h ( i.e. , compared to their respective ne groups ) and at 1.2 km / h ( i.e. , compared to their respective 0.8 km / h groups ) . in addition , these molecules were higher in young rats compared to middle - aged rats at the 0.8 km / h training intensity but were not different at the 1.2 km / h training intensity . these findings suggest that middle - aged rats will present the same molecular responses as young rats when the training intensity is high . in accordance with the elegant study of koltai et al . , we demonstrated that treadmill training may reverse the negative effects of aging on pivotal molecules that are associated with the mitochondrial control of skeletal muscle of rats . in fact , koltai et al . also verified that treadmill training increased the sirt-1 activity in both young and aged rats ( 26 months of age ) . the elegant study of cant et al . showed that ampk activation precedes and determines the changes in sirt-1 activity in situations of energy stress . the regulation of the acetylation levels of transcriptional regulators through the ampk / sirt-1 axis provides a mechanism by which mitochondrial and lipid oxidation genes can be rapidly and selectively controlled in response to energy levels [ 21 , 34 , 36 ] . in the present investigation , we observed that the phosphorylation of ampk and the protein levels of sirt-1 and pgc-1 decreased in the quadriceps of ne middle - aged rats compared to the ne young rats . on the other hand , these proteins increased when the experimental groups were trained at 0.8 km / h compared to their respective ne groups , although the results of the young rats were higher compared to the middle - aged rats at 0.8 km / h training intensity . however , these proteins remained unchanged between young and middle - aged rats at 1.2 km / h training intensity . cited that ampk is activated by the decrease in the atp / amp ratio and phosphocreatine ( cp ) and glycogen levels . on the other hand , the cp and glycogen depletion are dependent on the exercise intensity [ 37 , 38 ] . although we did not measure the cp and glycogen concentrations in the present paper , it is possible to hypothesize that the higher training intensity led to higher depletion of these substrates and was responsible for the higher ampk activation . interestingly , suwa et al . observed that the protein expressions of sirt-1 but not pgc-1 increased in response to training . the differences between our data and the results of suwa et al . may be related to the differences in the exercise protocols and age of the studied rats . the mitochondrial respiratory chain is composed of four respiratory complexes ( complexes i iv ) , and each one is capable of catalyzing electron transfers in a partial reaction of the respiratory chain . it is known that citrate synthase and succinate dehydrogenase activities can be used to estimate mitochondrial content and mitochondrial complexes can reflect the mitochondrial oxidative capacity . in accordance with the results of ampk phosphorylation , sirt-1 and pgc-1 protein levels , the activity and protein levels of citrate synthase , and the activities of the mitochondrial complexes i , ii , ii - iii , and iv also increased in both groups ( i.e. , young and middle - aged rats ) at the 1.2 km / h training intensity . studying ampk knockout mice , jrgensen et al . reported significant reductions in mitochondrial markers ( i.e. , citrate synthase activity and protein contents of one or more complexes in the mitochondrial respiratory chain ) . on the other hand , sirt-1 played a crucial role in the ability of ampk increasing the mitochondrial respiration , once the long - term aicar effects on cellular oxygen consumption were blunted by knocking - down sirt-1 . in addition , the authors also observed a decrease in the lipid oxidation and an increase in alternative substrate oxidation . in fact , the direct oleate oxidation measurement confirmed that the aicar chronic effects on lipid oxidation were blunted in myotubes when the sirt-1 expression was knocked down . therefore , the ampk and sirt-1 signaling pathways have similar effects on lifespan , aging , and metabolism . like sirt-1 , ampk has been considered as one of several molecules involved in the mammalian longevity regulation . other investigations have verified the training effects on the current studied parameters in aged rats [ 22 , 43 ] . for example , ljubicic and hood examined a very extreme form of muscle activation , chronic electrical stimulation , in rats ( i.e. , 36 months of age ) , and they verified that the mitochondrial biogenesis signaling response was compromised . these data indicate that high exercise intensity ( or muscle activation ) in aged rats is not sufficient to correct the age - related decline in the muscle aerobic plasticity . recently , bayod et al . observed that treadmill training during 36 weeks increased the protein content and activity of sirt-1 and the protein expression of pgc-1 in the heart , muscle , and liver of aged rats . however , no changes in ampk activation or mitochondrial biogenesis were found after 36-week treadmill training . for example , the ampk activation by aicar or exercise is blunted in skeletal muscles of old rats ( i.e. , 28 months of age ) . furthermore , the mitochondrial biogenesis was also reduced after chronic activation of ampk with -guanidinopropionic acid ( -gpa ) . on the other hand , sirt-1 protein levels are diminished in mouse embryonic fibroblasts that exhibit premature senescence . however , as previously mentioned , there is a considerable variability of aging impact on mitochondrial function [ 26 ] . the different types of studied rodents , their respective aging phases , and the analyzed parameters may be considered by the discrepancies mentioned above , justifying the new investigations to elucidate the relationship between aging and its molecular consequences . in addition , the selection of middle - aged rats to study molecular and physiological changes related to mitochondrial function is justified because this life period still allows the use of preventive actions that can lead to healthy aging . in summary , we conclude that age - related increases in tnf- , il-1 , and nf-b and age - related declines in the phosphorylation of ampk and in the protein levels of sirt-1 and pgc-1 in skeletal muscle can be reversed and largely improved by treadmill exercise training . in addition , the present data demonstrated that to achieve changes in muscle of middle - aged rats that are similar to young rats , high training intensities are necessary .

the present study investigated the effects of running at 0.8 or 1.2 km / h on inflammatory proteins ( i.e. , protein levels of tnf- , il-1 , and nf-b ) and metabolic proteins ( i.e. , protein levels of sirt-1 and pgc-1 , and ampk phosphorylation ) in quadriceps of rats . male wistar rats at 3 ( young ) and 18 months ( middle - aged rats ) of age were divided into nonexercised ( ne ) and exercised at 0.8 or 1.2 km / h . the rats were trained on treadmill , 50 min per day , 5 days per week , during 8 weeks . forty - eight hours after the last training session , muscles were removed , homogenized , and analyzed using biochemical and western blot techniques . our results showed that : ( a ) running at 0.8 km / h decreased the inflammatory proteins and increased the metabolic proteins compared with ne rats ; ( b ) these responses were lower for the inflammatory proteins and higher for the metabolic proteins in young rats compared with middle - aged rats ; ( c ) running at 1.2 km / h decreased the inflammatory proteins and increased the metabolic proteins compared with 0.8 km / h ; ( d ) these responses were similar between young and middle - aged rats when trained at 1.2 km . in summary , the age - related increases in inflammatory proteins , and the age - related declines in metabolic proteins can be reversed and largely improved by treadmill training .

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

PMC5255575

the era of precision gravitational - wave astrophysics is at our doorstep . with it , a plethora of previously unavailable information will flood in , allowing for unprecedented astrophysical measurements and tests of fundamental theories . nobody would question the importance of more precise astrophysical measurements , but one may wonder whether fundamental tests are truly necessary , considering the many successes of einstein s theory of general relativity ( gr ) . indeed , gr has passed many tests , including solar system ones , binary pulsar ones and cosmological ones ( for a recent review , see [ 438 , 359 ] ) . what all of these tests have in common is that they sample the quasi - stationary , quasi - linear weak field regime of gr . that is , they sample the regime of spacetime where the gravitational field is weak relative to the mass - energy of the system , the characteristic velocities of gravitating bodies are small relative to the speed of light , and the gravitational field is stationary or quasi - stationary relative to the characteristic size of the system . a direct consequence of this is that gravitational waves emitted by weakly - gravitating , quasi - stationary sources are necessarily extremely weak . to make this more concrete , let us define the gravitational compactness as a measure of the strength of the gravitational field : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal c } = { { \mathcal m } \over { \mathcal r}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{m}$\end{document } is the characteristic mass of the system , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{r}$\end{document } is the characteristic length scale associated with gravitational radiation , and henceforth we set g = c = 1 . for binary systems , the strength of gravitational waves and the mutual gravitational interaction between bodies scale linearly with this compactness measure . let us also define the characteristic velocities of such a system \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{v}$\end{document } as a quantity related to the rate of change of the gravitational field in the center of mass frame . we can then more formally define the weak field as the region of spacetime where the following two conditions are simultaneously satisfied : 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bf{weak } } \,\ , { \bf{field}}:\quad { \mathcal c } \ll 1,\quad { \mathcal v } \ll 1.$$\end{document } by similarity , the strong field is defined as the region of spacetime where both conditions in eq . ( 2 ) are not valid simultaneously1 . for the earth - sun system , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{m}$\end{document } is essentially the mass of the sun , while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{r}$\end{document } is the orbital separation , which leads to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{c}\approx 9.8\times 10^{-9}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{v}\approx 9.9\times 10^{-5}$\end{document}. even if an object were in a circular orbit at the surface of the sun , its gravitational compactness would be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{o}(10^{-6})$\end{document } and its characteristic velocity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{o}(10^{-3})$\end{document}. thus , we conclude that all solar - system experiments are necessarily sampling the weak field regime of gravity . similarly , for the binary pulsar j07373039 [ 298 , 274 ] , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{c}\approx 6\times 10^{-6}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{v}\approx 2\times 10^{-3}$\end{document } , where we have set the characteristic length \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{r}$\end{document } to the orbital separation via \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{r}\approx [ mp^{2}/(4\pi^{2})]^{1/3}\approx 10^{6}$\end{document } km , where p = 0.1 days is the orbital period and m 3 m is the total mass . although neutron stars are sources of strong gravity ( the ratio of their mass to their radius is of order one tenth ) , binary pulsars are most sensitive to the quasi - static part of the post - newtonian effective potential or to the leading - order ( newtonian piece ) of the radiation - reaction force . on the other hand , in compact binary coalescence the gravitational compactness and the characteristic speed can reach values much closer to unity . therefore , although in much of the pulsar - timing literature binary pulsar timing is said to allow for strong - field tests of gravity , gravitational information during compact binary coalescence would be a much - stronger field test . even though current data does not give us access to the full non - linear and dynamical regime of gr , solar - system tests and binary - pulsar observations have served ( and will continue to serve ) an invaluable role in testing einstein s theory . solar - system tests effectively cured an outbreak of modified gravity theories in the 1970s and 1980s , as summarized for example in . binary pulsars were crucial as the first indirect detectors of gravitational waves , and later to kill certain theories , like rosen s bimetric gravity , and heavily constrain others that predict dipolar energy loss , as we see in sections 2 and 5 . binary pulsars are probes of gr in a certain sector of the strong field : in the dynamical but quasi - linear sector , verifying that compact objects move as described by a perturbative , post - newtonian analysis to leading order . binary pulsars can be used to test gr in the strong field only in the sense that they probe non - linear stellar - structure effects , but they say very little to nothing about non - linear radiative effects . similarly , future electromagnetic observations of black - hole accretion disks may probe gr in another strong - field sector : the non - linear but fully stationary regime , verifying that black holes are described by the kerr metric . as of this writing , only gravitational waves will allow for tests of gr in the full strong - field regime , where gravity is both heavily non - linear and inherently dynamical . no experiments exist to date that validate einstein s theory of gr in the highly - dynamical , strong - field region . due to previous successes of gr however , as most scientists would agree , the role of science is to predict and verify and not to assume without proof . moreover , the incompleteness of gr in the quantum regime , together with the somewhat unsatisfactory requirement of the dark sector of cosmology ( including dark energy and dark matter ) , have prompted more than one physicist to consider deviations from gr more seriously . gravitational waves will soon allow us to verify einstein s theory in a regime previously inaccessible to us , and as such , these tests are invaluable . however , in many areas of physics gr is so ingrained that questioning its validity ( even in a regime where einstein s theory has not yet been validated ) is synonymous with heresy . dimensional arguments are usually employed to argue that any quantum gravitational correction will necessarily and unavoidably be unobservable with gravitational waves , as the former are expected at a ( planck ) scale that is inaccessible to gravitational - wave detectors . this rationalization is dangerous , as it introduces a theoretical bias in the analysis of new observations of the universe , thus reducing the potential for new discoveries . for example , if astrophysicists had followed such a rationalization when studying supernova data , they would not have discovered that the universe is expanding . dimensional arguments suggest that the cosmological constant is over 100 orders of magnitude larger than the value required to agree with observations . when observing the universe for the first time in a completely new way , it seems more conservative to remain agnostic about what is expected and what is not , thus allowing the data itself to guide our efforts toward theoretically understanding the gravitational interaction . when testing gr , one considers einstein s theory as a null hypothesis and searches for generic deviations . on the other hand , when testing alternative theories one starts from a particular modified gravity model , develops its equations and solutions and then predicts certain observables that then might or might not agree with experiment . similarly , one may define a bottom - up approach versus a top - down approach . in the former , one starts from some observables in an attempt to discover fundamental symmetries that may lead to a more complete theory , as was done when constructing the standard model of elementary particles . on the other hand , a top - down approach starts from some fundamental theory and then derives its consequence . both approaches possess strengths and weaknesses . in the top - down approach one has complete control over the theory under study , being able to write down the full equations of motion , answer questions about well - posedness and stability of solutions , and but , as we see in section 2 , carrying out such an approach can be quixotic within any one model . what is worse , the lack of a complete and compelling alternative to gr makes choosing a particular modified theory difficult . given this , one might wish to attempt a bottom - up approach , where one considers a set of principles one wishes to test without explicit mention of any particular theory . one usually starts by assuming gr as a null - hypothesis and then considers deformations away from gr . the hope is that experiments will be sensitive to such deformations , thus either constraining the size of the deformations or pointing toward a possible inconsistency . but if experiments do confirm a gr deviation , a bottom - up approach fails at providing a given particular action from which to derive such a deformation . in fact , there can be several actions that lead to similar deformations , all of which can be consistent with the data within its experimental uncertainties . nonetheless , both approaches are complementary . the bottom - up approach draws inspiration from particular examples carried out in the top - down approach . given a plausible measured deviation from gr within a bottom - up approach , one will still need to understand what plausible top - down theories can lead to such deviations . from this standpoint , then , both approaches are intrinsically intertwined and worth pursuing . perhaps one of the most important differences is the spacetime regime gravitational waves sample . indeed , as already mentioned , gravitational waves have access to the most extreme gravitational environments in nature . moreover , gravitational waves travel essentially unimpeded from their source to earth , and thus , they do not suffer from issues associated with obscuration . gravitational waves also exist in the absence of luminous matter , thus allowing us to observe electromagnetically dark objects , such as black - hole inspirals . this last point is particularly important as gravitational waves from inspiral black - hole binaries are one of the cleanest astrophysical systems in nature . in the last stages of inspiral , when such gravitational waves would be detectable by ground - based interferometers , the evolution of a blackhole binary is essentially unaffected by any other matter or electromagnetic fields present in the system . as such , one does not need to deal with uncertainties associated with astrophysical matter . unlike other tests of gr , such as those attempted with accretion - disk observations , black - hole binary gravitational - wave tests may well be the cleanest probes of einstein s theory . of course , what is an advantage here , can also be a huge disadvantage in another context . gravitational waves from compact binaries are intrinsically transient ( they turn on for a certain amount of time and then shut off ) . this is unlike binary pulsar systems , for which astrophysicists have already collected tens of years of data . moreover , gravitational wave tests rely on specific detections that can not be anticipated beforehand . this is in contrast to earth - based laboratory experiments , where one has complete control over the experimental setup . finally , the intrinsic weakness of gravitational waves makes detection a very difficult task that requires complex data - analysis algorithms to extract signals from the noise . as such , gravitational - wave tests are limited by the signal - to - noise ratio and affected by systematics associated with the modeling of the waves , issues that are not as important in other loud astrophysical systems . this review article focuses only on ground - based detectors , by which we mean both gravitational - wave interferometers , such as the laser interferometer gravitational observatory ( ligo ) [ 3 , 2 , 217 ] , virgo [ 5 , 6 ] and the einstein telescope ( et ) [ 361 , 377 ] , as well as pulsar - timing arrays ( for a recent review of gravitational - wave tests of gr with space - based detectors , see [ 183 , 446 ] ) . ground - based detectors have the limitation of being contaminated by man - made and nature - made noise , such as ground and air traffic , logging , earthquakes , ocean tides and waves , which are clearly absent in space - based detectors . ground - based detectors , however , have the clear benefit that they can be continuously upgraded and repaired in case of malfunction , which is obviously not possible with space - based detectors . as far as tests of gr are concerned , there is a drastic difference in space - based and ground - based detectors : the gravitational - wave frequencies these detectors are sensitive to . for various reasons that we will not go into , space - based interferometers are likely to have million kilometer long arms , and thus , be sensitive in the milli - hz band . on the other hand , ground - based interferometers are bound to the surface and curvature of the earth , and thus , they have kilometer - long arms and are sensitive in the deca- and hecta - hz band . different types of interferometers are then sensitive to different types of gravitational - wave sources . for example , when considering binary coalescences , ground - based interferometers are sensitive to late inspirals and mergers of neutron stars and stellar - mass black holes , while space - based detectors will be sensitive to supermassive black - hole binaries with masses around 10 m. the impact of a different population of sources in tests of gr depends on the particular modified gravity theory considered . when studying quadratic gravity theories , as we see in section 2 , the einstein hilbert action is modified by introducing higher - order curvature operators , which are naturally suppressed by powers of the inverse of the radius of curvature . thus , space - based detectors will not be ideal at constraining these theories , as the radius of curvature of supermassive black holes is much larger than that of stellar - mass black holes at merger . moreover , space - based detectors will not be sensitive to neutron - star - binary coalescences ; they are sensitive to supermassive black - hole / neutron - star coalescences , where the radius of curvature of the system is controlled by the supermassive black hole . on the other hand , space - based detectors are unique in their potential to probe the spacetime geometry of supermassive black holes through gravitational waves emitted during extreme - massratio inspirals . these inspirals consist of a stellar - mass compact object in a generic decaying orbit around a supermassive black hole . such inspirals produce millions of cycles of gravitational waves in the sensitivity band of space - based detectors ( in fact , they can easily out - live the observation time ! ) . therefore , even small changes to the radiation - reaction force , or to the background geometry , can lead to noticeable effects in the waveform observable and thus strong tests of gr , albeit constrained to the radius of curvature of the supermassive black hole . for recent work on such systems and tests , see [ 23 , 370 , 371 , 263 , 196 , 50 , 289 , 182 , 390 , 471 , 31 , 297 , 184 , 116 , 93 , 183 ] . space - based detectors also have the advantage of range , which is particularly important when considering theories where gravitons do not travel at light speed . space - based detectors have a horizon distance much larger than ground - based detectors ; the former can see black - hole mergers to redshifts of order 10 if there are any at such early times in the universe , while the latter are confined to events within redshift 1 . gravitational waves emitted from distant regions in spacetime need a longer time to propagate from the source to the detectors . thus , theories that modify the propagation of gravitational waves will be best constrained by space - based type systems . of course , such theories are also likely to modify the generation of gravitational waves , which ground - based detectors should also be sensitive to . ground - based detectors , as we see in section 3 , can not separate between the two possible scalar modes ( the longitudinal and the breathing modes ) of metric theories of gravity , due to an intrinsic degeneracy in the response functions . space - based detectors in principle also possess this degeneracy , but they may be able to break it through doppler modulation if the interferometer orbits the sun . pulsar - timing arrays , on the other hand , lack this degeneracy altogether , and thus , they can in principle constrain the existence of both modes independently . pulsar - timing arrays differ from interferometers in their potential to test gr mostly by the frequency space they are most sensitive to . the latter can observe the late inspiral and merger of compact binaries , while the former is restricted to the very early inspiral . this is why pulsar timing arrays do not need very accurate waveform templates that account for the highly - dynamical and non - linear nature of gravity to detect gravitational waves ; leading - order quadrupole waveforms are sufficient . in turn , this implies that pulsar timing arrays can not constrain theories that only deviate significantly from gr in the late inspiral , while they are exceptionally well - suited for constraining low - frequency deviations . therefore , we see a complementarity emerging : different detectors can test gr in different complementary regimes : ground - based detectors are best at constraining higher - curvature type modified theories that deviate from gr the most in the late inspiral and merger phase.space-based detectors are best at constraining modified graviton dispersion relations and the geometry of supermassive compact objects.pulsar-timing arrays are best at independently constraining the existence of both scalar modes and any deviation from gr that dominates at low orbital frequencies . through the simultaneous implementation of all these tests , gr can be put on a much firmer footing in all phases of the strong - field regime . ground - based detectors are best at constraining higher - curvature type modified theories that deviate from gr the most in the late inspiral and merger phase . space - based detectors are best at constraining modified graviton dispersion relations and the geometry of supermassive compact objects . pulsar - timing arrays are best at independently constraining the existence of both scalar modes and any deviation from gr that dominates at low orbital frequencies . we mainly follow the notation of , where greek indices stand for spacetime coordinates and spatial indices in the middle of the alphabet ( i , j , k , ) for spatial indices . parenthesis and square brackets in index lists stand for symmetrization and antisymmetrization respectively , e.g. , a( ) = ( a + a)/2 and a[ ] = ( a a)/2 . partial derivatives with respect to spacetime and spatial coordinates are denoted a = a, and ia = a , i respectively . = , and the curved spacetime dalembertian a = a. the determinant of the metric g is g , r is the riemann tensor , r is the ricci tensor , r is the ricci scalar and g is the einstein tensor . the levi - civita tensor and symbol are and respectively , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \varepsilon ^{0123 } } = + 1$\end{document } in an orthonormal , positively - oriented frame . we use geometric units ( g = c = 1 ) and the einstein summation convention is implied . we will mostly be concerned with metric theories , where gravitational radiation is only defined much farther than a gravitational - wave wavelength from the source . in this far or radiation zone , the metric tensor can be decomposed as 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } = { \eta _ { \mu \nu } } + { h_{\mu \nu}},$$\end{document } with the minkowski metric and h the metric perturbation . if the theory considered has additional fields , these can also be decomposed in the far zone as 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi = { \phi _ 0 } + \psi , $ $ \end{document } with 0 the background value of the field and a perturbation . with such a decomposition , the field equations for the metric will usually be wave equations for the metric perturbation and for the field perturbation , in a suitable gauge . in this section , we discuss the many possible alternative theories that have been studied so far in the context of gravitational - wave tests . we begin with a description of the theoretically desirable properties that such theories must have . we then proceed with a review of the theories so far explored as far as gravitational waves are concerned . we will leave out the description of many theories in this chapter , especially those which currently lack a gravitational - wave analysis . we will conclude with a brief description of unexplored theories as possible avenues for future research . the space of possible theories is infinite , and thus , one is tempted to reduce it by considering a subspace that satisfies a certain number of properties . although the number and details of such properties depend on the theorist s taste , there is at least one fundamental property that all scientists would agree on : precision tests . the theory must produce predictions that pass all solar system , binary pulsar , cosmological and experimental tests that have been carried out so far . the theory must produce predictions that pass all solar system , binary pulsar , cosmological and experimental tests that have been carried out so far . there must exist some limit , continuous or discontinuous , such as the weak - field one , in which the predictions of the theory are consistent with those of gr within experimental precision.existence of known solutions . the theory must admit solutions that correspond to observed phenomena , including but not limited to ( nearly ) flat spacetime , ( nearly ) newtonian stars , and cosmological solutions.stability of solutions . the special solutions described in property ( 1.b ) must be stable to small perturbations on timescales smaller than the age of the universe . for example , perturbations to ( nearly ) newtonian stars , such as impact by asteroids , should not render such solutions unstable . of course , these properties are not all necessarily independent , as the existence of a weak - field limit usually also implies the existence of known solutions . on the other hand , the mere existence of solutions there must exist some limit , continuous or discontinuous , such as the weak - field one , in which the predictions of the theory are consistent with those of gr within experimental precision . the theory must admit solutions that correspond to observed phenomena , including but not limited to ( nearly ) flat spacetime , ( nearly ) newtonian stars , and cosmological solutions . the special solutions described in property ( 1.b ) must be stable to small perturbations on timescales smaller than the age of the universe . for example , perturbations to ( nearly ) newtonian stars , such as impact by asteroids , should not render such solutions unstable . in addition to these fundamental requirements , one might also wish to require that any new modified gravity theory possesses certain theoretical properties . these properties will vary depending on the theorist , but the two most common ones are listed below : 2.well-motivated from fundamental physics . there must be some fundamental theory or principle from which the modified theory ( effective or not ) derives . this fundamental theory would solve some fundamental problem in physics , such as late - time acceleration or the incompatibility between quantum mechanics and gr.3.well-posed initial value formulation . a wide class of freely specifiable initial data must exist , such that there is a uniquely determined solution to the modified field equations that depends continuously on this data . the second property goes without saying at some level , as one expects modified - gravity - theory constructions to be motivated from some ( perhaps yet incomplete ) quantum - gravitational description of nature . as for the third property , the continuity requirement is necessary because otherwise the theory would lose predictive power , given that initial conditions can only be measured to a finite accuracy . moreover , small changes in the initial data should not lead to solutions outside the causal future of the data ; that is , causality must be preserved . there must be some fundamental theory or principle from which the modified theory ( effective or not ) derives . this fundamental theory would solve some fundamental problem in physics , such as late - time acceleration or the incompatibility between quantum mechanics and gr . freely specifiable initial data must exist , such that there is a uniquely determined solution to the modified field equations that depends continuously on this data . one might be concerned that property ( 2 ) automatically implies that any predicted deviation to astrophysical observables will be too small to be detectable . any quantum gravitational correction to the action will naturally introduce at least one new scale , and this , by dimensional analysis , must be the planck scale . since this scale is usually assumed to be larger than 1 tev in natural units ( or 10 m in geometric units ) , gravitational - wave observations will never be able to observe quantum - gravitational modifications ( see , e.g. , for a similar argument ) . although this might be true , in our view such arguments can be extremely dangerous , since they induce a certain theoretical bias in the search for new phenomena . for example , let us consider the supernova observations of the late - time expansion of the universe that led to the discovery of the cosmological constant . the above argument certainly fails for the cosmological constant , which on dimensional arguments is over 100 orders of magnitude too small . if the supernova teams had respected this argument , they would not have searched for a cosmological constant in their data . today , we try to explain our way out of the failure of such dimensional arguments by claiming that there must be some exquisite cancellation that renders the cosmological constant small ; but this , of course , came only after the constant had been measured . one is not trying to argue here that cancellations of this type are common and that quantum gravitational modifications are necessarily expected in gravitational - wave observations . rather , we are arguing that one should remain agnostic about what is expected and what is not , and allow oneself to be surprised without suppressing the potential for new discoveries that will accompany the new era of gravitational - wave astrophysics . one last property that we wish to consider for the purposes of this review is : 4.strong field inconsistency . the theory must lead to observable deviations from gr in the strong - field regime . many modified gravity models have been proposed that pose infrared or cosmological modifications to gr , aimed at explaining certain astrophysical or cosmological observables , like the late expansion of the universe . such modified models usually reduce to gr in the strong - field regime , for example via a vainshtein - like mechanism [ 413 , 140 , 45 ] in a static spherically - symmetric context . extending this mechanism to highly - dynamical strong - field scenarios gravitational - wave tests of gr , however , are concerned with modified theories that predict deviations in the strong - field , precisely where cosmological modified models do not . clearly , property ( 4 ) is not necessary for a theory to be a valid description of nature . this is because a theory might be identical to gr in the weak and strong fields , yet different at the planck scale , where it would be unified with quantum mechanics . property ( 4 ) is a desirable feature if one is to test this theory with gravitational wave observations . strong field inconsistency . the theory must lead to observable deviations from gr in the strong - field regime . many modified gravity models have been proposed that pose infrared or cosmological modifications to gr , aimed at explaining certain astrophysical or cosmological observables , like the late expansion of the universe . such modified models usually reduce to gr in the strong - field regime , for example via a vainshtein - like mechanism [ 413 , 140 , 45 ] in a static spherically - symmetric context . extending this mechanism to highly - dynamical strong - field scenarios gravitational - wave tests of gr , however , are concerned with modified theories that predict deviations in the strong - field , precisely where cosmological modified models do not . clearly , property ( 4 ) is not necessary for a theory to be a valid description of nature . this is because a theory might be identical to gr in the weak and strong fields , yet different at the planck scale , where it would be unified with quantum mechanics . however , property ( 4 ) is a desirable feature if one is to test this theory with gravitational wave observations . property ( 3 ) not only requires the existence of an initial value formulation , but also that it be well posed , which is not necessarily guaranteed . for example , the cauchy - kowalewski theorem states that a system of n partial differential equations for n unknown functions i of the form i , tt = fi(x ; j , ; j , ti ; j , ik ) , with fi analytic functions has an initial value formulation ( see , e.g. , ) . however , this theorem does not guarantee continuity or the causal conditions described above . for this , one has to rely on more general energy arguments , for example constructing a suitable energy measure that obeys the dominant energy condition and using it to show well - posedness ( see , e.g. , [ 225 , 425 ] ) . one can show that second - order , hyperbolic partial differential equations , i.e. , equations of the form 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla ^\mu}{\nabla _ _ \mu}\phi + b\phi + c = 0,$$\end{document } where a is an arbitrary vector field and ( b , c ) are smooth functions , have a well - posed initial value formulation . moreover , the leray theorem proves that any quasilinear , diagonal , second - order hyperbolic system also has a well - posed initial value formulation . proving the well - posedness of an initial - value formulation for systems of higher - than - second - order , partial differential equations is much more difficult . in fact , to our knowledge , no general theorems exist of the type described above that apply to third , fourth or higher - order , partial , nonlinear and coupled differential equations . usually , one resorts to the ostrogradski theorem to rule out ( or at the very least cast serious doubt on ) theories that lead to such higher - order field equations . ostrogradski s theorem states that lagrangians that contain terms with higher - than - first - time derivatives possess a linear instability in the hamiltonian ( see , e.g. , for a nice review).2 as an example , consider the lagrangian density 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal l } = { m \over 2}{\dot q^2 } - { { m{\omega ^2 } } \over 2}{q^2 } - { { gm } \over { 2{\omega ^2}}}{\ddot q^2},$$\end{document } whose equations of motion , 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ddot q + { \omega ^2}q = - { g \over { { \omega ^2}}}{\overset{\ldots.}{q}},$$\end{document } obviously contain higher derivatives . the exact solution to this differential equation is 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q = { a_1}\cos { k_1}t + { b_1}\sin { k_1}t + { a_2}\cos { k_2}t + { b_2}\sin { k_2}t,$$\end{document } where ( ai , bi ) are constants and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k_{1,2}^2/{\omega ^2 } = ( 1 \mp \sqrt { 1 - 4g)/(2g)}$\end{document}. the on - shell hamiltonian is then 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = { m \over 2 ) - { m \over 2}\sqrt { 1 - 4 g } k_2 ^ 2(a_2 ^ 2 + b_2 ^ 2),$$\end{document } from which it is clear that mode 1 carries positive energy , while mode 2 carries negative energy and forces the hamiltonian to be unbounded from below . if interactions are present , then an empty state would instantaneously decay into a collection of positive and negative energy particles , which can not describe the universe we live in . ( 6 ) describes an effective theory , i.e. , a theory that is a truncation of a more general or complete theory . let us reconsider the particular example above , assuming now that the coupling constant g is an effective theory parameter and eq . ( 6 ) is only valid to linear order in g. one approach is to search for perturbative solutions of the form qpert = x0 + gx1 + , which leads to the system of differential equations 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\ddot x_n } + { \omega ^2}{x_n } = - { 1 \over { { \omega ^2}}}{\overset{\ldots.}{x}}_{n - 1},$$\end{document } with x1 = 0 . solving this set of n differential equations and resumming , one finds 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_{{\rm{pert } } } } = { a_1}\cos { k_1}t + { b_1}\sin { k_1}t.$$\end{document } notice that qpert contains only the positive ( well - behaved ) energy solution of eq . ( 8) , i.e. , perturbation theory acts to retain only the well - behaved , stable solution of the full theory in the g 0 limit . one can also think of the perturbative theory as the full theory with additional constraints , i.e. , the removal of unstable modes , which is why such an analysis is sometimes called perturbative constraints [ 117 , 118 , 466 ] . another way to approach effective field theories that lead to equations of motion with higherorder derivatives is to apply the method of order reduction . in this method , one substitutes the low - order derivatives of the field equations into the high - order derivative part , thus rendering the resulting new theory usually well posed . one can think of this as a series resummation , where one changes the non - linear behavior of a function by adding uncontrolled , higher - order terms . to lowest order in g , the equation of motion is that of a simple harmonic oscillator , 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ddot q + { \omega ^2}q = { \mathcal o}(g),$$\end{document } which is obviously well posed . doing so , one obtains the order - reduced equation of motion 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ddot q + { \omega ^2}q = g\ddot q + { \mathcal o}({g^2}),$$\end{document } which now clearly has no high - order derivatives and is well posed , provided g 1 . the solution to this order - reduced differential equation is qpert once more , but with k1 linearized in g 1 . therefore , the solutions obtained with a perturbative decomposition and with the order - reduced equation of motion are the same to linear order in g. of course , since an effective field theory is only defined to a certain order in its perturbative parameter , both treatments are equally valid , with the unstable mode effectively removed in both cases . however , such a perturbative analysis can say nothing about the well - posedness of the full theory from which the effective theory derives , or of the effective theory if treated as an exact one ( i.e. , not as a perturbative expansion ) . in fact , a well - posed full theory may have both stable and unstable solutions . the arguments presented above only discuss the stability of solutions in an effective theory , and thus , they are self - consistent only within their perturbative scheme . a full theory may have non - perturbative instabilities , but these can only be studied once one has a full ( non - truncated in g ) theory , from which eq . ( 6 ) derives as a truncated expansion . lacking a full quantum theory of nature , quantum gravitational models are usually studied in a truncated low - energy expansion , where the leading - order piece is gr and higher - order pieces are multiplied by a small coupling constant . one can perturbatively explore the well - behaved sector of the truncated theory about solutions to the leading - order theory . however , such an analysis is incapable of answering questions about well - posedness or non - linear stability of the full theory . in this subsection we briefly describe the theories that have so far been studied in some depth as far as gravitational waves are concerned . in particular , we focus only on those theories that have been sufficiently studied so that predictions of the expected gravitational waveforms ( the observables of gravitational - wave detectors ) have been obtained for at least a typical source , such as the quasi - circular inspiral of a compact binary . scalar - tensor theories in the einstein frame [ 82 , 129 , 166 , 165 , 181 , 197 ] are defined by the action ( where we will restore newton s gravitational constant g in this section ) 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{st}}}^{({\rm{e } } ) } = { 1 \over { 16\pi g}}\int { { d^4}x\sqrt { - g } [ r - 2{g^{\mu \nu}}({\partial _ \nu}\varphi ) - v(\varphi ) ] + { s_{{\rm{mat}}}}[{\psi _ { { \rm{mat}}}},{a^2}(\varphi){g_{\mu \nu}}],}$$\end{document } where is a scalar field , a( ) is a coupling function , v( ) is a potential function , mat represents matter degrees of freedom and g is newton s constant in the einstein frame . for more details on this theory , we refer the interested reader to the reviews [ 438 , 435 ] . of course , one can consider more complicated scalar - tensor theories , for example by including multiple scalar fields , but we will ignore such generalizations here . the einstein frame is not the frame where the metric governs clocks and rods , and thus , it is convenient to recast the theory in the jordan frame through the conformal transformation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde g_{\mu \nu } } = { a^2}(\varphi){\tilde g_{\mu \nu}}$\end{document } : 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s_{{\rm{st}}}^{({\rm{j } } ) } = { 1 \over { 16\pi g}}\int { { d^4}x\sqrt { - \tilde g } \left [ { \phi \tilde r - { { \omega ( \phi ) } \over \phi}{{\tilde g}^{\mu \nu}}({\partial _ \mu}\phi)({\partial _ \nu}\phi ) - { \phi ^2}v } \right ] + { s_{{\rm{mat}}}}[{\psi _ { { \rm{mat}}}},{{\tilde g}_{\mu \nu}}],}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde g_{\mu \nu}}$\end{document } is the physical metric , the new scalar field is defined via = a , the coupling field is ( ) = ( 3)/2 and = a,/a . when cast in the jordan frame , it is clear that scalar - tensor theories are metric theories ( see for a definition ) , since the matter sector depends only on matter degrees of freedom and the physical metric ( without a direct coupling of the scalar field ) . when the coupling ( ) = bd is constant , then eq . ( 15 ) reduces to the massless version of jordan - fierz - brans - dicke theory . the modified field equations in the einstein frame are 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \square\varphi = { 1 \over 4}{{dv } \over { d\varphi } } - 4\pi g{{\delta { s_{{\rm{mat } } } } } \over { \delta \varphi}},}\\ { { g_{\mu \nu } } = 8\pi g\left({t_{\mu \nu}^{{\rm{mat } } } + t_{\mu \nu}^{(\varphi ) } } \right),}\\ \end{array}$$\end{document } where 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{\mu \nu}^{(\varphi ) } = { 1 \over { 4\pi}}\left [ { { \varphi _ { , \mu}}{\varphi _ { , \nu } } - { 1 \over 2}{g_{\mu \nu}}{\varphi _ { , \delta}}{\varphi ^{,\delta } } - { 1 \over 4}{g_{\mu \nu}}v(\varphi ) } \right]$$\end{document } is a stress - energy tensor for the scalar field . the matter stress - energy tensor is not constructed from the einstein - frame metric alone , but by the combination a()g. in the jordan frame and neglecting the potential , the modified field equations are 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \tilde \square\phi = { 1 \over { 3 + 2\omega ( \phi)}}\left({8\pi { t^{{\rm{mat } } } } - { { d\omega } \over { d\phi}}{{\tilde g}^{\mu \nu}}{\phi _ { , \mu}}{\phi _ { , \nu } } } \right),\quad \quad \quad \quad \quad \quad \quad \quad \quad}\\ { { { \tilde g}_{\mu \nu } } = { { 8\pi g } \over \phi}t_{\mu \nu}^{{\rm{mat } } } + { \omega \over { { \phi ^2}}}\left({{\phi _ { , \mu}}{\phi _ { , \nu } } - { 1 \over 2}{{\tilde g}_{\mu \nu}}{{\tilde g}^{\sigma \rho}}{\phi _ { , \sigma}}{\phi _ { , \rho } } } \right ) + { 1 \over \phi}({\phi _ { , \mu \nu } } - { { \tilde g}_{\mu \nu}}\tilde \square\phi),}\\ \end{array}$$\end{document } where t is the trace of the matter stress - energy tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_{\mu \nu}^{{\rm{mat}}}$\end{document } constructed from the physical metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde g_{\mu \nu}}$\end{document}. the form of the modified field equations in jordan frame suggest that in the weak - field limit one may consider scalar - tensor theories as modifying newton s gravitational constant via g g( ) = g/. using the decompositions of eqs . ( 3)(4 ) , the field equations of massless jordan - fierz - brans - dicke theory can be linearized in the jordan frame to find ( see , e.g. , ) 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\square_\eta}{\theta ^{\mu \nu } } = - 16\pi { \tau ^{\mu \nu}},\quad { \square_\eta}\psi = - 16\pi s,$$\end{document } where is the dalembertian operator of flat spacetime , we have defined a new metric perturbation 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\theta ^{\mu \nu } } = { h^{\mu \nu } } - { 1 \over 2}{\eta ^{\mu \nu}}h - { \psi \over { { \phi _ 0}}}{\eta ^{\mu \nu}},$$\end{document } i.e. , the metric perturbation in the einstein frame , with h the trace of the metric perturbation and 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tau ^{\mu \nu } } = \phi _ 0^{- 1}t_{{\rm{mat}}}^{\mu \nu } + { t^{\mu \nu}},$$\end{document } 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = - { 1 \over { 6 + 4{\omega _ { bd}}}}\left({{t^{{\rm{mat } } } } - 3\phi { { \partial { t^{{\rm{mat } } } } } \over { \partial \phi } } } \right)\left({1 - { \theta \over 2 } - { \psi \over { { \phi _ 0 } } } } \right ) - { 1 \over { 16\pi}}\left({{\psi _ { , \mu \nu}}{\theta ^{\mu \nu } } + { 1 \over { { \phi _ 0}}}{\phi _ { , \mu}}{\psi ^{,\mu } } } \right),$$\end{document } with cubic remainders in either the metric perturbation or the scalar perturbation . the quantity t/ arises in an effective point - particle theory , where the matter action is a functional of both the jordan - frame metric and the scalar field . the quantity t is a function of quadratic or higher order in or . these equations can now be solved given a particular physical system , as done for quasi - circular binaries in [ 441 , 374 , 336 ] . given the above evolution equations , jordan - fierz - brans - dicke theory possesses a scalar ( spin-0 ) mode , in addition to the two transverse - traceless ( spin-2 ) modes of gr , i.e. , jordan - fierz - brans - dicke theory is of type n3 in the e(2 ) classification [ 161 , 438 ] . let us now discuss whether scalar - tensor theories satisfy the properties discussed in section 2.1 . massless jordan - fierz - brans - dicke theory agrees with all known experimental tests provided bd > 4 10 , a bound imposed by the tracking of the cassini spacecraft through observations of the shapiro time delay . massive jordan - fierz - brans - dicke theory has been recently constrained to bd > 4 10 and ms < 2.5 10 ev , with ms the mass of the scalar field [ 348 , 20 ] . of course , these bounds are not independent , as when ms 0 one recovers the standard massless constraint , while when ms , bd can not be bounded as the scalar becomes non - dynamical . observations of the nordtvedt effect with lunar laser ranging observations , as well as observations of the orbital period derivative of white - dwarf / neutron - star binaries , yield similar constraints [ 131 , 132 , 20 , 177 ] . neglecting any homogeneous , cosmological solutions to the scalar - field evolution equation , it is clear that in the limit one recovers gr , i.e. , scalar - tensor theories have a continuous limit to einstein s theory , but see for caveats for certain spacetimes . moreover , [ 375 , 278 , 425 ] have verified that scalar - tensor theories with minimal or non - minimal coupling in the jordan frame can be cast in a strongly - hyperbolic form , and thus , they possess a well - posed initial - value formulation . therefore , scalar - tensor theories possess both properties ( 1 ) and ( 3 ) . scalar - tensor theories also possess property ( 2 ) , since they can be derived from the low - energy limit of certain string theories . the integration of string quantum fluctuations leads to a higher - dimensional string theoretical action that reduces locally to a field theory similar to a scalar - tensor one [ 189 , 176 ] , the mapping being = e , with one of the string moduli fields [ 133 , 134 ] . moreover , scalar - tensor theories can be mapped to f(r ) theories , where one replaces the ricci scalar by some functional of r. in particular , one can show that f(r ) theories are equivalent to brans - dicke theory with bd = 0 , via the mapping = df(r)/dr and v( ) = r df ( r)/dr f(r ) [ 104 , 396 ] . for a recent review on this topic , see . stellar configurations are modified from their gr profile [ 441 , 131 , 214 , 215 , 410 , 132 , 394 , 139 , 393 , 235 ] , while black holes are not , provided one neglects homogeneous , cosmological solutions to the scalar field evolution equation . indeed , hawking [ 224 , 159 , 222 , 98 , 244 , 363 ] has proven that brans - dicke black holes that are stationary and the endpoint of gravitational collapse are identical to those of gr . this proof has recently been extended to a general class of scalar - tensor models . that is , stationary black holes radiate any excess hair , i.e. , additional degrees of freedom , after gravitational collapse , a result sometimes referred to as the no - hair theorem for black holes in scalar - tensor theories . this result has recently been extended even further to allow for quasi - stationary scenarios in generic scalar - tensor theories through the study of extreme - mass - ratio inspirals ( small black hole in orbit around a much larger one ) , post - newtonian comparable - mass inspirals and numerical simulations of comparable - mass black - hole mergers [ 230 , 67 ] . damour and esposito - farse [ 129 , 130 ] proposed a different type of scalar - tensor theory , one that can be defined by the action in eq . ( 15 ) but with the conformal factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a(\varphi ) = { e^{\alpha \varphi + \beta { \varphi ^2}/2}}$\end{document } or the coupling function ( ) = 3/2 2g/( log ) , where and are constants . when = 0 one recovers standard brans - dicke theory . when 4 , non - perturbative effects that develop if the gravitational energy is large enough can force neutron stars to spontaneously acquire a non - trivial scalar field profile , to spontaneously scalarize . through this process , a neutron - star binary that initially had no scalar hair in its early inspiral would acquire it before merger , when the binding energy exceeded some threshold . binary pulsar observations have constrained this theory in the ( , ) space ; very roughly speaking > 4 and < 10 [ 131 , 132 , 177 ] as for property ( 4 ) , scalar tensor theories are not built with the aim of introducing strong - field corrections to gr.3 instead , they naturally lead to modifications of einstein s theory in the weak - field , modifications that dominate in scenarios with sufficiently weak gravitational interactions . although this might seem strange , it is natural if one considers , for example , one of the key modifications introduced by scalar - tensor theories : the emission of dipolar gravitational radiation . such dipolar emission dominates over the general relativistic quadrupolar emission for systems in the weak to intermediate field regime , such as in binary pulsars or in the very early inspiral of compact binaries . therefore , one would expect scalar - tensor theories to be best constrained by experiments or observations of weakly - gravitating systems , as it has recently been explicitly shown in . massive graviton theories are those in which the gravitational interaction is propagated by a massive gauge boson , i.e. , a graviton with mass mg 0 or compton wavelength g = h/(mgc ) < . einstein s theory predicts massless gravitons and thus gravitational propagation at light speed , but if this were not the case , then a certain delay would develop between electromagnetic and gravitational signals emitted simultaneously at the source . fierz and pauli were the first to write down an action for a free massive graviton , and ever since then , much work has gone into the construction of such models . for a detailed review , see , e.g. , . gravitational theories with massive gravitons are somewhat well - motivated from a fundamental physics perspective , and thus , one can say they possess property ( 2 ) . indeed , in loop quantum cosmology [ 42 , 77 ] , the cosmological extension to loop quantum gravity , the graviton dispersion relation acquires holonomy corrections during loop quantization that endow the graviton with a mass mg = (/c ) , with the barbero - immirzi parameter , the area operator , and and c the total and critical energy density respectively . in string - theory - inspired effective theories , such as dvali s compact , extra - dimensional theory , such massive modes also arise massive graviton modes also occur in many other modified gravity models . in rosen s bimetric theory , for example , photons and gravitons follow null geodesics of different metrics [ 438 , 435 ] . in visser s massive graviton theory , the graviton is given a mass at the level of the action through an effective perturbative description of gravity , at the cost of introducing a non - dynamical background metric , i.e. , a prior geometry . a recent re - incarnation of this model goes by the name of bigravity , where again two metric tensors are introduced [ 349 , 346 , 219 , 220 ] . in bekenstein s tensor - vector - scalar ( teves ) theory , the existence of a scalar and a vector field lead to subluminal gravitational - wave propagation . massive graviton theories have a theoretical issue , the van dam - veltman - zakharov ( vdvz ) discontinuity [ 418 , 475 ] , which is associated with property 1.a , i.e. , a gr limit . the problem is that certain predictions of massive graviton theories do not reduce to those of gr in the mg 0 limit . this can be understood qualitatively by studying how the 5 spin states of the graviton behave in this limit . another two become helicity states of a massless vector that decouples from the tensor perturbations in the mg 0 limit . however , the last state , the scalar mode , retains a finite coupling to the trace of the stress - energy tensor in this limit . therefore , massive graviton theories in the mg 0 limit do not reduce to gr , since the scalar mode does not decouple . however , the vdvz discontinuity can be evaded , for example , by carefully including non - linearities . vainshtein [ 413 , 269 , 140 , 45 ] showed that around any spherically - symmetric source of mass m , there exists a certain radius \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r < { r_v } \equiv { ( { r_s}\lambda _ g^4)^{1/5}}$\end{document } , with rs the schwarzschild radius , where linear theory can not be trusted . since rv as mg 0 , this implies that there is no radius at which the linear approximation ( and thus vdvz discontinuity ) can be trusted . of course , to determine then whether massive graviton theories have a continuous limit to gr , one must include non - linear corrections to the action ( see also an argument by ) , which are more difficult to uniquely predict from fundamental theory . recently , there has been much activity in the development of new , non - linear massive gravity theories [ 60 , 136 , 211 , 61 , 137 , 138 ] . lacking a particular action for massive graviton theories that modifies the strong - field regime and is free of non - linear and radiatively - induced ghosts , it is difficult to ascertain many of its properties , but this does not prevent us from considering certain phenomenological effects . if the graviton is truly massive , whatever the action may be , two main modifications to einstein s theory will be introduced : ( i)modification to newton s laws;(ii)modification to gravitational wave propagation . modifications of class ( i ) correspond to the replacement of the newtonian potential by a yukawa type potential ( in the non - radiative , near - zone of any body of mass m ) : v = ( m / r ) ( m / r ) exp ( r/g ) , where r is the distance to the body . tests of such a yukawa interaction have been proposed through observations of bound clusters , tidal interactions between galaxies and weak gravitational lensing , but such tests are model dependent . modification to newton s laws ; modification to gravitational wave propagation . modifications of class ( ii ) are in the form of a non - zero graviton mass that induces a modified gravitational - wave dispersion relation . such a modification to the dispersion relation was originally parameterized via 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{v_g^2 } \over { { c^2 } } } = 1 - { { m_g^2{c^4 } } \over { { e^2}}},$$\end{document } where g and mg are the speed and mass of the graviton , while e is its energy , usually associated to its frequency via the quantum mechanical relation e = hf . this modified dispersion relation is inspired by special relativity , a more general version of which , inspired by quantum gravitational theories , is 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{v_g^2 } \over { { c^2 } } } = 1 - { \lambda ^\alpha},$$\end{document } where is now a parameter that depends on the theory and represents deviations from light - speed propagation . for example , in rosen s bimetric theory , the graviton does not travel at the speed of light , but at some other speed partially determined by the prior geometry . in metric theories of gravity , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda = am_g^2{c^4}/{e^2}$\end{document } , where a is some amplitude that depends on the metric theory ( see discussion in ) . either modification to the dispersion relation has the net effect of slowing gravitons down , such that there is a difference in the time of arrival of photons and gravitons . moreover , such an energy - dependent dispersion relation would also affect the accumulated gravitational - wave phase observed by gravitational - wave detectors , as we discuss in section 5 . given these modifications to the dispersion relation , one would expect the generation of gravitational waves to also be greatly affected in such theories , but again , lacking a particular healthy action to consider , this topic remains today mostly unexplored . from the structure of the above phenomenological modifications , it is clear that gr can be recovered in the mg 0 limit , avoiding the vdvz issue altogether by construction . such phenomenological modifications have been constrained by several types of experiments and observations . using the modification to newton s third law and precise observations of the motion of the inner planets of the solar system together with kepler s third law , found a bound of g > 2.8 10 km . such a constraint is purely static , as it does not sample the radiative sector of the theory . dynamical constraints , however , do exist : through observations of the decay of the orbital period of binary pulsars , found a bound of g > 1.6 10 km;4 by investigating the stability of schwarzschild and kerr black holes , placed the constraint g > 2.4 10 km in fierz - pauli theory . new constraints that use gravitational waves have been proposed , including measuring a difference in time of arrival of electromagnetic and gravitational waves [ 126 , 266 ] , as well as direct observation of gravitational waves emitted by binary pulsars ( see section 5 ) . although massive gravity theories unavoidably lead to a modification to the graviton dispersion relation , the converse is not necessarily true . a modification of the dispersion relation is usually accompanied by a modification to either the lorentz group or its action in real or momentum space . such lorentz - violating effects are commonly found in quantum gravitational theories , including loop quantum gravity and string theory [ 107 , 403 ] , as well as other effective models [ 58 , 59 ] . in doubly - special relativity [ 26 , 300 , 27 , 28 ] , the graviton dispersion relation is modified at high energies by modifying the law of transformation of inertial observers . modified graviton dispersion relations have also been shown to arise in generic extra - dimensional models , in hoava - lifshitz theory [ 233 , 234 , 412 , 76 ] and in theories with non - commutative geometries [ 186 , 187 , 188 ] . none of these theories necessarily requires a massive graviton , but rather the modification to the dispersion relation is introduced due to lorentz - violating effects . one might be concerned that the mass of the graviton and subsequent modifications to the graviton dispersion relation should be suppressed by the planck scale . [ 111 , 110 ] have suggested that lorentz violations in perturbative quantum field theories could be dramatically enhanced when one regularizes and renormalizes them . this is because terms that vanish upon renormalization due to lorentz invariance do not vanish in lorentz - violating theories , thus leading to an enhancement . modified quadratic gravity is a family of models first discussed in the context of black holes and gravitational waves in [ 473 , 447 ] . the 4-dimensional action is given by 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { s \equiv \int { { d^4}x\sqrt { - g } \left\{{\kappa r + { \alpha _ 1}{f_1}(\vartheta){r^2 } + { \alpha _ 2}{f_2}(\vartheta){r_{\mu \nu}}{r^{\mu \nu } } + { \alpha _ 3}{f_3}(\vartheta){r_{\mu \nu \delta \sigma}}{r^{\mu \nu \delta \sigma } } } \right.}}\\ { \left . \mu}\vartheta { \nabla ^\mu}\vartheta + 2v(\vartheta ) ] + { { \mathcal l}_{{\rm{mat } } } } } \right\}.\quad \quad \quad \quad \quad \quad \quad \quad}\\ \end{array}$$\end{document } the quantity * r = ( 1/2) r is the dual to the riemann tensor . the quantity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{l}_{\mathrm{mat}}$\end{document } is the external matter lagrangian , while fi( ) are functionals of the field , with ( i, ) coupling constants and = ( 16g). clearly , the two terms second to last in eq . ( 25 ) represent a canonical kinetic energy term and a potential . at this stage , one might be tempted to set = 1 or the i = 1 via a rescaling of the scalar field functional , but we shall not do so here . ( 25 ) is well - motivated from fundamental theories , as it contains all possible quadratic , algebraic curvature scalars with running ( i.e. , non - constant ) couplings . the only restriction here is that all quadratic terms are assumed to couple to the same field , which need not be the case . for example , in string theory some terms might couple to the dilaton ( a scalar field ) , while others couple to the axion ( a pseudo scalar field ) . nevertheless , one can recover well - known and motivated modified gravity theories in simple cases . for example , dynamical chern - simons modified gravity is recovered when 4 = cs/4 and all other i = 0 . einstein - dilaton - gauss - bonnet gravity is obtained when 4 = 0 and ( 1 , 2 , 3 ) = ( 1 , 4 , 1)edgb.5 both theories unavoidably arise as low - energy expansions of heterotic string theory [ 203 , 204 , 12 , 89 ] . as such , modified quadratic gravity theories should be treated as a class of effective field theories . moreover , dynamical chern - simons gravity also arises in loop quantum gravity [ 43 , 366 ] when the barbero - immirzi parameter is promoted to a field in the presence of fermions [ 41 , 16 , 406 , 311 , 192 ] . one should make a clean and clear distinction between the theory defined by the action of eq . one could think of the subclass of f(r ) theories with f(r ) = r as the limit of modified quadratic gravity with only 1 = 0 and f1( ) = 1 . in that very special case , one can map quadratic gravity theories and f(r ) gravity to a scalar - tensor theory . another important distinction is that f(r ) theories are usually treated as exact , while the action presented above is to be interpreted as an effective theory truncated to quadratic order in the curvature in a low - energy expansion of a more fundamental theory . thus , when studying such an effective theory one should also order - reduce the field equations and treat all quantities that depend on perturbatively , the small - coupling approximation . one can show that such an order reduction removes any additional polarization modes in propagating metric perturbations [ 390 , 400 ] that naturally arise in f(r ) theories . in analogy to the treatment of the ostrogradski instability in section 2.1 , one would also expect that order - reduction would lead to a theory with a well - posed initial - value formulation . this family of theories is usually simplified by making the assumption that coupling functions fi( ) admit a taylor expansion : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_i}(\vartheta ) = { f_i}(0 ) + f_i^{{\prime}}(0)\vartheta + \mathcal{o}({\vartheta ^2})$\end{document } for small , where fi(0 ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f_i^{{\prime}}(0)$\end{document } are constants and is assumed to vanish at asymptotic spatial infinity . reabsorbing fi(0 ) into the coupling constants \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ i^{(0 ) } \equiv { \alpha _ i}{f_i}(0)$\end{document } into the constants \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f_i^{{\prime}}(0)$\end{document } , eq . ( 25 ) becomes s = sgr + s0 + s1 with 26a\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{gr } } } } \equiv \int { { d^4}x\sqrt { - g } \{\kappa r + { { \mathcal l}_{{\rm{mat}}}}\ } , } $ $ \end{document } 26b\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{\rm{0 } } } \equiv \int { { d^4}x\sqrt { - g } \left\{{\alpha _ 1^{(0)}{r^2 } + \alpha _ 2^{(0)}{r_{\mu \nu}}{r^{\mu \nu } } + \alpha _ 3^{(0)}{r_{\mu \nu \delta \sigma}}{r^{\mu \nu \delta \sigma } } } \right\},}$$\end{document } 26c\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s_{\rm{1 } } } \equiv \int { { d^4}x\sqrt { - g } \left\{{\alpha _ 1^{(1)}\vartheta { r^2 } + \alpha _ 2^{(1)}\vartheta { r_{\mu \nu}}{r^{\mu \nu } } + \alpha _ 3^{(1)}\vartheta { r_{\mu \nu \delta \sigma}}{r^{\mu \nu \delta \sigma } } } \right.}}\\ { \left . { + \alpha _ 4^{(1)}\vartheta { r_{\mu \nu \delta \sigma}}^{\ast}{r^{\mu \nu \delta \sigma } } - { \beta \over 2}[{\nabla _ \mu}\vartheta { \nabla ^\mu}\vartheta + 2v(\vartheta ) ] } \right\}.\quad \quad \quad}\\ \end{array}$$\end{document } here , sgr is the einstein - hilbert plus matter action , while s0 and s1 are corrections . the former is decoupled from , where the omitted term proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 4^{(0)}$\end{document } does not affect the classical field equations since it is topological , i.e. , it can be rewritten as the total 4-divergence of some 4-current . similarly , if the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ i^{(0)}$\end{document } were chosen to reconstruct the gauss - bonnet invariant , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\alpha _ 1^{(0)},\alpha _ 2^{(0)},\alpha _ 3^{(0 ) } ) = ( 1 , - 4,1){\alpha _ { { \rm{gb}}}}$\end{document } , then this combination would also be topological and not affect the classical field equations . on the other hand , s1 is a modification to gr with a direct ( non - minimal ) coupling to such that as the field goes to zero , the modified theory reduces to gr . another restriction one usually makes to simplify modified gravity theories is to neglect the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ i^{(0)}$\end{document } terms and only consider the s1 modification , the restricted modified quadratic gravity . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ i^{(0)}$\end{document } terms represent corrections that are non - dynamical . the term proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 1^{(0)}$\end{document } resembles a certain class of f(r ) theories . as such , it can be mapped to a scalar - tensor theory with a complicated potential , which has been heavily constrained by torsion - balance et - wash experiments to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 1^{(0 ) } < 2 \times { 10^{- 8}}{{\rm{m}}^2}$\end{document } [ 237 , 259 , 62 ] . moreover , these theories have a fixed coupling constant that does not run with energy or scale . in restricted modified gravity , the scalar field is effectively forcing the running of the coupling . then , let us concentrate on restricted modified quadratic gravity and drop the superscript in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ i^{(1)}$\end{document}. the modified field equations are 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { g_{\mu \nu } } + { { { \alpha _ 1}\vartheta } \over \kappa}{\mathcal h}_{\mu \nu}^{(0 ) } + { { { \alpha _ 2}\vartheta } \over \kappa}{\mathcal i}_{\mu \nu}^{(0 ) } + { { { \alpha _ 3}\vartheta } \over \kappa}{\mathcal j}_{\mu \nu}^{(0 ) } + { { { \alpha _ 1 } } \over \kappa}{\mathcal h}_{\mu \nu}^{(1 ) } + { { { \alpha _ 2 } } \over \kappa}{\mathcal i}_{\mu \nu}^{(1 ) } + { { { \alpha _ 3 } } \over \kappa}{\mathcal j}_{\mu \nu}^{(1 ) } + { { { \alpha _ 4 } } \over \kappa}{\mathcal k}_{\mu \nu}^{(1)}}\\ { = { 1 \over { 2\kappa}}\left({t_{\mu \nu}^{{\rm{mat } } } + t_{\mu \nu}^{(\vartheta ) } } \right),\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad}\\\end{array}$$\end{document } where we have defined 28a\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal h}_{\mu \nu}^{(0 ) } \equiv 2r{r_{\mu \nu } } - { 1 \over 2}{g_{\mu \nu}}{r^2 } - 2{\nabla _ { \mu \nu}}r + 2{g_{\mu \nu}}\square r,$$\end{document } 28b\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal i}_{\mu \nu}^{(0 ) } \equiv \square{r_{\mu \nu } } + 2{r_{\mu \delta \nu \sigma}}{r^{\delta \sigma } } - { 1 \over 2}{g_{\mu \nu}}{r^{\delta \sigma}}{r_{\delta \sigma } } + { 1 \over 2}{g_{\mu \nu}}\square r - { \nabla _ { \mu \nu}}r,$$\end{document } 28c\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal j}_{\mu \nu}^{(0 ) } \equiv 8{r^{\delta \sigma}}{r_{\mu \delta \nu \sigma } } - 2{g_{\mu \nu}}{r^{\delta \sigma}}{r_{\delta \sigma } } + 4\square { r_{\mu \nu } } - 2r\;{r_{\mu \nu } } + { 1 \over 2}{g_{\mu \nu}}{r^2 } - 2{\nabla _ { \mu \nu}}r,$$\end{document } 28d\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal h}_{\mu \nu}^{(1 ) } \equiv - 4({\nabla _ { ( \mu}}\vartheta){\nabla _ { \nu)}}r - 2r{\nabla _ { \mu \nu}}\vartheta + { g_{\mu \nu}}[2r\square\vartheta + 4({\nabla ^\delta}\vartheta){\nabla _ \delta}r],}$$\end{document } 28e\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \mathcal j}_{\mu \nu}^{(1 ) } \equiv - ( { \nabla _ { ( \mu}}\vartheta){\nabla _ { \nu)}}r - 2{\nabla ^\delta}\vartheta { \nabla _ { ( \mu}}{r_{\nu)\delta } } + 2{\nabla ^\delta}\vartheta { \nabla _ \delta}{r_{\mu \nu } } + { r_{\mu \nu}}\square\vartheta \,\quad } \\ { - 2{r_{\delta ( \mu}}{\nabla ^\delta}{\nabla _ { \nu)}}\vartheta + { g_{\mu \nu}}\left({{\nabla ^\delta}\vartheta { \nabla _ \delta}r + { r^{\delta \sigma}}{\nabla _ { \delta \sigma}}\vartheta } \right),\quad \,\,\ , } \\ \end{array}$$\end{document } 28f\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal j}_{\mu \nu}^{(1 ) } \equiv - 8({\nabla ^\delta}\vartheta)\left({{\nabla _ { ( \mu}}{r_{\nu)\delta } } - { \nabla _ \delta}{r_{\mu \nu } } } \right ) + 4{r_{\mu \delta \nu \sigma}}{\nabla ^{\delta \sigma}}\vartheta , $ $ \end{document } 28g\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal k}_{\mu \nu}^{(1 ) } \equiv - 4({\nabla ^\delta}\vartheta){\epsilon _ { \delta \sigma \chi ( \mu}}{\nabla ^\chi}r_{\nu)}^\sigma + 4{({\nabla _ { \delta \sigma}}\vartheta)^{\ast}}{r_{(\mu}}{\delta _ { \nu)}}\sigma .$$\end{document } the stress - energy tensor is 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{\mu \nu}^{(\vartheta ) } = \beta \left [ { ( { \nabla _ \mu}\vartheta)({\nabla _ \nu}\vartheta ) - { 1 \over 2}{g_{\mu \nu}}\left({{\nabla _ \delta}\vartheta { \nabla ^\delta}\vartheta - 2v(\vartheta ) } \right ) } \right].$$\end{document } the field equations for the scalar field are 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta \square\vartheta - \beta { { dv } \over { d\vartheta } } = - { \alpha _ 1}{r^2 } - { \alpha _ 2}{r_{\mu \nu}}{r^{\mu \nu } } - { \alpha _ 3}{r_{\mu \nu \delta \sigma}}{r^{\mu \nu \delta \sigma } } - { \alpha _ 4}{r_{\mu \nu \delta \sigma}}^{\ast}{r^{\mu \nu \delta \sigma}}.$$\end{document } notice that unlike traditional scalar - tensor theories , the scalar field is here sourced by the geometry and not by the matter distribution . this directly implies that black holes in such theories are likely to be hairy . from the structure of the above equations , it should be clear that the dynamics of guarantee that the modified field equations are covariantly conserved exactly . if one neglected the kinetic and potential energies of in the action , as was originally done in , the theory would possess preferred - frame effects and would not be covariantly conserved . moreover , such a theory requires an additional constraint , i.e. , the right - hand side of ( 30 ) would have to vanish , which is an unphysical consequence of treating as prior structure [ 470 , 207 ] . one last simplification that is usually made when studying modified quadratic gravity theories is to ignore the potential v( ) , i.e. , set v( ) = 0 . this potential can in principle be non - zero , for example if one wishes to endow with a mass or if one wishes to introduce a cosine driving term , like that for axions in field and string theory . first , a mass for will modify the evolution of any gravitational degree of freedom only if this mass is comparable to the inverse length scale of the problem under consideration ( such as a binary system ) . this could be possible if there is an incredibly large number of fields with different masses in the theory , such as perhaps in the string axiverse picture [ 40 , 268 , 303 ] . however , in that picture the moduli fields are endowed with a mass due to shift - symmetry breaking by non - perturbative effects ; such masses are not expected to be comparable to the inverse length scale of binary systems . second , no mass term may appear in a theory with a shift symmetry , i.e. , invariance under the transformation + const . such symmetries are common in four - dimensional , low - energy , effective string theories [ 79 , 204 , 203 , 92 , 89 ] , such as dynamical chern simons and einstein - dilaton - gauss - bonnet theory . similar considerations apply to other more complicated potentials , such as a cosine term . given these field equations , one can linearize them about minkowski space to find evolution equations for the perturbation in the small - coupling approximation . doing so , one finds 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \square_\eta}\vartheta = - { { { \alpha _ 1 } } \over \beta}{{\left({{1 \over { 2\kappa } } } \right)}^2}t_{{\rm{mat}}}^2 - { { { \alpha _ 2 } } \over \beta}{{\left({{1 \over { 2\kappa } } } \right)}^2}t_{{\rm{mat}}}^{\mu \nu}t_{\mu \nu}^{{\rm{mat}}}\quad \quad } \\ { - { { 2{\alpha _ 3 } } \over \beta}({h_{\alpha \beta , \mu \nu}}{h^{\alpha [ \beta , \mu ] \nu } } + { h_{\alpha \beta , \mu \nu}}{h^{\mu [ \nu , \alpha ] \beta } } ) } \\ { - { { 2{\alpha _ 4 } } \over \beta}{\epsilon ^{- \alpha \beta \mu \nu}}{h_{\alpha \delta , \gamma \beta}}{h_\nu}^{[\gamma , \delta ] } \mu , \quad \quad \quad \quad } \\ \end{array}$$\end{document } where we have order - reduced the theory where possible and used the harmonic gauge condition ( which is preserved in this class of theories [ 390 , 400 ] ) . the corresponding equation for the metric perturbation is rather lengthy and can be found in eqs . since these theories are to be considered effective , working always to leading order in i , one can show that they are perturbatively of type n2 in the e(2 ) classification , i.e. , in the far zone , the only propagating modes that survive are the two transverse - traceless ( spin-2 ) metric perturbations . however , in the strong - field region it is possible that additional modes are excited , although they decay rapidly as they propagate to future null infinity . lastly , let us discuss what is known about whether modified quadratic gravities satisfy the requirements discussed in section 2.1 . as it should be clear from the action itself , this modified gravity theory satisfies the fundamental requirement , i.e. , passing all precision tests , provided the couplings i are sufficiently small . this is because such theories have a continuous limit to gr as i 0.6 dynamical chern simons gravity is constrained only weakly at the moment , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ 4^{1/4 } < { 10 ^ 8}$\end{document } km , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\xi _ 4 } \equiv \alpha _ 4 ^ 2/(\beta \kappa)$\end{document } , only through observations of lense thirring precession in the solar system . the einstein - dilaton - gauss bonnet gravity coupling constant \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\xi _ 3 } \equiv \alpha _ 3 ^ 2/(\beta \kappa)$\end{document } , on the other hand , has been constrained by several experiments : solar system observations of the shapiro time delay with the cassini spacecraft placed the bound \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ 3^{1/4 } < 1.3 \times { 10 ^ 7}$\end{document } km [ 73 , 29 ] ; the requirement that neutron stars still exist in this theory placed the constraint \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ 3^{1/4 } \underset{\sim}{<}26$\end{document } km , with the details depending somewhat on the central density of the neutron star ; observations of the rate of change of the orbital period in the low - mass x - ray binary a062000 [ 358 , 255 ] has led to the constraint \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ 3^{1/4 } < 1.9$\end{document } km . however , not all sub - properties of the fundamental requirement are known to be satisfied . one can show that certain members of modified quadratic gravity possess known solutions and these are stable , at least in the small - coupling approximation . simons gravity , spherically - symmetric vacuum solutions are given by the schwarzschild metric with constant to all orders in i [ 245 , 470 ] . such a solution is stable to small perturbations [ 319 , 190 ] , as also are non - spinning black holes and branes in anti de sitter space . on the other hand , spinning solutions continue to be elusive , with approximate solutions in the slow - rotation / small - coupling limit known both for black holes [ 466 , 272 , 345 , 455 ] and stars [ 19 , 342 ] ; nothing is currently known about the stability of these spinning solutions . in einstein - dilaton - gauss bonnet theory even spherically - symmetric solutions are modified [ 473 , 345 ] and these are stable to axial perturbations . the study of modified quadratic gravity theories as effective theories is valid provided one is sufficiently far from its cut - off scale , i.e. , the scale beyond which higher - order curvature terms can not be neglected anymore . one can estimate the magnitude of this scale by studying the size of loop corrections to the quadratic curvature terms in the action due to n - point interactions . simple counting requires that the number of scalar and graviton propagators , ps and pg , satisfy the following relation in terms of the number of vertices v : 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_s } = { v \over 2},\quad { p_g } = ( n - 1){v \over 2}.$$\end{document } thus , loop corrections are suppressed by factors of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ i^vm_{{\rm{pl}}}^{(2 - n)v}{\lambda ^{nv}}$\end{document } , with mp1 the planck mass and the energy scale introduced by dimensional arguments . the cut - off scale above which the theory can not be treated as an effective one can be approximated as the value of at which the suppression factor becomes equal to unity : 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda _ c } \equiv m_{{\rm{pl}}}^{1 - 2/n}\alpha _ i^{1/n},$$\end{document } this cut - off scale automatically places a constraint on the magnitude of i above which higher - curvature corrections must be included . setting the largest value of c to be equal to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{o}(10\mu\mathrm{m})$\end{document } , thus saturating bounds from table - top experiments , and solving for i , we find 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ i^{1/2 } < { \mathcal o}({10 ^ 8}\;{\rm{km}}).$$\end{document } current solar system bounds on i already require the coupling constant to be smaller than 10 km , thus justifying the treatment of these theories as effective models . as for the other requirements discussed in section 2.1 , it is clear that modified quadratic gravity is well - motivated from fundamental theory , but it is not clear at all whether it has a well - posed initial - value formulation . from an effective point of view , a perturbative treatment in i naturally leads to stable solutions and a well - posed initial - value problem , but this is probably not the case when it is treated as an exact theory . in fact , if one were to treat such a theory as exact ( to all orders in i ) , then the evolution system would likely not be hyperbolic , as higher - than - second time derivatives now drive the evolution . although no proof exists , it is likely that such an exact theory is not well - posed as an initial - value problem . notice , however , that this says nothing about the fundamental theories that modified quadratic gravity derives from . this is because even if the truncated theory were ill posed , higher - order corrections that are neglected in the truncated version could restore well - posedness . as for the last requirement ( that the theory modifies the strong field ) , modified quadratic theories are ideal in this respect . this is because they introduce corrections to the action that depend on higher powers of the curvature . in the strong - field moreover , since the curvature scales inversely with the mass of the objects under consideration , one expects the largest deviations in systems with small total mass , such as stellar - mass black - hole mergers . on the other hand , deviations from gr should be small for small compact objects spiraling into a supermassive black hole , since here the spacetime curvature is dominated by the large object , and thus it is small , as discussed in . variable g theories are defined as those where newton s gravitational constant is promoted to a spacetime function . such a modification breaks the principle of equivalence ( see ) because the laws of physics now become local position dependent . in turn , this implies that experimental results now depend on the spacetime position of the laboratory frame at the time of the experiment . many known alternative theories that violate the principle of equivalence , and in particular , the strong equivalence principle , predict a varying gravitational constant . a classic example is scalar - tensor theory , which , as explained in section 2.3.1 , modifies the gravitational sector of the action by multiplying the ricci scalar by a scalar field ( in the jordan frame ) . in such theories , one can effectively think of the scalar as promoting the coupling between gravity and matter to a field - dependent quantity g g( ) , thus violating local position invariance when varies . another example are bimetric theories , such as that of lightman lee , where the gravitational constant becomes time - dependent even in the absence of matter , due to possibly time - dependent cosmological evolution of the prior geometry . a final example are higher - dimensional , brane - world scenarios , where enhanced hawking radiation inexorably leads to a time - varying effective 4d gravitational constant , whose rate of change depends on the curvature radius of extra dimensions . for example , consider the f(r ) model 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = \int { { d^4}x\sqrt { - g } \;\kappa r } \left [ { 1 + { \alpha _ 0}\ln \left({{r \over { { r_0 } } } } \right ) } \right ] + { s_{{\rm{mat}}}},$$\end{document } where = ( 16g ) , 0 is a coupling constant and r0 is a curvature scale . the field equations are 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } = { 1 \over { 2\bar \kappa}}t_{\mu \nu}^{{\rm{mat } } } - { { { \alpha _ 0 } } \over { \bar \kappa}}{r_{\mu \nu } } - 2{\kappa \over { \bar \kappa}}{{{\alpha _ 0 } } \over { { r^2}}}{\nabla _ { ( \mu}}r{\nabla _ { \nu)}}r - { 1 \over 2}{{{\alpha _ 0}\kappa } \over { \bar \kappa}}{g_{\mu \nu}}\square r,$$\end{document } where we have defined the new constant 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\bar \kappa : = \kappa \left [ { 1 + { { { \alpha _ 0 } } \over \kappa}\ln \left({{r \over { { r_0 } } } } \right ) } \right].$$\end{document } clearly , the new coupling constant \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar \kappa$\end{document } depends on the curvature scale involved in the problem , and thus , on the geometry , forcing g to run to zero in the ultraviolet limit . an important point to address is whether variable g theories can lead to modifications to a vacuum spacetime , such as a black - hole binary inspiral . in einstein s theory , g appears as the coupling constant between geometry , encoded by the einstein tensor g , and matter , encoded by the stress energy tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_{\mu \nu}^{{\rm{mat}}}$\end{document}. when considering vacuum spacetimes , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_{\mu \nu}^{{\rm{mat } } } = 0$\end{document } and one might naively conclude that a variable g would not introduce any modification to such spacetimes . in fact , this is the case in scalar - tensor theories ( without homogeneous , cosmological solutions to the scalar field equation ) , where the no - hair theorem establishes that black - hole solutions are not modified . on the other hand , scalar - tensor theories with a cosmological , homogeneous scalar field solution can violate the no - hair theorem , endowing black holes with time - dependent hair , which in turn would introduce variability into g even in vacuum spacetimes [ 246 , 236 , 67 ] . in general , newton s constant plays a much more fundamental role than merely a coupling constant : it defines the relationship between energy and length . for example , for the vacuum schwarzschild solution , g establishes the relationship between the radius r of the black hole and the rest - mass energy e of the spacetime via r = 2 ge / c . similarly , in a black - hole binary spacetime , each black hole introduces an energy scale into the problem that is quantified by a specification of newton s constant . therefore , one can treat variable g modifications as induced by some effective theory that modifies the mapping between the curvature scale and the energy scale of the problem , as is done for example in theories with extra dimensions . superstring theory suggests that physics should be described by 4 large dimensions , plus another 6 that are compactified and very small [ 354 , 355 ] . however , braneworld models , where a certain higher - dimensional membrane is embedded in a higher - dimensional bulk spacetime , can evade this constraint as only gravitons can interact with the bulk . the add model [ 32 , 33 ] is a particular example of such a braneworld , where the bulk is flat and compact and the brane is tensionless with ordinary fields localized on it . since gravitational - wave experiments have not yet constrained deviations from einstein s theory in the strong field , the size of these extra dimensions is constrained to micrometer scales only by table - top experiments [ 259 , 7 ] . what is relevant to gravitational - wave experiments is that in many of these braneworld models black holes may not remain static [ 163 , 405 ] . the argument goes roughly as follows : a five - dimensional black hole is dual to a four - dimensional one with conformal fields on it by the ads / cft conjecture [ 301 , 9 ] , but since the latter must evolve via hawking radiation , the black hole must be losing mass . the hawking mass loss rate is here enhanced by the large number of degrees of freedom in the conformal field theory , leading to an effective modification to newton s laws and to the emission of gravitational radiation . effectively , one can think of the black - hole mass loss as due to the black hole being stretched away from the brane into the bulk due to a universal acceleration , that essentially reduces the size of the brane - localized black hole . for black - hole binaries , one can then draw an analogy between this induced time dependence in the black - hole mass and a variable g theory , where newton s constant decays due to the presence of black holes . of course , this is only analogy , since large extra dimensions would not predict a time - evolving mass in neutron - star binaries . [ 170 , 172 , 171 ] numerically found stable solutions that do not require a radiation component . if such solutions were the ones realized in nature as a result of gravitational collapse on the brane , then the black hole mass would be time independent , up to quantum correction due to hawking evaporation , a negligible effect for realistic astrophysical systems . unfortunately , we currently lack numerical simulations of the dynamics of gravitational collapse in such scenarios . many experiments have been carried out to measure possible deviations from a constant g value , and they can broadly be classified into two groups : ( a ) those that search for the present or nearly present rate of variation ( at redshifts close to zero ) ; ( b ) those that search for secular variations over long time periods ( at very large redshifts ) . examples of experiments or observations of the first class include planetary radar ranging , surface temperature observations of low - redshift millisecond pulsars [ 249 , 362 ] , lunar ranging observations and pulsar timing observations [ 260 , 143 ] , the latter two being the most stringent . examples of experiments of the second class include the evolution of the sun and big - bang nucleosynthesis ( bbn ) calculations [ 119 , 47 ] , again with the latter being more stringent . for either class , the strongest constraints are about / g 10 yr , varying somewhat from experiment to experiment . lacking a particularly compelling action to describe variable g theories , one is usually left with a phenomenological model of how such a modification to einstein s theory would impact gravitational waves . given that the part of the waveform that detectors are most sensitive to is the gravitational wave phase , one can model the effect of variable g theories by studying how the rate of change of its frequency would be modified . assuming a taylor expansion for newton s constant one can derive the modification to the evolution equation for the gravitational wave frequency , given whichever physical scenario one is considering . solving such an evolution equation then leads to a modification in the accumulated gravitational - wave phase observed at detectors on earth . in section 5 the fundamental property can be satisfied if the rate of change of newton s constant is small enough , as variable g theories usually have a continuous limit to gr ( as all derivatives of g go to zero ) . whether variable theories are well - motivated from fundamental physics ( property 2 ) depends somewhat on the particular effective model or action that one considers . but in general , property 2 is usually satisfied , considering that such variability naturally arises in theories with extra dimensions , and the latter are also natural in all string theories . however , variable theories usually fail at introducing modifications in the strong - field region . usually , such variability is parameterized as a taylor expansion about some initial point with constant coefficients . that is , the variability of is not usually constructed so as to become stronger closer to merger . the well - posed property and the sub - properties of the fundamental property depend somewhat on the particular effective theory used to describe varying g modifications . in the f(r ) case , one can impose restrictions on the functional form f( ) such that no ghosts ( f > 0 ) or instabilities ( f > 0 ) arise . this , of course , does not guarantee that this ( or any other such ) theory is well posed . a much more detailed analysis would be required to prove well - posedness of the class of theories that lead to a variable newton s constant , but such is currently lacking . non - commutative geometry is a gravitational theory that generalizes the continuum riemannian manifold of einstein s theory with the product of it with a tiny , discrete , finite non - commutative space , composed of only two points . although the non - commutative space has zero spacetime dimension , as the product manifold remains four dimensional , its internal dimensions are 6 to account for weyl and chiral fermions . this space is discrete to avoid the infinite tower of massive particles that would otherwise be generated , as in string theory . through this construction , one can recover the standard model of elementary particles , while accounting for all ( elementary particle ) experimental data to date . of course , the simple non - commutative space described above is expected to be replaced by a more complex model at planckian energies . thus , one is expected to treat such non - commutative geometry models as effective theories . essentially nothing is currently known about the full non - commutative theory of which the theories described in this section are an effective low - energy limit . before proceeding with an action - principle description of non - commutative geometry theories , we must distinguish between the spectral geometry approach championed by connes , and moyal - type non - commutative geometries [ 389 , 206 , 322 ] . in the former , the manifold is promoted to a non - commutative object through the product of a riemann manifold with a non - commutative space . in the latter , instead , a non - trivial set of commutation relations is imposed between operators corresponding to position . , we will concentrate only on the former , as it is the only type of non - commutative gr extension that has been studied in the context of gravitational - wave theory . the effective action for spectral non - commutative geometry theories ( henceforth , non - commutative geometries for short ) is 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = \int { { d^4}x\sqrt { - g } ( \kappa r + { \alpha _ 0}{c_{\mu \nu \delta \sigma}}{c^{\mu \nu \delta \sigma } } + { \tau _ 0}{r^{\ast}}{r^{\ast } } - { \xi _ 0}r\vert h\vert ^{2 } ) + { s_{{\rm{mat}}}},}$$\end{document } where h is related to the higgs field , c is the weyl tensor , ( 0 , 0 , 0 ) are couplings constants and we have defined the quantity 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r^{\ast}}{r^{\ast } } : = { 1 \over 4}{\epsilon ^{\mu \nu \rho \sigma}}{\epsilon _ { \alpha \beta \gamma \delta}}{r_{\mu \nu}}^{\alpha \beta}{r_{\rho \sigma}}^{\gamma \delta}.$$\end{document } notice that this term integrates to the euler characteristic , and since 0 is a constant , it is topological and does not affect the classical field equations . the last term of eq . ( 38 ) is usually ignored as h is assumed to be relevant only in the early universe . finally , the second term can be rewritten in terms of the riemann and ricci tensors as 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${c_{\mu \nu \delta \sigma}}{c^{\mu \nu \delta \sigma } } = { 1 \over 3}{r^2 } - 2{r_{\mu \nu}}{r^{\mu \nu } } + { r_{\mu \nu \delta \sigma}}{r^{\mu \nu \delta \sigma}}.$$\end{document } notice that this corresponds to the modified quadratic gravity action of eq . ( 26 ) with all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ i^{(1 ) } = 0$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 1^{(0)},\alpha _ 2^{(0)},\alpha _ 3^{(0 ) } = ( 1/3 , - 2,1)$\end{document } , which is not the gauss bonnet invariant . notice also that this model is not usually studied in modified quadratic gravity theory , as one usually concentrates on the terms that have an explicit scalar field coupling . ( 27 ) , but we repeat them here for completeness : 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } - { { 2{\alpha _ 0 } } \over \kappa}[2{\nabla ^{\kappa \lambda } } + { r^{\lambda \kappa}}]\;{c_{\mu \lambda \nu \kappa } } = { 1 \over { 2\kappa}}t_{\mu \nu}^{{\rm{mat}}}.$$\end{document } one could in principle rewrite this in terms of the riemann and ricci tensors , but the expressions become quite complicated , as calculated explicitly in eqs . ( 2 ) and ( 3 ) of . due to the absence of a dynamical degree of freedom coupling to the modifications to the einstein ( 41 ) does not vanish in vacuum , thus violating the weak - equivalence principle and leading to additional equations that might over - constrain the system . in the presence of matter , the equations of motion will not be given by the vanishing of the covariant divergence of the matter stress - energy alone , but now there will be additional geometric terms . given these field equations , one can linearize them about a flat background to find the evolution equations for the metric perturbation [ 326 , 325 ] 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(1 - { \beta ^{- 2}}{\square_\eta})\;{\square_\eta}{h_{\mu \nu } } = - 16\pi t_{\mu \nu}^{{\rm{mat}}},$$\end{document } where the term proportional to = ( 320 ) acts like a mass term . here , one has imposed the transverse - traceless gauge ( a refinement of lorenz gauge ) , which can be shown to exist [ 326 , 325 ] . clearly , even though the full non - linear equations are not covariantly conserved , its linearized version is , as one can easily show that the divergence of the left - hand side of eq . because of these features , if one works perturbatively in , then such a theory will only possess the two usual transverse - traceless ( spin-2 ) polarization modes , i.e. , it is perturbatively of type n2 in the e(2 ) classification . let us now discuss whether such a theory satisfies the properties discussed in section 2.1 . non - commutative geometry theories clearly possess the fundamental property , as one can always take 0 0 ( or equivalently 0 ) to recover gr . therefore , there must exist a sufficiently small 0 such that all precision tests carried out to date are satisfied . as for the existence and stability of known solutions , [ 326 , 325 ] have shown that minkowski spacetime is stable only for 0 < 0 , as otherwise a tachyonic term appears in the evolution of the metric perturbation , as can be seen from eq . he calculated the relativistic shapiro time - delay and light deflection about a massive body in this theory and found that observations of the cassini satellite place constraints on |0| < 5.7 km . regarding black holes , all solutions that are ricci flat ( vacuum solutions of the einstein equations ) are also solutions to eq . this is because by the second bianchi identity , one can show that 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla ^{\kappa \lambda}}{r_{\mu \lambda \nu \kappa } } = { \nabla ^\kappa}_\nu { r_{\mu \kappa } } - \square{r_{\mu \nu}},$$\end{document } and the right - hand side vanishes in vacuum , forcing the entire left - hand side of eq . ( . however , this is not so for neutron stars where the equations of motion are likely to be modified , unless they are static . moreover , as of now there has been no stability analysis of black - hole or stellar solutions and no study of whether the theory is well posed as an initial - value problem , even as an effective theory . thus , except for the fundamental property , it is not clear that non - commutative geometries satisfy any of the other criteria listed in section 2.1 . parity , the symmetry transformation that flips the sign of the spatial triad , has been found to be broken in the standard model of elementary interactions . only the combination of charge conjugation , parity transformation and time inversion ( cpt ) still remains a true symmetry of the standard model . experimentally , it is curious that the weak interaction exhibits maximal parity violation , while other fundamental forces seem to not exhibit any . theoretically , parity violation unavoidably arises in the standard model [ 55 , 8 , 21 ] , as there exist one - loop chiral anomalies that give rise to parity - violating terms coupled to lepton number . in certain sectors of string theory , such as in heterotic and type i superstring theories , parity violation terms are also generated through the green schwarz gauge anomaly - canceling mechanism [ 204 , 355 , 12 ] . finally , in loop quantum gravity , the scalarization of the barbero immirzi parameter coupled to fermions leads to an effective action that contains parity - violating terms [ 406 , 90 , 311 , 192 ] . even without a particular theoretical model , one can show that effective field theories of inflation generically contain non - vanishing , second - order , parity - violating curvature corrections to the einstein alternatively , phenomenological parity - violating extensions of gr have been proposed through a scalarization of the fundamental constants of nature . one is then naturally led to ask whether the gravitational interaction is parity invariant in the strong field . a violation of parity invariance would occur if the einstein hilbert action were modified through a term that involved a levi - civita tensor and parity invariant tensors or scalars . let us try to construct such terms with only single powers of the riemann tensor and a single scalar field : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { ( { \rm{ia}})\,{r_{\alpha \beta \gamma \delta}}\,{\epsilon ^{\alpha \beta \gamma \delta}},\,\,\quad \quad \,\ , } & { ( { \rm{ib}})\,{r_{\alpha \beta \gamma \mu}}\,{\epsilon ^{\alpha \beta \gamma \nu}}{\nabla ^\mu}_\nu \vartheta , \,\quad } \\ { ( { \rm{ic}})\,{r_{\alpha \beta \gamma \mu}}\,{\epsilon ^{\alpha \beta \delta \nu}}\,{\nabla ^{\mu \gamma}}_{\nu \delta}\vartheta , } & { ( { \rm{id}})\,{r_{\alpha \zeta \gamma \mu}}\,{\epsilon ^{\alpha \beta \delta \nu}}\nabla _ { \,\,\,\,\,\,\beta \nu{\delta ^\zeta}\vartheta}^{\mu \gamma}. } \\ \end{array}$$\end{document } option ( ia ) and ( ib ) vanish by the bianchi identities . options ( ic ) and ( i d ) include the commutator of covariant derivatives , which can be rewritten in terms of a riemann tensor , and thus it leads to terms that are at least quadratic in the riemann tensor . therefore , no scalar can be constructed that includes contractions with the levi - civita tensor from a single riemann curvature tensor and a single field . one can try to construct a scalar from the ricci tensor 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$({\rm{iia}})\;{r_{\alpha \beta}}\;{\epsilon^{\alpha \beta \gamma \delta}}\;{\nabla _ { \gamma \delta}}\vartheta , \quad ( { \rm{iib}})\;{r_{\alpha \beta}}\;{\epsilon^{\alpha \mu \gamma \delta}}\;{\nabla _ { \gamma \delta \mu}}^\beta \vartheta , $ $ \end{document } but again ( iia ) vanishes by the symmetries of the ricci tensor , while ( iib ) involves the commutator of covariant derivatives , which introduces another power of the curvature tensor . obviously , the only term one can write with the ricci scalar would lead to a double commutator of covariant derivatives , leading to extra factors of the curvature tensor . one is then forced to consider either theories with two mutually - independent fields or theories with quadratic curvature tensors . of the latter , the only combination that can be constructed and that does not vanish by the bianchi identities is the pontryagin density , i.e. , r * r , and therefore , the action [ 245 , 17 ] 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = \int { { d^4}x\sqrt { - g } \left({\kappa r + { \alpha \over 4}\vartheta \;{r^{\ast}}r } \right),}$$\end{document } is the most general , quadratic action with a single scalar field that violates parity invariance , where we have rescaled the prefactor to follow historical conventions . simons modified gravity , initially proposed by jackiw and pi [ 245 , 17 ] . notice that this is the same as the term proportional to 4 in the quadratic gravity action of eq . ( 26 ) , except that here is prior geometry , i.e. , it does not possess self - consistent dynamics or an evolution equation . such a term violates parity invariance because the pontryagin density is a pseudo - scalar , while is assumed to be a scalar . the field equations for this theory are7 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } + { \alpha \over { 4\kappa}}{\mathcal k}_{\mu \nu}^{(1 ) } = { 1 \over { 2\kappa}}t_{\mu \nu}^{{\rm{mat}}},$$\end{document } which is simply eq . ( 27 ) with ( 1 , 2 , 3 ) set to zero and no stress - energy for . clearly , these field equations are not covariantly conserved in vacuum , i.e. , taking the covariant divergence one finds the constraint 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha { r^{\ast}}r = 0.$$\end{document } this constraint restricts the space of allowed solutions , for example disallowing the kerr metric . therefore , it might seem that the evolution equations for the metric are now overconstrained , given that the field equations provide 10 differential conditions for the 10 independent components of the metric tensor , while the constraint adds one additional , independent differential condition . ( 47 ) , is satisfied , matter fields will not evolve according to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\nabla ^\mu}t_{\mu \nu}^{{\rm{mat } } } = 0$\end{document } , thus violating the equivalence principle . from the field equations , we can derive an evolution equation for the metric perturbation when linearizing about a flat background , namely 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\square_\eta}{h_{\mu \nu } } + { \alpha \over \kappa}({\vartheta _ { , \gamma}}{\epsilon_{(\mu}}^{\gamma \delta \chi}{\square_\eta}{h_{\nu)\delta , \chi } } - { \vartheta _ { , \gamma}}^\zeta { \epsilon_{(\mu}}^{\gamma \delta \chi}{h_{\vert \delta \zeta \vert , \nu)\chi } } + { \vartheta _ { , \gamma}}^\zeta { \epsilon_{(\mu}}^{\gamma \delta \chi}{h_{\nu)\delta , \chi \zeta } } ) = - { 2 \over \kappa}t_{\mu \nu}^{{\rm{mat}}}$$\end{document } in a transverse - traceless gauge , which can be shown to exist in this theory [ 11 , 460 ] . the constraint of eq . ( 47 ) is identically satisfied to second order in the metric perturbation . however , without further information about one can not proceed any further , except for a few general observations . as is clear from eq . ( 48 ) , the evolution equation for the metric perturbation can contain third time derivatives , which generically will lead to instabilities . in fact , as shown in the general solution to these equations will contain exponentially growing and decaying modes . however , the theory defined by eq . ( 45 ) is an effective theory , and thus , there can be higher - order operators not included in this action that may stabilize the solution . regardless , when studying this theory order - reduction is necessary if one is to consider it an effective model . because of the structure of the modification to the field equations , one can always choose a sufficiently small value for such that all solar system tests are satisfied . in fact , one can see from the equations in this section that in the limit 0 , one recovers gr . simons gravity leads to modifications to the non - radiative ( near - zone ) metric in the gravitomagnetic sector , leading to corrections to lense thirring precession [ 14 , 15 ] . this fact has been used to constrain the theory through observations of the orbital motion of the lageos satellites to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\alpha/\kappa){\dot \vartheta ^{- 1 } } < 2 \times { 10 ^ 4}$\end{document } km , or equivalently \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\kappa/\alpha){\dot \vartheta ^{- 1 } } \underset{\sim}{>}{10^{- 14}}{\rm{ev}}$\end{document}. however , much better constraints can be placed through observations of the binary pulsar [ 472 , 18 ] : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\alpha _ . some of the sub - properties of the fundamental requirement are satisfied in non - dynamical chern simons gravity . on the one hand , all spherically - symmetric metrics that are solutions to the einstein equations are also solutions in this theory for a canonical scalar field ( t ) . on the other hand , axisymmetric solutions to the einstein equations are generically not solutions in this theory . moreover , although spherically - symmetric solutions are preserved , perturbations of such spacetimes that are solutions to the einstein equations are not generically solutions to the modified theory . what is perhaps worse , the evolution of perturbations to non - spinning black holes have been found to be generically overconstrained . this is a consequence of the lack of scalar field dynamics in the modified theory , which , via eq . such a conclusion also suggests that this theory does not posses a well - posed initial - value problem . simons gravity is well - motivated from fundamental theories , except that in the latter , the scalar field is always dynamical , instead of having to be prescribed a priori . thus , perhaps the strongest motivation for such a model is as a phenomenological proxy to test whether the gravitational interaction remains parity invariant in the strong field , a test that is uniquely suited to this modified model . in fact , there are many fascinating theories that we have chosen to leave out because they have not yet been analyzed in the gravitational wave context in detail . examples of these include the following : einstein - aether theory and hoava lifshitz theory ; standard model extension ; eddington - inspired born infeld gravity ; new massive gravity [ 60 , 136 ] and bi - gravity theories [ 349 , 346 , 219 , 220 ] . we will update this review with a description of these theories , once a detailed gravitational - wave study for compact binaries or supernovae sources is carried out and the predictions for the gravitational waveform observables are made for any physical system plausibly detectable by current or near future gravitational - wave experiments . einstein - aether theory and hoava lifshitz theory ; standard model extension ; eddington - inspired born infeld gravity ; new massive gravity [ 60 , 136 ] and bi - gravity theories [ 349 , 346 , 219 , 220 ] . kilometer - scale gravitational - wave interferometers have been in operation for over a decade . these types of detectors use laser interferometry to monitor the locations of test masses at the ends of the arms with exquisite precision . gravitational waves change the relative length of the optical cavities in the interferometer ( or equivalently , the proper travel time of photons ) resulting in a strain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = { { \delta l } \over l},$$\end{document } where l is the path length difference between the two arms of the interferometer . fractional changes in the difference in path lengths along the two arms can be monitored to better than 1 part in 10 . it is not hard to understand how such precision can be achieved . for a simple michelson interferometer , a difference in path length of order the size of a fringe for the typically - used , infrared lasers of wavelength 1 m , and interferometer arms of length l = 4 km , the minimum detectable strain is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \sim { \lambda \over l } \sim 3 \times { 10^{- 10}}.$$\end{document } this is still far off the 10 mark . in principle , however , changes in the length of the cavities corresponding to fractions of a single fringe can also be measured provided we have a sensitive photodiode at the dark port of the interferometer , and enough photons to perform the measurement . this way we can track changes in the amount of light incident on the photodiode as the lengths of the arms change and we move over a fringe . the rate at which photons arrive at the photodiode is a poisson process and the fluctuations in the number of photons is n/ , where n is the number of photons . therefore we can track changes in the path length difference of order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta l\sim{\lambda \over { { n^{1/2}}}}.$$\end{document } the number of photons depends on the laser power p , and the amount of time available to perform the measurement . for a gravitational wave of frequency f , we can collect photons for a time t 1/f , so the number of photons is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n\sim{p \over { f{h_p}\nu}},$$\end{document } where hp is planck s constant and = c/ is the laser frequency . for a typical laser power p 1 w , a gravitational - wave frequency f = 100 hz , and 1 m the number of photons is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n\sim{10^{16}},$$\end{document } so that the strain we are sensitive to becomes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\sim{10^{- 18}}.$$\end{document } the sensitivity can be further improved by increasing the effective length of the arms . in the ligo instruments , for example , each of the two arms forms a resonant fabry - prot cavity . for gravitational - wave frequencies smaller than the inverse of the light storage time , the light in the cavities makes many back and forth trips in the arms , while the wave is traversing the instrument . for gravitational waves of frequencies around 100 hz and below , the light makes about a thousand back and forth trips while the gravitational wave is traversing the interferometer , which results in a three - orders - of - magnitude improvement in sensitivity , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\sim{10^{- 21}}.$$\end{document } for frequencies larger than 100 hz the number of round trips the light makes in the fabry - prot cavities while the gravitational wave is traversing the instrument is reduced and the sensitivity is degraded . the proper light travel time of photons in interferometers is controlled by the metric perturbation , which can be expressed as a sum over polarization modes 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_{ij}}(t,\vec x ) = \sum\limits_a { h_{ij}^a } ( t,\vec x),$$\end{document } where a labels the six possible polarization modes in metric theories of gravity . the metric perturbation for each mode can be written in terms of a plane wave expansion , 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{ij}^a(t,\vec x ) = \int\nolimits_{- \infty}^\infty { df } \int\nolimits_{{s^2 } } { d\hat \omega } \;{e^{i2\pi f(t - \hat \omega \cdot \vec x)}}{\tilde h^a}(f,\hat \omega)\epsilon _ { ij}^a(\hat \omega){.}$$\end{document } here f is the frequency of the gravitational waves , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vec k = 2\pi f\hat \omega$\end{document } is the wave vector , is a unit vector that points in the direction of propagation of the gravitational waves , is the ath polarization tensor , with i , j = x , y , z spatial indices . the metric perturbation due to mode a from the direction \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \omega$\end{document } can be written by integrating over all frequencies , 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{ij}^a(t - \hat \omega \cdot \vec x ) = \int\nolimits_{- \infty}^\infty { df } \;{e^{i2\pi f(t - \hat \omega \cdot\vec x)}}{\tilde h^a}(f,\hat \omega)\epsilon_{ij}^a(\hat \omega){.}$$\end{document } by integrating eq . ( 50 ) over all frequencies we have an expression for the metric perturbation from a particular direction \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \omega$\end{document } , i.e. , only a function of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t - \hat \omega \cdot \vec x$\end{document}. the full metric perturbation due to a gravitational wave from a direction \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \omega$\end{document } can be written as a sum over all polarization modes 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_{ij}}(t - \hat \omega \cdot \vec x ) = \sum\limits_a { { h^a } } ( t - \hat \omega \cdot \vec x)\epsilon_{ij}^a(\hat \omega).$$\end{document } the response of an interferometer to gravitational waves is generally referred to as the antenna pattern response , and depends on the geometry of the detector and the direction and polarization of the gravitational wave . to derive the antenna pattern response of an interferometer for all six polarization modes for a gravitational wave propagating in the z direction , the polarization tensors are as follows 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \epsilon _ { ij}^ + = \left({\begin{array}{*{20}c } 1 & 0 & 0 \\ 0 & { - 1 } & 0 \\ 0 & 0 & 0 \\ \end{array } } \right),\epsilon _ { ij}^ \times = \left({\begin{array}{*{20}c } 0 & 1 & 0 \\ 1 & 0 & 0 \\ 0 & 0 & 0 \\ \end{array } } \right ) , } \\ { \epsilon _ { ij}^x = \left({\begin{array}{*{20}c } 0 & 0 & 1 \\ 0 & 0 & 0 \\ 1 & 0 & 0 \\ \end{array } } \right),\epsilon _ { ij}^y = \left({\begin{array}{*{20}c } 0 & 0 & 0 \\ 0 & 0 & 1 \\ 0 & 1 & 0 \\ \end{array } } \right ) , } \\ { \epsilon _ { ij}^b = \left({\begin{array}{*{20}c } 1 & 0 & 0 \\ 0 & 1 & 0 \\ 0 & 0 & 0 \\ \end{array } } \right),\epsilon _ { ij}^\ell = \left({\begin{array}{*{20}c } 0 & 0 & 0 \\ 0 & 0 & 0 \\ 0 & 0 & 1 \\ \end{array } } \right ) , } \\ \end{array}$$\end{document } where the superscripts + , , x , y , b , and correspond to the plus , cross , vector - x , vector - y , breathing , and longitudinal modes . suppose that the coordinate system for the detector is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat x = ( 1,0,0),\,\hat y = ( 0,1,0),\,\hat z = ( 0,0,1)$\end{document } , as in figure 1 . relative to the detector , the gravitational - wave coordinate system is rotated by angles \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\theta , \phi),\,\hat x{\prime } = ( \cos \theta \cos \phi , \cos \theta \sin \phi , - \sin \theta),\,\hat y{\prime } = ( \sin \phi , \cos \phi , 0)$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat z{\prime}(\sin \theta \cos \phi , \sin \theta \sin \phi , \cos \theta)$\end{document}. we still have the freedom to perform a rotation about the gravitational - wave propagation direction , which introduces the polarization angle , 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \hat m = \hat x{\prime}\cos \psi + \hat y{\prime}\sin \psi , } \\ { \hat n = - \hat x{\prime}\sin \psi + \hat y{\prime}\cos \psi , } \\ { \hat \omega = \hat z{\prime}.\quad \quad \quad \quad \quad \quad \;\;}\\ \end{array}$$\end{document } the coordinate systems \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\hat x,\hat y,\hat z)$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\hat m,\hat n,\hat \omega)$\end{document } are also shown in figure 1 . to generalize the polarization tensors in eq . ( 53 ) to a wave coming from a direction \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \omega$\end{document } , we use the unit vectors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat m,\hat n$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \omega$\end{document } as follows 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \epsilon ^ + } = \hat m \otimes \hat m - \hat n \otimes \hat n,}\\ { { \epsilon ^ \times } = \hat m \otimes \hat n + \hat n \otimes \hat m,}\\ { { \epsilon ^x } = \hat m \otimes \hat \omega + \hat \omega \otimes \hat m,}\\ { { \epsilon ^y } = \hat n \otimes \hat \omega + \hat \omega \otimes \hat n,}\\ { { \epsilon ^b } = \hat m \otimes \hat m + \hat n \otimes \hat n,}\\ { { \epsilon ^\ell } = \hat \omega \otimes \hat \omega .\quad \quad \quad}\\ \end{array}$$\end{document } for ligo and virgo the arms are perpendicular so that the antenna pattern response can be written as the difference of projection of the polarization tensor onto each of the interferometer arms , 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^a}(\hat \omega , \psi ) = { 1 \over 2}({\hat x^i}{\hat x^j } - { \hat y^i}{\hat y^j})\epsilon _ { ij}^a(\hat \omega , \psi){.}$$\end{document } this means that the strain measured by an interferometer due to a gravitational wave from direction \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \omega$\end{document } and polarization angle takes the form 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h(t ) = \sum\limits_a { { h_a}(t - \hat \omega \cdot x){f^a } } ( \hat \omega , \psi){.}$$\end{document } explicitly , the antenna pattern functions are , 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { f^ + } ( \theta , \phi , \psi ) = { 1 \over 2}(1 + { { \cos}^2}\theta)\cos 2\phi \cos 2\psi - \cos \theta \sin 2\phi \sin 2\psi , } \\ { { f^ \times}(\theta , \phi , \psi ) = - { 1 \over 2}(1 + { { \cos}^2}\theta)\cos 2\phi \sin 2\psi - \cos \theta \sin 2\phi \cos 2\psi , } \\ { { f^x}(\theta , \phi , \psi ) = \sin \theta ( \cos \theta \cos 2\phi \cos \psi - \sin 2\phi \sin \psi),\quad \quad \quad \quad \;\;}\\ { { f^y}(\theta , \phi , \psi ) = - \sin \theta ( \cos \theta \cos 2\phi \sin \psi + \sin 2\phi \cos \psi),\quad \quad \quad \quad}\\ { { f^b}(\theta , \phi ) = - { 1 \over 2}{{\sin}^2}\theta \cos 2\phi , \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad}\\ { { f^\ell}(\theta , \phi ) = { 1 \over 2}{{\sin}^2}\theta \cos 2\phi .\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad}\\ \end{array}$$\end{document } the dependence on the polarization angles reveals that the + and polarizations are spin-2 tensor modes , the x and y polarizations are spin-1 vector modes , and the b and polarizations are spin-0 scalar modes . figure 2 shows the antenna patterns for the various polarizations given in eq . ( 58 ) with = 0 . the color indicates the strength of the response with red being the strongest and blue being the weakest . figure 2antenna pattern response functions of an interferometer ( see eqs . ( 58 ) ) for = 0 . panels ( a ) and ( b ) show the plus ( f+ ) and cross ( f ) modes , panels ( c ) and ( d ) the vector x and vector y modes ( fx and fy ) , and panel ( e ) shows the scalar modes ( up to a sign , it is the same for both breathing and longitudinal ) . color indicates the strength of the response with red being the strongest and blue being the weakest . antenna pattern response functions of an interferometer ( see eqs . ( 58 ) ) for = 0 . panels ( a ) and ( b ) show the plus ( f+ ) and cross ( f ) modes , panels ( c ) and ( d ) the vector x and vector y modes ( fx and fy ) , and panel ( e ) shows the scalar modes ( up to a sign , it is the same for both breathing and longitudinal ) . color indicates the strength of the response with red being the strongest and blue being the weakest . if the rotational and magnetic axes are not aligned , the beams sweep through space like the beacon on a lighthouse . if the line of sight is aligned with the magnetic axis at any point during the neutron star s rotation the star is observed as a source of periodic radio - wave bursts . such a neutron star is referred to as a pulsar . due to their large moment of inertia pulsars are very stable rotators , and their radio pulses arrive on earth with extraordinary regularity . pulsar timing experiments exploit this regularity : gravitational waves are expected to cause fluctuations in the time of arrival of radio pulses from pulsars . the effect of a gravitational wave on the pulses propagating from a pulsar to earth was first computed in the late 1970s by sazhin and detweiler [ 378 , 145 ] . gravitational waves induce a redshift in the pulse train 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z(t,\hat \omega ) = { 1 \over 2}{{{{\hat p}^i}{{\hat p}^j } } \over { 1 + \hat \omega \cdot \hat p}}\delta { h_{ij}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat p$\end{document } is a unit vector that points in the direction of the pulsar , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \omega$\end{document } is the unit vector of gravitational wave propagation , and 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { h_{ij } } \equiv { h_{ij}}({t_{\rm{e}}},\hat \omega ) - { h_{ij}}({t_{\rm{p}}},\hat \omega),$$\end{document } is the difference in the metric perturbation at the pulsar when the pulse was emitted and the metric perturbation on earth when the pulse was received . the inner product in eq . ( 59 ) is computed with the euclidean metric . in pulsar timing experiments the times of arrival of pulses are measured and the timing residual is produced by subtracting off a model that includes the rotational frequency of the pulsar , the spin - down ( frequency derivative ) , binary parameters if the pulsar is in a binary , sky location and proper motion , etc . the timing residual induced by a gravitational wave , r(t ) , is just the integral of the redshift 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r(t ) \equiv \int\nolimits_0^t { dt{\prime}z(t{\prime})}.$$\end{document } times - of - arrival ( toas ) are measured a few times a year over the course of several years allowing for gravitational waves in the nano - hertz band to be probed . currently , the best timed pulsars have residual root - mean - squares ( rms ) of a few 10 s of ns over a few years . the equations above ( ( 59)ff ) can be used to estimate the strain sensitivity of pulsar timing experiments . for gravitational waves of frequency f the expected induced residual is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r \sim { h \over f},$$\end{document } so that for pulsars with rms residuals r 100 ns , and gravitational waves of frequency f 10 hz , gravitational waves with strains \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\sim rf\sim { 10^{- 5}}$$\end{document } would produce a measurable effect . to find the antenna pattern response of the pulsar - earth system , we are free to place the pulsar on the z - axis . the response to gravitational waves of different polarizations can then be written as 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^a}(\hat \omega , \psi ) = { 1 \over 2}{{{{\hat z}^i}{{\hat z}^j } } \over { 1 + \cos \theta}}\epsilon _ { ij}^a(\hat \omega , \psi),$$\end{document } which allows us to express the fourier transform of ( 59 ) as 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde z(f,\hat \omega ) = \left({1 - { e^{- 2\pi ifl(1 + \hat \omega \cdot \hat p ) } } } \right)\sum\limits_a { { { \tilde h}_a}(f,\hat \omega){f^a}(\hat \omega),}$$\end{document } where the sum is over all possible gravitational - wave polarizations : a = + , ,x , y , b , explicitly , 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^ + } ( \theta , \psi ) = { \sin ^2}{\theta \over 2}\cos 2\psi , $ $ \end{document } 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^ \times}(\theta , \psi ) = - { \sin ^2}{\theta \over 2}\sin 2\psi , $ $ \end{document } 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^x}(\theta , \psi ) = - { 1 \over 2}{{\sin 2\theta } \over { 1 + \cos \theta}}\cos \psi , $ $ \end{document } 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^y}(\theta , \psi ) = { 1 \over 2}{{\sin 2\theta } \over { 1 + \cos \theta}}\sin \psi , $ $ \end{document } 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^b}(\theta ) = { \sin ^2}{\theta \over 2},$$\end{document } 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f^\ell}(\theta ) = { 1 \over 2}{{{{\cos}^2}\theta } \over { 1 + \cos \theta}}.$$\end{document } just like for the interferometer case , the dependence on the polarization angle , reveals that the + and polarizations are spin-2 tensor modes , the x and y polarizations are spin-1 vector modes , and the b and polarizations are spin-0 scalar modes . unlike interferometers , the antenna pattern responses of the pulsar - earth system do not depend on the azimuthal angle of the gravitational wave , and the scalar modes are not degenerate . in the literature , it is common to write the antenna pattern response by fixing the gravitational - wave direction and changing the location of the pulsar . in this case the antenna pattern responses are [ 284 , 22 , 99 ] 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde f^ + } ( { \theta _ p},{\phi _ p } ) = { \sin ^2}{{{\theta _ p } } \over 2}\cos 2{\phi _ p},$$\end{document } 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde f^ \times}({\theta _ p},{\phi _ p } ) = { \sin ^2}{{{\theta _ p } } \over 2}\sin 2{\phi _ p},$$\end{document } 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde f^x}({\theta _ p},{\phi _ p } ) = { 1 \over 2}{{\sin 2{\theta _ p } } \over { 1 + \cos { \theta _ p}}}\cos { \phi _ p},$$\end{document } 73\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde f^y}({\theta _ p},{\phi _ p } ) = { 1 \over 2}{{\sin 2{\theta _ p } } \over { 1 + \cos { \theta _ p}}}\sin { \phi _ p},$$\end{document } 74\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde f^b}({\theta _ p } ) = { \sin ^2}{{{\theta _ p } } \over 2},$$\end{document } 75\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde f^\ell}({\theta _ p } ) = { 1 \over 2}{{{{\cos}^2}{\theta _ p } } \over { 1 + \cos { \theta _ p}}},$$\end{document } where p and p are the polar and azimuthal angles , respectively , of the vector pointing to the pulsar . up this is because fixing the gravitational - wave propagation direction while allowing the pulsar location to change is analogous to fixing the pulsar position while allowing the direction of gravitational - wave propagation to change there is degeneracy in the gravitational - wave polarization angle and the pulsar s azimuthal angle p . for example , changing the polarization angle of a gravitational wave traveling in the z - direction is the same as performing a rotation about the z - axis that changes the pulsar s azimuthal angle . the color indicates the strength of the response , red being the largest and blue the smallest . the plus mode is shown in ( a ) , breathing modes in ( b ) , the vector - x mode in ( c ) , and longitudinal modes in ( d ) , as computed from eq . the cross mode and the vector - y mode are rotated versions of the plus mode and the vector - x mode , respectively , so we did not include them here . the gravitational wave propagates in the positive z - direction with the earth at the origin , and the antenna pattern depends on the pulsar s location . the color indicates the strength of the response , red being the largest and blue the smallest . the plus mode is shown in ( a ) , breathing modes in ( b ) , the vector - x mode in ( c ) , and longitudinal modes in ( d ) , as computed from eq . the cross mode and the vector - y mode are rotated versions of the plus mode and the vector - x mode , respectively , so we did not include them here . the gravitational wave propagates in the positive z - direction with the earth at the origin , and the antenna pattern depends on the pulsar s location . the color indicates the strength of the response , red being the largest and blue the smallest . gravitational waves emitted during the inspiral , merger and ringdown of compact binaries are the most studied in the context of data analysis and parameter estimation . in this section , we will review some of the main data analysis techniques employed in the context of parameter estimation and tests of gr . we begin with a discussion of matched filtering and fisher theory ( for a detailed review , see [ 173 , 103 , 125 , 174 , 248 ] ) . we then continue with a discussion of bayesian parameter estimation and hypothesis testing ( for a detailed review , see [ 387 , 205 , 123 , 294 ] ) . when the detector noise n(t ) is gaussian and stationary , and when the signal s(t ) is known very well , the optimal detection strategy is matched filtering . for any given realization , such noise can be characterized by its power spectral density sn(f ) , defined via 76\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle \tilde n(f)\tilde n^\ast ( f{\prime})\rangle = { 1 \over 2}{s_n}(f)\delta ( f - f{\prime}),$$\end{document } where the tilde stands for the fourier transform , the asterisk for complex conjugation and the brackets for the expectation value . the detectability of a signal is determined by its signal - to - noise ratio or snr , which is defined via 77\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rho ^2 } = { { ( s\vert h ) } \over { \sqrt { ( h\vert n)(n\vert h)}}},$$\end{document } where h is a template with parameters and we have defined the inner product 78\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(a\vert b ) \equiv 4\re \int\nolimits_0^\infty { { { \tilde a\ast\tilde b } \over { { s_n}}}df.}$$\end{document } if the templates do not exactly match the signal , then the snr is reduced by a factor of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{m}$\end{document } , called the match : 79\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\bar { \mathcal m } \equiv { { ( s\vert h ) } \over { \sqrt { ( s\vert s)(h\vert h)}}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1 - \bar{\mathcal{m } } = \mathcal{m}\mathcal{m}$\end{document } is the mismatch . for the noise assumptions made here , the probability of measuring s(t ) in the detector output , given a template h , is given by 80\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p \propto { e^{- ( s - h\vert s - h)/2}},$$\end{document } and thus the waveform h that best fits the signal is that with best - fit parameters such that the argument of the exponential is minimized . for large snr , the best - fit parameters will have a multivariate gaussian distribution centered on the true values of the signal \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \lambda ^i}$\end{document } , and thus , the waveform parameters that best fit the signal minimize the argument of the exponential . the parameter errors will be distributed according to 81\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(\delta { \lambda ^i } ) \propto { e^{- { 1 \over 2}{\gamma _ { ij}}\delta { \lambda ^i}\delta { \lambda ^j}}},$$\end{document } where ij is the fisher matrix 82\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { ij } } \equiv \left({{{\partial h } \over { \partial { \lambda ^i}}}\left\vert { { { \partial h } \over { \partial { \lambda ^j } } } } \right . } \right).$$\end{document } the root - mean - squared ( 1 ) error on a given parameter is then 83\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt { \langle { { ( \delta { \lambda ^{\bar i}})}^2}\rangle } = \sqrt { { \sigma ^{\overline { ii } } } } , $ $ \end{document } where ( ij ) is the variance - covariance matrix and summation is not implied in eq . this root - mean - squared error is sometimes referred to as the statistical error in the measurement of . one can use eq . ( 83 ) to estimate how well modified gravity parameters can be measured . put another way , if a gravitational wave were detected and found consistent with gr , eq . ( 83 ) would provide an estimate of how close to zero these modified gravity parameters would have to be . the fisher method to estimate projected constraints on modified gravity theory parameters is as follows . first , one constructs a waveform model in the particular modified gravity theory one wishes to constrain . usually , this waveform will be similar to the gr one , but it will contain an additional parameter , , such that the template parameters are now plus . let us assume that as 0 , the modified gravity waveform reduces to the gr expectation . then , the accuracy to which can be measured , or the accuracy to which we can say is zero , is approximately ( ) , where the fisher matrix associated with this variance - covariance matrix must be computed with the non - gr model evaluated at the gr limit ( 0 ) . such a method for estimating how well modified gravity theories can be constrained was pioneered by will in [ 436 , 353 ] , and since then , it has been widely employed as a first - cut estimate of the accuracy to which different theories can be constrained . the fisher method described above can dangerously lead to incorrect results if abused [ 414 , 415 ] . one must understand that this method is suitable only if the noise is stationary and gaussian and if the snr is sufficiently large . how large an snr is required for fisher methods to work depends somewhat on the signals considered , but usually for applications concerning tests of gr , one would be safe with 30 or so . in real data analysis , the first two conditions are almost never satisfied . moreover , the first detections that will be made will probably be of low snr , i.e. , 8 , for which again the fisher method fails . in such cases , let us then assume that a detection has been made and that the gravitational wave signal in the data can be described by some model \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{m}$\end{document } , parameterized by the vector . using bayes theorem , the posterior distribution function ( pdf ) or the probability density function for the model parameters , given data d and model \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{m}$\end{document } , is 84\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p({\lambda ^i}\vert d,{\mathcal m } ) = { { p(d\vert { \lambda ^i},{\mathcal m})p({\lambda ^i}\vert { \mathcal m } ) } \over { p(d\vert { \mathcal m})}}.$$\end{document } obviously , the global maximum of the pdf in the parameter manifold gives the best fit parameters for that model . the prior probability density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p({\lambda ^i}\vert \mathcal{m})$\end{document } represents our prior beliefs of the parameter range in model \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{m}$\end{document}. the marginalized likelihood or evidence , is the normalization constant 85\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(d\vert { \mathcal m } ) = \int { d{\lambda ^i}p } ( d\vert { \lambda ^i},{\mathcal m})\;p({\lambda ^i}\vert { \mathcal m}),$$\end{document } which clearly guarantees that the integral of eq . the quantity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p(d\vert { \lambda ^i},\mathcal{m})$\end{document } is the likelihood function , which is simply given by eq . in that equation we used slightly different notation , with s being the data d and h the template associated with model \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{m}$\end{document } and parameterized by . the marginalized pdf , which represents the probability density function for a given parameter ( recall that is a particular element of ) , after marginalizing over all other parameters , is given by 86\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p({\lambda ^{\bar i}}\vert d,{\mathcal m } ) = \int\nolimits_{i \neq \bar i } { d{\lambda ^i}p({\lambda ^i}\vert { \mathcal m})p(d\vert { \lambda ^i},{\mathcal m } ) } , $ $ \end{document } where the integration is not to be carried out over . , one wishes to determine whether the data is more consistent with hypothesis a ( e.g. , that a gr waveform correctly models the signal ) or with hypothesis b ( e.g. , that a non - gr waveform correctly models the signal ) . using bayes theorem , the pdf for model a given the data is 87\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(a\vert d ) = { { p(d\vert a)p(a ) } \over { p(d)}}.$$\end{document } as before , p(a ) is the prior probability of hypothesis a , namely the strength of our prior belief that hypothesis a is correct . the normalization constant p(d ) is given by 88\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(d ) = \int { d{\mathcal m}\;p(d\vert { \mathcal m})\;p({\mathcal m}),}$$\end{document } where the integral is to be taken over all models . thus , it is clear that this normalization constant does not depend on the model . when hypothesis a and b refer to fundamental theories of nature we can take different viewpoints regarding the priors . if we argue that we know nothing about whether hypothesis a or b better describes nature , then we would assign equal priors to both hypotheses . if , on the other hand , we believe gr is the correct theory of nature , based on all previous experiments performed in the solar system and with binary pulsars , then we would assign p(a ) > p(b ) . this assigning of priors necessarily biases the inferences derived from the calculated posteriors , which is sometimes heavily debated when comparing bayesian theory to a frequentist approach . however , this biasing is really unavoidable and merely a reflection of our state of knowledge of nature ( for a more detailed discussion on such issues , please refer to ) . ( 88 ) can never be calculated in practice , simply because we do not know all models . thus , one is forced to investigate relative probabilities between models , such that the normalization constant p(d ) cancels out . the odds ratio is defined by 89\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal o}_{a , b } } = { { p(a\vert d ) } \over { p(b\vert d ) } } = { { p(a ) } \over { p(b)}}{{\mathcal b}_{a , b}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal{b}_{a , b } } \equiv p(d\vert a)/p(d\vert b)$\end{document } is the bayes factor and the prefactor p(a)/p(b ) is the prior odds . the odds - ratio is a convenient quantity to calculate because the evidence p(d ) , which is difficult to compute , actually cancels out . recently , vallisneri has investigated the possibility of calculating the odds - ratio using only frequentist tools and without having to compute full evidences . the odds - ratio should be interpreted as the betting - odds of model a over model b. for example , an odds - ratio of unity means that both models are equally supported by the data , while an odds - ratio of 10 means that there is a 100 to 1 odds that model a better describes the data than model b. the main difficulty in bayesian inference ( both in parameter estimation and model selection ) is sampling the pdf sufficiently accurately . several methods have been developed for this purpose , but currently the two main workhorses in gravitational - wave data analysis are markov chain monte carlo and nested sampling . in the former , one samples the likelihood through the metropolis - hastings algorithm [ 314 , 221 , 122 , 367 ] . this is computationally expensive in high - dimensional cases , and thus , there are several techniques to improve the efficiency of the method , e.g. , parallel tempering . once the pdf has been sampled , one can then calculate the evidence integral , for example via thermodynamic integration [ 420 , 167 , 419 ] . in nested sampling , the evidence is calculated directly by laying out a fixed number of points in the prior volume , which are then allowed to move and coalesce toward regions of high posterior probability . with the evidence in hand , one can then infer the pdf . as in the previous case , were the first to carry out a bayesian implementation of model selection in the context of tests of gr . their analysis focused on tests of a particular massive graviton theory , using the gravitational wave signal from quasi - circular inspiral of non - spinning black holes . cornish et al . [ 124 , 376 ] extended this analysis by considering model - independent deviations from gr , using the parameterized post - einsteinian ( ppe ) approach ( section 5.3.4 ) . 290 , 291 ] , who carried out a similar analysis on a large statistical sample of advanced ligo ( aligo ) detections using simulated data and a restricted ppe model . all of these studies suggest that bayesian tests of gr are possible , given sufficiently - high snr events . of course , whether deviations from gr are observable will depend on the strong - field character and strength of the deviation , as well as the availability of sufficiently - accurate gr waveforms . the model selection techniques described above are affected by other systematics present in data analysis . in general , we can classify these into the following : mismodeling systematic , caused by inaccurate models of the gravitational - wave template.instrumental systematic , caused by inaccurate models of the gravitational - wave response.astrophysical systematic , caused by inaccurate models of the astrophysical environment . mismodeling systematics are introduced due to the lack of an exact solution to the einstein equations from which to extract an exact template , given a particular astrophysical scenario . inspiral templates , for example , are approximated through post - newtonian theory and become increasingly less accurate as the binary components approach each other . cutler and vallisneri were the first to carry out a formal and practical investigation of such a systematic in the context of parameter estimation from a frequentist approach . mismodeling systematic , caused by inaccurate models of the gravitational - wave template . mismodeling systematics will prevent us from testing gr effectively with signals that we do not understand sufficiently well . for example , when considering signals from black hole coalescences , if the the total mass of the binary is sufficiently high , the binary will merge in band . the higher the total mass , the fewer the inspiral cycles that will be in band , until eventually only the merger is in band . since the merger phase is the least understood phase , it stands to reason that our ability to test gr will deteriorate as the total mass increases . of course , we do understand the ringdown phase very well , and tests of the no - hair theorem would be allowed during this phase , provided a sufficiently - high snr . on the other hand , for neutron star binaries or very - low - mass black - hole binaries , the merger phase is expected to be essentially out of band for aligo ( above 1 khz ) , and thus , the noise spectrum itself may shield us from our ignorance . instrumental systematics are introduced by our ignorance of the transfer function , which connects the detector output to the incoming gravitational waves . through sophisticated calibration studies with real data , one can approximate the transfer function very well [ 4 , 1 ] . however , this function is not time - independent , because the noise in the instrument is not stationary or gaussian . thus , un - modeled drifts in the transfer function can introduce systematics in parameter estimation that are as large as 10% in the amplitude and the phase . instrumental systematics can affect tests of gr , if these are performed with a single instrument . however , one expects multiple detectors to be online in the future and for gravitational - wave detections to be made in several of them simultaneously . instrumental systematics should be present in all such detections , but since the noise will be mostly uncorrelated between different instruments , one should be able to ameliorate its effects through cross - correlating outputs from several instruments . astrophysical systematics are induced by our lack of a priori knowledge of the gravitational wave source . as explained above , matched filtering requires knowledge of a waveform template with which to filter the data . for example , when considering inspiral signals , we ignore any third bodies , electric or magnetic fields , neutron star hydrodynamics , the expansion of the universe , etc . fortunately , however , most of these effects are expected to be small : the probability of finding third bodies sufficiently close to a binary system is very small ; for low redshift events , the expansion of the universe induces an acceleration of the center of mass , which is also very small ; electromagnetic fields and neutron - star hydrodynamic effects may affect the inspiral of black holes and neutron stars , but not until the very last stages , when most signals will be out of band anyways . for example , tidal deformation effects enter a neutron - star - binary inspiral waveform at 5 post - newtonian order , which therefore affects the signal outside of the most sensitive part of the aligo sensitivity bucket . perhaps the most dangerous source of astrophysical systematics is due to the assumptions made about the astrophysical systems we expect to observe . for example , when considering neutron - star - binary inspirals , one usually assumes the orbit will have circularized by the time it enters the sensitivity band . moreover , one assumes that any residual spin angular momentum that the neutron stars may possess is very small and aligned or counter - aligned with the orbital angular momentum . these assumptions certainly simplify the construction of waveform templates , but if they happen to be wrong , they would introduce mismodeling systematics that could also affect parameter estimation and tests of gr . in alternative theories of gravity , gravitational - wave sources such as core collapse supernovae may result in the production of gravitational waves in more than just the plus and cross - polarizations [ 384 , 380 , 216 , 334 , 333 , 369 ] . indeed , the near - spherical geometry of the collapse can be a source of scalar breathing - mode gravitational waves . however , the precise form of the waveform is unknown because it is sensitive to the initial conditions . when searching for un - modeled bursts in alternative theories of gravity , a general approach involves optimized linear combinations of data streams from all available detectors to form maximum likelihood estimates of the waveforms in the various polarizations , and the use of null streams . in the context of ground - based detectors and gr , these ideas were first explored by grsel and tinto and later by chatterji et al . with the aim of separating false - alarm events from real detections . the main idea was to construct a linear combination of data streams received by a network of detectors , so that the combination contained only noise . of course , in gr one need only include h+ . and h polarizations , and thus a network of three detectors suffices . this concept can be extended to develop null tests of gr , as originally proposed by chatziioannou et al . and recently implemented by hayama et al . . let us consider a network of d 6 detectors with uncorrelated noise and a detection by all d detectors . for a source that emits gravitational waves in the direction \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat \omega$\end{document } , a single data point ( either in the time - domain , or a time - frequency pixel ) from an array of d detectors ( either pulsars or interferometers ) can be written as 90\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d = fh + n.$$\end{document } here 91\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d \equiv \left [ { \begin{array}{*{20}c } { { d_1 } } \\ { { d_2 } } \\ \vdots \\ { { d_d } } \\ \end{array } } \right],\quad h \equiv \left [ { \begin{array}{*{20}c } { { h _ + } } \\ { { h _ \times } } \\ { { h_x } } \\ { { h_y } } \\ { { h_b } } \\ { { h_\ell } } \\ \end{array } } \right],\quad n \equiv \left [ { \begin{array}{*{20}c } { { n_1 } } \\ { { n_2 } } \\ \vdots \\ { { n_d } } \\ \end{array } } \right],$$\end{document } where n is a vector with the noise . the antenna pattern functions are given by the matrix , 92\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[{f^ + } { f^ \times}{f^x}{f^y}{f^b}{f^\ell } ] \equiv \left [ { \begin{array}{*{20}c } { f_1^ + } & { f_1^ \times } & { f_1^x } & { f_1^y } & { f_1^b } & { f_1^\ell}\\ { f_2^ + } & { f_2^ \times } & { f_2^x } & { f_2^y } & { f_2^b } & { f_2^\ell}\\ \vdots & \vdots & \vdots & \vdots & \vdots & \vdots \\ { f_d^ + } & { f_d^ \times } & { f_d^x } & { f_d^y } & { f_d^b } & { f_d^\ell}\\ \end{array } } \right].$$\end{document } for simplicity we have suppressed the sky - location dependence of the antenna pattern functions . ( 69 ) . for interferometers , since the breathing and longitudinal antenna pattern response functions are degenerate , and even though f is a 6 d matrix , there are only five linearly - independent vectors [ 81 , 80 , 102 , 228 ] . if we do not know the form of the signal present in our data , we can obtain maximum likelihood estimators for it . for simplicity , let us assume the data are gaussian and of unit variance ( the latter can be achieved by whitening the data ) . just as we did in eq . ( 80 ) , we can write the probability of obtaining datum d , in the presence of a gravitational wave h as 93\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(d\vert h ) = { 1 \over { { { ( 2\pi)}^{d/2}}}}\exp \left [ { - { 1 \over 2}\vert d - fh\vert ^{2 } } \right].$$\end{document } the logarithm of the likelihood ratio , i.e. , the logarithm of the ratio of the likelihood when a signal is present to that when a signal is absent , can then be written as 94\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l \equiv \ln { { p(d\vert h ) } \over { p(d\vert 0 ) } } = { 1 \over 2}\left [ { \vert d\vert ^{2 } - \vert d - fh\vert ^{2 } } \right].$$\end{document } if we treat the waveform values for each datum as free parameters , we can maximize the likelihood ratio 95\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 = { \left . { { { \partial l } \over { \partial h } } } \right\vert _ { h = { h_{{\rm{max}}}}}},$$\end{document } and obtain maximum likelihood estimators for the gravitational wave , 96\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_{{\rm{max } } } } = { ( { f^t}f)^{- 1}}{f^t}d.$$\end{document } we can further substitute this solution into the likelihood , to obtain the value of the likelihood at the maximum , 97\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{{\rm{sl } } } } \equiv 2l(h_{{\rm{max } } } ) = { d^t}{p^{{\rm{gw}}}}d,$$\end{document } where 98\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^{{\rm{gw } } } } \equiv f{({f^t}f)^{- 1}}{f^t}.$$\end{document } the maximized likelihood can be thought of as the power in the signal , and can be used as a detection statistic . p is a projection operator that projects the data into the subspace spanned by f. an orthogonal projector can also be constructed , 99\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^{{\rm{null } } } } \equiv ( i - { p^{{\rm{gw}}}}),$$\end{document } which projects the data onto a sub - space orthogonal to f. thus one can construct a certain linear combination of data streams that has no component of a certain polarization by projecting them to a direction orthogonal to the direction defined by the beam pattern functions of this polarization mode 100\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${d^{{\rm{null } } } } = { p^{{\rm{null}}}}d.$$\end{document } this is called a null stream and , in the context of gr , it was introduced as a means of separating false - alarm events due , say , to instrumental glitches from real detections [ 212 , 101 ] . with just three independent detectors , we can choose to eliminate the two tensor modes ( the plus and cross - polarizations ) and construct a gr null stream : a linear combination of data streams that contains no signal consistent within gr , but could contain a signal in another gravitational theory , as illustrated in figure 4 . with such a gr null stream , one can carry out null tests of gr and study whether such a stream contains any statistically - significant deviations from noise . notice that this approach does not require a template ; if one were parametrically constructed , such as in , more powerful null tests could be applied . in the future , we expect several gravitational wave detectors to be online : the two aligo ones in the united states , advanced virgo ( advirgo ) in italy , ligo - india in india , and kagra in japan . given a gravitational - wave observation that is detected by all five detectors , one can then construct three gr null streams , each with power in a signal direction . figure 4schematic diagram of the projection of the data stream d orthogonal to the gr subspace spanned by f and f , along with a perpendicular subspace , for 3 detectors to build the gr null stream . schematic diagram of the projection of the data stream d orthogonal to the gr subspace spanned by f and f , along with a perpendicular subspace , for 3 detectors to build the gr null stream . for pulsar timing experiments where one is dealing with data streams of about a few tens of pulsars , waveform reconstruction for all polarization states , as well as numerous null streams , can be constructed . much work has been done on the response of ground - based interferometers to non - tensorial polarization modes , stochastic background detection prospects , and data analysis techniques [ 299 , 323 , 191 , 329 , 121 ] . in the context of pulsar timing , the first work to deal with the detection of such backgrounds in the context of alternative theories of gravity is due to lee et al . , who used a coherence statistic approach to the detection of non - einsteinian polarizations . they studied the number of pulsars required to detect the various extra polarization modes , and found that pulsar timing arrays are especially sensitive to the longitudinal mode . here we follow the work in [ 329 , 99 ] that deals with the ligo and pulsar timing cases using the optimal statistic , a cross - correlation that maximizes the snr . in the context of the optimal statistic , the derivations of the effect of extra polarization states for ground - based instruments and pulsar timing are very similar . we begin with the metric perturbation written in terms of a plane wave expansion , as in eq . if we assume that the background is unpolarized , isotropic , and stationary , we have that 101\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle \tilde h_a^{\ast}(f,\hat \omega){\tilde h_{a{\prime}}}(f{\prime},\hat \omega { \prime})\rangle = { \delta ^2}(\hat \omega , \hat \omega { \prime}){\delta _ { aa{\prime}}}\delta ( f - f{\prime}){h_a}(f),$$\end{document } where ha(f ) is the gravitational - wave power spectrum for polarization a. ha(f ) is related to the energy density in gravitational waves per logarithmic frequency interval for that polarization through 102\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ a}(f ) \equiv { 1 \over { { \rho _ { { \rm{crit}}}}}}{{d{\rho _ a } } \over { d\ln f}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rho _ { { \rm{crit } } } } = 3h_0 ^ 2/8\pi$\end{document } is the closure density of the universe , and 103\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rho _ a } = { 1 \over { 32\pi}}\langle { \dot h_{aij}}(t,\vec x)\dot h_a^{ij}(t,\vec x)\rangle$$\end{document } is the energy density in gravitational waves for polarization a. it follows from the plane wave expansion in eq . ( 103 ) , that 104\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_a}(f ) = { { 3h_0 ^ 2 } \over { 16{\pi ^3}}}\vert f\vert ^{{- 3}}{\omega _ a}(\vert f\vert),$$\end{document } and therefore 105\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle \tilde h_a^{\ast}(f,\hat \omega){\tilde h_{a{\prime}}}(f{\prime},\hat \omega { \prime})\rangle = { { 3h_0 ^ 2 } \over { 16{\pi ^3}}}{\delta ^2}(\hat \omega , \hat \omega { \prime}){\delta _ { aa{\prime}}}\delta ( f - f{\prime})\vert f\vert ^{{- 3}}{\omega _ a}(\vert f\vert).$$\end{document } for both ground - based interferometers and pulsar - timing experiments , an isotropic stochastic background of gravitational waves appears in the data as correlated noise between measurements from different instruments . the data set from the a instrument is of the form 106\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${d_a}(t ) = { s_a}(t ) + { n_a}(t),$$\end{document } where sa(t ) corresponds to the gravitational - wave signal and na(t ) to noise . the noise is assumed in this case to be stationary and gaussian , and uncorrelated between different detectors , 107\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle { n_a}(t)\rangle = 0,$$\end{document } 108\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle { n_a}(t){n_b}(t)\rangle = 0,$$\end{document } for a b. since the gravitational - wave signal is correlated , we can use cross - correlations to search for it . the cross - correlation statistic is defined as 109\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{ab } } = \int\nolimits_{- t/2}^{t/2 } { dt } \int\nolimits_{- t/2}^{t/2 } { dt } { \prime}{d_a}(t){d_b}(t{\prime}){q_{ab}}(t - t{\prime}),$$\end{document } where qab(t t ) is a filter function to be determined . henceforth , no summation is implied on the detector indices ( a , b , ) . at this stage it is not clear why qab(t t ) depends on the pair of data sets being correlated . the optimal filter is determined by maximizing the expected snr 110\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{snr = } } { { { \mu _ { ab } } } \over { { \sigma _ { ab}}}}.$$\end{document } here ab is the mean sab, and ab is the square root of the variance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sigma _ { ab}^2 = \left\langle { s_{ab}^2 } \right\rangle - { \left\langle { s_{ab}^2 } \right\rangle ^2}$\end{document}. the expressions for the mean and variance of the cross - correlation statistic , ab and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mu _ { ab}}$\end{document } respectively , take the same form for both pulsar timing and ground - based instruments . in the frequency domain , ( 109 ) becomes 111\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{ab } } = \int\nolimits_{- \infty}^\infty { df } \int\nolimits_{- \infty}^\infty { d{f\prime } } { \delta_t}(f - f{\prime})\tilde d_a^{\ast}(f){\tilde d_b}(f{\prime}){\tilde q_{ab}}(f{\prime}),$$\end{document } by the convolution theorem , and the mean is then 112\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mu _ { ab } } \equiv \langle { s_{ab}}\rangle = \int\nolimits_{- \infty}^\infty { df } \int\nolimits_{- \infty}^\infty { d{f\prime } } { \delta _ t}(f - f{\prime})\langle \tilde s_a^{\ast}(f){\tilde s_b}(f{\prime})\rangle { \tilde q_{ab}}(f{\prime}),$$\end{document } where t is the finite time approximation to the delta function , t(f ) = sinft/(f ) . with this in hand , the mean of the cross - correlation statistic is 113\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mu _ { ab}}{{3h_0 ^ 2 } \over { 16{\pi ^3}}}t\sum\limits_a { \int\nolimits_{- \infty}^\infty { df } } \vert f\vert ^{{- 3}}{\tilde q_{ab}}(f){\omega _ a}(f)\gamma _ { ab}^a(f),$$\end{document } and the variance in the weak signal limit is 114\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \sigma _ { ab}^2 \equiv \langle s_{ab}^2\rangle - { { \langle { s_{ab}}\rangle}^2 } \approx \langle s_{ab}^2\rangle \quad \quad \quad \quad \quad \quad \;}\\ { \approx { t \over 4}\int\nolimits_{- \infty}^\infty { df } { p_a}(\vert f\vert){p_b}(\vert f\vert){{\left\vert { { { \tilde q}_{ab}}(f ) } \right\vert}^2},}\\ \end{array}$$\end{document } where the one - sided power spectra of the noise are defined by 115\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle \tilde n_a^{\ast}(f){\tilde n_a}(f{\prime})\rangle = { 1 \over 2}\delta ( f - f{\prime}){p_a}(\vert f\vert),$$\end{document } in analogy to eq . ( 76 ) , where pa plays here the role of sn(f ) . the mean and variance can be rewritten more compactly if we define a positive - definite inner product using the noise power spectra of the two data streams 116\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${(a , b)_{ab } } \equiv \int\nolimits_{- \infty}^\infty { df } a^\ast(f)b(f){p_a}(\vert f\vert){p_b}(\vert f\vert),$$\end{document } again in analogy to the inner product in eq . \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mu _ { ab } } = { { 3h_0 ^ 2 } \over { 16{\pi ^3}}}t{\left({{{\tilde q}_{ab}},{{{\sigma _ a}{\omega _ a}(\vert f\vert)\gamma _ { ab}^a(\vert f\vert ) } \over { \vert f\vert ^{3}{p_a}(\vert f\vert){p_b}(\vert f\vert ) } } } \right)_{ab}},$$\end{document } 118\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sigma _ { ab}^2 \approx { t \over 4}{\left({\tilde q,\tilde q } \right)_{ab}},$$\end{document } where we recall that the capital latin indices ( a , b , ) stand for the polarization content . from the definition of the snr and the schwartz s inequality , it follows that the optimal filter is given by 119\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde q_{ab}}(f ) = n{{{\sigma _ a}{\omega _ a}(\vert f\vert)\gamma _ { ab}^a(\vert f\vert ) } \over { \vert f\vert ^{3}{p_a}(\vert f\vert){p_b}(\vert f\vert)}},$$\end{document } where n is an arbitrary normalization constant , normally chosen so that the mean of the statistic gives the amplitude of the stochastic background . the differences in the optimal filter between interferometers and pulsars arise only from differences in the overlap reduction functions , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma _ { ab}^a(f)$\end{document}. for ground - based instruments , the signal data sa are the strains given by eq . the overlap reduction functions are then given by 120\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma _ { ab}^a(f ) = \int\nolimits_{{s^2 } } { d\hat \omega f_a^a(\hat \omega)f_b^a(\hat \omega ) } { e^{2\pi if\hat \omega \cdot ( { { \vec x}_a } - { { \vec x}_b})}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec x_a}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec x_b}$\end{document } are the locations of the two interferometers . the integrals in this case all have solutions in terms of spherical bessel functions , which we do not summarize here for brevity . for pulsar timing arrays , the overlap reduction functions are then given by 121\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma _ { ab}^a(f ) = { 3 \over { 4\pi}}\int\nolimits_{{s^2 } } { d\hat \omega \left({{e^{i2\pi f{l_a}(1 + \hat \omega \cdot { { \hat p}_a } ) } } - 1 } \right)\left({{e^{- i2\pi f{l_b}(1 + \hat \omega \cdot { { \hat p}_b } ) } } - 1 } \right)f_a^a(\hat \omega)f_b^a(\hat \omega ) } , $ $ \end{document } where la and lb are the distances to the two pulsars . for all transverse modes pulsar timing experiments are in a regime where the exponential factors in eq . ( 121 ) can be neglected [ 30 , 99 ] , and the overlap reduction functions effectively become frequency independent . for the + and mode the overlap reduction function becomes 122\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma _ { ab}^ + = 3\left\{{{1 \over 3 } + { { 1 - \cos { \xi _ { ab } } } \over 2}\left [ { \ln \left({{{1 - \cos { \xi _ { ab } } } \over 2 } } \right ) - { 1 \over 6 } } \right ] } \right\},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\xi _ { ab } } = { \rm{co}}{{\rm{s}}^{- 1}}({\hat p_a } \cdot { \hat p_b})$\end{document } is the angle between the two pulsars . , the overlap reduction function takes the closed form expression : 123\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma _ { ab}^b = { 1 \over 4}(3 + \cos { \xi _ { ab}}){.}$$\end{document } for the vector and longitudinal modes the overlap reduction functions remain frequency dependent and there are no general analytic solutions . the result for the combination of cross - correlation pairs to form an optimal network statistic is also the same in both ground - based interferometer and pulsar timing cases : a sum of the cross - correlations of all detector pairs weighted by their variances . the detector network optimal statistic is , 124\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{opt } } } } = { { { \sigma _ { ab}}\sigma _ { ab}^{- 2}{s_{ab } } } \over { { \sigma _ { ab}}\sigma _ { ab}^{- 2}}},$$\end{document } where ab is a sum over all detector pairs . in order to perform a search for a given polarization mode one ( 120 ) or ( 121 ) ) for that mode . with that in hand and a form for the stochastic background spectrum a(f ) , one can construct optimal filters for all pairs in the detector network using eq . it is important to point out that the procedure outlined above is straightforward for ground - based interferometers . however , pulsar timing data are irregularly sampled , and have a pulsar - timing model subtracted out . this needs to be accounted for , and generally , a time - domain approach is more appropriate for these data sets . the procedure is similar to what we have outlined above , but power spectra and gravitational - wave spectra in the frequency domain need to be replaced by auto - covariance and cross - covariance matrices in the time domain that account for the model fitting ( for an example of how to do this see ) . interestingly , nishizawa et al . show that with three spatially - separated detectors the tensor , vector , and scalar contributions to the energy density in gravitational waves can be measured independently . and alves and tinto showed that pulsar timing experiments are especially sensitive to the longitudinal mode , and to a lesser extent the vector modes . chamberlin and siemens showed that the sensitivity of the cross - correlation to the longitudinal mode using nearby pulsar pairs can be enhanced significantly compared to that of the transverse modes . for example , for the nanograv pulsar timing array , two pulsar pairs separated by 3 result in an enhancement of 4 orders of magnitude in sensitivity to the longitudinal mode relative to the transverse modes . the main contribution to this effect is due to gravitational waves that are coming from roughly the same direction as the pulses from the pulsars . in this case , the induced redshift for any gravitational - wave polarization mode is proportional to fl , the product of the gravitational - wave frequency and the distance to the pulsar , which can be large . when the gravitational waves and the pulse direction are exactly parallel , the redshift for the transverse and vector modes vanishes , but it is proportional to f l for the scalar - longitudinal mode . ( 59 ) to account for graviton dispersion , and found the modified overlap reduction functions ( i.e. , modifications to the hellings - downs curves eq . they conclude that a large number of stable pulsars ( 60 ) are required to distinguish between the massive and massless cases , and that future pulsar timing experiments could be sensitive to graviton masses of about 10 ev ( 10 km ) . this method is competitive with some of the compact binary tests described later in section 5.3.1 ( see table 2 ) . in addition , since the method of lee et al . only depends on the form of the overlap reduction functions , it is advantageous in that it does not involve matched filtering ( and therefore prior knowledge of the waveforms ) , and generally makes few assumptions about the gravitational - wave source properties . in this section , we discuss gravitational wave tests of gr with signals emitted by compact binary systems . we then proceed to describe the many direct or top - down tests and generic or bottom - up tests that have been proposed once gravitational waves are detected , including tests of the no - hair theorems . we concentrate here only on binaries composed of compact objects , such as neutron stars , black holes or other compact exotica . we will not discuss tests one could carry out with electromagnetic information from binary ( or double ) pulsars , as these are already described in . we will also not review tests of gr with accretion disk observations , for which we refer the interested reader to . gravitational - wave tests of einstein s theory can be classed into two distinct subgroups : direct tests and generic tests . direct tests employ a top - down approach , where one starts from a particular modified gravity theory with a known action , derives the modified field equations and solves them for a particular gravitational wave - emitting system . on the other hand , generic tests adopt a bottom - up approach , where one takes a particular feature of gr and asks what type of signature its absence would leave on the gravitational - wave observable ; one then asks whether the data presents a statistically - significant anomaly pointing to that particular signature . direct tests have by far been the traditional approach to testing gr with gravitational waves . the prototypical examples here are tests of jordan - fierz - brans - dicke theory . as described in section 2 , one can solve the modified field equations for a binary system in the post - newtonian approximation to find a prediction for the gravitational - wave observable , as we will see in more detail later in this section . other examples of direct tests include those concerning modified quadratic gravity models and non - commutative geometry theories . the main advantage of such direct tests is also its main disadvantage : one has to pick a particular modified gravity theory . because of this , one has a well - defined set of field equations that one can solve , but at the same time , one can only make predictions about that modified gravity model . unfortunately , we currently lack a particular modified gravity theory that is particularly compelling ; many modified gravity theories exist , but none possess all the criteria described in section 2 , except perhaps for the subclass of scalar - tensor theories with spontaneous scalarization . lacking a clear alternative to gr , it is not obvious which theory one should pick . given that the full development ( from the action to the gravitational wave observable ) of any particular theory can be incredibly difficult , time and computationally consuming , carrying out direct tests of all possible modified gravity models once gravitational waves are detected is clearly unfeasible . given this , one is led to generic tests of gr , where one asks how the absence of specific features contained in gr could impact the gravitational wave observable . for example , one can ask how such an observable would be modified if the graviton had a mass , if the gravitational interaction were lorentz or parity violating , or if there existed large extra dimensions . from these general considerations , meta-observable , i.e. , one that does not belong to a particular theory , but that interpolates over all known possibilities in a well - defined way . this model has come to be known as the parameterized post - einsteinian framework , in analogy to the parameterized post - newtonian scheme used to test gr in the solar system . given such a construction , one can then ask whether the data points to a statistically - significant deviation from gr . the main advantage of generic tests is precisely that one does not have to specify a particular model , but instead one lets the data select whether it contains any statistically - significant deviations from our canonical beliefs . such an approach is , of course , not new to physics , having most recently been successfully employed by the wmap team . the intrinsic disadvantage of this method is that , if a deviation is found , there is no one - to - one mapping between it and a particular action , but instead one has to point to a class of possible models . of course , such a disadvantage is not that limiting , since it would provide strong hints as to what type of symmetries or properties of gr would have to be violated in an ultra - violet completion of einstein s theory . let us first concentrate on jordan - fierz - brans - dicke theory , where black holes and neutron stars have been shown to exist . in this theory , the gravitational mass depends on the value of the scalar field , as newton s constant is effectively promoted to a function , thus leading to violations of the weak - equivalence principle [ 160 , 434 , 441 ] . the usual prescription for the modeling of binary systems in this theory is due to eardley .8 he showed that such a scalar - field effect can be captured by replacing the constant inertial mass by a function of the scalar field in the distributional stress - energy tensor and then taylor expanding about the cosmological constant value of the scalar field at spatial infinity , i.e. , 125\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_a } \rightarrow { m_a}(\phi ) = { m_a}({\phi _ 0})\left\{{1 + { s_a}{\psi \over { { \phi _ 0 } } } - { 1 \over 2}(s_a{\prime } - s_a^2 + { s_a}){{\left({{\psi \over { { \phi _ 0 } } } } \right)}^2 } + { \mathcal o}\left [ { { { \left({{\psi \over { { \phi _ 0 } } } } \right)}^3 } } \right ] } \right\},$$\end{document } where the subscript a stands for a different sources , while 0 1 and the sensitivities sa and sa are defined by 126\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_a } \equiv - { \left [ { { { \partial ( \ln { m_a } ) } \over { \partial ( \ln g ) } } } \right]_0},\quad \quad { s_a}\prime \equiv - { \left [ { { { { \partial ^2}(\ln { m_a } ) } \over { \partial { { ( \ln g)}^2 } } } } \right]_0},$$\end{document } where we remind the reader that g = 1/ , the derivatives are to be taken with the baryon number held fixed and evaluated at = 0 . these sensitivities encode how the gravitational mass changes due to a non - constant scalar field ; one can think of them as measuring the gravitational binding energy per unit mass . the internal gravitational field of each body leads to a non - trivial variation of the scalar field , which then leads to modifications to the gravitational binding energies of the bodies . in carrying out this expansion , one assumes that the scalar field takes on a constant value at spatial infinity 0 , disallowing any homogeneous , cosmological solution to the scalar field evolution equation [ eq . ( 19 ) ] . with this at hand , one can solve the massless jordan - fierz - brans - dicke modified field equations [ eq . ( 19 ) ] for the non - dynamical , near - zone field of n compact objects to obtain 127\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\psi \over { { \phi _ 0 } } } = { 1 \over { 2 + { \omega _ { { \rm{bd}}}}}}\sum\limits_a { ( 1 - 2{s_a } ) } { { { m_a } } \over { { r_a } } } + \ldots , $ $ \end{document } 128\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{00 } } = - 1 + \sum\limits_a { \left({1 - { { { s_a } } \over { 2 + { \omega _ { { \rm{bd } } } } } } } \right ) } { { 2{m_a } } \over { { r_a } } } + \ldots , $ $ \end{document } 129\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{0i } } = - 2(1 + \gamma)\sum\limits_a { { { { m_a } } \over { { r_a}}}v_a^i } + \ldots , $ $ \end{document } 130\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{ij } } = { \delta _ { ij}}\left [ { 1 + 2\gamma \sum\limits_a { \left({1 + { { { s_a } } \over { 1 + { \omega _ { { \rm{bd } } } } } } } \right){{{m_a } } \over { { r_a } } } } + \ldots } \right],$$\end{document } where a runs from 1 to n , we have defined the spatial field point distance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_a } \equiv \vert { x^i } - x_a^i\vert$\end{document } , the parameterized post - newtonian quantity = ( 1 + bd)(2 + bd) and we have chosen units in which g = c = 1 . this solution is obtained in a post - newtonian expansion , where the ellipses represent higher - order terms in va / c and ma / ra . from such an analysis , one can also show that compact objects follow geodesics of such a spacetime , to leading order in the post - newtonian approximation , except that newton s constant in the coupling between matter and gravity is replaced by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$g \to { { \mathcal g}_{12 } } = 1 - ( { s_1 } + { s_2 } - 2{s_1}{s_2}){(2 + { \omega _ { { \rm{bd}}}})^{- 1}}$\end{document } , in geometric units . as is clear from the above analysis , black - hole and neutron - star solutions in this theory generically depend on the quantities bd and sa . the former determines the strength of the correction , with the theory reducing to gr in the bd limit . the latter depends on the compact object that is being studied . for neutron stars , first , neglecting scalar corrections to neutron - star structure and using the tolman - oppenheimer - volkoff equation , one notes that the mass m n g , for a fixed equation of state and central density , with n the total baryon number . ( 126 ) , one has that 131\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_a } \equiv { 3 \over 2}\left [ { 1 - { { \partial ( \ln { m_a } ) } \over { \partial { { ( \ln n)}_g } } } } \right],$$\end{document } where the derivative is to be taken holding g fixed . in this way , given an equation of state and central density , one can compute the gravitational mass as a function of baryon number , and from this , obtain the neutron star sensitivities . eardley , will and zaglauer , and zaglauer have shown that these sensitivities are always in the range sa ( 0.19 , 0.3 ) for a soft equation of state and sa ( 0.1,0.14 ) for a stiff one , in both cases monotonically increasing with mass in ma ( 1.1,1.5 ) m. recently , gralla has found a more general method to compute sensitivities is generic modified gravity theories . will and zaglauer have argued that the no - hair theorems require sa = 1/2 for all black holes , no matter what their mass or spin is . as already explained in section 2 , stationary black holes that are the byproduct of gravitational collapse ( i.e. , with matter that satisfies the energy conditions ) in a general class of scalar - tensor theories are identical to their gr counterparts [ 224 , 408 , 159 , 398].9 this is because the scalar field satisfies a free wave equation in vacuum , which forces the scalar field to be constant in the exterior of a stationary , asymptotically - flat spacetime , provided one neglects a homogeneous , cosmological solution . if the scalar field is to be constant , then by eq . such an argument formally applies only to stationary scenarios , so one might wonder whether a similar argument holds for binary systems that are in a quasi - stationary arrangement . will and zaglauer and mirshekari and will extended this discussion to quasi - stationary spacetimes describing black - hole binaries to higher post - newtonian order . they argued that the only possible deviations from = 0 are due to tidal deformations of the horizon due to the companion , which are known to arise at very high order in post - newtonian theory , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\psi = \mathcal{o}[{({m_a}/{r_a})^5}]$\end{document}. recently , yunes et al . extended this argument further by showing that to all orders in post - newtonian theory , but in the extreme mass - ratio limit , black holes can not have scalar hair in generic scalar - tensor theories . finally , healy et al . have carried out a full numerical simulation of the non - linear field equations , confirming this argument in the full non - linear regime . the activation of dynamics in the scalar field for a vacuum spacetime requires either a non - constant distribution of initial scalar field ( violating the constant cosmological scalar field condition at spatial infinity ) or a pure geometrical source to the scalar field evolution equation . the latter would lead to the quadratic modified gravity theories discussed in section 2.3.3 . as for the former , horbatsch and burgess have argued that if , for example , one lets = t , which clearly satisfies = 0 in a minkowski background,10 then a schwarzschild black hole will acquire modifications that are proportional to . alternatively , scalar hair could also be induced by spatial gradients in the scalar field , possibly anchored in matter at galactic scales . such cosmological hair , however , is likely to be suppressed by a long time scale ; in the example above must have units of inverse time , and if it is to be associated with the expansion of the universe , then it would be natural to assume \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mu = \mathcal{o}(h)$\end{document } , where h is the hubble parameter . therefore , although such cosmological hair might have an effect on black holes in the early universe , it should not affect black hole observations at moderate to low redshifts . scalar field dynamics can be activated in non - vacuum spacetimes , even if initially the stars are not scalarized provided one considers a more general scalar - tensor theory , like the one introduced by damour and esposito - farse [ 129 , 130 ] . as discussed in section 2.3.1 , when the conformal factor takes on a particular functional form , non - linear effects induced when the gravitational energy exceeds a certain threshold can spontaneously scalarize merging neutron stars , as demonstrated recently by barausse , et al . therefore , neutron stars in binaries are likely to have hair in generic scalar - tensor theories , even if they start their inspiral unscalarized . what do gravitational waves look like in jordan - fierz - brans - dicke theory ? as described in section 2.3.1 , both the scalar field perturbation and the new metric perturbation satisfy a sourced wave equation [ eq . ( 19 ) ] , whose leading - order solution for a two - body inspiral is 132\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\theta ^{ij } } = 2(1 + \gamma){\mu \over r}\left({v_{12}^{ij } - { { \mathcal g}_{12}}m{{{x^{ij } } } \over { { r^3 } } } } \right),$$\end{document } 133\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\psi \over { { \phi _ 0 } } } = ( 1 - \gamma){\mu \over r}\left [ { \gamma { { ( { n_i}v_{12}^i)}^2 } - { { \mathcal g}_{12}}\gamma { m \over { { r^3}}}{{({n_i}{x^i})}^2 } - { m \over r}({{\mathcal g}_{12}}\gamma + 2\lambda ) - 2s{n_i}v_{12}^i } \right],$$\end{document } where r is the distance to the detector , n is a unit vector pointing toward the detector , r is the magnitude of relative position vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${x^i } \equiv x_1^i - x_2^i$\end{document } , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$x_a^i$\end{document } the trajectory of body a , = m1m2/m is the reduced mass and m = m1 + m2 is the total mass , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$v_{12}^i \equiv v_1^i - v_2^i$\end{document } is the relative velocity vector and we have defined the shorthands 134\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma \equiv 1 - 2{{{m_1}{s_2 } + { m_2}{s_1 } } \over m},\quad s \equiv { s_2 } - { s_1},$$\end{document } 135\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda \equiv { \mathcal{g}_{12}}(1 - { s_1 } - { s_2 } ) - { ( 2 + { \omega _ { { \text{bd}}}})^ { - 1}}[(1 - 2{s_1}){s'_2 } _ { } + ( 1 - 2{s_2}){s'_1}_{}].$$\end{document } we have also introduced multi - index notation here , such that a = aa . such a solution is derived using the lorenz gauge condition , = 0 and in a post - newtonian expansion , where we have left out subleading terms of relative order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$v_{12}^2$\end{document } or m / r . given the new metric perturbation , one can reconstruct the gravitational wave h metric perturbation , and from this , the response function , associated with the quasi - circular inspiral of compact binaries . after using kepler s third law to simplify expressions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$[\omega = { ( { \mathcal{g}_{12}}m/{r^3})^{1/2}}$\end{document } , where is the orbital angular frequency and m is the total mass and r is the orbital separation ] , one finds for a ground - based l - shaped detector : 136\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { h(t ) = - { { { { \mathcal m}_c } } \over r}{{(2\pi { { \mathcal m}_c}f)}^{2/3}}{e^{- 2i\phi}}\left\{{[{f _ + } ( 1 + { { \cos}^2}\iota ) + 2i{f _ \times}\cos \iota ] \left [ { 1 - { { 1 - \gamma } \over 2}\left({1 + { 4 \over 3}{s^2 } } \right ) } \right ] } \right.}\\ { \left . { - { { 1 - \gamma } \over 2}\gamma { f_{\rm{b}}}{{\sin}^2}\iota } \right\ } - { \eta ^{1/5}}{{{{\mathcal m}_c } } \over r}{{(2\pi { { \mathcal m}_c}f)}^{1/3}}{e^{- i\phi}}s(1 - \gamma){f_{\rm{b}}}\sin \iota \quad \quad \quad \quad}\\ { - { { { { \mathcal m}_c } } \over r}{{(2\pi { { \mathcal m}_c}f)}^{2/3}}{{1 - \gamma } \over 2}{f_{\rm{b}}}(\gamma + 2\lambda),\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad}\\ \end{array}$$\end{document } where /m is the symmetric mass ratio , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal{m}_c } \equiv { \eta ^{3/5}}m$\end{document } is the chirp mass , is the inclination angle , and where we have used the beam - pattern functions in eq . ( 58 ) . in eq . ( 136 ) and henceforth , we linearize all expressions in 1 1 . jordan - fierz - brans - dicke theory predicts the generic excitation of three polarizations : the usual plus and cross polarizations , and a breathing , scalar mode . we see that the latter contributes to the response at two , one and zero times the orbital frequency . one should note that all of these corrections arise during the generation of gravitational waves , and not due to a propagation effect . in fact , gravitational waves travel at the speed of light ( and the graviton remains massless ) in standard jordan - fierz - brans - dicke theory . the quantities and f are the orbital phase and frequency respectively , which are to be found by solving the differential equation 137\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{df } \over { dt } } = ( 1 - \gamma){s^2}{{{\eta ^{2/5 } } } \over \pi}{\mathcal m}_c^{- 2}{(2\pi { { \mathcal m}_c}f)^3 } + { { 48 } \over { 5\pi}}{\mathcal m}_c^{- 2}{(2\pi { { \mathcal m}_c}f)^{11/3}}\left [ { 1 - { { 1 - \gamma } \over 2}\left({1 - { { { \gamma ^2 } } \over 6 } + { 4 \over 3}{s^2 } } \right ) } \right ] \ldots , $ $ \end{document } where the ellipses stand for higher - order terms in the post - newtonian approximation . in this expression , and henceforth , we have kept only the leading - order dipole term and all known post - newtonian , gr terms . if one wished to include higher post - newtonian - order brans - dicke terms , one would have to include monopole contributions as well as post - newtonian corrections to the dipole term . the first term in eq . ( 137 ) corresponds to dipole radiation , which is activated by the scalar mode . that is , the scalar field carries energy away from the system modifying the energy balance law to [ 436 , 379 , 440 ] 138\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\dot e_{{\rm{bd } } } } = - { 2 \over 3}{\mathcal g}_{12}^2{\eta ^2}{{{m^4 } } \over { { r^4}}}(1 - \gamma){s^2 } - { { 32 } \over 5}{\mathcal g}_{12}^2{\eta ^2}{\left({{m \over r } } \right)^5}\left [ { 1 - { { 1 - \gamma } \over 2}\left({1 - { { { \gamma ^2 } } \over 6 } } \right ) } \right ] + \ldots , $ $ \end{document } where the ellipses stand again for higher - order terms in the post - newtonian approximation . solving the frequency evolution equation perturbatively in 1/bd 1 , one finds 139\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{256 } \over 5}{{{t_c } - t } \over { { { \mathcal m}_c } } } = { u^{- 8}}\left [ { 1 - { 1 \over { 12}}(1 - \gamma){s^2}{\eta ^{2/5}}{u^{- 2 } } + \ldots } \right],$$\end{document } 140\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi = - { 1 \over { 64\pi}}{\left({{{256 } \over 5}{{{t_c } - t } \over { { { \mathcal m}_c } } } } \right)^{5/8}}\left [ { 1 - { 5 \over { 224}}(1 - \gamma){s^2}{\eta ^{2/5}}{{\left({{{256 } \over 5}{{{t_c } - t } \over { { { \mathcal m}_c } } } } \right)}^{1/4 } } + \ldots } \right],$$\end{document } where we have defined \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$u \equiv { ( 2\pi { \mathcal{m}_c}f)^{1/3}}$\end{document}. in deriving these equations , we have neglected the last term in eq . ( 137 ) , as this is a constant that can be reabsorbed into the chirp mass . notice that since the two definitions of chirp mass differ only by a term of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{o}(\omega _ { { \rm{bd}}}^{- 1})$\end{document } , the first term of eq . ( 137 ) is not modified . one of the main ingredients that goes into parameter estimation is the fourier transform of the response function . this can be estimated in the stationary - phase approximation , for a simple , non - spinning , quasi - circular inspiral . in this approximation , one assumes the phase is changing much more rapidly than the amplitude [ 56 , 125 , 153 , 457 ] . one finds 141\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h(f ) = { { \mathcal a}_{{\rm{bd}}}}{(\pi { { \mathcal m}_c}f)^{- 7/6}}\left [ { 1 - { 5 \over { 96}}{{{s^2 } } \over { { \omega _ { { \rm{bd}}}}}}{\eta ^{2/5}}{{(\pi { { \mathcal m}_c}f)}^{- 2/3 } } } \right]{e^{- i\psi _ { { \rm{bd}}}^{(2 ) } } } + { \gamma _ { { \rm{bd}}}}{(\pi { { \mathcal m}_c}f)^{- 3/2}}{e^{- i\psi _ { { \rm{bd}}}^{(1)}}}$$\end{document } where we have defined the amplitudes 142\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal a}_{{\rm{bd } } } } \equiv { \left({{{5\pi } \over { 96 } } } \right)^{1/2}}{{{\mathcal m}_c^2 } \over r}{\left [ { f _ + ^2{{(1 + { { \cos}^2}\iota)}^2 } + 4f _ \times ^2{{\cos}^2}\iota - { f _ + } { f_{\rm{b}}}(1 - { { \cos}^4}\iota){\gamma \over { { \omega _ { { \rm{bd } } } } } } } \right]^{1/2}},$$\end{document } 143\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { { \rm{bd } } } } \equiv - { \left({{{5\pi } \over { 48 } } } \right)^{1/2}}{{{\mathcal m}_c^2 } \over r}{\eta ^{1/5}}{s \over { { \omega _ { { \rm{bd}}}}}}{f_{\rm{b}}}\sin \iota , $ $ \end{document } and the fourier phase 144\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \psi _ { { \rm{bd}}}^{(\ell ) } = - 2\pi f{t_c } + \ell \phi _ c^{(\ell ) } + { \pi \over 4 } - { { 3\ell } \over { 256}}{{\left({{{2\pi { { \mathcal m}_c}f } \over \ell } } \right)}^{- 5/3}}\sum\limits_{n = 0}^7 { { { \left({{{2\pi { { \mathcal m}_c}f } \over \ell } } \right)}^{n/3}}(c_n^{{\rm{pn } } } + l_n^{{\rm{pn}}}\ln f)}}\\ { + { { 5\ell } \over { 7168}}{{{s^2 } } \over { { \omega _ { { \rm{bd}}}}}}{\eta ^{2/5}}{{\left({{{2\pi { { \mathcal m}_c}f } \over \ell } } \right)}^{- 7/3}},\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad}\\ \end{array}$$\end{document } where the brans - dicke correction is kept only to leading order in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega _ { { \rm{bd}}}^{- 1}$\end{document } and , while \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(c_n^{{\rm{pn}}},l_n^{{\rm{pn}}})$\end{document } are post - newtonian gr coefficients ( see , e.g. , ) . in writing the fourier response in this way , we had to redefine the phase of coalescence via 145\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi _ c^{(\ell ) } = { \phi _ c } - { \delta _ { \ell , 2}}\left\{{\arctan \left [ { { { 2\cos \iota \;{f _ \times } } \over { ( 1 + { { \cos}^2}\iota){f _ + } } } } \right ] + { \gamma \over { { \omega _ { { \rm{bd}}}}}}{{\cos \iota ( 1 - { { \cos}^2}\iota){f _ \times}{f_{\rm{b } } } } \over { { { ( 1 + { { \cos}^2}\iota)}^2}f _ + ^2 + 4{{\cos}^2}\iota f _ \times ^2 } } } \right\},$$\end{document } where ,m is the kronecker delta and c is the gr phase of coalescence ( defined as an integration constant when the frequency diverges ) . of course , in this calculation we have neglected amplitude corrections that arise purely in gr , if one were to carry out the post - newtonian approximation to higher order . many studies have been carried out to determine the level at which such corrections to the waveform could be measured or constrained once a gravitational wave has been detected . the first such study was carried out by will , who determined that given a ligo detection at snr = 10 of a ( 1.4 , 3 ) m black - hole / neutron - star non - spinning , quasi - circular inspiral , one could constrain bd > 10 . scharre and will carried out a similar analysis but for a lisa detection with = 10 of a ( 1.4 , 10 ) m intermediate - mass black - hole / neutron - star , non - spinning , quasi - circular inspiral , and found that one could constrain bd > 2.1 10 . such an analysis was then repeated by will and yunes but as a function of the classic lisa instrument . they found that the bound is independent of the lisa arm length , but inversely proportional to the lisa position noise error , if the position error noise dominates over laser shot noise . all such studies considered an angle - averaged signal that neglected the spin of either body , assumptions that were relaxed by berti et al . they carried out monte - carlo simulations over all signal sky positions that included spin - orbit precession to find that the projected bound with lisa deteriorates to bd > 0.7 10 for the same system and snr . this was confirmed and extended by yagi et al . , who in addition to spin - orbit precession allowed for non - circular ( eccentric ) inspirals . in fact , when eccentricity is included , the bound deteriorates even further to bd > 0.5 10 . the same authors also found that similar gravitational - wave observations with the next - generation detector decigo could constrain bd > 1.6 10 . similarly , for a non - spinning neutron - star / black - hole binary , the future ground - based detector , the einstein telescope ( et ) , could place constraints about 5 times stronger than the cassini bound , as shown in . all such projected constraints are to be compared with the current solar system bound of bd > 4 10 placed through the tracking of the cassini spacecraft . table 1 presents all such bounds for ease of comparison,11 normalized to an snr of 10 . as should be clear , it is unlikely that ligo observations will be able to constrain bd better than current solar system bounds . in fact , even lisa would probably not be able to do better than the cassini bound . table 1 also shows that the inclusion of more complexity in the waveform seems to dilute the level at which bd can be constrained . this is because the inclusion of eccentricity and spin forces one to introduce more parameters in the waveform , without these modifications truly adding enough waveform complexity to break the induced degeneracies . one would then expect that the inclusion of amplitude modulation due to precession and higher harmonics should break such degeneracies , at least partially , as was found for massive black - hole binary [ 279 , 280 ] . however , even then it seems reasonable to expect that only third - generation detectors will be able to constrain bd beyond solar - system levels . reference binary massbd properties x4solar system(1.4 , 3 ) m0.1ligo , fisher , ang . circular , non - spinning(1.4,10 ) m0.7lisa , fisher , monte - carlo circular , w / spin - orbit(1.4,10 ) m0.5lisa , fisher , monte - carlo eccentric , spin - orbit(1.4,10 ) m160decigo , fisher , monte - carlo eccentric , spin - orbit(1.4,10 ) m10et , fisher , ang . the main reason that solar - system constraints of jordan - fierz - brans - dicke theory can not be beaten with gravitational - wave observations is that the former are particularly well - suited to constrain weak - field deviations of gr . one might have thought that scalar - tensor theories constitute strong - field tests of einstein s theory , but this is not quite true , as argued in section 2.3.1 . one can see this clearly by noting that scalar - tensor theory predicts dipolar radiation , which dominates at low velocities over the gr prediction ( precisely the opposite behavior that one would expect from a strong - field modification to einstein s theory ) . however , one should note that all the above analysis considered only the inspiral phase of coalescence , usually truncating their study at the innermost stable - circular orbit . the merger and ringdown phases , where most of the gravitational wave power resides , have so far been mostly neglected . one might expect that an increase in power will be accompanied by an increase in snr , thus allowing us to constrain bd further , as this scales with 1/snr . moreover , during merger and ringdown , dynamical strong - field gravity effects in scalar - tensor theories could affect neutron star parameters and their oscillations , as well as possibly induce spontaneous scalarization . all of these non - linear effects could easily lead to a strengthening of projected bounds . however , to date no detailed analysis has attempted to determine how well one could constrain scalar - tensor theories using full information about the entire coalescence of a compact binary . the subclass of scalar - tensor models described by jordan - fierz - brans - dicke theory is not the only type of model that can be constrained with gravitational - wave observations . in the extreme - mass - ratio limit , for binaries consisting of a stellar - mass compact object spiraling into a supermassive black hole , yunes et al . have recently shown that generic scalar - tensor theories reduce to either massless or massive jordan - fierz - brans - dicke theory . of course , in this case the sensitivities need to be calculated from the equations of structure within the full scalar - tensor theory . the inclusion of a scalar field mass leads to an interesting possibility : floating orbits . such orbits arise when the small compact object experiences superradiance , leading to resonances in the scalar flux that can momentarily counteract the gravitational - wave flux , leading to a temporarily - stalled orbit that greatly modifies the orbital - phase evolution . these authors showed that if an extreme mass - ratio inspiral is detected with a template consistent with gr , this alone allows us to rule out a large region of ( ms,bd ) phase space , where ms is the mass of the scalar ( see figure 1 in ) . this is because if such an inspiral had gone through a resonance , a gr template would be grossly different from the signal . such bounds are dramatically stronger than the current most stringent bound bd > 4 10 and ms < 2.5 10 ev obtained from cassini measurements of the shapiro time - delay in the solar system . even if resonances are not hit , berti et al . have estimated that second - generation ground - based interferometers could constrain the combination ms/(bd)/ 10 ev with the observation of gravitational waves from neutron - star / binary inspirals at an snr of 10 . these bounds can also be stronger than current constraints , especially for large scalar mass . lastly one should mention possible gravitational - wave constraints on other types of scalar tensor theories . let us first consider brans - dicke type scalar - tensor theories , where the coupling constant is allowed to vary . will has argued that the constraints described in table 1 go through , with the change 146\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{2{{\mathcal g}_{1,2 } } } \over { 2 + { \omega _ { { \rm{bd } } } } } } \to { { 2{{\mathcal g}_{1,2 } } } \over { 2 + { \omega _ { { \rm{bd}}}}}}{\left [ { 1 + { { 2{{\omega \prime}_{{\rm{bd } } } } } \over { { { ( 3 + 2{\omega _ { { \rm{bd}}}})}^2 } } } } \right]^2},$$\end{document } where bd dbd / d,/d. in the bd 1 limit , this implies the replacement \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \text{bd } } } } \to { \omega _ { { \text{bd}}}}[1 + { \omega ' _ { bd}}/2\omega _ { { \text{bd}}}^2){]^ { - 2}}$\end{document}. of course , this assumes that there is neither a potential nor a geometric source driving the evolution of the scalar field , and is not applicable for theories where spontaneous scalarization is present . another interesting scalar - tensor theory to consider is that studied by damour and esposito - farse [ 129 , 130 ] . as explained in section 2.3.1 , this theory is defined by the action of eq . ( 14 ) with the conformal factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a(\psi ) = { e^{\beta { \psi ^2}}}$\end{document}. in standard brans - dicke theory , only mixed binaries composed of a black hole and a neutron star lead to large deviations from gr due to dipolar emission . this is because dipole emission is proportional to the difference in sensitivities of the binary components . for neutron - star binaries with similar masses , this difference is close to zero , while for black holes it is identically zero ( see eqs . however , in the theory considered by damour and esposito - farse , when the gravitational energy is large enough , as in the very late inspiral , non - linear effects can lead to drastic modifications from the gr expectation , such as spontaneous scalarization . unfortunately , most of this happens at rather high frequency , and thus , it is not clear whether such effects are observable with current ground - based detectors . black holes exist in the classes of modified quadratic gravity that have so far been considered . in non - dynamical theories ( when = 0 and the scalar - fields are constant , refer to eq . ( 25 ) ) , stein and yunes have shown that all metrics that are ricci tensor flat are also solutions to the modified field equations ( see also ) . this is not so for dynamical theories , since then the field is sourced by curvature , leading to corrections to the field equations proportional to the riemann tensor and its dual . in dynamical chern - simons gravity , stationary and spherically - symmetric spacetimes are still described by gr solutions , but stationary and axisymmetric spacetimes are not . instead , they are represented by [ 466 , 272 ] 147\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ds_{{\rm{cs}}}^2 = ds_{{\rm{kerr}}}^2 + { 5 \over 4}{{\alpha _ { { \rm{cs}}}^2 } \over { \beta \kappa}}{a \over { { r^4}}}\left({1 + { { 12 } \over 7}{m \over r } + { { 27 } \over { 10}}{{{m^2 } } \over { { r^2 } } } } \right){\sin ^2}\theta \;d\theta \;dt + { \mathcal o}({a^2}/{m^2}),$$\end{document } with the scalar field 148\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\vartheta _ { { \rm{cs } } } } = { 5 \over 8}{{{\alpha _ { { \rm{cs } } } } } \over \beta}{a \over m}{{\cos \theta } \over { { r^2}}}\left({1 + { { 2 m } \over r } + { { 18{m^2 } } \over { 5{r^2 } } } } \right ) + { \mathcal o}({a^3}/{m^3}),$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ds_{{\rm{kerr}}}^2$\end{document } is the line element of the kerr metric and we recall that cs = 44 in the notation of section 2.3.3 . these expressions are obtained in boyer - lindquist coordinates and in the small - rotation / small - coupling limit to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{o}(a / m)$\end{document } in [ 466 , 272 ] and to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{o}({a^2}/{m^2})$\end{document } in . the linear - in - spin corrections modify the frame - dragging effect and they are of 3.5 post - newtonian order . the quadratic - in - spin corrections modify the quadrupole moment , which induces 2 post - newtonian - order corrections to the binding energy . however , the stability of these black holes has not yet been demonstrated . in einstein - dilaton - gauss - bonnet gravity , stationary and spherically - symmetric spacetimes are described , in the small - coupling approximation , by the line element 149\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ds_{{\rm{edgb}}}^2 = - { f_{{\rm{schw}}}}(1 + h)d{t^2 } + f_{{\rm{schw}}}^{- 1}(1 + k)d{r^2 } + { r^2}d{\omega ^2},$$\end{document } in schwarzschild coordinates , where d is the line element on the two - sphere , fschw = 1 2m / r is the schwarzschild factor and we have defined 150\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = { { \alpha _ 3 ^ 2 } \over { \beta \kappa { m^4}}}{1 \over { 3{f_{{\rm{schw}}}}}}{{{m^3 } } \over { { r^3}}}\left({1 + 26{m \over r } + { { 66 } \over 5}{{{m^2 } } \over { { r^2 } } } + { { 96 } \over 5}{{{m^3 } } \over { { r^3 } } } - 80{{{m^4 } } \over { { r^4 } } } } \right),$$\end{document } 151\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k = - { { \alpha _ 3 ^ 2 } \over { \beta \kappa { m^4}}}{1 \over { { f_{{\rm{schw}}}}}}{{{m^2 } } \over { { r^2}}}\left [ { 1 + { m \over r } + { { 52 } \over 3}{{{m^2 } } \over { { r^2 } } } + 2{{{m^3 } } \over { { r^3 } } } + { { 16 } \over 5}{{{m^4 } } \over { { r^4 } } } - { { 368 } \over 3}{{{m^5 } } \over { { r^5 } } } } \right],$$\end{document } while the corresponding scalar field is 152\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\vartheta _ { { \rm{edgb } } } } = { { { \alpha _ 3 } } \over \beta}{2 \over { mr}}\left({1 + { m \over r } + { 4 \over 3}{{{m^2 } } \over { { r^2 } } } } \right).$$\end{document } this solution is not restricted just to einstein - dilaton - gauss - bonnet gravity , but it is also the most general , stationary and spherically - symmetric solution in quadratic gravity . this is because all terms proportional to 1,2 are proportional to the ricci tensor , which vanishes in vacuum gr , while the 4 term does not contribute in spherical symmetry ( see for more details ) . although the stability of these black holes has not yet been demonstrated , other dilatonic black hole solutions obtained numerically ( equivalent to those in einstein - dilaton - gauss - bonnet theory in the limit of small fields ) have been found to be stable under axial perturbations [ 258 , 409 , 343 ] . neutron stars also exist in quadratic modified gravity . in dynamical chern - simons gravity , the mass - radius relation remains unmodified to first order in the slow - rotation expansion , but the moment of inertia changes to this order [ 469 , 19 ] , while the quadrupole moment and the mass measured at spatial infinity change to quadratic order in spin . this is because the mass - radius relation , to first order in slow - rotation , depends on the spherically - symmetric part of the metric , which is unmodified in dynamical chern - simons gravity . in einstein - dilaton - gauss - bonnet gravity , , these functions must be solved for numerically and they depend on the equation of state . gravitational waves are also modified in quadratic modified gravity . in dynamical chern - simons gravity , garfinkle et al . have shown that the propagation of such waves on a minkowski background remains unaltered , and thus , all modifications arise during the generation stage . in einstein - dilaton - gauss - bonnet theory , yagi et al . studied the generation mechanism in both theories during the quasi - circular inspiral of comparable - mass , spinning black holes in the post - newtonian and small - coupling approximations . they found that a standard post - newtonian analysis fails for such theories because the assumption that black holes can be described by a distributional stress - energy tensor without any further structure fails . they also found that since black holes acquire scalar hair in these theories , and this scalar field is anchored to the curvature profiles , as black holes move , the scalar fields must follow the singularities , leading to dipole scalar - field emission . during a quasi - circular inspiral of spinning black holes in dynamical chern - simons gravity , the total gravitational wave energy flux carried out to spatial infinity ( equal to minus the rate of change of a binary s binding energy by the balance law ) is modified from the gr expectation to leading order by 153\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \dot e_{{\rm{spin}}}^{{\rm{cs } } } } \over { { { \dot e}_{{\rm{gr } } } } } } = { \zeta _ 4}{\eta ^{- 2}}\left\{{{{25 } \over { 1536}}[\bar \delta + 2\langle ( \bar \delta \cdot { { \hat v}_{12}})\rangle ] + { { 75 } \over { 16}}{{{a_1}{a_2 } } \over { { m^2}}}\left\langle { \hat s_1^i\hat s_2^j\left({2\hat v_{12}^i\hat v_{12}^j - 2\hat n_{12}^i\hat n_{12}^j } \right ) } \right\rangle } \right\}v_{12}^4,$$\end{document } due to scalar field radiation and corrections to the metric perturbation that are of magnetic - type , quadrupole form . in this equation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\dot e_{{\rm{gr } } } } = ( 32/5){\eta ^2}v_{12}^{10}$\end{document } is the leading - order gr prediction for the total energy flux , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\zeta _ 4 } = \alpha _ 4 ^ 2/(\beta \kappa { m^4})$\end{document } is the dimensionless chern - simons coupling parameter , 12 is the magnitude of the relative velocity with unit vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat v_{12}^i,{\bar \delta ^i } = ( { m_2}/{m_1})({a_1}/m)\hat s_1^i - ( { m_1}/{m_2})({a_2}/m)\hat s_2^i$\end{document } , where aa is the kerr spin parameter of the ath black hole and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat s_a^i$\end{document } is the unit vector in the direction of the spin angular momentum , the unit vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat n_{12}^i$\end{document } points from body one to two , and the angle brackets stand for an average over several gravitational wave wavelengths . if the black holes are not spinning , then the correction to the scalar energy flux is greatly suppressed 154\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \dot e_{{\rm{no - spin}}}^{{\rm{cs } } } } \over { { { \dot e}_{{\rm{gr } } } } } } = { 2 \over 3}\delta _ m^2{\zeta _ 4}v_{12}^{14},$$\end{document } where we have defined the reduced mass difference m ( m1 m2)/m . notice that this is a 7 post - newtonian - order correction , instead of a 2 post - newtonian correction as in eq . , the dynamical chern - simons correction to the metric tensor induces a 6 post - newtonian - order correction to the gravitational energy flux , which is consistent with the numerical results of . on the other hand , in einstein - dilaton - gauss - bonnet gravity , the corrections to the energy flux are 155\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \dot e_{{\rm{no - spin}}}^{{\rm{edgb } } } } \over { { { \dot e}_{{\rm{gr } } } } } } = { 5 \over { 96}}{\eta ^{- 4}}\delta _ m^2{\zeta _ 3}v_{12}^{- 2},$$\end{document } which is a 1 post - newtonian correction . ( 152 ) ) , and when such a scalar monopole is dragged by the black hole , it emits electric - type , dipole scalar radiation . any hairy black hole with monopole hair will thus emit dipolar radiation , leading to 1 post - newtonian corrections in the energy flux carried to spatial infinity . such modifications to the energy flux modify the rate of change of the binary s binding energy through the balance law , = b , which in turn modify the rate of change of the gravitational wave frequency and phase , = ( deb / df). for dynamical chern - simons gravity ( when the spins are aligned with the orbital angular momentum ) and for einstein - dilaton - gauss - bonnet theory ( in the non - spinning case ) , the fourier transform of the gravitational - wave response function in the stationary phase approximation becomes [ 447 , 454 ] 156\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde h_{{\rm{dcs , edgb } } } } = { \tilde h_{{\rm{gr}}}}{e^{i{\beta _ { { \rm{dcs , edgb}}}}{u^{{b_{{\rm{dcs , edgb}}}}}},}}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde h_{{\rm{gr}}}}$\end{document } is the fourier transform of the response in gr , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$u \equiv { ( \pi { \mathcal{m}_c}f)^{1/3}}$\end{document } with f the gravitational wave frequency and [ 447 , 454 ] 157\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { { \rm{dcs } } } } = { { 1549225 } \over { 11812864}}{{{\zeta _ 4 } } \over { { \eta ^{14/5}}}}\left [ { \left({1 - { { 47953 } \over { 61969}}\eta } \right)\chi _ s^2 + \left({1 - { { 199923 } \over { 61969}}\eta } \right)\chi _ a^2 - 2{\delta _ m}{\chi _ s}{\chi _ a } } \right],\quad { b_{{\rm{dcs } } } } = - 1,$$\end{document } 158\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { { \rm{edgb } } } } = - { 5 \over { 7168}}{\zeta _ 3}{\eta ^{- 18/5}}\delta _ m^2,\quad { b_{{\rm{edgb } } } } = - 7,$$\end{document } where we have defined the symmetric and antisymmetric spin combinations s , a ( a1/m1 a2/m2)/2 . we have here neglected any possible amplitude correction , but we have included both deformations to the binding energy and kepler s third law , in addition to changes in the energy flux , when computing the phase correction . however , in einstein - dilaton - gauss - bonnet theory the binding energy is modified at higher post - newtonian order , and thus , corrections to the energy flux control the modifications to the gravitational - wave response function . from the above analysis , it should be clear that the corrections to the gravitational - wave observable in quadratic modified gravity are always proportional to the quantity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\zeta _ { 3,4 } } \equiv { \xi _ { 3,4}}/{m^4 } = \alpha _ { 3,4}^2/(\beta \kappa { m^4})$\end{document}. thus , any measurement that is consistent with gr will allow a constraint of the form 3,4 < n , where n is a number of order unity , and is the accuracy of the measurement . solving for the coupling constants of the theory , such a measurement would lead to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { 3,4}^{1/4 } < { ( n\delta)^{1/4}}m$\end{document } . therefore , constraints on quadratic modified gravity will weaken for systems with larger characteristic mass . this can be understood by noticing that the corrections to the action scale with positive powers of the riemann tensor , while this scales inversely with the mass of the object , i.e. , the smaller a compact object is , the larger its curvature . such an analysis then automatically predicts that ligo will be able to place stronger constraints than lisa - like missions on such theories , because ligo operates in the 100 hz frequency band , allowing for the detection of stellar - mass inspirals , while lisa - like missions operate in the mhz band , and are limited to supermassive black - holes inspirals . yagi et al . predicted , based on the results of cornish et al . , that a sky - averaged ligo gravitational - wave observation with snr of 10 of the quasi - circular inspiral of non - spinning black holes with masses ( 6,12)m would allow a constraint of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ 3^{1/4 } \underset{\sim}{<}20$\end{document } km , where we recall that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\xi _ 3 } = \alpha _ 3 ^ 2/(\beta \kappa)$\end{document}. a similar sky - averaged , elisa observation of a quasi - circular , spin - aligned black - hole inspiral with masses ( 10,3 10m ) would constrain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ 3^{1/4 } < { 10 ^ 7}$\end{document } km . the loss in constraining power comes from the fact that the constraint on 3 will scale with the total mass of the binary , which is six orders of magnitude larger for space - borne sources . these constraints are not stronger than current bounds from the existence of compact objects ( 3 < 26 km ) and from the change in the orbital period of the low - mass x - ray binary a062000 ( 3 < 1.9 km ) , but they are independent of the nature of the object and sample the theory in a different energy scale . in dynamical chern - simons gravity , one expects similar projected gravitational - wave constraints on 4 , namely \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ 4^{1/4 } < \mathcal{o}(m)$\end{document } , where m is the total mass of the binary system in kilometers . therefore , for binaries detectable with ground - based interferometers , one expects constraints of order \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ 4^{1/4 } < 10$\end{document } km . in this case , such a constraint would be roughly six orders of magnitude stronger than current lageos bounds . dynamical chern - simons gravity can not be constrained with binary pulsar observations , since the theory s corrections to the post - keplerian observables are too high post - newtonian order , given the current observational uncertainties . however , the gravitational wave constraint is more difficult to achieve in the dynamical chern - simons case , because the correction to the gravitational wave phase is degenerate with spin . however , yagi et al . argued that precession should break this degeneracy , and if a signal with sufficiently high snr is observed , such bounds would be possible . one must be careful , of course , to check that the small - coupling approximation is still satisfied when saturating such a constraint . black holes exist in non - commutative geometry theories , as discussed in section 2.3.5 . what is more , the usual schwarzschild and kerr solutions of gr persist in these theories . this is not because such solutions have vanishing weyl tensor , but because the quantity c happens to vanish for such metrics . similarly , one would expect that the two - body , post - newtonian metric that describes a black - hole - binary system should also satisfy the non - commutative geometry field equations , although this has not been proven explicitly . similarly , although neutron - star spacetimes have not yet been considered in non - commutative geometries , it is likely that if such spacetimes are stationary and satisfy the einstein equations , they will also satisfy the modified field equations . much more work on this is still needed to establish all of these concepts on a firmer basis . their generation for a compact binary system in a circular orbit was analyzed by nelson et al . they began by showing that a transverse - traceless gauge exists in this theory , although the transverse - traceless operator is slightly different from that in gr . they then proceeded to solve the modified field equations for the metric perturbation [ eq . ( 42 ) ] via a green s function approach : 159\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{ik } } = 2\beta \int { { { dt{\prime } } \over { \sqrt { { { ( t - t{\prime})}^2 } - \vert r\vert ^{2}}}}{{\ddot i}^{ik}}(t{\prime}){{\mathcal j}_1}(\beta \sqrt { { { ( t - t{\prime})}^2 } - \vert r\vert ^{2 } } ) } , $ $ \end{document } where recall that = ( 320 ) acts like a mass term , the integral is taken over the entire past light cone , 1( ) is the bessel function of the first kind , |r| is the distance from the source to the observer and the quadrupole moment is defined as usual : 160\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${i^{ik } } = \int { { d^3 } } x\;t_{{\rm{mat}}}^{00}{x^{ik}},$$\end{document } where t is the time - time component of the matter stress - energy tensor . of course , this is only the first term in an infinite multipole expansion . although the integral in eq . ( 159 ) has not yet been solved in the post - newtonian approximation , nelson et al . [ 326 , 325 ] did solve for its time derivative to find 161a\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\dot h^{xx } } = - { \dot h^{yy } } = 32\beta \mu r_{12}^2{\omega ^4}\left [ { \sin ( 2\phi){f_c}\left({\beta \vert r\vert , { \omega \over \beta } } \right ) + \cos ( 2\phi){f_s}\left({\beta \vert r\vert , { { 2\omega } \over \beta } } \right ) } \right],$$\end{document } 161b\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\dot h^{xy } } = - 32\beta \mu r_{12}^2{\omega ^4}\left [ { \sin \left({2\phi - { \pi \over 2 } } \right){f_c}\left({\beta \vert r\vert , { \omega \over \beta } } \right ) + \cos \left({2\phi - { \pi \over 2 } } \right){f_s}\left({\beta \vert r\vert , { { 2\omega } \over \beta } } \right ) } \right],$$\end{document } where = 2f is the orbital angular frequency and we have defined 162\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f_s}(x , z ) = \int\nolimits_0^\infty { { { ds } \over { \sqrt { { s^2 } + { x^2}}}}{{\mathcal j}_1}(s)\sin \;(z\sqrt { { s^2 } + { x^2}}),}$$\end{document } 163\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${f_c}(x , z ) = \int\nolimits_0^\infty { { { ds } \over { \sqrt { { s^2 } + { x^2}}}}{{\mathcal j}_1}(s)\sin \;(z\sqrt { { s^2 } + { x^2}}).}$$\end{document } and one has assumed that the binary is in the x - y plane and the observer is on the z - axis . however , if one expands these expressions about = , one recovers the gr solution to leading order , plus corrections that decay faster than 1/r . this then automatically implies that such modifications to the generation mechanism will be difficult to observe for sources at astronomical distances . given such a solution , one can compute the flux of energy carried by gravitational waves to spatial infinity . stein and yunes have shown that in quadratic gravity theories , this flux is still given by 164\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot e = { \kappa \over 2}\int { d\omega { r^2}\left\langle { { { \dot \bar h}_{\mu \nu}}{{\dot \bar h}^{\mu \nu } } } \right\rangle } , $ $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar h_{\mu v}}$\end{document } is the trace - reversed metric perturbation , the integral is taken over a 2-sphere at spatial infinity , and we recall that the angle brackets stand for an average over several wavelengths . given the solution in eq . ( 161 ) , one finds that the energy flux is 165\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot e = { 9 \over { 20}}{\mu ^2}r_{12}^2{\omega ^4}{\beta ^2}\left [ { \vert r\vert ^{2}f_c^2\left({\beta \vert r\vert , { { 2\omega } \over \beta } } \right ) + \vert r\vert ^{2}f_s^2\left({\beta \vert r\vert , { { 2\omega } \over \beta } } \right ) } \right].$$\end{document } the asymptotic expansion of the term in between square brackets about = is 166\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert r\vert ^{2}\left [ { f_c^2\left({\beta \vert r\vert , { { 2\omega } \over \beta } } \right ) + f_s^2\left({\beta \vert r\vert , { { 2\omega } \over \beta } } \right ) } \right ] \sim \vert r\vert ^{2}\left\{{{1 \over { { \beta ^2}\vert r\vert ^{2}}}\left [ { 1 + { \mathcal o}\left({{1 \over { \vert r\vert ^{2 } } } } \right ) } \right ] } \right\},$$\end{document } which then leads to an energy flux identical to that in gr , as any subdominant term goes to zero when the 2-sphere of integration is taken to spatial infinity . in that case , there are no modifications to the rate of change of the orbital frequency . of course , if one were not to expand about = , then the energy flux would lead to certain resonances at = 2 , but the energy flux is only well - defined at future null infinity . [ 326 , 325 ] to compute the rate of change of the orbital period of binary pulsars , in the hopes of using this to constrain . using data from the binary pulsar , they stipulated an order - of - magnitude constraint of 10 m. however , such an analysis could be revisited to relax a few assumptions used in [ 326 , 325 ] . first , binary pulsar constraints on modified gravity theories require the use of at least three observables . these observables can be , for example , the rate of change of the period , the line of nodes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot \omega$\end{document } and the perihelion shift . any one observable depends on the parameters ( m1 , m2 ) in gr or ( m1 , m2 , ) in non - commutative geometries , where m1,2 are the component masses . therefore , each observable corresponds to a surface of co - dimension one , i.e. , a two - dimensional surface or sheet in the three - dimensional space ( m1 , m2 , ) . if the binary pulsar observations are consistent with einstein s theory , then all sheets will intersect at some point , within a certain uncertainty volume given by the observational error . the simultaneous fitting of all these observables is what allows one to place a bound on . the analysis of [ 326 , 325 ] assumed that all binary pulsar observables were known , except for , but degeneracies between ( m1 , m2 , ) could potentially dilute constraints on these quantities . moreover , this analysis should be generalized to eccentric and inclined binaries , since binary pulsars are known to not be on exactly circular orbits . but perhaps the most important modification that ought to be made has to do with the calculation of the energy flux itself . ( 164 ) in terms of derivatives of the metric perturbation derives from the effective gravitational - wave stress - energy tensor , obtained by perturbatively expanding the action or the field equations and averaging over several wavelengths ( the isaacson procedure [ 241 , 242 ] ) . in modified gravity theories , the definition of the effective stress - energy tensor in terms of the metric perturbation is usually modified , as found for example in . in the case of non - commutative geometries , stein and however , the analysis of [ 326 , 325 ] evaluated this energy flux at a fixed distance , instead of taking the r limit . the balance law relates the rate of change of a binary s binding energy with the gravitational wave flux emitted by the binary , but for it to hold , one must require the following : ( i ) that the binary be isolated and possess a well - defined binding energy ; ( ii ) the total stress - energy of the spacetime satisfies a local covariant conservation law . if ( ii ) holds , one can use this conservation law to relate the rate of change of the volume integral of the energy density , i.e. , the energy flux , to the volume integral of the current density , which can be rewritten as an integral over the boundary of the volume through stokes theorem . since in principle one can choose any integration volume , any physically - meaningful result should be independent of the surface of that volume . this is indeed the case in gr , provided one takes the integration 2-sphere to spatial infinity . presumably , if one included all the relevant terms in , without taking the limit to i , one would still find a result that is independent of the surface of this two - sphere . therefore , the analysis of [ 326 , 325 ] should be taken as an interesting first step toward understanding possible changes in the gravitational - wave metric perturbation in non - commutative geometries . not much beyond this has been done regarding non - commutative geometries and gravitational waves . in particular , one lacks a study of what the final response function would be if the gravitational - wave propagation were modified , which of course depends on the time - evolution of all propagating gravitational - wave degrees of freedom , and whether there are only the two usual dynamical degrees of freedom in the metric perturbation . several massive graviton theories have been proposed to later be discarded due to ghosts , nonlinear or radiative instabilities . thus , little work has gone into studying whether black holes and neutron stars in these theories persist and are stable , and how the generation of gravitational waves is modified . such questions will depend on the specific massive gravity model considered , and of course , if a vainshtein mechanism is employed , then there will not be any modifications . one of them is that the non - dynamical ( near - zone ) gravitational field will be corrected , leading to yukawa - like modifications to the gravitational potential 167\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } g}}},\quad { \rm{or}}\quad { v_{{\rm{mg}}}}(r ) = { m \over r}\left({1 + { \gamma _ { { \rm{mg}}}}{e^{- r/{\lambda _ g } } } } \right),$$\end{document } where r is the distance from the source to a field point . such corrections lead to a fifth force , which then in turn allows us to place constraints on mg through solar system observations . nobody has yet considered how such modifications to the near - zone metric could affect the binding energy of compact binaries and their associated gravitational waves . another generic consequence of a graviton mass is the appearance of additional propagating degrees of freedom in the gravitational wave metric perturbation . in particular , one expects scalar , longitudinal modes to be excited ( see , e.g. , ) . this is , for example , the case if the action is of pauli - fierz type [ 169 , 148 ] . such longitudinal modes arise due to the non - vanishing of the 2 and 3 newman - penrose scalars , and can be associated with the presence of spin-0 particles , if the theory is of type n in the e(2 ) classification . the specific form of the scalar mode will depend on the structure of the modified field equations , and thus , it is not possible to generically predict its associated contribution to the response function . a robust prediction of massive graviton theories relates to how the propagation of gravitational waves is affected . if the graviton has a mass , its velocity of propagation will differ from the speed of light , as given for example in eq . ( 23 ) . will showed that such a modification in the dispersion relation leads to a correction in the relation between the difference in time of emission te and arrival ta of two gravitons : 168\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { t_a } = ( 1 + z)\left [ { \delta { t_e } + { d \over { 2\lambda _ g^2}}\left({{1 \over { f_e^2 } } + { 1 \over { f_e^{\prime2 } } } } \right ) } \right],$$\end{document } where z is the redshift , g is the graviton s compton wavelength , fe and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f_e^{\prime}$\end{document } are the emission frequencies of the two gravitons and d is the distance measure 169\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d = { { 1 + z } \over { { h_0}}}\int\nolimits_0^z { { { dz{\prime } } \over { { { ( 1 + { z{\prime}})}^2}{{[{\omega _ m}{{(1 + { z{\prime}})}^3 } + { \omega _ \lambda}]}^{1/2}}}},}$$ \end{document } where h0 is the present value of the hubble parameter , m is the matter energy density and is the vacuum energy density ( for a zero spatial - curvature universe ) . even if the gravitational wave at the source is unmodified , the graviton time delay will leave an imprint on the fourier transform of the response function by the time it reaches the detector . this is because the fourier phase is proportional to 170\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\psi \propto 2\pi \int\nolimits_{{f_c}}^f { [ t(f ) - { t_c}]df{\prime},}$$\end{document } where t is now not a constant but a function of frequency as given by eq . carrying out the integration , one finds that the fourier transform of the response function becomes 171\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde h_{{\rm{mg } } } } = { \tilde h_{{\rm{gr}}}}{e^{i{\beta _ { { \rm{mg}}}}{u^{{b_{{\rm{mg}}}}}}}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde h_{{\rm{gr}}}}$\end{document } is the fourier transform of the response function in gr , we recall that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$u = { ( \pi { \mathcal{m}_c}f)^{1/3}}$\end{document } and we have defined 172\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { { \rm{mg } } } } = - { { { \pi ^2}d{{\mathcal m}_c } } \over { \lambda _ g^2(1 + z)}},\quad { b_{{\rm{mg } } } } = - 3.$$\end{document } such a correction is of 1 post - newtonian order relative to the leading - order , newtonian term in the fourier phase . the most stringent solar system constraint is g > 2.8 10 km and it comes from observations of kepler s third law ( mainly mars orbit ) , which if the graviton had a mass would be modified by the yukawa factor in eq . observations of the rate of decay of the period in binary pulsars [ 174 , 53 ] can also be used to place the more stringent constraint > 1.5 10 km . similarly , studies of the stability of kerr black holes in pauli - fierz theory have yielded constraints of g > 2.4 10 km . gravitational - wave observations of binary systems could also be used to constrain the mass of the graviton once gravitational waves are detected . one possible test is to compare the times of arrival of coincident gravitational wave and electromagnetic signals , for example in white - dwarf binary systems . will was the first to consider constraints on g from gravitational - wave observations only . he considered sky - averaged , quasi - circular inspirals and found that ligo observations of 10 m equal - mass black holes would lead to a constraint of g > 6 10 km with a fisher analysis . such constraints are improved to g > 6.9 10 km with classic lisa observations of 10 m , equal - mass black holes . this increase comes about because the massive graviton correction accumulates with distance traveled ( see eq . since classic lisa would have been able to observe sources at gpc scales with high snr , its constraints on g would have been similarly stronger than what one would achieve with ligo observations . will s study was later generalized by will and yunes , who considered how the detector characteristics affected the possible bounds on g . they found that this bound scales with the square - root of the lisa arm length and inversely with the square root of the lisa acceleration noise . yagi and tanaka , arun and will , stavridis and will and berti et al . to allow for non - sky - averaged responses , spin - orbit and spin - spin coupling , higher harmonics in the gravitational wave amplitude , eccentricity and multiple detections . although the bound deteriorates on average for sources that are not optimally oriented relative to the detector , the bound improves when one includes spin couplings , higher harmonics , eccentricity , and multiple detections as the additional information and power encoded in the waveform increases , helping to break parameter degeneracies . however , all of these studies neglected the merger and ringdown phases of the coalescence , an assumption that was relaxed by keppel and ajith , leading to the strongest projected bounds g > 4 10 km . moreover , all studies until then had computed bounds using a fisher analysis prescription , an assumption relaxed by del pozzo et al . , who found that a bayesian analysis with priors consistent with solar system experiments leads to bounds stronger than fisher ones by roughly a factor of two . all of these results are summarized in table 2 , normalizing everything to an snr of 10 . in summary , projected constraints on g are generically stronger than current solar system or binary pulsar constraints by several orders of magnitude , given a lisa observation of massive black - hole mergers . even an aligo observation would do better than current solar system constraints by a factor between a few to an order of magnitude , depending on the source . reference binary mass g[10 km ] properties x0.0028solar - system dynamicsx1.6 10binary pulsar orbital period in visser s theory x0.024stability of black holes in pauli - fierz theory ( 10 , 10 ) m0.006ligo , fisher , ang . circular , non - spinning[281 , 126](0.5 , 0.5 ) m0.03lisa , wd - wd , coincident with electromagnetic signal(10 , 10 ) m50lisa , fisher , ang . circular , non - spinning(10 , 10)m10lisa , fisher , monte - carlo circular , w / spin - orbit(10,10)m10lisa , fisher , ang . higher - harmonics , circular , non - spinning(10 , 10)m22lisa , fisher , monte - carlo eccentric , spin - orbit(10 , 10)m2.4decigo , fisher , monte - carlo eccentric , spin - orbit(10 , 10)m50lisa , fisher , monte - carlo circular , w / spin modulations(10 , 10 ) m400lisa , fisher , ang . circular , non - spinning , w / merger(13 , 3)m0.0060.014ligo , bayesian , ang . circular , non - spinning(13 , 3)m30elisa , fisher , monte - carlo multiple detections , circular , non - spinning comparison of proposed tests of massive graviton theories . stands for an angular average over all sky locations . before proceeding , we should note that the correction to the propagation of gravitational waves due to a non - zero graviton mass are not exclusive to binary systems . in fact , any gravitational wave that propagates a significant distance from the source will suffer from the time delays described in this section . binary inspirals are particularly useful as probes of this effect because one knows the functional form of the waveform , and thus , one can employ matched filtering to obtain a strong constraint . but , in principle , one could use gravitational - wave bursts from supernovae or other sources . we have so far concentrated on massive graviton theories , but , as discussed in section 2.3.2 , there is a strong connection between such theories and lorentz violation . modifications to the dispersion relation are usually a result of a modification of the lorentz group or its action in real or momentum space . for this reason , it is interesting to consider generic lorentz - violating - inspired , modified dispersion relations of the form of eq . ( 24 ) , or more precisely 173\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{v_g^2 } \over { { c^2 } } } = 1 - a{e^{{\alpha _ { { \rm{lv } } } } - 2}},$$\end{document } where lv controls the structure of the modification and a its amplitude . when lv = 0 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a = m_g^2{c^2}$\end{document } one recovers the standard modified dispersion relation of eq . ( 173 ) introduces a generalized time delay between subsequent gravitons of the form 174\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { t_a } = ( 1 + z)\left [ { \delta { t_e } + { { { d_{{\alpha _ { { \rm{lv } } } } } } } \over { 2\lambda _ a^{2 - { \alpha _ { { \rm{lv}}}}}}}\left({{1 \over { f_e^{2 - { \alpha _ { { \rm{lv } } } } } } } - { 1 \over { f_e^{\prime2 - { \alpha _ { { \rm{lv } } } } } } } } \right ) } \right],$$\end{document } where we have defined \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\lambda _ a } \equiv { h_p}{a^{1/({\alpha _ { lv } } - 2)}}$\end{document } , with hp planck s constant , and the generalized distance measure 175\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${d_{{\alpha _ { { \rm{lv } } } } } } = { { { { ( 1 + z)}^{1 - { \alpha _ { { \rm{lv } } } } } } } \over { { h_0}}}\int\nolimits_0^z { { { { { ( 1 + z{\prime})}^{{\alpha _ { { \rm{lv } } } } - 2 } } } \over { { { [ { \omega _ m}{{(1 + z{\prime})}^3 } + { \omega _ \lambda}]}^{1/2}}}}dz{\prime}.}$$\end{document } such a modification then leads to the following correction to the fourier transform of the response function 176\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde h_{{\rm{lv } } } } = { \tilde h_{{\rm{gr}}}}{e^{i{\beta _ { { \rm{lv}}}}{u^{{b_{{\rm{lv}}}}}}}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde h_{{\rm{gr}}}}$\end{document } is the fourier transform of the response function in gr and we have defined 177\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta _ { { \rm{lv}}}^{{\alpha _ { { \rm{lv } } } } \neq 1 } = - { { { \pi ^{2 - { \alpha _ { { \rm{lv } } } } } } } \over { 1 - { \alpha _ { { \rm{lv}}}}}}{{{d_{{\alpha _ { { \rm{lv } } } } } } } \over { \lambda _ a^{2 - { \alpha _ { { \rm{lv}}}}}}}{{{\mathcal m}_c^{1 - { \alpha _ { { \rm{lv } } } } } } \over { { { ( 1 + z)}^{1 - { \alpha _ { { \rm{lv}}}}}}}},\quad b_{{\rm{lv}}}^{{\alpha _ { { \rm{lv } } } } \neq 1 } = 3({\alpha _ { { \rm{lv } } } } - 1).$$\end{document } the case lv = 1 is special leading to the fourier phase correction 178\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \psi _ { { \alpha _ { { \rm{lv } } } } = 1 } } = { { 3\pi { d_1 } } \over { { \lambda _ a}}}\ln u.$$\end{document } the reason for this is that when lv = 1 the fourier phase is proportional to the integral of 1/f , which then leads to a natural logarithm . different lv limits deserve further discussion here . of course , when lv = 0 , one recovers the standard massive graviton result with the mapping \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\lambda _ g^{- 2 } \rightarrow \lambda _ g^{- 2 } + \lambda _ 2}$\end{document}. when lv = 2 , the dispersion relation is identical to that in eq . ( 23 ) , but with a redefinition of the speed of light , and should thus be unobservable . indeed , in this limit the correction to the fourier phase in eq . ( 176 ) becomes linear in frequency , and this is 100% degenerate with the time of coalescence parameter in the standard gr fourier phase . finally , relative to the standard gr terms that arise in the post - newtonian expansion of the fourier phase , the new corrections are of ( 1 + 3lv/2 ) post - newtonian order . then , if ligo gravitational - wave observations were incapable of discerning between a 4 post - newtonian and a 5 post - newtonian waveform , then such observations would not be able to see the modified dispersion effect if lv > 2 . mirshekari et al . confirmed this expectation with a fisher analysis of non - spinning , comparable - mass quasi - circular inspirals . they found that for lv = 3 , one can place very weak bounds on a , namely a < 10 ev with a ligo observation of a ( 1.4 , 1.4 ) m neutron star inspiral , a < 0.2 ev with an enhanced - lisa or ngo observation of a ( 10 , 10 ) m black - hole inspiral , assuming a snr of 10 and 100 respectively . a word of caution is due here , though , as these analyses neglect any lorentz - violating correction to the generation of gravitational waves , including the excitation of additional polarization modes . one would expect that the inclusion of such effects would only strengthen the bounds one could place on lorentz - violating theories , but this must be done on a theory by theory basis . the lack of a particular lagrangian associated with variable g theories , excluding scalar - tensor theories , or extra dimensions , makes it difficult to ascertain whether black - hole or neutron - star binaries exist in such theories . whether this is so will depend on the particular variable g model considered . in spite of this , if such binaries do exist , the gravitational waves emitted by such systems will carry some generic modifications relative to the gr expectation . most current tests of the variability of newton s gravitational constant rely on electromagnetic observations of massive bodies , such as neutron stars . as discussed in section 2.3.4 , scalar - tensor theories can be interpreted as variable - g theories , where the variability of g is really a variation in the coupling between gravity and matter . however , newton s constant serves the more fundamental role of defining the relationship between geometry or length and energy , and such a relationship is not altered in most scalar - tensor theories , unless the scalar fields are allowed to vary on a cosmological scale ( background , homogeneous scalar solution ) . for this reason , one might wish to consider a possible temporal variation of newton s constant in pure vacuum spacetimes , such as in black - hole - binary inspirals . such temporal variation would encode / g at the time and location of the merger event . thus , once a sufficiently large number of gravitational wave events has been observed and found consistent with gr , one could reconstruct a constraint map that bounds / g along our past light cone ( as a function of redshift and sky position ) . since our past - light cone with gravitational waves would have extended to roughly redshift 10 with classic lisa ( limited by the existence of merger events at such high redshifts ) , such a constraint map would have been much more complete than what one can achieve with current tests at redshift almost zero . big bang nucleosynthesis constraints also allow us to bound a linear drift in / g from z 10 to zero , but these become degenerate with limits on the number of relativistic species . moreover , these bounds exploit the huge lever - arm provided by integrating over cosmic time , but they are insensitive to local , oscillatory variations of g with periods much less than the cosmic observation time . thus , gravitational - wave constraint maps would test one of the pillars of gr : local position invariance . this principle ( encoded in the equivalence principle ) states that the laws of physics ( and thus the fundamental constants of nature ) are the same everywhere in the universe . let us then promote g to a function of time of the form 179\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g(t , x , y , z ) \approx { g_{\rm{c } } } + { \dot g_{\rm{c}}}({t_c } - t),$$\end{document } where gc = g(tc , xc , yc , zc ) and c = ( g/t)(tc , xc , yc , zc ) are constants , and the sub - index c means that these quantities are evaluated at coalescence . clearly , this is a taylor expansion to first order in time and position about the coalescence event \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({t_c},x_c^i)$\end{document } , which is valid provided the spatial variation of g is much smaller than its temporal variation , i.e. , |g| , and the characteristic period of the temporal variation is longer than the observation window ( at most , tobs 3 years for classic lisa ) , so that ctobs gc . similar parameterization of g(t ) have been used to study deviations from newton s second law in the solar system [ 149 , 430 , 427 , 411 ] . thus , one can think of this modification as the consequence of some effective theory that could represent the predictions of several different alternative theories . the promotion of newton s constant to a function of time changes the rate of change of the orbital frequency , which then directly impacts the gravitational - wave phase evolution . to leading order , yunes et al . find 180\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot f = { \dot f_{{\rm{gr } } } } + { { 195 } \over { 256\pi}}{\mathcal m}_c^{- 2}{x^3}{\eta ^{3/5}}({\dot g_c}{{\mathcal m}_c}),$$\end{document } where gr is the rate of change of the orbital frequency in gr , due to the emission of gravitational waves and x = ( 2mf)/. such a modification to the orbital frequency evolution leads to the following modification to the fourier transform of the response function in the stationary - phase approximation [ 56 , 125 , 153 , 457 ] 181\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h = { \tilde h_{{\rm{gr}}}}(1 + { \alpha _ { \dot g}}{u^{{a_{\dot g}}}}){e^{i{\beta _ { \dot g}}{u^{{b_{\dot g}}}}}},$$\end{document } where we recall again that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$u = { ( \pi { \mathcal{m}_c}f)^{1/3}}$\end{document } and have defined the constant parameters 182\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\alpha _ { \dot g } } = - { 5 \over { 512}}{{{{\dot g}_c } } \over { { g_c}}}({g_c}{{\mathcal m}_z}),\quad { \beta _ { \dot g } } = - { { 25 } \over { 65536}}{{{{\dot g}_c } } \over { { g_c}}}({g_c}{{\mathcal m}_z}),\quad a = - 8,\quad b = - 13,$$\end{document } to leading order in the post - newtonian approximation . we note that this corresponds to a correction of 4 post - newtonian order in the phase , relative to the leading - order term , and that the corrections are independent of the symmetric mass ratio , scaling only with the redshifted chirp mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal{m}_z}$\end{document}. due to this , one expects the strongest effects to be seen in low - frequency gravitational waves , such as those one could detect with lisa or decigo / bbo . given such corrections to the gravitational - wave response function , one can investigate the level to which a gravitational - wave observation consistent with gr would allow us to constrain c . . carried out such a study and found that for comparable - mass black - hole inspirals of total redshifted mass mz = 10 m with lisa , one could constrain c / gc 10 yr or better to redshift 10 ( assuming an snr of 10 ) . the constraint is strengthened when one considers intermediate - mass black - hole inspirals , where one would be able to achieve a bound of c / gc 10 yr . although this is not as stringent as the strongest constraints from other observations ( see section 2.3.4 ) , we recall that gravitational - wave constraints would measure local variations at the source , as opposed to local variations at zero redshift or integrated variations from the very early universe . the effect of promoting newton s constant to a function of time is degenerate with several different effects . one such effect is a temporal variability of the black hole masses , i.e. , if 0 . such time - variation could be induced by gravitational leakage into the bulk in certain brane - world scenarios , as explained in section 2.3.4 . for a black hole of mass m , the rate of black hole evaporation is given by 183\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{dm } \over { dt } } = - 2.8 \times { 10^{- 7}}{\left({{{1{m _ \odot } } \over m } } \right)^2}{\left({{\ell \over { 10\mu { \rm{m } } } } } \right)^2}{m _ as expected , such a modification to a black - hole - binary inspiral will lead to a correction to the fourier transform of the response function that is identical in structure to that of eq . ( 181 ) , but the parameters ( , b ) ( ed , bed ) change to 184\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { { \rm{ed } } } } = - 8.378 \times { 10^{- 8}}{\left({{\ell \over { { { \mathcal m}_c } } } } \right)^4}\left({1 - { { 26 } \over 3}\eta + 34{\eta ^2 } } \right),\quad { b_{{\rm{ed } } } } = - 13.$$\end{document } a similar expression is found for a neutron - star / black - hole inspiral , except that the -dependent factor in between parenthesis is corrected . given a gravitational - wave detection consistent with gr , one could then , in principle , place an upper bound on . yagi et al . carried out a fisher analysis and found that a 1-year lisa detection would constrain 10 m with a ( 10 , 10 ) mq binary inspiral at an snr of 100 . this constraint is roughly two orders of magnitude weaker than current table - top experiment constraints . moreover , the constraint weakens somewhat for more generic inspirals , due to degeneracies between and eccentricity and spin . however , a similar observation with the third generation detector decigo / bbo should be able to beat current constraints by roughly one order of magnitude . such a constraint could be strengthened by roughly one order of magnitude further , if one included the statistical enhancement in parameter estimation due to detection of order 10 sources by decigo / bbo . another way to place a constraint on is to consider the effect of mass loss in the orbital dynamics . when a system loses mass , the evolution of its semi - major axis a will acquire a correction of the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot a = - ( \dot m / m)a$\end{document } , due to conservation of specific orbital angular momentum . there is then a critical semi - major axis ac at which this correction balances the semi - major decay rate due to gravitational wave emission . mcwilliams argues that systems with a < ac are then gravitational - wave dominated and will thus inspiral , while systems with a > ac will be mass - loss dominated and will thus outspiral . if a gravitational wave arising from an inspiraling binary is detected at a given semi - major axis , then is automatically constrained to about \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{o}(20\mu { \rm{m}})$\end{document}. yagi et al . extended this analysis to find that such a constraint is weaker than what one could achieve via matched filtering with a waveform in the form of eq . the correction to the gravitational - wave phase evolution is also degenerate with cosmological acceleration . that is , if a gravitational wave is generated at high - redshift , its phase will be affected by the acceleration of the universe . to zeroth - order , however , if one allows the redshift to be a function of time 185\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z \sim { z_c } + { \dot z_c}(t - { t_c } ) \sim { z_c } + { h_0}\left [ { { { ( 1 + { z_c})}^2 } - { { ( 1 + { z_c})}^{5/2}}\omega _ m^{1/2 } } \right](t - { t_c}),$$\end{document } then the observed waveform at the detector becomes structurally identical to eq . ( 181 ) but with the parameters 186\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { \dot z } } = { { 25 } \over { 32768}}{\dot z_c}{{\mathcal m}_z},\quad { b_{\dot z } } = - 13.$$\end{document } however , using the measured values of the cosmological parameters from the wmap analysis [ 271 , 156 ] , one finds that this effect is roughly 10 times smaller than that of a possible correction at the level of the possible bounds quoted above . of course , if one could in the future constrain better by 3 orders of magnitude , possible degeneracies with would become an issue . a final possible degeneracy arises with the effect of a third body , accretion disk migration [ 267 , 462 ] and the interaction of a binary with a circumbinary accretion disk . all of these effects introduce corrections to the gravitational - wave phase of negative pn order , just like the effect of a variable gravitational constant . however , degeneracies of this type are only expected to affect a small subset of black - hole - binary observations , namely those with a third body sufficiently close to the binary , or a sufficiently massive accretion disk . as discussed in section 2.3.6 the simplest action to model parity violation in the gravitational interaction a generic feature of this theory is that parity violation imprints onto the propagation of gravitational waves , an effect that has been dubbed amplitude birefringence . such birefringence is not to be confused with optical or electromagnetic birefringence , in which the gauge boson interacts with a medium and is doubly - refracted into two separate rays . in amplitude birefringence , right-(left)-circularly polarized gravitational waves are enhanced or suppressed ( suppressed or enhanced ) relative to the gr expectation as they propagate [ 245 , 295 , 11 , 460 , 17 , 464 ] . one can understand amplitude birefringence in gravitational wave propagation due to a possible non - commutativity of the parity operator and the hamiltonian . the hamiltonian is the generator of time evolution , and thus , one can write 187\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left({\begin{array}{*{20}c } { { h_{+ , k}}(t)}\\ { { h_{\times , k}}(t)}\\ \end{array } } \right ) = { e^{- ift}}\left({\begin{array}{*{20}c } { { u_c}\;iv}\\ { - iv\;{u_c}}\\ \end{array } } \right)\;\left({\begin{array}{*{20}c } { { h_{+ , k}}(0)}\\ { { h_{\times , k}}(0)}\\ \end{array } } \right),$$\end{document } where f is the gravitational - wave angular frequency , t is time , and h+,,k are the gravitational wave fourier components with wavenumber k. the quantity uc models possible background curvature effects , with uc = 1 for propagation on a minkowski metric , and proportional to redshift for propagation on a friedman - robertson - walker metric . the quantity models possible parity - violating effects , with = 0 in gr . one can rewrite the above equation in terms of right and left - circular polarizations , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${h_{{\rm{r , l } } } } = ( { h _ + } \pm i{h _ \times})/\sqrt 2$\end{document } to find 188\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left({\begin{array}{*{20}c } { { h_{{\rm{r}},k}}(t)}\\ { { h_{{\rm{l}},k}}(t)}\\ \end{array } } \right ) = { e^{- ift}}\left({\begin{array}{*{20}c } { { u_c } + v } & 0\\ { \quad \;\;0 } & { { u_c } - v}\\ \end{array } } \right)\;\left({\begin{array}{*{20}c } { { h_{{\rm{r}},k}}(0)}\\ { { h_{{\rm{l}},k}}(0)}\\ \end{array } } \right).$$\end{document } amplitude birefringence has the effect of modifying the eigenvalues of the diagonal propagator matrix for right and left - polarized waves , with right modes amplified or suppressed and left modes suppressed or enhanced relative to gr , depending on the sign of . in addition to these parity - violating propagation effects , parity violation should also leave an imprint in the generation of gravitational waves . however , such effects need to be analyzed on a theory by theory basis . moreover , the propagation - distance - independent nature of generation effects should make them easily distinguishable from the propagation effects we consider here . the degree of parity violation , , can be expressed entirely in terms of the waveform observables via 189\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v = { 1 \over 2}\left({{{{h_{\rm{r } } } } \over { h_{\rm{r}}^{{\rm{gr } } } } } - { { { h_{\rm{l } } } } \over { h_{\rm{l}}^{{\rm{gr } } } } } } \right ) = { i \over 2}(\delta { \phi _ { \rm{l } } } - \delta { \phi _ { \rm{r}}}),$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{{\rm{r , l}}}^{{\rm{gr}}}$\end{document } is the gr expectation for a right or left - polarized gravitational wave . in the last equality we have also introduced the notation , where is the gr gravitational - wave phase and 190\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_{{\rm{r , l } } } } = { h_{0,{\rm{r , l}}}}{e^{- i[\phi ( \eta ) - { \kappa _ i}{\chi ^i}]}},$$\end{document } where h0,r , l is a constant factor , is the conformal wave number and ( , ) are conformal coordinates for propagation in a friedmann - robertson - walker universe . for example , in non - dynamical chern - simons gravity with a field = (t ) , and in an expansion about z 1 , one finds 191\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v = { \alpha \over \kappa}\pi fz\left({{{\dot \vartheta}_0 } - { { { { \ddot \vartheta}_0 } } \over { { h_0 } } } } \right ) = { \alpha \over \kappa}\pi fd\left({{h_0}{{\dot \vartheta}_0 } - { { \ddot \vartheta}_0 } } \right),$$\end{document } where 0 is the chern - simons scalar field at the detector , with the chern - simons coupling constant [ see , e.g. , eq . ( 45 ) ] , z is redshift , d is the comoving distance and h0 is the value of the hubble parameter today and f is the observed gravitational - wave frequency . when considering propagation on a minkowski background , one obtains the above equation in the limit as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot a \rightarrow 0$\end{document } , so the second term dominates , where a is the scale factor . to leading - order in a curvature expansion , the parity - violating coefficient will always be linear in frequency , as shown in eq . , will be proportional to ( fd)f , where is a coupling constant of the theory ( or a certain derivative of a coupling field ) with units of [ length ] ( in the previous case , a = 0 , so the correction was simply proportional to fd , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha \propto \ddot \vartheta$\end{document } ) . ( 188 ) one can easily show that the fourier transform of the response function becomes [ 11 , 460 , 464 ] 192\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde h_{{\rm{pv } } } } = ( { f _ + } - i\;v\;{f _ \times}){\tilde h _ + } + ( { f _ \times } + i\;v\;{f _ + } ) { \tilde h _ \times}.$$\end{document } of course , one can rewrite this in terms of a real amplitude correction and a real phase correction . expanding in 1 to leading order , we find 193\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde h_{{\rm{pv } } } } = { \tilde h^{{\rm{gr}}}}(1 + v\;\delta { q_{{\rm{pv}}}}){e^{i{v^2}\delta { \psi _ { { \rm{pv}}}}}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde h_{{\rm{gr}}}}$\end{document } is the fourier transform of the response function in gr and we have defined 194\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q_{{\rm{gr } } } } = \sqrt { f _ + ^2{{(1 + { { \cos}^2}\iota)}^2 } + 4{{\cos}^2}\iota f _ \times ^2 } , $ $ \end{document } 195\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { q_{{\rm{pv } } } } = { { 2(1 + { { \cos}^2}\iota)\cos \iota \;(f _ + ^2 + f _ \times ^2 ) } \over { q_{{\rm{gr}}}^2}},$$\end{document } 196\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \psi _ { \rm{pv } } = { 2\cos \iota ( 1 + \cos^{2}\iota)(1 - \cos^{2}\iota)^{2}(f_{+}^{2 } + f_{\times}^{2})f_{+}f _ { \times } \over q_{\rm{gr}}^{4}}.$$\end{document } we see then that amplitude birefringence modifies both the amplitude and the phase of the response function . using the non - dynamical chern - simons expression for in eq . ( 193 ) as 197\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde h_{{\rm{pv } } } } = { \tilde h^{{\rm{gr}}}}(1 + { \alpha _ { { \rm{pv}}}}{u^{{a_{{\rm{pv}}}}}}){e^{i{\beta _ { { \rm{pv}}}}{u^{{b_{{\rm{pv}}}}}}}},$$\end{document } where we have defined the coefficients 198\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\alpha _ { { \rm{pv } } } } = \left({{d \over { { { \mathcal m}_c } } } } \right)\left [ { { { 2(1 + { { \cos}^2}\iota)\cos \iota ( f _ + ^2 + f _ \times ^2 ) } \over { q_{{\rm{gr}}}^2 } } } \right]{\alpha \over \kappa}\left({{h_0}{{\dot \vartheta}_0 } - { { \ddot \vartheta}_0 } } \right),\;\;\;{a_{{\rm{pv } } } } = 3,$$\end{document } 199\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { { \rm{pv } } } } = { \left({{d \over { { { \mathcal m}_c } } } } \right)^2}\left [ { { { 2\cos \iota ( 1 + { { \cos}^2}\iota){{(1 - { { \cos}^2}\iota)}^2}(f _ + ^2 + f _ \times ^2){f _ + } { f _ \times } } \over { q_{{\rm{gr}}}^2 } } } \right]{\alpha \over \kappa}{\left({{h_0}{{\dot \vartheta}_0 } - { { \ddot \vartheta}_0 } } \right)^2},\;\;\;{b_{{\rm{pv } } } } = 6,$$\end{document } where we recall that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$u = { ( \pi { \mathcal{m}_c}f)^{1/3}}$\end{document}. the phase correction corresponds to a term of 5.5 post - newtonian order relative to the newtonian contribution , and it scales quadratically with the chern - simons coupling field , which is why it was left out in . the amplitude correction , on the other hand , is of 1.5 post - newtonian order relative to the newtonian contribution . since both of these appear as positive - order , post - newtonian corrections , there is a possibility of degeneracy between them and standard waveform template parameters . given such a modification to the response function , one can ask whether such parity violation is observable with current detectors . [ 11 , 460 ] argued that a gravitational wave observation with lisa would be able to constrain an integrated measure of , because lisa can observe massive - black - hole mergers to cosmological distances , while amplitude birefringence accumulates with distance traveled . for such an analysis , one can not taylor expand about its present value , and instead , one finds that 200\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{1 + v } \over { 1 - v } } = { e^{2\pi f\zeta ( z)}},$$\end{document } where we have defined 201\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\zeta ( z ) = { { \alpha { h_0 } } \over \kappa}\int\nolimits_0^z { dz } { ( 1 + z)^{5/2}}\left [ { { 7 \over 2}{{d\vartheta } \over { dz } } + ( 1 + z){{{d^2}\vartheta } \over { d{z^2 } } } } \right].$$\end{document } we can solve the above equation to find 202\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v = { { { e^{2\pi f\zeta ( z ) } } - 1 } \over { 1 + { e^{2\pi f\zeta ( z ) } } } } \sim \pi f\zeta ( z),$$\end{document } where in the second equality we have linearized about 1 and f 1 . [ 11 , 460 ] realized that this induces a time - dependent change in the inclination angle ( i.e. , the apparent orientation of the binary s orbital angular momentum with respect to the observer s line - of - sight ) , since the latter can be defined by the ratio hr / hl . they then carried out a simplified fisher analysis and found that a lisa observation of the inspiral of two massive black holes with component masses 10 m(1 + z ) at redshift z = 15 would allow us to constrain the integrated dimensionless measure < 10 to 1. one might worry that such an effect would be degenerate with other standard gr processes that induce similar time - dependencies , such as spin - orbit coupling . however , this time - dependence is very different from that of the parity - violating effect , and thus , alexander et al . [ another test of parity violation was proposed by yunes et al . , who considered the coincident detection of a gravitational wave and a gamma - ray burst with the swift and glast / fermi gamma - ray satellites , and the ground - based ligo and virgo gravitational wave detectors . if the progenitor of the gamma - ray burst is a neutron - star / neutron - star merger , the gamma - ray jet is expected to be highly collimated . therefore , an electromagnetic observation of such an event implies that the binary s orbital angular momentum at merger must be pointing along the line of sight to earth , leading to a strongly - circularly - polarized gravitational - wave signal and to maximal parity violation . if the gamma - ray burst observation were to provide an accurate sky location , one would be able to obtain an accurate distance measurement from the gravitational wave signal alone . moreover , since glast / fermi observations of gamma - ray bursts occur at low redshift , one would also possess a purely electromagnetic measurement of the distance to the source . therefore , if no discrepancy is found , the error ellipse on the distance measurement would allow us to place an upper limit on any possible gravitational parity violation . because of the nature of such a test , one is constraining generic parity violation over distances of hundreds of mpc , along the light cone on which the gravitational waves propagate . the coincident gamma - ray burst / gravitational - wave test compares favorably to the pure lisa test , with the sensitivity to parity violation being about 23 orders of magnitude better in the former case . this is because , although the fractional error in the gravitational - wave distance measurement is much smaller for lisa than for ligo , since it is inversely proportional to the snr , the parity violating effect also depends on the gravitational - wave frequency , which is much larger for neutron - star inspirals than massive black - hole coalescences . mathematically , the simplest models of gravitational parity violation will lead to a signature in the response function that is proportional to the gravitational - wave wavelength12 gw df . although the coincident test requires small distances and low snrs ( by roughly 12 orders of magnitude ) , the frequency is also larger by a factor of 56 orders of magnitude for the ligo - virgo network . the coincident gamma - ray burst / gravitational - wave test also compares favorably to current solar system constraints . using the motion of the lageos satellites , smith et al . have placed the 1 bound \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\dot \vartheta _ 0 } < 2000$\end{document } km assuming \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\ddot \vartheta _ 0 } = 0$\end{document}. a similar assumption leads to a 2 bound of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\dot \vartheta _ 0 } < 200$\end{document } km with a coincident gamma - ray burst / gravitational - wave observation . moreover , the latter test also allows us to constrain the second time - derivative of the scalar field . finally , a lisa observation would constrain the integrated history of along the past light cone on which the gravitational wave propagated . however , these tests are not as stringent as the recently proposed test by dyda et al . , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\dot \vartheta _ 0 } < { 10^{- 7}}$\end{document } km , assuming the effective theory cut - off scale is less than 10 ev and obtained by demanding that the energy density in photons created by vacuum decay over the lifetime of the universe not violate observational bounds . the coincident test is somewhat idealistic in that there are certain astrophysical uncertainties that could hamper the degree to which we could constrain parity violation . one of the most important uncertainties relates to our knowledge of the inclination angle , as gamma - ray burst jets are not necessarily perfectly aligned with the line of sight . if the inclination angle is not known a priori , it will become degenerate with the distance in the waveform template , decreasing the accuracy to which the luminosity could be extracted from a pure gravitational wave observation by at least a factor of two . even after taking such uncertainties into account , yunes et al . found that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\dot \vartheta _ 0}$\end{document } could be constrained much better with gravitational waves than with current solar system observations . one of the biggest disadvantages of a top - down or direct approach toward testing gr is that one must pick a particular theory from the beginning of the analysis . however , given the large number of possible modifications to einstein s theory and the lack of a particularly compelling alternative , it is entirely possible that none of these will represent the correct gravitational theory in the strong field . thus , if one carries out a top - down approach , one will be forced to make the assumption that we , as theorists , know which modifications of gravity are possible and which are not . the parameterized post - einsteinian ( ppe ) approach is a framework developed specifically to alleviate such a bias by allowing the data to select the correct theory of nature through the systematic study of statistically significant anomalies . for detection purposes but if gr happened to be wrong in the strong field , it is possible that a gr template would still extract the signal , but with the wrong parameters . that is , the best fit parameters obtained from a matched filtering analysis with gr templates will be biased by the assumption that gr is sufficiently accurate to model the entire coalescence . this fundamental bias could lead to a highly distorted image of the gravitational - wave universe . in fact , recent work by vallisneri and yunes indicates that such fundamental bias could indeed be present in observations of neutron star inspirals , if gr is not quite the right theory in the strong - field . one of the primary motivations for the development of the ppe scheme was to alleviate fundamental bias , and one of its most dangerous incarnations : stealth - bias . if gr is not the right theory of nature , yet all our future detections are of low snr , we may estimate the wrong parameters from a matched - filtering analysis , yet without being able to identify that there is a non - gr anomaly in the data . thus , stealth bias is nothing but fundamental bias hidden by our limited snr observations . vallisneri and yunes have found that such stealth - bias is indeed possible in a certain sector of parameter space , inducing errors in parameter estimation that could be larger than statistical ones , without us being able to identify the presence of a non - gr anomaly . the ppe scheme was designed in close analogy with the parameterized post - newtonian ( ppn ) framework , developed in the 1970s to test gr with solar system observations ( see , e.g. , for a review ) . in the solar system , all direct observables depend on a single quantity , the metric , which can be obtained by a small - velocity / weak - field post - newtonian expansion of the field equations of whatever theory one is considering . thus , will and nordtvedt [ 331 , 432 , 439 , 332 , 433 ] proposed the generalization of the solar system metric into a meta - metric that could effectively interpolate between the predictions of many different alternative theories . this meta - metric depends on the product of certain green function potentials and ppn parameters . for example , the spatial - spatial components of the meta - metric take the form 203\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{ij } } = { \delta _ { ij}}(1 + 2\gamma u + \ldots),$$\end{document } where ij is the kronecker delta , u is the newtonian potential and is one of the ppn parameters , which acquires different values in different theories : = 1 in gr , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma = ( 1 + { \omega _ { { \rm{bd}}}}){(2 + { \omega _ { { \rm{bd}}}})^{- 1}}\sim 1 - \omega _ { { \rm{bd}}}^{- 1}$\end{document } in jordan - fierz - brans - dicke theory , etc . therefore , any solar system observable could then be written in terms of system parameters , such as the masses of the planets , and the ppn parameters . an observation consistent with gr allows for a bound on these parameters , thus simultaneously constraining a large class of modified gravity theories . the idea behind the ppe framework was to develop a formalism that allowed for similar generic tests but with gravitational waves instead of solar system observations . [ 37 , 317 ] , who considered the quasi - circular inspiral of compact objects . they suggested the waveform template family 204\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde h_{{\rm{pnt } } } } = { \tilde h^{{\rm{gr}}}}{e^{i{\beta _ { { \rm{pnt}}u}}^{{b_{{\rm{pn}}}}}}}.$$\end{document } this waveform depends on the standard system parameters that are always present in gr waveforms , plus one theory parameter pnt that is to be constrained . the quantity bpn is a number chosen by the data analyst and is restricted to be equal to one of the post - newtonian predictions for the phase frequency exponents , i.e. , bpn = ( 5 , 3 , 2 , 1 , ) . ( 204 ) allows for post - newtonian tests of gr , i.e. , consistency checks of the signal with the post - newtonian expansion . for example , let us imagine that a gravitational wave has been detected with sufficient snr that the chirp mass and mass ratio have been measured from the newtonian and 1 post - newtonian terms in the waveform phase . one can then ask whether the 1.5 post - newtonian term in the phase is consistent with these values of chirp mass and mass ratio . put another way , each term in the phase can be thought of as a curve in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({m_c},\eta)$\end{document } space . if gr is correct , all these curves should intersect inside some uncertainty box , just like when one tests gr with binary pulsar data . from that standpoint , these tests can be thought of as null - tests of gr and one can ask : given an event , is the data consistent with the hypothesis rppe = 0 for the restricted set of frequency exponents bpn ? a fisher and a bayesian data analysis study of how well pnt could be constrained given a certain bpn was carried out in [ 317 , 240 , 290 ] . mishra et al . considered the quasi - circular inspiral of non - spinning compact objects and showed that aligo observations would allow one to constrain pnt to 6% up to the 1.5 post - newtonian order correction ( bpn = 2 ) . third - generation detectors , such as et , should allow for better constraints on all post - newtonian coefficients to roughly 2% . clearly , the higher the value of bpn , the worse the bound on pn because the power contained in higher frequency exponent terms decreases , i.e. , the number of useful additional cycles induced by the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\beta _ { pnt}}{u^{b{\rm{pn}}}}$\end{document } term decreases as bpn increases . huwyler et al . repeated this analysis but for lisa observations of the quasi - circular inspiral of black hole binaries with spin precession . they found that the inclusion of precessing spins forces one to introduce more parameters into the waveform , which dilutes information and weakens constraints on pnt by as much as a factor of 5 . . carried out a bayesian analysis of the odds - ratio between gr and restricted ppe templates given a non - spinning , quasi - circular compact binary inspiral observation with aligo and advirgo . they calculated the odds ratio for each value of bpn listed above and then combined all of this into a single probability measure that allows one to quantify how likely the data is to be consistent with gr . ( 204 ) is that it is not rooted on a theoretical understanding of modified gravity theories . to alleviate this problem , yunes and pretorius re - considered the quasi - circular inspiral of compact objects . they proposed a more general ppe template family through generic deformations of the = 2 harmonic of the response function in fourier space : 205\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h_{{\rm{ppe , insp,1}}}^{(\ell = 2 ) } = { \tilde h^{{\rm{gr}}}}(1 + { \alpha _ { { \rm{ppe}}}}{u^{{a_{{\rm{ppe}}}}}}){e^{i{\beta _ { { \rm{ppe}}}}{u^{{b_{{\rm{ppe}}}}}}}},$$\end{document } where now ( ppe , appe , ppe , bppe ) are all free parameters to be fitted by the data , in addition to the usual system parameters . this waveform family reproduces all predictions from known modified gravity theories : when ( ppe , ppe ) = ( 0,0 ) , the waveform reduces exactly to gr , while for other parameters one reproduces the modified gravity predictions of table 3 . table 3parameters that define the deformation of the response function in a variety of modified gravity theories . the notation means that a value for this parameter is irrelevant , as its amplitude is zero.theory ppe a ppe ppe b ppe jordan - fierz - brans - dicke \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { 5 \over { 96}}{{{s^2 } } \over { { \omega _ { { \rm{bd}}}}}}{\eta ^{2/5}}$\end{document } 2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { 5 \over { 3584}}{{{s^2 } } \over { { \omega _ { { \rm{bd}}}}}}{\eta ^{2/5}}$\end{document } 7dissipative einstein - dilaton - gauss - bonnet gravity0 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { 5 \over { 7168}}{\varsigma _ 3}{\eta ^{- 18/5}}\delta _ m^2$\end{document } 7massive graviton0 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { { { \pi ^2}d{{\mathcal m}_c } } \over { \lambda _ g^2(1 + z)}}$\end{document } 3lorentz violation0 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { { { \pi ^{2 - { \gamma _ { { \rm{lv } } } } } } } \over { ( 1 - { \gamma _ { { \rm{lv}}}})}}{{{d_{{\gamma _ { { \rm{lv } } } } } } } \over { \lambda _ { { \rm{lv}}}^{2 - { \gamma _ { { \rm{lv}}}}}}}{{{\mathcal m}_c^{1 - { \gamma _ { { \rm{lv } } } } } } \over { { { ( 1 + z)}^{1 - { \gamma _ { { \rm{lv}}}}}}}}$\end{document } 3lv 3g(t ) theory \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { 5 \over { 512}}\dot g{{\mathcal m}_c}$\end{document } 8 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { { 25 } \over { 65536}}{{\dot g}_c}{{\mathcal m}_c}$\end{document } 13extra dimensions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { { 75 } \over { 2554344}}{{dm } \over { dt}}{\eta ^{- 4}}(3 - 26\eta + 24{\eta ^2})$\end{document } 13non - dynamical chern - simons gravity pv 3 pv 6dynamical chern - simons gravity0 dcs 1 parameters that define the deformation of the response function in a variety of modified gravity theories . the notation means that a value for this parameter is irrelevant , as its amplitude is zero . in table 3 , recall that s is the difference in the square of the sensitivities and bd is the brans - dicke coupling parameter ( see section 5.2.1 ; we have here neglected the scalar mode ) , 3 is the coupling parameter in einstein - dilaton - gauss - bonnet theory ( see section 5.2.2 ) , where we have here included both the dissipative and the conservative corrections , d is a certain distance measure and g is the compton wavelength of the graviton ( see section 5.3.1 ) , lv is a distance scale at which lorentz - violation becomes important and lv is the graviton momentum exponent in the deformation of the dispersion relation ( see section 5.3.1 ) , c is the value of the time derivative of newton s constant at coalescence and dm / dt is the mass loss due to enhanced hawking radiation in extra - dimensional scenarios ( see section 5.3.2 ) , dcs is given in eq . although there are only a few modified gravity theories where the leading - order post - newtonian correction to the fourier transform of the response function can be parameterized by post - newtonian waveforms of eq . ( 204 ) , all such predictions can be modeled with the ppe templates of eq . in fact , only massive graviton theories , certain classes of lorentz - violating theories and dynamical chern - simons gravity lead to waveform corrections that can be parameterized via eq . ( 204 ) does not allow for tests of gravitational parity violation or non - dynamical chern - simons gravity . ( 205 ) can be understood as a single - parameter deformation away from einstein s theory . if the correct theory of nature happens to be a deformation of gr with several parameters ( e.g. , several coupling constants , mass terms , potentials , etc . ) , then eq . ( 205 ) will only be able to parameterize the one that leads to the most useful cycles . ( 205 ) assumes that the modification can be represented as a power series in velocity , with possibly non - integer values . such an assumption does not allow for possible logarithmic terms , which are known to arise due to non - linear memory interactions at sufficiently - high post - newtonian order . it also does not allow for interactions that are screened , e.g. , in theories with massive degrees of freedom . nonetheless , the parameterization in eq . ( 205 ) will still be able to signal that the detection is not a pure einstein event , at the cost of biasing their true value . ( 205 ) is motivated not only from examples of modified gravity predictions , but from generic modifications to the physical quantities that drive the inspiral : the binding energy or hamiltonian and the radiation - reaction force or the fluxes of the constants of the motion . considered generic modifications of the form 206\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = { \mu \over 2}{m \over r}\left [ { 1 + { a_{{\rm{ppe}}}}{{\left({{m \over r } } \right)}^{{p_{{\rm{ppe } } } } } } } \right],\quad \quad \dot e = { \dot e_{{\rm{gr}}}}\left [ { 1 + { b_{{\rm{ppe}}}}{{\left({{m \over r } } \right)}^{{q_{{\rm{ppe } } } } } } } \right],$$\end{document } where ( p , q ) , since otherwise one would lose analyticity in the limit of zero velocities for circular inspirals , and where ( a , b ) are parameters that depend on the modified gravity theory and , in principle , could depend on dimensionless quantities like the symmetric mass ratio . such modifications lead to the following corrections to the spa fourier transform of the = 2 time - domain response function for a quasi - circular binary inspiral template ( to leading order in the deformations and in post - newtonian theory ) 207\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \tilde h = a{{(\pi { { \mathcal m}_c}f)}^{- 7/6}}{e^{- i{\psi _ { { \rm{gr}}}}}}\left [ { 1 - { { { b_{{\rm{ppe } } } } } \over 2}{\eta ^{- 2{q_{{\rm{ppe}}}}/5 } } } \right.{{(\pi { { \mathcal m}_c}f)}^{2{q_{{\rm{ppe}}}}}}\quad \quad}\\ { \left . { + { { { a_{{\rm{ppe } } } } } \over 6}(6 + 4{p_{{\rm{ppe } } } } - 5p_{{\rm{ppe}}}^2){\eta ^{- 2{p_{{\rm{ppe}}}}/5}}{{(\pi { { \mathcal m}_c}f)}^{2{p_{{\rm{ppe } } } } } } } \right]{e^{- i\delta { \psi _ { { \rm{ppe}}}}}},}\\ \end{array}$$\end{document } 208\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \delta { \psi _ { { \rm{ppe } } } } = { 5 \over { 32}}a{{5p_{{\rm{ppe}}}^2 - 2{p_{{\rm{ppe } } } } - 6 } \over { ( 4 - { p_{{\rm{ppe}}}})(5 - { p_{{\rm{ppe}}}})}}{\eta ^{- 2{p_{{\rm{ppe}}}}/5}}{{(\pi { { \mathcal m}_c}f)}^{2{p_{{\rm{ppe } } } } - 5}}\quad \quad}\\ { + { { 15 } \over { 32}}{{{b_{{\rm{ppe } } } } } \over { ( 4 - { q_{{\rm{ppe}}}})(5 - 2{q_{{\rm{ppe}}}})}}{\eta ^{- 2{q_{{\rm{ppe}}}}/5}}{{(\pi { { \mathcal m}_c}f)}^{2{q_{{\rm{ppe } } } } - 5}}.}\\ \end{array}$$\end{document } of course , usually one of these two modifications dominates over the other , depending on whether q > p or p < q. in jordan - fierz - brans - dicke theory , for example , the radiation - reaction correction dominates as q < p. if , in addition to these modifications in the generation of gravitational waves , one also allows for modifications in the propagation , one is then led to the following template family 209\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h_{{\rm{ppe}},{\rm{insp,2}}}^{(\ell = 2 ) } = { \mathcal a}\;{(\pi { { \mathcal m}_c}f)^{- 7/6}}{e^{- i{\psi _ { { \rm{gr}}}}}}\left [ { 1 + c{\beta _ { { \rm{ppe}}}}{{(\pi { { \mathcal m}_c}f)}^{{b_{{\rm{ppe}}}}/3 + 5/3 } } } \right]{e^{2i{\beta _ { { \rm{ppe}}}}{u^{{b_{{\rm{ppe}}}}}}}}{e^{i{k_{{\rm{ppe}}}}{u^{{k_{{\rm{ppe}}}}}}}}.$$\end{document } here ( bppe,ppe ) and ( kppe , ppe ) are ppe parameters induced by modifications to the generation and propagation of gravitational waves respectively , where still ( bppe , kppe ) , while c is fully determined by the former set via 210\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${c_{{\rm{cons } } } } = - { { 16 } \over { 15}}{{(3 - b)(42b + 61 + 5{b^2 } ) } \over { 5{b^2 } + 46b + 81}},$$\end{document } if the modifications to the binding energy dominate , 211\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${c_{{\rm{diss } } } } = - { { 16 } \over { 15}}(3 - b)b,$$\end{document } if the modifications to the energy flux dominate , or 212\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${c_{{\rm{both } } } } = - { { 32 } \over { 15}}{{b(3 - b)(44b + 71 + 5{b^2 } ) } \over { 5{b^2 } + 46b + 81}},$$\end{document } if both corrections enter at the same post - newtonian order . noticing again that if only a single term in the phase correction dominates in the post - newtonian approximation ( or both will enter at the same post - newtonian order ) , one can map eq . ( 207 ) to eq . ( 205 ) by a suitable redefinition of constants . of course , one can introduce more ppe parameters to increase the complexity of the waveform family , and thus , eq . in fact , one expects any modified theory of gravity to introduce not just a single parametric modification to the amplitude and the phase of the signal , but two new functional degrees of freedom : 213\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\alpha _ { { \rm{ppe}}}}{u^{{a_{{\rm{ppe } } } } } } \rightarrow \delta { a_{{\rm{ppe}}}}({\lambda ^a},{\theta ^a};u),\quad { \beta _ { { \rm{ppe}}}}{u^{{b_{{\rm{ppe } } } } } } \rightarrow \delta { \psi _ { { \rm{ppe}}}}({\lambda ^a},{\theta ^a};u),$$\end{document } where these functions will depend on the frequency u , as well as on system parameters and theory parameters . in a post - newtonian expansion , one expects these functions to reduce to leading - order on the left - hand sides of eqs . ( 213 ) , but also to acquire post - newtonian corrections of the form 214\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { a_{{\rm{ppe}}}}({\lambda ^a},{\theta ^a};u ) = { \alpha _ { { \rm{ppe}}}}({\lambda ^a},{\theta ^a}){u^{{a_{{\rm{ppe}}}}}}\sum\limits_n { { \alpha _ { n,{\rm{ppe } } } } } ( { \lambda ^a},{\theta ^a}){u^n},$$\end{document } 215\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \psi _ { { \rm{ppe}}}}({\lambda ^a},{\theta ^a};u ) = { \beta _ { { \rm{ppe}}}}({\lambda ^a},{\theta ^a}){u^{{b_{{\rm{ppe}}}}}}\sum\limits_n { { \beta _ { n,{\rm{ppe } } } } } ( { \lambda ^a},{\theta ^a}){u^n},$$\end{document } where here the structure of the series is assumed to be of the form u with u > 0 . such a model , also suggested by yunes and pretorius , would introduce too many new parameters that would dilute the information content of the waveform model . recently , sampson et al . demonstrated that the simplest ppe model of eq . ( 205 ) suffices to signal a deviation from gr , even if the injection contains three terms in the phase . in fact , this is precisely one of the most important differences between the ppe and ppn frameworks . in ppn , it does not matter how many ppn parameters are introduced , because the observations are of very high snr , and thus , templates are not needed to extract the signal from the noise . on the other hand , in gravitational wave astrophysics , templates are essential to make detections and do parameter estimation . spurious parameters in these templates that are not needed to match the signal will deteriorate the accuracy to which all parameters can be measured because of an occam penalty . thus , in gravitational wave astrophysics and data analysis one wishes to minimize the number of theory parameters when testing gr [ 124 , 376 ] . one must then find a balance between the number of additional theory parameters to introduce and the amount of bias contained in the templates . at this junction , one must emphasize that frequency exponents in the amplitude and phase correction were above assumed to be integers , i.e. , ( appe , bppe , n ) . this must be the case if these corrections arise due to modifications that can be represented as integer powers of the momenta or velocity . we are not aware of any theory that predicts corrections proportional to fractional powers of the velocity for circular inspirals . moreover , one can show that theories that introduce non - integer powers of the velocity into the equations of motion will lead to issues with analyticity at zero velocity and a breakdown of uniqueness of solutions . in spite of this , modified theories can introduce logarithmic terms , that for example enter at high post - newtonian order in gr due to non - linear propagation effects ( see , e.g. , and references therein ) . moreover , certain modified gravity theories introduce screened modifications that become active only above a certain frequency . such effects would be modeled through a heaviside function , for example needed when dealing with massive brans - dicke gravity [ 147 , 94 , 20 , 465 ] . however , even these non - polynomial injections would be detectable with the simplest ppe model . in essence , one finds similar results as if one were trying to fit a 3-parameter injection with the simplest 1-parameter ppe model . of course , one can also generalize the inspiral ppe waveform families to more general orbits , for example through the inclusion of spins aligned or counter - aligned with the orbital angular momentum . ( 205 ) or ( 209 ) , but where the parameters ( ppe , ppe ) would now depend on the mass ratio , mass difference , and the spin parameters of the black holes . with a single detection , one can not break the degeneracy in the ppe parameters and separately fit for its system parameter dependencies . however , given multiple detections one should be able to break such a degeneracy , at least to a certain degree . such breaking of degeneracies begins to become possible when the number of detections exceeds the number of additional parameters required to capture the physical parameter dependencies of ( ppe , ppe ) . ppe waveforms can be extended to account for the merger and ringdown phases of coalescence . yunes and pretorius have suggested the following template family to account for this as well 216\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h_{{\rm{ppe , full}}}^{(\ell = 2 ) } = \left\{{\begin{array}{*{20}c } { { { \tilde h}_{{\rm{ppe}}}}\quad \quad \quad \quad \quad \quad \quad \quad } & { f < { f_{{\rm{im}}}},\quad \quad \quad \quad}\\ { \gamma { u^c}{e^{i(\delta + \epsilon u)}}\quad \quad \quad \quad \quad \quad } & { { f_{{\rm{im } } } } < f < { f_{{\rm{mrd}}}},\quad}\\ { \zeta { \tau \over { 1 + 4{\pi ^2}{\tau ^2}\kappa { { ( f - { f_{{\rm{rd}}}})}^d } } } } & { f > { f_{{\rm{mrd}}}},\quad \quad \quad \quad}\\ \end{array } } \right.$$\end{document } where the subscripts i m and mrd stand for inspiral merger and merger ringdown , respectively . the merger phase ( fim < f < fmrd ) is modeled here as an interpolating region between the inspiral and ringdown , where the merger parameters ( , ) are set by continuity and differentiability , and the ppe merger parameters ( c , ) should be fit for . in the ringdown phase ( f > fmrd ) , the response function is modeled as a single - mode generalized lorentzian , with real and imaginary dominant frequencies frd and , ringdown parameter also set by continuity and differentiability , and the ppe ringdown parameters ( , d ) are to be fit for . the transition frequencies ( fim , fmrd ) can either be treated as ppe parameters or set via some physical criteria , such as at light - ring frequency and the fundamental ringdown frequency , respectively . recently , there has been effort to generalize the ppe templates to allow for the excitation of non - gr gravitational - wave polarizations . modifications to only the two gr polarizations map to corrections to terms in the time - domain fourier transform that are proportional to the = 2 harmonic of the orbital phase . however , arun suggested that if additional polarizations are present , other terms proportional to the = 0 and = 1 harmonic will also arise . chatziioannou , yunes and cornish have found that the presence of such harmonics can be captured through the more complete single - detector template family 217\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \tilde h_{{\rm{ppe}},{\rm{insp}}}^{{\rm{all}}\;\ell}(f ) = { \mathcal a}\;{{(\pi { { \mathcal m}_c}f)}^{- 7/6}}{e^{- i{\psi ^{(2)}}{\rm{gr}}}}\left [ { 1 + c\;{\beta _ { { \rm{ppe}}}}{{(\pi { { \mathcal m}_c}f)}^{{b_{{\rm{ppe}}}}/3 + 5/3 } } } \right]{e^{2i{\beta _ { { \rm{ppe}}}}u_2^{{b_{{\rm{ppe}}}}}}}{e^{2i{k_{{\rm{ppe}}}}u_2^{{\kappa _ { { \rm{ppe}}}}}}}}\\ { + { \gamma _ { { \rm{ppe}}}}\;u_1^{- 9/2}{e^{- i{\psi ^{{{(1)}_{{\rm{gr}}}}}}}}{e^{i{\beta _ { { \rm{ppe}}}}u_1^{{b_{{\rm{ppe}}}}}}}{e^{2i{k_{{\rm{ppe}}}}u_1^{{\kappa _ { { \rm{ppe}}}}}}},\quad \quad \quad \quad \quad \quad \quad \quad \quad}\\ \end{array}$$\end{document } 218\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\psi _ { { \rm{gr}}}^{(\ell ) } = - 2\pi f{t_c } + \ell \phi _ c^{(\ell ) } + { \pi \over 4 } - { { 3\ell } \over { 256u_\ell ^5}}\sum\limits_{n = 0}^7 { u_\ell ^{n/3}(c_n^{{\rm{pn } } } + l_n^{{\rm{pn}}}\ln { u_\ell}),}$$\end{document } where we have defined \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${u_\ell } = { ( 2\pi { m_c}f/\ell)^{1/3}}$\end{document}. the ppe theory parameters are now \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vec \theta = ( { b_{{\rm{ppe}}}},{\beta _ { { \rm{ppe}}}},{k_{{\rm{ppe}}}},{\kappa _ { { \rm{ppe}}}},{\gamma _ { { \rm{ppe}}}},\phi _ c^{(1)})$\end{document}. of course , one may ignore ( kppe , ppe ) altogether , if one wishes to ignore propagation effects . such a parameterization recovers the predictions of jordan - fierz - brans - dicke theory for a single - detector response function , as well as arun s analysis for generic dipole radiation . one might worry that the corrections introduced by the = 1 harmonic , i.e. , terms proportional to ppe in eq . ( 217 ) , will be degenerate with post - newtonian corrections to the amplitude of the = 2 mode ( not displayed in eq . ( 217 ) ) . however , this is clearly not the case , as the latter scale as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${(\pi { \mathcal{m}_c}f)^{- 7/6 + n/3}}$\end{document } with n an integer greater than 0 , while the = 1 mode is proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${(\pi { m_c}f)^{- 3/2}}$\end{document } , which would correspond to a ( 0.5 ) post - newtonian order correction , i.e. , n = 1 . on the other hand , the ppe amplitude corrections to the = 2 mode , i.e. , terms proportional to ppe in the amplitude of eq . ( 217 ) , can be degenerate with such post - newtonian corrections when bppe is an integer greater than 4 . the latter contains a stronger prior ( that ppe frequency exponents be integers ) , and thus , it is ideal for fitting a particular set of theoretical models . on the other hand , eq . ( 205 ) with continuous ppe frequency exponents allows one to search for generic deviations that are statistically significant , without imposing such theoretical priors . that is , if a deviation from gr is present , then eq . ( 205 ) is more likely to be able to fit it , than eq . ( 205 ) with ( appe , bppe ) can still recover deviations from gr , even if the latter can not be represented as a correction proportional to an integer power of velocity . given these ppe waveforms , how should they be used in a data analysis pipeline ? the main idea behind the ppe framework is to match filter or perform bayesian statistics with ppe enhanced template banks to allow the data to select the best - fit values of . as discussed in [ 467 , 124 ] and then later in , one might wish to first run detection searches with gr template banks , and then , once a signal has been found , do a bayesian model selection analysis with ppe templates . the first such bayesian analysis was carried out by cornish et al . , who concluded that an aligo detection at snr of 20 for a quasi - circular , non - spinning black - hole inspiral would allow us to constrain ppe and ppe much better than existent constraints for sufficiently strong - field corrections , e.g. , bppe > 5 . this is because for lower values of the frequency exponents , the corrections to the waveform are weak - field and better constrained with binary pulsar observations . the large statistical study of li et al . uses a reduced set of ppe waveforms and investigates our ability to detect deviations of gr when considering a catalogue of aligo / advirgo detections . of course , the disadvantage of such a pipeline is that it requires a first detection , and if the gravitational interaction is too different from gr s prediction , it is possible that a search with gr templates might miss the signal all together ; we deem this possibility to be less likely . a built - in problem with the ppe and the ppn formalisms is that if a non - zero ppe or ppn parameter is detected , then one can not necessarily map it back to a particular modified gravity action . on the contrary , as suggested in table 3 , there can be more than one theory that predicts structurally - similar corrections to the fourier transform of the response function . for example , both jordan - fierz - brans - dicke theory and the dissipative sector of einstein - dilaton - gauss - bonnet theory predict the same type of leading - order correction to the waveform phase . however , if a given ppe parameter is measured to be non - zero , this could provide very useful information as to the type of correction that should be investigated further at the level of the action . the information that could be extracted is presented in table 4 , which is derived from knowledge of the type of corrections that lead to table 3 . a ppe b ppe interpretation1parity violation813anomalous acceleration , extra dimensions , violation of position invariance7dipole gravitational radiation , electric dipole scalar radiation3massive graviton propagation spin1magnetic dipole scalar radiation , quadrupole moment correction , scalar dipole force interpretation of non - zero ppe parameters . moreover , if a follow - up search is done with the ppe model in eq . ( 209 ) , one could infer whether the correction is one due to modifications to the generation or the propagation of gravitational waves . in this way , a non - zero ppe detection could inform theories of what type of gr modification is preferred by nature . however , much care must be taken to avoid confusing a ppe theory modification with some other systematic , such as an astrophysical , a mismodeling or an instrumental effect . instrumental effects can be easily remedied by requiring that several instruments , with presumably unrelated instrumental systematics , independently derive a posterior probability for ( ppe , ppe ) that peaks away from zero . astrophysical uncertainties can also be alleviated by requiring that different events lead to the same posteriors for ppe parameters ( after breaking degeneracies with system parameters ) . however , astrophysically there are a limited number of scenarios that could lead to corrections in the waveforms that are large enough to interfere with these tests . for comparable - mass - ratio inspirals , this is usually not a problem as the inertia of each binary component is too large for any astrophysical environment to affect the orbital trajectory . magnetohydrodynamic effects could affect the merger of neutron - star binaries , but this usually occurs outside of the sensitivity band of ground - based interferometers . however , in extreme - mass - ratio inspirals the small compact object can be easily nudged away by astrophysical effects , such as the presence of an accretion disk [ 462 , 267 ] or a third supermassive black hole . however , these astrophysical effects present the interesting feature that they correct the waveform in a form similar to eq . this is because the larger the orbital separation , the stronger the perturbations of the astrophysical environment , either because the compact object gets closer to the third body or because it leaves the inner edge of the accretion disk and the disk density increases with separation . such effects , however , are not likely to be present in all sources observed , as few extreme - mass - ratio inspirals are expected to be embedded in an accretion disk or sufficiently close to a third body ( 0.1 pc ) for the latter to have an effect on the waveform . perhaps the most dangerous systematic is mismodeling , which is due to the use of approximation schemes when constructing waveform templates . for example , in the inspiral one uses the post - newtonian approximation series , expanding and truncating the waveform at a given power of orbital velocity . moreover , neutron stars are usually modeled as test - particles ( with a dirac distributional density profile ) , when in reality they have a finite radius , which will depend on its equation of state . such finite - size effects enter at 5 post - newtonian order ( the effacement principle [ 227 , 128 ] ) , but with a post - newtonian coefficient that can be rather large [ 320 , 72 , 175 ] . ignorance of the post - newtonian series beyond 3 post - newtonian order can lead to systematics in the determination of physical parameters and possibly also to confusion when carrying out ppe - like tests . much more work is needed to determine the systems and snrs for which such systematics are truly a problem . another way to search for generic deviations from gr is to ask whether any gravitational - wave signal detected contains more than the two traditional polarizations expected in gr . a general approach to answer this concept was first studied by grsel and tinto and later by chatterji et al . with the aim to separate false - alarm events from real detections . proposed the extension of the idea of null streams to develop null tests of gr , which was proposed using stochastic gravitational wave backgrounds in [ 329 , 330 ] and recently implemented in to reconstruct the independent polarization modes in time - series data of a ground - based detector network . given a gravitational - wave detection , one can ask whether the data is consistent with two polarizations by constructing a null stream through the combination of data streams from 3 or more detectors . as explained in section 4.3 , such a null stream should be consistent with noise in gr , while it would present a systematic deviation from noise if the gravitational wave metric perturbation possessed more than two polarizations . notice that such a test would not require a template ; if one were parametrically constructed , such as in , more powerful null tests could be applied to such a null steam . in the future , we expect several gravitational wave detectors to be online : the two aligo ones in the united states , advirgo in italy , ligo - india in india , and kagra in japan . given a gravitational - wave observation that is detected by all five detectors , one can then construct three enhanced gr null streams , each with power in a signal null direction . neutron stars in the slow - rotation limit can be characterized by their mass and radius ( to zeroth - order in spin ) , by their moment of inertia ( to first - order in spin ) , and by their quadrupole moment and love numbers ( to second - order in spin ) . one may expect these quantities to be quite sensitive to the neutron star s internal structure , which can be parameterized by its equation of state , i.e. , the relation between its internal pressure and its internal energy density . since the equation of state can not be well - constrained at super - nuclear densities in the laboratory , one is left with a variety of possibilities that predict different neutron - star mass - radius relations . recently , however , yagi and yunes [ 453 , 452 ] have demonstrated that there are relations between the moment of inertia ( i ) , the love numbers ( ) , and the quadrupole moment ( q ) , the i - love - q relations that are essentially insensitive to the equation of state . figure 5 shows two of these relations ( the normalized i - love and q - love relations see caption ) for a variety of equations of state , including apr , sly [ 150 , 385 ] , lattimer - swesty with nuclear incompressibility of 220 mev ( ls220 ) [ 283 , 335 ] , shen [ 382 , 383 , 335 ] , the latter two with temperature of 0.01 mev and an electron fraction of 30% , and polytropic equations of state with indices of n = 0.6 , 0.8 and 1.13 the bottom panels show the difference between the numerical results and the analytical , fitting curve . observe that all equations of state lead to the same i - love and q - love relations , with discrepancies smaller than 1% for realistic neutron - star masses . these results have recently been verified in through the post - newtonian - affine approach [ 168 , 305 ] , which proves the i - love - q relations hold not only during the inspiral , but also close to plunge and merger . figure 5top : fitting curves ( solid curve ) and numerical results ( points ) of the universal i - love ( left ) and q - love ( right ) relations for various equations of state , normalized as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar i = i / m_{{\rm{ns}}}^3$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \lambda ^{({\rm{tid } } ) } } = { \lambda ^{({\rm{tid}})}}/m_{{\rm{ns}}}^5$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar q = - { q^{({\rm{rot}})}}/[m_{{\rm{ns}}}^3{(s / m_{{\rm{ns}}}^2)^2}]$\end{document } , mns is the neutron - star mass , is the tidal love number , q is the rotation - induced quadrupole moment , and s is the magnitude of the neutron - star spin angular momentum . the neutron - star central density is the parameter varied along each curve , or equivalently the neutron - star compactness . the top axis shows the neutron star mass for the apr equation of state , with the vertical dashed line showing mns = 1 m. bottom : relative fractional errors between the fitting curve and the numerical results . observe that these relations are essentially independent of the equation of state , with loss of universality at the 1% level . top : fitting curves ( solid curve ) and numerical results ( points ) of the universal i - love ( left ) and q - love ( right ) relations for various equations of state , normalized as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar i = i / m_{{\rm{ns}}}^3$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \lambda ^{({\rm{tid } } ) } } = { \lambda ^{({\rm{tid}})}}/m_{{\rm{ns}}}^5$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar q = - { q^{({\rm{rot}})}}/[m_{{\rm{ns}}}^3{(s / m_{{\rm{ns}}}^2)^2}]$\end{document } , mns is the neutron - star mass , is the tidal love number , q is the rotation - induced quadrupole moment , and s is the magnitude of the neutron - star spin angular momentum . the neutron - star central density is the parameter varied along each curve , or equivalently the neutron - star compactness . the top axis shows the neutron star mass for the apr equation of state , with the vertical dashed line showing mns = 1 m. bottom : relative fractional errors between the fitting curve and the numerical results . observe that these relations are essentially independent of the equation of state , with loss of universality at the 1% level . image reproduced by permission from , copyright by aps . given the independent measurement of any two members of the i - love - q trio , one could carry out a ( null ) model - independent and equation - of - state - independent test of gr [ 453 , 452 ] . for example , assume that electromagnetic observations of the binary pulsar j0737 - 3039 have measured the moment of inertia to 10% accuracy [ 282 , 273 , 274 ] . the slow - rotation approximation is perfectly valid for this binary pulsar , due to its relatively long spin period . assume further that a gravitational - wave observation of a neutron - star - binary inspiral , with individual masses similar to that of the primary in j0737 - 3039 , manages to measure the neutron star tidal love number to 60% accuracy [ 453 , 452 ] . these observations then lead to an error box in the i - love plane , which must contain the curve in the left - panel of figure 5 . a similar test could be carried out by using data from only binary pulsar observations or only gravitational wave detections . in the case of the latter , one would have to simultaneously measure or constrain the value of the quadrupole moment and the love number , since the moment of inertia is not measurable with gravitational wave observations . in the case of the former , one would have to extract the moment of inertia and the quadrupole moment , the latter of which will be difficult to measure . therefore , the combination of electromagnetic and gravitational wave observations would be the ideal way to carry out such tests . such a test of gr , of course , is powerful only as long as modified gravity theories predict i - love - q relations that are not degenerated with the general relativistic ones . yagi and yunes [ 453 , 452 ] investigated such a relation in dynamical chern - simons gravity to find that such degeneracy is only present in the limit cs 0 . that is , for any finite value of cs , the dynamical chern - simons i - love - q relation differs from that of gr , with the distance to the gr expectation increasing for larger cs . yagi and yunes [ 453 , 452 ] predicted that a test similar to the one described above could constrain dynamical chern - simons gravity to roughly \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\xi _ { { \rm{cs}}}^{1/4 } < 10{m_{ns}}\sim 15$\end{document } km , where recall that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\xi _ { { \rm{cs } } } } = \alpha _ { { \rm{cs}}}^2/(\beta \kappa)$\end{document}. the test described above , of course , only holds provided the i - love - q relations are valid , which in turn depends on the assumptions made in deriving them . in particular , yagi and yunes [ 453 , 452 ] assumed that the neutron stars are uniformly and slowly rotating , as well as only slightly tidally deformed by their rotational velocity or companion . these assumptions would not be valid for newly - born neutron stars , which are probably differentially rotating and doing so quickly . however , the gravitational waves emitted by neutron - star inspirals are expected to have binary components that are old and not rapidly spinning by the time they enter the detector sensitivity band . some short - period , millisecond pulsars may spin at a non - negligible rate , for which the normalized moment of inertia , quadrupole moment and love number would not be independent of the rotational angular velocity . however , if then the above tests should still be possible , since binary pulsar observations would also automatically determine the rotational angular velocity , for which a unique i - love - q relation should exist in gr . another important class of generic tests of gr are those that concern the no - hair theorems . since much work has been done on this area , we have decided to separate this topic from the main generic tests section ( 5.3 ) . in what follows , we describe what these theorems are and the possible tests one could carry out with gravitational - wave observations emitted by black - hole - binary systems . the no - hair theorems state that the only stationary , vacuum solution to the einstein equations that is non - singular outside the event horizon is completely characterized by three quantities : its mass m , its spin s and its charge q. this conclusion is arrived at by combining several different theorems . first , hawking [ 223 , 222 ] proved that a stationary black hole must have an event horizon with a spherical topology and that it must be either static or axially symmetric . israel [ 243 , 244 ] then proved that the exterior gravitational field of such static black holes is uniquely determined by m and q and it must be given by the schwarzschild or the reissner - nordstrm metrics . carter constructed a similar proof for uncharged , stationary , axially - symmetric black holes , where this time black holes fall into disjoint families , not deformable into each other and with an exterior gravitational field uniquely determined by m and s. robinson and mazur later proved that such black holes must be described by either the kerr or the kerr - newman metric . black - hole horizons can be tidally deformed in dynamical situations , and if so , hawking s theorems [ 223 , 222 ] about spherical horizon topologies do not apply . this then implies that all other theorems described above also do not apply , and thus , dynamical black holes will generically have hair . consider , for example , an axially - symmetric black hole in the presence of a non - symmetrical matter distribution outside the event horizon . one might naively think that this would tidally distort the event horizon , leading to a rotating , stationary black hole that is not axisymmetric . however , hawking and hartle showed that in such a case the matter distribution torques the black hole forcing it to spin down , thus leading to a non - stationary scenario . if the black hole is non - stationary , then again the no - hair theorems do not apply by the arguments described at the beginning of this paragraph , and thus non - isolated black holes can have hair . third , the theorems only apply within gr , i.e. , through the use of the einstein equations . therefore , it is plausible that black holes in modified gravity theories or in gr with singularities outside any event horizons ( naked singularities ) will have hair . the no - hair theorems imply that the exterior gravitational field of isolated , stationary , uncharged and vacuum black holes ( in gr and provided the spacetime is regular outside all event horizons ) can be written as an infinite sum of mass and current multipole moments , where only two of them are independent : the mass monopole moment m and the current dipole moment s. one can extend these relations to include charge , but astrophysical black holes are expected to be essentially neutral due to charge accretion . if the no - hair theorems hold , all other multipole moments can be determined from [ 195 , 194 , 213 ] 219\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_\ell } + { \rm{i}}{s_\ell } = m{({\rm{i}}a)^\ell},$$\end{document } where m and s are the th mass and current multipole moments . even if the black - hole progenitor was not stationary or axisymmetric , the no - hair theorems guarantee that any excess multipole moments will be shed - off during gravitational collapse [ 356 , 357 ] . eventually , after the black hole has settled down and reached an equilibrium configuration , it will be described purely in terms of m0 = m and s1 = s = ma , where a is the kerr spin parameter . an astrophysical observation of a hairy black hole would not imply that the no - hair theorems are wrong , but rather that one of the assumptions made in deriving these theorems is not appropriate to describe nature . as described above , the three main assumptions are stationarity , vacuum and that gr and the regularity condition hold . astrophysical black holes will generically be hairy due to a violation of the first two assumptions , since they will neither be perfectly stationary , nor exist in a perfect vacuum . astrophysical black holes will always suffer small perturbations by other stars , electromagnetic fields , other forms of matter , like dust , plasma or dark matter , etc , which will induce non - zero deviations from eq . however , in all cases of interest such perturbations are expected to be too small to be observable , which is why one argues that even astrophysical black holes should obey the no - hair theorems if gr holds . put another way , an observation of the violation of the no - hair theorems would be more likely to indicate a failure of gr in the strong - field , than an unreasonably large amount of astrophysical hair . tests of the no - hair theorems come in two flavors : through electromagnetic observations [ 250 , 251 , 253 , 254 ] and through gravitational wave observations [ 370 , 371 , 112 , 196 , 44 , 50 , 289 , 390 , 471 , 422 , 421 , 184 , 423 , 364 ] . the former rely on radiation emitted by accelerating particles in an accretion disk around black holes . however , such tests are not clean as they require the modeling of complicated astrophysics , with matter and electromagnetic fields . gravitational wave tests are clean in that respect , but unlike electromagnetic tests , they can not be carried out yet due to lack of data . other electromagnetic tests of the no - hair theorems exist , for example through the observation of close stellar orbits around sgr a * [ 312 , 313 , 373 ] and pulsar - black - hole binaries , but these can not yet probe the near - horizon , strong - field regime , since electromagnetic observations can not yet resolve horizon scales . see for reviews on this topic . gravitational wave tests of the no - hair theorems require the detection of either extreme mass - ratio inspirals or the ringdown of comparable - mass black - hole mergers with future space - borne gravitational - wave detectors [ 25 , 24 ] . extreme mass - ratio inspirals consist of a stellar - mass compact object spiraling into a supermassive black hole in a generic orbit within astronomical units from the event horizon of the supermassive object . these events outlive the observation time of future detectors , emitting millions of gravitational wave cycles , with the stellar - mass compact object essentially acting as a tracer of the supermassive black hole spacetime . ringdown gravitational waves are always emitted after black holes merge and the remnant settles down into its final configuration . during the ringdown , the highly - distorted remnant radiates all excess degrees of freedom and this radiation carries a signature of whether the no - hair theorems hold in its quasi - normal mode spectrum ( see , e.g. , for a recent review ) . both electromagnetic and gravitational wave tests need a metric with which to model accretion disks , quasi - periodic oscillations , or extreme mass - ratio inspirals . one can classify these metrics as direct or generic , paralleling the discussion in section 5.2 . direct metrics are exact solutions to a specific set of field equations , with which one can derive observables . examples of such metrics are the manko - novikov metric and the slowly - spinning black - hole metric in dynamical chern - simons gravity . when computing observables with these metrics , one usually assumes that all radiative and dynamical process ( e.g. , the radiation - reaction force ) are as predicted in gr . generic metrics are those that parametrically modify the kerr spacetime , such that for certain parameter choices one recovers identically the kerr metric , while for others , one has a deformation of kerr . generic metrics can be further classified into two subclasses , ricci - flat versus non - ricci - flat , depending on whether they satisfy r = 0 . the most studied direct metric is the manko - novikov one , which although an exact , stationary and axisymmetric solution to the vacuum einstein equations , does not represent a black hole , as the event horizon is broken along the equator by a ring singularity . just like the kerr metric , the manko - novikov metric possesses an ergoregion , but unlike the former , it also possesses regions of closed time - like curves that overlap the ergoregion . nonetheless , an appealing property of this metric is that it deviates continuously from the kerr metric through certain parameters that characterize the higher multiple moments of the solution . the first geodesic study of manko - novikov spacetimes was carried out by gair et al . . they found that there are two ring - like regions of bound orbits : an outer one where orbits look regular and integrable , as there exist four isolating integrals of the motion ; and an inner one where orbits are chaotic and thus ergodic . suggested that orbits that transition from the integrable to the chaotic region would leave a clear observable signature in the frequency spectrum of the emitted gravitational waves . however , they also noted that chaotic regions exist only very close to the central body and are probably not astrophysically accessible . the study of gair et al . was recently confirmed and followed up by contopoulos et al . . they studied a wide range of geodesics and found that , in addition to an inner chaotic region and an outer regular region , there are also certain birkhoff islands of stability . when an extreme mass - ratio inspiral traverses such a region , the ratio of resonant fundamental frequencies would remain constant in time , instead of increasing monotonically . such a feature would impact the gravitational waves emitted by such a system , and it would signal that the orbit equations are non - integrable and the central object is not a kerr black hole . the study of chaotic motion in geodesics of non - kerr spacetimes is by no means new . chaos has also been found in geodesics of zipoy - voorhees - weyl and curzon spacetimes with multiple singularities [ 391 , 392 ] and in general for zipoy - voorhees spacetimes in , of perturbed schwarzschild spacetimes , of schwarzschild spacetimes with a dipolar halo [ 286 , 288 , 209 ] of erez - rosen spacetimes , and of deformed generalizations of the tomimatsy - sato spacetime . one might worry that such chaotic orbits will depend on the particular spacetime considered , but recently apostolatos et al . and lukes - gerakopoulos et al . although the kolmogorov , arnold , and moser theorem [ 270 , 35 , 321 ] states that phase orbit tori of an integrable system are only deformed if the hamiltonian is perturbed , the poincare - birkhoff theorem states that resonant tori of integrable systems actually disintegrate , leaving behind a chain of birkhoff islands . these islands are only characterized by the ratio of winding frequencies that equals a rational number , and thus , they constitute a distinct and generic feature of non - integrable systems [ 31 , 297 ] . given an extreme mass - ratio gravitational - wave detection , one can monitor the ratio of fundamental frequencies and search for plateaus in their evolution , which would signal non - integrability . of course , whether detectors can resolve such plateaus depends on the initial conditions of the orbits and the physical system under consideration ( these determine the thickness of the islands ) , as well as the mass ratio ( this determines the radiation - reaction timescale ) and the distance and mass of the central black hole ( this determines the snr ) . another example of a direct metric test of the no - hair theorem is through the use of the slowly - rotating dynamical chern - simons black hole metric . unlike the manko - novikov metric , the dynamical chern - simons one does represent a black hole , i.e. , it possesses an event horizon , but it evades the no - hair theorems because it is not a solution to the einstein equations . sopuerta and yunes carried out the first extreme mass - ratio inspiral analysis when the background supermassive black hole object is taken to be such a chern - simons black hole . they used a semi - relativistic model to evolve extreme mass - ratio inspirals and found that the leading - order modification comes from a modification to the geodesic trajectories , induced by the non - kerr modifications of the background . because the latter correspond to a strong - field modification to gr , modifications in the trajectories are most prominent for zoom - whirl orbits , as the small compact object zooms around the supermassive black hole in a region of unstable orbits , close to the event horizon . these modifications were then found to propagate into the gravitational waves emitted , leading to a dephasing that could be observed or ruled out with future gravitational - wave observations to roughly the horizon scale of the supermassive black hole , as has been recently confirmed by canizares et al . . however , these studies may be underestimates , given that they treat the black hole background in dynamical chern - simons gravity only to first - order in spin . a final example of a direct metric test of the no - hair theorems is to consider black holes that are not in vacuum . studied extreme - mass - ratio inspirals in a kerr - black - hole background that is perturbed by a self - gravitating , homogeneous torus that is compact , massive and close to the kerr black hole . they found that the presence of this torus impacts the gravitational waves emitted during such inspirals , but only weakly , making it difficult to distinguish the presence of matter . . carried out a similar study , where this time they considered a small compact object inspiraling completely within a geometrically thin , radiation - pressure dominated accretion disk . they found that disk - induced migration can modify the radiation - reaction force sufficiently so as to leave observable signatures in the waveform , provided the accretion disk is sufficiently dense in the radiation - dominated regime and a gap opens up . however , these tests of the no - hair theorem will be rather difficult as most extreme - mass - ratio inspirals are not expected to be in an accretion disk . generic ricci - flat deformed metrics will lead to laplace - type equations for the deformation functions in the far - field since they must satisfy r = 0 to linear order in the perturbations . the solution to such an equation can be expanded in a sum of mass and current multipole moments , when expressed in asymptotically cartesian and mass - centered coordinates . these multipoles can be expressed via [ 112 , 422 , 421 ] 220\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_\ell } + { \rm{i}}{s_\ell } = m{({\rm{i}}a)^\ell } + \delta { m_\ell } + { \rm{i}}\delta { m_\ell},$$\end{document } where m and s are mass and current multipole deformations . ryan [ 370 , 371 ] showed that the measurement of three or more multipole moments would allow for a test of the no - hair theorem . generic non - ricci flat metrics , on the other hand , will not necessarily lead to laplace - type equations for the deformation functions in the far field , and thus , the far - field solution and eq . ( 220 ) will depend on a sum of and m multipole moments . the first attempt to construct a generic , ricci - flat metric was by collins and hughes : the bumpy black - hole metric . in this approach , the metric is assumed to be of the form 221\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } = g_{\mu \nu}^{({\rm{kerr } } ) } + \epsilon \delta { g_{\mu \nu}},$$\end{document } where < 1 is a bookkeeping parameter that enforces that g is a perturbation of the kerr background . this metric is then required to satisfy the einstein equations linearized in , which then leads to differential equations for the metric deformation . collins and hughes assumed a non - spinning , stationary spacetime , and thus g only possessed two degrees of freedom , both of which were functions of radius only : 1(r ) , which must be a harmonic function and which changes the newtonian part of the gravitational field at spatial infinity ; and 1(r ) which is completely determined through the linearized einstein equations once 1 is specified . one then has the freedom to choose how to prescribe 1 and collins and hughes investigate two choices that correspond physically to point - like and ring - like naked singularities , thus violating cosmic censorship . vigeland and hughes and vigeland then extend this analysis to stationary , axisymmetric spacetimes via the newman - janis method [ 327 , 151 ] , showing how such metric deformations modify eq . ( 220 ) , and computing how these bumps imprint themselves onto the orbital frequencies and thus the gravitational waves emitted during an extreme - mass - ratio inspiral . that the bumps represent unphysical matter should not be a surprise , since by the no - hair theorems , if the bumps are to satisfy the vacuum einstein equations they must either break stationarity or violate the regularity condition . gravitational wave tests with bumpy black holes must then be understood as null tests : one assumes the default hypothesis that gr is correct and then sets out to test whether the data rejects or fails to reject this hypothesis ( a null hypothesis can never be proven ) . unfortunately , however , bumpy black hole metrics can not parameterize spacetimes in modified gravity theories that lead to corrections in the field equations that are not proportional to the ricci tensor , such as for example in dynamical chern - simons or in einstein - dilaton - gauss - bonnet modified gravity . glampedakis and babak proposed a different type of stationary and axisymmetric bumpy black hole through the hartle - thorne metric , with modifications to the quadrupole moment . kludge extreme mass - ratio inspiral waveform and estimated how well the quadrupole deformation could be measured . however , this metric is valid only when the supermassive black hole is slowly - rotating , as it derives from the hartle - thorne ansatz . recently , johansen and psaltis proposed yet another metric to represent bumpy stationary and spherically - symmetric spacetimes . this metric introduces one new degree of freedom , which is a function of radius only and assumed to be a series in m / r . johansen and psaltis then rotated this metric via the newman - janis method [ 327 , 151 ] to obtain a new bumpy metric for axially - symmetric spacetimes . however , such a metric possesses a naked ring singularity on the equator , and naked singularities on the poles . as before , none of these bumpy metrics can be mapped to known modified gravity black hole solutions , in the glampedakis and babak case because the einstein equations are assumed to hold to leading order in the spin , while in the johansen and psaltis case because a single degree of freedom is not sufficient to model the three degrees of freedom contained in stationary and axisymmetric spacetimes [ 401 , 423 ] . the only generic non - ricci - flat bumpy black - hole metric so far is that of vigeland , yunes and stein . they allowed generic deformations in the metric tensor , only requiring that the new metric perturbatively retained the killing symmetries of the kerr spacetime : the existence of two killing vectors associated with stationarity and axisymmetry , as well as the perturbative existence of a killing tensor ( and thus a carter - like constant ) , at least to leading order in the metric deformation . such requirements imply that the geodesic equations in this new background are fully integrable , at least perturbatively in the metric deformation , which then allows one to solve for the orbital motion of extreme - mass - ratio inspirals by adapting previously existing tools . brink [ 83 , 84 , 85 , 86 , 87 ] studied the existence of such a second - order killing tensor in generic , vacuum , stationary and axisymmetric spacetimes in einstein s theory and found that these are difficult to construct exactly . by relaxing this exact requirement , vigeland , yunes and stein found that the existence of a perturbative killing tensor poses simple differential conditions on the metric perturbation that can be analytically solved . moreover , they also showed how this new bumpy metric can reproduce all known modified gravity black hole solutions in the appropriate limits , provided these have an at least approximate killing tensor ; thus , these metrics are still vacuum solutions even though r 1 , since they satisfy a set of modified field equations . although unclear at this junction , it seems that the imposition that the spacetime retains the kerr killing symmetries leads to a bumpy metric that is well - behaved everywhere outside the event horizon ( no singularities , no closed - time - like curves , no loss of lorentz signature ) . recently , gair and yunes studied how the geodesic equations are modified for a test - particle in a generic orbit in such a spacetime and showed that the bumps are indeed encoded in the orbital motion , and thus , in the gravitational waves emitted during an extreme - mass - ratio inspiral . one might be concerned that such no - hair tests of gr can not constrain modified gravity theories , because kerr black holes can also be solutions in the latter . this is indeed true provided the modified field equations depend only on the ricci tensor or scalar . in einstein - dilaton - gauss - bonnet or dynamical chern - simons gravity , the modified field equations depend on the riemann tensor , and thus , ricci - flat metric need not solve these modified set . moreover , just because the metric background is identically kerr does not imply that inspiral gravitational waves will be identical to those predicted in gr . all studies carried out to date , be it direct metric tests or generic metric tests , assume that the only quantity that is modified is the metric tensor , or equivalently , the hamiltonian or binding energy . inspiral motion , of course , does not depend just on this quantity , but also on the radiation - reaction force that pushes the small object from geodesic to geodesic . moreover , the gravitational waves generated during such an inspiral depend on the field equations of the theory considered . therefore , all metric tests discussed above should be considered as partial tests . in general , strong - field modified gravity theories will modify the hamiltonian , the radiation - reaction force and the wave generation . let us now consider tests of the no - hair theorems with gravitational waves emitted by comparable - mass binaries during the ringdown phase . gravitational waves emitted during ringdown can be described by a superposition of exponentially - damped sinusoids : 222\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h _ + } ( t ) + i\;{h _ \times}(t ) = { m \over r}\sum\limits_{\ell mn } { \left\{{{{\mathcal a}_{\ell mn}}{e^{i({\omega _ { \ell mn}}t + { \phi _ { \ell mn}})}}{e^{- t/{\tau _ { \ell mn}}}}{s_{\ell mn } } + { \mathcal a}_{\ell mn}^\prime{e^{i(- { \omega _ { \ell mn}}t + \phi _ { \ell mn}^\prime)}}{e^{- t/{\tau _ { \ell mn}}}}s_{\ell mn}^{\ast } } \right\},}$$\end{document } where r is the distance from the source to the detector , the asterisk stands for complex conjugation , the real mode amplitudes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal{a}_{\ell , m , n}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{a}_{\ell , m , n}^{\prime}$\end{document } and the real phases nm and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\phi _ { \ell , m , n}^{\prime}$\end{document } depend on the initial conditions , smn are spheroidal functions evaluated at the complex quasinormal ringdown frequencies nm = 2fnm + i/nm , and the real physical frequency fnm and the real damping times nm are both functions of the mass m and the kerr spin parameter a only , provided the no - hair theorems hold . these frequencies and damping times can be computed numerically or semi - analytically , given a particular black - hole metric ( see for a recent review ) . the fourier transform of a given ( , m , n ) mode is 223\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h _ + ^{(\ell , m , n)}(\omega ) = { m \over r}{\mathcal a}_{\ell m n}^ + \left [ { { e^{i\phi _ { \ell mn}^ + } } { s_{\ell mn}}{b _ + } ( \omega ) + { e^{- i\phi _ { \ell mn}^ + } } s_{\ell mn}^{\ast}{b _ -}(\omega ) } \right],$$\end{document } 224\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h _ \times ^{(\ell , m , n)}(\omega ) = { m \over r}{\mathcal a}_{\ell mn}^\times \left [ { { e^{i\phi _ { \ell mn}^\times}}{s_{\ell mn}}{b _ + } ( \omega ) + { e^{- i\phi _ { \ell mn}^\times}}s_{\ell mn}^{\ast}{b _ -}(\omega ) } \right],$$\end{document } where we have defined \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{a}_{\ell mn}^{+ , \times}{e^{i\phi _ { \ell mn}^{+ , \times } } } \equiv { \mathcal{a}_{\ell mn}}{e^{i{\phi _ { \ell mn } } } } \pm \mathcal{a}{\prime}{e^{- i\phi _ { \ell mn}^{\prime}}}$\end{document } as well as the lorentzian functions 225\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${b _ \pm}(\omega ) = { { { \tau _ { \ell mn } } } \over { 1 + \tau _ { \ell mn}^2{{(\omega \pm { \omega _ { \ell mn}})}^2}}}.$$\end{document } ringdown gravitational waves will all be of the form of eq . ( 222 ) provided that the characteristic nature of the differential equation that controls the evolution of ringdown modes is not modified , i.e. , provided that one only modifies the potential in the teukolsky equation or other subdominant terms , which in turn depend on the modified field equations . tests of the no - hair theorems through the observation of black - hole ringdown date back to detweiler , and it was recently worked out in detail by dreyer et al . . let us first imagine that a single complex mode is detected \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 1}{m_1}{n_1}}}$\end{document } and one measures separately its real and imaginary parts . of course , from such a measurement , one can not extract the measured harmonic triplet ( 1,m1,n1 ) , but instead one only measures the complex frequency \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 1}{m_1}{n_1}}}$\end{document}. this information is not sufficient to extract the mass and spin angular momentum of the black hole because different quintuplets ( m , a , , m , n ) can lead to the same complex frequency \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 1}{m_1}{n_1}}}$\end{document}. the best way to think of this is graphically : a given observation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega _ { { \ell _ 1}{m_1}{n_1}}^{(1)}$\end{document } traces a line in the complex \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 1}{m_1}{n_1 } } } = m\omega _ { { \ell _ 1}{m_1}{n_1}}^{(1)}$\end{document } plane ; a given ( , m , n ) triplet defines a complex frequency mn that also traces a curve in the complex mn plane ; each intersection of the measured line \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 1}{m_1}{n_1}}}$\end{document } with mn defines a possible doublet ( m , a ) ; since different ( , m , n ) triplets lead to different mn curves and thus different intersections , one ends up with a set of doublets s1 , out of which only one represents the correct black - hole parameters . we thus conclude that a single mode observation of ringdown gravitational waves is not sufficient to test the no - hair theorem [ 152 , 69 ] let us then imagine that one has detected two complex modes , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 1}{m_1}{n_1}}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 2}{m_2}{n_2}}}$\end{document}. each detection leads to a separate line \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 1}{m_1}{n_1}}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 2}{m_2}{n_2}}}$\end{document } the complex plane . as before , each ( n , , m ) triplet leads to separate curves mn which will intersect with both \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 1}{m_1}{n_1}}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 2}{m_2}{n_2}}}$\end{document } in the complex plane . each intersection between mn and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell m n}}}$\end{document } leads to a set of doublets s1 , while each intersection between mn and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\omega _ { { \ell _ 1}{m_1}{n_1}}}$\end{document } leads to another set of doublets s2 . however , if the no - hair theorems hold sets s1 and s2 must have at least one element in common . therefore , a two - mode detection allows for tests of the no - hair theorem [ 152 , 69 ] . however , when dealing with a quasi - circular black - hole - binary inspiral within gr one knows that the dominant mode is = m = 2 . in such a case , the observation of this complex mode by itself allows one to extract the mass and spin angular momentum of the black hole . then , the detection of the real frequency in an additional mode can be used to test the no - hair theorem [ 69 , 65 ] . although the logic behind these tests is clear , one must study them carefully to determine whether all systematic and statistical errors are sufficiently under control so that they are feasible . first , they found that a matched - filtering type analysis with two - mode ringdown templates would increase the volume of the template manifold by roughly three orders of magnitude . a better strategy then is perhaps to carry out a bayesian analysis , like that of gossan et al . [ 256 , 201 ] ; through such a study one can determine whether a given detection is consistent with a two - mode or a one - mode hypothesis . [ 69 , 65 ] also calculated that a snr of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal{o}({10 ^ 2})$\end{document } would be sufficient to detect the presence of two modes in the ringdown signal and to resolve their frequencies , so that no - hair tests would be possible . strong signals are necessary because one must be able to distinguish at least two modes in the signal . unfortunately , however , whether the ringdown leads to such strong snrs and whether the sub - dominant ringdown modes are of a sufficiently large amplitude depends on a plethora of conditions : the location of the source in the sky , the mass of the final black hole , which depends on the rest mass fraction that is converted into ringdown gravitational waves ( the ringdown efficiency ) , the mass ratio of the progenitor , the magnitude and direction of the spin angular momentum of the final remnant and probably also of the progenitor and the initial conditions that lead to ringdown . thus , although such tests are possible , one would have to be quite fortunate to detect a signal with the right properties so that a two - mode extraction and a test of the no - hair theorems is feasible . another way to test gr is to modify the matter sector of the theory through the introduction of matter corrections to the einstein - hilbert action that violate the assumptions made in the no - hair theorems . more precisely , one can study whether gravitational waves emitted by binaries composed of strange stars , like quark stars , or horizonless objects , such as boson stars or gravastars , are different from waves emitted by more traditional neutron - star or black - hole binaries . in what follows boson stars are a classic example of a compact object that is essentially indistinguishable from a black hole in the weak field , but which differs drastically from one in the strong field due to its lack of an event horizon . a boson star is a coherent scalar - field configuration supported against gravitational collapse by its self - interaction . one can construct several lagrangian densities that would allow for the existence of such an object , including mini - boson stars [ 178 , 179 ] , axially - symmetric solitons , and nonsolitonic stars supported by a non - canonical scalar potential energy . boson stars are well - motivated from fundamental theory , since they are the gravitationally - coupled limit of q - balls [ 108 , 276 ] , a coherent scalar condensate that can be described classically as a non - topological soliton and that arises unavoidably in viable supersymmetric extensions of the standard model . in all studies carried out to date , boson stars have been studied within gr , but they are also allowed in scalar - tensor theories . at this junction , one should point out that the choice of a boson star is by no means special ; the key point here is to select a straw - man to determine whether gravitational waves emitted during the coalescence of compact binaries are sensitive to the presence of an event horizon or the evasion of the no - hair theorems induced by a non - vacuum spacetime . of course , depending on the specific model chosen , it is possible that the exotic object will be unstable to evolution or even to its own rotation . for example , in the case of an extreme mass - ratio inspiral , one could imagine that as the small compact object enters the boson star s surface , it will accrete the scalar field , forcing the boson star to collapse into a black hole . alternatively , one can imagine that as two supermassive boson stars merge , the remnant might collapse into a black hole , emitting the usual gr quasinormal modes . what is worse , even when such objects are in isolation , they are unstable under small perturbations if their angular momentum is large , possibly leading to gravitational collapse into a black hole or possibly a scalar explosion [ 95 , 96 ] . since most astrophysical black hole candidates are believed to have high spins , such instabilities somewhat limit the interest of horizonless objects . even then , however , the existence of slowly spinning or non spinning horizonless compact objects can not be currently ruled out by observation . boson stars evade the no - hair theorems within gr because they are not vacuum spacetimes , and thus , their metric and quasinormal mode spectrum can not be described by just their mass and spin angular momentum ; one also requires other quantities intrinsic to the scalar - field energy momentum tensor , scalar hair . therefore , as before , two types of gravitational wave tests for scalar hair have been proposed : extreme - mass - ratio inspiral tests and ringdown tests . as for the former , . showed that stable circular orbits exist both outside and inside of the surface of the boson star , provided the small compact object interacts with the background only gravitationally . this is because the effective potential for geodesic motion in such a boson - star background lacks the schwarzschild - like singular behavior at small radius , instead turning over and allowing for a new minimum . gravitational waves emitted in such a system would then stably continue beyond what one would expect if the background had been a supermassive black hole ; in the latter case the small compact object would simply disappear into the horizon . . found that orbits inside the boson star exhibit strong precession , exciting high frequency harmonics in the waveform , and thus allowing one to easily distinguish between such boson stars from black - hole backgrounds . just as the inspiral phase is modified by the presence of a boson star , the merger phase is also greatly altered , but this must be treated fully numerically . a few studies have found that the merger of boson stars leads to a spinning bar configuration that either fragments or collapses into a kerr black hole [ 339 , 338 ] . of course , the gravitational waves emitted during such a merger will be drastically different from those produced when black holes merge . unfortunately , the complexity of such simulations makes predictions difficult for any one given example , and the generalization to other more complicated scenarios , such as theories with modified field equations , is currently not feasible . recently , pani et al . [ 340 , 341 ] revisited this problem , but instead of considering a supermassive boson star , they considered a gravastar . this object consists of a schwarzschild exterior and a de sitter interior , separated by an infinitely thin shell with finite tension [ 307 , 100 ] . calculated the gravitational waves emitted by a stellar - mass compact object in a quasi - circular orbit around such a gravastar background . in addition to considering a different background , pani et al . used a radiative - adiabatic waveform generation model to describe the gravitational waves [ 351 , 238 , 239 , 458 , 456 , 459 ] , instead of the kludge scheme used by kesden et al . pani el al . concluded that the waves emitted during such inspirals are sufficiently different that they could be used to discern between a kerr black hole and a gravastar . on the ringdown side of no - hair tests , chirenti and rezzolla studied the non - radial , axial perturbations of gravastars , and pani et al . the non - radial , axial and polar oscillations of gravastars . [ 309 , 310 ] considered the quasinormal ringdown spectrum of skyrmion black holes . in all cases , it was found that the quasi - normal mode spectrum of such objects could be used to discern between them and kerr black holes . of course , such tests still require the detection of ringdown gravitational waves with the right properties , such that more than one mode can be discerned and extracted from the signal ( see section 5.4.3 ) . gravitational waves hold the key to testing einstein s theory of general relativity ( gr ) to new exciting levels in the previously unexplored strong - field regime . depending on the type of wave that is detected , e.g. , compact binary inspirals , mergers , ringdowns , continuous sources , supernovae , etc , different tests will be possible . irrespective of the type of wave detected , two research trends seem currently to be arising : direct tests and generic tests . generic tests , on the other hand , ask whether the data is statistically consistent with our canonical beliefs . or put another way : are there any statistically - significant deviations present in the data from what we expected to observe ? this approach is currently used in cosmological observations , for example by the wmap team , and it is particularly well - suited when one tries to remain agnostic as to which is the correct theory of nature . given that we currently have no data in the strong - field , it might be too restrictive to assume gr is correct prior to verifying that this is the case . what one would like to believe is that gravitational waves will be detected by the end of this decade , either through ground - based detectors or through pulsar timing arrays . given this , there is a concrete effort to develop the proper formalism and implementation pipelines to test einstein s theory once data becomes available . the theory part of the research load is being carried out at a variety of institutions without a given focal point . the implementation part is being done mostly within the ligo scientific collaboration and the pulsar timing consortia . cross - communication between the theory and implementation groups has recently flourished and one expects more interdisciplinary work in the future . so many accomplishments have been made in the past 50 years that it is almost impossible to list them here . from the implementation side , perhaps one of the most important is the actual construction and operation of the initial ligo instruments at design sensitivity in all of their frequency domain . similarly , the construction of impressive pulsar timing arrays , and the timing of these pulses to nano - second precision is an instrumental and data analysis feat to be admired . without these observatories no waves would be detectable in the future , and of course , no tests of einstein s theory would be feasible . on the theory side , perhaps the most important accomplishment has been the understanding of the inspiral phase to really - high post - newtonian order and the merger phase with numerical simulations . the latter , in particular , had been an unsolved problem for over 50 years , until very recently . it is these accomplishments that then allow us to postulate modified inspiral template families , since we understand what the gr expectation is . this is particularly true if one is considering small deformations away from einstein s theory , as it would be impossible to perturb about an unknown solution . the main questions that are currently at the forefront are the following . on the theory side of things , one would wish to understand the inspiral to high post - newtonian order in certain strong - field modifications to gr , like dynamical chern - simons gravity or einstein - dilaton - gauss - bonnet theory . one would also like to investigate theories with preferred frames , such as einstein - aether theory or hoava - lifshitz gravity , which will lead to lorentz violating observables . understanding these theories to high post - newtonian order is particularly important for those that predict dipolar gravitational emission , such as einstein - dilaton - gauss - bonnet theory . of course , a full inspiral - merger - ringdown template is not complete unless we also understand the merger . this would require full numerical simulations , which are very taxing even within gr . once one modifies the einstein field equations , the characteristic structure of the evolution equations will also likely change , and it is unclear whether the standard evolution methods will continue to work . moreover , when dealing with the merger phase , one is usually forced to treat the modified theory as exact , instead of as an effective theory . without the latter , it is likely that certain modified theories will not have a well - posed initial value problem , which would force any numerical evolution to fail . of course , one could order - reduce these equations and then use these to evolve black - hole spacetimes . much work still remains to be done to understand whether this is feasible . on the implementation side of things , currently , efforts are only beginning on the implementation of bayesian frameworks for hypothesis testing . this seems today like one of the most promising approaches to testing einstein s theory with gravitational waves . current studies concentrate mostly on single - detectors , but by the beginning of the next decade we expect four or five detectors to be online , and thus , one would like to see these implementations extended . the use of multiple detectors also opens the door to the extraction of new information , such as multiple polarization modes , a precise location of the source in the sky , etc . moreover , the evidence for a given model increases dramatically if the event is observed in several detectors . one therefore expects that the strongest tests of gr will come from leveraging the data from all detectors in a multiply - coincident event . ultimately , research is moving toward the construction of robust techniques to test einstein s theory . a general push is currently observed toward the testing of general principles that serve as foundations of gr . this allows one to answer general questions , such as : does the graviton have a mass ? are compact objects represented by the kerr metric and the no - hair theorems satisfied ? does the propagating metric perturbation possess only two transverse - traceless polarization modes ? these are examples of questions that can be answered once gravitational waves are detected . the more questions of this type that are generated and the more robust the methods to answer them are , the more stringent the test of einstein s theories and the more information we will obtain about the gravitational interaction in a previously unexplored regime .

this review is focused on tests of einstein s theory of general relativity with gravitational waves that are detectable by ground - based interferometers and pulsar - timing experiments . einstein s theory has been greatly constrained in the quasi - linear , quasi - stationary regime , where gravity is weak and velocities are small . gravitational waves will allow us to probe a complimentary , yet previously unexplored regime : the non - linear and dynamical strong - field regime . such a regime is , for example , applicable to compact binaries coalescing , where characteristic velocities can reach fifty percent the speed of light and gravitational fields are large and dynamical . this review begins with the theoretical basis and the predicted gravitational - wave observables of modified gravity theories . the review continues with a brief description of the detectors , including both gravitational - wave interferometers and pulsar - timing arrays , leading to a discussion of the data analysis formalism that is applicable for such tests . the review ends with a discussion of gravitational - wave tests for compact binary systems .

Introduction Alternative Theories of Gravity Detectors Testing Techniques Compact Binary Tests Musings About the Future

PMC3654347

the devotion to one 's parents has been displayed in various cultures throughout time and , as such , has usually included the care of one 's parents in old age . in colonial times , the care of frail aged persons was primarily the responsibility of the family . today in the west , elderly people are primarily cared for in hospitals , nursing homes , or by their families . the united nations predicts that by 2100 , 28.1% of the world population will be aged 65 years or older , compared with 10.0% in 2000 and 6.9% in 1990 . the resulting increased demand on the health system , coupled with decreasing taxpayer support and fewer younger people to care for the elderly , will introduce significant pressure on aged care services . for example , the fifth highest cause of death for elderly people is falls , and while the falls themselves may not be easily preventable , in many circumstances the deaths following them are , with appropriate monitoring and support . consequently , significant research interest has been focused towards home - telecare solutions allowing elderly people to live safely and independently in their homes . in recent years , it has been suggested that sound signatures are well suited to automated telemonitoring of elderly people and superior to video cameras from the perspective of privacy . telemonitoring using sound signatures is a relatively less explored area in the literature , in comparison with other techniques such as gait parameters and posture and motion information . the main methods for monitoring elderly people are the health smart home , wearable devices , or a combination of the two . the first health smart home used magnetic switches between doors , infrared sensors , and sound sensors to monitor which room or part of a room the person was in and the activity they were undertaking . in , infrared sensors and magnetic switches were used to monitor the activities of the subject and compare them with expected behaviour . the main shortcomings of the health smart home are the restriction to monitoring only nonbiomechanical parameters , its inability to monitor elderly subjects outside their home , and the need to distinguish the elderly person from others in the home . wearable devices are designed to measure biomechanical / physiological parameters and can be attached to clothing , worn either as jewellery or as a separate device . pedometers , foot - switches , and heart rate measurements can monitor the level of a person 's daily activity and energy expenditure . accelerometers measure the acceleration along the x , y , and z axes and can measure dynamic and static activities over a short or long period of time . gyroscopes measure the coriolis acceleration from rotational angular velocity and can therefore measure changes in the wearer 's posture . the major disadvantage of wearable devices is that they are not able to be used by people who are incapable , physically or mentally , of operating and maintaining them . investigations into telemonitoring using sound sensors have been performed by a french consortium of the clips and timc laboratories , who developed a five - microphone system [ 3 , 7 , 8 ] . the main shortcoming of this work is the specialised wiring in the dedicated test apartment . in addition , sound files in their database were recorded in a controlled environment and the noise sources mixed in artificially . nevertheless , this group has demonstrated the feasibility of classifying a range of different household sounds [ 6 , 810 ] . source separation is a problem in which a sound sensor receives a number of component sources , and the goal is to determine the original component sources . one technique for single - microphone source separation is to express each separated source as a weighted sum of subbands ( refiltering ) . a newer algorithm , which uses sub - band independent component analysis , has been proposed where each of the frequency bins is composed of a linear combination of the corresponding frequency bins . this technique is combined with a beamformer which estimates the direction of arrival . in , an acoustic event classification system is described which applies temporal ica on a single - microphone audio signal to determine the temporal window during which features should be extracted , resulting in improved system performance . in this context , ica was used as a method segmenting different sound sources rather than for separating them in an acoustic sense using multiple microphones . a variant of the bionic wavelet transform , an adaptive wavelet transform derived from a nonlinear auditory model of the cochlear , has been applied to the task of single - microphone speech enhancement . a novel prefilter has also been developed and uses temporal and simultaneous masking thresholds to shape the speech spectrum in order to obtain a better estimate of the autoregressive coefficients that characterize the speech spectrum . in many instances , the objective of telemonitoring is to form a model of the activities of daily life , so that deviations from this model can be used to automatically prompt for help . in sound - based telemonitoring , the aim is similar : to distinguish between different sounds of daily life ( sdls ) , which may correspond to some or all of the activities of daily life . extraction of sound signature parameters occurs at the front - end , where information putatively useful for differentiating between sound signatures is obtained from the microphone signal . as a precursor to the front - end , clearly it would be desirable to detect the presence or absence of a relevant sdl . similar approaches have been adopted in [ 3 , 7 , 8 , 16 ] where the energy of the most discriminating bands from the discrete wavelet transform ( dwt ) were used to distinguish between an event and noise . for example , to distinguish between a kettle sound and a door slap , energy may be a useful parameter , as the energy in the kettle sound would be significantly lower than that of the door slap ( assuming the microphones were equidistant from each sound source ) . since both microphones will normally contain both signal and noise components , a two - stage system is proposed , with the first stage attempting to determine estimates for the signal and noise . in general , multimicrophone sensing of sounds of daily life in realistic environments is likely to produce individual microphone signals comprising the superposition of the sound of daily life and other background noises . a related problem is known as the cocktail party effect , in which a number of people are speaking and a listener desires to listen exclusively to one of them . in a telemonitoring application , the objective is somewhat different : to separate the sound source from the background noise source , in order to classify it more accurately . suppose that we define each microphone signal xi[n ] , i = 1,2 , , m , as a linear combination of the unknown sources sj[n ] , j = 1,2 , independent component analysis ( ica ) assumes that the input source distributions are non - gaussian and independent . ica estimates a matrix b , which is the inverse of a = ( aij)mn ( in the case m = n ) from ( 1 ) , to separate the sources . ica uses the central limit theorem result , which states that a linear combination of non - gaussian independent sources will converge to a gaussian distribution . therefore , the distributions of the sensor signals xi[n ] will be more gaussian than those of the sources si[n ] . ica estimates the matrix b , which when applied to the sensors xi[n ] attempts to minimise the gaussianness of the estimated separated sources . various metrics are available for measuring a source 's gaussianness , including kurtosis and negentropy . in the context of this work , the sensor signals are recorded by n = 2 microphones m1[n ] and m2[n ] . each microphone is composed of a linear combination of the sound of daily life source ssdl[n ] and noise source snoise[n ] : ( 2)[m1[n]m2[n]]=[a11a12a21a22][ssdl[n]snoise[n ] ] . ica estimates the matrix b , which minimizes the gaussianness of ssep1[n ] and ssep2[n ] and determines estimates for the separated sources : ( 3)[ssep1[n]ssep2[n]]=[b11b12b21b22][m1[n]m2[n ] ] . there are two problems encountered using ica : ica only determines the separated sources up to a scaling factor . the matrix b could be multiplied by a scaling factor without changing the gaussianness of ssep1[n ] and ssep2[n ] ; ica separates the sources , but does not identify which is the sdl and which is the noise source . the matrix b could be multiplied by a scaling factor without changing the gaussianness of ssep1[n ] and ssep2[n ] ; ica separates the sources , but does not identify which is the sdl and which is the noise source . the focus of this paper is the classification of sounds of daily life ( sdls ) from multiple microphones of unknown location , in the presence of noise . if a range of sdls can be detected , a distress situation such as a thud , which could correspond to a fall or a cry for help , could be discovered and appropriate medical care sent to the house . in addition , a model of elderly people 's daily activities can be developed from which important information about their functional health status derived . this work is more portable than previously reported systems that employ fixed microphones [ 3 , 610 ] , employing two microphones which can be easily installed in a private residence , and data has been recorded in realistic scenarios in the presence of typical background noises . in particular , we develop methods for addressing the problems of identifying the ssdl[n ] signal following source separation and sound activity detection , as shown in figure 2 , and evaluate these on a large and realistic new multi - microphone database . the work builds on and substantially extends earlier work on sound activity detection for robust sounds of daily life classification . due to the prevalence of independent component analysis among source separation problems , we also adopt this method . to overcome the problem of ica determining the sources only up to a scaling factor , the separated sounds of daily life are herein normalized to have a maximum value of 1 . there are a number of ways to overcome the problem of identifying the ssdl[n ] signal following source separation , each with its advantages . the main approach adopted herein is to determine which separated source has the least noise ; however , it may not always be straightforward to identify which signal components are sounds of daily life and which are noise . if prior knowledge is available about which microphone has the best snr , the separation matrix can be analysed to determine which separated source has the superior snr . without loss of generality , assume that m1[n ] has the best snr . if it does not , it can be swapped with m2[n ] to ensure it does . if |b11| > |b12| , the first separated source contains more data from the microphone with the best snr and therefore has the least noise in it . |b12| , the second separated source has the smaller amount of noise : ( 4)s^sdl_apriori[n]={ssep1[n]if |b11|2>|b12|2ssep2[n]if |b11|2<|b12|2 . the main shortcoming of this algorithm is that it requires prior information as to which microphone has the best snr . this method extends the first method and automates the process of determining which microphone has the least amount of noise . a related problem is the determination of whether a sound is present or not , which we term sound activity detection ( sad ) . for each of the microphone signals , the sad algorithm is applied ( see section 3 ) , the sdl and noise segments are found using a reliable sad algorithm we used csle and an estimate of the snr determined using the following formula : ( 5)sn^rbestmic=sdl+w2w2 , where sdl+w is the average sdl ( signal plus noise ) power and w is the average noise power , estimated from segments of the signals suspected to contain sdls and noise , respectively . in addition , due to the varying microphones , noise and sdl locations , the microphone with the least noise may change . however , the main shortcoming is that if the sad is not sufficiently accurate , the microphone with the least amount of noise could be incorrectly selected which will result in not choosing the sdl correctly . a variant of this method is ( 6)sn^rleastnoise=1w2 , where n is the average noise power . this means that the sad algorithm only needs to classify noise frames correctly ( its performance is unaffected by noise frames which are classified as sdl frames ) . the snr estimates from the previous subsection extract information from the input microphone signals to determine which separated source has the best snr . the methods detailed in this section use information from the separated sources to determine which has the least amount of noise . this approach obtains an snr estimate by applying ( 8) to each separated source and determines which has the best snr : ( 7)s^sdl_bestsnr[n ] = { ssep1[n ] if snr(ssep1[n])>snr(ssep2[n])ssep2[n ] if snr(ssep1[n])<snr(ssep2[n ] ) . this method is similar to that of section 2.3 , however only the noise segments are used . the snr estimate is determined using sn^rleastnoise , which is then used as the snr estimate in ( 7 ) , to produce a separated source s^sdl_leastnoise[n ] . as with the calculation of sn^rleastnoise , this technique means the sad classification does not need to be as stringent . as discussed in section 2.1 , in some situations it is possible to use sound activity detection to identify regions of the signal for which there is noise only or sound and noise together . for the special case of two - source separation , this information can be used to estimate improved estimates of the unmixing matrix entries . consider the separated sdl from ( 7 ) and without loss of generality , assume that ssep1[n ] is the separated sdl following source selection . we define the snr of the separated sdl as follows:(8)sn^rsdl_sa(b11,b12 ) = ( 1/|{nk , nk+1, ,nh}(b11m1[n]+b12m2[n])2 , where { nk , nk+1 , , ng } are the sdl and noisy sample indices , and { nl , nl+1 , , nh } are the noisy sample indices . as b11 and b12 the snr space may exhibit an arbitrary number of peaks if both parameters are varied . if one of the parameters is constant and the other is varied , only one peak should exist . figure 4 depicts the variation in snr for the two microphone signals in figure 3 as a function of b12 . the first plot shows m1[n ] set as the primary and m2[n ] set as the secondary microphone . the second swaps m1[n ] and m2[n ] ( which is equivalent to not swapping the microphones , but holding b12 constant and varying b11 ) . both plots yield the same peak snr , suggesting that the algorithm only needs to be performed for one combination of the microphone signals . attempting to find let mbest[n ] and mworst[n ] be the microphone signals with the best and worst snrs , respectively . if b11 = 1 and b12 = 0 then ( 9)ssdl[n]=b11mbest[n]+b12mworst[n ] . the algorithm can be initialized by b11 = 1 and b12 = 0 , that is , the initial separated sdl will have the same snr as that of the microphone with the best snr . the approach adopted is to vary the separated source by adding or subtracting a scaled version of the microphone with the worst snr for the purpose of improving the snr of the separated source , ( 10)b12=b12+snrsdlb12 , where ( 11)snrsdlb12=b12(1/n)ns(b11mbest[n]+b12mworst[n])2(1/m)nn(b11mbest[n]+b12mworst[n])2 = qr((1/m)nn(b11mbest[n]+b12mworst[n])2)2,q=1mnn(b11mbest[n]+b12mworst[n])2 21nns(b11mbest[n]+b12mworst[n])mworst[n],r=1nns(b11mbest[n]+b12mworst[n])2 21mnn(b11mbest[n]+b12mworst[n])mworst[n ] . since the vector ( 1 , snrsdl/b12 ) points in the direction of steepest ascent in the ( b11 , b12 ) space , adapting the parameter b12 in this direction will increase the snr of the separated source . in ( 10 ) , the adaptation parameter is constant and determines the convergence of the algorithm . if < 0 , b12 approaches a value that yields the smallest snr . additionally , if is small , the rate of convergence will be slow . if is large , there is possible instability in the result although both issues are mitigated through adapting , as outlined below . the snrs in the database of sdls are very diverse . as a result , for some values of , the algorithm will converge quickly , some will converge slowly , and others will overshoot and miss the peak completely . to overcome this , an adaptive is implemented . the value of begins with a high value and when overshoot is detected ( which is found by a negative change in snr ) , the algorithm backtracks by one iteration and is decreased . a suitable method for achieving this , determined empirically , is multiplying by a factor of 0.1 each time an overshoot is detected . figure 5 shows how the snr evolves in each iteration of the algorithm for the example recorded in figure 3 . it was investigated whether the algorithm would converge in less time than if the optimum snr was found through an exhaustive search . two recordings were chosen at random , for each of the eight sdls from the radio at pleasant listening level and male speaker databases , using microphones 1 and 3 . figure 6 shows a plot of the difference between the best snr ( which was determined by an exhaustive search across b12 [ 10,10 ] and the snr ) which evolves in the algorithm . figure 7 shows the difference between the value of b12 , corresponding to the best snr value , and the evolved value of b12 . in both cases , all sdls converge in under 30 iterations . to obtain the same snr maximum using an exhaustive search over the region b12 [ 10,10 ) ( assuming we knew which region to select ) with a resolution of 0.01 , the number of iterations needed would be 2000 . this method is identical to the ssa method described in section 2.4 , except that here the goal is to minimize the noise power in the pure noise segments . the purpose of sound activity detection ( sad ) is to identify the location of the sdl in the audio signal ( either a raw microphone signal or separated signal ) and attempts to remove silence or pure noise components that produce unwanted variability during classification . sad requires the extraction of features that can distinguish between signal and noise , followed by a classification rule . in these respects , sad is analogous to voice activity detection in speech processing , and indeed some methods discussed below can be traced to speech processing research . the spatial separation of the multiple microphones suggests that differences in the relative energies of the sound and background noise in each microphone signal can be exploited to detect the presence or absence of the sound . in array processing , where sensors are placed in a known spatial pattern , eigenvalue methods have been used with success to estimate the bearing of a signal of interest . in the current problem , the spatial pattern of microphone sensors is unknown ; however , changes in the composition of the microphone signals due to the presence or absence of a particular source ( i.e. , the sound of daily life ) can still be detected using similar techniques . motivated by similar considerations , we define the cross - spectral matrix as : ( 12)c[k]=[|m1[k]|2|m1[k]||m2[k]||m2[k]||m1[k]||m2[k]|2 ] , where m1[k ] and m2[k ] are the discrete fourier transforms ( dfts ) of the respective microphone signals m1[n ] and m2[n ] . under the assumption that the noise is uncorrelated with the signal of interest , it has been shown that the largest eigenvalue of c[k ] comprises signal variance and noise variance terms , while the remaining eigenvalues comprise only noise variance terms . since c[k ] is a rank one matrix , we consider instead a cross - spectral matrix ( 13)c[k]=m=1mcm[k ] that has been averaged over m consecutive frames , where cm[k ] is the cross - spectral matrix at the mth frame calculated using ( 13 ) , as suggested in . the eigenvalues of this matrix are now ( 14)1,2=12(|m1[k]|2+|m2[k]|2 ) 124|m1[k]m2[k]|2+(|m1[k]|2|m2[k]|2)2 . in the problem at hand , assumptions of uncorrelated signal and noise can not be made ( typical sounds of daily life and noises are discussed in section 4 ) ; however , the choice of frequency or frequency range over which c[k ] is computed can be chosen empirically to minimize the correlation , by selecting frequencies in which the signal and noise are likely to have maximally different spectral characteristics . the eigenvalues of the rank one matrix c[k ] are ( 15)1,2={0,|m1[k]|2+|m2[k]|2 } , which can be trivially determined from the dfts of the microphone signals therefore , ( 16)2={(|a21|2+|a22|2)|snoise[k]|2 if only noise source is active(|a21|2+|a22|2)|snoise[k]|2 + ( |a11|2+|a12|2)|skitchen[k]|2 + 2(|a11a12|+|a21a22|)|skitchen[k]||snoise[k]| if sdl is occurring . therefore , one would expect a significant change in 2 when the sdl is occurring . since 2 0 , 2 is the largest eigenvalue and the smallest eigenvalue is 1 = 0 ( in practice 1 0 but 1 2 ) . therefore , a significant change in the largest eigenvalue is expected when an sdl is occurring , in comparison to during the pure noise segments . as the cross - spectral matrix has rank one , it is averaged across several hamming windowed frames , making it have rank two , allowing the eigen decomposition to be performed . the largest eigenvalues are calculated for each windowed frame average for k = 0,1 , , ( n/2 ) 1 where n is the length of the dft . the quantities ( 1/2)(|m1[k]|2+|m2[k]|2 ) , 4|m1[k]m2[k]|2 , and ( |m1[k]|2-|m2[k]|2)2 , representing terms of ( 14 ) , were also investigated ; however , none of them in isolation matched the performance of 2 as a method for sound activity detection . figure 8 shows a time - frequency plot of the largest eigenvalues for a recording when a spoon was dropped . it was determined empirically that in the 3 - 4 khz band , the eigenvalues are approximately zero when only the noise source is active and nonzero when the sdl is occurring for all sdls in the databases . an example of the 3 - 4 khz band is shown for the spoon dropping recording in the presence of radio noise in figure 9 . an average of the eigenvalues is calculated across the 3 - 4 khz band , then converted to a db scale and normalized to the range [ 1,0 ] db . this yields an indicator which is small during the pure noise segments and much higher when the sdl is occurring . a threshold was then applied to this largest eigenvalue - based indicator , a method we refer to as baseline sad . an example result of the csle algorithm , showing where the sdl was found for a dropping spoon recording , is given in figure 10 . the estimated endpoints are within 6% of the true endpoints . in the sound files checked across the entire database described in section 4 , similar deviations from the true endpoints in previous work by the authors , a database of seven sounds of daily life was constructed , in which a single radio was located close to one microphone in the recording environment . while many noise reduction applications have a sound sensor almost completely dedicated to recording the noise , this is not realistic in a general practical environment , as the sound sensors and noise source might be located anywhere . the microphone positions in the database described herein were carefully chosen with neither microphone as close to the radio as in . in addition , the microphones were chosen , where possible , to be near either a power outlet , or the mains electricity ( e.g , a light ) , to realistically evaluate the system 's performance , as the microphones would be attached to the mains power if this were to become a commercial product . microphone 1 was attached to a light , microphones 2 and 3 were mounted near power outlets and microphone 4 was placed diagonally and horizontally opposite microphones 2 and 3 , respectively , as there were no more suitable positions near a possible entry to the mains electricity . the eight sdls recorded comprised a cupboard door slamming ( s1 ) , a cup dropping ( s2 ) , a spoon dropping ( s3 ) , running tap water ( s4 ) , chopping vegetables ( s5 ) , a phone ringing ( s6 ) , a mixmaster operating ( s7 ) , and a microwave door opening ( s8 ) . in each instance four channels were recorded to compare system performance in the same conditions for different microphone placements , but a maximum of two microphones were used in any evaluations reported herein . each recording had a duration of 5 seconds , and the total duration of the database across all conditions was 2.78 hours ( 90 training samples 8 sdls 5 seconds = 1 hour . 40 training samples 8 sdls 5 seconds 4 noise degradations = 1.78 hours ) . in addition to the radio at pleasant listening level ( as in ) , other noise types recorded were the radio at a very loud volume , a male speaker talking in the background , and data recorded in the presence of rain ( note that rain can not be considered a point source , so does not satisfy the assumptions of csle ) . the estimated snr ranges , across all conditions , are shown in table 1 . the overall system is shown in figure 2 . the noise - corrupted testing data was preprocessed before feature extraction , using either signal enhancement , sound activity detection , or both . features were then extracted and maximum a posteriori ( map ) adaptation was applied before using the adapted gaussian mixture model ( gmm ) to determine the most likely sdl . feature extraction was performed using mel frequency cepstral coefficients ( mfccs ) , following earlier work showing that importance of magnitude spectrum information in distinguishing between the sdls . shifted delta cepstral ( sdc ) features were also employed , to allow some modelling of the temporal variation of the magnitude spectrum , and based on previous results [ 21 , 22 ] . a gmm classifier with six mixture components was employed , following successes in related applications [ 21 , 23 , 24 ] . map adaptation of the gmm mean vectors was conducted , to enhance the discrimination between the different sounds of daily life classes over the simpler alternative configuration comprising gmms trained separately on data from each class . since , during gmm training , random initialization of gmm parameters was employed , the results reported are the averages across 10 different initializations . classifier training was performed using clean sounds of daily life data . if it is trained using a noisy set , it will be unrealistically optimized for a specific spectral and spatial configuration . in any case , the system was trained using data from two microphones . if only one microphone is used , the classifier is biased towards data from that microphone ( as it has a unique frequency response and additive noise characteristics ) . a noise - reduced signal will contain data from two microphones , as suggested by ( 2 ) . consequently , even though the snr of the signal is improved by training using the microphone with the best snr , the classification performance may not be improved because the unmixed signal passed to the classifier will contain data from both microphones . all experiments were performed in matlab and focused on the eight - class classification accuracy of various components of the proposed system . a comparison of the seven ica - based methods for source separation among the microphone sources was made in which noise was artificially mixed into clean sounds of daily life data . the objective of this experiment was to eliminate delay between microphones , due to sound propagation , as a source of uncertainty in the results . combinations of the source separation and sound activity detection methods were tested for one of the microphone configurations of the noisy sdl database , for four different noise configurations . in these experiments , csle was selected as the sound activity detection method of choice in most configurations , based on previous work . three of the most promising source separation methods were combined with csle and tested for all microphone configurations of the noisy sdl database across all four different noise configurations . a comparison of the seven ica - based methods for source separation among the microphone sources was made in which noise was artificially mixed into clean sounds of daily life data . the objective of this experiment was to eliminate delay between microphones , due to sound propagation , as a source of uncertainty in the results . combinations of the source separation and sound activity detection methods were tested for one of the microphone configurations of the noisy sdl database , for four different noise configurations . in these experiments , csle was selected as the sound activity detection method of choice in most configurations , based on previous work . three of the most promising source separation methods were combined with csle and tested for all microphone configurations of the noisy sdl database across all four different noise configurations . in all tests , the gmm was trained with 30 recordings per sound class ( with samples chosen uniformly across the database ) , and the system was tested using 40 noisy recordings per sound class across the different types of noise . as seen in table 2 , under conditions where the noise was artificially mixed into the sounds of daily life , there was a clear advantage to using the steepest descent methods , with the noise inverse steepest ascent method of improving the unmixing coefficients producing the highest accuracies for both conditions tested . this suggests that the ssa and nisa methods may have often been able to find the optimum unmixing coefficients as occurred during the example of figure 4 . the advantage of steepest ascent methods over other source separation methods did not seem to be preserved for experiments on the noisy sdl database , in which noises were acoustically mixed in a realistic manner . as seen in table 3 , although the snr steepest ascent method with csle performed best on the mics 1 and 2 loud radio noise condition , this was by a narrow margin , and various other configurations proved slightly better for other noise conditions . in general , the results of table 3 primarily demonstrate the importance and effectiveness of sound activity detection for the problem of multichannel sdl classification in noise . finally , in table 4 , the accuracies from a selection of the most promising configurations from all conditions are reported , with a view to recommending a good system configuration across all conditions tested . from table 4 , it is clear that sound activity detection using csle is the major single contributor to classification accuracy . however , further gains might be achieved using csle and ica based on a priori snr ( section 2.1 ) , whose accuracy either improved on or was roughly comparable with that of csle alone . this paper has reported the application of source separation and sound activity detection methods to the problem of classifying sounds of daily life for home telemonitoring under realistic noisy conditions using multiple arbitrarily positioned microphones . overwhelmingly , the experimental evidence shows that the inclusion of an effective sound activity detection method , such as the cross - spectral largest eigenvalue ( csle ) method , in an activities of daily life classification system produces large and consistent gains in accuracy , up to 90% in terms of relative reduction in error rate under a range of noisy conditions . source separation methods , including the ica variants considered herein , provided at best modest gains . among these , the icasdl_apriori method , which identifies the two unmixed signals using estimates of their snrs , seems to perform well , providing relative reductions in error rates of up to 10% in the best case . the overall classification performance on an eight - class task provided accuracies between 70%100% across three different microphone - pair conditions and three noise conditions , while the most challenging low - snr loud radio noise condition resulted in accuracies in 40%50% range , still well above chance level . future work will examine the long - term performance of this system in the homes of elderly subjects , detecting a range of sounds that are found to commonly occur , and will begin correlating their occurrence with clinical indicators .

despite recent advances in the area of home telemonitoring , the challenge of automatically detecting the sound signatures of activities of daily living of an elderly patient using nonintrusive and reliable methods remains . this paper investigates the classification of eight typical sounds of daily life from arbitrarily positioned two - microphone sensors under realistic noisy conditions . in particular , the role of several source separation and sound activity detection methods is considered . evaluations on a new four - microphone database collected under four realistic noise conditions reveal that effective sound activity detection can produce significant gains in classification accuracy and that further gains can be made using source separation methods based on independent component analysis . encouragingly , the results show that recognition accuracies in the range 70%100% can be consistently obtained using different microphone - pair positions , under all but the most severe noise conditions .

1. Introduction 2. Proposed Source Selection Methods 3. Sound Activity Detection 4. Evaluation 5. Results and Discussion 6. Conclusion

PMC3016006

the delayed setting , there is often significant scarring , and traditionally these injuries have been repaired through a standard open surgical approach . however , the expansion of laparoscopic and robotic pelvic surgery suggests a role for minimally invasive approaches to the delayed repair of ureteral injuries . we present , to our knowledge , the first case of delayed robotic - assisted ureteral deligation and ureterolysis following iatrogenic ureteral injury occurring during a robotic abdominal hysterectomy . a 57-year - old female underwent a seemingly uncomplicated robotic - assisted total hysterectomy and bilateral oophorectomy for symptomatic fibroids . on postoperative day number 8 , she presented for her postoperative follow - up appointment with a complaint of persistent low back discomfort and right flank pain . the same day , a ct scan with iv contrast was obtained that demonstrated severe right - sided hydroureteronephrosis consistent with a high - grade obstruction ( figure 1 ) . she was admitted , and the following day ( postoperative day 9 ) , she was taken back to the operating room for further evaluation and management . a retrograde uretero - pyelogram demonstrated an abrupt cutoff of the right distal ureter consistent with suture ligation ( figure 2 ) . a ureteral stent was successfully placed , but the ureter remained severely medially deviated , and it was elected to definitively relieve the obstruction . the patient was explored , and the ureteral obstruction repaired via a less - invasive approach using robotic assistance . ct with iv contrast showing severe right - sided hydroureteronephrosis consistent with a high - grade obstruction . retrograde uretero - pyelogram demonstrating an abrupt cutoff of the right distal ureter consistent with suture ligation . a 4-port format was used with a 12-mm supraumbilical camera port , two 8-mm robotic ports each placed lateral to the medial umbilical ligaments and 19 cm from the pubic symphysis , and a 12-mm right lower quadrant assistant port . a penrose drain was placed encircling the ureter , and the ureter was dissected sharply towards the bladder . during this dissection , it became apparent that the right ureter had been incorporated into the running suture used to close the vaginal cuff . the suture was cut and the vaginal closure taken down in its entirety . after releasing the suture , the ureter appeared healthy but encased in inflammatory tissue that was excised . the vaginal cuff was re - closed with a running 30 vicryl secured with lapraty clips ( ethicon , cincinnati , oh ) . her ureteral stent was removed 3 weeks later . a follow - up lasix renal scan 3-months postoperatively demonstrated normal renal function bilaterally without evidence of obstruction . a 4-port format was used with a 12-mm supraumbilical camera port , two 8-mm robotic ports each placed lateral to the medial umbilical ligaments and 19 cm from the pubic symphysis , and a 12-mm right lower quadrant assistant port . a penrose drain was placed encircling the ureter , and the ureter was dissected sharply towards the bladder . during this dissection , it became apparent that the right ureter had been incorporated into the running suture used to close the vaginal cuff . the suture was cut and the vaginal closure taken down in its entirety . after releasing the suture , the ureter appeared healthy but encased in inflammatory tissue that was excised . the vaginal cuff was re - closed with a running 30 vicryl secured with lapraty clips ( ethicon , cincinnati , oh ) . her ureteral stent was removed 3 weeks later . a follow - up lasix renal scan 3-months postoperatively demonstrated normal renal function bilaterally without evidence of obstruction . in females , the ureter courses medially at the base of the broad ligament , traversing just below the uterine vessels and then enters the bladder posterolaterally approximately 2.3 cm from the lateral edge of the cervix . half of all ureteral injuries involve suture ligation , and the majority of ureteral injuries are not identified intraoperatively . the typical management of iatrogenic obstruction of the ureter that is recognized postoperatively involves initial stenting followed by delayed open ureterolysis , repair , ureteral reimplantation , or both . the role of robotic hysterectomy has been expanding rapidly , and iatrogenic ureteral ligation during robotic and laparoscopic gynecologic surgery has been reported . because these complications are rarely found intraoperatively , there is usually great reluctance on the part of the surgeon or patient , or both , to manage these complications robotically . despite this , there is a potential role for robotic surgery in the delayed management of iatrogenic ureteral obstruction . as the role of robotic - assisted surgery continues to expand , complications from minimally invasive urologic and gynecologic surgery can now be managed robotically as well . recent reports have shown that with robotic - assisted techniques , it is feasible to perform upper and lower urinary tract reconstruction with such procedures as ureteral reimplantation and ureteroureterostomy . it is logical that these same techniques can be used for the management of ureteral complications . our experience supports a role for robotic repair of iatrogenic ureteral obstruction due to suture ligation . this role can potentially be expanded to include the repair of other iatrogenic ureteral injuries . this can potentially preserve the advantages to the patient of the initial minimally invasive surgery .

background and objectives : ureteral injuries , while rare , do occur during gynecologic procedures . the expansion of laparoscopic and robotic pelvic surgical procedures increases the risk of ureteral injury from these procedures and suggests a role for minimally invasive approaches to the delayed repair of ureteral injuries . we present , to our knowledge , the first case of delayed robotic - assisted ureteral deligation and ureterolysis following iatrogenic ureteral injury occurring during a robotic abdominal hysterectomy.methods:we present a case report and review of the literature.results:a 57-year - old female underwent a seemingly uncomplicated robotic - assisted laparoscopic total abdominal hysterectomy and bilateral oophorectomy for symptomatic fibroids . on postoperative day 8 , she presented with persistent right flank pain . imaging studies revealed high - grade ureteral obstruction consistent with suture ligation of the right ureter . she underwent successful robotic - assisted ureteral deligation and ureterolysis . her postoperative course was unremarkable , and she was discharged home on postoperative day 1 from the deligation.conclusion:robotic-assisted management of complications from urologic or gynecologic surgery is technically feasible . this can potentially preserve the advantages to the patient that are being seen from the initial less - invasive surgery .

INTRODUCTION CASE REPORT Description of Robotic Surgical Procedure DISCUSSION CONCLUSION

PMC5404171

femoroacetabular impingement ( fai ) is a common cause of hip pathology characterized by post activity groin pain and joint stiffness due to the abnormal repetitive contact between the femoral head / neck and the acetabular rim . extra - articular causes of hip impingement have been increasingly recognized with abnormal morphology of the anterior inferior iliac spine ( aiis ) considered an important source . deformity of the aiis may arise from a developmental abnormality or from secondary ossification following an acute injury to the rectus femoris tendon or its apophysis . open or arthroscopic resection of the aiis deformity may be indicated for chronic symptoms of impingement [ 2 , 3 , 4 ] . we present a case series of three active , male patients with distinct clinical features of extra - articular aiis impingement ; the diagnosis and the mid - term post - operative outcomes at a mean of 1.7 years ( 1.5 - 2 years ) following arthroscopic surgery , with treatment of concomitant intra - articular pathology , are discussed . a 27-year - old football player presented with a history of an acute - onset discomfort in his right thigh and hip when he went to kick a football during a competitive game . over a 12-month period , he continued to experience sharp pain in the groin particularly with twisting and turning movements . his symptoms failed to improve with conservative treatment including reduction in intensity of sporting activities , physiotherapy , and platelet rich plasma injections into the rectus femoris tendon footprint on the aiis ( the main location of his pain ) . physical examination revealed restriction of flexion ( 110 ) and internal rotation of the flexed hip ( 27 ) . anterior groin pain developed once the hip was flexed above 60 and increased with flexion , adduction , and internal rotation faddir maneuver . review of an x - ray series of the right hip including anteroposterior ( ap ) pelvis , dunn and false profile views revealed a large bony exostosis extending anteroinferiorly from the aiis ( fig . the hip joint was in good condition , however , evidence of mixed fai was present ; a crossover sign was present on the ap pelvis view with a lateral centre - edge angle ( cea ) of 34 ; a pincer deformity was clearly observed on the false profile views and an anterior labral tear was evident on magnetic resonance arthrography ( mra ) of the hip . there was no significant cam deformity observed at the femoral head neck region with an alpha angle of 53 measured on the dunn view . ( a ) pre - operative anteroposterior ( ap ) x - ray image showing large bony exostosis arising from the anterior inferior iliac spine ( white arrow ) and prominent acetabular rim ( black dotted line ) . ( b ) ap x - ray image showing complete resection of the exostosis ( white arrow ) and acetabular rim recession ( black arrows ) after arthroscopic surgery . the patient underwent arthroscopy of his right hip through standard anterolateral and modified mid - anterior portals and an anterolateral pincer deformity was removed using a 4 mm mechanical burr under image intensifier guidance . the torn labrum was repaired using two suture anchors and an interportal capsulotomy was repaired using three fiberwire stitches . the instruments were then repositioned adjacent to the exostosis which was subsequently removed using a 5.5 mm mechanical burr . at 3 months postsurgery the patient was able to run with an increased stride and freedom of hip movements . improvements were observed in flexion by 3 ( 110 to 113 ) and internal rotation by 11 ( 27 to 38 ) . post - operative x - rays revealed complete removal of the large bony aiis exostosis on all views , with a decrease in the lateral cea by 9 ( 34 to 25 ) with reversal of the crossover sign evident on the ap pelvis view ( fig . improvements , at 1.5 year postoperation were observed across a range of validated health questionnaires : modified harris hip score : 68 ( pre - operative ) - 100 ( post - operative ) ; ucla activity rating : 9 ( pre - operative ) - 10 ( post - operative ) ; short form-36 : 64.8 ( pre - operative ) - 97.5 ( post - operative ) ; womac osteoarthritic index : 28 ( pre - operative ) - 0 ( post - operative ) . a 35-year - old physically active male , presented with chronic anterior groin pain for over 5 years . despite receiving extensive physiotherapy , he had responded poorly with a continued decline in his level of activity due to recurrent symptoms . the patient recalled an injury to his proximal thigh when he was 18 years of age , which settled well with rest and physiotherapy . physical examination revealed significant restriction of hip flexion to 112 and internal rotation was limited to 18 ( with the hip flexed to 90 ) ; faddir testing resulted in pain particularly in the anterolateral region of the joint . x - rays of the right hip revealed mixed pincer and cam impingement in a good quality joint with a large exostosis of the aiis originating at the footprint of the direct tendon of rectus femoris . a loss of lateral head - neck offset was evident on ap pelvis x - rays with an alpha angle of 65 ( an alpha angle of 45 was observed on the dunn view ) ; lateral cea of 40 was measured and a crossover sign observed ; a pincer deformity was evident on the false profile views . the patient underwent arthroscopy of his right hip with removal of the pincer and lateral cam deformities . the labrum was found to have a chondrolabral detachment anterolaterally , which was repaired using two suture anchors . the aiis deformity was located using x - ray guidance and a complete resection of the exostosis , which was rigidly attached to the pelvis , was performed using a 5.5 mm mechanical burr ( fig . intraoperative fluoroscopic views ( a ) localizing the exostosis ; ( b ) arthroscopic resection of the bony exostosis from the anterior inferior iliac spine ( aiis ) without distraction ; ( c ) completion of resection of exostosis . ( d ) pre - operative 90 dunn view x - ray image showing a large bony exostosis arising from the aiis ( white arrow ) . ( e ) post - operative 90 dunn view x - ray image at 18-months showing complete resection of the exostosis following arthroscopy surgery . post - operative x - rays demonstrated an improvement in the lateral cea by 6 ( 40 to 34 ) and alpha angle by 8 ( 65 to 57 ) . 2e ) and the aiis bony exostosis ( all views ) was observed . at 18 months postoperation the patient was pain free : the range of hip flexion improved by 10 ( 112 to 122 ) and internal rotation ( of the flexed hip ) by 12 ( 18 to 30 ) . ucla improved from 7 ( pre - operative ) - 9 ( post - operative ) ; womac : 7 ( pre - operative ) - 3 ( post - operative ) ; the mhhs : 100 ( pre - operative ) - 100 ( post - operative ) and sf-36 : 92 ( pre - operative ) - 91 ( post - operative ) remained largely unchanged . a 53-year - old physically active groundskeeper , presented with a 5-year history of progressive , chronic hip and groin pain , particularly on forward bending or twisting and turning movements . physical examination revealed limited range of hip movement with restriction in flexion ( 106 ) and internal rotation of the flexed hip ( 28 ) ; a catching sensation and increased groin pain resulted on faddir maneuver . x - rays demonstrated an area of dense ossification from the rectus femoris origin on the aiis forming a large exostosis over the front of the hip . the quality of the hip joint was good , however , evidence of mixed cam and pincer impingement was evident : on an ap pelvis view , a crossover sign was observed with a lateral cea of 33 , an alpha angle of 57 and 50 was measured on the ap and dunn views , respectively , and a pincer deformity was clearly observed on the false profile view . the labrum was degenerative with an intrasubstance tear anteriorly . using a ( 5.5 mm ) mechanical burr , the localized pincer deformity beneath the aiis was removed and the femoral head - neck offset was improved ; the labrum was repaired using two suture anchors . a large 4 cm hardened exostosis originating from the aiis was resected using the mechanical burr . the exostosis was found to be relatively mobile and not fixed to the aiis footprint on the pelvis . post - operative x - rays demonstrated an improvement in alpha angle by 9 ( 57 to 48 : ap pelvis ) and by 4 ( 50 to 46 : dunn view ) and the lateral cea by 8 ( 33 to 25 ) . complete resection of the aiis bony exostosis ( all views ) and pincer deformity ( false profile ) was observed ( fig . 3 ) , with reversal of the crossover sign also evident ( ap pelvis ) . ( a ) pre - operative false profile view demonstrating a finger - like bony prominence extending from the bony origin of the rectus femoris tendon ( white arrow ) . ( b ) post - operative false profile x - ray following complete arthroscopic removal of prominent exostosis . at 2 years postsurgery , the patient remained fully engaged in physical activity free from pain with excellent outcome : mhhs - 93 ( pre - operative ) , 100 ( post - operative ) ; ucla 8 ( pre - operative ) , 9 ( post - operative ) ; sf-36 - 77.1 ( pre - operative ) , 93 ( post - operative ) ; womac - 37 ( pre - operative ) , 2 ( post - operative ) . a 27-year - old football player presented with a history of an acute - onset discomfort in his right thigh and hip when he went to kick a football during a competitive game . over a 12-month period , he continued to experience sharp pain in the groin particularly with twisting and turning movements . his symptoms failed to improve with conservative treatment including reduction in intensity of sporting activities , physiotherapy , and platelet rich plasma injections into the rectus femoris tendon footprint on the aiis ( the main location of his pain ) . physical examination revealed restriction of flexion ( 110 ) and internal rotation of the flexed hip ( 27 ) . anterior groin pain developed once the hip was flexed above 60 and increased with flexion , adduction , and internal rotation faddir maneuver . review of an x - ray series of the right hip including anteroposterior ( ap ) pelvis , dunn and false profile views revealed a large bony exostosis extending anteroinferiorly from the aiis ( fig . the hip joint was in good condition , however , evidence of mixed fai was present ; a crossover sign was present on the ap pelvis view with a lateral centre - edge angle ( cea ) of 34 ; a pincer deformity was clearly observed on the false profile views and an anterior labral tear was evident on magnetic resonance arthrography ( mra ) of the hip . there was no significant cam deformity observed at the femoral head neck region with an alpha angle of 53 measured on the dunn view . ( a ) pre - operative anteroposterior ( ap ) x - ray image showing large bony exostosis arising from the anterior inferior iliac spine ( white arrow ) and prominent acetabular rim ( black dotted line ) . ( b ) ap x - ray image showing complete resection of the exostosis ( white arrow ) and acetabular rim recession ( black arrows ) after arthroscopic surgery . the patient underwent arthroscopy of his right hip through standard anterolateral and modified mid - anterior portals and an anterolateral pincer deformity was removed using a 4 mm mechanical burr under image intensifier guidance . the torn labrum was repaired using two suture anchors and an interportal capsulotomy was repaired using three fiberwire stitches . the instruments were then repositioned adjacent to the exostosis which was subsequently removed using a 5.5 mm mechanical burr . at 3 months postsurgery the patient was able to run with an increased stride and freedom of hip movements . improvements were observed in flexion by 3 ( 110 to 113 ) and internal rotation by 11 ( 27 to 38 ) . post - operative x - rays revealed complete removal of the large bony aiis exostosis on all views , with a decrease in the lateral cea by 9 ( 34 to 25 ) with reversal of the crossover sign evident on the ap pelvis view ( fig . improvements , at 1.5 year postoperation were observed across a range of validated health questionnaires : modified harris hip score : 68 ( pre - operative ) - 100 ( post - operative ) ; ucla activity rating : 9 ( pre - operative ) - 10 ( post - operative ) ; short form-36 : 64.8 ( pre - operative ) - 97.5 ( post - operative ) ; womac osteoarthritic index : 28 ( pre - operative ) - 0 ( post - operative ) . a 35-year - old physically active male , presented with chronic anterior groin pain for over 5 years . despite receiving extensive physiotherapy , he had responded poorly with a continued decline in his level of activity due to recurrent symptoms . the patient recalled an injury to his proximal thigh when he was 18 years of age , which settled well with rest and physiotherapy . physical examination revealed significant restriction of hip flexion to 112 and internal rotation was limited to 18 ( with the hip flexed to 90 ) ; faddir testing resulted in pain particularly in the anterolateral region of the joint . x - rays of the right hip revealed mixed pincer and cam impingement in a good quality joint with a large exostosis of the aiis originating at the footprint of the direct tendon of rectus femoris . a loss of lateral head - neck offset was evident on ap pelvis x - rays with an alpha angle of 65 ( an alpha angle of 45 was observed on the dunn view ) ; lateral cea of 40 was measured and a crossover sign observed ; a pincer deformity was evident on the false profile views . the patient underwent arthroscopy of his right hip with removal of the pincer and lateral cam deformities . the labrum was found to have a chondrolabral detachment anterolaterally , which was repaired using two suture anchors . the aiis deformity was located using x - ray guidance and a complete resection of the exostosis , which was rigidly attached to the pelvis , was performed using a 5.5 mm mechanical burr ( fig . intraoperative fluoroscopic views ( a ) localizing the exostosis ; ( b ) arthroscopic resection of the bony exostosis from the anterior inferior iliac spine ( aiis ) without distraction ; ( c ) completion of resection of exostosis . ( d ) pre - operative 90 dunn view x - ray image showing a large bony exostosis arising from the aiis ( white arrow ) . ( e ) post - operative 90 dunn view x - ray image at 18-months showing complete resection of the exostosis following arthroscopy surgery . post - operative x - rays demonstrated an improvement in the lateral cea by 6 ( 40 to 34 ) and alpha angle by 8 ( 65 to 57 ) . 2e ) and the aiis bony exostosis ( all views ) was observed . at 18 months postoperation the patient was pain free : the range of hip flexion improved by 10 ( 112 to 122 ) and internal rotation ( of the flexed hip ) by 12 ( 18 to 30 ) . ucla improved from 7 ( pre - operative ) - 9 ( post - operative ) ; womac : 7 ( pre - operative ) - 3 ( post - operative ) ; the mhhs : 100 ( pre - operative ) - 100 ( post - operative ) and sf-36 : 92 ( pre - operative ) - 91 ( post - operative ) remained largely unchanged . a 53-year - old physically active groundskeeper , presented with a 5-year history of progressive , chronic hip and groin pain , particularly on forward bending or twisting and turning movements . physical examination revealed limited range of hip movement with restriction in flexion ( 106 ) and internal rotation of the flexed hip ( 28 ) ; a catching sensation and increased groin pain resulted on faddir maneuver . x - rays demonstrated an area of dense ossification from the rectus femoris origin on the aiis forming a large exostosis over the front of the hip . the quality of the hip joint was good , however , evidence of mixed cam and pincer impingement was evident : on an ap pelvis view , a crossover sign was observed with a lateral cea of 33 , an alpha angle of 57 and 50 was measured on the ap and dunn views , respectively , and a pincer deformity was clearly observed on the false profile view . the labrum was degenerative with an intrasubstance tear anteriorly . using a ( 5.5 mm ) mechanical burr , the localized pincer deformity beneath the aiis was removed and the femoral head - neck offset was improved ; the labrum was repaired using two suture anchors . a large 4 cm hardened exostosis originating from the aiis was resected using the mechanical burr . the exostosis was found to be relatively mobile and not fixed to the aiis footprint on the pelvis . post - operative x - rays demonstrated an improvement in alpha angle by 9 ( 57 to 48 : ap pelvis ) and by 4 ( 50 to 46 : dunn view ) and the lateral cea by 8 ( 33 to 25 ) . complete resection of the aiis bony exostosis ( all views ) and pincer deformity ( false profile ) was observed ( fig . 3 ) , with reversal of the crossover sign also evident ( ap pelvis ) . ( a ) pre - operative false profile view demonstrating a finger - like bony prominence extending from the bony origin of the rectus femoris tendon ( white arrow ) . ( b ) post - operative false profile x - ray following complete arthroscopic removal of prominent exostosis . at 2 years postsurgery , the patient remained fully engaged in physical activity free from pain with excellent outcome : mhhs - 93 ( pre - operative ) , 100 ( post - operative ) ; ucla 8 ( pre - operative ) , 9 ( post - operative ) ; sf-36 - 77.1 ( pre - operative ) , 93 ( post - operative ) ; womac - 37 ( pre - operative ) , 2 ( post - operative ) . abnormal morphology of the aiis is recognized as a source of extra - articular hip impingement [ 2 , 3 , 4 ] . in patients presenting with aiis impingement , there is often a history of previous trauma to the rectus femoris muscle or tendon during their adolescence . in most cases , a single explosive contraction of the rectus femoris during hyperextension of the hip joint at the same time as flexion of the knee results in apophyseal avulsion or tearing of the rectus athletes who participate in particular impact sports that involve kicking , rapid twisting , and turning or running / acceleration at high speed are at higher risk from this injury [ 5 , 6 , 7 ] . the injury responds well to nonoperative treatment including rest , nonsteroidal anti - inflammatory drugs and physiotherapy with a predicted timeline to full recovery expected anywhere from 3 weeks to 4 months . however , where heterotopic ossification of the injured soft tissues or malunion of the avulsed fragment becomes a source of chronic or delayed symptoms , surgical intervention may be indicated for relief of symptoms [ 5 , 9 ] . abnormal morphology of the aiis leading to extra - articular impingement may occur as a developmental anomaly without prior injury and may occur from traction hypertrophy by prolonged and excessive strain on the rectus femoris tendon ; a retroverted acetabulum with excessive anterior pelvic tilt and following acetabular orientation surgery [ 4 , 10 ] . in rarer circumstances and less well reported is the presence of the developmental anomaly known as the pelvic digit which may contribute to impingement - like symptoms and should be considered in instances of pain and functional impairment in the absence of any history of trauma [ 11 , 12 ] . in our case series , patients 1 and 2 were involved in high impact sports involving sprinting and forceful kicking motions . these athletes were able to identify a specific instance where injury to the rectus femoris was likely to have occurred . in both cases , a classical pattern of acute injury followed by a chronic or delayed increase in symptoms from extra - articular impingement was evident . in contrast , patient 3 had no history of trauma but developed a chronic progressive increase in symptoms . a distinct , symptomatic bony projection with a pseudoarticulation with the pelvis was observed , which may represent a developmental abnormality such as a pelvic digit or nontraumatic ossification . patients with symptomatic aiis impingement will generally present to the clinic with limitation of hip movement and associated anterior groin pain . clinical signs may be subtle and similar to that of cam / pincer fai , as such an intra - articular injection of local anesthetic may help differentiate the source of the primary symptoms [ 3 , 4 ] . imaging is an important resource in helping to confirm the diagnosis : ap pelvis , false profile , and dunn x - rays should be utilized to assess the hip joints for the presence of cam / pincer deformity and abnormal aiis morphology . a computed tomography scan in addition will provide a more detailed three - dimensional image analysis and measures of femoral and acetabular version . mra will provide information on the presence and location of articular cartilage and labral pathology . while the development of large exostosis in each of our cases was likely the primary cause of impingement - like pain and discomfort , the coexistence with intra - articular abnormalities may have been a contributing factor in each case . an arthroscopic technique was , therefore , utilized in this case series to perform the aiis deformity resection and to treat the concomitant fai and associated intra - articular pathology . good outcome following open aiis resection for impingement was originally described by pan et al . . a number of authors have reported good outcome following the arthroscopic resection of aiis deformity in conjunction with treatment of intra - articular pathology [ 3 , 4 , 9 , 14 ] . at a mean of 1.7 years ( 1.5 - 2.0 ) following impingement deformity correction , all patients in our series reported excellent functional results , improved range of hip motion , were asymptomatic , with full participation in training and recreational fitness reestablished . abnormal morphology of the aiis may result in symptomatic extra - articular hip impingement and may coexist with intra - articular bony and soft tissue hip pathology . this case series demonstrates the sustainable benefits of arthroscopic correction of aiis bony exostosis as an uncommon cause of extra - articular fai . this article demonstrates three distinct clinical presentations of extra - articular hip impingement from abnormal morphology of the aiis ( one post - traumatic case with a failure of conservative management , the second also a post - traumatic case with a delayed return of symptoms 5 years later and the third from a developmental nontraumatic source ) . excellent post - operative results can be expected utilizing an arthroscopic technique for resection of the bony deformity with a full return to normal activity up to 2 years postsurgery . the article highlights the need for a high index of suspicion in diagnosis and treatment of active patients with hip and groin pain so that extra - articular sources for impingement are not overlooked .

introduction : abnormal morphology of the anterior inferior iliac spine ( aiis ) is a rarely recognized but important source of extra - articular hip impingement . chronic progressive symptoms of stiffness and limitation of hip motion with persistent groin pain may place significant restriction on activity . concomitant femoroacetabular impingement is often present but recognition and effective treatment of the uncommon extra - articular component is important for successful outcome.case report : three cases of symptomatic extra - articular hip impingement secondary to aiis deformity and in conjunction with mild underlying femoroacetabular impingement ( fai ) are presented . they include two athletic caucasian males aged 27 and 35-years old with a history of prior rectus tendon injury and secondary bony exostosis formation and a 53-year - old caucasian male with a nontraumatic , developmental aiis deformity . in all cases , an excellent clinical outcome with a full return to pain free activity was achieved postoperation . their clinical presentation , diagnosis and post - operative outcome at 1.5 - 2 years ( mean 1.7 years ) following arthroscopic aiis resection are discussed.conclusion:arthroscopic management of aiis extra - articular hip impingement has been rarely reported and the longer - term outcome is unknown . we report the successful clinical outcome in a case series of three patients up to 2 years following arthroscopic aiis resection . this case series demonstrates the sustainable benefits of arthroscopic correction of aiis bony exostosis as a cause of extra - articular fai .

Introduction Case Report Patient 1 Patient 2 Patient 3 Discussion Conclusion

PMC3714511

the study population consisted of 11 male type 1 diabetic patients ( mean sem ; age 24 2 years , bmi 22.6 0.6 , duration of diabetes 15 2 years , hba1c 7.7 0.3% , 61 3.4 mmol / mol , vo2peak 53 1 ml kg min ) . patients were eligible for inclusion if they were between 18 and 35 years of age with a duration of diabetes > 2 years on enrollment . patients were treated with a stable basal - bolus insulin regimen composed of either insulin glargine ( n = 8) or detemir ( n = 3 ) and fast - acting insulin analogs aspart ( n = 10 ) or lispro ( n = 1 ) for a minimum of 6 months . patients were regularly and consistently active ( participating in aerobic - based exercise for at least 30 min at a time , three times per week ) , free of diabetes - related complications , including hypoglycemia unawareness , and receiving no additional medication other than insulin . patients attended a preliminary screening visit , in which a comprehensive medical history and physical examination wasconducted . additionally , patients completed a cardiopulmonary exercise stress test to assess cardiac function in response to exercise . all screening procedures complied with american college of sports medicine ( acsm ) guidelines for exercise testing and prescription ( 23 ) . eligible patients underwent randomization by a computer program to determine the sequence of three crossover arms . peak cardiorespiratory parameters were collected during the completion of an incremental - maximal treadmill run protocol ( 11,12 ) . the treadmill run started at a velocity of 8 km h and increased 1 km h every 3 min to volitional exhaustion , as per west et al . anthropometric variables were also collected ( body mass , stature , and bmi ) . visits 2 , 3 , and 4 were experimental trials . patients were fitted with a continuous glucose monitoring system ( cgms ; ipro2 , medtronic diabetes , northridge , ca ) a minimum of 24 h before attending the laboratory and replicated their diet and activity patterns for 24 h prior to main trials . the ipro2 ( medtronic diabetes , northridge , ca ) system consists of a needle - like sensor that is inserted into subcutaneous tissue . sensor readings are obtained and stored in the memory of the monitor , which is attached to the indwelling sensor . patients wore enlite sensors ( medtronic minimed , northridge , ca ) , which were placed in the postural - lateral abdominal region to minimize the physiological time lags between blood and interstitial glucose ( 24 ) . the sensor was inserted 24 h prior to each main trial , with the site of insertion replicated between trials . sensors were removed 24 h after leaving the laboratory . during sensor wear , patients self - recorded capillary blood glucose concentrations at least four times per day for calibration purposes using a testing meter ( glucomen lx ; menarini diagnostics , berkshire , u.k . ) . sensor data were retrospectively processed using carelink ipro software ( medtronic diabetes ) and ipro2 retrospective algorithm ( medtronic diabetes ) calibration routines . patients arrived at the laboratory after an overnight fast , having replicated their diet in the previous 24 h ( assessed using weighed dietary recording sheets ) . additionally , patients were instructed to maintain their normal insulin regimen , with basal insulin dose standardized ( dose , injection site , and time of injection ) across trials . , hoofddorp , the netherlands ) , which they were instructed to wear in the previous 24 h. patients were required to avoid strenuous activity in the previous 48 h and maintain similar activity patterns between trials . all main trials were conducted on a morning ( 08:00 a.m. ) , with a minimum of 7 days between trials ; patients replicated their trial start time across conditions . after arrival to the laboratory , patients were seated while a 20-gauge cannula ( vasofix ; b. braun melsungen ag , melsungen , germany ) was inserted into the antecubital vein of their nondominant arm . a 6-ml resting venous blood sample was taken , of which 20 l was used for the immediate quantification of glucose and lactate ( biosen c - line ; ekf diagnostic gmbh , london , u.k . ) and 10 l was analyzed for hemoglobin and hematocrit ( hemo control ; ekf diagnostic gmbh ) , which was used to correct for changes in plasma volume ( 25 ) . venous blood ( 5 ml ) was collected in a serum separation tube that was centrifuged for 15 min at 3,000 rev min and stored at 80c for later analysis of rapid - acting insulin ( invitron insulin assay ; invitron , monmouth u.k . ) and -hydroxybutyrate ( ranbut ; randox laboratories , london , u.k . ) . rapid - acting insulin was measured in patients using basal insulin glargine only . immediately after this sample , patients administered a 25% ( 1.8 0.1 iu ) dose ( i.e. , a 75% reduction ) of rapid - acting insulin into the abdomen , as recommended by rabasa - lhoret et al . all patients were familiar with carbohydrate counting and were administering 1.0 0.1 iu of rapid - acting insulin per 10 g of carbohydrate . the injection site was standardized across trials through administration at half the distance between the anterior - superior iliac crest and the naval . insulin aspart and lispro were administered using novopen3 ( novonordisk , bagsvaerd , denmark ) and humapen luxura ( eli lilly , london , u.k . ) , respectively . after administration of the reduced rapid - acting insulin dose , patients consumed a cereal - based breakfast meal ( frosted flakes , semiskimmed milk , and peaches ) equating to 1 g carbohydrate kg bm ( 380 10 kcal ) within a 5-min period . after consuming breakfast , patients remained rested , with blood samples collected at 30 and 60 min after the ingestion of the meal . sixty minutes after breakfast , patients performed 45 min of steady - state treadmill ( woodway , weil am rhein , germany ) running at 72.5 0.9% vo2peak , an intensity that falls within the recommendations of acsm ( 23 ) for exercising individuals with diabetes . breath - by - breath respiratory parameters ( metalyzer 3b ; cortex , leipzig , germany ) and heart rate ( s810 ; polar , kempele , finland ) were continuously recorded throughout exercise . after the cessation of exercise , blood samples were collected at 0 , 5 , 15 , 30 , and 60 min postexercise . at 60 min postexercise , in a randomized and counter - balanced fashion , patients administered a full ( 7.5 0.3 iu ) , 75% ( 5.6 0.2 iu ) , or 50% ( 3.7 0.1 iu ) rapid - acting insulin dose , which was injected into the contralateral abdominal site to the pre - exercise , rapid - acting insulin dose . patients then consumed a pasta - based lunch meal ( pasta , tomato - based sauce , cheddar cheese ) , equating to 1 g carbohydrate kg bm ( 660 21 kcal ) , and then remained at rest for 180 min postmeal . blood samples were collected every 30 min throughout the postmeal period ; patients could drink water ad libitum . hypoglycemia was defined as a blood or interstitial glucose concentrations of 3.9 mmol l ( 15 ) . if patients experienced hypoglycemia during the laboratory period , a 20-g carbohydrate bolus was administered ( lucozade ; glaxosmithkline , middlesex , u.k . ) . during cgms wear , patients were required to replicate their diet across trials through the use of a weighed food diary . dietary intake and insulin administration was self - recorded , with patients required to indicate if additional carbohydrate intake had been ingested to correct falling blood glucose . patients were required to keep meal times and rapid - acting insulin dose consistent across trials . patients continued to record a minimum of four capillary blood glucose concentrations daily for cgms calibration . statistical analysis was performed using pasw statistics software ( ibm pasw version 18 ; ibm , armonk , ny ) . a repeated - measures anova on three levels ( condition time ) was conducted , with bonferroni - corrected pairwise comparisons and paired samples student t test used to examine time and treatment effects , respectively . area under the curve was calculated using the methods described by wolever and jenkins ( 26 ) . the study population consisted of 11 male type 1 diabetic patients ( mean sem ; age 24 2 years , bmi 22.6 0.6 , duration of diabetes 15 2 years , hba1c 7.7 0.3% , 61 3.4 mmol / mol , vo2peak 53 1 ml kg min ) . patients were eligible for inclusion if they were between 18 and 35 years of age with a duration of diabetes > 2 years on enrollment . patients were treated with a stable basal - bolus insulin regimen composed of either insulin glargine ( n = 8) or detemir ( n = 3 ) and fast - acting insulin analogs aspart ( n = 10 ) or lispro ( n = 1 ) for a minimum of 6 months . patients were regularly and consistently active ( participating in aerobic - based exercise for at least 30 min at a time , three times per week ) , free of diabetes - related complications , including hypoglycemia unawareness , and receiving no additional medication other than insulin . patients attended a preliminary screening visit , in which a comprehensive medical history and physical examination wasconducted . additionally , patients completed a cardiopulmonary exercise stress test to assess cardiac function in response to exercise . all screening procedures complied with american college of sports medicine ( acsm ) guidelines for exercise testing and prescription ( 23 ) . eligible patients underwent randomization by a computer program to determine the sequence of three crossover arms . peak cardiorespiratory parameters were collected during the completion of an incremental - maximal treadmill run protocol ( 11,12 ) . the treadmill run started at a velocity of 8 km h and increased 1 km h every 3 min to volitional exhaustion , as per west et al . anthropometric variables were also collected ( body mass , stature , and bmi ) . visits 2 , 3 , and 4 were experimental trials . patients were fitted with a continuous glucose monitoring system ( cgms ; ipro2 , medtronic diabetes , northridge , ca ) a minimum of 24 h before attending the laboratory and replicated their diet and activity patterns for 24 h prior to main trials . the ipro2 ( medtronic diabetes , northridge , ca ) system consists of a needle - like sensor that is inserted into subcutaneous tissue . sensor readings are obtained and stored in the memory of the monitor , which is attached to the indwelling sensor . patients wore enlite sensors ( medtronic minimed , northridge , ca ) , which were placed in the postural - lateral abdominal region to minimize the physiological time lags between blood and interstitial glucose ( 24 ) . the sensor was inserted 24 h prior to each main trial , with the site of insertion replicated between trials . wear , patients self - recorded capillary blood glucose concentrations at least four times per day for calibration purposes using a testing meter ( glucomen lx ; menarini diagnostics , berkshire , u.k . ) . sensor data were retrospectively processed using carelink ipro software ( medtronic diabetes ) and ipro2 retrospective algorithm ( medtronic diabetes ) calibration routines . patients arrived at the laboratory after an overnight fast , having replicated their diet in the previous 24 h ( assessed using weighed dietary recording sheets ) . additionally , patients were instructed to maintain their normal insulin regimen , with basal insulin dose standardized ( dose , injection site , and time of injection ) across trials . , hoofddorp , the netherlands ) , which they were instructed to wear in the previous 24 h. patients were required to avoid strenuous activity in the previous 48 h and maintain similar activity patterns between trials . all main trials were conducted on a morning ( 08:00 a.m. ) , with a minimum of 7 days between trials ; patients replicated their trial start time across conditions . after arrival to the laboratory , patients were seated while a 20-gauge cannula ( vasofix ; b. braun melsungen ag , melsungen , germany ) was inserted into the antecubital vein of their nondominant arm . a 6-ml resting venous blood sample was taken , of which 20 l was used for the immediate quantification of glucose and lactate ( biosen c - line ; ekf diagnostic gmbh , london , u.k . ) and 10 l was analyzed for hemoglobin and hematocrit ( hemo control ; ekf diagnostic gmbh ) , which was used to correct for changes in plasma volume ( 25 ) . venous blood ( 5 ml ) was collected in a serum separation tube that was centrifuged for 15 min at 3,000 rev min and stored at 80c for later analysis of rapid - acting insulin ( invitron insulin assay ; invitron , monmouth u.k . ) and -hydroxybutyrate ( ranbut ; randox laboratories , london , u.k . ) . rapid - acting insulin was measured in patients using basal insulin glargine only . immediately after this sample , patients administered a 25% ( 1.8 0.1 iu ) dose ( i.e. , a 75% reduction ) of rapid - acting insulin into the abdomen , as recommended by rabasa - lhoret et al . all patients were familiar with carbohydrate counting and were administering 1.0 0.1 iu of rapid - acting insulin per 10 g of carbohydrate . the injection site was standardized across trials through administration at half the distance between the anterior - superior iliac crest and the naval . insulin aspart and lispro were administered using novopen3 ( novonordisk , bagsvaerd , denmark ) and humapen luxura ( eli lilly , london , u.k . ) , respectively . after administration of the reduced rapid - acting insulin dose , patients consumed a cereal - based breakfast meal ( frosted flakes , semiskimmed milk , and peaches ) equating to 1 g carbohydrate kg bm ( 380 10 kcal ) within a 5-min period . after consuming breakfast , patients remained rested , with blood samples collected at 30 and 60 min after the ingestion of the meal . sixty minutes after breakfast , patients performed 45 min of steady - state treadmill ( woodway , weil am rhein , germany ) running at 72.5 0.9% vo2peak , an intensity that falls within the recommendations of acsm ( 23 ) for exercising individuals with diabetes . breath - by - breath respiratory parameters ( metalyzer 3b ; cortex , leipzig , germany ) and heart rate ( s810 ; polar , kempele , finland ) were continuously recorded throughout exercise . after the cessation of exercise , blood samples were collected at 0 , 5 , 15 , 30 , and 60 min postexercise . at 60 min postexercise , in a randomized and counter - balanced fashion , patients administered a full ( 7.5 0.3 iu ) , 75% ( 5.6 0.2 iu ) , or 50% ( 3.7 0.1 iu ) rapid - acting insulin dose , which was injected into the contralateral abdominal site to the pre - exercise , rapid - acting insulin dose . patients then consumed a pasta - based lunch meal ( pasta , tomato - based sauce , cheddar cheese ) , equating to 1 g carbohydrate kg bm ( 660 21 kcal ) , and then remained at rest for 180 min postmeal . blood samples were collected every 30 min throughout the postmeal period ; patients could drink water ad libitum . hypoglycemia was defined as a blood or interstitial glucose concentrations of 3.9 mmol l ( 15 ) . if patients experienced hypoglycemia during the laboratory period , a 20-g carbohydrate bolus was administered ( lucozade ; glaxosmithkline , middlesex , u.k . ) . during cgms wear , patients were required to replicate their diet across trials through the use of a weighed food diary . dietary intake and insulin administration was self - recorded , with patients required to indicate if additional carbohydrate intake had been ingested to correct falling blood glucose . patients were required to keep meal times and rapid - acting insulin dose consistent across trials . patients continued to record a minimum of four capillary blood glucose concentrations daily for cgms calibration . statistical analysis was performed using pasw statistics software ( ibm pasw version 18 ; ibm , armonk , ny ) . a repeated - measures anova on three levels ( condition time ) was conducted , with bonferroni - corrected pairwise comparisons and paired samples student t test used to examine time and treatment effects , respectively . area under the curve was calculated using the methods described by wolever and jenkins ( 26 ) . patients displayed similar glycemic control during the 24 h before arriving to the laboratory , with similar mean ( full , 8.6 0.4 ; 75% , 7.7 0.5 ; 50% , 8.1 0.6 mmol l ; p = 0.451 ) and total interstitial glucose area under the curve ( full , 12,389 705 ; 75% , 10,986 765 ; 50% , 11,385 791 mmol l min ; p = 0.400 ) across trials . during this time , dietary patterns were similar , with no differences in total energy intake ( full , 2,249 110 ; 5% , 2,290 178 ; 50% , 2,130 128 kcal ; p = 0.750 ) , with percentage contribution from carbohydrate ( full , 52 3 ; 75% , 49 4 ; 50% , 49 4% ; p = 0.844 ) , fat ( full , 29 3 ; 75% , 30 3 ; 50% , 33 3% ; p = 0.958 ) , and protein ( full , 18 2 ; 75% , 21 3 ; 50% , 18 2% ; p = 0.843 ) similar between conditions . no differences were observed in activity patterns with similar total steps recorded across conditions ( full , 7,507 124 ; 75% , 8,028 119 ; 50% , 8,056 138 steps ; p = 0.627 ) . 1 . there was a significant time effect ( p < 0.001 , partial- = 0.643 ) and a significant condition time interaction for absolute blood glucose concentrations ( p < 0.001 , partial- = 0.290 ) . fasting blood glucose concentrations were similar between conditions ( full , 7.2 0.9 ; 75% , 7.1 0.7 ; 50% , 7.1 0.6 mmol l ; p = 0.594 ) . after the ingestion of the breakfast meal and reduced rapid - acting insulin dose , blood glucose peaked at 60 min and was similar across conditions ( fig . there were no differences in fasting serum insulin ( p = 0.989 ) or -hydroxybutyrate ( p = 0.962 ) concentrations across conditions ( table 1 ) . pre - exercise serum insulin and -hydroxybutyrate responses were similar across conditions ( p > 0.05 ) ( table 1 ) . transparent sample point within a condition indicates significant difference from premeal concentrations ( p 0.05 ) . * significantly different from full ( p 0.05 ) ; * * significantly different from full and 75% ( p 0.05 ) . note that test meal and insulin were administered immediately after rest and 60-min postexercise sample points . serum insulin and -hydroxybutyrate responses during the trials there were no differences in exercising rates of % vo2peak ( full , 73.8 0.01 ; 75% , 72.8 0.01 ; 50% , 70.8 0.01% ; p = 0.575 ) or peak heart rate ( full , 80 2 ; 75% , 79 3 ; 50% , 79 1% ; p = 0.631 ) . the decline in blood glucose with exercise was similar across conditions ( full , = 6.8 0.03 ; 75% , = 6.9 0.03 ; 50% , = 6.2 0.03 mmol l ; p = 0.891 ) ; moreover , patients elicited similar peak lactates immediately postexercise ( full , 3.7 0.6 ; 75% , 3.8 0.5 ; 50% , 3.7 0.5 mmol l ; p = 0.965 ) . there were no incidences of hypoglycemia during exercise , with all patients completing the exercise protocol on each occasion . after the postexercise meal and rapid - acting insulin dose , total serum insulin area under the curve was significantly lower under 50% ( 21,748 4,799 pmol l min , p = 0.004 ) and 75% ( 33,351 5,634 individual peak serum insulin concentrations were lowest under 50% ( 83 29 pmol l min ) compared with 75% ( 196 42 pmol l min , p = 0.017 ) and full ( 229 44 pmol l min , p = 0.020 ) . blood glucose concentrations decreased under full and 75% ; however , under 50% , blood glucose was preserved , eliciting similar concentrations to premeal ( fig . 1 ) . during this time , five patients experienced hypoglycemia under full and two under 75% , whereas under 50% , all patients were protected . furthermore , some participants experienced multiple bouts of hypoglycemia , with total episodes across each trial greatest under full ( full , n = 9 ; 75% , n = 6 ; 50% , n = 0 ) . on average , blood glucose concentrations were 2.8 0.2 mmol l before receiving carbohydrate supplementation . upon leaving the laboratory on 180 min postmeal , blood glucose concentrations were greater under 50% when compared with full and 75% ( fig . 1 ) . there were no conditional differences in blood lactate responses ( p = 0.996 ) or serum -hydroxybutyrate ( p = 0.076 ) ( table 1 ) . 50% prevented hypoglycemia for a further 4 h , meaning the first hypoglycemic episode under 50% occurred at 8 h postexercise . during the evening ( fig . 2 ) , total interstitial glucose area under the curve was significantly greater under 50% ( 2,709 245 mmol l min ) when compared with full ( 1,706 247 mmol l min , p < 0.001 ) and 75% ( 1,860 244 mmol l min , p < 0.001 ) . data presented as mean ( sem error bars have been removed for reader clarity ) . black line , full ; blue line , 75% ; red line , 50% . * * interstitial glucose area under the curve is significantly different from full and 75% ( p 0.05 ) . vertical dashed line breaks indicate evening , nocturnal , or morning periods . by 9 h postexercise , glycemia under 50% had decreased over time , with concentrations lower than those elicited upon leaving the laboratory ( fig . interstitial glucose concentrations became comparable across conditions and were lowest during the night ( fig . all patients experiencing hypoglycemia during the laboratory period also experienced further hypoglycemic episodes during sleep . the total number of hypoglycemic episodes during the postlaboratory phase was lowest under 50% ( table 2 ) . of the total hypoglycemic episodes across all trials , 82% occurred nocturnally ( table 2 ) . additionally , the number of patients who self - corrected blood glucose to prevent further declines in glycemia was less under 50% ( n = 2 ) when compared with 75% ( n = 6 ) and full ( n = 6 ) . the total number of patients experiencing hypoglycemia and the total number of hypoglycemic episodes during the postlaboratory period during the postlaboratory period , total energy consumed ( full , 1,588 234 ; 75% , 1,769 275 ; 50% , 1,731 141 kcal ; p = 0.836 ) , with contribution from carbohydrate ( full , 55 4 ; 75% , 47 5 ; 50% , 48 4% ; p = 0.516 ) , fat ( full , 30 4 ; 75% , 31 4 ; 50% , 34 4% ; p = 0.916 ) , and protein ( full , 16 2 ; 75% , 22 6 ; 50% , 18 2% ; p = 0.827 ) , was similar between conditions . moreover , there were no differences in the total amount of insulin administered or in the insulin to carbohydrate ratio ( p > 0.05 ) . no differences were observed in activity patterns during the 24-h postexercise period , with similar steps recorded ( full , 5,341 95 ; 75% , 4,786 87 ; 50% , 5,543 108 steps ; p = 0.877 ) across conditions . patients displayed similar glycemic control during the 24 h before arriving to the laboratory , with similar mean ( full , 8.6 0.4 ; 75% , 7.7 0.5 ; 50% , 8.1 0.6 mmol l ; p = 0.451 ) and total interstitial glucose area under the curve ( full , 12,389 705 ; 75% , 10,986 765 ; 50% , 11,385 791 mmol l min ; p = 0.400 ) across trials . during this time , dietary patterns were similar , with no differences in total energy intake ( full , 2,249 110 ; 5% , 2,290 178 ; 50% , 2,130 128 kcal ; p = 0.750 ) , with percentage contribution from carbohydrate ( full , 52 3 ; 75% , 49 4 ; 50% , 49 4% ; p = 0.844 ) , fat ( full , 29 3 ; 75% , 30 3 ; 50% , 33 3% ; p = 0.958 ) , and protein ( full , 18 2 ; 75% , 21 3 ; 50% , 18 2% ; p = 0.843 ) similar between conditions . no differences were observed in activity patterns with similar total steps recorded across conditions ( full , 7,507 124 ; 75% , 8,028 119 ; 50% , 8,056 138 steps ; p = 0.627 ) . 1 . there was a significant time effect ( p < 0.001 , partial- = 0.643 ) and a significant condition time interaction for absolute blood glucose concentrations ( p < 0.001 , partial- = 0.290 ) . fasting blood glucose concentrations were similar between conditions ( full , 7.2 0.9 ; 75% , 7.1 0.7 ; 50% , 7.1 0.6 mmol l ; p = 0.594 ) . after the ingestion of the breakfast meal and reduced rapid - acting insulin dose , blood glucose peaked at 60 min and was similar across conditions ( fig . there were no differences in fasting serum insulin ( p = 0.989 ) or -hydroxybutyrate ( p = 0.962 ) concentrations across conditions ( table 1 ) . pre - exercise serum insulin and -hydroxybutyrate responses were similar across conditions ( p > 0.05 ) ( table 1 ) . transparent sample point within a condition indicates significant difference from premeal concentrations ( p 0.05 ) . * significantly different from full ( p 0.05 ) ; * * significantly different from full and 75% ( p 0.05 ) . thatched area indicates exercise , and vertical dashed line break indicates postexercise intervention . note that test meal and insulin were administered immediately after rest and 60-min postexercise sample points . serum insulin and -hydroxybutyrate responses during the trials there were no differences in exercising rates of % vo2peak ( full , 73.8 0.01 ; 75% , 72.8 0.01 ; 50% , 70.8 0.01% ; p = 0.575 ) or peak heart rate ( full , 80 2 ; 75% , 79 3 ; 50% , 79 1% ; p = 0.631 ) . the decline in blood glucose with exercise was similar across conditions ( full , = 6.8 0.03 ; 75% , = 6.9 0.03 ; 50% , = 6.2 0.03 mmol l ; p = 0.891 ) ; moreover , patients elicited similar peak lactates immediately postexercise ( full , 3.7 0.6 ; 75% , 3.8 0.5 ; 50% , 3.7 0.5 mmol l ; p = 0.965 ) . there were no incidences of hypoglycemia during exercise , with all patients completing the exercise protocol on each occasion . after the postexercise meal and rapid - acting insulin dose , total serum insulin area under the curve was significantly lower under 50% ( 21,748 4,799 pmol l min , p = 0.004 ) and 75% ( 33,351 5,634 individual peak serum insulin concentrations were lowest under 50% ( 83 29 pmol l min ) compared with 75% ( 196 42 pmol l min , p = 0.017 ) and full ( 229 44 pmol l min , p = 0.020 ) . blood glucose concentrations decreased under full and 75% ; however , under 50% , blood glucose was preserved , eliciting similar concentrations to premeal ( fig . 1 ) . during this time , five patients experienced hypoglycemia under full and two under 75% , whereas under 50% , all patients were protected . furthermore , some participants experienced multiple bouts of hypoglycemia , with total episodes across each trial greatest under full ( full , n = 9 ; 75% , n = 6 ; 50% , n = 0 ) . on average , blood glucose concentrations were 2.8 0.2 mmol l before receiving carbohydrate supplementation . upon leaving the laboratory on 180 min postmeal , blood glucose concentrations were greater under 50% when compared with full and 75% ( fig . 1 ) . there were no conditional differences in blood lactate responses ( p = 0.996 ) or serum -hydroxybutyrate ( p = 0.076 ) ( table 1 ) . 50% prevented hypoglycemia for a further 4 h , meaning the first hypoglycemic episode under 50% occurred at 8 h postexercise . during the evening ( fig . 2 ) , total interstitial glucose area under the curve was significantly greater under 50% ( 2,709 245 mmol l min ) when compared with full ( 1,706 247 mmol l min , p < 0.001 ) and 75% ( 1,860 244 mmol l min , p < 0.001 ) . data presented as mean ( sem error bars have been removed for reader clarity ) . black line , full ; blue line , 75% ; red line , 50% . * * interstitial glucose area under the curve is significantly different from full and 75% ( p 0.05 ) . vertical dashed line breaks indicate evening , nocturnal , or morning periods . by 9 h postexercise , glycemia under 50% had decreased over time , with concentrations lower than those elicited upon leaving the laboratory ( fig . , interstitial glucose concentrations became comparable across conditions and were lowest during the night ( fig . all patients experiencing hypoglycemia during the laboratory period also experienced further hypoglycemic episodes during sleep . the total number of hypoglycemic episodes during the postlaboratory phase was lowest under 50% ( table 2 ) . of the total hypoglycemic episodes across all trials , 82% occurred nocturnally ( table 2 ) . additionally , the number of patients who self - corrected blood glucose to prevent further declines in glycemia was less under 50% ( n = 2 ) when compared with 75% ( n = 6 ) and full ( n = 6 ) . the total number of patients experiencing hypoglycemia and the total number of hypoglycemic episodes during the postlaboratory period during the postlaboratory period , total energy consumed ( full , 1,588 234 ; 75% , 1,769 275 ; 50% , 1,731 141 kcal ; p = 0.836 ) , with contribution from carbohydrate ( full , 55 4 ; 75% , 47 5 ; 50% , 48 4% ; p = 0.516 ) , fat ( full , 30 4 ; 75% , 31 4 ; 50% , 34 4% ; p = 0.916 ) , and protein ( full , 16 2 ; 75% , 22 6 ; 50% , 18 2% ; p = 0.827 ) , was similar between conditions . moreover , there were no differences in the total amount of insulin administered or in the insulin to carbohydrate ratio ( p > 0.05 ) . no differences were observed in activity patterns during the 24-h postexercise period , with similar steps recorded ( full , 5,341 95 ; 75% , 4,786 87 ; 50% , 5,543 108 steps ; p = 0.877 ) across conditions . the aim of this study was to examine the acute and 24-h glycemic responses to reductions in postexercise rapid - acting insulin dose , when the recommended pre - exercise insulin reductions were used , in patients with type 1 diabetes . the results demonstrate that despite a large reduction in the pre - exercise , rapid - acting insulin dose , it is important that the postexercise dose is also heavily reduced . we have demonstrated that a reduction in postexercise rapid - acting insulin of 50% best preserves glycemia and provides the greatest protection against hypoglycemia early after exercise ( 8 h ) . however , beyond this time , this strategy becomes less effective . a potential mechanism explaining the late fall in glycemia could be the administration of further unchanged doses of rapid - acting insulin and/or unchanged circulating basal insulin concentrations during a second period of elevated glucose uptake ( 22 ) . all patients completed a bout of moderate - intensity aerobic exercise ( 73% vo2peak ) , running at a velocity of 9.3 0.3 km h and expending 708 16 kcal , with no incidences of hypoglycemia during exercise . this complements the recommendations of prior literature ( 1012 ) and emphasizes the importance of reducing the rapid - acting insulin dose before exercise . furthermore , patients were , on average , within euglycemic ranges for 60 min after exercise , suggesting this strategy exposes patients to only transient periods of hyperglycemia prior to exercise ( fig . was best preserved with a 50% reduced rapid - acting insulin dose , which protected all patients from hypoglycemia throughout the postexercise meal period , whereas blood glucose concentrations declined under both full and 75% doses ( fig . moderate - intensity running exercise requires a contribution from muscle glycogen ( 27,28 ) , of which the replenishment is of a high metabolic priority ( 21 ) , with muscle glycogen depletion suggested to have a role in the development of postexercise hypoglycemia ( 29 ) . after exercise , glycogen synthesis is increased dramatically , thought to be driven primarily by depleted glycogen ( 30,31 ) , an upregulation of insulin signaling pathways ( 32 ) , and a prolonged increase in the permeability of the muscle cell membrane to glucose ( 33 ) . taken together , an increased rate of glucose transport into the muscle and an increased capacity to convert glucose to glycogen results in a window where there is likely an increased potency of the administered rapid - acting insulin ( 34 ) . to suggest that the glycemic responses of our patients under full and 75% are solely due to depleted muscle glycogen stores is difficult , as large carbohydrate boluses ( 1.0 g carbohydrate kg bm ) were ingested before and after exercise , which would have helped supplement glycogen reserves ( 21 ) . however , additional explanations for why hypoglycemia was experienced during the full and 75% conditions may be that the administration of larger doses of rapid - acting insulin created a milieu of relative hyperinsulinemia , in turn suppressing hepatic glucose production and enhancing glucose uptake ( 34 ) , potentially into nonexercised tissue ( 27 ) . irrespective of the mechanisms at play , we have demonstrated the importance of reducing the dose of rapid - acting insulin administered with the meal after exercise . in contrast to current recommendations that advocate a reduction of 30% ( 35 ) , we found a 50% reduction was necessary to prevent falls in blood glucose early after exercise . although serum -hydroxybutyrate throughout the postmeal period was , on average , greatest under 50% ( table 1 ) , these concentrations were of little clinical significance , with concentrations well below those deemed hyperketonemic ( > 1.0 mmol ) ( 36 ) . from a clinical viewpoint , a 50% reduction in rapid - acting insulin after exercise , when the pre - exercise dose was also heavily reduced , does not increase ketone body concentrations . interstitial glucose concentrations revealed a preservation of glycemia under 50% for a further 4 h after leaving the laboratory , meaning patients had been protected against hypoglycemia for 8 h postexercise . however , patients were exposed to hyperglycemia during this time . in comparison , glycemia during the evening ( between 4 and 9 h postexercise ) ( fig . 2 ) was significantly lower under both full and 75% conditions , with patients experiencing further hypoglycemic episodes under these conditions . however , at 7 h postexercise , there was a decline in interstitial glucose concentrations under 50% , such that at 9 h postexercise , glycemia was similar between conditions . patients replicated dietary habits ( i.e. , meal composition , time of consumption , and rapid - acting insulin dose ) , and their basal insulin regimen was standardized across trials . type 1 diabetic patients face particular difficulty in avoiding hypoglycemia 711 h postexercise , as the requirement for glucose to maintain euglycemia is increased ( 26,37 ) . considering that increased insulin sensitivity may persist for > 48 h after exercise , an additional strategy could be to reduce the basal insulin dose . however , this strategy requires planning exercise in advance and offers little flexibility regarding the timing of exercise ; if unforeseen circumstances prevent planned exercise from taking place , the patient is likely to be exposed to prolonged periods of hyperglycemia ( 38 ) . therefore , it may be more appropriate to further adjust the rapid - acting insulin dose during this time . of the total number of hypoglycemic episodes experienced across all trials during the postlaboratory period , 82% occurred nocturnally . therefore , sleep may be a contributing factor to the increased incidence of hypoglycemia during the night ( 3 ) . as well as recent exercise ( 5 , 20 ) , antecedent hypoglycemia ( 6 ) may blunt symptomatic and autonomic responses to hypoglycemia later in the day , particularly during sleep ( 39 ) . this is important , as those patients who experienced hypoglycemia in the laboratory eliciting blood glucose concentrations <3 mmol l also experienced hypoglycemia again during sleep . these data suggest that if patients experience hypoglycemia early after exercise , there is potential that they are at an increased risk of blood glucose falling again during the night . although only 18% of patients experienced nocturnal hypoglycemia under 50% , the late fall in glycemia means the risk , albeit less , may still be present for patients despite the large reductions in pre- and postexercise rapid - acting insulin dose . experiencing hypoglycemia during sleep is a real fear for type 1 diabetic patients ( 39 ) , which is likely to be exacerbated when exercise is performed during the day . therefore , there is a need to further refine current exercise recommendations for type 1 diabetic patients such that patients can engage in regular exercise with a reduced risk , and fear , of postexercise nocturnal hypoglycemia . sport and recreational activities vary greatly in terms of their physiological demands and metabolic responses . the effects of changing the intensity , duration , or mode of exercise may therefore necessitate manipulating the rapid - acting insulin dose accordingly to account for these changes . moreover , the feeding pattern used in this study ( 60 min before and after exercise ) may not be typical of the normal dietary habits of exercising patients . worthy of note , the patients examined in this study were in good glycemic control ( hba1c 7.7 0.3% , 61 3.4 mmol / mol ) and had a good level of aerobic fitness ( vo2peak 53 1 ml kg min ) . thus , there is potential that the glycemic response to pre- and postexercise rapid - acting insulin reductions may differ with patient age , fitness , and glycemic control . although it was not possible to address these issues within this study , this should not detract from the clinical application of our findings . to our knowledge , we are the first to investigate postexercise rapid - acting insulin dose reductions after running exercise in patients with type 1 diabetes , when using the recommended pre - exercise insulin dose reductions . we have demonstrated the importance of large reductions in the pre- and postexercise rapid - acting insulin dose in preserving glycemia early after exercise and , through investigation under free - living conditions , further highlight that the difficulties related to delayed and , in particular , nocturnal postexercise hypoglycemia are still existent despite the application of large rapid - acting insulin dose reductions before and after exercise . therefore , future research should further investigate strategies to normalize glycemia in the late postexercise period , preventing late - onset hypoglycemia . in summary , the results of the current study suggest that a 50% reduction in postexercise rapid - acting insulin dose , when combined with a large pre - exercise rapid - acting insulin reduction , preserves glycemia and prevents hypoglycemia for 8 h after exercise . however , patients should be made aware that they may be exposed to periods of hyperglycemia , but not hyperketonemia , after the postexercise meal , and there is still a risk of developing late - onset hypoglycemia . patients are therefore recommended to refine these strategies according to their own individual exercise routine and glycemic responses .

objectiveto examine the acute and 24-h glycemic responses to reductions in postexercise rapid - acting insulin dose in type 1 diabetic patients.research design and methodsafter preliminary testing , 11 male patients ( 24 2 years , hba1c 7.7 0.3% ; 61 3.4 mmol / mol ) attended the laboratory on three mornings . patients consumed a standardized breakfast ( 1 g carbohydrate kg1 bm ; 380 10 kcal ) and self - administered a 25% rapid - acting insulin dose 60 min prior to performing 45 min of treadmill running at 72.5 0.9% vo2peak . at 60 min postexercise , patients ingested a meal ( 1 g carbohydrate kg1 bm ; 660 21 kcal ) and administered a full , 75% , or 50% rapid - acting insulin dose . blood glucose concentrations were measured for 3 h postmeal . interstitial glucose was recorded for 20 h after leaving the laboratory using a continuous glucose monitoring system.resultsall glycemic responses were similar across conditions up to 60 min postexercise . after the postexercise meal , blood glucose was preserved under 50% , but declined under full and 75% . thence at 3 h , blood glucose was highest under 50% ( 50% [ 10.4 1.2 ] vs. full [ 6.2 0.7 ] and 75% [ 7.6 1.2 mmol l1 ] , p = 0.029 ) ; throughout this period , all patients were protected against hypoglycemia under 50% ( blood glucose 3.9 ; full , n = 5 ; 75% , n = 2 ; 50% , n = 0 ) . fifty percent continued to protect patients against hypoglycemia for a further 4 h under free - living conditions . however , late - evening and nocturnal glycemia were similar ; as a consequence , late - onset hypoglycemia was experienced under all conditions.conclusionsa 25% pre - exercise and 50% postexercise rapid - acting insulin dose preserves glycemia and protects patients against early - onset hypoglycemia ( 8 h ) . however , this strategy does not protect against late - onset postexercise hypoglycemia .

RESEARCH DESIGN AND METHODS Patients Prelaboratory phase CGMS Diet and activity replication Testing procedure Postlaboratory period Data analysis RESULTS Prelaboratory phase Laboratory phase Postexercise intervention Postlaboratory phase CONCLUSIONS

PMC4110149

the neurovascular compression of the fifth and seventh cranial nerves is known as the cause of trigeminal neuralgia ( tn ) and hemifacial spasm . the rate of success of surgical decompression for these two syndromes is satisfactory.1 2 however , it is still difficult to consider neurovascular compression of the eighth cranial nerve as a major cause of disabling vertigo and tinnitus.3 in the last decade , microvascular decompression ( mvd ) of the vestibulocochlear nerve was reported as an efficient treatment option with a high rate of success ( up to 80% ) for several vestibulocochlear compression syndromes.4 5 6 as an anatomical consideration , vascular anomalies may cause compression at the root entry or exit zone of the cranial nerves.7 in contrast to tn , which almost always results from a compression at the nerve root entry zone , vestibulocochlear compression syndrome results from the nerve 's conflict over its entire segment in the cerebellopontine angle.8 although a loop of the anterior interior cerebellar artery ( aica ) can frequently lead to the dislocation and compression of the eighth cranial nerve at the cerebellopontine angle,9 the intracanalicular neurovascular compression of the vestibulocochlear nerve has been reported in only two cases8 10 ( table 1 ) . we present an unusual case of an intrameatal aica loop compressing the vestibulocochlear nerve in a patient who developed the symptoms of disabling vertigo and tinnitus without a unilateral hearing loss , and we also consider the surgical issues . a 40-year - old woman was admitted to our clinic with a 4-year history of recurrent vertigo and nonpulsatile tinnitus in the left ear , along with left - sided aural fullness without any hearing loss . the episodes of vertigo lasted up to 2 hours ( which was workload dependent and not positional ) and had significantly impaired the patient 's ability to work for the past 2 years . the tinnitus episodes did not have any relationship to the vertigo . during the physical examination , her neurologic examination was normal . a preoperative auditory brainstem response ( abr ) examination showed latency of wave i and iii brainstem potentials . a balance examination of the patient did not show any imbalance tendencies to the right or the left , even though she had complained about subjective disequilibrium that was noted in her medical record . further medical treatment of the condition including intravenous infusions according to the protocol proposed by stennert10 11 did not show any improvement . due to the unfavorable results of the primary neurotologic examinations , the patient additionally underwent magnetic resonance imaging ( mri ) ( fig . 1 ) . the preoperative magnetic resonance imaging examination did not show any pathologic findings . the mri did not show any pathologic findings , but an additional magnetic resonance angiography ( mra ) examination revealed a loop of the aica extending into the left internal auditory meatus ( fig . 2 ) . an additional preoperative magnetic resonance angiography examination revealed a loop of the anterior interior cerebellar artery extending into the left internal auditory meatus . after a long discussion with the patient about the relative indication of vestibulocochlear decompression and explaining the details of our experiences as well as the surgical risks , the patient decided to go ahead with the operation due to the negative influence of the vertigo and tinnitus on her quality of life . as preparation for a lateral suboccipital retrosigmoidal craniotomy in a semisitting position , the left retrosigmoid craniotomy ( with cranioplasty ) was performed in the semisitting position with intraoperative monitoring of the facial and vestibulocochlear nerve ( abr ) ( fig . a left retrosigmoid craniotomy ( with cranioplasty ) was performed in the semisitting position with intraoperative monitoring of the facial and vestibulocochlear nerve . microsurgical exploration of the vestibulocochlear nerve after drilling the posterior wall of the internal auditory canal ( iac ) revealed the aica entering the internal auditory meatus and looping into the canal for 8 mm , as expected from the mra , thereby compressing the vestibulocochlear nerve . the aica contact to the vestibular nerve extended from the porus into the middle part of the iac . after dissection of the arachnoid membrane , the aica was gently mobilized from the vestibulocochlear nerve and removed from the iac into the cerebellopontine angle ( fig . , two pieces of muscle were interposed between the eighth cranial nerve and the aica . the intraoperative mobilization of the intrameatal anterior interior cerebellar artery from the internal auditory canal ( iac ) after drilling the posterior wall of the iac . finally , the posterior wall of the iac was closed with muscle and fibrin glue . facial and abr monitoring was performed continuously during the surgery and did not show any significant changes compared with the preoperative findings . notably , there was no change in hearing ability at the postoperative audiogram examination ( fig . follow - up tests were performed six times during the last 2 years without any recurrence of the symptoms or changes in the audiogram examinations . the vestibulocochlear parameters ( including the caloric test , posturography , and general balance ) were normal during direct postoperative as well as follow - up examinations . the preoperative ( left ) and postoperative ( right ) audiogram of the patient did not show any hearing loss before or after the operation , respectively . the labyrinthine artery ( auditory artery , internal auditory artery ) is a long slender branch of the aica ( 85100% of cases ) , basilar artery ( < 15% of cases ) , or vertebral artery ( 4.1% ) . it arises from the middle of the artery , accompanies the vestibulocochlear nerve through the internal acoustic meatus , and is distributed to the internal ear.12 as shown in fig . 6 , the labyrinthine artery of our patient arose from the intrameatal aica loop and entered the internal acoustic meatus . during surgery , the aica should be mobilized and removed from the iac into the cerebellopontine angle . any direct or indirect injury to the labyrinthine artery during the mobilization of aica would result in postoperative hearing loss.13 fig . ( a ) the preoperative magnetic resonance angiography of the patient shows the labyrinthine artery ( red arrow ) arising from the anterior interior cerebellar artery ( aica ) and entering into the internal auditory canal ( iac ) . ( b ) the intraoperative image shows the labyrinthine artery ( yellow arrow ) arising from the aica and entering the iac ( see surgical considerations in the text ) . the anatomical variations of the neurovasculature in the cerebellopontine angle result from the late development of the aica and the posterior inferior cerebellar artery ( pica ) from the primitive lateral basivertebral anastomosis.8 14 15 16 17 these vascular variations may cause dislocation and compression of the cranial nerves in the posterior cranial fossa . over the past decade , neurosurgical clinical and anatomical experience has attributed several symptoms to the microvascular compression of the vestibulocochlear nerve . several cases of disabling vertigo , tinnitus , hearing loss , and imbalance were noted to be caused by vascular compression of the eighth cranial nerve.3 4 5 however , significant controversy persists among clinicians in the neurotologic diagnosis of neurovascular compression of the eighth cranial nerve . abnormal abr , brief spells of vertigo , unilateral sensorineural hearing loss , abnormal vestibular findings , continuous tinnitus , hearing loss , abnormal electronystagmograms , and other findings have been reported to be diagnostic for neurovascular compression of the eighth cranial nerve.18 19 20 21 22 many reports in the literature explain the role of aica or pica or their branches , resulting in the microvascular compression of the vestibulocochlear nerve in the cerebellopontine angle that causes symptoms of disabling vertigo , tinnitus , and hearing loss.14 23 as a theory , curves and loops of vessels running near the cranial nerves may be created mostly by degenerative alterations of aging , and such vessels can compress surrounding neural elements.24 furthermore , the different results of mvd of the eighth cranial nerve , including the surgical treatment of tinnitus and disabling positional vertigo , have been reported in the literature.4 19 25 in 1984 , janetta et al introduced a new term , disabling positional vertigo ( dpv ) , and they identified a group of patients with vestibular disorders whose symptoms were totally relieved by mvd of the eighth nerve.19 in 1993 , mller et al reported the results of mvd of the eighth cranial nerve in 207 patients with dpv and reported a cure rate of 80%.4 these reports were subsequently supported by numerous studies on vertigo and tinnitus . in 1998 , ryu et al reported the results of neurovascular decompression of the eighth cranial nerve in 43 cases of vertigo and tinnitus , and they established diagnostic criteria and operative indications . they reported a complete recovery or marked improvement of subjective symptoms in 100% of the patients with vertigo and 65.5% of the patients with tinnitus . they demonstrated that the symptoms of neurovascular compression of the eighth cranial nerve depend on the part of the nerve compressed by blood vessels . for example , in patients with vertigo and tinnitus , there was vascular compression of the rostroventral and caudal surface of the nerve , respectively.26 this review of the literature shows that the neurovascular compression of the vestibulocochlear nerve is usually located in the cerebellopontine angle but not the intrameatal angle . to the best of our knowledge , only two case reports have presented intrameatal vascular compression as a cause of vestibulocochlear compression syndrome ( table 1 ) . our case along with the two cases just cited adds another avenue to explore in the diagnosis and treatment of vestibulocochlear compression syndrome . it is still debatable if the exploration of the vestibulocochlear nerve should continue into the iac if no clear neurovascular conflict in the cerebellopontine angle is found.8 such case reports may introduce a new concept to the intrameatal exploration of the vestibulocochlear nerve in such situations . however , there are articles reporting that the radiologic demonstration of contact between a vascular loop formed by the aica and the eighth cranial nerve on mri scans should be considered a normal anatomical finding and should not be used to support the diagnosis of a vascular compression syndrome.27 28 also , mvd of the vestibulocochlear nerve ( especially with regard to the improvement of tinnitus ) is not always successful . in a study regarding mvd treatment for a selected group of 72 patients with severe tinnitus , mller and mller showed that 11% of patients had only slight improvement , 45.8% had no improvement , and 2.8% became worse . they showed that the success of such surgery depends on the duration of symptoms before the operation.29 the diagnosis of vestibulocochlear compression based on symptoms , signs , and radiologic findings is thus not easy to make . in addition , the surgery should be done in the hands of an expert surgeon because the indication of such decompression surgery is not really absolute . in addition to other common causes , disabling vertigo and tinnitus can rarely be caused by intrameatal vascular compression of the vestibulocochlear nerve and may be treated successfully by mvd . because the results of mvd of the vestibulocochlear nerve in the literature are not always acceptable , the other common causes of vestibulocochlear compression syndrome must be excluded before the decision to operate is made .

microvascular compression of the vestibulocochlear nerve is known as a cause of tinnitus and vertigo in the literature , but our review of the literature shows that the compression is usually located in the cerebellopontine angle and not intrameatal.we present a case of intrameatal compression of the anterior inferior cerebellar artery ( aica ) on the vestibulocochlear nerve of a 40-year - old woman with symptoms of disabling vertigo and intermittent high - frequency tinnitus on the left side without any hearing loss for 4 years . magnetic resonance imaging of the brain did not show any abnormality , but magnetic resonance angiography showed a left intrameatal aica loop as a possible cause of the disabling symptoms . after the exclusion of other possible reasons for disabling vertigo , surgery was indicated . the intraoperative findings proved the radiologic findings . the large aica loop was found extending into the internal auditory canal and compressing the vestibulocochlear nerve . the aica loop was mobilized and separated from the vestibulocochlear nerve.the patient 's symptoms resolved immediately after surgery , and no symptoms were noted during 2 years of follow - up in our clinic . her hearing was not affected by the surgery.in addition to other common reasons , such as acoustic neuroma , disabling vertigo and tinnitus can occur from an intrameatal arterial loop compression of the vestibulocochlear nerve and may be treated successfully by drilling the internal acoustic meatus and separating the arterial conflict from the vestibulocochlear nerve .

Introduction Case Presentation Surgical Considerations Discussion Conclusion

PMC4500429

the main concern is to ensure the highest possible standard for the services provided and to meet the needs of individual service users and communities ( 1 ) . in 1997 , the uk department of health introduced clinical governance ( cg ) as a strategy for improving quality of health care services ( 2 ) . the classic definition of cg is provided by scally and donaldson as a system through which [ health ] organizations are accountable for continuously improving the quality of their services and safeguarding high standards of care by creating an environment in which excellence in clinical care will flourish ( 2 , 3 ) . the iranian ministry of health and medical education ( mohme ) has applied cg as a framework for improving quality and safety in all hospitals since 2009 . mohme used the definition cited above as a guide to implement the policy . the cg model developed in iran consists of seven interlocking components including : clinical effectiveness , clinical audit , risk management , patient and public involvement , education and training , staff and staff management , and use of information ( 4 ) . systems awareness , leadership , ownership , teamwork , and communication are considered as a foundation of this model . mohme required curative deputy of medical universities and hospital managers to work together to implement such initiatives in iranian hospitals . the deputies for curative affairs in each medical university have the role of leadership in planning , implementing , monitoring and following up ministry of health policies particularly in quality improvement programs including cg ( 5 ) . a number of studies have assessed the implementation of cg in different health systems and health care settings ( 611 ) . insufficient knowledge and attitude toward cg , lack of resources , inadequate information technology systems , resistance to change , necessity of cultural change and professional boundaries were the main factors explored by lathman et al . another study using qualitative research identified some barriers such as speed of cg implementation , workload and earmarked funding in cg implementation ( 7 ) . ( 2002 ) considered lack of adequate senior management support as well as resources , structural and cultural issues as obstacles ( 8) . the scarcity of resources was one of the most important barriers in implementation of cg noted by walsh et al . a number of factors were identified which ultimately could affect the success of quality improvement activities . raising awareness of cg among managers , supportive culture and sufficient resources declared that state level accountability in clinical governance implementation could be addressed by allocating proper resources and empowering policy implementers with proper performance control system ( 11 ) . previous literatures mainly provide inadequate understanding about senior managers viewpoint toward facilitators and barriers in implementation of cg . in the current study an effort was done to capture both facilitators and barriers in cg implementation from the viewpoint of curative deputies in iranian medical universities . to obtain a comprehensive understanding about senior managers viewpoint toward cg barriers and facilitators , a qualitative research was employed . to do so , two main information sources were used : face to face interviews and relevant document reviews . deputies for curative affairs of all types of iranian medical universities were purposefully selected to maximize the sample diversity and provide a comprehensive view toward cg implementation . the sampling continued until reaching data saturation . finally , forty three deputies were interviewed . in the first step , an interview topic guide was developed on the basis of findings of literature review and expert opinions ( table 1 ) . it covered the concept of clinical governance , key factors relating to clinical governance implementation process , facilitators and barriers that hospitals were experiencing . some relevant documents were also analyzed such as cg annual reports , audit reports and minutes of meetings . the qualitative thematic framework analysis was used to analyze the data with the assistance of the atlas - ti , qualitative data analysis software . data analysis process includes five stages : familiarization , developing a thematic framework , indexing , charting , and mapping and interpretation ( 12 ) . to increase the validity , it helped to ensure that the findings were congruent with participants perceptions , beliefs and opinions ( 13 ) . the five main themes were explored and presented according with their sub - themes in ( table 2 ) . most senior managers accepted that improving quality of health care should be integral to their role and essential to safeguard patient care . they believed that quality improvement is a strategic goal , to achieve central government targets . the findings demonstrated the willingness to support and positive attitude towards clinical governance .. senior managers also declared that having adequate knowledge about clinical governance also positive attitude toward the necessity of the program are the main factors affecting clinical governance implementation . the level of enthusiasm expressed by senior managers seemed to be related in particular to their knowledge and attitude about cg concept and components . those managers who had gained such knowledge were more optimistic toward success of the program . continuous training about clinical governance concept and principles can make a positive attitude toward the program . main themes of senior managers viewpoint toward cg barriers and facilitators it was stated that such quality improvement program may facilitates the development of a culture focusing on continuous improvement . most interviewees declared that appropriate culture for improving quality in the organization ; team work and readiness toward change are the main factors which influence cg implementation . culture of openness in which staffs are willing to bring ideas related to service quality development was the other important factor mentioned by participants . it was believed that culture which promotes alignment of clinical governance goals at both managerial and staff level should be developed . they also stated that clinical governance components needed to be embedded in day to day work . at first , we should culturalise cg in our work place . interviewees believed that adequate organizational commitment toward clinical governance should be created in order to increase the likelihood of program progress . when continuous meetings are hold in high managerial levels of medical university about reporting our progress in implementing cg , it makes me sure that there is an organizational commitment . effective organizational interactions both within and between departments was the other organizational key factor highlighted by senior managers . they emphasized that clinical governance activities would be more effective if all departments in organization accept the importance of cg and participate in the implementation process . everywhere in our organization you can see something about cg definition , process , implementation and so on . they stated that most of staff working in this program does not have official position in the organizational chart which negatively affects their work . they suggested that creation of such position can guarantee stability and legitimacy of staff in their workplace and have positive effect . senior managers accepted that allocating adequate staff to undertake the responsibilities required by program had a critical role in pushing forward the program . the other important factor recognized as an organizational factor was the necessity for development and dissemination of national standards . interviewees believe that establishment of such standards and presence of guidelines is prerequisites of such huge quality improvement program . in addition , it can help to conduct evaluation against determined standards which shows gaps in delivery of services . one of the factors was managerial commitment toward clinical governance and their executive ability in implementing the program . most of senior managers mentioned themselves ultimately accountable for clinical activities and quality of care in their organization . they also believed that successful outcome of quality activities depends on managers participation in quality procedures and the level of their commitment . interviewees added that senior managers themselves should demonstrate executive capabilities in order to motivate departments to implement clinical governance principles , provide adequate resources , facilitate staff training and remove any obstacle in the way . i feel positive about the future of clinical governance because of my involvement with the program . furthermore , managers should use appropriate incentive mechanisms to sustain staff motivation and participation . the matter is that how top managers will motivate us in implementing quality programs besides doing our regular organizational task . interviewees highlighted that stability in managerial and executive positions is crucial to maintain the consistency and continuity of the program . such unnecessary managerial position changes not only waste money and reduce the program progress , but also , ruin the managers motivation . major changes in top managerial positions threaten the stability of cg and other quality improvement programs . most of the senior managers agreed that shortage of staff and limited dedicated resources to implement clinical governance were main barriers in implementing clinical governance . they believed that such barriers leave many managers feel beleaguered and faced with problems to effectively perform the program . i think a major challenge in implementation of cg is lack of resources especially staff and money . senior managers mostly emphasized that fostering a sense of engagement among medical staff especially physicians is important . one of the reasons for resistance was that the staff is overwhelmed by high workload and different responsibilities . they are seeing the program as being imposed and as policing their performance , rather than supporting quality improvement . there is somehow resistance to clinical governance among some physicians and medical staff which i think it is because they are overwhelmed with the responsibilities they have regarding to care giving . some managers had a concern that this program might be temporary and act as a wave . in addition some issues such as lack of support from physicians and medical staff , legal challenges , parallel quality improvement models running in the hospitals , increased workload , parallel functions in different domains of curative deputy and inadequate supporting systems developed for the staff in the way of clinical governance implementation were mentioned as barriers . much of work i perform on clinical governance is done on my own limited time competing with other activities on my time . i have lots of responsibilities to undertake and this encounters me with lack of time . some activities were also addressed by senior managers in order to mitigate problems in five afore - mentioned domains . most senior managers accepted that improving quality of health care should be integral to their role and essential to safeguard patient care . they believed that quality improvement is a strategic goal , to achieve central government targets . the findings demonstrated the willingness to support and positive attitude towards clinical governance .. senior managers also declared that having adequate knowledge about clinical governance also positive attitude toward the necessity of the program are the main factors affecting clinical governance implementation . the level of enthusiasm expressed by senior managers seemed to be related in particular to their knowledge and attitude about cg concept and components . those managers who had gained such knowledge were more optimistic toward success of the program . continuous training about clinical governance concept and principles can make a positive attitude toward the program . it was stated that such quality improvement program may facilitates the development of a culture focusing on continuous improvement . most interviewees declared that appropriate culture for improving quality in the organization ; team work and readiness toward change are the main factors which influence cg implementation . culture of openness in which staffs are willing to bring ideas related to service quality development was the other important factor mentioned by participants . it was believed that culture which promotes alignment of clinical governance goals at both managerial and staff level should be developed . they also stated that clinical governance components needed to be embedded in day to day work . at first , we should culturalise cg in our work place . interviewees believed that adequate organizational commitment toward clinical governance should be created in order to increase the likelihood of program progress . when continuous meetings are hold in high managerial levels of medical university about reporting our progress in implementing cg , it makes me sure that there is an organizational commitment . effective organizational interactions both within and between departments was the other organizational key factor highlighted by senior managers . they emphasized that clinical governance activities would be more effective if all departments in organization accept the importance of cg and participate in the implementation process . everywhere in our organization you can see something about cg definition , process , implementation and so on . they stated that most of staff working in this program does not have official position in the organizational chart which negatively affects their work . they suggested that creation of such position can guarantee stability and legitimacy of staff in their workplace and have positive effect . senior managers accepted that allocating adequate staff to undertake the responsibilities required by program had a critical role in pushing forward the program . the other important factor recognized as an organizational factor was the necessity for development and dissemination of national standards . interviewees believe that establishment of such standards and presence of guidelines is prerequisites of such huge quality improvement program . in addition , it can help to conduct evaluation against determined standards which shows gaps in delivery of services . one of the factors was managerial commitment toward clinical governance and their executive ability in implementing the program . most of senior managers mentioned themselves ultimately accountable for clinical activities and quality of care in their organization . they also believed that successful outcome of quality activities depends on managers participation in quality procedures and the level of their commitment . interviewees added that senior managers themselves should demonstrate executive capabilities in order to motivate departments to implement clinical governance principles , provide adequate resources , facilitate staff training and remove any obstacle in the way . i feel positive about the future of clinical governance because of my involvement with the program . furthermore , managers should use appropriate incentive mechanisms to sustain staff motivation and participation . the matter is that how top managers will motivate us in implementing quality programs besides doing our regular organizational task . interviewees highlighted that stability in managerial and executive positions is crucial to maintain the consistency and continuity of the program . such unnecessary managerial position changes not only waste money and reduce the program progress , but also , ruin the managers motivation . major changes in top managerial positions threaten the stability of cg and other quality improvement programs . most of the senior managers agreed that shortage of staff and limited dedicated resources to implement clinical governance were main barriers in implementing clinical governance . they believed that such barriers leave many managers feel beleaguered and faced with problems to effectively perform the program . i think a major challenge in implementation of cg is lack of resources especially staff and money . senior managers mostly emphasized that fostering a sense of engagement among medical staff especially physicians is important . one of the reasons for resistance was that the staff is overwhelmed by high workload and different responsibilities . they are seeing the program as being imposed and as policing their performance , rather than supporting quality improvement . there is somehow resistance to clinical governance among some physicians and medical staff which i think it is because they are overwhelmed with the responsibilities they have regarding to care giving . some managers had a concern that this program might be temporary and act as a wave . in addition some issues such as lack of support from physicians and medical staff , legal challenges , parallel quality improvement models running in the hospitals , increased workload , parallel functions in different domains of curative deputy and inadequate supporting systems developed for the staff in the way of clinical governance implementation were mentioned as barriers . much of work i perform on clinical governance is done on my own limited time competing with other activities on my time . i have lots of responsibilities to undertake and this encounters me with lack of time . some activities were also addressed by senior managers in order to mitigate problems in five afore - mentioned domains . in the present study , we tried to explore senior managers viewpoint about the facilitators and barriers in cg implementation . we found that sufficient knowledge and clear understanding about the principles and practice of cg have major roles in achieving desired improvement in service quality and patient safety in health care settings . this study has also highlighted the importance of supporting culture , appropriate organizational structure and managerial commitment as perceived facilitators in cg implementation . there was also a strongly accepted view that staff at all levels should be consulted , involved in planning and implementation of cg programs . the main identified obstacles were lack of adequate managerial support as well as resource , structural and cultural issues and professional boundaries . our results are parallel with the findings of many other studies . a model developed by o brien et al the model outlines four dimensions in successfully implementation of clinical governance : cultural , technical , structural and strategic . the cultural dimension related to beliefs , values , norms and behaviors in the organization which either suppress or support quality improvement activities . an organization with strong and clear vision and goals , stable managerial leadership , supportive structures for team work , effective interactions and inter professional relationships and continuous learning culture is more likely to successfully implement clinical governance ( 14 ) . in our study it is clear that senior managers perception about the important factors in implementing clinical governance were classified in five main domains . the domains were knowledge and attitude , culture , organizational factors , managerial factors and barriers . most of the senior managers believed in knowledge and attitude toward clinical governance and culture as the most important factors in cg implementation . this was thought to be a major factor in achieving improvement in service quality and patient safety mentioning in another study . similar to o brien study , our findings showed that culture is also an important factor and many organizations expend much effort to shape their culture in a way to improve quality . a sense of ownership toward quality improvement and a positive attitude to contribute new ideas also provide an organizational climate necessary to allow alignment of attitudes and values with a continuous quality improvement . in hogan study about consultants attitudes to clinical governance , quality improvement was considered as an integral part of consultants role and they accepted that maintaining service standards , monitoring and improving outcomes for patients were activities they should undertake . there was also recognition about the importance of team based approaches to quality improvement ( 15 ) . this supports the findings of our study which focuses on the importance of being involved in quality improvement activities by all staff especially physicians and medical staff . these include structures and processes with clear vision and goals toward quality improvement , involving staff in the process of change , rewarding positive behaviors , improving the effectiveness of communication across the organization and providing opportunity for team work . campbell study on the role of cg as a strategy for quality improvement in primary care found significant barriers in the way of cg implementation . these included in appropriate culture , too few staff , limited resources , disengagement by some practices and staff , lack of time to perform quality activities ( 8) . our study supported the above findings and declared that some senior managers felt powerless with the volume of work and shortage of resources . meaningful engagement and commitment at all levels of managers and staff has been highlighted as a major factor in implementing cg . the managerial level needed to match its commitment to a program of change with realistic timetables to secure the cultural and organizational changes needed to improve quality of care . the need for top management support is the most frequently cited imperative for success of any program . wilkinson and witcher in an examination of factors important in successfully implementation of clinical governance stressed on the importance of quality committed senior managers and staff effectively involved in all levels of organization ( 16 ) . fenton o creevy suggests that the most consistently barrier to the success of every quality improvement program is resistance from managers ( 17 ) . dawson found that one of the major problems encountered in implementing quality program is lack of commitment at the middle and supervisory management . they suggest that many of the problems of survivor syndrome arise from the breakdown of traditional psychological contract where managers promised job security ( 18 ) . in our study , the necessity of physicians and medical staff participation in clinical governance program has been emphasized . lawler , mohrman and ledford have demonstrated the close relationship between success of quality programs and employee involvement initiatives ( 19 ) . another issue is the importance of having an employee recognition and rewards system with supporting mechanisms providing adequate salaries for the staff being involved in the implementation of clinical governance program . encouraging workers to become involved in continuous improvement activities is relatively an important factor ( 20 ) . wilkins and witcher suggest that employees who are highly skilled , with adequate salaries and incentives are typically more likely to accept the program ( 16 ) . master produced a list of eight barriers in the way of implementing a quality improvement program includinglack of management commitment , lack of training , inability to adopt organizational culture suitable for quality improvement , lack of employee involvement , lack of resources , improper planning , in compatible organizational structure and inadequate use of team work ( 21 ) . he declares that quality improvement activities , team work , effective communication and supporting the quality program in all organizational levels are of great importance ( 22 ) . sohal , samson and ramsay also investigated the barriers of successful implementation of quality plan from the viewpoint of organizational management . they categorized the barriers in a number of groups : organizational culture ( top management support and effective involvement , changing values and culture to align with quality improvement requirements ) , strategic planning issues ( lack of planning for quality , inappropriate organizational structural ) , resource management issues ( lack of resources , inadequate number of personnel and additions to normal working load ) ( 23 ) . another study conducted by terziovski , sohal and moss showed that a successful quality organization would include the following characteristics : managers and staff with positive attitude toward quality , employment of quality management practices , dissemination of responsibility of quality to all staff at all levels , , leadership commitment , having strategic planning , providing adequate resources , focusing on training and adoption of appropriate culture ( 24 ) . in our study , senior managers stated some recommendations for implementing clinical governance more effectively such as : creating a suitable culture for implementing quality programs , evaluating the quality of organization , determining the existing deficiencies , setting up appropriate strategic and functional plan to achieve determined objectives , following the programs , evaluating the results and encouraging quality improvement activities continuously ( table 3 ) . the interviews reflect only top managerial perspectives at medical university level and not managers working in hospitals who should play important role in cg implementation . in addition , due to the nature of qualitative studies the results can not be generalized although we are looking for theoretical generalization . although this study has provided the first evaluation of senior managers viewpoint about facilitators and barriers in cg implementation in iran , further research is required to track the progress of the cg policy as it unfolds over time . this qualitative paper explores main facilitators and barriers perceived by deputies in curative affairs of iranian medical universities . identifying facilitators and barriers from the viewpoint of senior managers can have an effective role in successful progress of cg program . the reason is that these managers are directly responsible for piloting such quality programs and are the most familiar with challenges existing in the way of implementing cg . the authors conclude that one of the possible solutions is developing educational courses and workshops with the purpose of raising staff awareness toward cg concept and practice . developing a supportive culture , having all levels of staff commitment and involvement , effective communication , developing clinical guidelines , using incentive tools and overcoming legal challenges are other resolutions mentioned in this regard . by successfully implementing the program , patients will benefit from quality services . also health professionals take an advantage of working in a safer and more supportive system . evidence suggests that governance needs to match its commitment to a program of change with realistic timetables to secure the cultural and organizational changes needed to improve quality of care . 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 .

abstractbackgroundhealth care systems should assign quality improvement as their main mission . clinical governance ( cg ) is a key strategy to improve quality of health care services . the iranian ministry of health and medical education ( mohme ) has promoted cg as a framework for safeguarding quality and safety in all hospitals since 2009 . the purpose of this study was to explore perceived facilitators and barriers to implementing cg by deputies for curative affairs of iranian medical universities.methodsa qualitative study was conducted using face to face interviews with a purposeful sample of 43 deputies for curative affairs of iranian medical universities and documents review . thematic analysis was used to analyze the dataresultsfive themes were explored including : knowledge and attitude toward cg , culture , organizational factors , managerial factors and barriers . the main perceived facilitating factors were adequate knowledge and positive attitude toward cg , supporting culture , managers commitment , effective communication and well designed incentives . pe rceived barriers were the reverse of facilitators noted above in addition to insufficient resources , legal challenges , workload and parallel quality programs.conclusionssuccessful implementation of cg in iran will require identifying barriers and challenges existing in the way of cg implementation and try to mitigate them by using appropriate facilitators .

Introduction Materials and Methods Results Knowledge and attitude toward clinical governance Culture Organizational Factors Managerial factors Barriers Discussion Limitation of the Study Conclusion Ethical considerations

PMC3124966

recent research defines mobility as where people move or travel , ( while ) taking into account the frequency of movement and degree of independence during such movement . mobility is critical to the physical and mental health , as well as independence , of older adults , 65 and older [ 2 , 3 ] . healthy people 2020 recognizes the impact of mobility loss through its inclusion of older adult objectives reducing the proportion of older adults who have moderate to severe functional limitations . loss of mobility is a predictor of physical disability and is associated with falling , institutionalization , and mortality [ 5 , 6 ] . a majority ( 68% ) of adults , this loss of mobility not only affects physical health directly , but also indirectly older adults experiencing a loss of mobility may be less able to access primary care and other health services [ 79 ] . loss of mobility and other forms of impeded access in older adults can lead to underutilization of primary care and preventive care services , which in turn may result in unnecessary hospitalizations , increased morbidity , and higher costs to the healthcare system than necessary . as older adults age , they are at greater risk for functional impairments that may hinder their ability to drive to primary care appointments [ 8 , 11 ] . poor elderly , who are 50% more likely to experience a loss of mobility , have the added barrier of having more difficulty affording public transportation services in order to get to their primary care providers . elderly living in rural areas may not have access to public transportation services , or their communities may lack those services altogether [ 12 , 13 ] . furthermore , older adults with access to a public transportation system may not possess the physical ability or stamina necessary to endure long bus rides or the mental acuity to follow route directions or learn transfer points . to minimize the impact of mobility loss ( i.e. , frequency of movement and degree of independence during such movement ) in older adults as it relates to access to quality health care , some federally qualified health centers ( fqhcs ) have turned to including enhanced transportation services as part of their primary care service delivery to older adult patients . fqhcs are supported by the health resources and services administration ( hrsa).(while the term fqhc is a cms designation , the term is also used to refer to fqhcs that receive a hrsa - supported grant , which is authorized under section 330 of the public health service act . ) fqhcs provide community - based and patient - directed primary care services , as well as necessary enabling and support services , to medically underserved communities and vulnerable populations regardless of individuals ' ability to pay for care . fqhcs currently serve 20 million patients , and through provisions passed as part of the affordable care act ( including $ 11 billion in dedicated funding for fqhc operation and construction ) , fqhcs stand to double in capacity to serve 40 million patients by 2015 [ 16 , 17 ] . fqhcs are required to provide high - quality , cost - effective health care to people of all ages residing within their service areas . however the number of older adult patients at fqhcs is expected to grow over the next five years as the total number of patients doubles in size from 20 million to 40 million patients [ 16 , 17 ] . over one - third of the 1,200 fqhcs nationwide serves a larger older adult patient population relative to that of the national average ; some of these fqhcs provide primary care specifically to older adults . although fqhcs are statutorily required to provide transportation as a required primary health service to all patients regardless of their ability to pay , ( public health service act . section 330(b)(1)(a)(iv ) ) this paper explores case reports of four fqhc transportation service models that are tailored specifically for older adults affected by mobility loss and that would not have access to primary care without the fqhcs providing transportation services . these services range from free taxi cab rides , to partnerships with local public transportation authorities , to fqhc - owned - and - operated transportation services . by providing these transportation services to their older adult patients , fqhcs minimize effects of mobility loss on their older adults ' abilities to access high - quality primary care . these case reports investigate the impact of access to primary health care through fqhc - affiliated transportation programs on older adults living with mobility loss in medically underserved areas . the case reports were drawn from in - depth , one - hour interviews with staff of the four featured fqhcs . the first interview consisted of questions surrounding the history of the fqhc and its transportation program ; the program 's structure and organization , costs associated with the program and ways in which the program is financed ; affiliations and partnerships with local public and private entities ; patient satisfaction with the program . if necessary a follow - up interview was conducted in order to clarify or otherwise fill gaps in the information . the case report participants were self - selected by responding to an e - mail sent out on behalf of the national association of community health centers ( nachc ) to all fqhcs that were members of nachc at the time and/or members of nachc 's health policy committee 's subcommittee on elderly . this ensured that all the respondents would serve a relatively large percentage of older adult patients as compared to fqhcs nationwide . four fqhcs responded to the e - mail alert and were also selected for the interview : lifelong medical care ( san francisco , ca ) ; hudson headwaters health network ( queensbury , ny ) ; north end community health center ( boston , ma ) ; aaron e. henry community health services center , inc ( clarksdale , ms ) . the four fqhc participants represent different geographic regions , both in urban and rural communities , and their patients reflect varying racial / ethnic backgrounds and socioeconomic status . the following case reports are focused on transportation programs owned , operated , or coordinated by an fqhc and utilized primarily by the fqhc 's older adult patients . these four case reports are not intended to be representative of all fqhcs and their transportation programs ; instead , they offer insight to policymakers and researchers about the ground - level experience of running an enhanced transportation program for older adult patients at an fqhc . while this report focuses on transportation programs , fqhcs participate in other kinds of social service programs that reduce the effects of mobility loss in seniors that are not detailed in this paper , including enhanced case management , food delivery , home health programs , and senior public housing . lifelong medical care has provided a healthcare home for seniors since 1976 , when community advocates from the gray panthers , a senior citizens ' advocacy organization , founded its first clinic : the over-60 health center in berkley , ca . in 1996 , over-60 health center merged with another clinic borne of citizen activism , berkeley primary care access clinic , to become the fqhc now known as lifelong medical care . one of those sites has retained the over-60 focus , and another focused on adult health is located in east oakland a low income , high crime area . lifelong medical care also operates two adult day health care ( adhc ) centers for frail seniors and adults with disabilities in east oakland and novato . about 30% of lifelong medical care 's patients are of age 55 and over ; about 21% of the patients are of age 65 and over . when lifelong founded its adhc center in oakland in 2004 , it provided transportation to and from the adhc center by purchasing vouchers from east bay paratransit , a public transit service for people unable to use regular city buses due to a disability or a disabling health condition , such as poststroke complications or memory loss . paratransit provides door - to - door service by specially trained drivers using sedans and wheelchair - accessible vans . however , the service has several significant limitations : there is an application process to qualify , so rides are not available to new clients immediately ; rides must be scheduled in advance , precluding last - minute trips ; rides are typically shared by several people ( with multiple pick - up locations and destinations ) , so even traveling short distances can take a long time ; strict rules regarding no shows can cause particularly frail , disorganized , or otherwise compromised clients to lose access to the service . for these reasons , lifelong medical care also contracted with a private taxi company to bring adhc participants to and from the center . in addition to transportation services for adhc participants , lifelong medical care also provides limited transportation assistance in the form of taxi vouchers to primary care patients who otherwise would not be able to make their medical appointments . the vouchers are available upon request for those who can not arrange their own transportation to appointments at one of the fqhc sites or who need to visit the emergency room . lifelong medical care also contracts with a community - based social services organization to provide rides for its elderly and disabled patients . older adults , including those who are experiencing mobility loss , are more likely to utilize high - cost ambulance services [ 18 , 19 ] . by offering vouchers to older adult patients for emergency room visits , lifelong medical care is able to control costs to the local health delivery system . hudson headwaters health network is a system of 13 fqhc sites in the frontier adirondack , lake george , and glens falls areas of upstate new york . founded by a physician in 1974 , hudson headwaters serves about 60,000 patients each year ; over 20% of those patients are low - income older adults at least 65 years of age . as it relates to median age , the adirondack area is one of the oldest in the united states ; by 2020 , only the west coast of florida will exceed the adirondack area as the oldest region in america . it is also one of the most isolated , characterized by natural geographic boundaries formed by the adirondack mountains . for the thousands of older adults living in the rural adirondack , accessing primary care rugged terrain and long distances are common obstacles , especially for older adults with physical mobility limitations . therefore in 2002 , hudson headwaters began providing transportation services free of charge , to its low - income , older adult patients . the program consists of a wheelchair - accessible bus that hudson headwaters uses to bring older adult patients to and from their medical and dental appointments at the fqhc 's sites . the bus also transports patients to the hospital and to subspecialty services in upstate new york and neighboring vermont . the bus program at hudson headwaters is a demand service and does not operate a daily schedule like a public transportation system . patients call the bus program in advance of their need for a ride , and designated staff schedules the rides and dispatches the bus . there is no cost for patients 60 and over to ride the bus . to fund the program , hudson headwaters collaborates with adirondack rural health network , which is a community partnership of public , private , and nonprofit entities in upstate new york that funds rural providers to improve access to care . hudson headwaters also collaborates with inter - lakes health , another healthcare provider , to provide older adults with access to the bus program . hudson headwaters also receives funding from hrsa and private donations to operate the bus program , which currently has an annual budget of $ 47,000 . although hudson headwaters has not completed a formal satisfaction survey of its program , the fqhc believes the numbers speak for themselves . since 2002 , the bus program has offered rides to 5,200 patients 60 years of age and over ; approximately 2,000 rides have been given to patients that utilize wheelchairs . since 2002 , the bus has travelled 160,456 miles to take patients to and from their health care appointments , with an average round trip of 44 miles . hudson headwaters occasionally receives small , private donations from current and former riders who would not have access to primary care without the bus program . north end community health center was founded 40 years ago with a focus on providing outpatient medical care to adults . over the past 20 years the fqhc has added dental and behavioral services , as well as optometry , ophthalmology , podiatry , neurology , and obstetrics / gynecology to patients of all ages . even with its expanded services , north end , still retains a focus on older adult patients . over 20% of its patients are 65 years of age and older , and seniors comprise 40% of the fqhc 's total patient visits . north end also operates an adult day health care ( adhc ) center and provides primary care services to older adult residents of senior public housing . the north end transportation program was initially developed as part of the adhc center ( transportation is a required service under medicaid regulations for adhc ) . the adhc center serves older adults in four urban boston neighborhoods : east boston , charleston , south end and north end ( for which the fqhc is named ) . the transportation program started with one van servicing the four neighborhoods and only taking adhc patients to and from the center . the transportation program has since expanded to include all older adults who are patients of north end community health center , not only those who utilize adhc . north end currently has three vans and two drivers , which take patients to and from primary care appointments at the fqhc and the adhc center , to the hospital , and even to run errands . about 98% of the patients who utilize the transportation program are older adults . in an urban area like boston , however , older adults experiencing mobility loss may not possess the stamina necessary to map out a route on public transit , walk to and from bus stops , stand for long periods of time waiting for connections , and so forth . because public transit operates on a schedule , direct routes both to and from locations is uncommon and increases total travel time . in addition , public transportation options for those with disabilities and other mobility losses are difficult to access . these are a few of the reasons why north end opted to not only expand its transportation program to all older adult patients in the area , but also operate the transportation program as an on - demand service . north end 's vans respond to requests made at least 24 hours in advance ( with same - day requests acknowledged if the schedule is permitting ) and transport older adult patients door to door . because the neighborhood of north end is so small , many patients end up needing to go to the same places . for more common destinations , for example , north end community health center worked with massachusetts general hospital to provide three scheduled trips to the hospital each day through the hospital 's shuttle service . north end 's van also makes one morning trip to massachusetts general hospital each day ; the van stops at all the fqhc 's senior housing sites and takes older adult patients to their hospital visits . the fqhc also provides one weekly trip to the local grocery store and scheduled service from the fqhc to the adhc center . funding for the transportation program comes from north end 's budget and also from patient revenues ; north end does charge its patients a nominal fee to ride the vans , but will still provide the service even if the patient is unable to pay . north end does not require payment at time of service ; the patients are billed , and if they are unable to pay , north end does not refuse them the transportation services . generally the fee to ride the bus to a health care appointment at the fqhc is $ 7 round trip . to get to massachusetts general hospital or another health care provider , social workers and case managers work with north end to coordinate the rides to the adhc center on behalf of patients who may live outside of north end 's immediate service area ; medicaid reimburses north end for rides to the adhc center . according to north end , older adult patients feel safe and comfortable on the vans , which is in contrast to how they feel when utilizing other public transportation . the patients know the drivers by first name , and the drivers maintain good relationships both with the patients and with their family member and caregivers . aaron e. henry community health services center provides primary care services to 7 rural counties stretching from southeast mississippi to the base of tennessee . the fqhc began its transportation program in 1989 as an effort to provide older adult patients living in very rural mississippi with access to subspecialty care . these patients would often need to travel 20 miles or more to receive more specialized care than was available at the fqhc . before the transportation program started , older adult patients would secure rides from neighbors in order to make it to their specialty appointments neighbors would charge between $ 15 and $ 30 for these rural seniors to make the trip . when aaron e. henry became aware of their older adult patients ' inabilities to pay their neighbors for rides , the fqhc applied for a grant from the health resources and services administration ( hrsa ) to purchase two 15-passenger vans to transport older adult patients to and from their specialty appointments . soon the fqhc was permitting patients of other primary care providers in the area to access the vans . in 1993 , aaron e. henry received a grant from the mississippi department of transportation ( mdot ) to expand the fqhc 's transportation services and assist with costs ; many of the riders were low - income individuals and could not afford to pay what it costs to ride the vans . however the mdot grant came with the following condition : rides would need to be opened up to the general public . aaron e. henry seized the opportunity to provide transportation services not only to its own older adult primary care patients with the need to access specialty care providers , but also to nonpatient members of its large , rural service area who needed transportation to and from work , shopping centers , and even schools . although some riders are not primary care patients at aaron e. henry , the fqhc chooses to provide rides to whoever may need one , regardless of that person 's ability to pay . however , almost all of the riders are able to pay the full cost of a local trip , which is between $ 2 and $ 5 . trips to memphis ( tennessee ) are more expensive about $ 25 round trip and occur less often . now known as the delta area rural transit system ( darts ) , the transportation program at aaron e. henry serves individuals of all ages and mobility levels . the fqhc is the largest transportation provider in the area with 28 multipassenger vehicles booking over 99,000 one - way trips in 2009 ; over 58% of those trips were for low - mobility riders . darts currently operates an annual budget of $ 1.5 million and recently received funding through the american reinvestment and recovery act of 2009 ( arra , also known as the stimulus ) to cover the costs of purchasing and operating new vehicles . this paper explored transportation programs in four fqhcs that are reducing the effects of mobility loss in their older adult populations and summarized results of these case reports . it is the author 's understanding , after a review of the literature , that this report is also the first of its kind . this paper concludes that by providing elderly patients access to primary care services , these fqhcs are increasing their patients ' access to affordable primary care services , increasing their patients ' independence and affording their older adult patients ' the opportunity to remain in the community rather than be institutionalized . only 7% of the 20 million patients fqhcs currently serve are over the age of 65 . however as the population ages and fqhcs double their current capacity to serve 40 million patients by 2015 , the number of older adults receiving primary care services at fqhcs will increase , and there will be a greater need for strategies to increase primary care access for older adults through transportation programs . these case reports are also examples of how fqhcs continue to be responsive to the specific needs of their communities while potentially bending the cost curve in primary care service delivery . other studies have found that by providing access to primary care services to their older adult patients , fqhcs reduce the need for unnecessary hospitalizations and emergency department utilization in this population , which greatly decreases costs to the health care system [ 2224 ] . a limitation to this report is that these four fqhcs are not representative of fqhcs nationwide . while all fqhcs are required by law to provide transportation to the patients within their service areas , the fqhcs featured in this report provide transportation services both to their own patients and to patients of other providers . these fqhcs have also tailored their transportation programs to meet the specific needs of older adults experiencing mobility loss , which is not reflective of fqhcs nationwide . however , these case reports shed new light on how fqhcs with transportation programs can reduce the effects of mobility loss in older adults by providing access to primary care services . also the case reports featured in this paper are only examples of how fqhcs are able to use their resources and build collaborations with external partners in an effort to reduce the effects of mobility loss in the older adults that utilize their services ; fqhcs interested in establishing their own enhanced transportation programs , whether by replicating the case reports featured in this paper or by developing their own innovative models , should obtain legal advice and other expert assistance prior to establishing and implementing their own programs .

loss of mobility in older adults ( 65 and older ) is associated with falling , loss of independence , and mortality . this paper , which to the author 's knowledge is the first of its kind , summarizes findings of federally qualified health center ( fqhc ) case reports and how fqhcs minimize the impacts of mobility loss in older adult patients ( who would not receive primary services without these transportation programs ) by providing access to primary care services through transportation programs . this paper features the transportation programs of four fqhcs located in both urban and rural united states areas : lifelong medical care ( oakland , ca ) ; hudson headwaters health network ( queensbury , ny ) ; north end community health center ( boston , ma ) ; aaron e. henry community health services center , inc . ( clarksdale , ms ) . this paper is beneficial to primary care providers and public health officials in outlining how transportation may be used to minimize the effects of mobility loss in older adult patients .

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

PMC3230191

isothermal titration calorimetry ( itc ) is a well established technique that can determine all the thermodynamic parameters ( affinity , enthalpy and stoichiometry ) of a binding interaction in one experiment . the signal measured is the heat released or absorbed upon interaction ( binding ) of the two reactants . a series of injections are performed and the heat signal will approach zero as the limiting reactant becomes saturated . several reviews are available that describe the instrumentation as well as the math of data collection and analysis . while other calorimeters are available ( most notably the itc200 with small volumes ) , here we describe a general protocol for the vp - itc manufactured by microcal ( now part of ge healthcare ) . in order to prepare the macromolecule and ligand in buffer accurate fits require proper concentrations of the macromolecule , the species that typically goes in the sample cell of the itc , and ligand , the species in the injection syringe . because some proteins aggregate at the high concentrations needed for the species in the injection syringe , most often the protein is loaded into the sample cell . the optimal macromolecule concentration is determined from " c " , the product of the predicted affinity of the system , which can be estimated using orthogonal methods prior to using itc , and the total macromolecule concentration , where c = ka*[m ] . optimal values of c range from 10 - 1000 , though it is possible to get accurate data for weak - binding systems under specific experimental conditions with c - values below the lower limit . thus , the macromolecule concentration must be determined with this range of c values in mind ( i.e. , for a ka of 10 m , macromolecule concentrations of 10 to 1000 m should be used ) . prior knowledge of the binding affinity of the system can help minimize the protein used for itc through better design of the itc experiment . the concentration of the ligand should be large enough ( 7 - 25 fold more concentrated than the kd for the weakest ligand binding site ) so that saturation occurs within the first third to half of the titration . accurate fitting of the data also requires saturation of the signal . for systems with higher binding affinity , a lower ligand concentration should be used to avoid saturation too early in the titration , which will give inaccurate fits . once the heat of dilution control ( i.e. titration of ligand into buffer ) has been subtracted from the titration , the enthalpy at saturation should approach zero . because small molecule impurities can give rise to artifactual signals in the itc measurements , it is best , if possible , that the macromolecule and ligand be exhaustively dialyzed against buffer . alternatively , column chromatography , desalting spin columns , or buffer exchange centrifugal filters ( for example , centricons ) can be used to change the buffer of the macromolecule . if the ligand is a small molecule , it can be prepared using the dialysis buffer after the macromolecule has been dialyzed or by dialyzing against dialysis membranes with cutoffs suitable for small molecules ( i.e. 100 - 500 da for a spectra / por float - a - lyzer ) . differences in the buffer composition between the ligand and macromolecule solutions can lead to signal artifacts from the heat of the dilution of impurities in the samples . after preparation , check to make sure the ph of the buffer , macromolecule and ligand match ( 0.05 ph units ) as artifacts in the enthalpy can arise due to buffer protonation effects . make sure to prepare enough of each species . for triplicate experiments and a dilution control but , for most itc instruments that have approximate 2 ml volume sample cells , at least 6 - 7 ml of macromolecule solution will be needed , and can be conveniently prepared in 15 ml falcon tubes . for 300 l injection syringes , 1 - 2 ml of solution should be adequate , and can be prepared in either falcon tubes or microcentrifuge tubes . dust , and other particulates , can cause artifacts in the baseline of the itc thermogram . after preparation of sample stock solutions , the samples should be centrifuged in microcentrifuge tubes for five minutes at 8000 to 14,000 rpm to pellet particles in the solution . remove the supernatant , being careful not to disturb the pellet , and place it in a new falcon / microcentrifuge tube . if reductants are needed to maintain reduced cysteines , use low concentrations and fresh stocks of -mercaptoethanol , tcep ( tris(2-carboxyl)phosphine ) or dithiothreitol to minimize any artifacts due to reductant oxidation . also , the presence of organic solvents in the buffer ( common for some small molecule ligands that may need methanol or dmso in order to be soluble ) can cause signal artifacts . if organic solvents are needed for the ligand , then the macromolecule solution must also contain the same concentration to avoid any signal arising from the heat of dilution of the organic solvent . slight differences in buffer composition between the ligand and macromolecule due to cosolvents , salts or ph are possible during preparation of the samples . it is best to check the dilution of the ligand into the sample buffer or the sample buffer into the macromolecule to ensure that heat signals arising from differences in the buffer content do n't cause data arising solely from artifacts . check the concentrations of the macromolecule and ligand carefully using techniques suitable to your system ( such as absorbance measurements , hplc , colorimetric assays , bca assays for proteins , etc ) to record their exact concentrations . differences in the actual concentration and the concentration used to fit the isotherm will cause errors in the stoichiometry , enthalpy and binding affinity determined from the experiment . it is common to degas the samples to avoid signal artifacts due to air bubbles or release of dissolved gases during the titration , particularly at higher temperatures . make sure the sample cell and injection syringe are cleaned according to the manufacturer 's protocol prior to loading the macromolecule and ligand . rinse the sample cell two or three times with 1.8 ml of distilled water using the hamilton syringe ( use care with the hamilton syringe as the barrel is easily broken or the needle bent ) . load the sample cell with 1.8 ml of macromolecule solution , being careful to avoid bubble formation . however , for buffers with particularly high ionic strengths or osmolality , it is better to use the buffer as a reference . gently move the needle of the syringe up - and - down the sides of the cell knocking any bubbles that may be at the bottom of the cell and attached to the well to the top of the cell . place the needle of the injection syringe into the ligand solution and draw the ligand solution into the injection syringe until the entire syringe is full . continue drawing a little excess volume ( approximately 50 l or more ) into the port and attached tubing . immediately close the fill port of the syringe and detach the tubing and plastic syringe . purge and refill the injection syringe two more times to remove any bubbles from the syringe . remove the syringe from the ligand solution and wipe the side with a kimwipe to remove any drops , being careful not to touch the syringe tip to the kimwipe as this may remove volume from the syringe . also , be careful not to knock or jar the syringe as this also can cause loss of volume from the syringe tip . for binding systems with strong heat signals , a large number of low volume injections will give more data points for fitting ( e.g. 75 injections of 3 l ) . for systems that have weak heat signals , a small number of large volume injections are preferable ( e.g. 33 injections of 8 l ) . it may take several titrations to optimize the conditions that are best for your system . it is important to note that the binding enthalpy can either be exothermic or endothermic , depending upon the system being studied . unfortunately , some systems have low heat signals , making the heat of reaction difficult to determine . problems with such systems can potentially be overcome by increasing the concentration of the macromolecule , changing the temperature of the experiment ( depending upon the heat capacity of the binding system ) , and/or altering the ph or ionic strength of the buffer . also consider the time spacing between each injection . it is imperative that after each injection of ligand , the system is given time to equilibrate and the heat signal returns to baseline before the next injection occurs . the time between injections should be increased for systems where the equilibration does n't occur within five minutes . because there will be some mixing between the macromolecule and ligand solutions in the injection syringe once it is inserted into the sample cell it is best to use small volumes ( e.g. 2 l ) for the first one or two injections ( so they can later be discarded ) and keep the subsequent injections at the desired values . choose the temperature of the experiment ( with 25 c being the most common , though temperatures between 2 and 80 c can be used ) . it is best to choose a temperature that matches other experiments ( binding , kinetics , etc . ) performed on the ligand - macromolecule system . the itc can be set up to equilibrate at a temperature different from the temperature of the experiment . if the experiment is going to be performed at a temperature more than 10 c away from room temperature , then it is best to set the equilibration temperature for the instrument within 5 c of the experimental temperature . this will decrease the time the instrument will take to reach the temperature of the experiment . stirring is necessary for adequate mixing of the ligand and macromolecule during the titration , but some proteins are destabilized by rapid stirring . for these cases , once all the experimental parameters have been set up , then the experiment can be started . once the experiment has finished , the itc can be cleaned according to the manufacturer 's protocol . the solution in the sample cell at the end of the experiment can be kept if additional experiments on the macromolecule - ligand complex mixture are desired . run a control where ligand is titrated into buffer in the sample cell to determine the heat of dilution for the ligand . for some systems where there is cooperative binding , additional information about the binding process can be gained by injecting the macromolecule into the ligand . the different injection orientations can give additional information , which may be helpful for global fitting . fitting of the data can be easily performed using macros in any data fitting program ( usually supplied by the manufacturer along with the instrument ) . check the raw thermogram for any signs of air bubbles or other artifacts in the signal . if there are any artifacts ( such as spikes in the baseline or peaks when there was no injection at that time point ) , then note these data points as they should be removed . next , load the ligand dilution control data , and subtract the ligand dilution data from the binding isotherm . at this time , remove all spurious data points , including the first one or two data points of the titration where dilution artifacts occur ( refer to step 7 ) . select the data fitting model ( one binding site , two / multiple binding sites , cooperative binding , etc ) to be used to fit the data . the data can be fit with initial guesses of the fitting parameters , stoichiometry ( n ) , enthalpy ( h ) and binding affinity ( ka ) . if prior knowledge of the binding affinity , or other parameters , is known from orthogonal experiments , then these values can be entered . this helps avoid the fit becoming trapped in local minima during fitting . once the data have been fit for the first titration , repeat steps 15 and 16 for the rest of the isotherms . alternatively , the data can be fit globally using programs such as sedphat . sedphat may be particularly helpful in global fitting of binary complex datasets ( i.e. molecules a+b ) in combination with ternary complex datasets ( molecules a+b+c ) . for example , in binding of molecule c to an a - b complex , it is not always clear whether there might be any signal due to binding of c to a or of b to a ( if a is not fully saturated ) . to ensure that the itc data are not artifactual , the binding affinity and stoichiometries obtained from itc additionally , the itc enthalpy value can be compared to the enthalpy from a van't hoff plot . it can also prove helpful to use different concentrations of the ligand or macromolecule as the absolute value of the heat signal should increase with increasing macromolecule concentration . artifacts are most likely to arise if the buffers in the cell and syringe are not matched . we recommend that the user start with simple binary complex titrations for which there is previous information available on kd and stoichiometry . then , if additional ligands bind , the user can try more complicated ternary complex titrations , varying the ligand concentrations , and perhaps switching the syringe and cell components . a comparison of enthalpies for both paths to ternary complex formation should be additive as enthalpy is a state function . a representative example of a well - behaved titration for the binding of the cofactor nadph to e. coli chromosomal dihydrofolate reductase ( ecdhfr ) . panel ( a ) shows the raw thermogram ; ( b ) , the binding isotherm from the integrated thermogram fit using the one - site model in the origin software ; and ( c ) the fit of the isotherm using the one binding site model from sedphat along with fit residuals . from the origin software , using the one site binding model , n = 1.09 0.02 , kd = 0.194 0.001 m , h = -22.7 0.4 kcal / mol , ts = -13.1 0.4 kcal / mol , and g = -9.16 0.01 kcal / mol . fits of the data using sedphat afford an n of 0.94 0.01 , a kd of 0.195 0.013 m , a h of -22.5 0.2 kcal / mol , ts of -13.39 kcal / mol and g of -9.15 kcal / mol . in order to prepare the macromolecule and ligand in buffer , some potential issues need to be addressed . accurate fits require proper concentrations of the macromolecule , the species that typically goes in the sample cell of the itc , and ligand , the species in the injection syringe . because some proteins aggregate at the high concentrations needed for the species in the injection syringe , most often the protein is loaded into the sample cell . the optimal macromolecule concentration is determined from " c " , the product of the predicted affinity of the system , which can be estimated using orthogonal methods prior to using itc , and the total macromolecule concentration , where c = ka*[m ] . optimal values of c range from 10 - 1000 , though it is possible to get accurate data for weak - binding systems under specific experimental conditions with c - values below the lower limit . thus , the macromolecule concentration must be determined with this range of c values in mind ( i.e. , for a ka of 10 m , macromolecule concentrations of 10 to 1000 m should be used ) . prior knowledge of the binding affinity of the system can help minimize the protein used for itc through better design of the itc experiment . the concentration of the ligand should be large enough ( 7 - 25 fold more concentrated than the kd for the weakest ligand binding site ) so that saturation occurs within the first third to half of the titration . accurate fitting of the data also requires saturation of the signal . for systems with higher binding affinity , a lower ligand concentration should be used to avoid saturation too early in the titration , which will give inaccurate fits . once the heat of dilution control ( i.e. titration of ligand into buffer ) has been subtracted from the titration , the enthalpy at saturation should approach zero . because small molecule impurities can give rise to artifactual signals in the itc measurements , it is best , if possible , that the macromolecule and ligand be exhaustively dialyzed against buffer . alternatively , column chromatography , desalting spin columns , or buffer exchange centrifugal filters ( for example , centricons ) can be used to change the buffer of the macromolecule . if the ligand is a small molecule , it can be prepared using the dialysis buffer after the macromolecule has been dialyzed or by dialyzing against dialysis membranes with cutoffs suitable for small molecules ( i.e. 100 - 500 da for a spectra / por float - a - lyzer ) . differences in the buffer composition between the ligand and macromolecule solutions can lead to signal artifacts from the heat of the dilution of impurities in the samples . after preparation , check to make sure the ph of the buffer , macromolecule and ligand match ( 0.05 ph units ) as artifacts in the enthalpy can arise due to buffer protonation effects . make sure to prepare enough of each species . for triplicate experiments and a dilution control but , for most itc instruments that have approximate 2 ml volume sample cells , at least 6 - 7 ml of macromolecule solution will be needed , and can be conveniently prepared in 15 ml falcon tubes . for 300 l injection syringes , 1 - 2 ml of solution should be adequate , and can be prepared in either falcon tubes or microcentrifuge tubes . dust , and other particulates , can cause artifacts in the baseline of the itc thermogram . after preparation of sample stock solutions , the samples should be centrifuged in microcentrifuge tubes for five minutes at 8000 to 14,000 rpm to pellet particles in the solution . remove the supernatant , being careful not to disturb the pellet , and place it in a new falcon / microcentrifuge tube . if reductants are needed to maintain reduced cysteines , use low concentrations and fresh stocks of -mercaptoethanol , tcep ( tris(2-carboxyl)phosphine ) or dithiothreitol to minimize any artifacts due to reductant oxidation . also , the presence of organic solvents in the buffer ( common for some small molecule ligands that may need methanol or dmso in order to be soluble ) can cause signal artifacts . if organic solvents are needed for the ligand , then the macromolecule solution must also contain the same concentration to avoid any signal arising from the heat of dilution of the organic solvent . slight differences in buffer composition between the ligand and macromolecule due to cosolvents , salts or ph are possible during preparation of the samples . it is best to check the dilution of the ligand into the sample buffer or the sample buffer into the macromolecule to ensure that heat signals arising from differences in the buffer content do n't cause data arising solely from artifacts . check the concentrations of the macromolecule and ligand carefully using techniques suitable to your system ( such as absorbance measurements , hplc , colorimetric assays , bca assays for proteins , etc ) to record their exact concentrations . differences in the actual concentration and the concentration used to fit the isotherm will cause errors in the stoichiometry , enthalpy and binding affinity determined from the experiment . it is common to degas the samples to avoid signal artifacts due to air bubbles or release of dissolved gases during the titration , particularly at higher temperatures . make sure the sample cell and injection syringe are cleaned according to the manufacturer 's protocol prior to loading the macromolecule and ligand . rinse the sample cell two or three times with 1.8 ml of distilled water using the hamilton syringe ( use care with the hamilton syringe as the barrel is easily broken or the needle bent ) . load the sample cell with 1.8 ml of macromolecule solution , being careful to avoid bubble formation . however , for buffers with particularly high ionic strengths or osmolality , it is better to use the buffer as a reference . gently move the needle of the syringe up - and - down the sides of the cell knocking any bubbles that may be at the bottom of the cell and attached to the well to the top of the cell . place the needle of the injection syringe into the ligand solution and draw the ligand solution into the injection syringe until the entire syringe is full . continue drawing a little excess volume ( approximately 50 l or more ) into the port and attached tubing . immediately close the fill port of the syringe and detach the tubing and plastic syringe . purge and refill the injection syringe two more times to remove any bubbles from the syringe . remove the syringe from the ligand solution and wipe the side with a kimwipe to remove any drops , being careful not to touch the syringe tip to the kimwipe as this may remove volume from the syringe . also , be careful not to knock or jar the syringe as this also can cause loss of volume from the syringe tip . place the injection syringe into the sample cell . set up the parameters for running the itc . for binding systems with strong heat signals , a large number of low volume injections will give more data points for fitting ( e.g. 75 injections of 3 l ) . for systems that have weak heat signals , a small number of large volume injections are preferable ( e.g. 33 injections of 8 l ) . it may take several titrations to optimize the conditions that are best for your system . it is important to note that the binding enthalpy can either be exothermic or endothermic , depending upon the system being studied . unfortunately , some systems have low heat signals , making the heat of reaction difficult to determine . problems with such systems can potentially be overcome by increasing the concentration of the macromolecule , changing the temperature of the experiment ( depending upon the heat capacity of the binding system ) , and/or altering the ph or ionic strength of the buffer . also consider the time spacing between each injection . it is imperative that after each injection of ligand , the system is given time to equilibrate and the heat signal returns to baseline before the next injection occurs . the time between injections should be increased for systems where the equilibration does n't occur within five minutes . because there will be some mixing between the macromolecule and ligand solutions in the injection syringe once it is inserted into the sample cell it is best to use small volumes ( e.g. 2 l ) for the first one or two injections ( so they can later be discarded ) and keep the subsequent injections at the desired values . choose the temperature of the experiment ( with 25 c being the most common , though temperatures between 2 and 80 c can be used ) . it is best to choose a temperature that matches other experiments ( binding , kinetics , etc . ) performed on the ligand - macromolecule system . the itc can be set up to equilibrate at a temperature different from the temperature of the experiment . if the experiment is going to be performed at a temperature more than 10 c away from room temperature , then it is best to set the equilibration temperature for the instrument within 5 c of the experimental temperature . this will decrease the time the instrument will take to reach the temperature of the experiment . stirring is necessary for adequate mixing of the ligand and macromolecule during the titration , but some proteins are destabilized by rapid stirring . for these cases , once all the experimental parameters have been set up , then the experiment can be started . once the experiment has finished , the itc can be cleaned according to the manufacturer 's protocol . the solution in the sample cell at the end of the experiment can be kept if additional experiments on the macromolecule - ligand complex mixture are desired . run a control where ligand is titrated into buffer in the sample cell to determine the heat of dilution for the ligand . for some systems where there is cooperative binding , additional information about the binding process can be gained by injecting the macromolecule into the ligand . the different injection orientations can give additional information , which may be helpful for global fitting . fitting of the data can be easily performed using macros in any data fitting program ( usually supplied by the manufacturer along with the instrument ) . check the raw thermogram for any signs of air bubbles or other artifacts in the signal . if there are any artifacts ( such as spikes in the baseline or peaks when there was no injection at that time point ) , then note these data points as they should be removed . next , load the ligand dilution control data , and subtract the ligand dilution data from the binding isotherm . at this time , remove all spurious data points , including the first one or two data points of the titration where dilution artifacts occur ( refer to step 7 ) . select the data fitting model ( one binding site , two / multiple binding sites , cooperative binding , etc ) to be used to fit the data . the data can be fit with initial guesses of the fitting parameters , stoichiometry ( n ) , enthalpy ( h ) and binding affinity ( ka ) . if prior knowledge of the binding affinity , or other parameters , is known from orthogonal experiments , then these values can be entered . once the data have been fit for the first titration , repeat steps 15 and 16 for the rest of the isotherms . sedphat may be particularly helpful in global fitting of binary complex datasets ( i.e. molecules a+b ) in combination with ternary complex datasets ( molecules a+b+c ) . for example , in binding of molecule c to an a - b complex , it is not always clear whether there might be any signal due to binding of c to a or of b to a ( if a is not fully saturated ) . to ensure that the itc data are not artifactual , the binding affinity and stoichiometries obtained from itc additionally , the itc enthalpy value can be compared to the enthalpy from a van't hoff plot . it can also prove helpful to use different concentrations of the ligand or macromolecule as the absolute value of the heat signal should increase with increasing macromolecule concentration . artifacts are most likely to arise if the buffers in the cell and syringe are not matched . we recommend that the user start with simple binary complex titrations for which there is previous information available on kd and stoichiometry . then , if additional ligands bind , the user can try more complicated ternary complex titrations , varying the ligand concentrations , and perhaps switching the syringe and cell components . a comparison of enthalpies for both paths to ternary complex formation should be additive as enthalpy is a state function . figure 1 . a representative example of a well - behaved titration for the binding of the cofactor nadph to e. coli chromosomal dihydrofolate reductase ( ecdhfr ) . panel ( a ) shows the raw thermogram ; ( b ) , the binding isotherm from the integrated thermogram fit using the one - site model in the origin software ; and ( c ) the fit of the isotherm using the one binding site model from sedphat along with fit residuals . from the origin software , using the one site binding model , n = 1.09 0.02 , kd = 0.194 0.001 m , h = -22.7 0.4 kcal / mol , ts = -13.1 0.4 kcal / mol , and g = -9.16 0.01 kcal / mol . fits of the data using sedphat afford an n of 0.94 0.01 , a kd of 0.195 0.013 m , a h of -22.5 0.2 kcal / mol , ts of -13.39 kcal / mol and g of -9.15 kcal / mol . itc has been used extensively in studying ligand macromolecule interactions , with studies looking at protein - ligand , dna - ligand and rna - macromolecule studies . itc can even be run with solid materials , such as nanoparticles , that form uniform suspensions . further , ternary systems , where one ligand is already bound to the macromolecule and a second ligand is titrated , can be used to determine the thermodynamics of , for example , binding of substrate to an enzyme - cofactor system . studies can also be performed for molecules with very high binding affinities that would normally exceed the detection limit of the itc by performing competition binding assays with weaker binding ligands . the role of water in binding can be explored by itc , along with the dependence of the enthalpy on solvent reorganization . recently , the thermodynamics of conformational change of dnak variants were measured upon binding of adp and atp . protein - protein association can be studied by itc , yielding information on hetero complexes as well as on homo - association . temperature effects on the binding enthalpy will give the heat capacity of the binding event . the number of protons absorbed or released upon binding can also be determined from experiments performed in buffers with different enthalpies of ionization . if additional itc studies are done at different ph values , then the pka of the group involved can potentially be determined . to summarize , accurate measurements of the concentration of the macromolecule and ligand are imperative for a good itc experiment . care should be taken with the buffer as small molecule impurities and ph mismatches will cause artifacts in the thermogram .

isothermal titration calorimetry ( itc ) is a useful tool for understanding the complete thermodynamic picture of a binding reaction . in biological sciences , macromolecular interactions are essential in understanding the machinery of the cell . experimental conditions , such as buffer and temperature , can be tailored to the particular binding system being studied . however , careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data . concentrations of the macromolecule and ligand need to be accurately determined for reliable results . care also needs to be taken when preparing the samples as impurities can significantly affect the experiment . when itc experiments , along with controls , are performed properly , useful binding information , such as the stoichiometry , affinity and enthalpy , are obtained . by running additional experiments under different buffer or temperature conditions , more detailed information can be obtained about the system . a protocol for the basic setup of an itc experiment is given .

Protocol 1. Preparing Samples 2. Setting up the Experiment 3. Analyzing the Data 4. Representative Results: Discussion Disclosures

PMC5087092

one barrier to evaluating the effectiveness of ipm programs is the difficulty in accurately measuring pesticide use . pesticide use is often estimated based on pesticide sales and other data . at the federal level , estimated pounds of active ingredient applied nationwide have declined from a high in the early 1980s , and the environmental persistence , rate of application , and toxicity of pesticides used has declined in comparison with the same measurements in the 1970s ( fernandez - cornejo et al . arizona and california have state pesticide - use reporting requirements , and therefore have high - quality use data . in arizona , many types of agricultural pesticide applications are reported to the state , as required by state law . this includes all for - hire applications ( i.e. , custom ) , all aerial applications , some applications of products in section 18 exemptions or 24c registrations , and applications of all pesticides to the soil that are listed on arizona s department of environmental quality s groundwater protection list ( arizona department of environmental quality 2013 ) . reported data including crop name , location ( township , range , and section ) , product applied , pounds applied , rates , and target pest are entered into the state pesticide use reporting database . the arizona pest management center ( apmc ) of the university of arizona environmental protection agency [ epa ] product information , pesticide label data , and mode of action tables ) and invests significant resources in verifying data and correcting errors . the result is the apmc pesticide use database , a historical database ( 1991 to present ) of arizona pesticide use records that is used for research , education , addressing pesticide registration questions and needs , and evaluating the impact of arizona ipm programs ( u.s . although submitted data do not represent 100% of agricultural applications , data are representative of most standard practices with respect to key insect pests ( p. c. ellsworth , a. fournier , and j. palumbo , unpublished data ) . pesticide risk in arizona cotton is lower than 1995 due to reduction in insecticide use and transition to more selective insecticides . the amount of insecticide active ingredient applied to arizona cotton has declined by 1.16 million pounds , down 90% compared to 1995 levels . by 2011 , 76% of all cotton insecticides used were selective , meaning they are safer to use and help preserve beneficial insects in the cotton system . arizona cotton growers have reduced use of broadly toxic insecticides use by 74% compared with pre-2005 levels ( arizona pest management center 2014 ) . pesticide risk in arizona lettuce is lower due to reduction in insecticide use and transition to more selective insecticides , and pesticide risk scores in ipm pesticide risk mitigation engine ( ipmprime ) quantify that risk reduction . in arizona lettuce , the amount of broad - spectrum insecticide applied has declined by 72% and the average number of pesticide applications decreased from an average of > 10 sprays in 1995 to an average of < 2.4 sprays in 2011 . the use of safer , reduced - risk insecticides in arizona lettuce has increased 14-fold over the same period . safety to aquatic and other organisms has been progressively and significantly improved by > 80% from 1991 to 2011 , based on a comprehensive spatial analysis of lettuce pesticide use and calculation of pesticide risk scores using the ipmprime ( arizona pest management center 2014 ) . in both arizona cotton and lettuce , insecticide risk has been reduced through reduction in frequency of insecticide sprays , pounds of insecticide applied and reduction in the toxicity of the pesticides used . in california , all agricultural pesticide use is reported to county agricultural commissioners for submission to the california department of pesticide regulation , which releases an annual report of all nonhomeowner pesticide use in the state . total pounds of pesticide active ingredient in agricultural production and postharvest fluctuates year to year based on weather patterns and shifts in crop production , but has decreased from 191 million pounds ( 86.6 million kg ) in 1995 to 175 million pounds ( 79.4 million kg ) in 2014 , an 8% decrease ( fig . 2 ; california department of pesticide regulation 2015a ) . however , that decrease occurred while the state s agricultural production increased sharply . california s agricultural gross cash income increased from us$22.1 billion on 29.3 million acres ( 11.8 million hectares ) in 1995 to us$53.5 billion on 25.5 million acres ( 10.3 million hectares ) in 2014a 142% increase in income and a 12.9% decrease in area ( california department of food and agriculture 2015 ) . for comparison , the cumulative rate of inflation during that period was 55% ( united states bureau of labor statistics 2016 ) . contributing to the increase in gross cash income were shifts in crops produced from lower - value to higher - value crops and increases in crop yields . examples of shifts in crop production were a decrease in cotton acreage from 1,175,800 acres ( 71,100 hectares ) in 1995 to 210,000 acres ( 85,000 hectares ) in 2014 and an increase in pistachio acreage from 60,300 acres ( 24,400 hectares ) in 1995 to 221,000 acres ( 89,400 hectares ) in 2014 ( california department of food and agriculture 2015 ) . an example of increasing crop yields is the increase in processing tomato yields from an average of 33.4 tons per acre ( 74.8 megagrams per hectare ) in 1995 to an average of 48.5 tons per acre ( 108.6 megagrams per hectare ) in 2014and yields of > 60 tons per acre ( 134.4 megagrams per hectare ) are not uncommon ( geisseler and horwath 2013 ) . an examination of the pesticide use data in relation to the value of agricultural production revealed that california growers applied 8.7 pounds ( 3.9 kg ) of pesticide active ingredients for every us$1,000 of agricultural product in 1995 . in 2014 , they applied 3.3 pounds ( 1.5 kg ) per us$1,000 of agricultural product ( fig . 3 ) . therefore , measured in terms of production value , pounds of pesticide active ingredient integration of pesticide data with crop production statistics indicates that pesticide use has decreased slightly while crop yield and economic value have increased significantly . fig . 2.total pounds of active ingredient applied in agricultural production and postharvest fumigation in california from 1995 to 2014 . 3.average pounds of active ingredient applied per us$1,000 value of agricultural production from 1995 to 2014 . data obtained from pesticide use annual summaries ( california department of pesticide regulation ) and california agricultural statistics review ( california department of food and agriculture ) . total pounds of active ingredient applied in agricultural production and postharvest fumigation in california from 1995 to 2014 . average pounds of active ingredient applied per us$1,000 value of agricultural production from 1995 to 2014 . data obtained from pesticide use annual summaries ( california department of pesticide regulation ) and california agricultural statistics review ( california department of food and agriculture ) . in 2009 , california began reporting pesticide use by human and environmental toxicity category and extended the data back to 2000 ( california department of pesticide regulation 2010 and 2015b ) . their assignment of pesticides to specific toxicity classes provides greater detail about pesticide risk than aggregate - use data . the data show pesticide use reductions in some risk categories and pesticide use increases in others . for chemicals known to cause reproductive toxicity , use has declined from 28.6 million pounds ( 13 million kg ) in 2000 to 8.2 million pounds ( 3.7 million kg ) in 2014 , a 71% decrease . use of cholinesterase - inhibiting pesticides has dropped from 11.6 million pounds ( 5.3 million kg ) in 2000 to 4.6 million pounds ( 2.1 million kg ) in 2014 , a 60% decrease . the use of pesticides designated as having the potential to pollute groundwater has declined from 2.5 million pounds ( 1.1 million kg ) in 2000 to 690,000 pounds ( 313,000 kg ) in 2014 , a 72% decrease . for pesticides identified by the u.s . epa as b2 carcinogens or on california s proposition 65 list of chemicals that are known to cause cancer , use has increased from 25.5 million pounds ( 11.6 million kg ) in 2000 to 30 million pounds ( 13.6 million kg ) in 2014 , an 18% increase . the california department of pesticide regulation notes that pesticide oils are classified as carcinogens and that their use is included in these figures , even though some of those oils may not be carcinogenic due to their high degree of refinement and oils displace use of other more toxic pesticides ( california department of pesticide regulation 2015 ) . the use of toxic air contaminants has increased from 38.5 million pounds ( 17.5 million kg ) in 2000 to 44.1 million pounds ( 20 million kg ) in 2014 , a 15% increase ( california department of pesticide regulation 2015 ) . for example , 1 - 3 dichloropropene , metam - potassium , and mancozeb are potential carcinogens and toxic air contaminants ; methyl - bromide is a reproductive toxin and toxic air contaminant ; and metam - sodium is a reproductive toxin , carcinogen , and toxic air contaminant ( california department of pesticide regulation 2015 ) . epstein and zhang ( 2014 ) analyzed california pesticide use report data for 49 active ingredients that were used in cumulative state - wide quantities of 22,000 pounds ( 10,000 kg ) or more in either 1993 or 2000 and appeared on at least one of the five toxicity lists : reproductive toxins , carcinogens or probable carcinogens , cholinesterase - inhibiting pesticides , groundwater protection program compounds , and toxic air contaminants . of the 49 active ingredients analyzed between 1993 and 2010 , three are no longer in use and 40 have declined in use , two have increased in use , and one was newly added to the toxicity list ( epstein and zhang 2014 ) . these data document that use of pesticides classified as reproductive toxins , cholinesterase inhibitors , and potential groundwater contaminants is decreasing and use of pesticides classified as carcinogens and toxic air contaminants is increasing . therefore , potential human and environmental risk based only on use is decreasing for some categories and is increasing for others . assignment of pesticides to toxicity categories provides additional information but does not address the potential for exposure , an important part of the risk equation . anecdotally , increased use of plastic tarps , and more recently totally impermeable film tarps , has reduced potential exposure ( fig . 4 ) . however , there are no data documenting adoption of these specific practices . fig . consumers are exposed to pesticide residues on food . to manage this risk and prevent misuse of pesticides the pesticide data program within the usda agricultural marketing service ( ams ) collects fresh and processed food from distribution centers and conducts pesticide residue analysis . the specific commodities change from year to year , and fresh produce samples are washed in running water for 1520 s to mimic consumer practices . samples are then analyzed for > 300 pesticides , metabolites , degradates , and isomers in an analysis designed to detect the smallest possible amount of pesticide residues with limits of detection in parts per billion ( usda ams 2014 ) . the percentage of samples with pesticide residue exceeding the epa - established tolerance was 0.5% or less in each of the past 10 yr , and the majority of samples exceeding pesticide tolerance levels were imported , not domestic . the percentage of samples with residue of a pesticide for which there is no tolerance on that commodity contamination that may be the result of pesticide drift was 5.2% or less in each of the past 10 yr , and the residues were present at very low levels that did not exceed the tolerances established for similar commodities ( usda ams 2014 ) . the pesticide residue monitoring program within the california department of pesticide regulation collects raw fruits and vegetables from the channels of trade for pesticide residue analysis . in contrast to the federal pesticide data program , the california program does not change commodities each year and does not include processed food . the pesticide residue monitoring program analysis methods are continually improved and now detect > 300 pesticides or breakdown products ( california department of pesticide regulation 2014 ) . most samples have either no detectable residue or pesticide residues within legal tolerances , usually < 10% of the legal tolerance level . the percentage of samples with pesticide residue exceeding the epa - established tolerance is 1% or less in each of the past 10 yr . the percentage of samples with residue of a pesticide for which there is no tolerance on that commodity is 4% or less in each of the past 10 yr , and most of the samples for which there is no tolerance on that commodity have residues in the fractions of parts per million . data from 2010 to 2013 documents that 97.8% or more of the fruit and vegetables grown in california are in compliance with epa - established pesticide residue tolerances ( california department of pesticide regulation 2014 ) . winter and katz ( 2011 ) examined the dietary exposure to pesticide residues from the commodities on the environmental working group s 2010 dirty dozen list ( celery , peaches , strawberries , apples , blueberries , nectarines , bell peppers , spinach , cherries , kale , potatoes , and grapes [ imported ] ) . they analyzed exposures to the 10 most common pesticide residues on each of the 12 commodities . one hundred nineteen of the 120 exposure estimates were 1% or lower than the chronic reference dose for the pesticide , with 113 at 0.1% or lower . chronic reference dose is an estimate of the amount of pesticide that a person could ingest every day for a lifetime without appreciable risk of harm . pesticide residue data from the pesticide data program at usda and the pesticide residue monitoring program in california document that most pesticide residues are below the pesticide tolerance established by epa . dietary exposure analysis documents that human risk from pesticide residue on food is low , even for those fruits and vegetables identified as most likely to have pesticide residue . environmental risk from pesticides can be considered based on residues in water and air resources . the u.s . geological survey compared pesticide residues in streams draining agricultural , mixed use and urban surface waterways for the years 2002 to 2011 against the same data for 1992 to 2001 ( stone et al . stream classification was based on the dominant land - use in the watershed drained , and 49 of the 182 streams in the report are in the western united states . the percentages of assessed streams with at least one pesticide that exceeded aquatic - life benchmarks decreased slightly for agricultural streams , from 69% in 19922001 to 61% in 20022011 . mixed - use streams had similar levels of contamination , 45% in 19922001 and 46% in 20012011 , while urban - stream contamination increased sharply from 53% in 1992 to 2001 to 90% in 2002 to 2011 ( stone et al . 2014 ) . during 1992 to 2001 , 17% of agricultural streams and 5% of mixed - use streams had pesticide concentrations that exceeded human - health benchmarks . during 2002 to 2011 , human - health benchmarks were exceeded for atrazine in one agricultural stream ( stone et al . , california department of pesticide regulation has conducted air monitoring to determine concentrations of pesticides likely to be found in air . sampling locations are near the towns of salinas , shafter , and ripon , which were selected based on proximity to pesticide use , demographic data , and availability of other exposure and health data . california department of pesticide regulation monitored for 32 pesticides and 5 pesticide breakdown products and conducted 5,966 analyses in 2014 . of these , 5,471 analyses ( 91.7% ) had no detectable concentration , 498 analyses ( 8.3% ) had detectable concentrations , and in 225 analyses ( 3.8% ) the concentrations were quantifiable . of the 23 pesticides and pesticide breakdown products detected , 12 were in trace concentrations just above the limits of detection and 11 were in quantifiable concentrations . of the 23 pesticides and pesticide breakdown products detected , 22 did not exceed screening targets . 1,3-dichloropropene at shafter was detected at average concentrations that are calculated to increase cancer risk 1.7 times over a 70-yr lifetime of exposure . the sampling locations , number of pesticides and pesticide breakdown products analyzed , number of analyses , percent detections , and quantifiable detections were similar in the preceding three years . the exceptions were chloropicrin detections in salinas and shafter in 2013 , which were at levels calculated to increase cancer risk 1.4 and 3.47 times , respectively , over a 70-yr lifetime of exposure . in the environment , risk from pesticides in surface water in agricultural watersheds is decreasing and is correlated with local pesticide use . in air , based on four years of monitoring in three california locations all selected as likely to have high airborne pesticide concentrations the risk from pesticides in air sources is low . ipm is a method of reducing economic , human health , and environmental risks from pests and pest management strategies . pesticide applications are a component of ipm programs , but total pounds of pesticide applied is a poor measure of the impact of ipm . ipm programs should be measured by evaluation of economic , human health , and environmental risks from pests and pest management practices . metrics of the impacts of ipm programs need to incorporate pesticide toxicity , potential for exposure , and environmental risks in the context of agricultural productivity . over the past two decades , ipm programs have contributed to increasing agricultural productivity and have reduced some human health and environmental risks from pesticide applications due to decreases in use in arizona and california . risks from potential carcinogens and toxic air contaminants may have increased due to increased applications , although data on exposure are lacking . additional research and extension focus on ipm programs for the pests targeted by these applications is warranted . continued support for ipm research and extension programs is necessary to address new invasive pests and changes in agricultural production systems .

integrated pest management ( ipm ) is a method of reducing economic , human health , and environmental risks from pests and pest management strategies . there are questions about the long - term success of ipm programs in relation to continued use of pesticides in agriculture . total pounds of pesticides applied is a mis - measure of the impact of ipm in agriculture . a more complete measurement of the long - term impact of ipm includes consideration of changes in agricultural production practices and productivity , toxicity of the pesticides used , risks from human exposure to pesticides , and environmental sampling for pesticides in air and water resources . in recent decades , agricultural ipm programs have evolved to address invasive pests , shifts in endemic pest pressures , reductions in pest damage tolerance in markets , and increases in crop yields . additionally , pesticide use data from arizona and california revealed reduced use of pesticides in some toxicity categories but increased use of pesticides in a couple of categories . data from federal and california programs that monitored pesticide residue on food have documented low pesticide risk to consumers . environmental monitoring programs documented decreased pesticide levels in surface water resources in agricultural watersheds in the western united states and low levels of pesticides in air resources in agricultural areas in california . the focus of ipm assessment should be on reducing economic , human health , and environmental risks , not on pounds of pesticides applied . more broadly , ipm programs have evolved to address changes in pests and agricultural production systems while continuing to reduce human health and environmental risk from pesticides .

Pesticide Use Data Pesticide Residues on Food Pesticides in the Environment: Water and Air Resources Possible Solutions

PMC3159202

starting with winter semester 2003/2004 , the university of cologne introduced the new degree course at the medical faculty called 4c ( competence - based curriculum concept cologne ) , , . apart from fulfilling the goals of the medical licensure act ( approbationsordnung fr rzte ) , the specific profile of the 4c model is defined in the so - called general teaching principles ( leitbild lehre ) , according to which cologne graduates : have the necessary knowledge and skills to identify important and common diseases and acutely life - threatening situations and to induce their treatment;show behavior and attitudes that are conducive to their acceptance by patients and medical staff and to improving the standing of physicians in society;are willing and capable to engage in independent and science - based cpd in general practice but also in a clinical discipline or a basic subject of their choice . have the necessary knowledge and skills to identify important and common diseases and acutely life - threatening situations and to induce their treatment ; show behavior and attitudes that are conducive to their acceptance by patients and medical staff and to improving the standing of physicians in society ; are willing and capable to engage in independent and science - based cpd in general practice but also in a clinical discipline or a basic subject of their choice . these goals are primarily achieved through the following new elements in the curriculum , which are combined with traditional subject and cross - subject teaching : interdisciplinary competence areas ( 88 in total , each with 5 - 12 hours of lectures on important and common topics of in- and out - patient treatment ; competence areas 1 - 24 are for undergraduate students)patients treatment in parallel to the course ( studipat ) in which medical students are brought into contact with one patient each at a gp surgery for the first four years of studyorganization of week - long block placement from the 5th semester onwardsoffering electives at the end of each semester via compulsory blocks of electivesscientific qualification through two projects in which the students immerse themselves into a scientific subjectskills training in the kis ( cologne inter - professional skills lab and simulation center ) . interdisciplinary competence areas ( 88 in total , each with 5 - 12 hours of lectures on important and common topics of in- and out - patient treatment ; competence areas 1 - 24 are for undergraduate students ) patients treatment in parallel to the course ( studipat ) in which medical students are brought into contact with one patient each at a gp surgery for the first four years of study organization of week - long block placement from the 5th semester onwards offering electives at the end of each semester via compulsory blocks of electives scientific qualification through two projects in which the students immerse themselves into a scientific subject skills training in the kis ( cologne inter - professional skills lab and simulation center ) . kis is a central institution of the deanery of student affairs and serves to provide training and independent practice of practical medical skills in all outcome - areas as defined by harden , . in contrast to the traditional process - based separation of knowledge , skills and attitudes in the german - speaking world , outcome - based trainings starts by defining outcomes ( elsewhere also vaguely termed three different levels for curriculum development are discussed ( which build on each other ) . the first is doing the right thing which refers to the knowledge level and the ability to handle the daily clinical routine . the second is doing the things right , the level of emotional , analytic and creative intelligence to use knowledge and skills appropriately . the third level focuses on the person and their future development - the right one doing it , the professional role in its social and societal reality . recently more and more curricula have been developed from this point of view and as a result , there are also calls for the adaptation to accreditation criteria from the english - speaking world . this is somewhat surprising , especially in view of the thin to non - existent proofs of efficacy , in particular with regard to the 3rd level attitudes and professionalism " . based on these development criteria , and bearing in mind the requirements of the medical licensure act , clinical skills are grouped into three fields in cologne : emergency skills , communication skills and technical skills . these elements transcend the entire degree course as a teaching and learning helix , with the educational objective of students reaching clerkship maturity after five semesters and the internship maturity ( pj - reife ) after ten semesters . 1 ) shows the teaching content of skills training taught up to clerkship maturity divided into semesters . in a subsequent formative osce ( objective structured clinical examination , cf harden & gleeson ) with six stations during the 5th semester , students receive feedback on their practical skills on a german - english bilingual certificate , famulaturreife - clerkship maturity . the program running from the first semester of study to clerkship maturity was developed in 2003/2004 by an expert task force of the advisory board of study affairs of the medical faculty of the university of cologne . starting with the winter semester 2003/2004 , and with broad participation from many different disciplines ( anesthesia , psychosomatic medicine , transfusion medicine , internal medicine ) and following the delphi - model the leitbild lehre was developed . to validate this procedure retrospectively , two questions were investigated : what skills are required by the physicians and gps supervising students on clerkships?how frequently were students able to perform these skills as part of their clinical clerkship and which advantages did the clerkship maturity examination bring them from their point of view ? what skills are required by the physicians and gps supervising students on clerkships ? how frequently were students able to perform these skills as part of their clinical clerkship and which advantages did the clerkship maturity examination bring them from their point of view ? to evaluate the opinions of the teaching staff , a semi - standardized , machine - readable questionnaire was designed . the questionnaire was tested using ten randomly selected students to ensure its comprehensibility and subsequently sent to the senior physicians of the cologne teaching hospitals , the gp surgeries and the senior physicians at the university clinic of cologne by post . to evaluate the opinion of the students a semi - standardized , machine - readable questionnaire based on the above described questionnaire was designed to reflect the reality of education . in this context the primary concern was not an evaluation of the importance of the skills training content by the students but data on the frequency of skills used ( empirical relevance ) as part of the clerkships ( for questionnaire excerpts see table 3 ( tab . both questionnaires looked at the teaching / learning on offer in the kiss skills lab and the skills actually required during the clerkships . in addition , the form collected socio - demographic data regarding the age , sex , year of study , number of completed weeks on clerkship and additional medical training that may have been gained . results of the teaching staff questionnaire : 36 of 80 ( 45% ) hospital - based physicians and 77 of 140 ( 55% ) gps returned completed questionnaires . 1 ) shows the evaluation of the necessity / importance of different skills for clerkship maturity as viewed by the teaching staff . while frequently needed skills such as disinfecting hands or taking blood pressure and pulse are rated quite high , more rarely needed skills or skills deemed as premature for this stage were rated low ( such as placing a stomach tube or a cvl ) . significant differences between the hospital - based physicians and gps were only seen with a few items such as taking blood samples , iv / im injections and suturing techniques ( see figure 2 ( fig . it became clear that skills desirable for the ward round where rated more highly by hospital - based physicians significantly more frequently . while empathy with 64% was the most frequently mentioned criterion by gps , only 36% of hospital - based physicians rated this skill as being important . with 20% , both groups equally mention the ability to listen / handle patients or interest in the patient as an important skill for students . additional suggestions for skills that should be included in the kis offer regarding clerkship maturity are only given sporadically ( e.g. computer skills , wound management or team skills ) and thus do not allow a consistent overview . results of the student questionnaire : the questionnaire was sent to 140 students of which 75 were returned completed , a return rate of ( 54% ) . 24 of the 75 students who responded were male , 45 female ( 6 did not specify ) . 59 students were attending the 7 semester , 7 the 8th semester and 3 higher semesters . 7 students listed a qualification in the medical field , including 3 paramedics ( most frequent mention ) . only 5 students who had returned their questionnaires were studying under the regular degree course . for this reason , there is no comparison between the regular and the model degree course responses . 1 ) contrasts the student responses regarding the frequency of skills needed during the clerkship and their importance as rated by the teaching staff . it is clear that the frequency and necessity ( importance ) of some skills diverge rather drastically ( especially in emergency care ) . while the teaching staff rate training in examination - skills as very important ( 1.40.9 ) whereas the students rate this as less important ( 2.91.7 ) . students suggested the following in the open questions regarding possible improvements of the skills training prior to clerkship maturity : better knowledge transfer ( 9 mentions)more opportunities for practicing ( 5 mentions)practicing with real patients ( 3 mentions)ability to practicing rather than watching the lecturer ( 3 mentions)smaller groups ( 2 mentions ) better knowledge transfer ( 9 mentions ) more opportunities for practicing ( 5 mentions ) practicing with real patients ( 3 mentions ) ability to practicing rather than watching the lecturer ( 3 mentions ) smaller groups ( 2 mentions ) although skills in taking patient histories are rated as relevant and frequently needed , as can be seen in figure 1 ( fig . 1 ) , ( note that 16% of the students stated they did not have the opportunity to take medical histories in their clerkship ) , students rate the learning effect of the course on taking histories and communication rather moderately ( 2.91.7 to 3.11.4 , cf question 7b in table 3 ( tab . this is consistent with requests by the teaching staff for more training in taking patient histories ( 1.71.1 , cf question 10 in table 2 ( tab . the students also assessed the certificate they received following the osce examination during the 5th semester . the feedback on their practical skills provided by the certificate is rated rather modestly ( 3.4 1.7 on a 6-point likert - scale , 1=very helpful and 6=not helpful at all ) . the certificate was seen as even less helpful when it comes to applying for clerkship placements ( 4.71.8 ) . this corresponds to statements made by the physicians of which only 6% stated to be familiar with the certificate . the aim of this study was to validate the planning of training on practical skills as designed by the experts for the first 5 semesters of study in the new degree course in human medicine at the university of cologne empirically . as a validation of training goals , both teaching staff and students were questioned and the data of these two groups was compared . the students were also asked for their subjective experience gain through the kis training . generally speaking , it was possible to confirm the validity of the clerkship maturity goal based on the data collected from the teaching staff and the students . skills explicitly taught in the skills lab for achieving clerkship maturity are usually indeed frequently needed in clinical clerkships . even the handling of drips which is not explicitly part of the skills training and which is taught quasi in passing through the teaching of placing winged infusion sets . interestingly , students are confronted with the topics ultrasound and ecg rather more frequently than suspected . it might be worthwhile considering if the program needs to be amended to reflect demand in the clerkships . on the other hand , this content is taught later on in the clinical section in more detail . this also corresponds to the opinion of the physicians who rate neither as very important skills for the clerkships ( ultrasound even less so than ecgs ) . these differences that were discovered between skills rated as necessary by the teaching staff on the one hand and the experienced frequency of emergency care components needed during the clerkships as reported by the students on the other hand can be easily explained . these skills extremely important to have in some situations ( in some cases life - saving ) but in reality these are rarely required of students on clerkships . there is a remarkable difference concerning the divergence between the frequency of applying skills for clinical examination as stated by the students and the perceived necessity as rated by the teaching staff . the teaching physicians rate this item in place 2 , whereas the students rank this skill in place 5 . the method of comparing the frequency of a clinical skill used by students on the one hand with a ranking of the importance by teaching staff on the other could be seen as problematic . but it is not unlikely that at this stage students ( in the first or second clinical semester ) only possess a limited ability to evaluate the necessity of a skill but are quite capable of reporting the frequency based on their own experiences . allowing the frequency of skills used during clerkships to be rated by the teaching physicians is also questionable as such an evaluation might not be valid due to the workload and the fact that students are supervised by different members of staff . however , all physicians are able to rate the necessity of certain skills for the clinical activities of students on clerkships . the differences between the rated necessity of the skills in taking blood samples , iv / im injections and suture techniques between hospital - based staff and gps is not surprising , given the different fields of activity . nevertheless , it must be borne in mind that the practical training has to prepare students both for clerkships at a hospital and in gp s surgery . although the use of the majority of skills taught in training was confirmed by the questionnaire , a small number of skills at the bottom end of the ranking suggest room for re - organization . for example catheterizing the bladder , which seems to have a rather low relevance in the clinical routine of a student on clerkship ( ranked 15th of 18 items of all skills , see figure 1 ( fig . is given twice as frequently in the open questions when asked about the soft skills students should have with hospital - based doctors citing this item twice as often as gps . a possible explanation of this is the fact that the qualitative content analysis summed up related words like humbleness ( in front of the patient ) under the umbrella term empathy . in any case this should give food for thought further down the line , in particular regarding the teaching of attitudes , as they are referred to in the general teaching principles ( see above ) , corresponding to the third level of the outcome - based learning ( professionalism ) ; because empathy is seen as a prerequisite skill for a successful interaction between the doctor and the patient . although it was shown in the past that , as a first step to a conscious doctor - patient interaction , the first semester tutorial on medical psychology introduction to bio - psycho - social medicine as an introduction to teaching communicative competences ) that the thinking of students could be directed towards a multidimensional understanding of health and disease , , , . however , this does not correspond to a complex modulation of attitudes as required by the general teaching principles ( see also ) . so the comments made by the physicians in the open answers can be interpreted as a sign of gaps in the efforts to date to teach adequate doctor - patient communication . in this context the evaluation of the model degree course by the students that they had not received enough training in doctor - patient communication can also allow the conclusion that being familiar with a procedure and its risks leads to more reflective actions . especially because the quality of the self - evaluation given by the students is questionable as a valid source , more research it should not be left unmentioned that there is a possible clue that more intensive practical training can ( initially ) lead to unexpected uncertainty . because a structured curriculum for skills training in germany is not part of standard education ( and not currently required by the appo ) , it is not surprising that the answers to the question on the usefulness of the osce certificate at the end of the training phase in the 5th semester is subjectively not seems as being advantageous by the students . based on our data we can not clarify if this is simply down to the fact that this certificate is not sufficiently well known in hospitals and surgeries to date or if students do not even mention it in their applications . interestingly , isolated reports of experiences by students applying for international clerkships present a different picture . even though there is room for improvement regarding the perceived bonus of the skills training during the pre - clinical phase with the aim of clerkship maturity ( the usefulness of the osce feedback could , if necessary , be increased by skills area relevant evaluations such as hygiene skills , fine motor skills , etc . ) , the construct itself seems valid and reliable . the success of this initiative overall and a positive view by the students can only come into its forte through the national debate on education and the nascent national competence - based catalog of learning targets . only time will tell if the concept of clerkship maturity ( and the internship maturity and professional development maturity based upon it ) can be transferred . the development of medical skills training which is adapted to local needs is still in its infancy in the german - speaking countries and must be further developed in many ways . in addition the questionnaire for the teaching staff included questions about the general trainability of medical skills and differences in the performance of students from the regular degree course ( 8 appo ) and the model degree course ( 9 appo ) . these questions are not discussed in this article as it apparently was not possible for the teaching staff to differentiate between students of the regular and the model degree course . the student questionnaire also asked for an evaluation of their own competences in comparison to students of the regular degree course ( students following the old curriculum i.e. 8 appo ) . the results are not discussed in this article due to the low response rate . see the training goals developed in the study guidelines at the medical faculty of the university of cologne , based on the general teaching principles ( 1 sect 3 attitudes ) : readiness to apply medical ethical principles in practice and researchrespect and honesty towards patients and colleaguesrealistic estimation of one s own skills , abilities and limits and readiness , to draw appropriate conclusions from this;readiness to take on responsibility and for accuracy . readiness to apply medical ethical principles in practice and research respect and honesty towards patients and colleagues realistic estimation of one s own skills , abilities and limits and readiness , to draw appropriate conclusions from this ; readiness to take on responsibility and for accuracy . special thanks to mrs imke wietoska , marlen e. sauer and charlotte m. schober who contributed to the creation of the questionnaire databases as part of their scientific project . jan matthes for his support in bringing this master thesis to fruition as part of the mme - d .

background : with the reformed curriculum 4c , the medical faculty of the university of cologne has started to systematically plan practical skills training , for which clerkship maturity is the first step . the key guidelines along which the curriculum was development were developed by experts . this approach has now been validated . materials and methods : both students and teachers were asked to fill in a questionnaire regarding preclinical practical skills training to confirm the concept of clerkship maturity . results and discussion : the cologne training program clerkship maturity can be validated empirically overall through the activities of the students awaiting the clerkship framework and through the evaluation by the medical staff providing the training . the subjective ratings of the advantages of the training by the students leave room for improvement . apart from minor improvements to the program , the most likely solution providing sustainable results will involve an over - regional strategy for establishing skills training planned as part of the curriculum .

Introduction Materials and methods Results Discussion Notes Acknowledgement

PMC3382944

o. ficus indica is a species of cactus that belongs to the cactaceae family , the order centrospermae , and the genus opuntia . this plant is associated with the semiarid zones of the world mainly in africa , mediterranean countries , southwestern united states , northern mexico , and other areas . in tunisia , the main studies on the opuntia fruits were the chemical analysis of pulp , skin , and seeds . the proximate composition of o. ficus indica l. has been investigated [ 1 , 3 ] . seeds constitute about 1015% of edible pulp and are usually discarded in the food industry as waste after pulp extraction . edible oil can be obtained from prickly pear seeds in yield of 5.813.6% [ 1 , 46 ] . the oil has a high degree of unsaturation in fatty acids ( 82% ) with a linoleic acid content of 73.4% . according to the investigation of ramadan and mrsel using a chloroform / methanol as extraction solvent procedure of total lipids in seeds oil of cactus pear ( o. ficus indica l. ) , linoleic acid was the dominating fatty acid ( 53.5% ) , followed by palmitic ( 20.1% ) and oleic acids ( 18.3% ) . two spices of prickly pear from tunisia , o. ficus indica and o. stricta , were investigated for fatty acid composition seeds by ennouri et al . with an exceptional level of linoleic acid ( up to 70% ) ; the oils were extracted with hexane in a soxhlet extractor . moreover , no data has been mentioned about the difference of fatty acids composition in the two spines ( wild ) and thornless ( cultivated ) forms of tunisian o. ficus indica seeds . the oil obtained by mechanical separation processes was of high quality , but , in most cases , the yield was lower . hexane extraction achieved almost complete recovery of the oil , and the resulting oil contains traces of solvent ; however , the solvent was dangerous and unacceptable as it was quite harmful to human health and the environment , which may restrict its use in food , cosmetic and pharmaceutical industries . supercritical fluid extraction ( sfe ) with sc - co2 represents an alternative method for the extraction of oils from natural products and has received considerable attention . sfes have been used as solvents for a wide varieties of applications such as essential oil extraction . sfe use increases in the last years because of legal limitations of solvent residues , and solvents make this process more economical . the co2 remained the most commonly used fluid for sfe applications thanks to its low critical parameters ( tc = 31.1c ; pc = 74 bar ) , it has non - toxic , nonflammable properties , and it was available in high purity with low cost . the oils obtained by sc - co2 extraction were of outstanding quality , and the yields were comparable with those obtained with organic solvent extraction method . the co2 can be used in many extractions and reactions and has been recognized as earth - compatible solvent . therefore , sfe may serve as a promising technology in food and pharmaceutical processing . the objective of the present research was to illustrate and to compare between the sc - co2 and the soxhlet n - hexane profiles fatty acids of two forms of tunisian o. ficus indica seeds . research on oil extracted from o. ficus indica seeds showed that this material was a potential source of food and feed . moreover , these oils have several applications in pharmacology , medicinal , and cosmetic fields . the fact that extraction with sc - co2 enables to have pure oil free of solvent makes it possible to better enhance these oils safety . fresh and mature pears fruits of two forms of tunisian o. ficus indica were collected in summer 2009 . the spiny wild form was from the region of al - ala in the centre of tunisia located at 35 36 n ( north ) latitude , 9 34 e ( east ) longitude , and 450 m ( meter ) altitude . the thornless cultivated form was from local pilot cultivar of bou argoub region in the north east of tunisia located at 36 32 n latitude , 10 33 e longitude , and 62 m altitude . the seeds were recovered , after mixing the pulp and filtration through a 2 mm sieve . the seeds were washed thoroughly with bidistilled water , dried in an oven at 35c for 48 h. the seeds were ground by a domestic coffee grinder ( seb prep line ) just before extraction . the moisture content of the seeds was determined by drying 50 g of seeds at 70c to a constant weight in an oven for 72 h. reference standards of caprylic acid ( c8 : 0 ) , capric acid ( c10 : 0 ) , lauric acid ( c12 : 0 ) , myristic acid ( c14 : 0 ) , palmitic acid ( c16 : 0 ) , palmitoleic acid ( c16 : 1 ) , heptadecanoic acid ( c17 : 0 ) , stearic acid ( c18 : 0 ) , oleic acid ( c18 : 1 ) , linoleic acid ( c18 : 2 ) , linolenic acid ( c18 : 3 ) , arachidic acid ( c20 : 0 ) , eicosenoic acid ( c20 : 1 ) , eicosadienoic acid ( c20 : 2 ) and behenic acid ( c22 : 0 ) that had a minimum purity of 99% , sodium methoxide , methanol ( hplc grade ) , hexane , and sulfuric acid ( 1n ) were purchased from sigma aldrich ( usa ) . the sc - co2 extractions were carried out with a high purity carbon dioxide ( 99.95% ) supplied by messer ( france ) . the extractions were conducted in a cylindrical stainless steel extractor ( 300 mm 23 mm , internal volume : 125 cm ) and a three cyclonic separators type . the ground seeds ( 50 g ) of o. ficus indica ( spiny : 92.49%thornless : 93.64% dry matter ) were loaded into steel cylinder , filled into the extractor vessel and extracted at temperatures of 35 , 40 , 45 , 50 , 60 , and 70c ( spiny ) and 40c ( thornless ) , 180 bar and the co2 flow rate of 15 mls for 135 min . the desired extraction temperature was achieved by heating the extraction vessel , and the temperature was monitored by a thermocouple and regulated by a controller . the extraction pressure was controlled by a back pressure regulator , and the flow rate of co2 was controlled by manual adjustment of a needle - metering valve . the extracts were collected in one tube throughout the 135 min , and the yields were calculated ( for each temperature ) . the final volume of extract was analyzed using gc - fid to determine the fatty acids profile as described below . soxhlet n - hexane extraction was carried in triplicate for 30 g of ground seed of o. ficus indica by 250 ml of n - hexane ( 99% ) for 8 h. a rotary evaporator was used for the efficient removal of solvent ( n - hexane ) from samples by evaporation at 68c . the extracts were stored in a glass jars in the deep freeze ( 20c ) pending for further analysis . the extract samples were methylated by sodium methoxide according to the method of carreau and dubacq . the methylation of fatty acids occurred in the presence of 0.5 ml sodium methoxide in methanol 3% and 2 ml of hexane . a known amount of methyl ester heptadecanoic acid ( c17 : 0 ) was used as internal standard to quantify the fatty acids . after stirring during 60 seconds and decantation for 2 min , the reaction was neutralized by the addition of 0.2 ml of h2so4 ( 1n ) . finally , 1.5 ml salt water ( 10% nacl ) was added to allow the separation of two phases . the organic higher phase containing dissolved methyl esters in hexane was retained . after a concentration under nitrogen current , the methyl - esterified fatty acids were analyzed by gc - fid to determine fatty acid constituents . the gc analysis was performed by using a hewlett - packard ( hp ) 6890 series equipped with a flame ionization detector ( fid ) and a polar capillary column : hp innowax ( 30 m length 0.25 mm internal diameter and 0.25 m film thickness).the column temperature was initially at 120c ( held for 1 min ) and then increased to 200c at 15c min and finally increased to 250c at 2c min . the operational conditions were injector temperature 250c ; detector temperature 275c ; carrier gas was nitrogen at a flow of 1.7 ml min with a split ratio of 40 : 1 . the fatty acids were identified by comparing their chromatograms to those of pure standards analyzed under the same conditions . the hp chemstation software allowed the end of each analysis to provide the surface of each peak and its percentage . the standard deviations of the extractions yields and the fatty acid constituents were based on triplicate measurements . in this study , we intend to compare the efficiency of the sfe by sc - co2 extract process with the solvent extraction by soxhlet n - hexane . efficiency is based on the extraction yield ( quantity ) with its relationship to composition of fatty acids ( quality ) . various parameters potentially influenced the sc - co2 extraction process , and the first step of sc - co2 extract knowledge was to optimize the temperature at fixed conditions : pressure = 180 bar , co2 flow rate = 15 ml s , and time = 135 min , to obtain a better yield of extract . the experimental results of sc - co2 extract yield of the spiny form were calculated and illustrated in figure 1 . the yields ( y ) of oil defined on 100 g dry seeds of o. ficus indica basis and the total extraction yield were calculated as follow : ( 1)y%=[(m2m1)100]m , where m2 was the mass of glass trap with extract , m1 was the mass of empty glass trap , and m was the initial mass of seeds in the extractor . the extract collected in the cold trap at atmospheric pressure consisted of a yellow solid mass and water . according to figure 1 , the extraction yields first increased from 1114.8 mg 34.5/50 g of ground seeds at t = 35c , reached a maximum value at 40c ( 1702.4 mg 40.1/50 g of ground seeds ) , and then decreased at higher temperatures 70c ( 122.3 mg 6.6/50 g of ground seeds ) . extraction with sc - co2 at t = 40c versus time of spiny and thornless forms of o. ficus indica seeds showed , respectively , an extraction percentage of 3.4% ( 1702.4 mg 40.1/50 g of ground seeds ) and 1.94% ( 970.7 mg 49/50 g of ground seeds ) ( figure 2 ) . in the same condition of our extraction ( t = 40c and fixed parameters ) , the sc - co2 extraction yields depended on the variability of plant material ( seeds ) of two opuntia forms . at t = 40c , the extraction yield was gradually increased with increasing time , for the two forms of o. ficus indica , the majority of the extract oil was recovered at an interval time of 15 to 75 min , and the extraction yields were 79.42% and 78.82% , respectively , for the spiny and the thornless form . the oils soxhlet n - hexane extract yield for the wild spiny form ( 10.32% ) and cultivated form ( 8.91% ) of tunisian o. ficus indica were higher than the sc - co2 extract . the performance of sc - co2 extraction was relatively low compared to soxhlet n - hexane extract and other extraction methods that exist in the literature , the extraction solvent chloroform / methanol recovers 9.88% . according to snyder et al . , moisture content between 3% and 12% has very little effect on the extractability of oils from seeds with sc - co2 . in the literature , it was reported that o. ficus indica contain a good quality of edible oil [ 4 , 5 , 15 , 16 ] such as fatty acids . the fatty acid profiles of two forms of o. ficus indica seeds were analyzed by gc - fid , and the results are summarized in table 1 . the results enabled us to identify and compare the constituent fatty acids of total lipids extracted from seeds of the o. ficus indica ( table 1 ) . the percentage of each compound was calculated from the peak areas given by the gc- fid . the sc - co2 extraction of fatty acids profile showed eleven peaks ( figure 3(a ) ) ; the soxhlet n - hexane extract of fatty acids profile illustrated only 8 peaks ( figure 3(b ) ) . the main components for two forms were linoleic acid : sc - co2 ( spiny 57.60% , thornless 59.98% ) and soxhlet n - hexane ( spiny 57.54% , thornless 60.66 ) , oleic acid : sc - co2 ( spiny 22.31% , thornless 22.40% ) and soxhlet n - hexane ( spiny 25.28% , thornless 20.58% ) and palmitic acid : sc - co2 ( spiny 14.3% , thornless 12.92% ) and soxhlet n - hexane ( spiny 11.33% , thornless 31.08% ) . according to the ramadan and mrsel work , solvent extraction using a chloroform / methanol showed that palmitic acid was the second compound after linoleic acid in terms of amount . reported that the oils extracted with hexane using a soxhlet extractor , linoleic acid contents were higher than our results ( 70.3% ) , oleic acid was very much lower ( 16.8% ) and palmitic acid ( 9.3% ) . cokuner and tekin reported that the seed oil extracted from turkish o. ficus indica ( mersin ) with petroleum ether by soxhlet extractor , palmitic acid content ( 12% ) , and linoleic ( 52% ) . the observed difference is probably due to extraction process and the maturation stage of fruits . cokuner and tekin suggested that there was an increase in saturated fatty acid content towards the end of fruit maturation . the solvent extraction of o. ficus indica seeds has been studied at different conditions previously , but , to the best of our knowledge , there is not any research about the sc - co2 extraction of tunisian o. ficus indica . the results of sc - co2 extract showed that the saturated fatty acids composition was slightly more important for wild spiny form ( 14.30% for palmitic acid and 3.12% for stearic acid ) than the cultivated thornless form ( 12.92% for palmitic acid and 2.38% for stearic acid ) . in contrast , the soxhlet n - hexane extract of palmitic acid was most important for the thornless form ( 13.08% for ) than the spiny form ( 11.33% ) . for the extraction process , the results showed that the oils were found to be highly unsaturated ( us = 4.445.25 ) . the soxhlet n - hexane extract showed an us more important for the spiny form ( 5.22 ) , the soxhlet n - hexane extract allowed a better extraction of unsaturated fatty acids for the wild form . the sc - co2 extract showed an us more important for the thornless form ( 5.25 ) , and the sc - co2 extract allowed a better extraction of unsaturated fatty acids for the cultivated form ( table 1 ) . compared to ramadan and mrsel results , the sc - co2 profiles fatty acids of tunisian o. ficus indica seeds showed the richness of this profile with other compounds such as myristic , arachidic , eicosenoic , eicosadienoic , and behenic fatty acid . compared to our soxhlet n - hexane extract results , the sc - co2 profiles fatty acids showed the richness of small proportion compounds such as eicosenoic , eicosadienoic , and behenic fatty acid . ( cactaceae ) by supercritical co2 revealed other compounds found in low percentage such as vaccenic and margaric fatty acid . these results showed that the supercritical co2 extraction ensures a better extraction of small proportion compounds . the composition of the spiny tunisian o. ficus indica seeds oils revealed the similar fatty acid constituents at different temperature ( table 2 ) . the temperature of sc - co2 extraction seemed has not effect on the selectivity of fatty acids . the increasing temperature from 40c ( u / s = 4.44 ) to 70c ( u / s = 4.14 ) tended to decrease the sc - co2 extraction yield but not affect the solubility of fatty acids . the sc - co2 and soxhlet n - hexane extraction was applied to the extraction of oil seeds from wild and cultivated tunisian o. ficus indica . the oil yields were higher by soxhlet n - hexane extraction , and the wild form gave the best results . the fatty acids profiles were investigated , and linoleic acid was the major compound followed by oleic acid and palmitic acid for the two forms and for the two extraction process . the sc - co2 extract allowed a better extraction of unsaturated fatty acids for the cultivated form , and oils revealed a better extraction of small proportion compounds . in contrast , the soxhlet n - hexane extract allowed a better extraction of unsaturated fatty acids for the wild form , and the fatty acids profile did not identify some compound of lower proportion . the low extractable compounds of fatty acids and the purity of the sc - co2 extract are giving a better quality of oil . we can extend our research on the various parameters of supercritical extraction for optimizing better extraction efficiency . with a better extraction of small proportion compounds of fatty acids seed oils , the sc - co2 process could be more advantageous than the soxhlet n - hexane . the fatty acids seed oil would seem to have a potential use and be valuable in different areas of bioindustrial implementation , nutraceutical and pharmaceutical . these results provide a better opportunity to improve and to enhance the natural heritage that characterizes the tunisian o. ficus indica .

the fatty acids profiles of tunisian opuntia ficus indica seeds ( spiny and thornless form ) were investigated . results of supercritical carbon dioxide ( sc - co2 ) and soxhlet n - hexane extract were compared . quantitatively , the better yield was obtained through soxhlet n - hexane : 10.32% ( spiny ) and 8.91% ( thornless ) against 3.4% ( spiny ) and 1.94% ( thornless ) by sc - co2 extract ( t = 40c , p = 180 bar , time = 135 mn , co2 flow rate = 15 mls1 ) . qualitatively , the main fatty acids components were the same for the two types of extraction . linoleic acid was the major compound , sc - co2 : 57.60% ( spiny ) , 59.98% ( thornless ) , soxhlet n - hexane : 57.54% ( spiny ) , 60.66% ( thornless ) , followed by oleic acid , sc - co2 : 22.31% ( spiny ) , 22.40% ( thornless ) , soxhlet n - hexane : 25.28% ( spiny ) , 20.58% ( thornless ) and palmitic acid , sc - co2 : 14.3% ( spiny ) , 12.92% ( thornless ) , soxhlet n - hexane : 11.33% ( spiny ) , 13.08% ( thornless ) . the sc - co2 profiles fatty acids showed a richness with other minority compounds such as c20:1 , c20:2 , and c22.the seeds oil was highly unsaturated ( us = 4.445.25 ) , and the rising temperatures donot affect the selectivity of fatty acids extract by sc - co2 : us = 4.44 ( t = 40c ) and 4.13 ( t = 70c ) .

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

PMC5091776

repeated assessment of the abundance and diversity of phytophagous and entomophagous arthropods in crops is needed for ecological studies of population dynamics and species interactions , including the study of biological control . rice is a major staple food , with a high potential for biocontrol by entomophagous arthropods , but which can be disrupted by insecticides . the diversity of arthropods in rice crops can be high , and arthropod species occupy various crop strata ( e.g. , ground , stem , canopy , flowers ) , differ in mode of movement ( e.g. , walking , jumping , flying ) and foraging strategy ( e.g. , sessile sucking insects , hunting predators and flower visiting pollinators ) . there is a wide range of arthropod sampling techniques , each with strengths and weaknesses . for instance , pitfall traps can be used to sample ground - dwelling arthropods , but provide activity - dependent relative population estimates . sweep nets can be used to sample fast - flying insects in the canopy , but give relative estimates of arthropod abundance . the beat sheet method can be used to sample the plant - dwelling arthropod community and provides absolute estimates of arthropod abundance , but it can not be used effectively in flooded crop fields such as rice paddies . suction sampling , when conducted in combination with an enclosure covering a standardized area of the field , provides absolute estimates of the densities of plant - dwelling arthropods . the dietrick vacuum ( d - vac ) is the first commercially developed suction sampler . although d - vacs are still widely used , they are relatively expensive , have a limited suction force and are relatively heavy , which makes them hard to handle in flooded rice fields . arida and heong developed a suction sampler using a petrol - driven leaf blower - vac , and advantages of the blower - vac suction sampler as compared to the d - vac are that it is much cheaper and easier to handle . although the blower - vac suction sampling method has been used in many ecological studies , the instructions for its modification and application have not been clearly described . here we present a video - based , detailed description of the modification and application of a petrol - powered leaf blower - vac for suction sampling of arthropod populations in flooded rice fields . the modification is inspired by arida and heong and domingo and schoenly , but the design has been further simplified compared to these original publications , facilitating construction and use . collect all parts listed in the materials list . connect all polyvinyl chloride ( pvc ) pipes with unplasticized polyvinyl chloride ( u - pvc ) glue ( pipes 1 - 2 - 3 - 4 - 5 and 6 - 7 ) . drill three holes that are evenly distributed around the suction mouth of the machine . connect the machine to the end 1 of pvc pipe 1 - 5 by inserting one screw into each of the three holes . do not use glue to connect pipe end 1 to the machine because the connection should be reversible to clean the fan . note : in case the diameter of the suction mouth of the machine differs from the model described here , the diameter of pipe end 1 should be adjusted to fit the machine seamlessly . put the piece of metal gauze between the hose and the mouth part ( pvc pipes 6 and 7 ) to prevent the sampling net from being sucked into the machine . connect the hose to pipe ends 5 and 6 with metal clamp hoops . remove the bottom from a plastic bucket ( 50 l , 40 cm bottom diameter ) . this size of the enclosure covers 2 - 4 rice hills in a transplanted rice field , depending on crop stage . attach a nylon mesh sleeve , with a length of 1 m , to the top of the bucket using a rubber band . for this sleeve , use a mesh size that is small enough to prevent the escape of the smallest target arthropods . use a diameter less than 0.5 mm . use two persons to operate the device in the field . one person operates the blower - vac and the other handles the bucket enclosure and the sampling nets . insert a sampling net into the mouth part of the blower - vac and fix it with a rubber band . for the net , use a mesh size that is small enough to catch the smallest target arthropods , but does not create obvious airflow resistance . use the light nylon material with a mesh diameter between 0.2 - 0.5 mm . place the enclosure quickly over the plants at a random location in the field and push the bottom of the bucket firmly into the soil . make sure that the sleeve is closed to prevent arthropods from escaping from the top of the enclosure . remove all arthropods from inside the enclosure in a top - down spiraling way , for a standardized sampling duration . for rice crops in the vegetative and reproductive stages , use sampling durations of 1 and 2 min , respectively . after finishing the sample , remove the rubber band from the sampling net , quickly close the net and take it out of the mouth part of the blower - vac and close it with a knot , while keeping the machine running . repeat steps 3.2 to 3.5 at random locations in the field for the next samples . the number of replications depends on the variation in the spatial distribution of arthropods in the field , the required accuracy of the estimate and the purpose of the study . typically , six replicates will give a good impression of the arthropod community and species abundances . collect all parts listed in the materials list . connect all polyvinyl chloride ( pvc ) pipes with unplasticized polyvinyl chloride ( u - pvc ) glue ( pipes 1 - 2 - 3 - 4 - 5 and 6 - 7 ) . drill three holes that are evenly distributed around the suction mouth of the machine . connect the machine to the end 1 of pvc pipe 1 - 5 by inserting one screw into each of the three holes . do not use glue to connect pipe end 1 to the machine because the connection should be reversible to clean the fan . note : in case the diameter of the suction mouth of the machine differs from the model described here , the diameter of pipe end 1 should be adjusted to fit the machine seamlessly . put the piece of metal gauze between the hose and the mouth part ( pvc pipes 6 and 7 ) to prevent the sampling net from being sucked into the machine . remove the bottom from a plastic bucket ( 50 l , 40 cm bottom diameter ) . this size of the enclosure covers 2 - 4 rice hills in a transplanted rice field , depending on crop stage . attach a nylon mesh sleeve , with a length of 1 m , to the top of the bucket using a rubber band . for this sleeve , use a mesh size that is small enough to prevent the escape of the smallest target arthropods . use a diameter less than 0.5 mm . one person operates the blower - vac and the other handles the bucket enclosure and the sampling nets . insert a sampling net into the mouth part of the blower - vac and fix it with a rubber band . for the net , use a mesh size that is small enough to catch the smallest target arthropods , but does not create obvious airflow resistance . use the light nylon material with a mesh diameter between 0.2 - 0.5 mm . place the enclosure quickly over the plants at a random location in the field and push the bottom of the bucket firmly into the soil . make sure that the sleeve is closed to prevent arthropods from escaping from the top of the enclosure . remove all arthropods from inside the enclosure in a top - down spiraling way , for a standardized sampling duration . for rice crops in the vegetative and reproductive stages , use sampling durations of 1 and 2 min , respectively . after finishing the sample , remove the rubber band from the sampling net , quickly close the net and take it out of the mouth part of the blower - vac and close it with a knot , while keeping the machine running . repeat steps 3.2 to 3.5 at random locations in the field for the next samples . the number of replications depends on the variation in the spatial distribution of arthropods in the field , the required accuracy of the estimate and the purpose of the study . typically , six replicates will give a good impression of the arthropod community and species abundances . a total of 295 arthropods were collected in 8 three - minute blower - vac samples from a rice crop in the ripening stage in jiangxi province , china , in september 2015 . to determine the relationship between relative yield ( proportion of the arthropods collected in the sample ) and sampling duration , the mean number of individuals per sample was 36.9 4.1 ( mean sem ) . a total of eight arthropod orders were found , with hemiptera ( 28.8% ) , araneae ( 27.5% ) and diptera ( 17.6% ) being dominant ( figure 1 ) . the relative yield , expressed as a percentage of the number of collected arthropods after three minutes , was 52.9% 5.1 , 92.4% 1.9 and 97.3% 0.9 after 30 seconds , 2 and 2.5 minutes , respectively ( figure 2 ) . figure 1.species composition ( at the order level ) of arthropod samples in the suburbs of nanchang city in jiangxi province , china . cumulative relative yield as a function of total sampling duration based on sub - samples of 30 seconds each . the total number of arthropods collected over a sampling duration of three minutes is set at 100% . suction sampling is one of many possible methods to sample arthropod communities in crops . for scientific research in rice systems , suction sampling is an appropriate option because the method provides absolute estimates of arthropod densities , it is non - destructive , and in contrast to visual counts allows collection and storage of samples for later processing . compared to the commercially available d - vac , the blower - vac is smaller , lighter and easier to handle in ( flooded ) rice fields and also easier to combine with an enclosure . for example , a blower - vac weighs about 6 kg , whereas the backpack d - vac model , presented as the international standard for insect sampling , has a weight 12 kg . more importantly , the sampling efficiency of the blower - vac is higher than the d - vac , while the cost of the blower - vac is less . the modification of a leaf blower - vac into a suction sampler does not require special skills or equipment and takes less than an hour after all additional parts have been collected . the blower - vac described here is easier to construct and operate than versions described previously in the literature , and the required parts ( table 1 ) are standard construction materials that are widely available . this makes the blower - vac also accessible to researchers with small budgets in developing countries . the power and displacement of the engine determines the suction force of the blower - vac . here we recommend a machine with a power between 0.7 - 1.2 kw and a displacement between 25 - 35 cc , which is adequate for sampling the plant - dwelling arthropod community in rice . the length of the flexible plastic hose and the diameter of the sucking mouth part ( pipe 7 ) are critical for a good sampling performance . a hose that is too long will reduce the suction power , whereas a hose that is too short will be inconvenient to use during sampling . similarly , a mouth part with too large a diameter will reduce the suction power , whereas a diameter that is too small will reduce the sampling efficiency due to the small surface . if sampling is conducted throughout the growing season , the sampling duration may have to be adjusted to the plant size , structure , and planting density to maintain a similar level of efficiency . sampling efficiency should be checked by careful visual inspection of the enclosed area after sampling . the recommended sampling durations for rice crops in the vegetative stage is 1 minute and in the reproductive and ripening stages it is 2 minutes . suction sampling with the blower - vac can be conducted in flooded fields , while alternative methods , such as pitfall and beat sheet sampling are not feasible in standing water . the blower - vac can also be used to sample the arthropod community on the water surface of flooded rice fields ( e.g. , predatory water bugs ) , as the machine is capable of sucking in some water . however , it is not recommended for sampling aquatic arthropods as the motor may stop running when the mouth part is inserted deeply into the water and the airflow is blocked . apart from rice , the blower - vac can also be used in other crops and non - crop habitat , as long as the height and structure of the vegetation allows proper placement of the enclosure . almost all arthropods collected in the sampling net survived , including those soft bodies such as mosquitos and damselflies . the blower - vac in the field will result in some disturbance , and therefore this method may underestimate disturbance - sensitive species such as grasshoppers . fast and abrupt placement of the enclosure in a relatively undisturbed area in the forward direction of movement may limit this potential bias . the loud noise of the blower - vac machine may also cause disturbance , and sampling at night in residential areas is not recommended . the method is not suitable for sampling highly mobile flying insects , such as predatory odonata or larger hymenopterous parasitoids . as with any sampling method , the combination of the blower - vac with other methods , such as sweep net sampling or destructive harvesting of plants , can provide a more complete and balanced assessment of the arthropod community .

rice fields host a large diversity of arthropods , but investigating their population dynamics and interactions is challenging . here we describe the modification and application of a leaf blower - vac for suction sampling of arthropod populations in rice . when used in combination with an enclosure , application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area . the sampling efficiency depends critically on the sampling duration . in a mature rice crop , a two - minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population . the device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies , but it is not suitable for sampling fast flying insects , such as predatory odonata or larger hymenopterous parasitoids . the modified blower - vac is simple to construct , and cheaper and easier to handle than traditional suction sampling devices , such as d - vac . the low cost makes the modified blower - vac also accessible to researchers in developing countries .

Introduction Protocol 1. Modification of a Leaf Blower-vac for Suction Sampling 2. Prepare the Sampling Enclosure 3. Field Application of the Modified Leaf Blower-vac for Suction Sampling Representative Results Discussion Disclosures

PMC3679558

basidiomycete fungi are attracting increased interest because of their plant pathogenic properties , along with an ability to produce enzymes , in particular glycoside hydrolases , which can degrade recalcitrant carbon sources , thereby increasing the range of substrates available for bioethanol production . although the genomes of a number of basidiomycete species have been sequenced and a number of proteomic studies have been carried out , one genera in particular , armillaria , has received little attention to date . yet , armillaria spp . are among the most serious plant root pathogens and cause major economic losses following infection of tree species of both agronomic importance and those grown for timber production purposes . the genera consists of at least 40 species , including a. mellea , a. gallica , a. tabescens , and a. borealis , with the former species attracting the most attention . in particular , a. mellea has been studied because of its virulence , bioluminescent properties , ability to produce unusual natural products and from a strain typing perspective , for definitive identification of the armillaria spp . recently , a novel infection model system for a. mellea has been established which deployed confocal microscopy and quantitative pcr for the estimation of fungal biomass produced during grape rootstock infection . this approach has facilitated a more quantitative assessment of fungal infection of plant roots , in addition to distinguishing between susceptible and resistant plant varieties . armillaria spp . also produce uncommon secondary metabolites , such as sesquiterpene aryl esters , diketopiperzines and sphingolipids . next generation sequencing has dramatically accelerated research into basidiomycete genomics , for example the genomes of over 50 basidiomycetes are currently available , including the maize and teosinte pathogen ustilago maydis , the ectomycorrhizal fungus , laccaria bicolor , white rots ( phanerochaete chrysosporium and schizophyllum commune(11 ) ) and the multicellular species coprinopsis cinerea . in initial basidiomycete proteomics studies , 2-d page coupled with maldi - tof or lc ms / ms led to identification of 25 extracellular proteins from p. chrysosporium , mainly those involved in carbohydrate polymer degradation , and noted difficulties with peptide identification caused by protein glycosylation . soon after , these techniques were used to assess the effects of vanillin , an intermediate in lignin degradation on the same fungus and revealed up- and down - regulation of the citric acid and glyoxylate cycles , respectively . a proteomic approach was also used to investigate the differential secretome composition of p. chrysosporium grown under ligninolytic conditions , where peroxidases were mainly detected . a greater diversity of fungal glycoside hydrolases was identified during culture in biopulped material including cellulases , hemicellulases , putative glucan 1,3--glucosidases and -glycosidases . this work , which was the first differential secretome characterization of p. chrysosporium , highlighted the power of high - throughput proteomic analysis to reveal culture - dependent proteome remodelling due to substrates used for fungal culture . although no genome sequences are available , proteomic analyses were also carried out on two economically important edible basidiomycetes , sparassis crispa and hericium erinaceum , yielding identification of 77 and 121 proteins from both species , respectively , 21 of which were found to be common in both species . l. bicolor is an ectomycorrhizal basidiomycete , which exists in either a saprotrophic phase in soil or an extended mutualistic interaction with tree roots . a recent proteomic study of the l. bicolor secretome in liquid culture identified 224 proteins by a combined electrophoretic fractionation and mass spectrometry - based shotgun approach . this work significantly extended knowledge of the secretome of this saprophytic organism and identified multiple glycoside hydrolases and proteases , hypothesized to be involved in either plant colonisation or basidiomycete defense . of interest , among proteins of unknown function , an induced small secreted protein , probably a putative effector , has been detected . functional assignment of unknown proteins produced by basidomycetes is set to become one of the major challenges in fungal biology in the coming years . however , despite such advances in other basidiomycete species , to date , only 16 distinct a. mellea proteins are curated in genbank including an atp synthase subunit 6 , gapdh isoform , putative zinc finger / binuclear cluster transcriptional regulator , rna polymerase ( two subunits ) , a laccase - like multicopper oxidase lcc2 and a metalloendopeptidase . this is especially problematic since so little is known at the molecular level about this plant pathogen . here , we reveal the first extensive genome sequence analysis of a. mellea , combined with a detailed proteomic characterization . in addition , we propose a novel infection model system that utilizes candida albicans as a target to investigate a. mellea virulence . a. mellea ( vahl : fr ) kummer dsm 3731 was purchased from dsmz ( braunschweig , germany ) and maintained on malt extract agar ( 50 liquid cultures ( 200 ml ) were undertaken in 500 ml erlenmeyer flasks using potato dextrose broth ( pdb ) ( 2.4% ( w / v ) ) or minimal media ( 5 mm ammonium tartarate as nitrogen - source , trace elements according to schrettl and colleagues and containing 1% ( w / v ) of one of the following carbon - sources : lignin , xylan , cellulose , rutin , yeast extract ) . ethanol ( 0.15% ( v / v ) ) was added to liquid cultures before inoculation with mycelia and incubation at 25 c in darkness for 26 weeks . mycelia from liquid cultures were harvested by filtration through miracloth , washed with distilled water , dried thoroughly with absorbent paper , and put immediately into liquid nitrogen and stored at 70 c . malt extract agar ( mea ) plates were inoculated with a. mellea mycelia and incubated at 25 c in darkness for 26 weeks . coculture experiments involving a. mellea and candida albicans were carried out on pdb agar . c. albicans ( 5 l ; 10 cells / ml ) was applied in quadruplicate around central mycelial cultures of a. mellea . after 52 days coculture , c. albicans plugs were collected for subsequent growth ( via reculture on pdb agar ) and viability assessment ( via fluorescein diacetate ( fda ) ) , in the absence of a. mellea . briefly , fda ( 2 g/100 l ) and propidium iodide ( pi : 0.6 g/30 l ) were added to 2 10c . after sequential washing with phosphate buffered saline ( pbs ) and 20% ( w / v ) bovine serum albumin ( bsa ) in pbs , fluorescence was determined using an olympus fluoview 1000 confocal microscope . viability was also assessed by replating serial dilutions of monoculture and coculture - derived c. albicans on mea . proteins of a. mellea produced during coculture were extracted by gently grinding mycelia and agar ( 25 ml ) to avoid mycelial lysis with 50 mm kh2po4 ph 7.0 . after overnight agitation and filtration , proteins were tca precipitated , resolubilized in 50 l of 10 mm ammonium hydrogen carbonate containing 10% ( v / v ) acetonitrile and trypsin ( 65 ng / ml ) and digested ( 37 c ) overnight . resultant peptides were diluted 1/4 in 0.1% ( v / v ) formic acid ( 20 l ) and analyzed directly by lc a. mellea dna was extracted using a zr fungal / bacterial dna kit ( zymo research group ) using a modified protocol . mycelia were ground into a fine powder in liquid n2 , and 300 mg were added to tubes containing glass beads ( 200 mg , 0.4 mm ) . lysis solution ( 750 l ) was added , specimens were bead - beaten for 2 min at 20 hz followed by centrifugation at 10000 g for 1 min . supernatants ( 400 l ) were transferred to zymo - spin iv spin filters and the manufacturer s instructions were then followed . dna was precipitated by adding 3 m sodium acetate ( 1/10 vol . ) and 3 vol . ice - cold ethanol ( 100% ( v / v ) ) . following incubation at 20 c for 3 h , specimens were centrifuged at 13000 g for 10 min and supernatants removed . pellets were washed again with ice - cold ethanol ( 75% ( v / v ) ; 180 l ) followed by centrifugation at 13000 g for 10 min . after supernatant removal , pellets were air - dried to remove traces of ethanol before resuspending in milli - q h2o ( 20 l ) . pcr to confirm a. mellea dna presence was carried out using two different primer pairs ; regf1 and regr1 primers and amel3 and its4 primers . a single no - pcr paired - end library was generated from a. mellea genomic dna according to the method of kozareva and turner with an estimated fragment size of 168 92 bp ( inferred by remapping the reads onto the assembly with maq software ) . the library was sequenced on three lanes of an illumina gaii at 76 bp read length producing a total of 6.72 gb q20 bases . the raw sequence data have been submitted to the european nucleotide archive ( ena : http://www.ebi.ac.uk/ena/ ) under accession : erp000894 . gene models were predicted using the ab initio gene finder augustus . based on a close phylogenetic relationship blast2go was also used to assign gene ontology ( go ) terms to all gene models . proteins that may be secreted via the nonclassical secretion pathway were located using the secretomep 2.0 server . a. mellea multigene families were located by performing an all against all blastp database search and the resulting hits were clustered into families using the mcl algorithm with an inflation value of 2 . numbers along branches represent bootstrap support values . two ascomycete species ( candida albicans and saccharomyces cerevisiae ) were chosen as outgroups . the initial genome assembly and predicted protein coding genes are available at http://www.armillariamellea.com . our representative genome species tree consisted of 23 basidiomycete species and 2 ascomycete outgroups ( supplementary table 1 , supporting information ) . genome data was obtained from the relevant sequencing centers ( supplementary table 1 , supporting information ) . in total , 8860 homologous gene families ( containing 4 or more genes ) were identified using a previously described randomized blastp approach . gene families were aligned using the multiple sequence alignment software muscle v3.7 with the default settings . misaligned or fast evolving regions of alignments were removed with gblocks . permutation tail probability ( ptp ) tests were performed on each alignment to ensure that the presence of evolutionary signal was better than random ( p < 0.05 ) . we found that 2924 gene families failed the ptp test , these were removed , leaving a data set of 5936 gene families . optimum models of protein evolution were selected using modelgenerator and these were used to reconstruct maximum likelihood phylogenies in phyml v3.0 . bootstrap resampling was performed 100 times on each alignment and majority rule consensus ( threshold of 50% ) trees were reconstructed . gene families were used to reconstruct supertrees using gene tree parsimony implemented in the software duptree version 1.48 . mycelial protein extraction was carried out at 4 c using two different methods . for 2-d page , mycelia were ground in liquid n2 and 1 g resuspended in 6 ml 10% ( w / v ) trichloroacetic acid ( tca ) ( 4 c ) and sonicated six times ( bandelin senoplus hd2200 sonicator , cycle 6 , 10 s , power 10% ) . samples were then centrifuged at 20200 g for 10 min ( 4 c ) , followed by washing in ice cold acetone , twice . pellets were finally resuspended in ief buffer ( 10 mm tris , 8 m urea , 2 m thiourea , 4% ( w / v ) chaps , 1% ( v / v ) triton x-100 , 65 mm dithiothreitol ( dtt ) and 0.8% ( w / v ) ipg buffer ) , centrifuged at 14000 g for 5 min ( 4 c ) and supernatants used for further analysis . proteins were quantified using the bradford assay and 300 g were used for ief using 13 cm strips ( ph range 310 ; ipgphorii ; ge healthcare ) ( step : 50 v 12 h ; step : 250 v 0.25 h ; gradient to 5000 v 2 h ; step : 5000 v 5 h ; gradient to 8000 v 2 h ; step : 8000 v 1 h ) followed by sds - page separation ( 60 v , 30 min ; 120 v , 1.5 h ) on protean plus dodeca cell ( bio - rad ) and colloidal coomassie blue staining . for shotgun analysis , 1 g of mycelial powder was resuspended in 6 ml of 25 mm tris - hcl , 6 m guanidine - hcl , 10 mm dtt , ph 8.6 and sonicated five times to generate whole cell lysates . dtt ( 1 m ; 10 l / ml lysate ) was added to supernatants and incubated at 56 c for 30 min . samples were cooled to room temperature and 55 l of 1 m iodoacetamide per milliliter of lysate was added followed by incubation ( in the dark ) for 20 min . samples were then dialyzed twice against cold 100 mm ammonium bicarbonate , ( 2 50 vol . ) , for at least 3 h with stirring . one - hundred microliters of denatured protein solution was transferred to an eppendorf tube and 5 l trypsin ( promega v5111 ) ( 0.4 g/l in 10% ( v / v ) acetonitrile in 10 mm ammonium bicarbonate ) was added . after overnight incubation at 37 c , the protein precipitates disappeared and protein digests were spin filtered ( agilent technologies , 0.22 m cellulose acetate ) prior to lc ms / ms analysis . for extracellular protein isolation , a modified version of that described by fragner et al . was used . culture supernatants ( 40 ml ) were frozen 20 c , thawed , centrifuged at 48000 g for 40 min , transferred to clean tubes and frozen in liquid n2 . samples were lyophilized to 4 ml , brought to 10% ( w / v ) tca and centrifuged 10000 g for 5 min before washing with ice cold acetone ( 3 times ) with centrifugation steps of 10000 g for 5 min . protein pellets were solubilized with 100 l of 10% ( w / v ) sodium dodecyl sulfate ( sds ) , 0.1 m dtt and 5 l of sds - page solubilization buffer and boiled for 10 min prior to sds - page . for protein extraction of fungal cultures grown on solid phase , mea plates ( 25 ml ) were briefly ground with a pestle , transferred to containers and brought to 50 ml with 50 mm kh2po4 ph 7.0 . samples were agitated overnight at 4 c , after which liquid was removed by gravity filtration through miracloth . polysaccharides were removed by centrifugation at 48000 g for 30 min , and supernatants were precipitated with 10% ( w / v ) tca under agitation overnight at 4 c . samples were centrifuged at 1700 g for 40 min , pellets were washed ( 3 times ) with 20% ( v / v ) 50 mm tris - hcl in ice cold acetone followed by washing in pure ice - cold acetone before air drying and storage 20 c . proteins were digested overnight at 37 c with 50 l of trypsin ( 65 ng/l in 10% ( v / v ) acetonitrile in 10 mm ammonium bicarbonate ) . gel digestions ( sds - page bands and 2-d page spots ) were carried out according shevchenko and colleagues with little modification . spots were excised and destained with 10% ( v / v ) acetonitrile in 100 mm ammonium bicarbonate . gel pieces were shrunk with 500 l of acetonitrile and digested with 50 l of trypsin ( 13 ng/l ) at 37 c overnight before filtration and lc ms / ms analysis . an agilent 6340 ion trap mass spectrometer running chemstation 0.01.03-sr2 ( 204 ) and a protiid - chip150 c18 150 mm ( g424062006 ) chip was used for peptide separation from in - gel protein digests , while a 160 nl 150 mm c18 chip ( g424062010 ) was used for tryptic peptides prepared by total protein digestion . solvent a : 0.1% ( v / v ) formic acid in water and solvent b : 0.1% ( v / v ) formic acid in 90% ( v / v ) acetonitrile / 10% ( v / v ) water . for protein identification following sds- or 2-d page , a flow rate of 0.6 l / min with a linear gradient of 5 to 70% solvent b over 7 min followed by 1 min increase to 100 % solvent b for 1 min and decrease to 5% solvent b for proteomic studies , a flow rate of 0.4 l / min with non - linear acetonitrile / water + 0.1% ( v / v ) formic acid gradients , optimised on a sample - specific basis from initial chromatographic analyses , were used . for shotgun proteomic studies , a flow rate of 0.4 l / min with a nonlinear acetonitrile / water + 0.1% ( v / v ) formic acid gradient was optimized on a sample - specific basis from initial chromatographic analyses . gradients ranging from 20 to 180 min were optimized for identification of the maximum number of proteins per sample : peptide identification from protein spots or bands required 20 min gradients , while shotgun ( complex mixtures ) gradients were generally up to 180 min duration . all lc ms spectra were analyzed using spectrum mill software ( rev a.03.03.084 sr4 ; agilent technologies ) to identify proteins against the translated a. mellea cdna database . ms / ms searches were carried out with the following settings : enzymatic trypsin digestion , carbamidomethylation as fixed modification and two variable modifications were set : deamination and oxidation of methionine . the minimum scored peak intensity was set to 70 , with precursor mass tolerance 2.5 da and product mass tolerance 0.7 . the maximum ambiguous charge was set to 3 with a sequence tag length > 3 and minimum detected peaks set to 4 . the genome of a. mellea ( vahl : fr ) kummer dsm 3731 was sequenced using an illumina genome analyzer ii . a total of 6.72 gb of sequence data was obtained from 3 single read lanes . on the basis of nuclear dna measurements by rflp analysis , this represents 57.25-fold coverage of the a. mellea genome . assembly of the illumina reads with the velvet assembler resulted in 58.35 mb of sequence data in 4377 scaffolds with an n50 size of 36.7 kb ( table 1 ) . the overall gc content of the genome is 49.1% , while in the coding regions it is 53.2% . a total of 14473 putative a. mellea open reading frames ( orfs ) were predicted ( table 2 ) which was subsequently used as the reference database for proteomic analyses . the average gene length was found to be 1575 bp , similar to what is observed in close relatives of a. mellea , such as l. bicolor and coprinopsis cinerea ( figure 1 and table 2 ) . the average exon / intron lengths were also comparable among these species , as were the average number of introns per gene ( 4.72 , 4.44 and 4.66 , respectively ) . blast2go successfully assigned a go term to 9514 ( 65.7% ) of the predicted genes ( data not shown ) . the signalp webserver identified 1903 proteins with secretion signal peptides , while phobius predicted 4035 proteins ( 27.8% ) with membrane regions , 2746 ( 19% ) with transmembrane regions and 246 with transmembrane regions plus signal peptide regions . homology searches followed by markovian clustering inferred that 9939 ( 68.7% ) of the a. mellea orfs belonged to multigene families . using the same approach we calculated that 68.5% of l. bicolor genes belong to multigene families . approximately 18.8% ( 2721 ) of the predicted a. mellea genes have no significant homologues in the ncbi database , a figure consistent with other newly sequenced basidiomycete genomes . we also successfully located 240 trna genes which compares favorably to l. bicolor which has 297 trna genes . given the paucity of a. mellea proteins curated and present in genbank ( n = 16 ) , it was necessary to interrogate the translated version of the a. mellea cdna database with peptide sequence data for complete and accurate de novo protein identification . as a consequence , a total of 921 proteins were identified in both mycelia and culture supernatants from a. mellea grown under a range of different culture conditions . these identifications were based on the detection of 3641 unique peptides , 2839 of which came from mycelia and 802 from the supernatant , respectively , with 197 common to both ( supplementary tables 2 and 3 , supporting information ) . mycelial proteins ( n = 739 ) were identified by using the a. mellea database obtained from the translation of the protein - coding genes . six - hundred twenty - nine proteins were uniquely identified in mycelia ( supplementary table 2 , supporting information ) , while others were also identified in the a. mellea secretome ( supplementary tables 3 and 4 , supporting information ) . of the mycelia - specific proteins , 509 were uniquely identified by a shotgun approach , with 98 and 9 proteins identified following prior 2-d page and sds - page fractionation , respectively ( supplementary figure 1 , supporting information ) . supplementary table 2 , supporting information , shows the actual peptide sequence with corresponding spectrum mill scores where values > 11 were deemed significant according to manufacturer s guidelines . sequence coverage ranged from 1 to 5% , 1 to 81% and 1 to 72% , respectively , using sds - page , 2-d page fractionation and shotgun analysis . functional analysis undertaken with blast2go and database searches classified proteins according to the biological process ( bp ) in which they are involved , the molecular function ( mf ) and the cellular component ( cc ) . the largest categories were catabolic processes ( n = 114 ) followed by translation ( n = 88 ) , carbohydrate metabolic processes ( n = 81 ) , precursor metabolites and energy ( n = 56 ) , response to stress ( n = 22 ) and secondary metabolic processes ( n = 19 ) ( figure 2a ) . mf of mycelial proteins revealed nucleotide binding ( n = 150 ) and structural molecule activity ( n = 70 ) as the largest categories , accounting for 53% of mycelial proteins identified . go identified 38 proteins in mf as having peptidase activity ( figure 2b ) . mycelial proteins cc mainly comprised ribosomal proteins ( n = 78 ) and protein complexes ( n = 77 ) . there were also 23 proteins classified as mitochondrial ( figure 2c ) and 14 proteins that have not been previously annotated and appear to be unique to a. mellea . one - hundred two proteins were detected that were previously reported as hypothetical or predicted ( table 3 ) . as expression of these proteins is now demonstrated , they can be reclassified as unknown function proteins . overall , 114 known domains were assigned to 55 proteins ( table 3 ) . eight proteins ( am15909 , am6889 , am18190 , am14843 , am14848 , am14568 , am13925 and am17640 ) were found to contain a nad(p)-binding domain , however no other domain was over - represented in our analysis . moreover , no go function was identifiable for 73 proteins , while 26 showed no blast description . among these are proteins with conserved domains which indicate a putative process , function or cellular location such as am12246 containing a start domain typical of proteins which transport ubiquinone during respiration or am14390 containing a 60 residue conserved bsd domain of unknown function usually located in btf2-like transcription factors , synapse - associated proteins and dos2-like . blast2go annotation of mycelial protein classification : ( a ) biological process ; ( b ) molecular function ; ( c ) cellular component . the total number of proteins in each category , as well as the overall % , are given for each subdivision ( no . , % ) . gravy score , grand average of hydropathy ( negative score indicates hydrophilicity while positive score indcates hydrophobicity ) . ms / ms analysis , 2-d proteins separated in two dimensions prior to lc source : m , mycelia ; sn , culture supernatant / secretome . regarding 2-d page analysis , a reference 2-d page map is shown in figure 3 and information ( pi and mr ) on proteins identified can be found in supplementary table 2 ( as 2d under column method , supporting information ) . possible protein isoforms were identified in different spots ( e.g. , spots 89 , 92 , 107 ( am10779 ) and 47 , 122 , 124 , 128 , 130 ( am16991 ) . one protein , am17600 , was identified in 17 spots ( nos . 1 , 3 , 11 , 58 , 88 , 89 , 90 , 97 , 103 , 104 , 107 , 117 , 118 , 119 , 131 , 132 , 133 ) probably due to a protease activity or degrading events . a total of 293 proteins were identified in culture supernatants derived from a. mellea , 183 of which were uniquely detected in the secretome , while 111 were also identified in mycelia ( supplementary table 3 , supporting information ) . to overcome the considerable difficulty of precipitating low abundance proteins from liquid culture , a. mellea was grown on agar , which was subsequently gently macerated and the liquid phase subject to tca precipitation followed by sds - page fractionation . a nonredundant protein search of the translated a. mellea gene set , identified 226 proteins . analysis of lc ms / ms data identified 157 unique proteins , while 37 were uniquely identified by a shotgun strategy without sds - page fractionation . sequence coverage ranged from 1 to 55% and 1 to 46% following sds - page and the shotgun approach , respectively , in the case of supernatant protein analyses ( supplementary table 3 , supporting information ) . eight proteins were identified in culture supernatants which were a. mellea - specific and included : am1874 , am2320 , am5632 , am9253 , am11032 , am11414 , am13545 and am13611 . ips revealed low - complexity segments in am11414 , am2320 , am5632 and am9253 . analysis revealed two sigp hits ( am11032 and am13611 ) , three secp signals ( am11414 , am13545 and am1874 ) and a tmhmm search predicted am11032 to be a hydrophobic membrane protein . the proportions of proteins involved in catabolic processes ( n = 29 ) and carbohydrate metabolism ( n = 38 ) was greater ( 55% ) than in mycelia ( 33% ) ( figure 4a ) . go analysis related to the molecular function of supernatant proteins showed peptidase activity as the largest functional category ( n = 15 ) followed by nucleotide binding proteins ( n = 14 ) . the class including carbohydrate binding proteins represented 14% in the secretome , yet in mycelia it accounted for only 1% . proteins classified as having antioxidant activity represent 11% of the secretome , while only 3% of mycelial proteins were similarly classified ( figures 2b , 4b ) . concerning go annotation of cc , the largest categories in the secretome comprised protein complexes ( n = 11 ) , and extracellular proteins ( figure 4c ) . ( a ) biological process ; ( b ) molecular function ; ( c ) cellular component . the total number of proteins in each category , as well as the overall % , are given for each subdivision ( no . , % ) . signalp analysis predicted signal peptide presence for 12% ( n = 90 ) and 50.2% ( n = 147 ) of mycelial and secretome proteins , respectively . furthermore secretomep indicated that 99 secreted proteins ( 34% ) contained a nonclassical secretion signal meaning that less than 20% ( 55 ) of those proteins do not contain a secretion signal . gravy and phobius evaluation of hydrophobicity and transmembrane domain presence indicated that 12.7% ( n = 93 ) of mycelial proteins were hydrophobic in nature , 10.5% ( n = 77 ) contained transmembrane regions , and 1.4% ( n = 10 ) contained both a transmembrane region and signal peptide . six proteins ( am9118 , 13744 , 6597 , 5593 , 17215 and 19909 ) contained greater than 10 transmembrane regions , while 71 proteins contained between 1 and 8 transmembrane domains . with respect to supernatant proteins , 26.3% ( n = 77 ) were hydrophobic in nature , 29 contained predicted transmembrane domains , and 9 of these were also predicted to possess signal sequences . although basidiomycetes have been considered as sources of glycoside hydrolases ( gh ) , with potential applications in recalcitrant carbohydrate digestion , no data whatsoever exists on gh complement in a. mellea . here , we reveal the presence of 226 gh genes in a. mellea ( supplementary table 5 , supporting information ) , and further demonstrate the expression of 52 of these at the protein level , in culture supernatants , under a range of culture conditions ( table 4 ) . specifically , gh genes belonging to 43 families are present ( supplementary table 5 , supporting information ) . the number of a. mellea gh genes is greater than that identified in either p. chrysosporium ( n = 177 ) or ceriporiopsis subvermispora ( n = 171 ) , both of which are white rot fungi , a finding that may be related to the pathogenic nature of a. mellea . gravy score , grand average of hydropathy ( negative score indicates hydrophilicity while positive score indcates hydrophobicity ) . ms / ms analysis , 2-d proteins separated in two dimensions prior to lc the a. mellea genome encodes 16 xyloglucan : xyloglucosyltransferases ( gh16 family , supplementary table 5 , supporting information ) . xyloglucan , a hemicellulose , is a major component of plant cell walls whereby its cellulose binding capacity provides structural support for the cell wall . gh 16 family enzymes cleave the 14 glycosidic backbone of xyloglucan and transfer the segment either to another xyloglucan or to a xyloglucan oligosaccharide ( xgo ) , thus restructuring and modifying the primary cell wall . xgos have been shown to activate plant cellulases and promote plant growth , but plant xgos do not activate fungal cellulase . two enzymes ( am17743 and am19909 ) in this class were also implicated in gtpase activity , which may indicate a signaling role . other gh families with increased or decreased representation in the a. mellea genome compared to lignolytic and cellulolytic fungi are reported in table 5 . contrary to expectation that lignolytic fungi would have similar frequencies of particular gh family proteins , a. mellea appears to contain more gh28 , gh61 , and gh76 family members than the other white or brown rot fungi ( table 5 ) . for example , 17 gh28 genes ( endo / exo-(rhamno)galacturonases , essential for pectin degradation ) are present in the a. mellea genome ( supplementary table 5 , supporting information ) in comparison to the 6 identified in l. bicolor and the 4 present in p. chrysosporium . moreover , gh109 , an -n - acetylgalactosaminidase present in a. mellea and the mycorrhizial l. bicolor ( supplementary table 5 , supporting information ) , has previously been also identified in prokaryotes and its crystal structure defined . chitinases , gh18 ( endo--n - acetylglucosaminidases ) ( n = 11 ) , are also encoded within the genome , and the expression of two of these was detectable in a. mellea culture supernatants ( am17468 and am17470 ) ( supplementary table 3 , supporting information ) . in addition to glycoside hydrolases , 72 glycosyltransferase and 19 polysaccharide lyases have been identified ( supplementary table 5 , supporting information ) . besides glycoside hydrolases , other cbm enzymes were discovered in the a. mellea genome . cbm proteins exhibit three functions encompassing ( i ) carbohydrate binding and concentration of enzymes on the substrate , ( ii ) binding to the polysaccharides that are the substrate targets of the catalytic moiety of the enzyme and ( iii ) substrate degradation . three cbm proteins were identified ( am18408 , am9123 and am11736 ) by lc ms / ms analysis , belonging to families 1 , 12 and 13 , respectively ( table 4 ) . cbm12 previously only described in bacteria , comprises 4060 residues , binds chitin , which is unusual among cbm proteins which have broad polysaccharide specificity . the c - terminus of the chitin binding domain ( chbd ) binds very specifically to insoluble chitin but not to chitin oligosaccharides or glucose . cbm13 is a 150 residue module that has been identified in galactose - binding plant lectins . predominantly found in bacteria , they have also been identified in eukaryotes , including some fungi ; cbm13 modules occur in glycoside hydrolases and glycosyltransferases and also bind xylan . compared to some basidiomycete and ascomycete species , a. mellea has increased representation of carbohydrate esterase ( ce ) families ( supplementary table 5 , supporting information ) , specifically ce4 , ce8 , ce9 and ce14 comprised 94 genes . ce1 is an acetyl xylan esterase ( axe ) which depolymerises xylan microfibrils and solubilizes lignocellulosic material and releases fermentable sugars . a ce1 enzyme ( am19807 ) was identified at the protein level in the growth condition we tested ( supplementary table 2 , supporting information ) . the ce4 family exhibits acetyl xylan , chitin , chitooligosaccharide and peptidoglycan deacetylase activity . four ce4 enzymes ( am18938 , am3987 , am3988 , and am6064 ) have been identified in a. mellea culture in both mycelia and secretome ( supplementary tables 2 and 3 , supporting information ) . these enzymes act on n - acetylglucosamine ( glcnac ) which is present in bacterial as well as fungal cell walls ; have very broad substrate specificity and catalyze the hydrolysis of acetyl groups from xylan and carbon chitin deacetylases ( cda ) are secreted during hyphal penetration of plants by colletotrichum lindemuthianum . tsigos and bouriotis hypothesized that chitin of invading pathogens triggers recognition by plant defenses inducing plant chitinase activity which gives rise to formation of lignin and callus . deacetylases modify plant chitinases thereby neutralizing plant defenses , and modify chitin to chitosan which plant deacetylases are unable to modify , thus enabling the fungus to invade the plant . pectin methylesterase ( pme ) are members of the ce8 family and catalyze the demethyloxidation of pectin , a major plant cell wall polysaccharide . pmes are important in vegetative and reproductive plant development , but pathogenic fungi can also degrade pectin in the cell wall during plant invasion . ce9 ( n - acetylglucosamine 6-phosphate deacetylase ) is another gene family detected in a. mellea which is involved in carbohydrate , specifically n - acetyglucosamine , catabolism . surprisingly , a glcnac-6-phosphate deacetylase ( dac1 ) has been linked to c. albicans virulence . the ce14 deacetylase family has a conserved lmbe domain which catalyzes the n - deacetylation of appropriate substrates . tanaka et al . identified a deacetylase from thermococcus kodakarensis kod1 which degrades diacetylchitobiose ( glcnac2 ) to glcn the a. mellea genome encodes 6 laccases ( am380 , am12382 , am16732 , am16804 , am17018 and am19478 ) ( figure 5 ) , all of which contain the canonical l1-l4 signature sequences , characteristic of this multicopper oxidase ( mco ) family of enzymes . overall , 15 laccase - like enzymes are encoded within the a. mellea genome . ms / ms analysis ( supplementary tables 2 and 3 , supporting information ) , while expression of any other laccase was undetectable under the condition tested . expression of two laccase - like enzymes ( lcc5 : am2859 and am4666 ) was also evident in the a. mellea secretome ( supplementary table 3 , supporting information ) . notably , the genome of the white rot fungus p. chrysosporium does not encode any laccase , while those of c. subvermispora(51 ) and l. bicolor(69 ) encode 7 and 9 laccase gene models , respectively . one - hundred bootstrap replicates were applied with the appropriate protein model using the software program phyml ( v3.0 ) and summarized using the majority - rule consensus method . monophyletic laccases , canonical ferroxidases ( fet3 ) and other multicopper oxidases ( mco ) are highlighted . basidiomycete peroxidases are a class of oxidoreductase involved in lignin biodegradation in white rot fungi , ligninolytic haem peroxidases have been shown to cleave the main linkage types in lignin due to their high redox - potential and specialized catalytic mechanisms . to date , three ligninolytic haem peroxidases have been isolated and characterized in white - rot fungal species . these include lignin peroxidase ( lip ) , characterized by its ability to oxidize high redox - potential compounds such as alcohol and methoxybenzenes and veratryl , manganese peroxidase ( mnp ) which exhibits an ability to oxidize mn to mn and versatile peroxidase ( vp ) which can oxidize both lip and mnp substrates . our genome analysis and subsequent homology modeling infers that a. mellea contains nine putative ligninolytic peroxidases . in an attempt to elucidate the function of the a. mellea peroxidases we reconstructed a phylogenetic tree using representative mnp , lip and vp sequences from c. subvermispora and p. chrysosporium . our phylogeny infers that the a. mellea peroxidases are the result of a species - specific expansion as they are clustered together in a monophyletic clade ( figure 6 ) . interestingly the a. mellea - specific clade is clustered beside c. subvermispora mnp13 , and this gene has previously been described as a short mnp . the length of the a. mellea genes is comparable to typical mnps however ( not shown ) . the phylogeny also infers that the a. mellea genes are more closely related to p. chrysosporium lip genes than the typical c. subvermispora mnp genes ( figure 6 ) . however , close sequence inspection shows that the a. mellea peroxidases do not contain the conserved aromatic substrate oxidation sites required for lip enzymatic activity . furthermore , five of the nine a. mellea peroxidases contain the three acidic residues ( two glutamates and an aspartate ) required for the mn binding site of mnps . based on this observation we hypothesize that at least five of the peroxidases found in a. mellea have mnp activity . one hundred bootstrap replicates were used with the appropriate protein model using the software program phyml ( v3.0 ) and summarized using the majority - rule consensus method . comparative growth studies of c. albicans recovered from either mono- or coculture conditions demonstrates that significant killing ( p = 0.0004 ) of c. albicans by a. mellea occurs in cocultures ( figure 7 ) . this observation can be associated with the detection of 30 a. mellea proteins , not identified under any other culture condition ( table 6 ) including redox - active , degradative and attack - type proteins . interestingly , cys-2 peroxiredoxin ( am10593 ) and thioredoxin ( am7929 ) were both uniquely detected in fungal cocultures . cys-2 peroxiredoxin converts peroxides to water , with concomitant formation of intramolecular disulfide linkages . these disulfide linkages are in turn reduced by thioredoxin to regenerate the active form of cys-2 peroxiredoxin . we speculate that this system may be induced in a. mellea as an antifungal response to c. albicans presence . alternatively , peroxide dismutation may be required to attenuate products originating from c. albicans cell wall degradation . in this regard , it is notable that a class ii chitinase ( am13890 ) was also uniquely detected in fungal cocultures which may be deployed by a. mellea to degrade candida cell wall material . two proteins classified as prohibitins ( termed am14001 and am20304 ) , which have been proposed to be involved in the negative regulation of cell proliferation , have also been identified elsewhere . these serine - threonine - rich membrane proteins are anchored by glycosylphosphatidylinositol ligands . several gpi - anchored proteins have been characterized in fungi as having multiple functions including cell wall organization in a. fumigatus , fruiting body formation in lentinus edodes , and interaction with mapk , where they may have a signaling role . am3423 belongs to the same protein family of drmip , hesp-379 , which is a haustorially expressed secreted protein involved in host tissue penetration in l. edodes . in addition , a number of oxidoreductases , monooxygenases , reductases and dehydrogenases of basidiomycete origin were detected in the fungal cocultures ( table 6 ) . similarly , metabolite extracts from a. tabescens have also been observed to exhibit antifungal activity . apart from demonstrating the power of proteomic investigations to dissect fungal fungal interactions , the coculture system we have developed for a. mellea and c. albicans may represent a model system for the further investigations of a. mellea pathogenicity . ( a ) culture plates : ( i ) c. albicans only ; ( ii ) coculture of c. albicans and a. mellea for up to 52 days . reculture of c. albicans was carried out from multiple plugs taken from inoculation points ( arrows ) . ( b ) comparative viable cell count of recultured c. albicans from single and coculture plates shows that coculture with a. mellea results in significant ( p = 0.0004 ) killing of c. albicans . simultaneous fda ( live ) and pi ( dead ) cell staining of c. albicans . 1 . c. albicans live culture ( 24 h ; positive control ) ; 2 . monoculture of c. albicans and 4 . c. albicans following coculture with a. mellea ( a. ) . ms / ms analysis , 2-d proteins separated in two dimensions prior to lc ms / ms has , for the first time , revealed new insights into the proteome of the important fungal plant pathogen armillaria mellea , including the identification of multiple glycodegradative enzyme systems which can be useful for recalcitrant carbon compound degradation in biotechnology applications . moreover , proteomics tools shed light on potent antifungal properties of a. mellea , which in part may be mediated by basidiomycete enzymes .

armillaria mellea is a major plant pathogen . yet , no large - scale -omics data are available to enable new studies , and limited experimental models are available to investigate basidiomycete pathogenicity . here we reveal that the a. mellea genome comprises 58.35 mb , contains 14473 gene models , of average length 1575 bp ( 4.72 introns / gene ) . tandem mass spectrometry identified 921 mycelial ( n = 629 unique ) and secreted ( n = 183 unique ) proteins . almost 100 mycelial proteins were either species - specific or previously unidentified at the protein level . a number of proteins ( n = 111 ) was detected in both mycelia and culture supernatant extracts . signal sequence occurrence was 4-fold greater for secreted ( 50.2% ) compared to mycelial ( 12% ) proteins . analyses revealed a rich reservoir of carbohydrate degrading enzymes , laccases , and lignin peroxidases in the a. mellea proteome , reminiscent of both basidiomycete and ascomycete glycodegradative arsenals . we discovered that a. mellea exhibits a specific killing effect against candida albicans during coculture . proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive a. mellea proteins ( n = 30 ) . overall , our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms .

Introduction Experimental Procedures Results and Discussion Conclusions

PMC4210398

clinical diagnosis of fibromyalgia syndrome ( fms ) is based on many accompanying subjective symptoms such as fatigue , sleep disorders , stiffness , irritable bowel syndrome , depression , and anxiety , in addition to chronic widespread musculoskeletal pain1 . according to the american college of rheumatology ( acr ) 1990 classification criteria , fms is defined as > 3 months of continuous tenderness determined by a finger pressure of around 4 kg at at least 11 out of 18 predefined sites and widespread musculoskeletal pain2 . recently , new acr diagnostic criteria including fatigue , non - restorative sleep , abdominal discomfort and cognitive symptoms have been proposed3 , 4 . although it is reported that a multifactorial condition with various biological and psychosocial factors may play a role in the initiation and maintenance of pain , the etiopathogenesis of pain is not yet fully understood5 , 6 . it is reported that the available treatment modalities provide relief of symptoms in less than 50% of patients6 . aerobic exercise programs have been shown to improve the physical capacity of patients with fms1 . additionally , it has been suggested that aerobic exercise has modest effects on some symptoms of fms and physical function6 , 7 . researchers have investigated the effectiveness of various exercises programs including bicycle ergometer , side step in the pool , jogging , walking outdoors and walking on a treadmill6 , 8 . some studies have reported that aerobic exercise has positive effects on pain , disability and mood8 , 9 . in a meta - analysis , it was mentioned that the effectiveness of aerobic and strengthening exercises on general well - being in fms is contradictory . in the assessment of functional condition , the positive effects of exercise have been reported in some studies using the fibromyalgia impact questionnaire ( fiq ) , but the absence of a statistically significant difference has also been reported10 . although there might be many reasons behind these differing results , one possibility is the personality characteristics of the patients . it has been showed that the profiles of individuals with fms include difficulties regarding physical , functional and psychological status . additionally , it has been emphasized that odyophobia lies at the bottom of psychological and behavior responses in the subgroups comprised of individuals with fms and a self - management approach should be included in the treatment for chronic pain11 . according to the biopsychosocial model of health , it has been reported that when education programs developing self - managements for individuals are supported by exercise programs , there are positive effects on physical function and fms symptoms12 . a previous study also reported that some personality parameters assessed by the minnesota multiphasic personality inventory ( mmpi ) differ with pain management13 . besides fms is a disease characterized by widespread pain and tender points , therefore it is important to consider the psychological aspect of fms14 . it has been reported that it is necessary to focus on dysfunction in both physical and emotional aspects of treatment together9 . evaluation of personality characteristics and the role of personality characteristics in treatment planning are important in fms . in this study , we aimed to evaluate the personality characteristics of patients with fms and to assess the pain , quality of sleep , physical function , and depression parameters after a 6-week home - based aerobic exercise program . eighteen female patients admitted to the physical medicine and rehabilitation department of bakirkoy dr . sadi konuk training and research hospital and diagnosed with fms according to 1990 acr classification criteria were included in this study . the exclusion criteria were as follows : psychotic disorder ; suicidal ideation ; presence of neurological disease ; use of neurological and psychiatric tricyclic antidepressant , antipsychotics , selective serotonin - norepinephrine reuptake inhibitors , or medicines similar to anticonvulsants ; presence of a systemic disease that would interfere with doing exercise ; pregnancy or abnormality in routine tests . sadi konuk training and research hospital and written informed consent was received from each subject prior to their participation . demographic characteristics , fms symptoms and the number of tender points of the patients were identified . pain and quality of sleep were assessed using visual analog scale ( vas ) . physical function was assessed with the fiq , depression was assessed with the beck depression inventory ( bdi ) , and personality characteristics were assessed with the mmpi . the structured clinical interview for dsm - iv axis i disorders ( scid - i ) was used to determine whether the patients had dsm - iv axis i disorders or not . a home - based exercise program was performed three times a week for 6 weeks . each exercise session lasted 60 minutes and consisted of a warming - up period of 10 minutes , moderate intensity aerobic exercises period of 40 minutes and a cooling down period of 10 minutes . the training program included exercises for strength , endurance , and elasticity , and stretching of both the upper and lower extremities . the intensity of exercise was performed at a heart rate ( hr ) of between 60% and 80% of the maximal heart rate , which was calculated by karvonen formula ( target hr= ( ( 220-age)-resting hr ) 0.60 + resting hr5 ) . the training program was checked by a physical therapy and rehabilitation specialist every 2 weeks . in this study , we assessed whether there were significant differences in the pain , quality of sleep , physical function and depression parameters of the patients with fms before and after the exercise program . additionally , features of the psychiatric diagnosis and personality profiles of the patients were evaluated . ncss ( number cruncher statistical system ) 2007 statistical software ( utah , usa ) program was used for the statistical analysis performed in this study . the paired t - test was used for comparisons of descriptive statistics ( mean , standard deviation ) as well as for comparisons of data before and after treatment . mcnemar s test was used for the comparison of qualitative data , and pearson s correlation test was used for the comparison of the correlations among the variables . four of 18 patients included in our study dropped out of the study because they did not want to maintain the exercise program . therefore , the data of only 14 patients with fms used in the analysis . the mean age of the patients with fms was 435.33 years ( minimum : 25 , maximum : 43 ) , and their mean body mass index ( bmi ) was 24.213.3 ( minimum : 17.99 , maximum : 30.11 ) the other demographic characteristics are shown in table 1table 1.demographic characteristics of the patients with fibromyalgia syndromevariablemean sdminimummaximumage ( year)35.43 5.332543bmi ( kg / m)24.21 3.317.9930.11number of children1.5 1.1603variable n%educationprimary school214.3secondary school321.4high school535.7university428.6marrial statussingle214.3married1178.6divorced17.1occupationhousewives857.14employees321.43public official17.14private sector214.9smokingyes321.4no1178.6bmi : body mass index , sd : standard deviation . bmi : body mass index , sd : standard deviation after exercise , the symptoms accompanying fms ( fatigue , morning stiffness , disturbance of sleep , morning fatigue , paresthesia , headache , irritable bowel syndrome and soft tissue edema ) were determined to be significantly lower than before exercise ( p<0.05 ) ( table 2table 2.comparison of accompanying fibromyalgia syndrome before and after exercisebefore exerciseafter exercisevariablen%n%fatigue*1392.9642.9morning stiffness*1392.9535.7sleep disorder*1285.7428.6morning tiredness*1392.9857.1paresthesia*1178.6321.4headache*1392.977raynaud s phenomenon00.000irritable bowel syndrome*750.017.1sicca symptoms321.417.1female urethral syndrome535.717.1soft tissue edema*1178.617.1dysmenorrhea857.1221.4*p<0.05 ) . the number of tender points , vas values for pain , and quality of sleep after exercise were found to be significantly lower than their respective values before exercise ( p<0.05 ) ( table 3table 3.comparison of clinical parameters before and after exercisevariablebefore exerciseafter exercisemean sdmean sdnumber of tender points by palpation*14.79 1.378.36 3,05vas pain*7.71 2.164.29 2.43vas sleep*6.14 2.983.64 2.68fiq physical function*5.21 1.544.34 2.47fiq job loss0 00 0fiq working5.4 3.443.8 0.84fiq pain*7.79 2.044.64 2.31fiq fatigue*8.21 1.536.71 2.81fsesf resting*9 1.246.71 2.81fiq stiffness*7.29 2.555.14 3.37fiq anxiety*7.93 2.165.5 2.9fiq depression*7.29 1.984.57 2.9total fiq*70.58 14.6450.57 20.53bdi*17.57 7.8210.64 7.52*p<0.05 , vas : visual analog scale , fiq : fibromyalgia impact questionnaire , bdi : beck depression inventory , sd : standard deviation ) . * p<0.05 , vas : visual analog scale , fiq : fibromyalgia impact questionnaire , bdi : beck depression inventory , sd : standard deviation in the patients with fms , mean values of physical function , feeling good , pain , fatigue , resting , stiffness , anxiety , depression parameters and total fiq scores after the exercise program , except the working parameter , ( p>0.05 ) were determined to be significantly improved compared to before the exercise program ( p<0.05 ) ( table 3 ) . mean bdi after exercise was found to be significantly lower than mean bdi before exercise ( p<0.05 ) ( table 3 ) . when scid - i was investigated , major depression , dysthymia or depressive disorder not otherwise specified were determined in 5 patients ( 35.7% ) . social phobia , generalized anxiety disorder , or obsessive compulsive disorder were determined in 6 patients ( 42.9% ) , and specific phobia in 3 patients ( 21.4% ) . anxiety disorder not otherwise specified was determined in 9 patients ( 64.3% ) , and somatoform disorders in 8 patients ( 57.1% ) . diagnosis of panic disorder , post trauma stress disorder , adjustment disorder or other dsm iv disorders were not seen in any patient . major depression , dysthymia or depressive disorder not otherwise specified according to scid - i lifetime values were determined in 13 patients ( 92.9% ) . obsessive compulsive disorder was determined in 6 patients ( 42.9% ) and anxiety disorder not otherwise specified in 9 patients ( 64.3% ) . adjustment disorder was determined in 4 patients ( 28.6% ) , social phobia or generalized anxiety disorder in 12 patients ( 85.7% ) and specific phobia in 7 patients ( 50% ) . somatoform disorders were determined in 8 patients ( 57.1% ) but post - traumatic stress disorder , panic disorder or other dsm iv disorders were not found in any patient . according to the mmpi personality inventory , only the hysteria subscale score ( 71.218.84 ) was determined to be above the significance level ( table 4table 4.assessment of the personality characteristics of the patients with fibromyalgia syndrome by the minnesota multiphasic personality inventorymmpimean sdminimummaximumlie55.43 11.374181frequency50.64 7.473865correction52 10.363772hypochondriasis63.86 6.625071depression58.57 10.974278hysteria71.21 8.845286psychotic deviation53.86 9.73573male - female48.21 10.283466paranoia51 9.663162psychasthenia56.93 9.474273schizophrenia52.86 7.613863hypomania48.43 7.63764social introversion58.64 8.714574mmpi : the minnesota multiphasic personality inventory , sd : standard deviation ) . non - pharmacological approaches to treatment of fms have gradually gained in importance in recent years . a physical exercise program is an important treatment option either alone or combined with the other treatment components in the treatment of fms due to its relatively ease of applicability , low risk and low cost10 , 15,16,17 . it has been reported that a home exercise program is effective at easing the level of pain and improving functional status18 . in addition researchers have suggested that active individual exercise programs improve muscle function19 . in the literature , the positive effects of aerobic exercise on pain , physical function and quality of life have been reported9 , 10 , 15 . a meta - analysis reported a significant improvement in the number of tender points by palpation after aerobic exercise alone or in combination with strengthening exercises , and its authors emphasized that improvement in the number of tender points through exercise supports the reduction of widespread pain and tenderness in female patients with fms20 . jones et al.21 reported positive effects of submaximal aerobic exercise on pain , fatigue and the quality of life . huser et al.16 reported positive effects of aerobic and mixed exercise on physical function . in contrast , king et al.22 concluded that aerobic exercise was ineffective in the treatment of physical function as assessed by fiq in the patients with fms . on the other hand , in the systematic review performed by busch et al.23 the positive effects of aerobic exercise on fms symptoms again emphasized the positive effects of aerobic exercise on pain , the number of tender points , physical function and fms symptoms , albeit with the caveat that more comprehensive studies of exercise prescription and compliance of patients with the exercise program were necessary24 . according to the results of our study , a significant improvement was seen in the symptoms accompanying fms , the number of tender points by palpation , vas score of pain and quality of sleep in the patients with fms after the home - based aerobic exercise program . according to the assessment of physical function by the fiq the symptoms of the patients with fms , and the type and levels of physical exercise affect the response to fms treatment9 . most authors have recommended gradually increasing the intensity of exercise and the start low and go slow approach and moderate intensity exercise programs are especially recommended . a reduction in post - exercise pain and fatigue complaints with these approaches suggest that this type of approach is most suitable9 , 25 . the authors of a previous study tried to develop a formula to calculate target heart rate for aerobic exercise program using a mathematical model . according to this model , the recommended exercise intensity for patients with fms is within the range of 5260% of heart rate reserve or 7585% of hr at the anaerobic threshold26 . in our present study , in addition to fms symptoms , and characteristics of physical exercise , the importance of psychological characteristics has also been mentioned in relation to fms treatment9 . the correlation between chronic pain and emotional , physical and social function was shown in two studies10 , 27 . in a meta - analysis , it was reported that aerobic exercise interventions had positive effects on depression16 . mannerkorpi et al.12 used the hospital depression scale to assess the psychological status before and after exercise and reported that there was a mild improvement in the scale . in our study , post - exercise mean bdi was significantly lower than the pre - exercise mean bdi . some studies have reported poor patient compliance with exercise regimes and high dropout rates28 , 29 . in our study , 4 patients dropped out because they did not want to continue the exercise program . although it was reported that the personality characteristics of patients with fms were heterogeneous in a review , studies performed on this subject have been very few . assessment tools and the characteristics of the patients included in studies might have contributed to these differences30 . in our study , we evaluated the personality characteristics of the patients with fms using the mmpi . according to the mmpi personality inventory the hysteria scale of the mmpi is a subset of individuals with higher scores who mainly use denial , who are childishly egoistic , and have anxiety - related somatic complaints . it is known that personality plays a major role in the etiology of psychosomatic disorders31 . taking account of this , multidisciplinary approaches should be planned to increase the participation of patients in an exercise program by evaluation of personality characteristics and psychological status of the patients before the exercise program . some studies have reported the effectiveness of home - based programs including exercise program . for example , in a controlled study , it was reported that targeted individual training increases the effectiveness of exercise in the treatment of fms34 . another study mentioned that frequent visits during a training program had significant effects on the psychosocial status of patients with fms35 . in our study , the 6-week home - based aerobic exercise programs of the patients with fms were checked by a physical therapy and rehabilitation specialist every two weeks . in a meta - analysis performed by huser et al.36 , results were assessed over a period of 115 months . moreover , the assessment was performed according to whether the total exercise period was shorter or longer than 30 hours . huser et al . emphasized that short - term and long - term aerobic exercise program had positive effects on physical function and depression but that multicomponent treatment programs are necessary to increase compliance with an exercise program during long - term home care . with the development of individually focused psychological strategies for patients with fms , it is generally accepted that maintaining compliance with an aerobic exercise program increases the success of the treatment . fms is a highly complicated syndrome and its etiopathogenesis is not yet well understood . however , many studies indicate that it is a neurobiological disease and emphasize its association with central sensitization , endocrine factors , sleep disorders , psychosocial , physical stress and physical trauma37 . it has also been reported that physical function , anxiety , pain , stiffness , general fatigue and morning tiredness of fms patients are significantly improved by psychosocial training33 . psychosocial training programs for personality profiles and the psychological status of the patients with fms can be included in aerobic exercise programs . in this study , we investigated the personality characteristics of the patients with fms while assessing the effectiveness of aerobic exercise . we wanted to confirm that aerobic exercise is effective in the treatment of fms , and that emotional status and personality characteristics should be considered during the planning process of the treatment for fms . we think that many more studies with a larger number of patients are required on this subject .

[ purpose ] the aim of this study was to assess the effectiveness of a 6-week aerobic exercise program on pain , physical function , and psychological status , and to evaluate the personality characteristics of fibromyalgia syndrome ( fms ) patients . [ subjects and methods ] fourteen women with fms were enrolled . they were trained for a 6-week home - based aerobic exercise program . the fibromyalgia impact questionnaire , the beck depression inventory , the visual analog scale of pain and sleep quality were measured at baseline and at the end of week 6 . the personality profiles were evaluated using the minnesota multiphasic personality inventory ( mmpi ) . [ results ] after the exercise program , significant improvements were determined in pain , sleep quality , physical function , depression and fms symptoms compared to baseline . in addition , the hysteria item ( 71.218.84 ) of the mmpi was significantly higher in fms . [ conclusion ] our findings indicate that home - based aerobic exercise may be a useful treatment in the management of fms . personality characteristics should be considered during the planning process of the treatment of fms . personality is a filter between life events and psychological responses . it is defined to be the integration of effective and behavioral patterns . long - term studies involving larger clinical samples are needed to define the role of personality characteristics in fms .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3057488

expression in e. coli , purification , and liposome reconstitution of clc - ec1 ( swiss - prot i d p37019 ) were performed as described14 , as were cl and h flux assays , except that we used lipid mixtures of egg phosphatidylcholine and 1-palmitoyl , 2-oleoyl phosphatidylyglycerol , 3/1 weight ratio . ion flux rates in these lipids are 510-fold lower than observed in the e. coli phospholipids that we customarily use . liposomes were formed at 20 mg / ml lipid , 1 g protein / mg lipid by dialysis or by centrifugation of 0.1 ml samples through 3-ml sephadex g-50 columns . clh exchange stoichiometry was determined as the ratio of initial transport rates16 , with cl efflux and h uptake recorded via cl and h electrodes using liposomes loaded with 300 mm kcl , 40 mm citrate - naoh ph 5.0 suspended in solutions of 1 mm kcl , 300 mm k - isethionate , 2 mm citrate - naoh ph 5.2 . for crystallography , clc protein ( 915 mg / ml ) in 100 mm nacl , ~40 mm dm,10 mm tris - hcl ph 7.5 was mixed with an equal volume of 100 mm lino3 , 4145% ( w / v ) peg400 , 100 mm glycine - naoh ph 9.5 , with ~10 mm 4-cyclohexyl-1-butyl--d - maltoside added to the 2 l drop . crystals grown by vapor diffusion in sitting drop trays for 24 weeks at 20c were frozen in liquid nitrogen , and data were collected remotely at beamline 8.2.1 , advanced light source eastern annex , waltham , mass . molecular replacement was done in phaser using residues 30450 of a single subunit of clc - ec1 as search model , and refinement was carried out in refmac5 .

channels and transporters of the clc family bring about transmembrane movement of inorganic anions in service of a variety of biological tasks , from the arcane - generating the kilowatt pulses by which electric fish parboil their prey - to the quotidian - acidification of endosomes , vacuoles , and lysosomes1 . the homodimeric architecture of clc proteins ( fig 1 ) , initially inferred from single - molecule studies of an elasmobranch cl channel2 and later confirmed by crystal structures of bacterial cl/h+ antiporters3,4 , appears to be universal . moreover , the basic machinery enabling ion movement through these proteins - the aqueous pores for anion diffusion in the channels and the ion - coupling chambers that coordinate cl and h+ antiport in the transporters - are contained wholly within each subunit of the homodimer . the near - normal function of a bacterial clc transporter strait - jacketed by covalent crosslinks across the dimer interface and the behavior of a concatameric human homologue argue that the transport cycle resides within each subunit and does not require rigid - body rearrangements between subunits5,6 . however , this evidence is only inferential , and since examples are known in which quaternary rearrangements of extramembrane clc domains that contribute to dimerization modulate transport activity7 , we can not declare as definitive a parallel pathways picture in which the homodimer consists of two single - subunit transporters operating independently . a strong prediction of such a view is that it should in principle be possible to obtain a monomeric clc . in this study , we exploit the known structure of a clc cl/h+ exchanger , clc - ec1 from e. coli , to design mutants that destabilize the dimer interface while preserving both structure and transport function of individual subunits . the results demonstrate that the clc subunit alone is the basic functional unit for transport and that cross - subunit interaction is not required for cl/h+ exchange in clc transporters .

Methods Summary Supplementary Material

PMC3437071

fever is the most appreciated manifestation of disease.1 although nonspecific , it may indicate the presence of a spectrum of illnesses ranging from a self - limiting viral syndrome to life - threatening bacterial disease of malignancy.2 fever is one of the most frequent complaints encountered in an emergency department . people identify fever as a sign of illness more readily than they recognize the importance of most other symptoms . patients come to the emergency department ( ed ) because they think they are sick and feverish . if they feel that they are sick but not feverish , they may not come to ed . in addition to causing concern , the presence of fever usually raises high therapeutic expectations.3 medical staff may over or underestimate the patient 's complaint of fever . overestimation of subjective fever by medical staff , even if not proven on examination , may influence them to order unnecessary investigations or medications.4 as some of the endemic diseases such as malaria , brucellosis and tuberculosis ( tb ) prevalent in our area may present early as intermittent fever , the patient may feel feverish at night or at some point but be afebrile on presentation at the ed . fever in elderly persons even in the absence of other manifestations of disease , may indicate the presence of serious infection , most often caused by bacteria or a non - infectious disease ; such as connective tissue disorders or a malignancy.5 subjective fever may , therefore , be an important clue for further evaluation of the patient . it is therefore important that the patients ability to detect the presence or absence of fever be better characterized . since the reliability of patients in saudi arabia to predict absence or presence of fever has not been studied before , the objective of this study is to assess the reliability of subjective fever in triage of adult patients in the ed and to compare their assessment with temperature measurement on the ivac digital machine . a prospective study was carried out over a three - week period on all ambulatory adult patients ( above 12 years of age ) of both genders , attending a triage clinic in the ed of king fahd hospital of the university in al - khobar , saudi arabia . injured and trauma patients , those with a respiratory rate above 30 per minute , those with altered mental status and all patients attending obstetric and gynecology triage room were excluded from the study . the physician in the triage room asked each patient whether or not he or she had fever or felt feverish before oral temperature was taken with the ivac digital machine . two sets of temperature readings were taken to define fever as 37.8c or greater and 38.0c or greater . the clinical diagnosis of the patient 's complaints was also recorded and grouped by gender for 996 patients . a single standard 22 table was constructed according to the patient 's subjective sensation of fever and the measured oral temperature . sensitivity , specificity and positive and negative predictive values with 95% confidence intervals ( 95% ci ) were calculated . data was entered into a personal computer using spss / pc version 6.0 statistical package . the method of chi - square was applied to calculate significant and non - significant values . the total number of adult patients in this study was 1241 patients , 540 ( 43.5% ) males and 701 ( 56.5% ) females . the mean age ( sd ) of males was 30.75 14.1 years and the mean age of females was , 32.9 13.6 years . the sensitivity of male and female patient detection of fever by subjective means was 89.6% and 90.0% respectively , while the specificity was 94.5% and 94.8% respectively ( table 1 ) . sensitivity and specificity of adult patients in subjectively predicting oral temperature increase sensitivity , specificity , positive predictive value , negative predictive value and accuracy rate of ability of adult patients to subjectively predict objective oral temperature increase the positive predictive value ( ppv ) and negative predictive value ( npv ) with 95% ci for male patients was 69.8% ( 63.5 - 76.1 ) and 98.5% ( 97.5 99.6 ) and for the female patients was 52.1% ( 36.8 67.4 ) and 99.4% ( 98.6 99.9 ) respectively . the prevalence of objective fever was 8.7% yielding general positive and negative predictive values of 80.9% and 98.9% with an accuracy rate of 94.2% ( table 2 ) . other calculations were made when fever was defined as a temperature of 37.8c or above . table 2 shows sensitivity , specificity , positive and negative predictive values and accuracy rate in subjectively predicting objective oral temperature of 37.8c or greater in comparison with temperature of 38.0c or greater . the normal body core temperature ranges between 36.2c and 37.8c and is influenced by several factors : age ( infants less than one year have higher baseline temperature ) , times of day ( temperature generally elevates in the afternoon ) , exercise , metabolic rate , environmental temperature and bundling . fever is generally , albeit arbitrarily , considered as a rectal temperature of 38.0c or greater.2 usually , documentation of actual recording of elevated temperature is required before a patient is considered feverish and further action taken . nevertheless , a history of subjective fever may influence the differential diagnosis and the plan of action drawn by the treating physicians . in this study , the reliability of our adult patients attending triage clinic in assessing subjective fever was tested . their ability to predict the presence or absence of an objective fever of 38.0c or greater the ability of our patients to predict the presence or absence of an objective fever seems to be better than the us patients studied by buckley.4 the sensitivity and specificity in us patients were 83% each , while in our study , the sensitivity and specificity were 89.9% and 94.7% respectively . the denials of subjective fever by the patient in our study and buckley 's study4 were almost the same , where it was highly predictive of not having an objective increase in oral temperature ( npv 98.9% and 99.0% respectively ) . on the other hand , the ppv of the complaints of subjective fever was only 25.0% in buckley's4 study compared to 80.9% in our study . this means , four out of five of our patients who believed they had a fever were actually found to have objective temperature increase , compared to one out of four patients in buckley's4 study . medical staff should therefore , take the complaint of subjective fever in our population more seriously . the results of our study showed that the ability of females to predict presence or absence of subjective fever ( 38c or greater ) is higher than males ( 92.1% for females and 69.8% for males ) . this could be explained by the fact that women make better nurses then men . on the other hand , further investigation may be required on this difference . when the set of definition of an objective fever is put at 37.8c or above , the sensitivity and ppv of the ability of the patient to predict presence or absence of fever drops from 89.9% and 80.9% to 81.4% and 73.8% respectively and the specificity increases from 94.7% to 96.4% . though the difference in objective temperature between the two readings was only 0.2c it is still statistically significant ( p - value < 0.01 ) . the clinical diagnosis of 996 adult patients out of the whole sample was recorded as seen in table 3 . the written diagnoses indicate the diagnosis of the current encounter for this particular visit to ed . ear , nose and throat ( ent ) and musculoskeletal problems were the most frequent presentations encountered in the triage clinic ( 19.4% and 16.7% respectively ) . since this study was carried out towards the end of june and early july , the high presentation of both diagnoses can not be attributed to cold weather or winter viral illnesses . there were significant differences between male and female in the overall clinical diagnoses ( chi = 29.92 p - value = 0.0078 ) . more female patients presented with cardiovascular and gastrointestinal diseases than the male patients ( p - value = 0.0208 and 0.0069 respectively ) . on the other hand , more males presented with dermatological disease , trauma and injury than females ( p - value = 0.0188 and 0.0124 respectively ) . these differences may be due to the fact that males in our community at work or at leisure are more exposed than females to the risk of contact with different chemical and allergic agents , to trauma and injury . in conclusion , this study showed that adult patients were reasonably reliable in their assessment of subjective fever . therefore , medical staff should take the complaints of subjective fever in our community more seriously .

introduction : fever is the most appreciated manifestation of disease which usually raises high therapeutic expectations . patients seek medical advice because they think they are sick and feverish . if they feel that they are sick but not feverish , they may not seek medical advice . subjective fever may also be an important clue to further evaluation of the patient.objective:the aim of this study was to assess the reliability of adult patients to predict absence or presence of fever in emergency department triage clinic.methods:a prospective study of 1241 ambulatory adult patients ( above 12 years of age ) was carried out over a three - week period . all patients were asked whether or not they had fever or felt they had fever or were running a temperature before oral temperature was taken with an ivac digital machine . two sets of temperature readings were taken to define fever as 37.8c or greater , and 38.0c or greater.results:the sensitivity and specificity of detecting fever by subjective means was 89.6% and 94.5% for male and 90.0% and 94.8% for female . the accuracy rates were 93.9% and 94.6% respectively . the prevalence of objective fever was 8.7% yielding general positive and negative predictive values of 80.9% and 98.9% with an accuracy rate of 94.2%.conclusion : the reliability of adult patients attending triage clinic in assessing subjective fever was found to be good . four out of five of our patients who believed they had a fever were actually found to have an objective temperature increase ( 38.0c or greater ) . this means that medical staff should take a complaint of subjective fever in our population more seriously .

INTRODUCTION METHODS RESULTS DISCUSSION

PMC3405641

the promotion of physical activity ( pa ) is a major public health initiative in many countries due to its protective effect on numerous major health problems , including canada and the us where public health bodies recommend 30 to 60 minutes of moderate - to - vigorous activities per day . however , there has long been a concern that such promotion could lead to a rise in hip and knee oa , the major public health problem in musculoskeletal medicine and a leading cause of chronic disability . while there is a broad agreement that pa is an important determinant of joint health , it is unclear what amount and type of pa are beneficial or pose a risk . in short , despite numerous studies , the association between pa and joint health is complex and poorly understood . while different study designs , case definitions , sampling frames , and size play a role , the wide variation in how pa is defined is the most probable reason for the uncertainty . there is a lack of valid , reliable , and standardized instruments across studies , substantial measurement error , variation in the period and nature of pa measured , and failure to measure the most relevant aspect of pa - joint load . where accurate and precise measures are available , they are impractical for use in population - based research . the goals of this study were to address these gaps by ( 1 ) measuring historic pa , a key variable given the long latency and asymptomatic induction period of oa that , while potentially imprecise , is likely more important than accurate measure of an irrelevant variable such as current activity , ( 2 ) examining loads applied to the joint using quantitative joint loading units that allowed for assessment of cumulative force from pa , including dose response , and ( 3 ) including household activity ( in addition to occupation and sport / recreation ) to capture all three primary domains of activity . the purpose of this study was to evaluate the association between levels of lifetime knee joint force and knee oa in a large canadian sample of community dwelling adults . data source and study populationthe source population was community - dwelling members of the canadian association of retired persons , canada 's largest 50-plus advocacy group with 350,000 members . briefly , recruitment was via direct email to 28,000 members and to 100,000 additional members via an advertisement in an online newsletter , appearing in two consecutive newsletters . all e - mails and newsletters contained hyperlinks or banner advertisements directing subjects to the study website . through these methods , after completing an electronic consent form , subjects were given password access to the questionnaire . all data collection was web - based and used skip logic technology that allowed subjects to follow individualized paths through the survey , moving forward based on responses to previous questions . extensive pre- and pilot - testing was carried out to ascertain best recruitment methods , survey duration , navigation , and to ensure that respondents could understand items , retrieve information , and make appropriate estimations . the baseline questionnaire , carried out from june to september 2005 , took 60 to 90 minutes to be completed.response rates have been previously described and are presented in figure 1 . individuals who completed the baseline survey were contacted by email and letter for follow - up surveys at approximately one ( may 2006 ) and two years ( june 2007 ) . follow - up surveys inquired about knee joint health using the same questions as the baseline survey . the source population was community - dwelling members of the canadian association of retired persons , canada 's largest 50-plus advocacy group with 350,000 members . briefly , recruitment was via direct email to 28,000 members and to 100,000 additional members via an advertisement in an online newsletter , appearing in two consecutive newsletters . all e - mails and newsletters contained hyperlinks or banner advertisements directing subjects to the study website . through these methods , after completing an electronic consent form , subjects were given password access to the questionnaire . all data collection was web - based and used skip logic technology that allowed subjects to follow individualized paths through the survey , moving forward based on responses to previous questions . extensive pre- and pilot - testing was carried out to ascertain best recruitment methods , survey duration , navigation , and to ensure that respondents could understand items , retrieve information , and make appropriate estimations . the baseline questionnaire , carried out from june to september 2005 , took 60 to 90 minutes to be completed . individuals who completed the baseline survey were contacted by email and letter for follow - up surveys at approximately one ( may 2006 ) and two years ( june 2007 ) . follow - up surveys inquired about knee joint health using the same questions as the baseline survey . a period prevalence study using a cross - sectional design was utilized , as there were an insufficient number of incident knee oa cases to power the study to meet the objectives . to compile the dataset , subjects reporting oa at any of the three time points ( figure 1 ) were identified as cases . while the inclusion of prevalent cases could potentially limit the ability to delineate cause and effect , the approach was justified on several grounds , and steps were taken to guard against reverse causality . first , increasing the number of cases by combining prevalent and incident cases allowed for greater study power to assess gender - specific relationships between pa and oa while including a number of covariates , and assessing dose - response relationship of pa with oa . second , a new measure of exposure quantitative lifelong joint force from work , sport , and household activity measured in joint loading units was investigated for the first time . third , the relationship between the historic pa measured in this study ( up to age 50 , as discussed in methods ) and the time of diagnosis for the vast majority of cases is separated in time , providing some protection against reverse causality . in addition , the highest levels of lifetime pa for most people occur prior to the age of 50 . lastly , since oa is not a curable condition , prevalence is a relatively stable reflection of disease frequency . lifetime physical activity was assessed using the lifetime physical activity questionnaire ( l - paq ) , whose development and validation has been described previously . in summary , the l - paq is a self - administered , web - based questionnaire that was based on existing instruments [ 810 ] , developed using the principles of construct validation , adapted for self - administration over the internet , incorporated skip logic technology , and expanded to capture more detailed information including bodily movements involving the knee . using a subsample of the current study , intraclass coefficients for reliability ranged from 0.65 to 0.89 ; convergent validity testing against two validated lifetime questionnaires resulted in spearman correlation coefficients ranged from 0.41 to 0.71 . the lpaq measures lifetime pa across three domains : sport / recreation , occupation , and household and had been described previously [ 4 , 5 ] . sample questions in each domain are shown in appendix a. briefly , in the sports / recreational section , respondents were provided with a list of 64 possible sports and were permitted to add other sports . data collected included the duration , frequency , and average length of time per occasion , and time spent per hour ( none , 15 , 515 , 1530 , 3045 , and 4560 minutes per hour ) in eight bodily positions or movements . the occupational section used an open format in which respondents , for each occupation ever held , provided details on job title or type , duration , average hours per week , and whether the job was full time , part time , or seasonal and time spent per eight - hour segment in nine bodily positions or movements . household activity covered four areas : ( 1 ) caring for children ; ( 2 ) caring for elderly or disabled individuals ; ( 3 ) gardening ; ( 4 ) housework . for each household area of activity , participants were prompted to provide detailed information on duration and average number of hours per week of participation and were required to report time spent in an 8-hour period for each of the bodily movements as per the occupational section ( appendix b list of activities and assigned joint force ) . to obtain a measure of cumulative joint force at the knee , a cumulative peak force index ( cpfi ) score was estimated at the knee and has been described previously as the product of time spent in a specific activity ( total lifetime hours ) , bodyweight ( bw ) , and typical peak joint force for that activity ( % bw ) , ( i.e. , cpfi score ( bodyweight - hours ) = total lifetime hours bodyweight typical peak joint force , per each activity ) . as previously described [ 4 , 5 ] , the cpfi is a newly proposed measure , and steps were taken to validate and/or ensure the greatest precision in the components that comprise it . in addition to validation of the l - paq , self - reported bodyweight and height were utilized , measures with established validity properties used in numerous epidemiologic studies . we improved on a single self - report of bodyweight by asking about it at three time points ( baseline survey , age 20 , maximum lifetime ) , and deriving a lifetime bodyweight trajectory , interpolated using a lowess ( nonparametric smooth ) curve . the third component of the measure was the typical peak joint force assigned to each activity ( appendix b ) . these values were determined after an extensive literature [ 1240 ] ( full bibliography available on request ) that prioritized in vivo studies and incorporated judgments about data quality and study rigor . data were synthesized and a consensus achieved by a panel of experts from biomechanical engineering , rheumatology , physiotherapy , and musculoskeletal epidemiology . the main reason for this was to capture the primary pa exposure prior to the diagnosis of oa ( and first symptoms ) for the vast majority of cases , and to minimize the effects of subclinical , undiagnosed , or early oa on pa patterns . support for this approach was also drawn from the previous study on lifetime trajectories that revealed that peak exposure window for pa is prior to 50 , being at its highest lifetime levels from ages 3045 . an algorithm was used to ascertain knee oa cases , requiring that subjects report both health - professional - diagnosed knee oa and pain , aching , or stiffness on most days at any of the 3 time points ( baseline , 1st and 2nd followups ) . the questionnaire item on health - professional - diagnosed oa informed subjects that oa was distinct from other msk diseases ( e.g. , rheumatoid arthritis and osteoporosis ) , and a response confirmation to this question was required in a follow - up item . the reliability and validity of self - reported knee oa was carried out in a substudy and has been previously published . briefly , using the american college of rheumatology ( acr ) clinical classification criteria for knee oa as a diagnostic referent , sensitivity was 0.76 , specificity 0.98 , positive predictive value 0.87 , and negative predictive value 0.95 . our findings are consistent with other studies comparing self - report medically diagnosed oa and clinical oa [ 4446 ] . the baseline questionnaire measured known knee health risk factors and included gender , age , body mass index ( bmi ) , ethnicity ( asian , black , caucasian , first nations , hispanic , and others ) , and education ( elementary , high school , postsecondary , and trade / technical ) . the following question from the baseline questionnaire was used to determine the presence of significant knee injury : have you ever had a knee injury that required you to use a walking aid ( e.g. , cane or crutch ) for at least one week ? follow - up questions included the age at injury ( if more than one injury , the time of first injury was requested ) . only injuries that occurred before the diagnosis of oa cpfi values for each activity were summed for sport , occupation , and household domains for each 5-year period of a person 's lifetime to factor in changes in bodyweight over time , and these domain values were then summed to give a total cpfi value . for the total force ( cpfi ) variable , subjects were categorized into quintiles of exposure for the overall distribution , prior to stratification by gender . for the domain specific analysis , the quintiles were based on the relative distribution within each domain ( occupation , household , and sport ) , again prior to stratification by gender ( e.g. , quintile 5 for occupation was at the same joint loading level for both sexes ) . crude odds ratios were calculated for the relationships between knee oa and joint force variables and other study covariates . covariates were selected based on scientific knowledge and the conceptual framework of causal pathways to knee oa . factors associated with an increased risk of knee oa , which also could be confounders , such as age , previous injury , and bmi were adjusted for in all the analyses . the potential effect of one domain on another ( e.g. , occupational activity when assessing the sport - oa relationship ) was also potentially confounding and included . men and women were examined separately because of known gender differences in disease prevalence , physical activity profiles , injury rates , and bmi . reference categories were the lowest cpfi , youngest age tertile ( < 58 ) , normal bmi ( 20.024.9 ) , and no previous injury . test for trends was obtained by treating the categories ( quintiles ) of the cpfi variable as continuous and testing the slope for significance ; the models contained all relevant adjustment covariates . multiple logistic regression was used to examine if levels of total knee cpfi were associated with a risk of knee oa , controlling for age , previous injury , and bmi . an additional adjusted analysis was carried out investigating the separate effect of occupational , sport , and household cpfi ( adjusted for the other domains ) . subject characteristics for the sample are presented in table 1 . frequencies of outcome , exposure , and covariates for this study are provided in tables 2 and 3 . the prevalence of knee oa was 22.4% for the sample overall17.8% for men and 25.1% for women ( table 2 ) . twenty - six percent of the sample were of normal bmi , with approximately 72% being either overweight ( 39.9% ) or obese ( 31.5% ) . more men than women ( 47.7 to 36.1% ) were overweight , and more women than men were obese ( 33.4 to 29.5% ) . twenty percent had a history of previous knee joint injury ( 24% in men and 18% in women ) . the prevalence of subjects in occupational and household quintiles varied substantially by gender ( table 3 ) . for example , in the male household strata , the largest prevalence ( 36.3% ) was in the lowest ( referent ) quintile of exposure , while the smallest prevalence ( 6.2% ) was in the highest quintile of exposure . for women , the largest prevalence ( 27.5% ) was in the highest quintile of exposure and the smallest prevalence in the referent quintile ( 11.1% ) . for occupational force , these relative proportions were reversed by gender , though the percentages were slightly different ( table 3 ) . crude ors provided evidence that older age , previous knee injury , obesity in men , and overweight and obesity in women were associated with knee oa ( table 2 ) . compared with the lowest category of total lifetime knee force the highest category ( 5th quintile ) , in men and 3rd , 4th , and 5th quintiles in women were crudely related to knee oa ( table 2 ) . for domain - specific lifetime force , the 5th quintile of occupational force in men and both occupational and household activity in and women were crudely related to oa ( table 3 ) . adjusted ors for risk factors on knee oa obtained from multiple logistic regression oa and total lifetime force remained significant for the highest force quintile in both men ( 1.70 ; 95% ci1.06 , 2.70 ) and women ( 1.52 ; 95% ci1.15 , 2.02 ) . in domain - adjusted models , occupational force in men and household force in women were related to knee oa and tests for trend in these domains , as well as in total lifetime force ( both men and women ) , were significant ( p < 0.001 ) . sport / recreational force was not related to knee oa . after adjustment , older age , injury , and high bmi remained being overweight ( bmi 2529.9 ) increased the risk of knee oa for women ( or 1.69 ; 95% ci1.31 , 2.19 ) but was not related to knee oa in men . obesity ( bmi > 30 ) was a greater than two - fold risk for men and three - fold risk for women . this prevalence study on a large sample of canadian adults presents a newly proposed measure of lifetime mechanical knee joint force based on hours in pa , bodyweight , and typical knee joint force for specific activities and relates it to self - reported knee oa . we provide evidence that while lifelong pa may generally be safe for the knee joint , that very high force from lifelong total force , and from high levels of total occupational force ( men and women ) and household activity ( women ) , is a potential risk for knee oa . our results are consistent with previous studies that show overweight / obesity , age , female sex , and previous injury are significant risk factors for knee oa [ 4755 ] . the results of this study must be compared cautiously with previous studies due to its cross - sectional design , how subjects were assembled , and how exposures and outcomes were measured . for example , no other studies evaluating pa and knee oa have used the internet for data collection or have completely classified pa in terms of a joint loading variable over the long term , perhaps partly explaining inconsistent results from these past studies [ 44 , 5661 ] . of note , the cross - sectional design may have resulted in reverse causality potentially attenuating risk estimates . conversely , subjects with oa may have overreported prior pa exposure because they perceive that pa caused their oa ( recall bias ) , potentially increasing the risk estimates . since this type of bias threatens most prevalence studies , emphasis should be placed on recent high - quality cohort studies evaluating the association between pa and oa . a recent prospective cohort study with a 22-year - followup using physician - diagnosed oa , reported an adjusted or for the heaviest category of physical demands at work compared with the lightest category of 18.3 for knee during 12 years of followup , recently reported that 463 of 1678 respondents ( 28% ) developed clinical knee oa , and that a high mechanical strain score was associated with an increased risk of knee oa ( hr 1.43 , 95% ci 1.151.77 ) after adjustment for a number of covariates . , in a prospective cohort study of approximately 40,000 australians with an average 5 years of followup , reported a composite sport and occupational exposure ( past 6 months , measured at baseline ) and found a risk for total knee joint arthroplasty for the vigorous level of activity ( hr 1.42 , 95% ci 1.081.86 ) . several studies from the framingham cohort suggest that job activities may cause as much as 15% to 30% of knee oa in men [ 65 , 66 ] . felson et al . reported that elderly persons ( average age 70 ) in the highest quartile of pa at a baseline examination had over three times the risk of developing radiographic knee oa nine years later , when compared with those in the lowest quartile . mcalindon et al . using longitudinal framingham data reported that the number of hours per day of heavy physical activity was associated with the risk of incident radiographic knee oa ( or = 7.0 for 4 + hours heavy physical activity / day ) . in contrast , a study by hannan et al . in the same cohort found no increase in the risk of knee oa with increasing physical activity . in the highest quartile of pa compared to the least active , the or was 1.3 for men and 1.1 for women ( both nonsignificant ) . using data from the chingford study , followed 715 women ( mean age : 54 years ) for 4 years with no radiographic knee oa at baseline and included the pa categories of walking , occupation , and sport / recreation . they found no relationship between incident knee oa and pa , while walking protected against joint space narrowing ( or = 0.4 , 95% ci 0.20.9 ) . it is evident from these often - cited reports that , despite the longitudinal cohort designs , large samples and lengthy followups , and estimates for the risk of pa on knee oa vary extensively . while differences in eligibility criteria , covariates included in multivariable models and small samples may account for some of the disparity , the most likely reason is the wide variation in pa exposure measurement . of note , most studies have not measured the joint - force aspects of pa nor attempted to completely classify pa ( including historic pa ) from all three major activity domains . apart from the verweij et al 's study , none of the above studies considered pa from all three major activity domains or attempted to estimate the effect of activities in terms of joint force . was an or of 1.43 ( 95% ci 1.151.77 ) for a high knee mechanical strain score , close to our reported or 's from the highest quintiles of total knee force , occupational force , and household force in women . results are not directly comparable since apart from differences in design , the mechanical strain score was a ranking ( 1 to 4 ) of certain physical activities over the past 2 weeks ( taken at baseline ) and did not look at sex - specific differences in occupational and household activity . a number of longitudinal and case control studies [ 5557 , 6875 ] , we did not find a relationship between sport / recreational activity and knee oa . studies that have shown a relationship between sport and knee oa have generally been in populations of athletes in specific sports with high knee forces [ 54 , 7681 ] and not from population - based studies , or the association has been explained by joint injury . in population - based studies of lifetime activity , the highest sport / recreation rates typically occurs at a relatively young age , as it did in the current cohort , prior to the age of 25 . thus , high forces from sport later in life , when the joint may be more vulnerable , were not well - represented in this sample and may contribute to the lack of association here . the prevalence of knee oa in our study was 22.4% , 17.8% in men and 25.1% in women . these gender differences in prevalence are consistent with previous large population - based north american studies for this age group [ 8284 ] . even though there is probably some misclassification , our definition , which required a medical diagnosis and the presence of pain on most days , is important since pain is usually the most important aspect of disease to patients and may precede x - ray change , potentially capturing earlier disease . we report the results of a validity study in a subsample of the current study comparing self - reported oa to clinical oa . it was important to measure and simultaneously adjust for pa - related force from all three major activity domains . most previous pa - oa studies have investigated one or two domains ( usually sport and/or occupation ) . given the high levels of household and occupational pa reported in our previous paper , omitting one or both of these domains leaves these studies vulnerable to confounding from the unmeasured domain(s ) . in studying all three domains separately by sex , we also observed relationships of pa with very high occupational force in men and household force in women . questionnaires used in many previous studies did not assess the frequency , duration , and intensity of pa actually performed by women . the majority of women 's exposure to pa , particularly in older cohorts such as the current one , is due to accumulation of regular household activities [ 8588 ] . while household activity may generally not be considered vigorous from an energy expenditure perspective and is often ignored in epidemiologic study of oa , there are many repetitive motions ( e.g. , stair climbing , squatting , and kneeling ) and activities ( e.g. , gardening , lifting , and carrying ) that are associated with high knee joint forces [ 20 , 21 , 28 , 36 ] but have low energy expenditure . this is only the second study to measure lifelong household load at the knee joint and relate it to knee oa , and the first to quantify household knee joint force from historic activity for the assessment of dose response . in the previous study by sandmark et al . , exposure to physically demanding tasks at home was significantly associated with knee oa among women ( but not men ) and was the strongest risk factor for women among the physical load variables that were investigated in that study . given that women have been shown to have higher pa than men when including household together with occupational and sporting activities , and that reasons for the higher prevalence of knee oa in women are not clearly elucidated , our findings provide preliminary evidence that the role of historic household pa requires further investigation . the role of occupational activity has received much more study , and is better understood in men than in women , in part because previous studies were based historically on male - dominated workforce cohorts . even though women often spend forty or more hours a week at a full - time job and from twenty to forty - five hours per week working in the home , questionnaires used in many previous studies do not assess the frequency , duration , and intensity of pa actually performed by women . it has been shown that , when the definition of regular physical activity measured in surveys is expanded to include household activity , pa levels rise and associations with health outcomes are more evident including protective relationships with cardiovascular disease and myocardial infarction , cancer , and an inverse relationship with all - cause mortality [ 85 , 90 ] . our finding of an increased risk of knee oa for the top quintile of occupational joint force is generally consistent with previous studies [ 62 , 64 ] and several systematic reviews [ 9294 ] . the cpfi , a quantitative joint force measure , together with a large sample allowed for evaluation of a dose - response relationship between lifelong force and knee oa . in the models where a significant relationship with knee oa was found ( total force , occupation , and household ) , there was an increasing , significant trend in the ors from lower to higher levels of cpfi , though only the ors for the highest ( 5th ) quintile reached statistical significance . while this requires confirmation and further delineation in future studies , the presence of dose response strengthens evidence for a causal relationship . this study had several strengths , including a large sample drawn from the population and a sufficient number of cases to adjust for a number of covariates , the separate analyses of men and women ( equivalent to including interactions with gender for all variables ) , and assessment of dose - response . another strength was the use of detailed information on the duration , frequency , and joint loading aspects of historic activities , from all three main physical activity domains , allowing for relatively complete classification of the total volume of pa . historic pa is a potentially important exposure in oa etiology given the lengthy induction and asymptotic latency period . measuring current or recent levels of pa does not capture long - term joint forces , may miss etiologically important periods of exposure and is a poor proxy for cumulative lifetime exposure . lastly , many studies have used advanced disease markers , such as total joint arthroplasty or moderate - to - marked radiographic change as the outcome in assessing the role of pa . it is not clear whether the relationship with pa for early , symptomatic cases is the same as that observed for advanced or radiographically defined oa . this may be important in understanding modifiable risk factors that could play a role in a prevention strategy for oa , something not currently available . there are a number of limitations that are important in interpreting the results of this study . self - report of knee oa may lead to misclassification . in our examination of the measurement properties of our case definition this is critical for studies of risk factors , since low specificity ( inclusion of many false positives among cases ) causes a greater attenuation of effect than low sensitivity . ppv was also high , another important measure indicating that the vast majority of the cases identified in the survey were true cases . nevertheless , we did not use radiography as part of the classification criteria for knee oa . radiographic oa in the presence of symptoms is thought to represent the best definition of oa . however , x - ray change is associated largely with moderate - to - advanced disease , and there is only moderate agreement between pain and symptoms and x - ray changes . wu et al . , in a study using a validated outcome instrument for knee oa based on arthroscopic visualization , suggest that the acr clinical classification criteria can be used to identify patients with early articular cartilage loss , before any radiographic changes are evident . however , it is probable that the false positives include not only subjects with early oa not captured by the acr criteria , but also other causes of knee symptoms the subjects were fairly well - educated , predominantly caucasian canadians with access to public health care and internet users . this method of data collection may not be as effective in low - income populations , and those with decreased access to medical care important since we asked about medically - diagnosed osteoarthritis as part of the case definition . further , since recruitment and enrolment of subjects was via the internet , subjects were largely self - selected . self - selection implies that the nature of the bias can not be known with certainty . studies of subjects who participate in online research reveal that they are more likely to be older , females and have higher socioeconomic status . many more individuals potentially view invitations to participate in research , with most declining to participate , making validity of results more challenging to interpret . in online surveys , there is no single response rate - multiple metrics for calculating a response rate have been defined such as the participation rate and completion rate . however , the goal of this study was not to describe characteristics of the population at large , but to assemble subjects to test hypotheses about pa - related knee joint force and knee oa in a large sample of individuals who met criteria for a disease and those who did not , sampled in the same way ( internal validity ) . the limitations of our measure of self - report of pa measures and construction of the cpfi variable have been discussed elsewhere [ 4 , 5 ] . in short , self - reported pa measures require cautious interpretation because of large within - person variability and problems with recall [ 102104 ] , that may lead to nondifferential misclassification and attenuation of the effect size in analytic studies using the exposure . in particular , this attenuation may have contributed to a lack of a significant finding from the sport domain , since the highest sport levels occurred in the distant past ( prior to age 25 ) for most subjects , are not part of the generic memory pattern ( shown to have better recall ) and thus may been imprecisely recalled . despite these limitations , it has been repeatedly shown that pa questionnaires are both practical and valid when used appropriately for large - scale epidemiologic studies [ 86 , 104 , 106 , 107 ] . the cpfi , a time - force - bodyweight product , was a stronger predictor of knee oa than any of its component parts alone and is a new measure of pa - related force measured in joint loading units . however , the cpfi does not separately and specifically capture elements of activity - related force that may be most injurious such as shear , rapid deceleration , or high - impulse loads . activities where those elements of force occur ( e.g. , cutting and pivoting sports , jumping sports , and carrying heavy loads ) were captured , indirectly measuring these harmful types of load , but the strength of a potential signal from these forces may have been blunted . another potential limitation related to recall is the possibility of recall bias , where the ability to recall past exposure is dependent on outcome status . of note , subjects with oa at baseline this could lead to increased risk estimates , and while justified for the reasons outlined previously , the results remain vulnerable to this type of bias . however , risk estimates for sport and occupational exposure as well as other covariates were generally in the expected direction and consistent with the literature including prospective data [ 4755 ] , lending validity to the findings . regardless , the possibility of this bias must be acknowledged , and study results interpreted in light of this . although this study provides evidence of an association between high levels of lifelong joint force , overweight / obesity , previous injury , bmi , and knee oa , the cross - sectional design makes the determination of a cause and effect more challenging . however , the time window used for the main pa exposure ( prior to age 50 ) captures the ages [ 3045 ] with the highest level of lifetime force and is separated in time from knee oa diagnosis for the vast majority of cases . supporting this , most of the risk estimates for covariates reported in this study were in the expected direction and effect sizes consistent with the literature [ 4755 ] , and lastly as this study is the first attempt to examine the effect of a new exposure measure ( quantitative lifelong joint load from all three primary activity domains ) on knee oa , a cross - sectional approach is reasonable . the finding that most pa - related force is not related to knee oa , but that the highest levels of joint force are , is biologically plausible and fits within the conceptual framework of causation . under normal physiological conditions , the transmission and distribution of joint loads can occur for decades with little or no wear . however , when normal joint physiologic mechanisms are overwhelmed via excessive local mechanical force , biologic events are triggered which destabilize the normal coupling of degradation and synthesis of articular cartilage and subchondral bone . animal studies clearly illustrate that high joint force from pa affects cartilage metabolism and plays a role in the development of oa [ 110112 ] . in summary , a newly proposed measure of lifetime mechanical knee force was used to estimate the risk of self - reported knee oa . while it must be interpreted cautiously because of the cross - sectional design and the possibility of recall bias , this study suggests that lifelong physical activity is generally safe . high levels of lifetime knee force from occupational activity in men and women , and household activity in women were associated with knee oa . prevention efforts may best be directed at occupations requiring high physical demands , at weight - control programs and injury prevention . future research should further investigate the potential role of household activity , improve the estimation and validity of knee force measurement in new populations , and apply these measures in longitudinal studies .

purpose . to investigate the association of cumulative lifetime knee joint force on the risk of self - reported medically - diagnosed knee osteoarthritis ( oa ) . methods . exposure data on lifetime physical activity type ( occupational , household , sport / recreation ) and dose ( frequency , intensity , duration ) were collected from 4,269 canadian men and women as part of the physical activity and joint heath cohort study . subjects were ranked in terms of the cumulative peak force index , a measure of lifetime mechanical knee force . multivariable logistic regression was conducted to obtain adjusted effects for mean lifetime knee force on the risk of knee oa . results . high levels of total lifetime , occupational and household - related force were associated with an increased in risk of oa , with odds ratio 's ranging from approximately 1.3 to 2 . joint injury , high bmi and older age were related to risk of knee oa , consistent with previous studies . conclusions . a newly developed measure of lifetime mechanical knee force from physical activity was employed to estimate the risk of self - reported , medically - diagnosed knee oa . while there are limitations , this paper suggests that high levels of total lifetime force ( all domains combined ) , and occupational force in men and household force in women were risk factors for knee oa .

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

PMC4139190

changes in environmental conditions can have a significant impact on the development and function of the brain . neural stem cells ( nscs ) integrate both local and systemic signals to modulate their rate and extent of proliferation to meet the needs of the organism ( kokovay et al . , 2008 ) . these quiescent nscs are reactivated in response to a variety of metabolic stimuli ( rafalski and brunet , 2011 ) . understanding how systemic and metabolic signals are sensed by the brain and converted into specific neural stem cell behaviors is essential to deciphering how the brain adapts to a changing environment . in drosophila , nscs enter quiescence at the end of embryogenesis and are reactivated during early larval life in response to feeding ( britton and edgar , 1998 ; truman and bate , 1988 ; figure 1a ) . amino acid availability is sensed by the fat body , the functional equivalent of the mammalian liver and adipose tissue ( andersen et al . , 2013 ; colombani et al . , 2003 ) . the fat body sends an as - yet - unidentified signal , or signals , to the brain to induce the production and secretion of insulin - like peptides ( dilps ) by blood - brain barrier ( bbb ) glial cells . dilps act locally to trigger the insulin / insulin - like growth factor receptor pathway in underlying nscs ( chell and brand , 2010 ; sousa - nunes et al . , 2011 ) . nsc reactivation occurs synchronously in all neurogenic zones of the cns , suggesting that bbb glial cells and/or nscs are linked by an intercellular signaling mechanism . gap junctions are intercellular channels formed by the juxtaposition of connexin hexamers ( segretain and falk , 2004 ) . gap junctions are found throughout the mammalian brain and are important regulators of stem cell behavior , controlling self - renewal , survival , and aging ( kar et al . , 2012 ; wong et al . , we show that gap junction proteins play a key role in the nutrient - dependent reactivation of dormant neural stem cells in the drosophila brain . interestingly , gap junction proteins are required in the bbb glia , but not in neural stem cells , for reactivation . we show that gap junction proteins coordinate nutrient - dependent calcium oscillations within the bbb and are required for the production and secretion of insulin - like peptides . gap junction proteins thus enable the synchronous reactivation of quiescent stem cells throughout the cns . to assess whether gap junctions play a role in nsc reactivation , we systematically targeted each of the eight members of the innexin ( inx ) family ( bauer et al . , 2005 ) , the drosophila functional equivalents of connexins and pannexins ( phelan , 2005 ; figure s1a available online ) , by rnai in either nscs or glia ( table s1 ) . however , knockdown of innexin 1 ( inx1 ) or innexin 2 ( inx2 ) in glia gave a striking phenotype in which brain size is dramatically reduced ( figures 1b1d ) without affecting overall body size ( data not shown ) . this suggests that the inx phenotype is not the result of a systemic growth defect but that inx1 and inx2 have a specific role in the brain . we checked the specificy of inx1 and inx2 using in silico methods ( naito et al . , 2005 ) , which predict no off - targets . , 2013 , who showed that targeted rnai against inx1 ( ogre ) or inx2 in glia disrupts development of the larval nervous system and leads to adult behavioral phenotypes . we demonstrate that innexins are not required to link nscs either to each other or to glial cells . instead , inx1 and inx2 are required within the glial population alone for brain development . to understand how glial gap junctions regulate growth in the cns , we first examined nsc behavior after inx1 or inx2 knockdown at different time points during the process of nsc reactivation . knockdown of inx1 or inx2 in glia did not affect the number of nscs in the ventral nerve cord ( vnc ; figure 1e ) , demonstrating that the phenotype is not due to the loss of nscs prior to nsc reactivation ( 0 hr after larval hatching , alh0 ) . next we assessed cell diameter because one of the earliest events in nsc exit from quiescence is cell enlargement ( chell and brand , 2010 ; sousa - nunes et al . , we found that nsc diameter is markedly reduced ( alh24 ) after inx1 or inx2 knockdown in glia ( figure 1f ) . we assayed the mitotic marker phosphohistone h3 ( ph3 ) before nsc reactivation ( alh0 ) , just after reactivation ( alh48 ) and at a time when wild - type nscs are cycling actively ( alh72 ) . knockdown of either inx1 or inx2 in glial cells resulted in a severe reduction in the number of dividing nscs at all times ( figure 1 g ) . we found that nsc enlargement and entry into mitosis were also dramatically impaired in inx1 and inx2 mutants ( inx1 and inx2 , respectively , see figures s1b and s1c ) , and that reactivation could be rescued in inx2 mutants by glial expression of inx2 ( figures s1d s1e ) . we conclude that inx1 and inx2 are required in the glia for nsc exit from quiescence . gap junction proteins ( connexins , pannexins , or innexins ) are classically involved in forming intercellular channels or hemi - channels , which enable exchange between the cytoplasm and the extracellular medium . evidence also exists for channel - independent roles , such as cell adhesion and direct gene regulation ( reviewed in dbouk et al . , 2009 ; elias and kriegstein , 2008 ) . to test if channel function is important for nsc reactivation , we treated brains in culture with carbenoxolone , a classic blocker of gap junction channels and hemi - channels ( giaume and theis , 2010 ; see supplemental experimental procedures ) . carbenoxolone completely blocked nsc reactivation ( figures 2a2c ) , implying a channel role for inx1 and/or inx2 , the only innexins required for nsc reactivation . we also found that protein fusions that interfere with the folding of the innexin n - terminal domain ( gfp - inx1 and rfp - inx2 ) , which is essential for channel formation ( nakagawa et al . , 2010 ) , act as dominant - negative mutants ( figures s2a s2c ; data not shown for inx2 ) . this suggests that the function of inx1 and inx2 in glia is channel - based . inx1 and inx2 could be part of the same channel or form two distinct channels , performing different functions that are both required for nsc reactivation . gap junction channels are formed by the apposition of connexons ( innexons in drosophila ) on adjacent cells . connexons can be homomeric , formed from six molecules of a single subtype of connexin , or heteromeric , formed from different subtypes . in the larval vnc prior to nsc reactivation ( alh7 ) , inx1 and inx2 were strongly expressed in glia and colocalized in plaques typical of gap junctions ( segretain and falk , 2004 ; figure 2d ) . super - resolution microscopy ( dobbie et al . , 2011 ) further demonstrated the tight association of inx1 and inx2 , using tagged fusion proteins ( figures 2e2e ) . this close association is present from hatching and is not lost under starvation conditions ( figures s2d and s2e ) , demonstrating that formation of the complex is not driven by nutrition . we found that most inx1 staining was lost after knockdown of inx2 in glia , and vice versa . this suggests that inx1 and inx2 localization is interdependent ( figures s2f s2h ) and that inx1 and inx2 form heteromeric innexons ( segretain and falk , 2004 ) rather than independent gap junction channels ( lehmann et al . , 2006 ) . inx1 and inx2 have been shown to form functional heteromeric channels in paired xenopus oocytes ( holcroft et al . , 2013 ) . we conclude , therefore , that inx1 and inx2 form heteromeric channels or hemi - channels in the glia . by alh24 , when reactivation has taken place , inx1 and inx2 are still expressed but they no longer colocalize ( figure 2f ) , suggesting that formation of inx1/inx2 channels is temporally regulated . consistent with this observation , we discovered that the temporal requirement for inx1 and inx2 function in nsc reactivation is between alh0 and alh24 ( figure 2 g , table s2 , and data not shown ) . therefore , the formation and maintenance of inx1 and inx2 heteromeric channels are developmentally regulated and coincide with the time when the innexins are required for nsc reactivation . inx1/inx2 channels are required in glia to transmit nutritional stimuli to quiescent nscs , they are likely to be found , therefore , in cells situated between the nscs and the exterior of the brain . to determine in which glial cells inx1/inx2 are required , we knocked down inx1/inx2 in different glial populations using subtype - restricted gal4 drivers to drive rnai or express dominant - negative constructs ( table s3 ) . we found that inx function is necessary within the subperineurial glia because knockdown in this glial subtype alone phenocopies knockdown in the entire glial population ( table s3 and figures s3a and s3b ) , preventing nsc reactivation ( figures 3a3d ) . the subperineurial glia and the perineurial glia constitute the drosophila bbb ( stork et al . , 2008 ) . in vertebrates , the bbb consists of a single layer of vascular endothelium closely associated with astrocytic glia ( daneman , 2012 ) . the bbb shields the brain from the external environment owing to tight junctions between endothelial cells . it acts as a selective sieve to reject potentially neurotoxic factors but allow the passage of nutrients , ions , or other signals to maintain brain homeostasis ( daneman , 2012 ) . the drosophila bbb exhibits similar neuroprotective strategies to its vertebrate counterpart , including a layer that limits the diffusion of neurotoxic factors , and an array of conserved transporters that regulates bbb permeability ( daneman and barres , 2005 ; desalvo et al . , 2011 ; mayer et al . , the subperineurial glia are large , flat polyploid ( unhavaithaya and orr - weaver , 2012 ) cells ( figures 3e3e ) that envelop the brain ( figure 3f ) and are closely apposed to the nscs ( figures 3f and 3f ) . they isolate the brain from the hemolymph ( the drosophila equivalent of blood ) by virtue of lateral septate junctions ( figures 3e and 3e ; schwabe et al . , 2005 ; stork et al . , knockdown of inx did not disrupt the septate junctions ( figures s3c and s3d ) , and we were not able to see a change in dextran dye penetration ( data not shown ; pinsonneault et al . , 2011 ; schwabe et al . , although weak permeability defects are difficult to detect at this stage and can not be excluded , they do not prevent nsc reactivation ( moody mutant ( bainton et al . , 2005 ) , data not shown ) . these data suggest that the inx mutant phenotype is not due to an impaired , leaky bbb . using super - resolution microscopy , we detected inx1 ( and inx2 , data not shown ) along the bbb membranes and the septate junctions lining the lateral cell membranes ( figures 3 g and s3e ) . we conclude that inx1/inx2 channels are required autonomously in the bbb glial cells for nsc reactivation ( figure 3h ) . nsc reactivation requires the expression and secretion of insulin - like peptides , dilps , by bbb glial cells ( chell and brand , 2010 ) . of the eight identified insulin - like peptides in drosophila , dilp6 transcription was shown to increase dramatically in the cns upon feeding . furthermore , when larvae were starved , forced expression of dilp6 in the glia was able to rescue nsc reactivation ( chell and brand , 2010 ) . dilp6 binds to the insulin receptor ( inr ) on nscs , activating the pi3k / akt pathway and inducing exit from quiescence ( chell and brand , 2010 ) . we found a significant decrease in dilp6 transcription after knocking down both inx1 and inx2 in the glia ( figure s4a ) . , we expressed a tagged , functional version of dilp6 ( dilp6-flag ) in the bbb glia . we found that dilp6 secretion from the bbb glia was strongly impaired in inx1 loss - of - function mutants ( figures 4a , 4b , and 4d ) . secretion of dilp6 was similarly impaired upon starvation ( figures 4a , 4c , and 4e ) . therefore , both the expression and secretion of dilp6 are regulated by nutrition and depend on gap junction proteins . nsc reactivation in inx mutants was rescued by forced expression of dilp6 in glia ( figures 4f4h , see arrowheads ) , as shown by the recovery of brain volume ( 80% of the brains , figure 4i ) and of nsc diameter ( figure 4j ) . direct activation of the pi3k / akt pathway in nscs also resulted in rescue of brain volume and nsc enlargement and entry into mitosis ( figures s4b s4c ) . we conclude that gap junction proteins in the bbb glia are required to activate insulin signaling and induce nsc reactivation . secretion of insulin by the pancreas is induced by glucose , leading to synchronized calcium oscillations within gap junction - coupled beta cells and insulin exocytosis ( macdonald and rorsman , 2006 ) . gap junctions enable the passage of secondary messengers that either trigger the release of calcium from intracellular stores or the influx of calcium from the extracellular environment ( laude and simpson , 2009 ; leybaert and sanderson , 2012 ; orellana et al . inhibits coordinated intercellular calcium signaling ( leybaert and sanderson , 2012 ; orellana et al . , 2012 ) . gap junction proteins are thus an important means of transmitting calcium waves . to investigate whether calcium signaling plays a role in gap junction - mediated nsc reactivation , we expressed a calcium sensor , gcamp3 ( tian et al . , 2009 ) , in the bbb glia ( figure 5a ; figures s5a the bbb glia of feeding larvae exhibited clear calcium oscillations ( movie s1 ; figure 5b ) . the bbb glia pulsed simultaneously , suggesting that calcium oscillations are coordinated across the entire cns . individual cell tracking showed that glial calcium oscillations exhibited striking synchrony ( figures 5b and 5b ) in all brains analyzed ( n = 15 , six are displayed ; figure s5d ) . to further assess the extent of calcium oscillation coordination within the bbb glia under these conditions , we performed correlation analysis ( see experimental procedures ) for 20 regions of interest ( roi ) chosen at random within the bbb glial layer ( figures 5c , s5e , and s5e ) . in fed larvae before reactivation , the central correlation peak ( coefficient 1 ) demonstrates the synchronicity of calcium oscillations within the bbb layer . additional peaks on each side reveal that this synchronicity repeats ( see experimental procedures for details ) . none of the mutant brains ( n = 11 ) showed coordinated calcium oscillations ( movie s2 ; figure 5d ; figure s5f ) . instead , bbb glial cells pulse independently , with no coordination between neighboring cells ( figures 5d and 5d ) . we conclude that inx1/inx2 gap junctions are required to coordinate synchronous calcium oscillations within the bbb glia . in accordance with our observation , the graph of correlation coefficient for inx1 mutants established the total absence of synchronicity between bbb glial cells ( figures 5e , s5 g , and s5g ) , showing that gap junctions are required for propagating calcium oscillations within the bbb . to assess whether calcium oscillations in the bbb are induced by a nutritional stimulus , we first assayed calcium dynamics in the bbb glia of newly hatched larvae ( alh0 ) , before they started to feed . the calcium oscillations differed both in extent and frequency from those seen in fed larvae ( n = 9 ; movie s3 ; figures 6a6a ; figure s6a ) . correlation analysis revealed a partial coordination within the bbb glia ( central correlation peak with a coefficient of 0.5 , figures 6b , s6b , and s6b ) , strengthening the idea that nutrition is important for extending and establishing robust calcium synchronicity in the bbb . next , we assessed calcium dynamics after starvation , specifically the absence of essential amino acids . calcium dynamics in the bbb of starved larvae resembled inx1 mutant brains ( movie s4 ; figure 6c ) . synchronous calcium oscillations were completely abolished in all brains examined ( n = 19 , figure s6c ) . individual bbb cells displayed some calcium pulses ( figures 6c and 6c ) , but with different profiles to those seen in fed larvae . in addition , correlation analysis of starved larvae showed very weak synchronicity ( figures 6d , s6d , and s6d ) . this suggests that the synchronous , nutrition - dependent , calcium oscillations are lost upon starvation . importantly , neither inx1 nor inx2 is lost under starvation conditions ( figure s2e ) . we conclude that nutrition , in particular essential amino acids , shape calcium dynamics . upon feeding , we assayed whether glial calcium oscillations arise from the release of intracellular calcium , or from the influx of extracellular calcium , and how these influence nsc reactivation . first , we assessed the importance of the inositol - triphosphate ( ip3 ) pathway . stimulation of g - coupled receptors by a wide range of signals activates phospholipase c , leading to the production of ip3 from cleaved pip2 . ip3 then binds to its receptor ( ins3pr ) , a ligand - gated ca channel found on the surface of the er , releasing intracellular calcium . we knocked down ins3pr ( itpr in drosophila ) in the bbb glia by rnai ( chorna and hasan , 2012 ) . both nsc enlargement and proliferation were strongly impaired ( figures 7a7d ) . membrane depolarization triggers the entry of extracellular calcium via voltage - gated calcium channels ( catterall , 2011 ; leybaert and sanderson , 2012 ) , whereas hyperpolarization prevents it . we hyperpolarized bbb membranes by expressing the inward - rectifying potassium channel , kir2.1 ( baines et al . , bbb hyperpolarization blocks nsc reactivation dramatically , as revealed by the complete failure of both enlargement and mitotic re - entry ( figures 7e7h ) . interestingly , the mushroom body neuroblasts , a small group of central brain nscs that do not undergo quiescence and reactivation , are not affected by bbb hyperpolarization , suggesting that nutrition - dependent nsc reactivation is specifically affected ( figures s7a and s7b , see arrowheads ) . bbb hyperpolarization both decreases dilp6 mrna levels ( figure s7c ) and dilp6 secretion ( figures s7d and s7e ) , similar to what is seen during starvation or gap junction loss - of - function ( see figure s4a and figures 4a4e ) . in support of the role of calcium oscillations in reactivating nscs , we found that overexpressing the calcium - binding protein , calmodulin , prevents nsc reactivation ( figures s7c s7f ) . the nutrient - dependent reactivation of nscs in the drosophila brain demonstrates how nscs can adapt to environmental changes to fulfil the needs of the organism . here , we show that gap junction proteins within the bbb glia are required for insulin expression and secretion , a prerequisite for nsc exit from quiescence ( figure 7i ) . we demonstrate that gap junction proteins coordinate glial calcium oscillations that are required for nsc reactivation . membrane depolarization is known to regulate exocytosis via calcium signaling ( fridlyand and philipson , 2011 ; stojilkovic , 2012 ; sdhof , 2012 ) , which controls stimulus - secretion coupling in secretory cells , such as in endocrine cells ( dolenek et al . , 2011 ; gminard et al . , 2009 ; stojilkovic , 2012 ; sdhof , 2012 ) . we show that conditions that block calcium oscillation in the bbb glia ( the loss of gap junction proteins , starvation ) also impair insulin secretion . the sequence of events leading to glial secretion of insulin bears a striking resemblance to the diet - induced release of insulin by the beta cells of the pancreas ( macdonald and rorsman , 2006 ) . in the pancreas , a nutritional stimulus is sensed by gap junction - coupled beta cells , inducing depolarization resulting in synchronized calcium oscillation and insulin secretion . loss of gap junction coupling results in uncoordinated calcium pulses . in drosophila inx loss of function mutants , individual subperineurial bbb compared to starvation , in which the nutritional signal is absent and nsc reactivation can not occur , the scattered signals from individual bbb cells are able to induce delayed , asynchronous , reactivation in a small number of nscs ( see ph3 positive nscs in figures 1c1d ) . we propose that gap junction function within the bbb enables glial insulin release to reach a threshold high enough to trigger nsc reactivation throughout the central nervous system . in both beta cells and bbb cells , however , sustained depolarization of cells can lead to desensitization and a decline in insulin release ( willenborg et al . , interestingly , we find that forced depolarization of bbb glia only mildly enhances nsc reactivation ( data not shown ) . this could be due to desensitization or it may be that the system is already maximally active . insulin mrna levels are decreased after gap junction knockdown , both in pancreatic islets ( bosco et al . it remains to be determined if calcium oscillations can directly affect gene expression , as has been shown in other systems ( alonso and garca - sancho , 2011 ) . insulin produced by the pancreas is distributed via the circulatory system , whereas glial insulin is secreted locally , directly to underlying nscs . glial insulin signaling is thus contained within the brain , enabling local , differential regulation of this organ . the bbb acts both as a niche and as a protective barrier , providing specific factors directly to the stem cell while shielding the brain from unwelcome systemic regulation . in the context of nsc reactivation , these two roles are conveniently complementary . in the vertebrate bbb , similar functions may be split between endothelial cells and astrocytic glia . the vascular endothelium provides the barrier function , while astrocytic glia have a regulatory role in sensing and adjusting barrier permeability to various stimuli ( daneman , 2012 ) . bbb endothelial cells can secrete cytokines , chemokines , and prostaglandins , suggesting that the bbb behaves like an endocrine tissue ( banks , 2012 ) . interestingly , calcium oscillations have been observed in cultured vertebrate bbb endothelial cells , but their function is largely unknown ( de bock et al . , 2013 ) . gap junction communication can influence stem cell behavior by directly coupling stem cells to each other or to supporting cells , such as found in a stem cell niche . in the brain , connexon - mediated communication has been reported to occur between progenitor cells , within astrocytic networks , and between radial glia and neurons or progenitor cells and astrocytes ( giaume et al . , 2010 ; lacar et al . , 2011 ; nakase and naus , 2004 ; lo turco and kriegstein , 1991 ) . the proliferation of neural progenitors and the formation of cortical layers in the mouse brain depend on an intercellular gap junction network ( malmersj et al . , 2013 ) , and grafted human nscs integrate into organotypic cultures through connexin coupling ( jderstad et al . , we show that gap junction function within a niche , the bbb , can also influence nsc behavior . the drosophila bbb is a protective and selective barrier as well as a signaling center that orchestrates major developmental and physiological events . here we show that gap junction communication enables cells within the bbb to act as a concerted unit , leading to coordinated calcium signaling and insulin release . similarities between the bbb in vertebrates and invertebrates suggest that our findings are likely to have broader significance . the rnai and gal4 drivers used in this study are listed in tables s1 and s3 . inx1 and moody - gal4 were kind gifts of p. phelan and r. bainton , respectively . all glial knockdowns were performed with repo - gal4 as driver , unless stated otherwise . the bbb driver was either moody - gal4 or mdr65-gal4 ( see for each experiment ) . center values are averages . student s t test was performed using a threshold of p < 0.05 ( confidence interval of 95% ) , represented by . all tests performed in this study were two - sided and with samples of same variance , unless stated otherwise . for rescue of inx1 mutant by glial expression of dilp6 , we used a one - way anova test , with p < 0.05 represented by . whisker plots were drawn using the minimum , quartile 1 , median , quartile 3 , and maximum of each condition s sample . in addition , a student s t test based on a sample s average and sd was used to generate p values with the same criteria as above . we used tub - gal80 , inx1 ; repo - gal4 to achieve conditional expression of inx1 . at 25c ( permissive temperature for gal80 ) , larvae showed a wild - type phenotype , but at 30c ( restrictive temperature for gal80 ) , all larvae showed a mutant phenotype : lack of nsc reactivation and clear reduction in brain volume . in further experiments , we scored mutant phenotypes by brain volume only in third instar larvae . twelve larvae were scored for each condition . in single temperature - shift experiments , 1 hr embryos or 1 hr first instar larvae we coexpressed a tagged version of dilp6 ( dilp6-flag ) and a membrane marker ( mcd8-rfp ) in the bbb glia alone , in controls ( genotype moody - gal4 , uas - mcd8-rfp , uas - dilp6-flag ) and inx1 mutants ( genotype inx1/y ; moody - gal4 , uas - mcd8-rfp , uas - dilp6-flag ) . all brains were imaged using the same confocal settings for dilp6-flag . for each vnc , the total dilp6 intensity ( t ) retained dilp6 intensity ( r ) was then measured as the dilp6 signal ( same threshold as previously ) colocalizing with the bbb membrane ( rfp signal ) . the threshold for the rfp signal was determined manually for each brain , to ensure selection of the whole membrane . due to the flatness of the bbb glia , the membrane signal represents most of the cells , although some cytoplasmic signal could be missed . the percentage of secreted dilp6 was calculated as percent secreted dilp6 = ( 1 r / t ) 100 . moody - gal4 , uas - kir2.1-gfp ; tubulin - gal80 flies were grown at permissive temperature until hatching , then switched to 30c from alh0 to alh24 . two different conditions were analyzed : control ( genotype fm7 , dfd gmr yfp / y ; moody - gal4 , uas - mcd8-rfp , uas - gcamp3 ) and inx1 mutant ( genotype inx1/y ; moody - gal4 , uas - mcd8-rfp , uas - gcamp3 ) . larvae were raised and staged at 29c , then fed or starved as described in the supplemental experimental procedures . larvae were placed in a drop of voltalef oil on a 22 mm diameter welco dish , ventral side down . a 13 mm diameter round coverslip was placed tilted on top and lowered until the larva was immobilized . mounted larvae were imaged with a 40 oil immersion objective on an olympus inverted fv1000 . gcamp and mcd8-rfp were imaged simultaneously because we did not detect any bleed - through . to measure the mean intensity of the whole plane over time , the mean intensity was measured for each point both for gcamp3 ( channel 1 , c1 ) and mcd8-rfp signals ( channel 2 , c2 ) . we normalized changes in gcamp3 intensity against changes in mcd8-rfp intensities ( accounting mainly for loss of focal plane due to larval movement ) , and then normalized the ratio by its minimum over the whole movie , so that the baseline for all movies was always 1 . the normalized gcamp3 signal is then : i(gcamp ) norm = ( c1/c2 ) / ( c1/c2)min . this normalized ratio was plotted over time using spreadsheet software . to follow the behavior of calcium in different regions of the brain to check for oscillation coordination , rois were selected manually in the first frame of the movie , and their central coordinates recorded . a 64 64 pixel tracking window was centered on each roi , and a 32 32 pixel subwindow in the middle of each tracking window was used for measuring intensity values . the tracking operations were performed on the relatively stable red channel ( cell membrane ) rather than the more dynamic green channel ( the calcium signal to be measured ) . the script moves the tracking window by calculating the correlation between the window region from one frame to the next . the location of the peak of the correlation function indicates how far and in which direction to move the tracking window to keep it centered on the roi . to reduce tracking drift due to rounding errors , times - two upsampling was used . to further improve tracking accuracy , the image was low pass filtered to remove noise . the upsampling and filtering were accomplished simultaneously by taking the product of the fast fourier transforms of the tracking window and extracting the low - frequency coefficients then zero padding before taking the inverse fast fourier transforms . the intensity measurements were performed by summing the pixel values within the 32 32 pixel measuring window for both the red and green channels . normalization was then applied as described above . the intensity signal for each roi ( computed and normalized as described above ) the baseline is calculated using a 50 sample running average filter and then subtracted out from the signal . the remaining signal is then scaled so that the sum of the squares of the samples is one . each remaining roi signal is correlated with the reference for shifts of 50 to + 50 samples . if a signal is identical to the reference , the correlation coefficient will peak at + 1 for a shift of 0 . if a signal is uncorrelated with the reference , then the correlation coefficient will take small random values .

summaryneural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions . how do stem cells sense and respond to metabolic changes ? in the drosophila cns , quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus . feeding triggers insulin production by blood - brain barrier glial cells , activating the insulin / insulin - like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation . here we show that gap junctions in the blood - brain barrier glia mediate the influence of metabolic changes on stem cell behavior , enabling glia to respond to nutritional signals and reactivate quiescent stem cells . we propose that gap junctions in the blood - brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion .

Introduction Results Discussion Experimental Procedures

PMC3015612

ureteropelvic junction ( upj ) obstruction is a condition where urine flow and transport from the kidney to the upper ureter is impaired , due to a variety of causes ( intrinsic obstruction several management options are available for this problem , including open surgery ( op ) , antegrade and retrograde endourologic techniques , and laparoscopic repair ( lp ) . to our knowledge , no study has evaluated and compared an individual surgeon 's initial experience with laparoscopic pyeloplasty , with and without robotic assistance . recently , ahlering et al reported their initial experience with robot - assisted laparoscopic radical prostatectomy and concluded that robotic skills are quickly acquired , even by a laparoscopically inexperienced surgeon . we aimed to compare laparoscopic pyeleoplasty , with and without robotic assistance , during the initial experience of a laparoscopically experienced surgeon , to determine whether robotic assistance has distinct advantages over the pure laparoscopic technique . between 1999 and 2003 , patients diagnosed with upj obstruction and subsequently treated with lp or rp by a single surgeon ( cps ) were identified , retrospectively reviewed , and compared . the expected results following endopyelotomy and laparoscopic pyeloplasty given the preoperative characteristics of the individual patient were discussed with each patient before selecting the treatment modality . the lps were performed in 1999 through 2002 by using interrupted sutures for the anastomosis . objective data collected from the chart review included demographics and preoperative data ( age , sex , weight , height , body mass index ) , intraoperative data ( total operative time and estimated blood loss ) and postoperative data ( length of hospitalization , analgesic requirements , complications , follow - up radiographic images , and clinical subjective improvement ) . a definition of favorable outcomes included a patent ureteropelvic junction confirmed by a radionuclide diuretic renogram as well as subjective clinical response , which was ascertained from review of follow - up clinic appointments . subjective results were determined from the improvement in the analog pain scores of the patient at the last follow - up . cystoscopy and ureteral stent ( 6 french ) placement under fluoroscopic guidance is first achieved with the patient in the standard lithotomy position . the length of the upj obstruction , the size of the pelvis , and the presence of a crossing vessel helps determine the type of upj repair . the upper end of the ureteral stent is positioned in the upper calyx of the kidney to allow for movement of the stent during manipulation of the upj without dislodgment of the lower end of the stent into the distal ureter . the patient is repositioned and the laparoscopic technique is performed with the patient in the flank position and a mild degree of flexion of the operative table . pneumoperitoneum is established in the ipsilateral midclavicular line at the level of the umbilicus , and an 8-mm da vinci trocar is placed . the second 8-mm da vinci trocar is placed 8 cm to 10 cm above the umbilicus in the midline . a 12-mm trocar is inserted 6 cm to 8 cm below the umbilicus in the midline . this port is used by the assistant for suction / irrigation and for insertion of the sutures and needle . on the right side , a 5-mm trocar is used in the subcostal region in the midline to help with anterior retraction of the liver , using a locking grasper at the lateral abdominal sidewall . the position of the trocar is selected depending on the location of the lower edge of the liver . the upper end of the stent is removed from the renal pelvis and the pelvis spatulated . if an anterior crossing vessel is present , the renal pelvis is transposed anterior to the vessel and the posterior anastomosis performed with a running 4 0 polyglactin suture , 6 inches in length . the proximal coil of the stent is then replaced into the renal pelvis and the anterior anastomosis completed with a second running suture . a nondismembered fengerplasty is performed if no crossing vessel is present and there is focal stenosis . a 10 fr or 15 fr drain is inserted through an 8-mm lateral trocar site . flexible cystoscopy or fluoroscopy confirms proper positioning of the distal coil of the stent , and a foley urethral catheter is inserted . the urethral catheter and the drain follow - up appointments are scheduled at 8 weeks to 12 weeks after stent removal , at 6 months , and annually thereafter . cystoscopy and ureteral stent ( 6 french ) placement under fluoroscopic guidance is first achieved with the patient in the standard lithotomy position . the length of the upj obstruction , the size of the pelvis , and the presence of a crossing vessel helps determine the type of upj repair . the upper end of the ureteral stent is positioned in the upper calyx of the kidney to allow for movement of the stent during manipulation of the upj without dislodgment of the lower end of the stent into the distal ureter . the patient is repositioned and the laparoscopic technique is performed with the patient in the flank position and a mild degree of flexion of the operative table . pneumoperitoneum is established in the ipsilateral midclavicular line at the level of the umbilicus , and an 8-mm da vinci trocar is placed . the second 8-mm da vinci trocar is placed 8 cm to 10 cm above the umbilicus in the midline . a 12-mm trocar is inserted 6 cm to 8 cm below the umbilicus in the midline . this port is used by the assistant for suction / irrigation and for insertion of the sutures and needle . on the right side , a 5-mm trocar is used in the subcostal region in the midline to help with anterior retraction of the liver , using a locking grasper at the lateral abdominal sidewall . the position of the trocar is selected depending on the location of the lower edge of the liver . the divided ureteral end is spatulated laterally for 1 cm . the upper end of the stent is removed from the renal pelvis and the pelvis spatulated . if an anterior crossing vessel is present , the renal pelvis is transposed anterior to the vessel and the posterior anastomosis performed with a running 4 0 polyglactin suture , 6 inches in length . the proximal coil of the stent is then replaced into the renal pelvis and the anterior anastomosis completed with a second running suture . a nondismembered fengerplasty is performed if no crossing vessel is present and there is focal stenosis . a 10 fr or 15 fr drain is inserted through an 8-mm lateral trocar site . flexible cystoscopy or fluoroscopy confirms proper positioning of the distal coil of the stent , and a foley urethral catheter is inserted . the urethral catheter and the drain follow - up appointments are scheduled at 8 weeks to 12 weeks after stent removal , at 6 months , and annually thereafter . fourteen patients underwent laparoscopic pyeloplasty during the study period , 7 with and 7 without robotic assistance . the two patients in the lp group had small anastomotic leaks ( diagnosed by elevated drain fluid creatinine levels ) that resolved with conservative treatment with a drain and a urethral catheter for a week . one patient in the rp group had a febrile postoperative urinary tract infection that required intravenous antibiotics . another patient in the rp group was readmitted 2 days after discharge with hematuria due to minor bleeding from the anastomotic site . follow - up was 10 months ( range , 5 to 15 ) in the rp group and 24 months ( range , 22 to 30 ) in the lp cohort . all 14 patients have done well with improvement in renal function ( 30% to 44% ) and normalization of t1/2 on lasix renography . the perceived advantages of rp are improved dexterity and precision with suturing , shorter learning curve for surgeons , improved visualization of the operative field , and shorter operative times , with equivalent outcomes as those for conventional laparoscopy . nevertheless , disadvantages to robotic - assisted laparoscopic surgery are no tactile feedback , increased setup time , and cost . gettman et al retrospectively compared their initial 6 patients after rp with 6 age - matched lp controls and concluded that operative times were improved with robotic assistance ( 140 min vs 235 min ) as was suturing time ( 70 min vs 120 min ) . hospital stay ( 4 days ) , estimated blood loss ( < 50 ml ) , and complications ( none ) were equivalent between the 2 groups . in another review with 9 patients , in which a 4-port rp technique was used , operative time was 139 minutes , suturing time was 62 minutes , hospitalization was 5 days , and blood loss was minimal . in our study , the times reported included the preoperative cystoscopy and stent placement as well as the time for setting up the robotic system . furthermore , our overall experience with the robotic system had been minimal , before this series of patients . because more surgeries , such as the laparoscopic radical prostatectomies , are performed with robotic assistance , the operating times will significantly decrease . we have noted a significant decrease in our robot setup time since this series of patients underwent surgery . the lack of tactile feedback with the robotic system during the early experience may adversely effect delicate dissection in the region of the crossing vessels . during procedures in the first 2 of our patients in the robotic series , we used traditional laparoscopy for initial dissection of the ureter and the crossing vessel . however , with experience , the improved 3d visualization allowed the surgeon to perceive tissue consistency and the tension on the sutures during knot tying . the anastomotic leaks that occurred with the lp patients could be related to the relative inexperience of the surgeon with the procedure . however , the impression is that the anastomosis is less challenging with robotic assistance and can be reliably performed with less experience . first , the number of patients in the study was small . also , this was a retrospective review with the entire lp cohort performed initially , and the rp performed after the surgeon had gained more experience with the procedure . lastly , the types of repair and suturing techniques were not similar in the 2 groups . nevertheless , it is our impression that outcomes in the rp are equivalent to those in the lp group . longer follow - up and a larger patient cohort would certainly solidify our conclusions and determine other differences not elucidated in this small series . because robotic - assisted pyeloplasty is a new technique , evidenced by fewer than 10 patients in most series , more experience is needed to better determine the relative advantages of the rp procedure . from this review , in the surgeon 's initial learning curve , a significant difference in perioperative variables or outcome does not appear to exist .

objectives : the benefits of laparoscopic surgery with robotic assistance ( da vinci robotic surgical system , intuitive surgical , sunnyvale , ca ) includes elimination of tremor , motion scaling , 3d laparoscopic vision , and instruments with 7 degrees of freedom . the benefit of robotic assistance could be most pronounced with reconstructive procedures , such as pyeloplasty . we aimed to compare laparoscopic pyeloplasty , with and without robotic assistance , during a surgeon 's initial experience to determine whether robotic assistance has distinct advantages over the pure laparoscopic technique.methods:we retrospectively compared the first 7 laparoscopic pyeloplasties with the first 7 robotic pyeloplasties performed by a single surgeon . all patients were preoperatively evaluated with computed tomographic angiography with 3d reconstruction to image crossing vessels at the ureteropelvic junction . all patients were followed up by lasix renograms and routine clinic visits.results:patients were similar with respect to mean age ( 34 in laparoscopic pyeloplasty group vs 32 in the robotic pyeloplasty group ) , operative time ( 5.2 hours vs 5.4 hours ) , estimated blood loss ( 40 ml vs 60 ml ) , and hospital stay ( 3 days vs 2.5 days ) . two patients in the laparoscopic pyeloplasty group had small anastomotic leaks managed conservatively , and one patient in the robotic pyeloplasty group had a febrile urinary tract infection necessitating treatment with intravenous antibiotics . another patient in the robotic pyeloplasty group was readmitted with hematuria that was treated conservatively without transfusion . no recurrences were detected in either group.conclusions:operating times and outcomes during the learning curve for laparoscopic pyeloplasty were similar to those for robotic pyeloplasty . long - term data with greater experience is needed to make definitive conclusions about the superiority of either technique and to justify the expense of robotic pyeloplasty .

INTRODUCTION METHODS Operative Technique with Robotic Assistance RESULTS DISCUSSION CONCLUSION

PMC3582872

this was a retrospective study . among 412 patients who underwent surgical cuff repair for full - thickness rotator cuff tears confirmed by arthroscopic findings at the authors ' institution between january 2005 and july 2008 , 191 patients who underwent both preoperative mri and postoperative mdct at least one year after the operation were included . the postoperative mdct was performed at a mean of 13.2 3.8 months after operation ( range , 12 to 16 months ) , without special indication . we excluded patients with isolated subscapularis tear ( n = 5 ) , previous operation on the same shoulder joint ( n = 4 ) , and incomplete repair ( n = 6 ) . patients who did not have preoperative mri ( n = 8) or postoperative mdct ( n = 166 ) , and those with mri or mdct images that were not usable due to poor quality or absence of appropriate y - view were also excluded ( n = 32 ) . most of the patients without the postoperative mdct underwent ultrasonography as a postoperative imaging modality instead of mdct ( 136 of 166 ) , and thirty patients refused to undergo either ultrasonography or mdct . there were no special criteria for the selection of postoperative imaging modality ; however , ultrasonography was usually performed for those with financial difficulties . the average age at the time of operation was 59.7 7.9 years ( range , 39 to 80 years ) , and the postoperative mdct was performed at a mean of 13.2 3.8 months after operation . biceps tenotomy or tenodesis was performed for a symptomatic biceps tear involving greater than 50% of the tendon and for a symptomatic degenerative superior labral anterior and posterior ( slap ) lesion according to age or activity level of the patient . the operative technique was arthroscopic repair , mini - open repair , or open repair according to the arthroscopy learning curve and arthroscopical reparability . repair technique was either single or double row according to the tear size , tendon status , and tear configuration and reparability . immobilization after cuff repair was maintained with an abduction brace , and duration of immobilization was based on tear size as measured at the time of operation , from 4 weeks to 6 weeks . passive motion was performed immediately after the operation , except in cases of large - to - massive tears . active - assisted range of motion exercise was allowed after the weaning of the brace . muscle strengthening exercises began between 9 and 12 weeks , and sports activities were permitted at 6 months after the operation . even though mri still remains the standard preoperative imaging modality for evaluation of rotator cuff , mdct has been widely used recently for its advantages , such as cost - effectiveness , short examination time , and absence of foreign body artifact in the postoperative evaluation for patients who have undergone rotator cuff repair surgery . our previous study showed that mdct arthrography ( mdcta ) is a very valuable method to confirm postoperative integrity after rotator cuff repair using suture anchors.15 ) as such , we have used mri as a preoperative imaging modality and mdct as a postoperative imaging modality . mri was performed on the gyroscan intera 1.5-t system ( philips medical systems , utrecht , the netherlands ) . from the mri examination , we obtained t1-weighted and t2-weighted spin - echo oblique sagittal images parallel to the joint surface of the glenoid . ct was also performed using a 16-multidetector ct system ( mx8000 idt , philips medical systems ) . we generated oblique sagittal reconstruction images as well as oblique coronal and axial images at a three - dimensional workstation . the oblique sagittal images were reconstructed parallel to the glenohumeral joint surface , same as the mri oblique sagittal images . the patient positioning for mri and mdct examinations was also same with the arm placed alongside the body in a neutral supine position . the supraspinatus muscle atrophy was measured on the same scapular y - view of the oblique sagittal images of mri or mdct at the location where the body of the scapula , scapular spine , and medial border of the coracoids process form a y - shape ( fig . 1 ) . we defined the atrophy by calculating occupation ratio according to the method of thomazeau et al.,11 ) the ratio between the cross - sectional area of the supraspinatus muscle and the supraspinatus fossa on the scapular y - view . the boundary of the supraspinatus fossa was defined as the area from the inner margins of the y - shape to the inner limit of the clavicle and the acromion . originally , the occupation ratio is measured on the picture archiving and communications systems ( pacs ) workstation by drawing the boundary of the supraspinatus muscle and supraspinatus fossa.11 ) however , this method is limited in its accuracy and reliability for measuring occupation ratio , because the outlining process is conducted by subjective manual hand drawing . for that reason , in our previous study,12 ) we introduced a new measuring method for the supraspinatus muscle atrophy using the photoshop software ( magic selection tool , adobe systems inc . , san jose , ca , usa ) , which is the method that automatically designates the regions of interest and calculates the number of pixels in the selected area . with the photoshop method , we solved the reliability issue for the measurement of the atrophy , showing very high intra- and interobserver reliability : interclass correlation coefficient ( icc ) of 0.89 to 0.96 , 0.90 to 0.98 , and 0.85 to 0.97 for intraobserver correlation on mdct and both mr t1- and mr t2-weighted images ( mrt1w and mrt2w ) ; and icc of 0.89 , 0.92 , and 0.91 for interobserver correlation , respectively ( all p < 0.001 ) . further , we demonstrated a significant correlation between mri and mdct with pearson 's correlation coefficient ( pcc ) of 0.78 to 0.87 between mdct and mrt1w and 0.73 to 0.82 between mdct and mrt2w ( all p < 0.001 ) . we showed it was possible to compare the occupation ratio between mri and mdct by using very good - fitting regression equation derived from linear regression analysis , y = 0.03 + 0.98x ( x is the occupation ratio of mri ; and y is that of mdct , r = 0.76 ) . using this photoshop method , we measured the cross - sectional area of the supraspinatus muscle and supraspinatus fossa on the y - view , and by dividing the cross - sectional area of the supraspinatus muscle by that of the supraspinatus fossa , we calculated the occupation ratio ( fig . a ratio above 0.6 was considered to be normal or mild atrophy ; between 0.4 and 0.6 , moderate atrophy ; and below 0.4 , severe atrophy.16 ) correlation between mri and mdct was excellent and reliable in both mrt1w and mrt2w ; however , since the correlation of mrt1w was slightly better than mrt2w,12 ) we used the measurements of mrt1w in this study . for the comparison of pre- and postoperative atrophic change , we first calculated the modified occupation ratio after inputting the preoperative occupation ratio measured on mrt1w into the regression equation , y = 0.03 + 0.98x ( r = 0.76 ) ( fig . 3).12 ) for example , an occupation ratio of 0.48 based on mrt1w is converted into 0.50 ( modified occupation ratio ) based on mdct . then , we compared this modified occupation ratio with the occupation ratio measured on postoperative mdct . although the measurement of occupation ratio using the photoshop method is very accurate and reliable , because it designates the regions of interest automatically with excellent mrt1w - mdct correlation,12 ) there may be some errors with the measurement . thus , we set a possible error range of plus or minus 10% ; that is , we defined the improvement of atrophy as more than a 10% increase in occupation ratio and the worsening of atrophy as more than a 10% decrease . we decided that plus or minus 10% was enough to take the measurement error into account , as the mean value of intraobserver relative error was 3.03% . therefore , if the modified occupation ratio was 0.50 , only an occupation ratio measured on postoperative mdct of higher than 0.55 or lower than 0.45 was regarded as meaningful and then further classified into either the improved atrophy group or worsened atrophy group , respectively . we evaluated the postoperative change of the atrophy , and performed stratification analysis according to age , retraction length , and initial degree of atrophy . the age was stratified into < 55 years , 55 to 65 years , and > 65 years ; retraction length into small ( < 1 cm ) , medium ( 1 to 3 cm ) , and large - to - massive ( > 3 cm ) size ; and initial degree of atrophy into the occupation ratio below 0.4 ( severe atrophy ) , between 0.4 and 0.6 ( moderate atrophy ) , and above 0.6 ( mild atrophy or normal ) . in addition , we assessed various factors such as age , gender , side of involvement , tear size of both anteroposterior ( ap ) dimension and retraction , quality of rotator cuff muscles , symptom duration , smoking , diabetes , steroid injection , level of sports activity and demand of shoulder activity which may affect to the change of the atrophy , and the association between the change of the atrophy and cuff integrity after surgical cuff repair . the tear size in the ap dimension was measured at the lateral edge of the footprint , and retraction length was estimated by the distance from the apex of the tear to the footprint . the quality of rotator cuff muscles was evaluated by global fatty degeneration index , which is the mean grade of fatty degeneration of the supraspinatus , infraspinatus , and subscapularis described by goutallier et al.6 ) the level of sports activity and the demand of shoulder activity were recorded as high , medium , and low . high level of sports activity was defined as enjoying dynamic or contact sports ( e.g. , boxing , rugby , basketball , football , volleyball , tennis ) ; medium level , enjoying static sports ( e.g. , golf , yoga , swimming , bicycle , running ) ; and low level , rarely playing sports . high demand of shoulder activity during work was defined as heavy manual labor ; medium demand , manual labor with less activity ; and low demand , sedentary worker . in the mdct interpretation for the integrity of the repaired supraspinatus tendon , a successful healing was defined as maintenance of the insertion into the footprint , whereas failed healing was taken as a discontinuity at the footprint . the contrast media leakage with maintenance of the cuff insertion into the footprint was not considered a failure of healing , as other authors presented.17 ) experienced musculoskeletal radiologists , who were unaware of the present study , performed and interpreted the mdct , and further evaluated the rotator cuff healing to the greater tuberosity . descriptive statistics were used to report the frequency of the improvement or worsening of the supraspinatus muscle atrophy after surgical cuff repair . the paired t - test was used to compare the change of the atrophy between pre- and postoperative occupation ratios . the pre- and postoperative occupation ratios followed normal distribution ( p = 0.191 and 0.100 , respectively , in kolmogorov - smirnov test ) . to determine the differences between the improved atrophy group and the worsened atrophy group , student 's t - test was used for the continuous variables , and or fisher 's exact test was used for the categorical variables . for all statistical analyses , spss ver . 12.0 ( spss inc . , chicago , il , usa ) was used , and p < 0.05 was considered statistically significant . this was a retrospective study . among 412 patients who underwent surgical cuff repair for full - thickness rotator cuff tears confirmed by arthroscopic findings at the authors ' institution between january 2005 and july 2008 , 191 patients who underwent both preoperative mri and postoperative mdct at least one year after the operation were included . the postoperative mdct was performed at a mean of 13.2 3.8 months after operation ( range , 12 to 16 months ) , without special indication . we excluded patients with isolated subscapularis tear ( n = 5 ) , previous operation on the same shoulder joint ( n = 4 ) , and incomplete repair ( n = 6 ) . patients who did not have preoperative mri ( n = 8) or postoperative mdct ( n = 166 ) , and those with mri or mdct images that were not usable due to poor quality or absence of appropriate y - view were also excluded ( n = 32 ) . most of the patients without the postoperative mdct underwent ultrasonography as a postoperative imaging modality instead of mdct ( 136 of 166 ) , and thirty patients refused to undergo either ultrasonography or mdct . there were no special criteria for the selection of postoperative imaging modality ; however , ultrasonography was usually performed for those with financial difficulties . the average age at the time of operation was 59.7 7.9 years ( range , 39 to 80 years ) , and the postoperative mdct was performed at a mean of 13.2 3.8 months after operation . biceps tenotomy or tenodesis was performed for a symptomatic biceps tear involving greater than 50% of the tendon and for a symptomatic degenerative superior labral anterior and posterior ( slap ) lesion according to age or activity level of the patient . the operative technique was arthroscopic repair , mini - open repair , or open repair according to the arthroscopy learning curve and arthroscopical reparability . repair technique was either single or double row according to the tear size , tendon status , and tear configuration and reparability . immobilization after cuff repair was maintained with an abduction brace , and duration of immobilization was based on tear size as measured at the time of operation , from 4 weeks to 6 weeks . passive motion was performed immediately after the operation , except in cases of large - to - massive tears . active - assisted range of motion exercise was allowed after the weaning of the brace . muscle strengthening exercises began between 9 and 12 weeks , and sports activities were permitted at 6 months after the operation . even though mri still remains the standard preoperative imaging modality for evaluation of rotator cuff , mdct has been widely used recently for its advantages , such as cost - effectiveness , short examination time , and absence of foreign body artifact in the postoperative evaluation for patients who have undergone rotator cuff repair surgery . our previous study showed that mdct arthrography ( mdcta ) is a very valuable method to confirm postoperative integrity after rotator cuff repair using suture anchors.15 ) as such , we have used mri as a preoperative imaging modality and mdct as a postoperative imaging modality . mri was performed on the gyroscan intera 1.5-t system ( philips medical systems , utrecht , the netherlands ) . from the mri examination , we obtained t1-weighted and t2-weighted spin - echo oblique sagittal images parallel to the joint surface of the glenoid . ct was also performed using a 16-multidetector ct system ( mx8000 idt , philips medical systems ) . we generated oblique sagittal reconstruction images as well as oblique coronal and axial images at a three - dimensional workstation . the oblique sagittal images were reconstructed parallel to the glenohumeral joint surface , same as the mri oblique sagittal images . the patient positioning for mri and mdct examinations was also same with the arm placed alongside the body in a neutral supine position . the supraspinatus muscle atrophy was measured on the same scapular y - view of the oblique sagittal images of mri or mdct at the location where the body of the scapula , scapular spine , and medial border of the coracoids process form a y - shape ( fig . 1 ) . we defined the atrophy by calculating occupation ratio according to the method of thomazeau et al.,11 ) the ratio between the cross - sectional area of the supraspinatus muscle and the supraspinatus fossa on the scapular y - view . the boundary of the supraspinatus fossa was defined as the area from the inner margins of the y - shape to the inner limit of the clavicle and the acromion . originally , the occupation ratio is measured on the picture archiving and communications systems ( pacs ) workstation by drawing the boundary of the supraspinatus muscle and supraspinatus fossa.11 ) however , this method is limited in its accuracy and reliability for measuring occupation ratio , because the outlining process is conducted by subjective manual hand drawing . for that reason , in our previous study,12 ) we introduced a new measuring method for the supraspinatus muscle atrophy using the photoshop software ( magic selection tool , adobe systems inc . , san jose , ca , usa ) , which is the method that automatically designates the regions of interest and calculates the number of pixels in the selected area . with the photoshop method , we solved the reliability issue for the measurement of the atrophy , showing very high intra- and interobserver reliability : interclass correlation coefficient ( icc ) of 0.89 to 0.96 , 0.90 to 0.98 , and 0.85 to 0.97 for intraobserver correlation on mdct and both mr t1- and mr t2-weighted images ( mrt1w and mrt2w ) ; and icc of 0.89 , 0.92 , and 0.91 for interobserver correlation , respectively ( all p < 0.001 ) . further , we demonstrated a significant correlation between mri and mdct with pearson 's correlation coefficient ( pcc ) of 0.78 to 0.87 between mdct and mrt1w and 0.73 to 0.82 between mdct and mrt2w ( all p < 0.001 ) . we showed it was possible to compare the occupation ratio between mri and mdct by using very good - fitting regression equation derived from linear regression analysis , y = 0.03 + 0.98x ( x is the occupation ratio of mri ; and y is that of mdct , r = 0.76 ) . using this photoshop method , we measured the cross - sectional area of the supraspinatus muscle and supraspinatus fossa on the y - view , and by dividing the cross - sectional area of the supraspinatus muscle by that of the supraspinatus fossa , we calculated the occupation ratio ( fig . a ratio above 0.6 was considered to be normal or mild atrophy ; between 0.4 and 0.6 , moderate atrophy ; and below 0.4 , severe atrophy.16 ) correlation between mri and mdct was excellent and reliable in both mrt1w and mrt2w ; however , since the correlation of mrt1w was slightly better than mrt2w,12 ) we used the measurements of mrt1w in this study . for the comparison of pre- and postoperative atrophic change , we first calculated the modified occupation ratio after inputting the preoperative occupation ratio measured on mrt1w into the regression equation , y = 0.03 + 0.98x ( r = 0.76 ) ( fig . 3).12 ) for example , an occupation ratio of 0.48 based on mrt1w is converted into 0.50 ( modified occupation ratio ) based on mdct . then , we compared this modified occupation ratio with the occupation ratio measured on postoperative mdct . although the measurement of occupation ratio using the photoshop method is very accurate and reliable , because it designates the regions of interest automatically with excellent mrt1w - mdct correlation,12 ) there may be some errors with the measurement . thus , we set a possible error range of plus or minus 10% ; that is , we defined the improvement of atrophy as more than a 10% increase in occupation ratio and the worsening of atrophy as more than a 10% decrease . we decided that plus or minus 10% was enough to take the measurement error into account , as the mean value of intraobserver relative error was 3.03% . therefore , if the modified occupation ratio was 0.50 , only an occupation ratio measured on postoperative mdct of higher than 0.55 or lower than 0.45 was regarded as meaningful and then further classified into either the improved atrophy group or worsened atrophy group , respectively . we evaluated the postoperative change of the atrophy , and performed stratification analysis according to age , retraction length , and initial degree of atrophy . the age was stratified into < 55 years , 55 to 65 years , and > 65 years ; retraction length into small ( < 1 cm ) , medium ( 1 to 3 cm ) , and large - to - massive ( > 3 cm ) size ; and initial degree of atrophy into the occupation ratio below 0.4 ( severe atrophy ) , between 0.4 and 0.6 ( moderate atrophy ) , and above 0.6 ( mild atrophy or normal ) . in addition , we assessed various factors such as age , gender , side of involvement , tear size of both anteroposterior ( ap ) dimension and retraction , quality of rotator cuff muscles , symptom duration , smoking , diabetes , steroid injection , level of sports activity and demand of shoulder activity which may affect to the change of the atrophy , and the association between the change of the atrophy and cuff integrity after surgical cuff repair . the tear size in the ap dimension was measured at the lateral edge of the footprint , and retraction length was estimated by the distance from the apex of the tear to the footprint . the quality of rotator cuff muscles was evaluated by global fatty degeneration index , which is the mean grade of fatty degeneration of the supraspinatus , infraspinatus , and subscapularis described by goutallier et al.6 ) the level of sports activity and the demand of shoulder activity were recorded as high , medium , and low . high level of sports activity was defined as enjoying dynamic or contact sports ( e.g. , boxing , rugby , basketball , football , volleyball , tennis ) ; medium level , enjoying static sports ( e.g. , golf , yoga , swimming , bicycle , running ) ; and low level , rarely playing sports . high demand of shoulder activity during work was defined as heavy manual labor ; medium demand , manual labor with less activity ; and low demand , sedentary worker . in the mdct interpretation for the integrity of the repaired supraspinatus tendon , a successful healing was defined as maintenance of the insertion into the footprint , whereas failed healing was taken as a discontinuity at the footprint . the contrast media leakage with maintenance of the cuff insertion into the footprint was not considered a failure of healing , as other authors presented.17 ) experienced musculoskeletal radiologists , who were unaware of the present study , performed and interpreted the mdct , and further evaluated the rotator cuff healing to the greater tuberosity . descriptive statistics were used to report the frequency of the improvement or worsening of the supraspinatus muscle atrophy after surgical cuff repair . the paired t - test was used to compare the change of the atrophy between pre- and postoperative occupation ratios . the pre- and postoperative occupation ratios followed normal distribution ( p = 0.191 and 0.100 , respectively , in kolmogorov - smirnov test ) . to determine the differences between the improved atrophy group and the worsened atrophy group , student 's t - test was used for the continuous variables , and or fisher 's exact test was used for the categorical variables . for all statistical analyses , spss ver . 12.0 ( spss inc . , chicago , il , usa ) was used , and p < 0.05 was considered statistically significant . the atrophy measured as occupation ratio significantly improved from 0.44 0.17 ( range , 0.11 to 0.89 ) preoperatively to 0.52 0.17 ( range , 0.13 to 0.91 ) postoperatively ( p < 0.001 ) ( fig . the number of patients whose postoperative occupation ratio increased by more than a 10% ( improved atrophy group ) was 81 ( 42.4% ) , and the number of patients with decreased occupation ratio of more than a 10% ( worsened atrophy group ) was 33 ( 17.3% ) ( table 2 ) . the overall composition of the patients with respect to the occupation ratio was changed from low occupation ratio ( severe atrophy ) to high occupation ratio ( mild atrophy - to - normal , p < 0.001 ) ( table 3 ) . even after stratification , the atrophy improved regardless of age , retraction length , or initial degree of the atrophy in all cases , although small retraction ( < 1 cm ) and mild - to - normal initial atrophy ( occupation ratio > 0.6 ) did not reach significant levels . however , with respect to the repair integrity , the occupation ratio was slightly decreased ( atrophy was worsened ) in patients whose cuffs were failed to heal after surgical repair , even though the change was not significant ( table 4 ) . there was no significant difference in every demographic and clinical factor between improved and worsened atrophy group ; however , the proportion of unhealed cuffs was significantly higher in the worsened atrophy group ( 48.5% , 16 of 33 ) compared with the improved atrophy group ( 22.2% , 18 of 81 , p = 0.007 ) ( table 5 ) . one of the main causes of the atrophy of the cuff muscles is rotator cuff tear.10,18 ) the degree of the atrophy depends on several factors such as the size of the tear , aging , and disuse , and it can lead to the loss of shoulder function including decrease in strength and failure of cuff healing after surgical repair.8,19,20 ) many authors have shown that muscular atrophy is the most important prognostic factor for arthroscopic repairs of rotator cuff tendons.5,6,10,14 ) unfortunately , the atrophy progresses throughout the nonoperative treatment of rotator cuff tears,7 ) and even after structurally successful surgical tendon repair , it has been suggested that the rotator cuff does not recover from the atrophy.5,13,14 ) these studies asserted that in grade 2 or higher fatty degeneration , using the goutallier classification,2 ) the muscle condition undergoes irreversible change , resulting in permanent loss of muscle function including decrease in muscle strength and an increased rate of secondary tear . however , two studies have suggested the possibility of the improvement of the muscular atrophy . gerber et al.9 ) showed that the muscular atrophy in cases of massive rotator cuff tear was at least stopped and might be reversed in successfully repaired supraspinatus musculotendinous units at least within two years . they showed that , for patients with successful repair , the increase in cross - sectional area of the supraspinatus almost reached significance ( p = 0.054 ) . in addition , thomazeau et al.11 ) reported a reversal of supraspinatus atrophy in half of the successfully repaired cuffs . these two studies support the possibility of the improvement of the atrophy after effective tendon repair . however , the significance of both is limited due to a small number of included cases : 29 cases for the study of gerber et al.9 ) ; and 30 cases for thomazeau et al.11 ) for that reason , we investigated the change of the supraspinatus muscle atrophy with a large number of cases ( 191 cases ) using a very accurate and reliable measuring method12 ) in the present study , and detected a high percentage of the improvement of the atrophy ( 42.4% ) , assessed at a mean of 13.2 3.8 months after operation . the atrophy improved regardless of age , retraction length , or initial degree of atrophy , although a small retraction and initial mild - to - normal atrophy ( occupation ratio > 0.6 ) did not reach a significant level . we are quite convinced of the results , as we used a highly accurate method for the measurement of the supraspinatus muscle atrophy . the method was nearly automated and showed high interobserver ( icc , 0.89 and 0.92 on mdct and mrt1w , respectively ) and intraobserver ( icc , 0.89 to 0.96 and 0.90 to 0.98 on mdct and mrt1w , respectively ) reliabilities , with an excellent mdct - mrt1w correlation ( pcc , 0.78 to 0.87 between mdct and mrt1w , r = 0.76 , p < 0.001).12 ) we also applied very strict criteria for the definitions of the improvement and worsening of the atrophy ( more than a 10% difference in occupation ratio ) . we consider that the mechanical reloading may play a role for the improvement of the muscular atrophy after surgical cuff repair . torn cuffs , without effects of mechanical stress from the proximal humerus , will consequently lead to fatty infiltration or atrophy of cuff muscles.18,21 ) the prolonged load - deprivation status of torn cuff muscles may be similar to that of disuse or immobilization , and skeletal muscle atrophy in such conditions has been well demonstrated in many studies.22,23 ) on the contrary , cuff muscles may be mechanically reloaded after surgical cuff repair , possibly stimulating protein synthesis in atrophied cuff muscles and eventually resulting in recovery of muscular mass and decrease of fatty infiltration.24 ) many factors have been suggested to be related to the initiation of protein synthesis from mechanical loading of the muscle . recent data have shown that mechanical loading of the muscle membrane may induce activation of the mammalian target of rapamycin ( mtor ) , which stimulates the initiation of protein synthesis.25 ) in addition , a number of cytokines including leukemia inhibitory factor , insulin - like growth factor , and integrin have been suggested to have the ability to contribute to loading - induced muscle hypertrophy.26 - 28 ) also , we think that the change in the pennation angle after surgical cuff repair may be another mechanism for the improvement of the muscular atrophy . some authors have suggested that retraction of the muscle belly as a result of full - thickness rotator cuff tear could lead to a change in the pennation angle between the muscle fibers , and this altered angle might allow fat to infiltrate between the fibers of muscle and produce atrophy of the rotator cuff muscle.29 ) therefore , it is possible that the recovery of pennation angle via surgical cuff repair may induce a decrease in the amount of fatty tissue between the muscle fibers and an improvement of muscular atrophy . gerber et al.29 ) demonstrated that continuous elongation and repair of a retracted and atrophied rotator cuff muscle can lead to a restoration of the normal pennation angle , partial reversal of muscle atrophy , and arrest of the progression of fatty infiltration in the animal study using sheep . the failure rate of cuff healing was significantly higher in patients whose atrophy was worsened postoperatively ( 48.5% ) , compared with those with improved atrophy ( 22.2% ) in this study . initial atrophy of the cuff muscles is a well - known prognostic factor for the anatomical results following cuff repair.6,9,30 ) however , it is interesting that not only initial atrophy but also postoperative change of the atrophy is a prognostic factor for structural healing after cuff repair . moreover , while the postoperative atrophy improved distinctly in every other condition except the failure of cuff healing , patients with unhealed cuffs showed worsened atrophy postoperatively , though it was not statistically significant . thus , we can say that not only do the unhealed cuffs show more severe atrophy than healed cuffs ( the mean occupation ratios were 0.39 0.14 and 0.59 0.14 in this study , respectively ; p < 0.001 ) , as many other authors have demonstrated,6,9,14 ) but also the cuffs themselves become more atrophied or at least not improved postoperatively if the cuffs fail to heal after surgical repair . such is consistent with the results of previous studies that stressed the importance of successful cuff repair with the more severe atrophy and poorer function in patients with unhealed cuffs.6,9,13 ) there are several limitations of the current study . first , we evaluated the change of the supraspinatus muscle atrophy alone . in our previous study , we revealed that the infraspinatus muscle atrophy was the most independent prognostic factor for anatomic outcome.10 ) there have also been other studies which stress the influence of the infraspinatus muscle atrophy on the functional and anatomic outcomes after rotator cuff repair.6 ) therefore , it is necessary to evaluate other cuff muscles such as the infraspinatus muscle , in addition to the supraspinatus muscle . however , the occupation ratio we have measured is defined only for the supraspinatus muscle , and it is almost impossible to measure the occupation ratio accurately in the other cuff muscles , as they are not surrounded by bony structure and the outer margin of the muscles is not distinct . we wanted to focus on the change of the supraspinatus muscle atrophy , which we can evaluate accurately by using our method . in addition , the fact that tear retraction can influence occupation ratio measured at one cross - sectional image is a known limitation regarding the measuring method currently used.14 ) while our measuring method using photoshop overcame the weakness of previous methods with regard to accuracy and reliability,12 ) it also measured the occupation ratio with one cross - sectional image of the y - view , as in other current methods which measure the atrophy of cuff muscles.11,16 ) such may not well represent the condition of the entire muscle . this one cross - sectional area of the supraspinatus muscle may be influenced by retraction of the torn supraspinatus muscle.11 ) however , there is no widely accepted volumetric measuring method for the atrophy of rotator cuff muscles , and all studies asserting the atrophy of the rotator cuff muscles to be irreversible and worsening after successful repair have measured and compared it at a scapular y - view , same as our study.5,14 ) as such , our results revealing a significant improvement of the supraspinatus muscle atrophy in a large proportion after successful cuff repair , assessed at the same scapular y - view , may be valuable . another limitation of the study may be the difference in the occupation ratio measured in two different imaging tools . we have compared the modified values derived from the linear regression equation and applied strict criteria of above or below 10% change of the occupation ratio . finally , not all patients who underwent surgical repair of full - thickness rotator cuff tear were included , as we limited the patients to those who underwent both preoperative mri and postoperative mdct with an appropriate y - view . some patients refused mdct postoperatively , because they did not want the rather expensive and time - consuming procedure . this drop - out from the cohort may cause the risk of selection bias of which we are not aware . a well - organized , randomized prospective trial may be needed to confirm our results .

backgroundatrophy of rotator cuff muscles has been considered an irreversible phenomenon . the purpose of this study is to evaluate whether atrophy is truly irreversible after rotator cuff repair.methodswe measured supraspinatus muscle atrophy of 191 patients with full - thickness rotator cuff tears on preoperative magnetic resonance imaging and postoperative multidetector computed tomography images , taken at least 1 year after operation . the occupation ratio was calculated using photoshop cs3 software . we compared the change between pre- and postoperative occupation ratios after modifying the preoperative occupation ratio . in addition , possible relationship between various clinical factors and the change of atrophy , and between the change of atrophy and cuff integrity after surgical repair were evaluated.resultsthe mean occupation ratio was significantly increased postoperatively from 0.44 0.17 to 0.52 0.17 ( p < 0.001 ) . among 191 patients , 81 ( 42.4% ) showed improvement of atrophy ( more than a 10% increase in occupation ratio ) and 33 ( 17.3% ) worsening ( more than a 10% decrease ) . various clinical factors such as age tear size , or initial degree of atrophy did not affect the change of atrophy . however , the change of atrophy was related to repair integrity : cuff healing failure rate of 48.5% ( 16 of 33 ) in worsened atrophy ; and 22.2% ( 18 of 81 ) in improved atrophy ( p = 0.007).conclusionsthe supraspinatus muscle atrophy as measured by occupation ratio could be improved postoperatively in case of successful cuff repair .

METHODS Demographics Surgical Procedures MRI and MDCT Measurement of the Atrophy Using Photoshop on MRI and MDCT Evaluation of the Change of the Atrophy Using Regression Equation Evaluation of the Factors Related with the Change of the Atrophy Statistics RESULTS DISCUSSION

PMC3164153

happy mapping , the analysis of approximately haploid dna samples using the polymerase chain reaction , is a genome mapping method based on random dna breakage and determination of linkage 1 - 2 . this approach is essentially analogous to classical linkage mapping , but the chromosome breakage and segregation are generated by in vitro analogues with gamma - irradiation or shearing . genes / markers are then segregated by diluting the resulting fragments to give aliquots that contain approximately 1 haploid genome equivalent . first , it allows construction of gene / marker maps without cloning , thus avoiding many potential errors and artifacts 3 . second , the approach can also be easily adapted to any desired level of resolution , in particular , to a high resolution of genome maps 4 . third , unlike the radiation hybrid mapping approach , a happy panel contains no carrier dna , which eases specific pcr amplification of markers and makes multiplexing more amenable . lastly , happy mapping does not require any polymorphic markers so any piece of dna can be mapped to a genome region . therefore , the happy mapping approach is applicable to all species , from human 5 , to plant 6 and even to unicellular eukaryotes 7 . however , such a simple and powerful happy mapping method has not yet come into general use even though it was developed by dear and cook as early as 1989 . up to date , only eight maps have been generated using the happy approach and all of them were contributed by the inventors ' group 1 - 8 . the bottleneck of the method as far as we can see , is the lack of faithful amplification of the whole dna that provides enough material for genotyping a large number of markers . it seems that this problem should be now overcome by using a well - developed whole genome amplification method , termed multiple displacement amplification ( mda ) 9 . mda can yield about 20 - 30 ug of product from as few as 1 - 10 copies of genomic dna . in comparison to other whole genome amplification methods , mda provides the most reliable genotypes , highest call rates , best genomic coverage , and lowest amplification bias 10 . therefore , improving the happy approach by solving the bottleneck will help promote its application in genome mapping of many species . amphibians have been used since the 19th century as vertebrate models for investigating many important aspects of biological sciences 11 . in particular , the study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development 12 . in order to meet the xenopus research community 's needs , the national institutes of health xenopus initiative is supporting the development of genetic and genomic resources , such as ( 1 ) complementary deoxyribonucleic acid ( cdna ) libraries and expressed sequence tag ( est ) sequences , ( 2 ) unigene clusters , ( 3 ) full - insert cdna sequences , ( 4 ) a genetic map , ( 5 ) genomic libraries , ( 6 ) a physical map , ( 7 ) genome sequence , ( 8) microarrays , ( 9 ) mutagenesis and phenotyping , and ( 10 ) bioinformatics 13 - 14 . while genomic resources for x. tropicalis have advanced significantly in recent years as described above , tough challenges lie ahead , especially with respect to high - quality assembly of the whole genome for the species . the department of energy 's joint genome institute produced about 1.33 gbp of high quality dna sequences using a seventh - generation inbred nigerian female 15 , but these sequences need to be accurately ordered on 10 chromosomes . here we present our pilot study to demonstrate the feasibility of the happy approach for construction of whole genome maps as reference for a high quality chromosome - based long - range assembly in x. tropicalis . development of a happy panel for genome mapping of x. tropicalis was carried out by following a protocol developed by dear and colleagues 5 for construction of a high resolution metric happy map of human chromosome 14 , but with modifications . a blood sample from an inbred f10 nigerian x. tropicalis animal was collected into 0.9x ssc ( sodium chloride - sodium citrate buffer ) on ice . after collection , the blood cells were resuspended by inverting the tube , and counted using a " bright line " hemocytometer ( hausser scientific , horsham , pa ) . the suspension was then centrifuged at 453 x g for 3 minutes at 4c in a tabletop centrifuge , the supernatant was poured off , and the cells were resuspended in pbsg ( phosphate - buffered saline + 1% glucose ) in aliquots at 6 x 10 cells / ml and 5 x 10 cells / ml , the latter being for high - density controls . each cell suspension was mixed 1:1 with pbsg + 2% agarose ( kept at 37c in a water bath ) , for final concentrations of 3 x 10 cells / ml and 2.5 x 10 cells / ml , respectively , in pbsg + 1% agarose . these mixtures were taken up into approximately 46 or 20 ( high density controls ) 100 l calibrated glass pipets ( vwr international ) and cooled to 4c . each set of agarose strings was allowed to fall by gravity into 150 ml of lysis solution ( 10 mm tris - cl , 1 mm edta , 1% lithium dodecyl sulfate , ph 7.5 ) and incubated on a rotator at 4c . the lysis solution was replaced after intervals of 15 minutes , 30 minutes , 3 hourly intervals , and overnight , and the strings were stored in lysis buffer at 4c until use . the dna contained in the cell - agarose strings was separated in a 0.8% chromosomal grade agarose gel under 1x tae buffer . the cell - agarose strings were cut to fit the length of an electrophoresis well with a razor blade , placed into the well with a spatula and pressed to front of the well such that less than 90% of the height of the well was occupied . the sample plugs were finally sealed into the well with 0.8% low melt agarose in 1x tae . the electrophoresis was performed using chef - dr iii pulsed field electrophoresis system ( bio - rad , hercules , ca ) using s. pombe chromosomes ( new england biolabs , ipswich , ma ) as molecular ladder . 1 ) 24h with a 96 reorientation angle and 1200 sec switch time ; block 2 ) 24h with a 100 reorientation angle and 1500 sec switch time ; and block 3 ) 24h with a 106 reorientation angle and 1800 sec switch time . after electrophoresis was completed , the sides of gel containing yeast standards and high concentration of dna fragments were excised and stained with ethidium bromide and visualized with uv light . a total of 383 plugs were collected with capillary tubes across the gel lane at sizes ranging from ~0.5 mb to ~6 mb . in addition , 7 plugs of agarose were also collected from locations outside of the running lane and were used as controls . whole genome amplification ( wga ) and dna measurement . each plug was then transferred to an individual pcr tube and the first round of wga was performed using the illustra genomiphi v2 dna amplification kit ( ge healthcare lifesciences , piscataway , nj ) . briefly , 9 l of sample buffer was added to each agarose plug and dna was denatured by heating to 95c for 3 min , followed by cooling to 4c on ice . next , 9 l of reaction buffer was mixed with 1 l of enzyme mix on ice and added to the cooled sample . amplification was performed by incubating the sample at 30c for 2 h , after which time the reaction was terminated by heating to 65c for 10 min , followed by cooling to 4c . the second round of wga was accomplished by taking 1 l of amplified dna from the first wga round and repeating each step as described above . samples from the second round of wga were purified by ethanol precipitation with 1.5 m sodium acetate ( ph > 8)/250 mm edta buffer . purified dna pellets were resuspended in te and quantity and quality determined with a nanodrop spectrophotometer . these amplified dna happy lines were tested for a total of 58 x. tropicalis genes , which have the human orthologs representing all autosomes and x chromosome ( see supplementary material : table s1 ) . putative single nucleotide polymorphisms ( snps ) were identified for each frog gene based on comparison between cdna and genomic dna sequences and only one of these sequences were selected to genotype these 383 happy lines using the sequenom genotyping assay ( see supplementary material : table s2 ) . four frog original whole genome dna samples served as positive controls and four blanks were used as negative controls . the genotype scores were converted to 1 when there was a call for a genotype or 0 when there was no call for a genotype and the data were then used for a pair - wise similarity analysis . the genotyping data of 58 markers were considered as character string variables , so there are 58 characters for each happy line . the similarity between two character strings was calculated using a vba ( visual basic for application ) program by considering both character and the order of the characters in strings . only the happy lines that were < 0.85 similar to others were selected to form a 146-sample working panel . a megablast program was used to identify ultraconserved elements ( uces ) between frog scaffolds ( v5.1 ) and the human chromosomes . we selected a region from 0 mb to ~10 mb on human chromosome 1 as our target to test the feasibility of the happy panel for map construction . several pseudo - snps were randomly assigned to each of 48 selected uces and the sequenom assay designed picked 29 of them for multiplex genotyping ( see supplementary material : table s2 ) . like the conventional radiation hybrid ( rh ) mapping , we genotyped the same set of markers twice on the 146-sample working frog happy panel with 5 ng of dna as template . we scored the genotypes by two different methods . in a simple approach , we assigned 1 or 0 to cases where the call rate of replicates was either = 100% or 0% , while ? if call rate = 100% and penalty / yield / skew were acceptable , then 1 was assigned as genotype present . if call rate 50% and best penalty / best yield were acceptable , then ? the dna plugs sampled from the pulsed field electrophoresis gel were the starting materials used in the preparation of the frog happy panel . based on the s. pombe chromosome standard ( new england biolabs , ipswich , ma ) , we were able to collect a total of 383 dna plugs of various sizes , including 56 plugs ranging from 0.5 mb to 1 mb , 71 from 1 mb to 2 mb , 48 from 2 mb to 3 mb , 71 from 3 mb to 4 mb , 32 from 4 mb to 5 mb and 8 from 5 mb to 6 mb , respectively . in addition , 97 were randomly sampled with sizes of less than 3 mb . in order to provide enough dna for the community to map the x. tropicalis genome later , we decided to keep our first round of wga products as stock . only 1 l of the stock was used for a second round of wga as described above . the second round of wga was performed on all 383 happy lines , which produced an average of 64.26 ng dna/l , varying from 13.21 ng dna/l to 133.37 ng dna/l . since the total reaction volume was 20 l , wga resulted in a total average yield of 1,285 ng dna per happy line . 17 observed an average yield of 225 - 350 ng/ l with 50 - 500 pg of input dna after using the genomiphi amplification kit ( amersham biosciences ) . first , we did not carry out gamma - irradiation or ultrasonic shearing to break chromosomes into fragments . chromosome breakage might be only induced during lysis and pulsed field electrophoresis , thus limiting the amount of dna released from the strings and causing low dna flows in the gel . this might also provide initial evidence to support that each happy line contains a random subset of frog genome , rather than whole genome content . second , we used only 1 l of dna from the first round of wga and proceeded to the second round of wga . as such , we did n't really know how much dna was input - it could be much lower than 50 pg . lastly , we used ethanol precipitation to purify wga products , which may have resulted in loss of some amplified dna during the purification process . as shown in figure 1a , happy lines sampled between 2 - 3 mb in size yielded an average 1,737 ng / line , which was the highest ( p<0.05 ) amount of amplified dna produced , followed by sample sizes of 3 - 4 mb ( 1,307 ng / line ) , < 1 mb ( 1,289 ng / line ) and 1 - 2 mb ( 1,256 ng / line ) . the happy lines sampled at 4 - 5 mb and random <3 mb in size gave the lowest ( p<0.05 ) yields of wga dna with 1,097 ng / line and 1,132 ng / line , respectively . samples collected between 5 - 6 mb resulted in an average of 1,239 ng wga dna per line . the authors performed two separate wga amplifications of 10 ng of dna each of one clone of their canine panel rhdf5000 and analyzed the presence in the amplified dna of 74 markers known to be present and 18 known to be absent from the original dna . among 74 positive markers , the authors found 73 present in one of the amplified dna samples , while none of the 18 negative markers were detected . in the second amplified dna sample , the 74 positive markers were all present , whereas all of the 18 negative markers were absent . as such , we feel confident to claim that the second round of wga on our happy panel would not generate any significant loss or bias from the original round of wga amplifications . marker retention rate . a total of 58 gene markers were genotyped on these 383 happy lines using the sequenom assay in two multiplex sets . as shown in supplementary material : table s1 , most of these genes are well distributed among human autosomes and x chromosome and are more than 10 mb apart , except for two genes on human chromosome 14 , three genes on 17 and two genes on x that are less than 6 mb apart . fortunately , we were able to find orthologs of all of these human genes in the newest assembly of the x. tropicalis genome ( v7.1 ) , including 12 on scaffold 1 , 9 on scaffold 2 , 7 on scaffold 6 , 5 on scaffold 4 , 4 each on scaffolds 5 , 7 , 8 and 9 , 3 on scaffold 3 , and only one each on scaffolds 10 , 35 , 83 , 181 , 532 and 656 , respectively . the scaffolds 1 to 10 on the newest assembly are very likely to represent 10 chromosomes in x. tropicalis . only two genes ( pnn and mga ) had a physical distance of less than 0.5 mb , while the remaining genes are separated by at least 3 mb up to 74 mb ( supplementary material : table s1 ) . the happy retention rate was calculated as the percentage of markers per line . among 383 happy lines genotyped on these 58 markers , only one line had a marker retention rate of 0% . for the remainder of the happy lines , the rate varied from 1.72% to 87.93% , but averaged 14.90% . a total of 157 happy lines had a retention rate of < 10% and 20 samples had a retention rate of > 40% . as shown in figure 1b , the sampled fragment size had a significant effect on the marker retention rate . in particular , the happy lines sampled with fragments of 4 - 5 mb in length had a more than double the marker retention rate ( 27.2% ) compared to lines less than 3 mb in size ( 10.2% to 12% ) ( p<0.05 ) and nearly twice the figure compared to lines with 3 - 4 mb in size ( 13.8% ) ( p<0.05 ) . the marker retention rate further increased to 45.5% when the happy lines were sampled with fragments ranging from 5 mb to 6 mb in size ( figure 1b ) ( p<0.05 ) . marker retention rate remained very high ( over 96% ) for most of the 58 markers genotyped on four frog positive controls , because they represent whole genome dna ( figure 2a ) . in contrast , 51 of these 58 genes had a < 20% call rate in the happy lines ( figure 2b ) . the remaining 7 genes include one close to 90% ( gans345 ) and six between 20% and 45% the overall retention rate was 21.7% for all 58 genes in our original happy panel . hukriede and colleagues 19 formed a total of 93 rh lines to map the zebrafish genome . among them , 81 were derived from a 5,000-rad irradiation dose and 12 from a 4,000-rad dose . after genotyping a total of 1,055 markers on the rh panel , the authors observed an overall retention rate of 22% . therefore , we would speculate that our happy panel would be equivalent to a rh panel with a 5,000-rad irradiation dose . based on the genotyping characters ( 1 as presence or 0 as absence ) of 58 markers , we calculated the marker similarity among 383 happy lines using a vba ( visual basic for application ) program , which considers both characters and their orders between a pair of strings / happy lines . we then selected 146 lines that were < 0.85 similar to others and formed a working panel assuming that they randomly represent subsets of the frog whole genome . in order to have enough dna for mapping a large number of markers on the happy working panel , dna amplification was achieved again using wga with the illustra genomiphi v2 dna amplification kit ( ge healthcare lifesciences , piscataway , nj ) . briefly , 2 individual reactions each with 1l of stock dna were separately amplified following the manufacturer 's instructions . after amplification , products were pooled in order to balance any bias that could have occurred during the amplification process . these pooled samples were subsequently purified with the dna clean & concentrator kit ( zymo research , irvine , ca ) . the frog working panel of 146 lines was then used in construction of a pilot happy map in the present pilot study described below . ultraconserved elements ( uces ) are nucleotide sequences that show extreme evolutionary conservation between two or more distinct species . in order to identify uces in the current x. tropicalis genome assembly the human build 37.1 contains 219 contigs ( including 29 unassigned in the assembly ) , while frog v5.1 consists of 4,297 assembled scaffolds . as such , a local megablast was set up to run each of these 4,297 frog scaffolds against all human 22 autosome , x and y chromosome assemblies . we used the aligned length x sequence similarity ( % ) = 25 as an arbitrary cut - off score for collecting the uces between human and frog , which requires at least an aligned length of 25 bp with 100% identity . after removal of uce repeats , 51,498 putative uce orthologs were established between human and frog ( data not shown ) . we selected 29 uces that span a region of 860,330 - 9,416,416 bp on human chromosome 1 as markers for this pilot study to test the feasibility of our working happy panel for map construction of x. tropicalis genome . the locations of uce markers on human chromosome 1 as well as on frog scaffolds are listed in table 1 . these 29 uce markers were genotyped on the working panel of 146 happy lines with one multiplex set using the sequenom assay . among them , seven markers had a retention rate of < 10% and one marker reached a retention rate of > 80% ( table 1 ) . as such , they were excluded from further analysis . therefore , the remaining 21 uce markers were used for map construction . in frog assembly v5.1 , these 21 uces were distributed in scaffolds 34 , 57 , 119 , 160 , 266 , 296 , 341 , 342 , 689 and 830 , respectively ( table 1 ) . statistical analysis using the rh2pt program of the rhmap 3.0 package 20 assigned these 21 uce markers to 2 linkage groups on the basis of a two - locus lod score of at least 4.0 , including 20 markers in group 1 and one marker in group 2 . interestingly enough , these uce markers in group 1 were assigned to scaffold 7 , while the marker in group 2 was assigned to scaffold 5 ( table 1 ) . the results indicate that our happy assignment of markers supports the newest assembly of x. tropicalis genome v7.1 , which integrated 9 scaffolds in v5.1 into a large scaffold in v7.1 . the target region between human chromosome 1 and frog scaffold 7 rearranged during evolution based on these 20 uce markers linked in group 1 ( figure 3 ) . genome assembly comparison of marker locations and orders revealed ten putative conserved segments between human ( build 37.1 ) and frog ( v7.1 ) in the region . in the present study , a conserved segment is defined as a genome region in which uce content and order are parallel , either in the same or in the opposite orientation between frog and human . using the rhmaxlik program of the rhmap 3.0 package 20 , we ordered the same set of 20 uce markers into a first frog happy map with a total length of 12.45 centihaps ( ch ) ( figure 3 ) , which should be equivalent to centirays ( cr ) in radiation hybrid mapping . interestingly , alignment of our frog happy map with the human genome assembly found only eight tentative conserved segments between them instead of ten identified above based on the comparison of genome assemblies . we believed that our happy map might have resulted in superior marker order , because they are consistent with the locations on each scaffold assembled in v5.1 ( table 1 and figure 3 ) . , the whole genome amplification technique and the sequenom snp genotyping assay further improved the conventional happy method and enhanced its capability to map various genomes . the former technique amplified enough dna materials for genotyping a large number of markers , while the latter allowed for relatively high throughput marker genotyping with multiplex assays on the happy lines . such an improved happy technique made it possible for us to develop a first happy panel of the x. tropicalis genome and establish a pilot happy map for the species . in the near future , we anticipate that genotyping of markers on a happy panel should be replaced by next generation sequencing , contributing to formation of happy pipeline for whole genome sequencing , mapping and assembly 22 . table s1 : genes selected for genotyping on the x. tropicalis original happy panel . table s2 : putative snps and their flanking sequences of the gene markers genotyped on the original happy panel of x. tropicalis.table s3 . pseudo - snps and their flanking sequences of uce markers genotyped on the working happy panels of x. tropicalis .

happy mapping was designed to pursue the analysis of approximately random haploid dna breakage samples using the polymerase chain reaction for mapping genomes . in the present study , we improved the method and integrated two other molecular techniques into the process : whole genome amplification and the sequenom snp ( single nucleotide polymorphism ) genotyping assay in order to facilitate whole genome mapping of x. tropicalis . the former technique amplified enough dna materials to genotype a large number of markers , while the latter allowed for relatively high throughput marker genotyping with multiplex assays on the happy lines . a total of 58 x. tropicalis genes were genotyped on an initial panel of 383 happy lines , which contributed to formation of a working panel of 146 lines . further genotyping of 29 markers on the working panel led to construction of a happy map for the x. tropicalis genome . we believe that our improved happy method described in the present study has paved the way for the community to map different genomes with a simple , but powerful approach .

Introduction Materials and Methods Results and Discussion Supplementary Material

PMC2193133

mice expressing transgenes for anti hen egg lysozyme ( hel ) igm / igd b cell antigen receptor ( bcr ) contain large homogeneous populations of b cells that are functionally naive . when anti - hel transgenic mice are crossed to transgenic mice expressing soluble hel , the b cells in the double - transgenic offspring are constantly exposed to low levels of antigenic stimulation and become functionally tolerant ( anergic ) . the tolerant cells lose some responses to antigen , notably bcr - induced proliferation , but retain other responses , including bcr - induced apoptosis , changes in follicular homing , and inhibition of plasmacyte differentiation 123 . binding of hel to the bcr on these cells results in only a subset of the biochemical responses that are triggered by hel in naive anti - hel transgenic b cells . for example , the bcr on tolerant b cells continually activates low intracellular calcium oscillations and extracellular signal regulated kinase ( erk ) , and nuclear factor of activated t cells ( nfat)c pathways but does not produce high calcium elevations or activate the c - jun nh2-terminal kinase ( jnk ) and nuclear factor ( nf)-b pathways 4 . how the bcr is selectively uncoupled from some signaling pathways but not others is unclear . antigen - induced protein tyrosine phosphorylation of the bcr and other molecules is greatly reduced in hel - tolerant b cells 5 . however , tolerant b cells express at normal levels and with normal basal activity proteins important for bcr - induced signal transduction , such as syk , lyn , cd45 , and src homology 2 domain containing protein tyrosine phosphatase ( shp)-1 . here we present evidence for an early , regulated step in bcr signaling that may resolve this paradox . we find that in naive cells , the stimulated bcr becomes associated with a detergent - insoluble fraction containing lyn , and that in tolerant cells , the bcr does not efficiently associate with this fraction . partitioning of the naive bcr occurs in less than 6 s and does not require src family tyrosine kinase activation . this suggests a novel mechanism of maintaining tolerance by physically segregating the bcr from downstream signaling components . mice transgenic for the md4 anti - hel specific bcr were bred either to b6 mice or to mice expressing the soluble hel ml5 transgene 1 . spleens from transgenic animals were harvested and washed in room temperature rpmi media containing 10 mm hepes , ph 7.6 , and 1% fcs . red blood cells were lysed at room temperature with tris ammonium chloride . in experiments using pure b cells , spleen cells were incubated with 10 l anti - b220 microbeads ( miltenyi biotec ) per 10 cells and purified on a minimacs column ( miltenyi biotec ) to purity of > 95% b220 . cells were resuspended at 10 cells per 200-l sample in rpmi , 10 mm hepes . cells were warmed to 37c for 3 min and then stimulated as indicated . in some experiments , pp1 6 was added to cells for 10 min before stimulation at the indicated concentration ( see fig . 4 ) . cells were fractionated to isolate signaling domains essentially as described by marano et al . 7 . cells were centrifuged for 3 min at 800 g after stimulation and resuspended at 10 cells per 200 l of 4c lysis buffer ( 0.5% triton x-100 , 20 mm hepes , ph 7.6 , 10.3% sucrose , 20 mm nacl , 5 mm mgcl2 , 0.1 mg / ml bsa , 2 mm sodium orthovanadate , 2.5 mm pmsf , 500 g / ml aprotinin , 500 g / ml leupeptin , 100 m phenylarsine oxide , 5 m pp1 ) . in time course experiments , 1.3 lysis buffer containing no nacl was added directly to the stimulated cells instead of centrifuging and resuspending . lysed cells were centrifuged at 400 g for 10 min at 4c , and the detergent - soluble fraction was removed from the pellet . the pellet was washed in the same buffer and then resuspended in the same buffer containing 150 mm nacl instead of 20 mm and centrifuged again . salt extract. samples were added to 5 sample buffer , boiled , and resolved on 10% sds - page gels . in all cases , three times as many cell equivalents of salt extract were loaded as compared with corresponding lanes of the detergent - soluble fraction . gels were transferred to polyvinylidene difluoride nylon membranes and immunoblotted with antiphosphotyrosine mab 4g10 ( upstate biotechnology , inc . ) , rabbit anti - syk ( a gift from j. richards and a. defranco , university of california san francisco , san francisco , ca ) , rabbit anti - cd22 ( gift from p. crocker , oxford university , oxford , uk ) , rabbit anti - cd45 , or with a mixture of anti - lyn , anti - ig , anti - ig 8 , and anti- l chain ( southern biotechnology associates , inc . ) . cholera toxin reactive gm1 sphingolipids were detected on the dye front using cholera toxin horseradish peroxidase ( sigma chemical co. ) . for quantitation , blots were developed with protein a i ( icn ) , imaged on a molecular dynamics phosphorimager , and quantitated with imagequant software ( molecular dynamics ) . for fig . 1 e , images of chemiluminescent blots were scanned and analyzed with quantityone ( bio - rad labs . ) . statistical analysis was performed by first standardizing the densitometric values of ig phosphorylation in each experiment by dividing them by the value for the extract fraction in that experiment and expressing them as percent of phosphorylation in extract ( i.e. , extract phosphorylation = 100% ) . the values for relative phosphorylation induced by antigen in the supernatant were then analyzed by one - tailed t test , with h0 : = 100% and h1 : < 100% . mice transgenic for the md4 anti - hel specific bcr were bred either to b6 mice or to mice expressing the soluble hel ml5 transgene 1 . spleens from transgenic animals were harvested and washed in room temperature rpmi media containing 10 mm hepes , ph 7.6 , and 1% fcs . red blood cells were lysed at room temperature with tris ammonium chloride . in experiments using pure b cells , spleen cells were incubated with 10 l anti - b220 microbeads ( miltenyi biotec ) per 10 cells and purified on a minimacs column ( miltenyi biotec ) to purity of > 95% b220 . cells were resuspended at 10 cells per 200-l sample in rpmi , 10 mm hepes . cells were warmed to 37c for 3 min and then stimulated as indicated . in some experiments , pp1 6 was added to cells for 10 min before stimulation at the indicated concentration ( see fig . cells were fractionated to isolate signaling domains essentially as described by marano et al . 7 . cells were centrifuged for 3 min at 800 g after stimulation and resuspended at 10 cells per 200 l of 4c lysis buffer ( 0.5% triton x-100 , 20 mm hepes , ph 7.6 , 10.3% sucrose , 20 mm nacl , 5 mm mgcl2 , 0.1 mg / ml bsa , 2 mm sodium orthovanadate , 2.5 mm pmsf , 500 g / ml aprotinin , 500 g / ml leupeptin , 100 m phenylarsine oxide , 5 m pp1 ) . in time course experiments , 1.3 lysis buffer containing no nacl was added directly to the stimulated cells instead of centrifuging and resuspending . lysed cells were centrifuged at 400 g for 10 min at 4c , and the detergent - soluble fraction was removed from the pellet . the pellet was washed in the same buffer and then resuspended in the same buffer containing 150 mm nacl instead of 20 mm and centrifuged again . samples were added to 5 sample buffer , boiled , and resolved on 10% sds - page gels . in all cases , three times as many cell equivalents of salt extract were loaded as compared with corresponding lanes of the detergent - soluble fraction . gels were transferred to polyvinylidene difluoride nylon membranes and immunoblotted with antiphosphotyrosine mab 4g10 ( upstate biotechnology , inc . ) , rabbit anti - syk ( a gift from j. richards and a. defranco , university of california san francisco , san francisco , ca ) , rabbit anti - cd22 ( gift from p. crocker , oxford university , oxford , uk ) , rabbit anti - cd45 , or with a mixture of anti - lyn , anti - ig , anti - ig 8 , and anti- l chain ( southern biotechnology associates , inc . ) . cholera toxin reactive gm1 sphingolipids were detected on the dye front using cholera toxin horseradish peroxidase ( sigma chemical co. ) . for quantitation , blots were developed with protein a i ( icn ) , imaged on a molecular dynamics phosphorimager , and quantitated with imagequant software ( molecular dynamics ) . for fig 1 e , images of chemiluminescent blots were scanned and analyzed with quantityone ( bio - rad labs . ) . statistical analysis was performed by first standardizing the densitometric values of ig phosphorylation in each experiment by dividing them by the value for the extract fraction in that experiment and expressing them as percent of phosphorylation in extract ( i.e. , extract phosphorylation = 100% ) . the values for relative phosphorylation induced by antigen in the supernatant were then analyzed by one - tailed t test , with h0 : = 100% and h1 : < 100% . to track the subcellular location of bcr and signaling molecules on naive and tolerant b cells , we adapted a fractionation technique described by marano et al . 7 . spleen b cells lysed in 0.5% triton x-100 , sucrose , and low salt buffer at 4c release most of their bcr into the detergent - soluble supernatant . however , after stimulation with hel antigen , a portion of the bcr from anti - hel transgenic b cells becomes insoluble in this buffer but can be released by increasing the nacl concentration to 150 mm . igm and igd bcr complexes containing h chain , l chain , ig , and ig become detergent insoluble and salt extractable after stimulation ( fig . the detergent - soluble and the salt extract fractions , ig in the salt extract fraction was more highly phosphorylated ( fig . the salt extract fraction of unstimulated and stimulated cells contains 1030% of cellular lyn ( 15% in the experiment shown in fig . 1 b ) . a similar fraction of gm1 sphingolipids in the cell , detected by cholera toxin western blotting , was also present in the salt - extracted fraction . the compartmentalization of the bcr to the detergent - insoluble fraction is consistent with the idea that to generate antigen - induced signals , kinase activity must be separated from phosphatase activity 9 . to determine whether there was segregation of the bcr from other membrane proteins , other membrane - associated molecules were examined by western blotting . the salt extract fraction contains < 0.3% of cellular cd45 and no detectable cd22 ( fig . only 2.5% of cellular ig and ig is present in the salt extract fraction before stimulation ( fig . these data show that the salt extract fraction is enriched for certain positive signaling molecules and depleted for certain negative signaling molecules . the exclusion of cd45 from the detergent - insoluble fraction is particularly significant , as this transmembrane tyrosine phosphatase represents most of the phosphatase activity at the membrane and could serve to inhibit signal transduction 9 . as early signaling events are inhibited in tolerant b cells , we analyzed whether or not bcrs on tolerant cells could partition into the salt extract fraction after stimulation . the salt extract fraction of tolerant b cells appeared similar to unstimulated naive cells in that it contained similar amounts of lyn , gm1 sphingolipids , and basal amounts of the bcr . however , only a small increase in the amount of bcr in the salt extract was detected after stimulation of tolerant b cells ( fig . quantitation of i - developed western blots showed a sevenfold increase in the amount of the ig complex in the salt extract of naive cells after stimulation , whereas no increase was detectable in the amount of the ig complex in the salt extract of tolerant cells after stimulation ( fig . spleen b cells from some tolerant mice do exhibit a slight increase ( less than twofold ) in the amount of ig in the salt extract after stimulation , consistent with weak residual signals seen in tolerant b cells from some double - transgenic mice 5 . this experiment was done seven times with similar results , and experiments using pure b cells ( > 95% b220 ) showed the same results . because inducibly phosphorylated bcr was preferentially associated with the salt extract fraction , we wanted to determine whether partitioning into this domain was an early step after antigen binding . in particular , does partitioning precede or follow an increase in ig phosphorylation by src family kinases ? both partitioning and increase in ig phosphorylation in response to antigen binding occur within 6 s of antigen binding at 37c ( fig . however , when stimulation is performed at 4c , partitioning still occurs within 6 s , whereas little tyrosine phosphorylation of ig is induced until 30 s. some increase in phosphorylation of ig and ig is detected in the salt extract fraction at 6 s , but this may be due to the increased amount of ig and ig in the fraction rather than any increase in phosphorylation levels . the phosphorylation of this material increases between 6 and 30 s , with no additional increase in amount of ig and ig. to examine the role of src kinases in partitioning of the bcr into detergent - insoluble domains , src family kinase activity was blocked using the kinase inhibitor pp1 10 . pp1 eliminated both the basal and induced tyrosine phosphorylation of most detergent - soluble cellular substrates , including ig and ig receptor subunits , but did not inhibit partitioning of bcr into the detergent - insoluble salt extract fraction ( fig . these experiments show that partitioning into the salt extract does not require src family kinase activation and may precede the activation of these kinases . the importance of spatial localization of both receptors and signaling molecules into subcellular structures is becoming appreciated in many biological systems . for example , proteins containing pdz domains concentrate ion channels with downstream effector molecules 11 . in lymphocytes , the adapter proteins lat ( linker for activation of t cells ; reference 12 ) and blnk ( b cell linker protein ; reference 13 , 14 ) are necessary to bring together molecules such as grb2 and phospholipase c . on the surfaces of activated t cells , the tcr and certain signaling molecules form an immunological synapse structure surrounded by adhesion molecules 1516 . the data presented here suggest that the localization of antigen receptors on b lymphocytes into signaling domains is an important step during lymphocyte activation . the block in partitioning of tolerant cells ' bcrs may underlie the changes in tolerant bcr signaling that lead to different biological responses from naive b cells . many studies have documented bcr association with the cytoskeleton or reorganization of the cytoskeleton after b cell stimulation 17181920 . the cytoskeleton is an important part of lymphocyte activation and is clearly necessary for the formation of the ordered structure of the tcr described above 152122 . partitioning of bcr into signaling domains shown here may be an early step in the association with the cytoskeleton , as the buffer used stabilizes the cytoskeleton of the lysed cells 23 . the detergent - insoluble salt extracts may correspond to membrane rafts , glycolipid- and gpi - linked , protein - rich domains of the cell surface also known as detergent - resistant membranes , detergent - insoluble glycolipid domains , or glycolipid - enriched membranes 24 . consistent with this hypothesis , the salt extracts are enriched for lyn , a doubly acylated src kinase , and gm1 sphingolipids , both known to be raft components 24 . in leukocytes , the fcr on mast cells was the first receptor shown to enter membrane rafts after stimulation 2526 . in addition to src kinases , rafts also contain the t cell adapter protein lat 29 , consistent with the hypothesis that rafts concentrate signaling molecules together with antigen receptors . basophilic leukemia mast cells and jurkat t cells , clustering of raft components into patches reveals high concentrations of actin microfilaments underlying the patch 3031 . partitioning of antigen receptors into membrane rafts could represent the first step in their cytoskeletal association and in the formation of more ordered structures . partitioning of bcrs into the detergent - insoluble signaling domains described here may represent the first step in bcr signaling , allowing efficient phosphorylation of the bcr by src family kinases . concentration of src kinases and antigen receptors , along with exclusion of tyrosine phosphatases from the same sites , may be necessary to allow efficient phosphorylation of immunoreceptor tyrosine - based activation motif ( itam)-containing receptor subunits . cd45 is required to dephosphorylate the inhibitory site in src family kinases , a necessary step in antigen receptor signaling . however , cd45 can also inhibit src family kinases , and it may therefore be necessary to sequester kinase activity from phosphatase activity to propagate signals through the bcr 9 . the failure to sequester kinase activity from phosphatase activity in anergic b cells may explain their inability to generate signals efficiently through the bcr . the diminished partitioning of bcrs into signaling domains in tolerant b cells may explain their ability to generate only a subset of the normal signals in response to antigen . signaling events that are blocked in tolerant cells , such as protein tyrosine phosphorylation , high intracellular calcium flux , nf-b translocation , and jnk activation 45 , may depend on efficient partitioning into signaling domains . by contrast , activation of the erk cascade and the low calcium oscillations responsible for continued nfatc shuttling to the nucleus are not blocked in tolerant cells 4 . it will be important to distinguish whether the latter signaling events can be activated without entry of the bcr into signaling domains , or whether the small amount of bcr that is found in signaling domains in tolerant b cells is sufficient to activate these pathways . in summary , we have shown that the bcr becomes associated with a fraction of the cell enriched for signaling molecules such as lyn and depleted for molecules such as cd45 . partitioning of the bcr occurs within seconds of antigen binding and appears to precede tyrosine kinase activation . partitioning is blocked in tolerant b cells , suggesting that bcr partitioning is an early , regulated step in antigen receptor signaling . future work will need to determine what mediates the partitioning of the bcr into detergent - insoluble domains . since this event appears to precede src kinase activity , it may result from another enzyme or from a physical change in the bcr that changes its affinity for lipid or protein membrane constituents .

signal transduction through the b cell antigen receptor ( bcr ) is altered in b cells that express a receptor that recognizes self - antigen . to understand the molecular basis for the change in signaling in autoreactive b cells , a transgenic model was used to isolate a homogeneous population of tolerant b lymphocytes . these cells were compared with a similar population of naive b lymphocytes . we show that the bcr from naive b cells enters a detergent - insoluble domain of the cell within 6 s after antigen binding , before a detectable increase in bcr phosphorylation . this fraction appears to be important for signaling because it is enriched for lyn kinase but lacks cd45 tyrosine phosphatase and because the bcr that moves into this domain becomes more highly phosphorylated . partitioning of the bcr into this fraction is unaffected by src family kinase inhibition . tolerant b cells do not efficiently partition the bcr into the detergent - insoluble domain , providing an explanation for their reduced tyrosine kinase activation and calcium flux in response to antigen . these results identify an early , regulated step in antigen receptor signaling and self - tolerance .

Introduction Materials and Methods Mice. Stimulation. Fractionation. Western Blotting. Results Discussion

PMC5324595

participants were drawn from a national institute of mental healthfunded study of risk factors for the development of ptsd in a low socioeconomic , primarily african american urban population . participants were recruited from waiting rooms in the diabetes , gynecology , and primary care medical ( nonpsychiatric ) clinics at grady memorial hospital , a publicly funded hospital in atlanta , georgia . interviewers approached participants waiting for appointments . to be eligible for participation , subjects had to be at least 18 years old and able to give informed consent . the investigation was carried out in accordance with the latest version of the declaration of helsinki and informed consent of the participants was obtained after the nature of the procedures had been fully explained . after signing the informed consent approved by the emory institutional review board , an initial interview was administered with questionnaires regarding trauma history and psychological variables . trained research assistants administered this interview by reading each question aloud to participants ; interviews took 4575 minutes to complete ( duration largely dependent on the participant 's trauma history and symptoms ) . a more detailed and comprehensive assessment of psychological functioning was conducted in an associated study , which included a subset of participants who completed the initial assessment ( see gillespie et al . , 2009 , for full details regarding study procedures ) . the current study includes 260 females with a mean age of 39.76 years ( standard deviation [ sd ] = 11.73 years ) who completed both the initial and secondary assessments . the majority of the participants selfidentified as african american ( 96.9% , n = 252 ) . the remaining ethnicities represented included caucasian ( 1.1% , n = 3 ) and mixed or other ( 2.0% , n = 5 ) . this was a highly traumatized sample , with all participants reporting the experience of at least one type of trauma in their lifetime ( mean [ m ] = 4.97 , sd = 2.96 ) . the ders ( gratz & roemer , 2004 ) is a psychometrically validated , selfreport , 36item measure of emotion regulation difficulties . it measures several aspects of emotion regulation , including awareness and understanding of one 's emotions , acceptance of negative emotions , the ability to successfully engage in goaldirected behavior and control impulsive behavior when experiencing negative emotions , and the ability to use situationally appropriate emotion regulation strategies . for the present study , the internal consistency of the ders total scale was high ( = 0.92 ) . the caps is an intervieweradministered , psychometrically validated diagnostic instrument measuring ptsd ( blake et al . , 1990 , 1995 ) . it includes items that rate social and occupational functioning , global ptsd symptom severity , and the validity of a participant 's responses . the caps assesses current ptsd and yields a continuous measure of the severity of overall ptsd and the three dsmivtr symptom clusters ( i.e. , reexperiencing , avoidance and numbing , and arousal , or criterion b , c , and d , respectively ) . the frequency and intensity scores for each of the 17 diagnostic criteria are summed to arrive at a total severity score , and each criterion also has a total severity score based on the individual items for the respective criterion ( five for criterion b , seven for criterion c , and five for criterion d ) . the mini ( sheehan et al . , 1998 ) is a structured diagnostic interview that assesses mood , anxiety , substance use , and psychotic disorders based on dsmivtr criteria . the mini has shown good reliability and validity across different samples ( lecrubier et al . , 1997 ; for the present study , only the current ad section was used . within our sample , 24.2% ( n = 63 ) affirmed to drinking three or more alcoholic drinks in a 6hour period on three or more occasions within the past year , and 6.9% met full dsmiv criteria for ad . for all analyses , we created a continuous variable to measure ad in an effort to capture overall severity of ad symptoms . the tei ( gillespie et al . , 2009 ; schwartz et al . , 2005 ) is a 14item screening instrument for lifetime history of traumatic events witnessed or experienced . the tei was used in this investigation to assess total number of types of traumas exposed to in one 's lifetime ; therefore , the types of traumatic events either witnessed or experienced were summed to obtain an overall trauma load score . the overall analytic approach was to examine the predictive utility of current ptsd symptoms and ed on reported ad symptoms . . however , the level of skewness ( range : 0.421.66 ) as well as the level of kurtosis ( range : 0.631.43 ) in this sample fell within acceptable parameters for the sample size on all variables ( tabachnick & fidell , 2000 ) . descriptive statistics of the sample and all variables of interest were computed and are presented in table 1 . sd = standard deviation ; tei = traumatic events inventory ; mini = mini international neuropsychiatric interview ; ders = difficulties in emotion regulation scale ; caps = clinicianadministered ptsd scale . then , based on the results of the correlational analyses , two hierarchical linear regression models were used to examine the unique predictive value of ptsd symptoms and ed ( total and separated by the individual dimensions of ed ) on ad symptoms . both because it will provide a more nuanced understanding of associations between variables and because results have differed by ptsd symptom cluster in previous research ( debell et al . , 2014 ; saladin , brady , dansky , & kilpatrick , 1995 ) , we analyzed the three symptom clusters of ptsd separately . because research has shown that rates of ad vary by age and trauma exposure ( kessler , berglund , et al . , 2005 ; turner & lloyd , 1995 ) , these variables were included as covariates in analyses . finally , mediation analyses were performed with indirect ( preacher & hayes , 2008 ) for spss to test whether ed had an indirect effect on the relationship between current avoidance and numbing ptsd symptoms and ad symptoms . bootstrapping with 95% confidence intervals ( cis ) was used to determine significance of mediation effects . bootstrapping is a particularly useful method for interpreting mediation analyses because it does not assume that sampling distributions of the indirect effects are normally distributed ( preacher & hayes , 2008 ) . reexperiencing and hyperarousal symptoms were also controlled for in an effort to determine the specific association between avoidance and numbing and ad symptoms . participants were drawn from a national institute of mental healthfunded study of risk factors for the development of ptsd in a low socioeconomic , primarily african american urban population . participants were recruited from waiting rooms in the diabetes , gynecology , and primary care medical ( nonpsychiatric ) clinics at grady memorial hospital , a publicly funded hospital in atlanta , georgia . interviewers approached participants waiting for appointments . to be eligible for participation , subjects had to be at least 18 years old and able to give informed consent . the investigation was carried out in accordance with the latest version of the declaration of helsinki and informed consent of the participants was obtained after the nature of the procedures had been fully explained . after signing the informed consent approved by the emory institutional review board , an initial interview was administered with questionnaires regarding trauma history and psychological variables . trained research assistants administered this interview by reading each question aloud to participants ; interviews took 4575 minutes to complete ( duration largely dependent on the participant 's trauma history and symptoms ) . a more detailed and comprehensive assessment of psychological functioning was conducted in an associated study , which included a subset of participants who completed the initial assessment ( see gillespie et al . , 2009 , for full details regarding study procedures ) . the current study includes 260 females with a mean age of 39.76 years ( standard deviation [ sd ] = 11.73 years ) who completed both the initial and secondary assessments . the majority of the participants selfidentified as african american ( 96.9% , n = 252 ) . the remaining ethnicities represented included caucasian ( 1.1% , n = 3 ) and mixed or other ( 2.0% , n = 5 ) . this was a highly traumatized sample , with all participants reporting the experience of at least one type of trauma in their lifetime ( mean [ m ] = 4.97 , sd = 2.96 ) . the ders ( gratz & roemer , 2004 ) is a psychometrically validated , selfreport , 36item measure of emotion regulation difficulties . it measures several aspects of emotion regulation , including awareness and understanding of one 's emotions , acceptance of negative emotions , the ability to successfully engage in goaldirected behavior and control impulsive behavior when experiencing negative emotions , and the ability to use situationally appropriate emotion regulation strategies . for the present study , the internal consistency of the ders total scale was high ( = 0.92 ) . the caps is an intervieweradministered , psychometrically validated diagnostic instrument measuring ptsd ( blake et al . it includes items that rate social and occupational functioning , global ptsd symptom severity , and the validity of a participant 's responses . the caps assesses current ptsd and yields a continuous measure of the severity of overall ptsd and the three dsmivtr symptom clusters ( i.e. , reexperiencing , avoidance and numbing , and arousal , or criterion b , c , and d , respectively ) . the frequency and intensity scores for each of the 17 diagnostic criteria are summed to arrive at a total severity score , and each criterion also has a total severity score based on the individual items for the respective criterion ( five for criterion b , seven for criterion c , and five for criterion d ) . the mini ( sheehan et al . , 1998 ) is a structured diagnostic interview that assesses mood , anxiety , substance use , and psychotic disorders based on dsmivtr criteria . the mini has shown good reliability and validity across different samples ( lecrubier et al . , 1997 ; for the present study , only the current ad section was used . within our sample , 24.2% ( n = 63 ) affirmed to drinking three or more alcoholic drinks in a 6hour period on three or more occasions within the past year , and 6.9% met full dsmiv criteria for ad . for all analyses , we created a continuous variable to measure ad in an effort to capture overall severity of ad symptoms . the tei ( gillespie et al . , 2009 ; schwartz et al . , 2005 ) is a 14item screening instrument for lifetime history of traumatic events witnessed or experienced . the tei was used in this investigation to assess total number of types of traumas exposed to in one 's lifetime ; therefore , the types of traumatic events either witnessed or experienced were summed to obtain an overall trauma load score . the ders ( gratz & roemer , 2004 ) is a psychometrically validated , selfreport , 36item measure of emotion regulation difficulties . it measures several aspects of emotion regulation , including awareness and understanding of one 's emotions , acceptance of negative emotions , the ability to successfully engage in goaldirected behavior and control impulsive behavior when experiencing negative emotions , and the ability to use situationally appropriate emotion regulation strategies . for the present study , the internal consistency of the ders total scale was high ( = 0.92 ) . the caps is an intervieweradministered , psychometrically validated diagnostic instrument measuring ptsd ( blake et al . , 1990 , 1995 ) . it includes items that rate social and occupational functioning , global ptsd symptom severity , and the validity of a participant 's responses . the caps assesses current ptsd and yields a continuous measure of the severity of overall ptsd and the three dsmivtr symptom clusters ( i.e. , reexperiencing , avoidance and numbing , and arousal , or criterion b , c , and d , respectively ) . the frequency and intensity scores for each of the 17 diagnostic criteria are summed to arrive at a total severity score , and each criterion also has a total severity score based on the individual items for the respective criterion ( five for criterion b , seven for criterion c , and five for criterion d ) . the mini ( sheehan et al . , 1998 ) is a structured diagnostic interview that assesses mood , anxiety , substance use , and psychotic disorders based on dsmivtr criteria . the mini has shown good reliability and validity across different samples ( lecrubier et al . , 1997 ; for the present study , only the current ad section was used . within our sample , 24.2% ( n = 63 ) affirmed to drinking three or more alcoholic drinks in a 6hour period on three or more occasions within the past year , and 6.9% met full dsmiv criteria for ad . for all analyses , we created a continuous variable to measure ad in an effort to capture overall severity of ad symptoms . the tei ( gillespie et al . , 2009 ; schwartz et al . , 2005 ) is a 14item screening instrument for lifetime history of traumatic events witnessed or experienced . the tei was used in this investigation to assess total number of types of traumas exposed to in one 's lifetime ; therefore , the types of traumatic events either witnessed or experienced were summed to obtain an overall trauma load score . the overall analytic approach was to examine the predictive utility of current ptsd symptoms and ed on reported ad symptoms . . however , the level of skewness ( range : 0.421.66 ) as well as the level of kurtosis ( range : 0.631.43 ) in this sample fell within acceptable parameters for the sample size on all variables ( tabachnick & fidell , 2000 ) . descriptive statistics of the sample and all variables of interest were computed and are presented in table 1 . sd = standard deviation ; tei = traumatic events inventory ; mini = mini international neuropsychiatric interview ; ders = difficulties in emotion regulation scale ; caps = clinicianadministered ptsd scale . then , based on the results of the correlational analyses , two hierarchical linear regression models were used to examine the unique predictive value of ptsd symptoms and ed ( total and separated by the individual dimensions of ed ) on ad symptoms . both because it will provide a more nuanced understanding of associations between variables and because results have differed by ptsd symptom cluster in previous research ( debell et al . , 2014 ; saladin , brady , dansky , & kilpatrick , 1995 ) , we analyzed the three symptom clusters of ptsd separately . because research has shown that rates of ad vary by age and trauma exposure ( kessler , berglund , et al . , 2005 ; turner & lloyd , 1995 ) , these variables were included as covariates in analyses . finally , mediation analyses were performed with indirect ( preacher & hayes , 2008 ) for spss to test whether ed had an indirect effect on the relationship between current avoidance and numbing ptsd symptoms and ad symptoms . bootstrapping with 95% confidence intervals ( cis ) was used to determine significance of mediation effects . bootstrapping is a particularly useful method for interpreting mediation analyses because it does not assume that sampling distributions of the indirect effects are normally distributed ( preacher & hayes , 2008 ) . reexperiencing and hyperarousal symptoms were also controlled for in an effort to determine the specific association between avoidance and numbing and ad symptoms . to determine the extent of association between our predictor variables and current ad symptoms , we first calculated pearson correlation coefficients . as shown in table 2 , of the current ptsd variables measured , only ptsd symptom cluster c ( avoidance and numbing ) was significantly positively correlated with current ad symptoms ( p < 0.01 ) . total ed and all six dimensions of ed measured by the ders also were significantly positively correlated with current ad symptoms ( p 0.001 ) . furthermore , overall ed and all six dimensions of ed were significantly correlated with each ptsd symptom cluster ( p < 0.01 ) , except for a nonsignificant association between lack of emotional awareness and ptsd symptom cluster b. a hierarchical linear regression model was first run to test the differential associations of ptsd symptoms and ed with current ad symptoms . age and overall trauma load were entered in the first step of each regression as covariates . as shown in table 3 , when the three ptsd symptom clusters were entered into the second step of the regression model , they were significantly predictive of ad symptoms ( p < 0.05 ) . of the three symptom clusters included in the model , only avoidance and numbing symptoms were predictive ( p < 0.01 ) of ad symptoms above and beyond the effects of age and trauma load . when overall ed was included in the model in step 3 , the effect of avoidance and numbing symptoms was no longer significant . overall , ed was significantly predictive of ad symptoms ( p < 0.001 ) , suggesting that ed may mediate the relationship between avoidance and numbing symptoms of ptsd and current ad symptoms . age and trauma exposure were not significantly predictive of ad symptoms at any step . linear regression model predicting current alcohol dependence symptom severity from current ptsd symptoms and overall ed note . ptsd = posttraumatic stress disorder ; ed = emotion dysregulation ; tei = traumatic events inventory ; ders = difficulties in emotion regulation scale . we also examined the effects of the six dimensions of ed independently to determine if there were differential effects ( see table 4 ) . similar results emerged in step 2 of this model ; more specifically , only avoidance and numbing symptoms were significantly predictive of ad symptoms ( p < 0.01 ) . in step 3 , ed dimensions were significantly predictive of current ad symptoms , accounting for 14% of additional variance in explaining alcohol symptoms independent of age , trauma exposure , and current ptsd symptoms . as shown in table 4 , the only ed dimension significant at step 3 was difficulty with goal directed behavior in the presence of strong emotions after controlling for the effects of age , trauma exposure , ptsd symptoms , and the other dimensions of ed . again , avoidance and numbing ptsd symptoms were no longer significant when ed dimensions were included in the model , suggesting a potential mediation effect . linear regression model predicting current alcohol dependence symptom severity from current ptsd symptoms and the six dimensions of ed note . ptsd = posttraumatic stress disorder ; ed = emotion dysregulation ; tei = traumatic events inventory ; ders = difficulties in emotion regulation scale . we used preacher and hayes ( 2008 ) indirect to formally test the mediation model suggested by the linear regression analyses . age , trauma load , and caps subscales for reexperiencing symptoms ( criterion b ) and hyperarousal symptoms ( criterion d ) were entered in the model as covariates . as shown in figure 1 , overall ed showed a full indirect effect on the relationship between avoidance and numbing ptsd symptoms and current ad symptoms ( overall model : r = 0.15 , f = 7.49 , p < 0.001 ; bias corrected 95% ci [ 0.01 , 0.04 ] ) . a similar full indirect effect was found when running the mediation model using the difficulty with goaldirected behavior dimension of the ders ( overall model : r = 0.13 , f = 6.42 , p < 0.001 ; bias corrected 95% ci [ 0.01 , 0.03 ] ) . again , age , trauma load , and caps subscales for reexperiencing symptoms ( criterion b ) and hyperarousal symptoms ( criterion d ) were entered in the model as covariates and none of the covariates were significant . in support of previous research ( e.g. , brady et al . , 2004 ; kessler et al . , 1995 ; kessler , chiu , et al . , 2005 ; mcfarlane , 1998 ; stewart et al . , 1998 ; swendsen et al . , 2010 ) , we found a significant association between ptsd symptoms and ad symptoms in this sample of urban , minority females . when examining the specific ptsd symptom clusters , only avoidance and numbing ptsd symptoms were significantly correlated to ad symptoms . lifetime trauma load was not significantly related to ad , suggesting that within this sample it was not the number of traumatic events experienced but the presence of anxiety symptoms attributable to ptsd that contributed to ad . this adds to research suggesting that alcohol is used as an avoidance strategy in ptsd patients ( brady et al . , 2004 ; hayes et al . , 1996 ) and highlights that trauma load itself may not increase risk for dependence , although it may increase risk for the development of ptsd which might then increase risk for alcohol use problems . our results also showed that all dimensions of ed were positively significantly correlated with ad , supporting previous findings that ed and ad are associated ( bonnmiller et al . , we also found that ed had a full indirect effect on the relationship between current ptsd avoidance and numbing symptoms and current ad symptoms among traumatized females . moreover , difficulties engaging in goaldirected behaviors had an indirect effect on this relationship above and beyond the other dimensions of ed in this sample . these findings suggest that poor emotion regulation in general , and an inability to stay focused on goaldirected behaviors when faced with strong emotions in particular , may help explain why women with ptsd are driven to ad . individuals with ptsd may experience greater negative emotions , and if they do not have adequate emotion regulation capabilities , then they might use alcohol to avoid those negative emotions ( cooper , russell , skinner , frone , & mudar , 1992 ; hayes et al . , 1996 ; khantzian , 1997 ; stewart , zvolensky , & eifert , 2002 ; tull et al . , 2007 ) . the specific use of alcohol as an avoidance strategy may help explain why there was only a significant association between ad symptoms and the avoidance and numbing symptom cluster , although that can not be determined from the current study based on its crosssectional nature . it is unclear why having difficulties with goaldirected behaviors in the presence of strong emotions in particular was related to ad in these women . one possibility is that if negative emotions repeatedly resurface while attempting to focus and engage in goaldirected behavior , then women with a tendency to try to control rather than modulate emotions may choose alcohol as a way to numb themselves from the negative situation . this behavior pattern would be analogous to the selfmedication theory , whereby individuals suffering from ptsd turn to alcohol abuse as a way to avoid fearful memories and to dampen negative emotions brought on by those memories ( brady et al . , 2004 ; khoury , tang , bradley , cubells , & ressler , 2010 ; leeies , pagura , sareen , & bolton , 2010 ; quitkin , rifkin , kaplan , & klein , 1972 ; read , merrill , griffin , bachrach , & khan , 2014 ) . selfmedicating with alcohol among women with ptsd may involve ed processes . lehavot , stappenbeck , luterek , kaysen , and simpson ( 2014 ) found that coping motives ( i.e. , drinking to avoid negative affect ) were associated with amount of alcohol use in women . additional research is needed to determine how in fact ed does relate to the selfmedication theory in traumatized females with ptsd . future studies of urban females suffering from ptsd would benefit from including direct measures of selfmedication and drinking motives in addition to ed dimensions to further delineate how these constructs are interrelated with each other and with alcohol problems . the results of this study provide further support for targeting emotion regulation skills in therapy for traumatized individuals . interventions for individuals suffering from ptsd and ad symptoms should be broad enough to support the needs of comorbidities and underlying areas of dysfunction ( e.g. , ed ) . the most common and effective therapy for ptsd treatment is prolonged exposure therapy ( see mrkved et al . , 2014 , for review ) . while early studies warned against antagonistic effects of exposure therapy in people with comorbid addiction disorders ( e.g. , becker , zayfert , & anderson , 2004 ; pitman et al . , 1991 ) , studies that are more recent have shown successful treatment in populations with comorbid ptsd and ad using various types of exposure therapy ( e.g. , berenz , rowe , schumacher , stasiewicz , & coffey , 2012 ; nosen , littlefield , schumacher , stasiewicz , & coffey , 2014 ) . it is possible that targeting emotion regulation skills in addition to traumafocused treatment would be beneficial for traumatized females . theoretically , improving emotion regulation abilities in women suffering from ptsd and ad may lessen ad symptoms , allowing for a more successful intervention of ptsd symptoms later in treatment . this has been shown in other populations , such as those with exposure to childhood sexual abuse ( cloitre , koenen , cohen , & han , 2002 ; cloitre , stovallmcclough , miranda , & chemtob , 2004 ) , and researchers should continue to examine how best to incorporate emotion regulation training into treatment protocols . we can not say for certain that the onset of ptsd and ed symptoms preceded the onset of ad symptoms in every participant , and therefore we can not determine directional causality . prospective , longitudinal studies are required to examine the temporal onset of ed , ptsd , and ad symptoms . also , although the ders is a validated measure of ed , it is inherently biased by its selfreport nature . finally , our sample was largely lowincome african american females , which limits the generalizability of our findings to other populations . however , this weakness is balanced by the public health importance of studying these variables in an often underresearched and underserved population with such high rates of trauma exposure as well as mental health problems . we can not say for certain that the onset of ptsd and ed symptoms preceded the onset of ad symptoms in every participant , and therefore we can not determine directional causality . prospective , longitudinal studies are required to examine the temporal onset of ed , ptsd , and ad symptoms . also , although the ders is a validated measure of ed , it is inherently biased by its selfreport nature . finally , our sample was largely lowincome african american females , which limits the generalizability of our findings to other populations . however , this weakness is balanced by the public health importance of studying these variables in an often underresearched and underserved population with such high rates of trauma exposure as well as mental health problems . this study contributes to the research on ptsd and ad comorbidity by identifying a potential underlying factor that may help explain this wellestablished relationship . in a highly traumatized , low socioeconomic , urban sample of women , we found that ed had a full indirect effect on the association between avoidance and numbing ptsd symptoms and ad symptoms ; therefore , ed may be an important component of what drives this relationship . the current findings provide support for growing literature suggesting the addition of therapeutic interventions for emotion regulation difficulties in traumatized patient populations , especially those with comorbid ptsd and ad .

objectivethe purpose of this study was to examine how emotion dysregulation ( ed ) might help explain the relationship between posttraumatic stress disorder ( ptsd ) and alcohol dependence ( ad ) symptoms in females.methodparticipants included 260 women from primary , diabetes , and gynecological clinics of an urban public hospital . this is a primarily african american sample ( 96.9% ) , including individuals reporting exposure to at least 1 traumatic event . we examined the associations and predictability patterns between severity of ptsd symptoms , ed , and ad symptoms.resultsusing linear regression analyses , ptsd avoidance and numbing symptoms and ed were significant predictors of ad symptoms . when looking at specific dimensions of ed , one 's inability to engage in goaldirected behavior under strong emotional influences showed a full indirect effect on the relationship between ptsd avoidance and numbing symptoms and ad symptoms.conclusionour findings suggest that having poor emotion regulation skills may help explain why females with ptsd become dependent on alcohol .

Method Procedure Participants Measures Difficulties in Emotion Regulation Scale (DERS) ClinicianAdministered PTSD Scale MINI International Neuropsychiatric Interview Traumatic Events Inventory (TEI) Statistical Analysis Results Discussion Limitations Conclusion

PMC3137837

keeping in mind the medical and surgical aspects , the nerve supply of arm is very important . the brachial plexus is the network formed by the communication between the anterior rami of the fifth to eighth cervical nerve roots and first thoracic nerve root . almost every muscle of the upper limb is supplied by nerves derived from the brachial plexus , as is most of its overlying skin . the radial nerve is a terminal branch of the posterior cord of the brachial plexus . it is the largest branch of the plexus , supplying majority of the posterior and extensor components of the arm and forearm . the medial cutaneous nerve of forearm arises from the medial cord of the brachial plexus , initially lying between the axillary artery and vein . travelling distally , it lies medial to the brachial artery , and pierces the deep fascia at the midpoint of the upper arm adjacent to the basalic vein . here it divides into two branches - the anterior branch passes anterior to the median cubital vein , lays anteromedially in the forearm and supplies skin to the wrist . the posterior branch has an oblique course anteriorly , lying medial to the basalic vein , becoming posterior distal to the medial epicondyle of the humerus to descend on the posteromedial border of the forearm towards the wrist ; it supplies a smaller area of the posteromedial forearm . although communication between the nerves in the arm are rare , communication between median nerve and musculocutaneous nerve is being described since the 19 century . anatomical variations of peripheral nerves are important to medical personnel , especially to orthopedic surgeons , neurophysicians , physiotherapist and radiologists . the present variation was observed during routine dissection at the anatomy department of medical college and rural hospital . in a 58-year - old male cadaver , the brachial plexus was exposed after excising pectoralis major and pectoralis minor muscles . a communication between the radial and medial cutaneous nerves of forearm on left side was found [ figure 1 ] . the communicating nerve was found to be arising from the medial cutaneous nerve of forearm . the communicating nerve was seen crossing the third part of the axillary artery on its medial side and running downward backward , laterally , to join the radial nerve . this nerve was related medially with the axillary vein and laterally to the axillary artery and ulnar nerve . the dissection was continued to expose the entire brachial plexus from the origin and was found to be normal . ( aa : axillary artery ; bbr : biceps brachii ; cbr : coracobrachialis ; ld : latissimus dorsi ; mc : medial cord ; mcn : musculocutaneous nerve ; mn : median nerve ; rn : radial nerve ; sc : subscapularis muscle ; un : ulnar nerve ; communication between radial nerve and medial cutaneous nerve of forearm ; 1 : medial cutaneous nerve of forearm ; 2 : nerve to latissimus dorsi ; 3 : lower subscapular nerve ; 5 : subscapular region ) in the present case report , we report the communication between the radial and medial cutaneous nerves of forearm on the left side in an adult male cadaver . the surgeon should keep such variations in mind as while performing operations of the region of axilla ( specifically constructive shoulder arthroplasty and treating shoulder dislocations ) . report communication between the radial and axillary nerve in eight of the 602 brachial plexus . report the presence of communication between the radial and ulnar nerves on the dorsal surface of the hand . das s and paul s report an unusual communication between ulnar and medial cutaneous nerves of the forearm . the existence of this variation , described in our case report , may be attributed to the random factors influencing the mechanism of formation of limb muscles and the peripheral nerves during embryonic life . in the context that ontogeny recapitulates phylogeny ; it is possible that the variation seen in the current study is the result of developmental anomaly . in human beings , forelimb muscles develop from the mesenchyme of paraxial mesoderm in the fifth week of intrauterine life . the radial extensor muscles of the forearm have a different origin from other extensor muscles of the forearm . regional expression of five hox d ( hox d 1 to hox d 5 ) genes is responsible for upper limb development . the motor axons arrive at the base of limb bud ; they mix to form brachial plexus in upper limb . the guidance of the developing axons is regulated by the expression of chemo - attractants and chemo - repulsant in highly coordinated sight specific fission . the tropic substances attract the correct growth cones or support the viability of the growth cones that happen to take the right path . tropic substances include brain - derived neurotropic growth factor , c - kit ligand , neutrin-1 , neutrin-2 etc . significant variations in nerve pattern may be result of altered signaling between mesenchymal cells and neuronal growth cones or circulatory factors at the time of fission of brachial plexus cords . meticulous knowledge of possible variations of medial cutaneous nerve of the forearm and the radial nerve may provide valuable help in the management of traumatology of shoulder joint and arm ; help the surgeon in circumventing iatrogenic damage during repair operations of these regions . meticulous knowledge of possible variations of medial cutaneous nerve of the forearm and the radial nerve may provide valuable help in the management of traumatology of shoulder joint and arm ; help the surgeon in circumventing iatrogenic damage during repair operations of these regions .

radial nerve is usually a branch of the posterior cord of the brachial plexus . it innervates triceps , anconeous , brachialis , brachioradialis , extensor carpi radialis longus muscles and gives the posterior cutaneous nerve of the arm , lower lateral cutaneous nerve of arm , posterior cutaneous nerve of forearm ; without exhibiting any communication with the medial cutaneous nerve of forearm or any other nerve . we report communication between the radial nerve and medial cutaneous nerve of forearm on the left side in a 58-year - old male cadaver . the right sided structures were found to be normal . neurosurgeons should keep such variations in mind while performing the surgeries of axilla and upper arm .

Introduction Case Report Discussion Clinical significance

PMC4247952

although the overall incidence of breast cancer is rising worldwide , the mortality rate has been decreasing in the united states . the improved survival rate is likely to be a result of the success in early detection and better treatment in patients with positive estrogen receptors ( er ) , progesterone receptors ( pr ) , or human epidermal growth factor receptor 2 ( her2/neu ) breast cancers . triple negative breast cancers ( tnbc ) by default have been grouped together because of the lack of er , pr , and her2/neu markers [ 4 , 5 ] . compared to the other subtypes of breast cancer , these tumors are frequently more aggressive , manifested by a higher distant relapse rate with more frequent visceral as well as central nervous system metastases and higher mortality rate despite chemotherapy [ 68 ] . the heterogeneous biology and histopathology of tnbc underlie the unpredictable responses to chemotherapy and diverse clinical outcomes seen in these patients . the majority of tnbc with relapse is multidrug resistant and ultimately becomes refractory to all therapies [ 9 , 10 ] . to improve treatment , it is important to develop novel therapeutic strategies to predict and overcome drug resistance . in the last two decades , proteomics has emerged as a powerful tool in biomarker discovery and mechanism understanding . using these tools proteomics has been used as a tool to identify new disease related biomarkers in tnbc [ 11 , 12 ] . protein phosphorylation , one of the most ubiquitous posttranslational modifications ( ptms ) , is a key event in regulating many vital functions in cells including proliferation , survival , apoptosis , and signal transduction [ 1315 ] . protein phosphorylation , involved in signal transduction of the cells , requires a coherent activation of protein kinases and phosphatases , which leads to the defined functions . the basal level of the phosphor - proteins may also represent the characters of the cells . reported that the stable tyrosine phosphorylation of the il-10 receptor may increase timp-1 levels to block tumor cell invasion in modified boyden chamber invasion assays . brner et al . reported that the stable phosphorylation of the inhibitory tyr-505 of the leukocyte - specific protein tyrosine kinase ( lck ) may arrest lck in its inhibited form . in recent years , the advances in phosphoproteomics research have allowed discovery of many important functions operating in cancer progression . performed quantitative phosphoproteome and transcriptome analysis on ligand - stimulated mcf-7 breast cancer cells to study the mechanism of tamoxifen resistance . they found that gsk3 and ap-1 transcription factors might be involved in the tamoxifen resistance in mcf-7 cells . used a phosphoproteomic approach to study lapatinib - resistance of her2-overexpressing human breast cancer cell lines and found that the increased src kinase activity was a mechanism of lapatinib resistance . oliveras - ferraros et al . also reported a study on tnbc cell lines using low throughput phosphoproteomic approaches . however , there has been no study focusing on dissecting tnbc drug resistance using large - scale phosphoproteomic tools . in this study , we used high throughput technologies to study changes in phosphorylated proteins to uncover important pathways involved in tnbc drug resistance . for the purpose of this study , tnbc cell lines responding to multiple chemotherapeutic drugs were studied and were compared . twelve established tnbc cell lines were tested against four chemodrugs and the half maximal inhibitory concentrations ( ic50s ) were calculated . the phosphorylated peptides of four resistant and two sensitive cell lines were analyzed using lc - ms / ms to discover important pathways related to chemodrug resistance of tnbc . our study may lead to identification of useful prognostic biomarkers and therapeutic pathways for tnbc treatment . triple negative breast cancer cell lines mda - mb-231 , mda - mb-468 , mda - mb-436 , hcc1187 , hcc1806 , and hcc1937 ( all of which stain negative for er , pr , and lack her2/neu amplification ) were obtained from american tissue type culture collection ( atcc , manassas , va , usa ) [ 22 , 23 ] . cells were maintained in dulbecco 's minimal essential medium ( invitrogen , carlsbad , ca , usa ) or rpmi 1640 ( invitrogen ) with 10% heat - inactivated fetal bovine serum ( thermo fisher scientific , san jose , ca , usa ) , 100 units / ml penicillin , and 100 g / ml streptomycin , at 37c in 5% co2 . cell lines were treated with docetaxel , doxorubicin , gemcitabine , and cisplatin in vitro to determine the half maximal inhibitory concentration ( ic50 ) of each drug . the cells were treated with dmso or twenty predetermined doses of each drug for two days . cell viabilities were determined by celltiter - glo luminescent cell viability assay ( promega , madison , wi , usa ) . triplicated experiments were performed twice and the ic50s were calculated using graphpad prism 5 software . cultured cells were lysed in lysing buffer ( 8 m urea , 4% chaps , 40 mm tris - base , 65 mm dtt , 1% sds ) and the supernatant was collected into 1.5 ml tubes . protein concentration of the lysate was determined using the pierce bca protein assay ( thermo fisher scientific ) . 20 l of 1 m dtt ( thermo fisher scientific ) was added to samples containing 1 mg of proteins and incubated at 56c for 1 hour and followed by an incubation at room temperature for 40 min in darkness with 80 l of 1 m iaa added into the buffer . each sample was treated with 0.11 volumes of ice - cold 100% trichloroacetic acid ( tca ) ( sigma aldrich , st . louis , mo , usa ) on ice for 10 min and 500 l of ice - cold 10% tca on ice for 20 min and then was spun down at 20,000 g for 30 min . the pellet was washed with 500 l of acetone and was centrifuged again at 20,000 g for 10 min . 500 l of 100 mm ammonium bicarbonate ( sigma aldrich ) and 20 g of trypsin ( promega , san luis obispo , ca , usa ) were added to the sample in each tube at 37c for 2 hours . an additional 20 g of trypsin was added to the sample and subsequently incubated for 16 hours . the peptides were then filtered through 10 kda filter columns ( emd millipore , billerica , ma , usa ) and dried via vacuum evaporator . 400 l of loading buffer ( 80% acetonitrile ( acn ) and 2% trifluoroacetic acid ( tfa ) ) was added to 1 mg of peptides . the mixture was incubated with 4 mg of tio2 beads ( gl sciences , torrance , ca , usa ) for 1 hour . the samples were centrifuged at 3,000 g for 5 min and the supernatant was discarded . tio2 beads were collected and washed with 1 ml wash buffer i ( 30% acn , 2% tfa ) followed by 1 ml of wash buffer ii ( 80% acn , 0.1% tfa ) , each for 20 min at 4c with rotation and centrifuged at 3,000 g for 5 min . the phosphopeptides were eluted first with 400 l elution buffer i ( 400 mm nh4oh , 50% acn , ph 11 ) and was followed by 400 l elution buffer ii ( 500 mm nh4oh , 60% acn , ph 11 ) . nest group microspin strong cation exchange solid phase extraction tubes ( the nest group inc . , southborough , ma , usa ) were used as a separation technique before mass spectrometry to reduce the complexity of samples and enhance the identification rate . all peptide fractions were desalted before analysis using c18 tips made from the empore c18 90 mm disk ( 3 m corporate , st . nanoliquid chromatography and tandem mass spectrometry ( nlc - ms / ms ) with collision induced dissociation ( cid ) was performed on a ltq - orbitrap ( thermo fisher scientific ) integrated with an eksigent nano - lc ( eksigent technologies , monmouth junction , nj , usa ) . the flow rate for reverse - phase chromatography was 500 nl / min for the loading and analytical separation ( buffer a : 0.1% formic acid , 3% acn ; buffer b : 0.1% formic acid , 100% acn ) . the orbitrap was operated in data - dependent mode with a full precursor scan at high - resolution ( 60000 at m / z 400 ) and ten ms / ms experiments at low resolution on the linear trap while the full scan was completed . for cid mass spectra were searched against the uniprot human database using proteome discoverer software ( version 1.4 , thermo fisher sceintific ) , utilizing the sequest ( thermo fisher scientific ) , mascot ( matrix science , london , uk ) , and x ! tandem ( http://www.thegpm.org/tandem/ ) algorithms , while running a target decoy search strategy to increase protein identity confidence . mascot and sequest were searched with a fragment ion mass tolerance of 0.80 da and a parent ion tolerance of 10.0 ppm and tolerated up to two missed trypsin cleavages . glu or gln to pyro - glu of the n - terminus , oxidation of methionine , and phosphorylation of serine , threonine , and tyrosine were specified as variable modifications . the proteome discoverer search results were loaded to scaffold ( version 4.1.1 , proteome software inc . , portland , or , usa ) to quantify and validate the ms / ms peptide and protein identifications . identifications were accepted if they had a greater than 95% peptide probability and contained at least one identifiable phosphopeptide . phosphosite localization probabilities were calculated using the ascore probability based scoring technique and only sites that met the stringent minimum of 99% were accepted . the spectral count data of the phosphopeptides were acquired through scaffold ptm and were compared between grouped cell lines : ( 1 ) all four resistant cell lines were compared with the two sensitive cell lines hcc1806 and mda - mb-468 ; ( 2 ) resistant cell lines mda231 and mda - mb-436 were compared to the two sensitive cell lines ; ( 3 ) resistant cell lines hcc1187 and hcc1937 were compared to the two sensitive cell lines . a t - test of each peptide between the groups of the three comparisons was performed and the peptides with p values < 0.05 and a fold change of at least 2.0 ( 2.0 or 0.5 ) were considered differential peptides . from the comparison ( 1 ) , all changed phosphopeptides were further analyzed using online database string which is a database of known and predicted protein interactions including direct ( physical ) and indirect ( functional ) associations . hierarchical clustering analysis was performed using euclidean distance formulation and complete linkage criteria for linkage of normalized phosphopeptide spectrum counts . based on the comparison ( 1 ) , only the peptide spectrum counts with p values less than 0.05 were imported to permutmatrix software . protein phosphorylation is an important posttranslational modification that governs many of the signaling changes in cancer cells . the current study was performed to screen the signaling pathway changes , through the comparison of phosphorylated proteins in tnbc cell lines with extreme responses to the four chemotherapy drugs . the half maximal inhibitory concentrations ( ic50s ) of twelve tnbc cell lines against four chemotherapy drugs were determined and four resistant and two sensitive cell lines were selected for further analysis . cell lines were ranked according to their ic50s against each drug ( table 1 ) . no response is defined as the inability to reach a 50% of inhibition when the highest dose of chemotherapeutic drug was administered . coefficient of determination r values of the drug sensitivity curves ( data not shown ) less than 0.7 were also considered as no response . hcc1187 , mda - mb-436 , mda - mb-231 , and hcc1937 had the highest ic50s for at least three of the four drugs and were thus considered chemotherapy - resistant . hcc1806 and mda - mb-468 were ranked lowest on the ic50 scale for at least three of the four drugs and were considered chemotherapy - sensitive . these six cell lines were selected for phosphorylated protein analysis . to increase the accuracy of identification , the spectra were cross - validated using three searching algorithms ( sequest , mascot , and x ! tandem ) against the uniprot - human database with the application of a decoy database . a total of 1,645 phosphoprotein groups were identified by scaffold software across all six tnbc cell lines , using peptide probability thresholds of 95% with a minimum of one unique peptide ( figure 1(a ) ) . the peptide false discovery rates ( fdr ) accordingly , a total of 9,585 unique phosphopeptides with 10,091 unique phosphosites were identified ( see supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/390781 ) . instances where the same peptide sequence had different phosphosites were counted as one peptide and ultimately 3,062 peptide sequences were identified ( figure 1(b ) ) . the overall class profiles of the phosphoproteins specifically for resistant and sensitive cell lines were also expressed according to their gene ontology ( go ) annotation . when compared to each other , more phosphoproteins involved in cellular adhesion processes were found in resistant cell lines while in sensitive cell lines more phosphoproteins were associated with multiorganism processes , cell killing , pigmentation , and rhythmic processes ( figures 1(c ) and 1(d ) ) . when the cellular compartments of these proteins were studied , the resistant cell lines showed more phosphoproteins in extracellular regions , mitochondrion , and plasma membrane whereas in sensitive cell lines more phosphoproteins were found in golgi apparatus and cytoskeleton ( figures 1(e ) and 1(f ) ) . figure 2 shows the representative ms / ms spectra for three phosphopeptides marked with phosphosites . these phosphopeptides differed dramatically between drug resistant and sensitive cell lines and will be discussed below . the high quality spectra maps were very helpful in identifying peptides as well as phosphosites on them . in the current study , scaffold ptm was used to further confirm the phosphosites identified and to construct a final quantification report . scaffold ptm uses the ascore algorithm to verify the presence of the phosphorylation sites . with the condition of a 99% ascore certainty and 99% minimum localization probability threshold , a total of 1,340 phosphoproteins groups , 2,760 unique phosphopeptides , and 4,549 unique phosphosites were identified across all 6 cell lines ( supplementary table 2 ) . the mass spectra quantification data of these phosphopeptides were exported and further analyzed to determine the variations between chemotherapy - resistant and chemotherapy - sensitive cell lines ( supplementary table 2 ) . the spectrum counts of phosphorylated peptides were evaluated by computing the p value and fold change between the two groups of cell lines . only phosphopeptides with a p value less than 0.05 and a fold change above 2 or less than 0.5 were considered . from three sets of comparisons ( seen in section 2 ) , three sets of numbers were obtained ( shown in supplementary table 3 , sheets 1 , 2 , and 3 ) . all significantly changed phosphopeptides were further analyzed using the online string database for predicted protein interactions ( see below ) . four drug resistant cell lines and two drug sensitive cell lines selected by ic50 ranking were subjected to phosphoproteome analysis in the current study . to confirm that these cell lines can be separated by the differentially expressed phosphopeptides into two groups : chemotherapy - resistant and chemotherapy - sensitive tnbc , the clustering analysis was performed . figure 3 shows that the four drug - resistant cell lines shared much more similarity with each other than the two sensitive cell lines and they could be clearly segregated from the two sensitive cell lines . this result further supported the ic50 data and suggested that the phosphopeptide identification and quantification methods are valuable in characterizing drug sensitivity of tnbc . the changed phosphopeptides described above were then analyzed with string database . all the changed phosphopeptides from supplementary table 3 were analyzed . the corresponding genes of these phosphopeptides were loaded to string to construct a network showing the associations between them . the genes with most connections in the network were shown in figure 4 and table 2 . as shown in figure 4 , the changed phosphoproteins have strong associations and intricate interactions with one another . the pathways associated with the most prominent changes in drug - resistant tnbc are schematically summarized in figure 5 . over the last few decades there has been a steady decrease in breast cancer mortality rate largely due to the improvements in the treatment of breast cancer . despite the significant advancement made in breast cancer therapy in recent years , much of this progress is limited to hormone receptor positive and her2/neu positive breast cancers . specific targeted agents are still lacking for tnbc tumors leaving cytotoxic chemotherapy as the only therapeutic choice . though cytotoxic chemotherapy is effective for many of these patients , the absence of a therapeutically targetable molecule or pathway limits the progress in treating these patients , which is manifested by rapid relapse and death when the tumors are resistant to the conventional chemotherapy . the identification of novel biomarkers indicative of drug sensitivity is imperative for the effective treatment of this exceptionally aggressive type of breast cancer . in our previous study we utilized a hydrophobic fractionation protocol in an effort to detect novel membrane proteins in triple negative breast cancer . although the identification of membrane biomarker is valuable , the heterogeneity of tnbc tumors commands the identification of a variety of biomarkers and signaling pathways in order to arrive at the best treatment strategy . it is important to identify aberrations in tnbc subtypes that cause these tumors to be resistant and unresponsive to chemotherapeutic treatments . protein phosphorylation plays a crucial role in cellular signal transduction pathways . when activated , protein kinase binds and phosphorylates a specific substrate to mediate preprogrammed protein function [ 29 , 30 ] . to find potential biomarkers that could predict the patient 's response to chemotherapy and potential targets to improve tnbc treatment , we profiled phosphorylated peptides in tnbc cell lines with different sensitivities to a variety of chemotherapy drugs . label - free quantification was favored in this study since it has the largest dynamic range and highest proteome coverage - a prerequisite for the objective of this study as protein phosphorylation is a transient process and can be found with varying concentrations [ 31 , 32 ] . fractionation via scx was also utilized in this study to decrease the sample complexity and thus improve the identification . software as well as several computer - based algorithms was also used to correct any inconsistencies and to increase reliability , thus allowing for more accurate results . as shown in figure 3 , the phosphopeptide clustering data perfectly matched the drug sensitivity data ( table 1 ) . these results gave additional proof that the quantification and identification system used was reliable and potentially had predictive value . because different phosphosites in a protein may trigger either protein activation or inactivation , we used phosphosite instead of phosphoprotein as unit for quantification . the functions of proteins will be discussed according to the changes of phosphosites . among the dramatically altered phosphoproteins identified in the current study , many our results further support the roles of these proteins in tnbc drug resistance and offer new insights for future studies . some new phosphorylated sites found in this study together with several of the previously reported protein phosphosites were connected for the first time to tnbc drug resistance . transcription factor ap-1 ( ap-1 ) is a multiprotein transcription factor and a member of the jun and fos protoontogenetic family . previous studies have linked the activated ap-1 and its family members to increased tumorigenesis , metastasis , and invasion [ 33 , 34 ] as well as drug resistance [ 35 , 36 ] . overexpression of phosphorylation at ser73 of c - jun was reported to be responsible for the development of multidrug resistance in colorectal cancer cells . in the current study phosphorylated ser73 of c - jun was only found in the resistant cell lines , suggesting ap-1 is important in tnbc drug resistance . the overexpression and activation of promyelocytic leukemia protein ( pml ) is known to induce the transcriptional activation of ap-1 [ 38 , 39 ] . in this study , we also found an upregulation of ser583 on pml in drug resistant cell lines , a new phosphosite which has not been previously described . our data suggests that this phosphosite provokes the activation of pml , which may lead to tnbc drug resistance through the activation of ap-1 . heat shock factor 1 ( hsf-1 ) is a master regulator for the transcription and heat shock proteins ( hsps ) and was reported to induce a multidrug resistance phenotype through constitutive activation of the multidrug resistance gene 1 ( mdr-1 ) . in the current study phosphorylation of ser303 on hsf-1 was identified to be upregulated in the resistant cell lines . proposed that phosphorylation of ser303 induced a slow repression of hsf-1 allowing for the accumulation of the hsps that are crucial for cell growth and recovery . the above evidence suggested that ser303 phosphorylation of hsf-1 might play an important role in tnbc drug resistance . in addition , both hsf-1 and ap-1 were capable of activating vimentin gene and could be responsible for its overexpression [ 4244 ] . vimentin is a well - recognized biomarker in epithelial to mesenchymal transition ( emt ) and has been associated with metastasis , poorer prognosis , and cell motility in various types of cancer . in the current study , ser73 and ser56 on vimentin were found to be phosphorylated in the resistant tnbc cell lines but were absent in the sensitive cell lines . interestingly , cyclin - dependent kinase 5 ( cdk5 ) was reported to mediate phosphorylation at ser56 of vimentin . ser23 of lamin b1 ( lmnb1 ) was also identified as an upregulated phosphosite in the resistant cell lines in this study . taken together , cdk5 might be activated in drug resistant tnbc cell lines and might phosphorylate lmnb1 and vimentin , leading to emt and drug resistance in tnbc . it has been reported that epidermal growth factor receptor ( egfr ) plays a significant role in hepatocyte growth factor receptor ( hgf / c - met ) mediated biological activities . activation of hgf is indicative of aggressive tumor pathology , including enhanced proliferation and invasion . egfr might also promote the multiple drug resistance ( mdr ) phenotypes in breast cancer cells via accelerating the g1/s transition . furthermore , bagowski et al . reported that egfr phosphorylation at thr654 and thr669 by prkd1 inhibits egf - induced mapk8/jnk1 activation . their studies showed that activation of egfr and hgf might contribute to drug resistance of breast cancer through the downregulation of phosphosites such as thr654 and thr669 of egfr . in the current study , the phosphosites thr-693 , tyr-1197 , and tyr-1110 on egfr and thr977 on hgf were found to be downregulated in resistant cell lines , suggesting a release of negative regulation of egfr and hgf functions in these cells . downregulation of these phosphosites might increase egfr and hgf activity and led to drug resistance of tnbc cell lines . it is noticeable that many of the changed phosphoproteins found in the current study were involved in mrna processing including heterogeneous nuclear ribonucleoprotein a2/b1 , serine / arginine repetitive matrix 1 and matrix 2 , eukaryotic translation initiation factor 4 gamma-1 , and several others . as reviewed by eblen , alternative splicing is a normal cellular process that can be manipulated by a cancer cell to enhance survival in response to chemotherapeutic treatment . we also found the changes of phosphorylation status of some microtubule related proteins and dna binding proteins in the current study ( figure 4 and table 2 ) . the current study has shown that proteomic analysis is a powerful tool for profiling of the phosphorylation patterns and may help better understand drug resistant tnbc cells . our data suggested that pml , ap-1 , and hsf-1 were preferentially activated in resistant tnbc cells and might promote downstream signals including vimentin activation . in addition , our study also suggested that cdk5 might also promote vimentin and lmnb1 activation leading to an increased emt in the resistant tnbc cells . we are also reporting on the potential roles of egfr and hgf in the promotion of a multiple drug resistance ( mdr ) phenotype in tnbc . we have identified several signaling pathways that may work together to mediate the drug resistance in tnbc ( figure 5 ) .

because of the absence of a clear therapeutic target for triple negative breast cancer ( tnbc ) , conventional chemotherapy is the only available systemic treatment option for these patients . despite chemotherapy treatment , tnbc patients still have worse prognosis when compared with other breast cancer patients . the study is to investigate unique phosphorylated proteins expressed in chemoresistant tnbc cell lines . in the current study , twelve tnbc cell lines were subjected to drug sensitivity assays against chemotherapy drugs docetaxel , doxorubicin , gemcitabine , and cisplatin . based on their half maximal inhibitory concentrations , four resistant and two sensitive cell lines were selected for further analysis . the phosphopeptides from these cells were enriched with tio2 beads and fractionated using strong cation exchange . 1,645 phosphoprotein groups and 9,585 unique phosphopeptides were identified by a high throughput lc - ms / ms system ltq - orbitrap . the phosphopeptides were further filtered with ascore system and 1,340 phosphoprotein groups , 2,760 unique phosphopeptides , and 4,549 unique phosphosites were identified . our study suggested that differentially phosphorylated cdk5 , pml , ap-1 , and hsf-1 might work together to promote vimentin induced epithelial to mesenchymal transition ( emt ) in the drug resistant cells . egfr and hgf were also shown to be involved in this process .

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

PMC3606764

obesity abets several life style diseases such as diabetes type ii , atherosclerosis , or impaired wound healing . these adverse effects can be avoided or , if already present , reduced by physical activity . exercise training and increased catecholamine levels affect adipocyte metabolism , on one hand by influencing insulin secretion and on the other hand by additionally channelling the adipocyte metabolism towards lipolysis . adenylyl cyclase transforms atp into cyclic amp ( camp ) , which in turn increases the activity level of camp - dependent protein kinase a ( pka ) [ 3 , 4 ] . activation of the insulin receptor results in decreased lipolysis , increased lipogenesis and uptake of fatty acids . lipolysis is decreased by insulin - receptor - induced phosphodiesterase-3b ( pde-3b ) and 14 - 3 - 3beta - dependent reduction of pka activity [ 3 , 5 ] . furthermore , the insulin receptor activates several lipogenic enzymes by tyrosine phosphorylation and promotes glut-4 recruitment to the cell membrane . protein tyrosine phosphatase interacting protein 51 ( ptpip51 ) is expressed in different organs with a multitude of functions [ 7 , 8 ] . as previously shown for mouse adipose tissue , ptpip51 acts as an effector in insulin signalling . ptpip51 expression was correlated to the insulin resistance of the animals , according to the expression of its interaction partner protein tyrosine phosphatase 1b ( ptp1b ) . in addition to that , ptpip51 is connected to the lipolytic mapk pathway by its interaction with raf-1 mediated by 14 - 3 - 3beta . 14 - 3 - 3beta also interacts with phosphodiesterase 3b , resulting in reduced intracellular camp levels and decreased pka activity . as 14 - 3 - 3beta and ptp1b are important for adipocyte metabolism control , the role of its interaction partner ptpip51 needs to be disclosed . the interaction of ptpip51 with the insulin receptor and pka was investigated in adipose tissue from animals kept under different defined experimental conditions to search for a potential role of ptpip51 in adipocyte metabolism . the experiments were performed with male c57bl/6 mice ( n = 40 ) and approved by the local animal care and use committee ( gi 20/24 nr . the animals were kept under standard conditions ( 12 h light / dark cycle ) with no access to running wheels . the experiments were run for 14 weeks , with a four - week period of fat feeding followed by a ten - week training period . control group ( sd ) : the animals were fed a standard diet ( altromin standard diet no . 1324 , altromin , lage , germany ) . endurance training control group ( sdt ) : the animals were fed a standard diet ( altromin standard diet no . 1324 , altromin , lage , germany ) and trained for a period of ten weeks . the training was performed on a motorized treadmill for 35 min / day at 12 m / min , at 12% grade , 5 times per week , for ten weeks . running speed was 0.27 m / s 0.05 m / s corresponding to 80% of maximal oxygen consumption . high - fat - diet group ( hfd ) : the animals were fed a high - fat diet containing 45% fat for 14 weeks of the experimental period . high - fat - diet , training group ( hfdt ) : the animals were fed a high - fat - diet during the complete experimental time period . the endurance training period started four weeks after the beginning of high - fat - diet feeding and was similar to the training described above . after 14 weeks , the animals were sacrificed in the morning between 10 and 12 o'clock and the abdominal adipose tissue was frozen in liquid nitrogen precooled isopentan , and transferred to 80c till further analysis . a glucose tolerance test was performed to determine the insulin sensitivity of the mice [ 911 ] . for testing the glucose tolerance of the animals , they were injected intraperitoneally with 2 g of 20% d - glucose dissolved in sterile 0.9% nacl solution per kg body weight . the test was performed in the morning ; last feeding of the animals took place the previous day . an antibody against a defined peptide sequence at the c - terminus of ptpip51 ( sequence : iqkdleelevilrd , exon 13 ) was produced ( biolux , stuttgart , germany ) . identity and purity of the synthesized peptide were approved by esi - ms and uv - analysis . the primary ptpip51 antibody to the c - terminus was used in 1 : 800 dilutions and visualized by alexa 555 ( molecular probes , darmstadt , germany , cat . primary monoclonal mouse antibodies were used for double staining experiments with pka ( ab58187 , abcam plc , 330 cambridge science park , cambridge , uk ) and the beta - subunit of the ir ( clone ct-3 , millipore , 28820 single oak drive , temecula , ca , usa ) . to avoid unspecific binding of the primary mouse antibodies , samples were preincubated with biotin - coupled anti - mouse - antibodies for one hour . the reaction of the primary monoclonal mouse antibody was visualized using alexa fluor 488 secondary antibodies ( molecular probes , darmstadt , germany , cat . confocal images of cells were obtained with a leica confocal laser scanning microscope ( clsm , 5 tcs sp2 , leica , bensheim , germany ) . confocal images of alexa fluor 555 fluorescence were acquired using 6 plan - apochromat 40/1.4 oil objective , 548 nm excitation wavelengths ( helium - neon laser ) , and a 560585 nm band - pass filter . the pinhole diameter was set to yield optical sections of 1 airy unit . for the detection of alexa fluor 488 , we used a plan - apochromat 40/1.4 oil objective , the 488 nm excitation wavelength of an argon laser , and a 505530 nm band - pass filter . t3605 ) ( nuclear staining ) were acquired using plan - apochromat 40/1.4 oil objective , 633 nm excitation wavelengths ( helium - neon laser ) , and the 650670 nm bandpass filter . the pinhole diameter was set to yield optical sections of 1 airy unit . acquired dic and confocal images acquired images were subsequently processed by imagej ( v1.43 m ; rasband , w.s . , imagej , us national institutes of health , bethesda , md , usa , http://imagej.nih.gov/ij/ , 19972011 ) using an iterative deconvolution plug - in by bob dougherty ( http://www.optinav.com/imagej.html , iterative deconvolution ) . options were set for all confocal acquired images as follows : 8 numbers of iteration and 2.0 pixels of lp filter diameter . point spread function was calculated for each channel separately by the imagej plug - in created by bob dougherty ( http://www.optinav.com/imagej.html , diffraction limit psf ) . intensity correlation analysis ( ica ) was carried out using imagej ( v1.43 m ; rasband , w.s . , imagej , us national institutes of health , bethesda , md , usa , http://imagej.nih.gov/ij/ , 19972011 ) and an appropriate plug - in for ica included in the plug - in package of the wright cell imaging facility ( http://www.uhnres.utoronto.ca/facilities/wcif/fdownload.html ) . interaction of ptpip51 with either pka or beta - subunit of the ir was detected by the proximity ligation assay kit duolink ( olink |bioscience , uppsala , sweden , pla probe anti - rabbit minus for the detection of the rabbit ptpip51 antibody , cat . 90602 ; pla probe anti - mouse plus for the detection of the mouse anti pka or ir antibody , cat . the dpla was performed according to the manufacturer 's protocol ; negative controls were included [ 9 , 14 , 15 ] . for the principle of the dpla , see figure 1 . the axioplan 2 fluorescence microscope equipped with plan - apochromat objectives ( carl zeiss jena , germany ) was used for photo documentation . for visualization of the dpla , an excitation filter with a spectrum of 530560 nm and an emission filter with a spectrum of 572647 nm were used . the duolink image tool ( olink biosciences , uppsala , sweden ) was used for quantification of the detected dpla signals . we accomplished a database research using the gps 2.1 database to identify kinases within adipocyte metabolism that possess the ability to phosphorylate ptpip51 . the observed data were analysed by nonparametrical tests to determine the significance of the results . the glucose tolerance was determined by a standard glucose tolerance test at the end of the experimental period . sd and sdt animals displayed a slight increase of blood glucose levels 30 minutes after application of glucose which was almost normalized after 60 minutes . in hfd animals , blood glucose levels strongly increased from the basal level of 100 mg / dl to 400 mg / dl 60 minutes after application . blood glucose levels did not return to normal values within 120 minutes . in hfdt animals , blood glucose levels increased to 270 mg / dl 30 minutes after application and almost returned to normal values after 120 minutes ( figure 2 ) . the values of hfd , hfdt , and animals fed a standard diet ( sd and sdt ) are significantly different after 30 and 60 minutes . after 120 minutes , the differences between standard diet and high fat diet animals remained significant , whereas there was no significant difference between standard diet and trained high - fat - diet animals . mean body weight was measured at different time points throughout the test ( see table 2 ) . statistical analysis showed that there was no significant difference between the four groups at 10 weeks of age . from the beginning of the training , the difference between standard diet and high - fat - diet - fed groups was highly significant , but there was no significant difference between the sd and sdt and , respectively hfd and hfdt groups . ptpip51 immunoreactivity was detected within the cytoplasm and at the plasma membrane ( figure 3 ) . the ir revealed a similar expression pattern when compared to ptpip51 . in the cytoplasm , co - localization is displayed in yellow to orange and non - co - localized parts are shown in blue . the duolink proximity ligation assay ( dpla ) was performed to detect interactions between ptpip51 and the beta - subunit of the ir . the interaction of ptpip51 and ir was verified as seen in figure 4 , where each dot indicates the interaction of both proteins . this interaction of ptpip51 and the ir was found in the adipocytes of animals from all experimental groups . the grade of interaction in adipose samples from sd animals corresponds to the level in hfdt animals . the assumption of an interaction of ptpip51 and pka based on the database research was verified ( figure 4 ) . the interaction levels of sd , hfd , and hfdt mice did not show significant differences . to sum up the results , adipocytes from mice subjected to different diets and activity levels displayed a connection between ptpip51 and the insulin receptor , and ptpip51 and pka respectively . the glucose - tolerance test revealed a glucose - intolerant status in hfd and hfdt animals , whereas the glucose tolerance of hfdt animals improved compared to hfd animals . immunofluorescence data displayed colocalization of ptpip51 with ir and pka suggesting an interaction between ptpip51 and ir , and ptpip51 and pka respectively . search results displayed ptpip51 to be phosphorylated at tyrosine 176 by the insulin receptor and at serine 46 by pka . these two phosphorylation sites are possible regulatory sites for the interaction of ptpip51 with 14 - 3 - 3beta , thus being able to influence the mapk pathway by interaction with raf-1 . phosphorylation of serine 46 in ptpip51 results in augmented interaction with raf-1 via 14 - 3 - 3 and thus in increased erk1/2 phosphorylation , promoting the mapk pathway [ 12 , 17 ] . pka is known to induce lipolysis through the mapk pathway [ 18 , 19 ] . the insulin receptor induces tyrosine phosphorylation of ptpip51 at tyrosine 176 , which leads to reduced interaction of ptpip51 with 14 - 3 - 3beta and raf-1 , thereby decreasing the mapk pathway activity . analysis of the ptpip51 interaction profile of adipocytes displayed high interaction levels with the insulin receptor in hfd animals . interaction levels were reduced by 30% in sd and hfdt and by 50% in sdt animals , compared to hfd animals . high - fat feeding increases the lipogenic activity in adipose tissue [ 21 , 22 ] . the reduced ptpip51-ir interaction in trained groups compared to the corresponding untrained groups suggests that ptpip51 is especially involved in the processes of insulin induced lipogenesis or in its antilipolytic action . interaction levels between ptpip51 and pka did not show significant differences in sd , hfd , and hfdt animals . in sdt animals , but , as pka also interacts with the hormone - sensitive lipase ( hsl ) , a change of the interaction partner of pka away from ptpip51 under these preconditions is assumable . the activities of both pka and hsl are increased in endurance - trained animals [ 24 , 25 ] . our experiments suggest that ptpip51 could mediate between lipogenesis and lipolysis by switching between the lipogenic insulin pathway and the lipolytic pka pathway . further experiments need to be conducted in order to prove our hypothesis and to determine the magnitude in which ptpip51 engages in adipocyte metabolism .

aims . our previous experiments revealed an association of ptpip51 ( protein tyrosine phosphatase interacting protein 51 ) with the insulin signalling pathway through ptp1b and 14 - 3 - 3beta . we aimed to clarify the role of ptpip51 in adipocyte metabolism . methods . four groups of ten c57bl/6 mice each were used . two groups were fed a standard diet ; two groups were fed a high - fat diet . two groups ( one high - fat diet and one standard diet ) were submitted to endurance training , while the remaining two groups served as untrained control groups . after ten weeks , we measured glucose tolerance of the mice . adipose tissue samples were analyzed by immunofluorescence and duolink proximity ligation assay to quantify interactions of ptpip51 with either insulin receptor ( ir ) or pka . results . ptpip51 and the ir and ptpip51 and pka , respectively , were colocalized in all groups . standard diet animals that were submitted to endurance training showed low ptpip51-ir and ptpip51-pka interactions . the interaction levels of both the ir and pka differed between the feeding and training groups . conclusion . ptpip51 might serve as a linking protein in adipocyte metabolism by connecting the ir - triggered lipogenesis with the pka - dependent lipolysis . ptpip51 interacts with both proteins , therefore being a potential gateway for the cooperation of both pathways .

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

PMC2896103

when conducting a scientific research project , a considerable amount of time and effort is often expended in the process of designing effective protocols or methodology . this process comprises a larger proportion of the work load in hypothesis - free or hypothesis - generating disciplines such as bioinformatics and computational genomics ( 1 ) , which generally involve knowledge discovery and data mining based on heuristic approaches . research in these areas often requires the comparative testing of numerous software tools with the use of hundreds of parameters , and a multitude of input sequences from a variety of data sources . the g - language genome analysis environment ( g - language gae ; http://www.g-language.org/ ) ( 2,3 ) has been developed as a workbench to expedite routine heuristic research processes in computational genomics . it provides a set of tools , software libraries and interfaces for use primarily in bacterial genome analysis . because the g - language gae is comprised of a set of perl libraries , it is compatible with bioperl , but it is 732 times faster than bioperl at parsing genbank files ( see http://www.g-language.org/wiki/benchmark for benchmarking details ) , and it has a more intuitive gateway interface . for example , the g - language gae can parse and load sequence data from local flat files in numerous file formats , or from remote databases by using accession numbers , or from data objects created with bioperl ( 4 ) , all with a single gateway function with automatic interpretation of the data location and type . moreover , g - language gae is equipped with an interactive perl / unix shell , which supports a persistent workspace , so a user does not need to write or edit a script in every trial - and - error routine , but instead can interactively test one method at a time . the software package also contains more than 100 original analysis programs , including those for the identification of binding sites with the use of information theory , analysis of nucleotide composition bias , analysis of the distribution of characteristic oligonucleotides , analysis of codons and prediction of expression levels , and visualization of genomic information [ for a comprehensive review describing the detailed algorithms for each of these tools , see ref . some of the analysis capabilities are trivial , such as the tools for global alignment and for basic statistics , and for some of the programs similar functionality is available in comprehensive suites like emboss ( 6 ) . on the other hand , the g - language gae contains a more comprehensive collection of tools for codon analysis than that found in most popular packages , such as codonw ( http://codonw.sourceforge.net/ ) , and a number of published tools are only available in this system : for example , accurate prediction of the origin and terminus of replication in bacteria using noise - reduction filtering with the fast fourier transform algorithm ( 7 ) , quantification of replication - related mutation or selection bias on bacterial chromosomes and plasmids ( 8,9 ) , measurement of codon bias by using the weighted sum of relative entropy ( 10 ) , accurate detection of the long - term host of a plasmid by measuring the mahalanobis distance of the genomic signatures ( 11 ) , interactive and zoomable chaos game representation ( 12 ) , and several visualization tools for genomic information using the google maps application programming interface ( api ) ( 13 ) . with the availability of thousands of software tools and an ever increasing wealth of genomic resources the availability of tools as web services provides interoperability , as well as other advantages such as a lack of cost for installation and maintenance ( 14 ) . the two major web service technologies that are widely adopted in the bioinformatics community are soap ( http://www.w3.org/tr/soap/ ) with web services interoperability ( ws - i ; http://www.ws-i.org ) guidelines and representational state transfer ( rest ; http://www.ics.uci.edu/fielding/pubs/dissertation/rest_arch_style.htm ) . soap is a traditional web service protocol that exchanges structured information using xml - based messages . soap requires the use of dedicated software libraries , and hence the messages are not meant to be human - readable . numerous bioinformatics services are already available as soap - based web services , and a number of graphical client software packages for the construction of workflows utilize these services : for example , the taverna workbench ( 16 ) . in contrast , rest is laissez faire approach without strict definitions for its protocol , that is recently revived in light of its advantages over soap . basically , rest provides universal resource locator ( url)-based access over hypertext transfer protocol ( http ) , and therefore can be easily utilized with any tools that can access the web , such as web browsers . moreover , each rest operation has a unique universal resource identifier ( uri ) , which can be used to represent the service as an online resource that can be utilized from other web sites as a hyperlink . in short , soap is suited for operation - centric access by programming languages , and rest is suited for quick resource - centric access without the requirement of specialized tools . therefore , we provide a total of 145 services encompassing 21 categories ( table 1 ) , in soap and/or rest interfaces . in the following sections , we describe the system architecture and provide usage examples of these services . table 1.available servicesservice class ( number of web apis)descriptiong ( 39 ) , primitive ( 7 ) , utils ( 6)basic sequence manipulation and data accessalignment ( 2)sequence alignmentaminoacid ( 3)calculation of properties of amino acidscodon ( 19)calculation of codon usage and prediction of expression levelsconsensus ( 6)prediction of sequence motifs using molecular information theorygcskew ( 13)analysis of skews in genome composition , prediction of replication origin and terminusgenomemap ( 7 ) , graph / gmap ( 2)visualization of genomic informationoverlapping ( 1)identification of overlapping genespatsearch ( 15)analysis of oligomer frequencies , calculation of genomic signatureshelp ( 2)documentationstatistics ( 13)basic statisticswebservices ( 3 ) , eutils ( 2 ) , blast ( 1 ) , emboss ( 1 ) , cog ( 1 ) , operon ( 1 ) , pec ( 1)simplified interface to external web servicestotal ( 145 ) rest services are suited for resource - centric access and are extremely easy to use without the need for programming or specialized tools . we therefore provide basic data parsing functionality as well as all 145 functions of the g - language gae in our set of rest services . while the use of xml as an exchange format is sometimes recommended even for rest services , all of our services return the results in plain text for human - readability and simplicity , and status and error reports comply with the http status code : 200 for successful request , 404 for invalid request and 302 for url transfer to result image files . each service operation is represented by two unique urls following a simple syntax , with the base url being either http://rest.g-language.org/ or http://useg.jp/. these two urls point to an identical server , and in the following descriptions we use the latter . an overview of the url syntax is presented in figure 1 . parsing a flat file containing a dna sequence requires three input fields : [ sequence ] , [ gene ] and [ feature name ] ( figure 1a ) . for example , in order to obtain the start position of the reca gene in the escherichia coli k-12 mg1655 genome ( accession number refseq : nc_000913 or embl : u00096 ) , a user can access the corresponding unique url http://useg.jp/nc_000913/reca/start to obtain the position 2 820 730. the first field [ sequence ] can be ( i ) an accession number of one of the genbank files available on our server ( 1247 files containing complete genome and plasmid sequences , at the time of writing ; see http://useg.jp/organism_list/ for a comprehensive listing ) , ( ii ) built - in genomes in the g - language gae , such as ecoli , mgen , bsub , cyano , pyro , corresponding to e. coli , mycoplasma genitalium , bacillus subtilis , synechococcus sp . and pyrococcus furiosus , respectively , ( iii ) a uniform sequence address ( usa ) in the form of [ database name]:[accession number ] such as refseq : nc_000913 to automatically retrieve a dna or protein sequence from uniprot ( swiss ) , genbank ( genbank ) , genpept ( genpept ) , embl ( embl ) and refseq ( refseq ) , or ( iv ) a temporary six digit hexadecimal i d given to an imported sequence that is in a format supported by bioperl , such as genbank , embl , fasta , fastq and swissprot , and has been uploaded using the function at http://useg.jp / upload/. therefore , input of ecoli , refseq : nc_000913 , embl : u00096 , or the temporary six - digit i d in the field [ sequence ] leads to identical results . the second input field [ gene ] can be the common gene symbol ( e.g. reca ) , the canonical gene i d ( e.g. b2699 ) , or the feature i d in the g - language gae ( e.g. feature5804 or cds2646 ) . to obtain multiple entries of interest , the [ sequence ] input can also be a wild - card ( * ) or a search field - value pair ; for example , to search for the keyword when multiple genes are specified in this way , results are given in multi - fasta format with gene names as sequence ids . the last field [ feature name ] is the name of the feature key in genome flat files ( such as start , end , direction , translation , db_xref ) . , users can easily utilize the 145 analysis programs of the g - language gae by specifying the program name in the field [ method ] , and by appending optional parameters when necessary in the field [ option = value ] ( figure 1b and c ) . for example , the retrieval of all the 100-bp nt sequences that are immediately upstream of the start codon of genes in e. coli , in a multi - fasta format , can be achieved using http://useg.jp/nc_000913/*/before_startcodon , gc skew analysis can be performed using http://useg.jp/nc_000913/gcskew/ , and a cumulative gc skew with a 1 kb window can be calculated using http://useg.jp/nc_000913/gcskew/cumulative = 1/window = 1000/. the genome sequence analysis programs often return the results in graphical form , so that the user can readily interpret the results ( figure 2 ) . ( a ) gc skew graph calculated in all regions of the entire genome ( red ) , coding regions ( green ) , intergenic regions ( blue ) and third nucleotide in codons ( purple ) with genomicskew function in b. subtilis genome . ( c ) nucleotide frequency in regions of 100 bp around start and stop codons using all genes within the genome of b. subtilis using view_cds function . strong conservation of nucleotides at start and stop codons , as well as conserved upstream shine ( d ) chaos game representation visualized for synechococus sp . examples of graphical analysis results . ( a ) gc skew graph calculated in all regions of the entire genome ( red ) , coding regions ( green ) , intergenic regions ( blue ) and third nucleotide in codons ( purple ) with genomicskew function in b. subtilis genome . ( c ) nucleotide frequency in regions of 100 bp around start and stop codons using all genes within the genome of b. subtilis using view_cds function . strong conservation of nucleotides at start and stop codons , as well as conserved upstream shine ( d ) chaos game representation visualized for synechococus sp . here we have implemented our rest web service to directly return result text and images in arbitrary text and urls , mostly for intuitive usage by biologists from web browsers . on the other hand , restful resources should ideally be marked up with resource uris for semantic interoperability ( 17 ) , which will be our future work . the g - language soap services include 77 operation - centric analyses , and exclude simple data access and manipulation services that are better handled with rest . all services return the results synchronously without polling , and take either single scalar values ( 64 methods ) or lists ( 13 methods ) as their main input ( named according to the input type ) , followed by a hash of optional parameters ( named params ) , as demonstrated in the following perl program . as is the case for rest services , the input sequence can be a refseq or g - language gae i d , a usa of remote data , or a raw dna or protein sequence in a file format supported in bioperl . the types of output values are scalar , list , or a url of the result file . these services are based on soap 1.1 , and service descriptions are listed in web services definition language ( wsdl ) 1.1 at http://soap.g-language.org/g-language.wsdl . the following is an example of a perl program that utilizes our soap service to calculate and visualize cumulative gc skew in the e. coli genome ; it accesses the $ soap = soap::lite->service(http://soap.g - language.org / g - language.wsdl ) ; $ in0 = soap::data->new(name=>'in0 ' , value=>ecoli ) ; % param = ( -cumulative=>1 ) ; $ inputparams = soap::data->name(params')->type(map=>\%param ) ; # run the web service gcskew print $ soap->gcskew($in0 , $ inputparams ) ; one of the primary advantages of soap services is their ability to integrate with hundreds of other bioinformatics services that are already available with soap technology , in the form of workflows . because the wsdl file for our soap service includes the description of all 77 services within one file , users can readily incorporate all these services into the taverna workbench by loading this wsdl file . moreover , nine example workflows utilizing g - language soap services have been submitted to the myexperiment web site ( http://www.myexperiment.org/ ) ( 18,19 ) that can be loaded in taverna , and users can download and customize these workflows according to their specific needs ( http://www.myexperiment.org/search?query=g-language&type=all ) . figure 3 shows one of the workflows loaded in taverna , namely the bacteria analysis system ( id:779 in myexperiment ) , which runs 12 analysis services based on a given bacterial genome . this workflow calculates and graphs the dna sequence conservation around the start codon in terms of relative entropy ( base_relative_entropy ) , entropy ( base_entropy ) and information content ( base_information_content ) ( 20 ) , calculates the codon usage ( codon_usage ) , graphs the gc skew ( gcskew ) , cumulative gc skew ( gcskew_cumulative ) , gc skew in multiple regions of genome ( genomicskew ) ( 21 ) , visualizes the nucleotide composition around the coding regions ( view_cds ) , calculates the codon adaptation index and predicts gene expression levels ( cai ) ( 22 ) , visualizes the gc content ( gcwin ) , calculates the gc skew index ( gcsi ) ( 8) , and predicts the origin and terminus of replication ( find_ori_ter ) ( 7 ) . this workflow calculates and graphs the dna sequence conservation around the start codon in terms of relative entropy ( base_relative_entropy ) , entropy ( base_entropy ) and information content ( base_information_content ) ( 20 ) , calculates the codon usage ( codon_usage ) , graphs the gc skew ( gcskew ) , cumulative gc skew ( gcskew_cumulative ) , gc skew in multiple regions of genome ( genomicskew ) ( 21 ) , visualizes the nucleotide composition around the coding regions ( view_cds ) , calculates the codon adaptation index and predicts gene expression levels ( cai ) ( 22 ) , visualizes the gc content ( gcwin ) , calculates the gc skew index ( gcsi ) ( 8) , and predicts the origin and terminus of replication ( find_ori_ter ) ( 7 ) . the perl api for the g - language gae provides powerful features that aid programmatic sequence manipulation ; however , installation of the api needs expert knowledge because the api requires many external modules . therefore , a more lightweight version of the g - language gae api has been developed as a wrapper around the rest services . this module , named bio::glite , is available from the comprehensive perl archive network ( cpan ; http://www.cpan.org/ ) , and can be easily installed using the command users of this module can utilize the g - language rest services with the same usability as the original perl api . complete documentation about each methods and services are available at ajax document center ( http://ws.g-language.org/gdoc/ ) . a list of all 145 g - language rest services is available at http://useg.jp/method_list/gb/ and http://useg.jp/method_list/ mainly for programmatic access , and detailed documentation about each of the services , including service description , example usage , optional parameters and their default values can be viewed at http://useg.jp/help/[method ] , where [ method ] is the input field for the function name . further documentation and usage examples are available at http://rest.g - language.org/. for soap services , example scripts in perl , ruby and python languages and detailed documentation , are available at http://www.g - language.org / wiki / soap/. the soap services have also been registered in the biocatalogue(http://www.biocatalogue.org / services/2623-glangsoapservice_651637 ) . we have designed and implemented a set of web service interfaces to the g - language gae , which allow access to 145 analysis programs through rest and soap web service technologies . the rest services enable quick and easy access to all programs from any web browser through unique urls , and thus the services can be accessed , in the form of hyperlinks , from other online resources . the generic handling of sequence data , with automatic interpretation of multiple identifiers and file formats , allows easy linking to a wide variety of resources . the 77 soap services enable programmatic access to the g - language gae by programs written in languages other than perl , such as ruby , python and java . by using workflow management software such as taverna , all g - language soap services can be readily integrated with other bioinformatics web services that have rich graphical user interfaces . 20710158 from the japan society for the promotion of science ( jsps ) , and by funds from the yamagata prefectural government and tsuruoka city . funding for open access charge : funds from the yamagata prefectural government and tsuruoka city .

g - language genome analysis environment ( g - language gae ) contains more than 100 programs that focus on the analysis of bacterial genomes , including programs for the identification of binding sites by means of information theory , analysis of nucleotide composition bias and the distribution of particular oligonucleotides , calculation of codon bias and prediction of expression levels , and visualization of genomic information . we have provided a collection of web services for these programs by utilizing rest and soap technologies . the rest interface , available at http://rest.g-language.org/ , provides access to all 145 functions of the g - language gae . these functions can be accessed from other online resources . all analysis functions are represented by unique universal resource identifiers . users can access the functions directly via the corresponding universe resource locators ( urls ) , and biological web sites can readily embed the functions by simply linking to these urls . the soap services , available at http://www.g-language.org/wiki/soap/ , provide language - independent programmatic access to 77 analysis programs . the soap service web services definition language file can be readily loaded into graphical clients such as the taverna workbench to integrate the programs with other services and workflows .

INTRODUCTION REST SERVICES SOAP SERVICES APPLICATIONS DOCUMENTATION CONCLUSIONS FUNDING

PMC2570077

the ability to analyse and learn new information and , if necessary , modify ongoing behaviour , based on a combination of previous and recent memories , is an important factor for the survival of organisms in an ever - changing world . creating and managing these memory traces , comprise core functions of the central nervous system , which rely on its ability to engage in synaptic plasticity . they can be monitored at different levels , ranging from the subcellular to the behavioural . since its discovery in the late 1960s ( andersen and lmo , 1967 ; bliss and lmo , 1973 ; see for review : lmo , 2003 ) , long - term potentiation ( ltp ) has been the subject of intense study , using a plethora of induction protocols , species , and various preparations both in vivo and in vitro . nowadays , ltp , along with long - term depression ( ltd ) , is widely used as a model of synaptic information storage and are believed to represent the processes of learning and memory in neuronal networks ( abraham , 2003 ; bear , 1996 ; bliss and collingridge , 1993 ; kemp and manahan - vaughan , 2007 ; lynch , 2004 ; whitlock et al . , synaptic plasticity in the form of ltp , typically results in both functional and structural reorganisation of the synapse , that evolves over time and comprises different phases , namely ltp - induction ( post - tetanic potentiation ) , short - term potentiation , ltp expression ( early ltp ) and maintenance ( late ltp ) ( abraham , 2003 ; bliss and collingridge , 1993 ; frey et al . , 1993 ; furthermore , each of these phases is characterised by the different involvement of neurotransmitter systems acting via both ionotropic and metabotropic receptors . for the induction phase , several lines of evidence underlie the important role of the activation of n - methyl - d - aspartate ( nmda ) receptors , at least for the hippocampal ca1 region and dentate gyrus ( abraham and mason , 1988 ; collingridge et al . , 1983 ; errington et al . , 1987 ; fox et al . , 2006 ; frey et al . , 1996 ; manahan - vaughan , 1997 ; niewoehner et al . , 2007 ) . for early ltp , activation of the metabotropic glutamate receptors ( mglurs ) , particularly group i mglurs , plays a critical role ( aiba et al . , 1994 ; anwyl , 1999 ; manahan - vaughan , 1997 ; manahan - vaughan and braunewell , 2005 ; manahan - vaughan and reymann , 1996 ; manahan - vaughan et al . , 1998 ; richter - levin et al . , 1994 ) . late - ltp ( maintenance phase ) requires de novo protein synthesis ( frey et al . , 1996 ; krug et al . , 1984 ; otani and abraham , 1989 ) and activation of several immediate - early genes and late - response genes ( abraham et al . , 1991 ; brakeman et al . , 1997 ; cole et al . , 1989 additionally , various neuromodulators can either increase or decrease the probability , magnitude and/or longevity of changes in synaptic transmission , hence affecting the outcomes of patterned synaptic activation ( granado et al . , 2008 ; kemp and manahan - vaughan , 2005 , 2008 ; kulla and manahan - vaughan , 2002 ; lemon and manahan - vaughan , 2006 ; li et al . , 2003 , 2007 ; manahan - vaughan and kulla , 2003 ; swanson - park et al . , 1999 ) . besides the different relative involvement of neurotransmitter systems , the induction and/or expression of ltp , as a reflection of central synapses to acquire , process and subsequently store new information , depends upon several other factors , including environmental enrichment , various forms of stress , etc . bruel - jungerman et al . , 2005 ; duffy et al . , 2001 ; , 1999 ; xu et al . , 1998 ) , which couple the sensory input to a current behavioural state and provide the contextual frame for processing of sensory stimuli . thus , taking into account the plethora of mechanisms underlying the phenomenon of ltp , it is not surprising that various factors and/or experimental manipulations can markedly affect its different phases . however , for most forms of ltp , and particularly for electrically induced ltp , the state of the animal ( synaptic and behavioural ) during a short period of time , both during and immediately after tetanisation , appears decisive in determining the persistence of potentiation ( abraham , 2003 ) . in our study ( bikbaev and manahan - vaughan , 2007 ) , we applied high - frequency tetanisation ( hft ) in freely behaving adult rats and monitored field excitatory synaptic potentials ( fepsps ) in parallel with the intrahippocampal electroencephalogram over a period of 24 h. despite the fact that an identical stimulation protocol was employed for all animals , substantial variation in the outcomes of tetanisation was found . in the first group of animals , a robust ltp occurred that was stable for at least 24 h ( ltp group ) , in the second group short - term potentiation that endured for up to 3 h was induced ( stp group ) , whereas in the third group a failure of potentiation occurred , i.e. no persistent increase in synaptic transmission was seen ( failure group ) . given this range of tetanisation results , we analysed them in the context of the changes in network activity that occurred during and within first 300 s after tetanisation . oscillatory activity is an integral part of functional neuronal networks , and significant variability in patterns of oscillatory activity can be registered under certain conditions both in vivo and in vitro . modification of synaptic weights occurs very fast during the activated state of the hippocampus ( buzski , 1996 ) , with entorhinal hippocampal network oscillations at theta frequency playing a crucial role in this process ( kamondi et al . , 1998 ) . particularly in the hippocampus , theta oscillations ( theta rhythm , rhythmic slow activity ) have been proposed to play a role in a wide variety of hippocampal functions ( see for review : buzski , 2005 ) . in rodents , periods of prominent theta oscillations occur during exploratory behaviour and phases of rapid eye movement rem ( sleep ) ; conditions which are generally termed theta - associated behaviour ( bland , 1986 ; buzski , 2002 , 2005 ; vanderwolf , 1969 ) . these behavioural states , and particularly exploratory behaviour , are characterised by neocortico - hippocampal transfer of new spatial information when acquisition and/or encoding of sensory information should be facilitated , whereas transfer of the information from the hippocampus to neocortex is associated with other patterns , such as sharp waves and ripples ( bragin et al . , 1995 ; buzski , 1996 ; buzski et al . , 2002 ; gamma oscillatory activity represents another pattern of network activity , which is characteristic for cortical locations , and is proposed to be involved in various cognitive functions ( buzski and chrobak , 1995 ; fell et al . , 2001 ; fries et al . , 2001 ; gray and singer , 1989 ; hermann et al . , 2004 ; hopfield , 1995 ; lisman , 1999 ; lisman and idiart , 1995 ; montgomery and buzski , 2007 ; singer , 1993 ) . in the hippocampus of freely moving animals , the amplitude of gamma oscillations is higher in the dentate gyrus than in other hippocampal regions expressing gamma oscillations ( bragin et al . , 1995 ) , and hence , hippocampal theta and gamma oscillations comprise functionally - associated patterns of network activity that emerge as a consequence of the combination of intrinsic oscillatory properties of principal cells and interneurons , the rhythmic activation of which is driven by intra- and extrahippocampal connections ( bartos et al . , 2007 ; bragin et al . , 1995 ; csicsvari et al . , 2003 ; klausberger et al . , 2003 ; penttonen et al . , 1998 ; the results of our study demonstrated that the successful potentiation of synaptic transmission ( ltp and stp groups ) in freely moving rats , was preceded by a higher relative theta power in the period of 300 s after hft , when compared with the pre - tetanisation period ( figure 1 ) . in contrast , a gradual recovery of theta power to pre - tetanisation values , but not an increase , was found in the ltp - failure group . in other words , successful induction of potentiation ( either in the form of stp or ltp ) , resulted in an enhancement of theta oscillatory activity after tetanisation , whereas no such enhancement occurred in rats that showed failure of ltp . the mean amplitude of a single theta cycle was also significantly higher within the post - tetanisation period in the ltp and stp groups , when compared to the group of failed synaptic potentiation . taken together , these data suggest that a transient increase of theta power is necessary for the proper handling of sensory input activation ( mimicked here by hft ) . this , in turn , may play a permissive role in ltp induction , and , in general , in the formation of memory trace(s ) of newly acquired information . transient enhancement of theta and gamma power in the post - tetanisation period correlates with potentiation of synaptic transmission in the dentate gyrus of freely moving rats . ( a ) examples of eeg epochs , which comprise 100 s long periods of tetanisation ( 200 hz , 10 trains : first train is marked by arrow ) and following 300 s , recorded in rats that showed either ltp ( left panel ) , stp ( middle panel ) or failure of potentiation ( right panel ) , respectively . asterisk denotes the period immediately after hft , when the amplitude of network oscillations is higher in ltp and stp cases , in comparison with failed potentiation . scale bar : 20 s. ( b ) successful potentiation ( ltp and stp ) of synaptic transmission is associated with a prominent increase of the relative theta and gamma power particularly in the period encompassing 100 s after tetanisation . the results were pooled for ltp , stp and failure groups and presented as mean s.e.m . ( modified from bikbaev and manahan - vaughan , 2007 ) . however , the differences between ltp , stp and failed potentiation groups were not limited in our study by theta oscillations . we found that within the first 100 s after hft the increase in theta power , and the mean amplitude of a single theta cycle , was accompanied by higher gamma power in the ltp group , and higher amplitude of gamma oscillations within a single theta cycle in both the ltp and stp groups , when compared with cases of failed potentiation ( figure 1b ) . this indicates that not the increase of theta power per se , but a correlated increase of both theta and gamma power is important for enabling or mediating the plastic changes in synaptic weights . furthermore , our results show that these enhancements of oscillatory activity should take place within a relatively short period of time for synaptic plasticity to occur . taking into account that the amplitude of gamma oscillations in the dentate gyrus , but not in the ca3 or ca1 regions , is significantly higher in the presence of theta oscillations than in non - theta states ( csicsvari et al . , 2003 ) , these findings suggest that the tetanisation - induced facilitation of theta activity can play a causal role for the enhancement of gamma oscillations . in the dentate gyrus of the intact brain , the power of both theta and gamma activity is driven by and strongly depends upon entorhinal input , and is higher during exploratory behaviour ( bragin et al . , 1995 ; csicsvari et al . , 2003 ) . furthermore , behavioural data indicating enhanced eyeblink conditioning in rabbits that received pairings of stimuli during epochs of prominent theta activity , when compared to those stimulated during non - theta periods ( seager et al . , 2002 ) , provide additional support for a facilitatory role of theta oscillations in learning . however , one should emphasise that the increase of theta power occurred in our study in the period after tetanisation . thus , the differences in theta power that we observed , did not comprise an endogenous pre - condition for synaptic plasticity , but rather occurred as a consequence of perforant path stimulation . accordingly , if natural theta rhythm is necessary for the acquisition or processing of sensory stimuli , one can presume that the artificial activation of sensory inputs ( in the form of hft ) would require and/or induce an enhancement of oscillatory activity in the theta frequency range , in order to temporally organise neuronal activity and favour synaptic plasticity . the depolarisation of dendritic compartments of dentate gyrus granule cells via activation of nmda receptors and mglurs , after strong activation of the glutamatergic perforant path , is likely to contribute to such an enhancement . perforant path stimulation of the dentate gyrus , in conjunction with subsequent firing of mossy fiber collaterals , will also activate parvalbumin - expressing basket cells ( kneisler and dingledine , 1995 ) , which via ionotropic -amino - butyric acid ( gabaa ) receptors provide rhythmic inhibition of somata and perisomatic region of principal cells and play a pivotal role in the generation of both theta ( buzski , 2002 ; klausberger et al . 1995 ) and gamma ( bartos et al . , 2001 , 2007 ; penttonen et al . , 1998 ; vida et al . , additionally , activation of gabaa receptors has been reported to be critical also for the generation of transient tetanically induced gamma oscillations in vitro ( traub et al . , 2004 ; hence , the combination of nmdar - induced dendritic excitation and gabaar - mediated somatic inhibition results in current flow through distal dendrites , which is important for the generation and maintenance of extracellular theta currents ( buzski , 2002 ) . this could also effectively trigger and/or enhance theta oscillations in post - hft period . in turn , theta oscillations may dynamically modulate the probability of nmdar activation , which is highest on the peak of the theta cycle and the lowest on the trough ( vertes , 2005 ) . the increase of gamma power in the post - tetanisation period may rely on the activation of group i mglurs , which was reported to induce gabaar - dependent gamma oscillations in vitro ( whittington et al . , we have found recently that prolonged inhibition of mglur5 results in a significant impairment of ltp associated with a marked suppression of gamma oscillations in the dentate gyrus of freely moving rats ( bikbaev et al . , 2008 ) . taken together , correlated activation of granule cells and interneurons , via fast and slow glutamatergic excitation , not only can contribute to long - term synaptic potentiation , but , complemented with fast rhythmic gabaergic inhibition , can affect network oscillations on both short- and long - term time - scales ( figure 2 ) . in this respect , pharmacological modulation of selected neurotransmitter systems , including the glutamatergic , gabaergic and cholinergic systems , coupled with correlation analysis of network activity and long - term changes in synaptic transmission , would be necessary for a more conclusive support of this suggestion . for instance , such analysis after activation of the cholinergic system , which can facilitate oscillatory activity in the theta frequency band , and simultaneous inhibition of gabaar- and/or mglur - mediated signalling , which affects hippocampal gamma activity , could help to dissect the roles of theta and gamma oscillations in the shaping of synaptic plasticity . a schematic representation of the consequences of high - frequency tetanisation that occur on the network and cellular levels , and lead towards long - term potentiation of synaptic transmission . ( a ) strong afferent stimulation results in an enhancement of both theta and gamma oscillations , which occurs within 5 min interval after tetanisation and can be mediated via gabars , nmdars and mglurs . ( b ) tetanisation - triggered activation of both ionotropic and metabotropic glutamate receptors is followed by the expression of plasticity - related immediate - early genes ( iegs ) and protein synthesis , which can underlie structural synaptic reorganisation and long - term increase in synaptic efficacy . experimental procedures that inhibit the induction , expression or maintenance of ltp can result in impairment of post - tetanic potentiation , stp , early ltp or late ltp ( 14 , respectively ) . grey arrows denote possible links between nmdar and mglur activation and facilitation of network activity in theta and gamma frequency bands . the apparent relationship between theta activity and certain types of behaviour , and the relative regularity and stability of this oscillatory pattern suggests that theta rhythm may serve as an internal timing mechanism for individual elements of spatially distributed neuronal ensembles ( buzski , 2005 ) . in other words , enhanced theta oscillations may provide an appropriate time - window for the firing of individual neurons within currently activated ensemble(s ) and , therefore , optimise the encoding of the acquired signal . indeed , the firing rate of both principal cells and several classes of interneurons in the hippocampus has been found to be theta phase - locked , i.e. to depend upon the phase of the ongoing theta cycle ( csicsvari et al . , 1999 ; an important link between hippocampal theta oscillations and the probability of potentiation of synaptic transmission is provided by observations with regard to increases or decreases of synaptic efficacy , which were caused by trains of high - frequency stimulation delivered either at the peak or the trough of theta cycle , and which resulted in ltp or ltd respectively ( huerta and lisman , 1993 , 1995 ; mccartney et al . , 2004 ; pavlides et al . , 1988 ) . during naturally occurring , locomotion - induced theta oscillations , ltp , that persists for at least 48 h , is preferentially induced by stimuli delivered near the local theta peak in behaving animals ( hyman et al . , 2003 ; orr et al . , 2001 ) , whereas in anesthetised animals a long - lasting enhancement of fepsp is elicited , if hippocampal afferents are stimulated synchronously on the peak of theta oscillations ( hlscher et al . , 1997 ; pavlides et al . , 1988 ) this demonstrates that not only the parameters of the stimulation protocol , but also the timing of tetanisation relative to the phase of theta cycle , can change the direction of synaptic plasticity ( ltd vs. ltp ) . thus , tetanisation - driven increases of theta and gamma oscillations , such as those observed in our study ( bikbaev and manahan - vaughan , 2007 ) , seem to be intrinsically interconnected , and play complementary roles in synaptic potentiation . the heightened theta power in the period after tetanisation is likely to temporally organise the firing of principal cells within theta time - windows , hence supporting the increase of the amplitude of gamma oscillations . bearing in mind that the higher amplitude of gamma oscillations reflects a higher degree of synchronisation in the network ( see for review : axmacher et al . , 2006 ) , the increase of gamma power in the period immediately after tetanisation may indicate that , during the facilitated theta activity , the firing of principal cells and interneurons is more synchronised ( or precise ) and/or recruits more elements into the network activity . accordingly , enhanced gamma oscillations synchronise neuronal firing within narrower gamma time windows and elevate the probability for afferent stimuli to coincide with the peak of theta cycle , where the gamma amplitude is highest , hence resulting in the strengthening of the activated synaptic pathway . the outcomes of studies of spike - timing dependent plasticity ( see for review : dan and poo , 2004 ) demonstrate that such synchronisation of neuronal spiking , within a 1030 ms window : which precisely matches to a single gamma cycle , can dramatically increase the probability of potentiation of activated synaptic connections . moreover , in in vitro recordings from the primary visual cortex , where theta activity is generally not so prominent as in the hippocampus , pairing the stimuli with either the peak or trough of oscillations in the beta and gamma frequency ranges has been found to effectively drive synaptic plasticity towards potentiation or depression , respectively ( wespatat et al . , 2004 ) . in this context , one can conclude that the transient enhancement of theta and gamma oscillations in the post - tetanisation period reflects the engagement of the system in encoding of acquired information . this can result in a subsequent reconfiguration of the network , coupled with the strengthening of currently activated connections , and the weakening of the others . furthermore , facilitated theta activity can support a tighter timing control of the firing of principal cells and interneurons in the network , which serves to optimise information processing . tetanisation - triggered enhancement of gamma oscillations is associated , on the other hand , with higher synchronisation and reflects the coordinated ensemble activity aimed to encode / retrieve relevant memories . accordingly , the absence or inconsistency of such changes in network activity can be related to the lack , or insufficiency , of activation of required neurotransmitter systems , sub - optimal conditions for the processing of sensory information and , therefore , a lower probability for ltp . for several decades , theta rhythm was regarded in rodents as an electrographic hallmark of exploratory behaviour , that occurs when the hippocampus is engaged in the acquisition and processing of sensory stimuli , which can lead in turn to experience - based modification of ongoing behaviour . gamma oscillations were also proposed to play a role in various cognitive processes both in human and animals . at the neuronal level , the acquisition and formation of memory traces are believed to rely on activity - related changes in synaptic efficacy , and ltp of synaptic transmission after patterned afferent stimulation is considered as an adequate experimental model of these processes . however , despite the existence of extensive experimental data showing the involvement of hippocampal theta and gamma oscillations in cognitive processes , a relationship between the coordinated enhancement of both theta and gamma power immediately after tetanisation and the consequences of induction of synaptic plasticity in freely moving rats was not , to our knowledge , described earlier . we found that changes in theta and gamma oscillatory activity precede electrically induced potentiation of synaptic transmission . importantly , the correlated tetanisation - driven enhancement of both theta and gamma oscillations is associated with successful short- and ltp , but not with its failure . these findings link ltp with the oscillatory activity of rather large assemblies of hippocampal principal cells and interneurons , and provide additional support for the role of hippocampal theta and gamma oscillations in information processing and the formation of new memories . 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 .

in the hippocampus in vivo , both synaptic plasticity and network activity are closely interdependent . we have found that immediately after an attempt to induce long - term potentiation ( ltp ) , changes in theta ( 510 hz ) and gamma ( 30100 hz ) activity correlate tightly with the occurrence of ltp , suggesting that tetanisation - driven activation of sensory inputs synchronises the activity of granule cells and interneurons , and thus , facilitates the encoding of acquired stimuli . this results in increase of theta and gamma power , and elevates the probability that afferent stimuli both coincide with the peak of theta cycle and reach their post - synaptic target within the gamma time - window ( of 1030 ms ) . both these mechanisms can effectively shift the direction , of tetanisation - induced changes in synaptic weight , towards potentiation and induction of ltp . here , we discuss our findings in the context of possible mechanisms that link theta and gamma oscillations with ltp induction , as well as their role in information processing and formation of memories .

Long-Term Potentiation of Synaptic Transmission as a Model of Synaptic Plasticity Tetanisation-Triggered Facilitation of Theta and Gamma Activity Favours LTP The Direction of Change in Synaptic Weight Correlates with the Phase of Theta Cycle Summary Conflict of Interest Statement

PMC4691630

one of the prominent hallmarks of obesity - induced inflammation is a shift in the phenotype of macrophage and t - cell populations present in adipose tissue ( at ) reflecting in a unique set of pro- and anti - inflammatory cytokines . free fatty acids and saturated fatty acids ( sfa ) are suggested to induce inflammation by activation of toll - like receptors ( tlrs ) 4 and 2 in adipose tissue and macrophages , culminating in an increased activation of nuclear factor kappa - b ( nf-b ) thus increasing the expression in proinflammatory cytokines like tnf- or il-6 [ 2 , 3 ] . apart from sfa , a deregulated ceramide synthesis has also been suggested to play an important role in obesity related inflammation [ 4 , 5 ] . aerobic exercise is known to have triglyceride - lowering effects accompanied by a reduction of visceral and abdominal adipose tissue in overweight and obese subjects [ 6 , 7 ] . these metabolic effects of exercise training are regularly accompanied by a reduction of the inflammatory status [ 8 , 9 ] . thereby , exercise training was followed by a reduced expression of both tlrs and several proinflammatory cytokines [ 9 , 10 ] . however , most studies on exercise effects on obesity investigated the effect of aerobic / endurance training [ 3 , 11 ] . resistance training received less attention , but there are several evidences that it also affects metabolic disorders and inflammation in obesity [ 1216 ] . insulin resistance is a common complication of obesity - induced inflammation and the reduction of blood glucose a desirable outcome of weight loss programs . in recent years , the nucleotide - binding domain and leucine - rich - repeat and pyrin domain containing 3 ( nlrp-3 ) inflammasome has been identified as a major contributor to the development of systemic inflammation and insulin resistance . nlrp-3 assembly is initiated upon tlr activation , extracellular atp , or active interleukin 1 ( il-1 ) . expression of proil-1 and proil-18 is nf-b dependent which in turn depends on activation through tnf-. the maturation of nlrp-3 renders caspase 1 active that cleaves inactive proil-1 and proil-18 into their active forms which are then secreted into the surrounding tissue . it was also found that ceramides induced the nlrp-3 inflammasome , driving the secretion of il-1 followed by the development of insulin resistance . ceramides induce insulin resistance through the dephosphorylation of serine / threonine kinase akt / pkb , hence disabling the translocation of glut4 to the cell membrane and reducing the uptake of glucose [ 1921 ] . however , the effects of differentiated exercise regimes on ceramide levels , at inflammation , and systemic cytokine levels in mice have so far not been investigated in one cohesive study . therefore , we investigated whether different exercise regimes affect glucose tolerance , serum fatty acids and ceramides , inflammatory parameters in at , and several inflammatory cytokines in serum of hfd fed mice . moreover , these effects might be achieved most likely via modifying serum ceramide levels as well as local and systemic inflammatory markers . whereas endurance training addresses mainly type i and type iia muscle fibers , resistance training is followed by a hypertrophy of types ii and iix [ 23 , 24 ] . finally , it is suggested that endurance and resistance exercise might address different targets of these pathophysiological signaling cascades . male c57bl/6 mice ( n = 36 ) , 10 weeks of age , were kept at 21 1c in a reverse dark - light cycle ( light on from 9 pm to 9 am ) . mice were randomly assigned to 6 groups ( 5 to 8 mice per group ) : standard chow / sedentary ( st / s , altromin 1320 , altromin spezialfutter gmbh & co. , germany ) , standard chow / endurance training ( st / et ) , standard chow / resistance training ( st / rt ) , high fat / sedentary ( hfd / s , td 03584 , harlan teklad , germany ) , high fat / endurance training ( hfd / et ) , and high fat / resistance training ( hfd / rt ) . the assigned diet was fed for 4 weeks before exercise protocols were implemented . a more detailed specification of the fatty acid composition can be found in table 1 . the animals were put on their diet 4 weeks before the 10-week exercise protocols commenced , which means a total of 14 weeks on the diet . the animals were handled according to the guide for the care and use of laboratory animals . the experimental design was approved by the animal welfare officer of the justus - liebig - university and the regional council of the city of giessen ( number 94/2010 ) . before et commenced , each mouse assigned to the endurance group was trained on an electrical treadmill ( custom - made ) at 80% vo2max for 30 min / day , 5 times / week for 10 weeks . briefly , by using a treadmill spiroergometry ( custom - made ) , maximal oxygen uptake ( vo2max ) and maximal running speed ( vmax ) of mice were determined . the treadmill was placed in a metabolic chamber where air was led through at a rate of 0.5 l / min . samples of 200 ml / min of gas were extracted to the paramagnetic oxygen analyzer ( type 1155 , servomex ) and the carbon dioxide analyzer ( lair 12 , m&c instrument ) . all animals were acclimated to the treadmill before vo2max and vmax were tested during a continuous , progressive test on the treadmill ergometer until exhaustion . after 10 min of acclimatization in the treadmill chamber the test uptake started at 0.15 m / s , increasing every 3 min by 0.05 m / s . the mice gripped with their front and their back paws on a horizontal wire of the metal mesh and the plate was placed in a vertical position . strength training was performed for 5 times / week for 3 minutes and 3 series . it was previously demonstrated that this type of isometric strength training was followed by an increase of maximum holding time in isometric strength test , a type ii fiber hypertrophy , and an increased glut4 protein content in the membrane fraction . glucose tolerance tests were performed at the end of the 10-week experimental period as follows : after an overnight 12-hour fast , blood was drawn from the tail vein of all animals ( approximately 10 l ) and glucose concentration measured ( glucometer , roche diagnostics , mannheim , germany ) . glucose ( 2 g / kg ) was applied to the mice via intraperitoneal injection . blood was collected in edta - coated tubes ( sarstedt , germany ) by cardiac puncture with heparin solution - washed 27 g syringes ( sarstedt , germany ) and centrifuged for 10 min at 4c and 1200 g . we used real - time quantitative pcr to determine tnf- , nlrp-3 , il-1 , and il-18 in isolated at . the plasma was used for a multiplex elisa approach ( mouse inflammation map , myriad rbm , germany ) as well as for determining circulating total fatty acids and ceramides . serum was tested for the following cytokines : cd40 ( cd40 ) , cd40 ligand ( cd40-l ) , c - reactive protein ( crp ) , endothelin-1 ( et-1 ) , eotaxin , epidermal growth factor mouse ( egf mouse ) , factor vii , fibrinogen , fibroblast growth factor 9 ( fgf-9 ) , fibroblast growth factor basic ( fgf - basic ) , granulocyte chemotactic protein-2 mouse ( gcp-2 mouse ) , macrophage colony - stimulating factor ( m - csf ) , growth - regulated alpha protein ( kc / gro ) , haptoglobin , immunoglobulin a ( iga ) , interferon gamma ( ifn - gamma ) , interferon gamma - induced protein 10 ( ip-10 ) , interleukin- ( il- ) 1 alpha ( il-1 alpha ) , il-1 beta , il-10 , il-11 , il-12 subunit p70 ( il-12p70 ) , il-17a , l-18 , il-2 , il-3 , il-4 , il-5 , il-6 , il-7 , leukemia inhibitory factor ( lif ) , lymphotactin , macrophage colony - stimulating factor-1 ( m - csf-1 ) , macrophage inflammatory protein-1 alpha ( mip-1 alpha ) , macrophage inflammatory protein-1 beta ( mip-1 beta ) , macrophage inflammatory protein-1 gamma ( mip-1 gamma ) , macrophage inflammatory protein-2 ( mip-2 ) , macrophage inflammatory protein-3 beta ( mip-3 beta ) , macrophage - derived chemokine ( mdc ) , matrix metalloproteinase 9 ( mmp9 ) , monocyte chemotactic protein-1 ( mcp-1 ) , monocyte chemotactic protein 3 ( mcp-3 ) , monocyte chemotactic protein-5 ( mcp-5 ) , myeloperoxidase ( mpo ) , myoglobin , oncostatin - m ( osm ) , serum amyloid p - component ( sap ) , serum glutamic oxaloacetic transaminase ( sgot ) , stem cell factor ( scf ) , t - cell - specific protein , rantes , thrombopoietin ( tpo ) , tissue factor ( tf ) , tissue inhibitor of metalloproteinase 1 mouse ( timp-1 mouse ) , tumor necrosis factor alpha ( tnf - alpha ) , vascular cell adhesion molecule-1 ( vcam-1 ) , vascular endothelial growth factor a ( vegf - a ) , and von willebrand factor ( vwf ) by a multiplexed fluorescent bead - based immunoassay ( luminex , myriad rbm , austin , texas ) . adipose tissue ( at ) was isolated from mice ( for groups see above ) and immediately frozen with liquid nitrogen and stored at 80c until use . rna was isolated by a combination of trifast ( peqlab , erlangen , germany ) and rneasy mini kit ( qiagen , hilden , germany ) . respective tissue was homogenized with a precellys 24 ( peqlab , erlangen , germany ) homogenizer in the presence of trifast , followed by 5 min incubation on ice and centrifugation at 4c and 12000 g for 10 min . the supernatant was mixed with 200 l chloroform , followed by 10 min incubation at room temperature . after centrifugation at full speed for 15 min , the upper phase was mixed with an equal amount of 70% ethanol . the mix was loaded on an rneasy mini spin column and rna isolated according to the manufacturer 's instructions . the rna concentrations were measured with a nanodrop nd-1000 ( kisker - biotech , steinfurt , germany ) . the isolated rna ( 500 ng , each ) was converted to cdna using the iscript cdna synthesis kit ( biorad , munich , germany ) . the conditions for the reverse transcription were as follows : 1 cycle at 25c for 5 min ; 1 cycle at 42c for 30 min ; 1 cycle at 85c for 5 min . relative quantification of il-1 , tnf- , nlrp-3 , and il-18 was performed by quantitative real - time pcr with the iq sybr green supermix in duplets according to the manufacturer 's instructions ( biorad , munich , germany ) . per reaction , a 25 l mixture was used containing 12.5 l iq sybr green supermix , 0.5 l forward and reverse primer ( rpl32-f : 5-tggaggtgctgctgatgtg-3 ; rpl32-r : 5-gcgttgggattggtgactct-3 ( product size : 127 bp ) ; il-1-f : 5-tggtgtgtgacgttcccatt-3 ; il-1- : 5-tcgttgcttggttctccttg-3 ( product size : 172 bp ) ; nlrp-3-f : 5-tccagcacccaggctgtaac-3 ; nlrp-3-r : 5-tgcagagcaggtgcttcagt-3 ( product size : 189 bp ) ; tnf--f : 5-agggctgtgggacctaaatgt-3 ; tnf--r : 5-atgggatgagtatggggcagc-3 ( product size : 199 bp ) ; il-18-f : 5-gccgacttcactgtacaaccg-3 ; il-18-r : 5-gagggtcacagccagtcctc-3 ( product size : 182 bp ) ) , 9.5 l sterile water , and 2 l of the 1 : 5 diluted complementary dna template . the conditions of the real - time pcr ( mx3000p , stratagene , heidelberg , germany ) were as follows : 1 cycle at 95c for 10 min and then 40 cycles at 95c for 10 s , 59c for 10 s , and 72c for 10 s , followed by a dissociation curve . the intron - spanning primers were designed by using sequence information from the ncbi database . the threshold cycle ( ct ) values were normalized to the endogenous control ( ribosomal protein l32 , rpl32 ) , n = 3 - 4 . the specificity of the primer pair products was tested by melting curves and agarose ( 1.6% ) gel electrophoresis . the expression of the following enzymes was investigated : ceramide synthase 2 ( cers2 ) , ceramide synthase 4 ( cers4 ) , fatty acid elongase 1 ( elovl1 ) , fatty acid elongase 3 ( elovl3 ) , alkaline ceramidase 2 ( acer2 ) , serine palmitoyltransferase , long chain base subunit 2 ( sptlc2 ) , sphingomyelin phosphodiesterase 1 acid lysosomal ( smpd1 ) , and stearoyl - coa desaturase ( delta-9-desaturase , scd1 ) . rna isolation , cdna synthesis , and qpcr analysis were performed as described recently in detail . in brief , total rna was isolated from 10 mg liver aliquots using trizol reagent ( invitrogen , karlsruhe , germany ) , and rna concentration and purity were estimated from the optical density at 260 and 280 nm ( infinite 200 m microplate reader , tecan , mnnedorf , switzerland ) . rna integrity was assessed by confirming intact bands corresponding to the 18s and 28s ribosomal rna subunits using 1% agarose gel electrophoresis . following cdna synthesis within one week after rna isolation using dt18 primer and m - mulv reverse transcriptase ( mbi fermentas , st . leon - rot , germany ) , qpcr runs were performed with a rotorgene 2000 system ( corbett research , mortlake , australia ) as described recently in detail . the used primers were as follows : ceramide synthase 2-f : caaagctggaccaaggttcc , ceramide synthase 2-r : gagagggaggcagtgagatc , ceramide synthase 4-f : ctgtgggtgtccttgtagtcc , ceramide synthase 4-r : tcctctggctttggtttctg , fatty acid elongase 1-f : tcccatcccttctcccagcc , fatty acid elongase 1-r : ccagccctttcagcctccag , fatty acid elongase 3-f : agacctaccggctgtcctcc , fatty acid elongase 3-r : gaaccgagtgagcgtccagg , alkaline ceramidase 2-f : agcccgccatcaacaata , alkaline ceramidase 2-r : ccagcagaagagagccagag , serine palmitoyltransferase , long chain base subunit 2-f : cctgtcagcagctcatacca , serine palmitoyltransferase , long chain base subunit 2-r : cacactgtcctgggaggaat , sphingomyelin phosphodiesterase 1 , acid lysosomal - f : gccagtcagccgtccatc , sphingomyelin phosphodiesterase 1 , acid lysosomal - r : cccagaagcaagccacaag , stearoyl - coa desaturase ( delta-9-desaturase)-f : cgtggcttcttcttctctctca , and stearoyl - coa desaturase ( delta-9-desaturase)-r : cttctcggctttcaggtcag . all primer pairs were designed to have melting temperatures of about 60c , and if possible , both primers of a primer pair were designed to be located in different exons . ct values of target genes and reference genes were obtained using rotorgene software 5.0 ( corbett research ) . for determination of relative expression levels relative quantities were calculated using genorm normalization factor . in order to calculate the normalization factor , all ct values were transformed into relative quantification data by using the 2 equation , and the highest relative quantities for each gene were set to 1 . from these values the normalization factor was calculated as the geometric mean of expression data of the three most stable out of six tested potential reference genes ( atp5b , canx , cyc1 , eif4 , gapdh , and rps9 ) . reference gene stability across samples from each tissue and each cell line was determined by performing genorm analysis . after normalization of gene expression data using the calculated genorm normalization factor , means and sd were calculated from normalized expression data for samples of the same treatment group . lipids were quantified by direct flow injection electrospray ionization tandem mass spectrometry ( esi - ms / ms ) in positive ion mode using the analytical setup and strategy described previously . in brief , plasma samples were extracted according to the method by bligh and dyer in the presence of nonnaturally occurring lipid species used as internal standards . a fragment ion of m / z 264 was applied for sphingosine - based ceramides ( cer ) and hexosylceramides ( hexcer ) with n - heptadecanoyl - sphingosine as internal standard . fatty acid methyl ester was formed by methanolic acetyl - chloride derivatization and extracted with hexane . total fatty acid ( fa ) analysis was carried out by gas chromatography coupled to mass spectrometry ( gc - ms ) . two - way anovas with bonferroni post hoc testing were performed in order to analyze differences between treatments . for the analysis of fa and ceramides a bonferroni correction was applied with significance levels of p < 0.002 ( fatty acids ) and p < 0.005 ( ceramides ) . we present the proinflammatory panel measured by multiplex elisa as a heat map . to compare the cytokine production between the groups we used a two - way anova with bonferroni correction and set the new significance cut - off at p < 0.001 . in order to calculate the corrected p value we divided p < 0.005 by the number of compared parameters . furthermore , we calculated percentage changes of the cytokine levels of the hfd groups relative to the mean of the st / s group . for the analysis of the rt - pcr results from at , the comparative ct method was used . data are shown as 2 ( ct ) that were calculated by subtracting the mean ct ( ct ( reference gene ) ct ( gene of interest ) ) of control sample from the ct of each examined sample . the statistical analysis of hepatic mrna expression was performed as a two - way anova on the relative mrna levels calculated as described above . diet and exercise had significant effects on blood glucose ( p < 0.01 ) after glucose challenge . compared to st / s group blood glucose levels in the hfd / s group were significantly higher at all - time points after glucose challenge ( p < 0.001 ) . both types of exercise led to a significant reduction of glucose intolerance in the hfd animals when compared with the sedentary group . however , the effect was more pronounced after et ( p < 0.001 ; figure 1 ) . mice fed the hfd gained significantly more weight than the mice fed the st diet ( p < 0.001 , figure 2(a ) ) . within the high fat group , both et ( p < 0.05 ) and rt ( p < 0.001 ) attenuated the weight gain when compared with the sedentary group . vo2max change was measured at baseline before exercise commenced as well as after 10 weeks on exercise . the percent changes in vo2max for each diet and exercise group are shown in figure 2(b ) . et but not rt led to significant increases of vo2max in both st and hfd groups ( p < 0.001 ) . the hfd significantly increased most of the total fatty acids and ceramides in plasma of mice from hfd / s group ( p < 0.005 ; tables 2 and 3 ) . neither et nor rt was able to reduce significantly any fatty acid compared to the enhanced levels of sedentary mice . eight out of 10 ceramides analyzed increased significantly in plasma from hfd / s mice compared to st / s mice ( table 3 ) . et and rt affected ceramide levels slightly different in hfd mice . while et decreased cer d23:0 and d24:1 , rt was followed by a significant decrease of cer d24:1 and hexcer d24:1 ( p < 0.005 ; table 3 ) . endurance training significantly decreased the relative mrna levels of cers2 , elovl1 , and elovl3 as well as smpd1 ( p < 0.05 ) in comparison with the sedentary st group ( figures 5(a ) , 5(c ) , 5(d ) , and 5(g ) ) . furthermore , et increased the relative mrna level of cers2 ( p < 0.05 ) in the hfd in comparison to the corresponding st fed mice . in contrast , elovl3 was strongly reduced in the et group due to the hfd feeding . however , elovl3 was reduced by rt only in the st group ( figures 5(c ) and 5(d ) ) . elovl1 and elovl3 mrna expression decreased in the sedentary group due to the high fat diet . scd1 and acer2 were not significantly changed in the st groups . however , we observed a trend towards a reduction of mrna expression due to exercise in the hfd groups that did not reach statistical significance ( figures 5(e ) and 5(h ) ) . the comparison within the hfd groups revealed that et significantly reduced cers4 and sptlc2 mrna . resistance training had a similar effect on sptlc2 but did not reach significance for cers4 ( figure 5(f ) ) . plasma concentrations of eotaxin , haptoglobin , mcp-1 , mcp-3 , mpo , cd40 , kc / gro , sap , vcam-1 , vwf , timp-1 , ip-10 , il-18 , and mip-1 were significantly increased in hfd / s group compared to the st / s ( p < 0.001 ) . again , exercise regimes differently affect cytokine expression in the hfd groups ( figure 3(b ) ) . while et significantly reduced cytokine levels of mip-1 , rt was followed by a significant reduction of cd40 , il-18 , mip-1 , and timp-1 ( p < 0.001 ) . in addition , we show in figure 3(a ) , as a supplementary graph , the percentage change of selected cytokines levels after both et and rt in the hfd groups which represent the most prominent effects . this should illustrate the effect of exercise on the cytokine level of high fat diet fed mice . notably , mice fed the hfd showed a decrease of plasma immunoglobulin a ( p < 0.001 ) which was not affected by any exercise intervention . feeding the hfd was followed by a significant increase of il-18 , tnf- , and nlrp-3 mrna expression in at , while the enhanced mrna expression of il-1 did not reach significance ( figures 4(a)4(d ) ) . et was accompanied by a significant reduced expression of tnf- and il-1 compared to hfd / s group . in contrast , rt significantly reduced only the nlrp-3 expression ( figure 4(d ) ) . a secondary finding was that both exercise treatments were effective in enhancing il-18 expression in at of st fed mice . we showed that hfd - induced obesity was associated with a substantial increase in body weight , a decrease of physical performance , elevated levels of serum ceramides and fatty acids , both a local , in at , and a systemic inflammatory response , and finally glucose intolerance . both training interventions , endurance as well as resistance exercise , were effective in reversing or in attenuating the diet - induced glucose intolerance . however et reduced blood glucose more efficiently than rt ( p < 0.001 ) probably due to higher energy expenditure in the et group . it is speculated that the exercise effect might be mediated via metabolic and inflammatory pathways as indicated by decreased levels of serum ceramides and reduced levels of tissue and serum inflammatory markers . obesity - associated tissue inflammation is assumed to be an important factor in the activation of the innate immune system leading to a systemic chronic low grade inflammation . it has been suggested that the starting point for at inflammation might be the cell stress - associated accumulation of metabolites which have been shown to dramatically increase in response to hfd feeding . in this regard , ceramides may represent a potential upstream initiating event for obesity - induced inflammation . current data demonstrated that high fat feeding induced an increase of several ceramides in serum , an observation that is in line with previous studies . likewise , ceramide transported in ldl has been shown to be elevated in the plasma of obese patients with type 2 diabetes . incubating cultured myotubes with reconstituted low density lipoprotein ceramide 24:0 promoted ceramide accrual in cells and was accompanied by reduced insulin - stimulated glucose uptake . moreover , in lean mice the infusion of ldl - ceramide reduced insulin - stimulated glucose uptake , and this was due to impaired insulin action specifically in skeletal muscle . ceramide seems to induce insulin resistance by inhibiting insulin signal transduction , principally at akt . ceramide is also postulated to activate proinflammatory pathways in macrophages , perhaps via amplification of tlr 4-mediated inflammation . therefore , it has been suggested that changes in plasma long chain ceramides might link the effects of hfd on the inflammatory status of adipose tissue . we did not determine macrophage phenotype in adipose tissue but showed that an increased expression of tnf- , il-18 , and nlrp-3 which was detected in adipose tissue of hfd / s group might indicate adipose tissue inflammation . these findings are in line with previous studies which found an increased inflammatory status in at in response to chronic high fat feeding [ 34 , 37 ] . while et significantly reduced the expression of tnf- , il-1 , and il-18 , rt reduced nlrp-3 expression . thereby , a reduced expression of tnf- and il-1 might be interpreted as a reduction of macrophage and lymphocyte activation . although we did not determine caspase activity , the reduced expression of il-1 might represent a decrease in the maturation of il-1 by a decrease in nlrp-3 dependent caspase 1 activity [ 18 , 38 ] . however , a reduction of caspase 1 activity has been shown to be linked with lower levels of ceramides in serum , which has been demonstrated in our study after the exercise interventions . therefore , our study suggests that a starting point for the anti - inflammatory effects of exercise training might be the reduction in ceramide levels . these results are in line with previously published research that demonstrated the essential involvement of ceramides in the development of insulin resistance [ 39 , 40 ] . exercise significantly decreased cer d18:1/23:0 and d18:1/24:1 in the hfd / et group and d18:1/24:1 and hexcer d18:1/24:1 in the hfd / rt group when compared to the hfd / s group . it has been shown that cer 24:1 and 24:0 make up about 80% of all measured plasma ceramides . ceramides like reconstituted low density lipoprotein ceramide 24:0 were found to increase tnf- , il-1 , il-6 , and mcp-1 in vitro and to induce insulin resistance in lean mice . it is therefore assumed that the exercise - induced reduction of plasma long chain ceramides is at least one reason for the reduced expression of inflammatory markers in serum and at as well as the increased glucose tolerance . because of the reduced levels of ceramides in plasma , we measured whether exercise affects the expression of genes involved in the synthesis of ceramides . cers2 and elovl1 have been shown to be essential for the synthesis of cer d24:0 and cer d24:1 . the hepatic mrna expression levels of cers2 and cers4 do not change significantly in the sedentary hfd mice groups which might suggest adequate substrate availability under condition of our hfd protocol . elovl1 and elovl3 decreased significantly in the hfd / s group in comparison with the st / s group . since the hfd feeding led to a significant increase in long chain fas , this downregulation of elongases might indicate a feedback loop mechanism where a high pool of fa downregulates mrna expression . in murine hepatocytes , the expression of cers4 is far below that of cers2 , which is the most abundantly expressed ceramide synthase in mammals . cers2 and cers4 share some substrate specificity ; for example , cers4 utilizes c18-c22 fa , whereas cers2 utilizes c20-c26 fa [ 42 , 43 ] . furthermore , the synthesis rate of cers in vitro has been shown to be dose - dependent and that ceramide synthesis is governed by the availability of fa [ 44 , 45 ] . in addition , our results showed that sptlc2 expression was significantly reduced by exercise which might also point towards a decline of available fa for ceramide synthesis . therefore , we can speculate that exercise affects blood ceramide profile mostly through an alteration of substrate availability for ceramide synthesis rather than a change in expression of gene involved in the ceramide pathway . another reason for the reduced expression of inflammatory markers in serum and at might be the effects of both exercise and diet on the expression of il-18 . alterations in il-18 might have been responsible for both the downregulation of glucose tolerance in hfd / s group and the increased glucose sensitivity after exercise training . these assumptions are supported by previous studies which found that obese individuals had higher il-18 mrna content in the abdominal adipose tissue than nonobese subjects and that exercise training lowered the elevated il-18 mrna levels in obese subjects . activation of the nlrp-3 inflammasome in course of diet - induced obesity in mice led to an increase in il-18 secretion and was shown to affect insulin signaling . in addition , it has been shown that il-18 mrna and plasma il-18 correlated with insulin resistance , suggesting that training - induced reduction in il-18 expression may be a contributing mechanism to improve insulin sensitivity after training [ 18 , 46 , 47 ] . we found that il-18 mrna expression in at was increased in all high fat groups but was not changed by exercise . the levels of plasma il-18 , however , were significantly increased in the hfd / s group and were reduced by endurance as well as by resistance training . the results of our study therefore suggest that the reduction of nlrp-3 levels in at was not effective enough in reducing il-18 mrna expression in at . however , we speculate that it affected posttranslational modification leading to a reduced maturation of il-18 protein in at or a reduced mobilization of il-18 into the blood stream . the role of il-18 during development of obesity seems to be ambivalent as it has been shown that deficiency of either il-18 or il-18 receptor triggers obesity and hyperinsulinemia in mice and that treatment with recombinant il-18 reversed these effects . additionally , il-18 elevated adenosine monophosphate - activated protein kinase in skeletal muscle which is known to increase energy expenditure and endurance in mice [ 48 , 49 ] . these studies indicate that il-18 might play an essential role in metabolic regulation and exercise adaptation . in the current study the expression of il-18 , in at , was significantly increased in the exercised standard fed mice when compared with the st / s animals . additionally , we did not detect elevated glucose levels in the exercising st groups . these results suggest that the role of il-18 in at is that of a metabolic key cytokine which might also be involved in the adaptation to exercise . in addition to il-18 , exercise was effective in reducing tnf- mrna expression which might represent another pathway of action . regarding the connection between inflammation and insulin resistance , it is known that tnf- induces insulin resistance in vitro and in mice . however , in contrast to il-18 a decrease of tnf- was found in at after exercise while no changes of peripheral tnf- were detected . the localized inflammatory environment inside the adipose tissue seemed to have systemic consequences as reflected by enhanced levels of several proinflammatory serum cytokines . a cluster of proinflammatory cytokines increased in the hfd / s group might indicate a state of chronic low grade inflammation . thereby , notable increases of cd40 might be the result of an activation of lymphocytes , while cytokines like il-18 , mip-1 , and timp-1 might point towards an increased activation of macrophages . while rt mainly affected cd40 , il-18 , mip-1 , and timp-1 expression , et effectively reduced mip-1. since both exercise types reduced body weight , we can not exclude the possible confounding effect of this weight loss and potential lower body fat content on the reduction of inflammatory markers . a major limitation of this study is that our hfd model contained 34% lipids by weight , which is a moderate challenge . however , we propose that exercise diminishes both lymphocyte and macrophage activity which is in accordance with previously published data . taken together , we show that hfd - induced obesity is accompanied by an increase of ceramides , activation of inflammasome in at , and a secretion of several proinflammatory cytokines including il-18 that all are known to affect glucose sensitivity . both et and rt are effective therapeutic strategies to improve both metabolic dysfunction and inflammatory status resulting in an improved glucose tolerance . however , the identification of definitive molecular targets for both training regimes , which seem to address slightly different signaling pathways , has to be elucidated in future studies .

the study aimed to investigate the effects of differentiated exercise regimes on high fat - induced metabolic and inflammatory pathways . mice were fed a standard diet ( st ) or a high fat diet ( hfd ) and subjected to regular endurance training ( et ) or resistance training ( rt ) . after 10 weeks body weight , glucose tolerance , fatty acids ( fas ) , circulating ceramides , cytokines , and immunological mediators were determined . the hfd induced a significant increase in body weight and a disturbed glucose tolerance ( p < 0.05 ) . an increase of plasma fa , ceramides , and inflammatory mediators in adipose tissue and serum was found ( p < 0.05 ) . both endurance and resistance training decreased body weight ( p < 0.05 ) and reduced serum ceramides ( p < 0.005 ) . while rt attenuated the increase of nlrp-3 ( rt ) expression in adipose tissue , et was effective in reducing tnf- and il-18 expression . furthermore , et reduced levels of mip-1 , while rt decreased levels of il-18 , mip-1 , timp-1 , and cd40 in serum ( p < 0.001 ) , respectively . although both exercise regimes improved glucose tolerance ( p < 0.001 ) , et was more effective than rt . these results suggest that exercise improves hfd - induced complications possibly through a reduction of ceramides , the reduction of inflammasome activation in adipose tissues , and a systemic downregulation of inflammatory cytokines .

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

PMC5430260

ankle sprains are the most common musculoskeletal injuries1 , and athletes with excessive movement and activities are the most common victims . soccer players perform many running , jumping , and turning movements , and these movements cause excessive range of motion at the ankle joint . this excessive motion range is the most likely cause of ankle sprain in soccer players2 . a person with an acute ankle sprain has functional limitations due to severe pain , a limited range of motion , and increased instability of the ankle . many treatments are available for ankle sprain depending on the severity of the injury , but the rices method in the acute phase is the most representative one4 . after such a treatment process is applied in the acute phase , the functional movement of the ankle is improved through treatments including electrostimulation , ultrasound therapy , and manual therapy5 . the taping method is frequently used to prevent ankle sprain in athletes , and is widely used for therapeutic purposes . according to lee et al.6 applied to young soccer players with functionally instable ankle , kinesiology taping method increase dynamic balance ability . hong et al.7 reported that application of kinesiology taping to a football player with injury is an effective adjunct therapy . this study evaluates the effect of ankle balance taping on dynamic and static balance of soccer players with acute ankle sprain . sixteen subjects were recruited and all met the inclusion criteria for study procedure . subjects were recruited from a high school soccer team , after providing informed , written consent . written informed consent according to the ethical standards of the declaration of helsinki was provided by all subjects prior to participation , and all agree to participate in this study . the mean age of the participants was 17.6 0.7 years , height was 175.1 5.2 cm , weight was 77.2 9.1 kg , bmi was 25.0 1.7 , cait score was 10.8 4.1 , and days since onset 8.2 3.2 days . the enrollment criteria applied were as follows : ( 1 ) suffered ankle sprain within one month , ( 2 ) cumberland ankle instability tool ( cait ) score < 248 , ( 3 ) not participating in any ankle treatment program , ( 4 ) no muscular - skeletal disorder and other severe lower extremity injury , ( 5 ) no history of ankle fracture and ankle surgery , ( 6 ) no ankle edema , ( 7 ) no history of neurological disease , vestibular or visual disturbance , or any other pathology , ( 8) no taping side effects such as skin redness . a cross - over randomized design was used . subjects were randomly assigned to a taping , placebo taping , and no taping . subject characteristics and all outcome measures obtained before and after treatment were assessed by physician 1 , who was blinded to treatment allocations . intervention was performed in a closed room by physician 2 , who was not involved in subject assessment . both physicians were instructed not to communicate with subjects about study goals or treatments . the patient is seated in a comfortable position on a table that is high enough to prevent the feet from touching the ground , while the therapist applies the tape on the damaged ankle stretched with a tension of 3040%9 . the first stage is posterior talar gliding taping to increase the dorsiflexion of the ankle . since the patients have a disability in the inversion of feet due to lateral ankle sprain , taping is done twice to reinforce the inversion of ankle through eversion taping application . the fourth stage applies the posterior talar gliding taping conducted in the first stage twice . the first stage begins from the inner malleolus , and it is applied up to the inner middle point of the pelvic limb . the second stage begins from the outer malleolus up to the outer middle point of the pelvic limb . for postural adjustment ability , center of pressure ( cop ) was measured using biorescue ( rm ingenierie , rodes , france ) . the elements measured included the sway length and sway speed to evaluate the static balance ability ; low value indicated good balance ability . also , the element measured included the limit of stability ( los ) to evaluate the dynamic balance ability ; high value indicated good balance ability . limit of stability ( los ) , sway length , and sway speed were measured for one minute . general characteristics were analyzed using descriptive statistics and results are reported as means and standard deviations . one - way repeated anova was used for the group analysis , and the post - hoc tukey test was used to correct for multiple comparisons . null hypotheses of no difference were rejected if p - values were less than 0.05 . abt showed significantly difference of limit of stability , sway length , and sway speed than nt and pt ( p<0.05 ) ( table 1table 1.comparison of the limit of stability ( los ) , sway length , sway speed among the three conditions ( n=16)mean sdpost - hocntptabtlos ( mm)5,943.0 3,565.86,304.7 3,516.88,537.0 3,472.4abt > pt abt > ntsway length ( cm)31.8 9.629.3 7.722.7 5.8abt > pt abt > ntsway speed ( cm / s)0.5 0.10.4 0.10.3 0.1abt > pt abt > ntnt : no taping ; pt : placebo taping ; abt : ankle balance taping ) . nt : no taping ; pt : placebo taping ; abt : ankle balance taping this study investigated the effect of ankle balance taping on the improvement of balance ability of soccer players with acute ankle sprains . after applying ankle balance taping , sway length and sway speed ( measures of static balance ) , and limits of stability ( los ) ( a measure of dynamic balance ) , improved significantly compared to the other two interventions ( placebo taping and no taping ) . nicole et al.11 reported that kinesiology taping reduced chronic low back pain ( clbp ) . lee et al.12 reported that the kinesiology taping badminton players with achilles tendon pain reduced achilles tendon pain . fouladi et al.13 proposed that dynamic posture stability is improved if kinesiology taping is applied to the foot . first , the small sample size may have influenced certain variables and influenced the results . therefore , these results can not be generalized to all subjects who have ankle instability . second , the current experiment is a crossover design , which makes it difficult to observe the learning effect of taping . further studies , including a long - term follow - up assessment , are needed to evaluate the long - term benefits of balance taping . in conclusion , the application of ankle balance taping that uses kinesiology tape instantly increased the dynamic and static balance ability of soccer player with an ankle sprain . therefore abt can be a useful alternative to prevent and treat ankle sprain of soccer players .

[ purpose ] this study aimed to evaluate the immediate effect of ankle balance taping on balance ability of soccer players with acute ankle sprain . [ subjects and methods ] this study was conducted with 16 subjects who were diagnosed with ankle sprain . a cross - over randomized design was used . each subject performed three interventions in a random order . subjects were randomly assigned to an ankle balance taping , placebo taping , and no taping . for dynamic and static balance , ability was measured using biorescue ( rm ingenierie , rodes , france ) . limit of stability , sway length and sway speed for one minute were measured . [ results ] the limit of stability , sway length and sway speed differed significantly among the three different taping methods . [ conclusion ] in this study , we found that ankle balance taping was effective in terms of improving balance ability of soccer players with an ankle sprain .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC1615254

the lengthy process and expertise required to isolate and identify potential microsatellite markers often precludes the use of this valuable technique in studies to determine genetic variation in natural populations . if microsatellite markers identified and developed from one biological source could be applied to other similar species , the usefulness of these genetic markers could be broadened . fortunately , a number of microsatellites markers have been developed to study the population genetic variation in the old world bollworm , helicoverpa armigera , a serious insect pest of several agriculturally important grain and fiber crops ( tan et al . , 2001 ; ji et al . , 2003 ; scott et al . , 2004 ) . we therefore undertook a survey of some of the available lepidopteran species that are used routinely in our laboratory , namely helicoverpa zea , heliothis virescens , and heliothis subflexa , to determine if previously designed microsatellite markers for h. armigera from several published sources could be applied to these closely related lepidopteran species . selection of these species for study was also contingent on their importance as field crop pests . the host range of h. zea , the corn earworm , includes over 100 plants with the most significant crops being corn , cotton and tomato . occasional hosts include bean , broccoli , cabbage , chrysanthemum , eggplant , head cabbage , green bean , lettuce , okra , pea , pepper , soybean , strawberry and watermelon . the tobacco budworm , h. virescens , is also principally a field crop pest , attacking such crops as alfalfa , clover , cotton , flax , soybean , corn , and tobacco . however , it sometimes feeds on such vegetables as cabbage , cantaloupe , lettuce , pea , pepper , pigeon pea , squash , and tomato , especially when cotton or other favored crops are not abundant . h. virescens is a common pest of geranium and other flower crops such as ageratum , bird of paradise , chrysanthemum , and gardenia , to name a few . in contrast , h. subflexa is of minor agricultural importance feeding on a few plant species such as solanum nigrum and physalis spp , but serves as a unique laboratory subject in studies to determine and compare host range infectivity and genetic resistance to baculoviruses . based on a previously published protocol ( mcintosh et al . , 1996 ) , genomic dna was extracted from 2 or early 3 instar h. zea and h. virescens larvae obtained from the north carolina state university- entomology insectary , raleigh , north carolina , whereas 2 or 3 instar h. subflexa larvae were obtained in - house at the usda , ars , biological control of insects research laboratory , columbia , missouri . fourteen published primer sets designed to amplify the following microsatellite loci of h. armigera were employed in this study : ( 1 ) ( hab60 ) -- ( ctg)2 ( ttg)3 ( ctg)5 ( ttg)2 , ( 2 ) ( hac14 ) -- ( attt)5 , ( 3 ) ( had47 ) --(ca)5 ( tca)4 , ( 4 ) ( hac87 ) - ( tc)5 ( scott et al . , 2004 ) ; ( 5 ) ham2 -- ( ttttga)9 , ( 6 ) ham3 --(taaa)2 ( taaat)4 , ( 7 ) ham4 -- ( tctg)6 tctt ( tctg)6 , ( 8) ham5 -- ( t)n ( g)n , ( 9 ) ham6 --(gat)2 tt ( gat)2 tt (aata)5 ( tan et al . , 2001 ) ; ( 10 ) harssr1 - ( tgc)2gat ( tgy)4gat(tgy)35(tga)2 agc(tgy)8 ( 11 ) harssr2 - ( atg)7 , ( 12 ) harssr3 - ( tca)6 , ( 13 ) harssr4 - ( gyt)25 , and ( 14 ) harssr5 - [ t(t)aa]6 ( ji et al . , 2003 ) . dna microsatellite amplification was conducted under the following two polymerase chain reaction conditions using a hybaid omnigene thermal cycler ( midwest scientific , www.midsci.com ) in 25 l of puretaq ready - to - gotm pcr bead reaction mixture ( amersham biosciences , www.apbiotech.com ) , including 100 - 200 ng of genomic dna template . first , after initial denaturing at 94 c for 5 min , the reaction mixture underwent 35 cycles at 94 c for 1 min , variable annealing temperature ( see tables 1 and 2 ) for 30 sec , 72 c for 40 sec , and a final extension at 72 c for 5 min ( tan et al . , 2001 ) . second , after initial denaturing at 940 c for 1 min , the reaction mixture underwent 35 cycles at 94 c for 1 min , 50 c for 1 min , 73 c for 1 min , and a final extension at 72 c for 5 min ( scott et al . , 2004 ) . these two previously published pcr conditions with their respective primers were used to establish a comparative baseline for the three lepidopteran species examined in this study . however , if the expected fragment size(s ) was not detected under the original pcr conditions for a particular microsatellite , empirical studies with various annealing temperatures were conducted in an attempt to resolve these problematic microsatellite markers ( table 1 ) . a 10 l aliquot of each amplified sample was run on a 2.5% metaphor agarose gel ( 10 mm sodium hydroxide - boric acid buffer , ph 8.5 ) for ca . 1 h at a constant 120 v using a bio - rad wide mini - sub cell - gel system . initially , annealing temperatures previously published for the various microsatellites detected in h. armigera were employed in this study with resulting mixed success . therefore , as indicated in table 1 , several annealing temperatures were tested for each locus in all three species in an attempt to determine the optimal running conditions for successful microsatellite amplification . table 2 shows the microsatellite loci that failed to show distinct single - copy bands under the various pcr amplification conditions tested . of the fourteen loci surveyed , only four loci ( hac14 , hab60 , hac87 , and harssr1 ) consistently demonstrated scorable single - copy microsatellite bands that might lead to the potential detection of population polymorphism in subsequent studies ( fig.1 ) . potential detection must be emphasized since the samples tested were limited to only laboratory reared insects . of the four loci that consistently demonstrated scorable single - copy microsatellite bands , length polymorphism was identified only in the hab60 marker ( 160 bp and 140 bp ) . the remaining microsatellites investigated showed multiple banding patterns , which have typically been observed in a number of lepidopteran species during the process of microsatellite clone development , and further indicate the repetitive nature of the flanking regions of microsatellites throughout the genome of lepidoptera ( zhang , 2004 ) . the hac14 270 bp band ( fig . 2 ) detected among all three species , and first thought to be a microsatellite repeat variation , appears to be actually caused by a duplication of the downstream primer sequence used to amplify the microsatellite ( sequence data not shown ) . to obtain a more accurate picture of the nucleotide base composition of some of the detected single - copy microsatellites that showed either the expected allele size or a variant , direct dna sequencing of pcr products was performed at the university of missouri dna core facility , columbia , missouri using an applied biosystems ( www.appliedbiosystems.com ) 3730 dna analyzer . because of the known potential for amplification errors during the pcr reaction due the inherent nature of the taq polymerase , 2 - 3 replicate samples of each locus were sequenced from individual insects and a single consensus sequence was generated employing viscose ( spitzer et al . in addition to the generated sequence alignments , the t - coffee program also provides an index of consistency of the overall residue evaluation ( core ) , an objective measure that identifies which regions of the compared sequences are correctly aligned by averaging the scores of each of the aligned pairs involving a base within a column ( notredame et al . , 2000 ) . a core value > = 3 would indicate a properly aligned base position and is considered the best compromise between a level of sensitivity and specificity required for proper base alignment . all of the aligned portions of the expected microsatellites showed reasonably high core scores for their individual alleles ( 70% for hac14 ; 70% , 51% for hab60 ( 160 bp , 140 bp , respectively ) ; 91% for hac87 ( 118bp ) ; and 71% for harssr1 ( 240bp ) , indicating at least for the most part a good portion of the base positions were properly aligned . based on the aligned regions generated by the t - coffee program ( www.ch.embnet.org/software/tcoffee.html ) , the identity of the nucleotide sites of the partially sequenced microsatellites relative to h. armigera was found to be 78% for all three species at the hac14 160 allele , 83% for all three species at the hab60 160 allele , 41% for h. virescens and h. zea at the hab60 140 allele , and 84% for h. zea and h. virescens at the hac87 118 allele , and 76% for h. zea and h. virescens at the harssr1 240 allele . as indicated in fig . 3 ( a - d ) only the downstream primer used in pcr amplification for each locus appeared in the sequence along with the microsatellite marker . however , the upstream primer that would typically be included as part of the 5-end of the microsatellite marker was not sequenced during the automatic analysis . several reports have shown that comparing allele sizes can result in inaccurate allele size differences for microsatellites ( estoup et al . , 1995 ; haberl and tautz , 1999 ) . one can approach this potential problem of size homoplasy by either employing single - strand conformation polymorphism analysis ( sscp ) or sequence analysis of the dna fragments . ( 2001 ) has shown that even sequenced , aligned microsatellites can show differences in repeat numbers occurring among clones and samples from the same individual . their solution was to split the analyzed complex locus into two new loci . nevertheless , we chose sequence analysis to determine if our unknown fragments contained not only the microsatellite but also to obtain an overall view of the alignment patterns of the fragments relative to the h. armigera markers . overall , the alignments of the four microsatellite loci detected in the three species , but with the occurrence of some inversions at hab60 , a substitution in hac87 , and deletions in hac14 and harssr1 , showed a high number of identical nucleotide sites with the h. armigera repetitive motifs ( table 3 ) . the length polymorphism detected in h. virescens and h. subflexa at the hab60 locus revealed a large deletion of the repetitive array in the 140 bp allele of both species . however , with the complete sequence of one primer and a partial of the other 5-end primer contained in the sequence read , it was still deemed to be a factual allele ( fig.3c ) . the occurrence of null alleles in microsatellites is known to be an impediment to their successful application as markers in population genetic studies ( pemberton et al . , 1995 ; schltterer and pemberton , 1998 ; liewlaksaneeyanawin et al . , 2002 ) , and have been implicated as a possible cause for the low levels of heterozygosity found in lepidoptera ( meglecz et al . , 2004 ) . since only samples collected from laboratory populations were employed in this study , we probably restricted ourselves from determining some level of polymorphism , if any , in the loci studied from the three species , though the number of polymorphic microsatellites to date has been found to be typically low in lepidoptera ( ji and zhang , 2004 ) . given the inherent variability of the microsatellite flanking regions in lepidoptera , further work , in particular controlled mating studies , will be needed to elucidate the frequency of null alleles in these species . the specific repetitive nature of the microsatellite flanking regions found in lepidoptera demonstrates the difficulty of isolating similar microsatellites from closely related species . however , some of the data presented here extends the utility of previously developed microsatellites of one species to closely related members , and has the potential to be used as population genetic markers in other related lepidopteran species . mention of trade names or commercial product in the publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s . an assortment of pcr amplifications depicting several potential microsatellite primer pairs . ( a ) pcr amplification of three single - copy microsatellites from 10 individual heliothis subflexa larvae ; ( b ) pcr amplification of two single - copy microsatellites from eight heliothis virescens larvae ; ( c ) three single - copy microsatellites detected in h. zea , the more closely related of the three species to helicoverpa armiger a. base pair markers are indicated on the left of each gel . the size of specific bands that were sequenced is indicated for each of the microsatellite loci . successful identification of pcr amplified single - copy microsatellites from sampled individuals of the three species . ( a ) hac14 ; ( b ) hab60 ; ( c ) hac87 ; and ( d ) harssr1 . the size of specific bands that were sequenced is also indicated for each of the microsatellite loci . microsatellite alleles are shown for ( a ) hac14 , ( b , c ) hab60 , ( d ) hac87 and ( e ) harssr1 . bold letters indicate the location of the simple sequence repeat and the box - shaded regions indicate identities . a core index for each base position is indicated in the outlined box below each alignment . microsatellite markers previously published for helicoverpa armigera found to successfully amplify similar microsatellite loci in three other related lepidopteran species . microsatellite markers previously published for helicoverpa armigera found to unsuccessfully amplify similar microsatellite loci in three other related lepidopteran species . comparison between four h. armigera microsatellites and the repetitive sequences identified in three related lepidopteran species .

primers previously designed to amplify microsatellite dna markers in the old world bollworm , helicoverpa armigera , larvae were tested in three closely related species : the corn earworm , helicoverpa zea , tobacco budworm , heliothis virescens , and heliothis subflexa . of the fourteen loci surveyed , only four loci ( hab60 , hac14 , hac87 , harssr1 ) consistently demonstrated scorable single - copy microsatellite bands . of these four , length polymorphism was identified only in the hab60 marker ( 160 bp , 140 bp ) of the h. virescens and h. subflexa sampled laboratory populations . partial dna sequences of all the identified single - copy microsatellites are presented as well as alignments to their respective h. armigera microsatellite .

Introduction Materials and Methods Results Disclaimer Figures and Tables

PMC4236627

the course of weathering of exposed rock surfaces depends on both internal factors , such as mineral composition , structure and texture of the rock considered , and external ones , for instance , climate and anthropogenic pressure . the changes affect particularly the porous rocks whose minerals are relatively less resistant to weathering and lead to the development of surface crusts . for a certain time , the crust may protect the subsurface layer of the rock , but it can also be destructive , boosting exfoliation of the rock surface . the impact of anthropogenic factors , particularly of air pollution , increases the intensity of weathering and the formation of secondary minerals . these are primarily salts that form black crust and efflorescences on the rock surfaces and accumulate in subsurface layers ( thomachot and jeannette 2004 ; charola et al . such changes affect various rock types , as limestones ( bede 2000 ; marszaek 2000 ; belafiore et al . 2013 ) , granitoids ( labus 1998 ; schiavon 2000 ) , sandstones ( bai et al . 2003 ) and also masonry walls ( gentilini et al . 2012 ) . the mechanism and path of these processes have been well known and explained ( e.g. goudie and viles 1997 ; charola 2000 ; doehne 2002 ; espinoza - marzal and scherer 2010 ; adamovi et al . 2011 ; ludovico - marques and chastre 2012 ; navrtil et al . 2013 ) . in the case of sandstones , the salt weathering usually leads to their disintegration due to an increase in intergranular pressure caused by growing salt crystals , hydration of salts ( goudie and viles 1997 ; winkler 1997 ) and dissolution of silicates by saline fluids . on the other hand , in fine - grained polymictic sandstones rich in clay minerals , case hardening may occur as well ( adamovi et al . 2011 ) . only few studies , however , focused on comparing the structures and compositions of weathering crusts developing in various environments but on comparable rock substrates ( siedel and klemm 2001 ; bai et al . 2003 ; wilczyska - michalik 2004 ; pikryl 2007 ) . the present authors investigated secondary substances of crusts formed on sandstones influenced to various degrees by air pollutants . the aims of this paper are to study the damage mechanisms affecting lithologically and genetically similar sandstones in the natural outcrops and in the urban environment and to correlate the patterns and composition of the weathering crust with different components of atmospheric aerosol . the analyses were conducted on the outermost parts of weathering crusts , black or dark grey in colour , developed on the surfaces of natural sandstone tors occurring within the carpathian foothill of the polish outer ( flysch ) carpathians ( fig . 1 ) , and the crusts that grow on analogous sandstones used in historic buildings within the urban area of krakw , a town placed on the unesco world heritage list . unfortunately , krakw is one of the european towns with the most polluted atmosphere ( e.g. fenger 1999 ; mira - salama et al . 1sampling sites of the weathering crusts developed on sandstone surfaces of architectonic details in krakw and on natural tors in the carpathian foothill ( the latter marked in grey ) sampling sites of the weathering crusts developed on sandstone surfaces of architectonic details in krakw and on natural tors in the carpathian foothill ( the latter marked in grey ) the outer carpathians , whose age spans upper jurassic and neogene , are composed mainly of flysch , i.e. alternating complexes of sandstones , mudstones and shales ( oszczypko 2004 , 2006 ; poprawa and malata 2006 ) . among dominating thin- and medium - layered strata many outcrops of these rocks were shaped during quaternary into tors due to various denudation processes . particularly prone to such changes in the carpathian foothill are the complexes of the istebna sandstones ( upper cretaceous lower eocene ) and the cikowice sandstones ( lower eocene ) , which reveal similar lithological and sedimentary features , typical of submarine flows ( fluxoturbidites ) ( alexandrowicz 1978 ; leszczyski 1981 ; oszczypko 2004 ) . these sandstones are usually coarse - grained , in places conglomeratic , poorly sorted , developed as quartz - rich oligomictites , but also arkoses and greywackes ( kamieski et al . their cement is scarce , composed of clay minerals , sometimes of silica , occasionally of calcite . the sandstone complexes mentioned consist of very thick beds ( often above several meters ) and are characterised by high water absorbability , low or medium compression strength and very high frost resistance ( bromowicz et al . this lithological type of sandstones has been quarried within the carpathian foothill since the early middle ages . the carpathian sandstones have been used in constructing works , as the rocks perfectly suitable for building purposes because of their good physical and chemical parameters . other reasons are of the logistic nature ( rajchel 2004 , 2008 ; bromowicz 2009 ; bromowicz and magiera 2010 , 2013 ) : the stones were quarried chiefly within the carpathian foothill , just in a short distance south of krakw ( fig . 1 ) , from where could be transported in large blocks ( fig . therefore , they were widely used in krakw in the romanesque , gothic , renaissance and nineteenth - century constructions ( rajchel 2004).fig . 2 a the brodziski stones natural monument in the carpathian foothill . photos by z. alexandrowicz a the brodziski stones natural monument in the carpathian foothill . photos by z. alexandrowicz current quarrying of these sandstones is limited . of around 1,000 quarries of various sandstones that have been recorded in the carpathians ( rajchel 2004 ) , currently active are 36 operations ( brzeziski 2013 ) . on the other hand , some of the quarries and most of natural exposures of sandstone tors are subject of protection as examples of various types of sandstones , elements of the natural landscape , and also due to the presence of their specific sedimentary structures and weathering processes ( alexandrowicz 2008 , 2009 ) . they have been protected separately as nine reserves and 38 monuments of the inanimate nature ( fig . 2a , b ) , and additionally , a number of such tors are situated within national parks and landscape parks ( alexandrowicz and poprawa 2000 ) . the crust samples occurring in their natural environment were obtained from the tors comprised the istebna and cikowice sandstones of the silesian unit ( upper cretaceous lower eocene ) of the outer ( flysch ) carpathians . in terms of lithology , their surface is covered by a weathering crust , usually 12-cm thick ( it can reach a maximum of 10 cm ) , characterised by lamination , either continuous or developed in patches , with zones of variable colour and thickness running parallel to the rock surface ( alexandrowicz and pawlikowski 1982 ; rzepa et al . the lamination is chiefly associated with variable proportions and mineralogy of iron compounds and is a separate issue discussed in detail by alexandrowicz et al . the outermost part of the weathering crust is often developed as a thin hard , black or dark grey layer . because of its properties , chiefly the hardness associated with the presence of , e.g. amorphous silica , it closely resembles the varnish type covers described primarily on rocks exposed in deserts and only sometimes on the surfaces of sandstone tors in europe ( dorn 1984 ; robinson and williams 1986 ; clek 1997 ; thiry 2005 ; dorn and krinsley 2011 ) . it is also termed patina , whereas its analogues formed as a result of pollution on stony architectonic elements in urban areas are called false patinas ( manecki et al . the present authors have focused on the external , black or grey layers of sandstone weathering crusts from sites with rocky forms occurring within forest enclaves in the carpathian foothill : the brodziski stones , the stone town nature reserve , the mushroom stone , kobyla gra and szczyyc . a total of 30 samples were collected for the study , and the results of investigating 14 of them have been presented here . the weathering crusts on natural rocks have mostly the area not exceeding 25 cm and are several centimetres thick . the samples selected reveal a distinctly developed black or dark grey outer layer affected by atmospheric factors and are usually clearly laminated underneath . the samples of grey and black weathering crusts on sandstone architectonic elements were collected from three historical buildings situated near the centre of krakw : the ethnographic museum , the czartoryski museum and the czapski palace ( fig . 3general view and examples of black crust development of : a , d the ethnographic museum , b , e the czartoryski museum and c , f the czapski palace . photos : d by t. sokal , e by a. bohan - strumiska , f by a. orzechowska general view and examples of black crust development of : a , d the ethnographic museum , b , e the czartoryski museum and c , f the czapski palace . photos : d by t. sokal , e by a. bohan - strumiska , f by a. orzechowska the history of the first of the buildings , which has been housing the ethnographic museum since 1947 , dates back to 1414 , when construction of the city hall began in kazimierz , a satellite town of krakw into which it was incorporated at the beginning of the nineteenth century . in the last two centuries , the building has been repeatedly rebuilt and extended , assuming its recent shape in 19621966 as a result of a major restoration ( roek 2000 ) . the czartoryski museum ( a branch of the national museum ) is the oldest institution of this type in poland , established in 1801 in puawy ( central poland ) by princess izabela czartoryska ( ne flemming ) , who belonged to an eminent polish aristocratic family ( roek 2000 ) . transferred to krakw in the mid - nineteenth century , the collection was placed in building of the municipal arsenal , remodelled in the same time into a small castle - mansion in the early - renaissance style . the collection was also housed in neighbouring buildings : part of an old piarist monastery and three tenement houses that were merged in the nineteenth century into a single czartoryski palace building . the arsenal , or so - called the small monastery , and the palace are linked by corridor - like , overhead passages . the single most valuable piece of art in the museum is the lady with an ermine by leonardo da vinci . the czapski palace ( also a branch of the national museum ) was erected in 1884 . its owner , emeryk hutten - czapski , placed his valuable numismatic collection there , which he donated next to the city of krakw and its national museum ( roek 2000 ) . at present , the building houses the european centre of polish numismatics . the samples of sandstone weathering crusts from the ethnographic museum ( three specimens ) were collected from the portal of the main entrance to the building and from the plinth ( at a height of up to 1 m above the ground ) . the samples from the czartoryski museum ( seven specimens ) were collected from plinths and window sills at a similar height , whereas those from the czapski palace ( four specimens ) from columns of a balcony . the samples were collected during the dry season , in june and july of 2011 , utilizing a restoration campaign . although the study was to be centred on the outermost sandstone layers , i.e. black crusts , also the altered sandstone zones located directly below were investigated as the so - called weathering zones . the black crust and the weathering zone depending on the local conditions , samples were collected with a hammer or a knife ; their volume ranged from single to several tens cubic centimetres . sample descriptions and localisations are given in table 1.table 1list of samples , sampling sites and methods of analysissample locationsamplessite and height above the ground ( m)analysis scopeomsemxrdtachemical analysiscarpathian foothillthe mushroom stonekg-1sandstone tor ; 1.2+++kobyla grach-1sandstone tor ; 1+++the brodziski stoneskb-1sandstone tor ; 1.5+++kb-2sandstone tor ; 2.5++++kb-6sandstone tor ; 1.7++kb-8sandstone tor ; 1.5++kb-9sandstone tor ; 1+++kb-10sandstone tor ; 1+++++szczyycsz-1sandstone tor ; 1.3++the stone town nature reservesmc-1sandstone tor ; 1.5+++smc-11sandstone tor ; 1+++++smc-12sandstone tor ; 1.5+++smc-13sandstone tor ; 1+++++smc-14sandstone tor ; 1.5+++++krakwthe ethnographic museumme-81portal fragment ; 0.6+++++me-82plinth fragment ; 0.3+++me-83plinth fragment ; 0.1++++the czartoryski museummc-001plinth fragment ; 0.7++mc-008plinth fragment ; 0.5++mc-009plinth fragment ; 0.7++++mc-010plinth fragment ; 1.3+mc-011window still fragment ; 1.7++mc-012window still fragment ; 1.7++mc-013window still fragment ; 1.7+the czapski palacepc-01balcony column ; 0.3+++++pc-03balcony column ; 0.4++pc-04balcony column ; 0.6+++pc-05balcony column ; 0.8 + om optical microscopy , sem sem - eds , ta thermal analysis ( tg / dta ) , + indicate that the analyses have been done list of samples , sampling sites and methods of analysis om optical microscopy , sem sem - eds , ta thermal analysis ( tg / dta ) , + indicate that the analyses have been done microscopic observations ( including petrographic descriptions of natural unaltered rocks as well ) were carried out using optical transmission microscopy with an olympus bx-51 instrument and an olympus dp-12 digital camera . detailed studies of the components of weathered crusts were performed by the scanning electron microscopy ( sem ) , using a fei 200 quanta feg microscope with an eds / edax spectrometer . the excitation voltage was 20 kv and the pressure 60 pa ( low vacuum , the samples were not coated ) . the phase composition was determined using x - ray diffractometry ( xrd ) with a philips apd pw 3020 xpert instrument . the measurement conditions were as follows : anode cuk , generator settings 35 kv and 30 ma , recording range 3702 , step size 0.05 , counting time 1 s per step . the xrd patterns were evaluated by an xrayan software using a diffraction pattern database of international centre for diffraction data ( the powder diffraction file pdf-42013 ) . the thermal analyses ( thermogravimetry and differential thermal analysis -tg / dta ) were conducted with a derivatograph c apparatus made by mom budapest . the powdered sample with a weight of about 100 mg was heated in the air in the range 201,000 c with the constant heating rate of 10 c / min . the chemical compositions were determined by applying different analytical methods at the activation laboratories ltd . concentrations of major compounds ( sio2 , al2o3 , fe2o3 , mno , mgo , cao , na2o , k2o , tio2 and p2o5 ) . were measured using inductively coupled plasma optical emission spectrometry ( icp - oes ) method after fusion of the ground sample with a mixture of lithium metaborate and lithium tetraborate in an induction furnace followed by dissolution of the melt in 5 % nitric acid . trace element concentrations were determined either with inductively coupled plasma mass spectrometry ( icp - ms ) ( be , bi , cd , cu , mo , ni , pb , sr , v , y , zn , zr ) or with instrumental neutron activation analysis ( inaa ) ( au , ag , as , ba , br , co , cr , cs , hf , hg , ir , rb , ree , sb , sc , se , ta , th , u , w ) . the samples for icp - ms analyses were prepared by the digestion of a material with a mixture of concentrated hclo4 , hno3 , hcl and hf at 200 c to fuming and then dilution with aqua regia . inaa analyses were carried out as follows ( hoffmann 1992 ) : a 1 g aliquot of the sample was encapsulated in a polyethylene vial and irradiated with flux wires and an internal standard at a thermal neutron flux of 7 10 n cm s. after a 7-day period , the samples were counted on a high purity ge detector with resolution of better than 1.7 kev for the 1,332 kev co-60 photo peak . using the flux wires , the decay - corrected activities were compared to a calibration developed from multiple certified international reference materials . optical and scanning electron observations were made on universal polished thin sections cut perpendicular to stone surfaces . additionally , broken surfaces of outer parts were studied using the sem method . the phase and chemical composition were established for samples of outermost parts of the sandstones ( layer reaching to ca . . 2 cm below the surface ) . due to a limited size of samples therefore , full range of analyses was carried out only for selected , representative samples ( table 1 ) . their grain framework comprises quartz , feldspars , rock fragments , micas ( biotite and muscovite ) and accessory minerals ( e.g. rutile , ilmenite , zircon , tourmaline and opaques , including pyrite ) . the contact - porous cement is composed chiefly of clay minerals and iron ( oxyhydr)oxides . it is locally enriched in carbonates , while glauconite aggregates were also found in some places . the xrd analysis has revealed among clay components kaolinite , illite and probably illite - smectite mixed - layer minerals ( fig . 4xrd patterns weathering crusts from natural outcrops ( smc-13 , smc-14 ) and architectonic details of the ethnographic museum ( me-81 ) and the czapski palace ( pc-01 ) . ilt illite , kln kaolinite , pl plagioclase , gp gypsum , fsp feldspar , kfs k - feldspar , qz quartz , cal calcite , dol dolomite , hl halite xrd patterns weathering crusts from natural outcrops ( smc-13 , smc-14 ) and architectonic details of the ethnographic museum ( me-81 ) and the czapski palace ( pc-01 ) . ilt illite , kln kaolinite , pl plagioclase , gp gypsum , fsp feldspar , kfs k - feldspar , qz quartz , cal calcite , dol dolomite , hl halite the outer , altered rock crusts ( a few centimetres thick ) are covered by a thin , hard , often discontinuous dark grey to black layer of variable morphology . as the identification of the components of this layer under an optical microscope was not possible because of their cryptocrystalline nature , the electron scanning microscope observations provided more information ( fig . the framework minerals of the outer zone are covered by a thin ( several to several dozen micrometers thick ) , amorphous film of a variable chemical composition , which includes c , si , al , fe , p , cl and k. amorphous silica predominates among its components , accompanied by organic matter in various morphological forms , occasionally found grains of iron oxides and spherical particles of aluminosilicate glass from industrial emissions . of salt minerals , euhedral crystals of gypsum ( caso4 2h2o ) , barite ( baso4 ) and potassium alum [ kal(so4)2 12h2o ] were observed ( fig . gypsum was also found in deeper layers of the crust ( up to 1 cm below the surface ) in the intergranular spaces and pores , together with clay minerals and iron compounds . another sulphate , jarosite [ kfe3(oh)8(so4)2 ] , sporadically occurs in the intergranular spaces , whereas on the crust surface , single crystals of halite ( nacl ) and syngenite [ k2ca(so4)2 h2o ] were found.fig . 5scanning electron microscope image of the weathering crust developed on the sandstone tor surface ( sample kb-9 ) . bc black crust , fe iron ( oxyhydr)oxides incrustation between quartz grains , qz quartzfig . 6scanning electron microscope images of the sandstone surface : a , b from natural outcrops ( sample smc-13 ) and c , d from architectonic details ( sample me-81 ) . gp gypsum , alum-(k ) potassium alum , hl halite , partial dissolution of mineral grains is also visible ( d ) scanning electron microscope image of the weathering crust developed on the sandstone tor surface ( sample kb-9 ) . bc black crust , fe iron ( oxyhydr)oxides incrustation between quartz grains , qz quartz scanning electron microscope images of the sandstone surface : a , b from natural outcrops ( sample smc-13 ) and c , d from architectonic details ( sample me-81 ) . gp gypsum , alum-(k ) potassium alum , hl halite , partial dissolution of mineral grains is also visible ( d ) immediately beneath the above described hard and black crust , there is a zone of sandstone altered by weathering ( fig . 7a , b ) , which differs from the inner parts of the rock due to the presence of variably coloured zones running parallel to the surface . ( 2012 , 2014 ) have found that the darker layers contain more iron compounds than the lighter ones , whereas their colour depends on the prevailing type of the fe - bearing minerals . the red and pink zones contain mostly an oxide hematite ( -fe2o3 ) , whilst the yellow and brown zones are characterised by the presence of an oxyhydroxide goethite ( -feooh ) . the layers closer to the surface of the weathering crust contain often major hematite and are richer in iron ( fig . iron released during the decomposition of primary aluminosilicates , and to a lesser extent of carbonates and sulphides , is transported towards the rock surface , where it precipitates and can undergo further transformations ( alexandrowicz et al . additionally , physical changes of rock - forming minerals have been noted in the weathering crust in the form of cracks in quartz grains and fissures running along the cleavage surfaces of feldspars . these cracks and fissures are filled with iron compounds , principally goethite and hematite , whose particles were also found within other minerals ( fig . 7microphotographs : sandstone from natural outcrops a outer layer covered by ferruginous compounds ( sample smc-14 , 1p ) , b iron oxides between mineral grains ( sample smc-12 , 1p ) , and c iron compounds occurring in cleavage fissures of feldspar ( sample smc-14 , xp ) ; sandstone from architectonic detail d black crust composed of gypsum covering the outer layer of the detail ( sample mc-001 , 1p ) , e gypsum crystals filling pores and cracks , replacing stone cement ( sample mc-001 , 1p ) , and f gypsum filling cleavage on feldspar ( sample mc-011 , xp ) . 1p one polar , xp crossed polars microphotographs : sandstone from natural outcrops a outer layer covered by ferruginous compounds ( sample smc-14 , 1p ) , b iron oxides between mineral grains ( sample smc-12 , 1p ) , and c iron compounds occurring in cleavage fissures of feldspar ( sample smc-14 , xp ) ; sandstone from architectonic detail d black crust composed of gypsum covering the outer layer of the detail ( sample mc-001 , 1p ) , e gypsum crystals filling pores and cracks , replacing stone cement ( sample mc-001 , 1p ) , and f gypsum filling cleavage on feldspar ( sample mc-011 , xp ) . 1p one polar , xp crossed polars the xrd analyses confirmed the presence of quartz , feldspars , clay and carbonate minerals ( fig . 4 ) . no salt minerals identified in the sem - eds examinations were noted , except for gypsum but only in single samples . it is a proof of minor amounts of the salt - group phases in the overall composition of the crust . the tg / dta thermal examinations have been rather inconclusive as they indicated , obviously , major , natural sandstone components : quartz , clay and carbonate minerals . the thermal curves obtained for the surface - closest crust samples point to a higher content of iron oxyhydroxides and a lower content of clay minerals than those of the inner parts of the rock ( alexandrowicz et al . 2014 ) . the precipitation of amorphous silica ( silicification ) leads to a hardening and sealing of the rock surfaces . the hydrolytic decomposition of less resistant aluminosilicate minerals , such as feldspars and biotite , which to a variable degree have been replaced by clay minerals and iron compounds , is the main source of the silica . dissolution of aluminosilicates is favoured by an acidic reaction within the rock , low ph being a result of weathering of sulphides and precipitating iron ( oxyhydr)oxides , as well as intensive microbial activity or infiltration of acid waters from atmospheric precipitation ( mcarthur et al . the first two processes seem to be more significant in the sandstones studied ( alexandrowicz et al . the dark grey to black colour of the outer layer can be associated with the presence of atmospheric dusts of industrial origin and also an activity of microorganisms , such as cyanobacteria , bacteria oxidising fe and mn , and melanin - producing fungi and actinomycetes ( gaylarde et al . 2007 ; corenblit et al . 2011 ; dorn and krinsley 2011 ; dakal and cameotra 2012 ) . the presence of salts ( e.g. gypsum , barite , potassium alum , jarosite , syngenite and halite ) results from evaporation . the majority of anions necessary to form these minerals are probably associated with the effect of air pollutants ( e.g. lentschig - sommer 1960 ; jaboska et al . the decomposition of sulphides ( pyrite ) present in sandstones is a probable source of sulphate ions ( soukupov et al . the last process must be particularly important in the case of jarosite , whose formation requires a highly acidic environment ( bingham and nordstrom 2000 ) . the alteration of aluminosilicate ( e.g. feldspars , micas , glauconite and clay minerals ) and carbonate rock components is the chief source of cations : k , ca , na , ba and fe ( e.g. steiger 2003 ; wilczyska - michalik 2004 ) . however , a connection of these cations with atmospheric particulate pollutants is also probable ( wilczyska - michalik 2004 ; kamh 2005 ; pikryl et al . their surface is covered by a discontinuous , grey or black crust up to 0.5-mm thick . optical microscopy has revealed only opaque and/or translucent grains of variable morphology , probably iron ( oxyhydr)oxides and carbon particles , and transparent gypsum crystals that are usually very fine . gypsum also fills the cracks in quartz grains and the fissures along the cleavage surfaces of feldspars , as well as spaces between flakes of micas . in some places the near - surface framework minerals of sandstones are covered by a thin layer rich in c , si , al , fe , p , cl , k and ba . grain components include iron oxides , along with local occurrences of barite and carbon particles ( soot ) . above all , however , discontinuous accumulations , containing chiefly salt minerals , mainly gypsum and halite ( fig . 6c , d ) , spherical particles of aluminosilicate glass and spherules of iron oxides ( magnetite and hematite ) , were observed . the properties and composition of the latter components indicate them to originate from industrial emissions ( marszaek 2008 ) . another significant component of this layer is organic matter ( confirmed in tg / dta analyses ) . sem observations corroborated that numerous laths of gypsum cover the crust surface and also occur within pores of the sandstones , where they replace a primary cement ( fig . sometimes , however , the intergranular spaces after leaching the cement remain empty ( fig . 6d ) . in samples from the czartoryski museum and the ethnographic museum collected at a small distance from the ground level , halite and gypsum halite is absent , however , in the samples collected from the balcony columns of the czapski palace.fig . 8scanning electron microscope images of sandstone samples from architectonic detail : a gypsum replacing cement of the stone ( sample mc-010 ) and b empties after leaching the sandstone cement ( sample me-81 ) scanning electron microscope images of sandstone samples from architectonic detail : a gypsum replacing cement of the stone ( sample mc-010 ) and b empties after leaching the sandstone cement ( sample me-81 ) the presence of these salt minerals was also confirmed by xrd analyses , which is a proof of their significant presence in the composition of black crust ( x - ray detection limit of salt minerals is relatively high , being around 3 % ) . the dark grey to black colour of the crust surfaces results chiefly from the presence of atmospheric dusts . however , an impact of microorganisms , similar to that on natural outcrops , can not be excluded ( saiz - jimenez 2003 ) . intensive destruction of sandstones indicates that natural weathering is enhanced by processes of anthropogenic nature . significant amounts of gypsum and halite are , among others , effects of such pollution and due to easy crystallisation and re - crystallisation of salt minerals lead to granular disintegration of rocks ( alexandrowicz and pawlikowski 1982 ; rodriguez - navarro and doehne 1999 ; saby et al . 2000 ; sabbioni 2003 ; rembi and smoleska 2010 ) . the sulphates result from the presence of so2 , a common component of air pollutants . its two - pronged deposition , wet one ( periodic effect of , e.g. acid rains ) and so - called dry one ( long - term impact of so2 on the surface of stones , where sulphuric acid immediately forms in the reaction with the moisture present in the rock ) , is the cause of gypsum precipitation ( manecki et al . the sources of chloride ions may be represented by air pollutants and chemicals used in removing ice from streets and pavements ( chiefly nacl and cacl2 ) . an impact of chloride aerosols brought by winds from the nearby vistula river , polluted by salty mine waters from the upper silesian coal basin , was also possible in previous decades . calcium , barium and sodium ions originate from the decomposition of primary aluminosilicates ( calcium also from dissolution of carbonates ) of the sandstones . nevertheless , significant amounts of these elements may also be linked with particulate air pollutants ( sabbioni and zappia 1992 ; marszaek 2000 ; jaboska et al . 2001 ; wilczyska - michalik 2004 ; toniolo et al . 2006 ) and capillary transport from the ground . with regard to the sodium ion in halite ( nacl ) present in the sandstone samples collected low above the pavement level ( the plinths at the ethnographic museum and the czartoryski museum ) , nevertheless , the most indicative of the effects of anthropogenic pollution are the spherical particles of aluminosilicate glass and iron oxides observed on the stone surface . comparing the chemical compositions of crusts on the sandstones from natural outcrops and those from architectonic elements , higher concentrations of mgo , cao , na2o , s , cu and zn were found in the latter ( table 2 ) . thick , and may contain a certain admixture of a material from below as it was not always possible to separate the crust layer alone . in the case of samples collected in krakw , these results do not differ considerably from those of the layers situated deeper , ca . 2 cm below the crust surface , which probably reflects advanced changes reaching deep into the sandstones . nevertheless , in the inner layers of the sandstones from the architectonic elements the assays for cao , na2o and losses on ignition ( loi ) , and also the contents of sulphur and some trace elements ( zn , cu ) slightly decrease , whereas the contents of sio2 , al2o3 , fe2o3 , mgo and k2o slightly increase in comparison with the outer layer ( fig . such a trend manifests the accumulation of secondary salt minerals ( gypsum and halite ) as well as particulate pollutants ( the latter are carriers of heavy metals ) on sandstone surfaces and their lower amounts in subsurface layers . similar tendencies were described , e.g. by thomachot and jeannette ( 2004 ) , wilczyska - michalik ( 2004 ) and kamh ( 2005).table 2major ( wt% ) and some trace ( ppm ) element concentrations ( combined icp - oas , icp - ms and inaa analyses ) in the weathering crust of the sandstones ( kb-10 , smc-11 , smc-13 , smc-14samples from carpathian foothill ; me-81the ethnographic museum , pc-01the czapski palace)kb-10smc-11smc-13smc-14me-81pc-01sio2 87.8390.6089.6186.6487.8390.10tio2 0.150.270.260.270.010.06al2o3 4.094.284.184.474.393.67fe2o3 3.401.081.423.491.471.50mno0.010.010.010.010.010.01mgo0.090.080.080.080.210.18cao0.060.080.090.190.380.11na2o0.370.330.330.391.430.44k2o1.971.831.791.901.831.37p2o5 0.150.050.800.150.2760.248loi1.341.151.282.182.301.38sum99.4699.7699.8599.77100.6799.95s ( wt%)0.0240.0150.0420.0380.1950.190cu ( ppm)91115102633zn ( ppm)3410162641154sr ( ppm)383536434536ba ( ppm)406356353437289261pb ( ppm)201520301054 loi loss on ignitionfig . 9bulk chemical composition ( combined icp - oes , icp - ms and inaa analyses ) of inner and outer layers of the sandstones crusts from architectonic details ( major elements and sulphur in wt% , trace metals in ppm ) . me-81 the ethnographic museum , pc-01 the czapski palace , i inner layer , o outer layer major ( wt% ) and some trace ( ppm ) element concentrations ( combined icp - oas , icp - ms and inaa analyses ) in the weathering crust of the sandstones ( kb-10 , smc-11 , smc-13 , smc-14samples from carpathian foothill ; me-81the ethnographic museum , pc-01the czapski palace ) loi loss on ignition bulk chemical composition ( combined icp - oes , icp - ms and inaa analyses ) of inner and outer layers of the sandstones crusts from architectonic details ( major elements and sulphur in wt% , trace metals in ppm ) . me-81 the ethnographic museum , pc-01 the czapski palace , i inner layer , o outer layer in natural outcrops , spatial differences of the chemical composition between the weathering crust and an inner sandstone layer are associated , first of all , with the accumulation of iron compounds and the prevailing type of fe - bearing minerals ( rzepa et al . 2011 ; alexandrowicz et al . the phase and chemical composition of weathering crusts indicate that their development is controlled by the environmental conditions combined , in particular , with climate . the maopolska voivodeship , in which the study region is situated , is one of the most polluted areas in poland ( wilczyska - michalik 2004 ; pajk 2011 ) , and the krakw conurbation is characterised by very high levels of gaseous and particulate pollution . they inherently result from adverse atmospheric conditions of krakw , situated in the vistula river valley surrounded by hills , as well as from a proximity of large industrial centres , and further aggravated by distant industrial emissions . the annual average concentrations of total dust particulate matter < 10 m ( pm10 ) particles , nitrogen dioxide and sulphur dioxide recorded in a measuring station situated in the city centre are high . as regards pm10 and nox , their concentrations markedly exceed permissible standards ( table 3 ) . the excessive concentrations of nox , so2 and pm10 are recorded most often during the heating season , which usually lasts from october till march . local boiler houses and household furnaces that use solid fuels ( coal and/or coke ) of variable ( often inferior ) quality are additional sources of pollution . road transport also contributes its share , quite significant in case of any old town with its dense , structural layout.table 3average so2 , no2 and pm10 concentrations ( g / m ) in the air in krakw and cikowice ( carpathian foothill ) in 2010cityaverage concentrations ( g / m)so2 no2 pm10annualsummer seasonheating seasonannualsummer seasonheating seasonannualsummer seasonheating seasonkrakw114.518707170.57948.691.8city centre , average so2 , no2 and pm10 concentrations ( g / m ) in the air in krakw and cikowice ( carpathian foothill ) in 2010 from ogar et al . the area of the carpathian foothill ( the region where the samples from the natural outcrops were collected ) is affected by industrial emissions from krakw and neighbouring towns such as tarnw and nowy scz ( lach and wilczyska - michalik 1996 ; gabaa and khne 2002 ; gobiowska et al . the state of its atmosphere is worsened by local pollution sources associated with industrial activities , combustion of fuels ( during heating season ) and road transport . nevertheless , the overall air quality is much better there than in the krakw urban agglomeration . the annual average concentrations of sulphur dioxide , nitrogen oxides and pm10 suspended dust stay on much lower levels than those within krakw ( table 3 ) . in the study area , the load of acidifying compounds introduced via atmospheric precipitation : so4 > n > ca > cl > na > k > mg > zn > p > fe > cu > mn > h > pb > ni > cd > cr is high , although the last decade shows a declining trend . the deposition of sulphate and nitrate ions in the maopolska region is the highest in poland ( pajk 2011 ) . the different level of the environmental pollution in the two subareas studied is reflected in the concentrations of salts , size of their crystals and the state of preservation of sandstone surfaces . gypsum , which fairly sporadically occurs on the rock surfaces in natural outcrops , develops into tabular , relatively large and well - formed crystals ( fig . the crystals of this sulphate found on the architectonic details of historic buildings in krakw are , as a rule , finer and lath - shaped , and occur in aggregates densely covering the surface ( fig . . such differentiation of the size and morphology probably reflects the conditions prevailing during precipitation : high concentrations of acid air pollutants ( including so2 ) result in gypsum crystallising from more saturated solutions , thereby producing a higher number of relatively small crystals ( maurice 2009 ) . on the surfaces of the tors in the carpathian foothill , crystallisation is slower and proceeds from less concentrated solutions , which results in the formation of large , euhedral gypsum crystals . nevertheless , the airborne particles observed there ( aluminosilicate glass and the spherical forms of iron oxides ) provide an evidence of a far - reaching industrial pollution . thus , this contamination must be a reason of the sulphate salts and chlorides precipitating on the surfaces of the sandstone tors . it seems , however , that the main source of the cations ( k , ca , na , ba and fe ) necessary to the formation of these salts must rather be contributed to the weathering of mineral rock components . there is a certain difference in the mineral composition of weathering crusts from both subareas studied : those developed on the sandstone architectonic elements contain only gypsum and halite , whereas those from the sandstone in natural exposures in addition to gypsum and halite also traces of other mineral phases : alum , barite or jarosite . the most probable explanation is that these minor salt phases have other cations than the na and ca composing gypsum and halite . the additional cations must have been released in the natural environment during weathering of aluminosilicates , for instance , potassium from micas and feldspars . in an urban environment , the anthropogenic supply of sodium and calcium is so high that the two cations dominate porous solutions in sandstones . another factor must also be taken into considerations : the sulphates present in the crusts from natural exposures crystallise under distinctly acid conditions . jarosite is formed at very low ph values ( bingham and nordstrom 2000 ) , and barite also favours acid conditions . in turn , the al - containing sulphates precipitate when aluminium is released into weathering solutions ( navrtil et al . 2013 ) . this element is mobile when the solutions are either strongly acidic or strongly basic ; otherwise , it tends to precipitate ( fest et al . urban environments have high concentrations of dusts , most of which are alkaline and neutralise the acid reaction of airborne deposits . in the distant , natural environments of the tors , the amounts of the industrial particulates are distinctly lower and , as a result , insufficient to neutralise acid depositions and to prevent other processes , e.g. weathering of pyrite , which leads to the formation of sulphuric acid . another acidifying factor may be represented by organic acids produced by moss and lichens colonizing the surfaces of natural exposures of sandstones . the factors mentioned combined with a low acid neutralisation capacity of waters developed within the sandstones of a low calcium content ( table 2 ) result in an acid reaction of the porous waters ( navrtil et al . it should also be emphasised that the sandstones forming the tors occur in their natural geological environment . on the other hand , the sandstones used as therefore , in contrast to those in the natural outcrops , they do not have immediate contact with the parental substrate , and stone blocks are often arranged in various positions and additionally cemented with calcareous or clayey material . therefore , masonry may also affect future weathering processes . water circulation , essential in weathering in the tors , proceeds both upwards and downwards , is usually limited in building stones , which intensifies evaporation and precipitation of secondary salts . the recurrent precipitation stress acting along the borders of external ( affected ) and internal ( less affected or not affected at all ) parts of any rock , particularly of a building stone , is a main factor of its damaging by exfoliation . in sandstones that form natural outcrops , they facilitate hardening of rock surfaces via silification and cementation with iron compounds and protect the rock from damage exerted by atmospheric factors . despite of this , with the passage of time , the stress caused by the differences in the coefficients of thermal expansion , in moisture contents between the rock surface and its inner parts , and in the composition of rock cement , result in natural loosening of the rock and exfoliation of weathering crust fragments . in the process , the crust substrate is exposed , which in a further course of chemical weathering gradually transforms and develops a new protective crust layer . the outer , altered rock crusts ( a few centimetres thick ) are covered by a thin , hard , often discontinuous dark grey to black layer of variable morphology . as the identification of the components of this layer under an optical microscope was not possible because of their cryptocrystalline nature the framework minerals of the outer zone are covered by a thin ( several to several dozen micrometers thick ) , amorphous film of a variable chemical composition , which includes c , si , al , fe , p , cl and k. amorphous silica predominates among its components , accompanied by organic matter in various morphological forms , occasionally found grains of iron oxides and spherical particles of aluminosilicate glass from industrial emissions . of salt minerals , euhedral crystals of gypsum ( caso4 2h2o ) , barite ( baso4 ) and potassium alum [ kal(so4)2 12h2o ] were observed ( fig . gypsum was also found in deeper layers of the crust ( up to 1 cm below the surface ) in the intergranular spaces and pores , together with clay minerals and iron compounds . another sulphate , jarosite [ kfe3(oh)8(so4)2 ] , sporadically occurs in the intergranular spaces , whereas on the crust surface , single crystals of halite ( nacl ) and syngenite [ k2ca(so4)2 h2o ] were found.fig . 5scanning electron microscope image of the weathering crust developed on the sandstone tor surface ( sample kb-9 ) . bc black crust , fe iron ( oxyhydr)oxides incrustation between quartz grains , qz quartzfig . 6scanning electron microscope images of the sandstone surface : a , b from natural outcrops ( sample smc-13 ) and c , d from architectonic details ( sample me-81 ) . gp gypsum , alum-(k ) potassium alum , hl halite , partial dissolution of mineral grains is also visible ( d ) scanning electron microscope image of the weathering crust developed on the sandstone tor surface ( sample kb-9 ) . bc black crust , fe iron ( oxyhydr)oxides incrustation between quartz grains , qz quartz scanning electron microscope images of the sandstone surface : a , b from natural outcrops ( sample smc-13 ) and c , d from architectonic details ( sample me-81 ) . gp gypsum , alum-(k ) potassium alum , hl halite , partial dissolution of mineral grains is also visible ( d ) immediately beneath the above described hard and black crust , there is a zone of sandstone altered by weathering ( fig . 7a , b ) , which differs from the inner parts of the rock due to the presence of variably coloured zones running parallel to the surface . ( 2012 , 2014 ) have found that the darker layers contain more iron compounds than the lighter ones , whereas their colour depends on the prevailing type of the fe - bearing minerals . the red and pink zones contain mostly an oxide hematite ( -fe2o3 ) , whilst the yellow and brown zones are characterised by the presence of an oxyhydroxide goethite ( -feooh ) . the layers closer to the surface of the weathering crust contain often major hematite and are richer in iron ( fig . iron released during the decomposition of primary aluminosilicates , and to a lesser extent of carbonates and sulphides , is transported towards the rock surface , where it precipitates and can undergo further transformations ( alexandrowicz et al . additionally , physical changes of rock - forming minerals have been noted in the weathering crust in the form of cracks in quartz grains and fissures running along the cleavage surfaces of feldspars . these cracks and fissures are filled with iron compounds , principally goethite and hematite , whose particles were also found within other minerals ( fig . a outer layer covered by ferruginous compounds ( sample smc-14 , 1p ) , b iron oxides between mineral grains ( sample smc-12 , 1p ) , and c iron compounds occurring in cleavage fissures of feldspar ( sample smc-14 , xp ) ; sandstone from architectonic detail d black crust composed of gypsum covering the outer layer of the detail ( sample mc-001 , 1p ) , e gypsum crystals filling pores and cracks , replacing stone cement ( sample mc-001 , 1p ) , and f gypsum filling cleavage on feldspar ( sample mc-011 , xp ) . 1p one polar , xp crossed polars microphotographs : sandstone from natural outcrops a outer layer covered by ferruginous compounds ( sample smc-14 , 1p ) , b iron oxides between mineral grains ( sample smc-12 , 1p ) , and c iron compounds occurring in cleavage fissures of feldspar ( sample smc-14 , xp ) ; sandstone from architectonic detail d black crust composed of gypsum covering the outer layer of the detail ( sample mc-001 , 1p ) , e gypsum crystals filling pores and cracks , replacing stone cement ( sample mc-001 , 1p ) , and f gypsum filling cleavage on feldspar ( sample mc-011 , xp ) . 1p one polar , xp crossed polars the xrd analyses confirmed the presence of quartz , feldspars , clay and carbonate minerals ( fig . 4 ) . no salt minerals identified in the sem - eds examinations were noted , except for gypsum but only in single samples . it is a proof of minor amounts of the salt - group phases in the overall composition of the crust . the tg / dta thermal examinations have been rather inconclusive as they indicated , obviously , major , natural sandstone components : quartz , clay and carbonate minerals . the thermal curves obtained for the surface - closest crust samples point to a higher content of iron oxyhydroxides and a lower content of clay minerals than those of the inner parts of the rock ( alexandrowicz et al . the precipitation of amorphous silica ( silicification ) leads to a hardening and sealing of the rock surfaces . the hydrolytic decomposition of less resistant aluminosilicate minerals , such as feldspars and biotite , which to a variable degree have been replaced by clay minerals and iron compounds , is the main source of the silica . dissolution of aluminosilicates is favoured by an acidic reaction within the rock , low ph being a result of weathering of sulphides and precipitating iron ( oxyhydr)oxides , as well as intensive microbial activity or infiltration of acid waters from atmospheric precipitation ( mcarthur et al . the first two processes seem to be more significant in the sandstones studied ( alexandrowicz et al . the dark grey to black colour of the outer layer can be associated with the presence of atmospheric dusts of industrial origin and also an activity of microorganisms , such as cyanobacteria , bacteria oxidising fe and mn , and melanin - producing fungi and actinomycetes ( gaylarde et al . 2007 ; corenblit et al . the presence of salts ( e.g. gypsum , barite , potassium alum , jarosite , syngenite and halite ) results from evaporation . the majority of anions necessary to form these minerals are probably associated with the effect of air pollutants ( e.g. lentschig - sommer 1960 ; jaboska et al . the decomposition of sulphides ( pyrite ) present in sandstones is a probable source of sulphate ions ( soukupov et al . . the last process must be particularly important in the case of jarosite , whose formation requires a highly acidic environment ( bingham and nordstrom 2000 ) . the alteration of aluminosilicate ( e.g. feldspars , micas , glauconite and clay minerals ) and carbonate rock components is the chief source of cations : k , ca , na , ba and fe ( e.g. steiger 2003 ; wilczyska - michalik 2004 ) . however , a connection of these cations with atmospheric particulate pollutants is also probable ( wilczyska - michalik 2004 ; kamh 2005 ; pikryl et al . 2007 ; their surface is covered by a discontinuous , grey or black crust up to 0.5-mm thick . optical microscopy has revealed only opaque and/or translucent grains of variable morphology , probably iron ( oxyhydr)oxides and carbon particles , and transparent gypsum crystals that are usually very fine . gypsum also fills the cracks in quartz grains and the fissures along the cleavage surfaces of feldspars , as well as spaces between flakes of micas . in some places , it also replaces rock cement ( fig . the near - surface framework minerals of sandstones are covered by a thin layer rich in c , si , al , fe , p , cl , k and ba . grain components include iron oxides , along with local occurrences of barite and carbon particles ( soot ) . above all , however , discontinuous accumulations , containing chiefly salt minerals , mainly gypsum and halite ( fig . 6c , d ) , spherical particles of aluminosilicate glass and spherules of iron oxides ( magnetite and hematite ) , were observed . the properties and composition of the latter components indicate them to originate from industrial emissions ( marszaek 2008 ) . another significant component of this layer is organic matter ( confirmed in tg / dta analyses ) . sem observations corroborated that numerous laths of gypsum cover the crust surface and also occur within pores of the sandstones , where they replace a primary cement ( fig . sometimes , however , the intergranular spaces after leaching the cement remain empty ( fig . 6d ) . in samples from the czartoryski museum and the ethnographic museum collected at a small distance from the ground level , halite and gypsum halite is absent , however , in the samples collected from the balcony columns of the czapski palace.fig . 8scanning electron microscope images of sandstone samples from architectonic detail : a gypsum replacing cement of the stone ( sample mc-010 ) and b empties after leaching the sandstone cement ( sample me-81 ) scanning electron microscope images of sandstone samples from architectonic detail : a gypsum replacing cement of the stone ( sample mc-010 ) and b empties after leaching the sandstone cement ( sample me-81 ) the presence of these salt minerals was also confirmed by xrd analyses , which is a proof of their significant presence in the composition of black crust ( x - ray detection limit of salt minerals is relatively high , being around 3 % ) . the dark grey to black colour of the crust surfaces results chiefly from the presence of atmospheric dusts . however , an impact of microorganisms , similar to that on natural outcrops , can not be excluded ( saiz - jimenez 2003 ) . intensive destruction of sandstones indicates that natural weathering is enhanced by processes of anthropogenic nature . significant amounts of gypsum and halite are , among others , effects of such pollution and due to easy crystallisation and re - crystallisation of salt minerals lead to granular disintegration of rocks ( alexandrowicz and pawlikowski 1982 ; rodriguez - navarro and doehne 1999 ; saby et al . 2000 ; sabbioni 2003 ; rembi and smoleska 2010 ) . the sulphates result from the presence of so2 , a common component of air pollutants . its two - pronged deposition , wet one ( periodic effect of , e.g. acid rains ) and so - called dry one ( long - term impact of so2 on the surface of stones , where sulphuric acid immediately forms in the reaction with the moisture present in the rock ) , is the cause of gypsum precipitation ( manecki et al . the sources of chloride ions may be represented by air pollutants and chemicals used in removing ice from streets and pavements ( chiefly nacl and cacl2 ) . an impact of chloride aerosols brought by winds from the nearby vistula river , polluted by salty mine waters from the upper silesian coal basin , was also possible in previous decades . calcium , barium and sodium ions originate from the decomposition of primary aluminosilicates ( calcium also from dissolution of carbonates ) of the sandstones . nevertheless , significant amounts of these elements may also be linked with particulate air pollutants ( sabbioni and zappia 1992 ; marszaek 2000 ; jaboska et al . 2001 ; wilczyska - michalik 2004 ; toniolo et al . with regard to the sodium ion in halite ( nacl ) present in the sandstone samples collected low above the pavement level ( the plinths at the ethnographic museum and the czartoryski museum ) , the latter process is probably of significant importance . nevertheless , the most indicative of the effects of anthropogenic pollution are the spherical particles of aluminosilicate glass and iron oxides observed on the stone surface . comparing the chemical compositions of crusts on the sandstones from natural outcrops and those from architectonic elements , higher concentrations of mgo , cao , na2o , s , cu and zn were found in the latter ( table 2 ) . thick , and may contain a certain admixture of a material from below as it was not always possible to separate the crust layer alone . in the case of samples collected in krakw , these results do not differ considerably from those of the layers situated deeper , ca . 2 cm below the crust surface , which probably reflects advanced changes reaching deep into the sandstones . nevertheless , in the inner layers of the sandstones from the architectonic elements the assays for cao , na2o and losses on ignition ( loi ) , and also the contents of sulphur and some trace elements ( zn , cu ) slightly decrease , whereas the contents of sio2 , al2o3 , fe2o3 , mgo and k2o slightly increase in comparison with the outer layer ( fig . such a trend manifests the accumulation of secondary salt minerals ( gypsum and halite ) as well as particulate pollutants ( the latter are carriers of heavy metals ) on sandstone surfaces and their lower amounts in subsurface layers . similar tendencies were described , e.g. by thomachot and jeannette ( 2004 ) , wilczyska - michalik ( 2004 ) and kamh ( 2005).table 2major ( wt% ) and some trace ( ppm ) element concentrations ( combined icp - oas , icp - ms and inaa analyses ) in the weathering crust of the sandstones ( kb-10 , smc-11 , smc-13 , smc-14samples from carpathian foothill ; me-81the ethnographic museum , pc-01the czapski palace)kb-10smc-11smc-13smc-14me-81pc-01sio2 87.8390.6089.6186.6487.8390.10tio2 0.150.270.260.270.010.06al2o3 4.094.284.184.474.393.67fe2o3 3.401.081.423.491.471.50mno0.010.010.010.010.010.01mgo0.090.080.080.080.210.18cao0.060.080.090.190.380.11na2o0.370.330.330.391.430.44k2o1.971.831.791.901.831.37p2o5 0.150.050.800.150.2760.248loi1.341.151.282.182.301.38sum99.4699.7699.8599.77100.6799.95s ( wt%)0.0240.0150.0420.0380.1950.190cu ( ppm)91115102633zn ( ppm)3410162641154sr ( ppm)383536434536ba ( ppm)406356353437289261pb ( ppm)201520301054 loi loss on ignitionfig . 9bulk chemical composition ( combined icp - oes , icp - ms and inaa analyses ) of inner and outer layers of the sandstones crusts from architectonic details ( major elements and sulphur in wt% , trace metals in ppm ) . me-81 the ethnographic museum , pc-01 the czapski palace , i inner layer , o outer layer major ( wt% ) and some trace ( ppm ) element concentrations ( combined icp - oas , icp - ms and inaa analyses ) in the weathering crust of the sandstones ( kb-10 , smc-11 , smc-13 , smc-14samples from carpathian foothill ; me-81the ethnographic museum , pc-01the czapski palace ) loi loss on ignition bulk chemical composition ( combined icp - oes , icp - ms and inaa analyses ) of inner and outer layers of the sandstones crusts from architectonic details ( major elements and sulphur in wt% , trace metals in ppm ) . me-81 the ethnographic museum , pc-01 the czapski palace , i inner layer , o outer layer in natural outcrops , spatial differences of the chemical composition between the weathering crust and an inner sandstone layer are associated , first of all , with the accumulation of iron compounds and the prevailing type of fe - bearing minerals ( rzepa et al . 2011 ; alexandrowicz et al . 2014 ) . the phase and chemical composition of weathering crusts indicate that their development is controlled by the environmental conditions combined , in particular , with climate . the maopolska voivodeship , in which the study region is situated , is one of the most polluted areas in poland ( wilczyska - michalik 2004 ; pajk 2011 ) , and the krakw conurbation is characterised by very high levels of gaseous and particulate pollution . they inherently result from adverse atmospheric conditions of krakw , situated in the vistula river valley surrounded by hills , as well as from a proximity of large industrial centres , and further aggravated by distant industrial emissions . the annual average concentrations of total dust particulate matter < 10 m ( pm10 ) particles , nitrogen dioxide and sulphur dioxide recorded in a measuring station situated in the city centre are high . as regards pm10 and nox , their concentrations markedly exceed permissible standards ( table 3 ) . the excessive concentrations of nox , so2 and pm10 are recorded most often during the heating season , which usually lasts from october till march . local boiler houses and household furnaces that use solid fuels ( coal and/or coke ) of variable ( often inferior ) quality are additional sources of pollution . road transport also contributes its share , quite significant in case of any old town with its dense , structural layout.table 3average so2 , no2 and pm10 concentrations ( g / m ) in the air in krakw and cikowice ( carpathian foothill ) in 2010cityaverage concentrations ( g / m)so2 no2 pm10annualsummer seasonheating seasonannualsummer seasonheating seasonannualsummer seasonheating seasonkrakw114.518707170.57948.691.8city centre , i. krasiskiego average so2 , no2 and pm10 concentrations ( g / m ) in the air in krakw and cikowice ( carpathian foothill ) in 2010 from ogar et al . the area of the carpathian foothill ( the region where the samples from the natural outcrops were collected ) is affected by industrial emissions from krakw and neighbouring towns such as tarnw and nowy scz ( lach and wilczyska - michalik 1996 ; gabaa and khne 2002 ; gobiowska et al . 2002 ; karnia et al . 2007 ; ogar et al . 2011 ) . the state of its atmosphere is worsened by local pollution sources associated with industrial activities , combustion of fuels ( during heating season ) and road transport . nevertheless , the overall air quality is much better there than in the krakw urban agglomeration . the annual average concentrations of sulphur dioxide , nitrogen oxides and pm10 suspended dust stay on much lower levels than those within krakw ( table 3 ) . in the study area , the load of acidifying compounds introduced via atmospheric precipitation : so4 > n > ca > cl > na > k > mg > zn > p > fe > cu > mn > pb > ni > cd > cr is high , although the last decade shows a declining trend . the deposition of sulphate and nitrate ions in the maopolska region is the highest in poland ( pajk 2011 ) . the different level of the environmental pollution in the two subareas studied is reflected in the concentrations of salts , size of their crystals and the state of preservation of sandstone surfaces . gypsum , which fairly sporadically occurs on the rock surfaces in natural outcrops , develops into tabular , relatively large and well - formed crystals ( fig . the crystals of this sulphate found on the architectonic details of historic buildings in krakw are , as a rule , finer and lath - shaped , and occur in aggregates densely covering the surface ( fig . . such differentiation of the size and morphology probably reflects the conditions prevailing during precipitation : high concentrations of acid air pollutants ( including so2 ) result in gypsum crystallising from more saturated solutions , thereby producing a higher number of relatively small crystals ( maurice 2009 ) . on the surfaces of the tors in the carpathian foothill , crystallisation is slower and proceeds from less concentrated solutions , which results in the formation of large , euhedral gypsum crystals . nevertheless , the airborne particles observed there ( aluminosilicate glass and the spherical forms of iron oxides ) provide an evidence of a far - reaching industrial pollution . thus , this contamination must be a reason of the sulphate salts and chlorides precipitating on the surfaces of the sandstone tors . it seems , however , that the main source of the cations ( k , ca , na , ba and fe ) necessary to the formation of these salts must rather be contributed to the weathering of mineral rock components . there is a certain difference in the mineral composition of weathering crusts from both subareas studied : those developed on the sandstone architectonic elements contain only gypsum and halite , whereas those from the sandstone in natural exposures in addition to gypsum and halite also traces of other mineral phases : alum , barite or jarosite . the most probable explanation is that these minor salt phases have other cations than the na and ca composing gypsum and halite . the additional cations must have been released in the natural environment during weathering of aluminosilicates , for instance , potassium from micas and feldspars . in an urban environment , the anthropogenic supply of sodium and calcium is so high that the two cations dominate porous solutions in sandstones . another factor must also be taken into considerations : the sulphates present in the crusts from natural exposures crystallise under distinctly acid conditions . jarosite is formed at very low ph values ( bingham and nordstrom 2000 ) , and barite also favours acid conditions . in turn , the al - containing sulphates precipitate when aluminium is released into weathering solutions ( navrtil et al . this element is mobile when the solutions are either strongly acidic or strongly basic ; otherwise , it tends to precipitate ( fest et al . urban environments have high concentrations of dusts , most of which are alkaline and neutralise the acid reaction of airborne deposits . in the distant , natural environments of the tors , the amounts of the industrial particulates are distinctly lower and , as a result , insufficient to neutralise acid depositions and to prevent other processes , e.g. weathering of pyrite , which leads to the formation of sulphuric acid . another acidifying factor may be represented by organic acids produced by moss and lichens colonizing the surfaces of natural exposures of sandstones . the factors mentioned combined with a low acid neutralisation capacity of waters developed within the sandstones of a low calcium content ( table 2 ) result in an acid reaction of the porous waters ( navrtil et al . 2013 ) , which favours crystallisation of the sulphates mentioned above . it should also be emphasised that the sandstones forming the tors occur in their natural geological environment . on the other hand , the sandstones used as therefore , in contrast to those in the natural outcrops , they do not have immediate contact with the parental substrate , and stone blocks are often arranged in various positions and additionally cemented with calcareous or clayey material . water circulation , essential in weathering in the tors , proceeds both upwards and downwards , is usually limited in building stones , which intensifies evaporation and precipitation of secondary salts . the recurrent precipitation stress acting along the borders of external ( affected ) and internal ( less affected or not affected at all ) parts of any rock , particularly of a building stone , is a main factor of its damaging by exfoliation . in sandstones that form natural outcrops , they facilitate hardening of rock surfaces via silification and cementation with iron compounds and protect the rock from damage exerted by atmospheric factors . despite of this , with the passage of time , the stress caused by the differences in the coefficients of thermal expansion , in moisture contents between the rock surface and its inner parts , and in the composition of rock cement , result in natural loosening of the rock and exfoliation of weathering crust fragments . in the process , the crust substrate is exposed , which in a further course of chemical weathering gradually transforms and develops a new protective crust layer . the weathering crusts developed on the carpathian sandstones exposed to various conditions of air pollution show differences in their development . the black crusts collected in highly ( krakw ) and low - polluted ( carpathian foothill ) areas differ in both their fabric and composition . the sandstone black crust from tors in the carpathian foothill is rich in organic matter and composed of amorphous , opal - type silica . sulphate incrustations ( mainly gypsum ) accompanied by spherical iron oxide or aluminosilicate glass particles from power plant emissions have been sometimes observed . in trace amounts the black crust is underlain by a zone coloured by iron compounds ( goethite and/or hematite ) . the enrichment of the surface crust in silica and iron ( oxyhydr)oxides hardens its surface and lowers its porosity , thus protecting the rock interior from atmospheric agents . in contrast , the sandstones from architectonic details in monuments are loosened and covered in places by a thin black or dark grey crust , enriched in organic matter . spherical particles of aluminosilicate glass and iron oxides ( hematite and magnetite ) from industrial emissions are common here . on the rock surface and also in intergranular spaces , numerous salts crystallise , mainly gypsum and halite . in contrary to the weathering crust developed on natural sandstone outcrops , these occurring on monuments enhance deterioration of the rock and contribute to mechanical disintegration of the stone . different levels of environmental pollution in the two subareas studied are also reflected in the concentrations and size of salt crystals . gypsum , sporadically occurring on the rock surfaces in natural outcrops , develops into large and well - formed crystals , because the precipitation of this is slower and proceeds from less concentrated solutions . the crystals found on the architectonic details are finer and densely cover the surface , because high concentrations of acid air pollutants result in gypsum crystallising from more saturated solutions . the mechanism of developing weathering crust is similar on the surfaces of sandstone tors and of architectonic sandstone elements . the course of weathering of rock surfaces depends besides inherent petrographic features ( mineral composition , texture , structure ) also upon environmental conditions , being particularly affected by atmospheric pollution . in the regions of low pollution , the sandstone surfaces become covered with the natural weathering crust that provides a long - term protection of inner rock layers from further alterations . the weathering crust developed as an effect of pollution in urban areas contains considerable amounts of secondary salt minerals , mainly gypsum , reduces the resistance of surface stone layers to weathering and , as a side effect , lowers the stone aesthetic quality . the crust layer additionally sealed with airborne particulates hampers a free exchange of stone moisture with the air , a migration of soluble salts from inner parts of stones outwards and their crystallisation on the surface . all this leads to the loosening of the rock structure and finally , to the granular disintegration of the stone . the difference in the type of salt minerals found in weathering crusts formed on the carpathian sandstones used as architectonic elements ( only gypsum and halite ) and those occurring in natural outcrops ( where gypsum and halite are occasionally accompanied by alum , barite or jarosite ) may also result from various amounts of airborne dusts and local colonisation of natural rock exposures by moss and lichens . these factors affect the reaction of solutions migrating within the rocks , and due to that change , the composition of salt precipitates . a certain impact on the process of weathering can also be exerted by differences in water circulation in the sandstones remaining in their natural ( geological ) environment and those used as building materials in towns . the sandstones of natural outcrops are characterised by easier circulation of capillary or atmospheric waters . in contrast , a block of building stone shows limited , usually one - directional water circulation with the prevalence of surface moisturising and rapid evaporation . as a consequence , the growth of weathering crusts on tors is slower than that on weathering sandstones of urban buildings .

this work presents mineralogical and chemical characteristics of weathering crusts developed on sandstones exposed to various air pollution conditions . the samples have been collected from sandstone tors in the carpathian foothill and from buildings in krakw . it has been stated that these crusts differ in both fabric and composition . the sandstone black crust from tors is rich in organic matter and composed of amorphous silica . sulphate incrustations accompanied by dust particles have been only sometimes observed . beneath the black crust , a zone coloured by iron ( oxyhydr)oxides occurs . the enrichment of the surface crust in silica and iron compounds protects the rock interior from atmospheric impact . the sandstones from architectonic details are also covered by a thin carbon - rich black crust , but they are visibly loosened . numerous salts , mainly gypsum and halite , crystallise here , thus enhancing deterioration of the rock . moreover , spherical particles originated from industrial emissions are much more common . gypsum in natural outcrops , forms isolated and well - developed crystals , whilst these found on the architectonic details are finer and densely cover the surface . such diversity reflects various concentrations of acid air pollutants in solutions .

Introduction Materials Methods Results and discussion Weathering crusts on sandstone tors Weathering crusts on architectonic details Chemical composition of the crusts Conclusions

PMC4070575

despite the importance of physical activity in maintaining and improving health and quality of life in those individuals over the age of 65 , research suggests that less than 3% of elderly americans comply with the american college of sports medicine 's physical activity recommendations [ 2 , 3 ] . given the correlation between physical activity and increased health , it is of interest to accurately measure physical activity levels within a population . however , the ability to quantify physical activity in individuals can be a problematic task . in addition , because the current recommendations for physical activity include both intensity and duration , there is a compulsory need that measurement tools do the same . criterion methods , such as doubly labeled water and open - circuit spirometry , may not be plausible when examining large populations due to cost and/or an inability to obtain frequency and intensity of physical activity . furthermore , the commonly used self - report instruments are subjective and rely on recall information , which may be limited when applied to an older population . this information can be used to provide estimates of time spent in different levels of physical activity intensities and overall patterns of physical activity . unfortunately , few applications to the elderly population have been made and the raw output data can be difficult to interpret . moreover , accelerometer activity count cut - points used to determine physical activity intensity levels have been established from studies testing young participants [ 69 ] . to date , there are only very few studies that have sought to determine an activity count cut - point for determination of moderate physical activity intensity for elderly adults [ 8 , 10 , 11 ] and only one study has evaluated the relationship between oxygen consumption and activity counts of younger and older adults . furthermore , since investigations have shown that elderly individuals require a greater metabolic cost for walking at the same absolute intensity as young individuals [ 12 , 13 ] , it seems necessary to make such a comparison . lastly , evidence suggests that the standard 3.5 mlkgmin may not represent the resting metabolic rate of the general population [ 14 , 15 ] and that older adults may have a lower resting metabolic rate compared to younger individuals [ 14 , 16 ] . due to those differences at rest and during exercise between younger and older adults , the researchers hypothesized that a lower resting metabolic rate and higher oxygen consumption during exercise with similar accelerometer derived activity counts may result in a lower activity count threshold associated with moderate exercise intensity for older adults . therefore , the first purpose of this investigation was to compare accelerometer derived activity counts and oxygen consumption , measured during treadmill walking , between young and elderly participants . secondly , we sought to determine the relationship between metabolic equivalent ( met ) and accelerometer derived activity counts for each group of participants by using estimated and measured met levels . thirty - two individuals , 16 elderly ( age 66.6 2.9 yrs ) and 16 young ( age 21.3 2.5 yrs ) , recruited from the local community agreed to participate in this study . participants were recruited from flyers posted at recreational facilities ( e.g. , senior centers ) and bulletin boards at various locations ( e.g. , university , grocery stores ) . only participants that were healthy , free of injury within the past six months , and not taking medication ( e.g. , antidepressants , beta - blockers ) or stimulants ( e.g. , caffeine , smoking ) known to influence energy expenditure or exercise capacity were tested . all participants provided written informed consent and approval was obtained by the central washington university human subjects review committee . participants attended a familiarization session prior to testing . during the familiarization session , participants rested for 10 minutes in a seated position while breathing through a mouthpiece and then walked at the various intensities to familiarize them with the walking speeds and grade on the treadmill . at least two days and no more than seven days after the familiarization session , a testing session was scheduled . even though the majority ( 27 out of 32 ) of the tests were performed in the morning ( before 9 a.m. ) , there were some tests that were performed at a later time mainly due to participant availability . participants were instructed to refrain from caffeine and exercise on the day of testing . also , participants were asked to withhold food consumption 3 - 4 hours prior to testing . participants reported to the laboratory and after 10 min of rest to determine compliance with pretest instructions and answer last minute questions ; height was recorded to the nearest centimeter ( med art , st . louis , mo ) and weight was measured to the nearest 0.5 kg ( detecto - medic , brooklyn , ny ) . body density and percent body fat were determined from skinfold measurements ( lange skinfold calipers , cambridge scientific instruments inc . , cambridge , md ) as described by pollack et al . . indirect calorimetry ( true max 2400 , parvo medics inc . , sandy , utah ) was used for the measurement of resting metabolic rate . gas analyzers were calibrated before each test with a gas tank of known concentration ( 16% o2 , 4% co2 ) . respiratory flows were measured with a pneumotachometer calibrated before each test with a syringe of known volume ( 3.0 l ) . participants were then fitted with a mouthpiece that incorporates a two - way breathing valve apparatus ( hans rudolph inc . , initially participants were in a supine position but during pilot testing older participants indicated that they were uncomfortable and therefore all participants remained awake in a seated position for 30 min while expired gases were collected . expired gases were sampled in 30 s intervals and averaged oxygen uptake values ( vo2 ) , both relative ( mlkgmin ) and absolute ( lmin ) , obtained from minutes 15 to 25 were used to calculate an individual 's resting metabolic rate . in addition , heart rate ( polar electro , usa ) was monitored throughout the entire resting period and averaged during minutes 1525 and this value was considered as the individual 's resting heart rate . similar to the procedures of copeland and esliger , the treadmill bout consisted of 6 min stages with 5 min seated rest periods between stages . the four treadmill stages included walking at speeds of 27 , 54 , and 80 mmin ( 1.0 , 2.0 , and 3.0 mph , resp . ) and 0% grade , performed in random order , and a final stage of 94 mmin ( 3.5 mph ) and 2% grade . these intensities were selected to induce light , moderate , and vigorous physical activity . based on pilot testing , the 94 mmin and 2% grade stage was excluded from the random selection because this intensity was too difficult for some individuals to begin with , particularly in the absence of a warm - up and could have resulted in higher oxygen consumption on subsequent stages . all trials were performed on the same quinton q65 treadmill ( quinton instruments company , seattle , wa ) . oxygen consumption during the treadmill bout was calculated by averaging the 30 s sampling rate values obtained in the final 3 min of each treadmill stage completed . in order to determine measured met value per stage , averaged oxygen consumption per stage was divided by the individual 's resting metabolic rate ( measured vo2/measured resting metabolic rate ) and by the standard ( 3.5 mlkgmin ) metabolic rate ( measured vo2/3.5 ) . prior to the treadmill bout , participants were fitted with two actigraph ( pensacola , florida ) accelerometers ( one gt1 m model and one gt3x model ) . the gt1 m ( mass , 27 g ; width , 38 mm ; height , 37 mm ; thickness , 18 mm ) is a newer smaller version of the discontinued actigraph model 7164 . this updated model has demonstrated similar activity counts per minute , when compared with the actigraph 7164 , at treadmill speeds of 54 , 80 , and 107 mmin . unlike the gt1 m model , which senses vertical accelerations , the gt3x detects accelerations along three planes of motion . according to the manufacturer , both activity monitors were initialized before each trial using the same laptop computer and using the actilife low frequency extension . in addition , the activity monitors were marked so that the same units were used for each participant . accelerometers were securely mounted side by side along the anterior - axillary line of the right hip with an adjustable nylon belt . the activity counts recorded by the gt1 m and the vertical axis activity counts from the gt3x were summed using 1 min sampling intervals ( epochs ) and averaged for the final 3 min of each treadmill stage . for the purposes of this investigation , the average activity counts from the gt1 m and gt3x were used in the statistical analysis . body weight , height , and body composition were evaluated for significant differences between groups using a two - tailed independent t - test . likewise , two - tailed independent t - tests were used to determine differences in heart rate and oxygen consumption between groups at rest . a two - way repeated measures ( rm ) analysis of variance ( anova ) with one within factor ( exercise intensity ) and one between factor ( elderly versus young ) was used to evaluate differences between groups in averaged activity counts , oxygen consumption , and met values for each of the intensities . to examine for differences between activity counts derived from the gt1 m and gt3x actigraph models , a linear regression analysis was used to determine the relationship between measured mets and averaged vertical axis activity counts and between estimated mets and averaged vertical axis activity counts for each group ( young and elderly ) . the prediction equation was determined from the entire data set for each group . finally , activity counts corresponding to moderate intensity ( 3 mets ) exercise were calculated from the developed regression equations . data are expressed as means sd , and statistical significance was set at p < 0.05 . thirty - two individuals participated in the investigation ( 16 elderly and 16 young ; table 1 ) . the young and elderly groups were similarly matched for gender ( 9 males and 7 females ; 8 males and 8 females , resp . ) . however , two of the elderly participants were unable to perform at the 94 mmin and 2% grade . in one case , the participant declined to begin the final stage due to fatigue . in the other case , the participant began the final stage but requested to stop because of difficulty maintaining pace . activity counts measured by the gt1 m and gt3x actigraph models are presented in table 2 . there were no differences in activity counts between accelerometers for any of the exercise intensities , although the two - way rm anova revealed significant difference for activity counts among the different intensities ( table 2 ) . at rest , oxygen uptake relative to body mass among the elderly was significantly lower than the young group ( 3.49 0.50 and 3.89 0.28 mlkgmin , resp . ) . there were no differences between groups in relative oxygen uptake ( mlkgmin ) and heart rate at rest ( table 3 ) . however , the two - way rm anova showed a significant interaction between groups and exercise intensities . bonferroni post hoc analysis indicated that oxygen uptake , measured met , and heart rate values were significantly greater for the elderly participants at each of the intensities performed ( table 3 ) . measured met values were significantly greater among the elderly participants for each of the treadmill intensities ( figure 1 ) . a strong correlation ( r = 0.89 ) between averaged activity counts and measured met values existed among the young group ( figure 2(a ) ) . activity counts and estimated met values were also strongly correlated ( r = 0.91 ) for the young group ( figure 2(b ) ) . the relationships between activity counts and measured mets ( figure 3(a ) ) were moderate ( r = 0.72 ) as it was the relationship ( r = 0.76 ) between averaged activity counts and estimated mets ( figure 3(b ) ) for the older participants . based on the linear regression equations accelerometer activity counts of 824 ( 95% ci : 2261719 ) and 2207 ( 95% ci : 17303008 ) countsmin were associated with moderate ( 3 mets ) physical activity intensity for the elderly and young participants , respectively , when using measured resting metabolic rate . using the standard 3.5 mlkgmin , linear regression equations indicated that moderate ( 3 mets ) intensity physical activity was associated with 784 ( 95% ci : 1041859 ) and 2009 ( 95% ci : 12672320 ) countsmin for the elderly and young participants , respectively . the purpose of this investigation was to compare treadmill derived activity counts , oxygen consumption , and met values between young and elderly participants . despite the widespread use of accelerometers for obtaining objective measures of physical activity , few applications to older populations have been made . consequently , this study provides valuable information pertaining to the plausibility of the use of accelerometers within an elderly population . the main finding of the study is that despite no significant differences in accelerometer derived activity counts between older and younger participants at various walking intensities , there were significant differences in relative oxygen consumption and met values at rest and all exercise intensities indicating that moderate intensity physical activity for older adults may be determined at lower accelerometer derived activity counts than younger adults . oxygen uptake values obtained during treadmill exercise from the younger individuals in this study are in agreement with data reported in the literature . reported oxygen uptake values in a group of young adults ( age 23.7 4 ) , who performed treadmill walking exercises at speeds of 54 , 80 , and 94 mmin , respectively . these values closely parallel those measured from the younger participants in our study ( table 3 ) . furthermore , oxygen consumption values associated with walking at the respective speeds in elderly individuals in this study are consistent with findings from previous investigations [ 10 , 13 ] . in this investigation , elderly participants had significantly higher oxygen uptake values than the younger participants during treadmill walking across the range of intensities . similar findings have demonstrated a 12% and 22% increase , among 65- and 80-year - old participants , respectively , in the energy cost of walking at speeds of 80 and 94 mmin , compared with 25-year - olds . energy expenditure per mile walked , among elderly , has been shown to be 21% greater than younger individuals . others have found 31% greater energy cost of walking in older ( age 74 3 ) compared to younger adults ( age 27 3 ) . the average oxygen consumption demonstrated by the elderly participants in this investigation ranged from 14 to 19% greater than the younger participants across the selected speeds . the data in our study support the idea of a greater energy cost in the elderly when compared to a younger cohort for varying walking intensities . nonetheless , the underlying reasons for the increased energy cost among elderly individuals are not fully understood . interestingly , activity counts ( countsmin ) did not differ between groups at any of the treadmill intensities performed . several other studies have reported actigraph activity counts per minute similar to ours for equivalent treadmill walking speeds [ 19 , 2123 ] , although these studies have been specific to younger populations . unfortunately , data concerning actigraph activity counts produced during treadmill walking in older populations is limited . therefore , few comparisons to this study can be made . however , an investigation similar to ours reported mean activity counts of 515 , 1041 , and 2504 countsmin at speeds of 33.5 , 54 , and 80 mmin , respectively , in a group of elderly participants ( age 69.7 3.5 ) . although these values closely resemble ours , the activity counts obtained from our sample of elderly individuals tend to be slightly higher . this difference can be attributed to different models of actigraph accelerometer used to determine activity counts . copeland and esliger used the 7164 model whereas in the present study we used the gt1 m and gt3x models . a study by kozey et al . has shown that activity counts from the gt1 m model are slightly but significantly higher than the 7164 model . research has indicated that the standard 3.5 mlkgmin may not represent the resting metabolic rate of the general population and that resting metabolic rate may be lower in older individuals . since previous studies by copeland and esliger and miller et al . have not measured resting metabolic rate in older adults , this study aimed to determine measured met values for each of the exercise intensities . to our knowledge , this attempt to identify the relationship between measured met values and activity counts for a sample of elderly participants had not previously been performed . in agreement with previous research [ 14 , 16 ] , resting metabolic rate in our group of elderly individuals was significantly less than the younger participants . however , the average resting metabolic rate for the older participants was similar to the standard met value of 3.5 mlkgmin whereas the average resting metabolic rate for the younger adults was significantly higher than the standard met value of 3.5 mlkgmin . the average resting metabolic rates for both older and younger participants in this study are higher than other studies [ 1416 ] . it is possible that the differences between the present study and previous studies are due to different positions during resting periods . while the mean measured met value among the older individuals approached but was not equivalent to 6 mets at a walking speed of 94 mmin 2% grade , five of them obtained values greater than 6 mets . at the intermediate treadmill speeds of 54 and 80 mmin , all of the elderly participants were exercising at or above a moderate intensity . in contrast , the younger participants did not reach a level of moderate physical activity intensity until a treadmill speed of 80 mmin . furthermore , even at the fastest speeds , younger individuals were exercising well below a vigorous intensity . translating accelerometer activity counts to determine intensity of physical activity has proven to be a difficult task and has been questioned by some studies [ 25 , 26 ] . however , based on the data , we were able to determine an activity count threshold that is associated with moderate ( 3 mets ) physical activity for this group of younger and older adults and compare this activity count threshold with current literature . the commonly used actigraph activity count threshold for moderate physical activity intensity ( 1952 countsmin ) , developed by freedson et al . , was derived from exercise tests involving young adults . similarly , actigraph activity count cut - points for moderate physical activity intensity ranging from 1810 to 2260 countsmin have been shown in other studies testing adults aged 2045 . in agreement with previous studies of younger adults , the present study found activity count cut - points for moderate physical activity intensity to be 2207 and 2009 countsmin depending on either measured or standard resting metabolic rate . in contrast to those studies , copeland and esliger and miller et al . have indicated a moderate intensity threshold value of 1041 and 1566 countsmin , respectively , for their group of elderly individuals . in this investigation , the threshold value , equivalent to 3 mets , was 824 countsmin when using measured resting metabolic rate and 784 countsmin when using the standard resting metabolic rate . the activity count threshold associated with moderate intensity activity reported in this study is lower than the ones reported by previous research . copeland and esliger did not attempt to determine cut - points of various intensities . the researchers simply used the activity counts that corresponded to a mean oxygen consumption of 13 mlkgmin . in addition , miller et al . used a different methodology than previous research . participants exercised at various intensities for five minutes and rested between intensities for five or six minutes . it is therefore difficult to compare the activity counts reported in the present study and the one by miller et al . . the results of the present study , along with the results of previous research , may represent the need to establish agreeable metabolic equivalents for respective age groups to effectively determine physical activity intensity level activity count cut - points . these findings are of significant importance because recent epidemiological investigation pertaining to physical activity behaviors among elderly united states citizens reported a substantially low amount of time spent in moderate intensity activity . however , the activity count threshold employed for identifying moderate physical activity in adults aged 1860 was 2020 countsmin . it is possible that previous epidemiological reports may be greatly underestimating the amount of time elderly populations spend performing moderate physical activity . this sample size of younger and older adults is small and therefore the activity count threshold that is associated with moderate ( 3 mets ) physical activity for this group of participants might not be generalizable to all older and younger adults . in addition have shown that accelerometry counts per minute do not change between ages of 18 and 59 . in addition , the activity count thresholds established by freedson et al . were based on similar age group as ours . therefore , the purpose of this study was to examine the differences in oxygen consumption and activity counts between a group of younger individuals ( similar to the one by freedson et al . ) and a group of older individuals above 60 years . the majority of the participants exercised below vigorous intensity ( < 6 mets ) . the intensity of 94 mmin and 2% grade was used to elicit vigorous intensity . it is difficult to elicit vigorous intensity for older adults due to either their lack of fitness , fear , or functional limitations . despite similarity in accelerometer activity counts produced during treadmill walking across a range of treadmill speeds among elderly and young participants , an increased oxygen uptake among elderly participants was evident . therefore , accelerometers appear to be valid in their detection of exercise intensity across age groups . moreover , translating activity counts into categorical levels of physical activity intensity can not be done in the same manner for young and elderly individuals . measurement of resting metabolic rate may offer the most effective way for determining the relationship between activity counts and the intensity level of physical activity performed . further research is necessary in order to better understand the relationship between accelerometer activity counts and physical activity intensity levels for an elderly population .

the purpose of this investigation was to compare accelerometer activity counts and oxygen consumption between young and elderly individuals . sixteen young ( 21.3 2.5 yrs ) and sixteen elderly ( 66.6 2.9 yrs ) participants completed 30 minutes of resting oxygen consumption to determine resting metabolic rate and four 6 min walking intensities ranging from 27 to 94 mmin1 . resting oxygen uptake was significantly lower for the older participants . exercise oxygen consumption was significantly higher for the elderly group . there were no significant differences in activity counts between groups at each of the exercise intensities . when using measured resting metabolic rate , activity counts of 824 and 2207 countsmin1 were associated with moderate ( 3 mets ) physical activity intensity for the older and young participants , respectively . however , using standard resting metabolic rate ( 3.5 mlkg1min1 ) , activity counts of 784 and 2009 countsmin1 were associated with moderate physical activity intensity for the elderly and young participants , respectively . these findings indicate that activity counts are similar across age groups even though the oxygen consumption of exercise is greater among elderly individuals .

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

PMC4710219

the fbi is reading our poster ! granted , this is not a typical refrain heard at the annual meetings of the american society for cell biology , but it is heard frequently at other research meetings , for example , in the field of synthetic biology . i admit that it was head turning when i first heard these words spoken a few years ago at the international genetically engineered machines ( igem ) jamboree , which is an annual , global , intercollegiate synthetic biology competition . in format , the igem jamboree is much like the annual meeting of any major scientific society . but why , in the aisles , were there suited people with badges ? perhaps a new age has dawned upon the research community . the contents of this special issue of molecular biology of the cell , with an emphasis on big data , certainly suggest that this is true . nonetheless , overt governmental examination of research beyond the standard purview of granting agencies and its program officers can only raise questions . to answer some of these questions , i invited , on behalf of molecular biology of the cell , supervisory special agent ( ssa ) edward you , who heads the biological countermeasures unit ( bcu ) at federal bureau of investigation ( fbi ) headquarters in washington , d.c . , and frequently addresses the synthetic biology community , to have a conversation on biosecurity , especially with respect to big data ( figure 1 ) . this conversation was recorded on july 17 , 2015 , and is presented here , abridged and edited for clarity and considerations of space . in addition to heading the fbi s bcu , he is a working group member of the national security council interagency policy committee on countering biological threats and an ex officio member of the nih national science advisory board for biosecurity . he also serves on two national academies committees : the institute of medicine s forum on microbial threats and the committee on science , technology , and law s forum on synthetic biology . ssa you also serves on the strategic advisory board for the synthetic biology and engineering research center and as an instructor for the united nations interregional crime and justice research institute mboc : agent you , before you talk about big data , please tell our readers about your scientific background and path to the fbi . ssa you : i got my bachelor s degree in the biological sciences from the university of california at irvine , then a master s degree in biochemistry and molecular biology at the university of southern california . all that has served me well ; it does show that there is life without a phd . before joining the bureau , i had six years of graduate research in human gene therapy , with a focus on retrovirology , and three years in the biotech sector at amgen , where i did oncology research . ssa you : i sit at headquarters at the weapons of mass destruction ( wmd ) directorate in the biological countermeasures unit . it was born out of the events of september 11 , 2001 . on the heels of that terrorist event , we had the anthrax mailings . since then , as a law enforcement service , our priority has become one of prevention rather than being reactive , just going in and investigating a crime or incident . now our number one priority is to prevent in particular a 9/11 from happening again . part of that is reaching out proactively to different members of the scientific community , ranging from the private sector , biotech and the pharmaceutical industry ; to universities ; to the igem ; and even to the amateur community , the sprawling do - it - yourself bio community , showing how members of law enforcement and the life science community have a shared responsibility of safeguarding the development and very beneficial applications of the life sciences . i find myself in a unique position , where i have one foot in national security and another in the life sciences . i seek very hard to ensure that we are able to support both at the same time . however , it seems more recently that the fbi has been showing more overt concern toward the security of big data in the life sciences . ssa you : if you take my consideration of how to protect the life sciences in a proactive manner , it is our responsibility to identify emerging areas . that is why you have seen all this activity and outreach occurring , especially at igem . the reason why big data has become very significant is that it is the next evolutionary step that synthetic biology will take , meaning that all applications and technologies coming out of this field will be completely dependent upon data all the various omics . a very good example is precision personalized medicine , where you are seeing tremendous investments in drug development , particularly in cancer research and metabolic disease , where very large data sets are leveraged . if you are looking at an individual s genome , it is just one snap shot . what are needed are data over time , during exposure to the environment , for example . from the human standpoint , it all goes into helping determine potential health vulnerabilities and appropriate therapies . if you set that as a stage and then look at potential policy aspects , there is a lot of activity looking at privacy , but not a whole lot looking specifically at security so , back in april 2014 , i partnered with the american association for the advancement of science ( aaas ) and the united nations interregional crime and justice research institute ( unicri ) . we kicked off a meeting where the theme was national and transnational security implications of big data in the life sciences . we really wanted to tackle some of the security implications in the area of big data , where biology has almost a complete overlap with the digital world . at this meeting i had representatives from microsoft , intel , ibm , google , and amazon , the entities leveraging this big data bio - innovation future , and challenged them at the outset to identify potential security issues . we did find some significant issues and published some reports that are now publically available . mboc : you had an incredible lineup of expertise contributing to the aaas report national and transnational security implications of big data in the life sciences ( berger and roderick , 2014 ) . was there any specific event that motivated the fbi to launch this reflection on biosecurity or was this entirely a proactive endeavor ? security discussions in the past tried to overlay security structures that were used in the nuclear or chemical realm . completely locking down certain areas of expertise or materiel if our mission is one of preventing the misuse , exploitation , or abuse of the life sciences , how do we approach security without becoming a hindrance to the life science enterprise ? over the last two years , we have had the issues with regard to the centers for disease control and prevention ( cdc ) and department of defense ( dod ) . a lot of discussion also came when the j. craig venter institute synthesized that bacterial genome . there were a lot of calls and discussions about the scientific community needing more ethics training and the need to develop a greater culture of responsibility . what has been lacking is the scientific community being provided security awareness something that augments how they approach the life sciences . individuals , no matter where they are in the world or when they enter the life sciences , always start with the premise , do no harm , taking a page from the hippocratic oath . unfortunately there are groups and individuals who do not subscribe to the same ethics and norms and agreements to integrity that we all take for granted and are almost innate for us . how do we graduate from do no harm to not on my watch ? it means you take an active role in being sentinels for what you are doing and preventing the abuse , misuse , and exploitation of the life sciences . if you are not fully aware what the security vulnerabilities are , then that becomes a true vulnerability for all of us . it is amazing to me that we have an international treaty to which we are all beholden , yet there are very few programs , if any , in which incoming biology students are exposed to it or the fact that the bwc exists because biology had been absolutely used and exploited for offensive purposes , even by the united states . if we do not teach that little bit of history and other security aspects , then it becomes really a challenge in the future on how to better protect biology . it is not about ethics ; it is not about responsibility ; it really is about having a healthy appreciation of some of the security concerns . mboc : how real is the threat ? the aforementioned aaas report read , very little , if any , information exists about the theft , manipulation , or exploitation of big data in the life sciences . one of the goals of generating this report was to galvanize people to start thinking about security because quite honestly i do not think we really appreciate how deep or how wide the security vulnerabilities are in leveraging these large data sets or big data in general . referring to my prior comments about precision medicine , it all hinges on genetic information and longitudinal data over time . you need a large data set because when you do an analysis you need statistical significance to know whether your results are right . as you think about that , let me walk you back to some of the most significant cyber - intrusions this past year . in august 2014 , there was a community health systems hack with 4.5 million patient records accessed ; a few months after that was the large anthem blue cross hack with 80 million individuals impacted ; and then a month after that , the premera blue cross hack in which 11 million patient records were hit . this is when it keyed off for me . in the premera blue cross hack , clinical data were accessed too . across the government , with these particular intrusions , the focus has been only on the potential loss of personal identifying information , the risk of fraud , and identity theft . i do not want to give that short shrift , because we are talking about tens , hundreds of millions of dollars in potential loss . however , if you think about the critical data a beautiful longitudinal data set , containing an individual s demographics , disease state , drugs administered , and treatment received unfortunately , all of those hacks were allegedly attributed to a hacking group based out of china . there is a much broader security vulnerability , the potential loss of our ability to stay globally competitive in the new drug market . now somebody out there has the brass ring this gigantic data set , where the only limitation is deriving the analytical tools to make all that data useful . we have to get beyond the paradigm of just looking at the financial loss . in the area of big data with specific applications to the life sciences a second is that , with the analytical tools that are coming online today , it will be almost impossible to deidentify information in the future . this was a key takeaway from the meeting with the aaas - unicri last year . if you have any short genetic sequence of an individual , you can effectively deanonomize it in fewer than three steps with publically available tools . if you are part of an institutional review board , you are in big trouble in maintaining compliance and keeping up with protecting human subject information . mboc : where is the greatest security threat to big data through hacking ? is it through the lone wolf , companies engaged in industrial espionage , or is it from state - sponsored activities ? mboc : are the threats to big data greater for private big data , for example pharma , or for academic big data ? our aaas meeting came to the crux of it : whoever has the largest and most diverse data set is going to win . that means we really need to start thinking in a more holistic manner what security means with a data set . mboc : does the fbi define big data in terms of volumes of data or analytical functions ? i do not want to go too far into definitions because one of the issues is how to define big data . from a life sciences standpoint finally , ask whether we have to come up with novel ways to address security in this bio - future . the power of the life sciences is open source , open sharing , but in it there is the added dimension of an individual s very intimate information . so there may be a call to redefine how we address security in the future . it may not be building up secure walls , whether they are physical or virtual , that protect data like our financial data . in this world of the life sciences , which is inherently open , we are going to have to rethink security . mboc : how should life scientists , faculty members at universities , respond to the worries of the fbi in terms of biosecurity ? what do you see people doing to improve the situation ? ssa you : to me , the strategy is that once we build trusted partnerships with the scientific community , first with the fbi reaching out and providing the security awareness and education , something really profound happens . you see the scientific community doing their own assessments of their technologies , self - identifying potential security vulnerabilities and then providing notification to the fbi to my unit or other partners at the fbi . they do us a favor , helping us to be better informed to better protect the life sciences , universities , and communities . even better , the community will then develop security solutions based on their expertise , which is the best of both worlds . how powerful would that be when the experts , who are developing these powerful tools and applications of the future , immediately , on the front end , start developing and implementing security measures within these applications ? that is where we want to be ; that is where the future has got to be . so there is absolutely a very necessary and important partnership between law enforcement and the scientific community . take , for example , the scientific papers regarding crispr / cas9 and gene drives and most recently the genetically modified yeast producing opioids . scientists drafted the scientific manuscript and a companion editorial piece calling out the potential security vulnerabilities . that is powerful ; that is a home run . we have successfully empowered the scientific community to understand security and then to take some proactive actions of their own . mboc : it seems one of the concerns of your unit , the bcu , is dual use of data . does the bcu have formal relationships or work with the national institutes of health ( nih ) national science advisory board for biosecurity or the cdc ? one of the cornerstone aspects of our program is the really important position called the wmd coordinator . these are men and women , special agents , trained in chemical , biological , radiological , and nuclear matters . we have at least one stationed in each of our 56 field offices across the united states . the wmd coordinator s role , as the name implies , is to coordinate and lead the notification protocols with state and local law enforcement , public health , partner with other federal entities , and then build relationships with universities , companies , and institutions within their jurisdiction . so if anything did occur , a local university , for example , would then know they have a local federal representative that can respond . if there is ever a biological incident or actual bio - crime , then those wmd coordinators become critical in the response . they actually have been a big part of the action over the years with the dod and cdc events , the discovery of smallpox at nih at a food and drug administration laboratory , the two high - profile ricin mailings almost two years ago , and the incident at georgetown university where a student was manufacturing ricin in his dorm room . in all of these different incidents , those wmd coordinators were called in and were part of the response . no matter where in the government , the coordinator is there to help and assist with either preventive training or , if anything did occur , the response . ssa you : they can just call the fbi field office in their jurisdiction and ask to be referred to the wmd coordinator . should any suspicious or criminal activity be observed that puts personnel , institutions , or materials at risk , contact your local fbi wmd coordinator to help with any assessments . think of them as being a resource to the scientific community . if you call them , it is not immediately the opening of an investigation . they are someone specifically within the fbi who is familiar with the life sciences community and with whom you can just touch base to see if something passes the sniff test . mboc : although many readers of molecular biology of the cell are gaining a greater awareness of big data , their own research does not take them into the realm of big data . for those readers , does the fbi have biosecurity concerns that lie with small data or is the focus really on big data ? ssa you : the focus on big data is because it is an emerging area . if you see all of our activities , the overall theme is safeguarding science , whether you are working in large data analytics , with select agents , yeast , or escherichia coli . we are not honing in on a specific subgroup or subtopic of the life sciences . mboc : the fbi s primary role is safeguarding the homeland , the united states . is there a separate , special , or additional message for people doing life science research , especially big data research , outside the boundaries of the united states ? i hope for a future when biologists are working as wmd coordinators in other law enforcement agencies around the world . the 21st century will see the same leaps and bounds with the life sciences that we saw in the 20th century with the internet and personal computing . if there is going be a global impact from the life sciences , there is absolutely a call to action for biologists wherever they are in the world to be guardians of science . however , we need to come to a realization first that there will be issues . we have to start discussing these things now before it is too late , before any attempts at security will be too little , too late . i hate to say it , but , if we are not careful and there is a complete overlap of the life sciences and the digital world , we might see ourselves with our security as we are facing cyber - security right now , and we do not want to be in that position . mboc : it is clear there is a lot of work ahead , not just for the scientific community , but for the fbi as well . what are the career opportunities for cell biologists in the fbi , whether they have big data experience or not ? you can be a special agent like me , or there are support positions such as the scientists who work in our laboratory division . a key piece of our mission is looking at intelligence ; that is an analyst position . i will be completely candid , upfront our hiring is a very competitive process . prior to 9/11 , the fbi s focus was on hiring individuals with law enforcement or military experience , lawyers , or accountants because the primary mission was tackling organized crime . in this day , when our number one priority is prevention , there is an absolute critical need for hiring individuals with background in computer science , foreign languages , and especially the natural sciences . the minimal criteria are a bachelor degree and at least three years of real - world experience . more than anything else , if you can articulate and show how you excelled in your specific field , then you are a good candidate you need to be passionate about what you do because in doing so , you inherently excel . the key is to set yourself in a position where you can really excel so when we begin talking to your coworkers and managers about who you really are , you have put them in a position where they can say you are an integral part of the team and made significant contributions . ssa you : partnerships between the fbi and the scientific community to build security awareness are essential . big data in the life sciences is taking the biosecurity discussion beyond pathogens and toxins . historically , the conversation almost always fell on pathogens and almost exclusively on select agents . we have to widen the aperture of what we mean by biosecurity in the future .

new scientific frontiers and emerging technologies within the life sciences pose many global challenges to society . big data is a premier example , especially with respect to individual , national , and international security . here a special agent of the federal bureau of investigation discusses the security implications of big data and the need for security in the life sciences .

INTRODUCTION ONE FOOT IN NATIONAL SECURITY; ONE FOOT IN THE LIFE SCIENCES BIG DATA WORRIES AT THE FBI FROM BENCH TO BADGEARE YOU HIRING? THE TAKE-AWAY

PMC3798153

urine is composed of numerous harmful metabolites , which are stored for prolonged time in the urinary bladder.1 for that , the primary function of urinary bladder is to prevent internalization of metabolites from bladder lumen into the underlying tissues and blood.2 this blood - urine permeability barrier has to remain functional during the stretching - contraction of the urinary bladder.3,4 the permeability barrier is formed by the apical plasma membrane of superficial urothelial cells , tight junctions , and inhibited endocytosis.4 the apical plasma membrane of normal superficial urothelial cells is characterized by the formation of urothelial plaques , which are detergent - resistant membrane domains made of ordered transmembrane proteins known as uroplakins.511 urothelial plaques cover 70%90% of the apical plasma membrane , where they contribute to the transcellular permeability barrier for ie , urea , water , ammonia or small non - electrolytes,2,12 but do not prevent co2 transport through the urothelium.13 in the urothelium , where superficial cells do not reach their terminal differentiation , as is the case during urothelial carcinogenesis,1417 the permeability barrier function is decreased.4,18 urothelial plaques also cover cytoplasmic fusiform vesicles ( fvs ) , which are implicated in the transport and incorporation of the urothelial plaques into the apical plasma membrane.3,4,19 nevertheless , involvement of fvs in the endocytosis of urothelial plaques from the apical plasma membrane is controversially discussed.3,4,19 if fvs are involved in the endocytosis from the luminal side of the bladder , they would inevitably capture some urine despite their fattened shape.20,21 therefore , demonstrating whether fvs are involved or not in the internalization of urine constituents would enhance our understanding of the permeability barrier function of the urothelium . a physiological approach to study the internalization of urine would be the introduction of nanoparticles as urine constituents . nanoparticles are small enough to be filtered through the kidneys and are accumulated in the bladder lumen , they are biologically inert , and they do not influence endocytotic processes.22,23 in this study , we tested 1.9 nm ultra - small gold nanoparticles ( aunps ) , covered by a highly water soluble organic shell , as markers for studying endocytosis in normal and neoplastic urothelium under in vivo conditions . using light and electron microscopy , we show that intravenously injected aunps reach the urinary bladder and act as physiological constituents of urine . experiments were approved by the veterinary administration of the slovenian ministry of agriculture and forestry ( permit no 34401 - 29/2007/3 ) in compliance with the animal health protection act and the instructions for granting permits for animal experimentation for scientific purposes . adult male mice , strain a / j ola hsd , were housed in plastic cages at 23c 2c , at 50%60% relative humidity and at 12-hour light / dark circadian cycle . ten animals were divided into three groups by simple random sampling : two animals were fed normal diet and were not injected with aunps , but were injected with a 0.9% nacl physiological solution ( control animals ) , four animals were fed normal diet and were then injected with aunps ( aunps - n animals ) , and four animals were n - butyl - n-(4-hydroxybutyl)nitrosamine ( bbn ) treated and were then injected with aunps ( aunps - bbn animals ) . bbn ( tokyo chemical industry co , ltd tokyo , japan ) was diluted to 0.05% with tap water and this mixture was provided as drinking water ( ad libitum ) to aunps - bbn animals for 10 weeks . animals were anesthetized with 2% chanazine ( chanelle pharmaceuticals manufacturing ltd loughrea , ireland ; 5 l/100 g body weight ) and 10% bioketan ( vetoquinol biowet , gorzw , poland ; 10 l/100 g body weight ) . once sedated aunps - n and aunps - bbn animals were injected with 200 l of the aunps solution into the tail vein . the aunps solution was prepared as follows : forty milligrams of 1.9 nm aurovist gold nanoparticles ( nanoprobes , yaphank , ny , usa ) were diluted in 400 l of physiologic solution according to manufacturer recommended protocol to obtain the final concentration of approximately 1.4 g au / kg body mass . the animals from the control group were injected with 200 l of physiologic solution into the tail vein . the urinary bladders were exposed with abdominal incision and fixed with 4% paraformaldehyde in phosphate saline buffer without potassium ( pbs - k ) for 3 minutes , applied from the serosa side in situ . next bladders were removed from the animals and dissected into multiple pieces in the same fixative . samples from each of the bladders were further processed for light , transmission ( tem ) , and scanning ( sem ) electron microscopy . urine was collected ; a 5 l drop of urine was put on gold electron microscopy grids , dried and either directly observed or processed for aunps intensification . hq silver enhancement ( nanoprobes ) and gold enhanced ( nanoprobes ) were used to enlarge gold nanoparticles . both silver and gold enhanced solutions were prepared and used according to manufacturer recommended protocols with enhancement times of 4 and 5 minutes , respectively . the urinary bladder samples were fixed in 4% paraformaldehyde in pbs ( k ) at 4c overnight , dehydrated and embedded in paraffin . paraffin blocks were cut into serial sections and counterstained with hematoxylin and eosin and examined by a uropathologist . histological urothelial changes of aunps - bbn animals were classified according to world health organization ( who ) classification of tumors of the urinary tract.24 the urinary bladder samples were fixed in 4% paraformaldehyde in pbs ( k ) at room temperature for a total of 45 minutes . after fixation , the samples were washed in pbs ( k ) , then in deionized water , and finally in the citrate buffer . the samples were then incubated in the hq silver enhancement solution ( nanoprobes ) and prepared according to manufacturer protocol in the dark for 5.5 minutes . after washing in deionized water , the samples were post - fixed in 0.2% oso4 for 75 minutes , washed in 0.1 m cacodylate buffer , dehydrated in graded ethanol , and embedded in epon . semi - thin sections ( thickness 1 m ) , were stained with 1% toluidine and examined with an eclipse te300 inverted microscope ( nikon , tokyo , japan ) . from the regions of interest , the ultrathin sections ( 50 nm ) were cut and counterstained with lead citrate and uranyl acetate . next , they were examined with a cm100 tem ( philips ) running at 80 kv the urinary bladder samples from aunps - bbn animals were also prepared using the modified tokuyasu method.25 briefly , urothelium , cut into < 1 mm pieces , was fixed in 4% paraformaldehyde in pbs ( k ) at room temperature for 2 hours , washed in pbs ( k ) , embedded into 12% gelatine , and cryoprotected by incubation in 2.3 m saharose at 4c overnight . subsequently , 300 nm thick cryosections were cut with fcs cryo - ultramicrotome ( leica microsystems , wetzlar , germany ) at 100c and collected on glass cover slips . nanoparticles in cryo - sections were silver enhanced with the hq silver enhancement solution ( nanoprobes ) in the dark for 6 minutes , washed in deionized water , and counterstained with toluidine and examined with an eclipse te300 inverted microscope ( nikon ) . from the regions of interest the cryo - ultrathin sections ( 50 nm ) were cut and counterstained with uranil acetate - methyl cellulose mixture . energy dispersive x - ray spectroscopy ( edxs ) analysis was performed using a transmission electron microscope 2100 ( jeol , tokyo , japan ) equipped with an edx system ex-23003bu ( jeol ) . the tem was operated at 200 kv the urinary bladder samples were fixed in 4% paraformaldehyde plus 2% glutaraldehyde in 0.1 m cacodylate buffer at 4c for 3 hours , washed overnight in cacodylate buffer , dehydrated in a series of ethanol , and critical - point dried ( cpd030 baltec , balzers , liechtenstein ) . after sputter - coating with gold ( balzers union scd040 , balzers , liechtenstein ) , samples were examined with a jsm840a scanning electron microscope ( jeol ) at 15 kv . the cultures of normal porcine urothelial cells ( 5th passage ) were established on 12-well culture inserts with 0.4 m porous membranes ( bd falcon , san jose , ca , usa ) and maintained in urom media as described previously.26,27 when cultures reached confluence , they were divided into two groups by simple random sampling : treated cells and control cells . aurovist aunps ( nanoprobes ) , in the same concentration as were injected into experimental animals , were added to the urom media of treated cells . control cells were kept in the urom media without aunps . after 90 minutes , cells were detached by using triple select ( gibco , life technologies , carlsbad , ca , usa ) , centrifuged , and re - suspended in fresh urom media . the survival of cells treated with aunps is presented as the percentage of the trypan blue - labeled cells versus non - labeled cells ; average , standard deviation ( sd ) , and student s t - test were performed with excel 2010 software ( microsoft corporation , redmond , wa , usa ) . experiments were approved by the veterinary administration of the slovenian ministry of agriculture and forestry ( permit no 34401 - 29/2007/3 ) in compliance with the animal health protection act and the instructions for granting permits for animal experimentation for scientific purposes . adult male mice , strain a / j ola hsd , were housed in plastic cages at 23c 2c , at 50%60% relative humidity and at 12-hour light / dark circadian cycle . ten animals were divided into three groups by simple random sampling : two animals were fed normal diet and were not injected with aunps , but were injected with a 0.9% nacl physiological solution ( control animals ) , four animals were fed normal diet and were then injected with aunps ( aunps - n animals ) , and four animals were n - butyl - n-(4-hydroxybutyl)nitrosamine ( bbn ) treated and were then injected with aunps ( aunps - bbn animals ) . bbn ( tokyo chemical industry co , ltd tokyo , japan ) was diluted to 0.05% with tap water and this mixture was provided as drinking water ( ad libitum ) to aunps - bbn animals for 10 weeks . animals were anesthetized with 2% chanazine ( chanelle pharmaceuticals manufacturing ltd loughrea , ireland ; 5 l/100 g body weight ) and 10% bioketan ( vetoquinol biowet , gorzw , poland ; 10 l/100 g body weight ) . once sedated aunps - n and aunps - bbn animals were injected with 200 l of the aunps solution into the tail vein . the aunps solution was prepared as follows : forty milligrams of 1.9 nm aurovist gold nanoparticles ( nanoprobes , yaphank , ny , usa ) were diluted in 400 l of physiologic solution according to manufacturer recommended protocol to obtain the final concentration of approximately 1.4 g au / kg body mass . the animals from the control group were injected with 200 l of physiologic solution into the tail vein . the urinary bladders were exposed with abdominal incision and fixed with 4% paraformaldehyde in phosphate saline buffer without potassium ( pbs - k ) for 3 minutes , applied from the serosa side in situ . next bladders were removed from the animals and dissected into multiple pieces in the same fixative . samples from each of the bladders were further processed for light , transmission ( tem ) , and scanning ( sem ) electron microscopy . urine was collected ; a 5 l drop of urine was put on gold electron microscopy grids , dried and either directly observed or processed for aunps intensification . hq silver enhancement ( nanoprobes ) and gold enhanced ( nanoprobes ) were used to enlarge gold nanoparticles . both silver and gold enhanced solutions were prepared and used according to manufacturer recommended protocols with enhancement times of 4 and 5 minutes , respectively . the urinary bladder samples were fixed in 4% paraformaldehyde in pbs ( k ) at 4c overnight , dehydrated and embedded in paraffin . paraffin blocks were cut into serial sections and counterstained with hematoxylin and eosin and examined by a uropathologist . histological urothelial changes of aunps - bbn animals were classified according to world health organization ( who ) classification of tumors of the urinary tract.24 the urinary bladder samples were fixed in 4% paraformaldehyde in pbs ( k ) at room temperature for a total of 45 minutes . after fixation , the samples were washed in pbs ( k ) , then in deionized water , and finally in the citrate buffer . the samples were then incubated in the hq silver enhancement solution ( nanoprobes ) and prepared according to manufacturer protocol in the dark for 5.5 minutes . after washing in deionized water , the samples were post - fixed in 0.2% oso4 for 75 minutes , washed in 0.1 m cacodylate buffer , dehydrated in graded ethanol , and embedded in epon . semi - thin sections ( thickness 1 m ) , were stained with 1% toluidine and examined with an eclipse te300 inverted microscope ( nikon , tokyo , japan ) . from the regions of interest , the ultrathin sections ( 50 nm ) were cut and counterstained with lead citrate and uranyl acetate . next , they were examined with a cm100 tem ( philips ) running at 80 kv the urinary bladder samples from aunps - bbn animals were also prepared using the modified tokuyasu method.25 briefly , urothelium , cut into < 1 mm pieces , was fixed in 4% paraformaldehyde in pbs ( k ) at room temperature for 2 hours , washed in pbs ( k ) , embedded into 12% gelatine , and cryoprotected by incubation in 2.3 m saharose at 4c overnight . subsequently , 300 nm thick cryosections were cut with fcs cryo - ultramicrotome ( leica microsystems , wetzlar , germany ) at 100c and collected on glass cover slips . nanoparticles in cryo - sections were silver enhanced with the hq silver enhancement solution ( nanoprobes ) in the dark for 6 minutes , washed in deionized water , and counterstained with toluidine and examined with an eclipse te300 inverted microscope ( nikon ) . from the regions of interest the cryo - ultrathin sections ( 50 nm ) were cut and counterstained with uranil acetate - methyl cellulose mixture . energy dispersive x - ray spectroscopy ( edxs ) analysis was performed using a transmission electron microscope 2100 ( jeol , tokyo , japan ) equipped with an edx system ex-23003bu ( jeol ) . the urinary bladder samples were fixed in 4% paraformaldehyde plus 2% glutaraldehyde in 0.1 m cacodylate buffer at 4c for 3 hours , washed overnight in cacodylate buffer , dehydrated in a series of ethanol , and critical - point dried ( cpd030 baltec , balzers , liechtenstein ) . after sputter - coating with gold ( balzers union scd040 , balzers , liechtenstein ) , samples were examined with a jsm840a scanning electron microscope ( jeol ) at 15 kv . the cultures of normal porcine urothelial cells ( 5th passage ) were established on 12-well culture inserts with 0.4 m porous membranes ( bd falcon , san jose , ca , usa ) and maintained in urom media as described previously.26,27 when cultures reached confluence , they were divided into two groups by simple random sampling : treated cells and control cells . aurovist aunps ( nanoprobes ) , in the same concentration as were injected into experimental animals , were added to the urom media of treated cells . after 90 minutes , cells were detached by using triple select ( gibco , life technologies , carlsbad , ca , usa ) , centrifuged , and re - suspended in fresh urom media . the survival of cells treated with aunps is presented as the percentage of the trypan blue - labeled cells versus non - labeled cells ; average , standard deviation ( sd ) , and student s t - test were performed with excel 2010 software ( microsoft corporation , redmond , wa , usa ) . all animals survived the injection of aunps or physiologic solution into the tail vein and fully recovered after anesthesia . aunps were filtered through the kidneys and reached the urinary bladder after 90 minutes , which was evaluated both macro- and microscopically ( figure 1 ) . macroscopically , lumen of the urinary bladders turned black in the aunps - n ( figure 1a and b ) and aunps - bbn animals , while in the control animals they remained translucent . the presence of aunps was confirmed in the urine ( figure 1c ) by tem ( figure 1d ) . the concentration of aunps in the urine was high ( figure 1d ) , so that it was not a limiting factor for detection . to make aunps easily detected by tem , we tested if they were suitable for silver or gold enhancement . the results showed that the gold core of nanoparticles can be enlarged by both methods ( figure 1e and f ) . gold enhancement provided a more uniform size of enlarged nanoparticles than silver enhancement , but on the other hand , many particles remained unenhanced . therefore , silver enhancement was used in further experiments . under light microscopy ( lm ) , the reaction product of silver enhancement was brown . to prove that the silver enhancement reaction specifically enlarges aunps in the tissue , results have confirmed that nanoparticles seen as accumulation of electron - dense material in tem contained elementary gold ( figure 1 g ) . analysis of a large number of the individual nanoparticles always showed co - localization of ag ( silver ) and au , whereas in areas of the samples without the nanoparticles , au could not be detected . to verify the low toxicity of aunps , they were added to the medium of urothelial cell culture at the same concentration as the aunps - n and aunps - bbn in experimental animals . after 90 minutes , the viability of control cells was 90.3% 12% , while the treated cells had a viability of 78.2% 5% ; the difference was statistically significant , p < 0.001 . to test if aunps are internalized by the superficial urothelial cells , we analyzed urinary bladder samples under light and electron microscopes . under lm , the urothelia of control and aunps - n animals consisted of three cell layers : small basal , intermediate , and large superficial cells ( figure 2a ) . brown - labeled product of silver enhancement was not seen in any layer of the urothelial or in the blood vessels of the urinary bladder wall ( figure 2a ) . under electron microscopes , the superficial urothelial cells of control and aunps - n animals were observed as large , homogenous , and polygonal shaped ( figure 2b ) , and contained numerous fvs in their cytoplasm ( figure 2c ) . in the aunps - n animals , aunps were not detected in approximately three out of four superficial cells ( figure 2c ) . in one fourth of the superficial urothelial cells , aunps were observed in membrane compartments with 3001200 nm diameter , which were presumably endosomes ( figure 2d and e ) . epithelial intracellular spaces , lamina propria , blood vessels , and bladder muscles were also unlabeled ( figure 2f ) . occasionally , the regions of exfoliated urothelium were observed in semi - thin sections of the normal urothelium of control and aunps - n animals . these regions were limited to one or a few superficial cells ( figure 3a ) . in the aunps - n animals , regions of exfoliated urothelium were surrounded by apically labeled cells ( figure 3a and b ) . labeling was seen only in the superficial cell layer and was present in an all - or - nothing manner ; eg , by observing two cells , one contained a relatively constant amount of brown labeling while the neighboring superficial cell contained no labeling at all ( figure 3b ) . sem analysis of the apical urothelial surface revealed cells of different sizes ; relatively small cells next to exfoliated regions and large polygonal cells further away from these areas ( figure 3c ) . under tem , labeled cells showed fvs , which are characteristic of highly differentiated urothelial cells ( figure 3d and e ) . these cells were heavily loaded with aunps with the majority of them found in the cytosol ( figure 3d and e ) . the sharp boundary between labeled and non - labeled cells was seen ( figure 3e ) . in order to follow the internalization of aunps into the neoplastic urothelium after 10 weeks of bbn administration , the urothelium of aunps - bbn animals developed flat hyperplasia with moderate dysplasia . in these regions , the brown labeling was observed in the majority of superficial cells , in the significant portion of intermediate cells , and in some basal cells ( figure 4a ) . relatively small superficial cells of dysplastic urothelium were covered with microvilli and ropy ridges ( figure 4b ) and their apical cytoplasm contained no fvs ( figure 4c ) . the labeling was seen in the vesicular compartments , presumably endosomes , as well as in the cytosol ( figure 4d ) . all animals survived the injection of aunps or physiologic solution into the tail vein and fully recovered after anesthesia . aunps were filtered through the kidneys and reached the urinary bladder after 90 minutes , which was evaluated both macro- and microscopically ( figure 1 ) . macroscopically , lumen of the urinary bladders turned black in the aunps - n ( figure 1a and b ) and aunps - bbn animals , while in the control animals they remained translucent . the presence of aunps was confirmed in the urine ( figure 1c ) by tem ( figure 1d ) . the concentration of aunps in the urine was high ( figure 1d ) , so that it was not a limiting factor for detection . to make aunps easily detected by tem , we tested if they were suitable for silver or gold enhancement . the results showed that the gold core of nanoparticles can be enlarged by both methods ( figure 1e and f ) . gold enhancement provided a more uniform size of enlarged nanoparticles than silver enhancement , but on the other hand , many particles remained unenhanced . therefore , silver enhancement was used in further experiments . under light microscopy ( lm ) , the reaction product of silver enhancement was brown . to prove that the silver enhancement reaction specifically enlarges aunps in the tissue , results have confirmed that nanoparticles seen as accumulation of electron - dense material in tem contained elementary gold ( figure 1 g ) . analysis of a large number of the individual nanoparticles always showed co - localization of ag ( silver ) and au , whereas in areas of the samples without the nanoparticles , au could not be detected . to verify the low toxicity of aunps , they were added to the medium of urothelial cell culture at the same concentration as the aunps - n and aunps - bbn in experimental animals . after 90 minutes , the viability of control cells was 90.3% 12% , while the treated cells had a viability of 78.2% 5% ; the difference was statistically significant , p < 0.001 . to test if aunps are internalized by the superficial urothelial cells , we analyzed urinary bladder samples under light and electron microscopes . under lm , the urothelia of control and aunps - n animals consisted of three cell layers : small basal , intermediate , and large superficial cells ( figure 2a ) . brown - labeled product of silver enhancement was not seen in any layer of the urothelial or in the blood vessels of the urinary bladder wall ( figure 2a ) . under electron microscopes , the superficial urothelial cells of control and aunps - n animals were observed as large , homogenous , and polygonal shaped ( figure 2b ) , and contained numerous fvs in their cytoplasm ( figure 2c ) . in the aunps - n animals , aunps were not detected in approximately three out of four superficial cells ( figure 2c ) . in one fourth of the superficial urothelial cells , aunps were observed in membrane compartments with 3001200 nm diameter , which were presumably endosomes ( figure 2d and e ) . epithelial intracellular spaces , lamina propria , blood vessels , and bladder muscles were also unlabeled ( figure 2f ) . occasionally , the regions of exfoliated urothelium were observed in semi - thin sections of the normal urothelium of control and aunps - n animals . these regions were limited to one or a few superficial cells ( figure 3a ) . in the aunps - n animals , regions of exfoliated urothelium were surrounded by apically labeled cells ( figure 3a and b ) . labeling was seen only in the superficial cell layer and was present in an all - or - nothing manner ; eg , by observing two cells , one contained a relatively constant amount of brown labeling while the neighboring superficial cell contained no labeling at all ( figure 3b ) . sem analysis of the apical urothelial surface revealed cells of different sizes ; relatively small cells next to exfoliated regions and large polygonal cells further away from these areas ( figure 3c ) . under tem , labeled cells showed fvs , which are characteristic of highly differentiated urothelial cells ( figure 3d and e ) . these cells were heavily loaded with aunps with the majority of them found in the cytosol ( figure 3d and e ) . the sharp boundary between labeled and non - labeled cells was seen ( figure 3e ) . in order to follow the internalization of aunps into the neoplastic urothelium , we induced urothelial carcinogenesis with 0.05% bbn in drinking water . after 10 weeks of bbn administration , the urothelium of aunps - bbn animals developed flat hyperplasia with moderate dysplasia . in these regions , the brown labeling was observed in the majority of superficial cells , in the significant portion of intermediate cells , and in some basal cells ( figure 4a ) . relatively small superficial cells of dysplastic urothelium were covered with microvilli and ropy ridges ( figure 4b ) and their apical cytoplasm contained no fvs ( figure 4c ) . the labeling was seen in the vesicular compartments , presumably endosomes , as well as in the cytosol ( figure 4d ) . great care should be devoted to the choice of endocytotic markers and the method of their application in order to study endocytosis in vivo under physiological conditions . we have selected aunps that fulfill three requests : ( 1 ) biocompatibility , ( 2 ) size , and ( 3 ) ability to detect . aunps have a relatively long history in biological applications and have been confirmed biocompatible by various in vitro and in vivo experiments.23,2832 in our study , the viability of cultured cells exposed to the highest recommended concentration of aunps was lower than of control cultured cells ( 78.2% 5% versus 90.3% 12% ) ; however , the survival rate was still acceptable and well above ld50 ( median lethal dose ) . moreover , all animals survived the tail vein injection of aunps and fully recovered after 90 minutes of anesthesia . . size of nanoparticles . based on the standard definition of the american society for testing and materials , nanoparticles are particles with lengths that range from 1 to 100 nm in two or three dimensions . the aunps we used , with their gold core 1.9 nm in diameter , are on the smaller side of the nanoparticle definition scale that is a prerequisite for substances to pass the macromolecular filter in the kidney . in our study , intravenously injected aunps were detected in the urine , which suggests that aunps are filtered in the kidneys and become constituents of the urine under physiological conditions . colloidal gold with diameters above 5 nm have sufficient electron density to be visualized in ultrathin sections of biological samples by tem . aunps used in our study can be seen under tem when directly applied to a tem grid , but are too small to be easily recognized in the ultrathin sections of the tissues . however , after silver or gold enhancement,3336 nanoparticles are clearly seen . we confirmed specificity of the enhancement reaction by tem and edxs analysis and showed that silver enhancement enlarges a higher percentage of nanoparticles than gold enhancement . with the three basic requests fulfilled , the aunps can be used as a potential marker to follow internalization into urothelial cells from the luminal side of the urinary bladder under physiological conditions in vivo . since there are contradicting results regarding endocytosis in urothelial cells,3,19,37,38 we used the aunps to follow internalization into superficial urothelial cells of normal and neoplastic urothelium . in the terminally differentiated superficial urothelial cells of normal urothelium , internalization of the aunps was minimal , which is in agreement with our previous study on urothelial cells in vitro.38 despite the high concentration of nanoparticles in the bladder lumen , only one quarter of cells contained a few labeled endosomes , which shows extremely low dynamics of endocytosis compared to other cell types.38 nanoparticles were not found in the fvs , demonstrating that fvs are not involved in the removal of urothelial plaques from the apical plasma membrane . limited internalization of nanoparticles into terminally differentiated superficial urothelial cells hence supports the idea that internalization of urine and its constituents is minimized , which contributes to the barrier function of the urothelium.4 in the normal urothelium , terminally differentiated superficial cells have an extremely long life time;39,40 however , they are occasionally removed by exfoliation and the exfoliated areas are rapidly resealed.4144 in our study , we observed regions of exfoliated urothelium in control and in aunps - n animals , and in either case exfoliated regions were surrounded by differentiated , but smaller urothelial cells . in aunps - n animals , these cells contained nanoparticles indicating that what is observed are regenerating cells , which have not yet established a fully functional permeability barrier . to further explore the functionality of the permeability barrier during alternative differentiation , we applied aunps to aunps - bbn animals . bbn is widely used in the urothelial carcinogenesis model systems and prevents urothelial cells reaching their terminal differentiation.45,46 a consequence of low cellular differentiation is leaky epithelium,38,47 which was also observed in our study . the increased internalization into superficial cells of dysplastic urothelium in comparison to normal superficial cells reveals the potential that nanoparticles could be used in improved diagnostic and optimized treatment of urinary bladder cancer.31,4851 however , the increased nanoparticle internalization is not an explicit property of the partially differentiated cells found in the neoplastically transformed urothelium , but is also seen in normal urothelial cells located next to the exfoliated regions of the urothelium . therefore , the aunps above can all be used to follow endocytosis as well as be used as a sensitive marker of differentiation and functionality in urothelial cells . our experiments were intended to determine the physiological conditions for studying internalization in superficial urothelial cells in vivo . we provide evidence that the aunps we used are an excellent choice for the given studies , both on normal and in neoplastic urinary bladders . our results support the hypothesis that endocytosis is very limited in terminally differentiated urothelial cells , while the internalization of urine constituents increases significantly when the urothelial cells are at a lower differentiation stage . the conception of the latter provides solid ground for the developing novel , more effective bladder cancer diagnostic and treatment tools using nanoparticles .

backgroundurothelial bladder is the reservoir of urine and the urothelium minimizes the exchange of urine constituents with this tissue . our aim was to test 1.9 nm biocompatible gold nanoparticles as a novel marker of internalization into the urothelial cells under physiological conditions in vivo.methodswe compared normal and neoplastic mice urothelium . neoplastic lesions were induced by 0.05% n - butyl - n-(4-hydroxybutyl)nitrosamine ( bbn ) in drinking water for 10 weeks . nanoparticles , intravenously injected into normal and bbn - treated mice , were filtered through the kidneys and became constituents of the urine within 90 minutes after injection.resultsgold nanoparticles were densely accumulated in the urine , while their internalization into urothelial cells depended on the cell differentiation stage . in the terminally differentiated superficial urothelial cells of normal animals , nanoparticles were occasionally found in the endosomes , but not in the fusiform vesicles . regions of exfoliated cells were occasionally found in the normal urothelium . superficial urothelial cells located next to exfoliated regions contained gold nanoparticles in the endosomes and in the cytosol beneath the apical plasma membrane . the urothelium of bbn - treated animals developed fat hyperplasia with moderate dysplasia . the superficial cells of bbn - treated animals were partially differentiated as demonstrated by the lack of fusiform vesicles . these cells contained the gold nanoparticles distributed in the endosomes and throughout their cytosol.conclusiongold nanoparticles are a valuable marker to study urine internalization into urothelial cells in vivo . moreover , they can be used as a sensitive marker of differentiation and functionality of urothelial cells .

Introduction Material and methods Animals and treatment Urine Histology Ultrastructure and internalization of AuNPs Apical surface Cell viability Results Gold nanoparticles become urine constituents and accumulate in the urinary bladder Gold nanoparticles are not toxic for urothelial cells Internalization of gold nanoparticles is minimal in terminally differentiated urothelial cells Gold nanoparticles penetrate urothelial cells bordering exfoliated regions Internalization of gold nanoparticles is increased during urothelial carcinogenesis Discussion Conclusion

PMC3096766

arthroscopic debridement and bone marrow stimulation is considered the primary treatment of osteochondral talar defect ( ocd ) and yields 85% success . in case of failure of the primary treatment , current secondary treatment options include repeat arthroscopic debridement and bone marrow stimulation , osteochondral autograft transfer , autogenous cancellous bone graft , and autologous chondrocyte implantation [ 4 , 8 , 9 , 20 , 23 ] . however , these techniques are sometimes associated with donor - site morbidity or involve two - stage surgery [ 2 , 3 , 13 , 14 ] . in order to treat ocds of the medial talar dome after failed primary treatment , a contoured articular inlay implant ( hemicap , arthrosurface inc . , franklin , ma , usa ) with a diameter of 15 mm was developed . fifteen articular component offset sizes are available , based on the surface anatomy of the medial talar dome . in a cadaveric study , the offset sizes were found appropriate for a variety of talar specimens . furthermore , it was shown that a slightly recessed implantation level prevents high contact pressures . clinical goals of the implant are to offer relief of pain and swelling , return to activity , and prevent ( further ) cyst formation . a two - year result of the first patient who was treated with the novel implant is reported . the last procedure was 1 year and 2 months before her presentation . at presentation , the patient s predominant symptom was pain deep in the ankle joint during and after activity . she used to play korfball at competitive level but had stopped due to the pain . korfball is a mixed gender team ball game , similar to mixed netball and basketball , and is very popular in the netherlands . at physical examination , leg alignment was neutral , the range of motion was normal and symmetric , and both ankle joints were stable . radiographs of the right ankle revealed a large cystic osteochondral defect of the medial talar dome ( fig . 1 ) . a computed tomography scan confirmed the osteochondral defect , measuring 17 8 8 mm , accompanied by some small subchondral cysts and a loose fragment . 1preoperative anteroposterior mortise view and lateral radiographs of the affected ankle showing a radiolucent osteochondral defect in the medial talar dome ( arrow ) preoperative anteroposterior mortise view and lateral radiographs of the affected ankle showing a radiolucent osteochondral defect in the medial talar dome ( arrow ) a curved skin incision was made over the medial malleolus . after two screw holes were predrilled in the medial malleolus , a medial malleolar osteotomy was made at an angle of 30 degrees relative to the long tibial axis , exposing the osteochondral defect ( fig . a the talar lesion was exposed through an oblique medial malleolar osteotomy , and the necrotic fragment was excised ( arrow ) . b after the insertion of a screw and the determination of the appropriate offset sizes , a trial articular component ( arrow ) was placed . c the final articular component ( a ) was orientated in the correct plane and placed on the screw ( s ) . d final view of the talus after engagement of the articular component ( arrow ) . note that the edges of the implant are slightly recessed compared to the adjacent cartilage level intraoperative pictures of the operative technique . a the talar lesion was exposed through an oblique medial malleolar osteotomy , and the necrotic fragment was excised ( arrow ) . b after the insertion of a screw and the determination of the appropriate offset sizes , a trial articular component ( arrow ) was placed . c the final articular component ( a ) was orientated in the correct plane and placed on the screw ( s ) . d final view of the talus after engagement of the articular component ( arrow ) . note that the edges of the implant are slightly recessed compared to the adjacent cartilage level the defect was debrided . utilizing a drill guide , a guide pin was placed into the center of the defect , perpendicular to the curvature of the medial talar dome . a contact probe was used to determine the radius of curvature ; an offset size of 0.5 3.5 mm was measured . the osteotomy was fixated with two 3.5-mm lag screws , and the wound was closed . the postoperative management was initiated with a plaster cast for 1 week and continued with a functional brace ( walker , ssur , son en breugel , the netherlands ) for 5 weeks . after this 6-week period , radiographs of the operated ankle confirmed consolidation of the malleolar osteotomy . initially , there was a small area of numbness about the scar but this had resolved at 3 months follow - up . the patient was able to play korfball at the preinjury level after 1 year and continued to play at this level at 2 years follow - up . the sagittal range of motion arc remained slightly reduced : 15 of dorsiflexion to 45 of plantar flexion compared to 20 of dorsiflexion to 45 of plantar flexion in the left ankle . using numeric rating scales , the patient preoperatively rated the pain as 0 when at rest , 8 when walking , and 10 when running . these numbers improved to 0 , 0 , and 1 at final follow - up , respectively . the foot and ankle outcome score improved from preoperatively to final follow - up on four of five subscales ; the subscale other symptoms decreased due to subjective inability to fully straighten and bend the ankle . the american orthopaedic foot and ankle society ankle - hindfoot score improved from 74 preoperatively to 90 at 1 and 2 years . the implant remained in position , and there were no signs of osteolysis or progressive degenerative changes in the ankle joint ( fig . there are no implant - related complications or progressive degenerative changes of the ankle joint when compared to preoperatively radiographs after 2 years follow - up . there are no implant - related complications or progressive degenerative changes of the ankle joint when compared to preoperatively after two screw holes were predrilled in the medial malleolus , a medial malleolar osteotomy was made at an angle of 30 degrees relative to the long tibial axis , exposing the osteochondral defect ( fig . a the talar lesion was exposed through an oblique medial malleolar osteotomy , and the necrotic fragment was excised ( arrow ) . b after the insertion of a screw and the determination of the appropriate offset sizes , a trial articular component ( arrow ) was placed . c the final articular component ( a ) was orientated in the correct plane and placed on the screw ( s ) . d final view of the talus after engagement of the articular component ( arrow ) . note that the edges of the implant are slightly recessed compared to the adjacent cartilage level intraoperative pictures of the operative technique . a the talar lesion was exposed through an oblique medial malleolar osteotomy , and the necrotic fragment was excised ( arrow ) . b after the insertion of a screw and the determination of the appropriate offset sizes , a trial articular component ( arrow ) was placed . c the final articular component ( a ) was orientated in the correct plane and placed on the screw ( s ) . d final view of the talus after engagement of the articular component ( arrow ) . note that the edges of the implant are slightly recessed compared to the adjacent cartilage level the defect was debrided . utilizing a drill guide , a guide pin was placed into the center of the defect , perpendicular to the curvature of the medial talar dome . a contact probe was used to determine the radius of curvature ; an offset size of 0.5 3.5 mm was measured . the osteotomy was fixated with two 3.5-mm lag screws , and the wound was closed . the postoperative management was initiated with a plaster cast for 1 week and continued with a functional brace ( walker , ssur , son en breugel , the netherlands ) for 5 weeks . after this 6-week period , radiographs of the operated ankle confirmed consolidation of the malleolar osteotomy . initially , there was a small area of numbness about the scar but this had resolved at 3 months follow - up . the patient was able to play korfball at the preinjury level after 1 year and continued to play at this level at 2 years follow - up . the sagittal range of motion arc remained slightly reduced : 15 of dorsiflexion to 45 of plantar flexion compared to 20 of dorsiflexion to 45 of plantar flexion in the left ankle . using numeric rating scales , the patient preoperatively rated the pain as 0 when at rest , 8 when walking , and 10 when running . these numbers improved to 0 , 0 , and 1 at final follow - up , respectively . the foot and ankle outcome score improved from preoperatively to final follow - up on four of five subscales ; the subscale other symptoms decreased due to subjective inability to fully straighten and bend the ankle . the american orthopaedic foot and ankle society ankle - hindfoot score improved from 74 preoperatively to 90 at 1 and 2 years . the implant remained in position , and there were no signs of osteolysis or progressive degenerative changes in the ankle joint ( fig . there are no implant - related complications or progressive degenerative changes of the ankle joint when compared to preoperatively radiographs after 2 years follow - up . there are no implant - related complications or progressive degenerative changes of the ankle joint when compared to preoperatively this prospective case report is the first clinical report of a metal implant for ocds of the talus and gives an insight into the surgical technique and clinical follow - up . treatment of osteochondral lesions or osteonecrosis by means of metal resurfacing implants is relatively new . during the past 3 years , promising clinical results were reported for the treatment of the femoral and humeral head , as well as the first metatarsal and patellar surface . two biomechanical cadaveric studies provided foundations for use of the talus implant in the ankle joint [ 1 , 22 ] . the ideal indications for treatment with this implant are not yet known . the authors consider its use in patients with a large ocd on the medial talar dome who have clinically significant pain more than 1 year after primary surgical treatment . the following are regarded as contraindications : age < 18 years , ocd size > 20 mm , ankle osteoarthritis grade ii or iii , concomitant ankle pathology , advanced osteoporosis , infection , diabetes , or a known allergy to implant material . the surgical approach is an important part of the implantation technique because the accuracy of implantation of this device strongly depends on the approach and quality of vision . for adequate exposure the osteotomy is ideally directed toward the intersection between the tibial plafond and medial malleolus at an angle of 30 relative to the long tibial axis . although there are other osteotomy options , including anterior tibial , step - cut , and inverted v or u osteotomies , the oblique osteotomy is our preferred technique because it is relatively simple , exposure of the talus is excellent , and congruent reduction is well possible [ 12 , 17 , 21 ] . the surface of the prosthetic device should be placed slightly recessed relative to the surrounding surface of the talar cartilage because talar cartilage deforms during weight bearing while the implant does not . wan et al . measured a peak cartilage deformation of 34.5 7.3% under full body weight in persons with a medial talar dome cartilage thickness of 1.42 0.31 mm . we therefore aim at an implantation level of 0.5 mm below the adjacent cartilage . this implantation level was found appropriate in a previous cadaveric study . when the prosthetic device is correctly implanted , excessive contact pressures of the implant are avoided . while there are various offset sizes , each articular component has a diameter of 15 mm . this is based on the finding that primary arthroscopic treatment is generally successful for lesions up to 15 mm , while this treatment is less successful for larger lesions . the part of the ocd that was not covered by the implant is expected to be filled by fibrocartilaginous tissue . alternative current treatment methods for this patient are osteochondral autograft transfer system ( oats ) , cancellous bone grafting , an allograft , ankle arthrodesis or prosthesis . oats and cancellous bone grafting carry the risk of donor - site pain and are available in limited amounts [ 19 , 2 ] . ankle arthrodesis or prosthesis is definite solutions for a recurrent ocd but are rather not used in young patients . recently reported other treatment options are matrix - induced autologous chondrocyte implantation ( maci ) and engineered osteochondral grafts [ 5 , 16 ] . the metallic implantation technique appears to be a promising treatment for osteochondral defects of the medial talar dome after failed primary treatment . although the clinical and radiological results of this prospective case report with 2 years follow - up are promising , more patients and longer follow - up are needed to draw any firm conclusions and determine whether the results continue with time .

the primary treatment of most osteochondral defects of the talus is arthroscopic debridement and bone marrow stimulation . there is no optimal treatment for large lesions or for those in which primary treatment has failed . we report a 20-year - old female patient with persistent symptoms after two previous arthroscopic procedures . computed tomography showed a cystic defect of the medial talar dome , sized 17 8 8 mm . the patient was treated with a novel contoured metal implant . at 1 and 2 years after surgery , the patient reported considerable reduction in pain and had resumed playing korfball at competitive level.level of evidence iv .

Introduction Case report Operative technique Outcome Discussion Conclusion

PMC2626940

a sequence of nh2-aaaagaaacp - cooh was used as the switch - tag ( st ) ( munoz and serrano 1994 ) . we have previously observed that c - terminal cysteines , which have thiol and carboxylic acid groups in close proximity , can be self - proteolytic and thus added a protecting c - terminal proline residue which does not hydrogen bond to the helix and , in in silico modeling , orientates the sh group of the cysteine towards the target binding surface ( figure 1a ) . a synthetic st with additional n - terminal residues ( kydd ) to aid solubility and concentration measurements showed a coil to helix transition in increasing concentrations of the hydrophobic solvent tri - fluoroethanol ( tfe ) ( figure 1b ) . specific binding of the peptide to gold from aqueous solution was assessed by spr ( biacore - x ) with bare gold ( au - chip ) surfaces and a synthetic switch tag peptide incorporating the flag antibody - binding motif ( dykddddkgg ) at its n - terminus ( flag - st ) . to reduce nonspecific adhesion specific binding was determined by reference to peptides in which the cys sh group was blocked by iodoacetamide . this showed that 75% of surface mass is specifically bound via the cys after washing . after infilling the remaining surface by injection of peg - thiol solution , to inhibit nonspecific binding ( nsb ) ( seigel et al 1997 ) , anti - flag antibody was injected . following a wash with 0.1% sodium dodecyl sulfate ( sds ) solution to remove nonspecifically bound antibody , 145 resonance units ( r.u . = 0.0001 degree shift in spr minimum position ) remained bound to the iodoacetamide - blocked peptide - treated surface , whereas 1250 r.u . were bound to the nonblocked peptide surface , clearly demonstrating the thiol - directed assembly of flag - peptides ( figure 2a ) . the background is reduced still further if a longer ex situ assembly of peg - thiol is performed ( data not shown ) . the st sequence was genetically engineered onto the c - terminus of the bacterial protein tolaiii , fusions to which are known to be well expressed and readily purified from escherichia coli . the two naturally occurring cysteine residues in the protein were mutated to serine , leaving the st peptide cysteine as the only thiol group . the complete plasmid allows for insertion of proteins between the tolaiii and st modules and subsequent proteolytic cleavage to remove the stabilizing tola fusion partner . this was achieved with a multiple cloning site ( mcs ) to allow the simple insertion of any chosen protein sequence between the tola - iii and st coding regions . a thrombin protease cut site was also included between the tola - iii and mcs sequences to allow the tola - iii protein to be cleaved from the tagged fusion protein and a flag epitope inserted in tolaiii . the tolaiii- flag - st fusion protein was expressed in e. coli bl-21 cells and purified by ni affinity chromatography as described ( anderluh et al 2003 ) . far uv - cd analysis showed that it was soluble and correctly folded and spr revealed that 510 r.u the surfaces were briefly in - filled in situ with peg - thiol , and probed with anti - flag antibody , as described previously ; 1580 r.u . of antibody remained bound after washing to the normal surface whereas only 265 r.u . a longer peg - thiol incubation would decrease this still further since errors in the sam are sites for nonspecific binding . next , green fluorescent protein ( gfp ) was used as a model for an inserted protein domain as it is easily visualized on surfaces . the coding dna sequence of gfp was cloned into the plasmid and protein expressed in bl-21 e. coli . microcontact printing ( xia and whitesides 1998 ) was used to pattern arrays of 5-m diameter spots of a tolaiii - gfp - switch - tag fusion protein later in filled with 1 mm peg - thiol solution in 100% ethanol overnight to assemble a peg - terminated sam both in and around the spots . the protein was easily visualized by fluorescence microscopy and thus remains folded ( figure 3a ) . rat primary osteoblast cells incubated on the surface adhered to the islands of protein but were unable to bind to the surrounding peg - thiol coated surface , as shown in figure 3b . the cells are much larger than the pattern and adjust their contact points to avoid the peg in - filled areas . gfp has no known specific cell adhesion properties but provides clear anchor points within the peg - layer . this illustrates that the st is able to specifically immobilize recombinant proteins as defined patterns within an otherwise homogeneous sam . the stamped surface was backfilled with peg - thiol and incubated with primary osteoblast cells which avoided the protein - free region ( figure 3c ) . the ability of phospholipid layers to limit nonspecific binding has been exploited in combination with patterned arrays of soluble proteins ( kung et al 2000 ) , whilst other approaches have combined the peptide with the lipid itself ( svedhem et al 2003 ) or printed proteins onto a reactive sam ( rozkiewicz et al 2006 ) . incorporation of functional synthetic peptides into sam is straightforward and patterning has been achieved by micro - contact printing ethylene glycol - terminated sam in patterns leaving exposed gold for subsequent peptide assembly ( zhang et al 1999 ) . the system described here provides a generic method to co - assemble proteins with classical sam . we have designed a plasmid which attaches a short peptide sequence which can insert into an 11 carbon chain thick sam ( figure 3d ) . it was previously shown that hydrophobic helical peptides can be assembled into dense sam and retain a helical structure ( kitagawa et al 2004 ) . the current system avoided altering the protein s hydrophobicity by allowing the peptide to be random coil in solution and helical in the sam . although the behavior of the peptide and modified proteins confirmed the first property of water solubility , it was unfortunate that the relatively low immobilization density prevented ftir confirmation of helix formation in the sam ( kitagawa et al 2004 ) . nevertheless the tolaiii - st expression system is able to produce engineered proteins that show specific adhesion without compromising water solubility or expression levels in e. coli . we have previously shown that by pre - treating the surface with -mercapto - ethanol surface interactions are reduced and proteins keep their 3d structure ( terrettazet al 2002 ; keegan et al 2005 ) the subsequent assembly with lipids stabilizes the array and further contributes to retaining protein structure ( terrettaz et al 2002 ; holt et al 2005 ; cisneros et al 2006 ) . this expression system has the potential to increase the range and complexity of proteins that can be specifically immobilized on surfaces designed to modify cellular growth ( shah et al 2007 ) or the development of new detector elements for spr , saw , or qcm . the st sequence was developed using agadir ( munoz and serrano 1994 ) ( http://www.embl-heidelberg.de/services/serrano/agadir/agadir-start.html ) . the helix - disrupting glycine residue was added to a poly - alanine sequence at different positions and the resulting helix propensities calculated . a sequence with a central glycine retained sufficient helical propensity that an increase in environmental hydrophobicity would cause this sequence to rearrange from random coil to -helix . molecules based on this sequence , with the addition of a c - terminal cysteine and proline residues and n - terminal sequences of either kydd to aid solubility and concentration measurements or dykddddkgg , a flag epitope , were synthesised by f - moc chemistry and purified to > 95% as assessed by high - pressure liquid chromatography and maldi - tof mass spectrometry ( invitrogen , evotrack , paisley , uk ) . helical structure and thio alkane monolayer were modeled and energy minimized using the quanta v4.0 package ( accelrys , orsay cedex , france ) . solutions of 0.2 mg / ml peptide in 50 mm nah2po4 , ph 7.0 were made with increasing concentrations of the hydrophobic solvent tfe ( sigma - aldrich , gillingham , uk ) up to 80% tfe , the limit of peptide solubility . all measurements were made with 0.2 mm path length quartz suprasil cuvettes ( hellma uk , southend on sea , uk ) using a jasco j-810 spectropolarimeter , over a wavelength range of 250195 nm at a scan speed of 20 nm min . the mean spectrum derived from 10 scans was corrected by subtraction of a background buffer blank . surface plasmon resonance measurements of sam formation used a biacore x system with au gold sensor chips . the gold surface was cleaned with piranha solution ( kitagawa et al 2004 ) for 30 minutes , rinsed with h2o , 1% sds and finally extensively with h2o before drying with air and immediate docking into the biacore system . a buffer of 200 mm nacl , 0.5 mm edta , and 20 mm tris ph 8.0 was used for all solutions and also as the running buffer . peptide and protein solutions at a concentration of 0.1 mg / ml were used in all experiments . the peg - thiol ( 11-mercapto-1-triethyleneglycol - undecane , hsc11-eg3 ) ( prochimia surfaces , sopot , poland ) was used at a concentration of 0.5 mm . the solutions used for thiol binding steps were supplemented with 0.1 mm tcep ( tris(2-carboxyethyl)phosphine hydrochloride ) for 1 h before use . anti - flag monoclonal antibodies were obtained from sigma - aldrich and used at a dilution concentration of 0.1 mg / ml prepared daily in running buffer . tolaiii - switch tag fusion protein expression constructs were based on the ptol - t vector ( anderluh et al 2003 ) . they were expressed in e. coli bl-21 cells and purified as detailed previously ( anderluh et al 2003 ) . micro - contact printing using pdms stamps was used to transfer patterns of st protein onto gold substrates ( xia and whitesides 1998 ) . the stamps were inked using the relevant alkanethiol or protein and dried in a stream of compressed air , placed on the gold and left for 15 seconds . the stamps were removed and the surface air dried for 10 minutes . if required the surface of the gold was then immersed in the relevant infill solution and left for a minimum of 4 hours to allow formation of a sam . primary osteoblastic cells were isolated from the calvariae of five - day old rats ( bokhari et al 2005 ) they were maintained in a culture medium of dulbecco s modified eagle s medium ( dmem , sigma aldrich ) supplemented with 1% penicillin / streptomycin solution , 1% l - glutamine solution , and 10% fetal calf serum . solutions of 0.2 mg / ml peptide in 50 mm nah2po4 , ph 7.0 were made with increasing concentrations of the hydrophobic solvent tfe ( sigma - aldrich , gillingham , uk ) up to 80% tfe , the limit of peptide solubility . all measurements were made with 0.2 mm path length quartz suprasil cuvettes ( hellma uk , southend on sea , uk ) using a jasco j-810 spectropolarimeter , over a wavelength range of 250195 nm at a scan speed of 20 nm min . the mean spectrum derived from 10 scans was corrected by subtraction of a background buffer blank . surface plasmon resonance measurements of sam formation used a biacore x system with au gold sensor chips . the gold surface was cleaned with piranha solution ( kitagawa et al 2004 ) for 30 minutes , rinsed with h2o , 1% sds and finally extensively with h2o before drying with air and immediate docking into the biacore system . a buffer of 200 mm nacl , 0.5 mm edta , and 20 mm tris ph 8.0 was used for all solutions and also as the running buffer . peptide and protein solutions at a concentration of 0.1 mg / ml were used in all experiments . the peg - thiol ( 11-mercapto-1-triethyleneglycol - undecane , hsc11-eg3 ) ( prochimia surfaces , sopot , poland ) was used at a concentration of 0.5 mm . the solutions used for thiol binding steps were supplemented with 0.1 mm tcep ( tris(2-carboxyethyl)phosphine hydrochloride ) for 1 h before use . anti - flag monoclonal antibodies were obtained from sigma - aldrich and used at a dilution concentration of 0.1 mg / ml prepared daily in running buffer . tolaiii - switch tag fusion protein expression constructs were based on the ptol - t vector ( anderluh et al 2003 ) . they were expressed in e. coli bl-21 cells and purified as detailed previously ( anderluh et al 2003 ) . micro - contact printing using pdms stamps was used to transfer patterns of st protein onto gold substrates ( xia and whitesides 1998 ) . the stamps were inked using the relevant alkanethiol or protein and dried in a stream of compressed air , placed on the gold and left for 15 seconds . if required the surface of the gold was then immersed in the relevant infill solution and left for a minimum of 4 hours to allow formation of a sam . primary osteoblastic cells were isolated from the calvariae of five - day old rats ( bokhari et al 2005 ) they were maintained in a culture medium of dulbecco s modified eagle s medium ( dmem , sigma aldrich ) supplemented with 1% penicillin / streptomycin solution , 1% l - glutamine solution , and 10% fetal calf serum .

surface biology aims to observe and control biological processes by combining bio- , surface , and physical chemistry . self - assembled monolayers ( sam ) on gold surfaces have provided excellent methods for nanoscale surface preparation for such studies . however , extension of this work requires the specific immobilization of whole protein domains and the direct incorporation of recombinant proteins into sam is still problematic . in this study a short random coil peptide has been designed to insert into thioalkane layers by formation of a hydrophobic helix . surface plasmon resonance ( spr ) studies show that specific immobilization via the internal cysteine is achieved . addition of the peptide sequence to the terminus of a protein at the genetic level enables the production of a range of recombinant fusion - proteins with good yield . spr shows that the proteins display the same gold - binding behavior as the peptide . it is shown that cell growth control can be achieved by printing the proteins using soft lithography with subsequent infilling with thioalkanes the expression plasmid is constructed so that any stable protein domain can be easily cloned , expressed , purified and immobilized .

Results and discussion Materials and methods Circular dichroism spectroscopy Surface plasmon resonance measurements Micro-contact printing Eukaryotic cell culture

PMC4662977

although diabetes is highly prevalent worldwide , its presence among american indians and alaska natives ( ai / ans ) is particularly alarming . adjusting for age , ai / ans suffer from type 2 diabetes mellitus at rates greater than two times those of non - hispanic whites and exhibit the highest prevalence of this disease of any racial group in the united states [ 1 , 2 ] . given the sharp increase in incident diabetes among ai / ans over the last 20 years , these circumstances seem unlikely to change without substantial intervention [ 35 ] . the special diabetes program for indians diabetes prevention ( sdpi - dp ) demonstration project has been implemented over the past decade to address this problem using a well - established , evidence - based preventive intervention . the sdpi - dp initiative was developed based upon the national institute of diabetes , digestive and kidney disease 's ( niddk ) diabetes prevention program ( dpp ) , which was a large - scale clinical trial that demonstrated that lifestyle interventions ( e.g. , changing diet and exercise habits ) can be effective in delaying or preventing the onset of diabetes in individuals who are at increased risk for developing this disease . the dpp outcomes did not differ significantly for various ethnic groups , including american indians ; however , the dpp was conducted as a highly controlled clinical trial , which did not allow for evaluating the effectiveness of lifestyle interventions in preventing the onset of diabetes in community - based settings with underserved populations . ai / an communities often face a lack of health care resources and a highly mobile population , thereby making it particularly difficult to implement large - scale prevention programs . therefore , the sdpi - dp worked with experts in a variety of ai / an communities to implement cultural adaptations to the original dpp lifestyle curriculum ( e.g. , the use of indigenous foods , drumming during class sessions ) , in order to make the program more relevant to ai / an individuals and more transferrable to a geographically , culturally , and organizationally diverse array of settings in tribal communities . the sdpi - dp demonstration program resulted in reduced diabetes incidence among high risk ai / ans at a rate comparable to the results for ai / ans in the original dpp study . in addition , improvements in weight , blood pressure , and lipid levels were detected following the intervention . however , despite the overall effectiveness with which the intervention was delivered to sdpi - dp participants , several participant characteristics were related to retention in the program ; participants who were younger , were male , had less education , and had lower income were more likely not to complete the core intervention . these initial findings regarding the relationship between sociodemographic factors and retention led program staff to question the potential additional impact of individual - level psychosocial factors on participant engagement , ability to grasp the knowledge conveyed , and mastery of skills related to the behavioral changes associated with the desired outcomes . therefore , it was determined that further analyses were warranted in order to evaluate the extent to which program outcomes were related to individual - level psychosocial characteristics . the observation of a potential impact of psychosocial factors on self - management of medical illnesses is not unique . for example , the influence of depression and anxiety on intervention outcomes for individuals with prediabetes was examined in at least one previous study , and more positive baseline mood was correlated with increased physical activity . specifically , there is evidence that diabetes may increase the likelihood of depressive episodes and that depression may increase the risk of developing diabetes [ 1013 ] . furthermore , psychological distress in general has also been shown to be associated with many chronic health conditions , including obesity [ 14 , 15 ] , which is a significant risk factor for the development of type 2 diabetes . other studies have identified increased odds of diabetes among ai / ans with a history of trauma and significant life stressors [ 16 , 17 ] . conversely , strong coping skills and other positive emotional attributes have been found to enhance metabolic control among those with diabetes . in addition , increased spirituality has been associated with improved self - management among african americans who suffer from diabetes , lower stress and higher quality of life in persons afflicted by chronic illness , and decreased likelihood of developing depression . although the relationship between spirituality and diabetes has not been studied specifically in ai / an populations , previous research has highlighted the importance of religious and spiritual practices for ai / an individuals struggling to overcome other health issues , such as the problematic use of alcohol . additionally , family support has been correlated with increased weight loss in the prevention of diabetes among arab americans . similarly , positive family support was correlated with improved diet in a study of older hispanic adults with diabetes . furthermore , active family nutritional support was linked to improved control of diabetes - related factors ( i.e. , triglycerides , cholesterol , and hba1c ) among navajo tribal members . finally , several psychological and behavioral factors , including increased self - efficacy , were associated with improved weight loss for dpp participants . given this prior body of evidence supporting significant relationships between a variety of psychosocial characteristics and multiple health outcomes , the correlation of psychosocial factors ( psychological distress , trauma exposure , coping skills , spirituality , and family support ) with a key clinical indicator of diabetes risk ( weight ) among ai / ans participating in the sdpi - dp demonstration project was assessed in the present study . resulting insights could suggest enhancements targeting such factors in the core components of sdpi - dp that hold promise for increasing its effectiveness . eligibility criteria for participating in the sdpi - dp demonstration projects were being ai / an ( based on eligibility to receive ihs services ) , being at least 18 years of age , and having either impaired fasting glucose ( ifg ) ( i.e. , a fasting blood glucose ( fbg ) level of 100125 mg / dl and an oral glucose tolerance test ( ogtt ) result < 200 mg / dl ) or impaired glucose tolerance ( igt ) ( i.e. , an ogtt result of 140199 mg / dl two hours after a 75 g oral glucose load and an fbg level < 126 mg / dl ) . exclusion criteria included a previous diagnosis of diabetes ( not including those who only have had gestational diabetes ) , pregnancy , end - stage renal disease on dialysis , and any condition that would affect successful participation based on provider judgment ( e.g. , cardiac concerns given the physical activity element of the program , severe substance use , and undergoing treatment for cancer ) . participants attended a 16-session educational curriculum , a series of lifestyle coaching sessions , and community - based exercise programs focused on reducing the risk of developing type 2 diabetes through moderate weight loss , increased physical activity , and healthy eating habits . clinical measurements and participant surveys were obtained at baseline , within 30 days of completing the 16-session curriculum , and annually thereafter . participants were enrolled at one of 36 tribal , indian health service ( ihs ) or urban indian health care programs serving 80 tribes between 2006 and 2010 . seventy - eight percent of participants were from a rural geographic setting , and 22% were from an urban area . to be included in the current study , participants minimally completed a baseline clinical assessment and a baseline survey ( n = 3,135 ) . the 193 individuals who completed a baseline clinical assessment but did not complete any participant surveys were excluded from these analyses . these individuals did not differ significantly from those included in the study with regard to age , gender , and baseline weight . the sdpi - dp protocol was approved by the institutional review board of the university of colorado denver and the national ihs institutional review board . when required , grantees obtained approval from other entities overseeing research in their programs ( e.g. , tribal review boards ) . the study sample was 74% female and had a mean age at baseline of 46.7 years . sixty - three percent of participants attended at least some college courses ; 72% of participants reported annual household incomes of less than $ 50,000 . sociodemographic variables including participant gender , age , educational status , and annual household income were collected through a survey at baseline . frequency of participants ' experience of various symptoms of depression and anxiety during the previous 30 days was assessed using this scale . item scores ranged from 1 ( none of the time ) to 5 ( all the time ) . participants ' ability to cope with life stressors was measured using the brief resilient coping scale . this 4-item scale asked participants to rate descriptions of coping reactions ( e.g. , approaching difficult situations in creative ways , focusing on the positive growth that can come from dealing with adversity ) , using a scale ranging from 1 ( does not describe me at all ) to 5 ( describes me exactly ) . a modified 6-item version of the diabetes family behavior checklist was used to measure participants ' perceptions of positive and negative family support in regard to their efforts to prevent the onset of diabetes . sdpi - dp research staff modified the original checklist slightly by removing items that referred to specific activities for individuals with diabetes ( e.g. , family providing suggestions about taking insulin on time ) that would not have been relevant to a program focusing on diabetes prevention . participants rated how often their family members provided positive support on 4 items ( e.g. , exercising with them ) and negative support on 2 items ( e.g. , criticizing them for not exercising regularly ) . item scores on the six items ranged from 1 ( less than once a month ) to 5 ( at least once a day ) . no items were reverse - scored , as items were phrased in either a positive or negative manner , consistent with the two scored dimensions . two additional psychosocial variables ( trauma experience and spirituality ) were assessed by participant surveys only at baseline . these two particular variables were not collected at follow - up due to the expectation of their high stability across a relatively short period of time . a single dichotomous variable from a posttraumatic stress disorder ( ptsd ) screener captured whether participants had ever experienced a significant traumatic event ( e.g. , being the victim of a violent crime or domestic violence , being in a disaster like a flood or fire , being in combat , being seriously injured in an accident , being sexually assaulted , and witnessing someone else being seriously injured or killed ) . this variable was coded either 0 ( no trauma ) or 1 ( history of trauma ) . spirituality was assessed via a 7-item scale designed specifically to capture the culturally relevant components of spirituality for ai / ans ; item scores ranged from 1 ( strongly disagree ) to 5 ( strongly agree ) . the items on this scale were developed through consultation with tribal leaders to reflect american indian cultural views of the connectedness of humans to all other physical and transcendental entities . the seven items were as follows : ( 1 ) i am in harmony with all living things , ( 2 ) i feel connected with other people in life , ( 3 ) i follow my tribal path , ( 4 ) when i need to return to balance , i know what to do , ( 5 ) i feel like i am living the right way , ( 6 ) i give to others and receive from them in turn , and ( 7 ) i am a person of integrity . confirmatory factor analyses were conducted at the item level for each psychosocial scale in order to establish measurement invariance across the two time points [ 32 , 33 ] . subsequently , latent difference scores were created to measure change over time in the outcome variable ( weight ) and applicable psychosocial variables [ 33 , 34 ] . latent difference scores are not subject to the restrictive assumptions of traditional anova approaches and permit the measurement of change without error by including multiple indicators of each construct at each of two time points . this modeling approach decomposes the data from the second time point into two components : ( 1 ) variance associated with time 1 and ( 2 ) variance associated with the difference from time 1 . therefore , latent difference scores allow for the estimation of baseline variance as well as variance regarding change in a construct over time . following these initial steps , a series of bivariate analyses were conducted within a structural equation modeling framework , which separately evaluated the relationships between each psychosocial variable and weight . for psychosocial variables that were measured at both baseline and follow - up , three parameters of primary interest were estimated : ( 1 ) the correlation between the psychosocial characteristic and weight at baseline , ( 2 ) the predictive relationship of the baseline psychosocial characteristics on change in weight , and ( 3 ) the association of change in the psychosocial characteristic with change in weight . for psychosocial variables that were measured only at baseline ( i.e. , trauma and spirituality ) , after evaluating the bivariate relationships , a multivariate model estimated the three parameters described above simultaneously for all psychosocial variables . this model also controlled for baseline sociodemographic characteristics , including gender , age , education , and income . psychosocial variables were eliminated from the multivariate model in a stepwise manner if they reached a p value greater than 0.2 for all three primary parameters , in order to arrive at a final model . biostatisticians have suggested that a p value greater than 0.2 is a reasonable cutoff to eliminate variables that are clearly nonsignificant in regression models . an effect size measure for the final model ( r ) was computed as the proportion of variance of change in weight that was explained by the predictor variables . confirmatory factor analysis and structural equation models macs analyses allow for the inclusion of mean - level information in addition to the covariance structures information of standard structural equation modeling techniques , which is necessary for the interpretation of latent difference scores . macs analyses also provide a particular advantage over ordinary least - squares regression approaches , namely , the fact that the unreliability of instruments / scales is taken into account and that corrections are made for measurement error . when employing structural equation modeling techniques , it is important to assess the degree to which the specified model fits the actual data in order to determine the appropriateness of a particular model . in the present study , the root mean square error of approximation ( rmsea(90% confidence interval ) ; less than .08 is adequate fit and less than .05 is good fit ) , the comparative fit index ( cfi ; greater than .90 is adequate fit and greater than .95 is good fit ) , and the tucker - lewis index ( tli ; greater than .90 is adequate fit and greater than .95 is good fit ) were used as indices of model fit . in all models , full information maximum likelihood ( fiml ) was implemented in all analyses in order to address potential bias and decreased power due to missing data [ 37 , 38 ] . furthermore , although there was very little variation across programs in class attendance for the participants included in the current study ( 95% of participants who completed a follow - up assessment had completed at least 14 of the 16 recommended curriculum classes ) , other elements of the program may have varied slightly across sites . therefore , in order to control for the clustering of participants into 36 separate health care programs , standard errors that are robust to nonnormality and nonindependence of observations were computed using a sandwich estimator . descriptive statistics for all clinical and psychosocial variables are presented in table 2 . between baseline and follow - up , a significant decrease of 8.58 lbs was found with regard to average weight ( = 8.58 , p < .001 ) . with regard to the change in psychosocial factors , general psychological distress decreased over time ( = 0.14 , p < .001 ) , while coping abilities increased ( = 0.07 , p < .001 ) . furthermore , positive family support , as perceived by participants , was higher at follow - up ( = 0.27 , p < .001 ) , whereas negative family support remained stable ( = 0.03 , p = .28 ) . at baseline , 48% of sdpi - dp participants reported a lifetime history of at least one significant trauma , and the average level of reported cultural spirituality at baseline was 3.81 ( on a scale from 1 to 5 ) . confirmatory factor analyses of the kessler distress , coping , and family support measures supported invariance of factor loadings and intercepts across the two measurement time points , which indicates that the measures exhibited similar structures and measured the same constructs across time . strong factorial invariance was established for both the kessler distress and coping measures , with all factor loadings and intercepts constrained to be equal across time . partial measurement invariance was established for the family support measure , as one of the intercepts for the positive family support scale was not invariant across time . it is generally acceptable to use measures with partial invariance in further structural models , if at least two indicators ( scale items ) have an invariant factor loading and intercept [ 32 , 33 ] . in the case of the family support measure , all six items exhibited loading invariance , and all but one item exhibited intercept invariance . it was important to establish that these psychosocial measures had identical or near identical structures at both time points in order to calculate reliable and valid difference scores . bivariate analyses were performed prior to running the multivariate model in order to evaluate the strengths of individual predictor / outcome relationships ( see table 3 ) . all estimates ( = covariance ; = regression coefficient ) are provided in an unstandardized metric in order to allow meaningful interpretation based upon the original scale ranges . all bivariate models exhibited good model fit ( rmsea < .05 ; cfi > .95 ; tli > .95 ) , and several significant correlations were found between psychosocial characteristics and weight at baseline . greater psychological distress at baseline was related to higher baseline weight ( = 2.44 , p < .001 ) . in addition , greater negative family support was significantly correlated with higher baseline weight ( = 4.55 , p < .001 ) , whereas greater identification with culturally relevant spirituality was associated with lower baseline weight ( = 3.13 , p < .001 ) . baseline levels of coping , positive family support , and trauma experience were not significantly related to baseline weight . in addition to investigating the relationships between psychosocial characteristics and weight at baseline , regression analyses were conducted in order to elucidate the predictive relationships between psychosocial variables ( independent variables ) and change in weight from baseline to follow - up ( dependent variable ) . the results of these analyses underscore the importance of psychological distress and family support in predicting weight loss in ai / ans with prediabetes . greater psychological distress at baseline predicted less successful weight loss between baseline and follow - up ( = 1.85 , p < .001 ) , and an increase in psychological distress between baseline and follow - up was also significantly related to less successful weight loss ( = 3.23 , p < .001 ) . participants who reported an increase in positive family support after the intervention were more successful in losing weight ( = 3.56 , p < .001 ) . conversely , higher negative family support at baseline as well as an increase in negative family support after the intervention was significantly associated with less weight loss ( = 1.38 , p = .003 and = 3.13 , p < .001 , resp . ) . coping , trauma experience , and cultural spirituality were not significantly related to weight change . after conducting the bivariate analyses , all of the psychosocial variables were included in a single multivariate model . sociodemographic variables ( gender , age , education status , and annual household income ) that have previously been shown to be related to participant engagement and retention in the sdpi - dp program were entered as covariates in the model , in order to determine the effect of the psychosocial factors on weight change above and beyond any potential effect of sociodemographic characteristics . using the stepwise procedure described above , coping and trauma experience were dropped from the final model , as they were neither correlated with baseline weight nor predictive of weight change . results of the final multivariate model are presented in figure 1 , which mirror the results of the bivariate models , with one exception . after controlling for sociodemographic factors and other psychosocial variables , baseline psychological distress was no longer predictive of weight loss from baseline to follow - up . however , the correlations between psychological distress , negative family support , and cultural spirituality with weight at baseline remained significant . in addition , change in psychological distress , positive family support , and negative family support over the course of the intervention , as well as levels of negative family support at baseline , remained significantly associated with change in weight . overall , the final multivariate model exhibited good model fit ( rmsea = .026(.025.028 ) ; cfi = .966 ; tli = .958 ) and accounted for 11% of the variability in weight change . this proportion of variance explained is not large , but it does represent a medium effect size . the importance of psychosocial characteristics as sources of diabetes risk and resilience has been demonstrated previously among ai / ans [ 16 , 17 , 25 , 41 ] . the present study is a critical first step in moving from research focused primarily on individuals with diabetes to examining factors related to successfully preventing incident diabetes among native people at high risk of the disease . although the influence of depression and anxiety on intervention outcomes for prediabetic individuals was examined in at least one previous study , the present study is the first to focus on determining which psychosocial factors successfully predict a specific outcome of a large - scale initiative aimed at preventing the onset of diabetes in the ai / an population . moreover , the statistical approach employed in the current study made it possible to simultaneously examine the relative contributions of the various psychosocial factors to successful health changes in a single model , unlike previous studies that have analyzed psychosocial factors in isolation of one another . specifically , structural equation modeling provides the ability to simultaneously examine the relationships of the psychosocial variables to a key clinical outcome with regard to baseline levels and change over time . as expected , when analyzing such relationships in a bivariate manner , several psychosocial factors were related to baseline levels of weight . higher levels of psychological distress and negative family support were associated with higher weight , whereas greater spirituality was correlated with lower weight . the same pattern of correlations of these three psychosocial variables with weight at baseline also was supported in the multivariate model when controlling for sociodemographic factors . psychosocial factors were also related to the degree of weight change following participants ' completion of the sdpi - dp intervention . greater psychological distress at baseline and increased psychological distress over the course of the intervention both contributed to less weight loss . similarly , greater negative family support at baseline and increased negative family support over the course of the intervention were associated with a smaller reduction in weight . increased positive family support , on the other hand , predicted greater weight loss . controlling for sociodemographic factors within a multivariate model , change in psychological distress , negative family support , and positive family support , as well as baseline levels of negative family support , continued to significantly affect weight reduction . the results of the present study are consistent with prior research on psychological distress as a risk factor with regard to chronic illness [ 14 , 15 , 18 ] and with previous findings regarding the role of positive family support in both reducing the risk of and successfully managing diabetes [ 2325 ] . the results underscore the importance of regularly assessing the psychosocial status and functioning of ai / ans at high risk of diabetes . prevention programs will be well served by developing the capacity to evaluate and monitor participants ' mental health status , including the presence of depression and anxiety , the nature and extent of their spirituality , and the adequacy of their family support . these personalized assessments , combined with the knowledge of the general effects of psychosocial factors uncovered in the present study , will allow program staff to know which adjunctive interventions may maximize participant benefit with respect to the desired outcomes ( e.g. , weight loss ) . for example , by increasing the focus on mental health components within the core curriculum , one could strengthen participants ' strategies for decreasing depressive and stress - related symptoms , which then may make it more likely that the participants will be more engaged in the intervention and experience more successful weight loss . offering self - management techniques , simple cognitive - behavioral skills , and referral to local support groups or treatment options is a logical extension of the goals , process , and structure of an intensive lifestyle balance intervention . additionally , knowing that a participant has a strong preexisting spiritual focus may be helpful information for program staff who then may be able to use a participant 's connectedness to the natural world as a pathway to increase motivation to engage in a healthier lifestyle . likewise , given the strong relationship between family support and program outcomes , a greater effort should be made to incorporate close family members into various aspects of the prevention program . sdpi - dp demonstration projects have begun to do so , guided by their initial impressions of the potential gains . for example , some programs encourage participants to identify a support person to attend curriculum classes and other program - related activities with the participant . the present study has several limitations , which suggest directions for future research . data specific to the psychosocial characteristics were collected solely by self - report , thereby possibly increasing shared method variance and artificially strengthening the relationships among these variables . future studies may benefit from using a variety of methods to operationalize and assess similar constructs . for example , family support could be measured through multiple informants , including close family members , and levels of depression and anxiety could be assessed through interview - based rating scales . nevertheless , the primary relationships of interest were between self - reported psychosocial characteristics and an objective clinical measure ( weight ) , which were not subject to problems of shared method variance . the relationships between psychosocial factors and program outcomes may wax and wane over a longer follow - up period , or certain interactions may occur over time that are not evident within a relatively short follow - up period . in addition , sdpi - dp participants were more likely to be female , be older , have a higher level of education , and have a higher household income than the general ai / an adult population . though previous research has shown similar trends when comparing clinical populations to the general population [ 43 , 44 ] , the generalizability of the present findings to individuals with widely differing sociodemographic backgrounds may be limited . for example , it is possible that weight change for males may not be as strongly related to psychological distress or family support as it was for this largely female sample . in addition , individuals with less education and lower household income than the participants in the current study may be more likely to have suffered a greater number of significant traumas . future studies should attempt to enroll individuals with broader sociodemographic characteristics and should include a measure of the number of traumas experienced , which would provide the opportunity to analyze a possible additive effect of repeated trauma upon successful weight loss that was not possible with the dichotomous trauma item used in the present investigation . similarly , although both rural and urban participants were included in the study , the majority of participants lived in rural settings , which may further limit the generalizability of the results . although it would be difficult to extend these results to the mainstream american population without further research , the current findings may also be applicable to other populations that share similar structures and values with ai / an communities ( e.g. , a greater emphasis on extended family support as opposed to individualism ; a spiritual emphasis on connectedness to others and nature ) . furthermore , although a medium effect size for the prediction of weight change within the multivariate model was observed ( 11% of outcome variability explained ) , additional factors are likely at work and will need to be addressed to more comprehensively improve the effectiveness of such prevention programs . some additional factors may include lack of access to healthy food selections , high levels of family and caregiver stress that make it difficult to follow through with healthy eating and exercise routines , and lack of transportation to attend program classes . moreover , characteristics of the treatment team and health care program in general previously have been shown to be related to participant retention , which in turn predicts program outcomes [ 7 , 8 ] . therefore , it likely will be critical to incorporate a multifaceted approach to crafting additional components that promise to enhance the intervention . for example , rather than just adding a stand - alone mental health screening module , a program might consider addressing barriers to participation ( e.g. , lack of transportation ) in concert with increasing positive family support and thereby decrease the isolation that can lead to psychological distress . finally , a more precise and comprehensive assessment of mental health status would enable a program to determine the most appropriate approach for decreasing symptoms likely to interfere with participation in the preventive intervention . in light of the relationship between depression and diabetes [ 1013 , 45 , 46 ] , referral to a mental health professional is a logical option to be pursued , although other possibilities , such as a group treatment model , should also be considered given the limited numbers of mental health providers within tribal , ihs , and urban indian health care programs . the present study demonstrates the importance of psychosocial factors for maximizing the potential benefits to participants in preventive interventions such as the sdpi - dp demonstration project . the challenge now becomes how to incorporate the lessons learned into the fabric of these programs . augmentation of the current intervention may be achieved either directly by incorporating adjunctive components or indirectly through referral to relevant local resources . the overall goal of these program additions would be to maximize participants ' engagement , their ability to grasp the knowledge conveyed , and their mastery of the skills related to the behavioral changes associated with the desired outcomes .

the association of psychosocial factors ( psychological distress , coping skills , family support , trauma exposure , and spirituality ) with initial weight and weight loss among american indians and alaska natives ( ai / ans ) in a diabetes prevention translational project was investigated . participants ( n = 3,135 ) were confirmed as prediabetic and subsequently enrolled in the special diabetes program for indians diabetes prevention ( sdpi - dp ) demonstration project implemented at 36 indian health care programs . measures were obtained at baseline and after completing a 16-session educational curriculum focusing on weight loss through behavioral changes . at baseline , psychological distress and negative family support were linked to greater weight , whereas cultural spirituality was correlated with lower weight . furthermore , psychological distress and negative family support predicted less weight loss , and positive family support predicted greater weight loss , over the course of the intervention . these bivariate relationships between psychosocial factors and weight remained statistically significant within a multivariate model , after controlling for sociodemographic characteristics . conversely , coping skills and trauma exposure were not significantly associated with baseline weight or change in weight . these findings demonstrate the influence of psychosocial factors on weight loss in ai / an communities and have substantial implications for incorporating adjunctive intervention components .

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

PMC3196962

a 80-year - old man presented with a recent onset of constipation , anal discomfort , and anal incontinence . several months ago , he experienced a sensation of something passing through the anus at defecation , that he manually reinserted , accompanied by tenesmus and rectal bleeding , for which he had a diagnosis of hemorrhoids . these symptoms worsened in the following months , since the mass began to prolapse independently of defecation , became painful when standing or walking , made defecation obstructed , and caused anal incontinence , for which he was referred with a presumptive diagnosis of neoplasia . inspection of anal region revealed a large , soft , ulcerated mass protruding through the anus that was suspected for a submucosal neoplasia . , the mass was pulled through the anal canal , and it was noted that it arises in the posterior wall of the rectum , 3 centimetres above the dentate line , where it developed a pseudostalk , presumably as a result of the repeated prolapses . lipomas of the large intestine are relatively uncommon in clinical practice , although they are considered to be the benign tumours that occur most frequently , adenomas excluded , and the commonest nonepithelial tumours . some authors have reported a female predominance while others found a nearly equal sex incidence . in general , most lipomas are silent , and most series in the literature are autoptic reports in which their incidence ranges from 0.35 to 4.4% . the number of large bowel lipomas found incidentally at necropsy far exceed the number of surgically treated cases . the symptomatic lipomas are of clinical importance because of the many diagnostic and therapeutic problems they may cause . clinical signs are reported in a poor percentage of patients and are related to tumour location and size . symptoms , that include bleeding , constipation , changing bowel habits , abdominal pain , intestinal obstruction , or intussusception of the mass [ 37 ] , are more common when the lipoma is greater than 2 cm in diameter . lesions are sessile or polypoid and are submucosal , in about 90% of cases , or subserosal . in size , the tumours range from a few millimetres to several centimetres ( 0.58 cm in the serie of castro and stearns ; 421 in that of michowitz et al . ; 0.510 in that of rogy et al . ) . in some percentage , the lipomas are two or more ( 24.4% ) and not rarely associated with other pathologies , namely , colorectal cancer . anyway , the high incidence of this association in some reports ( 33.3% ) does not reflect the actual incidence in the general population , but it reflects that of a cancer - patient population . the most common site of origin is the right colon [ 3 , 9 ] . there is no explanation for the predilection of lipomas of the large bowel to occur in the right side . lipomas of the rectum are quite rare , and , in the various studies , are reported in a small number of patients . in the serie of castro and stearns , only 5 out of 45 lipomas of the large bowel ( 11.1% ) were in the rectum and only one of these was palpable . the size of the lipomas of the rectum was recorded in 4 cases : 3 were less than 2 cm , and one was 24 cm in diameter . haller and roberts reviewed 29 lipomas of the large bowel in 20 patients , none of which was in the rectum . , found only one lipoma of the rectosigmoid ( 4.54% ) in his series of 22 cases . in an 18-year study , rogy et al . reported 17 patients with lipomas of the large bowel , and only three out of 21 were in the rectum . reported 11 patients with large bowel lipomas , all located proximal to the sigmoid colon . the prolapse of a rectal lipoma through the anus is a rare event , and a few cases are reported in the literature [ 13 , 14 ] . taylor and wolff , in a review of the mayo clinic experience of 91 surgically managed colonic lipomas from 1976 to 1985 , report no rectal location . ryan et al . , in a review of the surgical pathology files , over a 50-year period , found 13 cases of colonic lipomas , none of these were in the rectum . identified 5 patients with symptomatic lipoma of the large bowel , searching the computerized histopathologic records of the leicester general hospital and leicester royal infirmary , in a 11-year period , located , respectively in proximal colon ( 2 ) , sigmoid colon ( 2 ) , and rectum ( 1 ) . the patient with the rectal lipoma was a 35-year - old female presented as an emergency with lower abdominal pain and prolapsed rectal polyp , 6 centimetres in diameter ( see figures 1 and 2 ) . in a review of 11 patients treated for symptomatic lipomas of the gastrointestinal tract , during the period 19661972 , ackerman and chughtai found the tumour located in the stomach ( 3 cases ) , in the ileocaecal region ( 4 ) , and in the colon ( 3 ) ; the more distal lipoma was found in the sigmoid colon .

submucosal lipomas of the large bowel are uncommon . occasionally , they occur in the rectum and may cause aspecific symptoms ; presentation with rectal prolapse is very unusual and may lead to a misdiagnosis of simple mucosal prolapse . the paper describes an additional case of a prolapsing rectal mass that led to diagnosis and surgical treatment of a rectal lipoma under local anesthesia .

1. Case Report 2. Discussion

PMC4895090

a 15-year - old female slipped down during gymnastics practice and had a right knee pain localizing to the lateral side . she went to a hospital 5 days after the injury and was diagnosed with ocd of the lfc and anterior defect with posterocentral shift of the discoid lateral meniscus . the patient underwent arthroscopic partial meniscectomy with repair of the lateral meniscus and multiple drilling at the lfc 2 weeks after the injury at the hospital . during 3 months after the surgery , however , her pain had worsened and she could not walk well . eventually , the patient was transferred to our clinic at 4 months after the first surgery . physical examination revealed tenderness on the lateral joint line of the right knee and positive mcmurray test on internal rotation of the tibia . plain radiographs of the right knee showed an obvious ocd lesion with a large bony defect in the lfc and narrowing of the lateral joint space ( fig . standing alignment of the lower legs demonstrated about 2 degrees of slight valgus of the right knee compared to the left knee ( fig . 2 ) . magnetic resonance imaging ( mri ; intera achieva , philips , amsterdam , the netherlands ) of the right knee revealed a 2.5 cm3 cm sized lesion mainly in the posterolateral area of the lfc ( fig . the lesion progressed more compared with the preoperative initial mri obtained 5 months ago ( fig . the mri also demonstrated a complex tear of the lateral meniscus where flaps adhered to the ocd lesion of the lfc ( fig . the patient underwent arthroscopic total meniscectomy of the lateral meniscus and debridement of the ocd lesion first . at that time , since the torn lateral meniscus showed severe plastic deformation , we considered there was no regrowth potential of the remnant lateral meniscus . the ocd lesion treated with multiple drilling at another hospital showed a large osteochondral defect without any regeneration ( fig . 4 ) . therefore , we planned to perform simultaneous osteoperiosteal autologous iliac crest graft implantation for the massive ocd lesion and lateral meniscus allograft transplantation for deficiency of the lateral meniscus with joint space narrowing , considering the patient was a high - level gymnast and wanted early return to previous level of sports activities . at one month after the first arthroscopic surgery performed at our hospital ( 5.5 months after the surgery performed at another hospital ) , she underwent osteoperiosteal autologous iliac crest graft implantation and lateral meniscus allograft transplantation as a one - stage procedure . the ocd lesion was identified and completely debrided , then multiple drilling using a fine 1.21.5-mm diameter k - wire was performed toward the lesion to break down the sclerotic avascular zone ( fig . 5 ) . in the next step , the size and depth of the defect was measured as 1.6 cm2.4 cm and 0.8 cm , respectively using various - sized osteotomes . the anterior iliac crest was then accessed by making an incision 23 cm posterior to the anterior - superior iliac spine and an osteochondral fragment that matches the size of the lesion was harvested without damaging periosteum ( fig . the defect in the iliac crest was backfilled with allograft bone from the lateral meniscus allograft . the harvested iliac crest plug with periosteum preservation was inserted into the lesion of the lfc and impacted by adjusting to the adjacent cartilage level ( fig . the periosteum layer was placed slightly below the cartilage level . the following step for lateral meniscus allograft transplantation was performed using a " key - hole technique . " but , the depth of the bone bridge was made to be 6 mm not to harm the growth plate of the tibia . knee joint motion was performed to check the stability of the inserted iliac crest plug and the transplanted lateral meniscus allograft . after completion of the procedure , occurrence of bleeding from the healthy underlying bone resulting from multiple drilling was checked . the knee was immobilized in a cast during the first 3 weeks after surgery and protected using a brace for 12 weeks . a 60 range of motion ( rom ) was allowed at 3 weeks and 90 rom at 6 weeks after surgery . light running was allowed at 3 months after surgery and return to sports at 6 months after surgery , although strenuous contact sports were prohibited . plain radiography and 3-dimensional computed tomography ( 3d ct ; aquilion 64 , toshiba , tokyo , japan ) were carried out on the first postoperative day , which showed a well integrated bone plug in the lfc and an allograft in the lateral tibial plateau ( fig . the lysholm score improved from 47 to 85 , the international knee documentation committee subjective knee form improved from 57 to 75 , and the tegner activity score improved from 4 to 8 . there was no donor - site complication such as pain and sensory loss as well as growth deformity . isokinetic muscle strength test , follow - up mri , and second - look arthroscopy were performed at 30 months after surgery . the deficit of isokinetic muscle strength ( flexor peak torque / body weight ) of the involved side was 9.2% and the hamstring - quadriceps ratio was 59% . on follow - up mri and second - look arthroscopy , the iliac crest plug was well incorporated with the lfc , and the ocd lesion healed with fibrous cartilage regeneration as well as stabilized lateral meniscus allograft ( fig . she could return to previous level of gymnastic activities without any swelling or pain at final evaluation at 24 months after surgery . at one month after the first arthroscopic surgery performed at our hospital ( 5.5 months after the surgery performed at another hospital ) , she underwent osteoperiosteal autologous iliac crest graft implantation and lateral meniscus allograft transplantation as a one - stage procedure . an anterolateral incision was used to expose the lateral compartment of the knee joint . the ocd lesion was identified and completely debrided , then multiple drilling using a fine 1.21.5-mm diameter k - wire was performed toward the lesion to break down the sclerotic avascular zone ( fig . the size and depth of the defect was measured as 1.6 cm2.4 cm and 0.8 cm , respectively using various - sized osteotomes . the anterior iliac crest was then accessed by making an incision 23 cm posterior to the anterior - superior iliac spine and an osteochondral fragment that matches the size of the lesion was harvested without damaging periosteum ( fig . the defect in the iliac crest was backfilled with allograft bone from the lateral meniscus allograft . the harvested iliac crest plug with periosteum preservation was inserted into the lesion of the lfc and impacted by adjusting to the adjacent cartilage level ( fig . the periosteum layer was placed slightly below the cartilage level . the following step for lateral meniscus allograft transplantation but , the depth of the bone bridge was made to be 6 mm not to harm the growth plate of the tibia . knee joint motion was performed to check the stability of the inserted iliac crest plug and the transplanted lateral meniscus allograft . after completion of the procedure , occurrence of bleeding from the healthy underlying bone resulting from multiple drilling was checked . the knee was immobilized in a cast during the first 3 weeks after surgery and protected using a brace for 12 weeks . a 60 range of motion ( rom ) was allowed at 3 weeks and 90 rom at 6 weeks after surgery . light running was allowed at 3 months after surgery and return to sports at 6 months after surgery , although strenuous contact sports were prohibited . plain radiography and 3-dimensional computed tomography ( 3d ct ; aquilion 64 , toshiba , tokyo , japan ) were carried out on the first postoperative day , which showed a well integrated bone plug in the lfc and an allograft in the lateral tibial plateau ( fig . the lysholm score improved from 47 to 85 , the international knee documentation committee subjective knee form improved from 57 to 75 , and the tegner activity score improved from 4 to 8 . there was no donor - site complication such as pain and sensory loss as well as growth deformity . isokinetic muscle strength test , follow - up mri , and second - look arthroscopy were performed at 30 months after surgery . the deficit of isokinetic muscle strength ( flexor peak torque / body weight ) of the involved side was 9.2% and the hamstring - quadriceps ratio was 59% . on follow - up mri and second - look arthroscopy , the iliac crest plug was well incorporated with the lfc , and the ocd lesion healed with fibrous cartilage regeneration as well as stabilized lateral meniscus allograft ( fig . she could return to previous level of gymnastic activities without any swelling or pain at final evaluation at 24 months after surgery . this case report describes osteoperiosteal autologous transplantation for massive ocd combined with lateral meniscus allograft transplantation for deficiency of the lateral meniscus as a " one - step " technique . the patient could return to high - level sports activities as well as full daily activities without symptoms . in addition , a good morphological restoration with fibrous cartilage covering of the massive ocd lesion including osseous defect and well - maintained lateral meniscus allograft were shown on the follow - up mri and second - look arthroscopy . a discoid lateral meniscus sometimes results in ocd of the lfc , which has been reported in co - presence with discoid meniscus in up to 19%1 ) . the discoid meniscus , regardless of tear , could be an important risk factor for ocd , because it might produce repetitive abnormal stress on the immature osteochondral bone in patients who are still growing and participate in sports activities1 ) . these phenomena would be more accelerated after total resection of the discoid meniscus as in our patient2 ) . hence , we performed lateral meniscus allograft transplantation concomitantly , despite the patient 's young age . although there has been paucity of literatures on the outcomes of meniscus transplantation in children or juveniles and it has not yet been considered as an optimal treatment for the meniscus - deficient knee in patients with immature skeleton , meniscus preservation or restoration has been suggested as an alternative treatment for young patients with use of improved techniques of meniscus allograft transplantation3 ) . andrish3 ) noted that the best candidate for meniscus transplantation could be young patients who had minimal degenerative joint disease . we could perform lateral meniscus transplantation with a bone block technique without any violation of the physis of the tibia by making a shallow bone hole after failure of meniscectomy with repair at another hospital . several techniques such as multiple drilling , osteochondral autograft transplantation , and osteochondral allograft transplantation for ocd of the knees have been evolved . osteochondral autograft transplantation can be used to treat osteochondral lesions with a large defect providing stability between cartilage and osseous levels2 ) . however , obtaining large plugs in the osteochondral autograft transplantation , so called mosaicplasty , could result in significant donor - site morbidity4 ) . for this reason , there have been several attempts to obtain autologus osteochondral grafts from other sites . espregueira - mendes et al.5 ) developed autologous osteochondral grafting from the upper tibio - fibular joint for the treatment of knee cartilage lesions . even though the upper tibiofibular joint does not play a significant role , there are still problems of donor site morbidity such as disruption of the proximal joint or risk of peroneal nerve injury through the lateral approach . leumann et al.2 ) designed a modified mosaicplasty for severe ocd of the talus using bony periosteum - covered iliac crest plug transplantation and showed good radiological results in 82% at a mean follow - up of 25 months . the anterior part of the iliac crest has been widely used for autologous bone grafting including cancellous bone and structured bone2 ) . in addition , the periosteum of membrane bone was identified to have abundant stem cells with the chondrogenic capacity and to provide mechanical stability and biological interaction6 ) . matsushima et al.6 ) reported that the ileum represented relatively high gene expression of type i collagen and type ii collagen compared with cranial or mandibular bone . o'driscoll and fitzsimmons7 ) demonstrated that the chondrocyte precursor cells exist in the cambium layer of periosteum and they could meet the requirements as a source of cells , a scaffold , and a source of growth factors and revealed that dynamic fluid pressure increases cartilage formation . most of these articles were laboratory studies , not clinical studies , except for the study of leumann et al.2 ) . peterson et al.8 ) reported that 91% of 58 patients with advanced ocd lesions treated with autologous chondrocyte transplantation showed good or excellent clinical results after a mean follow - up of 5.6 years . in 7 of 58 patients with a deep defect over 8 mm , the defect was filled with cancellous bone after removal of the subchondral bone , and autologous cultured chondrocyte transplantation was performed using a double - layered " sandwich technique " . this technique could be considered as a treatment option in our current patient who had a large bony defect with cartilage loss . however , autologous chondrocyte implantation requires two or three operative procedures and it takes long time for one stage operation with lateral meniscus allograft transplantation . hence , we determined to perform " one - step " osteoperiosteal autologous iliac crest graft and lateral meniscus allograft transplantation . although hyaline cartilage has been known to be superior to fibrous cartilage in terms of biomechanical property and longevity , there are some reports showing the regenerated fibrous cartilage is comparable to the grafted hyaline cartilage . knutsen et al.9 ) performed a comparison study of clinical outcomes between the fibrous cartilage and the hyaline cartilage after microfracture and aci , respectively and concluded that there were no significant differences in clinical and radiological results in the mid - term follow - up . gobbi et al.10 ) also showed no significant differences in terms of clinical results between them in the ankle joint . it is critical to provide a good congruency of articular cartilage in osteochondral autograft transplantation avoiding step off , which strongly related to better clinical outcomes in the long - term follow - up . in our patient , we used press - fitting mortise , which is wider and deeper at the articular surface , assuming that it could have stronger pullout strength ( fig . although the short - term follow - up and lack of cases were the most significant limitations of this report , the modified osteochondral autograft transplantation demonstrated several advantages such as reproducibility , lower risk of donor site morbidity , and provision of fibrous cartilage coverage as well as filling of the bone defect . in conclusion , this osteoperiosteal autologous iliac crest graft transplantation could be a strategy for a joint salvage procedure with restoration of the osteochondral defect , reproduction of the fibrous cartilage , and mechanical stability as well as biological healing for massive ocd lesions . additional aggressive treatments for combined torn discoid lateral meniscus , especially for deficiency of the meniscus , should be considered to prevent failure of the treatment of ocd . the successful results of our " one - step " technique should be validated in further studies with long - term evaluation .

the optimal treatment for combined osteochondritis dissecans ( ocd ) with considerable bony defect of the lateral femoral condyle ( lfc ) and torn discoid lateral meniscus is unclear . we present a case of a 15-year - old female who was a gymnast and had a large ocd lesion in the lfc combined with deficiency of the lateral meniscus . the patient underwent the " one - step " technique of osteoperiosteal autologous iliac crest graft and lateral meniscus allograft transplantation after a failure of meniscectomy with repair at another hospital . twenty - four months postoperatively , clinical results were significantly improved . follow - up imaging tests and second - look arthroscopy showed well incorporated structured bone graft and fibrous cartilage regeneration as well as stabilized lateral meniscus allograft . she could return to her sport without any pain or swelling . this " one - step " surgical technique is worth considering as a joint salvage procedure for massive ocd lesions with torn discoid lateral meniscus .

Case Report 1. Surgical Technique 2. Postoperative Rehabilitation 3. Follow-up Evaluation Discussion

PMC3177383

dental ceramics are considered chemically inert restorative materials . however , many factors such as the composition , microstructure , chemical properties of the ceramic materials , erosive or acidic agents , exposure time , and the temperature , may influence the durability of dental ceramics.1 hence , previous studies have reported degradation of dental ceramics when exposed to aqueous solutions or acidic agents.29 this condition results from selective releasing of alkaline ions,68 which are far less stable in the glassy phase than in the crystalline phase of dental ceramics.2 the consequences of ceramic degradation are coarseness of the exposed surface,810 increase in plaque accumulation2681011 and wear to antagonist materials or teeth.2 in addition , an increase in surface roughness of ceramics may decrease strength1213 and affect the clinical success and failure of ceramic restorations.14 dental ceramics have different microstructures , chemical compositions , and properties . feldspathic ceramics enclose 19 weight percentage ( wt% ) of leucite crystals ( k2o al2o3 4sio2 ) after incongruent melting of a mixture of glass and potassium feldspar.15 aluminous ceramics , the feldspathic - base ceramics , has increased the amounts of 40 to 50 wt% aluminum oxide crystals.16 feldspathic and aluminous ceramics are used as laminate veneers , inlays , onlays and as covering material for ceramic restorations . the high leucite ceramics contain up to 40 - 50 wt% leucite crystals.17 recently , fluorapatite ceramics are composed of dispersed fluorapatite crystals [ ca10(po4)6f2 ] in a feldspathic glassy matrix that results in a microstructure unlike that of any other commercially available dental ceramic.18 in general , the behavior of eating and chewing sour fruits , such as green mangoes , pineapples , and limes are most commonly found in tropical countries such as australia , cuba and also countries in southeast asia.1922 people who frequently consume these foods often have a high incidence of dental erosion.2324 the potential erosive effect of these acidic food and beverages on enamel are affected primarily by the dissolution of apatite crystals.2425 however , little is known about their effect on ceramic restorations . the present study was conducted to evaluate changes of surface roughness and surface characteristics of 4 types of dental ceramics after being immersed in acidic agents ( citrate buffer solution , green mango juice , pineapple juice , and 4% acetic acid ) for 168 hours . the null hypothesis was that there would be no significant changes of surface roughness and surface characteristics of dental ceramics after immersion in acidic agents . in this study , four types of dental ceramics were selected as representatives of the various ceramic types available . the studied ceramics were vmk 95 ( lot no . 7530 , vita zanhfabrik , bad sckingen , germany ) feldspathic ceramic , vitadur alpha ( lot no.7435 , vita zanhfabrik ) aluminous ceramic , ips empress esthetic ( lot no . jm0817 , ivoclar vivadent ag , schaan , liechtenstein ) high leucite ceramic , ips e.max ceram ( lot no . 583614 , ivoclar vivadent ) fluorapatite ceramic . a total of 83 disc specimens ( 12 mm in diameter and 2 mm in thickness ) from each ceramic were fabricated . the sintered ceramic specimens were made by a silicone mold ( provil , heraeus kulzer , wehrheim , germany ) and then , cured according to the manufacturer 's instructions . for ips empress esthetic , subsequently , the specimens were polished ( phoenix 4000 , buehler gmbh , dsseldorf , germany ) under running water using 600 and 1,200-grit silicon carbide paper ( 3 m espe , st.paul , mn , usa ) and were submitted to self - glazing according to the manufacturer 's instructions . the exclusion criteria of the specimens were the presence of defects , pores , or cracks on the surfaces as evaluated by visual inspection and under a stereoscope ( smz 1500 m , nikon instech , kanagawa , japan ) at 40 magnification . all of the specimens were then ultrasonically cleaned in distilled water for 10 min and dried in a stream of oil - free compressed air and kept at room temperature before testing . fifty ceramic disks of each type of the studied ceramics were divided into 5 groups ( n=10 ) . surface roughness of the specimens was measured by a profilometer ( surfcorder se-2300 , kosaka laboratory ltd . , the cut - off value for surface roughness was 0.8 mm and the traversing distance of the stylus was 4 mm . the radius of the tracing diamond tip was 5 m , and the measuring force and speed were 4 mn and 0.5 m / s , respectively . the surface roughness values ( ra , rmax , rz , and sm26 ; see table 1 ) of each specimen were obtained in five different positions ( 1.5 mm apart ) . four groups were immersed in 25 ml of three acidic agents ( citrate buffer solution , ph 4.99 ; green mango juice , ph 2.37 ; and pineapple juice , ph 3.65 ) and deionized water ( control ) at 37c for 168 hours . one group was immersed in 4% acetic acid , ph 2.45 at 80c for 168 hours ( as modified from iso 687227 ) . the original ph of each acidic agent was measured using a ph meter ( orion model 900a , orion research inc . , subsequently , the specimens were rinsed with deionized water , blotted dry and subjected to surface roughness testing at intervals of 24 , 96 , and 168 hours . surface roughness parameters26 three specimens of each ceramic before immersion and three specimens from each of the acidic agents at 96 and 168 hours immersion ( n = 33 ) were examined by a scanning electron microscopy ( jsm 5800lv , jeol , tokyo , japan ) to evaluate the surface topography . the specimens were rinsed with distilled water for 5 minutes , dried and fixed onto an aluminum cylinder ( 13 mm in diameter and 10 mm in height ) . consequently , the specimens were sputter - coated with a gold - palladium alloy ( spi - module sputter , spi supplies , west chester , pa , usa ) and evaluated under sem . a two - way repeated analysis of variance ( anova ) was performed for each of the roughness parameters to assess the influence of different storage agents and ceramics on the surface roughness . tukey 's hsd multiple comparison was used in each of the parameters for comparison among the five storage agents . within - group analysis was compared between the baseline value and each time point ( = 0.05 ) . in this study , four types of dental ceramics were selected as representatives of the various ceramic types available . the studied ceramics were vmk 95 ( lot no . 7530 , vita zanhfabrik , bad sckingen , germany ) feldspathic ceramic , vitadur alpha ( lot no.7435 , vita zanhfabrik ) aluminous ceramic , ips empress esthetic ( lot no . jm0817 , ivoclar vivadent ag , schaan , liechtenstein ) high leucite ceramic , ips e.max ceram ( lot no . 583614 , ivoclar vivadent ) fluorapatite ceramic . a total of 83 disc specimens ( 12 mm in diameter and 2 mm in thickness ) from each ceramic were fabricated . the sintered ceramic specimens were made by a silicone mold ( provil , heraeus kulzer , wehrheim , germany ) and then , cured according to the manufacturer 's instructions . for ips empress esthetic , subsequently , the specimens were polished ( phoenix 4000 , buehler gmbh , dsseldorf , germany ) under running water using 600 and 1,200-grit silicon carbide paper ( 3 m espe , st.paul , mn , usa ) and were submitted to self - glazing according to the manufacturer 's instructions . the exclusion criteria of the specimens were the presence of defects , pores , or cracks on the surfaces as evaluated by visual inspection and under a stereoscope ( smz 1500 m , nikon instech , kanagawa , japan ) at 40 magnification . all of the specimens were then ultrasonically cleaned in distilled water for 10 min and dried in a stream of oil - free compressed air and kept at room temperature before testing . fifty ceramic disks of each type of the studied ceramics were divided into 5 groups ( n=10 ) . surface roughness of the specimens was measured by a profilometer ( surfcorder se-2300 , kosaka laboratory ltd . , the cut - off value for surface roughness was 0.8 mm and the traversing distance of the stylus was 4 mm . the radius of the tracing diamond tip was 5 m , and the measuring force and speed were 4 mn and 0.5 m / s , respectively . the surface roughness values ( ra , rmax , rz , and sm26 ; see table 1 ) of each specimen were obtained in five different positions ( 1.5 mm apart ) . four groups were immersed in 25 ml of three acidic agents ( citrate buffer solution , ph 4.99 ; green mango juice , ph 2.37 ; and pineapple juice , ph 3.65 ) and deionized water ( control ) at 37c for 168 hours . one group was immersed in 4% acetic acid , ph 2.45 at 80c for 168 hours ( as modified from iso 687227 ) . the original ph of each acidic agent was measured using a ph meter ( orion model 900a , orion research inc . , subsequently , the specimens were rinsed with deionized water , blotted dry and subjected to surface roughness testing at intervals of 24 , 96 , and 168 hours . surface roughness parameters26 three specimens of each ceramic before immersion and three specimens from each of the acidic agents at 96 and 168 hours immersion ( n = 33 ) were examined by a scanning electron microscopy ( jsm 5800lv , jeol , tokyo , japan ) to evaluate the surface topography . the specimens were rinsed with distilled water for 5 minutes , dried and fixed onto an aluminum cylinder ( 13 mm in diameter and 10 mm in height ) . consequently , the specimens were sputter - coated with a gold - palladium alloy ( spi - module sputter , spi supplies , west chester , pa , usa ) and evaluated under sem . a two - way repeated analysis of variance ( anova ) was performed for each of the roughness parameters to assess the influence of different storage agents and ceramics on the surface roughness . tukey 's hsd multiple comparison was used in each of the parameters for comparison among the five storage agents . within - group analysis was compared between the baseline value and each time point ( = 0.05 ) . in this study , four types of dental ceramics were selected as representatives of the various ceramic types available . the studied ceramics were vmk 95 ( lot no . 7530 , vita zanhfabrik , bad sckingen , germany ) feldspathic ceramic , vitadur alpha ( lot no.7435 , vita zanhfabrik ) aluminous ceramic , ips empress esthetic ( lot no . jm0817 , ivoclar vivadent ag , schaan , liechtenstein ) high leucite ceramic , ips e.max ceram ( lot no . 583614 , ivoclar vivadent ) fluorapatite ceramic . a total of 83 disc specimens ( 12 mm in diameter and 2 mm in thickness ) from each ceramic were fabricated . the sintered ceramic specimens were made by a silicone mold ( provil , heraeus kulzer , wehrheim , germany ) and then , cured according to the manufacturer 's instructions . for ips empress esthetic , subsequently , the specimens were polished ( phoenix 4000 , buehler gmbh , dsseldorf , germany ) under running water using 600 and 1,200-grit silicon carbide paper ( 3 m espe , st.paul , mn , usa ) and were submitted to self - glazing according to the manufacturer 's instructions . the exclusion criteria of the specimens were the presence of defects , pores , or cracks on the surfaces as evaluated by visual inspection and under a stereoscope ( smz 1500 m , nikon instech , kanagawa , japan ) at 40 magnification . all of the specimens were then ultrasonically cleaned in distilled water for 10 min and dried in a stream of oil - free compressed air and kept at room temperature before testing . fifty ceramic disks of each type of the studied ceramics were divided into 5 groups ( n=10 ) . surface roughness of the specimens was measured by a profilometer ( surfcorder se-2300 , kosaka laboratory ltd . , the cut - off value for surface roughness was 0.8 mm and the traversing distance of the stylus was 4 mm . the radius of the tracing diamond tip was 5 m , and the measuring force and speed were 4 mn and 0.5 m / s , respectively . the surface roughness values ( ra , rmax , rz , and sm26 ; see table 1 ) of each specimen were obtained in five different positions ( 1.5 mm apart ) . four groups were immersed in 25 ml of three acidic agents ( citrate buffer solution , ph 4.99 ; green mango juice , ph 2.37 ; and pineapple juice , ph 3.65 ) and deionized water ( control ) at 37c for 168 hours . one group was immersed in 4% acetic acid , ph 2.45 at 80c for 168 hours ( as modified from iso 687227 ) . the original ph of each acidic agent was measured using a ph meter ( orion model 900a , orion research inc . , subsequently , the specimens were rinsed with deionized water , blotted dry and subjected to surface roughness testing at intervals of 24 , 96 , and 168 hours . three specimens of each ceramic before immersion and three specimens from each of the acidic agents at 96 and 168 hours immersion ( n = 33 ) were examined by a scanning electron microscopy ( jsm 5800lv , jeol , tokyo , japan ) to evaluate the surface topography . the specimens were rinsed with distilled water for 5 minutes , dried and fixed onto an aluminum cylinder ( 13 mm in diameter and 10 mm in height ) . consequently , the specimens were sputter - coated with a gold - palladium alloy ( spi - module sputter , spi supplies , west chester , pa , usa ) and evaluated under sem . the data were statistically analyzed using spss version 16 . a two - way repeated analysis of variance ( anova ) was performed for each of the roughness parameters to assess the influence of different storage agents and ceramics on the surface roughness . tukey 's hsd multiple comparison was used in each of the parameters for comparison among the five storage agents . within - group analysis was compared between the baseline value and each time point ( = 0.05 ) . the results of two - way repeated anova for each of the surface roughness parameters revealed significant differences among five storage agents and among four ceramic materials ( p < 0.001 ) . all of the roughness parameters had no significant interactions between the type of storage agents and ceramics . the surface roughness values for ra , rz , rmax and sm parameters of the studied ceramics in deferent storage media and storage times were shown in tables 25 . baseline values ( before immersion ) were recorded in order to verify the initial surface roughness which found not to be any significant differences among all groups . ra , rz and rmax were significantly increased after 168 h immersion in all acidic storage agents ( p < 0.05 ) . there were significant differences for ra , rmax , rz and sm after 168 h in 4% acetic acid from other groups ( p < 0.001 ) of all of the ceramics evaluated . there was no significant difference for sm in 4% acetic acid and green mango juice of vmk 95 , empress esthetic , and ips e.max ceram . among all studied ceramics , vitadur alpha showed significantly the greatest values of all surface roughness parameters after immersion in 4% acetic acid ( p < 0.001 ) while in other acidic agents , there was no significant difference for all surface roughness parameters among all studied ceramics . mean ( standard deviation ) values of roughness ( ra , rmax , rz , and sm ) of vita vmk95 ceramic immersed in various storage agents at different times mean ( standard deviation ) values of roughness ( ra , rmax , rz , and sm ) of vitadur alpha ceramic immersed in various storage agents at different times roughness ( ra , rmax , rz , and sm ) , mean and standard deviation values of ips empress esthetic ceramic immersed in various storage agents at different times roughness ( ra , rmax , rz , and sm ) , mean and standard deviation values of ips e.max ceram ceramic immersed in various storage agents at different times sem photomicrographs of the studied ceramics , before immersion and after 168 hours of immersion periods in different storage agents are presented in figures 14 . before immersion , all of the ceramics tested showed mostly smooth surfaces , with small and little porosities . after exposure to all acidic agents and even deionized water , there were surface changes in various degrees . immersion in 4% acetic acid of all of the ceramics tested showed the most roughening patterns . the gradual degradation of specimen surfaces was observed ; an increase of porosities corresponded with increasing immersion times . the largest defects were found when vitadur alpha was immersed in 4% acetic acid ( figure 2f ) . for ips e.max ceram , fluorapatite crystals could be observed before immersion , but disappeared after immersion ( figure 4 ) . scanning electron microscope photomicrographs of vita vmk95 ( a ) before immersion , and after immersion in ( b ) deionized water , ( c ) citrate buffer solution , ( d ) green mango juice , ( e ) pineapple juice , and ( f ) 4% acetic acid for 168 hours . scanning electron microscope photomicrographs of vitadur alpha ( a ) before immersion , and after immersion in ( b ) deionized water , ( c ) citrate buffer solution , ( d ) green mango juice , ( e ) pineapple juice , and ( f ) 4% acetic acid for 168 hours . scanning electron microscope photomicrographs of ips empress esthetic ( a ) before immersion , and after immersion in ( b ) deionized water , ( c ) citrate buffer solution , ( d ) green mango juice , ( e ) pineapple juice , and ( f ) 4% acetic acid for 168 hours . original magnification 2000 . scanning electron microscope photomicrographs of ips e.max ceram ( a ) before immersion , and after immersion in ( b ) deionized water , ( c ) citrate buffer solution , ( d ) green mango juice , ( e ) pineapple juice , and ( f ) 4% acetic acid for 168 hours . the results of the present study support rejecting the null hypothesis since the acidic agents used and even deionized water caused rough surfaces to the ceramics tested . in oral cavity , the ceramic restorative materials would be exposed to the various temperature and acidic - base changes from food and beverages . hence , the ceramic materials should resist or have only little changes in these environments . previous studies have reported a clinical service life of metal - ceramic restorations up to 20 years.2829 the speed of the degradation process was accelerated by increasing the aggressiveness of the agent compared with intraoral conditions . therefore , a long immersion time was used in this study as an alternative for demonstrating the extensive effect of acidic solutions . the 168 hours used here is comparable to 22 years of immersion in artificial saliva at 22c.8 the results of roughness values in the present study demonstrated that none of the ceramics evaluated were found to be chemically inert . a very low level of roughness or degradation occurred even in a neutral aqueous condition ( the deionized water ) as observed on sem photomicrographs , although there was no significant difference in surface roughness . the two dominant mechanisms have been explained for this degradation process.2 firstly , the selective leaching of alkali ions , and secondly , the dissolution of the ceramic silicate network ( si - o - si ) . these mechanisms are controlled by the diffusion of hydrogen ions or hydronium ions ( h3o ) from an aqueous solution into the ceramic and loss of alkali ions from the ceramic surface into an aqueous solution to maintain electrical neutrality.2 the results in rough surfaces are seen on sem photomicrographs and roughness values in this study . contact and non - contact methods are currently used for surface roughness measurement.30 non - contact methods use a light beam or a laser beam to obtain a surface profile . one of the disadvantages of this method is that shiny surfaces are sometimes difficult to measure due to the scattering effect of the reflected light . this can cause false values being documented.30 therefore , a contact method with a profilometer was used in the present study . although it has been claimed that when using this contact method , the stylus tip may damage or alter the surfaces tested,26 the results of this study as seen on the sem showed that this method did not cause any damage to ceramic surfaces . there were no scratches observed on the sem because the measuring force applied by the stylus tip was 4 mn , which was very little . the ra value is the most commonly used roughness parameter in both dentistry and engineering.31 nevertheless , the ra value is limited by the two - dimensional aspect , providing only information on the average roughness height , and also giving no information at all on the surface profile.26 to clearly demonstrate given this limitation , this study used additional roughness parameters , rmax , rz , and sm , including photomicrographs of sem . rmax and rz represented the amplitude parameter in the vertical axis which showed how the depth of degradation was . sm showed the average spacing between the peaks which presented how the width of alteration was . the combination of quantitative measurements and qualitative data by microscopy supports a qualitative value in three dimensions of the surface tested.3032 as seen from the roughness results of the present study along with sem photographs , it was clearly showed that 4% acetic acid significantly caused rough surfaces to the ceramics evaluated in all dimensions ( p < 0.05 ) . in addition , roughness measurements achieved from relatively short scans may not be representative of the whole surface . it was shown in the present study that the roughness values of the ceramics corresponded with those of other studies.303133 regarding the types of studied ceramics , vitadur alpha revealed the greatest degradation after immersion in 4% acetic acid . this result seemed to show that alumina crystals in vitadur alpha had the least durability compared with leucite crystals in ips empress esthetic and vmk 95 and fluorapatite crystals in ips e.max ceram . previous studies have documented that increasing surface roughness of ceramics may decrease strength.1213 another study also reported that the critical mean ra for the adhesion and colonization of bacteria on restorative materials was 0.2 m.34 this value is equivalent to the ra value of ceramics evaluated in this study before immersion . the increasing surface roughness of the studied ceramics may cause bacterial colonization , strength reduction of the ceramics evaluated , and would result in clinical failure of ceramic restorations . they consist of citric acid , which has been reported as a harmful acid that can cause dental erosion.2325 this citric acid might affect a change to the surface roughness of ceramics due to its chelating effect.8 this effect takes place by complex binding of citrate molecules ( as chelating acids ) to dissolved metal ions of ceramics in acidic agents , and results in more ion dissolution of ceramics to maintain electrical neutrality.1 acetic acid is the acid used for chemical stability testing in accordance with iso standard 6872.27 acetic acid is a weak organic acid , however , it is fairly corrosive to ceramics because of its chelating effect.1 a similar effect has been found in citric acid . this investigation was an in vitro study and the different oral conditions such as saliva,35 water , the ph level , and temperature changes may affect the results . therefore , further studies are required to elaborate the effect of acidic agents on dental ceramics in vivo . within the limitations of this in vitro study , it can be concluded that the surface roughness of the evaluated ceramics were negatively affected by the acidic agents tested , citrate buffer solution , green mango juice , pineapple juice , and 4% acetic acid at 80c for 168 hours . this fact should be taken into consideration when restoring the affected tooth with ceramic restorations in patients who have a high risk of erosive conditions .

background : an increase in surface roughness of ceramics may decrease strength and affect the clinical success of ceramic restorations . however , little is known about the effect of acidic agents on ceramic restorations . the aim of this study was to evaluate the surface roughness of dental ceramics after being immersed in acidic agents.methods:eighty-three ceramic disk specimens ( 12.0 mm in diameter and 2.0 mm in thickness ) were made from four types of ceramics ( vmk 95 , vitadur alpha , ips empress esthetic , and ips e.max ceram ) . baseline data of surface roughness were recorded by profilometer . the specimens were then immersed in acidic agents ( citrate buffer solution , pineapple juice and green mango juice ) and deionized water ( control ) at 37c for 168 hours . one group was immersed in 4% acetic acid at 80c for 168 hours . after immersion , surface roughness was evaluated by a profilometer at intervals of 24 , 96 , and 168 hours . surface characteristics of specimens were studied using scanning electron microscopy ( sem ) . data were analyzed using two - way repeated anova and tukey 's multiple comparisons ( = 0.05).results : for all studied ceramics , all surface roughness parameters were significantly increased after 168 hours immersion in all acidic agents ( p < 0.05 ) . after 168 hours in 4% acetic acid , there were significant differences for all roughness parameters from other acidic agents of all evaluated ceramics . among all studied ceramics , vitadur alpha showed significantly the greatest values of all surface roughness parameters after immersion in 4% acetic acid ( p < 0.001 ) . sem photomicrographs also presented surface destruction of ceramics in varying degrees.conclusion:acidic agents used in this study negatively affected the surface of ceramic materials . this should be considered when restoring the eroded tooth with ceramic restorations in patients who have a high risk of erosive conditions .

Introduction Materials and Methods None Specimen preparations Storage agent immersions and surface roughness measurements Surface topography analysis Statistical analysis Results Discussion Conclusion

PMC3195815

the increased prevalence of food allergies is a significant clinical and public health problem in the usa . it is estimated that in the usa alone , food - allergic reactions accounted for over 20,000 emergency department visits , 2,300 episodes of anaphylaxis , and over 500 hospitalizations over just a 2-month period in 2003 [ 1 , 2 ] . despite the prevalence of food allergy , diagnosis and treatment eight types of food account for over 90% of allergic reactions in affected individuals : milk , eggs , peanuts , tree nuts , fish , shellfish , soy , and wheat [ 3 , 4 ] . reactions to these foods by an allergic person can range from a tingling sensation around the mouth and lips and hives to death , depending on the severity of the allergy . additionally , allergic reactions to various foods often develop into allergy - related respiratory diseases such as asthma , allergic rhinitis , and atopic dermatitis . several studies provide strong evidence that both host genetic susceptibility and environmental factors determine the complex regulation of ige - mediated food allergies , however , the mechanisms by which a person develops an allergy to specific foods are largely unknown . as the ige - mediated , immediate type food hypersensitivity reactions account for significant morbidity and mortality , further understanding of these mechanisms that contribute to the allergenicity could improve the prediction , diagnosis , and management of severity food allergy in individuals . food allergies are caused by ige - dependent or ige - independent immunologic reactions , which then lead to an inflammatory reaction , in which mast cells , eosinophilic granulocytes , and other cells are involved . food intolerances or nonimmunologic food incompatibilities are often caused by specific enzyme deficiencies and must be diagnostically differentiated from food allergies . constant allergen exposure and other environmental factors determine whether a sensitised individual will become chronically allergic and experience persistent symptoms . therapeutics for the treatment of allergy are focused on prevention or diminishing the specific serum immunoglobulin e ( ige ) responsible for the appearance of the allergic reaction [ 5 , 6 ] . proteomic technologies play an important role in drug discovery , diagnostics , and molecular medicine because they are the link between genes , proteins , and disease states . identifying unique patterns of protein expression or biomarkers associated with ige - mediated food allergy genomics provides only a partial picture , while proteomics identifies specific proteins responding to gene expression . progress in protein annotation and in our understanding of protein - protein interactions will undoubtedly lead to diagnostic and therapeutic advances in the treatment of food allergies . proteomics research technologies are rapidly changing our understanding of complex and dynamic biological systems by providing information relevant to functionally associated changes in protein abundances , protein protein interactions , and posttranslational modifications [ 710 ] . 2-dimensional fluorescence difference gel electrophoresis ( 2d - dige ) has emerged as a robust method to study protein expression profiling in clinical samples . this method separates proteins in 2 dimensions according to their isoelectric point and their molecular size . fluorescent , 2-d dige [ 1113 ] allows the multiplex analysis of 3 sample proteomes on the same gel . data from proteomic analysis provide enormous amount of biological information and can help establish multimodal markers for early diagnosis and prognosis . additionally , these proteomic data can provide an opportunity for identifying molecular pathways that underlie various clinical allergic phenotypes . identifying unique biomarkers associated with certain allergic phenotypes , and potentially cross - reactive proteins that can be identified through bioinformatics analyses will provide enormous insight into the mechanisms that underlie allergic response in patients with food allergies . the current study examines the proteomic profile of a group of rast subjects with severe food allergies and compares their proteomic profile with rast subjects . our goal is to not only identify biomarkers that are specific to food allergies that result in ige - mediated atopy but also to identify molecular mechanism that underlie the allergic response in patients with food allergies . both pediatric and adult patients with mild to severe food allergies who were evaluated in our allergy clinic were informed about the research study and some patients volunteered to participate . patients were consented based on the hsirb guidelines ; in case of minors , parental consent was obtained as per the irb guidelines . a total of 10 ml of blood sample was drawn from the patient , of which a portion of the sample approx 3 ml was used for a rast ( radioallergosorbent test ) as a routine clinical procedure and the remaining sample was used for isolation of peripheral blood mononuclear cells ( pbmc ) which were used for the proteomics analysis . further , gene expression studies were done using rna which was extracted from these cells . rast is a blood test used to detect specific ige antibodies to suspected or known allergens , in this case , specific to food allergens . the ige antibody is associated with type i allergic response . based on the results of the rast test , the patients were classified into rast positive ( rast ) or rast negative ( rast ) . a total of n = 9 rast and n = 12 rast patients were recruited for the study . table 1 provides demographic and clinical information for the food rast and rast subjects . comparative analysis was done between rast and rast patient groups using 2d - dige proteomic analysis . three independent sample pools from rast versus rast patient samples were analyzed by 2d - dige for identifying the differentially expressed proteins . each pooled sample consisted of 3 rast and 3 rastpatient samples , respectively . peripheral blood leukocytes were isolated from whole blood using ficoll - hpaque gradient ( amersham - pharmacia , piscataway , nj , cat number 17 - 1440 - 03 ) . samples were centrifuged for 30 min at 700 g and 20c without applying a brake . the pbmc interface was carefully removed by pipetting and was washed twice with pbs / edta and resuspended in 2 ml of complete rpmi media and total number of cells counted using a hemocytometer . cytoplasmic rna is extracted using trizol reagent ( invitrogen- life technologies , carlsbad , calif ) . the amount of rna is quantitated using a nanodrop nd-1000 spectrophotometer ( nanodrop wilmington , del ) and isolated rna is stored at 80c until used . rna is reverse transcribed to cdna using the reverse transcriptase kit from promega ( promega inc , madison , wis , usa ; cat number a3500 ) . relative abundance of each mrna species is quantitated by real time quantitative pcr for specific primers using the brilliant sybr green qpcr master mix from stratagene ( stratagene inc , la jolla , calif , usa ; cat number 600548 - 51 ) . all data are controlled for quantity of rna input by performing measurements on an endogenous reference gene , -actin . in addition , results on expression levels of different genes from rast samples are normalized to results obtained from rast samples . pbmc isolated from rast and rast subjects were washed twice with 1x pbs ( invitrogen , grand island , ny , usa ) . total protein was extracted using standard cell lysis buffer ( 30 mm triscl ; 8 m urea ; 4% ( w / v ) chaps , adjusted to ph 8.5 ) for 10 min on ice . the cell lysate was centrifuged at 4c for 10 min at 12000 g and was further purified by precipitation with chloroform / methanol as described . protein concentrations were determined using the coomassie protein reagent ( bio - rad , hercules , calif , usa ) prior to dige analysis . the ettan dige technique developed by ge healthcare ( piscataway , nj , usa ) was used to detect differences in protein abundance between the rast and rast samples . the ettan dige system uses 3 cydye dige fluors ( cy2 , cy3 , cy5 ) , each with a unique fluorescent wavelength , matched for mass and charge . cydyes form a covalent bond with the free epsilon amino group on lysine residues of the sample proteins . this system allows in each set of gel 2 experimental samples and an internal standard to be simultaneously separated on the same gel . the internal standard is comprised a pool of an equal amount of all the experimental samples . the use of an internal standard facilitates accurate inter - gel matching of spots , and allows for data normalization between gels to minimize gel to gel experimental variability . all reagents used were from ge healthcare ( amersham biosciences , piscataway , nj , usa ) . three independent ( n = 3 ) 2d - dige experiments were done comparing rast and rast patient samples . briefly , six samples ( 3 rast and 3 rast ) , each of 50 g cell lysate were labeled with 400 pmol of either cy3 or cy5 , kept on ice for 30 min and then quenched with a 50-fold molar excess of free lysine . 50 g of protein pooled from equal amounts of protein from all the six experimental samples were labeled with cy2 and used as an internal standard in each of the three sets of cy3/cy5 gels . cy3- , cy5- , and cy2-labeled samples and unlabelled protein ( 5001000 g ) were pooled . unlabeled protein was added to enhance the protein staining and hence a better image quality of the sypro ruby staining . an equal volume of 2x sample buffer ( 8 m urea ; 2% ( v / v ) pharmalytes 310 ; 2% ( w / v ) dithiothreitol ( dtt ) ; 4% ( w / v ) chaps ) was added and incubated on ice for 10 min . the total volume of sample was adjusted to 450 l with rehydration buffer ( 4% ( w / v ) chaps ; 8 m urea ; 1% ( v / v ) pharmalytes 310 nonlinear ( nl ) ; 13 mm dtt ) . samples were applied to immobilized ph gradient ( ipg ) strips ( 24 cm , ph 310 nl ) and absorbed by active rehydration at 30 v for 13 hr . isoelectric focusing was carried out using an ipgphor ief system with a 3-phase program ; first phase at 500 v for 1 hr , second phase at 1000 v for 1 hr , and third phase ( linear gradient ) 8000 v to 64000 v for 2 hr ( 50 ua maximum per strip ) . prior to separation in the second dimension , strips were equilibrated for 15 min in equilibration buffer i ( 50 mm tris - hcl , 6 m urea , 30% ( v / v ) glycerol , 2% ( w / v ) sds , 0.5% ( w / v ) dtt ) . the strips were again equilibrated for 15 min in equilibration buffer ii ( 50 mm tris - hcl , 6 m urea , 30% ( v / v ) glycerol , 2% ( w / v ) sds , 4.5% ( w / v ) iodoacetamide ) and the equilibrated ipg strips were transferred onto 18 20 cm , 12.5% uniform polyacrylamide gels poured between low fluorescence glass plates . gels were bonded to inner plates using bind - silane solution ( promega , madison , wis , usa ) according to the manufacturer . strips were overlaid with 0.9% agarose in 1x running buffer containing bromophenol blue and were run for 16 hr ( 1.8 w / gel overnight ) at 15c in an ettan dalt electrophoresis system . after the run was completed , the 2d gels were scanned 3 times with a typhoon 9410 imager , each time at different excitation wavelengths ( cy3 , 532 nm ; cy5 , 633 nm ; cy2 , 488 nm ) . images were cropped with imagequant v5.2 software and then imported into decyder differential in - gel analysis ( dia ) v5.0 software from ge healthcare for spot identification and normalization of spot intensities within each gel . gels were fixed in 30% ( v / v ) methanol , 7.5% ( v / v ) acetic acid for 3 hr and stained with sypro - ruby dye ( molecular probes , eugene , ore , usa ) overnight at room temperature . decyder software ( ge healthcare ) was specifically developed for use with the ettan dige system . decyder software allows for automatic detection of spots , background subtraction , quantitation , normalization , internal standardization , and integral matching . the differential in - gel analysis ( dia ) component of decyder software draws boundaries around spots in a composite gel image obtained from the intragel overlap of the cy2- , cy3- , and cy5- scanned images and normalizes the data from each cydye to account for differences in dye fluorescence intensity and scanner sensitivity . for all the three sets of experiments ( six images ) , the abundance difference between samples ( rast and rast ) run on the same gel was then analyzed . the biological variation analysis ( bva ) component of decyder software was then used to match all image comparisons from individual cy3/cy5 gel sets for a cross - gel statistical analysis . decyder bva initially calculates normalized intensities ( standard abundance ) for all spots by comparison to the internal standard , and from this , an average volume ratio and a student 's paired t - test derived p value were calculated for each spot . spots were excised from 2d - gels loaded with 500 to 1000 g of total protein and stained with sypro ruby ( molecular probes ) . the gel plugs were incubated in 200 l of 200 mm ammonium bicarbonate with 50% acetonitrile for 30 min at 37c and then dried with a speed vac . samples were then incubated for 1 h at room temperature in 3.5 l of 20 g / ml trypsin ( promega ) in 40 mm ammonium bicarbonate . an additional 30 l of 40 mm ammonium bicarbonate with 10% acetonitrile was added and samples were incubated overnight at 37c . the samples were desalted using c18 ziptips ( millipore , bedford , mass , usa ) and eluted directly onto a mtp ground steel 384-target ( bruker daltonics , billerica , mass , usa ) with the matrix solution ( a saturated solution of -cyano-4-hydroxy - cinnamic acid [ chca : bruker daltonics ] dissolved in 0.1% trifluoroacetic acid:75% acetonitrile ) . maldi spectra in positive ion mode were obtained using a bruker daltonics biflex iv maldi - tof mass spectrometer operating in the reflectron mode . the spectra in the range of 5003200 da were obtained by the summation of 50200 laser shots . external calibration was carried out using the peptide calibration standard ii ( 7004000 da ) ( bruker daltonics , number 222570 ) . data acquisition was executed using the flex control program ( build 2.4.0.0 ) and data processing was performed using the bruker xtof 5 software ( version 5.1.5 ) . the signatures of proteins of interest formed by the masses of the products of tryptic digestion were analyzed using an on - line search engine mascot ( http://www.matrixscience.com/ ) to compare the calculated masses of a theoretical tryptic digestion . searches were conducted for matching masses from theoretical tryptic peptides from the ncbi protein databases . we used the following search parameters : monoisotopic masses , with a tolerance of 200 ppm or less ( typically 75100 ppm or 0.10.5 da ) , one missed tryptic cleavage , a carbamidomethyl modification of cysteine ( to account for iodoacetamide treatment ) and variable modifications of oxidation of methionine and phosphorylation of serine , threonine or tyrosine . a protein identification was considered significant for a mowse score with p < 0.05 . protein profiles of rast and rast pbmcs were analyzed by dige to examine the differential expression of proteins among the two groups . protein samples from rast and rast pbmc cell lysates were labeled with fluorescent cydyes , pooled and analyzed in triplicate by 2d - dige . the pooled mixture of equal amounts of protein from each of the six experimental samples labeled with cy2 and run on every gel provided an internal standard to ensure that every protein in each sample appears on all of the gels . each cy3- or cy5-labeled sample was then compared to the same internal standard and measurements were taken relative to the internal cy2 standard , thereby reducing gel - to - gel variation and increasing statistical confidence . the dia ( differential in - gel analysis ) module of the differential analysis software decyder ( ge healthcare ) was used to calculate protein spot volumes and the normalized volume ratio for each differentially labeled comigrated protein . on an average of about 1000 spots were detected and matched successfully in three rast and rast cydye - labeled proteome gels ( figure 1 ) . the ratio of spot intensity of the two groups of protein spots is then calculated . gel to gel matching of the standard spot maps from each gel was performed using the decyder bva software module . this allowed for the statistical analysis of changes in protein abundance between samples . statistical analysis ( paired t - test ) was performed for the difference between the abundance of proteins from rast cells versus proteins from and rast cells . a total of 52 protein spots showed a significant change in abundance with p < 0.05 . figure 2 is the sypro ruby - stained 2d gel image showing all protein spots . differentially expressed protein spots showing significant modulation were selected for subsequent identification and are shown as numbered outlines . the maximum increase in protein expression was 1.65-fold ( spot number 1302 in figure 2 ) and the maximum decrease observed was 10.7-fold ( spot # 242 , figure 2 ) . proteomic evaluation of differential expression of proteins in pbmcs of rast and rast subjects by 2d - dige and maldi - tof resulted in a short list of 37 proteins that were found to be significantly modulated ( table 2 ) . the table lists these proteins and includes their symbols , % sequence coverage of the identified peptide , number of matching peptides , theoretical mass , and calculated pi of the identified protein . having shortlisted these key proteins that are differentially expressed in the rast and rast subjects , we evaluated if the gene expression levels of these proteins were significantly different in these two groups . based on an exhaustive literature review and on comparative analysis using gene expression studies by quantitative pcr , we have highlighted a group of key proteins that play a role in food allergenicity . these include chondroitin sulfates , zinc finger proteins , c - type lectins , retinoic acid - binding proteins , heat shock proteins , membrane proteins such as myosin and cytokines such as il-17f , certain mast cell expressed proteins , and signal transduction molecules such as the family of map kinases . almost all of these proteins play a role in immune tolerance and hypersensitivity [ 1822 ] , and they modulate cytokine patterns that favor a th2 response that typically results in ige - mediated allergic response . the following are the gene expression levels of the key proteins that were significantly lower in rast subjects as compared to rast subjects . heat shock protein-40 homolog ( djc15 ) ( 19% decrease , p = 0.001 ) ; c - type lectins ( 15% decrease ; p = 0.006 ) ; mapk ( 24% decrease ; p = 0.0001 ) ; retinoic acid - binding protein ( 18% decrease ; p = 0.002 ) ; chondroitin transferase ( 15% decrease ; p = 0.006 ) ; zinc finger protein ( 10% decrease ; p = 0.05 ) ; il-17f protein ( 12% decrease ; p = 0.02 ) ; and mcem1 mast cell expressed membrane protein 1 ( 29% decrease ; p = 0.0001 ) . the gene expression levels of cytokine il-10 were significantly higher ( 50% increase ; p = 0.0001 ) in the rast subjects as compared to the rast subjects . no significant differences in the gene expression levels of myosin and ikaros were observed between the 2 patient groups . figures 3 , 4 , and 5 show the fold regulation in the gene expression levels of these key proteins . although the fold changes in gene and protein changes in these key molecules are not of similar magnitude , the trends in their expression are in concordance and therefore suggests that these proteins may play a significant role in the development of food allergies . given the high through put data that emerges from proteomic analysis , additional data analysis using novel software tools , can be used to interrogate the biological context of lists of genes and proteins . we used one such software tool , metacore , which uses a proprietary , manually curated database of human protein - protein , protein - dna , and protein compound interactions , metabolic and signaling pathways which is supported by proprietary ontologies . the transcription regulation networks of differentially expressed proteins in the rast and rast groups were generated by metacore software using a transcription regulation algorithm . the list of significantly regulated proteins emerged after 2d - dige / maldi - tof analysis of the rast and rast samples , highlighted in table 2 , was entered in to metacore . the metacore software has in - built algorithms that create biological networks from the uploaded protein list and assign biological processes to the generated networks . algorithm of metacore which is useful in providing additional information that is not present in the original list of root nodes . 37 differentially expressed proteins identified on comparison between the rast and rast groups were mapped into the software and subsequently analyzed . networks were generated as per p value , z - score and g - score . p value is based on the saturation of each sub - network with proteins from the user selected list . z - score ranks the subnetworks according to saturation with the objects from the initial list of seed objects . the z - score ranks the sub - networks of the analyze network algorithm with regard to their saturation with proteins from the experiment . the g - score modifies the z - score based on the number of canonical pathways used to build the network . if a network has a high g - score , it is saturated with expressed proteins ( from z - score ) and it contains many canonical pathways . we chose the top 3 networks from the search results as ranked by both g - score and p values . the significance of association between the biological processes and the differentially expressed proteins were represented by p values . the list of most significant go processes associated with the network is shown in table 3 along with the respective p values . the top 3 processes , the percentage of target nodes involved and the corresponding p values are positive regulation of immune system process ( 35% , p - value 2.2e 50 ) , myd88-independent toll - like receptor signaling pathway ( 19.32% , 2.51e 49 ) and toll - like receptor 3 signaling pathway ( 19.32% , 4.1e 49 ) . figure 6 illustrates the close clustering and interconnectedness of biochemical process networks of the identified proteins which are differentially regulated . allergic responses to food antigens involve a state of immediate hypersensitivity to certain food proteins . the mechanism underlying the initiation and development of allergic responses involves cytokine activation which is believed to directly induce the differentiation of effector th2 lymphocytes ; these th2 responses play a pivotal role in the development of allergic responses to specific food antigenic triggers . in this paper , we utilized a high throughput proteomic approach , 2d - dige , coupled with maldi - tof protein identification methods to study the differential expression of proteins in two groups of rast and rast subjects with the goal to identify key proteins that may play a role in the development of food allergenicity . in contrast to traditional biochemical approaches that can only study one or a few particular protein species , proteomics analysis allows the simultaneous analysis of thousands of proteins . this is the first comprehensive differential proteomic analysis of pbmc obtained from rast and rast subjects . over 1,000 distinct proteins were identified of which about 52 proteins displayed significant differential expression between the rast and rast groups . of these 52 significantly regulated proteins , 37 were successfully identified ( table 2 ) . a large proportion of these proteins have direct or indirect biological relevance to food allergy . food allergenicity is characterized by abnormal ige production , peripheral eosinophilia , mast cell activation , and induction of th2 lymphocytes expressing cytokines such as il-4 and il-10 [ 23 , 24 ] . even though we could not observe any differential expression of il-4 and il-10 in the 2d - dige analysis , metacore analysis indicated a very active involvement of these molecules in the networks . a further qpcr analysis showed a significant increase in il-10 levels in rast patients as compared to rast patients . il-10 is a cytokine that downregulates both th1 and th2 cytokine production , and the fact is that its levels are significantly elevated in rast patients and may be useful in identifying human clinical tolerance to foods . il-10 is also believed to help differentiation of t helper cells into the th2 phenotype , thereby contributing to the development of allergic responses to food antigens . il-4 levels in rast patients were lower but not statistically significant as compared to rast patients . a decrease in il-4 is believed to impair the development of food allergy and the aversion to antigen . production of il-4 and levels of specific ige / igg1 antibodies correlate with aversion to antigen induced by food allergy in mice . the mechanism underlying the initiation and development of allergic responses involves il-4 that directly induces the differentiation of committed effector th2 lymphocytes , and th2 responses play a pivotal role in the development of allergic responses . il-4 inhibits the induction of foxp3 and the generation of inducible regulatory t cells and may also function as anti - inflammatory cytokine . il 17f is reported to be involved in inflammation and a study on effect of il-17/il-17f deficiency in an allergic asthma il-17 ko mice model , exhibited reduced th2 cytokine expression , whereas il-17f ko mice showed elevated type 2 cytokines and eosinophil functions compared with wt mice . this indicates that il-17 and il-17f may have opposite functions in chronic allergic airway diseases . the precise role of il-17 and a new population of il-17-producing th cells ( th17 ) in allergic inflammation is unclear , but th17 cells are characterized by the production of inflammatory cytokines such as il-17a , il-17f , il-22 , and il-26 . the highest percentage of il-17-producing cells has been found in severe atopic dermatitis , suggesting a direct correlation between the presence of th17 cells and severity of the disease , indicating that there is possible cross - talk between th17 cells and eosinophils . our observation of a significant decrease in il-17 in rast patients is contradictory to what we expected based on its role as a promoter of allergic inflammation . this observation leads us to believe that il-17 may be associated with patterns of cytokine dysregulation that are associated with specific atopic phenotypes , though the precise mechanism of its modulation is unclear . mast cells are believed to be the key effector cells of ige - mediated anaphylactic reaction , therefore , a modulation in the expression of the mast cell expressed protein mcem1 in rast patients is anticipated . we observed a significant decrease in mcem1 expression in rast patients . the mapk pathway and its downstream effectors play an important role in the regulation of cytokine expression in mast cells . signals emanating from many cell surface antigenic receptors and environmental stimuli can converge on the mapk pathway , which in turn phosphorylates and activates various transcriptional factors and molecular effectors , thereby modulating allergic response . proteins such as zinc finger proteins , retinoic acid - binding proteins , chondroitin transferase , c - type lectins and heat shock proteins in the soluble microenvironment are believed to be involved in the conditioning of antigen presenting cells , thereby promoting tolerance to dietary antigen primarily via the induction of regulatory t cells . this family of proteins plays an important role in hematopoietic development and is believed to modulate the levels of an immunosuppressive compound called bis(tributyltin)oxide ( tbto ) that is present in the diet and which can contribute significantly to the development of food allergy to peanut or ovalbumin . tbto can decrease allergen - specific th2 cytokine production , number of eosinophilic and basophilic granulocytes in the blood and production of mast cell proteases after an oral food challenge . to understand the relevance of these proteins and their role in allergy or overall inflammation and to ascertain their interaction with other proteins in known networks the identified proteins of the total 37 identified proteins imported to metacore , 20 were successfully mapped in the networks ( figure 6 ) . the proteins differentially regulated in rast subjects were shown in red circles in the network . in protein network generated using the uploaded , differentially expressed proteins in this study showed significant links to inflammatory and immunological responses . among the upregulated proteins all except globin-1 could be mapped on to the network . the current investigation has helped to identify key proteins involved in food allergenicity that provide evidence for a relationship between th2 immune response and food allergy and suggests that these responses play an important role in the development of food allergy . this study provides important information about global protein expression in rast patients with food allergies . although we could not subdivide the patients with food allergies into specific food type , namely , peanut , egg , shell fish , and so forth , the identification of these key proteins provides clues to elucidate mechanisms of the structures that contribute to allergenicity , which thus , in turn , would help alleviate food allergens . further , these key proteins can be utilized as diagnostic markers that will not only help in the diagnosis and management of food allergy but also may be used in future immunotherapy .

proteomic profiles of rast+ subjects with severe food allergies and rast subjects were compared using 2d - dige analysis to obtain candidate biomarkers specific to food allergies . our analysis highlighted 52 proteins that were differentially expressed between the rast+ and rast groups of which 37 were successfully identified that include chondroitin sulfates , zinc finger proteins , c - type lectins , retinoic acid binding proteins , heat shock proteins , myosin , cytokines , mast cell expressed proteins , and map kinases . biological network analysis tool metacore revealed that most of these regulated proteins play a role in immune tolerance , hypersensitivity and modulate cytokine patterns inducing a th2 response that typically results in ige - mediated allergic response which has a direct or indirect biological link to food allergy . identifying unique biomarkers associated with certain allergic phenotypes and potentially cross - reactive proteins through bioinformatics analyses will provide enormous insight into the mechanisms that underlie allergic response in patients with food allergies .

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

PMC4131328

the hue samples were designed to represent perceptually equal steps of hue and to form a natural hue circle . they are numbered on the back according to the correct color order of the hue instruction manual and scoring sheets are provided . the examiner prearranges the caps in random order on the upper lid of the box . the observer is instructed to arrange the caps in order according to color in the lower tray [ with a see - through bottom ] , where the two fixed caps appear . the box is presented for each eye at a comfortable distance under illuminate c providing at least 270 lux of natural light . the generally recommended time for arranging each panel is 2 minutes . for each panel , the time spent on it is recorded , depending on the first panel , second panel , etc . score of a cap is the sum of the differences between the number of that cap and the numbers of the caps adjacent to it on either side , e.g. , if cap number 50 is wrongly positioned say between 55 and 56 , then the score of this cap is 55 - 50 + 56 50 = 5 + 6 = 11 . 50 had been correctly positioned , then the score of that cap would have been 50 49 + 51 -50 = 2 : and with 2 subtracted , its error score would have been zero ] . sum of the error scores of the entire set of caps goes to make the total error score ( tes ) . by plotting the scores manual scoring of error scores and plotting graphs are of course extremely time consuming and very tedious . to overcome this , our attempt was to save needless time - wastage during precious hospital working hours , and increase maximum patient turnover in the limited time available to the professional specialist . the test consists of 85 movable color samples arranged in four opaque boxes of 22 colors in the first box and 21 colors in each of the remaining 3 [ no . 85 - 21 , 22 - 42 , 43 - 63 , and 64 - 84 ] . the hue samples were designed to represent perceptually equal steps of hue and to form a natural hue circle . they are numbered on the back according to the correct color order of the hue instruction manual and scoring sheets are provided . the examiner prearranges the caps in random order on the upper lid of the box . the observer is instructed to arrange the caps in order according to color in the lower tray [ with a see - through bottom ] , where the two fixed caps appear . the box is presented for each eye at a comfortable distance under illuminate c providing at least 270 lux of natural light . the generally recommended time for arranging each panel is 2 minutes . for each panel , the time spent on it is recorded , depending on the first panel , second panel , etc . score of a cap is the sum of the differences between the number of that cap and the numbers of the caps adjacent to it on either side , e.g. , if cap number 50 is wrongly positioned say between 55 and 56 , then the score of this cap is 55 - 50 + 56 50 = 5 + 6 = 11 . 50 had been correctly positioned , then the score of that cap would have been 50 49 + 51 -50 = 2 : and with 2 subtracted , its error score would have been zero ] . sum of the error scores of the entire set of caps goes to make the total error score ( tes ) . by plotting the scores manual scoring of error scores and plotting graphs are of course extremely time consuming and very tedious . to overcome this , our attempt was to save needless time - wastage during precious hospital working hours , and increase maximum patient turnover in the limited time available to the professional specialist . the original fm 100-hue carrier box consists of a wooden cover with 4 elongated boxes inside it . this is an opaque outer cover , so there is obvious difficulty in photo - documentation . one had to manually record the scores of caps on sheets , which hugely prolonged the time spent per patient [ fig the original fm 100-hue elongated boxes with an opaque wooden cover we modified the fm 100-hue procedure by converting the outer opaque box into a transparent clear plastic carrier container that allowed a ready photo - documentation of the arranged open trays from the top and bottom without even opening the box or inverting the caps [ fig . 2 ] . ( the inner trays already have a flip - top opaque cover , but a see - through bottom ) [ figs . 3 and 4 ] . we screened 200 reportedly normal and 50 known color defectives using this simply modified fm 100 -hue test , first od then os . after capturing the digital images from both the bottom and top of the arranged fm 100-hue box with patient i d , the [ used ] box was promptly handed over to the next subject for rearrangement . our study followed the tenets of the declaration of helsinki and was duly approved by our institutional ethics committee . our modified transparent carrier box for the fm-100 hue test the caps arranged by a reportedly normal subject , according to hue perceived [ photographed through the transparent plastic top cover ] - the arrangement is almost normal but slightly defective showing arranged numbers imaged from the bottom through the transparent plastic [ along with the patient i d ] - note the slight defects the immediate test running time for each subject including the technician 's measuring and plotting time was reduced from 60 - 75 min to ~15 min with no waste of invaluable lab hours . the box is relatively cheap and easy to maintain , and also replaces whenever required [ figs . 3 and 4 ] . these numbers on the caps are normally not visible because of the original opaque cover evidently , the investigator is standing by to observe that the patient does not turn over the entire box to peep at the numbers of the color buttons . nowadays , the farnsworth munsell 100-hue test often includes windows based scoring software , which calculates a numerical score and provides a graphic display and printout of the subject 's score and status . our simple modification and adjustment of the original box of the fm 100-hue test alone proved to be not just cost - effective but also permitted rapid assessment of color vision and quick patient turnover with easy data storage , without the need of any expensive scoring software with a requirement of a routine camera , preferably digital , for easy retrieval .

purpose : the farnsworth - munsell ( fm ) 100-hue test is well known but is also time consuming , especially its analytical component . to reduce this needless time - waste during precious working hours , a simple modification was devised.design:prospective , comparative , observational study.materials and methods : a transparent clear plastic carrier box replaced the opaque one , allowing ready digital photodocumentation of top and bottom without even opening the box , or handling / inverting the caps -200 reportedly normals and 50 known color vision defectives could be easily tested on this modified - fm and results stored , allowing rapid turnover . the captured scores with patient i d were analyzed , at leisure , outside hospital time , saving 45 - 60 minutes / patient . after recording , the box was promptly handed over to the next subject for rearrangement . times taken for test / patient were recorded.results:running time was reduced from 60 - 75 min to ~15 min / patient with no waste of invaluable lab hours . turnover time is limited to capturing two photographs ( ~60 sec ) . the box is relatively cheap and easy to maintain.conclusions:our simplified fm 100-hue test allowed rapid assessment of color visions with easy data storage of both top and bottom .

None Administration Scoring Materials and Methods Results Conclusion

PMC2915873

trabeculectomy has been the surgery of choice in eyes with glaucoma since it was first described by cairns in 1968.1 antifibrotic drugs , such as 5-fluorouracil and mitomycin c ( mmc ) , have been commonly used adjunctively to prevent scar formation , and they have increased the success rate of trabeculectomy . however , there is still a low but unacceptable rate of failure of trabeculectomy because of progressive scarring of the subconjunctival tissues . patients with secondary glaucoma , aphakia , and prior surgery to the conjunctiva are reported to have poorer success rates following trabeculectomy.2 when filtration surgery fails to reduce the intraocular pressure ( iop ) , a revision of the bleb is one of the options . several investigators have advocated bleb revision with adjunctive 5-fluorouracil or mmc,3 while others have advocated the use of an amniotic transplant.47 the amniotic membrane is the innermost layer of the three - layered fetal membrane , and has anti - inflammatory , antifibrotic , and antiangiogenic properties.8,9 amniotic membranes have been transplanted to reconstruct the ocular surface after pterygium excision10 and in nonhealing corneal epithelial defects.11 nagai - kusuhara et al reported that the use of an amniotic membrane for reconstructing a filtration bleb is useful for late - onset bleb leaks.12 because of its ability to suppress wound healing , amniotic membrane transplantation has been used in glaucoma - filtering surgery to prevent subconjunctival fibrosis.47 the purpose of this study was to assess the effectiveness of conventional surgical revision using adjunctive mmc with that of bleb revision using adjunctive mmc and transplantation of an amniotic membrane in lowering iop . the medical charts of 36 eyes of 36 patients who had undergone trabeculectomy and bleb revision by a single surgeon ( yk ) at the otemae hospital from february 2003 to july 2006 were reviewed . this study was approved by the otemae hospital institutional review board , and the procedures used conformed to the tenets of the declaration of helsinki . two groups were compared ; there were 24 eyes in the nonamnion - transplanted groups and 12 eyes in the amnion - transplanted group . the patients in the nonamnion group had conventional bleb revision with adjunctive mmc between february 2003 and july 2005 . the remaining 12 patients had bleb revision with amniotic membrane transplantation and mmc between august 2005 and july 2006 . the surgical procedures were slightly modified from those reported by drolsum et al.6 for the surgery , the conjunctiva was anesthetized with a 1 ml injection of 2% xylocaine , and a conjunctival incision was made approximately 9 mm from the limbus or at the limbus . for this , the conjunctiva was separated from the sclera , and the scar tissue was dissected from the scleral surface both anteriorly and posteriorly . after applying 0.4 mg / ml mmc to the sclera for five minutes , the surgical area was irrigated with 100 ml of balanced salt solution . the wall of the bleb or fibrotic membrane on the scleral flap was reopened as confirmed by the leakage of aqueous humor . the amniotic membrane was obtained under sterile conditions after a cesarean delivery and cryopreserved at 80c in dimethyl sulfoxide until immediately before use . in the amnion group , a piece of amniotic membrane measuring approximately 7 10 mm was placed with the epithelial side over the scleral flap . six of 12 cases ( 50% ) in the amnion group had limbus - based surgery , and the remaining six cases had fornix - based surgery . the amniotic membrane was sutured to the sclera at the corneoscleral limbus using 8 - 0 virgin silk . the fornix side of the amniotic membrane was inserted beneath the healthy conjunctiva posterior to the conjunctival incision line or scar line . then two to three sutures were placed 5 mm from the limbus to fix the amniotic membrane to the sclera . then the limbus - based conjunctival flap was closed , as was done in the nonamnionic group . postoperatively , all patients received similar topical medical regimen ; 0.1% topical betamethasone and 0.5% levofloxacin for six weeks to three months which was reduced within three months , depending on the degree of inflammation . patients were examined monthly for the first 3 months , and every 23 months thereafter . success was defined as a 30% reduction of the iop from the preoperative iop and maintenance below 21 mmhg with or without the use of antiglaucoma agents . when additional surgery was performed , the iop control was judged as a failure regardless of iop at the time of the decision . the significance of the differences in success was determined using the log rank test . when the iop was higher than the targeted pressure in two consecutive examinations , the bleb was considered to have failed . when additional glaucoma surgery was performed , the iop control was judged as a failure regardless of the iop level at the time of the decision . the significance of the differences in the demographic characteristics of the subjects and the incidence of surgical complications were determined using mann - whitney u tests and fisher s exact probability tests . the iop changes were evaluated using mann - whitney tests and wilcoxon signed - rank tests . a p value of < 0.05 was considered statistically significant . there were no significant differences in age , duration of follow - up , preoperative iop , number of prior surgeries , number of prescribed antiglaucoma medications , and type of glaucoma between the nonamnion amnion groups . the pretrabeculectomy ( former trabeculectomy before revision ) iops were 21.2 4.0 mmhg in the nonamnion group and 21.8 3.8 mmhg in the amnion group . the mean prebleb revision iop was 21.4 6.5 mmhg in the nonamnion group and 21.8 4.6 mmhg in the amnion group . the postoperative iop at the last visit in the nonamnion group was 12.1 5.5 mmhg and that in the amnion group was 16.0 3.7 mmhg . in each group , the iop was reduced significantly , although the iop in the nonamniotic group was significantly lower than that in the amniotic group ( p = 0.025 ) . the number of postoperative antiglaucoma eye drops was 0.24 0.60 in the nonamnion group and 0.58 0.79 in the amnion group ( p = 0.105 ) . in the amnion group , kaplan - meier lifetable analysis showed that the success rate was 58.3% after six months and 21.9% after one year . the success rate in the nonamnion group was 74.8% from six months to one year after surgery . this difference in the surgical outcome was not significant ( p = 0.109 , see figure 1 ) . at the final visit , two had a fornix - based conjunctival flap , and three had a limbus - based flap . two cases of hyphema were seen in the nonamnion group and one in the amnion group . leakage at the wound edge developed in one case in the nonamnion group and three cases in the amnion group . late complications of trabeculectomy such as hypotonic maculopathy , late bleb leak , bleb - associated infection , and corneal decompensation were not found in any of our patients . although appearance of the bleb is an important factor that predicts iop results and bleb - related complications , we did not evaluate bleb appearance in this study . kaplan - meier lifetable analyses showed that the success rate in the nonamniotic group was 74.8% from six months to one year after the surgery . the mean iop was reduced significantly from 21.4 6.5 mmhg to 12.1 5.5 mmhg in the nonamnion group ( p < 0.001 ) . these findings are in good agreement with those of hara and shirato13 who reported that an iop under 21 mmhg was achieved in 53.4% without medication and in 60.2% with and without medication at 60 months following conventional bleb revision surgery . anand and arora14 reported that the probability of maintaining an iop between 5 and 18 mmhg or a 20% decrease from the preoperative iop was 64% at three years after conventional revision surgery . earlier studies reported that the success rate of bleb revision was around 60% although there were some differences in the criteria for success . thus , our results in the nonamnion group are in good agreement with these earlier findings . while bleb revision with adjunctive mmc is an effective treatment in cases of bleb failure , glaucoma surgeons have been recently examining the effectiveness of amnion membrane transplantation . the amniotic membrane is made of strong and stable collagen , and has good anti - inflammatory , antifibrotic , and antiangiogenic properties . in glaucoma filtration surgery , the healing response is characterized by fibroblast outgrowth , and the fibrosis has been reported to be delayed more by an amniotic membrane than with conventional conjunctival closure.5 for amniotic membrane transplantation , fujishima et al4 transplanted the amniotic membrane under a scleral flap and sutured it to the sclera . for the 14 eyes of the 13 patients with treatment - resistant glaucoma , the iop was reduced to < 20 mmhg after surgery in 13 eyes . drolsum et al6 used a large amniotic membrane , and sutured it to the scleral surface , and one end was positioned under the scleral flap and the other end was secured to subconjunctival tissue . the mean preoperative iop was 32.2 mmhg , and the mean postoperative iop was 16.4 mmhg . although there were differences in the methods and definition of success , they reported that amniotic membrane transplantation was good for the revision of failed glaucoma filtering blebs.46 these reports encouraged us to try to use the amniotic membrane for bleb revision . one year after starting the use of amniotic membranes , we evaluated the efficacy of amniotic membrane transplantation to repair failed filtration blebs , and compared that with conventional bleb revision . kaplan - meier lifetable analyses showed that bleb revision with amniotic membrane transplantation was not better than conventional bleb revision with mmc in our cases . in fact , the postoperative iop was significantly higher in the amnion - transplanted group than in the nonamnion - transplanted group . these results suggest that a subconjunctival placement of amniotic membrane does not improve the filtration in high - risk eyes . these poorer results using amniotic membranes for bleb revision discouraged us from recruiting new patients . so the number of the amnion group is smaller than the nonamnion group . in the amnion group , six cases ( 50% ) were fornix - based , and all of the cases in the nonamnion group were limbus - based because a fornix - based conjunctival flap allowed us to fix the amniotic membrane at the edge easier than a limbus - based flap . earlier studies15,16 reported that a fornix - based conjunctival flap and a limbus - based flap did not significantly alter the iop - lowering effect of trabeculectomy . in our small amnion group , the success rates in the limbus - based group and fornix - based group were not significantly different . the iop control results seemed to be unrelated to the surgical technique of handling the conjunctiva . budenz et al17 stated that amniotic membrane transplantation did not offer an effective alternative to conjunctival advancement for repair of a leaking filtering bleb . the postoperative iop in their amniotic membrane transplantation group was relatively higher than in the conjunctival advancement group , although the difference was not statistically significant . demir et al18 concluded that amniotic membrane transplantation can depress wound healing in a rabbit model of glaucoma . li et al19 described the usefulness of amniotic membrane transplantation for the repair of conjunctival buttonholes during trabeculectomy with mmc . they concluded that amniotic membrane transplantation was useful when used as a platform for re - epithelialization as opposed to tissue replacement for conjunctiva after bleb excision . they reported on the importance of the way the amniotic membrane was handled to obtain good surgical results . recently , sheha et al7 reported that amniotic transplantation in trabeculectomy with mmc for refractory glaucoma had achieved better iop reduction than trabeculectomy with mmc alone . although we sutured the amniotic membrane over the scleral flap , they placed a single layer of amniotic membrane under and around the scleral flap . all of these findings indicate that improvements in the surgical procedures for handling and placing the amniotic membrane are needed . for example , if the amniotic membrane is placed beneath the scleral flap , our outcome might have been improved . amniotic membranes have antifibrotic effects . however , the antifibrotic effects are not as potent as that of mmc.18 the ability of amniotic membrane transplantation to suppress the fibroblast proliferation of the conjunctiva in eyes with refractory glaucoma may not be strong enough to maintain a functional bleb . this can be another reason for the mixed results of amniotic membrane transplantation in glaucoma - filtering surgery . to use the amniotic membrane for bleb revision before the bleb becomes severely scarred there were no severe complications such as hypotonic maculopathy , late bleb leaks , and bleb - associated infection in our cases . the revision of a failed filtering bleb with adjunctive mmc is an effective way of lowering iop . the use of amniotic membrane transplantation did not enhance the surgical outcome in our cases . establishing better ways to handle amniotic membrane may also be a key factor in improving surgical results .

purpose : to compare the effectiveness of standard revision surgery using mitomycin c ( mmc ) with revision using amniotic membrane transplantation and mmc for elevated intraocular pressure ( iop ) after trabeculectomy.patients and methods : a retrospective , nonrandomized comparative study of 36 eyes of 36 patients with a failed trabeculectomy . patients were divided into two groups , ie , a nonamnion - transplanted group and an amnion - transplanted group . the amniotic membrane was placed on the scleral flap under the conjunctiva in the amnion - transplanted group . both groups recovered filtration of aqueous humor from the surgical site with the adjunctive use of mmc . the changes in iop and cumulative survival rate were compared for the two groups . success was defined as a 30% reduction in iop from the preoperative iop and maintenance below 21 mmhg with or without the use of antiglaucomatous agents.results:the mean preoperative iop was not significantly different in the two groups . the mean postoperative iop in the nonamnion group , 12.1 5.5 mmhg , was significantly lower than the iop in the amnion group , 16.0 3.7 mmhg . survival curves in the two groups did not reach significantly different levels.conclusions:conventional surgical bleb revision with mmc can significantly reduce the elevated iop associated with a failed filtration bleb . the use of an amniotic membrane transplant did not improve the surgical outcome in our cases .

Introduction Material and methods Results Discussion Conclusion

PMC5088003

we performed sequential cross - sectional assessments from a prospective cohort study of andean children in peru ( 3 ) . during 20092011 , children <3 years of age from the district of san marcos , cajamarca , peru , were assessed for aris during weekly household visits . the population was rural and had low incomes and limited access to healthcare ( 3,4 ) . institutional review boards of vanderbilt university ( nashville , tn , usa ) and the instituto de investigacion nutricional ( lima , peru ) approved the study . an ari episode was defined as the length of time a child had cough or fever ( 5,6 ) . if a child was ill during a household visit , we assessed for pneumonia or lower respiratory tract infection using imci - who ( integrated management of childhood illness world health organization ) criteria ( 5,7 ) . if the child had an ari during the preceding 7 days , we collected a nasal swab sample and tested it for respiratory viruses by reverse transcription pcr at vanderbilt university ( 6,811 ) . nasopharyngeal swab samples were collected monthly without regard to ari and tested at emory university ( atlanta , ga , usa ) by using quantitative pcr for pneumococcal density determinations . for this study , we used samples collected in 2009 and 2011 , representing periods before and after routine pcv7 use ( 12 ) ( technical appendix ) . nasopharyngeal samples were classified according to their collection time surrounding aris : peri - ari periods included pre - ari ( 814 or 17 days before an ari ) and post - ari ( 17 or 814 days after an ari ) . we compared log - transformed pneumococcal nasopharyngeal densities of samples from ari , peri - ari , and non - ari periods by using multivariable quantile regression with robust ses and adjusting for relevant covariates . in secondary analyses , we assessed the role of respiratory viruses on pneumococcal density in children with aris . because detection of nonrhinovirus respiratory viruses in nasal swabs was infrequent , we grouped samples into 4 distinct groups : rhinovirus only , rhinovirus and other viruses , other viruses only , and negative for any viruses . we examined the role of pneumococcal acquisition on pneumococcal density using pneumococci - positive nasopharyngeal samples from children who had a sample collected within the preceding 60 days . samples were categorized as 1 ) new colonization if the prior sample was negative , 2 ) serotype persistence if the prior sample was the same serotype , and 3 ) serotype replacement if the prior sample was a different serotype . we assessed 3,579 nasopharyngeal samples from 833 children : 450 ( 12.6% ) were collected during aris , 956 ( 26.7% ) during peri - ari periods , and 2,173 ( 57.8% ) during non - ari periods . the median duration for aris was 8 days ( interquartile range [ iqr ] 513 days ) . according to imci - who criteria , 33 samples were associated with pneumonia or severe pneumonia ( 13 ) ( table ) . overall , 36.7% of nasopharyngeal samples were from children who had received > 2 pcv7 doses and were considered vaccinated . approximately 5.0% of samples were from children who had received aminopenicillins , cotrimoxazole , chloramphenicol , or furazolidone within the 7 days preceding sample collection . quantitative pcr detected s. pneumoniae in 68.9% of nasopharyngeal samples ; 78.9% of ari and 65.3% of non - ari samples were positive ( p = 0.06 ) . adjusted analyses showed that densities peaked during aris . in post hoc adjusted comparisons , densities were higher during the 17 days pre - ari ( p<0.0001 ) , ari ( p<0.0001 ) , 17 days post - ari ( p<0.0001 ) , and 814 days post - ari ( p = 0.007 ) than during the non - ari period ( figure 1 ) . estimated median pneumococcal densities with 95% cis ( vertical bars ) by acute respiratory illness ( ari ) period . estimates derived from a quantile regression model that accounted for sex , age , daycare attendance , electricity , water supply , housing materials , kitchen type , smokers at home , vaccination , antimicrobial drug use , season , and altitude of residence . asterisk indicates significantly different from ari samples ; dagger indicates significantly different from non - ari samples . of 450 ari nasopharyngeal samples , 435 ( 97% ) had corresponding nasal swab samples available for identification of respiratory viruses ; 299 ( 68.7% ) tested positive for at least 1 virus . rhinovirus , which was detected in 44.6% ( 194/435 ) of samples , was the most common virus ( technical appendix ) . the median log - transformed pneumococcal densities of 299 virus - positive samples and 136 virus - negative samples were not significantly different ( 4.73 vs 3.94 , respectively ; adjusted p = 0.06 ) . during ari , the median log - transformed pneumococcal densities varied among virus groups : virus - negative ( 3.94 , iqr 0.005.67 ; n = 136 ) , nonrhinovirus ( 4.49 , iqr 3.125.48 ; n = 105 ) , rhinovirus - only ( 4.91 , iqr 3.436.23 ; n = 147 ) , and rhinovirus detected with other viruses samples ( 5.03 , iqr 3.286.53 ; n = 47 ) . in multivariable analyses , the only significant difference was between rhinovirus - only and virus - negative samples ( p = 0.02 ) ( figure 2 ) . pneumococcal densities of current acute respiratory illness samples subdivided by reverse transcription pcr detection of respiratory viruses . each circle represents a single bacterial density measurement . the median for the samples of each subgroup asterisk indicates significantly different from virus - negative samples . for the colonization patterns assessment , 2,479 ( 69.3% ) nasopharyngeal samples had another sample collected < 60 days before the current sample ; the median time between samples was 28 days . the median log - transformed pneumococcal densities among samples that represented new colonizations ( 5.14 , iqr 3.566.24 ; n = 411 ) , serotype replacement ( 5.49 , iqr 4.536.44 ; n = 322 ) , and serotype persistence ( 5.79 , iqr 4.826.47 ; n = 489 ) were compared . in multivariable analysis , serotype - replacement ( p = 0.005 ) and serotype - persistence ( p = 0.0003 ) samples had higher density than new colonization samples . the difference between serotype replacement and serotype persistence was not significant ( p = 0.2 ) . our findings demonstrate a dynamic evolution of pneumococcal densities before , during , and after ari episodes among young children . we observed a gradual increase in pneumococcal density leading up to an ari episode , peak density during symptomatic ari , and a decrease in density post - ari to levels similar to those in baseline non - ari periods . our observations of higher densities during ari than non - ari episodes align with those in studies from vietnam and south africa ( 14,15 ) and complement those assessments by illustrating the dynamic evolution of pneumococcal densities and the role of virus co - infections and pneumococcal colonization patterns . unlike other studies that focused on hospitalized children , our community - based study showed relatively modest variations in nasopharyngeal pneumococcal density . rhinovirus detection was associated with increased pneumococcal density during ari . although we observed an even higher median pneumococcal density in samples co - infected with rhinovirus and other respiratory viruses , the number of observations was small and statistical power to demonstrate significant differences was limited . compared with new colonization in our study , serotype persistence and replacement were associated with higher pneumococcal density . because many new colonizations might ultimately succumb to host mechanisms and fail to establish stable colonization ( 2 ) , the observed lower densities might reflect a decline of pneumococcal populations as clearance evolved . nevertheless , although statistically significant , the differences in density were relatively modest , and we can not establish the precise time of colonization or clearance in our samples . ari identification depended on the presence of cough or fever , which are subjective but widely used for routine ari surveillance ( 57 ) . because our study used household - based rather than health facility based surveillance , severe disease was infrequent , precluding detailed assessments of disease severity . due to small numbers , we could not study serotype - specific pneumococcal densities . in addition , because the study was conducted in rural communities of peru , caution is warranted when extrapolating our findings to other settings . our findings demonstrated that , among young children , nasopharyngeal pneumococcal density started increasing before the onset of ari symptoms , peaked during symptomatic ari , and decreased after symptoms subsided . rhinovirus co - infection , serotype persistence , and serotype replacement were associated with increased nasopharyngeal pneumococcal density . nasopharyngeal pneumococcal density is dynamic surrounding ari episodes and likely driven by complex virus bacteria host interactions . expanded description of methods and results for a study of the role of nasopharyngeal pneumococcal density in the evolution of acute respiratory illnesses in young children , peru , 20092011 .

we examined nasopharyngeal pneumococcal colonization density patterns surrounding acute respiratory illnesses ( ari ) in young children in peru . pneumococcal densities were dynamic , gradually increasing leading up to an ari , peaking during the ari , and decreasing after the ari . rhinovirus co - infection was associated with higher pneumococcal densities .

The Study Conclusions None

PMC4936457

chronic obstructive pulmonary disease ( copd ) is one of the most frequent causes of mortality in the world . copd is usually associated with a number of secondary impairments such as cardiac , metabolic , peripheral muscle , and psychosocial dysfunction . a major problem among these co - morbidities is skeletal muscle dysfunction , which is typically characterized by reduced muscle strength and exercise intolerance . muscular dysfunction in copd patients mainly concerns the lower limbs , but upper limb muscle function is reduced as well . reduced muscle strength and endurance significantly deteriorate functional capacity , health - related quality of life , and even mortality in copd patients . however , muscle weakness and increased fatigability are not the only limitations of skeletal muscle function . coupling or coordination means modulation of the temporal patterns of both respiration and the additional motor process due to central nervous interactions . ideally , these interactions result in the adoption of a common rhythm or a stable integer frequency ratio . motor respiratory coordination ( mrc ) can be found during each kind of voluntary motor activity , particularly during rhythmical movements . it typically occurs during locomotion , especially during physical activities at higher workloads such as running , cycling , or rowing . however , it has been observed even during fine motor actions at lowest workloads such as hand , finger , head , and eye movements . in mrc , however , mrc is a true mutual interaction ; this means that the respiratory rhythm can modulate the time course of a non - respiratory movement . this may compromise motor performance , particularly in fine motor actions such as precision movements . in patients with ventilatory disorders such as copd , skeletal muscle dysfunction might result not only from reduced muscle strength and endurance but additionally , from impaired motor performance and movement precision due to neural effects from the respiratory rhythm . as precision movements become more and more important both in professional life and in leisure activities ( e.g. , for using smartphones or computers ) we designed a study to investigate whether increased effort and workload of breathing due to increased breathing resistance would affect the performance of a simple rhythmical flexion extension movement of the right forearm . as the workload of this movement is low , reduced working capacity of the muscles is not probable to impair task performance . to have a direct comparison , the experiments were carried out in healthy subjects under normal conditions and under the condition of a simulated obstruction ( so ) . in this pilot study , we investigated the effects of a transiently increased breathing resistance on the temporal patterns of both breathing and the rhythmical forearm movement . we hypothesized that the increased breathing resistance would affect the time course of breathing and consequently , that of the forearm movement . experiments were performed in 21 voluntary healthy subjects ( 10 females and 11 males , aged 23.5 4.8 years ) . all subjects but one were right - handed ; the left - handed person regularly used her right hand for writing . this study was approved by the local ethics committee and was performed in accordance with the ethical standards laid down in the declaration of helsinki . subjects were seated at a table looking onto a 17 monitor ( philips 107e20 ) at a distance of 70 cm . to guarantee a fixed and comfortable head position , the chin was supported and the forehead was leaned against a horizontal bar . they were instructed to perform rhythmical flexion extension movements with their right forearm at a rate ranging between 0.2 and 0.4 hz . when subjects put their forearm straight on the table , the angle between the upper arm and the forearm ranged between 130 and 150. their elbow was supported on a small upholstered pad on the table . the angles between the forearm and the table surface were 18 in the maximum extended position and 36 in the maximum flexed position of the forearm movement . the limits of the required movement amplitude were presented on the screen by two horizontal lines . additionally , the forearm movement was displayed on the monitor . forearm movements were measured using a goniometer that was attached to the radial side of the upper arm and the forearm . the goniometer signal was digitalized using dasylab 3 software ( datalog , mnchengladbach , germany ) . this software also generated the movement signal and the movement limits presented on the screen . breathing was recorded using a fleisch pneumotachograph ( g. hertel , lengenfeld , germany ) . the breathing signal was also digitalized with dasylab but was not visualized to the subjects . at the beginning , subjects were informed in detail on the experimental course , in particular , on the condition of so . then , spirometry was conducted twice under normal conditions ( ctrl ) using a masterscope pc spirometer ( viasys healthcare , hoechberg , germany ) . , a plug of 2 cm of length with a narrow hole with a diameter of 4 mm was inserted between the face mask and pneumotachograph . we analyzed inspiratory vital capacity ( ivc ) and forced expiratory volume in 1 s ( fev1 ) . after subjects were seated and equipped with measuring facilities , breathing at rest was recorded for 34 min under the ctrl condition and thereafter , further 34 min under the so condition . after a 1530 min interval , subjects performed a pre - test consisting in a visually guided tracking procedure with flexion extension movements of their right forearm for about 5 min under ctrl conditions . both tracking signal and forearm movement were displayed on the screen as vertical bars in different colors . the tracking signal bar moved upwards and downwards according to a sinus function at a frequency of 0.3 hz . at this target rate , most stable entrainment patterns with movement - to - breathing frequency ratios of 1:1 have been observed . these tracking tests served to familiarize the subjects with the required movement rate . in the main experiment , subjects had to perform a similar flexionextension movement at the same amplitude but without a visual tracking signal . movement amplitude should be kept exactly in accordance with the limits displayed on the screen . subjects were instructed to keep the movement rate approximately in the same range as during the pre - test . they did not receive any cues unless their movement rate fell below 0.2 hz or exceeded 0.4 hz . the main experiment consisted of two trials lasting 56 min each with a resting interval of 3 min in between . the first trial was performed under ctrl condition and the second one under the condition of so . subjects were instructed to maintain their movement rate and amplitude according to the prior tests . after completion of the tests , subjects were questioned whether or not they paid attention to their forearm movements and breathing , and whether they felt discomfort with breathing . the goniometer signal and the pneumotachogram were digitalized at a sample rate of 100 hz . we assessed time of inspiration ( ti ) , time of expiration ( te ) , time of a total breath ( ttot ) , expired tidal volume ( vt ) , and ventilation . for each movement period , we determined time of flexion ( tflex ) , time of extension ( text ) , and time of a total period ( tmove ) . in addition , we calculated the mean cycle time ratio between movement and breathing ( mbctr ) . differences between the conditions were tested for significance using a wilcoxon matched pairs signed rank test ( statgraphics plus 4.1 for windows ; statistical graphics corporation , rockville , maryland , usa ) . experiments were performed in 21 voluntary healthy subjects ( 10 females and 11 males , aged 23.5 4.8 years ) . all subjects but one were right - handed ; the left - handed person regularly used her right hand for writing . this study was approved by the local ethics committee and was performed in accordance with the ethical standards laid down in the declaration of helsinki . subjects were seated at a table looking onto a 17 monitor ( philips 107e20 ) at a distance of 70 cm . to guarantee a fixed and comfortable head position , the chin was supported and the forehead was leaned against a horizontal bar . they were instructed to perform rhythmical flexion extension movements with their right forearm at a rate ranging between 0.2 and 0.4 hz . when subjects put their forearm straight on the table , the angle between the upper arm and the forearm ranged between 130 and 150. their elbow was supported on a small upholstered pad on the table . the angles between the forearm and the table surface were 18 in the maximum extended position and 36 in the maximum flexed position of the forearm movement . the limits of the required movement amplitude were presented on the screen by two horizontal lines . forearm movements were measured using a goniometer that was attached to the radial side of the upper arm and the forearm . the goniometer signal was digitalized using dasylab 3 software ( datalog , mnchengladbach , germany ) . this software also generated the movement signal and the movement limits presented on the screen . breathing was recorded using a fleisch pneumotachograph ( g. hertel , lengenfeld , germany ) . the breathing signal was also digitalized with dasylab but was not visualized to the subjects . at the beginning , subjects were informed in detail on the experimental course , in particular , on the condition of so . then , spirometry was conducted twice under normal conditions ( ctrl ) using a masterscope pc spirometer ( viasys healthcare , hoechberg , germany ) . , a plug of 2 cm of length with a narrow hole with a diameter of 4 mm was inserted between the face mask and pneumotachograph . we analyzed inspiratory vital capacity ( ivc ) and forced expiratory volume in 1 s ( fev1 ) . after subjects were seated and equipped with measuring facilities , breathing at rest was recorded for 34 min under the ctrl condition and thereafter , further 34 min under the so condition . after a 1530 min interval , subjects performed a pre - test consisting in a visually guided tracking procedure with flexion extension movements of their right forearm for about 5 min under ctrl conditions . both tracking signal and forearm movement were displayed on the screen as vertical bars in different colors . the tracking signal bar moved upwards and downwards according to a sinus function at a frequency of 0.3 hz . at this target rate , most stable entrainment patterns with movement - to - breathing frequency ratios of 1:1 have been observed . these tracking tests served to familiarize the subjects with the required movement rate . in the main experiment , subjects had to perform a similar flexionextension movement at the same amplitude but without a visual tracking signal . movement amplitude should be kept exactly in accordance with the limits displayed on the screen . subjects were instructed to keep the movement rate approximately in the same range as during the pre - test . they did not receive any cues unless their movement rate fell below 0.2 hz or exceeded 0.4 hz . the main experiment consisted of two trials lasting 56 min each with a resting interval of 3 min in between . the first trial was performed under ctrl condition and the second one under the condition of so . subjects were instructed to maintain their movement rate and amplitude according to the prior tests . after completion of the tests , subjects were questioned whether or not they paid attention to their forearm movements and breathing , and whether they felt discomfort with breathing . the goniometer signal and the pneumotachogram were digitalized at a sample rate of 100 hz . we assessed time of inspiration ( ti ) , time of expiration ( te ) , time of a total breath ( ttot ) , expired tidal volume ( vt ) , and ventilation . for each movement period , we determined time of flexion ( tflex ) , time of extension ( text ) , and time of a total period ( tmove ) . in addition , we calculated the mean cycle time ratio between movement and breathing ( mbctr ) . differences between the conditions were tested for significance using a wilcoxon matched pairs signed rank test ( statgraphics plus 4.1 for windows ; statistical graphics corporation , rockville , maryland , usa ) . ivc was 4.76 0.21 l ( 102% of predicted value ) and fev1 was 4.08 0.17 l ( 106% of predicted value ) . while ivc was almost unaffected by so ( 101% of predicted value ) , fev1 was reduced by 53% of ctrl ( 55% of predicted value , p = 4 * 10 ; 45.3% of ivc ) . thus , our simulation corresponded to a moderate degree of obstruction according to the gold classification of airflow limitation in copd . in the resting ctrl condition , breathing rate was 15.7 breaths per min ( ttot = 3.82 0.21 s ) on average , with ti and te being 1.56 0.08 s and 2.23 0.13 s , respectively . tidal volume was 0.59 0.03 l and ventilation was 9.46 0.32 l / min . so induced a significant prolongation of breath duration ( ttot = 4.17 0.2 s , p = 0.047 ) that was mainly based on an increase in ti ( 1.74 0.09 s , p = 0.015 ) . this was associated with a significant reduction in vt ( 0.53 0.03 l , p = 0.002 ) and ventilation ( 7.60 0.30 l / min , p = 7 * 10 ) ; see figure 1 . in the movement experiment , breathing rate and vt increased slightly to 16.6 breaths per minute and 0.61 0.03 l in the ctrl condition . breath duration decreased to 3.61 0.14 s with ti = 1.50 0.06 s and te = 2.10 0.09 s. although these changes were not significant , ventilation was significantly higher than at rest ( 10.19 0.36 l / min , p = 0.005 ) . however , in the so condition , no change in the breathing pattern occurred while movements were performed . consequently , with so breath duration was longer ( ti = 1.74 0.08 s , p = 0.0003 ; te = 2.35 0.12 s , p = 0.006 ; ttot = 4.14 0.19 s , p = 0.002 ) and vt and ventilation were lower ( 0.55 0.03 l , p = 0.003 and 7.97 0.29 l / min , p = 9 * 10 , respectively ) than in the ctrl condition ( figure 1 ) . in the ctrl condition , the average movement period tmove was 3.70 0.12 s. durations of flexion and extension periods were almost equal ( tflex = 1.88 0.06 s ; text = 1.82 0.07 s ) . in so , movement period was significantly longer with tmove = 3.93 0.11 s ( p = 0.039 ) . this was mainly due to an increase in tflex to 2.02 0.06 s ( p = 0.024 ) while the extension period was less prolonged ( text = 1.91 0.06 s ; p = 0.034 ) ; see figure 2 . consequently to this prolongation in tmove , mbctr was maintained at 0.99 0.06 . in the final interview , 14 subjects stated that they had noticed to adopt a common rhythm between forearm movement and breathing . four of them inhaled with flexion and exhaled during forearm extension ; the others did not specify the phase relation . ivc was 4.76 0.21 l ( 102% of predicted value ) and fev1 was 4.08 0.17 l ( 106% of predicted value ) . while ivc was almost unaffected by so ( 101% of predicted value ) , fev1 was reduced by 53% of ctrl ( 55% of predicted value , p = 4 * 10 ; 45.3% of ivc ) . thus , our simulation corresponded to a moderate degree of obstruction according to the gold classification of airflow limitation in copd . in the resting ctrl condition , breathing rate was 15.7 breaths per min ( ttot = 3.82 0.21 s ) on average , with ti and te being 1.56 0.08 s and 2.23 0.13 s , respectively . tidal volume was 0.59 0.03 l and ventilation was 9.46 0.32 l / min . so induced a significant prolongation of breath duration ( ttot = 4.17 0.2 s , p = 0.047 ) that was mainly based on an increase in ti ( 1.74 0.09 s , p = 0.015 ) . this was associated with a significant reduction in vt ( 0.53 0.03 l , p = 0.002 ) and ventilation ( 7.60 0.30 l / min , p = 7 * 10 ) ; see figure 1 . in the movement experiment , breathing rate and vt increased slightly to 16.6 breaths per minute and 0.61 0.03 l in the ctrl condition . breath duration decreased to 3.61 0.14 s with ti = 1.50 0.06 s and te = 2.10 0.09 s. although these changes were not significant , ventilation was significantly higher than at rest ( 10.19 0.36 l / min , p = 0.005 ) . in the so condition , no change in the breathing pattern occurred while movements were performed . consequently , with so breath duration was longer ( ti = 1.74 0.08 s , p = 0.0003 ; te = 2.35 0.12 s , p = 0.006 ; ttot = 4.14 0.19 s , p = 0.002 ) and vt and ventilation were lower ( 0.55 0.03 l , p = 0.003 and 7.97 0.29 l / min , p = 9 * 10 , respectively ) than in the ctrl condition ( figure 1 ) . in the ctrl condition , the average movement period tmove was 3.70 0.12 s. durations of flexion and extension periods were almost equal ( tflex = 1.88 0.06 s ; text = 1.82 0.07 s ) . in so , movement period was significantly longer with tmove = 3.93 0.11 s ( p = 0.039 ) . this was mainly due to an increase in tflex to 2.02 0.06 s ( p = 0.024 ) while the extension period was less prolonged ( text = 1.91 0.06 s ; p = 0.034 ) ; see figure 2 . consequently to this prolongation in tmove , mbctr was maintained at 0.99 0.06 . in the final interview , 14 subjects stated that they had noticed to adopt a common rhythm between forearm movement and breathing . four of them inhaled with flexion and exhaled during forearm extension ; the others did not specify the phase relation . the main finding of this pilot study was that simulation of moderate obstruction modulated the temporal pattern of a rhythmical forearm movement performed without any additional workload . although subjects were instructed to move their forearm in the same rhythm as they did just before under ctrl condition , their movement was slowed down in the so condition . while the normal pulmonary resistance is about 1 cm h2o l , it can increase in copd to 515 cm h2o roughly calculated , increasing the inspiratory resistance by 10-fold during breathing at rest would increase the resistive work of breathing also by about 10-fold . our subjects reported that they felt discomfort and greater effort of breathing in the so condition . due to the elevated inspiratory resistance in this condition , they reduced their tidal volume and prolonged their breaths by about 10% even in the absence of any additional activity . the start of the forearm movement was accompanied by a slight increase in breathing rate by about 6% in the ctrl condition . in the so condition , no change in the breathing pattern was observed with the start of the movements . we assume that the increased respiratory work due to the elevated breathing resistance was an additional drive to breathing exceeding that from motor control . a previous study with finger tracking movements under hypercapnic conditions showed that at the onset of movement , breathing rate increased only half as much as under normocapnic conditions . although subjects were instructed to perform the rhythmical forearm movement both in the ctrl and in the so condition at the same rate , their movement rate was lower in the so condition . it is well documented that resistive loaded breathing can modulate the electromyogram pattern of limb muscles in a differential way . inspiratory resistive loading significantly prolonged latencies in somatosensory evoked potentials of the median nerve in humans . central respiratory activation by increased respiratory drive also modulates the temporal pattern of motor functions . we observed that hypercapnia increased the latency of finger tracking movements , predominantly during expiration . these findings suggest that increased breathing resistance may amplify neural effects from respiration onto the rhythm of a non - respiratory movement . during rhythmic movements rhythmical forearm tracking movements , 1:1 coordination was observed between 0.2 and 0.6 hz with a maximum at 0.3 hz . within this range , breathing rate increased with the movement rate , though maintenance of a 1:1 rate ratio became more and more unstable . at higher movement rates , however , breathing rate did not further increase but coordination switched to higher integer ratios such as 2:1 , 3:1 , and more . modulation of the respiratory time course by additional motor activities is typical , but there are also effects in the opposite direction . in walking , modulation of the stepping rate by the breathing rhythm has been observed in periods of mrc , particularly when the variability of breathing was restricted by acoustic pacing of the respiratory rhythm . we assume that in the so condition , variability of the respiratory rhythm has been reduced by the additional breathing effort . this might help to reduce discomfort of breathing , to avoid further inefficiency of breathing , and to ensure adequate gas exchange . the respiratory rhythm , in turn , slowed down the rhythm of the forearm movement so that the 1:1 rate ratio was maintained . the majority of subjects noticed that their breathing and movement rhythms were equal but most of them did not clearly state which phases of forearm movement and breathing were coupled to each other . coordination is associated with the adoption of a certain phase relationship , which often consists in the coincidence of so - called anchor points , that is , points of maximum angular excursion . in mrc , the start of a movement phase preferably coincides with the start of inspiration or expiration . in a recent study with rhythmical forearm tracking movements when subjects started a single finger movement in the first half of inspiration or expiration , they performed it faster and more precisely than a movement started late in inspiration or expiration . likewise , breath duration was modulated in a phase - dependent manner : when the finger movement started early in inspiration , both the current inspiration and the related expiration were shortened . in contrast , when the movement started late in expiration , duration of the current expiration did not change or even increase but the subsequent breath was shortened . these phase - dependent differences were even more pronounced in hypercapnia indicating that mrc became stronger with increased respiratory drive . this may also count for the changes in breathing and movement duration observed in the present study with increased breathing resistance . the findings of the present study clearly demonstrate that mrc can considerably modulate the temporal patterns of both respiration and non - respiratory movements . with increased respiratory resistance such as in copd patients , this may particularly impair fine motor skills and thus , significantly compromise the daily life of copd patients .

background and objectivesskeletal muscle dysfunction is a major problem among the co - morbidities associated with chronic obstructive pulmonary disease ( copd ) . however , muscle weakness and increased fatigability are not the only limitations of skeletal muscle function . motor respiratory coordination ( mrc ) may occur even during movements at lowest workloads . mrc modifies the temporal pattern of motor actions , thus probably impairing motor performance and movement precision . little attention has been paid to the question of whether motor functions may be compromised in copd patients independent of workload and required muscle strength and endurance . the present pilot study was designed to investigate the effects of a simulated obstruction ( so ) in healthy subjects on their breathing pattern and the timing of a rhythmical forearm movement.methodstwenty-one subjects performed flexion extension movements with their right forearm at a self - chosen rate within a range between 0.2 and 0.4 hz . after a control experiment with normal breathing , a plug with a narrow hole was inserted between face mask and pneumotachograph to simulate obstruction . subjects were required to repeat the rhythmical forearm movement at the same rate as in the control experiment.resultsthe condition of so significantly prolonged breath duration but reduced tidal volume and ventilation . in addition , period duration of the forearm movement increased significantly under this condition while the movement - to - breathing frequency ratio remained almost constant . increased breathing resistance was considered to cause prolonged breath duration accompanied by an increase in movement period duration . the constant near - integer ratio between movement and breathing rates indicates that the change in movement period duration resulted from mrc.conclusionsthe findings of this pilot study demonstrate that increased breathing resistance may compromise motor performance even at lower workloads . this means that in copd patients , not only muscle strength and endurance are reduced but , moreover , fine motor skills may be impaired . this aspect has particular importance for many everyday activities as reduced fine motor performance substantially contributes to a progressive inability of the patients to manage their daily life .

INTRODUCTION METHODS Subjects Experimental set-up Study protocol Parameters and statistical analysis RESULTS Spirometry Breathing at rest Breathing during rhythmical forearm movement Temporal pattern of forearm movement and MBCTR DISCUSSION CONCLUSION

PMC3684126

hormonal changes at the moment of parturition may constitute an important regulatory mechanism for myometrial contraction . in some species ( e.g. , sheep ) , sudden changes in circulating cortisol , oestrogen , and progesterone appear to be important factors for the onset of labour , whereas in humans , these signs remain unknown [ 110 ] . therefore , we investigated the concentration of oestrogen receptors ( ors ) and progesterone receptors ( prs ) in pregnant human women because local modifications of these receptors may be associated with the myometrial conditions responsible for the mechanism of parturition [ 110 ] . prior studies on hormone concentrations during labour have failed to demonstrate significant changes indicating their direct involvement in labour ; however , local changes in hormone receptors are believed to be responsible for parturition [ 4 , 5 , 11 ] . close to term , the effect of progesterone may be inhibited by a genetic mechanism involving one or more agents not associated with receptors [ 1 , 2 ] . this genetic mechanism may lead to the attenuation of progesterone 's effects by , for example , a specific gene that interrupts its action at a molecular , biochemical , and/or cellular level . corroborating these studies in a review , mendelson postulates that an increase in inflammatory response occurs as a result of the activation of the nuclear factor ( kappa ) beta , leading to uterine contractility in two ways : ( 1 ) the direct activation of contractile genes ( e.g. , cox-2 , oxytocin receptors , and connexin 43 ) and ( 2 ) a blockade of the ability of progesterone receptors ( prs ) to maintain uterine quiescence . according to previous reports , a decrease in the concentration of prs is observed in patients during labour [ 412 ] . nonetheless , the inhibition of the effects of gestagenic agents does not result in the immediate onset of labour , although this inhibition sensitises the cells of the myometrium to agents that induce uterine contractility . in contrast , oestrogen enables the myometrium to prepare for parturition by inducing oxytocin receptors , which is perhaps the first step of parturition [ 510 , 12 ] . recent studies have also suggested that the withdrawal of progesterone is the initiator of parturition , and they have demonstrated the importance of the concentrations of prs a and b and their relationship in the mechanism of parturition [ 510 , 12 ] . currently , other aspects are also known to be required for the onset of parturition . cervical changes , for example , are associated with the expression of receptors that modify local mechanisms such as the remodelling of collagen , the migration of inflammatory cells , and changes in the extracellular matrix as a whole [ 9 , 1315 ] . to quantify the concentrations of ors and prs in the myometrium during labour , a prospective study including 21 patients was designed to evaluate the concentrations of these receptors on the surfaces of myometrial cells in tissue biopsies collected during a caesarean section of full - term pregnant women in relation to the presence or absence of labour . myometrial tissue was obtained by biopsies performed during caesarean sections of 21 full - term pregnant women ( 37 to 41 weeks ) . six women were in labour ( mean 39 weeks , range 37 to 41 ) , and 15 were not in labour ( mean 39 weeks , range 37 to 41 weeks ) . the study was evaluated and approved by the research ethics committee of the federal university of so paulo ( unifesp ) . gestational age was confirmed by the date of the last menstrual period and/or by ultrasonography in the first and/or second trimesters . women were considered parturient based on the assessment of the signs and/or symptoms of labour : a bishop score greater than or equal to 5 , two or more uterine contractions lasting more than 40 seconds in 10 minutes , cervical dilation of 2 cm in primiparous women and 3 cm in multiparous women , and insinuated foetal presentation . the indications of cesarean section in patients during the labour were as follows : two or more previous cesarean sections , fetal distress observed during the evolution of the labour , and primiparous women in breech . the exclusion criteria were patients with obstructed labour , clinical , and/or obstetrical complications , and patients undergoing induction / conduction of labour with oxytocin or prostaglandin , using medication except multivitamins . during the caesarean section ( after placental expulsion ) , a small piece of the myometrium was removed at the upper middle portion of the incision ( hysterotomy ) by the researcher ( jsa ) , always using the same technique : a resection of 0.5 cm of the myometrium with the aid of a scalpel , excluding the endometrium and visceral peritoneum , and then proceeding to a normal suture of the hysterectomy . the material obtained from each biopsy was placed in 10% buffered formaldehyde for a maximum of 48 hours and subsequently processed in an autotechnicon ( autotechnicon 2 , chauncey , ny , usa ) for 12 hours ( overnight ) , using alcohol , xylene , and paraffin . after processing , the specimens were placed in cassettes and embedded in paraffin ( soldan cytological products ltd . , porto alegre , rs , brazil ) . the paraffin blocks containing the histological processed materials were cooled in a freezer and cut on a rotary microtome to obtain 4-micron - thick histological sections using disposable razors ( leica ) suitable for this purpose . the paraffin ribbons obtained from the microtome sectioning process were carefully placed in a histological water bath , and the sections were deposited on precleaned slides coated with a special adhesive that improves tissue adhesion ( ver - sa plus , euriegas ) . ( 1 ) preparation of the histological sections : washing in xylene at room temperature for 15 minutes , twice , followed by 100% ethanol ( 3 times ) for 30 seconds each , 95% ethanol for 30 seconds , 80% ethanol for 30 seconds , and 70% ethanol for 30 seconds , with a final washing in running and distilled water . ( 2 ) antigen retrieval : the slides were incubated in moist heat and incubated in citrate buffer ( 10 nm , ph 6.0 ) in a steamer ( t - fal ) for 20 minutes . the immunohistochemical visualisation consisted of the following steps in a reaction using the streptavidin - biotin peroxidase complex ( streptabc ) . ( 1 ) the slides were incubated with a specific antibody ( monoclonal er ( 1b)/pr ( 2b ) ) for 30 minutes in a moist chamber . ( 2 ) the slides were washed in tris - buffered saline ( dako tbs ) , and the buffer was exchanged every 3 minutes . ( 3 ) the slides were then incubated with biotinylated secondary antibody ( anti - immunoglobulin from the animal species from which the specific antibody was obtained ) in a moist chamber for 30 minutes at 37c . ( 4 ) the slides were again washed in buffer with three exchanges , once every 3 minutes . ( 5 ) revelation and mounting : the slides were incubated in a substrate solution ( diaminobenzidine ; dab , 60 mg% ) for 3 minutes at 37c in the dark . the positive control slides were then observed through the microscope for the development of a golden - brown precipitate , the final product of the reaction ; the slides were washed in running and distilled water for 3 minutes , counterstained with harris haematoxylin for 30 seconds , and washed in running and distilled water . the slides were then dehydrated in 50% ethanol , 80% ethanol , 95% ethanol , and 100% ethanol ( 3x ) followed by xylene ( 3x ) , mounted in entellan cover slips ( merck 1.07961 ) and examined under an optical microscope by three pathologists to confirm the suitability of the tissue sample . the pathologists also verified whether scarring from previous caesarean sections would interfere with the results , which was avoided by observing specific areas for study . the quantitative analysis of the presence of ors and prs was performed using a computerised image - analysis program ( imagelab ) . for the quantification of the areas occupied by the two hormone receptors , microscopic fields of 422 m 322 m were captured at a magnification of 100x while maintaining several standardised settings of the optical microscope ( leitz carbelux model 6mbh ) . a leitz wetzlar-160/0.17 plan 10/0.45 lens was used at maximum light intensity with the maximum condenser aperture at a distance of 6.5 cm from the slide . the digitalised fields had a horizontal resolution of 640 pixels and a vertical resolution of 480 pixels in 24-bit colour ( 16 million colours ) , and the same range of colours for the area to be quantified was used in each of the fields . the colour range was adjusted for isolation in areas that represented the ors and prs in the image . the averaged values obtained from both analysed fields in each slide were determined by considering two parameters : the percentage of the area occupied by hormone receptors and the optical density of the light reflected by each of the receptors . to avoid possible subjectivity , all examined materials were identified by each patient 's medical record number , and the collected data and the results obtained were compared only at the end of the study . the pathological anatomy laboratory technician numbered the patients according to the order of entry of the biopsy material in the laboratory , enabling the statistical analysis of patients numbered 1 to 21 . the name and number of the medical record were retained , allowing for easy identification . therefore , the patients in labour and not in labour could readily be compared at the end of the study following the examination of the slides . redmond , wa , usa ) spreadsheet and analyzed by the statistical package for the social sciences ( spss ) program , version 19.0 for windows ( spss inc . , chicago , il , usa ) . the mann - whitney test was used to compare the pregnant women in each study group with respect to the numbers of ors and prs . the wilcoxon test was used to compare the numbers of ors with the number of prs in each group . spearman 's correlation coefficients ( r ) were used to study the possible relationships between ors and prs in each group separately . the medians of prs and ors in pregnant women in labour ( group i ) were 29.3 2.38 ( range , 24.630.2 ) and 32.3 10.92 ( range , 22.949.0 ) , respectively . the medians of prs and ors in pregnant women without labour ( group ii ) were 43.6 14.94 ( range , 23.670 ) , and 43.9 13.17 ( range , 18.362.6 ) , respectively . we did not observe significant differences of the number of ors and prs in both groups by the mann - whitney test ( p = 0.13 and 0.37 , resp . ) . the z critical between both groups was 1.96 ( oestrogen - z calculated = 1.56 and progesterone - z calculated = 0.93 ) , proving difference in the concentration of both receptors in the myometrium of lower uterine segment of pregnant women during and without labour ( table 1 ) . the number of ors was statistically more than that of prs in the myometrium of lower uterine segment of group ii ( group i z calculated = 0.94 versus group ii z calculated = 16.00 ) . we observed that the prs and ors concentrations were smaller in the myometrium of lower uterine segment of group i , and these concentrations were higher in group ii ( group i r calculated = 0.49 versus group ii r calculated = 0.74 ) ( table 2 ) . knowledge of the mechanism of parturition should allow patients in preterm labour and patients with postterm pregnancy to be properly treated , minimising the complications of such conditions [ 2 , 3 , 14 , 16 ] . recent works have suggested that a decrease in the number of progesterone b receptors , favouring an increase in the expression of progesterone receptor a , results in a triggering of the mechanism that initiates labour [ 57 , 9 , 10 ] . thus , these authors suggest a functional withdrawal of progesterone . the results of the present study suggest that the downregulation of the concentration of prs may be associated with human parturition , or at least with its initiation . because no significant changes were noted in ors numbers in either labour or nonlabour conditions , oestrogen action may be held constant . a constant level of oestrogen may also maintain the status of both ors and prs , given the well - known upregulation of both hormone receptors by oestrogen and the downregulation of these receptors by progesterone [ 1 , 2 , 4 , 11 ] . another implication of the sustained or levels may be explained by the observation that oestrogen excites the myometrium , increases the levels of oxytocin receptors , and induces the inflammatory process currently known to be involved in parturition [ 5 , 6 , 9 ] . the present study confirmed the findings of several previous studies , particularly from the study that served as our model , that is , the upregulation of ors and prs . when pr numbers decrease , there is a less expressive decrease in or numbers , and prs apparently tend to decrease in patients in labour [ 8 , 10 ] . we note that the present study was conducted in the myometrium of lower uterine segment and , similarly to the studies conducted by rezapour et al . and , we observed a low concentration of prs in the myometrium of lower uterine segment . the study by merlino et al . required a correction of the receptor analysis due to the small number of myometrial cells in the uterine cervix , which is also consistent with findings of the present study . similarly to the study of winkler et al . , in the present study , a tendency for a decrease in the number of prs was observed in pregnant women at the beginning of labour with a bishop score equal to or less than 5 . according to study , the fact that lower concentrations of prs were found may have been due to a missing analysis of a specific receptor type . the authors showed that the ratio of pr - a to pr - b increased during labour . they also studied pregnant women subjected to elective caesareans , in both term and preterm pregnancies , although not in labour . their results showed that a pr - a / pr - b ratio of 0.5 was maintained in the term and preterm pregnancies , whereas during labour , the ratio doubled in favour of pr - a , with a value of 2.5/1 . these findings are consistent with previous studies and support the hypothesis that human parturition involves a specific increase in the ratio of pr - a / pr - b in the myometrium due to an increased expression of pr - a . this result drew special attention to the difference in hormone concentrations in pregnant women who had no signs or symptoms of labour . significant differences were found between the concentrations of hormone receptors in nonparturient women , whereas no difference was noted in parturient women ; that is , there appeared to exist a constancy in the numbers of steroid receptors when the myometrium was clearly in labour activity , which may be explained by the postulate of mendelson stated above . the differences in the methodologies used to assess the concentrations of the studied hormone receptors in our study and others allowed for a corroboration of the results obtained in other analyses using different methods . thus , it is worth noting that the study and quantification of or and pr is less laborious and costly using the methodology developed in the present study . the similarities between this study and the present study are the following : the patients were subjected to undergo elective caesarean sections ; that is , they were not in labour ; parturients were subjected to caesarean section due to obstetric requirements and distributed according to clinical criteria for the assessment of the presence or absence of labour ; biopsies were obtained from the myometrium of lower uterine segment , at the upper portion of the hysterotomy incision ; and we attempted to remove the whole tissue that was not myometrium . however , the authors note that , despite the rigour adopted for myometrial tissue removal , there may have been some contamination with other tissues . in an other study on the myometrium , merlino et al . investigated the expression of the nuclear progesterone receptor in foetal membranes and decidua at term , before and after the onset of labour . their results suggested that the decidua , rather than the foetal membranes , is the direct target for the nuclear pr during pregnancy because the receptor was only detected in the decidua . our hard criteria to include only pregnant women with spontaneous labour ( group i ) and to exclude the dystocia as indication of caesarean section contributed to this small sample . the present study is in accordance with the literature , showing the changes in steroid receptor sites and assisting in understanding the possible mechanism of labour and how they can intervene in this situation if necessary , for example , in preterm and prolonged gestation . finally , the need for an animal model with a parturition physiology similar to the human system is also required , allowing more detailed studies to be performed in vivo .

objective . to assess the concentration of progesterone ( prs ) and oestrogen ( ors ) receptors of myometrium of full - term pregnant women in the myometrium of lower segment of the uterus in relationship with presence or absence of labour . methods . this was a cross - sectional prospective study with 21 pregnant women , being 6 in labour ( group i ) and 15 without labour ( group ii ) . the biopsy of myometrium was realized during caesarian section , and the excised tissue was stained using immunohistochemical techniques for the quantification of the receptors , and with the aid of image - analysis software , the numbers of receptors for each hormone were determined spectrophotometrically . the mann - whitney test was used to compare the pregnant women in each study group with respect to the numbers of ors and prs . the wilcoxon test was used to compare the concentration of ors and prs in each group separately . results . the mean of gestational age was 39 weeks , ( range , 37 to 41 weeks ) . the medians of prs and ors in pregnant women in labour ( group i ) were 29.3 ( range , 24.630.2 ) and 32.3 ( range , 22.949.0 ) , respectively . the medians of prs and ors in pregnant women without labour ( group ii ) were 43.6 ( range , 23.670 ) and 43.9 ( range , 18.362.6 ) , respectively . we did not observe significant differences of the number of ors and prs in both groups ( p = 0.13 and 0.37 , resp . ) . the number of ors was statistically more than that of prs in group ii ( z calculated = 16.00 ) . conclusion . the concentrations of prs and ors were similar in the myometrium of the lower uterine segment of pregnant women during and without labour , but the concentration of ors was more than that of prs in the myometrium of the lower uterine segment of pregnant women without labour .

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

PMC5094528

cardiovascular prevention and rehabilitation improves functional and clinical status , health - related quality of life , and prognosis in patients with coronary heart disease ( chd ) recovering from acute events and invasive interventions.1 for more than a decade , cardiac rehabilitation ( cr ) has been recommended for chd as part of integrated cardiac care.2,3 despite this , studies from the us and europe , including denmark , have documented that less than half of eligible patients participate in cr.4 systematic monitoring of delivery and outcomes of cr is recommended to meet this challenge.5,6 thus , the national working group on preventive cardiology and rehabilitation , under the danish society of cardiology , initiated a national clinical quality registry on cr approved by the danish health and medicines authority , in 2011 . although danish cardiac rehabilitation database ( dhrd ) was initially implemented on september 1 , 2013 , as a result of a breakdown of the database system , data collection ceased until august 14 , 2015 , at which point the database was implemented once again . the overall aim of the dhrd is to monitor and improve the quality of phase ii cr throughout denmark , in order to improve the outcome for patients recovering from cardiac events . phase ii cr in denmark refers to the initial 812 weeks of outpatient cr performed at hospitals and municipalities . data are collected at a patient level as well as program level ( figure 1 ) , as recommended by scientific associations in europe and the us.5,6 furthermore , the database aims to support research within the field of cr . it is mandatory for all hospitals delivering phase ii cr to register all patients onto the dhrd , who are discharged alive and receiving cr following hospitalization for chd ( ie , acute coronary syndrome , or stable angina pectoris with proven stenosis on coronary angiography treated with percutaneous coronary intervention , coronary artery bypass grafting , or medication alone ) . all patients discharged alive from hospitals are identified from the national patient register7 using the who s international classification of diseases , 10th edition ( icd-10 ) coding system : acute coronary syndrome ( st - elevation myocardial infarction , non st - elevation myocardial infarction , and unstable angina ) as primary diagnosis , or secondary diagnoses ( di210di219 , di248 , di240 , di249 ) and stable angina pectoris as primary diagnoses ( di209 , di251 , di251b , di251c ) . furthermore , the nordic classification of surgical procedures is used to identify coronary angiograms and invasive procedures ( coronary angiograms : uxac40 , uxac85 , uxac90 , uxuc85 , uxuc86 , uxuc87 , ufya20 ; percutaneous coronary intervention : kfng00 , kfng02 , kfng02a , kfng05 , kfng05a , kfng10 , kfng12 , kfng20 , kfng22 , kfng30 , kfng40 , kfng96 , kzfx01 ; and coronary artery bypass grafting : kfna , kfnb , kfnc , kfnd , kfne , kfnf , kfnh , kfnj , kfnk , kfnw ) . the database will include information on approximately 14,000 patients annually . based on administrative data on ambulatory visits ( ie , data from the danish national patient register7 cross - checked with procedure codes referring to cr services ) , 75% of eligible patients are receiving one or more outpatient rehabilitation services from all 35 hospitals in the five regions of denmark that were delivering cr in denmark by 2013.8 the database is also capable of including other patient groups receiving cr . patient - level data are collected by the clinical team and registered directly onto an online system at the time of entry into phase ii cr . the team comprises multidisciplinary professionals involved in the rehabilitation intervention ( ie , nurses , medical doctors , physiotherapists , and dieticians ) . to minimize the registration workload and double registration , simultaneous linkage to national administrative patient registers has been established ( the danish civil registration system,9 the danish national patient register,7 and the danish national database of reimbursed prescriptions).10 linkage to the clinical laboratory information system11 is also planned . at baseline , data are captured from the aforementioned registers and then validated and approved by the clinical staff , by reading and validating the captured information . registration of data into the dhrd began on august 14 , 2015 , which led to the collection of data on 3,846 patients by march 2016 . the quality of data is assessed as part of an annual data audit with feedback to clinicians . patient - level data are collected by the clinical team and registered directly onto an online system at the time of entry into phase ii cr . the team comprises multidisciplinary professionals involved in the rehabilitation intervention ( ie , nurses , medical doctors , physiotherapists , and dieticians ) . to minimize the registration workload and double registration , simultaneous linkage to national administrative patient registers has been established ( the danish civil registration system,9 the danish national patient register,7 and the danish national database of reimbursed prescriptions).10 linkage to the clinical laboratory information system11 is also planned . at baseline , data are captured from the aforementioned registers and then validated and approved by the clinical staff , by reading and validating the captured information . registration of data into the dhrd began on august 14 , 2015 , which led to the collection of data on 3,846 patients by march 2016 . the quality of data is assessed as part of an annual data audit with feedback to clinicians . the variables recorded in the dhrd are related to patient characteristics at baseline , for example , smoking status , exercise capacity , height , weight , blood pressure , and pharmacological therapy . also , data on performed diagnostic tests ( eg , diabetes and depression ) are recorded in the database , along with the individual plan for rehabilitation ( eg , training sessions , dietary treatment , and/or smoking cessation ) . data on physical capacity , lifestyle , and patient - reported outcome measures are reentered at follow - up , in order to monitor progression and outcome of cr . the data collected are aligned with the european core cr dataset ( mcgee , unpublished data , 2004 ) and the variables of european cardiac rehabilitation database project , in order to make sure that data from denmark can be compared to data from other european countries . data at a program level are collected every third year through surveys targeted at cr staff . these surveys gather information about the structure and provision of cr , and provide program - level information that can be compared with patient outcome over time . a set of outcome variables has been defined and approved nationally , in order to provide a set of benchmark quality indicators . the performance on the quality indicators is published in annual feedback reports at a national , regional , and hospital level as part of the annual audit of cr . the indicator list and benchmarks are subject to regular adjustments , in order to keep the database in line with current evidence . the database has been implemented since the summer of 2015 ; consequently , the first patient - level data will be available by autumn 2016 . applications for access to use data are reviewed by the chair and executive committee upon application to the danish clinical registries . in the short term , program - level data and administrative data have been presented as part of an annual report from dhrd8 and will be analyzed as part of an implementation research program investigating the gap between guidelines , clinical practice , and use of a clinical registry.12 data are collected and maintained according to danish data protection laws and regulations , without the need to obtain patient consent . the dhrd was initiated by the national working group on preventive cardiology and rehabilitation , under the danish society of cardiology , and was approved as a national clinical quality registry on cr by the danish health and medicines authority in 2011 . the dhrd is supported by the cross - disciplinary professional associations : the danish society of cardiology , the danish nurses organisation , the association of danish physiotherapists , and the danish association of clinical dieticians . the database is available at www.danheart.dk ; however , this web site can only be accessed through the danish hospital internet networks . the danish clinical registries , which constitutes the infrastructure of the danish national clinical quality registries , has responsibility for the maintenance of majority of the data and data protection , as well as seeking funding . the database is headed by a steering committee with an elected chair , an executive committee , and an academic secretariat . data are collected and maintained according to danish data protection laws and regulations , without the need to obtain patient consent . the dhrd was initiated by the national working group on preventive cardiology and rehabilitation , under the danish society of cardiology , and was approved as a national clinical quality registry on cr by the danish health and medicines authority in 2011 . the dhrd is supported by the cross - disciplinary professional associations : the danish society of cardiology , the danish nurses organisation , the association of danish physiotherapists , and the danish association of clinical dieticians . the database is available at www.danheart.dk ; however , this web site can only be accessed through the danish hospital internet networks . the danish clinical registries , which constitutes the infrastructure of the danish national clinical quality registries , has responsibility for the maintenance of majority of the data and data protection , as well as seeking funding . the database is headed by a steering committee with an elected chair , an executive committee , and an academic secretariat . the national dhrd , aimed at monitoring and improving the quality of cr programs , has recently been established in denmark . the dhrd is unique due to the direct and simultaneous linkages to the administrative hospital registers . this resource makes follow - up on clinically important outcomes , such as the number of hospitalizations and mortality rate , directly accessible . accreditation by use of clinical quality registries has been pointed out as an important tool to improve clinical practice within cr and clinical follow - up care13 ( figure 2 ) , and referral to cr has been added as a performance measure following acute coronary syndrome by the european society of cardiology and the american heart association.14,15 the growing literature indicates that clinical quality registries can provide vital information that can be used to facilitate improvements in practice16 with positive effects primarily on the processes of care , whereas measures on outcome of care are less often positively affected.16 findings , however , are heterogeneous , indicating that neither a registry itself nor the reporting of data initiates change as quality improvement requires comprehensive approaches,17 including successful implementation of the registry and continuous data quality assurance , as well as transparency and timely feedback.18 based on experiences from the field of stroke care , embracing a comprehensive quality assurance approach , including a culture with focus on quality , thorough data entry , and availability of feedback as well as willingness to act on feedback , is critical in order to achieve improved quality of care.18 this can not be achieved by dhrd alone , but is a shared responsibility of the cr team and the health care sector . the dhrd includes all hospitals offering cr and will , therefore , reflect all hospital - based cr services at a national level . since 2007 , the municipalities in denmark have had the overall responsibility for prevention and rehabilitation following discharge . cr services were , hence , transferred from hospitals to the municipalities ( either partly or fully ) in 2007 . quality assurance and accreditation of municipality - based cr will , therefore , become an important focus for dhrd in the future . a high reporting and coverage rate of patients with chd is , therefore , essential . coverage of the database will be closely monitored and routinely evaluated by matching data to the administrative data in the danish national patient register.7 the aim is to achieve > 90% coverage of cr activities in all cr programs . dhrd is an online , clinical quality database that aims toward providing higher quality cr for patients with chd in denmark . data can be assessed as a part of research related to both the outcome and organization of cr .

aim of databasethe danish cardiac rehabilitation database ( dhrd ) aims to improve the quality of cardiac rehabilitation ( cr ) to the benefit of patients with coronary heart disease ( chd).study populationhospitalized patients with chd with stenosis on coronary angiography treated with percutaneous coronary intervention , coronary artery bypass grafting , or medication alone . reporting is mandatory for all hospitals in denmark delivering cr . the database was initially implemented in 2013 and was fully running from august 14 , 2015 , thus comprising data at a patient level from the latter date onward.main variablespatient - level data are registered by clinicians at the time of entry to cr directly into an online system with simultaneous linkage to other central patient registers . follow - up data are entered after 6 months . the main variables collected are related to key outcome and performance indicators of cr : referral and adherence , lifestyle , patient - related outcome measures , risk factor control , and medication . program - level online data are collected every third year.descriptive databased on administrative data , approximately 14,000 patients with chd are hospitalized at 35 hospitals annually , with 75% receiving one or more outpatient rehabilitation services by 2015 . the database has not yet been running for a full year , which explains the use of approximations.conclusionthe dhrd is an online , national quality improvement database on cr , aimed at patients with chd . mandatory registration of data at both patient level as well as program level is done on the database . dhrd aims to systematically monitor the quality of cr over time , in order to improve the quality of cr throughout denmark to benefit patients .

Aim of database Study population Data collection Main variables Follow-up Examples of research Ethics, funding, and administrative issues Discussion Conclusion

PMC4499983

fibromyalgia syndrome ( fms ) , which is characterized by widespread pain and fatigue , is a disease that affects the connective tissues and locomotor system1,2,3 . patients with fms also suffer from weakness , fatigue , lack of energy , sleep disturbances , waking up tired in the morning ( non - restorative sleep ) , and stiffness . while complaints especially in cold weather increase with the change of seasons , patientswork is affected and they experience , a decrease in their quality of life4 . it is reported that the incidence of fibromyalgia is 3.4% among women and 0.5% among men , and 7080% of patients are women5 . although it may be observed at any age groups6 , fms is often observed in sedentary women between 3050 years of age7 . it is reported that muscle oxygenation changes , and psychological , biochemical , hormonal and immunological factors are involved in the pathogenesis of fms8 . in addition , irritable bowel syndrome , tension - type headaches , perfectionist personality , premenstrual syndrome , sicca , female urethral syndrome , and raynaud like symptoms are all associated with fms10,11,12 . the american college of rheumatology ( acr ) suggested some criteria for the classification of fibromyalgia in 1990 . according to the criteria , widespread pain lasting longer than 3 months ( pain in the right and the left side and in the lower , and upper back must be located in the axial skeleton ) and tenderness in at least in 11 of 18 tender points are symptomatic of fms5 . in addition , hyperemia and skin discoloration are observed at the tender points of patients with fms10,11,12,13 . there is no suggested cure for fibromyalgia , as in the treatment of common chronic illnesses , so informing and educating the patients about the illness should be the focus of treatment14 . numerous studies have shown that low intensity physical activity and exercise including stretching , swimming , walking , jogging , and cycling are beneficial for patients with chronic pain and fms , increasing fitness levels , reducing the spread of pain and tenderness , improving the quality of life , and reducing depression levels15,16,17,18,19,20,21,22 . muscular strength , endurance , and the aerobic fitness levels of fms patients are lower than those of healthy controls23,24,25 . thus , it is important to provide well - structured exercise programs and physical activity for fms patients in order to reduce their sedentary life style . in the study of gowans , 20 female patients were assigned to pool exercise twice a week for 30 minutes , while a control group did not do any exercise . there were significance changes in fibromyalgia impact questionnaire ( fiq ) , fatigue , pain , and improvement in disease symptoms between the control and experimental groups . alina investigated the relationship between functional capacity and a self - report of quality of life with the practice of regular exercise by patients with fms , and found that intervention elicited26 significant differences in the quality of life and functional capacity between the regular exercise group and the no - regular exercise group . a significantly lower score for functional capacity in the health assessment questionnaire ( haq ) was found in the no - regular exercise group , while higher values were observed in the regular exercise group for the physical functioning ( p<0.001 ) and bodily pain ( p<0.05 ) domains of sf-36 . there were no significant differences between two the groups in the physical , general health , vitality , social functioning , emotion and mental health domains , and alina concluded that regular exercise had a positive impact on the functional capacity and quality of life of patients with fms26 . each session consisted of 45 minutes and the fms patients in the exercise group were followed for 6 months . their fiq anxiety scores showed a significant improvement compared to the control group7 . in a study of 50 patients with fms by altan et al . , the patients were divided into experimental and control groups and performed balneotherapy pool workouts . the experimental group performed a two - week training program of 2 35 minutes sessions / week consisting of aerobics , flexibility and relaxation exercises . the control group did not participate in any exercise program and were only treated with spa therapy . the experimental group showed a significant level of depression . in both groups , significant improvements in pain intensity stiffness , and fatigue were observed27 . in the studies conducted by verstappen fms patients were tested after exercise treatments including aerobics and pool exercises10 , 11 . the exercise groups showed improvements in overall well - being compared to the control group , and both study groups reported that the exercise program had not only a corrective effect on posture during sitting or standing upright , due to strengthening of the muscles , but also decreased pain through more balanced muscle contraction12 . various exercise modalities such as ground - based , water - based , aerobic , pool or aquatic balnotherapy , and hot water spa are used in the treatment of patients with fms28,29,30,31 . especially , balnotherapy ( hot water therapy ) , isokinetic strength stretching , aerobic and strengthening exercises are commonly used for the reduction of pain29 , 31 . however , the implementation of these exercises are generally based upon the availability of facilities , so water - based exercises are very rare in turkey . this study compared the effects of home - based isometric strength and stretching , gym - based aerobic and pool - based aquatic exercises programs on the physical and psychological parameters of patients with fms . the data were collected from fibromyalgia patients who were diagnosed by a specialist in the clinic of physical medicine and rehabilitation department , faculty of medicine , cukurova university . among the 107 patients recruited for the study , 32 were excluded because : 9 were postmenopausal , 10 were over 50 years , refused to participate in the study , one had a cardiovascular problem , one had cushing syndrome , and one was under 18 years . the study was approved by the local ethics committee . after informing all the subjects about the design and the risks of this study , the patients were randomly assigned to one of three exercise program groups and matched by age and their symptoms of fms . the exercise training programs comprised aerobic gymnastic , isometric strength - stretching , and aquatic exercises , all of which aimed to increase flexibility , and muscular and aerobic endurance based on graded activity . seventy - five female patients with fms according to the acr definition between the ages of 1850 ( 35.08.8 ) were randomized into three groups . the first group ( n : 25 ) performed a home - based isometric strength and stretching exercise program ( issep ) lasting 15 minutes per day . the second group ( n : 25 ) attended a gymnastic - based aerobic exercise program ( aep ) with group therapy two times per week32 . the third group ( n : 25 ) attended a pool - based aquatic aerobic exercise program ( aaep ) with group therapy two times per week . the durations of the exercise programs were 40 minutes for aep and aaep in first month , 45 minutes in the second month and 50 minutes in the third month . the secondary outcome measures were : health status fiq , the six - minute walk test ( 6mwt ) for endurance33 , quality of life scores represented by sf-36 physical and mental component scores34 and depression as assessed by the beck depression inventory ( bdi)35 . the intensity of exercise was determined according to the karvonen method and was generally 6080% of a subject s maximal heart rate . heart rates were measured eight times at regular intervals during the exercise program to monitor loading level during aerobic exercise programs . three - minute loadings with 30 seconds rest between three sets of low to moderate intensity were repeated in the first month of the exercise program ( 3 3 ) . in the second month 30 seconds high intensity loadings with 30 seconds rest period between four sets were performed after three minutes of low intensity loading ( 3 4 ) . with reducing rest intervals from 30 to 10 seconds , three - minute loadings of five sets were repeated in the third month of the exercise program ( 3 5 ) . spss ( statistical package for social sciences ) for windows version 13.0 was used for the statistical evaluation of the data . descriptive statistics were calculated and the chi square test , mann - whitney u test and kruskal - wallis test were used for two - and three - group comparisons . the wilcoxon test was also used for pairwise comparisons of pre- and post - test . demographic characteristics of the patients including age , weight , height , mean symptom duration , mean tender point count and educational level were similar . baseline values of the patients vas , bdi , fiq , 6mwt and sf-36 scores were also similar among the three groups . the results are presented in table 1table 1.comparison of the primary and secondary outcome scores at baseline and after 12 weeks of trainingvariablesgroupsnbaselineafter 12 weeksmean rankmean sdmean rankmean sdvas / mm**issep2535.768.211.857.670.412.5aep2537.570.012.928.448.28.8aaep2540.771.513.128.148.09.3bdi**issep2541.919.410.156.422.610.0aep2539.920.512.333.59.96.2aaep2532.115.79.024.16.17.8fiq**issep2539.659.116.556.862.916.1aep2535.358.313.429.636.615.7aaep2539.160.112.127.634.917.16mwt / m**issep2534.1541.553.319.7540.453.8aep2546.7569.548.349.9628.855.5aaep2533.2543.456.444.4619.461.8mcs*issep2533.431.69.019.232.029.4aep2536.832.59.742.745.27.0aaep2543.735.27.952.249.48.3pcs**issep2534.537.37.618.036.88.4aep2546.441.88.451.253.65.4aaep2533.136.48.544.850.37.4*significant at 0.05 level , * * significant at 0.01 level , sd : standard deviation . issep : home - based isometric strength and stretching exercise program aep : gym - based aerobic exercise program , aaep : pool - based aquatic aerobic exercise program vas : visual analog scale , bdi : beck depression inventory , fiq : fibromyalgia impact questionnaire , 6mwt : six - minute walk test , mcs : mental component scores , pcs : physical component scores . the comparisons of the pre - and post - test results of each group are presented in table 2table 2.the comparison of pre- post- test results of each groupvariablesgroupsnbaselineafter 12 weeksmean rankmean sdmean rankmean sdvas / mmissep**2535.768.211.857.670.412.5aep**2537.570.012.928.448.28.8aaep**2540.771.513.128.148.09.3bdiissep**2541.919.410.156.422.610.0aep**2539.920.512.333.59.96.2aaep**2532.115.79.024.16.17.8fiqissep2539.659.116.556.862.916.1aep**2535.258.213.429.636.615.7aaep**2539.160.912.127.634.917.16mwt / missep2534.1541.453.319.7540.453.8aep**2546.7569.548.349.9628.855.5aaep**2533.2543.356.444.4619.461.8mcsissep2533.431.69.019.232.09.4aep**2536.832.59.742.745.37.0aaep**2543.735.27.952.249.48.3pcsissep2534.537.37.618.036.88.4aep**2546.441.88.451.253.65.4aaep**2533.136.48.544.850.37.4*significant at 0.05 level , * * significant at 0.01 level , sd : standard deviation . issep : home - based isometric strength and stretching exercise program , aep : gym - based aerobic exercise program , aaep : pool - based aquatic aerobic exercise program vas : visual analog scale , bdi : beck depression inventory , fiq : fibromyalgia impact questionnaire 6mwt : six - minute walk test , mcs : mental component scores , pcs : physical component scores . * significant at 0.05 level , * * significant at 0.01 level , sd : standard deviation . issep : home - based isometric strength and stretching exercise program aep : gym - based aerobic exercise program , aaep : pool - based aquatic aerobic exercise program vas : visual analog scale , bdi : beck depression inventory , fiq : fibromyalgia impact questionnaire , 6mwt : six - minute walk test , mcs : mental component scores , pcs : physical component scores * significant at 0.05 level , * * significant at 0.01 level , sd : standard deviation . issep : home - based isometric strength and stretching exercise program , aep : gym - based aerobic exercise program , aaep : pool - based aquatic aerobic exercise program vas : visual analog scale , bdi : beck depression inventory , fiq : fibromyalgia impact questionnaire 6mwt : six - minute walk test , mcs : mental component scores , pcs : physical component scores for vas , the issep group had the lowest mean score before the exercise program , while the aaep group had the lowest score after the 12-week exercise program . for bdi , the aaep group had the lowest mean value before and after 12-week exercise program . for fiq , the aep group had the lowest mean score , while the aaep group had the lowest value after the 12-week exercise program . for 6mwt , the aep group had the highest mean value before and after the exercise program . for mental component scores of sf-36 ( mcs ) , the aaep group had the highest mean score before and after the exercise program . for physical component scores of sf-36 ( pcs ) , the aep group had the highest mean score before and after the exercise program . vas and bdi values showed significant differences in their pre - and post - test values in all three groups , while fiq , 6mwt , mcs and pcs values showed no significant differences between pre - and post - test in the issep group . on the other hand , the results of both the aep and aaep groups showed significant differences in fiq , 6mwt , mcs and pcs between pre - and post - tests fibromyalgia syndrome is a chronic musculoskeletal disorder with generalized muscular pain characterized by fatigue and tenderness at specific tender points along with commonly associated symptoms such as altered sleep , cognitive and emotional disturbances ( anxiety and depression ) , increase of disability and decrease of quality of life . researchers suggest that the effective treatment for fibromyalgia should include medication with lifestyle changes and alternative treatments28 . thus , this study aimed to determine which exercise program among isometric strength and stretching , and aerobic and aquatic exercises programs is the best at reducing the symptoms of fibromyalgia36 , 37 . vas was compared among the three groups and , both the aquatic ( aaep ) and gym ( aep ) groups showed statistically significant improvements compared to the isometric strength - stretching exercise ( issep ) group . in addition , no statistically significant difference was found between the aaep group and the aep exercise group . when the intra - group results were compared , only the issep group showed an increase in the mean value of vas , whereas both the aaep and aep exercise programs improved the pain threshold with only slight differences between them . no significant difference in vas was found when aep and aaep groups were compared38 . in the 48-week study conducted by ramsey et al . , one group performed aerobic exercises , while the other group participated in a home - based stretch - strength exercise program , and no improvement in the values of vas was found between the two groups . besides , no statistically significant difference between the groups was found16 . contrary to several previous studies reporting similar treatment effect of issep and aep , our present study found no difference in vas between aep and aaep16 . in addition , bircan et al . reported that aerobic exercise and strength exercise were similarly effective at improving symptoms , tender point count , fitness depression , and quality of life in fms39 . when bdi was compared among the three groups , no significant difference before the exercise program however , a significant decrease was found after the exercises in the mean values of the aaep and aep groups . the aep group showed more significant improvement than the aaep group in the level of depression . conversely , assis et al . found no significant improvement in bdi after the exercise program38 . in our study both the aaep and aep exercise programs had a significant effect on depression levels , but aep was more effective than aaep at decreasing the depression level . similar to our results , sanudo et al.40 reported bdi scores decreased by 8.58 ( p<0.001 ) and 6.44 points ( p<0.001 ) in a supervised aerobic exercise group and a combined program of supervised aerobic , muscle strengthening , and flexibility exercises , respectively . similarly , santiago and lacomba reported that aquatic and combined exercise , and alternative activities seemed to be better for the treatment of depression41 . among the exercise groups , both the aaep and aep exercise groups showed more improvement than the issep group in the mean value of fiq . also , no statistically significance difference was found between the aaep and aep exercise groups . reported that the level of improvement in the aaep group was higher than that of the aep group38 . in the study of mannerkorpi , the effect of aquatic training on fiq of fms patients was investigated with a combination of six sessions education , which resulted in significant differences in the mean value of fiq as compared to the control42 . found a significant difference in fiq between the groups in their studies17 . in the present study there was a significant improvement in fiq in both the aaep and aep groups , but there was no difference between the two groups . this result suggests that both of the exercise programs were equally effective at improving physical functioning , increasing quality of the life , and decreasing of pain . , who compared an exercise program of exclusively aerobic exercise , with another of combined exercise , and found similar improvements of around 14% in fiq40 . in previous studies , better results were obtained by the aquatic program , however , in the present study no significant difference was observed17 . showed that aquatic and home - based land exercise therapy was beneficial for pain reduction at the end of treatment , and after a long - term follow up of 3 months . additional benefits of aquatic exercises may be related to the water itself , as well as the benefits derived from the social aspects of exercising with others outside of the home43 . the comparison of our three groups revealed that both the aaep and aep groups showed statistically significant improvements in 6mwt as compared to the issep exercise group . no statistically significant difference was found between the aaep and aep groups , indicating that both aaep and aep had equally positive effects on walking ability . although the 6mwt is a simple , safe and low cost test that has gained much attention in the clinical and scientific fields33 , 44 , 45 , researchers have reported that a substantial number of women with fms show increased pain intensity and perceived effort compared to controls46 . combined six sessions of education investigated the effect of aquatic training on the 6mwt distance of fms patients . the 6mwt distance of the aquatic training group was significantly longer than that of the controls42 , suggesting that patients with fibromyalgia might benefit from aerobic fitness training . the pcs of sf-36 was compared across the three groups . both the pool - based aquatic and gym - based aerobic exercise groups showed more improvement than the home - based isometric strength - stretching exercises group . however , no statistically significant difference was found between the pool and gym groups . found no statistically significant difference groups in the sf-36 physical health scores of two groups38 . in the literature only the study conducted by jentoft et al . reported a result similar and comparable to that of our study . they did not find statistically significant differences in the physical health scores when the groups were compared17 . both the pool and gym exercise programs provided similar increases in the quality of life . both the pool - based and gym - based groups showed statistically significant differences , but the home - based exercise group did not . when the pool and gym groups were compared , a more significant difference was found in the pool group than in the gym group . two studies presented similar results , reporting no significant difference in terms of mcs17 , 38 . the results of our present study suggest that gym exercise programs lead to improvement of the mcs of fms patients , and that , different from other studies , the aquatic program was more efficient than the other programs in this regard . in a study conducted by van santen et al . , 37 fms patients were divided into two groups and one group with 19 members exercised with higher intensity while the other group with 18 members performed aerobic exercises of lower intensity18 . their showed low intensity exercises provided a medium - level improvement in the quality of life and physical health after 20 weeks , but it had no effect on the general health and psychological state of the patients ; also , the high intensity exercises had no statistically significant effect on the quality of life or physical health18 . in the study of salek et al . , patients were assigned to two groups , and one group of patients did exercises while the other group was given antidepressants and analgesics . when the pre - treatment and the post - treatment measurements after 16 weeks were compared , it was found that the level of sore points and pain had improved 48% in the group that exercised regularly , and 39% in the group that did not exercise . water - based exercises are not only an important effective treatment for fms7 , 9 , 27 , but they are also important and effective tools for improving isometric , isotonic , and isokinetic strength in young women47 , 48 . significant improvements in isokinetic knee extensor and flexor torques in the elderly after a water - based program were reported49 . it was also reported that isokinetic muscle contraction in the water provided excellent rate of strength gain over range of motion and with a low possibility of muscle injury compared to isometric and isotonic muscle contractions28 , 31 . the results of our present study show that the issep group demonstrated only slight improvements between pre - and post - test in fiq , 6mwt , mcs and pcs , while significant improvements ( p<0.01 ) in all variables were observed in the aep and aaep groups with respect to their baseline values . the issep group showed significant improvements ( p<0.01 ) only vas and bdi between pre - and post - test ( table 2 ) . the aaep group had statistically better results than the aep and issep groups for all variables . significant differences among the three groups were derived from the low improvements of the issep group , issep with the exception of 6mwt distance . both the aaep and aep groups had significantly higher scores of mcs and pcs than the issep group . in conclusion , aaep also had additional effects on the sf-36 mental health score and the bdi . aaep was also significantly more effective in the treatment of fms than to aep and issep . the superiority of aaep as a treatment method for fms may originate from its lack of impact force on the joints , and the use of only concentric or isokinetic contractions during shallow and deep water exercises28 . further research is required to assess the advantages and disadvantages of issep , aaep and aep in the treatment of fms using experimental groups matched for health status , symptoms , body structure , physical fitness and age , and a long - term follow - up .

[ purpose ] there are various treatment modalities for fibromyalgia syndrome ( fms ) , which is characterized by widespread pain and fatigue . the aim of this study was to investigate the effects of aquatic , aerobic and isometric strength - stretching exercises on the physical and psychological parameters of patients with fms . [ subjects and methods ] seventy five female patients with fms were randomly selected and divided into three groups . patients ( 1850 years ) were treated for 3 months using one of three methods : a home - based isometric strength and stretching exercise program ( issep ) , a gym - based aerobic exercise program ( aep ) , and a pool - based aquatic aerobic exercise program ( aaep ) . items evaluated were : the number of tender points , visual analog scale ( vas ) , fibromyalgia impact questionnaire ( fiq ) , the six - minute walk test ( 6mwt ) , sf-36 physical and mental health scores , and the beck depression inventory ( bdi ) . [ results ] the results revealed that aaep was the most effective treatment of the three . all of the groups showed significant improvements in all variables between pre - and post - test , except the mean values of vas and bdi in issep . [ conclusion ] the results suggest that aquatic aerobic exercise program is more effective than aep and issep in the treatment of fms .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

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birds reproduce within electromagnetic fields ( emfs ) from transmission lines . melatonin influences physiologic and behavioral processes that are critical to survival , and melatonin has been equivocally suppressed by emfs in mammalian species . we examined whether emfs affect photophasic plasma melatonin in reproducing adult and fledgling american kestrels ( falco sparverius ) , and whether melatonin was correlated with body mass to explain previously reported results . captive kestrel pairs were bred under control or emf conditions for one ( short - term ) or two ( long - term ) breeding seasons . emf exposure had an overall effect on plasma melatonin in male kestrels , with plasma levels suppressed at 42 days and elevated at 70 days of emf exposure . the similarity in melatonin levels between emf males at 42 days and controls at 70 days suggests a seasonal phase - shift of the melatonin profile caused by emf exposure . melatonin was also suppressed in long - term fledglings , but not in short - term fledglings or adult females . melatonin levels in adult males were higher than in adult females , possibly explaining the sexually dimorphic response to emfs . melatonin and body mass were not associated in american kestrels . it is likely that the results are relevant to wild raptors nesting within emfs.imagesfigure 1

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PMC4311565

karen lawson , md , abihm , an assistant professor of university of minnesota 's family medicine and community health , is director of integrative health coaching at the center for spirituality & healing ( www.csh.umn.edu ) . she is a physician , board - certified in both family medicine and integrative and holistic medicine . she was the 2003 to 2005 president of the american holistic medical association and a founding diplomate of the american board of integrative and holistic medicine ( abihm ) . she is an executive leader of the national consortium for credentialing health and wellness coaches ( www.ncchwc.org ) . gahmj : how have health coaching , coach training , and education changed in the last 10 years ? karen : health coach training and education have really been evolving over almost 2 decades . while we 've had coaching training in general for probably between 30 and 40 years , health and wellness coaching got started about 20 years ago , beginning with entrepreneurs with small , private foundations and organizations doing educational programs sometimes private one - on - ones but also within private corporations and for large employers . in the past 10 to 12 years , that field has really taken off , with more players coming into the field . probably the largest - volume trainer of health coaches we currently have is wellcoaches ( wellesley , massachusetts ) , and they 've been on the job for 12 years now . one of the biggest gains that we 've seen is with credibility and recognition in the marketplace . i 'm at the university of minnesota , and we began our program 10 years ago . there is an evolving spectrum from smaller , more limited , directed , and focused programs done for groups in corporate settings , insurance settings and with different kinds of employers all the way to graduate degree programs at major universities . there is a very wide spectrum , and that 's one of the gifts in the field but also one of the challenges : how do we come to common terrain and agree upon what it means when someone says that he 's a health and wellness coach ? karen : there is a large and growing swell of interest from people of many , many diverse backgrounds and areas of professional practice who are coming to health and wellness coaching as a new profession . there is also a natural flow of those from different healthcare professional backgrounds nurses , psychologists , social workers , allied health workers , nutritionists , even physicians . some of those people are adding onto the existing training that they have in whatever is their established area of expertise . augmenting those skill sets with coaching training the fascinating thing to me has been watching the people from widely varying fields who are coming to this saying , this is what i dreamt of doing my whole life . i have architects , engineers , lawyers who have had a personal calling in the area of health and wellness for years and have never worked in the field professionally and are now seeing an opportunity to take the passion that they have personally and to retrain to be able to work with people in a way that 's very health - focused . health coaching can be very prevention - focused ; it does n't have the disease framework that dissuaded many people from going into the healthcare field in the first place . that 's raising interesting challenges for those of us who are in the education realm . that 's really an area of the field for which we 're trying to gain clarity . i think there 's a pretty strong agreement from across the consortium and the training schools that people need a solid background with at least a bachelor 's degree or , if not a bachelor 's , then a licensed healthcare profession of some kind . the bachelor 's degree could be in a different discipline , but then we need to add further education on top of that . the irony is that sometimes i find the absolute best health and wellness coaches haven't come out of the ranks of conventional healthcare because they bring fresh eyes and they haven't become jaded by the pathology perspective that is so prevalent in most of healthcare . so while medicine in general and healthcare more broadly have done some wonderful things , looking through the lens of health and wellness has not been the orientation of those fields . therefore , there is now an opportunity for these people to bring their passions to work in new ways , and they 'll need additional training and different skill sets for creating those new ways of practicing , but i think it 's going to create a fascinating new face for healthcare . gahmj : do you see a master 's degree as a path for future coaches ? karen : in academic settings , there are three different ways people can obtain educational training in the field of health and wellness coaching . the first path is to come in through professional continuing education ; that 's a common path that some students take . the second is the route that we started with at the university of minnesota and several other schools have adopted as well , which is to offer what we call a graduate certificate . it 's not a full graduate program , but it 's a good fit for people who have existing , particularly graduate - level , education already . if you already have a master 's or a doctorate or a specific degree in the healthcare field and you obtain a health and wellness coaching certificate , that gives you the content that you need to do the work , and you already have a degree that 's at a sufficient level to get you into the job market . the third path , which is something that we just started this year at the university of minnesota , is to offer a master 's degree in integrative health coaching . one is we have new and excited graduates who are coming out of undergraduate training with bachelor 's degrees already knowing that this is the professional field in which they want to work . however , they recognize that in this competitive job market they need a master 's degree to get into many different kinds of positions in healthcare . the second reason is that there are those people who have backgrounds in other areas and are switching fields . they need to gain some greater understanding in the areas of lifestyle medicine , mind - body skills , mind - body science , chronic conditions , foundational nutrition , and movement practices . for those people , it 's a natural fit to come back to graduate school and get a full master 's degree in the area of health coaching so that they have adequate skill sets to be able to go out into the job market , work in healthcare , and understand the system into which they 're entering . gahmj : how can the national consortium for credentialing of health & wellness coaches reform coach training and education ? karen : one of the reasons the national consortium for credentialing of health & wellness coaches was formed was to set standards for credentialing education and training because with any new field , when a new profession is evolving , it 's a little bit like the wild west . you have mom - and - pop shops ; you have people that are doing 1 day of training and putting the credential on their business card and saying , hi , i 'm a health and wellness coach . one of the primary tasks for the consortium is defining necessary standards around the minimal criteria of what 's needed for education and what 's needed for training . when a new profession is evolving , when it 's first getting started , it 's widely variant . you have people who have done very little training or have purchased a certificate without any education , and then you have people who have studied for decades . so it 's important to bring together all of the stakeholders in the field , to combine our voices , to stand up and be heard , and say , we believe , as professionals in the field , that this is where the entry - level bar is set . this is the minimum that people need to have in their knowledge base , in their applied skills , and their abilities to interface with clients in the existing healthcare system . by having an organization like the consortium , we have the ability to bring all those voices together and to be able to let employers , third - party payers , and government officials hear our voices and recognize that there is a credible force for standards . once the standards exist , educational programs need to step up to meet those standards . that 's a natural progression . by making clear the items that we 've agreed upon in a way that is recognized and accepted nationally , new schools and new training programs that are launched will need to step up to a recognized standard . people running these entities will know where they need to go in order to understand the expectations for offering a recognized and eventually accredited training program . new students will seek out such accredited educational opportunities in order to meet employer and consumer expectations . linda bark , phd , rn , mcc , nc - bc , is founder and president of the wisdom of the whole coaching academy , which teaches a holistic / integral approach to coaching for health and wellness practitioners . dr bark 's model also works effectively in leadership and management arenas , and she trains leaders internationally , especially in india . the wisdom of the whole , the textbook for her courses , won first place in professional development from the american journal of nursing . a book she coauthored , the art and science of nurse coaching , was published by the american nursing association and has set standards internationally for a coaching role in nursing . linda : from the very early days of forming the consortium , we realized there were 2 groups that were using coaches and coaching . one was stand - alone coaches who were just doing coaching , so they were working with patients and behavior change . then there were the people who already were trained in clinical roles and were adding coaching to their patient skills that they already had . in healthcare , there is a wide variety of people saying they 're coaches and that 's one of the things that the consortium and our standards will address . one of the problems that we have in healthcare is that there are people calling themselves health coaches when they have had maybe an hour of training or sometimes no training . they 're really asked to ensure compliance : they 're not helping patients get clear about their goals and how they 're going to achieve them but rather to make the patient comply with the doctor 's orders . besides these 2 groups , there is another place that coaching is burgeoning , and that is in healthcare leadership . over the past years of training , i have been fascinated by the impact i have seen coaching have in leadership training . there is a time for the expert role in patient care and also in management , but when it comes to intrinsic motivation and empowerment , coaching comes to the fore - front in both areas . they become excited when they are truly valued and able to offer solutions and next steps and move toward important goals . most people like working at their potential and feeling successful so they stay and contribute to their team or organization in new ways . for example , one of my students is a new nurse manager , and she is amazed at how her coaching skills are helping her transform her unit into a place where people are collaborating more on how to address problems that have been around for years . she is listening to her employees , and they are beginning to listen to each other more and also the patients . the awareness of this is increasing because of outcomes and pay - for - performance measures . we 've known for a while that it is n't working , but because of changes in laws and healthcare , we 're seeing now that people are n't changing behaviors . healthcare systems are realizing they 're not getting money for things that they need to be paid for because people are n't following through and achieving the outcomes they should . the changes that are being made involve health coaching because from the research , it 's becoming very clear that people become motivated internally , yet when they 're supported , when they have autonomy , when they have confidence , they really do make necessary changes . health coaching is becoming more popular at the very time when leaders in healthcare systems are saying , oh my gosh , we haven't done this correctly ; we 're not doing well . it 's a perfect storm in a good way when health coaching has an opportunity to move in a way that can help people make lasting changes . one of the myths about coaching in healthcare is that a health practitioner needs a lot of time to do coaching . really , i haven't found that to be true , so coaching is popping up in many areas of practice . a powerful question can take less than a minute to ask and a minute or so to answer and yet have a great impact on patients and the changes they want to make . one of my students began to ask her orthopedic patients what they wanted to do in the summer . she did this as she was taking care of them so it was not a sit down kind of interaction . they realized that they wanted to go on vacation and take care of young children in their family and as they talked , they got clear about their own motivation . this changed their behavior , and they started to work harder in physical therapy and rehab . the outcomes were so obvious that other nurses asked her what she was doing , and she explained the coaching process to them . one of the trends that i 've seen in health coaching is that some health coaches tend to come from a whole - person perspective . there is a trend now to focus on the patient experience , to see the whole person , and this bigger picture is helping patients make lasting changes . it is my belief that when patients integrate feelings , thoughts , images , body feedback , and values , they arrive at decisions or actions that are more congruent to the whole self , and they move faster and make lasting change . they 're more holistic , and that is really a gift , i think , that health coaching could give to coaching in general . gahmj : how can the national consortium for credentialing of health & wellness coaches help set the standards and guidelines to help those using coaching in a clinical role ? linda : as we 've mentioned , there are so many people calling themselves health coaches , so we want to set a minimum standard to ensure that the coaching will be successful . employers have been calling me for years asking , what kind of training do health coaches need ? what shall i include in the job description ? how do i measure outcomes ? this is another area in which the standards and the guidelines that we 're setting can truly help healthcare . coach meg , a 17-year veteran of the biotechnology industry , founded the wellcoaches school of coaching ( wellesley , massachusetts ) for health professionals in 2002 in collaboration with the american college of sports medicine . margaret is co - founder and co - director of the institute of coaching at mclean hospital , a harvard medical school affiliate , and co - director of the annual coaching in leadership & healthcare conference offered by harvard medical school . she co - founded and co - leads the national consortium for credentialing of health & wellness coaches , which is developing national standards , certification , and collaborative research . she is coauthor of the first coaching textbook in healthcare , the coaching psychology manual , published by lippincott , williams & wilkins , and a harvard health book entitled organize your mind , organize your life . gahmj : how did the national consortium for credentialing of health & wellness coaches get started ? margaret : the national consortium for credentialing health & wellness coaches which is a mouthful got started because a number of us had come together several years ago and were focused on creating a new professional role in healthcare and corporate / consumer wellness , and we had all come to the same conclusion : that we needed a standard , we needed to set a bar in order to move the field forward , to do good research so that we could bring credibility to the field in order to support reimbursement and dissemination . we were finding interest , in different ways and in different places . dr mehmet oz and his network were really interested developing national standards for health and wellness coaches , as was the integrative medicine field ; there were a number of coaching schools in the private sector , too . in the end , we all came together and realized that we have a common mission , which is to build this field , and we needed a standard . the consortium brought together thought leaders who were passionate about setting high standards for the field , and up until that point , we were not collaborators . the way the marketplace works , we were competing , but we also all realized that collaboration was going to be the answer to move the field forward . it was at that point that about a dozen or so leaders came together , and probably the biggest accomplishment that we made in the early years was and it did take years was getting us to all agree on there being one code , one standard for coaches across the full spectrum , from the clinical world to the corporate and consumer worlds . we knew it would really confuse the marketplace if we had different standards and different baskets of competency . so we pulled that all together , and once we had decided that we wanted to build this together , then we had to raise money , we had to create a legal entity , we had to build a website . and now it 's moving at a rapid pace because we created a legal entity , we raised donations , we completed the first steps that are required to create a new profession . it took years for us to gather enough practicing coaches in enough areas to have a large and diverse cadre that could then define the job , define the tasks , and figure out the knowledge and skills that are needed . now we have achieved the first stage : we have defined what a coach is and what a coach does and what skills and knowledge a coach has to have to be able to practice this set of tasks . we 're very close to defining what it takes in terms of training and education and background to get there , and then the next step is to bring the field together to endorse and pressure - test and make sure we 've done all the right things . gahmj : in what year did the national consortium for credentialing of health & wellness coaches get started ? the first stage was team building , and then we held a summit with 70 stakeholders participating , which was an amazing experience because we used an appreciative inquiry process that led us to a common vision : that we really wanted to transform the healthcare system and be a catalyst for change . we could see that the way coaches work and participate in the patient - centered , client - centered agenda , we could be really pivotal in that experience from there , the next step was to define the path to a national credential , and there are lots of different ways to do it . we eventually landed on building a nonprofit entity , raising money , and following a best practices process to figure out the tasks , the skills , and knowledge that coaches needed to perform . so we built the legal structure to do that , we created a board of directors , and we were all pretty well coalesced around one agenda . the biggest accomplishment was we went from being competitors to having deep friendships and a collaborative agenda . i think we 've all transcended , we 've all moved past ego and self - interest and our own organizations and even personal and financial interest to say , this is for the betterment of the coaching industry and the world . and that energy , that collaboration of serving a larger cause , is now the life force behind this group . we 're not far off having a national certification : a year , maybe 2 or 3 years . but the energy is there , the resources are there , so we 're not far from the finish line now . margaret : the consortium has a mailing list ; people can sign up for that and keep up to date with what we 're doing . and second , making a donation is the best way to to show your support , to help fund the various activities that we 're needing to do to help drive this forward . it also helps to be an advocate through social networking and to talk about the value of standards . there are a lot of people calling themselves coaches who have not been through any formal training in coaching competencies . and and so we really need for people to help make the case for the field and for the betterment of the field . this is a team effort , and we 're hoping that more coaches and more organizations that are supporting coaching will get on board with us and make donations and spread the word because we are volunteers , and the more shoulders to carry this opportunity and project , the better . if you 're a coach or you 've been a client of a coach or coach trainer or you 're an organization that employs coaches or delivers coaching services , we 'd love to have your help because we 'll all be better off . the field will grow and flourish when all of us are together on this mission . margaret : we live in a world where evidence is essential when you 're offering new interventions and new programs in the healthcare system , so it 's very important to be thinking about how we 're going to generate the evidence that 's going to make the difference . the evidence used to trickle in , and now it 's a bit of a flood . there was a meta - analysis done a year ago , and we have nearly 300 studies of health and wellness coaching that would meet the definition of what a coach does which means we help people find their own way . we help people discover their goals , we help them be more autonomous and more confident and to improve their health and wellness in ways they 've not been able to do alone . now we have a growing literature base ; we had an article published recently in global advances in health and medicine on smoking cessation in a hospital in the midwest . it showed that multiple 40-minute coaching sessions over a few months had something like a 73% quit rate within a month , and within a year we were getting close to 60% , which is a pretty good predictor of long - term smoking cessation . that 's an example of how we can move the field forward . coaching addresses the self - care side , which is applicable to many , many diseases , so we have research that duke university has done on coaching in cases for heart disease and diabetes . we have studies on attention deficit hyperactivity disorder , with cancer survivors , in cases of obesity . there are case reports in primary care , employee wellness , and rehab situations , and i 'm involved in a clinical study on fibromyalgia that 's going very well , so we 're starting to see breadth and depth in the research . now the challenge is that in order to really evaluate the evidence , you have to have one population that 's homogeneous and one protocol . so what we 're seeing is an emergence of a lot of data across many different populations with many different protocols . randomized studies are so difficult to fund , and they 're often very narrow in terms of the population and the protocol . so i think the future is going to see all of us pooling all of our data together and going with this new format that the national institutes of health is supporting , which is practice effectiveness research , where you need tens of thousands of patients , or in our case , coaching clients , across a wide range of settings and a wide range of clinical indications . when we pull all of that together , we 're going to see significant improvements . we need to assemble a team of researchers , and i think we 're within a year or 2 of having enough data to say , this is moving the needle . the group is collaborative , and in the coming years we 're going to be able to see what worked and what did n't . we have new research that shows that there 's a statistical significant improvement in blood pressure as a result of health coaching . through the relationship , through the promotion of autonomous control and efficacy and agency you reduce stress , which likely brings blood pressure down , and then the natural desire to be well surfaces . i often say the thing we need to do most is to bring down stress levels so that the natural urge to be well will bubble up , and then people will have the inner resources to do the right things and become healthy . we 're on the way , but i think we need to be clear about the standard and then go out and pool all the data and get it all into the literature just the way global advances in health and medicine has been doing : through case reports , through more nontraditional ways of showing that this is working in practice . margaret : the national consortium has a websitencchwc.organd we have a page on it called research , and there we keep a power point deck that summarizes all the recent studies that add up to make the case for the positive outcomes based on both hard biometric data as well as more general wellbeing measures . it 's a joy to be a coach , to train and mentor coaches on a large scale , to collaborate with the leading health and wellness coach trainers and educators . to help manifest the life and world - changing potential of coaching in healthcare and wellness through standards and evidence

in september 2014 , global advances in health and medicine editor michele mittelman , rn , mph , interviewed four of the leaders in health and wellness coaching about trends in coaching and the progress of the national consortium for credentialing of health & wellness coaches . following are the transcripts of those interviews . additionally , videos of the interviews are available at www.gahmj.com .

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PMC4738911

the use of whole body vibration ( wbv ) has become an increasingly popular method of training for enhancing performance in sport . significant improvements in performance have been demonstrated across a wide spectrum of sporting activities with the use of wbv training . the most common improvements are seen in explosive activities such as weightlifting or vertical jump tests ( 2 ) . a review of vibration studies by issurin shows a clear trend of performance enhancement towards that of explosive strength and maximum dynamic force production with decreases being seen in endurance activities ( 8) . it appears that the effects of wbv are short lived and improvements are best seen immediately following vibration . it has also been shown that prolonged duration vibration can be damaging to health such as that seen in hand - arm vibration syndrome commonly seen in occupational health clinics as well as damaging to athletic performance ( 12 , 14 ) . short duration wbv ( 1 minute or less ) however , demonstrates significant improvement ( 1 , 7 , 9 , 10 ) . wbv has been described to produce gravitational acceleration changes similar to those of power and strength training and has been reported to improve muscular power of the upper and lower musculature , changes to hormonal profile , such as increases in testosterone and growth hormone levels ( 2 ) , and increases in cardiovascular responses ( 4 ) . one of the theories is that muscles have a particular resonant frequency that , when excited , stimulates the gamma motor neurons to cause muscle spindle contraction . it is also theorized that muscles are dynamically tuned to match the resonant properties of the entire musculoskeletal system ( 11 ) . muscle spindle contraction may cause an increase in the responsiveness of the extrafusal alpha fibers of the muscle which would allow for a stronger and quicker reflexive response . the muscle spindle s connections provide a means for rapidly executed reflex adjustments in muscle length following any stretching or change in muscle load . since the neural pathways for the muscle spindle are relatively short , the change in motor neuron firing and ultimately muscle adjustments can be made more rapidly than if intervention by the central nervous system of the brain were needed . if this theory is indeed the case , it would be expected to be a short - term effect as the muscle would rapidly return to a state of relaxation following stimulation . five weeks of wbv training in addition to conventional training of sprint - trained athletes showed no surplus value upon the conventional training program to improve speed - strength performance in sprint - trained athletes ( 5 ) . although several studies have analyzed the long term cumulative effects of wbv , very few studies have been aimed at wbv s immediate , acute effects in sport , especially on sprint starts in a sprint trained population . this study analyzed the effects of wbv on trained sprint athletes and specifically looked at force production off of the sprint blocks immediately following wbv . this study was approved by the university internal review board for use of human subjects . participation in the study was voluntary and , following an explanation of the study , each subject gave informed consent . all subjects were tested for the maximum resultant force they could produce during a sprint starts on two testing days one week apart . subjects were randomly assigned to groups that performed whole body vibration ( wbv ) on the first or second days of testing . subjects mean height was 18.0 0.04 m , mean mass was 66.6 6.3 kg and mean age was 21.92.5 years . on each testing day three trials were performed . on the vibration testing trials the wbv protocol included 60 seconds of vibration at 26 hz at an amplitude of 4 mm on a powerplate ( galileo 2000 , orthometrix , white plains , ny ) . subjects stood on the platform with the heels slightly off the ground and 45 degrees of knee flexion . subjects completed their traditional warm - up on both testing days and were asked not to vary their warm - up between days . a typical warm - up for these subjects included about 10 minutes of jogging , followed by static and dynamic stretching , then four 80 m strides . measurements were taken during the teams preseason training . a kistler force plate ( 9287ba , amherst , ny ) was under the surface of an indoor track and measured peak ground reaction forces at a vibration frequency of 1000 hz . subjects completed a start and ran 30 m at maximum effort . in order to replicate a normal sprint peak ground reaction force was recorded for each start after normalizing by body weight . a wireless timing light system ( brower , draper , ut ) with sensors placed at head height were interfaced with starting blocks were used to determine 30-m sprint time . a repeated measures analysis of the variance ( anova ) was completed to compare conditions with the alpha - level set at 0.05 . this study was approved by the university internal review board for use of human subjects . participation in the study was voluntary and , following an explanation of the study , each subject gave informed consent . all subjects were tested for the maximum resultant force they could produce during a sprint starts on two testing days one week apart . subjects were randomly assigned to groups that performed whole body vibration ( wbv ) on the first or second days of testing . subjects mean height was 18.0 0.04 m , mean mass was 66.6 6.3 kg and mean age was 21.92.5 years . the wbv protocol included 60 seconds of vibration at 26 hz at an amplitude of 4 mm on a powerplate ( galileo 2000 , orthometrix , white plains , ny ) . subjects stood on the platform with the heels slightly off the ground and 45 degrees of knee flexion . subjects completed their traditional warm - up on both testing days and were asked not to vary their warm - up between days . a typical warm - up for these subjects included about 10 minutes of jogging , followed by static and dynamic stretching , then four 80 m strides . measurements were taken during the teams preseason training . a kistler force plate ( 9287ba , amherst , ny ) was under the surface of an indoor track and measured peak ground reaction forces at a vibration frequency of 1000 hz . subjects completed a start and ran 30 m at maximum effort . in order to replicate a normal sprint peak ground reaction force was recorded for each start after normalizing by body weight . a wireless timing light system ( brower , draper , ut ) with sensors placed at head height were interfaced with starting blocks were used to determine 30-m sprint time . a repeated measures analysis of the variance ( anova ) was completed to compare conditions with the alpha - level set at 0.05 . acute effects of wbv showed an increased peak ground reaction force 6% greater than the control condition ( table 1 , p=0.013 ) . no significant differences in 30-m sprint time whole body vibration is effective for aiding sprinters in producing slightly greater peak forces during sprint starts . previous studies show mixed results in the benefits of short - term wbv in vertical jump performance ( 2 , 15 ) . short - term training effects on sprinting speed were non - significant in this and other studies ( 4 ) . other studies have shown 10% or greater improvements in explosive strength ( 8 , 18 ) . while an increase in peak resultant force is encouraging , there are other factors to consider . studies have not concluded an optimal direction of the resultant force during the sprint start . there may be no benefit to increased force production if the direction of the increased force is not optimized . increases in momentum can only be attained by increases in impulse , which is a combination of force and time of force application . since the hands were not on the force plate at any point of the start , impulse could not be calculated accurately . so , there may not be increased running speed or an improved start just because of increased peak ground reaction forces . however , increased peak force typically leads to increased impulse and greater changes in momentum in human movement . thus , further research looking into the above issues should finish answering the question of whether acute wbv has a benefit on improved performance in the sprint start . the above listed limitations to determining an increase in performance based upon greater peak ground reaction force were supported by the lack of any decrease in 30-m sprint time . the 30-m sprint times were so similar that it is unlikely that vibration platforms will make it to the warm - up area at track meets . furthermore , the cost and the necessity to travel with the large and heavy platform make it unlikely that wbv will be used in competitive track meets . there is also the possibility of the increases being due to other variables such as health on the day of testing or psychological impacts resulting in a placebo effect . none of the studies to date have looked at collegiate sprinters , and data for the acute effects of wbv on any trained athletes is limited to one study testing vertical jump on trained handball players ( 3 ) . significant increases have only been shown with the use of acute wbv for recreational athletes in leg press power and counter - movement jump ( 8) . furthermore , many of the previous studies on acute wbv examine the effects that long term ( more than a minute ) wbv has while the subject is on the plate ( 6 , 13 , 1517 ) . long term wbv is likely to have a fatiguing effect due to excessive contraction of the extrafusal fibers caused by constant muscle spindle firing . this fatiguing effect may be the reason that a decrease in actual race time is not observed . short term ( acute ) wbv may stimulate the gamma motor neurons just enough for an increase in spindle sensitivity resulting in an increase in peak force . further research is needed to determine the optimal duration and frequency of the wbv training protocol . however , improvements in 30-m sprinting time do not exist in this or previous studies . athletic performance is affected by wbv , yet it is still unknown the method by which this is happening . further investigations of vibration training should answer at least three major questions : how is the response occurring ? what are the most efficient training protocols and how can the health risks associated with whole body vibration be eliminated ?

whole body vibration ( wbv ) is characterized by a vibratory stimulus emitted throughout the body through the use of a vibrating platform on which the subject stands . studies have shown over 30% increases in maximal explosive strength such as maximal speed biceps curl as well as increases in maximum dynamic force such as maximal sitting bench pull as the result of vibration . the purpose of this study was to investigate the effects of short term whole - body vibration on sprint starts among collegiate track athletes . on the first day eleven subjects were randomly assigned to either a non - vibration or vibration group for initial testing . the vibration group used whole body vibration along with their normal warm - up routine while the non - vibration group did not . force measurements were taken where the starting blocks were placed using a force plate embedded under the track surface following the warm up . one week later the groups alternated . the results were then compared between vibration and non - vibration groups for individual athletes . the vibration protocol occurred for 60 s at 26 hz with an amplitude of 4 mm on a galileo 2000 platform . repeated measures analysis of the variance showed peak resultant force was 6% greater when the vibration platform was utilized prior to the start ( p=0.013 ) . further research is needed to determine whether any meaningful differences exist in sprint start velocity as a result of wbv . there were no observed differences in the 30 m sprint times .

INTRODUCTION METHOD Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC4669535

the human apolipoprotein a - i gene ( apoa1 ) belongs to the apoa1-ciii - aiv gene cluster , located in chromosome 11q23 . this cluster consists of evolutionarily related genes that regulate serum lipid and lipoprotein levels ( karathanasis , 1985 , elshourbagy et al . , 1986 ) . the nucleotide sequence of apoa1 is interspersed by three introns ( karathanasis et al . , 1983 ) . apoa1 codes for apolipoprotein a - i ( apo a - i ) . since apo a - i is the major protein component of high density lipoprotein ( hdl ) particles which offer protection against atherosclerosis , therefore , much of the scientific work on apo a - i has traditionally focussed on its atheroprotective role . but , recent studies have implicated derangements in serum apo a - i concentration in some other pathological conditions that are not conspicuously related to atherosclerotic disorders . thus , genetic variations of apoa1 that influence serum apo a - i levels are of considerable interest . the g-75a and c+83 t single nucleotide polymorphisms ( snps ) are two common variations of the apoa1 gene . the g-75a polymorphism , located in the promoter region 75 base pairs ( bp ) upstream from the transcription start site of apoa1 , is due to a guanine to adenine interchange ( pagani et al . , the position of this snp has been variously described as 75 bp , 76 bp and 78 bp . this difference in representation is due to three different transcription start sites being described for apoa1 by different studies ( higuchi et al . , 1988 , sastry et al . , 1988 ) . it is caused by a cytosine / thymine substitution in the position 83 bp downstream from the transcription initiation site ( wang et al . , 1995 ) . each of these snps alters the recognition site for the restriction endonuclease mspi , which facilitates their detection by a restriction fragment length polymorphism ( rflp ) based technique . historically , the g-75a and the c+83 t polymorphisms have been investigated in relation to coronary artery disease ( cad ) and cardiovascular risk factors ( reguero et al . , 1998 , jeenah et al . , 1990 , sigurdsson et al . , 1992 , talmud et al . , 1994 , wang et al . , 1996 , bai et al . , 1996 , chhabra et al . , 2003 , shanker et al . , 2008 , dawar et al . , 2010 , miroshnikova et al . , 2011 , however , it is increasingly believed that these polymorphisms may have important roles to play in a host of other disorders such as alzheimer 's disease ( vollbach et al . , 2005 , smach et al . , 2011 ) , multiple sclerosis ( koutsis et al . , 2009 ) , schizophrenia ( yang et al . , 2010 ) , breast cancer ( hamrita et al . , 2011 ) , parkinson 's disease ( swanson et al . , 2014 ) , cholelithiasis ( dixit et al . , 2007 ) , gout ( cardona et al . , 2005 ) , diabetes mellitus ( ma et al . , 2003 ) and acute lung injury following cardio - pulmonary bypass surgery ( tu et al . , 2013 ) the g-75a and c+83 t polymorphisms have been studied globally in different populations . however , no information is available on these polymorphisms from the north - eastern region of india . the distribution of these polymorphisms varies with the ethnicity of the studied population and geography as previous studies have indicated . geographically , the north - eastern region of india is a unique corridor that links the indian subcontinent to east asia and southeast asia . assam is the most populous and second largest ( in terms of land area ) of the eight north - eastern states . data about the distribution of a polymorphism in the healthy subjects are a prerequisite to investigating the associations of the polymorphism with the implicated disease . with a renewed interest in g-75a and c+83 t polymorphisms worldwide and a variety of diseases being described in relation to them , we attempted to describe these polymorphisms in a sample population from assam , northeast india for the first time . our aim was to study the distributions of g-75a and c+83 t polymorphisms of the apoa1 gene in a cross - section of healthy subjects from assam , and further compare their frequencies with those observed in other populations from india and elsewhere . additionally , we assessed the influence of these polymorphisms on the phenotype by determining the variation in fasting serum apo a - i concentrations across the detected genotypes . a total of 150 unrelated and apparently healthy individuals of either sex were recruited for the study . all the individuals were inhabitants of assam and identified themselves as natives of the state , with no history of migration from elsewhere at least in the past four generations . the study was approved by the institutional ethics committee of gauhati medical college and hospital , assam . all the subjects voluntarily provided informed written consent to participate in the study prior to enrolment . blood samples ( after at least 12 h of overnight fast ) were drawn from the median cubital vein in two sets from each subject under sterile conditions . one set consisted of 3 ml of blood anticoagulated in edta vials for isolation of dna and genotyping . the second set included 3 ml of blood collected in plain vials and the serum separated by centrifugation after clot formation for apo a - i estimation . genomic dna was isolated from peripheral blood leucocytes ( obtained from edta admixed whole blood ) following a rapid dna isolation protocol ( sambrook and russell , 2001 ) . the concentration and purity of the genomic dna was ascertained by measuring the optical density ( od ) values at 260 nm and 280 nm . the samples had acceptable purity with the od ratio ( 260 nm/280 nm ) in the range of 1.7 to 1.9 . a 435 bp fragment at the 5 end of apoa1 gene spanning the promoter region and the first intron was amplified by polymerase chain reaction ( pcr ) using the following primer pairs as described previously ( larson et al . , 2002 ) : forward : 5-agg gac aga gct gat cct tga act ctt aag-3 , reverse : 5-tta ggg gac acc tac ccg tca gga aga gca-3 ( metabion international ag , germany ) . the pcr reaction was performed with a 25 l reaction mixture ( 1 l dna template , 0.5 l each of forward and reverse primers , 12.5 l dreamtaq pcr master mix from thermofisher scientific , 10.5 l nuclease - free water ) in a thermal cycler ( bio - rad model s1000 , usa ) using the following cycling conditions : initial denaturation at 95 c for 5 min , denaturation at 94 c for 30 s , annealing at 62 c for 1 min , extension at 72 c for 30 s and final extension at 72 c for 10 min . the amplified products were run on 1% agarose gels along with 100 bp dna ladder ( invitrogen , usa ) , and subsequently photographed under uv light using gel doc xr + system ( bio - rad , usa ) ( fig . the presence of g-75a and c+83 t polymorphisms was ascertained using an rflp method by digesting the pcr product with mspi restriction enzyme . for this , 10 l of the pcr product was digested overnight at 37 c with mspi under conditions specified by the supplier ( new england biolabs inc . the digestion products were analysed by running them on 12% polyacrylamide gels simultaneously with 50 bp dna ladder ( thermofisher scientific , usa ) . as the 435 bp pcr product contains three mspi cutting sites at 75 , + 37 and + 83 loci , its complete digestion would produce 4 fragments of sizes 66 bp , 114 bp , 46 bp and 209 bp . + 83 loci are polymorphic and coincide with the g-75a and c+83 t snps respectively . molecular sizes of the restriction fragments according to the genotype are shown in table 1 . concentration of apolipoprotein a - i ( apo a - i ) in serum was determined by immunoturbidometry in vitros 5600 integrated system autoanalyser using commercially available reagents and kits ( vitros chemistry products , ortho - clinical diagnostics inc . quality control measures were undertaken using control material ( apoa1 performance verifier 1 ) to ensure precision and accuracy of the test results . all statistical analyses were performed using spss version 11.5 ( spss , chicago , usa ) . for continuous variables , the results were reported as means with standard deviation ( sd ) . the kolmogorov smirnov test was performed to ensure normal distribution of the data . for the qualitative variables , the allelic and genotype frequencies of the two snps and the linkage disequilibrium between them were determined using popgene 2.0 software ( yeh and boyle , 1997 ) . conformity to hardy weinberg equilibrium ( hwe ) was assessed by goodness of fit chi - square test ( ) and g - square test . the gender - wise frequencies of the polymorphisms were also calculated . besides , diplotype frequency of the snps was ascertained . allelic frequencies obtained in our population were compared to those reported by other studies using test . comparison of continuous variables across genotypes was carried out by unpaired t - test or one way analysis of variance ( anova ) , whereas , categorical data were compared by test ( with yates correction wherever required ) . a p value of less than 0.05 was considered statistically significant and that less than 0.01 was considered highly significant . a total of 150 unrelated and apparently healthy individuals of either sex were recruited for the study . all the individuals were inhabitants of assam and identified themselves as natives of the state , with no history of migration from elsewhere at least in the past four generations . the study was approved by the institutional ethics committee of gauhati medical college and hospital , assam . all the subjects voluntarily provided informed written consent to participate in the study prior to enrolment . blood samples ( after at least 12 h of overnight fast ) were drawn from the median cubital vein in two sets from each subject under sterile conditions . one set consisted of 3 ml of blood anticoagulated in edta vials for isolation of dna and genotyping . the second set included 3 ml of blood collected in plain vials and the serum separated by centrifugation after clot formation for apo a - i estimation . genomic dna was isolated from peripheral blood leucocytes ( obtained from edta admixed whole blood ) following a rapid dna isolation protocol ( sambrook and russell , 2001 ) . the concentration and purity of the genomic dna was ascertained by measuring the optical density ( od ) values at 260 nm and 280 nm . the samples had acceptable purity with the od ratio ( 260 nm/280 nm ) in the range of 1.7 to 1.9 . a 435 bp fragment at the 5 end of apoa1 gene spanning the promoter region and the first intron was amplified by polymerase chain reaction ( pcr ) using the following primer pairs as described previously ( larson et al . , 2002 ) : forward : 5-agg gac aga gct gat cct tga act ctt aag-3 , reverse : 5-tta ggg gac acc tac ccg tca gga aga gca-3 ( metabion international ag , germany ) . the pcr reaction was performed with a 25 l reaction mixture ( 1 l dna template , 0.5 l each of forward and reverse primers , 12.5 l dreamtaq pcr master mix from thermofisher scientific , 10.5 l nuclease - free water ) in a thermal cycler ( bio - rad model s1000 , usa ) using the following cycling conditions : initial denaturation at 95 c for 5 min , denaturation at 94 c for 30 s , annealing at 62 c for 1 min , extension at 72 c for 30 s and final extension at 72 c for 10 min . the amplified products were run on 1% agarose gels along with 100 bp dna ladder ( invitrogen , usa ) , and subsequently photographed under uv light using gel doc xr + system ( bio - rad , usa ) ( fig . the presence of g-75a and c+83 t polymorphisms was ascertained using an rflp method by digesting the pcr product with mspi restriction enzyme . for this , 10 l of the pcr product was digested overnight at 37 c with mspi under conditions specified by the supplier ( new england biolabs inc . the digestion products were analysed by running them on 12% polyacrylamide gels simultaneously with 50 bp dna ladder ( thermofisher scientific , usa ) . as the 435 bp pcr product contains three mspi cutting sites at 75 , + 37 and + 83 loci , its complete digestion would produce 4 fragments of sizes 66 bp , 114 bp , 46 bp and 209 bp . the 75 and + 83 loci are polymorphic and coincide with the g-75a and c+83 t snps respectively . molecular sizes of the restriction fragments according to the genotype are shown in table 1 . concentration of apolipoprotein a - i ( apo a - i ) in serum was determined by immunoturbidometry in vitros 5600 integrated system autoanalyser using commercially available reagents and kits ( vitros chemistry products , ortho - clinical diagnostics inc . quality control measures were undertaken using control material ( apoa1 performance verifier 1 ) to ensure precision and accuracy of the test results . all statistical analyses were performed using spss version 11.5 ( spss , chicago , usa ) . for continuous variables , the results were reported as means with standard deviation ( sd ) . the kolmogorov smirnov test was performed to ensure normal distribution of the data . for the qualitative variables , the allelic and genotype frequencies of the two snps and the linkage disequilibrium between them were determined using popgene 2.0 software ( yeh and boyle , 1997 ) . conformity to hardy weinberg equilibrium ( hwe ) was assessed by goodness of fit chi - square test ( ) and g - square test . the gender - wise frequencies of the polymorphisms were also calculated . besides , diplotype frequency of the snps was ascertained . allelic frequencies obtained in our population were compared to those reported by other studies using test . comparison of continuous variables across genotypes was carried out by unpaired t - test or one way analysis of variance ( anova ) , whereas , categorical data were compared by test ( with yates correction wherever required ) . a p value of less than 0.05 was considered statistically significant and that less than 0.01 was considered highly significant . we found both the g-75a and the c+83 t loci to be polymorphic in the current population . the genotype and the allelic frequencies for the two polymorphisms are presented in table 3 . all the three possible genotypes , namely , gg , ga and aa were detected . the aa genotype was not found in females . on the other hand , for the c+83 t locus , the rare t allele frequency was 0.06 . the distributions of g-75a and c+83 t polymorphisms were consistent with a population at hardy weinberg equilibrium . the genotype and allelic frequencies for the g-75a site with respect to the a allele was apparently lower in females than that in males , but , it was not statistically significant ( p > 0.05 ) . however , for the c+83 t polymorphism , the frequencies were very similar in the two sexes . we encountered 5 combinations of the two polymorphisms in the studied population : gg / cc , gg / ct , ga / cc , ga / ct and aa / cc ( fig . overall , the gg / cc double homozygous variant was the most prevalent combination , while the double heterozygous ga / ct variant was the least common table 5 compares the allelic frequencies of the two snps obtained in our study with those reported by other workers . to assess the effect of the two polymorphisms on apo a - i concentrations in serum ( table 6 ) this was done to take into account the gender - specific effects . for the g-75a snp , no significant differences ( p > 0.05 ) in serum apo a - i values were noted across the gg , ga and aa genotypes . likewise , serum apo a - i levels were comparable between the cc and the ct variants of the c+83 t locus too . we further examined the combined influence of the two polymorphic sites by analysing the variation in serum apo a - i levels in a diplotype - specific manner . however , the apo a - i values did not differ significantly across the 5 diplotypes ( p > 0.05 ) . the current study was undertaken to investigate the g-75a and c+83 t polymorphic sites of the apoa1 gene in a sample population from assam , northeast india from where data was hitherto not available . both the polymorphisms were found to be in hardy weinberg equilibrium , indicating that they are normally distributed in the study population . for the 75 bp site , the gg genotype was the most common in the study subjects , followed by ga and aa variants respectively . however , it is unlikely that the aa variant is distributed in a sex - specific manner , and we attribute its absence in the female subjects of our sample to chance . as all the aa homozygotes were men , the a allele frequency appeared to be higher in males ( 0.26 ) than in females ( 0.16 ) ; though statistically the difference was not significant . for the + 83 bp locus , only the cc and the ct variants were detected . the rarity of the tt genotype seems to be a universal phenomenon as it was not detected in a number of other populations too ( kamboh et al . jia et al . , 2005 , dawar et al . , 2010 , biswas et al . , 2013 , when detected , the frequency of this variant was mostly found to be less than 1% ( sigurdsson et al . , 1992 , . , 2002 , miroshnikova et al . , 2011 , hamrita et al . , 2011 ) . a review of existing literature reveals that only certain samples from northern india have exhibited a comparatively higher prevalence for this genotype ( dixit et al . , 2007 , rai et al . , 2008 , khan et al . , 2012 ) . the a allele frequency for the g-75a locus in our sample was found to occupy an intermediate position with respect to the other populations of the world . nigerians had the lowest values ( ~ 0.1 ) ( kamboh et al . , 1999 ) , while chinese had the highest values ( ~ 0.3 ) ( heng et al . , 2001 , li et al . , 2008 ) in the spectrum . our values were considerably higher compared to those of certain caucasian populations from europe ( jeenah et al . , 1990 , sigurdsson et al . , 1992 , talmud et al . , 1994 , 1996 ) , north america ( larson et al . , 2002 , ordovas et al . , 2002 ) and brazil ( chen et al . , 2009 ) . when compared with populations from other asian countries in east asia and southeast asia regions , the malays ( heng et al . , 2001 ) and taiwanese ( chien et al . , 2008 ) were found to have a significantly higher frequency , whereas , the japanese ( bai et al . , 1996 ) had a significantly lower frequency of this allele . koreans on the other hand had comparable values ( yang et al . , 2010 ) . amongst the indian studies , the a allele frequency was seen to be higher in north indians from new delhi ( dawar et al . , 2010 ) and kashmir ( khan et al . , 2012 ) as compared to our sample . chandigarh ( rai et al . , 2008 ) were also found to have relatively higher values , though this was not statistically significant . in contrast to the g-75a polymorphism , comparatively fewer reports are available on the c+83 t polymorphism . the frequency of the rare t allele in our study was considerably lower than in nigerians ( kamboh et al . , 1999 ) and brazilians ( de franca et al . , 2005 , chen et al . , 2009 ) , and higher than in non - hispanic whites ( kamboh et al . , 1996 ) . it was also different from that reported in other indian populations from new delhi ( dawar et al , 2010 ) , kashmir ( khan et al . , 2012 ) , singapore ( heng et al . , 2001 ) , and mumbai and bangalore ( shanker et al . , 2008 ) . the similarities in the allelic frequencies of the g-75a and the c+83 t polymorphisms seen amongst some populations may be due to ancestral homology . likewise , the differences in the allelic frequencies observed across some other nationalities of the world are perhaps attributable to the diverse genetic background . this holds true for the different values reported from various parts of india as well , since the indian population is heterogeneous . the northeastern region of india is sandwiched between two major subcontinental regions the indian subcontinent in the west and the east / southeast asia region in the east . owing to the geographical proximity and strategic location , there have been multiple waves of migration to assam and the other northeastern states from the indian mainland and the neighbouring asian countries in the past . the region has thus acted as a melting pot for people of different stocks from the adjoining regions since the ancient times . by and large , the present day population of assam has an obvious affinity to the other indian populations , but with discernible mongoloid elements ( flatz et al . the region represents a sort of ethnological transition zone between india and neighbouring china , myanmar and thailand ( ali and das , 2003 ) . the rare allelic frequencies of the two apoa1 polymorphisms in the current population being recorded in the intermediate range between other indian ( chhabra et al . , 2003 , shanker et al . , 2008 , padmaja et al . , 2009 ) and southeast asian populations ( jia et al . , 2005 , heng et al . , 2001 , li et al . , 2008 , chien et al . , 2008 ) are perhaps a reflection of the above fact . indians in general have exhibited a lower frequency for the rare a allele as compared to their southeast asian counterparts . for instance , a very high frequency of the a allele was observed in the control subjects by dawar et al . but , this might not be truly representative because these control subjects were selected on the basis of absence of cardiovascular diseases , and hence may not necessarily represent the healthy population . some studies have suggested that the g-75a polymorphism influences the apo a - i levels in serum . the a allele has been found to be associated with significantly higher values of apo a - i in certain populations ( jeenah et al . , 1990 , talmud et al . , 1994 , sigurdsson et al . , 1992 , rai et al . , 2008 , dawar et al . , 2010 , kamboh et al . , 1996 ) . however , in the current population , the serum apo a - i levels were comparable across the gg , ga and aa genotypes in males as well as in females . our findings are in agreement with those observed in other populations where no effect of the a allele was noted on serum apo a - i levels ( dallinga - thie et al . , 1996 , heng et al . , 2001 , jia et al . , 2005 ) in contrast , in individuals belonging to the hei yi zhuang ethnic group in china , the gg homozygotes had higher serum apo a - i levels than the a allele carrying subjects ( li et al . , 2008 ) . the c+83 t snp , which is situated in the first intron of apoa1 has been relatively less investigated as opposed to the g-75a snp . although few population studies have shown this polymorphism to influence serum apo a - i concentrations ( kamboh et al . , 1996 , dawar et al . , 2010 , larson et al . , 2002 ) , we observed no such effect . the differences in serum apo a - i between the cc and ct genotypes were not significant in either sex of the current population . , 2001 , de franca et al . , 2005 , jia et al . , 2005 , padmaja et al . , 2009 , chen et al . , 2009 ) . the proposed mechanism by which g-75a snp alters apo a - i expression is that the g to a substitution alters the transcription efficiency of the gene and the apo a - i production rate ( tuteja et al this site is located in a gc rich sequence of the promoter which is subject to transcription regulation by binding of a 90 kda binding factor . it is believed that the g to a interchange at the 75 bp site alters the binding affinity of this binding factor to the gc rich region and consequently influences the rate of transcription ( angotti et al . , 1994 ) . as for the c+83 t snp which is located in the first intron of apoa1 , it is a part of the cpg dinucleotide ( shemer et al . , 1990 ) . the + 83 bp site and the 5-region of apoa1 are differentially expressed in tissues . they are methylated in non - expressing tissues , but hypo - methylated in tissues that express the gene ( liver ) . the cytosine to thymine transition at the + 83 bp site possibly results in further demethylation , and thus facilitates increased expression of apoa1 in the expressing tissues ( shemer et al . , 1990 , wang et al . , 1996 ) . due to the contradictory findings reported by different studies , it is increasingly believed that the g-75a and the c+83 t polymorphic sites are probably in linkage disequilibrium with other regulatory elements that actually control the production of apo a - i . for instance , strong linkage disequilibrium has been described between the g-75a polymorphism and the xmni polymorphism located in the 5-flanking region of the apoa1 gene ( paul - hayase et al . , 1992 ) . the x2-allele of the xmni polymorphism was found at a higher frequency in hyperlipidemia than in normolipidemia ( kessling et al . , 1985 ) . it may also be that the effects of these snps on the apo a - i values are seen only in certain ethnic groups . or the effects attributed to these snps in some of the earlier studies were actually due to gene another possibility is that they influence apo a - i production only in specific subgroups like females ( pagani et al . , 1990 , minnich et al . , 1995 ) . considering the possibility that these two polymorphisms may influence the apo a - i concentrations in a synergistic manner , we further examined the variation in apo a - i values across the 5 diplotypes encountered in our study population . still , none of the combinations seemed to be associated with higher apo a - i levels . thus , irrespective of the gender and irrespective of the combined effect of the g-75a and the c+83 t snps , these two snps did not influence the apo a - i levels in the current population . we described the distribution of these two polymorphisms in a healthy sample from assam , in northeast india for the first time . we documented the differences and similarities in the allelic frequencies in the study population with respect to the populations in the adjoining regions and the other parts of the world . we also observed that these polymorphisms do not directly affect the serum apo a - i levels in the studied population . however , with the recent spurt in interest on the role of these two polymorphisms in a plethora of clinical conditions , it remains to be seen how these allelic variants influence various clinical outcomes in this population . we hope our data will be useful for future studies investigating the role of these two polymorphisms in the implicated disorders in the northeast indian population . the authors declare that they have no conflicts of interest , financial or otherwise associated with the manuscript .

backgroundthere is a growing interest in the role of allelic variants of the apoa1 gene in relation to a number of disorders . we described two common polymorphisms of the apoa1 gene , g-75a and c+83 t and investigated their potential influence on the serum apolipoprotein a - i ( apo a - i ) levels in the native population of assam a region that is ethnically distinct and from where no information is hitherto available.methodsblood samples were collected from 150 healthy volunteers . apo a - i levels were estimated by immunoturbidometry . genotyping was done by a pcr - rflp method that involved dna extraction from whole blood , followed by polymerase chain reaction and digestion of the pcr product by mspi restriction enzyme , and analysis of fragment sizes in 12% polyacrylamide gel.resultsthe gg variant at g-75a locus and cc variant at c+83 t locus were the most prevalent . gg / cc was the most common combination . homozygous tt genotype was not detected in any of the subjects . the rare allele frequencies for the g-75a and c+83 t sites were found to be 0.22 and 0.06 respectively , which significantly differed from those reported in some other populations in neighbouring regions . serum apo a - i concentrations did not vary significantly across the detected genotypes . these findings were consistent in both sexes.conclusionwe described the distribution of the g-75a and c+83 t polymorphisms of the apoa1 gene in the population of assam for the first time . these polymorphisms were not found to directly influence apo a - i concentrations in this population either individually or synergistically .

Introduction Materials and methods Study subjects Blood sampling DNA isolation and genotyping Biochemical analysis Statistical analysis Results Discussion Conclusion Conflict of interest

PMC3015853

the term hepatic cyst usually refers to solitary nonparasitic cysts of the liver , also known as simple cysts . the precise frequency of liver cysts is not known because most do not cause symptoms , but liver cysts have been estimated to occur in 2.5% to 5% of the population . if the whole liver is involved , polycystic liver disease ( pcld ) is presumed to exist . the cysts are lined with epithelium , and because liver cysts seldom contain bile , the current hypothesis is that they fail to develop normal connections with the biliary tree . the majority of patients afflicted with adult polycystic liver disease are asymptomatic . for those who are symptomatic , the choice of surgical procedure is related to the severity of the disease and morphology of the cysts within the liver . most case series in the literature are relatively small , reporting fewer than 50 patients each . when the cysts become large and cause symptoms like pain , treatment is warranted . today we present our series of 12 patients who underwent laparoscopic surgery for symptomatic polycystic disease of the liver . we have managed 12 patients with large , nonparasitic , simple hepatic cysts laparoscopically , from 1996 to 2006 . there were 7 males and 5 females ; average age was 65 years ( range , 58 to 72 ) . eight patients had isolated cysts of both lobes of the liver , and the other 4 cases had associated ( asymptomatic ) polycystic renal disease . all the patients in this series had nonparasitic cysts in both lobes of the liver , the largest measuring 20149 cm in a 72-year - old lady . routine laboratory investigations including liver function tests and bleeding profile , chest radio - gram , and electrocardiogram were done in all patients . the initial imaging modality was the ultrasonogram in all cases , followed by ct scan . the 8 cases with the isolated multiple cysts underwent laparoscopic deroofing of the larger cysts and puncturing of the smaller cysts . pneumoperitoneum was established by veress needle , and pressure was maintained at 13 mm hg . the patient was positioned supine with both legs apart , the head - end raised to 30. the chief surgeon stood in between the legs , camera surgeon stood to the right of the patient , an assistant and scrub nurse stood on the left side . the monitor was placed on the head - end of the patient , directly opposite the chief surgeon . four ports were used : ( 1 ) a 10 mm port 2 cm above the umbilicus for the laparoscope ; ( 2 ) a 5 mm port in the left midclavicular line for right - hand working ; ( 3)a5 mm port in the right midclavicular line for left - hand working ; ( 4 ) and a 5 mm port in the epigastrium for liver retraction . in all the patients , there was always at least one large ( dominant ) cyst with other smaller cysts . the first step was to introduce the 5 mm left - hand working port directly into the largest cyst and suck out the contents without any spillage ( figure 1 ) . this causes the cyst to collapse ( figure 2 ) . through the cystotomy opening , a harmonic ace scalpel ( ethicon endosurgery , cincinnati , oh , usa ) was used to cut out the cyst wall ( figure 3 ) . in patients where the cysts were more exophytic , the laparoscope was turned to the left of the falciform ligament and the cysts excised ( figure 4 ) . complete resection of the cysts was achieved by keeping the dissection close to the cyst wall and was performed in 8 cases . the harmonic ace scalpel was used for these resections and found to be very effective . in 3 patients with pcld , the greater omentum was packed into the bigger cavities to obliterate dead space and prevent fluid collection . frozen section examination of tissue bits from the cyst wall revealed no evidence of cystadenoma or carcinoma in any case . liquids were started on the same evening as surgery , and a normal diet was started on the first postoperative day ( pod ) . parenteral analgesics were administered for 1 pod , and thereafter , oral analgesia was sufficient . fluid drainage was present for 8 days to 12 days . between 25 ml and 350 ml of fluid these 4 patients were discharged between the third and fifth pod with the drain tube and were advised to empty and measure the fluid daily . follow - up was for 12 months to 48 months ; 6 patients were followed up with regular ultrasonograms . up to 5% of the population has one or more liver cysts ; with a sharp rise in incidence with age . women ( age 50 to 60 years ) are more commonly affected than men are . most cysts are thought to arise as a congenital aberration of bile duct development and are termed simple cysts . several other small cysts within the liver usually accompany the dominant cyst , as we have seen in all the patients in this series . multiple hepatic cysts are usually considered to be a part of a polycystic disease complex . in our series , polycystic liver disease can arise in childhood or adult life , sometimes associated with polycystic kidney disease . liver cysts only rarely cause hepatic fibrosis and liver failure . occasionally , isolated polycystic liver disease has been reported . although it is easy to attribute symptoms to the presence of a large hepatic cyst , the possibility of coexisting pathology must be excluded . despite improved imaging techniques , asymptomatic patients do not require therapy because the likelihood of developing complications related to the lesion is lower than the risks associated with treatment . cyst aspiration with sclerosis , open or laparoscopic cyst fenestration , combined hepatic resection and fenestration , and liver transplantation are possible treatments . percutaneous aspiration of symptomatic hepatic cysts is a simple option , but recurrence is invariable , not to mention lifethreatening infection . several reports detail the efficacy of laparoscopic surgery for both multiple liver cysts and pcld . if the gallbladder is closely adherent to the cysts , cholecystectomy may be indicated ; one such case occurred in our study where we managed to preserve the gallbladder ( figure 5 ) . drainage of the operated site is not mandatory in all patients with large liver cysts ; it is advisable only if bile leakage is anticipated . only 4 of our patients needed drainage , out of which none had bile leakage . ultimately , it is ideal to tailor the procedure in each individual case . in our series , 8 patients underwent laparoscopic radical excision of the cysts to prevent recurrence , and liver resection was not done in any case . in the 5 ( 41.66% ) patients who developed recurrence in our series , only 1 had undergone radical excision . based on this , we are tempted to think radical excision of cysts could reduce recurrence rates , though more studies are needed to confirm this . martin et al11 compared the results of laparoscopic deroofing with open deroofing and open resection in 23 patients with simple cysts and 15 patients with polycystic disease . they found that laparoscopic de - roofing of simple cysts was associated with a 20% morbidity rate . the symptomatic recurrence rate was 8% for simple cysts and 71% for polycystic liver disease . recurrence of symptoms was due to growth of the untreated cysts and not to re - expansion of the treated cysts . lower recurrence rates were seen in a series of large dominant cysts in the anterior segment of the liver . additional omen - toplasty and oversewing of the margins by a running suture appear to reduce the recurrence rate to between 0% and 14% . liver transplantation has been performed in rare cases , especially when the above - mentioned interventions are not an option . collapsed cyst seen closely adherent to gall bladder . laparoscopic radical excision and omentoplasty for all large polycystic lesions and deroofing with simple puncture of all the cysts in cases of non - pcld are highly effective procedures .

background : liver cysts have been estimated to occur in 5% of the population . multiple liver cysts can also be part of the polycystic disease complex . only symptomatic or complicated cysts need surgery . traditionally , laparotomy is the procedure of choice . we present our experiences with laparoscopic management of both symptomatic multiple liver cysts and polycystic liver disease.methods:between 1995 and 2006 , we treated 12 patients with large , multiple liver cysts , including 4 cases of polycystic liver disease . most of the patients were elderly males . the lung and other organs were not involved in any case . laparoscopic deroofing or radical excision with omentoplasty was successfully performed in these patients.results:postoperatively , 4 patients had fluid draining through the drainage tube for an average of 10 days . one patient had ascites that resolved spontaneously . cysts recurred in 5 patients.discussion:there are not many reports in the literature regarding large series of patients , further confirming the rarity of the disease . liver cysts can occur as a part of polycystic renal and lung disease or isolated to the liver alone . laparoscopic deroofing is the ideal treatment for nonpolycystic liver disease , and laparoscopic radical excision is ideal for polycystic liver disease . simple needle aspiration or sclerotherapy is inadequate as recurrence is almost 100%.conclusion : currently , laparoscopy scores over laparotomy for the treatment of nonparasitic liver cysts as evidenced by this and other studies .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

PMC4987886

bulk segregant analysis ( bsa ) coupled with high - throughput sequencing is a widely used method to associate genomic regions to complex traits ( 1,2 ) . identifying these regions , commonly known as quantitative trait loci ( qtl ) , is a fundamental step toward understanding the genomic component of phenotypic variation ( 36 ) . bsa relies on crossing two parents which differ in a trait of interest , having one parent with a desired selectable trait . segregants displaying the desired phenotype are pooled , the pooled dna is extracted and subjected to pool sequencing . the sequencing data is mapped to a reference strain ( usually the known parent not exhibiting the desired phenotype ) and the allele frequency is calculated for all polymorphic marker sites . genomic regions for which the allele frequency of the marker sites significantly deviates from random segregation are prioritized as qtl for the trait ( 7,8 ) . current availability of whole genome sequencing ( wgs ) provides the capacity to infer population allele frequencies for nearly every site that is polymorphic between the parental lines . hence , the quality and length of the identified qtl is only affected by the number of crossovers during the development of the population which on its turn is determined by the recombination rate ( 9,10 ) . bsa has been particularly useful in identifying trait - linked alleles in eukaryotes with small genomes such as yeast , mainly because the smaller number of base pairs per centimorgan reduces the number of candidate genes within each predicted qtl ( 1113 ) . however , this method has also been successfully applied in rice ( 14,15 ) and other plants ( 16,17 ) . to efficiently differentiate true from spuriously linking qtls , previous work has shown that one of the approaches that largely contributes to the power of bsa data analysis is to exploit the properties of linkage disequilibrium ( ld ) ( 10,21,22 ) . the restricted number of recombination events cause proximal marker sites to be co - inherited , resulting in ld between neighboring marker sites : in a bsa set up , a causative mutation will therefore be embedded in a larger region of marker sites for which the variant counts all display a similar deviation from the allelic distribution one would expect if loci were not segregating with the trait . ld thus produces deviations of variant counts toward the alleles of the parent with the desired phenotype , not only at the genetic marker site(s ) causative to the phenotype of interest , but also in genetic marker sites closely located to these causative marker sites . although different algorithms for bsa exploiting ld have recently been implemented ( 10,21,22 ) in open source scripts and software tools , these tools remain difficult to operate for users . therefore we present explora - web , an intuitive web server that facilitates users performing data analysis of bsa experiments . explora - web is wrapped around our previously published bsa data analysis method that was shown to maximize power and accuracy in detecting qtls by exploiting the properties of ld . the details of the hidden markov model ( hmm ) and benchmarking with other tools are discussed in ( 10 ) . explora web is accessible using any internet browser ( google chrome , microsoft edge and firefox , among others ) . the web server 's help pages http://bioinformatics.intec.ugent.be/explora-web/help provide detailed guidelines on how to perform the analysis , tune the parameters and interpret the results . the explora web server is freely accessible and does not require login , although an optional account can be created to have easy access to the results of previously analyzed experiments . explora , which is the algorithm implemented in this web service , navigates the variable sites of the genome using an hmm that calculates the probability of allele frequencies at each marker site to be emitted by two possible states : a phenotype - linked state and a neutral state . while markers linked to the phenotype are expected to show predominantly the allele of the parent with the desired phenotype , neutral markers are expected to show the alleles of the two parents at a ratio that reflects random segregation . the effect of ld is modeled by the transition probabilities between two neighboring marker sites . the transition probability models the chance of a change of state between two neighbor sites . its distribution is described by a negative exponential function of the recombination rate and the physical distance between neighboring marker sites following haldane 's recombination model ( 23 ) . the model captures the fact that neighboring marker sites are likely to be in ld and hence the probability of a state change between them is small . emission probabilities of marker site states are modeled by two binomial distributions , one for the emission probabilities in phenotype linked states and another for emission probabilities in neutral states . the distribution describing the expected frequencies of the phenotype - linked variants is defined by the user selecting the and parameters . the ratio between and defines the degree to which the relative variant frequency at a marker site needs to differ from the one expected based on random segregation in order to be called linked to the phenotype. for full description of the model and algorithmic details we refer to ( 10 ) . the data necessary to run explora consists of the information on the experimental setup , the allele counts from the pooled sequencing and the selection of the method 's parameters . information related to the experimental setup consists of the number of segregrants that were pooled prior to sequencing and an approximation of the average recombination rate of the organism under study . the latter is required to take into account the effect of ld between neighboring markers . although we realize recombination rates are not constant across a genome , the average recombination works as a good approximation for our model : as we have shown in our previous work , small deviations from the recombination rate do not interfere with the performance of the algorithm in accurately detecting truly linked regions ( 10 ) . additional fields to name and describe the experiment are also provided ( figure 1a ) . ( a ) input experimental information . ( b ) upload count data . ( c ) parameter selection . the black line corresponds to the cumulative distribution of allele frequencies ( alternative read count / total read count ) derived from the uploaded data and is used to estimate the probability distribution that models the emission probability of the neutral state allele distribution . the blue lines represent the -distributions model for each of the three different ratios of / where line 1 corresponds to a setting reflecting high specificity and low sensitivity , line 2 medium specificity and sensitivity and line 3 low specificity and high sensitivity . the x - axis corresponds to the chromosomal positions and the y - axis to the posterior probabilities obtained for each marker site . the allele count at the marker sites derived from the pooled dna sequencing should be uploaded as a variant call format ( vcf ) file or as a simple tab delimited file in which rows correspond to the different marker sites ( figure 1b ) . for the simple text tab delimited file the columns correspond respectively to the chromosome at which the marker sites are located ( marker chromosome ) , the genomic position of the marker sites ( marker site position ) and two columns describing the read counts containing respectively the total read count at a marker site and the alternative allele read count ( which usually corresponds to the allele of the parent with the desired phenotype ) . vcf files containing these allele counts are produced from raw reads by analysis pipelines for high - throughput sequencing data such as ngsep ( 24 ) and gatk ( 25 ) , so the outputs of these pipelines can be directly uploaded to explora - web . alleles refer to polymorphisms relative to a reference genome and are thus defined when generating the vcf or counts file prior to using explora . besides the data and experimental information , explora also requires specifying the parameters that control the model . the main parameter to be selected is the / ratio that determines the shape of the distribution that models the emission probability for the phenotype - linked states ( figure 1c ) . changing the / ratio affects the probability with which an observed relative variant allele frequency is interpreted by the model as representative for a region linked to the trait of interest . increasing the / ratio makes the identification of phenotype - linked regions more stringent , meaning that a higher deviation of the relative allele frequency from the one expected under random segregation is needed before the region is considered linked . the higher the / ratio , the less phenotype - linked markers are called and the smaller the size of the called regions : identifying only the most pronouncedly linked regions results in more precisely pinpointing the linked region . however , this comes at the expense of potentially missing some truly linked markers / regions ( lower sensitivity ) . by default , the web server proposes three different / ratio 's corresponding to different trade - offs between sensitivity and specificity . a region that is identified with a stringent threshold and that thus corresponds to the most reliable signal in the data will by definition also be identified by the less stringent / ratio . providing the results obtained with different thresholds thus allows the user to assess the reliability of the predictions based on the stringency of the threshold at which the linked region was first detected . the effect of changes in the / ratio can be observed visually in the graphical interface . the emission probability of markers to be in the neutral state ( i.e. not linked to the trait of interest ) is directly estimated from the count data uploaded by the user , hereby assuming that most of the markers are not linked to the phenotype and their count data thus display the allele frequency distribution expected under random segregation . the web server also displays the distribution inferred from the real counts , allowing the user to select a set of running parameters in agreement with the data to be analyzed . the explora web service determines per / ratio the posterior probability of each marker site to be linked to the phenotype of interest . because of ld , neighboring markers on the chromosome will together display either high or low posterior probabilities . consecutive sets of neighboring markers displaying high posterior probabilities are thus used to identify the qtl . the server displays the marker - specific posterior probability scores graphically ( figure 1d ) along the chromosome for each / combination together with the observed allele frequencies at the variant sites and accordingly derives the genomic regions covered by the identified phenotype - linked markers . notice that when parameter settings are chosen too stringent the resulting posterior probabilities of finding a site being linked to the phenotype will become zero for all marker sites and no results can be found . results can be downloaded in two formats : ( i ) as a comma separated file , listing the posterior distributions per marker and parameter setting , and ( ii ) as a bed file containing the genomic information to identify the regions found to be linked to the phenotype . the bed file can be imported into other tools such as the integrative genomic viewer ( 26 ) . the web service was implemented in java and connects to a mysql database for information storage . the server runs on a 16-core , 64bit centos 6.2 system with 128 gb of memory . the data necessary to run explora consists of the information on the experimental setup , the allele counts from the pooled sequencing and the selection of the method 's parameters . information related to the experimental setup consists of the number of segregrants that were pooled prior to sequencing and an approximation of the average recombination rate of the organism under study . the latter is required to take into account the effect of ld between neighboring markers . although we realize recombination rates are not constant across a genome , the average recombination works as a good approximation for our model : as we have shown in our previous work , small deviations from the recombination rate do not interfere with the performance of the algorithm in accurately detecting truly linked regions ( 10 ) . additional fields to name and describe the experiment are also provided ( figure 1a ) . the black line corresponds to the cumulative distribution of allele frequencies ( alternative read count / total read count ) derived from the uploaded data and is used to estimate the probability distribution that models the emission probability of the neutral state allele distribution . the blue lines represent the -distributions model for each of the three different ratios of / where line 1 corresponds to a setting reflecting high specificity and low sensitivity , line 2 medium specificity and sensitivity and line 3 low specificity and high sensitivity . the x - axis corresponds to the chromosomal positions and the y - axis to the posterior probabilities obtained for each marker site . the allele count at the marker sites derived from the pooled dna sequencing should be uploaded as a variant call format ( vcf ) file or as a simple tab delimited file in which rows correspond to the different marker sites ( figure 1b ) . for the simple text tab delimited file the columns correspond respectively to the chromosome at which the marker sites are located ( marker chromosome ) , the genomic position of the marker sites ( marker site position ) and two columns describing the read counts containing respectively the total read count at a marker site and the alternative allele read count ( which usually corresponds to the allele of the parent with the desired phenotype ) . vcf files containing these allele counts are produced from raw reads by analysis pipelines for high - throughput sequencing data such as ngsep ( 24 ) and gatk ( 25 ) , so the outputs of these pipelines can be directly uploaded to explora - web . alleles refer to polymorphisms relative to a reference genome and are thus defined when generating the vcf or counts file prior to using explora . besides the data and experimental information , explora also requires specifying the parameters that control the model . the main parameter to be selected is the / ratio that determines the shape of the distribution that models the emission probability for the phenotype - linked states ( figure 1c ) . changing the / ratio affects the probability with which an observed relative variant allele frequency is interpreted by the model as representative for a region linked to the trait of interest . increasing the / ratio makes the identification of phenotype - linked regions more stringent , meaning that a higher deviation of the relative allele frequency from the one expected under random segregation is needed before the region is considered linked . the higher the / ratio , the less phenotype - linked markers are called and the smaller the size of the called regions : identifying only the most pronouncedly linked regions results in more precisely pinpointing the linked region . however , this comes at the expense of potentially missing some truly linked markers / regions ( lower sensitivity ) . by default , the web server proposes three different / ratio 's corresponding to different trade - offs between sensitivity and specificity . a region that is identified with a stringent threshold and that thus corresponds to the most reliable signal in the data will by definition also be identified by the less stringent / ratio . providing the results obtained with different thresholds thus allows the user to assess the reliability of the predictions based on the stringency of the threshold at which the linked region was first detected . the effect of changes in the / ratio can be observed visually in the graphical interface . the emission probability of markers to be in the neutral state ( i.e. not linked to the trait of interest ) is directly estimated from the count data uploaded by the user , hereby assuming that most of the markers are not linked to the phenotype and their count data thus display the allele frequency distribution expected under random segregation . the web server also displays the distribution inferred from the real counts , allowing the user to select a set of running parameters in agreement with the data to be analyzed . the explora web service determines per / ratio the posterior probability of each marker site to be linked to the phenotype of interest . because of ld , neighboring markers on the chromosome will together display either high or low posterior probabilities . consecutive sets of neighboring markers displaying high posterior probabilities are thus used to identify the qtl . the server displays the marker - specific posterior probability scores graphically ( figure 1d ) along the chromosome for each / combination together with the observed allele frequencies at the variant sites and accordingly derives the genomic regions covered by the identified phenotype - linked markers . notice that when parameter settings are chosen too stringent the resulting posterior probabilities of finding a site being linked to the phenotype will become zero for all marker sites and no results can be found . results can be downloaded in two formats : ( i ) as a comma separated file , listing the posterior distributions per marker and parameter setting , and ( ii ) as a bed file containing the genomic information to identify the regions found to be linked to the phenotype . the bed file can be imported into other tools such as the integrative genomic viewer ( 26 ) . the web service was implemented in java and connects to a mysql database for information storage . the server runs on a 16-core , 64bit centos 6.2 system with 128 gb of memory . we implemented explora web , a novel web service for the identification of qtl from whole genome resequencing data in bsa experiments . the web server relies on a highly accurate model developed previously ( 10 ) that maximizes the power to identify promising candidate genomic regions for further study of causal relationships with complex traits . explora was compared and shown to have superior accuracy to other recently published command line executable methods for qtl analysis on bsa experiments ( 18,21 ) . the use of standard formats such as the vcf allows users to upload directly the outputs of software pipelines to obtain allele counts and genotype calls from wgs data such as ngsep ( 24 ) or gatk ( 25 ) as inputs for qtl analysis . with explora we anticipate on the increasing use of bsa in combination with pooled sequencing both for fundamental and applied purposes and on the concomitant need for user friendly applications to facilitate its complex data analysis activities . wetenschappelijk onderzoek - vlaanderen ( fwo ) [ g.0329.09 , 3g042813 , g.0a53.15n ] ; agentschap voor innovatie door wetenschap en technologie ( iwt ) [ nemoa ] ; katholieke universiteit leuven [ pf/10/010 ] ( natar ) ; international center for tropical agriculture ( ciat ) . bioinformatics : from nucleotides to networks ; fonds wetenschappelijk onderzoek - vlaanderen ( fwo ) [ g.0329.09 , 3g042813 , g.0a53.15n ] .

identification of genomic regions associated with a phenotype of interest is a fundamental step toward solving questions in biology and improving industrial research . bulk segregant analysis ( bsa ) combined with high - throughput sequencing is a technique to efficiently identify these genomic regions associated with a trait of interest . however , distinguishing true from spuriously linked genomic regions and accurately delineating the genomic positions of these truly linked regions requires the use of complex statistical models currently implemented in software tools that are generally difficult to operate for non - expert users . to facilitate the exploration and analysis of data generated by bulked segregant analysis , we present explora - web , a web service wrapped around our previously published algorithm explora , which exploits linkage disequilibrium to increase the power and accuracy of quantitative trait loci identification in bsa analysis . explora - web provides a user friendly interface that enables easy data upload and parallel processing of different parameter configurations . results are provided graphically and as bed file and/or text file and the input is expected in widely used formats , enabling straightforward bsa data analysis . the web server is available at http://bioinformatics.intec.ugent.be / explora - web/.

INTRODUCTION EXPLoRA WEB Input Output Implementation DISCUSSION FUNDING

PMC270702

pulse oximetry is used almost universally in the management of critically ill patients in the intensive care unit and operating theatre . its uses include the detection of hypoxia , avoidance of hyperoxia , reduction in the frequency of blood gas analysis , titration of fractional inspired oxygen and for weaning from mechanical ventilation . an arterial oxygen saturation ( sao2 ) of 90% has been proposed as a target for adequate oxygenation during mechanical ventilation . previous studies investigating the use of the pulse oximeter oxygen saturation ( spo2 ) in intensive care patients have reported that the minimum spo2 levels to maintain sao2 at 90% range between 92% and 96 % . however , these studies have not answered the question of whether , after achieving a target sao2 , a subsequent change in spo2 predicts a corresponding change in sao2 in the critically ill . some studies have reported that anaemia reduces the precision of pulse oximetry by increasing the signal to noise ratio with low haemoglobin concentrations , whereas others failed to demonstrate this phenomenon . the in vitro method employed by the carbon monoxide ( co)-oximeter requires red blood cell lysis , whereas the pulse oximeter analyzes haemoglobin in whole blood . the difference between intracellular and extracellular hydrogen ion concentrations under normal physiological conditions has been incorporated into the pulse oximeter algorithms . however , the robustness of this adjustment has not been evaluated in the critically ill and acidotic patient . we therefore conducted a prospective observational study to test the hypothesis that a change in spo2 would predict an equivalent change in sao2 . such a relation , if it exists , would be invaluable in deciding when to titrate fractional inspired oxygen and/or repeat arterial blood gases in the individual patient . furthermore , we examined the effects of anaemia and acidosis on the precision of using the pulse oximeter to predict the sao2 in a heterogeneous group of critically ill patients . this study was considered by the local research and ethics committee and the need for informed consent was waived in view of the observational nature of the study . during a 2-month period all patients admitted to our intensive care unit ( icu ) who had an arterial line for the measurement of blood gases and who were being monitored by continuous pulse oximetry were recruited . the following patients were excluded : those with significant jaundice ( bilirubin > 40 mol / l ) or a history of smoke inhalation ; those with an inadequate spo2 trace ( as determined by visual analysis of a flat , absent , or irregular signal waveform ) ; and those in whom fewer than two arterial blood gas readings were taken . serial arterial blood gas samples were taken after 5 ml blood had been discarded when indicated as part of routine clinical care . no samples were taken solely for the study nor was any attempt made to vary inspired oxygen concentration or mechanical ventilation for the purposes of the study . arterial blood gas samples were analyzed using a co - oximeter ( abl725 , radiometer , copenhagen , denmark ) that was calibrated daily by laboratory staff and has a 2-hourly automatic internal calibration sequence . haemoglobin concentration ( g / dl ) , hydrogen ion concentration ( nmol / l ) , and percentage sao2 were recorded for each sample . precision and accuracy of a whole blood sample for sao2 , hydrogen ion concentration and haemoglobin concentration are 0.3 and 0% , 0.034 and 0.008 nmol / l , and 0.12 and 0.4 g / dl , respectively , within a haemoglobin range of 520 g / dl . pulse oximetry readings were recorded simultaneously with blood gas sampling using a nellcor ( puritan bennett , pleasanton , nj , usa ) finger probe attached to a hewlett packard ( palo alto , ca , usa ) merlin monitor . the pulse oximeter displays an average spo2 from the preceding 5-s beat by beat analysis . the measurements between healthy individuals ( n= 12 ) had a coefficient of variation of 0.4% at a spo2 of 97% . probes were attached to a finger , choosing the digit that gave the best trace and but not necessarily on the arm from which the arterial blood gas sample was drawn . data were stored using microsoft excel 97 and analyzed using sigmastat for windows 95 ( spss inc . chicago , il , usa ) and glim ( generalized linear interactive modeling ) version 4 , update 8 ( royal statistical society , london , uk ) , running on a dec alpha axp mainframe computer under the ultirx operating system ( osf/1 ) . the linear relations between differences in two successive measurements of spo2 and sao2 in all patients were analyzed using pearson correlation coefficient ( r ) , linear regression and goodness - of - fit ( adjusted r ) . the variations between and within the patients were examined using comparisons of the residual standard deviations ( sds ) between a single line from a common slope through all the changes for all 41 patients and a separate line to each patient . the effects of anaemia and acidosis on the agreement between the two measurement techniques were examined using a bland altman plot in which the difference between spo2 and sao2 was plotted against their average . bias was calculated as the mean of the differences between the co - oximeter and pulse oximeter readings ( sao2spo2 ) . positive bias indicated that the pulse oximeter underestimated the sao2 , whereas negative bias indicated that the pulse oximeter was overestimating the sao2 . the limits of agreement were taken as the bias ( 1.96 sd ) . approximately 95% of data fell within the haemoglobin concentration range 811.9 g / dl and the hydrogen ion concentration range 2562.9 nmol / l ( ph7.27.6 ) . therefore , haemoglobin concentrations 7.9 or 12 g / dl or hydrogen ion concentrations 63 the differences of biases between these three groups were analyzed using one - way , repeated measure analysis of variance . data were stored using microsoft excel 97 and analyzed using sigmastat for windows 95 ( spss inc . chicago , il , usa ) and glim ( generalized linear interactive modeling ) version 4 , update 8 ( royal statistical society , london , uk ) , running on a dec alpha axp mainframe computer under the ultirx operating system ( osf/1 ) . the linear relations between differences in two successive measurements of spo2 and sao2 in all patients were analyzed using pearson correlation coefficient ( r ) , linear regression and goodness - of - fit ( adjusted r ) . the variations between and within the patients were examined using comparisons of the residual standard deviations ( sds ) between a single line from a common slope through all the changes for all 41 patients and a separate line to each patient . the effects of anaemia and acidosis on the agreement between the two measurement techniques were examined using a bland altman plot in which the difference between spo2 and sao2 was plotted against their average . bias was calculated as the mean of the differences between the co - oximeter and pulse oximeter readings ( sao2spo2 ) . positive bias indicated that the pulse oximeter underestimated the sao2 , whereas negative bias indicated that the pulse oximeter was overestimating the sao2 . the limits of agreement were taken as the bias ( 1.96 sd ) . approximately 95% of data fell within the haemoglobin concentration range 811.9 g / dl and the hydrogen ion concentration range 2562.9 nmol / l ( ph7.27.6 ) . therefore , haemoglobin concentrations 7.9 or 12 g / dl or hydrogen ion concentrations 63 nmol / l were regarded in the study as extremes . the differences of biases between these three groups were analyzed using one - way , repeated measure analysis of variance . forty - one ( 22 male ) patients ( age [ mean sd ] 70 14 years ) were recruited into the study . a total of 1132 simultaneous arterial blood gas and pulse oximeter readings were taken ( mean [ range ] 27 [ 391 ] readings per patient ) . sequential readings in each patient were grouped together into pairs , which gave 1085 paired readings ( 47 readings were excluded because they were either not paired or unidentifiable to a particular patient , or the patient had fewer than two readings taken ) . these data were analyzed to determine the relation between changes in spo2 ( spo2 ) and changes in sao2 ( sao2 ) . the mean sd for spo2 was 94.6 2.7% and the mean for sao2 was 95.9 2.4% . in terms of predicting sao2 from spo2 , fitting a single line from all the 41 patients , gives a residual sd of 1.303 and fitting a separate line to each patient gives a residual sd of 1.288 ( p = 0.999 ) . although we found moderate correlation between spo2 and sao2 ( r = 0.606 ; p < 0.01 ; fig . 1 ) , only 36.7% of the variation in this relation was due to the association of changes in spo2 with changes in sao2 ( adjusted r= 0.367 ) . the prediction of sao2 from spo2 ( sao2 = 0.003 + 0.477 spo2 ) demonstrates that the pulse oximeter overestimates actual changes in sao2 . the 1085 simultaneous arterial blood gas and pulse oximeter readings from the 41 patients were analyzed to determine the effects of anaemia and acidosis on bias and limits of agreement . for the data altogether , the bias was 1.34 and the limits of agreement were -2.29 and + 4.97 ( fig . there were only small changes in bias with anaemia ( + 2.09 ) and acidosis ( + 0.38 ) , as shown in table 1 . the difference in bias between hydrogen ion concentrations of 2563 nmol / l and 63 nmol / l ( p < 0.01 ) , and between haemoglobin concentrations of < 8 g / dl , 811.9 g / dl and > 12 g / dl ( p < 0.01 ) all achieved statistical significance . the bias was not significantly different between haemoglobin concentrations of < 7.9 g / dl and 811.9 g / dl ( p= 0.24 ) . there were insufficient numbers in the group with hydrogen ion concentration < 24.9 nmol / l ( n= 10 ) for analysis to be done . the present study shows that changes in spo2 do not reliably predict equivalent changes in sao2 , with the pulse oximeter tending to overestimate actual changes in sao2 . we also showed that spo2 underestimates sao2 to a greater extent with progressive anaemia , whereas acidosis increases the spo2 estimate of sao2 . the titration of fractional inspired oxygen during weaning from mechanical ventilation is frequently adjusted with the goal of maintaining a target spo2 value . jubran , in a study involving 54 icu patients , reported that levels of spo2 of 92% in white patients and 95% in black patients maintained arterial oxygen tension at 8 kpa or greater in 92% and 85% of patients , respectively . seguin defined a minimum spo2 of 96% to ensure that no patients had a sao2 below 90% . this approach avoided hypoxia , but 15% of patients had a sao2 of 98% or greater . although target values can be helpful , it would be valuable to know whether a change in spo2 would predict a similar change in sao2 in critically ill patients over time . hypothetically , the relatively static patient factors that interfere with pulse oximetry ( skin colour , finger size , carboxyhaemoglobin , methaemoglobin ) do not change , and so the correlation between changes in sao2 and spo2 might be expected to be closer than that between absolute values from a mixed patient population . this could allow individualized target spo2 to be set , based on a single , one - off sao2 reading . only one small study has attempted to address this question in the intensive care setting . in a series of 45 patients ( 135 measurements ) , van de louw and coworkers recently reported that changes in spo2 could not accurately predict changes in sao2 . the prediction of sao2 from spo2 ( sao2 = 0.003 + 0.477spo2 ) demonstrates that , on average , the pulse oximeter overestimates actual changes in sao2 . this suggests that a similar degree of caution is required in interpreting changes in pulse oximetry in the critically ill as in one - off readings . progressive reductions in haemoglobin concentration may reduce the precision of the pulse oximeter as the signal : noise ratio from surrounding tissue increases . early studies examining the effects of anaemia on the precision of the pulse oximeter found reduced precision in association with anaemia . lee and coworkers demonstrated a deterioration in bias and precision in dogs with a haematocrit below 10% , and severinghaus and coworkers reported increased error in anaemic humans when the sao2 was less than 75% . in contrast , case reports have described cases in which the pulse oximeter remained precise at haemoglobin concentrations of 2.7 g / dl and 3.0 g / dl . a subsequent case series of 17 patients with acute anaemia due to haemorrhage ( haemoglobin concentration 2.38.7 g / dl ) did not detect any deterioration in the accuracy of measurements using the pulse oximeter in the absence of hypoxia . our study did not include sufficient numbers with hypoxia ( spo2 < 90% ) for the influence of anaemia on bias and precision to be studied in this patient group . however , under normal physiological conditions ( spo2 > 90% ) our results support and extend previous findings in demonstrating that anaemia has only a minor impact on the precision of measurements using the pulse oximeter . our data show that , in the presence of acidosis , the degree to which spo2 underestimates sao2 was reduced . one possible explanation for this finding may relate to the differences in the techniques used for measuring oxygen saturation . the pulse oximeter analyzes haemoglobin saturation in whole blood in vivo , whereas sao2 measured by co - oximetry requires red blood cell lysis prior to analysis . under normal physiological conditions , algorithms incorporated in the pulse oximeter account for this , although the validity of this adjustment has not been tested outside normal physiological ranges . alternatively , the effects of the complex interactions between cardiac output , systemic vascular resistance , temperature and vasoactive drugs on precision of measurements using the pulse oximeter might have contributed to this finding . a further study looking at the precise contribution of each of these factors would be required to elucidate the aetiology of these findings definitively . there are several potential confounding variables that were not controlled for in the study design . first , like in other studies , we did not analyze the influence of carboxyhaemoglobin and methaemoglobin concentrations on bias and precision . the pulse oximeter is unable to distinguish between these two forms of haemoglobin and oxyhaemoglobin , leading it to overestimate the actual sao2 if significant concentrations of either are present . we excluded patients with a history of smoke inhalation , in whom carboxyhaemoglobin levels may be high . in nonsmokers carboxyhaemoglobin levels are normally less than 2% and methaemoglobin levels are less than 1% levels that are already accounted for by the built - in algorithms of pulse oximeters . in cigarette smokers carboxyhaemoglobin is initially elevated ( average 4.78% ) but falls over time ( half life 56 hours ) . the clearance of carboxyhaemoglobin is also accelerated by ventilation . because most patients had been ventilated for several hours before entry into the study we excluded patients with significant jaundice a group known to have high carboxyhaemoglobin levels in order to minimize this potential error , and no patients were admitted following smoke inhalation during the study period . anaemia and acidosis have not been found to influence car - boxyhaemoglobin or methaemoglobin concentrations . although we believe that the influence of carboxyhaemoglobin levels in the study was minimal , we are unable to rule it out as a potential confounding variable . second , we did not classify patients according to skin colour or race , which may impact on accuracy of the pulse oximeter . because skin colour is constant , comparisons of changes in spo2 are unlikely to have been affected . data for the assessments for bias and precision caused at the extremes of anaemia and acidosis were collected from 19 and 14 patients , respectively , and there did not appear to be any systematic difference in the groups ' racial composition from that in the overall study population . no patients to our knowledge had sickle cell anaemia / trait , although this was not specifically tested for . third , the mean spo2 reading for the total data was 94.6% , with a corresponding sao2 value of 95.9% . this is consistent with previous investigators ' recommendations for minimal target values for spo2 during mechanical ventilation . however , less than 5% of data fell in the range of spo2 levels below 90% . this is consistent with a worsening of bias and precision with pulse oximetry when the sao2 is less than 90% . at lower saturations the effects of anaemia and acidosis may become more prominent , and our results should therefore be applied with caution in this situation . finally , the pulse oximeter presents spo2 data as integers whereas the co - oximeter presents sao2 data to 1 decimal place . with over 1000 data points , it is likely that the oximeter rounded up and rounded down a similar number of times , and so these differences will most likely cancel each other out . at most , the maximum differences due to the measurement of spo2 in integers will account for less than 1% of the observed bias as compared with sao2 . in conclusion , in a heterogeneous group of icu patients , we showed that changes in pulse oximetry do not reliably predict equivalent changes in sao2 . we also demonstrated that neither anaemia nor acidosis alters the precision of measurements between the nellcor pulse oximeter and co - oximeter to any clinically important extent . the pulse oximeter remains a valuable tool in the care of intensive care patients , but an awareness of its limitations is an important component of enhancing the quality of care . changes in spo2 do not reliably predict equivalent changes in sao2 in the critically ill anaemia and acidosis have only a minor influence on the precision of measurements of spo2 and sao2 icu = intensive care unit ; sao2 = arterial oxygen saturation ; sd = standard deviation ; spo2 = pulse oximeter oxygen saturation . linear relations between changes in pulse oximeter oxygen saturation ( spo2 ) and arterial oxygen saturation ( sao2 ) . bland and altman plot for bias and limits of agreement for total data . the effects of anaemia and acidosis on bias and limits of agreement p < 0.01 , versus * haemoglobin > 12 g / dl , haemoglobin < 8 g / dl , haemoglobin 812 g / dl and hydrogen 2563 nmol / l .

introductionthis study investigates the relation between changes in pulse oximeter oxygen saturation ( spo2 ) and changes in arterial oxygen saturation ( sao2 ) in the critically ill , and the effects of acidosis and anaemia on precision of using pulse oximetry to predict sao2.patients and methodsforty - one consecutive patients were recruited from a nine - bed general intensive care unit into a 2-month study . patients with significant jaundice ( bilirubin > 40 mol / l ) or inadequate pulse oximetry tracing were excluded.resultsa total of 1085 paired readings demonstrated only moderate correlation ( r= 0.606 ; p < 0.01 ) between changes in spo2 and those in sao2 , and the pulse oximeter tended to overestimate actual changes in sao2 . anaemia increased the degree of positive bias whereas acidosis reduced it . however , the magnitude of these changes was small.conclusionchanges in spo2 do not reliably predict equivalent changes in sao2 in the critically ill . neither anaemia nor acidosis alters the relation between spo2 and sao2 to any clinically important extent .

Introduction Patients and methods Statistical analysis Results Discussion Conclusion Competing interests Key messages Abbreviations Acknowledgement Figures and Tables

PMC3191730

nursing homes face challenges in the coming years due to an increased number of elderly . quality will be under pressure , expectations of the services will rise , and clinical complexity will grow . modern clinical information systems with decision support may be part of that . based on clinical experience and information about 513 long - term patients , we believed that there could be failure of the blood - thinning treatment of patients with atrial fibrillation . atrial fibrillation is an independent risk factor for developing a stroke , and in the elderly it is one of the most important causes of stroke . the current guidelines in force are that all over-75s with atrial fibrillation are to be anticoagulated , but increasing age often produces the opposite effect with decreased prescriptions to the oldest [ 3 , 4 ] . there is good evidence showing that inappropriate weight loss in the elderly materially impairs their quality of life and life prospects . a significantly increased risk has been shown for secondary complications like pressure sores , infection , depression , and functional impairment [ 8 , 9 ] as well as increased mortality [ 1014 ] . the high consumption of psychopharmaceuticals in nursing homes has been emphasized in a number of studies . 23% of the demented nursing home population in bergen was found to be on antipsychotics . the prevalence of psychoactive drugs has been well documented earlier , but some years ago [ 15 , 16 ] . double - blind studies have shown little symptom reduction connected with the use of antipsychotics as compared with placebo [ 17 , 18 ] . . often test results are not followed up . in a meta - analysis , kawamoto and colleagues dealt with studies on the effectiveness of decision - making support tools and found that 68% of the systems showed significant improvement in practice . a clinical decision support system ( cdss ) decision - making support is closely correlated with structured record information , because the decision - making support tools have to respond to correct information in the record for example , pathological test results . the traditional medical approach focuses on the individual , with particular emphasis on symptoms , signs and test results emerging from the encounter with individual patients . in order to ensure accessible and correct treatment for all patients , it is also possible to operate using population - directed control measures , for example , keeping track of the proportion of a patient population on a specific drug . this is especially applicable in nursing homes where about 75% of patients are demented and can not take responsibility for their own followup . the users ' views on data systems are important for the real - term prospects of disseminating such tools . nurses ' views on electronic patient records have been studied at a major hospital in florida . of 100 nurses , one - third felt that electronic patient records resulted in a reduced workload and roughly the same proportion preferred being able to provide documentation at the patient 's bedside , but said that this was often made difficult . a quarter thought that documentation was improved and the use of electronic patient records would improve patient safety . nurses with experience of information technology consistently had more positive views than those without such experience . in this project , we wanted to study whether decision support as part of an electronic , structured patient record system , together with targeted education , could improve the quality of medical treatment while avoiding negative impact on job satisfaction among users . we performed a prevalence study to quantify the proportion of long - term patients with atrial fibrillation that were being given warfarin , were taking neuroleptics , and had not been weighed for the last 30 days . then , a before - after intervention study was done . the before - after study was conducted as a double cross - sectional design . instead of randomization , we decided to use the proportion of patients on digitalis , thyroxin , and antidiabetics as internal controls ( participants exposed to the same study , but these drugs were not part of any concrete intervention and were unlikely to undergo any changes by external factors . ) . the proportion of patients on neuroleptics was in addition followed as a time series ( a sequence of data points , measured typically at successive times spaced at uniform time intervals ) [ 2628 ] in two nursing homes from 23 october 2008 to 15 july 2010 . the prevalence ( before ) survey was performed among long - term residents in seven nursing homes ( n = 513 ) . bergen , norway has approximately 250,000 inhabitants and 37 nursing homes ( 2,300 beds ) . medical students conducted the data collection , which consisted of copying medication cards , weighing by means of a local tool , but standardized clothing , and finding previously recorded weight / date . electrocardiography ( ecg ) to diagnose atrial fibrillation was performed and a standardized set of blood samples analysed at the laboratory of the bethany hospital . the recorded data were keyed into excel and the drugs coded according to the anatomic therapeutic chemical ( atc ) drug register . the nursing homes were selected based on approximately similar size , function , and public ownership . they were comparable in terms of nursing staff and financing , but some differences existed with regard to other variables ( table 1 ) . after quantifying clinical issues through the before - survey , we installed an electronic , structured patient record system which included decision support . the system had been developed and operating at lvsen educational nursing home ( developing institution ) for several years , funded by the norwegian medical association and the norwegian research council . installation and training were carried out in the last quarter of 2008 , and 6 of the 7 nursing homes were running the system on a daily basis from january 2009 for 12 months . after the active intervention period , the number of nursing homes running the system was reduced to three institutions , owing to contracts between bergen municipality and another vendor having been signed before the project started . lvsen nursing home and two of the study institutions continued and were the only institutions available for follow - up data . the system incorporated filters and alarms within several areas , but we decided to test 4 filters and 2 reminders . population filters were predefined filters that produced lists of patients according to criteria in seconds . ( list and proportion ) , ( b ) patients taking warfarin ( list and proportion ) , ( c ) patients with the diagnosis atrial fibrillation and their treatment status ( list and proportion ) , and ( d ) patients not weighed for last 30 days ( list and proportion ) . tested reminders visible in each patient 's record , if the criteria were met , were ( a ) patient has diagnosis of atrial fibrillation but is not on warfarin and ( b ) patient not weighed for last 30 days . figures 1 and 2 demonstrate how this was arranged within the application . results from the before - study were presented to the participating doctors during the first 12 months of intervention through three two - hour lectures . two institutions ( e and f in table 1 ) participated in the after - study . to collect the data we used the population filters in the medical record system to find the proportions of patients on endpoint drugs , internal controls , and patients not weighed for the last 30 days . before - data were extracted from the prevalence study for institutions e and f ( n = 183 ) for the before - after analysis . institution e participated with an additional department ( 22 patients ) in the intervention study ( n = 205 , after ) . to study user satisfaction , we performed an anonymous survey among the doctors , nurses , assistant nurses , and physiotherapists ( n = 504 ) . 16 questions about position and several aspects of their view on the functionality of the application were presented . questions were closed , with a score from 1 to 5 ( worse better ) . the survey was constructed by a group of study nurses from the institutions , with technical organization by the vendor . for the prevalence ( before ) survey , the recorded data were keyed into an excel worksheet and the drugs coded according to the anatomical therapeutic chemical ( atc ) drug register . to compare proportion of populations , pearson 's chi - square test was used . to compare distribution of continuous variables , kruskal - wallis ' nonparametric one - way analysis of variance was performed . straight counts were performed in excel , but jmp 8 was generally used for statistical analyses . in the prevalence study , all long - term patients in 7 nursing homes were examined ( n = 513 ) . the before - after study included all long - term patients in two institutions ( n = 183 before , 205 after ) , also no drop - outs . 272 ( 54% ) users responded to the questionnaire , tables 2 and 3 . in the prevalence study of 484 long - term patients ecg - ed , of 91 atrial fibrillation patients , 14.2% were anticoagulated with warfarin , that is , 2.5% of all patients . at the two institutions ( e and f ) participating in the before - after study , the proportion of patients taking neuroleptics was reduced from 33.0% to 21.5% ( n = 183 before/205 after , chi - square test , p = 0.015 ) , that is , a difference of 11.5% ( 95% ci : 2.3 to 20.6% ) . warfarin increased from 3.0% to 9.8% ( p = 0.013 ) , that is , a difference of 6.8% ( 95% ci : 1.6 to 12.1% ) . use of digitoxin did not increase significantly ( 8.0% versus 8.5% ; p = 0.1 ) , thyroxin was not reduced ( 10.0% versus 8.6% , p = 0.765 ) , and antidiabetics did not increase ( 10.0% versus 10.5% ; p = 0.996 ) , figure 4 . the proportion of patients not weighed for the last 30 days was reduced from 72.6% to 16.0% ( p < 0.001 ) , that is , a difference of 56.6% ( 95% ( ci : 47.5 to 64.5% ) . the time - series for proportions of patients using neuroleptics is presented in figure 3 . in the user survey ( n = 272 , 54% ) , 43% reported great or slightly better job satisfaction . endpoints changed significantly during intervention by increased use of warfarin , decreased use of neuroleptics , and a higher weighing rate . the role of nursing homes in the delivery of social and health care services differs between , as well as within , countries . nursing homes in many countries are managed as part of social care . in norway , such study designs should generally be used in a context where they build on appropriate theoretical , qualitative , and modelling work , particularly in the development of appropriate interventions . we feel that we have properly documented the fields in need of improvement and thus study objectives . baseline and follow - up data for the first cross - sectional sample were collected , by the prevalence study ( before ) and ( in the case of the second sample ) after the intervention , respectively . as it was not possible to collect follow - up data for everyone included in the first study ( for political reasons ) and the patients included in the second sample were not identical to those in the first due to deaths , the two samples were compared using methods for comparing unpaired data . this is a weakness , but to compensate for the 40% or so annual death rate in nursing homes , the sample size would have had to be much higher to be able to use patients as their own controls , and this may not have been advisable when testing front - end technology in the first study . our design must be seen in light of the immaturity of research in nursing homes in general . as far as we know , neither recent data exist on the prevalence of atrial fibrillation in a nursing home population and the use of warfarin , nor is information available on how well basic procedures like weighing are conducted . there was therefore a need to do a prevalence study , to define valid endpoints , and to do an intervention study . it may be claimed that we should have separated the tasks , but nursing homes are in great need of improvement strategies and time is short before the elderly wave is set to impact heavily on nursing home demands . financial and political limitations played a role too , obstructing any possibility of performing a full - scale follow - up study . but conversely , it would have been a challenge , in a randomized trial within one municipality , albeit with 37 institutions to choose from , to avoid study group pollution and the hawthorne effect [ 29 , 30 ] . starting out with a comprehensive and expensive randomized study would probably not have been ethical due to all the uncertainty and possible threats from new information technology systems , and hence probably difficult to fund . when we started , we did not know if it was possible to install the application , educate , and prepare for critical daily use in 7 institutions with 500 to 800 users within a timeframe of 4 months . looking back , this may be regarded as the most convincing result of the project . we used a design where we evaluated endpoints in a before - after study with internal controls [ 2628 ] . the stability of internal controls throughout the intervention reinforces the internal validity of the study . awareness of the performance process is responsible , through education , for the changes measured . yet without any performance figures , it is not possible to focus on changing processes , so the two ways of influencing results are not independent factors . the technology made it possible to monitor performance without time - consuming traditional studies and made performance feedback possible . this is a relatively new concept in medical research , made possible by structured medical input applicable to automatic and continuous analysis . consequently , the proportion of patients taking neuroleptics could also be followed using time - series , giving more detailed information about the changing process and strengthening the validity of the effect results . as a critical information element , drugs were punched into each patient record at an early stage of the implementation process and this gave us a sufficient before - point on the time - series . we discovered that the significant change in the before - after study was due to only one of the participating institutions and that this institution showed up a clear brake in the curve at the point of intervention startup . we see from the time - series on neuroleptics ( time - series are part of the patient record system ) that the tool also provides a deeper understanding of the change process . by observing the curves , figure 3 , we learn something about possible variation in the use of neuroleptics during the year . this makes one think that performance feedback , over time , may equal performance around a total average . other authors have concluded that evaluative studies should report on usage patterns and progression of outcomes over time . very few authors have reported on feedback at institutional level with performance comparisons . at vaccination clinics , however , it has been shown that performance feedback at institutional level can improve quality . logically , this may be one way of influencing both generalized over- and undertreatment in institutions . our own research ( same material , and publications to come ) has shown that differences between comparable nursing homes do exist , significantly . with regard to both the proportion of patients using cardiovascular and psychoactive medications , and weight loss and weighing routines , we found significant differences between the 7 nursing homes participating in the prevalence survey ( same study , different publications ) . we hope to continue our research based on this feedback method to test if this is a way to increase equality and quality among a bigger number of institutions . technology is a tool only , without any impact , if managers and practitioners are not reacting to warnings and are not exploiting the population filter function ! the structured electronic patient record system with decision support we tested can improve drug treatment and monitoring and better implementation of procedures . it can easily be installed for use in nursing homes . controlled studies on a broader spectrum of clinical and administrative parameters

background . nursing homes face challenges in the coming years due to the increased number of elderly . quality will be under pressure , expectations of the services will rise , and clinical complexity will grow . new strategies are needed to meet this situation . modern clinical information systems with decision support may be part of that . objectives . to study the impact of introducing an electronic patient record system with decision support on the use of warfarin , neuroleptics and weighing of patients , in nursing homes . methods . a prevalence study was performed in seven nursing homes with 513 subjects . a before - after study with internal controls was performed . results . the prevalence of atrial fibrillation in the seven nursing homes was 18.8% . after intervention , the proportion of all patients taking warfarin increased from 3.0% to 9.8% ( p = 0.0086 ) , neuroleptics decreased from 33.0% to 21.5% ( p = 0.0121 ) , and the proportion not weighed decreased from 72.6% to 16.0% ( p < 0.0001 ) . the internal controls did not change significantly . conclusion . statistics and management data can be continuously produced to monitor the quality of work processes . the electronic health record system and its system for decision support can improve drug therapy and monitoring of treatment policy .

1. Background 2. Material and Methods 3. Results 4. Discussion 5. Conclusions

PMC3362854

osteoarthritis ( oa ) is a cartilage degenerative disease and causes more disability with respect to mobility than any other single disease in the elderly . knee oa typically affects joints in a nonuniform manner ; the medial compartment of the knee is most frequently affected in both men and women . furthermore , the three most common areas of knee oa distribution are medial compartment , patellofemoral compartment , and medial / patellofemoral compartment overlap . the predominance of medial knee oa is likely due to the high medial forces generated during weight - bearing activities ( e.g. , walking ) [ 4 , 5 ] . it is believed that the increased knee varus loading is strongly associated with risk of medial knee oa progression . in fact , a radiographic study shows that varus knee alignment ( bowlegged ) increases medial knee oa progression in as little as 18 months . medial knee oa causes severe knee pain and knee stiffness , reduces knee function , and leads to disability [ 1 , 8 ] . the most common surgical treatment of medial knee oa is total knee replacement ( tkr ) . tkr has been the gold standard for treatment of knee oa because it results in excellent pain relief and has a long - term success rate ( 1015 years ) [ 10 , 11 ] . however , tkr alternates the entire knee articular geometry and sacrifices the cruciate ligaments , which often leads to abnormal knee kinematics [ 12 , 13 ] . it is ideal to only replace the affected knee compartments and reserve other intact portions and cruciate ligaments because the damage associated with medial knee oa is often limited to cartilage degeneration in the medial and patellofemoral compartments . one alternative to the tkr , especially for more active patients , is partial knee replacement ( pkr ) , which leaves the lateral knee compartment and cruciate ligaments intact while replacing the affected knee compartments ( e.g. , medial compartment and patellofemoral joint ) . pkr results in fast recovery times , less bone loss and normal knee mechanics [ 8 , 1215 ] . adequate fixation of pkr tibial tray in the bone is important for reproducing normal knee kinematics . fast - curing bone cement has been used to glue the knee implant to the bone for both tkr and pkr designs . however , the contact area between the pkr implant and bone is less than half of that of the tkr . due to the small contact area between the pkr implant and bone , it is not uncommon to observe implant loosening and migration after one or two years of usage . cementless fixation features in promoting bone growth into the implant - bone interface for stability . the implant - bone contact areas include bottom surface of the tibial tray and surface of the press - fit pegs . new bone tissue can easily grow into the coating and form a reliable long - term fixation . pandit et al . reported that the bone in - growth at the implant - bone interface is satisfactory at one year of postsurgery . when cementless fixation is in use , it normally takes two to three months for the bone to remodel and replace the damaged tissue at the implant - bone interface and fully grow into the implant surface . during this process , bone strength is weakened ; bone tissue lacks the ability of stabilizing the tibial tray . thus , mechanical loading from knee rehab exercises may result in early loosening of the tibia tray . migration of the tibial tray due to loosening may further lead to abnormal knee kinematics and eventually failure of the knee replacement . to date , the effect of mechanical loads from knee exercises on bone stress / strain at implant - bone interface has yet to be determined . quantifying bone strain at implant - bone interface is essential for designing better pkrs to resist implant loosening . normal knee flexion is associated with femoral rollback with regard to the tibia . during exercises involved knee flexion such as sit - to - stand , stair climbing , squat , and lunge , the femorotibial contact point is in the posterior region of the tibial plateau [ 18 , 19 ] . thus , axial mechanical loads during knee exercises are applied in the posterior region of the tibial plateau . as normal knee kinematics aree associated with pkr knees , the mechanical load from knee exercises is expected to be applied in the posterior region of the tibial tray . therefore , it is possible that the tibia could experience a large bending load and the anterior implant - bone interface could be subjected to high tensile forces , which may result in early implant loosening . therefore , introducing external fixation mechanisms to the pkr may reduce the risk of developing implant loosening during rehab exercises . one proposed method is to apply a screw in the anterosuperior region of the tibial tray to fix the tray to the bone . an alternative method is to include a frontal flange to the tibial tray and screw the flange on the anteromedial aspect of the proximal tibia . to date , little is known about the effectiveness of these two fixation techniques . it is yet to be determined whether these two fixation mechanisms could enhance stability of the pkr during rehab exercises requiring knee flexion . finite element analysis ( fea ) is ideal to analyze stresses and strains in complex biological structures . the application of this method requires developing a model which represents the real structure in essential features . the model is mathematically divided into little elements connected in nodal points . computer programs are used to calculate the stresses and strains in the elements and nodes after material properties are assigned and mechanical loads are applied . to date , advanced technologies make personal computers capable of running sophisticate fea programs and providing results in a matter of hours or days . thus , it is feasible to investigate the influence of implant design on stress and strain profile at the implant - bone interface via running fea on a personal computer . firstly , to use fea to determine whether a typical axial mechanical load applied in the posterior region of the pkr results in tension in the anterior implant - bone interface . secondly , to use fea to quantify the effects of external fixation mechanisms such as a screw design or a frontal flange design on mechanical stress / strain at the pkr implant - bone interface during weight - bearing knee exercise . it is hypothesized that a posterior axial load would lead to increased tensile loading in the anterior region of the implant - bone interface . it is further hypothesized that external fixation mechanisms could reduce the tensile loading in the anterior region of the implant - bone interface during weight - bearing knee exercise . the left tibia of a healthy male ( age = 21 yr . , body mass = 78 kg , body height = 181 cm ) was ct scanned ( ge light speed vct , ge corp , usa ) . slice thickness was 0.625 mm with a 15 cm 15 cm field of view . ct scans were segmented in mimics 14.0 ( materialise , leuven , belgium ) and a surface mesh was generated . the surface mesh was used as boundaries for the automatic formulation of a solid mesh using hexahedral elements in marc 2010 ( msc software , santa ana , ca , usa ) . the mesh consisted of 2 mm 2 mm 2 mm elements . young 's modulus and the shear elastic modulus of the bone were set at 17 gpa and 10 gpa , respectively . in addition , young 's modulus and the shear modulus of the bone were assumed to be transversely isotropic with values of 5 and 3.5 gpa , respectively [ 21 , 22 ] . poisson 's ratio was set at 0.3 , and bone density was set at 1.9 g / cm . after the 3d tibia was reconstructed , a typical surgical procedure was simulated in mimics ( materialise , leuven , belgium ) to remove the medial tibial plateau . the first model represented a pkr tibial tray with press - fit pegs ( figure 1 ) . the fe pkr model was glued to the tibial plateau to mimic the effect of the press - fit pegs . the second model was a modification of the first model with a superior screw ( located 10 mm posteriorly from the front edge of the tray ) for stabilizing the tray to the tibial plateau ( figure 2 ) . in this fe pkr model , rigid - body - element nodal ties ( rbe2 ) running from the superior - anterior part of the pkr to the tibial plateau were used to simulate the effect of a superior screw stabilizing the pkr on the bone . the third model was also a modification of the first model with a frontal flange design ( figure 3 ) . the other end of the flange was screwed to the medioanterior surface of the proximal tibia to externally stabilize the tray . in this fe pkr model , rbe2 nodal ties were used to simulate the effect of the anterior flange screwed on the anteromedial tibia surface . the tibial tray of each pkr was solid meshed in marc 2010 ( msc software , santa ana , ca , usa ) . the 3d model of the pkr tibial tray consisted of 4750 hexahedral elements and 5826 nodes . material properties of cobalt - chromium ( cocr ) were assigned to the tibial component . young 's modulus was set at 195 gpa ; poisson 's ratio was set at 0.3 . the gluing force at the pkr implant and bone interface simulated the effect of the press - fit pegs in a real setting . figure 4 shows the frontal view of a pkr tibial tray implanted on the left medial plateau . medial axial load of 1800 n was applied in the posterior region of the pkr , which was 15 mm posterior to the center of the pkr . lateral axial load of 1200 n was applied in the posterior aspect of the lateral tibial plateau , which was 15 mm posterior to the center of the lateral plateau . the axial loads chosen were typical loads during stair climbing , squat , or lunge activities [ 24 , 25 ] . static fea was performed to quantify the maximum principal stress / strain at the implant - bone interface when axial loads were applied . figure 5 shows the profile of maximum principal strain at the implant - bone interface during the fea simulation on the first pkr with no external fixations . two separate regions with high tensile strain can be identified in the anterior implant - bone interface . the average maximum principal strain in this region is 750 microstrains ; the maximum principal strain in this region ranges from 482 microstrains to 1265 microstrains . the second region is located in the anterior aspect of the implant - bone interface and is near the midline of the tibial plateau ( figure 5 ) . the average maximum principal strain in this region is 1198 microstrains ; the maximum principal strain in this region ranges from 493 microstrains to 2505 microstrains . figure 6 shows the profile of maximum principal strain at the implant - bone interface when the fea simulation was performed on the modified pkr with a superior screw design . in addition , the average maximum principal strain in the second region is 851 microstrains . the maximum principal strain in the second region ranges from 201 microstrains to 2305 microstrains . when compared to the pkr model without external fixations , the screwed pkr model exhibits 32% and 29% reductions in maximum principal strain in the first and the second regions , respectively . specifically , the strain reduction in the first region ranges from 18% to 55% , and the strain reduction in the second region ranges from 4% to 80% . figure 7 shows the profile of maximum principal strain at the implant - bone interface when the fea simulation was performed on a modified tibial implant with a frontal flange design . the maximum principal strain in the second region ranges from 313 microstrains to 1133 microstrains . when compared to the pkr design without external fixations , the flanged pkr demonstrates smaller maximum principal strain in the first region ( specifically , the strain reduction in the first region ranges from 42% to 95% , and the strain reduction in the second region ranges from 30% to 56% . the first design depended on press - fit pegs embedded into the tibial plateau for stability . the second design was a modification of the first design with an addition of a superior screw . the third design was also a modification of the first design with a frontal flange added . an fea was performed to examine the influence of the pkr design on the stress / strain at the anterior region of the implant - bone interface . the same loading conditions and boundary conditions were applied during each of the fea simulation . the tensile strains in the anterior region of the pkr implant - bone interface were then examined after the fea . we had hypothesized that when axial loads were applied in the posterior region of the first pkr model , there would be increased tensile strain in the anterior region of the implant - bone interface . indeed , we had identified that there were areas in the anterior region of the implant - bone interface that experienced large tensile strains . the first region showing large tensile strain was near the anterior edge of the implant - bone interface . the second region showing increased tensile strain was found to be posterior to the first region and near the midline of the tibial plateau . large tensile strains found in the anterior region of the implant - bone interface appear to be a result of the bending effect imposed on the tibia during the simulated weight - bearing exercise . we also hypothesized that a pkr design with a superior screw would effectively reduce the tensile strain at the anterior implant - bone interface . our fea simulation showed that the screw fixation resulted in decreases of tensile strain in the anterior implant - bone interface . in particular , when compared to the pkr model with no external fixations , the screwed pkr showed an average strain reduction of 32% in the first high - strain region and 29% in the second high - strain region . thus , it appears that the superior screw fixation can alleviate the tensile strain in the anterior region of the implant - bone interface during weight - bearing exercise . when a frontal flange is added to the tibial tray , a fixation is established between the tibial tray and the medioanterior aspect of the proximal tibia . we had hypothesized that the flanged pkr could effectively reduce the high tensile strain in the anterior region of the implant - bone interface during simulated weight - bearing exercises . this hypothesis was supported . in particular , when compared to the pkr model with no external fixations , the flanged pkr showed an average strain reduction of 64% in the first high - strain region and 47% in the second high - strain region . thus , it is clear that the frontal flange design can effectively decrease the tensile strain in the anterior region of the implant - bone interface . in this study , although both the screw design and the flange design can effectively reduce the tensile strain in the high - strain regions , the flange design leads to a greater strain reduction than the screw design . specifically , the flanged pkr resulted in a 62% strain reduction in the first region compared to a 32% reduction seen in the screwed pkr . also , the flanged pkr resulted in a 47% strain reduction in the second region compared to a 29% reduction seen in the screwed pkr . furthermore , the screw design exhibited various effectiveness of strain reduction in the second region . it is possible that stress concentration at the interface of the screw and bone may contribute to the small effect of strain reduction in some locations within the second region . interestingly , the flanged pkr demonstrated a uniform effect of tensile strain reduction in the second region . standard knee rehab programs for pkr patients include weight - bearing exercises such as squatting and lunging . depending on the type of exercises , the axial load can be as high as 57 times of the body weight . the medial compartment of the knee normally experiences higher loading than the lateral compartment [ 4 , 5 , 24 ] . it was found that during squat exercise , the axial loading in medial compartment is 1.5 times of the lateral compartment or 60% of the total axial load in the knee joint . in addition , during knee rehab exercises requiring knee flexion , the axial load is applied in the posterior region of the tibial tray [ 18 , 19 ] . thus , for patients with medial pkr , rehab exercises could result in high tensile strain in the anterior implant - bone interface . in this study , the press - fit pkr knee experiences a peak tensile strain of 2500 microstrain in the anterior region . the magnitude of the strain has exceeded the physiological tensile strain in human bone , which is below 15002000 microstrain [ 2830 ] . as bone can develop fatigue fracture with relatively few loading cycles when cyclic strains are large , it is possible that the cyclic loading introduced during knee rehab exercises may increase risk of bone microfracture at the implant - bone interface . thus , it is advisable for patients to avoid rehab exercises resulting in mechanical loading in the posterior region of the pkr knee . in addition , even after new bone tissue has fully grown into the implant surface , it is cautious for patients to regularly engage in exercises such as squatting and lunging , which would lead to large mechanical loads in the posterior region of the pkr . not surprisingly , the tensile strains at the implant - bone interface of the screwed pkr knee are mainly within the range of the physiological bone strain with an exception of one small area showing a relatively high strain of 2300 microstrains . this reflects that the effect of a superior screw on lowering bone strain varies from region to region . it is possible that the superior screw may not be effective in reducing strain in some areas of the implant - bone interface . thus , for the longevity of a pkr knee , patients with a screwed pkr may consider avoiding exercises requiring knee flexion with large posterior knee loading . in this study , we found that the flanged pkr could sustain typical posterior knee loading without having large strains . therefore , patients with a flanged pkr can safely engage in knee rehab exercises involving lunge and squats , which could help the surgical knee to regain function quickly . in the long run , with a flanged pkr , patients can regularly perform physical activities involving knee flexion and habitual posterior knee loading ( e.g. , squatting and lunging ) without risk of bone microdamage at the implant - bone interface . knee oa is often limited in medial knee compartment and patellofemoral joint . compared to tkr , pkr requires less bone cut and a shorter hospital stay . with a pkr , patients can retain cruciate ligaments and normal joint geometry to achieve satisfied knee mechanics [ 8 , 1215 ] . most importantly , patients can achieve fast recovery by engaging in aggressive rehab exercises to regain knee strength and good knee mechanics . however , in the first two to three months postsurgery , bone tissue is in the process of remodeling and new bone tissue is growing into the implant surface . during bone remodeling , thus , large mechanical load may result in bone microdamage and implant loosening . in this study , we determine that a typical axial load during knee exercises can lead to bending effect on the anterior aspect of the tibia and result in increased tensile strain in the anterior region of the implant - bone interface . if the implant - bone interface experiences repetitively large tensile strain during rehab exercise , implant loosening is likely to occur due to microdamage of weakened bone tissue at the implant - bone interface . thus , it is important to introduce external fixation mechanisms to stabilize the implant during knee rehab exercises . in this study , we confirmed that external fixation mechanisms enhance stability of the cementless pkr during simulated knee rehab exercises . both the superior screw design and the frontal flange design have demonstrated effectiveness of reducing tensile strain in the anterior region of the implant - bone interface . in particular , the flanged pkr showed lowered tensile strain within physiological strain range . therefore , to better alleviate tensile strain at the implant - bone interface , external fixation mechanisms such as a superior screw or a frontal flange should be incorporated into future cementless pkr designs . firstly , a phantom was not used during the ct scan . without phantom data , it was difficult to establish an accurate relationship between bone density and material properties for both the cortical and cancellous bone . although it would be ideal to model the tibia plateau with separate cancellous bone properties , it was acceptable to model the entire tibia with linear elastic and transversely isotropic material [ 21 , 22 ] . because the mechanical behavior of cancellous bone was found to be similar to that of cortical bone , fea studies have been conducted to analyze fatigue life of cancellous bone with cortical bone 's properties applied . furthermore , as comparisons were made among the three pkr designs based on the same tibial model , the trend of mechanical differences exhibited among the three designs is valid . the strain values from the fea simulation can only be applied to the tibia bone chosen . as the geometry of the tibia bone may vary from person to person , it is possible that for a given mechanical load , the strain level at the implant - bone interface may be different from person to person . however , this does highlight the importance of using subject - specific fe models for evaluating the mechanics of individual pkr knees . in summary , posterior axial loading could lead to increased tensile strain in the anterior interface between the pkr and tibia bone . both the screwed and flanged pkrs can reduce the tensile strain in the anterior region of the implant - bone interface . flange design demonstrates a more uniform effect of reducing tensile strain in the anterior region of the implant - bone interface than the screw design .

partial knee replacement ( pkr ) results in fast recovery and good knee mechanics and is ideal to treat medial knee osteoarthritis . cementless pkr depends on bone growing into the implant surface for long - term fixation . implant loosening may occur due to high tensile strain resulted from large mechanical loads during rehab exercises . the purpose of this study is to investigate whether external fixations such as superior screw and frontal flange could reduce the tensile strain at the implant - bone interface . three medial pkrs were designed . the first pkr had no external fixations . a superior screw and a frontal flange were then added to the first pkr to form the second and third pkr designs , respectively . finite element analysis was performed to examine the tensile strain at the implant - bone interface during weight - bearing exercises . the pkr with no external fixations exhibited high tensile strain at the anterior implant - bone interface . both the screwed and flanged pkrs effectively reduced the tensile strain at the anterior implant - bone interface . furthermore , the flanged pkr resulted in a more uniform reduction of the tensile strain than the screwed pkr . in conclusion , external fixations are necessary to alleviate tensile strain at the implant - bone interface during knee rehab exercises .

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

PMC4336661

angiogenesis or neovascularization is defined as the growth and development of new capillaries from pre - existing blood vessels , and is essential for tumors to grow beyond a minimal size.1 this could occur either by branching and extension of adjacent blood vessels or by recruitment of endothelial progenitor cells from the bone marrow . angiogenesis plays an essential part in a variety of physiological and pathological processes , including embryogenesis , wound healing , inflammation and tumor progression.2 this process depends on tightly controlled endothelial cell proliferation , migration , and differentiation , and is controlled by a finely tuned balance of pro and anti - angiogenic factors and their receptors , most of which are polypeptide growth factors . although the regulatory mechanisms in angiogenesis are not fully understood , evidence suggests that a cascade of events occur , and that intervention at one of the steps is sufficient to prevent or promote revascularization . considerable evidence exists to indicate that solid tumor growth is dependent on angiogenesis , a hypothesis first proposed by folkman in 1971.3 it is a well - accepted paradigm that tumors recruit new blood vessels from the existing circulation by secreting growth factors from the tumor cells . this has been shown in many human malignancies , especially lung , prostate and breast cancers . tumor cells secrete angiogenesis - stimulating proteins , such as vascular endothelial growth factor ( vegf ) , fibroblast growth factor ( fgf ) , thymidine phosphorylase , basic fgf , hepatocyte binding growth factor , and others , whose specific receptors are found on the endothelial cells . in addition , tumor - associated macrophages , lymphocytes , and fibroblasts of the tumor stroma can also produce angiogenic factors , promoting tumor growth and spread . though many growth factors exhibit angiogenic activity , most evidence points to a special role for vegf . vegf also known as vascular permeability factor , is a heparin - binding , dimeric glycoprotein with a selective mitogenic effect on vascular endothelial cells , and is the most potent and specific agent among the many molecules capable of initiating angiogenesis.4 actions of vegf include enhancement of both angiogenesis and vascular permeability . furthermore , expression levels of vegf correlate with tumor status and also a useful biomarker for the prediction of response to therapy.5 a tumor may persist in a diffusion - limited state , usually not more than 2 mm in diameter , with cell proliferation balanced by cell death , for many months or years . a tumor may , however , emerge from dormancy by inducing the growth of new blood vessels ( angiogenesis ) . this process allows the tumor to progress from the avascular ( lacking blood vessels ) to the vascular ( possessing a blood supply ) state . there are a large number of pro - angiogenic and anti - angiogenic factors , some of which are produced by the tumor and some of which are produced by host cells in response to the tumor . it is a shifting of the balance from the anti- to the pro - angiogenic factors ( the so - called angiogenic switch ) that causes the transition from the dormant to the angiogenic phase . odontogenic tumors and cysts comprise a major portion of the pathology occurring in the orofacial region . the odontogenic epithelium , which is responsible for tooth development can also give rise to these tumors and cysts.6 ameloblastoma is the most frequently encountered tumor arising from odontogenic epithelium and is characterized by a benign but locally invasive behavior with a high tendency to recur . on the other hand , odontogenic keratocyst ( okc ) is a developmental epithelial odontogenic cyst most commonly occurring in the jaws , which could arise from the dental lamina or its remnants or extensions of basal cells from the overlying oral epithelium ( basal cell off shoots ) . there has been a great deal of interest in the okc since it became apparent that it may grow to a larger size before it manifests clinically ( aggressive clinical behavior ) and that unlike other jaw cysts , it has a particular tendency to recur following surgical treatment ( high recurrence rate ) . there has been considerable evidence that has accumulated over the years that the okc may be a benign cystic neoplasm , which has gone to the extent of a change in terminology from okc to keratinizing cystic odontogenic tumor ( kcot).7,8 in the current study , we have compared the expression of vegf between ameloblastoma and kcot using immunohistochemistry in an attempt to analyze the property of angiogenesis in kcot . the study protocol was approved by srm institutional ethical committee . the samples diagnosed as multicystic ameloblastoma and solitary formalin - fixed paraffin - embedded biopsy material of ameloblastoma and sporadic kcot were retrospectively studied , the diagnosis in each case having been made on the basis of clinical , radiographic and histologic findings . a total number of 30 cases were selected , out of which 15 were cases of ameloblastoma ( plexiform type ) and 15 cases of kcot . hematoxylin and eosin sections of all cases were repeated to reconfirm the sign out diagnosis . sections were deparaffinized in xylene , hydrated through graded alcohol , and washed with tap water . antigen retrieval was performed by immersing the slides in citrate buffer , ph 6.1 and run for two cycles in the biogenex antigen retrieval system at 97c for 8 min each and then quenched in running tap water . sections were incubated with rabbit antihuman vegf polyclonal antibody for 1 h. following this the sections were treated with super enhancer and poly horse radish peroxidase reagent for 30 min each . each of these incubation procedures was preceded by 10-min rinse with phosphate buffer except before the addition of primary antibody . negative controls were obtained by omitting the primary antibody and were confirmed to be unstained . immunohistochemical reactivity for vegf was assessed according to the presence of positive staining and intensity of staining . based on this , the immunoreactivity of vegf was classified into four groups : ( ) negative ( none of the epithelial or neoplastic cells staining ) , ( ) weakly ( < 10% of epithelial or neoplastic cells ) positive , ( + ) moderately ( 10 - 50% of epithelial or neoplastic cells ) positive , and ( + + ) strongly ( more than 50% of epithelial or neoplastic cells ) positive . statistical analysis was performed using spss software and p < 0.05 was considered to indicate statistical significance . sections were deparaffinized in xylene , hydrated through graded alcohol , and washed with tap water . antigen retrieval was performed by immersing the slides in citrate buffer , ph 6.1 and run for two cycles in the biogenex antigen retrieval system at 97c for 8 min each and then quenched in running tap water . sections were incubated with rabbit antihuman vegf polyclonal antibody for 1 h. following this the sections were treated with super enhancer and poly horse radish peroxidase reagent for 30 min each . each of these incubation procedures was preceded by 10-min rinse with phosphate buffer except before the addition of primary antibody . negative controls were obtained by omitting the primary antibody and were confirmed to be unstained . immunohistochemical reactivity for vegf was assessed according to the presence of positive staining and intensity of staining . based on this , the immunoreactivity of vegf was classified into four groups : ( ) negative ( none of the epithelial or neoplastic cells staining ) , ( ) weakly ( < 10% of epithelial or neoplastic cells ) positive , ( + ) moderately ( 10 - 50% of epithelial or neoplastic cells ) positive , and ( + + ) strongly ( more than 50% of epithelial or neoplastic cells ) positive . statistical analysis was performed using spss software and p < 0.05 was considered to indicate statistical significance . immunoreactivity for vegf was mainly seen in the cytoplasm of neoplastic epithelial cells in cases of ameloblastoma and in the epithelial lining of kcot . endothelial cells and inflammatory cells were also reactive for vegf . strong reactivity for vegf was seen in the peripheral columnar cells in ameloblastoma and in cases of kcot all the epithelial cell layers were positive with the exception of the parakeratin layer . strong expression was seen in 60% of ameloblastoma ( figures 1 and 2 ) and in 46.67% of kcot ( figure 3 ) , while weak expression was seen in 6.67% of ameloblastoma and 20% of kcot . vegf expression was stronger in ameloblastoma when compared to kcot , but there was no statistically significant difference between ameloblastoma and kcot ( p = 0.345 , mann immunohistochemical staining with vascular endothelial growth factor in ameloblastoma ( 10 ) . staining of tumor cells and also blood vessels adjacent to the odontogenic epithelium . immunohistochemical staining with vascular endothelial growth factor in ameloblastoma ( 40 ) . staining of tumor cells . immunohistochemical staining with vascular endothelial growth factor in keratocystic odontogenic tumor ( 10 ) . staining of tumor cells except the parakeratin layer . the term okc was first introduced by philipsen over 50 years ago to describe a group of odontogenic cysts which showed a characteristic histological appearance . as compared with other types of odontogenic cysts , kcots appear to have an intrinsically higher growth potential.9 - 11 a propensity to recur following surgical treatment and the potential risk of neoplastic change place kcots in a unique position within the spectrum of odontogenic lesions and has been suggested that kcots should be regarded as benign neoplasms . numerous investigations have pointed this distinctive behavior of kcot toward the nature of its epithelium . the epithelium of the kcot has been reported to show a higher rate of proliferation than other cyst types indicated by strong expression of p53 , proliferating cell nuclear antigen and ki-67 in kcot.12 - 14 however , very little attention has been paid to the connective tissue of odontogenic cysts and stroma of odontogenic tumors . browne ( 1975 ) was one of the first to suggest that the connective tissue wall may have a significant role in the pathogenesis of kcot . interaction between the fibrous capsule and epithelium of this cyst has also been confirmed in other studies . a number of connective tissue elements such as tenascin and fibronectin ; rank , rankl and osteoprotegerin ; laminins and collagen 4 have also been studied in this lesion.15 - 18 li et al . studied the expression of epidermal growth factor receptor ( egfr ) in epithelial cells of odontogenic cysts , including kcot and determined that high levels of egfr expression in kcot supported the view that they have an intrinsic growth potential . it was reasoned that kcot epithelium is derived from dental lamina remnants and this may reflect the potential for epithelial - mesenchymal interactions and growth factor / receptor modulations . also transforming growth factor alpha , egf , egfr expression suggested involvement of growth factors in the pathogenesis of kcot , via autocrine and paracrine mechanism.19 angiogenesis is one of the best known stromal factors participating in tumor progression and has been extensively investigated in various lesions.20 - 22 angiogenesis is the physiological process involving the growth of new blood vessels from pre - existing vessels and , like cancer , is a complex multi - stage process including degradation of extracellular matrix , proliferation and migration of endothelial cells , capillary differentiation and anastomosis.21 among the very many angiogenic factors , vegf has a selective mitogenic effect on the vascular endothelium and is the most potent agent among the many molecules capable of initiating the angiogenesis.23,24 also the expression levels of vegf correlate with tumor status and prognosis . all solid tumors such as ameloblastoma are composed of the neoplastic cell compartment and the stromal component . the stroma is composed of new blood vessels , inflammatory cells , connective tissues , and a fibrin - gel matrix . tumor fibrin originates from the extravasation and extravascular clotting of plasma fibrinogen by tumor cell prothrombinase and is dependent on increased vascular permeability . hyperpermeability of capillaries and the macromolecular transvascular transport are facilitated by vegf produced by tumor cells.25,26 however , the mechanism by which vegf modulates the changes leading to an increase in capillary permeability is unknown . ultrastructural findings on blood vessel architecture including volume and surface densities of odontogenic cysts ( kcot and dentigerous cyst ) were compared based on a premise that angiogenesis is a feature of a benign neoplasm and evidence of this in kcot may account for its characteristic behavior . although there was no significant difference in the overall vascularity , there were differences in the ultrastructural findings between the two cysts . kcot connective tissue wall showed fenestrated capillaries and degeneration of the endothelial lining with associated thrombosis . it was also speculated that an increase in the number of platelets seen in the capillary thrombi of the kcot may stimulate epithelial cell growth and viability through unspecified growth factors.27 the present study showed that vegf is expressed strongly in ameloblastomas than in kcot . although ameloblastomas showed stronger expression of vegf than kcot , the results were statistically not significant , and there was a strong expression of vegf in kcot as well . this finding supports the fact that kcot has an aggressive behavior like tumors though being a cystic lesion of odontogenic origin . studied the microvessel densities ( mvd ) in odontogenic lesions and suggested that intratumoral mvd has a prominent role in growth and clinical behavior of odontogenic lesions and hypothesized that both ameloblastoma and kcot share similarities regarding angiogenesis accounting for its aggressive behavior.28 furthermore , kcot exhibited increase in the total vascular area and mean vascular area in comparison with other development cysts and suggested that this angiogenesis with dilated and tortuous blood vessels in kcot , which may be related to the increase in tissue metabolism , nutrition requirement of the proliferating epithelium and associated lymphatic drainage.29 elevated expressions of vegf was detected in benign and malignant ameloblastomas , suggesting that vegf production by odontogenic epithelial cells was up regulated in association with neoplastic changes , malignant transformation or both . in addition , it was also noted that vegf expression was markedly decreased in keratinizing , and granular cells and also vegf may act differently according to the architecture of tumor tissues . this could partly account for the difference in the levels of expression of vegf among ameloblastoma and kcot in our present study.21 in summary , the results suggest that vegf expression was strongly expressed in ameloblastoma than kcot . however , the immunoreactivity between these two lesions does not show a significant difference , suggesting that angiogenesis in part could explain the aggressive behavior of these cystic neoplasms . these results suggest that vegf is an important mediator of tumor angiogenesis and upregulation of vegf might be associated with tumorigenesis . the results and hypothesis achieved from the study , proved to be consistent , and augments the already existing hypothesis regarding the concepts of its aggressive behavior . nevertheless , angiogenesis is a complex process that involves the interaction between pro and anti - angiogenic factors and several agents directly or indirectly influence this event . hence , it is logical that other proteins associated with angiogenesis are concurrently evaluated in selected cases to arrive at a consensus . the role of vegf in the pathogenesis of odontogenic cysts and tumors should be further evaluated using more sophisticated methods and additional cases , as angiogenesis could be a potential target for the therapeutic management of these lesions .

background : odontogenic keratocyst ( okc ) is a developmental odontogenic cyst with an aggressive clinical behavior suggesting a change in its terminology from a cyst to a tumor and has now been renamed as keratocystic odontogenic tumor ( kcot ) . the purpose of this study was to assess and compare angiogenesis in ameloblastoma and okc.materials and methods : angiogenesis was assessed by studying the immunohistochemical expression of vascular endothelial growth factor ( vegf ) . the study samples included 15 ameloblastomas and 15 kcots . the immunoreactivity was statistically evaluated using mann whitney u-test.results:vegf expression was higher in ameloblastoma than kcots . however , a non - significant difference of vegf expression was noted between ameloblastoma and kcots ( p = 0.345).conclusion : the results suggest that tumor angiogenesis may play a significant role in aggressive biologic behavior of kcot . thus , angiogenesis could be a potent target for developing anatiangiogenic therapeutic strategies .

Introduction Materials and Methods Immunohistochemistry Evaluation of immunostaining Results Discussion Conclusion

PMC3671310

caloric restriction ( cr ) and exercise have been considered to have beneficial effects on human health , including reducing the risks for the development of diabetes , cardiovascular disease , and cancer [ 1 , 2 ] . studies have shown that cr and exercise can improve the metabolic conditions in obesity , but the underlying mechanism is still unclear . the amp - activated protein kinase ( ampk ) , as an important energy sensor , can be activated by the increased amp / atp ratio or adp in energy deprival state . ampk activation plays important roles in adjusting the metabolic pathways to restore the atp level in both short - term and long - term manner . because of this activity , it has been considered as a potential mediator of the effects of cr and exercise . however , whether ampk activation is induced in chronic cr or exercise is still an unresolved issue due to distinct observations on the ampk activity after long - term cr or exercise in mouse . autophagy is an intracellular recycling pathway that functions during basal conditions but can be induced under stress such as starvation . recent findings established the relationship between insulin resistance in obesity and the decreased autophagy activity in liver , and restoration of atg7 was shown to enhance the systemic glucose tolerance in mice . further , it has been shown that bcl2-regulated autophagy is indispensable in mediating the improvement of glucose homeostasis by either long- or short - term exercise . since ampk has already been known to participate in regulating autophagy activity by activating ulk1 through direct phosphorylation , we wonder if chronic cr and exercise can induce autophagy in obese mice and if the improvement in metabolic conditions is correlated with the autophagy activity . further , we also sought to investigate if there is any relationship between induced autophagy and ampk activity in obese mice . to study the relationship between the chronic cr , exercise , and autophagy and whether ampk activation is involved in this process , we used high fat diet to induce the obese mice models , and then we investigated the effects of different intervention on the improvement of mice metabolic conditions . then , we examined the level of lc3 proteins and ampk phosphorylation to investigate the relationship between metabolic improvement and ampk activation , as well as the relationship with autophagy activity . studies were conducted on 6-week - old male c57 mice , which were fed with high fat diet containing 58% fat ( kcal% , total calorie 5.56 kcal / g ) . mice in the control group were fed with normal 5% fat diet ( kcal% , total calorie 3.4 kcal / g ) . six mice , from the experimental and the control group , respectively , were taken and measured for body weight and 24-hour calorie intake every other week . high fat diet was switched to low fat diet , and the running speed of the exercise groups was 8 m / min , 10 min per day , and 7 days a week . the calorie intake of cr group is around 70% of that of the model control group , a level that has been reported to effectively protect against high fat diet induced obesity in mice . the running speed was gradually increased from 8 to 22 m / min within one week , 30 min / day , and 5 days / week , as described by reznick et al . , which is considered as sufficient to increase the amp / atp level and activate ampk . serum glucose levels were tested by using glucose ( hk ) assay kit ( sigma - aldrich ) . triglycerides levels were tested by triglyceride ( gpo trinder ) reagent a kit . to quantitatively determine the hdl cholesterol in serum , wako hdl - cholesterol e assay was used by phosphotungstate - magnesium salt precipitation . alt and ast tests kits ( american screening corporation ) were used to evaluate liver function of the mice . liver , skeletal muscle , and heart homogenates were resolved by sds - page , and proteins were transferred onto polyvinylidene difluoride membranes . rabbit polyclonal antibodies against phosphorylated ampk , total ampk , and lc3 and anti - rabbit secondary antibodies were purchased from cell signaling . data were analyzed by a two - tailed , independent student 's t - test . the calorie intake of the dio mice was consistently higher than that of the control group , and the body weight also increased more rapidly . the weight of control group became stable at week 10 , while that of the dio group continued increasing even at the end of this study . significant difference exists in the weight , liver , mesenteric fat , postperitoneal fat , and the total visceral fat between the two groups . the weight of the heart also had a significant increase , while no obvious difference was observed in the weight of skeletal muscle . similarly , the coefficient ( organ weight/100 g body weight ) of liver , mesenteric fat , postperitoneal fat , and the total visceral fat , increased as well , but that of heart and skeletal muscle had decreased significantly . by the end of this study , the dio mice had exhibited hyperinsulinemia , insulin resistance , and increase in cholesterol , triglycerol , hdl - cholesterol , and ldl - cholesterol . meanwhile , alt and ast in the serum also increased . these results showed the obese mice models were successfully induced by using high fat diet . the intake of the groups 3 and 6 , whose diet was switched from high fat to low fat , has decreased at the initial stage . after two - week adaption , the intake of these two groups has gradually recovered and got stable . the calorie intake of the exercise group with high fat diet was significantly lower than that of group 2 . at the end of this experiment , the body weights of groups 3~6 were all significantly lower compared with group 2 ( figure 1 ) . the level of leptin , which is an appetite - inhibiting adipokine , in dio mice significantly decreased after cr and/or exercise , and the level of leptin decreased to a similar extent in the cr treated groups regardless of hfd or lfd . the level of adiponectin was significantly reduced in dio mice but was recovered in the groups treated with lfd and cr , regardless of exercise . however , in hfd groups neither cr nor exercise had a significant effect on adiponectin level ( figure 2 ) . serum glucose slightly increased in dio mice and reduced to a small extent in mice with all the treatments , but most of the differences were not statistically significant . the insulin level also increased in dio mice , but to a higher degree , suggesting insulin resistance was induced in the obese model and can be recovered by cr or cr plus exercise . interestingly , however , lfd with cr did not improve the insulin resistance conditions as hfd plus cr did . this plausible result was probably caused by the large variation in this group 's insulin level , since the average actually exhibited a decreasing trend when compared with dio group ( figure 3 ) . further , when combined with lfd and/or exercise , the effects became even more prominent . lfd and cr can reduce the level of triglyceride either with or without exercise training ( figure 4 ) . alteration in ast and alt levels has been shown to be related with metabolic conditions and directly associated with insulin resistance . in addition , these markers can be also used to assess the extents of liver injury in metabolic disorders . therefore , we further examined their serum levels . while both ast and alt levels increased in dio mice , they can be reduced to normal level by either cr or exercise . in consistence with the hyperlipidemia conditions , different extents of hepatic steatosis were found in the he staining of dio liver sections . the finding of fatty livers in these mice also explained the increase in serum asl and alt levels . as expected , steatosis was obviously improved in the treatment groups , in line with the decrease in serum lipids and transaminase levels ( figures 5 and 6 ) . to investigate the relationship between the improvements in metabolic conditions and ampk activation , we examined the levels of ampk phosphorylation in liver , skeletal muscle , and cardiac muscle . the results showed that there was no significant difference in ampk phosphorylation among groups in any of these tissues ( figure 7 ) , implying the ampk activation does not contribute to the long - term benefits of caloric restriction . next , we evaluated the autophagy activity in cardiac muscle as represented by lc3 ii / lc3 i ratio . the results demonstrated that autophagy activity was significantly elevated in groups 4 and 6 , and such trend was also quite obvious in group 3 as compared with group 2 ( figure 7 ) . considering the facts that these three groups were treated with caloric restriction and showed consistent improvement in metabolic indices , it strongly indicates autophagy activity is responsible for the metabolic improvements in caloric restriction conditions . the activation of ampk has been considered as an important factor in adapting the body to the state of energy stress . upon sensing an increased level of amp / atp ratio , ampk can be fully activated by an increased phosphorylation level at thr . activated ampk can interact with numerous pathways including that of sirt , mtor , and autophagy and regulate the activities including mitochondrial biogenesis , glucose uptake , and lipid metabolism . the functions of ampk imply that its activation may participate in mediating the beneficial effects of chronic caloric restriction and exercise . however , whether ampk activation plays a part in caloric restriction and exercise is still controversial . in this study , high fat diet was used to induce the obese mice models , and then interventions of cr and exercise were used to improve the metabolic conditions . after that , we examined the level of ampk phosphorylation in liver , skeletal muscle , and heart tissues to investigate the relationship between metabolic improvement and ampk activation . as expected , two - month feeding of high fat diet successfully induced the obesity and altered the metabolic state of the model mice , which was characterized by increased fat accumulation , insulin resistance , and impaired liver function . however , to most of the indices , exercise alone failed to make a significant improvement , while the effects of caloric restriction were quite prominent . though it is plausible that exercise alone did not have an obvious effect , which may be caused by an insufficient dosage , it did have a synergistic effect when combined with cr . moreover , because we are more concerned with the correlation between metabolic improvement and levels of signaling proteins , instead of the effects of individual interventions , this result should not preclude us from getting a reasonable conclusion . our results indicate that the activation of ampk is not closely correlated with the improvement of the metabolic conditions of the diet induced obese mice . thus , the long - term beneficial effects of chronic cr and exercise may not be mediated through ampk activation . the result on ampk in our study is in consistence with that in the study of gonzalez et al . , which showed ampk activation did not respond to chronic caloric restriction or even fasting in skeletal muscle , cardiac muscle , or liver . however , these results appeared to be contradictory to some other studies , in which cr was shown to lead to ampk activation [ 1416 ] . in the study of edwards et al . , it was shown that a life - long cr was able to protect the myocardium against ischemia / reperfusion ( i / r ) , and this protective effect was demonstrated to involve ampk activation , because inhibition of ampk activation was able to abolish the protection . indeed , ampk can interact with multiple pathways that may assist in the adjustment of body to different energy metabolic states , and there is also pharmacologic evidence supporting the role of ampk in mediating cr effects , in which resveratrol was shown to activate ampk by decreasing atp production . therefore , the question why there are different observations concerning ampk activation in chronic cr and exercise still needs to be clarified . in a previous study , it was proposed that a lack of an effect of cr on ampk activity is probably due to that the energetic challenge presented by cr was not severe enough to cause the changes in atp and amp to activate ampk . this level of energy challenge brought by cr , however , should be sufficient to affect the metabolic states of mice as shown in both their and this study . therefore , this may be an explanation on why chronic cr and exercise did not induce the activation of ampk , and further , it suggests that the beneficial effects of chronic cr and exercise are mediated by some other pathways instead of ampk activation . it is possible that ampk only becomes activated in more severe and acute energy deprival conditions . for instance , in the study of edwards et al . , a life - long cr could indeed protect myocardium from i / r injuries accompanied by an increase in p - ampk level , but this does not indicate that ampk became activated in the basal condition . also , the cancelling of protection by araa only proves the ampk was activated in i / r , which represented an acute and a much more severe energy stress situation than that in chronic cr or exercise . however , the more active ampk during i / r after a life - long cr does imply a mechanism by which ampk was sensitized by long - term cr . if these effects are not mediated by ampk , then there must be other mechanisms that explain why chronic cr and exercise may improve the metabolic conditions . the study of yang et al . showed that loss of autophagy might be responsible for the insulin resistance in obesity , which was characterized by the downregulation of atg7 , and restoration of atg7 was able to improve the glucose tolerance in mice . in another research , both long - term and short - term exercises were shown to be able to improve the insulin sensitivity , and this effect depended on induced autophagy . in the light of these findings , we examined the level of conversion of unlipidated lc3 i to lc3 ii ( autophagosome - membrane - associated lapidated form ) in the heart tissues . the results clearly exhibited a trend which is consistent with the change in metabolic conditions in the interventional groups . specifically , a significant increase in autophagy activity was found in group 6 mice , and this group also showed a much more prominent trend in increased insulin level , which indicated an improvement in insulin sensitivity . also , significant increase in the antidiabetic adiponectin , decrease in serum lipids , and improvement in liver function were also exhibited . therefore , this may be biochemical evidence that an enhanced autophagy activity may participate in regulating the metabolic conditions during chronic cr and exercise , and the effect of autophagy may further extend beyond glucose homeostasis . moreover , although autophagy in living cells is under control of ampk , the induction in this context possibly does not depend on ampk activation . since autophagy in living cells is known to be under the regulation of multiple pathways including ampk , sirt , and mtor , it may be important to investigate which among these pathways is responsible for the activation of autophagy during chronic cr and exercise . in the study of he , the activation of autophagy in short - term exercise was shown to involve bcl2-beclin1 complex . upon dissociating with the antiautophagic bcl2 however , the long - term exercise might act through a different pathway according to our data , since the p - ampk level did not change significantly in the groups with long - term exercise . the further study on sirt and mtor pathway activity might be helpful to find the molecules responsible for autophagy activation during chronic cr and exercise . our results showed that ampk activation , which was represented by the level of p - ampk , did not correlate with the improvement of metabolic conditions in dio mice , implying ampk activation may not participate in mediating the beneficial effects of chronic cr or exercise . on the other hand , however , we found the autophagy activity might be related to the improved metabolic conditions but was not correlated with ampk activation . thus , we propose that autophagy may play a role in mediating the effects of chronic cr and exercise , but it is not regulated by ampk activity . however , this hypothesis based on the findings in our research still requires confirmation . for example , ampk knockout mice might be used to check if these interventions are still effective in the absence of ampk activation . finally , detecting sirt and mtor pathway activity in the samples may also provide clues on the regulation of autophagy activity in obese mice that received chronic cr or exercise treatment .

aim . to investigate the role of ampk activation and autophagy in mediating the beneficial effects of exercise and caloric restriction in obesity . methods . dietary - induced obesity mice were made and divided into 5 groups ; one additional group of normal mice serves as control . mice in each group received different combinations of interventions including low fat diet , caloric restriction , and exercise . then their metabolic conditions were assessed by measuring serum glucose and insulin , serum lipids , and liver function . ampk phosphorylation and autophagy activity were detected by western blotting . results . obese mice models were successfully induced by high fat diet . caloric restriction consistently improved the metabolic conditions of the obese mice , and the effects are more prominent than the mice that received only exercise . also , caloric restriction , exercise , and low fat diet showed a synergistic effect in the improvement of metabolic conditions . western blotting results showed that this improvement was not related with the activation of ampk in liver , skeletal muscle , or heart but correlates well with the autophagy activity . conclusion . caloric restriction has more prominent beneficial effects than exercise in dietary - induced obese mice . these effects are correlated with the autophagy activity and may be independent of ampk activation .

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

PMC3592707

our prototype project was described in a paper published in june , sex differences and organizational effects of androgen in spinal cord motor nuclei ( grisham et al . , 2003 ) , along with a collection of digital photomicrographs of snb spinal cord motor neurons ( see figure 2 ) . this collection of digital images came from a study that examined sex differences in these motor neurons and the effect of an anti - androgen drug on motor neuron development ( grisham et al . , the original study required a rat breeding colony , microtomes , hoods for staining , a good quality microscope , a digital camera , image - capturing software , and a year s work from two graduate students and a postdoc . in its current digital format , faculty using this image collection need only have computers at their disposal , so the costs of running this lab are minimal . this image collection allows students the opportunity not only to replicate a study , but also to extend the original study by investigating another motor neuron pool ( rdln see figure 2 ) . subsequent to the publication in june , this image collection was downloaded over a thousand times , indicating the need and interest for these types of teaching tools . the image library and relevant support materials ( including a pdf of the june article ) can be downloaded at http://mdcune.psych.ucla.edu/modules/ratscia . this success inspired a nsf - funded project , the modular digital course in undergraduate neuroscience education ( mdcune ) , which is a free , online resource for disseminating digital materials for laboratory instruction http://mdcune.psych.ucla.edu/. this project is generating three more modules of digital tools that will provide genuine , inquiry - based instruction in neuroscience : a virtual neurophysiology lab module , a neuroinformatics /bioinformatics module , and a bird song module . swimmy has been more thoroughly described in a previous issue of june ( grisham et al . , 2008b ) and was presented at the faculty for undergraduate neuroscience workshop at macalester college in july 2008 . swimmy was developed to circumvent the many problems in teaching neurophysiology as a wet lab . subsequently , students ascertain the neurons involved in the swimming behavior and determine their interconnections by examining the time relationships between neuronal events and by devising and executing virtual experiments . once students determine all the connections , they will arrive at a schematic that looks something like figure 3 . students then are challenged to discover which cells are the central pattern generator ( cpg ) via further experiments , and discover how the swimming rhythm is generated by the cpg . finally , students use an animation feature that reads out the output of the motor neurons to describe the behavior produced by various lesions , and to explain the behavioral phenomena via their understanding of the circuit ( figure 4 ) . thus , to arrive at a thorough understanding of the circuit , students must compare and contrast the results obtained from the experiments that they design to create well - reasoned , logical arguments supporting their deductions . there is a clear need for instruction at the undergraduate level in the use of bioinformatics tools , but few good resources are available . our module will remedy this situation by offering a cohesive package that integrates several bioinformatic resources in a way that is accessible to undergraduate students . this module brings together information gathered from brain anatomy and bioinformatics websites , including the mouse brain library ( rosen et al . , 2000 ) , genenetwork s webqtl ( wang et al . , 2003 ) , ucsc genome browser ( kent et al . , 2002 ) , the allen brain atlas ( lein et al . , 2007 ) , the national center for biotechnology information s entrez gene , and ncbi s pubmed . in this module , students quantify a phenotype using nih image ( see references for url ) to analyze digital images that are downloaded from the mouse brain library ( see references for url)figure 5 . the mouse brain library provides digitized images of sectioned and stained mouse brains from individuals of different recombinant inbred strains and allows the quantification of many brain phenotypes . we quantify olfactory bulb size since it is relatively easy to operationally define , and there is a published study with which to compare the data ( williams et al . , 2001 ) . after students clean - up their morphometric data by statistically controlling for variables , they enter their data into a web - based qtl analysis ( genenetwork s webqtl see references for url ) . qtl analysis relates variations in phenotype to loci on chromosomes where genes impacting the phenotype can be found ( figure 6 ) . genenetwork s webqtl provides a direct link to the university of california , santa cruz genome browser ( url in references ) , which provides a list of the genes that are in that portion of the chromosome ( see figure 7 ) . students can search through this list and find a gene that is highly expressed in the olfactory bulb from the data provided by the ucsc genome browser . once students identify a gene that is highly expressed , they also can link to the allen brain atlas ( url in references ) , which displays the expression pattern of that specific gene via in situ hybridization views along with an atlas ( c.f . thus , students can discover the cell layers where their gene of interest is expressed ( figure 8) . the ucsc genome browser also provides links to the national center for biotechnology information resources such as entrez gene and pubmed ( urls in references ) . these resources allow the students to discover more information about their highly expressed gene including its nucleotide and amino acid sequence , as well as find articles about their gene that provide a deeper intellectual involvement in this exercise . our website has already been populated with some of these materials http://mdcune.psych.ucla.edu/. a detailed instructor s manual should be available in summer 2009 . 1988 ) and dramatic brain sex differences ( nottebohm and arnold , 1976 ) were first described in this system , and it is still the focus of ongoing investigations on the neural basis of learning and memory ( nordeen and nordeen , 1988 ; troyer and doupe , 2000 ) , as well as on the genetic and hormonal bases of sexual differentiation in the nervous system ( cf . grisham and arnold , 1995 ) . the rich literature base on the song system , the robust difference between the sexes in the size of song nuclei ( grisham and arnold , 1995 ) , and the dramatic masculinizing effect of hormones on this system s development ( grisham and arnold , 1995 ) make the zebra finch song system ideal for use in undergraduate laboratories . nevertheless , studying this system requires significant investments : supporting a bird colony , purchasing good quality microscopes , microtomes , histological supplies for processing brains , and digital microscope cameras . such extensive facilities and equipment requirements make studying the bird song system out of reach for many institutions . we are surmounting these obstacles by providing a collection of images for undergraduate students to replicate a published experiment that examined the doses of hormone required to masculinize the song system ( grisham et al . further , students can extend this study -- we are adding images from untreated males , so that students can explore whether or not any of the treatments fully sex reverse the female song system at this point in time , the digital images are being collected , and we anticipate that they will be fully available in summer 2010 . so far , our experience with students using a prototype library of digital images has proved to be highly successful ( see figure 10 ) . anecdotally , the digital images are better at focusing students on the relevant aspects of the task relative to using tissue on slides . when we tried using the actual tissue , one student looked for the telencephalic song nuclei in the lower brainstem . swimmy has been more thoroughly described in a previous issue of june ( grisham et al . , 2008b ) and was presented at the faculty for undergraduate neuroscience workshop at macalester college in july 2008 . swimmy was developed to circumvent the many problems in teaching neurophysiology as a wet lab . subsequently , students ascertain the neurons involved in the swimming behavior and determine their interconnections by examining the time relationships between neuronal events and by devising and executing virtual experiments . once students determine all the connections , they will arrive at a schematic that looks something like figure 3 . students then are challenged to discover which cells are the central pattern generator ( cpg ) via further experiments , and discover how the swimming rhythm is generated by the cpg . finally , students use an animation feature that reads out the output of the motor neurons to describe the behavior produced by various lesions , and to explain the behavioral phenomena via their understanding of the circuit ( figure 4 ) . thus , to arrive at a thorough understanding of the circuit , students must compare and contrast the results obtained from the experiments that they design to create well - reasoned , logical arguments supporting their deductions . there is a clear need for instruction at the undergraduate level in the use of bioinformatics tools , but few good resources are available . our module will remedy this situation by offering a cohesive package that integrates several bioinformatic resources in a way that is accessible to undergraduate students . this module brings together information gathered from brain anatomy and bioinformatics websites , including the mouse brain library ( rosen et al . , 2000 ) , genenetwork s webqtl ( wang et al . , 2003 ) , ucsc genome browser ( kent et al . , 2002 ) , the allen brain atlas ( lein et al . , 2007 ) , the national center for biotechnology information s entrez gene , and ncbi s pubmed . in this module , students quantify a phenotype using nih image ( see references for url ) to analyze digital images that are downloaded from the mouse brain library ( see references for url)figure 5 . the mouse brain library provides digitized images of sectioned and stained mouse brains from individuals of different recombinant inbred strains and allows the quantification of many brain phenotypes . we quantify olfactory bulb size since it is relatively easy to operationally define , and there is a published study with which to compare the data ( williams et al . , 2001 ) . after students clean - up their morphometric data by statistically controlling for variables , they enter their data into a web - based qtl analysis ( genenetwork s webqtl see references for url ) . qtl analysis relates variations in phenotype to loci on chromosomes where genes impacting the phenotype can be found ( figure 6 ) . genenetwork s webqtl provides a direct link to the university of california , santa cruz genome browser ( url in references ) , which provides a list of the genes that are in that portion of the chromosome ( see figure 7 ) . students can search through this list and find a gene that is highly expressed in the olfactory bulb from the data provided by the ucsc genome browser . once students identify a gene that is highly expressed , they also can link to the allen brain atlas ( url in references ) , which displays the expression pattern of that specific gene via in situ hybridization views along with an atlas ( c.f . thus , students can discover the cell layers where their gene of interest is expressed ( figure 8) . the ucsc genome browser also provides links to the national center for biotechnology information resources such as entrez gene and pubmed ( urls in references ) . these resources allow the students to discover more information about their highly expressed gene including its nucleotide and amino acid sequence , as well as find articles about their gene that provide a deeper intellectual involvement in this exercise . our website has already been populated with some of these materials http://mdcune.psych.ucla.edu/. a detailed instructor s manual should be available in summer 2009 . 1988 ) and dramatic brain sex differences ( nottebohm and arnold , 1976 ) were first described in this system , and it is still the focus of ongoing investigations on the neural basis of learning and memory ( nordeen and nordeen , 1988 ; troyer and doupe , 2000 ) , as well as on the genetic and hormonal bases of sexual differentiation in the nervous system ( cf . agate et al . , 2003 ; grisham and arnold , 1995 ) . the rich literature base on the song system , the robust difference between the sexes in the size of song nuclei ( grisham and arnold , 1995 ) , and the dramatic masculinizing effect of hormones on this system s development ( grisham and arnold , 1995 ) make the zebra finch song system ideal for use in undergraduate laboratories nevertheless , studying this system requires significant investments : supporting a bird colony , purchasing good quality microscopes , microtomes , histological supplies for processing brains , and digital microscope cameras . such extensive facilities and equipment requirements make studying the bird song system out of reach for many institutions . we are surmounting these obstacles by providing a collection of images for undergraduate students to replicate a published experiment that examined the doses of hormone required to masculinize the song system ( grisham et al . further , students can extend this study -- we are adding images from untreated males , so that students can explore whether or not any of the treatments fully sex reverse the female song system at this point in time , the digital images are being collected , and we anticipate that they will be fully available in summer 2010 . so far , our experience with students using a prototype library of digital images has proved to be highly successful ( see figure 10 ) . anecdotally , the digital images are better at focusing students on the relevant aspects of the task relative to using tissue on slides . when we tried using the actual tissue , one student looked for the telencephalic song nuclei in the lower brainstem . digital labs are highly successful teaching tools . we have used swimmy for several terms , and students have invariably responded positively . we obtained data from a cohort of students that had experienced both in vivo electrophysiology labs as well as our digital lab , swimmy . students experiencing both conditions rated the virtual lab experience as having some definite advantages ( see figure 11 ) . students show gains in their knowledge of neuroscience and their ability to reason their way through problems based in neuroscience ( cf . we have also taught with the neuroinformatics module for several terms , and this has similarly proved to be a valuable tool for student learning . in addition to introducing them to bioinformatics , this module provides instruction in genetics , statistical analyses , neuroanatomy , and molecular techniques . notably , one of our students reported that the qtl module helped her choose and understand the statistical analyses for her senior thesis . the bird song module has been extensively piloted with undergraduates , and has been successful every time . further , students benefit not only in learning the facts about hormonal influences on sexual differentiation and comparative neuroanatomy , they also learn about statistical analyses , particularly anova and post - hoc comparisons . in our experience , digital labs can provide valuable educational experiences , but they do offer a different educational experience than traditional wet labs . digital labs typically sidestep procedure learning so students will not necessarily learn manual skills or technical problem solving required for bench science . instead , digital labs focus more on data collection , analysis and synthesis . digital labs restrict the types of procedural errors that can occur , so students lose the opportunity of learning from such mistakes . on the other hand , students sometimes make such disastrous errors in wet labs that their results are uninterpretable , and digital labs prevent such outcomes . also , mistakes are more reversible in the digital realm than in wet labs , which we find reduces student anxiety . neither wet labs nor our digital labs always produce the predicted data , which encourages an opportunity for valuable didactic discussion . nonetheless , our digital labs are likely to yield replicable and interpretable results that allow students to relate their own data to broader concepts discussed in the literature . having run both wet and digital labs , we also appreciate other strengths of digital labs . in wet labs , students often had to take turns waiting to share equipment , animals , etc . in contrast , digital labs both allow and demand that each and every student be engaged in all aspects of the process and through all of the instructional time . in addition , digital labs reduce the use of animals in teaching , thus side - stepping potentially thorny ethical objections some students may have . perhaps the greatest advantage of digital labs is that they can be adapted for and adopted in a variety of learning environments / communities such as a traditional classroom , in a lab setting , in blended instruction , or even distance learning . although these labs may be of most interest to institutional settings where traditional wet labs are not possible due to limitations on resources , they have utility in other contexts as well . we have augmented and amplified students laboratory experiences by using digital labs along with wet labs in our courses . digital labs also allow easy preservation of teaching materials and tools and easy access to these resources for other faculty . we are pleased to offer these labs to faculty and students at other institutions for free via our website : http://mdcune.psych.ucla.edu/.

we are providing free digital resources for teaching neuroscience labs at http://mdcune.psych.ucla.edu/. these resources will ultimately include materials for teaching laboratories in electrophysiology of neuronal circuits ( swimmy ) , a neuroinformatics / bioinformatics module , and two modules for investigating the effects of hormones on early cns development one focusing on the development of the song system and one focusing on sex differences in spinal cord motor neurons . all of these modules are inquiry based students gain from genuine experiences in doing actual studies rather than just simulations . these materials should provide instructors the ability to provide good quality laboratory experiences regardless of resource limitations . currently , modules on sex differences in the spinal cord and virtual neural circuits ( swimmy ) are available on our website . more will be available in summer 2009 and 2010 . swimmy was demonstrated at the faculty for undergraduate neuroscience ( fun ) workshop the undergraduate neuroscience education : interactions , interdisciplines , and curricular best practices at macalester college in july 2008 .

Prototype Project: RatSCIA Modular Digital Course in Undergraduate Neuroscience Education SWIMMY: Circuit Neurophysiology and Analyses of Virtual Central Pattern Generators Bioinformatics: QTL Analysis of Mouse Olfactory Bulb Morphology Bird Song System: Developmental Effects of Hormones STUDENT EXPERIENCES CONCLUSION

PMC4771196

fifty - four swiss webster mice aged 1 to 24 weeks ( both sexes ) were purchased from charles river laboratories ( wilmington , ma , usa ) and housed at the neuroscience center of excellence , louisiana state university health ( lsuh ; new orleans , la , usa ) . the animals were handled in compliance with the guidelines of the arvo statement for the use of animals in ophthalmic and vision research , and the experimental protocol was approved by the institutional animal care and use committee at lsuh . rabbit monoclonal antiiii - tubulin ( tuji1 , mrb-435p-100 ) antibody was purchased from covance antibody services , inc . ( berkeley , ca , usa ) ; rat monoclonal anti - substance p ( sp ) was purchased from millipore ( temecula , ca , usa ) ; and guinea pig polyclonal anti - cgrp was purchased from pierce antibody products ( thermo fisher scientific , inc . , rockford , il , usa ) . secondary antibodies alexafluor 488 goat anti - rabbit igg ( h+l ) , alexafluor 594 goat anti - guinea pig igg ( h+l ) , and alexafluor 594 goat anti - rat igg ( h+l ) were purchased from invitrogen ( carlsbad , ca , usa ) . mice were anesthetized by an intraperitoneal injection of 0.1 ml sodium pentobarbital ( 10 mg / ml ) . the position of the cornea was marked by three stitches at 3 , 9 , and 12 o'clock points with a nylon 10 - 0 monofilament ( ethilon ; ethicon , inc . , the mice were euthanized and the eyeballs were enucleated and fixed in freshly prepared 2% paraformaldehyde for 15 minutes . then the corneas were carefully excised along the sclerocorneal rim and fixed for an additional 45 minutes , followed by three washes with 0.1 m pbs containing 0.1% bovine serum albumin ( pbs - bsa ) . to block nonspecific binding , corneas were placed in a 96-well plate ( one cornea / well ) and then incubated with 10% normal goat serum plus the tissue was then incubated with rabbit monoclonal antiiii - tubulin antibody ( 1:1000 ) for 72 hours at 4c . after washing with pbs - bsa ( 3 10 minutes ) , the corneas were incubated with the corresponding secondary antibodies for 24 hours at 4c and then washed thoroughly with pbs - bsa . for double immunofluorescence , after labeling with the first set of antibodies ( iii - tubulin and corresponding secondary antibodies ) was completed , the tissue was incubated with a second primary antibody ( cgrp or sp ) for 72 hours and followed by the corresponding fluorescein isothiocyanate ( fitc)- or tetramethylrhodamine ( tritc)-conjugated secondary antibody and washings , as described above . four radial cuts were performed on each cornea , and the tissue was flatly mounted on a slide with the endothelium side up . images were acquired in time - lapse mode using a fluorescence microscope ( nikon eclipse te200 ; nikon corp . , tokyo , japan ) equipped with a digital camera ( coolsnap hq ; photometrics , tucson , az , usa ) using imaging software ( metavue ; molecular devices , sunnyvale , ca , usa ) . 1 ) were taken with a fluorescent microscope ( olympus ix71 ; olympus corp . , the images at the same layer recorded from one cornea were merged together to build an entire view of the corneal nerve network of both the epithelium and stroma . to obtain a transected view of the corneal nerves , the same cornea used above was embedded in optimal cutting temperature ( oct ) , and serial 15-m cryostat sections were cut and photographed with the same microscope . images in ( a h ) show representative whole mounts of entire corneal innervation from 32 mice aged 1 to 24 weeks ( 4 mice / time point ) . highlighted images in ( a h ) are close - ups showing the detailed structures of corneal nerves in the central area as framed in ( a h ) . corneas were labeled with anti-iii - tubulin antibody and images taken with a fluorescent microscope ( olympus corp . ) and with a 10 objective lens . to test the origin of the sensory neuropeptides cgrp and sp , mice were euthanized , the crania opened , and both left and right tg removed and processed as previously described . briefly , after fixing and washing the tissue , the whole tg were embedded in oct compound and serial 10-m cryostat sections were cut , dried at room temperature for 2 hours , and stored at 20c until use . for double immunofluorescence , the sections of the tg were washed , blocked , and permeabilized as already described and then incubated with primary antibodies against iii - tubulin ( 1:1000 ) plus cgrp ( 1:500 ) , iii - tubulin plus sp ( 1:500 ) , or cgrp plus sp in 0.1 m pbs containing 1.5% normal goat serum overnight at 4c . after washing with pbs - bsa ( 3 5 minutes ) , the sections were incubated with corresponding fitc- or tritc - conjugated secondary antibodies for 1 hour at room temperature . to exclude nonspecific labeling , the primary antibodies were replaced by serum igg of the same host species as the primary antibody . in controls without primary antibodies , there was no staining ( data not shown ) . the adult mouse corneas ( 22 mice ) have a radius of approximately 1.5 mm . to calculate the subbasal epithelial nerve densities , we divided the mouse cornea into central and peripheral zones . the central zone was defined by a radius of 0.5 mm starting at the apex , and the peripheral zone with a radius of 0.5 mm beginning at the limbus . to avoid overlap , approximately 0.5 mm of space between the two zones was left uncounted . to get a better contrast , the fluorescent images were changed to grayscale mode and placed against a white background using imaging software ( photoshop ; adobe systems , inc . the subbasal nerve fibers in each image were carefully drawn with 4-pixel lines following the course of each fiber by using the brush tool in the imaging software ( adobe systems , inc . ) . the nerve area and the total area of the image were obtained by using the histogram tool . the percentage of total nerve area was quantified for each image as described previously . to compare nerve densities in the central and peripheral areas , eight images for each zone were randomly chosen from each cornea ( two images / quadrant ) . a total of 80 images for each zone from 10 corneas of 10 mice ( 5 mice / sex ) were averaged . nerve terminals in the superficial epithelia within the central and peripheral zones were calculated by directly counting the number of terminals in each image . the terminal numbers in each image were counted directly . since each image comprised an area of 0.335 mm , the terminal numbers per square millimeter were calculated . to examine the relative content of neuropeptides in the subbasal nerves , 12 corneas that had been stained with anti-iii - tubulin were double - stained with cgrp or sp . for each neuropeptide , a total of 24 whole - mount images from the central zone ( one image / quadrant ) were taken , and then the same numbers of images were taken for iii - tubulin . in the same visual field , the percentage of iii - tubulin equaled that of the total nerve area , and the ratio of the peptide - positive nerve area against iii - tubulin represented the relative content . to calculate cgrp- and sp - positive neurons in the tg , 20 images were selected randomly from 10 mice ( 1 section / ganglion ) and counted in a blind fashion . differences in central and peripheral corneal nerve densities , terminal numbers , and the relative content of neuropeptides in the central cornea and tg were expressed as means sem and t - test was performed ; p < 0.05 was considered a statistically significant difference between two groups . fifty - four swiss webster mice aged 1 to 24 weeks ( both sexes ) were purchased from charles river laboratories ( wilmington , ma , usa ) and housed at the neuroscience center of excellence , louisiana state university health ( lsuh ; new orleans , la , usa ) . the animals were handled in compliance with the guidelines of the arvo statement for the use of animals in ophthalmic and vision research , and the experimental protocol was approved by the institutional animal care and use committee at lsuh . rabbit monoclonal antiiii - tubulin ( tuji1 , mrb-435p-100 ) antibody was purchased from covance antibody services , inc . ( berkeley , ca , usa ) ; rat monoclonal anti - substance p ( sp ) was purchased from millipore ( temecula , ca , usa ) ; and guinea pig polyclonal anti - cgrp was purchased from pierce antibody products ( thermo fisher scientific , inc . , rockford , il , usa ) . secondary antibodies alexafluor 488 goat anti - rabbit igg ( h+l ) , alexafluor 594 goat anti - guinea pig igg ( h+l ) , and alexafluor 594 goat anti - rat igg ( h+l ) were purchased from invitrogen ( carlsbad , ca , usa ) . mice were anesthetized by an intraperitoneal injection of 0.1 ml sodium pentobarbital ( 10 mg / ml ) . the position of the cornea was marked by three stitches at 3 , 9 , and 12 o'clock points with a nylon 10 - 0 monofilament ( ethilon ; ethicon , inc . , somerville , nj , usa ) on the sclerocorneal rim . the mice were euthanized and the eyeballs were enucleated and fixed in freshly prepared 2% paraformaldehyde for 15 minutes . then the corneas were carefully excised along the sclerocorneal rim and fixed for an additional 45 minutes , followed by three washes with 0.1 m pbs containing 0.1% bovine serum albumin ( pbs - bsa ) . to block nonspecific binding , corneas were placed in a 96-well plate ( one cornea / well ) and then incubated with 10% normal goat serum plus 0.3% triton x-100 solution in pbs - bsa for 60 minutes at room temperature . the tissue was then incubated with rabbit monoclonal antiiii - tubulin antibody ( 1:1000 ) for 72 hours at 4c . after washing with pbs - bsa ( 3 10 minutes ) , the corneas were incubated with the corresponding secondary antibodies for 24 hours at 4c and then washed thoroughly with pbs - bsa . for double immunofluorescence , after labeling with the first set of antibodies ( iii - tubulin and corresponding secondary antibodies ) was completed , the tissue was incubated with a second primary antibody ( cgrp or sp ) for 72 hours and followed by the corresponding fluorescein isothiocyanate ( fitc)- or tetramethylrhodamine ( tritc)-conjugated secondary antibody and washings , as described above . four radial cuts were performed on each cornea , and the tissue was flatly mounted on a slide with the endothelium side up . images were acquired in time - lapse mode using a fluorescence microscope ( nikon eclipse te200 ; nikon corp . , tokyo , japan ) equipped with a digital camera ( coolsnap hq ; photometrics , tucson , az , usa ) using imaging software ( metavue ; molecular devices , sunnyvale , ca , usa ) . 1 ) were taken with a fluorescent microscope ( olympus ix71 ; olympus corp . , the images at the same layer recorded from one cornea were merged together to build an entire view of the corneal nerve network of both the epithelium and stroma . to obtain a transected view of the corneal nerves , the same cornea used above was embedded in optimal cutting temperature ( oct ) , and serial 15-m cryostat sections were cut and photographed with the same microscope . images in ( a h ) show representative whole mounts of entire corneal innervation from 32 mice aged 1 to 24 weeks ( 4 mice / time point ) . highlighted images in ( a h ) are close - ups showing the detailed structures of corneal nerves in the central area as framed in ( a h ) . corneas were labeled with anti-iii - tubulin antibody and images taken with a fluorescent microscope ( olympus corp . ) and with a 10 objective lens . to test the origin of the sensory neuropeptides cgrp and sp , mice were euthanized , the crania opened , and both left and right tg removed and processed as previously described . briefly , after fixing and washing the tissue , the whole tg were embedded in oct compound and serial 10-m cryostat sections were cut , dried at room temperature for 2 hours , and stored at 20c until use . for double immunofluorescence , the sections of the tg were washed , blocked , and permeabilized as already described and then incubated with primary antibodies against iii - tubulin ( 1:1000 ) plus cgrp ( 1:500 ) , iii - tubulin plus sp ( 1:500 ) , or cgrp plus sp in 0.1 m pbs containing 1.5% normal goat serum overnight at 4c . after washing with pbs - bsa ( 3 5 minutes ) , the sections were incubated with corresponding fitc- or tritc - conjugated secondary antibodies for 1 hour at room temperature . to exclude nonspecific labeling , the primary antibodies were replaced by serum igg of the same host species as the primary antibody . in controls without primary antibodies , there was no staining ( data not shown ) . the adult mouse corneas ( 22 mice ) have a radius of approximately 1.5 mm . to calculate the subbasal epithelial nerve densities , we divided the mouse cornea into central and peripheral zones . the central zone was defined by a radius of 0.5 mm starting at the apex , and the peripheral zone with a radius of 0.5 mm beginning at the limbus . to avoid overlap , approximately 0.5 mm of space between the two zones was left uncounted . to get a better contrast , the fluorescent images were changed to grayscale mode and placed against a white background using imaging software ( photoshop ; adobe systems , inc . the subbasal nerve fibers in each image were carefully drawn with 4-pixel lines following the course of each fiber by using the brush tool in the imaging software ( adobe systems , inc . ) . the nerve area and the total area of the image the percentage of total nerve area was quantified for each image as described previously . to compare nerve densities in the central and peripheral areas , eight images for each zone were randomly chosen from each cornea ( two images / quadrant ) . a total of 80 images for each zone from 10 corneas of 10 mice ( 5 mice / sex ) were averaged . nerve terminals in the superficial epithelia within the central and peripheral zones were calculated by directly counting the number of terminals in each image . the terminal numbers in each image were counted directly . since each image comprised an area of 0.335 mm , the terminal numbers per square millimeter were calculated . to examine the relative content of neuropeptides in the subbasal nerves , 12 corneas that had been stained with anti-iii - tubulin for each neuropeptide , a total of 24 whole - mount images from the central zone ( one image / quadrant ) were taken , and then the same numbers of images were taken for iii - tubulin . in the same visual field , the percentage of iii - tubulin equaled that of the total nerve area , and the ratio of the peptide - positive nerve area against iii - tubulin represented the relative content . to calculate cgrp- and sp - positive neurons in the tg , 20 images were selected randomly from 10 mice ( 1 section / ganglion ) and counted in a blind fashion . differences in central and peripheral corneal nerve densities , terminal numbers , and the relative content of neuropeptides in the central cornea and tg were expressed as means sem and t - test was performed ; p < 0.05 was considered a statistically significant difference between two groups . when comparing the subbasal epithelial nerve densities in corneas of adult male and female mice ( n = 10 corneas / sex ) , no differences in epithelial nerve densities were found between sexes either in the central or in the peripheral epithelial nerves ( data not shown ) . therefore , the data presented represent a combination of both sexes . for this study , a total of 32 mice were used , with four mice for each time point . figure 1 contains representative images of corneal stroma and epithelial nerves from mice 1 to 24 weeks after birth . images were recorded with a 10 objective lens focusing on the subbasal layer . in the very young mice ( aged between 1 and 3 weeks ) , the more prominent nerves were the thick stromal nerves , which connected with each other , constituting a dense stromal nerve network . there was little change in the size of the stromal nerve bundles between the periphery and center . subbasal nerves originating from the branches of stromal nerves were few , short and thin , as can be seen in the images ( figs . whorl - like structures started forming at 4 weeks . from age 4 to 6 weeks , the density and branches of the corneal stromal nerves in the central cornea were gradually reduced ( compare figs . the stromal nerves looked like a tree ; their trunks around the limbal area were thicker , but their branches became gradually thinner from the corneal periphery to the center , in which a few stromal nerve branches were visible . on the contrary , the subbasal nerves , which mainly originated from the peripheral stromal nerves , were much longer and thicker . from 8 to 24 weeks , the vortex was well defined ( figs . the stromal nerves originate from four major nerve trunks , which pass through the attachment points of extraocular muscles and run from the episclera to the corneoscleral limbus , where they divide into several branches . a reconstructed image in figure 2a shows the fiber course of a medial ( nasal ) nerve trunk in an 8-week - old mouse . some of the stromal branches connected with each other to constitute a dense stromal nerve network around the limbal area , but most of the branches run forward and divide further into many subbranches . in the peripheral zone , these branches run upward to the subbasal layer to give place to subbasal bundles . figure 2b shows the entire whole - mount view of stromal nerves recorded from two mouse eyes . there was an average of 21.7 0.8 ( n = 20 eyes , means sem ) branches per eye , and based on qualitative observations , the density of the stromal nerves was higher in the periphery than in the center . mouse corneal nerves originate from four major nerve trunks that run through the attachments of extraocular muscles to the limbus , where they divide repeatedly into many branches . the branches connect with each other and constitute a stromal nerve network . in adult mice ( 8 weeks ) , the density of stromal nerves is higher in the periphery than that in the center . ( a ) reconstructed image showing the nerve course of a nasal major nerve trunk . ( b ) reconstructed whole - mount images showing the entire view of the stromal nerve architectures recorded from two eyes of a mouse . corneas were labeled with antiiii - tubulin antibody and images were recorded with a 10 objective lens , focusing on the subbasal layer . figure 3a shows the whole mount of the entire subbasal nerves recorded from the same mouse as in figure 2b . the epithelial nerves emerge mainly from the tips of the stromal nerve branches in the periphery and form the nerve bundles in the subbasal layer . long bundles run from the periphery and converge into the central area to form the whorl - like structure or vortex . these subbasal bundles project numerous divisions that connect to each other to constitute the subbasal nerve plexus . fine terminals or free endings derivate from the plexus and innervate the epithelial cells ( fig . 3b ) . highlighted images in figure 3c show the detailed nerve architecture of stromal nerves , subbasal bundles , and nerve terminals at the vortex area . the epithelial nerves derive from the stromal nerve branches that penetrate to the subbasal layer and give place to the subbasal bundles . in adult mouse corneas ( 8 weeks ) , the penetrating sites were located mainly in the peripheral zone , and it was generally found that one penetrating site gives origin to several nerve bundles . those bundles run centripetally and converge into the central area to form the whorl - like structure or vortex . these main bundles divided into branches , which connect to the bundles to form the subbasal nerve network . ( a , b ) whole - mount images of the entire architecture ; ( a ) focuses on the corneal subbasal nerves and ( b ) focuses on the superficial free endings recorded from the same mouse shown in figure 2b . ( c ) highlighted images , as framed in ( a ) , show the detailed nerve architectures in the vortex area . it is noteworthy that in the same mouse , the left eye has two vortexes that go in opposite directions . one interesting finding was that the patterns and numbers of whorl - like structures varied between corneas of 8-week - old mice ( fig . 3a ) . among the 60 corneas analyzed , 44 ( 73.3% ) showed one whorl - like structure per cornea , and 28 of those eyes had a clockwise pattern , as shown in figure 3a ( right eye ) , while the other 16 eyes had a counterclockwise pattern ( fig . eleven eyes ( 18.3% ) showed two whorl - like structures , as represented in figure 3c ( left eye , arrows ) ; in two eyes ( 3.3% ) , we observed three whorl - like structures ( fig . 4 ) . three other eyes ( 5% ) did not present these structures , but instead contained long bundles running across the central area and merging in the inferior - nasal corneoscleral area . furthermore , among the 30 mice analyzed , 18 mice showed one whorl - like structure in both eyes ; seven mice showed one whorl - like structure in one eye and two such structures in another eye . one mouse showed two vortexes in both eyes , and two mice showed one cornea with two whorl - like structures and three vortexes in the other cornea . whole - mount view of entire subbasal nerves in a cornea with three vortexes labeled with iii - tubulin antibody . the highlighted image shows the details of the three vortexes in which vortexes a and c go clockwise , while vortex b goes counterclockwise . the montage of the corneal sagittal sections labeled with iii - tubulin antibody showed the cross - sectional view of the whole corneal innervation ( fig . 5 ) . highlighted images from the center and periphery depict the detailed distribution of nerve terminals in the epithelia , revealing that the density of epithelial terminals was higher in the center than in the periphery . entire cross - sectional view of mouse corneal innervation labeled with iii - tubulin antibody . highlighted images show the detailed distribution of epithelial terminals at the center and periphery . a close - up image counterstained with dapi shows the location of subbasal bundles ( red arrow ) and the shape of epithelial terminals ( white arrows ) . the density of subbasal nerves and nerve terminals in the mouse cornea were recorded from 10 corneas , as explained in the methods section . the subbasal nerve density , calculated as the percentage of total area , was 28.2% 0.3% in the central area and 18.1% 0.4% in the peripheral area ( figs . similarly , nerve terminals , calculated from 48 images ( 24 images per zone ) of 6 eyes as the average number of terminals / mm were also greater in the center ( 3972 58 ) than in the periphery ( 1976 99 , p < 0.001 , figs . 6c , 6d ) . difference of corneal subbasal nerve density and nerve terminals between the central and the peripheral zones . ( a ) nerve density in mouse corneas was calculated as the percentage of the total area in each image . a total of 160 images ( 80 images / zone ) recorded with a 20 objective lens from 10 corneas ( male / female = 5/5 corneas ) were used . ( c ) number of epithelial nerve terminals ; 48 images ( 24 images / zone ) from 6 corneas were used . ( d ) representative images of nerve terminals recorded from the central and peripheral zones of the same cornea . whole corneas labeled with iii - tubulin were double stained with antibodies against cgrp or sp . representative images of mouse subbasal nerve bundles in the vortex area , terminals , and stromal trunks are shown in figures 7a and 7b . ( a , b ) representative images showing the expression of cgrp- and sp - positive nerves in the central subbasal nerve bundles , terminals , and limbal stromal trunks . ( c ) percentage calculated as ratios of cgrp- or sp - positive nerves versus total nerve area ( iii - tubulin nerves ) in each image . a total of 24 images for each neuropeptide and the same number of images for iii - tubulin were recorded from six corneas . cgrp - positive nerve fibers constituted 70.2% 2.4% of the total subbasal nerve content , while sp - positive nerves were 58.6% 1.4% ( n = 6 eyes ; p < 0.0001 ) . the percentage of cgrp - positive terminals was also higher ( 62% 1% ) than the percentage of sp - positive terminals ( 59.3% 0.8% ; p < 0.05 ) . similarly , in the main stromal trunks recorded within the limbus , cgrp - positive nerve bundles were more abundant ( 57% 1.2% ) than the sp - positive nerve bundles 51.2% 1.6% , ( n = 5 eyes ; p < 0.05 ) . to investigate the origin and relative contents of the sensory neuropeptides cgrp and sp in neurons of the tg , cross - sections were double stained with iii - tubulin . as shown in figure 8a , 31.9% 1.2% of the total neurons were cgrp positive while 26.2% 0.9% were sp - positive neurons . 8d ) demonstrate that all sp - positive neurons were also cgrp - positive , while there were some cgrp - positive neurons that were negative for sp . ( a ) relative density of cgrp- or sp - positive neurons . for each neuropeptide , data represent the average ratios ( mean sem ) of cgrp- or sp - positive cells versus iii - tubulin neurons . ( b , c ) representative images . ( d ) colocalization of cgrp- and sp - positive neurons . when comparing the subbasal epithelial nerve densities in corneas of adult male and female mice ( n = 10 corneas / sex ) , no differences in epithelial nerve densities were found between sexes either in the central or in the peripheral epithelial nerves ( data not shown ) . therefore , the data presented represent a combination of both sexes . for this study , a total of 32 mice were used , with four mice for each time point . figure 1 contains representative images of corneal stroma and epithelial nerves from mice 1 to 24 weeks after birth . images were recorded with a 10 objective lens focusing on the subbasal layer . in the very young mice ( aged between 1 and 3 weeks ) , the more prominent nerves were the thick stromal nerves , which connected with each other , constituting a dense stromal nerve network . there was little change in the size of the stromal nerve bundles between the periphery and center . subbasal nerves originating from the branches of stromal nerves were few , short and thin , as can be seen in the images ( figs . whorl - like structures started forming at 4 weeks . from age 4 to 6 weeks , the density and branches of the corneal stromal nerves in the central cornea were gradually reduced ( compare figs . the stromal nerves looked like a tree ; their trunks around the limbal area were thicker , but their branches became gradually thinner from the corneal periphery to the center , in which a few stromal nerve branches were visible . on the contrary , the subbasal nerves , which mainly originated from the peripheral stromal nerves , were much longer and thicker . from 8 to 24 weeks , the vortex was well defined ( figs . the stromal nerves originate from four major nerve trunks , which pass through the attachment points of extraocular muscles and run from the episclera to the corneoscleral limbus , where they divide into several branches . a reconstructed image in figure 2a shows the fiber course of a medial ( nasal ) nerve trunk in an 8-week - old mouse . some of the stromal branches connected with each other to constitute a dense stromal nerve network around the limbal area , but most of the branches run forward and divide further into many subbranches . in the peripheral zone , these branches run upward to the subbasal layer to give place to subbasal bundles . figure 2b shows the entire whole - mount view of stromal nerves recorded from two mouse eyes . there was an average of 21.7 0.8 ( n = 20 eyes , means sem ) branches per eye , and based on qualitative observations , the density of the stromal nerves was higher in the periphery than in the center . the origin and distribution of corneal stromal nerves . mouse corneal nerves originate from four major nerve trunks that run through the attachments of extraocular muscles to the limbus , where they divide repeatedly into many branches . the branches connect with each other and constitute a stromal nerve network . in adult mice ( 8 weeks ) , the density of stromal nerves is higher in the periphery than that in the center . ( a ) reconstructed image showing the nerve course of a nasal major nerve trunk . ( b ) reconstructed whole - mount images showing the entire view of the stromal nerve architectures recorded from two eyes of a mouse . corneas were labeled with antiiii - tubulin antibody and images were recorded with a 10 objective lens , focusing on the subbasal layer . figure 3a shows the whole mount of the entire subbasal nerves recorded from the same mouse as in figure 2b . the epithelial nerves emerge mainly from the tips of the stromal nerve branches in the periphery and form the nerve bundles in the subbasal layer . long bundles run from the periphery and converge into the central area to form the whorl - like structure or vortex . these subbasal bundles project numerous divisions that connect to each other to constitute the subbasal nerve plexus . fine terminals or free endings derivate from the plexus and innervate the epithelial cells ( fig . 3b ) . highlighted images in figure 3c show the detailed nerve architecture of stromal nerves , subbasal bundles , and nerve terminals at the vortex area . the epithelial nerves derive from the stromal nerve branches that penetrate to the subbasal layer and give place to the subbasal bundles . in adult mouse corneas ( 8 weeks ) , the penetrating sites were located mainly in the peripheral zone , and it was generally found that one penetrating site gives origin to several nerve bundles . those bundles run centripetally and converge into the central area to form the whorl - like structure or vortex . these main bundles divided into branches , which connect to the bundles to form the subbasal nerve network . ( a , b ) whole - mount images of the entire architecture ; ( a ) focuses on the corneal subbasal nerves and ( b ) focuses on the superficial free endings recorded from the same mouse shown in figure 2b . ( c ) highlighted images , as framed in ( a ) , show the detailed nerve architectures in the vortex area . it is noteworthy that in the same mouse , the left eye has two vortexes that go in opposite directions . one interesting finding was that the patterns and numbers of whorl - like structures varied between corneas of 8-week - old mice ( fig . 3a ) . among the 60 corneas analyzed , 44 ( 73.3% ) showed one whorl - like structure per cornea , and 28 of those eyes had a clockwise pattern , as shown in figure 3a ( right eye ) , while the other 16 eyes had a counterclockwise pattern ( fig . eleven eyes ( 18.3% ) showed two whorl - like structures , as represented in figure 3c ( left eye , arrows ) ; in two eyes ( 3.3% ) , we observed three whorl - like structures ( fig . 4 ) . three other eyes ( 5% ) did not present these structures , but instead contained long bundles running across the central area and merging in the inferior - nasal corneoscleral area . furthermore , among the 30 mice analyzed , 18 mice showed one whorl - like structure in both eyes ; seven mice showed one whorl - like structure in one eye and two such structures in another eye . one mouse showed two vortexes in both eyes , and two mice showed one cornea with two whorl - like structures and three vortexes in the other cornea . whole - mount view of entire subbasal nerves in a cornea with three vortexes labeled with iii - tubulin antibody . the highlighted image shows the details of the three vortexes in which vortexes a and c go clockwise , while vortex b goes counterclockwise . the montage of the corneal sagittal sections labeled with iii - tubulin antibody showed the cross - sectional view of the whole corneal innervation ( fig . 5 ) . highlighted images from the center and periphery depict the detailed distribution of nerve terminals in the epithelia , revealing that the density of epithelial terminals was higher in the center than in the periphery . entire cross - sectional view of mouse corneal innervation labeled with iii - tubulin antibody . highlighted images show the detailed distribution of epithelial terminals at the center and periphery . a close - up image counterstained with dapi shows the location of subbasal bundles ( red arrow ) and the shape of epithelial terminals ( white arrows ) . the density of subbasal nerves and nerve terminals in the mouse cornea were recorded from 10 corneas , as explained in the methods section . the subbasal nerve density , calculated as the percentage of total area , was 28.2% 0.3% in the central area and 18.1% 0.4% in the peripheral area ( figs . similarly , nerve terminals , calculated from 48 images ( 24 images per zone ) of 6 eyes as the average number of terminals / mm were also greater in the center ( 3972 58 ) than in the periphery ( 1976 99 , p < 0.001 , figs . 6c , 6d ) . difference of corneal subbasal nerve density and nerve terminals between the central and the peripheral zones . ( a ) nerve density in mouse corneas was calculated as the percentage of the total area in each image . a total of 160 images ( 80 images / zone ) recorded with a 20 objective lens from 10 corneas ( male / female = 5/5 corneas ) were used . ( c ) number of epithelial nerve terminals ; 48 images ( 24 images / zone ) from 6 corneas were used . ( d ) representative images of nerve terminals recorded from the central and peripheral zones of the same cornea . the stromal nerves originate from four major nerve trunks , which pass through the attachment points of extraocular muscles and run from the episclera to the corneoscleral limbus , where they divide into several branches . a reconstructed image in figure 2a shows the fiber course of a medial ( nasal ) nerve trunk in an 8-week - old mouse . some of the stromal branches connected with each other to constitute a dense stromal nerve network around the limbal area , but most of the branches run forward and divide further into many subbranches . in the peripheral zone , these branches run upward to the subbasal layer to give place to subbasal bundles . figure 2b shows the entire whole - mount view of stromal nerves recorded from two mouse eyes . there was an average of 21.7 0.8 ( n = 20 eyes , means sem ) branches per eye , and based on qualitative observations , the density of the stromal nerves was higher in the periphery than in the center . the origin and distribution of corneal stromal nerves . mouse corneal nerves originate from four major nerve trunks that run through the attachments of extraocular muscles to the limbus , where they divide repeatedly into many branches . the branches connect with each other and constitute a stromal nerve network . in adult mice ( 8 weeks ) , the density of stromal nerves is higher in the periphery than that in the center . ( a ) reconstructed image showing the nerve course of a nasal major nerve trunk . ( b ) reconstructed whole - mount images showing the entire view of the stromal nerve architectures recorded from two eyes of a mouse . corneas were labeled with antiiii - tubulin antibody and images were recorded with a 10 objective lens , focusing on the subbasal layer . figure 3a shows the whole mount of the entire subbasal nerves recorded from the same mouse as in figure 2b . the epithelial nerves emerge mainly from the tips of the stromal nerve branches in the periphery and form the nerve bundles in the subbasal layer . long bundles run from the periphery and converge into the central area to form the whorl - like structure or vortex . these subbasal bundles project numerous divisions that connect to each other to constitute the subbasal nerve plexus . fine terminals or free endings derivate from the plexus and innervate the epithelial cells ( fig . 3b ) . highlighted images in figure 3c show the detailed nerve architecture of stromal nerves , subbasal bundles , and nerve terminals at the vortex area . the epithelial nerves derive from the stromal nerve branches that penetrate to the subbasal layer and give place to the subbasal bundles . in adult mouse corneas ( 8 weeks ) , the penetrating sites were located mainly in the peripheral zone , and it was generally found that one penetrating site gives origin to several nerve bundles . those bundles run centripetally and converge into the central area to form the whorl - like structure or vortex . these main bundles divided into branches , which connect to the bundles to form the subbasal nerve network . ( a , b ) whole - mount images of the entire architecture ; ( a ) focuses on the corneal subbasal nerves and ( b ) focuses on the superficial free endings recorded from the same mouse shown in figure 2b . ( c ) highlighted images , as framed in ( a ) , show the detailed nerve architectures in the vortex area . it is noteworthy that in the same mouse , the left eye has two vortexes that go in opposite directions . one interesting finding was that the patterns and numbers of whorl - like structures varied between corneas of 8-week - old mice ( fig . 3a ) . among the 60 corneas analyzed , 44 ( 73.3% ) showed one whorl - like structure per cornea , and 28 of those eyes had a clockwise pattern , as shown in figure 3a ( right eye ) , while the other 16 eyes had a counterclockwise pattern ( fig . eleven eyes ( 18.3% ) showed two whorl - like structures , as represented in figure 3c ( left eye , arrows ) ; in two eyes ( 3.3% ) , we observed three whorl - like structures ( fig . did not present these structures , but instead contained long bundles running across the central area and merging in the inferior - nasal corneoscleral area . furthermore , among the 30 mice analyzed , 18 mice showed one whorl - like structure in both eyes ; seven mice showed one whorl - like structure in one eye and two such structures in another eye . one mouse showed two vortexes in both eyes , and two mice showed one cornea with two whorl - like structures and three vortexes in the other cornea . whole - mount view of entire subbasal nerves in a cornea with three vortexes labeled with iii - tubulin antibody . the highlighted image shows the details of the three vortexes in which vortexes a and c go clockwise , while vortex b goes counterclockwise . the montage of the corneal sagittal sections labeled with iii - tubulin antibody showed the cross - sectional view of the whole corneal innervation ( fig . 5 ) . highlighted images from the center and periphery depict the detailed distribution of nerve terminals in the epithelia , revealing that the density of epithelial terminals was higher in the center than in the periphery . entire cross - sectional view of mouse corneal innervation labeled with iii - tubulin antibody . highlighted images show the detailed distribution of epithelial terminals at the center and periphery . a close - up image counterstained with dapi shows the location of subbasal bundles ( red arrow ) and the shape of epithelial terminals ( white arrows ) . the density of subbasal nerves and nerve terminals in the mouse cornea were recorded from 10 corneas , as explained in the methods section . the subbasal nerve density , calculated as the percentage of total area , was 28.2% 0.3% in the central area and 18.1% 0.4% in the peripheral area ( figs . similarly , nerve terminals , calculated from 48 images ( 24 images per zone ) of 6 eyes as the average number of terminals / mm were also greater in the center ( 3972 58 ) than in the periphery ( 1976 99 , p < 0.001 , figs . 6c , 6d ) . difference of corneal subbasal nerve density and nerve terminals between the central and the peripheral zones . ( a ) nerve density in mouse corneas was calculated as the percentage of the total area in each image . a total of 160 images ( 80 images / zone ) recorded with a 20 objective lens from 10 corneas ( male / female = 5/5 corneas ) were used . ( c ) number of epithelial nerve terminals ; 48 images ( 24 images / zone ) from 6 corneas were used . ( d ) representative images of nerve terminals recorded from the central and peripheral zones of the same cornea . whole corneas labeled with iii - tubulin were double stained with antibodies against cgrp or sp . representative images of mouse subbasal nerve bundles in the vortex area , terminals , and stromal trunks are shown in figures 7a and 7b . ( a , b ) representative images showing the expression of cgrp- and sp - positive nerves in the central subbasal nerve bundles , terminals , and limbal stromal trunks . ( c ) percentage calculated as ratios of cgrp- or sp - positive nerves versus total nerve area ( iii - tubulin nerves ) in each image . a total of 24 images for each neuropeptide and the same number of images for iii - tubulin were recorded from six corneas . cgrp - positive nerve fibers constituted 70.2% 2.4% of the total subbasal nerve content , while sp - positive nerves were 58.6% 1.4% ( n = 6 eyes ; p < 0.0001 ) . the percentage of cgrp - positive terminals was also higher ( 62% 1% ) than the percentage of sp - positive terminals ( 59.3% 0.8% ; p < 0.05 ) . similarly , in the main stromal trunks recorded within the limbus , cgrp - positive nerve bundles were more abundant ( 57% 1.2% ) than the sp - positive nerve bundles 51.2% 1.6% , ( n = 5 eyes ; p < 0.05 ) . to investigate the origin and relative contents of the sensory neuropeptides cgrp and sp in neurons of the tg , cross - sections were double stained with iii - tubulin . as shown in figure 8a , 31.9% 1.2% of the total neurons were cgrp positive while 26.2% 0.9% were sp - positive neurons . ( n = 10 mice , p < 0.005 ) . images in figures 8b and 8c show neurons labeled with each neuropeptide . 8d ) demonstrate that all sp - positive neurons were also cgrp - positive , while there were some cgrp - positive neurons that were negative for sp . ( a ) relative density of cgrp- or sp - positive neurons . for each neuropeptide data represent the average ratios ( mean sem ) of cgrp- or sp - positive cells versus iii - tubulin neurons . ( b , c ) representative images . ( d ) colocalization of cgrp- and sp - positive neurons . we used a modified technique of immunofluorescence and imaging to show , for the first time , the entire mouse corneal nerve architecture including the nerve terminals , subbasal nerve bundles , and stromal nerve trunks . we found that in very young mice ( 13 weeks after birth ) , the cornea present a dense network of stromal nerves but that the epithelial nerve fibers that budded from the network are short , thin , and extend without a given direction . previous work using c57/b6 mice of 10 days and 4 weeks had shown a similar pattern in the central cornea . those nerve fibers gradually grow with the maturity of the mice , and a well - defined whorl - like structure appears at 4 weeks . from 4 to 6 weeks , the cornea size increases with aging . as we measured in this study , the radius increases approximately 2.6 times from newborn mice ( average : 0.58 mm ) to adult mice ( average : 1.5 mm ) . therefore , it is possible that a correlation exists between the length of the subbasal nerve bundles and the changes in corneal size as associated with the age of the mouse . at 8 weeks , the corneal nerves reach maturity with stromal nerves originating from four major trunks and fewer stromal nerves in the central cornea . one possibility is that there is a decreased release of growth factors from stromal keratocytes . early studies suggested there is cross - talk between the nerves and corneal resident cells . during postnatal eye development , corneal epithelial and stromal cells secrete growth factors such as nerve growth factor , ciliary neurotrophic factor , glial cell line derived neurotrophic factor , vascular endothelial growth factor , and pigment epithelium derived factor that may influence nerve fiber extension and survival . very young mouse corneas have a high density of keratocytes that decrease by age 12 days and change from active to quiescent keratocytes in normal adult corneas . this low metabolism may be unable to produce enough growth factors to support the survival of the dense stromal nerves in the central cornea observed at a young age . the mouse is considered to be an adult at 8 weeks ; however , in our experiments no noticeable changes in the nerve architecture were found up to 24 weeks . these results are in agreement with a recent study using in vivo confocal microscopy reporting that mouse subbasal nerve density is constant from age 8 to 52 weeks . the epithelial nerve bundles derived from the peripheral stromal branches ran centripetally and converged to form the whorl - like structure or vortex at the central cornea . the density of stromal nerves are lower in the center than in the periphery , while the density of epithelial nerves including the subbasal nerves and free endings are significantly higher in the center than in the periphery . however , there are some features in mouse corneal innervation that we have not found in humans . in human corneas , although the patterns and locations of the vortex differ among the samples , every cornea analyzed has only one vortex ; in mice , we found that 18% of the corneas have more than one whorl - like structure per cornea . early studies have postulated that the combined effect of the electric and magnetic fields on centripetally migrating epithelial cells lead to a clockwise converged pattern , and it is likely that similar effects are exerted on corneal subbasal nerves . however , this theory can not explain the phenomenon of counterclockwise pattern and the finding that some corneas show two vortexes running in opposing directions . another difference found was that mature mice corneas have higher epithelial nerve density than humans . compared with human corneas aged 40 to 57 years , there was a 28.2% 2.54% nerve density in the mouse central area versus an 18.8% 2.1% in humans , and an 18.1% 3.2% vs. 11.1% 2.3% in the peripheral area . coincidently , the number of terminals / mm of corneal epithelia was also greater in mice ( center , 3972 284 ; periphery , 1976 487 ) than in humans ( center , 525 72 ; periphery , 230 46 ) . although the reasons for this difference are unknown , we agree with yu and rosenblatt that a higher density of epithelial innervation may be needed by either anatomic or functional requirements specific to the mouse . sensory nerves originating from the tg innervate ocular tissues , including the cornea and iris , and loss of corneal sensory innervation can result in morphologic and metabolic epithelial disturbance . the expression of the sensory neuropeptides cgrp and sp in the corneas has been studied previously by both histochemical and immunofluorescence methods in a wide range of animal species , but their distribution in the entire tissue and their relative content in mouse corneas was not investigated . in the current study , we used double - labeling immunofluorescence to study the relative contents of these neuropeptides . our results showed that the corneas contain a large number of cgrp- and sp - positive nerve fibers . these two main sensory neuropeptides have been shown to induce epithelial cell proliferation , migration , and adhesion , facilitating corneal wound healing . they are involved in the regulation of tear production and mucus secretion from goblet cells and participate in the irritative and allergic response of the ocular surface . comparison of the proportions of cgrp- with sp - positive fibers shows that the content of cgrp is higher than that of sp in both epithelial and stromal innervation . this result is in agreement with our previous findings in the rabbit model in which the proportion of cgrp is significantly higher than that of sp - positive fibers in the corneal and iris innervations . an early study in the canine model has reported that both cgrp- and sp - positive nerve fibers take up 99% of the total corneal innervation . this result is much higher than those we have found in the rabbit and mouse corneas . the discrepancies may be due to the difference between the animal species , rather than the techniques of assessment used in the studies . the sensory nerves , which innervate the cornea and iris , mainly originate from the ophthalmic division of the trigeminal nerve , with a very small amount deriving from the superior cervical ganglion and ciliary ganglion . in a recent study , we have reported that in the rabbit tg , the number of cgrp - positive neurons significantly outnumber sp - positive neurons , which are also labeled with cgrp . our results are also in agreement with earlier studies in the rat and guinea pig , suggesting that those species share a similar expression pattern of sensory neuropeptides in the tg . in summary , using a modified technique of immunofluorescence and imaging , we provided a complete map of the entire nerve architecture and the total sensory neuropeptide distribution of the mouse cornea . the finding that the mouse corneal innervation has many similarities to human cornea makes the mouse an appropriate model to study pathologies in which corneal nerves are involved .

purposeto investigate the entire nerve architecture and content of the two main sensory neuropeptides in mouse cornea to determine if it is a good model with similarities to human corneal innervation.methodsmice aged 1 to 24 weeks were used . the corneas were stained with neuronal - class iii - tubulin , calcitonin gene related peptide ( cgrp ) , and substance p ( sp ) antibodies ; whole - mount images were acquired to build an entire view of corneal innervation . to test the origin of cgrp and sp , trigeminal ganglia ( tg ) were processed for immunofluorescence . relative corneal nerve fiber densities or neuron numbers were assessed by computer - assisted analysis.resultsbetween 1 and 3 weeks after birth , mouse cornea was mainly composed of a stromal nerve network . at 4 weeks , a whorl - like structure ( or vortex ) appeared that gradually became more defined . by 8 weeks , anatomy of corneal nerves had reached maturity . epithelial bundles converged into the central area to form the vortex . the number and pattern of whorl - like structures were different . subbasal nerve density and nerve terminals were greater in the center than the periphery . nerve fibers and terminals that were cgrp - positive were more abundant than sp - positive nerves and terminals . in trigeminal ganglia , the number of cgrp - positive neurons significantly outnumbered those positive for sp.conclusionsthis is the first study to show a complete map of the entire corneal nerves and cgrp and sp sensory neuropeptide distribution in the mouse cornea . this finding shows mouse corneal innervation has many similarities to human cornea and makes the mouse an appropriate model to study pathologies involving corneal nerves .

Methods Animals Antibodies Immunofluorescence Staining and Imaging Data Analysis Results Development of Mouse Corneal Innervation After Birth Corneal Nerve Architecture of Adult Mice Stromal Nerves. Epithelial Nerves. CGRP and SP Content of Mice Corneal Nerves CGRP- and SP-Positive Neurons in the Trigeminal Ganglion Discussion

PMC3563239

the process can be divided into four stages : hemostasis , inflammation , proliferation ( the formation of granulation , contraction , and reepithelialization ) , and remodeling . in hemostasis , as the blood components enter the site of injury , the platelets release essential growth factors and cytokines such as platelet - derived growth factor ( pdgf ) and transforming growth factor beta ( tgf- ) . in the inflammatory phase , neutrophils enter the wound and begin the critical task of phagocytosis to remove foreign materials , bacteria , and damaged tissue . macrophages appear and continue the process of phagocytosis as well as releasing more pdgf and tgf-. once the wound site is cleaned out , fibroblasts migrate in to begin the proliferative phase and deposit new extracellular matrix . some fibroblasts may correspond to myofibroblasts , which are distributed along the wound edge and wound bed and cooperate in wound contraction . thereafter , myofibroblasts increase in number , and collagen fibers are produced as well as myofibroblasts . the new collagen matrix then becomes cross - linked and organized during the final remodeling phase . many studies on honey produced in countries besides japan have been conducted , for example , indonesia ( indonesian honey ) , turkey ( chestnut honey , pure rhododendron honey , and pure blossom honey ) , malaysia ( gelam honey and tualang honey ) [ 58 ] , iran ( urmia honey ) , pakistan ( acacia honey ) , and nigeria ( jungle honey ) . in developing countries , honey has been used as a treatment for various wounds , while it is unfamiliar in japan . honey is reported to have a debriding effect [ 12 , 13 ] ; however , its mechanism of debriding action has not yet been explained , and it decreases infection because of its antibacterial activity : high osmotic effect , acidity , hydrogen peroxide , and phytochemical factors . in addition , it also decreases inflammation [ 5 , 16 ] and wound area [ 3 , 5 , 6 ] . the anti - inflammatory action of honey decreases edema and the high osmotic pressure of honey dehydrates tissue edema . wound area reduction in the inflammatory phase results from anti - inflammatory properties [ 9 , 12 , 14 ] and antibacterial activity by the hydrogen peroxidase in honey [ 12 , 18 ] . it also has a ph from 3 to 4 , and topical acidification causes oxygen release from hemoglobin ; in addition , the hydrogen peroxide contained at low levels in honey also stimulates angiogenesis and the growth of fibroblasts . honey enhances wound contraction by stimulating fibroblasts , myofibroblasts , and collagen deposition by providing a source of energy , namely , sugar [ 3 , 7 , 19 ] . acceleration of reepithelialization results from its high osmotic pressure , which dehydrates tissue edema and holds the wound edges together , and by the presence of hydrogen peroxide , which stimulates the growth of epithelial cells . honey is a natural product and its characteristics associated with wound healing may be affected by the species of bee , geographical location , and botanical origin , as well as processing and storage conditions . in general , pure commercial unheated honey is composed of approximately 40% glucose , 40% fructose , 20% water , amino acids , the vitamin biotin , aminonicotinic acid , folic acid , pantothenic acid , pyridoxine , thiamine , the enzymes diastase invertase , glucose oxidase , and catalase , and the minerals calcium , iron , magnesium , phosphorus and potassium ; honey also contains bee pollen enzymes and propolis , all of which can stimulate new tissue growth ; it may also contain other medicinal compounds , including essential oils , flavonoids , terpenes , and polyphenols , depending on the plant from which the pollen was taken [ 22 , 23 ] . on the other hand , concerning the three types of honey in this study , which are produced in japan and familiar to the japanese , acacia honey is composed of 70.8% glucose and fructose and 18.6% water , buckwheat flour honey is composed of 71.2% glucose and fructose and 17.2% water , and chinese milk vetch honey is composed of 71.0% glucose and fructose and 18.4% water ( yamada bee farm , okayama , japan ) . although more detailed information on their compositions is not known , since they contain a lot of sugar and water like honey produced in other countries , they seem to have the same effects on wound healing . however , to our knowledge , in japan , there have been no studies evaluating the use of japanese honey as a topical therapy in wound care both macroscopically and microscopically , so it is very important to clarify the effect of japanese honey on wound healing . if we identify that japanese types of honey have the same or better effects than hydrocolloid dressing or honey from other countries , we can use japanese honey as an alternative dressing for wound care ; such treatment using honey will enable cost reductions for both patients and institutions . against this background , we hypothesize that japanese honey also promotes wound healing as well as honey from other countries ; that is , it decreases inflammation and wound area , increases reepithelialization , contraction , and deposition of collagen , and promotes overall wound healing . therefore , the aim of this study is to clarify the effects of japanese types of honey on the wound healing process . seventy - two balb / ccrslc male mice aged 8 weeks ( sankyo lab service corporation , inc . , toyama , japan ) and weighing 21.326.0 g were used . they were caged individually in an air - conditioned room a t 25.0 2.0c with light from 08:45 to 20:45 hours . the experimental protocol and animal care were in accordance with the guidelines for the care and use of laboratory animals of kanazawa university , japan ( ap-112200 ) . three types of honey were used : acacia ( robinia pseudoacacia ) , buckwheat flour ( fagopyrum esculentum ) honey , and chinese milk vetch ( astragalus sinicus ) honey(yamada bee farm , okayama , japan ) . in accordance with previous studies [ 3 , 24 , 25 ] , the mice were anesthetized with an intraperitoneal ( ip ) injection of pentobarbital sodium ( 0.05 mg / g weight ) , and the dorsum was shaved . two circular ( 4 mm in diameter ) full - thickness skin wounds including the panniculus muscle on both sides of the dorsum of the mouse were made with a kai sterile disposable biopsy punch ( kai industries , gifu , japan ) . we chose two circular wounds on each mouse because this method decreases the number of mice required , as shown in our previous studies . wounds of the experimental groups , acacia honey , buckwheat flour honey , and chinese milk vetch honey groups , were treated with 0.1 ml of honey per wound . the wounds to which honey was applied were covered with gauze to prevent the honey from running off , and the mice were wrapped twice with a sticky bandage ( mesh pore tape ; nichiban , tokyo , japan ) . meanwhile , wounds of the control group were covered with hydrocolloid dressing ( tegaderm ; 3 m health care , tokyo , japan ) to maintain a moist environment . all control mice were wrapped twice with sticky bandages , the same as the experimental groups . the day when wounds were made was designated as day 0 , and the process of wound healing was observed from day 0 to 14 after wounding . the traces on the sheets were captured with a scanner onto a personal computer using adobe photoshop elements 7.0 ( adobe system inc . , tokyo , japan ) , and the areas of wounds were calculated using image analysis software scion image beta 4.02 ( scion corporation , frederick , maryland , usa ) . the mice were euthanized by a massive pentobarbital sodium ( 0.5 mg / g weight ) ip injection on days 3 , 7 , 11 , and 14 after wounding . the wounds and the surrounding intact skin were harvested , stapled onto transparent plastic sheets to prevent overcontraction of specimens , and fixed in 4% paraformaldehyde in 0.2 mol / l phosphate buffer ( ph 7.4 ) for 15 hours . specimens were dehydrated in an alcohol series , cleaned in xylene , and embedded in paraffin to prepare 5 m serial sections . sections of 5 m thickness were stained with hematoxylin - eosin ( h - e ) or subjected to azan staining and immunohistologically stained with anti - neutrophil antibody ( abcam japan , tokyo , japan ) for detecting neutrophils , anti - mouse mac-3 antibody ( bd pharmingen , tokyo , japan ) for detecting macrophages , or anti--smooth muscle actin ( -sma ) antibody , prediluted ( abcam kk , tokyo , japan ) , for detecting myofibroblasts . after deparaffinization and rehydration , antigen unmasking was accomplished by heating slides in a water bath followed by incubation in sodium citrate buffer ( 10 mm sodium citrate , 0.05% tween 20 , ph 6.0 ) for 20 minutes at approximately 100c . slides for anti - mouse mac-3 antibody and anti--sma were washed with phosphate - buffered saline ( pbs ) , and slides for anti - neutrophil antibody were washed with 0.3% triton x-100 in pbs . then , slides were incubated with anti - neutrophil antibody or mac-3 antibody at a concentration of 1 : 100 in pbs or anti--sma at 4c overnight . slides were again washed with pbs or 0.3% triton x-100 in pbs . for detection of primary antibodies , slides for anti - mouse mac-3 antibody and anti - neutrophil antibody were incubated with polyclonal rabbit anti - rat immunoglobulins / hrp ( dako north america , california , usa ) at a concentration of 1 : 300 in 0.3% mouse serum ( normal ) ( dako north america , california , usa ) in pbs for 30 minutes at 4c , and slides for anti--sma antibody were incubated with dako envision+ system - hrp labeled polymer anti - rabbit ( ready to use ) ( dako north america , california , usa ) for 30 minutes at room temperature . slides were again washed with pbs or 0.3% triton x-100 in pbs and then incubated in dako liquid dab+ substrate chromogen system ( dako north america , california , usa ) ( brown chromogen ) for 5 minutes or until staining was detected at room temperature . finally , slides were rinsed in distilled water , dehydrated , cleared , and mounted for analysis . we measured the ratio of reepithelialization ( % ) = length of new epithelium / length of wound between wound edges , counted the number of neutrophils , macrophages , myofibroblasts , and blood vessels by observation through a light microscope with 400x magnification , and then calculated the ratio of each parameter / mm granulation tissue . the ratio of collagen fibers in granulation tissue = number of pixels of collagen fibers / number of pixels of granulation tissue area using adobe photoshop element 7.0 . data are expressed as mean sd , analyzed using jmp 8.0.1 ( sas , usa ) ( anova , multiple comparison tukey - kramer ) . when we treated each wound with honey every day , honey remained on the wound surfaces , wounds were moist , and each gauze with honey covering wounds was easily removed . applied honey was viscid the next day , which could have been due to its absorption of exudate from the wound , and some of the honey leaked from the bandage covering the wound . on the other hand , hydrocolloid dressing absorbed so much of the exudate that it expanded , so the exudate did not spread out from the hydrocolloid . on days 3 to 5 after wounding , necrotic tissue clearly appeared on the surfaces of wound areas in the japanese honey groups , and it covered the entirety of wounds on day 7 , while necrotic tissue was not observed on wounds in the hydrocolloid dressing group ( figure 1 ) . the wound in the hydrocolloid dressing group was clearly covered with the deposition of exudate until day 5 or 6 . the exudate of the hydrocolloid dressing group decreased around day 7 , while that of the japanese honey groups was difficult to observe because honey , like exudate , is liquid ( figure 1 ) . since a lot of exudate was absorbed by honey and hydrocolloid dressing , it was unclear whether edema was present in the wound or the peripheral area of the wound in all groups . signs of infection were not observed in any wounds . on days 1 to 14 , the ratios of wound areas to the initial wound area on day 0 were calculated ( figure 2 and table 2 ) . in the hydrocolloid dressing group , the wound area increased gradually during the inflammatory and early proliferative phases , peaked on day 6 , then more rapidly decreased during the proliferative phase , and wounds healed with scarring on day 14 , being smaller in area than on day 0 ( p < 0.0001 ) . in the acacia honey group , the wound area decreased gradually until day 3 during the inflammatory phase , being smaller in area than on day 0 ( p = 0.0089 ) , increased gradually until day 7 during the proliferative phase , decreased again gradually until day 10 during the proliferative phase , increased gradually until day 13 , and then decreased on day 14 ( day 0 versus day 14 , p = 0.0400 ) during the remodeling phase . in the buckwheat flour honey group , the wound area decreased on day 1 , remained almost the same area until day 3 during the inflammatory phase , increased gradually until day 7 during the proliferative phase , being greater than the area on day 0 , decreased gradually until day 12 , and again increased until day 14 during the remodeling phase , being almost the same in terms of area as on day 0 ( p = 1.0000 ) . in the chinese milk vetch honey group , the wound area decreased until day 3 during the inflammatory phase , being smaller in area than on day 0 ( p = 0.0002 ) , increased gradually until day 6 during the proliferative phase , and then decreased gradually until day 14 during the late proliferative and remodeling phases , being smaller in area than on day 0 ( p = 0.0000 ) . on day 14 , when the wound of the hydrocolloid dressing group healed with scarring , there were no significant differences of wound area between the hydrocolloid dressing and the acacia and chinese milk vetch honey groups and between the acacia and chinese milk vetch honey groups , while there were significant differences between the buckwheat flour honey and chinese milk vetch honey groups ( p = 0.0019 ) and the buckwheat flour honey and hydrocolloid dressing groups ( p = 0.0031 ) and a trend between the acacia and buckwheat flour honey groups ( p = 0.0818 ) . the wounds of the acacia and chinese milk vetch honey groups seemed to almost heal with red soft scarring like granulation tissue on day 14 , and those of the buckwheat flour honey group did not seem to heal with red large granulation tissue on day 14 ( figure 1 ) . necrotic tissue covered almost all wound surfaces on days 3 and 7 in the japanese honey groups as determined by macroscopic observation . on day 3 after wounding , the ratio of reepithelialization covering wound surface was almost the same between all groups . thereafter , in only the hydrocolloid dressing group , new epithelium extended rapidly and covered about 77% of the wound surface until day 7 and then covered almost all of the wound surface on day 14 . on the other hand , the ratio of new epithelium in the japanese honey groups was much lower than that in the hydrocolloid dressing group on day 14 ( p < 0.0001 ) . the necrotic tissue covering the wound in the japanese honey groups seemed to prevent new blood vessels . the number of new blood vessels per mm in the wound in the japanese honey groups increased rapidly from day 3 to day 7 ( each p < 0.0001 ) and then decreased gradually . in the hydrocolloid dressing group , it peaked on day 7 and then decreased rapidly to day 14 . the numbers of blood vessels in the japanese honey groups were larger than in the hydrocolloid dressing group on days 7 , 11 , and 14 ( always p < 0.05 ) . since so many capillaries were observed in the granulation tissue in the japanese honey groups on day 14 , wounds did not seem to be scarring . the number of myofibroblasts in the japanese honey groups increased gradually from day 0 to day 14 , while that in the hydrocolloid dressing group peaked on day 11 and after that decreased drastically on day 14 . on day 7 , the number of myofibroblasts in the hydrocolloid dressing group was larger than that of the buckwheat flour honey and chinese milk vetch honey groups ( p = 0.0005 , p < 0.0001 , resp . ) , and the number of myofibroblasts in the acacia honey group was larger than those of the buckwheat flour honey and chinese milk vetch honey groups ( p = 0.0217 , 0.0032 , resp . ) . the ratio of collagen fibers stained with azan stain in the wound increased gradually from day 3 to 14 in all groups . on day 7 , the ratio of collagen fibers in the granulation tissue in the hydrocolloid dressing group was larger than those of the acacia honey and chinese milk vetch honey groups ( p = 0.026 , p = 0.005 , resp . ) . on day 14 , there was no significant difference between all groups , although the ratio of collagen fibers in the hydrocolloid dressing group seemed to be larger than in the japanese honey groups . numerous macrophages were observed in the wound on day 3 . on days 3 and 7 during the inflammatory and early proliferative phases , the number of macrophages per mm in the wound in the hydrocolloid dressing group was significantly larger than those in the japanese honey groups ( p < 0.0001 ) . the number of macrophages in the hydrocolloid dressing group remained almost the same on days 3 and 7 , and decreased gradually from day 7 to day 14 ( p = 0.0091 ) . however , those of the japanese honey groups until day 14 remained almost the same as on day 3 ( acacia : p = 0.6665 , buckwheat flour : p = 0.9736 , and chinese milk vetch : p = 0.1317 ) . numerous neutrophils appeared in the wound on day 3 at the inflammatory - phase , like macrophages . there were no significant differences in the number of neutrophils per mm in the wound between all groups on days 3 and 7 ; however , the buckwheat flour honey group tended to have fewer neutrophils than the hydrocolloid dressing group ( p = 0.0551 ) on day 3 . on day 11 , the buckweat flour honey group had a larger number than the hydrocolloid dressing group ( p = 0.0233 ) , and the buckweat flour honey group tended to have a large number than the hydrocolloid dressing group ( p = 0.0697 ) . on day 14 , the number of neutrophils in the chinese milk vetch honey group was larger than that of the hydrocolloid dressing group ( p = 0.0478 ) . the number of neutrophils in the hydrocolloid dressing group decreased rapidly from day 3 to day 14 ( p = 0.0008 ) , while the number of neutrophils in all japanese honey groups until day 14 remained almost the same , from day 3 to day 14 ( acacia : p = 0.9635 , buckwheat flour : p = 0.9668 , chinese milk vetch : p = 0.9444 ) . table 8 shows the differences between various types of honey in previous studies and japanese honey in the present study . this shows that japanese honey has some different effects from honey from other countries . the wound areas treated with the acacia and chinese milk vetch honey were almost the same as that of hydrocolloid dressing on day 14 , so the japanese honey may have an effect on wound healing , although the effect of buckwheat honey on the wound healing was not clear . however , it is very difficult to explain clearly the phenomenon that the wound area treated with japanese honey decreases during the inflammatory phase , increases during the proliferative phase , and then decreases during the remodeling phase . this may be due to the following phenomena observed in this study in the wounds treated with japanese honey : the small number of macrophages that produce factors for wound healing ; the delay of production of myofibroblasts that contract the wound ; the retention of numerous neutrophils in the proliferation and remodeling phases , which appear at the inflammatory phase and thereafter decrease rapidly ; the small amount of deposition of collagen fibers in granulation tissue ; the presence of numerous new blood vessels in granulation tissue on days 11 and 14 , which appear in large quantities at granulation tissue and decrease rapidly late in the proliferation and remodeling phase [ 3 , 4 ] . there are thus large differences between japanese honey and that from other countries ( table 8) . there is a need to clarify the reason for the differences between japanese honey and that from other countries ; both of which are composed of mainly water and sugar , as well as trace amounts of unknown , unique compounds that differ among each type of honey . in the present study , wound areas treated with japanese honey did not increase during the inflammatory phase , in comparison with hydrocolloid dressing , as well as in our previous study using indonesian and manuka honeys . the effect of honey on the contraction of wound area or protection against enlargement of wound area has been reported in other studies [ 4 , 10 , 21 ] . thus , contraction of wound area or suppression of the expansion of a wound during the inflammation phase is a very important effect of honey . it is likely that an increase of wound area in the inflammatory phase depends on the load for stretching , which pulls the wound edge by the breaking of collagen fibers , and the accumulation of exudate in the wound . although hydrocolloid dressing is suitable to absorb wound exudate , which is produced at a high level during the inflammatory phase , the wound area covered with the hydrocolloid dressing increased ; thus , it may not have such a good effect on inhibiting the production of exudate and the load for stretching of a wound . on the other hand , partly because honey has a high osmotic pressure produced by a high concentration of sugar , honey absorbs exudate like hydrocolloid dressing . in addition , partly because of anti - inflammatory action , which has been clarified by some reports [ 9 , 12 , 14 ] and recently by hussein et al . , which inhibits the production of exudate , and partly because the viscosity of honey covering the wound may keep the wound edge together by resisting the stretching of collagen fibers , honey may contract the wound area or suppress expansion of the wound during the inflammatory phase . the retention of necrotic tissue on the wound surface for a long time and the prevention of extension of new epithelium on the wound surface were observed during the wound healing process in the japanese honey groups . the former may have been due to the defect of debriding effect in japanese honey , which is reported for honey from other countries [ 13 , 20 ] . the latter may be due to the existence of necrotic tissue , which may physically prevent the migration of new epithelial cells , and the low number of macrophages at inflammatory and early proliferative phases , which secrete epidermal growth factor ( egf ) that generates epidermis [ 28 , 29 ] . these results indicate that the process of wound healing by japanese honey was very different from that by honey from other countries . therefore , we propose a combination treatment in which japanese honey is applied only in the inflammatory phase , followed by hydrocolloid dressing from the proliferative phase , which produces effective contraction for wound healing . we will conduct a study on this issue next , and examine whether such treatment is effective to promote contraction and healing with less scar formation . it was clarified that the process of wound healing by japanese honey was very different from that by honey from other countries : there was specific wound area transition that decreased the wound area initially , then increased , and decreased it without complete reepithelialization . therefore , it is suggested that japanese types of honey should be applied only in the inflammatory phase to reduce wound area and should then be exchanged for hydrocolloid dressing from the proliferative phase to promote the formation of granulation tissue and collagen fibers .

although many previous studies reported that honey promotes wound healing , no study has examined the effects of japanese honey . the aim of this study was to investigate the effects of three types of japanese honey , acacia , buckwheat flour , and chinese milk vetch honey , on wound healing in comparison with hydrocolloid dressing . circular full - thickness skin wounds were produced on male mice . japanese honey or hydrocolloid dressing was applied daily to the mice for 14 days . the ratio of wound area for the hydrocolloid dressing group increased initially in the inflammatory and early proliferative phases and then decreased rapidly to heal with scarring . however , the ratios of wound area for the japanese honey groups decreased in the inflammatory phase , increased in the proliferative phase , and decreased in the proliferative phase , and some wounds were not completely covered with new epithelium . these findings indicate that using japanese honey alone has limited benefit , but since it reduces wound size in the inflammatory phase , it is possible to apply a combined treatment in which japanese honey is applied only in the inflammatory phase , followed by hydrocolloid dressing from the proliferative phase , which would effectively contract the wound .

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

PMC3249984

trichomonas vaginalis , a flagellated protozoan parasite , is the causative agent of trichomoniasis , which is the commonest treatable sexually transmitted infection in industrialized countries . in women , trichomoniasis has a range of presentations , from asymptomatic infection to an acute inflammatory disease with a copious and malodorous vaginal discharge . as expected , the clinic - based studies report a higher prevalence than community - based studies . hemachandra reported a prevalence of 1% in a community - based study in the sabaragamuwa province , while herath reported a community prevalence of 0.6% in the colombo district . more recently , banneheke et al . have demonstrated a prevalence of 4.2% of trichomoniasis in women attending a sexually transmitted disease clinic . diagnosis of trichomoniasis is usually made by microscopic examination of a saline wet smear preparation of vaginal secretions . a positive wet smear is diagnostic because of its high specificity , whereas a negative test can not exclude trichomoniasis because of the low sensitivity of 3080%.[911 ] use of stained vaginal smears with giemsa stain to diagnose t. vaginalis infection has been compared to other diagnostic methods.[1214 ] in these studies , the vaginal swabs were smeared on the slides , air dried and fixed with methanol prior to staining with giemsa . the percentage of t. vaginalis infections detected by using giemsa stain has always been lower as compared to culture ( 19% vs 22% reported by ojuromi et al . , 21.7% vs. 29.8% reported by mason et al . , 5.5% vs. 10.5% by radonjic et al . the gold standard for the diagnosis of trichomoniasis is culture , which has a high sensitivity of 71100% . unfortunately , culture requires an incubator with a constant electricity supply , relatively expensive culture media , and an experienced microscopist . furthermore , it can take up to 57 days for results to be obtained ; these issues limit its use in resource - limited settings . this study compares the diagnostic yield of trichomoniasis by culture , wet smear examination , and giemsa stain . it also assesses the feasibility of employing the giemsa - stained smears ( after preparing the sample with a modified technique ) as a routine diagnostic tool in resource - limited settings . a clinic - based prospective study was carried out at the national std / aids control programme ( nsacp ) over a period of 18 months from october 2007 to april 2009 . this included symptomatic patients and asymptomatic patients who presented to the clinic to rule out a sexually transmitted disease . chronically ill women and those who did not give consent for speculum examination were excluded from the study . ethics approval for the study was obtained from the ethical review committee of the faculty of medicine , university of colombo . after obtaining consent , serology for syphilis and human immunodeficiency virus ( hiv ) , and vaginal / cervical samples to detect common reproductive tract infections in addition to these samples , at the time of collecting vaginal secretions , two extra swabs were obtained from the posterior fornix for diagnosis of trichomoniasis . the first swab was used to prepare a saline smear for examination at the nsacp clinic . the second swab was stored in a graduated test tube containing 5 ml of sterile physiological saline with three drops of 5% glucose solution at room temperature . the third swab was inoculated into a glass tube containing 5 ml of culture media . the second and third swabs were transferred to the department of parasitology , faculty of medicine , colombo within 68 h of collection . the three laboratory tests , namely , examination of a wet smear of vaginal secretions , examination of a vaginal smear stained with giemsa , and culture were carried out by three independent , trained personnel . the wet saline smear was examined immediately by an experienced microscopist as a part of the routine diagnostic procedure carried out at the nsacp . culture and the giemsa staining were carried out at the department of parasitology , faculty of medicine , colombo . culture for t. vaginalis was carried out using trichomonas medium ( oxoid code ; cmo 161b enriched with horse serum and incorporated with chloramphenicol ) based on manufacturer 's instructions . the tube containing the culture media was incubated at 37c for 3 days . afterwards , for the next 7 days , a fresh smear was prepared every 24 h from the medium taken from the bottom of the tube and examined microscopically for the presence of motile flagellates . the trichomonas medium used for culture in this study was a medium based on that of feinberg and whittington for the detection of t. vaginalis and candida species . currently , the trichomonas medium has been slightly modified by the incorporation of 0.1% w / v of agar which leads to reduced oxygen tension and consequently more prolific growth of trichomonads . the diagnostic yield for trichomoniasis varies depending on the type of culture medium used . a systemic review has shown that the sensitivity for diamond medium ( 11 studies ) varied from 88 to 99% while the same value for oxoid medium was 7689% . however , the oxoid medium was used in only three studies that were assessed in this review . diamond culture medium was not used in our study due to limited financial and laboratory resources . a modified technique was adopted to ensure efficiency in transport and preparation of specimens than the standard technique . instead of preparing vaginal smears and air drying it , this method was adopted from the procedure of preservation of samples for fine needle aspiration cytology where samples are not air dried but preserved in saline . prior to staining with giemsa , the tubes with the swabs were vigorously shaken to displace all the parasites into the saline solution . the swabs were then removed and the test tubes were centrifuged at a low speed ( 5001000 rpm ) for 30 min . the methanol supernatant was then removed leaving a thin layer of methanol as a liquid phase . the deposit which contains the parasites and the remaining layer of methanol was mixed thoroughly . one drop of this mixture was put on a clean dry slide and three such slides were prepared from a single sample . the slides were air dried and then incubated at 37c for 12 h. the slides were stained with giemsa diluted 1:9 with phosphate buffer solution ( ph 7.2 ) for 7 min . prevalence was calculated to reflect the relative frequency of trichomoniasis with the corresponding 95% confidence intervals ( ci ) . the variable measures were the numbers of true positives ( tp ) , true negatives ( tn ) , false positives ( fp ) , and false negatives ( fn ) . a clinic - based prospective study was carried out at the national std / aids control programme ( nsacp ) over a period of 18 months from october 2007 to april 2009 . this included symptomatic patients and asymptomatic patients who presented to the clinic to rule out a sexually transmitted disease . chronically ill women and those who did not give consent for speculum examination were excluded from the study . ethics approval for the study was obtained from the ethical review committee of the faculty of medicine , university of colombo . after obtaining consent , serology for syphilis and human immunodeficiency virus ( hiv ) , and vaginal / cervical samples to detect common reproductive tract infections are routinely collected in almost all clinic attendees at nsacp . in addition to these samples , at the time of collecting vaginal secretions , two extra swabs were obtained from the posterior fornix for diagnosis of trichomoniasis . the first swab was used to prepare a saline smear for examination at the nsacp clinic . the second swab was stored in a graduated test tube containing 5 ml of sterile physiological saline with three drops of 5% glucose solution at room temperature . the third swab was inoculated into a glass tube containing 5 ml of culture media . the second and third swabs were transferred to the department of parasitology , faculty of medicine , colombo within 68 h of collection . the three laboratory tests , namely , examination of a wet smear of vaginal secretions , examination of a vaginal smear stained with giemsa , and culture were carried out by three independent , trained personnel . the wet saline smear was examined immediately by an experienced microscopist as a part of the routine diagnostic procedure carried out at the nsacp . culture and the giemsa staining were carried out at the department of parasitology , faculty of medicine , colombo . culture for t. vaginalis was carried out using trichomonas medium ( oxoid code ; cmo 161b enriched with horse serum and incorporated with chloramphenicol ) based on manufacturer 's instructions . the tube containing the culture media was incubated at 37c for 3 days . afterwards , for the next 7 days , a fresh smear was prepared every 24 h from the medium taken from the bottom of the tube and examined microscopically for the presence of motile flagellates . the trichomonas medium used for culture in this study was a medium based on that of feinberg and whittington for the detection of t. vaginalis and candida species . currently , the trichomonas medium has been slightly modified by the incorporation of 0.1% w / v of agar which leads to reduced oxygen tension and consequently more prolific growth of trichomonads . the diagnostic yield for trichomoniasis varies depending on the type of culture medium used . a systemic review has shown that the sensitivity for diamond medium ( 11 studies ) varied from 88 to 99% while the same value for oxoid medium was 7689% . however , the oxoid medium was used in only three studies that were assessed in this review . diamond culture medium was not used in our study due to limited financial and laboratory resources . a modified technique was adopted to ensure efficiency in transport and preparation of specimens than the standard technique . instead of preparing vaginal smears and air drying it , this method was adopted from the procedure of preservation of samples for fine needle aspiration cytology where samples are not air dried but preserved in saline . prior to staining with giemsa , the tubes with the swabs were vigorously shaken to displace all the parasites into the saline solution . the swabs were then removed and the test tubes were centrifuged at a low speed ( 5001000 rpm ) for 30 min . the supernatant was carefully extracted using a pasteur pipette leaving the deposit . the methanol supernatant was then removed leaving a thin layer of methanol as a liquid phase . the deposit which contains the parasites and the remaining layer of methanol was mixed thoroughly . one drop of this mixture was put on a clean dry slide and three such slides were prepared from a single sample . the slides were air dried and then incubated at 37c for 12 h. the slides were stained with giemsa diluted 1:9 with phosphate buffer solution ( ph 7.2 ) for 7 min . prevalence was calculated to reflect the relative frequency of trichomoniasis with the corresponding 95% confidence intervals ( ci ) . the variable measures were the numbers of true positives ( tp ) , true negatives ( tn ) , false positives ( fp ) , and false negatives ( fn ) . however , speculum examination could not be carried out in 13 ( 4% ) women in the sample . the mean age of women was 32.9 years [ standard deviation ( sd ) 9.27 ] . fifty - four percent ( 188 ) was unemployed while a further 15% ( 53 ) were commercial sex workers . therefore overall , a majority in this cohort were young and middle aged , unemployed , married , and educated women . regarding the clinical symptoms , pruritus , vaginal discharge , and vulvovaginal soreness were significantly higher in women testing positive for trichomoniasis ( test , df2 , p<0.05 ) . other symptoms such as dysuria , post - coital bleeding , and visible inflammation of the cervix did not reach a level of statistical significance . using any one of the three methods , namely culture , wet smear examination or giemsa stain , the results of parasite detection by culture , giemsa stain , and wet smear are compared in table 2 . on examination of the smears prepared from the culture medium , 24 ( 6.9% ) individuals tested positive for t. vaginalis and 25 ( 7.2% ) tested positive with the giemsa stain . comparison of parasite detection by culture and wet smear examination comparison of parasite detection by culture and giemsa 's technique following the modifications in the procedure for transport and preparation of the specimen , the giemsa method was found to be highly sensitive ( 100% , 95% ci : 86.2 - 100 ) and 99.69% specific ( 95% ci : 98.26 - 99.95 ) for the diagnosis of t. vaginalis compared to culture with a positive predictive value of 96% and a negative predictive value of 100% [ table 3 ] . one false positive was found with giemsa staining ; a possible explanation is that the culture in this instance was falsely negative , since giemsa stain is based on morphological identification of the parasite . in contrast , the wet smear was found to have a sensitivity of 95.83% ( 95% ci : 79.7699.26 ) and a specificity of 100% ( 98.82100 ) when compared to diagnosis by culture with a ppv of 100% and a npv of 99.6% . performance of modified giemsa 's stain and wet smear examination for diagnosis of t. vaginalis relative to culture technique as effective therapeutic agents for the treatment of trichomoniasis are widely available , diagnostic difficulties have become the limiting factor in reducing the disease burden . wet preparation for microscopy is currently the preferred method for diagnosis of t. vaginalis infections in the sexually transmitted disease ( std ) clinics in sri lanka because of its low cost . even with skilled microscopists , this test shows a sensitivity of approximately 5080% compared to culture . furthermore , the specimens must be examined immediately ( within 20 min ) after collection to enable the visualization of viable , motile protozoa . the sensitivity of the wet mount for microscopy declines substantially with even short time delays between collection and examination . dry vaginal smears stained with giemsa stain have been used for the diagnosis of t. vaginalis infections.[1214 ] the optimum dilution of giemsa stain used in the current study ( 1:9 ) and the duration of staining ( 7 min ) were in accordance with the world health organization ( who ) criteria and different to that used earlier by mason et al . have demonstrated a low sensitivity of 52.4% for diagnosis of t. vaginalis with giemsa stain . in studies where dry vaginal smears were used for diagnosis , the smears were air dried and fixed immediately with absolute ethanol or methyl alcohol prior to staining in order to preserve the morphology of the parasites . this may not be feasible in clinical settings such as community - based clinics where there are no immediate laboratory facilities . in this study , the swabs were inserted into a transport medium containing 5% glucose solution which acted as an extracellular energy source to preserve the viability of the organisms in vitro . thereby , smears could be prepared up to 24 h later ( as established in two different field community studies carried out simultaneously ) . the fact that smears could be examined after a lag period is a definite advantage over the immediate examination of a wet smear preparation . the modifications to the preparation of the sample prior to staining make diagnosis more efficient as the sample are concentrated and morphology of the parasite is preserved . the examination of a centrifuged deposit has the added advantage of concentrating a larger number of parasites . further , centrifugation at a lower speed prevented the separation of the flagellum from the body of the parasite and preserved its structure for morphological studies . in addition , use of the modified technique allows general practitioners and even patients themselves to take blind vaginal swabs ( without speculum use ) and preserve the sample for later inspection . considering the cost of testing , in sri lanka the cost of a wet smear is usd 0.10 , the cost of giemsa staining is usd 0.30 , and the cost of culture is usd 2.00 . the modified method giemsa staining will cost approximately usd 0.10 extra . given the marginal extra cost , this makes it the most feasible and cost - effective test in our setting , considering its higher sensitivity when compared to the wet smear examination . they are more sensitive than wet preparation microscopy ( 78.5% and 72.4% , respectively ; p=0.04 ) but were less specific ( 98.6 and 100.0% , respectively ; p=0.01 ) . cost is a prohibitive factor for wide spread use of rapid diagnostics , but it may be of value in settings where microscopy is not possible . in a resource poor country such as sri lanka , where laboratory facilities and manpower are limited , and cost is a consideration , use of a low cost transport medium to preserve the high vaginal swabs and subsequent use of giemsa - stained smears to diagnose t. vaginalis may be used as an alternative to wet smear examination . when compared with culture , both methods were equally specific , but the giemsa stained smears were more sensitive in diagnosis .

objective : this study was designed to compare diagnosis of trichomoniasis by culture , wet smear examination , and giemsa stain . a modified technique was used to transport and prepare the specimen to ensure parasite viability prior to giemsa staining.materials and methods : a clinic - based prospective study was carried out in association with the national std / aids control programme over a period of 18 months . three swabs were collected from the posterior fornix of 346 newly registered female patients for diagnosis of trichomoniasis . a wet smear was prepared using the first swab . the second swab was placed in 5 ml of 0.9% saline with three drops of 5% glucose at room temperature and centrifuged twice at a low speed prior to preparation of a giemsa stained smear . the third swab was for culture . the three tests were performed independently . the specificity and sensitivity of the wet smear and giemsa stain were compared to culture.results:with culture , the prevalence of trichomoniasis was 6.9% ( 95% ci : 4.19.3% ) . the giemsa - stained smear was found to be highly sensitive ( 100% , 95% ci : 86.2100% ) and specific ( 99.69% , 95% ci : 98.2699.95% ) compared to culture . the wet smear was less sensitive ( 95.83% , 95% ci : 79.7699.26% ) but equally specific ( 100% , 95% ci : 98.82100%).conclusion : in developing countries , facilities for using culture are limited and wet smear examination in the field is also difficult due to the immediate need for laboratory facilities . our study demonstrated that , in this setting , using a transport medium prior to giemsa staining is a feasible alternative , with a high - diagnostic yield .

INTRODUCTION MATERIALS AND METHODS Study site and study population Specimen collection Laboratory tests Culture of Preparation of the sample for staining with Giemsa Data analysis RESULTS DISCUSSION CONCLUSIONS

PMC3551457

the use of titanium in dental implant materials is based on the low thermal conductivity of this metal , besides the fact that it does nt release toxic ions and has an appropriate elasticity module , which helps bone to heal when in close contact with the titanium implant surface.1 titanium is highly ductile and resistant to cyclic forces in the oral environment . these cyclic forces , known as fatigue , are produced by masticatory movements , which are capable of causing internal microcracks in titanium - based implants.2 titanium constantly undergoes mechanical and thermal stresses in an aggressive host oral environment . mouthwashes and toothpastes containing fluoride ions are among the most important products in the oral care market . in fact , fluoride has been considered the main method to reduce the enamel dissolution and prevent caries . fluoride ions have the ability to interact with the hydroxyapatite crystals forming fluoridated hydroxyapatite ( ca10(po4)6(oh)f ) or fluorapatite ( ca10(po4)6f2 ) . these minerals have greater lattice energy , higher crystallinity , and better resistance to dissolution than hydroxyapatite . fluoride also increases the seeding rate of apatite crystals.1 the in vivo treatment using fluoride toothpastes may lead to a partial substitution of the hydroxylic groups with fluoride ions in the native enamel hydroxyapatite.3,4 the bone - promoting activity of f has been extensively documented in the literature.16 titanium fluoride forms a stable layer on hydroxyapatite surfaces where titanium shares the oxygen atoms of phosphate , which becomes covalently bound to the hydroxyapatite surface . fluoride is essential in this reaction because it allows phosphate ions to react directly with titanium to form a firm and stable connection.1 the oxygen and phosphate in the tissue fluids may replace the fluoride and the phosphate becomes covalently bound to the titanium surface . such a reaction may induce a bone formation where the phosphate in the bone is firmly ( covalently ) bound to the titanium implant . this process may catalyze bone formation and facilitate deposition of particularly well - mineralized bone tissue close to the implant surface.5,6 oxidative processes can thicken and condense the titanium oxide layer on the surface , which improves the anticorrosion stability of the underlying titanium.7,8 on the other hand , reductive agents , such as fluoride ( f ) , may have the opposite effect and attack this layer.8 the corrosion process is caused by electrochemical reactions . titanium is known to present high resistance to corrosion against attack by solutions containing strong mineral acids , such as hydrochloric acid or sulfuric acid . however , when titanium is placed in contact with a fluorinated medium at ph below 3.5 , its titanium oxide layer is damaged and degraded . this phenomenon is macroscopically visible by the loss of surface brightness.9,10 the fluorides may also cause corrosion , which increases roughness on the surfaces exposed to the oral cavity , making it easier for microorganisms to grow where they are protected from oral hygiene mechanisms ( brushing , swallowing , and flow of crevicular fluid).2,11,12 anodic polarization and immersion tests indicated that the titanium corrosion in solutions containing f depends on the concentration of hydrofluoric acid ( hf ) . the passivation film on titanium was destroyed when the hf concentration in the solution was > 30 ppm . a rough surface favors plaque accumulation13 in the peri - implant crevices of the gingiva , which is an undesirable effect in this very sensitive region of the implant . accordingly , to avoid pathogenic plaque accumulation,14 the neck ( abutment ) of an implant must be polished . many studies shown the high risk for fluoride to produce fluorosis , especially in children , and bone diseases in the elderly.1521 the european food safety authority ( efsa ) scientific panel has advised that there is a risk of fluoride intake from ingested oral care products and toothpastes that can be fluoridated with a maximum level of 1500 mg / kg . the efsa considers that the maximum fluoride intake should be 0.1 mg fluoride / kg / day in children aged 18 years which is equivalent to 1.5 and 2.5 mg fluoride per day in children aged 13 years and 48 years , respectively.22,23 innovative commercial mouthwashes and toothpastes in the last decade have replaced fluoride with biomimetic hydroxyapatite nanocrystals as a remineralizing agent to avoid the dangerous effects of fluoride in human health , which have been ascribed to the amount of fluoride ingested daily.16 in the inorganic phase of bone and dental hard tissues , hydroxyapatite [ ca10(po4)6(oh)2 ] ( ha ) has been widely studied as a bone filler , prosthetic coating , and implantable biomaterial in dentistry and orthopedic surgery because of its biocompatibility , bioactivity , and osteoconductivity.17 biological ha is not stoichiometric according to the ideal formula ca10(po4)6(oh)2 , but at low quantities ca is replaced by other ions such as na , k , zn , mg , sr , while po4 and oh can be partially substituted by other anions like co3 , hpo4 , p2o7 , sio4.17,2426 carbonate is the prevalent foreign anion present into bone - hydroxyapatite structure and represents about 48 wt%.27,28 the substitution of co3 groups into the po4 sites ( type b carbonate apatite ) is prevalent in children , while the carbonate replacement to oh groups ( type a carbonate apatite ) increases with the age of the individual.29 biomimetic carbonate hydroxyapatite nanocrystals ( cha ) have been synthesized nearly stoichiometrically in bulk ca / p molar ratio of about 1.7 and contains 4 1 wt% of carbonate ions that replaced prevalently phosphate groups . biomimetic cha nanocrystals have been synthesized from 20 nm to 100 nm ( figure 1 ) with both acicular and plate - like morphology to deliver drugs3033 with a surface infused with amino acids and proteins.26,3436 biomimetic cha ( figure 2 ) can aggregate in microsized crystal clusters that have a nanostructured surface area of about 80 m / g and can be utilized as mineralized agents in toothpastes and mouthwashes.37 only recently , a chemical physical experimental approach has been followed to investigate in vitro37,38 and in vivo3,4 the capability of cha nanostructured crystals to produce in vivo biomimetic mineral deposition on enamel and dentin surfaces through daily use.11 demineralized enamel and dentine slabs have been treated in vitro with synthetic biomimetic hydroxyapatite nanocrystals for a few minutes and the surfaces chemically and physically characterized . cha induces surface remineralization , which forms a biomimetic apatite coating on enamel and dentin surfaces . enamel remineralization occurs quickly due to the specific chemical physical characteristics of innovative nanostructured hydroxyapatite particles , which closely resemble the natural hydroxyapatite present in enamel.39 therefore , our experimental results suggest that the deterioration of teeth can be prevented . cha synthesized with tailored biomimetic characteristics for composition , structure , size , and morphology can chemically bind themselves on the surfaces of hard tissues in teeth , filling in the scratches , and producing a bound biomimetic apatitic coating to protect the enamel surface structure.3,40,41 there are some commercial products for oral care , like toothpastes and mouthwashes , based on cha , which come into contact with titanium prostheses . the aim of this work is to evaluate the action of a nanostructured cha - based mouthwash on titanium implants , in comparison with a f - based mouthwash . in this work , we have performed an in vitro analysis on the effect of f and cha on the surface of titanium brackets , using scanning electron microscopy ( sem ) and atomic force microscopy ( afm ) , which allows a direct quantitative characterization of the surface roughness . we considered four types of titanium resulting from different processes : polished , blasted , machined , and acid - etched . all samples were titanium grade plates 3 mm thick and 6 mm diameter . in vitro tests were carried out using six samples for each type of titanium . polished titanium brackets were tested at room temperature in nanostructured microcrystal cha - based mouthwash for comparison to an amine fluoride f - based mouthwash with a ph ranging between 4.3 and 5.5 . the stoichiometric composition of biomimetic cha , which represents the active component in the used mouthwash , is ca(10x ) znx(po4)(6y)(co3)y ( oh)2 where x is 0.1 and y is 0.4 . each plate sample was dipped in mouthwash for 60 seconds , twice a day for 30 days to simulate a proper oral hygiene rinse . after each treatment , the titanium plates were washed under running water to replicate in vivo the usual teeth rinse procedure . sem observations were carried out by means of a carl - zeiss evo , 40 xvp ( oberkochen , germany ) equipped with an energy dispersive detector ( edax ) inca 250 ( oxford , ul ) , using secondary electrons at 25 kv and different magnifications . afm imaging was performed on a digital instruments nanoscope iiia multimode spm ( digital instruments , santa barbara , ca , usa ) . the advanced analog and digital circuit designs and innovative software and hardware provide superior multitasking control for scanning probe microscopes ( spms).42,43 the samples in contact mode using a j scanner and silicon nitride tips ( 200 lm long with nominal spring constant 0.06 n / m ) . we considered four types of titanium resulting from different processes : polished , blasted , machined , and acid - etched . all samples were titanium grade plates 3 mm thick and 6 mm diameter . in vitro tests were carried out using six samples for each type of titanium . polished titanium brackets were tested at room temperature in nanostructured microcrystal cha - based mouthwash for comparison to an amine fluoride f - based mouthwash with a ph ranging between 4.3 and 5.5 . the stoichiometric composition of biomimetic cha , which represents the active component in the used mouthwash , is ca(10x ) znx(po4)(6y)(co3)y ( oh)2 where x is 0.1 and y is 0.4 . each plate sample was dipped in mouthwash for 60 seconds , twice a day for 30 days to simulate a proper oral hygiene rinse . after each treatment , the titanium plates were washed under running water to replicate in vivo the usual teeth rinse procedure . sem observations were carried out by means of a carl - zeiss evo , 40 xvp ( oberkochen , germany ) equipped with an energy dispersive detector ( edax ) inca 250 ( oxford , ul ) , using secondary electrons at 25 kv and different magnifications . afm imaging was performed on a digital instruments nanoscope iiia multimode spm ( digital instruments , santa barbara , ca , usa ) . the advanced analog and digital circuit designs and innovative software and hardware provide superior multitasking control for scanning probe microscopes ( spms).42,43 the samples in contact mode using a j scanner and silicon nitride tips ( 200 lm long with nominal spring constant 0.06 n / m ) . the surface morphologies of different polished , blasted , machined , and acid - etched titanium plates have been characterized by sem and the images are reported in figure 3a d respectively . figure 3 shows that blasted , machine - treated , and acid - etched titanium plates have an inhomogeneous surface , while the polished plate surface ( figure 3a ) has low grooves and thus a smooth surface ( figure 3c ) . polished titanium plates that have a homogeneous surface with a very small roughness appear to be the more suitable titanium plates to be utilized in this study . comparable samples of polished titanium plates have been surface characterized by sem before and after a daily in vitro treatment with two different mouthwashes . a mouthwash containing an amine fluoride ( 100 ppm f ) and another containing zinc - substituted cha have been utilized to investigate their different effects on the titanium plate surface . after the in vitro treatment with the two different mouthwashes , sem analysis of the titanium plates showed an appreciable difference between the plates treated with amine f - based mouthwash ( figure 4a ) and those treated with the cha - based mouthwash ( figure 4c ) . in fact , the surface roughness present on titanium surface increases after the treatment with amine f - based mouthwash compared to that observed on the reference titanium plate ( figure 3a ) . contrarily , the cha - based mouthwash seems to cover valleys and grooves present on the titanium surface , which appreciably reduces the roughness present on the reference titanium plates . the edax elemental analysis performed on the surface of the titanium after treatment with a fluoride rinse showed only one peak related to titanium ( figure 4b ) . if the edax was performed after treatment with cha - based mouthwash , the same survey grade showed peaks attributable to the calcium and phosphate ions present in the right stoichiometric molar ratio of hydroxyapatite , 1.7 ( figure 4d ) . for more detailed results , titanium plates treated with the two different mouthwashes were investigated using titanium sections of different dimensions . we have examined titanium plate sections of 10 10 m and 100 100 m wide . figures 5 and 6 show afm images obtained on the surface of 10 10 m and 100 100 m sections , respectively . in fact , in the 10 10 m samples , afm measures the surface nanoroughness with high accuracy , while in the 100 100 m samples , it is possible to evaluate the roughness extension with high accuracy . in figures 5a and 6a , the afm images of the titanium reference surface are reported , in figures 5b and 6b , the afm images of f mouthwash - treated titanium surface are reported , while in figures 5c and 6c , the afm images show the effect of cha mouthwash on the titanium surface . the numerical analysis carried out on the afm images the root mean square ( rq ) , highest peak ( rp ) , and deepest valley ( rv ) were measured on 10 10 m sections of reference titanium surface and after treatment with mouthwashes containing f and cha ( table 1 ) . the rq , rp , and rv were measured on 100 100 m sections of reference titanium surface and after treatment with the mouthwashes containing f and cha ( table 2 ) . a morphological characterization was performed on the surface of polished , titanium brackets after in vitro treatment with mouthwashes containing amine f and cha . titanium plates treated with the two different mouthwashes were analyzed by sem and afm using titanium sections of two different dimensions ( 10 10 m and 100 100 m ) for a well - defined highly accurate evaluation of the modification of surface nanoroughness and its extension ( respect the value rq , rp and rv reported in table 2 ) induced by the treatment with the two different mouthwashes . the treatment with the f - based mouthwash induces a roughness characterized by streaking with higher peaks and deeper valleys on the titanium bracket surface compared with those observed in the reference polished titanium plates . contrarily , the treatment with the cha - based mouthwash induces a roughness characterized by streaking with smoothed peaks and less deep valleys on the titanium bracket surface compared with those observed in the reference polished titanium disc . these findings can be explained by the hydroxyapatite deposition filling the streaks on the titanium surface and therefore reducing surface roughness . the edax analysis revealed the presence of calcium and phosphorus in a molar ratio of 1.7 , which was characteristic of the cha present in the mouthwash . the increased roughness induced by the in vitro treatment with the mouthwash containing amine f ( tables 1 and 2 ) modify the mechanical behavior of the titanium brackets , which promotes a mechanical weakness of the metallic dental implant . the increased depth of the streaks encourage bacterial growth promoting infection and prosthesis contamination and mobility.44 contrarily , the in vitro treatment with the cha - based mouthwash reduces the surface roughness by filling in the streaks ( tables 1 and 2 ) . this hydroxyapatite deposition protects against the surface oxidative process , which can damage the mechanical behavior of the titanium brackets . the depositation of hydroxyapatite coating on titanium surface is able to prevent the bacterial growth contaminating the prosthesis . furthermore , when bacterial plaque , by its acidity , solubilizes the zinc - substituted cha present on the titanium implant.40,41,45 on one hand , our data shows that the presence of f in mouthwashes should be avoided to prevent any degradation of the metallic dental implant and subsequent contamination with bacteria . on the other hand , the cha - based mouthwash prevents the degradation of the metallic dental implants and the formation of bacterial film . the morphological characterization of the surface of polished titanium brackets after in vitro treatments with a mouthwash containing an amine f and another containing cha showed that the presence of f in a mouthwash induces mechanical weakness in the metallic dental implant and bacterial contamination . the presence of zinc - substituted cha in a mouthwash prevents the deterioration of the metallic dental implant and contamination with bacteria .

titanium plates treated in vitro with a mouthwash containing amine fluoride ( 100 ppm f ) and another containing zinc - substituted carbonate hydroxyapatite have been analyzed by scanning electron microscopy and atomic force microscopy to evaluate the modification of the surface roughness induced by treatment with these two different mouthwashes . the treatment with f-based mouthwash produces a roughness characterized by higher peaks and deeper valleys in the streaks on the titanium bracket surface compared with those observed in the reference polished titanium plates . this effect causes a mechanical weakness in the metallic dental implant causing bacterial growth and therefore promotes infection and prosthesis contamination . however , the in vitro treatment with a mouthwash containing zinc - substituted carbonate hydroxyapatite reduced the surface roughness by filling the streaks with an apatitic phase . this treatment counteracts the surface oxidative process that can affect the mechanical behavior of the titanium dental implant , which inhibits the bacterial growth contaminating prostheses .

Introduction Materials and method In vitro test Morphological characterization Results Discussion Conclusion

PMC3037191

high cost for dental services is a common reason for adults avoidance of dental visits.1,2 cost - sharing schemes as third - party payment , e.g. , health insurance systems , have attempted to reduce or remove cost barriers and to ease access to and use of dental services.3,4 in several studies , dental insurance has been addressed as one of the key factors affecting the use of dental services . insured people have been shown to use preventive and restorative services more than do those with no insurance.59 receiving dental services is mediated by a myriad of personal , cultural , and institutional factors , 10 being dependent on provider , receiver , and practice.1113 in addition to insurance status , other receiver - based factors should be taken into account . these include demographic factors,14 socioeconomic status,11 dental conditions,12 reason for visit , and attitudes towards health care.13 the ultimate goal of dental insurance is just to improve the oral health of its beneficiaries.15 depending on the social and political system of a country , the health policy , dental services , and their funding differ.16,17 consequently , the service panorama will usually differ among systems.18 in most developing countries dental services are provided mainly to relieve pain or harmful symptoms , 19,20 thus resulting in a service - mix different from those in developed countries . the aim of the present study was to assess the relationship between insurance status of dentate adults and types of service they reported as receiving in iran , a country with a developing oral health care system . in iran , dental service in both the public and private sector means responding to those who come to a dentist mostly for problem - related treatment.21,22 two dental insurance systems are available : public and commercial , their main function being to subsidize treatment costs . in public insurance , both the employer and employee pay compulsory premium , for employees it is deducted from their wages or income . in commercial insurance , public insurance covers examination , dental x - ray , tooth extractions , scaling , amalgam and composite fillings , and removable dentures with a subsidy of 100% at a clinic owned by and of 70% at a clinic contracted with the public insurance system . the dentist : population ratio in tehran is 1:1,800 , with about 4,500 practicing dentists serving the eight million inhabitants in the city . details of the iranian health insurance system have been described previously.21,23 the target population included dentate adults ( 18 years or older ) who were residents of tehran and had access to a fixed telephone line . of all tehran residents , 90% have fixed telephone lines.24 based on the 3-digit prefix codes and the 4-digit running numbers from a list of four million computerized options resembling real phone numbers , a total of 3,200 seven - digit numbers were randomly selected . a pilot study was carried out on 100 adults in february 2005 to determine the feasibility of the sampling method and relevance of the questionnaire . for each missed call , the reason for failure was recorded as busy , no answer , fax , or a non - existent line . after five attempts , a busy or non - answering line was omitted from the list . in total , 1,669 numbers were unavailable , most because of being a non - existent line or a fax . of the 1,531 subjects who answered the calls , 224 were outside the target age ( under 18 ) , leaving 1,307 subjects to be interviewed . the present study excluded those who said they were edentulous ( n=18 ) or had never visited a dentist ( n=67 ) ; the final sample thus comprised 1,001 subjects . sampling details have been published earlier.21 the questions in this interview were based on related studies,14,18,25,26 and after the pilot study were slightly modified . the questionnaire covered respondents insurance status , characteristics of dental attendance , and socio - demographic information . answers to the question about respondents dental insurance status were recorded as : no insurance , public insurance , or commercial insurance . in the present analyses , characteristics of dental attendance covered time elapsed since and reason for the most recent dental visit and items of service received . based on the respondent s answer to the question the interviewer marked one option on a list of seven options , later dichotomized into within the past 12 months and more than one year ago . answers to the question what was the reason for your last dental visit ? were recorded as : check - up , problem with teeth or gums , or continuing treatment . in the analyses , those ( n=17 ) who said that they were continuing their treatment were combined with the group having a problem with their teeth or gums . consequently , the reason for the dental visit was dichotomized as check - up or problem . oral health care behavior was assessed according to the frequency of tooth brushing and was categorized as : less than daily , once daily , and twice daily . respondents answered in their own words , the question what dental services did you receive during your most recent visit ? , and the interviewer marked each reason named from a list of 13 options . these were later classified into five types of services:27 1 ) diagnostics ( examination , prescription , and radiographs ) , 2 ) prevention ( scaling and dental prophylaxis ) , 3 ) restorations ( amalgam and resins fillings ) , 4 ) extractions , 5 ) high technique services ( surgical procedures , orthodontics , endodontics , crowns or bridges ) . socio - demographic information covered age , gender , level of education , and income . the respondent s date of birth was calculated to the nearest year . the respondent s education attained was recorded with eight options , later combined into three levels of education : low ( illiterate , primary or secondary school ) , medium ( high school or diploma ) , and high ( associate degree , bachelor s degree , master s degree ) . information about family income came from the open question how much is your monthly household income ? the answers were recorded in rials and then categorized as low ( under the poverty line ) , medium , and high . the strength of the factors related to each type of service was evaluated by fitting logistic regression models to the data and by calculating the corresponding odds ratios ( or ) and their 95% confidence intervals ( 95% ci ) . in iran , dental service in both the public and private sector means responding to those who come to a dentist mostly for problem - related treatment.21,22 two dental insurance systems are available : public and commercial , their main function being to subsidize treatment costs . in public insurance , both the employer and employee pay compulsory premium , for employees it is deducted from their wages or income . in commercial insurance , public insurance covers examination , dental x - ray , tooth extractions , scaling , amalgam and composite fillings , and removable dentures with a subsidy of 100% at a clinic owned by and of 70% at a clinic contracted with the public insurance system . the dentist : population ratio in tehran is 1:1,800 , with about 4,500 practicing dentists serving the eight million inhabitants in the city . the target population included dentate adults ( 18 years or older ) who were residents of tehran and had access to a fixed telephone line . of all tehran residents , 90% have fixed telephone lines.24 based on the 3-digit prefix codes and the 4-digit running numbers from a list of four million computerized options resembling real phone numbers , a total of 3,200 seven - digit numbers were randomly selected . a pilot study was carried out on 100 adults in february 2005 to determine the feasibility of the sampling method and relevance of the questionnaire . the reason for failure was recorded as busy , no answer , fax , or a non - existent line . after five attempts , a busy or non - answering line was omitted from the list . in total , 1,669 numbers were unavailable , most because of being a non - existent line or a fax . of the 1,531 subjects who answered the calls , 224 were outside the target age ( under 18 ) , leaving 1,307 subjects to be interviewed . the present study excluded those who said they were edentulous ( n=18 ) or had never visited a dentist ( n=67 ) ; the final sample thus comprised 1,001 subjects . the questions in this interview were based on related studies,14,18,25,26 and after the pilot study were slightly modified . the questionnaire covered respondents insurance status , characteristics of dental attendance , and socio - demographic information . answers to the question about respondents dental insurance status were recorded as : no insurance , public insurance , or commercial insurance . in the present analyses , characteristics of dental attendance covered time elapsed since and reason for the most recent dental visit and items of service received . based on the respondent s answer to the question the interviewer marked one option on a list of seven options , later dichotomized into within the past 12 months and more than one year ago . were recorded as : check - up , problem with teeth or gums , or continuing treatment . in the analyses , those ( n=17 ) who said that they were continuing their treatment were combined with the group having a problem with their teeth or gums . consequently , the reason for the dental visit was dichotomized as check - up or problem . oral health care behavior was assessed according to the frequency of tooth brushing and was categorized as : less than daily , once daily , and twice daily . respondents answered in their own words , the question what dental services did you receive during your most recent visit ? , and the interviewer marked each reason named from a list of 13 options . these were later classified into five types of services:27 1 ) diagnostics ( examination , prescription , and radiographs ) , 2 ) prevention ( scaling and dental prophylaxis ) , 3 ) restorations ( amalgam and resins fillings ) , 4 ) extractions , 5 ) high technique services ( surgical procedures , orthodontics , endodontics , crowns or bridges ) . socio - demographic information covered age , gender , level of education , and income . the respondent s date of birth was calculated to the nearest year . the respondent s education attained was recorded with eight options , later combined into three levels of education : low ( illiterate , primary or secondary school ) , medium ( high school or diploma ) , and high ( associate degree , bachelor s degree , master s degree ) . information about family income came from the open question how much is your monthly household income ? the answers were recorded in rials and then categorized as low ( under the poverty line ) , medium , and high . the strength of the factors related to each type of service was evaluated by fitting logistic regression models to the data and by calculating the corresponding odds ratios ( or ) and their 95% confidence intervals ( 95% ci ) . of the subjects ( n=1,001 ) , 58% were women and 61% were under age 35 . subjects mean age was 32.9 years ( sd=10.7 ; median 32.0 : 95% ci=32.233.5 ) , with no gender differences ( p=0.18 ) . level of education was high for 36% , medium for 47% and low for 17% , more women than men reporting a high level of education ( p=0.02 ) . regarding to household income ; 30% fell into the high - income , 31% into the medium , and 13% into the low category , and 26% refused to disclose their income being categorized as no answer . of those who responded to this question , more men than women reported a high household income ( p<.001 ) . of all , 56% , more women than men reported having had a dental visit within the past 12 months . the great majority ( 84% ) reported problems with their teeth as the reason for their most recent visit ; 16% had visited a dentist for a check - up . of all respondents , 43% reported twice daily , 50% once daily , and 7% less than daily tooth brushing . of the respondents , 71% reported having dental insurance ( 65% public , 6% , commercial insurance ) , 29% had no insurance . having dental insurance coverage was more likely for women ( or=1.5 ) , for those in oldest age group ( or=2.1 ) , and those with a high level of education ( or=2.5 ) . those respondents who did not disclose their income were less likely to have insurance coverage ( table 1 ) . restorative treatments were the most frequently and preventive care the least frequently reported type of services , regardless of subjects insurance status . the non - insured respondents reported tooth extractions almost twice as frequently as did the insured ones ( p<.001 ) ( figure 1 ) . tables 2 and 3 show distribution of the respondents according to the type of treatment reported by insurance status as well as to socio - demographic and dental attendance characteristics and the frequency of brushing . no gender or age differences emerged . among the insured subjects , diagnostic was most frequently reported by those with a high level of education , those who gave check - up as the reason for their most recent dental visit and those who reported twice daily tooth brushing . except for level of education these trends were similar among the non - insured respondents . among the insured subjects , preventive care was less frequently reported by those with medium or high household income , and those who reported less than daily brushing . a trend appeared among those non - insured subjects with a low level of education or income , those who visited a dentist more than last 12 months , and those who reported less than daily tooth brushing . tooth extractions were most frequently reported by those with a low level of education or income , by those who reported a problem - based visit to a dentist and those who reported never brushing . among the non - insured subjects , half of those with a low household income reported tooth extraction as their most recent treatment . the highest rate of extractions appeared among those with a low level of education , those who visited a dentist more than 12 months ago for problem - based reason , and those who reported less than daily tooth brushing . table 4 shows odds ratios for each type of service reported , as explained by subjects insurance status and socio - demographic and dental attendance characteristics . subjects in addition , reporting tooth extractions was more likely for those with a low level of education ( or=2.4 ) , or income ( or=2.2 ) , and for those who visited a dentist more than one year ago ( or=1.5 ) . diagnosis ( or=10.0 ) and preventive care ( or=5.0 ) were definitely more likely for those reporting check - up as their reason for the most recent dental visit . those reporting problem as their reason for the most recent dental visit were more likely to report restorative ( or=3.0 ) , extraction ( or=6.0 ) , and high technique services ( or=4.3 ) . reporting restorative service as received at their most recent visit was more likely for those subjects with medium ( or=1.8 ) or high ( or=1.7 ) level of education and those with medium income ( or=1.8 ) . subjects tooth brushing frequency made a difference only for diagnostic ; those who reported once daily tooth brushing ( or=1.6 ) were more likely to receive diagnostic service than those with less than daily brushing . preventive service was less likely for those with a medium income ( or=0.4 ) compared with those who did not answer to the question on income . table 5 shows odds ratios for each type of service reported as received by the insured subjects ( n=710 ) . tooth extractions were more likely for those reporting a problem - based dental visit ( or=6.0 ) or having a low level of education ( or=2.5 ) . receiving diagnostics ( or=8.0 ) or preventive care ( or=5.6 ) was more likely for those reporting a check - up visit . the present results reveal that insurance status made no difference regarding type of dental treatment received except for tooth extractions , which were more frequently reported by the noninsured subjects . these findings remained similar after controlling for subjects socio - demographic and dental attendance characteristics . further , reason for dental visit seemed to be a strong determinant for receiving each type of treatment , regardless of insurance status . the relationship between being insured and undergoing fewer tooth extractions is in line with recent reports from developed countries . in the us , only 7% of those with private insurance , but 27% with medicaid insurance have undergone tooth extractions.9 reports from australia have shown that 4% to 13% of the insured undergo tooth extractions compared with 10% to 31% of those receiving publicly funded support or being without insurance coverage.5,6,14,28 generally , fewer tooth extractions among those insured may reflect their better options for choice of treatment . in iran , those who are insured receive their dental treatments with a 70% to 100% subsidy , which may facilitate their choices as to maintaining dental health . the present results show that regardless of insurance status , restorative treatments dominated , and preventive care was rather infrequent . this leading role for restorative care is common in many developed and developing countries,6,7,26,31,32 even that some developed countries have placed higher emphasis upon preventive care.33,34 reorientation of oral health services towards prevention is one of the who s priority action areas for continuous improvement of oral health.35 insurance schemes should also support this emphasis on preventive care , especially in countries with a developing oral health service system , such as iran . providing a lesser amount of preventive care may mean lower fees income and thus affect dentists clinical decision - making . in iran , dentists have reported positive attitudes towards preventive care,36 which is a good starting point . however , the same dentists considered performing preventive measures not economically beneficial.36,37 this may have influenced on dentists willingness to provide such treatments for the present respondents as well . tehran has a population just over 8,000,000 , of which about 60% have health insurance . around 1,300 of all tehranian dentists work under insurance scheme , yielding a dentist : population ratio of 1:3,700 for the insured people . in respect to access to dental services this might not provide sufficient opportunity for dental care . on the other hand , remuneration for salaried dentists ( who are hired in insurance owned dental clinics ) , and for contracted dentists , which insurance paid them according to a fee - for - service payment , might lead to a lack of financial incentive and might influence their practice . in iran , the fee for each treatment item in insurance scheme is much lower than the one for the same treatment in the private sector . dental insurance has been reported to be associated with higher percentages visiting within the last year,23 and visiting for a check - up.21 such associations of insurance status with access to services may be expected to have an impact on the pattern of service received . in present study reason for dental visit had a strong impact on treatment - mix , a check - up indicating greater odds for receiving diagnostics and preventive care , in line with reports from australia.6,38 generally , people going for regular dental check - ups also have regular medical check - ups,9,39 have high levels of education or income,18,40 and usually have a good job , all of which may reflect their higher expectations of having a healthy dentition and , consequently lead them to avoid services resulting in loss of teeth . the present study revealed that tooth extraction was more likely for subjects with a low level of education or income , those with a longer time since the previous visit and reporting problem as a reason for it . this finding may reflect the variation in the attitudes or behaviour of insured and noninsured persons.32 several studies have shown that people with low income or education have higher rates of poor oral health and treatment need and also have less understanding of health value.42 the present data were based on interviews among adults living in tehran , iran , and with access to a fixed telephone line , representing more than 90% of the population . to increase the reliability of the data , each interview lasted on average of 15 minutes , which appeared to be sufficient for presenting the questions and answering to them . further , the population - based data and high response rate speaks for the generalisability of the present findings . in the present study , dental insurance showed a minor , if any impact , on receiving different types of dental treatment . the present findings indicate a need to modify insurance systems to encourage the preventive approach in oral health service provision , in particular in countries with a developing oral health care system .

objectives : to assess the relationship between insurance status and type of service received among dentate adults in a developing oral health care system.methods:a cross - sectional survey based on phone interviews in tehran , iran . four trained interviewers collected data using a structured questionnaire . of 1,531 subjects answering the phone call , 224 were < 18 years ; of the remaining 1,307 , 221 ( 17% ) refused to participate , and 85 ( 6% ) were excluded as edentate or reporting no dental visit , leaving 1,001 eligible subjects in the sample . the questionnaire covered insurance status , socio - demographics , frequency of tooth brushing , dental attendance as reasons for , and time since last dental visit , and dental service received then . data analysis included the chi - square test and logistic regression.results:of the subjects , 71% had a dental insurance . those with no insurance were more likely to report tooth extractions ( or=1.5 ) than those with an insurance coverage ; for all other treatments no differences according to the insurance status appeared . among the insured subjects , extractions were more likely for those reporting a problem - based dental visit ( or=6.0 ) or having a low level of education ( or=2.3).conclusions : in iran , with its developing oral health care system , dental insurance had only a minor impact on dental services reported .

INTRODUCTION MATERIALS AND METHODS Background Design and sampling Phone calls Interviewing and questions Statistical analysis RESULTS DISCUSSION CONCLUSIONS

PMC5172434

the aim of designing biras is to make full use of the retrieval function of entrez and timely retrieve the latest information of literatures and gene sequences . usually , when we retrieve a keyword or term by entrez web server in ncbi , we will first get a summary list of literatures and gene sequences . if we find the necessary literatures and sequences , we will click their links and read the detailed information . the first step is to send the keyword or term to the entrez web server and gets the identify ( i d ) list of the newly increased literatures and sequences . secondly , it retrieves each i d and saves the retrieval information to local disk . thus , biras effectively retrieved user - defined updating information of the literatures and gene sequences . the medline literatures , nucleotide sequences and protein sequences retrieval section of biras provides the complex term retrieval function of entrez ( http://www.ncbi.nlm.nih.gov/entrez/query/static/help/pmhelp.html ) . researchers can retrieve not only the simple single term but also the logical field composed of several terms with boolean operators and , or , not . furthermore , biras also provides some common download styles ( literatures : abstract , asn.1 , citation , medline , summary ; nucleotide sequences : asn.1 , fasta , genbank , report , summary ; protein sequences : asn.1 , fasta , genpept , report , summary ) . thereby , biras provides a quite convenient retrieval mode for the researchers who concern with the special literatures and sequences . besides , biras provides the simple way for browsing the updating literatures and sequences , and shows impact factor of each journal as an especial item to make provide various kinds of information for the user ( fig . to the researchers who are studying structure and function of novel genes , it is very important for them to know whether there is any new sequences with high homology to their - concerned genes in the public genbank database . such sequences often come from homology genes of different species or alternative splicing forms of the same gene . therefore , biras provides an information retrieval mode based on the user - defined sequence . in other words , biras automatically uses blast program from the web server at ncbi to find out related gene sequences or protein sequences that are homologous to the sequence in genbank . it is quite convenient for users to retrieve timely based on the homology of sequences . as far as the process of analysis , biras adopts the qblast s api to access the ncbi qblast system ( http://www.ncbi.nlm.nih.gov/blast/doc/urlapi.html ) , and uses the corresponding blast program of the ncbi qblast system for analysis . when the ncbi qblast process is finished , biras will automatically save the result to local disk . after then , biras does some necessary treatments with the analysis result and inform users of the latest increased homolog sequences in the genbank database . to automatically analyze sequence homology biras allows users to set four common analysis parameters : database , expect , descriptions , and alignments 4 . , 5 .. researchers can set these parameters according to their demands in order to get the valid analysis result . according to the above requirements , our designed biras has the following interface ( fig . users can adjust the parameters of update conditions in the menu set / update set , specify the server for sending e - mail in the menu set / mail set , and define the same or different e - mail addresses for any retrieval item in the menu set / mail address . thus , when the certain item retrieval finds out the updated information , biras can send update information as attachments in html format to the user - defined e - mail addresses , in order to make users receive the latest information as early as possible . the outstanding characteristic of biras program is that it can provide users with required information timely and dynamically according to defined requirements . users can get the valid information easily enough , but need not download the whole medline database or the whole nucleotide sequences and protein sequences database . consequently , it obviously conduces to promoting the efficiency of their studies . at present , biras merely concerns with the following contents : 1 ) medline literatures retrieval . namely , biras timely informs users of the latest related literatures according to the user - defined terms , and shows the impact factor of each journal . 2 ) nucleotide sequences and protein sequences retrieval , which means biras timely retrieves the concerned nucleotide sequences and protein sequences according to the user - defined terms . if it finds out the new sequences , it will inform users of the information immediately . 3 ) function analysis of special sequences . for this purpose , biras will use the latest database to do some analysis such as homology analysis , structure and function analysis with user - defined sequence . the analysis result was also sent to users by biras timely . to a certain extent if user does not initiatively retrieve the entrez web server at ncbi , the latest information will not be known by the users in time . therefore , our reported biras here is a quite important dynamic information retrieval and analysis system . in other words , biras seems to be a worker who works quietly and always transfers user - concerned latest information timely . therefore , biras could provide users with the maximum information retrieval services in the future . compared with other similar systems , the real - time feature of literatures and sequences retrieval ability of biras is quite outstanding . the medline workbench ( http://www.medbench.org/ ; medbench updates ver.3.1 ) allows users to order the information retrieval free service with the user - defined terms . however , in the practical test that respectively retrieves term apoptosis with biras and medline workbench in the same time , biras could find out 20 daily updated literatures in july 10 , 2002 . from then on biras has begun continuously retrieving the updating literatures every day . however , the medline workbench only sent a confirm e - mail to us within the first 24 hours , and did not contain any updating information . according to our experiences , in the daily using , the medline workbench merely sent about 1/4 number of literatures information at intervals . in fact , biras directly connects to the entrez web server of ncbi , and automatically retrieves updating information . therefore , its performance is superior to those of some existing literature retrieval systems . for the real - time literature retrieval , however , the mailing list service has no definite keywords filtering feature . in other words , the mailing list service of these magazines could only provide users the summary of the latest publications , but can not filter the information automatically according to user - defined terms . when the user reads the whole content of the journals , it will take a lot of time to judge what they really need or not . compared with this kind of time wasting , biras can completely retrieve and effectively gather the users concerned information from the databases of the ncbi according to the user - defined term . obviously , biras has the more practicability . in terms of new sequences and their functions retrieval based on the sequence homology analysis , for instance , the swiss shop server ( http://www.expasy.ch/swiss-shop/ ) allows user to specify the protein sequence and do homology analysis , and then returns the analysis result to user by e - mail . the embl database has ever provided the similar service , and then it stopped the service for some reasons . compared with these resources , our sequence homology analysis function of biras includes both the nucleotide sequences homology analysis and protein sequences homology analysis . furthermore , it can carry out analysis using all databases in ncbi including sequences databases derived from the patent division of genbank , database of expressed sequence tags ( est ) , and databases of high throughput genomic sequences ( htgs ) and so on . users can freely use these databases by setting different parameters . for realizing the characteristic of running in various operating systems ( windows/ linux / macintosh etc . ) , the core code of biras is written in standard c language , and then joins the corresponding management software based on the individual characteristic of the various operating systems . at present , of course , the computers installed with biras should have connected with the internet directly . in conclusion , through many strict tests , biras can retrieve the information of user - concerned literatures and sequences timely and effectively from the entrez server at ncbi . in fact , if you can retrieve and gain the information by using the entrez , biras can automatically retrieve them and inform users in time . thereby , it is believed that biras would be used to retrieve specific information from large amount of biological data in now days .

the aim of this study is to design a biological information retrieval and analysis system ( biras ) based on the internet . using the specific network protocol , biras system could send and receive information from the entrez search and retrieval system maintained by national center for biotechnology information ( ncbi ) in usa . the literatures , nucleotide sequence , protein sequences , and other resources according to the user - defined term could then be retrieved and sent to the user by pop up message or by e - mail informing automatically using biras system . all the information retrieving and analyzing processes are done in real - time . as a robust system for intelligently and dynamically retrieving and analyzing on the user - defined information , it is believed that biras would be extensively used to retrieve specific information from large amount of biological databases in now days . the program is available on request from the corresponding author .

The Scheme of Literatures, and Nucleotide Sequences and Protein Sequences Retrieval Based on Entrez Scheme of Sequence Analysis Based on Blast Program Design of BIRAS and the Interface Discussion

PMC4446890

left ventricular ( lv ) heart failure ( hf ) is a prevalent cardiac syndrome , characterized by altered lv size and shape , elevated filling pressure , increased biomarkers , specific risk factors , plus a wide variety of symptoms and signs.1 in hf often a distinction is made between patients on the basis of their ejection fraction ( ef ) . about half of all hf patients present with a reduced ef ( denoted as hfref ) , while the others show a preserved ef ( denoted as hfpef ) . in order to distinguish between both groups , the following criteria are commonly used2 : an ef larger than or equal to 50% and an end - diastolic ventricular volume index ( edvi ) smaller than 97 ml / m are required for hfpef , while an ef smaller than 50% is set for hfref . for both groups , the gold standard3 of an end - diastolic ventricular pressure ( edp ) larger than 16 mmhg ( millimeter mercury ) is an additional requirement . thus , the combination of lv pressure and volume plays a prominent role in the analysis of hf phenotypes . ef is the pivotal measure in the current classification scheme.1 however , the ef is both a quantitative measure that relates the ejected stroke volume ( sv ) to the edv , as a percentage , as well as a qualitative measure that summarizes the complex ventricular interplay with the venous system during the filling phase ( preload ) and the arterial system during the ejection phase ( afterload ) within that same single metric . as a result , the ef as a preferred measure of ventricular performance is often questioned.1,48 to quote robotham , for instance : thus , ef , although a relatively simple measure that is intuitively easily comprehended , is an extremely complex parameter describing the entire cardiovascular system and requires additional study.5 concentrating more specifically on hf , it is noted that despite recent insights into the recognition of the importance of lv reverse remodeling in hf , many clinicians do not consider simple measurements of lv structure ( ie , lv volume ) in their routine clinical decision - making process.9 instead , they often rely on ef when making decisions about medical and surgical treatment options.9 rather than combining the end - diastolic volume ( edv ) and the end - systolic volume ( esv ) of the ventricle in the single percentage of the ef , some authors advocate to study ventricular performance on the basis of both lv volumes by using a graph with the edv on the abscissa and the esv on the ordinate.1,10 recently , a linear relationship esv = q+sedv(1)with slope s and intercept q , was reported for a study concerning 34 hfpef patients and 29 hfref patients ( fig . each group clustered around its own regression line with a quite small overlap in the esv versus edv graph and , moreover , the slopes of these two lines were reported to be significantly different.8 to be specific , kerkhof reports a slope of 0.35 in hfpef versus a slope of 0.80 in hfref ( p < 0.0004 ) and an intercept of 6.97 ml / m in hfpef versus 19.24 ml / m in hfref.8 clearly , the ef and the slope of the regression line in the edv esv graph are related , namely ef = edvesvedv = edv(q+sedv)edv=1(s+qedv)(2 ) for a vanishing intercept ( q = 0 ) , this relation shows that ef equals 1 minus the slope s in the esv edv graph . as expected , figure 1 shows a steeper regression line for the hfref group ( ef < 50% ) than for the hfpef group ( ef 50% ) . however for a nonvanishing intercept ( q 0 ) , the relation between ef and s may be strongly influenced by edv . indeed , from figure 1 significant nonvanishing intercepts for both groups can be inferred , and hence , the presumed simple relation between ef and s is seriously distorted by prevailing values of edv . and , therefore , the ef and the esv edv graph must be considered as different and noncomparable metrics representing the volumes edv and esv . clearly , the ef and the esv edv graph appear to reflect quite differently the relationship between edv and esv and , with that , the ( patho-)physiological mechanisms of ventricular performance in the hf spectrum . therefore , this study aims to identify the dependence of the ef and the edv esv graph on the major determinants of ventricular performance , in particular , for the phenotypes hfpef and hfref , and preferable by using a simple mathematical model of the cardiovascular system . using a monoventricular model , first the major determinants are identified and second the dependence of the ef and the edv esv graph on these determinants is calculated . our model of the cardiovascular system consists of the systemic circulation only , with the atrium and ventricle being filled from a simple constant pressure source and the heart ejecting into a circulatory system . the model is discussed in detail in appendix a. in each simulation run , the model provides results on edv , esv , sv , ef , and end - diastolic pressure edp . the outcomes are classified as follows ( 2 ) : hfpef , if ef 50% , edv < 175 ml and edp > 16 mmhg; hfref , if ef < 50% and edp > 16 mmhg.and any result of a simulation run outside of these constraints was neglected . hfpef , if ef 50% , edv < 175 ml and edp > 16 mmhg ; hfref , if ef < 50% and edp > 16 mmhg . in order to be able to guide the model simulations , one aspect of the model needs explanation here . the single - sided cardiac unit consists of a left atrium and a lv together with the mitral and aortic valves . volume characteristics.11 this model reads as : p(t)=e(t){v(t)v0}(3)where p(t ) is the time - dependent ventricular pressure ( mmhg ) , v(t ) the time - dependent ventricular volume ( ml ) , v0 the ( extrapolated ) ventricular volume ( ml ) at zero ventricular pressure , and e(t ) the time - dependent ventricular elastance ( mmhg / ml ) . note that this relation holds for every point in time during the cardiac cycle and , in particular , for the end - systolic moment , in which case the relation is referred to as the end - systolic pressure volume relationship . for the lv , senzaki et al provide a formulation enabling to express the elastance as a function of time , namely in terms of a fourier series.12 we will use the suga sagawa elastance model to investigate the major determinants of edv and esv and , by the same token , those of the ef ( see below in this section ) . in order to identify the major determinates of ef and the edv esv graph , we reason as follows : ef = edvesvedv=1esvedv=1min{v(t)}max{v(t)}(4)where t represents time ( s ) and v(t ) is the time - dependent ventricular volume ( ml ) . clearly , edv is the maximum of v(t ) during diastole ( denoted as max{v(t ) } ) and esv is the minimum of v(t ) during systole ( denoted as min{v(t ) } ) . in order to relate the edv and esv to ventricular elastance , suga sagawa s pressure volume relation p(t ) = e(t ) { v(t ) v0 } is used.13 upon rewriting the suga sagawa s relation , the edv is edv=(1)max{v(t)}=(2)max{p(t)e(t)+v0}=(3)max{p(t)e(t)}+v0(4)max{p(t)}min{e(t)}+v0=(5)pfillemin+v0=(6)pfillemin(1+eminv0pfill)(5)with the following rationales at the numbered equal signs : ( 1 ) edv is the maximum ventricular volume during diastole ; ( 2 ) a rewritten form of suga sagawa s pressure volume relation ; ( 3 ) the maximum of the sum of a ratio and a constant equals the maximum of the ratio summed to the constant ; ( 4 ) by application of the general rule that the maximum of a ratio is smaller than or equal to the ratio of the maximum of the fraction s numerator divided by the minimum of the fraction s denominator ; ( 5 ) the maximum pressure of the ventricle during diastole is the filling pressure pfill ( mmhg ) ( neglecting the atrial kick ) and the minimum ventricular elastance is defined as emin ( mmhg / ml ) ; ( 6 ) rewriting in a more convenient form by factoring the term pfill / emin . likewise , and mutatis mutandis , the esv is found esv=(1)min{v(t)}=(2)min{p(t)e(t)+v0}=(3)min{p(t)e(t)}+v0(4)min{p(t)}max{e(t)}+v0=(5)pdiasemax+v0=(6)pdiasemax(1+emaxv0pdias)(6)with the following rationales at the numbered equal signs : ( 1 ) esv is the minimum ventricular volume during systole ; ( 2 ) a rewritten form of suga sagawa s pressure volume relation ; ( 3 ) the minimum of the sum of a ratio and a constant equals the minimum of the ratio summed to the constant ; ( 4 ) by application of the general rule that the minimum of a ratio is larger than or equal to the ratio of the minimum of the fraction s numerator divided by the maximum of the fraction s denominator ; ( 5 ) the minimum pressure of the ventricle during ejection is equal to the end - diastolic pressure in the aorta , ie , pdias ( mmhg ) and emax ( mmhg / ml ) is the maximum ventricular elastance and referred to as the maximum systolic elastance ; ( 6 ) rewriting in a more convenient form by factoring the term pdias / max . ( 4 ) : ef = edvesvedv=1esvedv1pdiasemax+v0pfillemin+v0=1(pdiasemax)(1+emaxpdiasv0)(pfillemin)(1+eminpfillv0)1(pdiasemax)(pfillemin)(8 ) in eqns . ( 7 ) and ( 8) the last approximation holds , only if v0 vanishes ( ie , v0 = 0 ) or if the numerical values of the ratios emax / pdias and emin / pfill are of equal size because , in either way , the terms ( 1 + v0emax / pdias ) and ( 1 + v0emin / pfill ) cancel . typical values are calculated from table 1 . in hfpef : emax / pdias = 0.11 ml and emin / pfill = 0.01 ; in hfref : emax / pdias = 0.02 ml and emin / pfill = 0.005 ml . notice that emax in table 1 is estimated assuming that v0 = 0 and that , therefore , these values are rough estimates only . so , these numerical values indicate that the terms ( 1 + v0emax / pdias ) and ( 1 + v0emin / pfill ) only cancel in the unlikely case of a vanishing v0 . therefore , the final approximations in eqns . ( 7 ) and ( 8) are somewhat questionable . to summarize these mathematical results : there is no reason to assume that v0 vanishes in this model simulation study.the major determinants of the edv ( see eq . ( 5 ) ) are the ratio of the filling pressure ( pfill ) over the elastance during diastole ( emin ) and the zero - pressure volume of the ventricle ( v0 ) . note that the ratio pfill / emin can be interpreted as the preload normalized to the diastolic ventricular elastance.the major determinants of the esv ( see eq . ( 6 ) ) are the ratio of the diastolic aortic pressure ( pdias ) over the elastance during systole ( emax ) and the zero - pressure volume of the ventricle ( v0 ) . note that the ratio pdias / emax can be interpreted as an indicator of the afterload normalized to the systolic ventricular elastance.because the ef and the esv edv graphs are defined in terms of the edv and esv , both the ef and the esv edv graphs share common determinants . note that this sharing of determinants does not imply the equivalence of the ef and the esv edv graph . ( 7 ) ) predicts a linear relation in the esv edv graph with a small or even vanishing intercept and slope smaller than 1 and , moreover , infers an elastance - normalized afterload pdias / emax smaller than the elastance - normalized preload pfill / emin . the major determinants of the edv ( see eq . ( 5 ) ) are the ratio of the filling pressure ( pfill ) over the elastance during diastole ( emin ) and the zero - pressure volume of the ventricle ( v0 ) . note that the ratio pfill / emin can be interpreted as the preload normalized to the diastolic ventricular elastance . ( 6 ) ) are the ratio of the diastolic aortic pressure ( pdias ) over the elastance during systole ( emax ) and the zero - pressure volume of the ventricle ( v0 ) . note that the ratio pdias / emax can be interpreted as an indicator of the afterload normalized to the systolic ventricular elastance . because the ef and the esv edv graphs are defined in terms of the edv and esv , both the ef and the esv edv graphs share common determinants . note that this sharing of determinants does not imply the equivalence of the ef and the esv edv graph . see the introduction for the relation between ef and the slope of the esv edv graph . ( 7 ) ) predicts a linear relation in the esv edv graph with a small or even vanishing intercept and slope smaller than 1 and , moreover , infers an elastance - normalized afterload pdias / emax smaller than the elastance - normalized preload pfill / emin . on the basis of these findings edv plot by varying the abovementioned determinants ( pdias , emax , pfill , emin , and v0 ) in order to identify major factors contributing to the distinct lv volume patterns manifested in hfpef relative to hfref ( cf . our model of the cardiovascular system consists of the systemic circulation only , with the atrium and ventricle being filled from a simple constant pressure source and the heart ejecting into a circulatory system . the model is discussed in detail in appendix a. in each simulation run , the model provides results on edv , esv , sv , ef , and end - diastolic pressure edp . the outcomes are classified as follows ( 2 ) : hfpef , if ef 50% , edv < 175 ml and edp > 16 mmhg; hfref , if ef < 50% and edp > 16 mmhg.and any result of a simulation run outside of these constraints was neglected . hfpef , if ef 50% , edv < 175 ml and edp > 16 mmhg ; hfref , if ef < 50% and edp > 16 mmhg . in order to be able to guide the model simulations , one aspect of the model needs explanation here . the single - sided cardiac unit consists of a left atrium and a lv together with the mitral and aortic valves . volume characteristics.11 this model reads as : p(t)=e(t){v(t)v0}(3)where p(t ) is the time - dependent ventricular pressure ( mmhg ) , v(t ) the time - dependent ventricular volume ( ml ) , v0 the ( extrapolated ) ventricular volume ( ml ) at zero ventricular pressure , and e(t ) the time - dependent ventricular elastance ( mmhg / ml ) . note that this relation holds for every point in time during the cardiac cycle and , in particular , for the end - systolic moment , in which case the relation is referred to as the end - systolic pressure volume relationship . for the lv , senzaki et al provide a formulation enabling to express the elastance as a function of time , namely in terms of a fourier series.12 we will use the suga sagawa elastance model to investigate the major determinants of edv and esv and , by the same token , those of the ef ( see below in this section ) . in order to identify the major determinates of ef and the edv esv graph , we reason as follows : ef = edvesvedv=1esvedv=1min{v(t)}max{v(t)}(4)where t represents time ( s ) and v(t ) is the time - dependent ventricular volume ( ml ) . clearly , edv is the maximum of v(t ) during diastole ( denoted as max{v(t ) } ) and esv is the minimum of v(t ) during systole ( denoted as min{v(t ) } ) . in order to relate the edv and esv to ventricular elastance , suga sagawa s pressure volume relation p(t ) = e(t ) { v(t ) v0 } is used.13 upon rewriting the suga sagawa s relation , the edv is edv=(1)max{v(t)}=(2)max{p(t)e(t)+v0}=(3)max{p(t)e(t)}+v0(4)max{p(t)}min{e(t)}+v0=(5)pfillemin+v0=(6)pfillemin(1+eminv0pfill)(5)with the following rationales at the numbered equal signs : ( 1 ) edv is the maximum ventricular volume during diastole ; ( 2 ) a rewritten form of suga sagawa s pressure volume relation ; ( 3 ) the maximum of the sum of a ratio and a constant equals the maximum of the ratio summed to the constant ; ( 4 ) by application of the general rule that the maximum of a ratio is smaller than or equal to the ratio of the maximum of the fraction s numerator divided by the minimum of the fraction s denominator ; ( 5 ) the maximum pressure of the ventricle during diastole is the filling pressure pfill ( mmhg ) ( neglecting the atrial kick ) and the minimum ventricular elastance is defined as emin ( mmhg / ml ) ; ( 6 ) rewriting in a more convenient form by factoring the term pfill / emin . likewise , and mutatis mutandis , the esv is found esv=(1)min{v(t)}=(2)min{p(t)e(t)+v0}=(3)min{p(t)e(t)}+v0(4)min{p(t)}max{e(t)}+v0=(5)pdiasemax+v0=(6)pdiasemax(1+emaxv0pdias)(6)with the following rationales at the numbered equal signs : ( 1 ) esv is the minimum ventricular volume during systole ; ( 2 ) a rewritten form of suga sagawa s pressure volume relation ; ( 3 ) the minimum of the sum of a ratio and a constant equals the minimum of the ratio summed to the constant ; ( 4 ) by application of the general rule that the minimum of a ratio is larger than or equal to the ratio of the minimum of the fraction s numerator divided by the maximum of the fraction s denominator ; ( 5 ) the minimum pressure of the ventricle during ejection is equal to the end - diastolic pressure in the aorta , ie , pdias ( mmhg ) and emax ( mmhg / ml ) is the maximum ventricular elastance and referred to as the maximum systolic elastance ; ( 6 ) rewriting in a more convenient form by factoring the term pdias / max . ( 4 ) : ef = edvesvedv=1esvedv1pdiasemax+v0pfillemin+v0=1(pdiasemax)(1+emaxpdiasv0)(pfillemin)(1+eminpfillv0)1(pdiasemax)(pfillemin)(8 ) in eqns . ( 7 ) and ( 8) the last approximation holds , only if v0 vanishes ( ie , v0 = 0 ) or if the numerical values of the ratios emax / pdias and emin / pfill are of equal size because , in either way , the terms ( 1 + v0emax / pdias ) and ( 1 + v0emin / pfill ) cancel . : emax / pdias = 0.11 ml and emin / pfill = 0.01 ; in hfref : emax / pdias = 0.02 ml and emin / pfill = 0.005 ml . notice that emax in table 1 is estimated assuming that v0 = 0 and that , therefore , these values are rough estimates only . so , these numerical values indicate that the terms ( 1 + v0emax / pdias ) and ( 1 + v0emin / pfill ) only cancel in the unlikely case of a vanishing v0 . therefore , the final approximations in eqns . ( 7 ) and ( 8) are somewhat questionable . to summarize these mathematical results : there is no reason to assume that v0 vanishes in this model simulation study.the major determinants of the edv ( see eq . ( 5 ) ) are the ratio of the filling pressure ( pfill ) over the elastance during diastole ( emin ) and the zero - pressure volume of the ventricle ( v0 ) . note that the ratio pfill / emin can be interpreted as the preload normalized to the diastolic ventricular elastance.the major determinants of the esv ( see eq . ( 6 ) ) are the ratio of the diastolic aortic pressure ( pdias ) over the elastance during systole ( emax ) and the zero - pressure volume of the ventricle ( v0 ) . note that the ratio pdias / emax can be interpreted as an indicator of the afterload normalized to the systolic ventricular elastance.because the ef and the esv edv graphs are defined in terms of the edv and esv , both the ef and the esv edv graphs share common determinants . note that this sharing of determinants does not imply the equivalence of the ef and the esv edv graph . ( 7 ) ) predicts a linear relation in the esv edv graph with a small or even vanishing intercept and slope smaller than 1 and , moreover , infers an elastance - normalized afterload pdias / emax smaller than the elastance - normalized preload pfill / emin . ( 5 ) ) are the ratio of the filling pressure ( pfill ) over the elastance during diastole ( emin ) and the zero - pressure volume of the ventricle ( v0 ) . note that the ratio pfill / emin can be interpreted as the preload normalized to the diastolic ventricular elastance . the major determinants of the esv ( see eq . ( 6 ) ) are the ratio of the diastolic aortic pressure ( pdias ) over the elastance during systole ( emax ) and the zero - pressure volume of the ventricle ( v0 ) . note that the ratio pdias / emax can be interpreted as an indicator of the afterload normalized to the systolic ventricular elastance . because the ef and the esv edv graphs are defined in terms of the edv and esv , both the ef and the esv edv graphs share common determinants . note that this sharing of determinants does not imply the equivalence of the ef and the esv edv graph . see the introduction for the relation between ef and the slope of the esv edv graph . ( 7 ) ) predicts a linear relation in the esv edv graph with a small or even vanishing intercept and slope smaller than 1 and , moreover , infers an elastance - normalized afterload pdias / emax smaller than the elastance - normalized preload pfill / emin . on the basis of these findings edv plot by varying the abovementioned determinants ( pdias , emax , pfill , emin , and v0 ) in order to identify major factors contributing to the distinct lv volume patterns manifested in hfpef relative to hfref ( cf . figure 2 shows the simulation results of pressures , volumes , and elastances as a function of time and , additionally , the lv pressure volume and elastance volume loops for a control and the hfpef and hfref groups . note that the model parameters are chosen such that the latter two groups represent values as given in table 1 . the control represents the values provided by katz,14 ie , filling pressure of 7 mmhg , maximum ventricular pressure of 120 mmhg , end - systolic pressure of 95 mmhg , end - diastolic pressure in the aorta of 80 mmhg , edv 120 ml including atrial kick of 20 ml , end - systolic volume 50 ml , and an ef of 60% . note in particular the large difference in the maximum ventricular elastance in the three simulations . the simulations closely resemble the data in table 1 and those reported by katz , with the exception of the pressures in hfref . note that , the elastance pressure plot and the pressure volume plot for the ventricle in figure 2 clearly show the constant value of the elastance during diastole and , consequently , a linear dependence of the pressure on the volume . figures 37 show the changes in the cardiovascular model when one of the model parameters is varied . to be specific , in figures 3 , 4 , 5 , 6 , and 7 , respectively , the variable varied is the diastolic elastance ( emin ) , the filling pressure , the systolic elastance , the capillary resistance ( rc ) , and finally , the zero - pressure ventricular volume ( v0 ) . in each of these figures , the following is shown : first and second rows show the lv pressure volume and elastance volume loops , respectively. third row shows the arterial and ventricular pressures and the ventricular volumes , the sv , and the ef as functions of the running variable. fourth row shows the esv edv plot ( with slope and intercept of regression line ) and the starling curve with sv as function of edv . first and second rows show the lv pressure volume and elastance volume loops , respectively . third row shows the arterial and ventricular pressures and the ventricular volumes , the sv , and the ef as functions of the running variable . fourth row shows the esv edv plot ( with slope and intercept of regression line ) and the starling curve with sv as function of edv . note that in figure 6 , the ventricular volume and ventricular ef ( third row , third and fourth graph ) are shown as a function of diastolic pressure in the aorta . the rationale for this exception is that end - diastolic pressure in the aorta ( pdias ) was identified as one of the determinants of esv but pdias is a variable calculated in a simulation with the model while the other running variables are parameters set in the model . in order to vary pdias , the parameter capillary resistance ( rc ) was varied instead with the results plotted with pdias on the abscissa . in figures 37 , the graphs with ventricular volume versus the varying parameter ( third row , third graph from left ) show that the edv and esv vary in correspondence with eqns . ( 5 ) and ( 6 ) ; ie , for edv a proportional relation with filling pressure ( pfill ) and an inverse proportional relation with diastolic elastance ( emin ) is found , and moreover , for esv a proportional relation with diastolic pressure in the aorta ( pdias ) and an inverse proportional relation with systolic elastance ( emax ) is found . finally , for both edv and esv a proportional relation with the zero - pressure ventricular volume ( v0 ) is found . clearly , the same line is found by varying the diastolic elastance ( emin ) and filling pressure ( pfill ) , the major determinants of edv ( eq . ( 5 ) ) , and also , another line is found by varying the systolic elastance ( emax ) and diastolic pressure in the aorta ( pdias ) , the major determinants of esv ( eq . ( 6 ) ) . thus , because of the differences in slopes and intercepts of these lines in the esv edv graph , it might be possible to infer the involvement of diastolic factors ( filling pressure or diastolic elastance ) or , in contrast , systolic factors ( the diastolic pressure in the aorta or the systolic elastance ) on the basis of the location of a patient s edv and in esv in the esv edv graph . the ef esv graphs in figures 37 show that , although the graphs are rather different , the results ( ie , lines ) overlap in a common region of the graph . so , from the position of the line in the ef esv graphs , it can not be interpreted which of the factors was involved . this is a disadvantage of the ef esv graph in comparison with the esv edv graph . by using a simple mathematical model of a monoventricular cardiovascular system , this study identified the major determinants of edv and esv and , by the same token , the major determinants in the graph of esv versus edv and the ef , in particular , for the different phenotypes hfpef and hfref . by using suga ( 3 ) and a simple mathematical reasoning ( eqns . ( 5 ) and ( 6 ) ) , the diastolic elastance and the filling pressure were identified as the major determinants of edv , while the systolic elastance and the end - diastolic aortic pressure were identified as the major determinants of the esv . simulations with the cardiovascular model , indeed , clearly confirmed these findings on the major determinants of esv and edv ( see figs . elsewhere , shoucri described theoretical aspects of the relationship between ef and emax in hfpef patients with emphasis on iso - volumic pressure ( ie , the active force of the myocardium).15 unfortunately , filling pressure and elastance during systole and diastole are difficult to measure in clinical practice . but , in contrast , edv and esv are far more easily measured in standard clinical routine using modern imaging modalities . thus , edv and esv can be regarded as measures that are closely related ( eqns . ( 5 ) and ( 6 ) ) to the major determinants of the filling and diastolic pressures and the diastolic and systolic elastances . in fact , by virtue of our findings in eqns . ( 5 ) and ( 6 ) , the edv is related to a normalized preload , ie , the filling pressure relative to the diastolic elastance ( neglecting the zero - pressure ventricular volume v0 ) , while the esv is related to as a normalized afterload , ie , the end - diastolic pressure in the aorta relative to the systolic elastance ( neglecting the v0 ) . so , edv and esv represent , in a normalized form , the classical concepts , of preload and afterload , respectively . in using esv and edv in clinically diagnostic decision making , a graph with the esv on the ordinate and edv on the abscissa ( the so - called esv edv graph ) allows the representation of esv and edv independently , and , with that , the normalized preload and afterload . this study showed that the hfpef and hfref are represented in this esv edv graph with different regression lines and , moreover , that the slope of the regression lines is related to the ef ( see eq . that is , the lower the ef , the steeper the regression line , although this relation is obscured considerably by the edv . so , the esv edv graph allows the differentiation between the phenotypes hfpef and hfref ( fig . this differentiation of hfpef and hfref in the esv edv graph , on the basis of the esv and edv , can be given a more ( patho-)physiological interpretation . table 1 shows that the major difference between hfpef and hfref as regards the maximum elastance ( ie , the systolic elastance ) is 7.98 mmhg / ml in hfpef and 1.13 mmhg / ml in hfref , while in both groups the left ventricular end - diastolic pressure ( lv edp ) , as a measure of the filling pressure , is elevated to around 22.5 mmhg ( much higher than 7 mmhg typically found in healthy controls ) and , finally , the diastolic pressure in the aorta is 73 mmhg ( around 7080 mmhg typically found in healthy controls ) . this finding shows that the normalized afterload ( end - diastolic pressure in the aorta relative to the systolic elastance ) is significantly increased from 9.2 ml in hfpef to 64 ml in hfref ( from table 1 by dividing diastolic pressure by emax ) , a finding that is reflected by the esv , ie , 27.66 ml in hfpef and 147.02 ml in hfref ( table 1 ) . note that the ratios are almost equal , ie , 9.2/64 = 0.15 fairly well compares with 27.66/147.02 = 0.19 . so , this confirms that the esv may be given a ( patho-)physiological interpretation as normalized afterload with end - diastolic aortic pressure and systolic elastance as major determinants . likewise , the pathophysiological interpretation of the normalized preload ( filling pressure relative to the diastolic elastance ) could be confirmed by experimental findings . but , from the reported filling pressure of around 22.5 mmhg in both groups , considerably higher than in healthy controls , and the significantly increased edv of 224.95 ml in hfref compared to 113.47 ml in hfpef ( table 1 ) , we calculate a diastolic elastance of 0.2 mmhg / ml in hrref and of 0.1 mmhg / ml in hfpef ( calculated from table 1 by dividing lvedp by edv ) . for an equally increased filling pressure and a two - fold larger diastolic elastance , the normalized preload in hfref is expected to be twice as large as in hfpef , a finding reflected by edv in hfref and hfpef . we infer that the edv may be given a ( patho-)physiological interpretation as normalized preload with filling pressure and diastolic elastance as major determinants . so , the esv edv graph differentiates between hfpef and hfref and , moreover , may be given a ( patho- ) physiological interpretation . in contrast to the esv edv graph that represents the esv and edv independently , the ef summarizes the esv and edv in one single metric and with that the ef summarized the complex interplay of cardiac filling and ejection in one metric . in terms of our model , the interplay of systolic and diastolic elastances as well as the filling and the end - diastolic aortic pressures , as the major determinants of esv and edv , are mapped into a single number with a size between 0 and 100% . ( 8) ) , it is highly implausible that each ( patho- ) physiological condition in one of the determinants is mapped into a unique numerical value of the ef and , consequently , it is equally implausible that a given value of the ef can be interpreted unambiguously in terms of the underlying ( patho- ) physiological mechanism . loss of insight is the price paid for summarizing too briefly . from these model - based results , one may infer that a plot of a patient s esv and edv in the esv edv graph allows a clinical differentiation between diastolic or systolic involvement while the ef does not offer such a distinction . so , the esv edv graph allows a better tool for the understanding of hfref and hfpef . however , this model - based result is a guidance for future clinical research that should establish the practical value of the esv edv graph over the single metric of ef.1 the advantage of mathematical modeling is the experimenter s full control of all parameters and conditions in the model and , with that , the simplicity to carry out a simulation experiment that is not so easily clinically performed . the validity of the modeling study needs , however , to be demonstrated by either the validity of model itself or by the validity of the obtained results . in this study , the validity of the model is based on the following considerations : ( 1 ) the model is built up of well - established components ( ie , suga sagawa s elastance model of the ventricle , senzaki et al s normalized elastance function , and the generally accepted three - element windkessel model of the arterial circulation).17 ( 2 ) by comparing the simulation results ( fig . 3 ) with the pressure and volumes in hfpef and hfref ( table 1 ) , we recognize the validity of the model . sagawa s elastance model , major determinants of the esv and edv were identified and these major determinants correspond with the simulations as well as the pathophysiological knowledge , ie , ( a ) diastolic elastance and filling pressure together mainly determine edv , while ( b ) systolic elastance and end - diastolic pressure in the aorta together mainly determine esv . in this perspective of the model s validity , we suggest to use our findings as a guidance in future clinical studies in order to provide supporting evidence . although the model mimics the characteristics of control , hfpef , and hfref patients quite accurately , this study clearly has its limitations because of the assumptions made in our mathematical model . to be more specific , ( 1 ) ventricular heart connected to a simple model of the filling system of the heart and a three - element windkessel extended with a venous part , to model the systemic circulation . ( 2 ) the system is an open loop with the simple model of a filling system that is not connected to the venous part of systemic circulation . ( 3 ) the model represents a denervated heart and a circulation without many control mechanisms , eg , neuro - humoral control of heart rate , flow resistance , filling pressure , and cardiac contractility . for some of the limitations , we could compensate by choosing adequate parameters eg , the different filling pressures , heart rate , maximum elastance in hfpef and hfref while other features will be incorporated in a future more advanced model eg , a model with a closed circulation of both the systemic and the pulmonary circulation including neuro - humoral mechanisms . it is to be expected that a more advanced model will reveal more details in the determining factors of esvs and edvs ( and with that in the esv nevertheless , it is hoped that the present insight will provide an adequate guidance for both clinical and modeling studies in the future . by using a simple mathematical model of the circulation , the filling pressure and diastolic elastance are identified as the major determinants of edv , while the end - diastolic aortic pressure in the aorta and the systolic elastance are identified as the major determinants of the esv . by mathematical analysis and with simulations , the dependence of the ef and the esv edv graph on the determinants was studied . it was found that the esv edv graph allows the separation between diastolic and systolic dysfunction , while these two pathologies are intricately summarized in the single metric of ef .

in left ventricular heart failure , often a distinction is made between patients with a reduced and a preserved ejection fraction ( ef ) . as ef is a composite metric of both the end - diastolic volume ( edv ) and the end - systolic ventricular volume ( esv ) , the lucidity of the ef is sometimes questioned . as an alternative , the esv edv graph is advocated . this study identifies the dependence of the ef and the edv esv graph on the major determinants of ventricular performance . numerical simulations were made using a model of the systemic circulation , consisting of an atrium ventricle valves combination ; a simple constant pressure as venous filling system ; and a three - element windkessel extended with a venous system . esv edv graphs and efs were calculated using this model while varying one by one the filling pressure , diastolic and systolic ventricular elastances , and diastolic pressure in the aorta . in conclusion , the esv edv graph separates between diastolic and systolic dysfunction while the ef encompasses these two pathologies . therefore , the esv edv graph can provide an advantage over ef in heart failure studies .

Introduction Methods Cardiovascular model Major determinants Results Discussion Limitations Conclusion

PMC4745549

bread is universally accepted as a very convenient form of food that is important to all populations . its origin dates back to the neolithic era and is still one of the most consumed and acceptable staple food products in all parts of the world . it is a good source of nutrients , such as macronutrients ( carbohydrates , protein , and fat ) and micronutrients ( minerals and vitamins ) that are essential for human health . in nigeria it has been hitherto produced from wheat as a major raw material . in nigeria , wheat production is limited and wheat flour is imported to meet local flour needs for bakery products . thus , huge amount of foreign exchange is used every year for import of wheat . efforts have been made to promote the use of composite flours in which flour from locally grown crops and high protein seeds replace a portion of wheat flour for use in bread , thereby decreasing the demand for imported wheat and helping in producing protein - enriched bread . most tropical cereal grains and some tubers have been used to make composite flour for bread making . although there is now a substantial amount of composite bread , such bread still requires at least 70% wheat flour to be able to rise because wheat contains gluten [ 4 , 5 ] . olaoye reported the use of composite flour of wheat , plantain , and soybeans in bread making . according to the authors , good quality and acceptable baked products could be derived from composite flours with up to certain levels of breadfruit flour substitution in wheat flour . in fact , several attempts have been made to produce cookies from different types of composite flours . in countries where malnutrition poses a serious problem especially among children , composite flours which have better nutritional quality it has also been reported that composite flour can be made from legumes and nuts and root and tubers such as yam , cassava , and sweet potatoes and sensory qualities of yam and sweet potatoes flours have been reported . potato is a food crop with potential for partial replacement of wheat in bread making . uncooked potato flour prepared by low - cost solar dehydration technology has a long shelf life and high nutritional quality , which could be valuable in cereal - based human diets , including bread . sweet potato flour can serve as a source of energy and nutrients ( carbohydrates , beta - carotene , and minerals ) and can add natural sweetness , color , flavor , and dietary fiber to processed food products . addition of various proportions of potato flour in wheat flour can increase the nutritive values in terms of fibre and carotenoids . the aim of this study was to replace part of the wheat flour in bread by potato flour in order to increase the fibre and other nutrients . the dry starchy yellow - fleshed cultivar of sweet potato ( ipomea batatas ) and irish potato ( solanum tuberosum ) and wheat flour were purchased from a local market in offa , kwara state , nigeria , and the central market , minna , niger state , nigeria , respectively , in polythene bags , and transported to the laboratory for processing . the potatoes were washed under running tap water ( to free them of adhering soil particles ) , air dried , and stored at 12c before use . the potato flours were prepared from solar - dried slices according to the method outlined in [ 9 , 11 ] . the baking formula was 50.4 g53.2 g wheat flour ( 9095% ) , 1.8 g5.6 g potato flour ( 510% ) , 36% water , 3.4% sugar , 1% skim milk powder , 1% salt and 1% yeast , similar to that of . all ingredients were mixed in a kenwood mixer ( model a 907d ) for 5 minutes . the dough was fermented in bowls , covered with wet clean muslin cloth for 55 minutes at warm temperature , punched , scaled to 250 g dough pieces , proofed in a proofing cabinet at 30c for 90 minutes and 85% relative humidity , and baked at 250c for 30 minutes . the baked bread samples were then depanned , cooled at ambient temperature and put in ziploc bags prior to analysis . total mesophilic ( total viable bacterial counts ) and fungi counts ( yeast and mould counts ) were carried out on the bread samples to determine the microbial load of the samples as described by american public health association . subsamples were diluted decimally and 0.1 ml aliquots were spread plated on nutrient agar ( na ) , macconkey agar ( mca ) , and potato dextrose agar ( pda ) for the enumeration of aerobic viable bacteria , coliforms , and fungi , respectively . the na and mca plates were incubated at 37c for 2448 hours while pda plates were incubated at room temperature ( 28 2c ) for 35 days . the colonies were then counted and expressed as colony forming units per gram ( cfu / g ) of samples . observed colonies were subcultured repeatedly on media used for primary isolation to obtain pure cultures . the bacterial isolates were characterized using gram reaction and biochemical tests and were identified by comparing their characteristics with those of known taxa as outlined in bergey 's manual of systematic bacteriology . the fungal isolates were characterized based on macroscopic and microscopic examination and identified using the scheme of . the concentration of protein in the digested sample was determined spectrophotometrically and calculated as ( 1)% crude protein=(titre of sampleblank)0.0114.0076.2510weight of sample100 . determination of the crude fibre and the ash content in the bread samples was carried out using the standard methods described by aoac . the crude fibre , crude protein , and the fat content were subtracted from organic matter ; the remainder accounted for carbohydrates : ( 2)% carbohydrate=100protein(%)+fat(%)+ash(% ) . sensory evaluation was preformed 24 hours after baking to evaluate loaf color , crust , aroma , crumb texture , taste , and overall acceptability of the bread sample . a panel of ten judges ( using a questionnaire ) of regular bread consumers using the hedonic scale product was set up . the panel was set up in three sets ( to obtain three replicates ) and the sensory scores were analyzed statistically . the bread samples were stored under ambient temperature ( 26c33c ) and observed for 10 days . difference in proximate composition and sensory scores was detected using one - way analysis of variance ( anova ) . the total bacterial counts of the bread samples ranged from 3.0 10 cfu / g to 1.0 10 cfu / g with the highest being recorded for bread made from 100% sweet potato flour ( spf ) while the lowest counts ( 3.0 10 cfu / g ) were obtained in bread made from 100% irish potato flour ( ipf ) . bacteria were not detected in bread baked from 100% wheat flour ( wf ) ; see wf / spf1 , wf / spf2 , and wf / ipf2 in table 2 . the fungi counts ranged from 8.0 10 cfu / g to 1.2 10 cfu / g with the highest counts recorded for bread baked from wf / spf2 while the lowest counts ( 8.0 10 cfu / g ) were observed in bread baked from spf / ipf ( table 2 ) . fungi were not detected in bread baked from wf , wf / spf1 , wf / spf2 , and wf / ipf2 . three species of bacteria were isolated which include bacillus subtilis , micrococcus sp . , and staphylococcus aureus . for the fungi , aspergillus niger , penicillium stolonifer , rhizopus nigricans , and mucor sp . were isolated . the highest moisture content of 16.0% was obtained in wf / ipf1 bread while the lowest moisture level ( 11.50% ) was obtained in spf / ipf . the respective values obtained in wf , spf , ipf , and wf / ipf bread were 15.15% , 15.31% , 14.945 , and 12.63% ( table 3 ) . the highest value for lipid ( 4.20% ) was obtained in wf / spf1 bread while the lowest value of 2.35% was recorded in spf / ipf bread . the crude fibres of 1.33% , 1.84% , 1.42% , 0.90% , 2.00% , 1.87% , 1.92% , and 2.0% were obtained in the wf , spf , ipf , sp / ipf , wf / spf1 , wf / ipf1 , wf / spf2 , and wf / ipf2 bread samples . the highest ash content ( 3.75% ) was obtained in ipf bread , while the lowest ash content ( 1.50% ) was obtained in sp / ipf bread . the carbohydrate content was higher in wf / spf1 ( 87.08% ) than in the rest of the flour blends , but it was , however , the least ( 70.10% ) in spf / ipf bread ( table 3 ) . the statistical analysis of the data revealed that the proximate composition of the various blends was significantly different ( p < 0.05 ) . the mean sensory scores for each quality attribute evaluated ( color , aroma , taste , crust , texture , and general acceptability ) of the bread samples prepared from the wheat / potato blends are presented in table 4 . the statistical analysis of the data showed that there were significant differences ( p < 0.05 ) among the wheat / potato blends with the exception of the wf and wf / spf2 for color , as well as wf / spf2 and wf / ipf2 for aroma . the scores also indicated that bread baked from wf was more acceptable than that from other blends . however , this was closely followed by bread baked from wf / ipf2 and wf / spf2 blends . the bread produced lasted for 68 days ( table 5 ) before obvious spoilage was noticed . wf , wf / spf2 , and wf / ipf2 lasted for 6 days while ipf and wf / ipf1 lasted for 7 days before spoilage occurred . it was also observed that spf , spf / ipf , and wf / spf1 lasted for 8 days before spoilage set in ( table 5 ) . spoilage was indicated by black , yellow , and green coloration on the bread ( suspected to be mold growth ) . when the black , yellow , and green coloring matters were stained and examined under the light microscope , they consisted of aspergillus flavus , penicillium sp . these are within the limit set by the standard organization of nigeria , which states that the counts of aerobic bacteria must not exceed 100 cfu / g and coliform growth must not be detected in bread samples . . the high bacteria population in spf ( 100% sweet potato flour ) could be due to the abundance of moisture and nutrient in the potato bread , which provide for a favorable condition for growth . the bacteria isolated from the bread samples included species of bacillus , micrococcus , and staphylococcus . the presence of the different bacterial species in the samples could have evolved during baking or from the raw ingredients used , for example , flour , sugar , and yeast . k. talaro and a. talaro reported that staphylococcus species are widely distributed in the environment and occur on the skin and nostrils of humans , from where the organisms can contaminate food . the fungi counts were higher in bread made from spf / ipf ( 50 : 50% ) than all other samples probably because of raw materials , processing , handling , and storage . this finding is in line with the study of daniyan and nwokwu who identified similar organisms in bread . the moisture content of food goes a long way in suggesting the shelf life of the product . the moisture content of bread made from wheat / irish potato flour ( wf / ipf , 90 : 10% ) was higher than that of the other flour blends . the values of the other blended samples fall within the acceptable moisture limit for dry products ( 15% ) . moisture is a very important factor in the keeping quality of bread and high moisture can have an adverse effect on storage stability . the bread sample having the highest moisture content may therefore have reduced shelf life in comparison with other samples . increase in the level of potato flour resulted in decrease in the protein content from 12.25% in 100% wheat flour bread ( wf ) to 3.75% in bread made from 50 : 50% sweet potato / irish potato flour ( spf / ipf ) . this may have been due to the low protein content of the potato flour which must have diluted the protein content of the wheat flour , thus reducing the protein level of the mixed flour . combination of wf and ipf ( 95 : 5% ) resulted in an increase in the protein content of the bread from 4.3% to 14.00% . this is similar to the earlier findings where protein content of snacks reduced with supplementation with starch based products for bread fruit flour , for plantain , and for soy flour . the sudden increase in the protein content with supplementation with 5% irish pf shows that pf can be incorporated into bread at high supplementation level and still retain its nutrient content similar to 100% wf bread . blending of wheat flour with 10% potato flour resulted in an increase in the fat content . this could be due to the fact that irish potato contains about 14% fat which could have been responsible for the slight increase in the fat content . the value of ash in bread made from wheat flour blended with potato flour was low compared to the ash content in bread made from wheat flour . the crude fibre was above the 1.5% maximum allowable fibre content of bread flour as stated by omole but fell within the 2.0% recommended by nigerian raw materials research and development council . similar results were observed by raji who also recorded low ash and crude fibre contents in cookies made by using potato flour . high ash and crude fibre contents in food depict that the material is difficult to digest in the human body . the carbohydrate content of bread increased with addition of potato flour to the wheat flour . cereals store starch as a source of energy and are low in protein , fat , and ash . the high level of carbohydrate is desirable in baked products because on heating starch granules in the presence of water , it swells and forms a gel which is important for the characteristic texture and structures of baked goods . the mean sensory scores of quality attributes of the products indicated that , generally , panelists expressed preference for three bread samples out of the eight presented . the bread samples were those made from wf ( 100% wheat flour ) , wf / spf2 ( wheat / sweet potato flour 95 : 5% ) , and wf / ipf2 ( wheat / irish potato flour , 95 : 5% ) . bread made from spf / ipf ( 50 : 50% sweet potato / irish potato flour ) , ipf ( 100% irish potato flour ) , and spf ( 100% sweet potato flour ) was highly rejected by the panelists . this shows that using either sweet or irish potato flour alone to bake bread will not be accepted , except if it is blended with wheat flour . the results of the storability of bread showed that bread made from 100% wheat flour , 10% sweet potato blended bread , and bread made from 50 : 50% irish / sweet potato could not stay longer than six days due to the presence of fungi , while other samples lasted 7 - 8 days . this shows that the use of 5% dehydrated potato flour in bread making reduces the rate of staling as a result of the ability to retain moisture in them . the incorporation of potato flour to wheat flour improved the nutritional value of the bread . bacteria and molds common in the environment contaminated the bread and led to its spoilage after 68 days ; however , sensory evaluation indicated that consumer acceptable bread could be substituted with wheat flour and dehydrated uncooked potato flour in commercial bread making without sacrificing consumer acceptability . with the use of potato flour in bread making ,

dehydrated uncooked potato ( irish and sweet ) flour was blended by weight with commercial wheat flour at 0 to 10% levels of substitution to make bread . comparative study of the microbial and nutritional qualities of the bread was undertaken . the total aerobic bacterial counts ranged from 3.0 105 cfu / g to 1.09 106 cfu / g while the fungal counts ranged from 8.0 101 cfu / g to 1.20 103 cfu / g of the sample . coliforms were not detected in the bread . bacteria isolated were species of bacillus , staphylococcus , and micrococcus while fungi isolates were species of aspergillus , penicillium , rhizopus , and mucor . the mean sensory scores ( color , aroma , taste , texture , and general acceptability ) were evaluated . the color of the bread baked from wf / ipf2 ( wheat / irish potato flour , 95 : 5% ) blend was preferred to wf ( wheat flour , 100% ) while wf / spf1 ( wheat / sweet potato flour , 100% ) and wf / ipf1 ( wheat / irish potato flour , 90 : 10% ) aroma were preferred to wf . however , the bread baked from wf , wf / ipf2 ( wheat flour / irish potato flour , 95 : 5% ) , and wf / spf2 ( wheat / sweet potato flour , 95 : 5% ) was more acceptable than other blends . the use of hydrated potato flour in bread making is advantageous due to increased nutritional value , higher bread yield , and reduced rate of staling .

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

PMC3013692

polyglutamine ( polyq ) repeats are implicated in several neurodegenerative diseases , including huntington s disease and several spinocerebellar ataxia s . it is commonly thought that a toxic gain - of - function mechanism is triggered by the presence of a polyq tract , involving a conformational change within the protein and the formation and deposition of -sheet rich amyloid - like fibrils ( 13 ) . the length of the polyq repeat is critical to pathogenesis ; however , there is evidence that other protein factors , including the location , type and number of flanking domains can modulate pathogenesis ( 410 ) . although there are many human polyq - containing proteins ( 11 ) , only nine polyq - containing proteins are implicated in pathogenesis , and the precise repeat threshold to pathogenesis varies within the disease subset , for example , a 37 glutamine repeat is sufficient to lead to huntington 's disease , while sca3 results only when the polyq repeat expands to 45 or greater ( 1214 ) . many other human , non - disease related proteins contain polyq repeats , which are intrinsically prone to expansion at the genetic level ( 11,15,16 ) . in fact , a 40 glutamine repeat is the normal allele present in forkhead box p2 transcription factor ; a protein that has not been found to be associated with a polyq disease ( 17,18 ) . this evidence has led to the hypothesis that protein characteristics modulate the propensity of polyq - containing proteins to aggregate and cause disease . to investigate the variable characteristics of polyq proteins we have performed a bioinformatics investigation of the protein context of polyglutamine repeats , and constructed a web - accessible database of all human proteins containing a polyq repeat greater than seven glutamines in length , termed polyq. the polyq database provides a tool to compare the polyq repeat location , the occurrence / type of domains and the number of domain repeats present across disease and non - disease proteins . polyq was created using open - source mysql relational database server software , version 5.0.82 ( http://www.mysql.com ) , running on an apple 8-core 3.0 ghz xeon / os x server ( version 10.5.8 ) . a web - based query interface to the database was developed using the php5 programming language , hosted via apache 2.2.14 . the user interface was developed with the utilisation of the jquery javascript library and jquery widgets . the polyq database was populated by extracting all human sequences from the ncbi non - redundant ( nr ) database that contained at least seven consecutive glutamine residues . we then performed a pfam ( 19 ) domain search to find protein domains within this subset of sequences . the ncbi nr contains many versions of the same protein , which created bias in the statistical analysis of polyq location data . we simplified the analysis by indentifying protein variants / isoforms and using only the longest protein isoforms ( which we termed multiple variants / isoforms of each protein were crudely identified by comparing the protein sequence following the polyq chains . the original sequences were then subjected to the blastclust ( 20 ) , force ( 21 ) , mcl ( 22 ) and homoclust algorithms ( 23 ) , and the variants / isoforms were adjusted as necessary . the crude identification used the 10 amino acids immediately after the polyq chain as a search string ; any sequence that had the 10 amino acids immediately following its own polyq chain was presumed to have homology with that sequence . this yielded a total of 128 master sequences , from an original data set of > 700 polyq - containing human protein sequences . the results of a typical search , shown in figure 1a , show both a graphical summary ( figure 1a , top ) and textual details ( figure 1a , bottom ) according to sequence classification ( see below ) . the graphical summary shows pie chart and bar chart representations of the results according to sequence classification ( figure 1a , top ) , pfam domain occurrence ( figure 1b ) and pfam domain repetition ( figure 1c ) . retrieved database entries are listed in table format with one row per protein , and three columns containing protein name ( with links to the genbank entry ) , pfam domains , and protein sequence ( with the polyq region annotated ) , respectively . homologs in the database can be included or excluded from the search . from this view , the domain and sequence context of the polyq sequence can be identified and further interrogated . to aid analysis figure 1.(a ) typical results of a simple search ( blank in this instance ) , showing graphical breakdown according to sequence classification ; results shown graphically according to domain occurrence ( b ) and domain repeats ( c ) using the tabs at top of page [ as seen in ( a ) ] . ( a ) typical results of a simple search ( blank in this instance ) , showing graphical breakdown according to sequence classification ; results shown graphically according to domain occurrence ( b ) and domain repeats ( c ) using the tabs at top of page [ as seen in ( a ) ] . the data are sorted and annotated according to the following sequence classifications : n - terminal polyqs sequences where the first polyq chain appears before all pfam domains ; c - terminal polyqs sequences where the polyq chains appear between the first pfam domain and the last pfam domain ; mid domain polyqs sequences in which the polyq chain appears in the middle of a pfam domain , or overlaps a pfam domain ; no significant domain polyqs sequences that do not contain any significant pfam domains ; unclassified polyqs sequences that did not fit into any of the above classifications . each group is readily accessed using the tabs in the web page ( figure 1a ) . we have also further reduced the redundancy in the data by clustering sequence homologs , and have also tagged known disease proteins . the website features pre - constructed pages that show the database entries sorted according to non - disease and disease - causing proteins respectively . this distinction is applied to the sequence classifications above , the domain occurrence ( e.g. listing all domains , figure 1b ) , and domain repeats ( figure 1c ) . this allows database entries to be grouped and examined according to whether the polyq tracts are found in non - disease or disease - causing proteins ( figure 2 ) . figure 2.selecting the stats menu text shows the entire database contents to be grouped into non - disease and disease causing proteins . to aid analysis specific entries can be selected ( indicated by a tickbox ) , using the examine button and grouped together . selecting the stats menu text shows the entire database contents to be grouped into non - disease and disease causing proteins . to aid analysis the data are sorted and annotated according to the following sequence classifications : n - terminal polyqs sequences where the first polyq chain appears before all pfam domains ; c - terminal polyqs sequences where the last polyq chain appears after all pfam domains ; interdomain polyqs sequences where the polyq chains appear between the first pfam domain and the last pfam domain ; mid domain polyqs sequences in which the polyq chain appears in the middle of a pfam domain , or overlaps a pfam domain ; no significant domain polyqs sequences that do not contain any significant pfam domains ; unclassified polyqs sequences that did not fit into any of the above classifications . each group is readily accessed using the tabs in the web page ( figure 1a ) . we have also further reduced the redundancy in the data by clustering sequence homologs , and have also tagged known disease proteins . the website features pre - constructed pages that show the database entries sorted according to non - disease and disease - causing proteins respectively . this distinction is applied to the sequence classifications above , the domain occurrence ( e.g. listing all domains , figure 1b ) , and domain repeats ( figure 1c ) . this allows database entries to be grouped and examined according to whether the polyq tracts are found in non - disease or disease - causing proteins ( figure 2 ) . figure 2.selecting the stats menu text shows the entire database contents to be grouped into non - disease and disease causing proteins . to aid analysis examine button and grouped together . selecting the stats menu text shows the entire database contents to be grouped into non - disease and disease causing proteins . to aid analysis specific entries can be selected ( indicated by a tickbox ) , using the examine button and grouped together . polyq is a valuable resource for theoreticians and experimentalists looking for insights into the context of polyq repeats in proteins and relationships with disease . although the query tool allows searching across much of the database , we are developing a custom interface that will allow user - configurable queries against the whole data set as well as user customization of how the results are displayed . we are also adding the structural information [ e.g. from the scop ( 24 ) , cath ( 25 ) and pdb databases ( 26 ) ] to the resources such that the structural context of polyq repeats can be investigated . this work is supported by national health and medical research council and the australian research council . are nhmrc senior research fellows . funding for open access charge : national health and medical research council ( australia ) .

the polyglutamine diseases are caused in part by a gain - of - function mechanism of neuronal toxicity involving protein conformational changes that result in the formation and deposition of -sheet rich aggregates . recent evidence suggests that the misfolding mechanism is context - dependent , and that properties of the host protein , including the domain architecture and location of the repeat tract , can modulate aggregation . in order to allow the bioinformatic investigation of the context of polyglutamines , we have constructed a database , polyq ( http://pxgrid.med.monash.edu.au/polyq ) . we have collected the sequences of all human proteins containing runs of seven or more glutamine residues and annotated their sequences with domain information . polyq can be interrogated such that the sequence context of polyglutamine repeats in disease and non - disease associated proteins can be investigated .

INTRODUCTION PolyQ DESCRIPTION AND USE Sequence classification Domain occurrence, repeats and disease statistics CONCLUSIONS AND FUTURE DIRECTIONS FUNDING

PMC5140815

although the feet occupy only 5% of the areas of the human body , they control postures through afferent information obtained through the sense of the soles , provide stability for maintenance of balance , and absorb impacts1 . the deformation into flatfoot is induced when the medial longitudinal arch ( mla ) has descended because the arch had been excessively relaxed to the extent that the arch can not be maintained and causes the feet to be excessively pronated compared to normal feet so that heel eversion appears and the weight load is shifted inward to compress the mla2 , 3 . when the mla has descended or has been completely lost leading to structural or functional deformation , the ability to absorb impacts will decrease and the sense of balance will be lost so that stability decreases during walking or running leading to walking difficulties and endurance decreases4 , 5 . foot wedges are clinically applied for correction of diverse foot diseases and for maintenance of balance6 , and customized insoles are used for correction of pronated feet7 . insoles widen the contact surface of the sole to improve stability during weight bearing , reduce turning actions when the foot has been pronated or supinated , and can be applied to the medial longitudinal arch of the foot to increase the sensory inputs of the sole8 . study results have been reported indicating that the conservative intervention methods of foot orthotics using support for the arch of the foot improve malformations of the foot , are effective for leg alignment and pain control , and improve gaits to become normal7 . the previous reported that sole stimulation could improve the motor sensation and postural movements to maintain proper postures9 . sensory - motor training applies the proprioceptive feedback of the body to activate the abductor pollicis muscle and the flexor hallucis brevis muscle , which are the intrinsic muscles of the feet important for maintenance of the mla , to help the formation of the arch and the maintenance of the balance of the body10 . in addition , sensory - motor training delivers proprioceptive sensory signals to the sensory cortex area of the brain and affects even the motor area to improve the asymmetric muscle tone of the sole and draw appropriate new movements thereby improving the motor sensation and postural disturbance to help maintaining the balance and stability of the body9 , 11 . reported that sensory stimulation training was helpful for flatfoot patients to form and support the arch12 . among exercise methods to improve flatfoot , toe bending exercises or towel - curl exercises mainly mobilize the extrinsic muscles of the foot such as the flexor digitorum longus muscle13 . however , short foot exercises ( sfe ) are sensory - motor training that activates the intrinsic muscles of the foot and actively forms the longitudinal arch and the horizontal arch14 , 15 . although studies utilizing foot orthotics such as therapeutic footwear , wedges , and insoles to improve the mla of flatfoot have been mainly conducted , studies for improvement of flatfoot that compared foot orthotics with sensory - motor training such as sfe are insufficient . therefore , the present study is intended to improve the mla of flatfoot and investigate the effects of the improvement on the dynamic balance of the feet and the lower limbs to identify effective application methods . the subjects of the present study were 14 university students ( males 10 , females 4 ) aged 21 to 26 years sampled from among students in the health college of s university in asan - si , chungcheongnam - do and the experiments were conducted from october to december 2015 . before the experiments , the overall details of the purpose and procedure of the present study were explained to all subjects pursuant to the ethical standard of the declaration of helsinki and the subject voluntarily agreed to participate in the experiments . the subjects that participated in the experiments were randomly assigned to an sfe group of seven subjects and an arch support insoles ( asi ) group of seven subjects . the selection criteria for participating subjects regarded the foot used to kick balls as the dominant foot to select subjects with flatfoot . navicular drop tests ( ndt ) were conducted to select those that had flexible flatfoot with a 10 cm or larger difference in the navicular tuberosity heights and those that were performing lower limb exercises separately , or had any foot hypoesthesia , fracture , dislocation , skin disease , or vascular disease were excluded . the subjects general characteristics are as shown in table 1table 1.general characteristics of the subjectssfe group ( n=7)asi group ( n=7)gender ( male / female)6/14/3age ( years)24.0 1.924.1 1.5height ( cm)172.2 6.9167.0 6.7weight ( kg)68.2 12.963.3 17.6mean sd . and the sfe group s mean age , height , and weight were 24.0 1.9 years , 172.2 6.9 cm , and 68.2 12.9 kg respectively . the asi group s mean age , height , and weight were 24.1 1.5 years , 167.0 6.7 cm , and 63.3 17.6 kg respectively . sfe was implemented as sensory - motor training for improvement in the flatfoot of the experimental group . before the intervention , thereafter , each subject was instructed to sit on a height - adjustable chair and bend the hip joint , knee joints , and ankle joints to 90 and a towel was placed below the feet . thereafter , the subject was instructed to pull the head of the first metatarsal bone toward the heel without bending the toes and maintain the state for 20 sec to form the mla . during the exercise , to prevent the forefoot and the heel from being lifted off the ground and smoothly induce the flexion of the head of the metatarsal bone , the measurer gently held the instep and the heel15 . the posture was maintained for 10 sec followed by 5 sec and this process was implemented for 30 minutes per time , three times per week for a total of five weeks . as a method for improvement in the flatfoot of the control group , insoles were applied as medial arch support structures . to make the insoles , the foot shape of each subject was sketched on 3.2 mm thick aquaplast - t ( sammons preston rolyan , usa ) , which is a thermoplastic material . thereafter , the aquaplast - t was cut out using scissors , wetted with 100 c water , and attached to the sole of the subject to copy the foot and the height of the arch . thereafter , the mla was made with a shore value of 20 and a height of at least 15 mm16 . the insoles made as such were given to the subjects and the subjects were instructed to put the insoles into their running shoes and walk on flatlands for 30 minutes per time , three times per week for a total of five weeks . ndts were conducted to measure changes in the height of the mla . in the ndt , each subject was instructed to sit on a chair with the knee joint bent to 90 and align the second toe and the knee so that the subtalar joint was placed on the neutral position and under a non - weight bearing condition , the distance from the ground to the navicular tuberosity was measured and marked . thereafter , the distance from the ground to the navicular tuberosity was measured in a standing position with the feet place at shoulder width and weight bearing by the two feet . using a plastic ruler and a 35 index card , the difference in the height of the navicular tuberosity between the non - weight bearing(sitting position ) position and the weight bearing ( standing position ) position was measured three times repeatedly and the average value was used . y - balance tests ( ybt ) ( move2perform , evansville , in , usa ) were conducted to measure the dynamic balance ability of the leg . the y - balance test ( ybt ) kit consists of three directional ( anterior , posteromedial , posterolateral ) pvc pipe graduated rulers . each subject stood on the platform located on the center on the ybt kit with the bare foot of the leg to be measured and pushed the reach indicator placed on the pvc pipe graduated ruler with the other leg and the maximum reached distance was measured . the pvc pipe graduated ruler has 0.5 cm grades and thus the distance was recorded in 0.5 cm units . during the measurement , considering the reaching ability depending on leg lengths , the leg length of each subject was measured and normalized and to minimize learning effects on measurement , reaching was practiced two times in each direction before measurement . the measurement was performed in the same order of directions and each distance was measured two times and the average value was calculated . normalized composite reach distance was computed for each leg as the sum of the maximum reach distances ( in centimeter ) in the 3 directions , divided by 3 times the limb length , and then multiplied by 10017 . the general characteristics of the subjects were presented as means and standard deviations using descriptive statistics ( table 1 ) . independent t - tests were conducted to compare the amounts of changes between the two groups and paired t - tests were used to measure differences before and after experiment in each group . changes in the ndt and ybt of the sfe group and the asi group are as shown in table 2table 2.comparison of changes in navicular drop height and dynamic balance between the groupsvariablesfe groupasi grouppre - testpost - testpre - testpost - testnavicular drop test ( mm)11.4 1.67.7 1.1*12.2 1.810.5 1.7y balance test ( % ) 74.3 8.382.4 7.472.4 7.174.2 7.2mean sd . * significant differences between the sfe and the asi groups ( p<0.05 ) . significant differences between pre- and post - tests ( p<0.05 ) . according to the results on inter - group ndt comparisons , the values of the sfe group significantly decreased after intervention compared to the asi group and according to the results of ybt comparisons , the values of the sfe group significantly decreased after intervention compared to the asi group ( p<0.05 ) . in intra - group ndt comparison between before and after intervention , whereas the sfe group showed significant decreases , the asi group showed no significant difference and in intra - group ybt comparison , both the sfe group and the asi group showed significant increases ( p<0.05 ) . in the case of flatfoot , as the pronated state of the heel is maintained , the talus bone is moved to the inside of the sole leading to the disappearance of the medial longitudinal arch so that the ability to accommodate and distribute the weight is reduced compared to normal persons3 . structural deformation of the feet leads to lesions in the ankle joint and the feet and problems in lower limb joints , results in early fatigue and pain due to the excessive compensating actions of the intrinsic muscles and the extrinsic muscles , and causes problems in the stability and balance of the feet during gaits18 . to improve these flatfoot conditions , methods that correct the mla using foot orthotics or therapeutic footwear are universally used15 other intervention methods include sfe for strengthening the intrinsic muscles and extrinsic muscles of the foot through sensory - motor training . presented sfe as the most positive method among methods of correcting the eversion of the subtalar articulation19 . the sfe induces the head of the metatarsal bone to approach the heel without bending the toes in a state where the weight is borne15 . in the present study , attempts were made to improve flatfoot conditions through changes in the height of the mla by applying sfe for six weeks . according to the results of measurement using ndt , the descending distance of the navicular bone decreased from 11.4 1.6 mm before intervention to 7.7 1.1 mm after intervention indicating that sfe was effective . however , in the asi group , changes in the medial longitudinal arch were not significant . in a study conducted by allen & glasoe , the mean descending distance of the mla this indicates that a flexible flatfoot can form ab arch close to that of normal feet through exercise through sfe for six weeks . reported that towel - curl exercises implemented for four weeks as foot intrinsic muscle strengthening exercises were effective21 and jung et al . reported that when toe curl exercises and arch formation exercises were applied , the rear foot angle significantly decreased while foot intrinsic muscle strength was improved15 . reported that changes in the structure of the arch of the sole affected balance ability and gait ability7 . in the present study , when dynamic balance was measured using ybt , both the sfe group and the asi group showed significant improvement between before and after intervention within each group . this is considered attributable to the fact that short foot exercises improved the functions and activity of the abductor pollicis muscle that plays the role of bearing the weight and pushing the body forward during push off in gaits and the flexor hallucis brevis muscle that maintains the mla during the terminal stance in gaits to maintain foot stability10 . the reason why dynamic balance ability was significantly improved in the group applied with arch support insoles is considered to be the fact that the support for the medial longitudinal arch reduced maximum load reactions to improve leg stability thereby brining about dynamic biomechanical effects11 . in the present study , short foot exercises and arch support insoles were applied to improve flatfoot conditions and it could be seen that as the medial longitudinal arch was improved , dynamic balance ability was improved . in addition , it could be seen that sensory - motor training such as short foot exercises was more effective than conservative treatment methods such as arch support insoles . in addition , it could be seen that the intervention period of six weeks was not sufficient to improve the medial longitudinal arch using arch support insoles . detailed comparison studies combining sensory - motor training of the foot and arch support insoles with more sufficient intervention periods are necessary .

[ purpose ] the purpose of the present study is to apply short foot exercises and arch support insoles in order to improve the medial longitudinal arch of flatfoot and compare the results to identify the effects of the foregoing exercises on the dynamic balance of the feet and the lower limbs . [ subjects and methods ] fourteen university students with flexible flatfoot were selected by conducting navicular drop tests and randomly assigned to a short foot exercise group of seven subjects and an arch support insoles group of seven subjects . the intervention in the experiment was implemented for 30 minutes per time , three times per week for five weeks in total . [ results ] in inter - group comparison conducted through navicular drop tests and y - balance tests , the short foot exercise group showed significant differences . among intra - group comparisons , in navicular drop tests , the short foot exercise group showed significant decreases . in y - balance tests , both the short foot exercise group and the arch support insoles group showed significant increases . [ conclusion ] in the present study , it could be seen that to improve flatfoot , applying short foot exercises was more effective than applying arch support insoles in terms of medial longitudinal arch improvement and dynamic balance ability .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3788816

structural changes in primary open - angle glaucoma ( poag ) occur in the optic nerve head as well as peripapillary retinal nerve fiber layer ( rnfl).14 while it has been assumed that structural loss of the rnfl anticipates functional visual field loss , the relationship between structure and function in glaucoma is not yet fully understood and remains the subject of intense research.2,59 while a close relationship between rnfl and the retinal pigment epithelium ( rpe ) is a prerequisite for normal vision,10,11 little is known about rpe involvement in patients suffering from glaucoma . a previously published study on dogs has shown pathologic abnormalities and rpe damage in glaucomatous eyes.12 few published studies on human glaucomatous eyes have shown correlations between peripapillary rpe abnormalities and optic nerve degeneration in glaucoma patients.1315 a recent study has shown that rpe atrophies seem to progress over the course of the disease.16 fundus autofluorescence ( faf ) has been used for the evaluation of degenerative , inflammatory , and neoplastic diseases of the posterior fundus.1720 the faf signal is supposedly derived primarily from fluorophores found in lipofuscin within the rpe layer mainly fluorophore n - retinylidene - n - retinyl ethanolamine ( a2e ) and indicates altered structure as a result of atrophic changes due to inflammation resulting in increased or decreased faf in the affected areas.2123 open - angle glaucoma ( oag ) is generally understood as a degenerative disease and remains one of the leading causes of blindness in the elderly population.3 poag is the most common subtype of oag , with glaucoma - typical structural as well as functional glaucomatous alterations.2 to date , there is only very limited evidence on altered rpe signal intensity in glaucoma patients . the recently developed ultra - wide - field scanning laser ophthalmoscope optomap panoramic 200tx ( optos , dunfermline , fife , scotland ) allows non - mydriatic wide - field imaging and provides an additional wide - field faf detection modality . the goal of this study was to investigate the relationship between rnfl thickness and rpe alterations as detected by wide - field faf in patients suffering from manifest poag . a consecutive , prospective series of 82 study eyes , mean age 64 years ( range : 3586 years ) , with poag and advanced glaucomatous visual field defects was included in this study ( 47 female and 35 male , 42 right and 40 left eyes ) . according to the definition of the european glaucoma society and a previous work by katz et al,24 a visual field defect suspicious for glaucoma is defined as a cluster of at least three or more contiguous non - edge points with significantly reduced sensitivity ( p < 0.05 ) , one of which with a significance of at least p < 0.01 on the same side of the horizontal meridian in the pattern deviation plot . all study participants underwent a full ophthalmic examination including best - corrected visual acuity ( bcva ) in decimals obtained with a snellen projection chart ; objective and subjective refraction ; slit - lamp biomicroscopy ; intraocular pressure measurement with goldmann applanation tonometry ; gonioscopy ; and fundus examination by indirect ophthalmoscopy . each included patient underwent visual field testing followed by spectral - domain optical coherence tomography ( sd - oct ) for peripapillary rnfl thickness and optos wide - field faf images . only eyes suffering from poag were included , defined by a documented history of elevated intraocular pressure , characteristic optic nerve head damage , and/or characteristic visual field defects . exclusion criteria were any evidence for optic nerve head degeneration derived from any other subtype of glaucoma than poag ; opticopathy of any reason other than glaucoma ; diabetic retinopathy ; macular disease ; posterior uveitis ; or retinal surgery . all research was conducted in accordance with institutional guidelines and board approval and conformed to the tenets of the world medical association declaration of helsinki . conventional predefined circular peripapillary optic nerve head ( onh ) cross - sectional scans were obtained from each included study eye with a spectralis optical coherence tomography device ( heidelberg engineering , heidelberg , germany ) with an excitation wavelength of 870 nm running at a speed of 40,000 a - scans per second . global and sectoral ( supero temporal , temporal , inferotemporal , inferonasal , nasal , and superonasal ) rnfl thickness values in m were calculated with the inbuilt heidelberg software . optomap imaging consisted of taking several images and saving the best image of each included eye for grading . total scanning time was about 35 minutes , which included patient positioning , and was performed by an experienced technician ( tm ) . basic operation of the optomap for ultra - wide - field composite color fundus images involves a scanning laser ophthalmoscope with two laser wavelengths scanning at 532 nm ( green laser separation ) and 633 nm ( red laser separation ) . wide - field faf images were obtained with an excitation wave length of 532 nm , and autofluorescence was detected by a broadband detector at 540 to 800 nm . for further analysis , the pattern grid with corresponding locations between functional visual field sectors and structural peripapillary rnfl thickness originally described by garway - heath et al25 was aligned to the faf images at corresponding locations ( figure 1 ) . faf brightness with values ranging between 0 ( black ) and 255 ( white ) was obtained for each faf sector ( superotemporal peripheral , superotemporal central , temporal central , inferotemporal central , inferotemporal peripheral , and nasal ; figure 1c and d ) using standalone software based on matlab ( 7.0 r14 ; mathworks inc , natick , ma , usa ) , as described in our previous works.19,27 mean faf intensity of each sector was standardized for each included study eye to eliminate individual faf intensity differences due to aging19 and correlated with the corresponding peripapillary rnfl thickness obtained with sd - oct . data were collected and analyzed using spss software ( version 19.0 ; ibm corporation , armonk , ny , usa ) . a p - value of < 0.05 was considered statistically significant . a consecutive , prospective series of 82 study eyes , mean age 64 years ( range : 3586 years ) , with poag and advanced glaucomatous visual field defects was included in this study ( 47 female and 35 male , 42 right and 40 left eyes ) . according to the definition of the european glaucoma society and a previous work by katz et al,24 a visual field defect suspicious for glaucoma is defined as a cluster of at least three or more contiguous non - edge points with significantly reduced sensitivity ( p < 0.05 ) , one of which with a significance of at least p < 0.01 on the same side of the horizontal meridian in the pattern deviation plot . all study participants underwent a full ophthalmic examination including best - corrected visual acuity ( bcva ) in decimals obtained with a snellen projection chart ; objective and subjective refraction ; slit - lamp biomicroscopy ; intraocular pressure measurement with goldmann applanation tonometry ; gonioscopy ; and fundus examination by indirect ophthalmoscopy . each included patient underwent visual field testing followed by spectral - domain optical coherence tomography ( sd - oct ) for peripapillary rnfl thickness and optos wide - field faf images . only eyes suffering from poag were included , defined by a documented history of elevated intraocular pressure , characteristic optic nerve head damage , and/or characteristic visual field defects . exclusion criteria were any evidence for optic nerve head degeneration derived from any other subtype of glaucoma than poag ; opticopathy of any reason other than glaucoma ; diabetic retinopathy ; macular disease ; posterior uveitis ; or retinal surgery . all research was conducted in accordance with institutional guidelines and board approval and conformed to the tenets of the world medical association declaration of helsinki . conventional predefined circular peripapillary optic nerve head ( onh ) cross - sectional scans were obtained from each included study eye with a spectralis optical coherence tomography device ( heidelberg engineering , heidelberg , germany ) with an excitation wavelength of 870 nm running at a speed of 40,000 a - scans per second . global and sectoral ( supero temporal , temporal , inferotemporal , inferonasal , nasal , and superonasal ) rnfl thickness values in m were calculated with the inbuilt heidelberg software . optomap imaging consisted of taking several images and saving the best image of each included eye for grading . total scanning time was about 35 minutes , which included patient positioning , and was performed by an experienced technician ( tm ) . basic operation of the optomap for ultra - wide - field composite color fundus images involves a scanning laser ophthalmoscope with two laser wavelengths scanning at 532 nm ( green laser separation ) and 633 nm ( red laser separation ) . wide - field faf images were obtained with an excitation wave length of 532 nm , and autofluorescence was detected by a broadband detector at 540 to 800 nm . for further analysis , the pattern grid with corresponding locations between functional visual field sectors and structural peripapillary rnfl thickness originally described by garway - heath et al25 was aligned to the faf images at corresponding locations ( figure 1 ) . faf brightness with values ranging between 0 ( black ) and 255 ( white ) was obtained for each faf sector ( superotemporal peripheral , superotemporal central , temporal central , inferotemporal central , inferotemporal peripheral , and nasal ; figure 1c and d ) using standalone software based on matlab ( 7.0 r14 ; mathworks inc , natick , ma , usa ) , as described in our previous works.19,27 mean faf intensity of each sector was standardized for each included study eye to eliminate individual faf intensity differences due to aging19 and correlated with the corresponding peripapillary rnfl thickness obtained with sd - oct . data were collected and analyzed using spss software ( version 19.0 ; ibm corporation , armonk , ny , usa ) . a p - value of < 0.05 was considered statistically significant . mean faf intensities and mean sectoral peripapillary rnfl thickness values for analyzed subfields 16 of each included study eye can be seen in table 3 . the highest mean faf intensity was observed in the peripheral infero temporal sector , while the central superotemporal sector showed the lowest mean faf intensity of all investigated subfields ( table 3 ) . mean rnfl thickness was highest in the peripheral inferotemporal sector and lowest in the nasal sector ( table 3 ) . correlation analyses between sectoral rnfl thickness and standardized faf intensity in the corresponding topographic retina segments revealed partly significant correlations ( table 4 ) . correlation coefficients ranged between 0.004 and 0.376 and were statistically significant in the temporal inferior central field ( r = 0.324 , p = 0.036 ) and the nasal field ( r = 0.376 , p = 0.014 ) . in retinal metabolism , a close symbiotic interaction between neurosensory retina and rpe exists.28 rpe has various support functions , including neuroprotection , and its damage results in functional alteration of the neurosensory retina , including the rnfl.2932 therefore , faf has become a valuable tool in monitoring the development and progression of atrophic rpe dysfunction and consecutive neurosensory degeneration , such as in age - related macula degeneration ( amd).18,19 a close correlation between rpe damage measured with faf intensity and visual deterioration has been de scribed.3336 in reverse , rnfl damage may be associated with rpe dysfunction . however , little is known as to whether rnfl damage , which has been demonstrated to correlate with visual field function,7,26,37 is connected with rpe malfunction or degeneration potentially detectable by faf . our observations of a certain correlation between sectoral rnfl thickness and faf intensity at corresponding locations in glaucomatous eyes are in accordance with published studies on dogs and reveal a close interaction between rpe and the neurosensory retina.12 the results of this study provide early evidence that peripapillary rnfl degeneration in glaucoma patients may be accompanied by corresponding rpe alterations . these findings apply especially for the temporal inferior and nasal sectors and thus are , at least in part , in agreement with results and observations of previously published data of structure function relationships in glaucomatous eyes , which suggest the temporal inferior peripapillary region in particular to be a predilection site for structural damage in glaucoma.38,39 those studies on evaluated functional visual field abnormalities in glaucoma patients correlated the observed functional abnormalities to structural rnfl changes and showed the temporal inferior sector , in particular , to be subject to structural changes in patients with visual field defects due to glaucoma . further studies are needed to give a better understanding of peripapillary sectors more susceptible to degenerative changes than others and to possibly address the question as to whether this observation is due to biomechanic or nutritive predisposing anatomic circumstances . significant correlations between nasal rnfl thickness and corresponding faf intensity in human poag eyes have not , to our knowledge , been published so far and remain our focus of continuing research . a limitation of our study is the fact that , due to its cross - sectional character , we do not have data from over the course of glaucoma disease and are not able to predict any of our observed developments . additionally , faf intensity changes during aging,19 so that interindividual comparison of measured faf intensity values should be done with caution . the presented early data from poag patients shows that , at first and from the structural point of view a certain correlation between faf , reflecting rpe function , and peripapillary rnfl thickness exists . as a next step , it will have to be explored whether these findings are in correlation to the actual function deficit measured by visual fields and whether our observation can be interpreted with regards to a potential predictive parameter for glaucomatous damage . in summary , we were able to correlate rpe abnormalities to corresponding peripapillary rnfl damage especially in the temporal inferior sector of patients with manifest glaucomatous visual field defects . a further evaluation of faf as a potential predictive parameter for glaucomatous damage is necessary .

purposeto investigate the relationship between retinal nerve fiber layer ( rnfl ) thickness and retinal pigment epithelium alterations in patients with advanced glaucomatous visual field defects.methodsa consecutive , prospective series of 82 study eyes with primary open - angle glaucoma and advanced glaucomatous visual field defects were included in this study . all study participants underwent a full ophthalmic examination followed by visual field testing with standard automated perimetry as well as spectral - domain optical coherence tomography ( sd - oct ) for peripapillary rnfl thickness and optos wide - field fundus autofluorescence ( faf ) images . a pattern grid with corresponding locations between functional visual field sectors and structural peripapillary rnfl thickness was aligned to the faf images at corresponding location . mean faf intensity ( range : 0 = black and 255 = white ) of each evaluated sector ( superotemporal , temporal , inferotemporal , inferonasal , nasal , superonasal ) was correlated with the corresponding peripapillary rnfl thickness obtained with sd-oct.resultscorrelation analyses between sectoral rnfl thickness and standardized faf intensity in the corresponding topographic retina segments revealed partly significant correlations with correlation coefficients ranging between 0.004 and 0.376 and were statistically significant in the temporal inferior central field ( r = 0.324 , p = 0.036 ) and the nasal field ( r = 0.376 , p = 0.014).conclusionretinal pigment epithelium abnormalities correlate with corresponding peripapillary rnfl damage , especially in the temporal inferior sector of patients with advanced glaucomatous visual field defects . a further evaluation of faf as a potential predictive parameter for glaucomatous damage is necessary .

Introduction Patients and methods Patients SD-OCT measurements Optomap imaging Quantification of FAF signal intensity and correlation with peripapillary RNFL thickness Statistical analysis Results Discussion Conclusion

PMC2737600

the samscl gene was inserted into a pet15b vector with a stop codon at glu 95 introduced by site - directed mutagenesis to generate the samscl - c26 mutant . the channel was overexpressed in bl21-gold cells and purified in the detergent ldao by immobilized metal affinity and gel filtration chromatographies . crosslinking was performed using n , n - dicyclohexylcarbodiimide at room temperature for 30 min-1 h and the products were analyzed by sds - polyacrylamide gel electrophoresis . the mjf465 ( mscl , mscs , msck ) ( de3 ) strain was used for the osmotic downshock assay . cells hosting ecmscl and an empty vector were included as positive and negative control sets , respectively . for the osmotic shock , the culture was diluted 1:500 into sterile double - distilled h2o . for the mock shock , lb mscl protein was reconstituted into giant azolectin liposomes via the dehydration / rehydration method and assayed by patch clamp electrophysiology . patches containing at least 1 - 10 channels were excised and currents measured at various pressures . recording and pipette solutions both contained 200 mm kcl , 40 mm mgcl2 , 5 mm hepes , ph 7.2 .

the ability of cells to sense and respond to mechanical force underlies diverse processes such as touch and hearing in animals , gravitropism in plants , and bacterial osmoregulation1 , 2 . in bacteria , mechanosensation is mediated by the mechanosensitive channels of large ( mscl ) , small ( mscs ) , potassium - dependent ( msck ) , and mini ( mscm ) conductances . these channels act as emergency relief valves protecting bacteria from lysis upon acute osmotic downshock3 . among them , mscl has been intensively studied since the original identification and characterization 15 years ago by kung and co - workers4 . mscl is reversibly and directly gated by changes in membrane tension . in the open state , mscl forms a nonselective 3 ns - conductance channel which gates at tensions close to the lytic limit of the bacterial membrane . an earlier crystal structure at 3.5 resolution of a pentameric mscl from mycobacterium tuberculosis ( tbmscl ) represents a closed - state or nonconducting conformation5 , 6 . mscl has a complex gating behaviour ; it exhibits several intermediates between the closed and open states , including one putative nonconductive expanded state and at least three sub - conducting states7 . although our understanding of the closed5 , 6 and open8 - 10 states of mscl has been increasing , little is known about the structures of the intermediate states despite their importance in elucidating the complete gating process of mscl . here we present the crystal structure of a truncation mutant ( 95 - 120 ) of mscl from staphylococcus aureus ( samscl - c26 ) at 3.8 resolution . strikingly , samscl - c26 forms a tetrameric channel with both transmembrane ( tm ) helices tilted away from the membrane normal at angles close to that inferred for the open state9 , likely corresponding to a nonconductive but partially expanded intermediate state .

Methods Summary Supplementary Material

PMC3539078

herniated lumbar disc is one of the most frequent medical problems18 ) , and for decades , lumbar discectomy has been one of the most common surgical practices performed by neurosurgeons22 ) . today , lumbar spinal fusion surgery has been commonly performed for patients who present with chronic back pain with neurological symptoms23 ) . at present , the advent of magnetic resonance imaging ( mri ) and the progressive increase in definition of this modality of imaging have considerably contributed to the knowledge of the natural evolution of a herniated lumbar disc . axial loaded mri can therefore give decisive information in dynamic spinal disorders by allowing simulation of an upright position4 ) . residual and recurring post - operative symptoms are still quite common in lumbar spinal surgeries12 ) . the reasons some people have this persistent pain after surgery remain unclear , so the surgical treatment of ruptured lumbar inter - vertebral discs is sometimes discouraging to both surgeon and patient . it is important to demonstrate the neurological and radiological correlation while determining clinical status in cases with lumbar complaints5 ) . any contribution to our knowledge of the cause of these operative failures is always welcome . since a purely experimental approach can not provide this information , retrospective studies are called for , and studies focusing on the accurate understanding of appropriate spinal parameters . the development of low back pain can be associated with certain alterations in the sagittal shape of the spine , some studies have analyzed spino - pelvic alignment in patients with lumbar degenerative disease15,16,25 ) , but the significance of the angle of the sacrum in the pathogenesis of low back pain has also received scant attention . sacral curvature ( sc ) , represented by the angle between the first and the last sacral vertebrae , is a feature that differentiates the human pelvis from that of other animals . in some sacra , the surface of the sacrum can be nearly straight with a very slight curvature affecting the lower end . in the others , the curvature is throughout the entire length of the sacrum , especially toward the middle . other sacra have a less marked curvature affecting only the lower third of the bone . it is unclear whether the degrees of vertical angle of sacral curvature are the same for all adult patients with disc herniations . we hypothesized that the mechanics of load transfer in the spine and in the intervertebral disc in particular is an important factor in understanding the patterns and mechanics of disc pain . the vertical angle of sacrum may have a pivotal role in etiology and post operative outcome of lumbar disc herniations . there is no publication quantitating human vertical angle of sacrum in lumbar disc herniations , so we analyzed this angle and and compared the results with patients without any lumbar disc disease . after informed consent , 185 patients who admitted to the department of neurosurgery of rize research and education hospital with low back pain between 2 april-10 july 2009 were analysed . to provide group homogenity , body mass index ( bmi ) all patients with bmi values less than 20 or more than 40 was excluded from the study as well as patients younger than 20 and older than 50 years of age . the exclusion criteria were as follows : bmi value lesser than 20 or greater than 40 , age younger than 20 and older than 50 , previous surgery , associated lumbar spine stenosis , foraminal or extraforaminal disc herniations , spondylolisthesis . of the 185 patients , 128 these patients were divided to two groups according to their sex . after giving informed consent , , ergn et al.7 ) analyzed differences in lumbosacral morphology associated with the tendency to develop intervertebral disc degeneration . sacral curvature ( sc ) , represented by the angle between the first and the last sacral vertebrae , and the vertical angle of sacral curvature ( vasc ) was defined as the angle created between the intersection of a line running centrum of sacral curvature and a vertical line ( fig . 1 , 2 ) . from sagittal plan of lumber mri , the vertical angle of sacral curvature . the normal orientation of lumbosacropelvic structure plays an important role in the determination of shear and compressive forces applied on the anterior ( corpus vertebrae and intervertebral discs ) and posterior ( facet joints ) elements of lumbar vertebral column19 ) . no publications in the literature were analyzed the relationship between intervertebral disc degeneration and the degree of vasc in females . data on age and sex , vasc were recorded . to show the uniformity of groups , data were analyzed with the statistical software package spss for windows ( spss inc . , ergn et al.7 ) analyzed differences in lumbosacral morphology associated with the tendency to develop intervertebral disc degeneration . sacral curvature ( sc ) , represented by the angle between the first and the last sacral vertebrae , and the vertical angle of sacral curvature ( vasc ) was defined as the angle created between the intersection of a line running centrum of sacral curvature and a vertical line ( fig . 1 , 2 ) . from sagittal plan of lumber mri , the vertical angle of sacral curvature . the normal orientation of lumbosacropelvic structure plays an important role in the determination of shear and compressive forces applied on the anterior ( corpus vertebrae and intervertebral discs ) and posterior ( facet joints ) elements of lumbar vertebral column19 ) . no publications in the literature were analyzed the relationship between intervertebral disc degeneration and the degree of vasc in females . data on age and sex , vasc were recorded . to show the uniformity of groups , data were analyzed with the statistical software package spss for windows ( spss inc . , chicago , il , usa ) . the comparisons between parameters were performed using wilkonson signed rank test . female patients comprised 60 lumbar disc herniation cases ( disc herniation group , dhg ) ; the mean age was of 38.3 years ( range 22 - 50 years ) . the second group comprised 34 cases without any vertebral disease ( non - disc herniation group ) , the mean age was of 35.02 years ( range 21 - 50 years ) . the first group of males comprised 25 subjects with lumbar disc herniation , the mean age was of 39.42 years ( range 21 - 50 years ) . the second group of male comprised 9 cases with free vertebral disease ; the mean age was of 35.5 years ( range 21 - 44 years ) . wilkonson signed rank test showed that the vertical angle of sacral curvature was statistically significantly bigger in female with lumbar disc herniations than the angles of control group , 28.32 and 25.4 , respectively ( p=0.034<0.05 ) . it is known that the human pelvis reaches its definite form at the age of 20 - 25 years . complete fusion of the sacrum has been reported to occur between years 25 and 33 of life and is related to the load - bearing aspects of this region . pathogenesis of the sacral morpho - topography changes could be easily explained by negative effects on human erect position . the sacrum of homo sapiens is not positioned posteriorly at birth and that it is during the first few years that the sacrum , in humans , moves dorsally in relation with the progressive acquisition of erect posture and the ontogeny of bipedal locomotion . the evolution of bipedal posture and ambulation in humans has transformed the horizontal vertebral column of vertebrates into a load - bearing erect spine that is required to efficiently transfer weight , provide stability and permit motion . due to the erect position , the pelvic bone system is subjected to new static and dynamic relations , which play a very important role in definite formation of this region6,8,14 ) . through the spinal column , the body load is shifted to the base of the sacrum and then , through the pelvic girdle to inferior extremities . the position of the sacrum relative to the vertical has been frequently mentioned but seldom measured . to express this verticality of the sacrum , newman drew a line from the posterior superior iliac spine to the symphysis pubis . the angle between this and a vertical line was the sacral inclination . this refers to the relationship of the sagittal plane of the sacrum to the vertical plane . vasc can be correlated with sacral tilt . in this study , we did not use the sacral inclination ( s1 : value of the angle between the posterior side of the body of the first sacral vertebra and the vertical ) and sacral slope ( ss ) parameter . both parameters are different from vasc . we think that vasc degree is more sufficient in showing the vertical or horizontal sacrum . orientation of s1 and sacrum itself may not be same degree , because the intervertebral disc between the fifth lumbar and the first sacral vertebrae is subject to more trauma , both acute and chronic , than any other intervertebral disc . this is due to the facts that it carries a heavier load and this disc is found by x - ray to be destroyed more frequently than any other . sacral biomechanics have an impact on the nerves exiting the spinal cord in the entire spine but particularly in the lower lumbar spine and from the sacrum itself . lumbar disc surgery is one of the most frequent procedures performed by spine surgeons22 ) . theories propose that this transformation in the mechanics of locomotion is the inciting evolutionary event that made the lumbar spine susceptible to degenerative disease . if the primary sources of disc pathology are these forces / stresses acting on the spine , why did the spine not evolve into a more rigid structure ? what is the purpose of having a vertebral column capable of torsion and compression , unless it is somehow linked to the demands of human gait ? this study investigates the relationship between lumbar disc herniation and the vertical angle of sacral curvature . this angle was found to be statistically significantly bigger in female with lumbar disc herniations when compared to subjects in control group , 28.32 and 25.4 , respectively ( p=0.034<0.05 ) . we supposed that there are different factors contributing to the formation of the vertical angle of sacral curvature . these factors include1 ) the effect of erect posture , which tilts the upper part of the sacrum dorsally and the lower part of the sacrum ventrally2 ) , and the influence of supine posture , which affects the development of the lower part of the sacrum . in addition to supine posture the levator ani , which is well developed in homo sapiens , also affects the lower part of the sacrum and coccyx and influences its ventral orientation . variations of the vasc can be result from differences in onset and frequency of supine posture . this is the first time that vertical angle of sacral curvature has been shown to play a role in female with lumbar disc herniation . the purpose of this study is to determine the reliability and validity of a goniometric measurement of the vertical angle of sacrum , found statistically significant difference in female with lumbar disc herniation and without intervertebral disc disease . if indeed one is the first to report something and that something is of value . we assumed that that this finding result in a higher gravitational compressive force which may , in turn , lead to progressive degeneration of the discs in female high vasc degree . men in their fourth to fifth decade appear to be the most prone to disc herniation with subsequent cauda equina and conus medullaris syndromes17,24 ) . because of insufficient number of males in this study , their angle was not analyzed . our study was performed in the eastern black sea region of turkey , and in this area women are exposed to more environmental and life issues than men . it was shown , that the vasc degree of females with lumbar disc herniations ( ldh ) is statistically significantly bigger than from the females without ldh . " the increased vasc in female found in the disc herniation group suggest that the spatial orientation of the sacrum is more vertical than in the normal group . even if it is difficult to determine the part of structural loss of lordosis and the postural part , we observed that increased vasc induces ldh in female , probably with anterior displacement of c7 plumbline and center of gravity , resulting in an increased flexion moment applied to the spine , here , increased vasc may be predisposed ldh in female . the gravity line of the body passes through many vertebrae and " leaves " the spine at the sacral promontory . we conducted this study with sacrum , because the sagittal sacral morphology is a constant anatomic variable specific to each individual and unaffected by the position of the patient in space and , found vasc was increased in female with ldh . discectomy is often used in the treatment of prolapsed intervertebral disc to relieve the associated leg pain . but conservative treatment should be considered when cauda equina syndrome or progressive motor weakness are absent in the acute stage of the lumbar herniated disc21 ) . although lumbar disc surgery is known to be able to relieve pain we have to make sure it improves the quality of life3,9 ) . the outcomes reported after both traditional techniques and microsurgery for lumbar disc herniation have generally been shown to be equal13,17 ) . however , between 10% and 40% of patients do not have a satisfactory outcome13 ) , and many in whom surgery is deemed successful continue to have some degree of low back pain . the reasons some people have persistent pain after surgery remain unclear , so the surgical treatment of ruptured lumbar intervertebral discs is sometimes discouraging to both surgeon and patient , because the operation fails to relieve the symptoms of back pain and/or sciatica , and the treatment of discogenic pain is always problematic for spine surgeons . there is an incessant search for the causes and the action mechanisms of operative failures of the lumbar disc herniation . any contribution to our knowledge of the cause of these operative failures is always welcome . increased vasc in females some authors also report that the female gender is a risk factor for failed back surgery2,20 ) . we copiously stress the fact that we are the first ones to report that such an increased vasc degrees in females with ldh . the recognition of this fact is of importance because it has to do with degenerative changes in the spine . if indeed one is the first to report something and that something is of value . this angle will not be changed in sagittal mri image or direct conventional radiographic x - rays ( fig . 1 , 2 ) . in the beginning of study , we measured the vas both in mri images and direct conventional lumbo - sacral lateral x - rays , and noted the vas degree is correlated with each other . in lying down , or standing x - rays , we found it was two times greater degree than sagittal mr images . we wanted to omit to give patient unnecessary radiation to by conventional x - ray , used mri images . the problem , here , may be important with regard with the vasc which is calculated on mri imaging in prone position and then referenced with a vertical line . this should not be problem , because sacral curve is the undistorted part of the spinal curves . regarding the second line which is the radius of the sacrum , it is positioning the centre of the sacral curvature . this angle will not be changed in sagittal mri image or direct conventional radiographic x - rays ( fig . 1 , 2 ) . in the beginning of study , we measured the vas both in mri images and direct conventional lumbo - sacral lateral x - rays , and noted the vas degree is correlated with each other . it was bigger in conventional x - rays than mri . in lying down , or standing x - rays we wanted to omit to give patient unnecessary radiation to by conventional x - ray , used mri images . the problem , here , may be important with regard with the vasc which is calculated on mri imaging in prone position and then referenced with a vertical line . this should not be problem , because sacral curve is the undistorted part of the spinal curves . regarding the second line which is the radius of the sacrum , it is positioning the centre of the sacral curvature . the sacral bone is an integral a part of the pelvis and constitutes the undistorted part of the spinal curves . we think that the development of the vertical angle of sacral curvature is related to the progressive acquisition of erect posture and the ontogeny of bipedal locomotion . as lumbosacral angle , this angle is almost nil in the nonprimate mammals , who only infrequently stand erect . organization of sagittal curves during growth can be followed up by looking at the sacrum and its vertical angle . this study suggested the impact of the sagittal sacral profile in pathogenesis of ldh in female . in this situation the compressive force component of gravity increases and these greater compressive forces accelerate the degeneration of the disc . this increase of the compressive forces is probably one of the many pathognomic conditions that lead to herniation . therefore , this pathology generally occurs after 20 - 25 years of age and rarely earlier in life . we suggest that the sacrum and also spine supported by the legs are balanced in the sagittal plane . the vertical angle of sacral curvature in female with ldh is bigger than the angle of female without ldh . the greater vasc or , anteriorly shifted the gravity line with increased vasc in females may be a risk factor for ldh . we think that increased vasc may have a direct influence on the development of lumbar disc herniation in females , but a thorough knowledge of the vertical angle of sacral curvature are needed to aid in understanding the mechanisms that lead to lumbar disc herniations in female and to provide rationale of management .

objectiveto characterize the importance of the vertical angle of the sacral curvature ( vasc ) in lumbar disc herniations.methodsmorphological data derived from lumbar sagittal mri imaging . the statistical significance of the findings are discussed . the angles of 60 female patients with lumbar disc herniations ( ldh ) were compared with the 34 female patients without ldh.results128 of the 185 patients met our inclusion criteria . the vertical angle of sacral curvature is statistically significantly bigger in females with lumbar disc herniations when compared to subjects in control group , 28.32 and 25.4 , respectively . ( p=0.034<0.05 ) . same difference was not seen in males.conclusionthe vertical angle of sagittal sacral curvature may be another risk factor in females with lumbar disc herniations .

INTRODUCTION MATERIALS AND METHODS Data collection Statistical analysis RESULTS DISCUSSION Why preferred sagittal MRI image to measure sacral curvature rate and vertical angle? CONCLUSION

PMC4574325

despite recent advances in endovascular techniques and technology for coil embolization of intracranial aneurysms , thromboembolic complications intra- and post - procedure are not uncommon , particularly in branches originating from the dome of the aneurysm . surgeons often face the dilemma of achieving complete coil obliteration of an arterial aneurysm while preserving blood flow to the branches . management decisions require a precise assessment of the risks of thromboembolic events tailored to individual aneurysms . the use of computational fluid dynamics ( cfd ) has helped to clarify patient - specific intra - aneurysmal flow patterns . here , we attempt to clarify the optimal coil configuration for endovascular embolization without compromising blood flow in the branching arteries , using cfd and a model based on an unruptured vertebral artery posterior inferior cerebellar artery ( va pica ) aneurysm . we selected a patient with an unruptured va pica aneurysm predicted to show impairment of flow in the pica during endovascular coil embolization . three - dimensional ct angiography was performed using an aquilion 64 ct scanner ( toshiba medical systems , tokyo , japan ) , and a patient - specific model was built using digital imaging and communications in medicine ( dicom ) data . next , a 3-dimensional voxel model was built ( case 0 ; figure 1 ) . this method is less time consuming compared with the method using an unstructured body - fitted grid , and aneurysm models , with or without modifications , can be easily built . we then built 6 models ( cases 16 ) of various idealized final coil configurations in the modeled aneurysm ( figure 2 ) . case 5 incorporated antegrade neck remodeling with a hyper - compliant balloon , and case 6 incorporated retrograde neck remodeling with a compliant balloon entering the pica from the va . finally , the outflow to the pica in each of the models was analyzed using cfd . . a : schema of a vertebral artery ( va)posterior inferior cerebellar artery ( pica ) aneurysm ( an ) . schema of 6 models with various final coil configurations in the modeled aneurysm ( case 0 ) . cfd of these models was carried out using the incompressible fluid analysis module in fujitsu -flow software ( fujitsu , tokyo , japan ) . we assumed that a non - newtonian fluid property for blood and an elastic property of the vessel walls could be neglected . the simulations were performed with the material constants for blood density = 1055 kg / m and blood dynamics viscosity = 0.0049 pas . each simulation was performed for 8 s. the boundary condition " velocity - inlet " was based on data from the patient with the aneurysm selected for modeling , acquired using a 3.0-tesla mr scanner ( signa infinity twinspeed with excite [ version 12 ] ; ge healthcare , milwaukee , wi , usa ) with a commercially available 8-channel head array coil . the grid points in the blood vessel and the aneurysm numbered about 500,000 in each simulation . the calculations were performed with the fujitsu hpc2500 at the information technology center of nagoya university . we selected a patient with an unruptured va pica aneurysm predicted to show impairment of flow in the pica during endovascular coil embolization . three - dimensional ct angiography was performed using an aquilion 64 ct scanner ( toshiba medical systems , tokyo , japan ) , and a patient - specific model was built using digital imaging and communications in medicine ( dicom ) data . next , a 3-dimensional voxel model was built ( case 0 ; figure 1 ) . this method is less time consuming compared with the method using an unstructured body - fitted grid , and aneurysm models , with or without modifications , can be easily built . we then built 6 models ( cases 16 ) of various idealized final coil configurations in the modeled aneurysm ( figure 2 ) . case 5 incorporated antegrade neck remodeling with a hyper - compliant balloon , and case 6 incorporated retrograde neck remodeling with a compliant balloon entering the pica from the va . finally , the outflow to the pica in each of the models was analyzed using cfd . . a : schema of a vertebral artery ( va)posterior inferior cerebellar artery ( pica ) aneurysm ( an ) . schema of 6 models with various final coil configurations in the modeled aneurysm ( case 0 ) . cfd of these models was carried out using the incompressible fluid analysis module in fujitsu -flow software ( fujitsu , tokyo , japan ) . we assumed that a non - newtonian fluid property for blood and an elastic property of the vessel walls could be neglected . the simulations were performed with the material constants for blood density = 1055 kg / m and blood dynamics viscosity = 0.0049 pas . each simulation was performed for 8 s. the boundary condition " velocity - inlet " was based on data from the patient with the aneurysm selected for modeling , acquired using a 3.0-tesla mr scanner ( signa infinity twinspeed with excite [ version 12 ] ; ge healthcare , milwaukee , wi , usa ) with a commercially available 8-channel head array coil . the grid points in the blood vessel and the aneurysm numbered about 500,000 in each simulation . the calculations were performed with the fujitsu hpc2500 at the information technology center of nagoya university . imaging and analysis of flow dynamics were successfully obtained for all models ( e.g. , case 0 ; figure 3 ) . the results of the outflow to the pica for each model are shown in the figure 4 and 5 . the average outflow to the pica was highest in case 0 and lowest in case 2 ( in descending order , case 0 , 5 , 4 , 6 , 1 , 3 , and 2 ) . there was better preservation of outflow to the pica in the balloon neck remodeling models than in the neck remnant models . therefore , a linear coil configuration did not necessarily improve preservation of outflow to the pica compared with a round coil configuration . the horizontal and vertical axis shows time ( second ) and the outflow velocity ( m / sec ) , respectively . this study reveals that the risk of thromboembolic complications in endovascular - treated aneurysms may be affected by subtle differences in the coil configuration around the aneurysmal neck . the geometry of some pica aneurysms precludes selective endovascular occlusion of the aneurysm with preservation of flow to the branch . however , direct surgery for these aneurysms is also challenging because of their location and close proximity to the medulla and lower cranial nerves . we used cfd in a modeled va pica aneurysm to determine outflow to the pica under a number of conditions . we have previously reported a successful method to make patient - specific aneurysmal models using dicom data in the rectangular coordinate system . in the present study , this aneurysm model was modified and various coil - embolized aneurysms were built . before running the models , we expected that cases 2 and 3 would have higher outflow than case 1 . moreover , we expected that case 2 would have higher flow than the pretreatment condition ( case 0 ) because of adjusted flow to the pica . however , it appears that when the branch originates at a sharp angle , as was the case with this aneurysm , the outflow is highest under the pretreatment condition . the finding of better preservation of outflow in the balloon neck remodeling models compared with the neck remnant models suggests that the use of a balloon to lift the coil mass around the aneurysmal neck was effective . in particular , balloon herniation with a hyper - compliant balloon ( case 5 ) preserved the highest amount of outflow to the pica . because surgical closure lines are generally linear , surgeons have to pay careful attention to preserve arterial branches . despite recent advances in computational flow analysis , the pulsating motion of vessels , peripheral vascular resistance , and differences among individual patients are also important factors in the accurate evaluation of flow patterns . previous reports have suggested that the effects of vessel wall elasticity and pulsation are not significant in relation to intra - aneurysmal flow . the present study shows a tendency towards different flow patterns in models with subtle changes in coil configuration in the neck of the aneurysm . further research , using patient - specific cfd , is needed to properly analyze the relationship between coil configuration in the neck of an arterial aneurysm and the outflow to the branches . in the future , cfd could be used to assess safety and to determine appropriate coil configurations for individual aneurysms . we identified considerable differences in the outflow to the branching artery with small changes in coil configuration for endovascular coil embolization . careful preoperative planning is important to minimize the risk of thromboembolic events during and after endovascular coil embolization . in the future , cfd could be applied to individual cases to determine a safe and adequate coil configuration for endovascular coil embolization .

abstractendovascular coil embolization of arterial aneurysms is often complicated by reduced blood flow to branching arteries . to determine the optimal coil configuration for safe embolization of endovascular aneurysms without compromising blood flow in branching arteries . a 3-dimensional voxel model , built based on an unruptured vertebral artery posterior inferior cerebellar artery ( va pica ) aneurysm , predicted to show impairment of flow in the pica during endovascular coil embolization ( case 0 ) . six different models of final coil configuration were generated and applied to this aneurysm . case 1 was a round coil mass . case 2 was designed with a stent assist . cases 3 , and 4 were designed with a neck remnant and cases 5 and 6 incorporated a balloon neck remodeling technique . computational fluid dynamics was used to analyze the flow in the pica in each model . the average outflow to the pica was highest in case 0 and lowest in case 2 ( in descending order , case 0 , 5 , 4 , 6 , 1 , 3 , and 2 ) . there was better preservation of outflow to the pica in the balloon neck remodeling models than in the neck remnant models . in a model of endovascular coil embolization , we found considerable differences in outflow to the branching artery with small changes in coil configuration . careful preoperative planning is important to minimize the risk of thromboembolic events during and after endovascular coil embolization .

INTRODUCTION MATERIAL AND METHODS Computational Models Computational Simulation RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS

PMC3035780

ten paid undergraduate students ( mean age 21 2 years ( sd ) ) participated in the experiment . three subjects were left - handed according to coren s test coren ( 1993 ) , and all others were right - handed . they were treated in accordance with the local guidelines and signed an informed consent form . they were naive as to the purpose of the experiments , and none of the subjects reported any known hand deficits . the stimuli consisted of varying numbers of brass shapes , which were suspended from flexible wires . the presented shapes were cubes ( edge length 1.5 cm ) , spheres ( radius 0.93 cm ) , and ellipsoids ( radii : 1.22 , 0.81 and 0.81 cm ) . to eliminate weight cues , these dimensions were chosen such that the volume , and thus the weight of the shapes , was constant . 1a.fig . 1stimuli and set - up . a shapes that were used in the experiment . the targets were always spheres , while the distractor was either a cube or an ellipsoid . the targets were always spheres , while the distractor was either a cube or an ellipsoid . b picture of a subject grasping the stimuli a custom - built device was used to measure response times . time measurement was started when the subject touched the stimulus activating the touch - sensitive contact of the device . the measurement was terminated by a vocal response registered with a headset microphone ( for details about this set - up see plaisier et al . before the experiment was started , the subjects were informed of which shapes were the target items and which shapes were distractor items . they were instructed to respond the correct number of target items as fast as possible . the subjects wore a blindfold and earplugs to avoid any sound cues . prior to a trial sets of shapes were suspended from flexible wires above the hand , and subjects were instructed to reach upwards to grasp all items simultaneously . after grasping all items , they were allowed to release items from their hand during the trial . it was emphasized that they should only release items from their hand if this seemed the most efficient strategy . subjects were instructed to respond the correct number of target items as fast as possible . error trials were repeated at the end of a block of trials , and only correct responses were included in the analysis . the two conditions were performed in separate blocks of trials on separate days in counter - balanced order . in one condition , a cube was added as distractor item , and in the other condition , an ellipsoid was used as distractor item . in both cases , spheres were the target items , and the number of spheres had to be judged . in half of the trials , a distractor item was present , and the distractor - present and distractor - absent trials were randomly interleaved . we measured up to 4 items , but did not include this numerosity in the analysis to exclude possible end - effects . each numerosity was repeated ten times with and without a distractor in the set leading to a total of ( 10 4 2 =) 80 trials per block . to each block of trials also 15 catch trials were added . in the catch trials , the presented set of items could contain up to 4 distractor items with the restriction that the total number of items in the set did not exceed 6 . this was done to prevent subjects from noticing there was always only one distractor item , but these trials were not included in the analysis . a similar procedure was used by trick and pylyshyn ( 1993 ) . prior to each block of trials , subjects performed at least 20 training trials , and trials it was never necessary to exceed 30 training trials . because subjects were instructed to respond as fast as possible , but also correct the error rate should be low in all conditions . therefore , the error rates are given as an indication that subjects could perform the task correctly . only the response times and item release rates weighted linear regression was performed on the response times averaged over subjects as well as on the single subjects data . the quality of fit to the data averaged over subjects will be better , but it is also important to show that the same trend is present for each subject individually . the last numerosity in the presented range of numerosities normally deviates from linearity and is therefore often excluded from the regression analysis ( e.g trick and pylyshyn 1993 ; watson et al . 2007 ; trick 2008 ) . the results from the present study were , however , not significantly different when the largest numerosity was included in the analysis . furthermore , repeated measures anova was performed on the single - subject regression slopes as well as on the item release rate . to determine the effect on the slope values , we compare the response times slopes to the values of response time slopes found in two previous studies performed without any distractors ( plaisier et al . 2009a , 2010 ) . these studies were performed using the same spheres as used in the present study , and the same response time measuring device was used . sets of up to 7 or 12 items were used , and a regression of a bi - linear function was used to determine the subitizing and counting slope values as well as the transition point between the two regimes . ten paid undergraduate students ( mean age 21 2 years ( sd ) ) participated in the experiment . three subjects were left - handed according to coren s test coren ( 1993 ) , and all others were right - handed . they were treated in accordance with the local guidelines and signed an informed consent form . they were naive as to the purpose of the experiments , and none of the subjects reported any known hand deficits . the stimuli consisted of varying numbers of brass shapes , which were suspended from flexible wires . the presented shapes were cubes ( edge length 1.5 cm ) , spheres ( radius 0.93 cm ) , and ellipsoids ( radii : 1.22 , 0.81 and 0.81 cm ) . to eliminate weight cues , these dimensions were chosen such that the volume , and thus the weight of the shapes , was constant . the targets were always spheres , while the distractor was either a cube or an ellipsoid . the targets were always spheres , while the distractor was either a cube or an ellipsoid . b picture of a subject grasping the stimuli a custom - built device was used to measure response times . time measurement was started when the subject touched the stimulus activating the touch - sensitive contact of the device . the measurement was terminated by a vocal response registered with a headset microphone ( for details about this set - up see plaisier et al . before the experiment was started , the subjects were informed of which shapes were the target items and which shapes were distractor items . they were instructed to respond the correct number of target items as fast as possible . the subjects wore a blindfold and earplugs to avoid any sound cues . prior to a trial sets of shapes were suspended from flexible wires above the hand , and subjects were instructed to reach upwards to grasp all items simultaneously . after grasping all items , they were allowed to release items from their hand during the trial . it was emphasized that they should only release items from their hand if this seemed the most efficient strategy . subjects were instructed to respond the correct number of target items as fast as possible . error trials were repeated at the end of a block of trials , and only correct responses were included in the analysis . the two conditions were performed in separate blocks of trials on separate days in counter - balanced order . in one condition , a cube was added as distractor item , and in the other condition , an ellipsoid was used as distractor item . in both cases , spheres were the target items , and the number of spheres had to be judged . in half of the trials , a distractor item was present , and the distractor - present and distractor - absent trials were randomly interleaved . we measured up to 4 items , but did not include this numerosity in the analysis to exclude possible end - effects . each numerosity was repeated ten times with and without a distractor in the set leading to a total of ( 10 4 2 =) 80 trials per block . to each block of trials also 15 catch trials were added . in the catch trials , the presented set of items could contain up to 4 distractor items with the restriction that the total number of items in the set did not exceed 6 . this was done to prevent subjects from noticing there was always only one distractor item , but these trials were not included in the analysis . a similar procedure was used by trick and pylyshyn ( 1993 ) . prior to each block of trials , subjects performed at least 20 training trials , and trials because subjects were instructed to respond as fast as possible , but also correct the error rate should be low in all conditions . therefore , the error rates are given as an indication that subjects could perform the task correctly . weighted linear regression was performed on the response times averaged over subjects as well as on the single subjects data . the quality of fit to the data averaged over subjects will be better , but it is also important to show that the same trend is present for each subject individually . the last numerosity in the presented range of numerosities normally deviates from linearity and is therefore often excluded from the regression analysis ( e.g trick and pylyshyn 1993 ; watson et al . 2007 ; trick 2008 ) . the results from the present study were , however , not significantly different when the largest numerosity was included in the analysis . furthermore , repeated measures anova was performed on the single - subject regression slopes as well as on the item release rate . to determine the effect on the slope values , we compare the response times slopes to the values of response time slopes found in two previous studies performed without any distractors ( plaisier et al . 2009a , 2010 ) . these studies were performed using the same spheres as used in the present study , and the same response time measuring device was used . sets of up to 7 or 12 items were used , and a regression of a bi - linear function was used to determine the subitizing and counting slope values as well as the transition point between the two regimes . the error rates were overall low . for the distractor - absent trials , error rates were 0.5% in the cube condition and 0.75% in the ellipsoid condition . in the distractor - present trials , the error rates were 3 and 4.25% , respectively . response times averaged over subjects as a function of the number of items with a cube as distractor are shown in fig . 2a , and response times with an ellipsoid as distractor are shown in fig . the response times for the distractor - absent trials are shown in black and those for the distractor - present trials are shown in gray . this yielded in the condition with a cube as distractor , a distractor - absent slope of 0.2 s / item and a distractor - present slope of 0.6 s / item . for the condition with the ellipsoid as distractor , the distractor - absent slope was found to be 1.1 s / items , and the distractor - present slope was 1.6 s / item.fig . 2response times averaged over subjects for the distractor - present and distractor - absent trials . a condition with a cube as distractor and b condition with an ellipsoid as distractor . the solid lines indicate linear regression to the response times , and the error bars indicate the between subjects standard deviation response times averaged over subjects for the distractor - present and distractor - absent trials . a condition with a cube as distractor and b condition with an ellipsoid as distractor . the solid lines indicate linear regression to the response times , and the error bars indicate the between subjects standard deviation linear regression was also performed on the single - subject data . the values from the single - subject slopes averaged again over subjects and the standard deviation between subjects are reported here . this yielded in the cube condition a distractor - absent slope of 0.2 0.1 s / item ( r = 0.8 0.1 ) and a distractor - present slope of 0.6 0.2 s / item ( r = 0.9 0.1 ) . for the ellipsoid condition , this yielded a distractor - absent slope of 1.1 0.3 s / item ( r = 0.99 0.02 ) and a distractor - present slope of 1.6 0.6 s / item ( r = 0.98 0.02 ) . a 2 2 ( distractor shape distractor presence ) repeated measures anova was performed on the single - subject slopes . there was an effect of distractor shape ( f(1 , 9 ) = 47 , p < 0.001 ) and of distractor presence ( f(1 , 9 ) = 22 , p = 0.001 ) . the percentage of trials in which items were released from the hand is shown in fig . a 2 2 ( distractor shape distractor presence ) repeated measures anova was performed on the item release rates . this showed an effect of distractor shape ( f(1 , 9 ) = 34 , p < 0.001 ) as well as distractor presence ( f(1 , 9 ) = 107 , p < 0.001 ) and an interaction effect ( f(1 , 9 ) = 7 , p = 0.025).fig . 3item release rate collapsed over all numerosities and averaged over subjects for distractor - present and distractor - absent trials in both conditions . the error bars indicate the between - subjects standard deviation item release rate collapsed over all numerosities and averaged over subjects for distractor - present and distractor - absent trials in both conditions . our results show that subitizing slopes increased when a distractor was added , regardless of the saliency of the distractor . when the distractor was a cube , the numerosity judgment slope increased from 0.2 to 0.6 s / item . when the distractor was an ellipsoid , slopes increased from 1.1 to 1.6 s / item . in two previous studies into haptic numerosity judgment , we measured the subitizing and counting slopes when the items were spheres ( plaisier et al . 2009a , 2010 ) . slope values were found to be 0.17 and 0.2 s / items in the subitizing regime and 0.8 s / item and 1.2 s / item in the counting regime . the slopes found in the present study for the ellipsoid condition have a value comparable to counting slopes . the slope values for the cube condition were , however , clearly below counting slope values . the distractor - absent slope was in this case in the same range as subitizing slopes that were found previously . this shows that adding a distractor increases numerosity judgement slopes , but subitizing was still possible in the cube condition . the distractor - absent slope in the cube condition was comparable to previously found subitizing slope values , but the distractor - absent slope in the ellipsoid condition was clearly in the range of generally found counting slopes . the stimulus was , however , the same in both cases . since a cube among spheres is highly salient subjects knew in the cube condition immediately after grasping , the set whether there were distractors present . when cubes were detected , they should be ignored while judging the numerosity , and this resulted in an increase in the slope . an ellipsoid among spheres , on the other hand , is not salient , and serial search is needed to find it . this is also indicated by the larger item release rate in the ellipsoid condition than in the cube condition . releasing items from the hand is normally an indication of serial processing ( plaisier et al . therefore , subjects visited each item subsequently regardless of whether a distractor was present in the ellipsoid condition . still , in this case , the slope was larger when a distractor was present . so , although items were processed in a serial fashion , the presence of a distractor increased the response time per item . note that this difference in slope can not be caused by the fact that in distractor - present trials , the total number of items was larger than in the distractor - absent case . this causes a larger offset of the response times , but does not affect the slopes . also in vision , it was found that the slope for small sets of items increased when a distractor was added , even if this was a highly salient distractor ( trick and pylyshyn 1993 ) . similar to our results , also the slope for distractor - absent trials differed depending on the type of distractor used . when a non - salient distractor was used , subitizing was possible for the distractor - absent trials , but the slope was larger than when a salient distractor was used . the effects reported here for haptic subitizing in the presence of a distractor are therefore comparable to those found in vision . the effect of adding a single distractor to the set was in the haptic case , however , more pronounced than in the visual case . our results suggest a modality - independent process underlying subitizing . trick and pylyshyn ( 1993 ) concluded from the fact that subitizing was only possible when the targets pop - out from among the distractors that there is a pre - attentive stage of item individuation in subitizing . they proposed that there are fingers of instantiation ( finst s ) that can be used to point to individual items in the visual field ( see pylyshyn 2001 for a review ) . fingers do not need to be attributed to item properties , such as color , shape or even position . although this visual theory might be extended to include the haptic modality , an alternative explanation which is it has been shown that humans can hold a limit number of chunks of information in their working memory . such a capacity limit would result in the same upper limit for subitizing in vision and touch which generally seems to be true ( see cowan 2001 for an overview ) . peterson and simon ( 2000 ) computationally showed that when varying numbers of dots are placed on a two - dimensional grid , there are many more recurrent patterns for small numerosities ( < 4 ) than for larger numerosities . they then showed that their neural network displayed subitizing - like behavior after a learning period . such a pattern recognition explanation is , however , less likely in the haptic case as the objects were not fixed in space . therefore , a capacity limit in short term memory would be the best candidate for a modality transcending explanation for the occurrence of subitizing . nor in the present study , nor in the study by ( trick and pylyshyn 1993 ) was the subitizing slope unaffected by adding distractors . it is clearly not caused by impairment of the subitizing process as the subitizing slopes were at their normal values in distractor - absent trials when pop - out targets were used . if first the targets were pre - attentively selected and then put into memory chunks , we would not expect the subitizing slope to be affected at all for pop - out targets . therefore , it seems that either distractors can not be completely ignored during subitizing or the total set size is determined first and the distractors are subtracted afterwards to arrive at the number of targets . either way , salient distractors impair subitizing in both vision and haptics although they do not prevent subitizing like non - salient distractors do .

subitizing refers to fast and error - free numerosity judgment for small ( < 4 ) sets of items . for larger sets , the slower process of counting is used . counting has a serial character , whereas subitizing is believed to have a parallel character . while subitizing was initially found in vision , it has been shown to exist in touch as well . in vision , it has been demonstrated that adding distractor items to a set of target items influences numerosity judgment of the target items . subitizing was in this case only possible if the distractor item is highly salient among the targets . in the present study , we investigated the effect of adding a distractor item on haptic judgement of a set of target items . to this end , we asked subjects to judge the number of spheres grasped in their hand . either a cube or an ellipsoid could be added to the set . a cube among spheres has been shown to be highly salient , while an ellipsoid among spheres is not . our results show that adding a distractor item led to an increase in the response time slopes regardless of the distractor shape . subitizing was , however , only possible in the case of a salient distractor . this is in agreement with results from vision .

Methods Participants Stimuli and apparatus Procedure Analysis Results Discussion

PMC4040383

the 95 ankles from 57 patients with achilles tendon tightness were included in the study . the first group ( idiopathic ) consists of the patients with no other specific disorders ( n = 26 ) or with disorders not involving the muscle and the tendon , such as flat foot ( n = 13 ) and tibial or femoral torsion ( n = 4 ) . the next group ( neuromuscular or other disorders ) consists of the patients with disorders involving the muscle and the tendon , such as the cerebral palsy ( n = 22 ) , muscular dystrophy ( n = 12 ) , recurred or residual clubfoot deformity ( n = 10 ) , and trauma ( n = 8) . the 84 ankles with mild ( less than 10 ) to moderate ( 10-20 ) equinus deformities were treated with the casts after the tendon lengthening , while 11 ankles with severe ( equinus more than 20 ) deformities received combination treatment of serial casts ( n = 6 ) and ilizarov gradual correction ( n = 5 ) . the mean age of the patients at the time of the surgery was 12.5 years ( range , 4 to 28 years ) . the mean follow - up period after the surgery was 2.2 years ( range , 1.1 to 5.4 years ) . we compared our modified technique ( 95 ankles ) to the percutaneous sliding lengthening ( 18 ankles ) and the z - lengthening with a medial longitudinal incision ( 19 ankles ) , in terms of the outcomes , the complications , the advantages , and the disadvantages , through the reviews of the patients ' medical records and the telephone interviews . the mean age of the patients at the time of surgery was 9.8 years ( range , 4 to 12 years ) for the percutaneous sliding technique and 18.2 years ( range , 6 to 34 years ) for the z - lengthening with a longitudinal incision technique . the first step is to identify two to three deep creases of the skin about 2 cm apart over the achilles tendon . when there is a mild to moderate equinus deformity , one skin crease of 2 to 3 cm above the tendon insertion is chosen ( fig . 1 ) . when the contracture is severe ( more than 20 ) , one or two more incisions on the proximal creases are needed for the adequate lengthening . step 2 . the skin and fascia are carefully incised with the number 15 blade avoiding an inadvertent transverse incision underneath the achilles tendon . the flares are used to separate the paratenon from the achilles tendon , distally and proximally . the plantaris tendon is identified on the medial border of the achilles tendon . with the small retractors placed under the paratenon , the distal longitudinal tendon incision must be done carefully , in order to avoid laceration of the distal part of the transverse skin incision ( fig . when the tip of the blade touches the calcaneus , the blade is slowly turned 90 medially and the medial 1/2 of the tendon is incised ( or a lateral 1/2 incision could be used when necessary , such as in the planovalgus with short achilles tendon ) . extra cautions must be taken to prevent damage to the tibialis posterior nerve and vessel and to the flexor hallucis longus tendon , which passes near the medial malleolus . the excessive advancement of the blade medially would increase the risk of damage to the neurovascular bundle . a small retractor is placed under the deep fascia and paratenon , and the ankle is dorsiflexed to advance the cut more proximally and to provide tension in the tendon , so the surgeon could feel when the tendon is completely cut . after 3 cm to 4 cm of a longitudinal cut proximally for mild to moderate equinus in children or 5 cm to 6 cm for moderate equinus in adolescents , the scissors are slowly turned laterally and the lateral 1/2 of the tendon is cut . extra caution is needed in order to prevent damaging the sural nerve . a thumb ( fig . 4 ) was placed over the tip of the surgical scissors to prevent them from advancing beyond the lateral ( to protect the sural nerve ) or medial border of the tendon . if unable to perform a dorsiflexion of the ankle after the proximal lateral cut , the plantaris tendon needs to be rechecked because it may not have been cut when the distal medial incision was done . when deformity is severe , the second and the third proximal longitudinal incisions of the tendon are made to create a longer lateral strip . the amount of lengthening required is determined with the medial and lateral strips of the tendon , held side by side with the thumb forceps while applying moderate tension after the ankle has been maximally dorsiflexed once or twice ( fig . , the level of the distal end of the medial strip , which is displaced proximally following the maximal ankle dorsiflexion , is marked on the lateral strip with the ankle in the neutral position ( fig . 6 ) . a simple 2 layer , anterior and posterior suture technique is used . the first is from the anterior side of the tendon , starting the suture and burying the knot inside the tendon ( fig . the consistent suturing is important to prevent a palpable lump on the skin after the surgery , especially on the posterior aspect of the tendon . step 6 . for the accurate suturing of the paratenon and fascia , it is important to identify them clearly . the first stitch has to be done carefully to avoid suturing these two structures together with the achilles tendon located below them . the suture binds the paratenon and the deep fascia together , and then a subcutaneous skin suture is applied . a short leg cast with the ankle at 90 is applied for 6 weeks in the patients under 10 years of age . for additional protection , either a second short leg cast or an ankle foot orthosis is applied for 4 to 6 weeks in the patients over 10 years of age . the gentle active and passive ankle dorsiflexion and heel lifting exercises are gradually recommended after immobilization . any participation in active sports we evaluated the dorsiflexion angle of the ankle before the surgery and at the latest follow - up . the time under anesthesia for the lengthening procedure and any of the postoperative complications were also noted . the surgical results were evaluated using the american orthopaedic foot & ankle society ( aofas ) ankle - hindfoot scale . in addition , to evaluate the patients ' satisfaction regarding the surgical scar , we allotted another 10 points to the cosmesis of surgical scar ( fig . the first step is to identify two to three deep creases of the skin about 2 cm apart over the achilles tendon . when there is a mild to moderate equinus deformity , one skin crease of 2 to 3 cm above the tendon insertion is chosen ( fig . when the contracture is severe ( more than 20 ) , one or two more incisions on the proximal creases are needed for the adequate lengthening . step 2 . the skin and fascia are carefully incised with the number 15 blade avoiding an inadvertent transverse incision underneath the achilles tendon . the flares are used to separate the paratenon from the achilles tendon , distally and proximally . the plantaris tendon is identified on the medial border of the achilles tendon . with the small retractors placed under the paratenon , the distal longitudinal tendon incision must be done carefully , in order to avoid laceration of the distal part of the transverse skin incision ( fig . when the tip of the blade touches the calcaneus , the blade is slowly turned 90 medially and the medial 1/2 of the tendon is incised ( or a lateral 1/2 incision could be used when necessary , such as in the planovalgus with short achilles tendon ) . extra cautions must be taken to prevent damage to the tibialis posterior nerve and vessel and to the flexor hallucis longus tendon , which passes near the medial malleolus . the excessive advancement of the blade medially would increase the risk of damage to the neurovascular bundle . a small retractor is placed under the deep fascia and paratenon , and the ankle is dorsiflexed to advance the cut more proximally and to provide tension in the tendon , so the surgeon could feel when the tendon is completely cut . after 3 cm to 4 cm of a longitudinal cut proximally for mild to moderate equinus in children or 5 cm to 6 cm for moderate equinus in adolescents , the scissors are slowly turned laterally and the lateral 1/2 of the tendon is cut . extra caution is needed in order to prevent damaging the sural nerve . a thumb ( fig . 4 ) was placed over the tip of the surgical scissors to prevent them from advancing beyond the lateral ( to protect the sural nerve ) or medial border of the tendon . if unable to perform a dorsiflexion of the ankle after the proximal lateral cut , the plantaris tendon needs to be rechecked because it may not have been cut when the distal medial incision was done . when deformity is severe , the second and the third proximal longitudinal incisions of the tendon are made to create a longer lateral strip . the amount of lengthening required is determined with the medial and lateral strips of the tendon , held side by side with the thumb forceps while applying moderate tension after the ankle has been maximally dorsiflexed once or twice ( fig . , the level of the distal end of the medial strip , which is displaced proximally following the maximal ankle dorsiflexion , is marked on the lateral strip with the ankle in the neutral position ( fig . 6 ) . a simple 2 layer , anterior and posterior suture technique is used . the first is from the anterior side of the tendon , starting the suture and burying the knot inside the tendon ( fig . the consistent suturing is important to prevent a palpable lump on the skin after the surgery , especially on the posterior aspect of the tendon . step 6 . for the accurate suturing of the paratenon and fascia , it is important to identify them clearly . the first stitch has to be done carefully to avoid suturing these two structures together with the achilles tendon located below them . the suture binds the paratenon and the deep fascia together , and then a subcutaneous skin suture is applied . a short leg cast with the ankle at 90 is applied for 6 weeks in the patients under 10 years of age . for additional protection , either a second short leg cast or an ankle foot orthosis is applied for 4 to 6 weeks in the patients over 10 years of age . the gentle active and passive ankle dorsiflexion and heel lifting exercises are gradually recommended after immobilization . we evaluated the dorsiflexion angle of the ankle before the surgery and at the latest follow - up . the time under anesthesia for the lengthening procedure and any of the postoperative complications were also noted . the surgical results were evaluated using the american orthopaedic foot & ankle society ( aofas ) ankle - hindfoot scale . in addition , to evaluate the patients ' satisfaction regarding the surgical scar , we allotted another 10 points to the cosmesis of surgical scar ( fig . the mean time under the anesthesia was 30.6 minutes ( range , 20 to 40 minutes ) ; the actual duration of the surgery was approximately 15 - 20 minutes . all of the patients showed improvement in gait pattern and they showed satisfactory cosmetic results . the mean aofas score improved from 56.1 to 81.8 . within this group of patients , those with neuromuscular or other disorders showed low aofas scores , which improved from 42.3 to 67.1 , compared to those with idiopathic achilles tendon tightness , which improved from 69.9 to 96.5 . as for the patients ' satisfaction with the transverse skin incisions , the mean score was 7.2 points out of 10 points ( fig . no patients had complications such as the tendon adhesion , total transection , excessive lengthening ( calcaneus deformity ) , or neurovascular damage . the recurrence of the tendon shortening was noticed in 4 ankles of the 2 cerebral palsy patients , and the second lengthening procedures were performed through the same incisions . the recurrence and other complications were noted for all 3 techniques ( table 1 ) . our new technique attempts to take the best points from both the percutaneous sliding lengthening and z - lengthening ( with a medial longitudinal incision ) techniques . the mean time under the anesthesia was 30.6 minutes ( range , 20 to 40 minutes ) ; the actual duration of the surgery was approximately 15 - 20 minutes . all of the patients showed improvement in gait pattern and they showed satisfactory cosmetic results . the mean aofas score improved from 56.1 to 81.8 . within this group of patients , those with neuromuscular or other disorders showed low aofas scores , which improved from 42.3 to 67.1 , compared to those with idiopathic achilles tendon tightness , which improved from 69.9 to 96.5 . as for the patients ' satisfaction with the transverse skin incisions , the mean score was 7.2 points out of 10 points ( fig . no patients had complications such as the tendon adhesion , total transection , excessive lengthening ( calcaneus deformity ) , or neurovascular damage . the recurrence of the tendon shortening was noticed in 4 ankles of the 2 cerebral palsy patients , and the second lengthening procedures were performed through the same incisions . the recurrence and other complications were noted for all 3 techniques ( table 1 ) . our new technique attempts to take the best points from both the percutaneous sliding lengthening and z - lengthening ( with a medial longitudinal incision ) techniques . the achillles tendon has three sources of blood : the posterior tibial artery in the distal section , the peroneal artery on the mid - section , and the posterior tibial artery in the proximal section.15,17,18 ) the arteries of the achilles tendon and its paratenon are oriented in three directions : longitudinal , transverse , and deep . in most cases , the larger arterial branches run on the surface of the tendon transversely , perpendicular to the direction of the tendon fibers . our transverse distal incision is made on a natural skin crease , with its length from 1.0 cm to 2.0 cm depending on the size of the patient . compared to the longitudinal incision , this minimizes the risk of damaging the arterial vessels running transversely on the posterior surface . the anatomical structures at the risk of damage , with our technique , are the posterior tibial nerve , the artery located in the posteromedial area of the ankle , and the sural nerve located laterally from the achilles tendon . at the level of the ankle joint in an adult , the posterior tibial nerve is located at an average of 11.8 2.4 mm and the posterior tibial artery at 16.7 3.8 mm , both anterior from the achilles tendon.19 ) these structures are , on average , less than 1 cm from the nearest margin of a given percutaneous triple - hemisection in the adult.11 ) the sural nerve crosses the lateral border of the tendon at an average of 9.8 cm ( range , 7 to 16 cm ) from the calcaneus in the adult.20,21,22 ) all of these distances are shorter in children . first , a small retractor is placed at the medial side of the tendon when the blade is turning 90 to cut the medial 1/2 of the tendon at the distal insertion site . second , the thumb or the middle finger tip , depending on which hand the physician uses to support the patient 's lower leg , is placed at the lateral margin of the tendon when the proximal lateral cut is performed . this will prevent the further advancement of the surgical scissors ' tip toward the lateral side , so the damage of the lateral sural nerve can be avoided . it is difficult to quantify the amount of corrections required.7,23 ) with our technique , preservation of the peritendinous attachments in the proximal tendon prevents the excessive proximal migration of the medial or lateral strips of the tendon , even with the maximal dorsiflexion of the ankle after z - lengthening , because only the posterior aspect of the tendon is exposed transversely . the foot is maximally dorsiflexed several times and then positioned in neutral , while the tendon is sutured under a moderate tension obtained by thumb forceps on both halves of the tendon . the toe flexors are carefully examined at the same time as the maximal ankle dorsiflexion to see whether they are also contracted . the flexor lengthening was performed when it was indicated , such as the flexor tenotomy at the proximal interphalageal joint in muscular dystrophy and trauma patients , and the tendon recession in the toe flexor and tibialis posterior at the level of the musculotendonous junction in cerebral palsy patients . our technique can provide the medial and lateral strips of the sufficient lengths with a single incision in the mild to moderate ankle equinus . it is desirable to have more than 1 cm in children or 1.5 cm in adults of overlap of the medial and lateral strips for a firm side by side suture with adequate correction . even suturing with the knots buried inside the tendon is essential to produce a smooth posterior surface . in a cross - section of the achilles tendon , the insertion of the tendon is flat and spread - out as it attaches to the tuberosity of the calcaneus , while the midsection is oval shaped and is narrower than its insertion and the origin.15 ) therefore , the partial excision of the thickened part of the distal tendon stump to match the proximal side is necessary to avoid a palpable lump after the suture . it is also important to suture the paratenon and the deep fascia together with the knot buried in the soft tissues , using an absorbable suture material . a special care must be taken with the first suture to avoid suturing these structures with the underlying achilles tendon . also , the suture should be even without crumpling the skin . in the case of a severe equinus deformity , we apply a cast with the ankle in a mild equinus position while making sure there is an adequate vascular supply in the skin . one week after the surgery , when the skin circulation is satisfactory , a second cast with the correct ankle position is applied . we also adopt the ilizarov gradual correction technique to correct a severe ankle equinus deformity . for this , before application of the ilizarov external fixator , we use 2 or 3 transverse incisions and lengthen the tendon through the second and third incisions without suturing from side to side . the lengthened tendons gradually slide against each other until the deformity is corrected with the ilizarov apparatus . the short leg cast after 4 weeks of the complete correction and the ankle foot orthosis during the night provide a good result and prevent recurrences , especially for the patients with neuromuscular disorders , club foot , and trauma . the correction happens quickly , without any significant pain or discomfort to the patient as the tendon lengthens . none of our patients , even those with severe deformity , have shown any skin problems due to the tension . we retrospectively studied the results of our new technique and compared them with the results of two other techniques we have used before . the patient attributes , which include the number of patients , ages , causes , severities , etc . , are not the same for the different techniques . our new technique seems to have a low rate of recurrence of equinus deformity ( 4 of 95 feet ) at the medium - term follow - up , compared to the other approaches ; and no patient with a crouch gait resulted from the over - lengthening . we believe that this was due to the short skin incisions , the preservations of the paratenon and the deep fascial tube , and the use of z - lengthening rather than the open or percutaneous sliding lengthening or lengthening the aponeurotic tendon of the gastrocnemius . with the new approach , we had fewer patients complaining of the pain compared to those who were operated with a longitudinal skin incision . the cases of recurrence were mostly limited to the cerebral palsy patients , who were less than 5 years old at the time of surgery , and they appeared at least 3 years after the surgery . although the range of the passive dorsiflexion was decreased during the period of rapid growth in these children , any recurrent equinus was easy to treat because there was little scarring at the operation site . in summary , our technique of achilles tendon lengthening has proven to be an improvement over the other techniques we have employed , in terms of surgical time , tendon healing , and complication rates . this is perhaps due to the small skin incision and the conservation of the paratenon and the deep fascia .

backgroundthe risk of various complications after achilles tendon lengthening is mainly related to the length of surgical exposure and the lengthening method . a comprehensive technique to minimize the complications is required.methodsthe treatment of achilles tendon tightness in 57 patients ( 95 ankles ) were performed by using a short transverse incision on a skin crease of the heel and by z - lengthening of the tendon . in the severe cases , two or three transverse incisions were required for greater lengthening of the tendon , and a serial cast or ilizarov apparatus was applied for the gradual correction . the results of these 95 ankles were compared to those of 18 ankles , which underwent percutaneous sliding lengthening , and to the 19 ankles , which received z - lengthening with a medial longitudinal incision.resultsthe functional and cosmetic satisfaction was achieved among those who underwent the tendon lengthening with the new technique . the mean american orthopaedic foot & ankle society ( aofas ) score improved from 56.1 to 81.8 . the second operations to correct recurrence were performed in the two cerebral palsy patients.conclusionsthe new technique has a low rate of complications such as scarring , adhesion , total transection , excessive lengthening , and recurrence of shortening . the excellent cosmesis and the short operation time are the additional advantages .

METHODS Surgical Technique After Treatment Assessment of Results RESULTS Results of Z-lengthening of the Achilles Tendon with a Transverse Skin Incision Comparison with Percutaneous Sliding Lengthening and Z-lengthening with a Medial Longitudinal Incision DISCUSSION

PMC3035808

muscular dystrophy includes loss of muscle strength and muscle mass and leads to gait disturbances resulting in compensatory kinematic and postural adaptation [ 13 ] . eventually , patients lose their ability to stand upright and walk . clinical gait analysis of subjects with muscular dystrophy is widely used to test the efficacy of new drugs because there are few reliable methods to determine function in muscular dystrophy . histopathological studies of animal models , including the delta - sarcoglycan - deficient dystrophic hamster , have played an important role in our understanding and treatment of muscular dystrophy . functional phenotypic evidence , however , of restoration of muscle strength with therapy in animal models , is becoming increasingly important in preclinical drug development [ 59 ] . a recent study of canine muscular dystrophy accounted for the slower walking speed in dystrophic subjects to more accurately quantify the kinematics of pathologic gait during overground walking . treadmill gait analysis provides the opportunity to have all subjects walk at the same speed to rule out differences in walking speed as being the most important confounder in the interpretation of gait disturbances . treadmill gait analysis has recently been applied to the characterization of mouse models of amyotrophic lateral sclerosis [ 11 , 12 ] , rat models of nerve injury [ 13 , 14 ] , and canine arthritis . quantitative kinematic gait analysis in a small animal model of muscular dystrophy , however , has not yet been reported . we , therefore , performed treadmill gait analysis in the bio to-2 strain of myopathic hamsters to fully characterize their posture and kinematics during walking . the bio to-2 [ to-2 ] hamster , with a deletion in the delta - sarcoglycan gene , is a widely studied animal model of muscular dystrophy [ 1620 ] . these animals show pathological signs of muscular dystrophy , including elevated serum creatine kinase and central nucleation and necrosis of muscle fibers . we compared gait in the myopathic hamsters to the gait in healthy bio f1b [ f1b ] control hamsters and found several functional indices that significantly and consistently deviate beginning early in life . the method described in this report provides quantitative functional metrics of gait associated with sarcoglycan deficiency in a readily available animal model . male bio f1b control and bio to-2 dystrophic hamsters were obtained from bio breeders , inc . , fitchburg , ma . handling and care of hamsters were consistent with federal guidelines and approved institutional protocols . gait dynamics were recorded using ventral plane videography , as previously described [ 21 , 22 ] . briefly , we devised a motor - driven treadmill with a transparent treadmill belt [ digigait imaging system , mouse specifics , inc . , quincy , ma ] . a high - speed digital video camera was positioned below the transparent belt to focus on the ventral view of subjects walking atop of the belt . an acrylic compartment , ~7 cm wide by ~30 cm long and adjustable for length and width , was mounted on top of the treadmill to maintain the hamsters within the view of the camera . figure 1(a ) depicts a 9-month - old to-2 hamster walking on the treadmill belt within the acrylic compartment . digital video images of the underside of hamsters were recorded at 125 frames per second . each image represents ~8 ms ; the paw area indicates the temporal placement of the paw relative to the treadmill belt . the color images ( figure 1(b ) , top panel ) were converted to their binary matrix equivalents ( figure 1(b ) , bottom panel ) and the areas of the approaching or retreating paws relative to the belt and camera were calculated throughout each stride . the plotted area of each digital paw print ( paw contact area ) imaged sequentially in time provides a dynamic gait signal , representing the temporal record of paw placement relative to the treadmill belt ( figure 1(c ) ) . each gait signal for each limb comprises a stride duration which includes the stance duration when the paw of a limb is in contact with the walking surface , plus the swing duration when the paw of the same limb is not in contact with the walking surface . stance duration was further subdivided into braking duration ( increasing paw contact area over time ) and propulsion duration ( decreasing paw contact area over time ) ( figure 1(c ) ) . stride length was calculated from the following equation : speed = stride frequency stride length . stance widths and paw placement angles at full stance were obtained by fitting ellipses to the paws and determining the centers , vertices , and major axes of the ellipses . forelimb and hindlimb stance widths were calculated as the perpendicular distance between the major axis of the left and right fore paw images and between the major axis of the left and right hindpaw images during peak stance . paw placement angle was calculated as the angle that the long axis of a paw makes with the direction of motion of the animal during peak stance . gait data were collected and pooled from both the left and right forelimbs , and the left and right hindlimbs . measures of stride - to - stride variability ( gait variability ) for stride length and stance width were determined as the standard deviation and the coefficient of variation ( cv ) . cv , expressed as a % , was calculated from the following equation : 100 standard deviation / mean value . each hamster was allowed to explore the treadmill compartment for ~1 minute to acclimatize it to the apparatus . we therefore randomly selected to-2 and f1b hamsters and subjected them to a range of walking speeds . we selected 16 cm / s for the 3-month- and 9-month - old hamsters since most animals walked comfortably at this speed and it was sufficiently fast to prevent the hamsters from rearing or turning around during videography . we selected a walking speed of 25 cm / s for the more active 1-month - old hamsters . we recorded ~3 seconds of video images for each hamster walking at a speed of 16 cm / s to provide more than 7 sequential strides . only video segments of hamsters walking with a regularity index of 100% were used for image analysis . for incline and decline walking , the treadmill , walking compartment , and camera system were pitched at an angle so that the animals walked up an incline of ~15 or down a decline of ~15 , dependent on the direction of the treadmill belt . the angle of treadmill incline and decline was based on published protocols [ 8 , 16 , 24 ] . the treadmill belt was wiped clean between studies if necessary . anova was used to test for statistical differences among to-2 and f1b hamsters at each age . when the f - score exceeded fcritical for = 0.05 , we used post - hoc unpaired student 's two - tailed t - tests to compare group means . gait indices between forelimbs and hindlimbs within groups were compared using paired student 's two - tailed t - tests . the ventral view of a 9-month - old healthy f1b control hamster walking on a transparent treadmill belt is shown in video 1 ( video 1 , available as supplemental material ) . f1b hamsters ( n = 9 ) walked ~2 steps every second , completed one stride within ~500 ms , and traversed ~8 cm with each step walking at a speed of 16 cm / s . the contributions of stance and swing duration to stride duration were ~75% ( stance / stride ) and ~25% ( swing / stride ) . differences in the posture and gait dynamics of the forelimbs and hindlimbs were quantified . forelimb stance width was significantly narrower than hindlimb stance width ( 2.4 0.1 cm versus 3.8 0.1 cm , p < .05 ) . stance width variability ( cv% ) of hindlimbs was lower than that of forelimbs in 9-month - old f1b hamsters walking at a speed of 16 cm / s . figure 2 illustrates the relative contributions of swing , stance , braking , and propulsion to the stride duration of forelimbs and hindlimbs in 9-month - old f1b hamsters . the braking phase was greater in forelimbs than in hindlimbs ( 34.3 2.1% versus 24.8 2.3% , p < .05 ) , and the propulsion phase was greater in hindlimbs than in forelimbs ( 75.2 2.3% versus 65.6 2.1% , p < .05 ) with comparable stance durations of forelimbs ( 367 8 ms ) and hindlimbs ( 361 8 ms ) . gait dynamics were significantly different in 9-month - old to-2 dystrophic hamsters ( n = 14 ) compared to age - matched f1b control hamsters ( n = 9 ) ( table 1 ) . figure 3(a ) illustrates the gait signal from the left hindlimb of a to-2 hamster superimposed over the gait signal of the left hindlimb of a f1b hamster . stride duration was significantly shorter in to-2 than that in f1b hamsters ( 443 4 ms versus 503 7 ms ; p < .05 ) . this was attributable to a shorter swing duration ( 112 2 versus 139 5 ms ; p < .05 ) , and a shorter stance duration ( 331 3 ms versus 364 6 ms ; p < .05 ) in to-2 compared to f1b hamsters . stride length was decreased in to-2 compared to f1b hamsters ( 7.1 0.1 cm versus 8.0 0.2 cm ; p < .05 ) at a walking speed of 16 cm / s . forelimb stance width was narrower in to-2 compared to f1b hamsters ( 2.1 0.1 cm versus 2.4 0.1 cm ; p < .05 ) . the paw placement angle of the hindlimbs was also more significantly everted in to-2 than in f1b hamsters ( 17.7 1.2 versus 8.7 1.6 ; p < .05 ) . forelimb stride length variability was higher in to-2 than in f1b hamsters ( 23.6 1.4% versus 17.6 1.6% ; p < .05 ) . the braking duration of the forelimbs , which is the time needed to decelerate the animal , was prolonged in to-2 compared to f1b hamsters ( 147 6 ms versus 126 8 ms ; p < .05 ) . the propulsion duration of the hindlimbs , which reflects the interval available for force generation , was shortened in to-2 compared to f1b hamsters ( 242 7 ms versus 272 11 ms ; p < .05 ) ( figure 3(a ) , arrows ) . figure 3(b ) illustrates the relative contributions of braking and propulsion to the stance duration of forelimbs and hindlimbs in 9-month - old f1b control hamsters and to-2 delta - sarcoglycan - deficient hamsters walking at 16 cm / sec . the relative contributions of forelimb braking to stance were significantly altered in to-2 dystrophic hamsters . most of the gait disturbances observed in 9-month - old dystrophic hamsters were also apparent at 3 months of age . stride length was decreased in 3-month - old to-2 dystrophic hamsters ( n = 12 ) compared to f1b control hamsters ( n = 6 ) ( 7.2 0.1 cm versus 8.4 0.2 cm ; p < hindlimb stance width was narrower in to-2 than in f1b hamsters ( 3.7 0.1 cm versus 4.0 0.1 cm ; p < .05 ) . forelimb stance width was significantly narrower in to-2 than in f1b hamsters when the animals were challenged to walk uphill or downhill ( figure 4 ) . as in older animals , the paw placement angle of the 3-month - old dystrophic to-2 hindlimbs was more significantly everted than that in f1b controls ( 16.4 1.2 versus 12.4 1.6 ; p < .05 ) . whereas the braking duration of the forelimbs was not different in 3-month - old to-2 from f1b hamsters walking horizontally , the propulsion duration of the hindlimbs was shortened in 3-month - old to-2 hamsters ( 241 6 ms versus 298 9 ms ; p < .05 ) . challenging the animals to walk uphill and downhill , moreover , exacerbated functional differences between 3-month - old to-2 ( n = 4 ) and f1b hamsters ( n = 4 ) ( figure 4 ) . even at 1 month of age , several gait indices deviated significantly in to-2 dystrophic hamsters . compared to 1-month - old f1b control hamsters ( n = 5 ) , 1-month - old to-2 dystrophic hamsters ( n = 4 ) were characterized by significantly shorter stride length ( 6.6 0.1 cm versus 7.1 0.1 cm ) , more eversion of the hindpaw ( 22.4 1.8 versus 17.9 1.8 ) , shortened hindlimb propulsion duration ( 125 5 ms versus 148 5 ms ) , and prolonged forelimb braking duration ( 81 3 ms versus 44 5 ms ) ( all p < .05 ) . forelimb stride length variability tended to be higher in 1-month - old to-2 compared to f1b hamsters ( 18.1 1.2% versus 15.1 1.4% ; p < .08 ) . figure 5 illustrates differences in stride length between f1b control and to-2 dystrophic hamsters across the three ages studied . this is the first comprehensive analysis of posture and gait in hamsters , and the first quantitative analysis of gait changes in a small animal model of muscular dystrophy . gait analysis of patients with muscular dystrophy is implemented to assess disease progression [ 13 ] . mouse and hamster models are widely used to better understand and develop therapies for muscular dystrophy ; yet , very little is known about how these animals walk . the postural and kinematic gait data we provide in f1b control hamsters and delta - sarcoglycan - deficient to-2 dystrophic hamsters may help to establish reference values for future studies aimed at reversing gait abnormalities with potential therapeutics tested in this animal model . loss of muscle strength is common to all types of muscular dystrophies ; age of onset , muscle groups involved , and progression vary . mutations in the sarcoglycan - gene result in limb - girdle muscular dystrophy ( lgmd ) , of which there are different types . the limb - girdle ( shoulders and hips ) muscles are usually the first affected , with muscle weakness becoming more progressive and spreading to more distal muscles , including tibialis anterior , wrists , and hands . most lgmd - type 2f patients have severe muscular dystrophy that presents early in life and progresses rapidly . the to-2 hamster , with a deletion in the delta - sarcoglycan gene and deficiency of alpha- , beta- , and gamma - sarcoglycan , has been widely studied [ 1620 ] and is considered a good animal model of human lgmd - type 2f . gross examination upon autopsy of the parental strain of to-2 hamsters indicated that shoulder muscles were the most affected . pathology has also indicated damage to the intercostal muscles , proximal limb muscles , muscles of the pelvic girdle , and muscles of the back and the abdominal wall . gastrocnemius , triceps , and tibialis muscles have also been shown to be affected [ 7 , 16 , 20 , 31 ] . all of these muscles are involved in quadrupedal gait . despite extensive pathology evidence of muscle damage , very little is known about muscle dysfunction in animal models of muscular dystrophy , including the to-2 hamster . vitiello et al . concluded that gene therapy ameliorated muscular dystrophy in hamsters based on their improved rotarod performance . our treadmill gait analysis study sought to reconcile systemic muscle weakness with the ability to walk , functionality most important to patients with muscular dystrophy . observation of to-2 hamsters in their cage does not suggest that they have gait disturbances , which is why we studied treadmill gait analysis in 9-month - old animals , an age of which the animals are known to have significant muscle weakness . the ~13% shorter stride length in the dystrophic hamsters is a very robust deficit ; in otherwise healthy patients with neuromuscular disease , ~10% reductions in stride length can be clinically relevant . because the treadmill speed was set to 16 cm / s for both to-2 dystrophic and f1b control animals , the shorter stride length in the to-2 animals required significantly increased stepping frequency . the paw placement angle of the hindlimbs was more open in to-2 animals , suggesting weakness of muscle in the hindpaws or lower hindlimbs . though the hindlimb propulsion duration was shorter , the percentage of contribution of the hindlimbs to propulsion was not different in to-2 from that of f1b hamsters , suggesting that the shorter propulsion duration was secondary to a shorter stride cycle . there were several important differences in forelimb gait in to-2 dystrophic compared to f1b control hamsters . this is in contrast to some animal models of neuromuscular diseases , such as the sod1 g93a mouse model of amyotrophic lateral sclerosis , in which the gait disturbances are more localized to the hindlimbs . the forelimb stance width was significantly narrower ; often , changes in base of support are compensatory to muscular and vestibular defects to maintain posture and balance . the forelimb braking duration was prolonged , but more significantly , the percentage contribution of forelimb deceleration was larger in to-2 hamsters . taken together , muscle weakness of the shoulder girdle and forelimbs might contribute substantively to the gait disturbances in to-2 hamsters . this shoulder girdle functional readout is notable given that the supraspinatus muscle was the most severely affected in autopsied dystrophic hamsters , the relatively larger load carried by the upper limbs in quadrupeds compared to humans , and the known involvement of the shoulder girdle in lgmd patients . the differences in gait between 3-month - old f1b and to-2 hamsters were strikingly similar to those observed in older animals . this may not be surprising , however , since histopathology indicates extensive muscle pathology at this age [ 16 , 17 , 19 , 31 , 34 ] . downhill walking especially increased the magnitude of differences in stride length , stance width , and propulsion duration . walking up an incline or down a decline is known to changes muscle load and force distribution [ 35 , 36 ] ; this is especially true for quadrupeds that use their forelimbs for weight support . downhill walking has been shown to increase loading on the shoulders and upper limbs of pongids , cats , and rats . the observations of exacerbated upper limb gait disturbances in the to-2 hamsters downhill walking suggest that the shoulder girdle may be more affected , at least functionally , than the hip girdle in the delta - sarcoglycan - deficient hamster model of muscular dystrophy . skeletal muscle pathology is already evident in to-2 animals one month old [ 17 , 19 , 30 , 34 ] . we observed significant differences in posture and gait between 1-month - old to-2 dystrophic and f1b control hamsters . several metrics , including shorter stride length and greater hindpaw eversion , were consistent findings in juvenile ( 1-month - old ) , young ( 3-month - old ) , and adult ( 9-month - old ) delta - sarcoglycan - deficient dystrophic hamsters . in summary , gait analysis in delta - sarcoglycan - deficient dystrophic bio to-2 hamsters provides early functional evidence of systemic muscle weakness that could accelerate the screening of potential therapies for muscular dystrophy . t. g. hampton is owner of mouse specifics , inc . , a company that has commercialized the gait analysis instrumentation . c. g. vandongen . is owner of biobreeders , inc .

the delta - sarcoglycan - deficient hamster is an excellent model to study muscular dystrophy . gait disturbances , important clinically , have not been described in this animal model . we applied ventral plane videography ( digigait ) to analyze gait in bio to-2 dystrophic and bio f1b control hamsters walking on a transparent treadmill belt . stride length was 13% shorter ( p < .05 ) in to-2 hamsters at 9 months of age compared to f1b hamsters . hindlimb propulsion duration , an indicator of muscle strength , was shorter in 9-month - old to-2 ( 247 8 ms ) compared to f1b hamsters ( 272 11 ms ; p < .05 ) . braking duration , reflecting generation of ground reaction forces , was delayed in 9-month - old to-2 ( 147 6 ms ) compared to f1b hamsters ( 126 8 ms ; p < .05 ) . hindpaw eversion , evidence of muscle weakness , was greater in 9-month - old to-2 than in f1b hamsters ( 17.7 1.2 versus 8.7 1.6 ; p < .05 ) . incline and decline walking aggravated gait disturbances in to-2 hamsters at 3 months of age . several gait deficits were apparent in to-2 hamsters at 1 month of age . quantitative gait analysis demonstrates that dystrophic to-2 hamsters recapitulate functional aspects of human muscular dystrophy . early detection of gait abnormalities in a convenient animal model may accelerate the development of therapies for muscular dystrophy .

1. Introduction 2. Methods 3. Results 4. Discussion Disclosure

PMC4914396

according to the studies of matsuba and shimamura in 1933 , respiratory frequency ( rf ) increased by 1.2 times ( all values compared to with those at rest ) when walking 200 m , by 1.5 times during a 200 m trot , and by 45-fold during a 5,0006,400 m canter . however , these numbers were obtained by determining the rf just after exercise by counting the movements of the ribcage and upper flank with naked eye of an observer based on the assumption that the rf immediately after exercise is equivalent to the rf during exercise . in 1944 , okabe and sugiyama measured rf continuously during exercise at a walk , trot , and canter by recording expiratory sounds with a microphone attached near the nostril that was connected to an amplifier and speaker pulled on a drafting vehicle . the rf and stride frequency ( stf ) during exercise were recorded for a total of 42 min consisting of a 30 min trot ( 170 m / min ) , 3 min canter ( 300400 m / min ) , and 9 min walk ( 100 m / min ) in 20 horses . as shown in fig . 1.changes in respiratory frequency ( rf ) , stride frequency ( stf ) , and velocity during exercise . the authors generated this graph using data from a table in a study by okabe and sugiyama . , the stf during trotting was about 84 str / min ( where str is strides ) 1 min after starting the trot and remained almost constant throughout , and the average stf for a 30 min trot was 83.1 str / min . the rf during the first 1 min of trotting was 73 br / min ( where br is breaths ) , then it increased to 100 br / min at 12 min and it increased to more than 110 br / min thereafter . the stf during cantering was 103 str / min 1 min after starting cantering and remained constant . the average stf during cantering was 105.3 str / min and the rf during cantering was synchronized with the stf , with expiratory sounds occurring during stance phase of the trailing forelimb . the stf during walikg just after finishing cantering was 63 str / min , and then it remained at 5759 str / min thereafter . the rf at the end of the canter was 108 br / min , it decreased to 97 br / min shortly after finishing cantering , and then it remained around at 110 br / min during walking . immediately after the horse stopped walking , the rf decreased dramatically to 80 br / min . the authors concluded that the rf immediately after exercise while standing was completely different from the rf during exercise because of the very rapid decrease in rf that occurred after finishing exercise . changes in respiratory frequency ( rf ) , stride frequency ( stf ) , and velocity during exercise . the authors generated this graph using data from a table in a study by okabe and sugiyama . it was also noted that several different respiratory sounds , such as snorting and booming , were heard during exercise , and a booming sound was recorded during the stance phase of the trailing forelimb . it was also noted that the tone of the respiratory sounds changed during exercise . during trotting , the strength , pitch , and length of respiratory sounds tended to become irregular 1620 min after starting trotting , but they became regular when the horse began cantering . compared the rfs under 4 different workloads including plowing , cultivating ( tilling with a cultivator ) , harrowing ( crushing soil with a harrow ) , and free walking ( as a control ) using the same equipment as okabe and sugiyama . according to their observations , the rf increased in a fluctuating manner and reached a steady state at each intensity of exercise . the average work resistance during plowing and harrowing ( 3035 kg ) was greater than that of cultivating ( 2025 kg ) . because horses have to walk on softer ground when harrowing compared with plowing , it is generally considered that harrowing is heavier work than plowing . because the rate of increase in rf tended to be higher when harrowing than plowing and higher in plowing than cultivating reported the rf during trotting at a small riding ground ( 380 m / lap ) with different exercise protocols : a , 300 m / min for 14 min ( or 20 min ) , and b , 200 m / min for 30 min ( or 45 min ) . the rf reached steady state in both protocols approximately 10 min after starting and remained constant thereafter . the rf in the steady state was higher during the 300 m / min trot ( 95100 br / min ) than the 200 m / min trot ( 7580 br / min ) . the authors suggested that there was a relationship between stf and rf , because rf was sometimes synchronized with stf systematically in 1:1 , 1:2 , or 1:3 ratios when trotting in a straight line . these systematic relationships tended to disappear when horses ran around a curve . in 1978 , attenburrow recorded both inspiratory and expiratory respiratory sounds with a radio - stethoscope during walking , trotting , cantering , and galloping . he also recorded respiratory and limb cycles simultaneously and found that the respiratory cycle and limb cycle were synchronized only at a canter and gallop . later , kai and kubo of the equine research institute of the japan racing association recorded respiratory and stride frequency simultaneously during galloping just after a start dash from the starting gate and reported that a 46 sec apneic pause occurred just after starting , after which respiration restarted and synchronized with the stride frequency . pulse rate immediately after exercise was measured by auscultation in the study of matsuba and shimamura . because direct measurement of heart rate ( hr ) during exercise was impossible at that time ( 1933 ) , pulse rate immediately after exercise was assumed to indicate hr during exercise . later , tatsumi et al . showed that there is a close relationship between oxygen consumption of horses during exercise as measured by the douglas bag method and the pulse rate immediately after exercise . researchers then recognized the importance of hr measurements during exercise and began developing methods for measuring hr during exercise . it was difficult to obtain an ecg necessary for calculating hr during exercise , although it could be recorded at rest [ 30 , 39 , 83 ] . in 1960 , nomura and tominaga recorded ecgs from a horse walking on a specially constructed treadmill at a speed of 70 m / min while drafting 5 different weights . the ecgs were obtained by transmitting the signal to a receiver using a 20 mhz carrier wave and reproducing the signal with an electromagnetic oscillograph . changes in hr during exercise and the recovery period were calculated by creating diagrams of r - r intervals from the ecg recording . heart rate increased immediately when the horse started exercising and returned to a steady state within a short time when the workload was light . even when the workload was heavy , the heart rate became to a steady state within 3 min , with a second increase in hr occurring about 10 min after the beginning of exercise . although recording of ecgs from a horse exercising on a treadmill had become possible by using a transmitter set near the horse and a carrier wave , it was still very difficult to record the ecg of a horse exercising under field conditions . a research project to record equine ecgs during exercise using radiotelemetry was performed as a collaborative study between nomura of the university of tokyo and the equine health laboratory of the japan racing association . for the research study , a custom - made radiotelemetry system that could record ecgs within 100 m was developed , and the performance of the system evaluated . during exercise at intensities ranging from walking to intense galloping , the investigators concluded that ecgs obtained by this system were clear enough to calculate hr or r - r intervals during exercise . ecgs were recorded using the newly developed system during both low - intensity exercise with a horse riding on the ground or jumping low hurdles and during high - intensity sprint - galloping on a track in order to calculate changes in hr during exercise . the major findings regarding changes in hr were as follows : 1 ) during low intensity exercise including the walk , trot , canter and extended canter , hr increased from 45 beat / min during pre - exercise to 150 beat / min at an extended canter , and ; 2 ) hr increased to 200 beat / min or more in most horses during 100 m high - intensity sprint galloping ( fig . the authors generated this graph using data from a table in a study by nomura et al . . ) . the first report of ecgs measured during field exercise in the horse was from this study by nomura et al . in 1964 [ 41 , 42 ] . a similar report by nomura was also published in 1966 in another journal . banister et al . in 1968 reported ecgs measured during field exercise in horses . in order to carry out a reliable recording of ecgs during exercise of a horse running on a large track , a high - power telemetric transmitter was constructed on special order and operated under a license from the radio regulatory bureau . because the high transmitting power of 1 w enabled an effective transmission range of 5 km or more , it became possible to record ecgs for horses exercising in large areas , e.g. , on an oval track at a racecourse . the authors generated this graph using data from a table in a study by nomura et al . . although it had become possible to measure instantaneous changes in an ecg during exercise using radiotelemetry , this system had certain disadvantages , such as the fact that it could only be used for recording from a single horse and could only be used within the effective transmission area of the radio waves . therefore , a portable electrocardiograph with a magnetic tape recording system for exercising horses was devised [ 2 , 29 ] . because the portable magnetic tape electrocardiograph allowed recording of ecgs from many horses exercising simultaneously , it became a useful tool for evaluating equine arrhythmias and hr during exercise tests performed at large race tracks . currently , digital holter ecg systems are widely used for exercise electrocardiography in racehorses . nomura also developed a beat meter to measure the hr of draft horses during heavy pulling work at slower speeds in the field . although this device could not record a raw ecg , it enabled hr to be measured by counting the popping sounds per unit time that were created when the meter detected larger amplitude signals in the ecg , e.g. , the qrs complex . matsuba and shimamura evaluated the fitness of racehorses by analyzing 20 parameters they considered as being important for exercise physiology and that were possible to measure at that time ( 19331939 ) . among them , 5 blood parameters - fragility of erythrocytes , blood viscosity , erythrocyte sedimentation rate , freezing point depression , and ph - changed with time during training , so they recommended that these parameters would be useful indexes for future training studies . in addition to the aforementioned parameters measured in their studies , other parameters such as the o2 concentration , co2 concentration , and blood lactate concentration were also measured . in 1937 , shijo reported that the o2 concentrations in arterial and venous blood were 20.81% and 12.08% , respectively , and showed that plasma co2 concentrations in arterial and venous blood were 46.60% and 57.85% , respectively , using 26 horses . shijo measured venous o2 and co2 concentrations weekly for about 3 months during a training period in 4 horses and showed that the average co2 concentration was 61.6% and almost the same in each horse . the o2 concentration was 12.7% , and there was relatively high correlation between the o2 concentration and erythrocyte count throughout the training period ( fig . 3fig . the authors generated this graph using data from a table in a study by shijo . ) . shijo also examined the effect of exercise on blood counts and showed that erythrocyte and leukocyte counts increased with intense exercise and numbers of neutrophils increased but that numbers of lymphocytes decreased . the authors generated this graph using data from a table in a study by shijo . although the blood lactate concentration was not measured in studies by matsuba et al . , the blood lactate concentration is considered to be one of the most important parameters in assessing performance in equine exercise physiology . in 1934 , yasuda measured blood lactate concentrations during 3 days of draft work ( total distance 111 km ) in 12 warhorses . because the exercise intensities were low , the authors generated this graph using data from a table in a study by yasuda . ( pre , before exercise ; post , just after exercise ; 30 min , 30 min after exercise ; and 60 min , 60 min after exercise ) ) . this was the only study that had measured the blood lactate concentration up to that time . if blood lactate had been measured in the studies of matsuba et al . the authors generated this graph using data from a table in a study by yasuda . ( pre , before exercise ; post , just after exercise ; 30 min , 30 min after exercise ; and 60 min , 60 min after exercise ) in 1967 , sakurai et al . compared the effects of exercise intensity ( 1,100 m at 6.7 m / s , 1,100 m at 11.1 m / s , and 1,600 m at 12.5 m / s ) on changes in blood characteristics and reported that the red blood count , white blood cell count , hemoglobin concentration , hematocrit , erythrocyte resistance , and blood lactate concentration increased but that eosinophils , erythrocyte sedimentation rate , and blood water content decreased . in this report , the blood lactate concentration was 0.7 mmol / l after 1,100 m ( at 6.7 m / s ) , 7.3 mmol / l after 1,100 m ( at 11.1 m / s ) , and 12.1 mmol / l after 1,600 m ( at 12.5 m / s ) . the glucose , pyruvate and lactate concentrations were also measured after 2 different kinds of exercise - relatively short distance cantering and long distance trotting - and the authors reported that both the lactate and pyruvate concentrations increased after cantering . the question as to whether or not changes in the thyroid hormone concentration in plasma ( t3 and t4 ) at rest might be useful as an indicator of physical fitness of a racehorse was investigated during the training period from september of the yearling year to may of the 2-year - old year . the concentration of t3 in plasma , which was used in that study as the t3 binding capacity index , decreased clearly during the early period of training and remained constant during the latter half of the training period . the authors found that greater function of the thyroid gland during training correlated with changes in t3 in the horses . therefore , they speculated that changes in t3 might be used to evaluate the physical fitness of active racehorses . the concentration of t4 in plasma decreased clearly in the middle stage of training , but it increased markedly in the final stage . because it was presumed that the horses were able to train more intensely in the final stage of their investigation , the authors considered that the elevation of t4 was useful for evaluating the reserve of strength in the horse . relationships between changes of some blood enzymes and physiological conditions in exercising horses were investigated using 3 horses subjected to long distance running . they ran 22,000 m / day for 5 consecutive days mainly at an extended trot but partially at a slow canter . blood samples were collected before , in the middle , immediately after , and 1 and 5 hr after exercise every day during the exercise period and at 1 , 2 , 3 , 5 , and 7 days following the exercise period to document the recovery process . in this experiment , changes in the activities of creatine kinase , aspartate amino transferase , and fructose diphosphate aldolase were measured . the magnitude of the rate of increase became larger as the exercise intensity increased . although aspartate amino transferase activity failed to increase markedly as the intensity of the exercise increased , it remained at a relatively high level and then increased gradually during the exercise period . fructose diphosphate aldolase activity changed in an intermediate manner between the two enzymes mentioned above . these results suggest that creatine kinase might be utilized as the most useful index of the severity of exercise and that aspartate aminotransferase might be utilized as an index for overtraining . however , the significance of changes in fructose diphosphate aldolase activity with exercise is unclear . the erythrocyte sedimentation rate ( esr ) is the rate at which erythrocytes form a sediment during a fixed period of time , typically 1 hr ; it considered to be a nonspecific parameter of inflammation . although this parameter has been used mainly as a nonspecific index of inflammation , it must be considered that it can be influenced by various factors . however , the relationship between the esr and exercise was previously unknown , especially in horses . yasuda and coworkers , who studied the exercise physiology of the warhorse , examined relationships between the esr and exercise and fatigue in warhorses . examined the effects on the esr of faster and slower speeds during exercise using 15 warhorses and 6 local horses . the faster exercise consisted of long distance riding for 3 days , and the total distance was 142.5 km . the slower exercise was draft exercise for 4 days , and the total distance was 116.5 km . the esr was calculated as the median value from the sedimentation at 1 hr and 2 hr ( ( 1 hr + 2 hr/2)/2 ) . it was found that the esr after faster exercise was 14.5 mm lower than the values before exercise . on the other hand , the esr after slower exercise did not differ between before and after exercise , and the esr tended to be low when the erythrocyte count was large . the authors suggested that measurement of the esr might be a useful tool for evaluating fatigue , especially with faster riding exercise . hiroe et al . also reported the effects of similar exercise on blood viscosity in 25 warhorses . blood viscosity was expressed as a relative value using distilled water as a reference ( 1.0 ) . the average blood viscosity at rest was 4.89 and increased to 12.6 after faster exercise , and recovery took about 21 hr . on the other hand , blood viscosity was not changed after slower exercise compared with the pre - exercise value . the authors suggested that blood viscosity might be a useful index for evaluating fatigue during intense exercise in conjunction with the esr . in 1937 , shijo examined relationships between both changes in esr after exercise and changes in blood characteristics ( blood o2 concentration , plasma co2 concentration , erythrocyte count , hemoglobin concentration , and plasma volume ) , and clinical findings ( pulse rate , respiratory frequency , and blood pressure ) . measurements were performed using 9 different exercise protocols and 4 racehorses as follows : 1 ) walk , 200 m ; 2 ) walk , 800 m ; 3 ) walk , 1,600 m ; 4 ) trot , 200 m ; 5 ) trot , 800 m ; 6 ) trot , 1,600 m ; 7 ) canter , 200 m ; 8) canter , 800 m ; and 9 ) canter , 1,600 m. the esr was calculated as a median value based on the values at 1 hr and 2 hr . the average esr at rest was 92.2 mm ( 84103 mm ) and decreased after exercise . the authors generated this graph using data from a table in a study by shijo . ( w , walk ; t , trot ; and c , canter ) ) . because the esr decreased when the erythrocyte count and o2 concentration were increased , the authors suggested that there were close relationships between the esr and erythrocyte count and o2 concentration . the authors generated this graph using data from a table in a study by shijo . ( w , walk ; t , trot ; and c , canter ) during the period from 1938 to 1941 , kikuchi published 4 papers on the esr in horses : one on the esr during normal status , one on the esr during pregnancy and the postnatal period , one on the esr with various diseases , and one on factors influencing the esr . in the first paper , 54 female horses ( 220 years old ) , 22 male horses ( 219 years old ) , 13 geldings ( 519 years old ) , and 37 foals ( 2170 days old ) were investigated , and showed that the esrs of each group were 66100 mm , 71100 mm , 71100 mm , and 2880 mm , respectively . the esr of foals 6 days after birth was the lowest and it increased with growth . in the final paper , the author reported that the esr decreased after exercise , as had been reported by hiroe et al . and shijo , and also reported that differences between pre - exercise and post - exercise values were smaller in trained horses than in untrained horses . in the same paper , kikuchi also investigated effects of various factors , such as temperature , congestion , concussion , feeding , and sodium citrate on the esr of horses . kikuchi wrote interpretive articles on measurement of the esr and its significance for clinical diagnosis . in these articles , he described several methods of measuring the esr , normal values of the esr in the horse , cattle , dog , goat , sheep , and pig , factors influencing the esr , the esr in various diseases , and the mechanism of the esr . ohi performed a series of experiments on species variation of the esr in domestic animals using horses , pigs , dogs , cattle , sheep , and goats to evaluate variations of the erythrocyte sedimentation curve , shape of the erythrocyte sedimentation curve , correlation between the esr and temperature of the laboratory , influence of measurement temperature on erythrocyte sedimentation , correlation between esr and inclination of the tube , influence of tube inclination on erythrocyte sedimentation , correlation between the esr and diameter of the tube , influence of tube diameter on erythrocyte sedimentation , correlation between esr and height of the blood column , and influence of the height of the blood column on erythrocyte sedimentation . . showed in 2-year - old thoroughbreds that the esr decreased as training advanced ( fig . 6.changes in erythrocyte sedimentation rate ( esr ) in relation to training in 2 horses . the authors generated this graph using data from a table in a study by matsuba et al . . ) . after working on equine exercise physiology with matsuba , sakurai started working on exercise physiology of racehorses trained at racecourses , mainly studying blood characteristics , especially the esr . sakurai examined the esr in 100 horses , including 46 racehorses purchased at sales ( referred to as yobiuma at that time ) , 31 racehorses purchased by an organization that held horse races ( owners of these horses were determined by lottery ; these horses were referred to as chusenba at that time ) , 10 trotters , and 13 riding horses . the chusenba lottery system was a unique system in japan . because the yobiuma horses were purchased at sales by owners , the yobiuma horses were generally considered to be higher quality horses than those acquired in the chusenba system . sakurai s measurements were carried out throughout the 26-day horse race meeting at tokyo racecourse in 1940 . the esrs of the yobiuma were the lowest , and they were progressively higher in chusenba horses and trotters . sakurai concluded that the esr of high - quality or high - performance horses tends to be lower . changes in erythrocyte sedimentation rate ( esr ) in relation to training in 2 horses . the authors generated this graph using data from a table in a study by matsuba et al . . changes in esr during the duration of a horse race meeting that included daily training and racing were investigated in 13 racehorses and compared to other parameters . the esr changed in relation to the state of the general condition of the horse , and it correlated highly with the erythrocyte count and hemoglobin concentration . this result was similar to the findings of shijo and matsuba et al . . sakurai measured the esr , plasma protein concentration , specific viscosity , and albumin content in 101 racehorses in order to examine relationships between the esr and the other variables , and he found that total plasma protein correlated with specific viscosity . the esr in horses with good racing performance was low and albumin content in these horses was higher . six horses were trained for about 1 month and able to perform a maximal speed run of 1,500 m at the end of the training period . while the general physical condition of the horses improved , the esr and body weight decreased . in contrast , when the condition of the horses by general inspection worsened , the esr increased . sakurai et al . performed intensive research throughout a long period of training from july to october using 110 racehorses trained at the tokyo racecourse . changes in esr , body weight , pulse rate , cardiac sounds , and nutritional status were observed , and the following results were reported : 1 ) the esr decreased when a horse s condition by general inspection improved and increased when a horse s condition by general inspection worsened ; 2 ) body weight decreased when the training intensity was higher and increased when the training intensity was reduced ; and 3 ) pulse rate increased and cardiac sounds became weaker when the horse s condition worsened , while pulse rate decreased and cardiac sounds strengthened when the horse s condition improved . they concluded that measurement of these 4 parameters were useful for evaluation of a horse s condition . sakurai also performed similar experiments regarding the effects of training over intervals ranging from several months to 1 year on clinical and blood parameters using 14 racehorses and concluded that changes in esr and body weight would be good parameters for evaluation of the condition of a racehorse . from these results , sakurai suggested that measurement of the esr , body weight , pulse rate , and cardiac sounds was useful for evaluation of a horse s condition . sakurai made similar observations on 18 riding horses trained for 2 months according to training regimens typically used for racehorses and reconfirmed that both the esr and body weight decreased as training advanced . in order to validate the usefulness of changes in esr and body weight for evaluation of fatigue , sakurai observed changes in esr and body weight once a week during a 6-week period of training using 2 horses . during the first 2 weeks of low - intensity training , the esr decreased and body weight increased . during the next 2 weeks , the exercise intensity was increased gradually , and the esr became much lower , and body weight decreased . during the final 2 weeks , higher - intensity training was carried out , and the horses showed signs of fatigue while esr increased and body weight decreased further . these results suggested that changes in esr and body weight were useful parameters for evaluation of fatigue or the condition of a racehorse . sakurai named this method as the esr - body weight method and concluded that it would be a useful index for screening tests of the effects of training on conditioning and fatigue . because previous research was conducted using adult horses trained at a racecourse or on retired racehorses , in 1961 , sakurai and ogawa observed effects of training and growth on the esr and body weight using 19 young racehorses trained at the utsunomiya yearling training farm of the japan racing association . measurement of blood characteristics was performed at the beginning of june of the yearling year ( 1st ) , the end of september of the yearling year ( 2nd ) , the beginning of december of the yearling year ( 3rd ) , and the beginning of march of the 2-year - old year ( 4th ) . the esr was measured as a 40 min value and converted to the value at 20c . the average esr for the 1st period was 74 mm , and it then decreased to 61 mm for the 2nd period , 40 mm for the 3rd period , and 39 mm for the 4th period . on the other hand , average body weight increased from 338 kg for the first period to 457 kg for the 4th period . in 1964 , sakurai et al . performed a more intense and longer study than in 1961 with 102 racehorses trained at the utsunomiya yearling training farm . table 1table 1.summary of training and testing ( ponying , that is , running on courses without rider with 2 lead horses with riders)agetrainingtestyearlingponying ( from september ) an breaking ( from december ) at the utsunomiya yearling training farm.1st : october to november ( n=102)2 years oldtraining at a racecourse ( from the end of march or the beginning of april ) . most horses could join races until the end of december.2nd : february to march ( n=103 ) 3rd : june ( n=49)3 years oldtraining at racecourses and participating in racing.4th : march ( n=39 ) shows the training protocols . these horses were trained with a method referred as oi - undo ( ponying , that is , running on a track without a rider , with 2 lead horses with riders ) until the horses were broken . breaking was carried out in december of the yearling year , and running exercise with a rider was started thereafter with the exercise intensity gradually increasing . the 2nd study was conducted in february and march of the 2-year - old year . the horses were moved to racecourses in march to april and were then trained with high - intensity exercise in their trainer s stable . the 3rd study was performed in june of the 2-year - old year . from july onward , horses began to join racing meets , and most of the horses could race until december , except for those horses with musculoskeletal disorders . the 4th and final study was performed in march of the 3-year - old year . although the number of horses examined was about 100 for the 1st and 2nd studies , the number decreased to 49 for the 3rd study and 39 for the 4th study . the average esr during the 1st study was 60 mm , and it decreased as training advanced to 11 mm by the 4th study ( fig . 7fig . 7.changes in erythrocyte sedimentation rate ( esr ) and body weight during the training periods from october to november of the yearling year ( 1st ) , february to march of the 2-year - old year ( 2nd ) , june of the 2-year - old year ( 3rd ) , and march of the 3-year - old year ( 4th ) . the authors generated this graph using data from a table in a study by sakurai et al . . ) . although individual variation was larger in the 1st survey , the variation became smaller and dispersion among horses decreased as training advanced , with 77% of the horses having values of 15 mm or less in the 4th study . the authors suggested that decrease of the esr in relation to time during training might be a response to the stress of training . on the other hand , although body weight increased when the horses were trained at utsunomiya yearling training farm , where the training intensity was low , body weight decreased after moving to racecourses where the training intensity was high ( fig . changes in erythrocyte sedimentation rate ( esr ) and body weight during the training periods from october to november of the yearling year ( 1st ) , february to march of the 2-year - old year ( 2nd ) , june of the 2-year - old year ( 3rd ) , and march of the 3-year - old year ( 4th ) . the authors generated this graph using data from a table in a study by sakurai et al . . in order to investigate relationships between the esr , fibrinogen , and packed cell volume ( pcv ) , 39 young horses were trained from september of the yearling year to may of the 2-year - old year , and changes in these parameters during the training period were measured . the esr during the early stage of training slowed , and fibrinogen decreased , as shown in fig . 8fig . 8.changes in fibrinogen concentration and erythrocyte sedimentation rate ( esr ) during the training period . the authors generated this graph using data from a study by takagi et al . . all the three factors , however , were unchanged during the latter half of the training period . additionally , there was a positive correlation between fibrinogen and the esr at each experiment . when the coefficient of correlation was compared among changing values , the esr appeared to be influenced mainly by the changes in fibrinogen , at least when the esr was greatly decreased , or by the racehorse s condition progressing to good condition . however , the authors considered that the esr might interact with the pcv and fibrinogen and result in the lack of observed changes . changes in fibrinogen concentration and erythrocyte sedimentation rate ( esr ) during the training period . the authors generated this graph using data from a study by takagi et al . . although the esr was first measured in a warhorse , esr recording in racehorses became popular following studies by matsuba , shijo , and sakurai as mentioned above . a small number of studies were being performed in foreign countries on the relationship between the esr and exercise in horses [ 7 , 81 ] . based on studies comparing the esr in various species [ 22 , 57 ] , it was reported that the esr of the horse was high and that the esr in ruminants were low , although the reason was unknown . it was also reported that the esr in active animals , such as pronghorn antelope , dog , and horse , was higher and that inactive animals , such as sheep and cattle , was lower . therefore , sakurai s finding that fit racehorses tend to have a low esr is an interesting phenomenon that contradicts other studies . several papers have evaluated factors influencing the esr [ 1 , 20 , 70 , 76 ] . sakurai indicated in a review article that the individual variation and dispersion of the esr among horses became smaller as training advanced , suggesting that this index would be useful for evaluation of the condition of individual horses but it would be difficult to use as the basis for comparison of racing performance between different individual horses . he had evaluating conditions of large numbers of racehorses according to criteria for evaluated conditions associated with fatigue as shown in table 2table 2.criteria for judgment of condition by using the erythrocyte sedimentation rate ( esr ) and body weightesrbody weightjudgment of condition1fastheavyuntrained2became fastbecame heavyso - called horse is heavy . it is ok to increase the training intensity.5became fastremarkably became lightneed to reduce training due to fatigue.6remarkably became slowbecame lightanorexia . intensive training will worsen condition.cited from sakurai .. although there is no clear relationship between the esr and a horse s condition , there may be an association between the two . cited from sakurai . the history of research on exercise physiology of the horse in japan began in the 1920s during a time when the role of the warhorse was still most important , and then studies on racehorses started . matsuba and shimamura of the university of tokyo played major roles in studies of racehorses performed during the 1930s . in particular , the first study they published in 1933 was a seminal study , as mentioned above , and what is surprising is that they already had tried to evaluate performance of racehorses in the 1930s at the dawn of modern horse racing in japan . although it seems that they evaluated the fitness of racehorses using many blood parameters that were considered to be important in exercise physiology and were possible to measure at that time , it was difficult to evaluate the performance of a racehorse and the effects of training at that time . however , based on their studies , new approaches for evaluating performance and conditioning of racehorses by using characteristics of the blood were developed by their successors [ 58,59,60,61,62,63,64,65,66,67,68 , 71,72,73,74 ] . additionally , techniques were developed for measuring cardiopulmonary variables such as respiration [ 56 , 69 , 70 ] , heart rate , and oxygen consumption during exercise [ 78 , 79 ] , leading to additional understanding of equine exercise physiology . in the present day , studies on cardiopulmonary function during exercise are mainly performed by measuring cardiopulmonary parameters such as oxygen uptake . although the numbers of studies on blood characteristics is not large in japan , studies on the fragility of erythrocytes were carried out intensively at the end of the 1990s . reported the effects of exercise and splenectomy on the osmotic fragility of circulating erythrocytes . in addition , studies of blood lactate changes during exercise have been carried out intensively . anaerobic work capacity estimated by the lactate accumulation rate has added new ideas regarding energy expenditure during exercise . studies of lactate metabolism during high - intensity exercise and of the monocarboxylate transporter as it relates to lactate metabolism are providing new perspectives and further insights into equine exercise physiology .

abstractafter publication of the epic report on equine exercise physiology by matsuba and shimamura in 1933 , papers on exercise physiology of the racehorse in japan began appearing in scientific journals and increased in number . in 1944 , respiration during exercise at a walk , trot , and canter was measured by recording expiratory sounds with a microphone attached near the nostril . respiratory frequency during cantering was synchronized with stride frequency , and expiratory sounds were found to occur during the stance phase of the trailing forelimb . development of a radiotelemetry system in 1964 for electrocardiogram recording enabled the first recording of an equine electrocardiogram during field exercise that included fast galloping and calculation of heart rate ( hr ) during exercise . during low intensity exercise including walking , trotting , cantering and extended cantering , hr increased from 45 beat / min during pre - exercise to 150 beat / min at an extended canter . hr increased to 200 beat / min or more in most horses during 100 m of high - intensity sprint galloping . when blood lactate was measured after 3 days of draft work in 12 warhorses in 1934 , no increase in blood lactate was found . the erythrocyte sedimentation rate ( esr ) was decreased by intense exercise and also decreased as training increased . it was suggested that measuring changes in esr and body weight in relation to training might become useful as a screening index of training , condition , and fatigue . this evaluation method was named the esr - body weight method .

Respiration during Exercise Heart Rate during Exercise Effect of Exercise on General Blood Characteristics Effect of Exercise on Erythrocyte Sedimentation Rate Conclusions

PMC2920404

first , research in the field of natural organic nanoparticles is new to material science . although natural nanostructures with biological functions have been found for many years , natural organic nanoparticles have not been investigated for material properties such as mechanical and optical properties until recent years . these nanoparticles formed of organic molecules and polymers offer variable optical properties due to the abundant alternative components . however , in the case of biological applications , organic nanoparticles formed in nature are also a promising alternative due to their compatibility and non - toxicity . natural nanoparticles can be used as the functional materials just as the artificial nanoparticles . optical absorption and light scattering the ingredients of modern sunscreens usually contain organic compounds , inorganic nanoparticles and organic nanoparticles . the inorganic nanoparticles ( such as zno and tio2 ) and organic nanoparticles can reflect , absorb and scatter the solar light . though inorganic nanoparticles have been widely used in cosmetic products , there are still concerns about the toxicity of these inorganic materials . tio2 has been reported to induce dna and rna damage through the process of chemical oxidation . considering these situations , if nature - derived harmless organic nanoparticles have strong ultraviolet absorption , they will be a potential promising alternative for sunscreen . in this paper , we reported the optical absorption and light scattering of ivy nanoparticles . the advantages of these nanoparticles in their application to sunscreen compared to inorganic nanoparticles were also demonstrated . fresh - grown rootlets of english ivy were collected around the university of tennessee , knoxville campus , before attaching themselves to solid surfaces . the collected rootlets were meshed using tweezers in 5-ml clean tube containing 500l of 20-nm - filtered water after washing them three times with ddh2o . the solution was then centrifuged at 3000 rpm for 5 min , and the supernatant was collected . two - thirds of the solution was dialyzed overnight at 4 degrees with ddh2o through a cellulose membrane ( sigma aldrich , d9277 ) allowing free pass of chemicals less than 12,500 daltons . size exclusion chromatography and high - performance liquid chromatography ( sec hplc ) was used to isolate the nanoparticles from the solution . the varian hplc system consists of three components including varian prostar 210 , the varian prostar 335 detector and the varian prostar 430 autosampler . next , 200 l of dialysis solution was loaded to the column ( phenomenex biosep - sec - s3000 column with an attached security guard cartridge system ) . the running speed of the mobile phase was set to 0.5 ml / min , and the back pressure of the column was 580 psi . these samples we investigated in this study were named sec-1 , sec-2 and sec-3 with the time sequence . the non - dialysis solutions were also prepared and named nd220 and nd20 , respectively . the morphologies of the ivy nanoparticles were observed by agilent 5500 atomic force microscope ( agilent technologies , santa clara , ca ) . the instrument was operated at room temperature ( 20c ) using picoview in ac mode , which means little or no contact between the tip and the sample . dynamic light scattering ( dls ) measurements were performed by a brookhaven instruments bi-200sm goniometer equipped with a pci bi-9000at digital correlator . the detector is located at the scattering angle of 90. the ultraviolet and visible ( uv vis ) extinction ( absorption and scattering ) spectra were measured by the thermo scientific evolution 600 uv - visible spectrophotometers . , size distributions of the ivy nanoparticles were observed in the samples of sec-1 , sec-2 , d220d1 and nd220 by dynamic light scattering ( dls ) measurement . this effect is believed to be due to the low concentration of ivy nanoparticles in sec-3 . the sizes of nanoparticles display the large ranges from tens of nanometers to several hundred nanoparticles . the average diameters of particles are 117.4 nm for sec-1 , 131.6 nm for sec-2 , 97.8 nm for d220d1 and 112.3 nm for nd220 . it is worth to note , in principle , dls gives the hydrodynamic sizes of the particles . the hydrodynamic sizes are usually greater than the sizes of dry particles obtained by afm . large particles scatter a significant amount of light so that the diameters given by dls intensity distributions will be greater than that obtained by afm even if there are only a few large particles fig . the morphologies of ivy nanoparticles observed by afm size distributions of ivy nanoparticles in water ( sec-1 and d220d1 ) obtained by dynamic light scattering the extinction spectra of samples from the sec technology and the filtration are shown in fig . the extinction spectra of ivy nanoparticles using the filtration method are obtained by subtracting the extinction spectra of samples filtered using 20-nm filters from those filtered by 220-nm filters . for example , the curve in fig . 3b with the symbol of high concentration denotes the difference between the extinction spectra found in d220d1 and d20d1 . the spectrum characteristics of those samples from the sec technology and the filtration are approximately consistent . these phenomena including the dls results mean that the sec and the filtration are the effective direct and indirect methods to investigate the optical properties of ivy nanoparticles . these nanoparticles display strong ultraviolet and weak visible light extinction and , interestingly , a sharp transition edge from the ultraviolet to the visible range . a significant extinction band and a shoulder can be found around the wavelengths of 280 nm and 325 nm for all the samples , respectively . vis extinction spectra of ivy nanoparticles in the samples prepared by a size exclusion chromatography technology and b the filtration . high concentration ( low concentration , non - dialysis ) represents the difference between the extinction spectra found in d220d1 ( d220d2 , nd220 ) and d20d1 ( d20d2 , nd20 ) to illustrate the content of the solution , the original spectra are shown before subtraction in fig . 4 . the extinction band and the shoulder are at the same positions not only for the samples filtered through the 220-nm filters but also the ones filtered through the 20-nm filters . this similarity implies that the components in the solutions are similar to that of the ivy nanoparticles . in addition , the non - dialysis samples show differences when compared to the dialysis samples in the original absorption spectra in fig . 4 . this may be due to the dialysis process in which some protein or other molecules less than 12,500 daltons are filtered out . the absorption peak at the wavelength of 280 nm is the characteristic for many proteins . in general , three of the amino acid side chains ( trp , tyr and phe ) contribute significantly to the uv absorption of a protein at 280 nm . those amino acids are believed to form ivy nanoparticles , but the actual types of proteins and amino acids are still under investigation . furthermore , the concentration of ivy nanoparticles in the sample of high concentration ( d220d1-d20d1 ) was measured to be 4.92 g / ml by the bradford protein assay . original extinction spectra of the filtered samples scattering ( just as the tyndall effects shown in dls experiments ) also makes considerable contribution to the total extinction besides the absorption . herein , we simulated extinction behaviors of the ivy nanoparticles by the mie scattering theory with the help of the software mietab 8.38 . while the refractive index of most organic compounds is between 1.3 and 1.73 , the refractive index of ivy nanoparticle was set to 1.5 . the refractive index of water from 280 nm to 400 nm was from the reported data . the fitting curve of the total extinction , scattering and absorption curves are shown in fig . the scattering effect is found to contribute to the extinction , although this contribution is much smaller than that of the absorption . 3b the characteristics of extinction spectra of ivy nanoparticle make a great advantage in the sunscreen application for blocking the ultraviolet light and maintaining the high visible transparence . ivy nanoparticles can use their optical absorption and scattering properties to eliminate the ultraviolet light , just as inorganic nanoparticles such as zno and tio2 do in sunscreen . in order to illustrate the relative ultraviolet visible extinction characteristics , solutions of zno nanoparticles ( nanoamor , 99.9% , 90200 nm ) and tio2 nanoparticles ( nanoamor , rutile , 99% , 50 nm ) were adjusted to be the same extinction value as sec-2 at 280 nm . this wavelength is the beginning of uv - b ( 280320 nm ) . sunscreens protect from both uv - b and uv - a ( 320400 nm ) irradiation . in fig . 6a , the ivy nanoparticles display a sharper edge from the ultraviolet range to the visible range . herein , the concentrations of tio2 and zno suspensions are 23 g / ml and 100 g / ml , respectively . after this , the extinction spectra of the above tio2 and zno suspensions were roughly converted to the spectra with the concentration of 4.92 g / ml , as shown in fig . 6b . the ivy nanoparticles ( high concentration ) exhibit a much stronger ultraviolet absorption and sharper decrease near the visible range . due to the non - toxicity and strong extinction property , ivy nanoparticles are a promising material for sunscreen application . comparison of the uv vis extinction spectra of tio2 , zno nanoparticles and ivy nanoparticles . a the relative uv vis extinction characteristics with the same extinction value at 280 nm . b the extinction behaviors at the same concentration ( 4.92 g / ml ) natural or biological nanoparticles have not been investigated in detail for their material properties . in this study , we investigate these type nanoparticles as the functional materials just like usual inorganic and organic nanoparticles . nanoparticles from the original tiny ones to the mature ones all exit in the rootlets of ivy . the sec technology isolated the nanoparticles from the mixed solutions including molecules , but it did not give us uniform size of nanoparticles . recently , in order to increase refractive index of organic polymers , surface modification methods , such as deep uv modification and ion - beam implantation , have been reported . if similar modifications are applied to ivy nanoparticles , the refractive index mismatch between ivy nanoparticles and the medium will become larger . this effect can increase the light scattering of ivy nanoparticles and , furthermore , benefit the extinction . in conclusion , ivy nanoparticles have been isolated by the sec technology and the filtration methods . optical absorption and scattering properties of these nanoparticles the extinction spectra of the ivy nanoparticles display strong ultraviolet and weak visible extinction with a sharp transition edge . compared to inorganic nanoparticles , ivy nanoparticles can be used in ultraviolet protection due to their optical properties and harmless nature . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .

though much research has been conducted for nanoparticles , naturally occurring nanoparticles have not yet been well explored for their diverse properties and potential applications . this paper reports the optical absorption and scattering properties of nanoparticles secreted by english ivy . both experimental and theoretical studies have been conducted . strong ultraviolet extinction and excellent visible transparency are observed , compared to the inorganic tio2 and zno nanoparticles at similar concentrations . the contributions of absorption and scattering to the total extinction are quantified by simulation of the mie scattering theory .

Introduction Experiments Results and Discussion Conclusions Acknowledgments Open Access

PMC3912782

autogenous , allogenous , or heterogenous bone can be used for contouring bony - defect sites in the oromaxillofacial area . autogenous bone is typically the best selection because of its osteogenic , osteoconductive , and osteoinductive properties1 - 3 . tibia can also be used as a bone graft due to its low complication rate , simple technique , and sufficient cancellous bone including cortical bone compared to any other site except the jaw . catone et al.4 stated that pain control was easy , and esthetic problems , neurological impairment , infection , hematoma , and fracture did not typically occur after tibial bone graft . even in cases of elderly patients , similar results were reported . however , as a tibial fracture can be evoked by intensive exercise or external force , they concluded that care must be taken to avoid fracture until three months after surgery . the subjects of this study were patients who underwent proximal - medial - tibial bone graft under general anesthesia between march 2004 and december 2011 , at the department of oral and maxillofacial surgery in inha university hospital ( incheon , korea ) , and the operations were performed by two surgeons . surgical procedures in all cases was performed as follows5,6 : ( 1 ) the location of the tibial tuberosity was palpated and identified on the anterior surface of the proximal end of the tibia , ( 2 ) a 1.0 cm or 1.5 cm horizontal line following skin creases was drawn just below the tibial tuberosity , ( 3 ) local anesthesia was infiltrated subcutaneously at the level of the periosteum , ( 4 ) a horizontal incision was made with a # 15 blade , and reflection of the periosteum was performed with a periosteal elevator in order to expose the tibial bone , ( 5 ) a circular bony window 1.0 cm in diameter was made with a fissure bur and osteotome , and ( 6 ) with a bone curette , cancellous bone was harvested as needed . a total of 105 subjects ( 30 females , 75 males ) , underwent surgery . the reason for operation was cystic lesion in 92 cases , benign tumor in 12 cases , and oral - maxillary sinus fistula in one case . we investigated the age , weight , circumstance , and graft timing in relation to tibial fracture in all patients . between march 2004 and december 2011 , tibial fracture occurred in four of 105 tibial bone grafts . there were no significant differences in graft region , shape , or scale between fractured and non - fractured patients on x - ray imaging before and after grafting . 2 ) noninvasive reduction and fixation were performed for 56 days ( figs . 3 , 4 ) , and the fracture was completely healed after six months.(fig . 9 ) patient 3 was a 53-year - old , 55.6 kg male who underwent tibial graft surgery due to a maxillary dentigerous cyst.(fig . 10 ) fracture occurred when he slipped and hit a stair edge 42 days after surgery.(fig . 11 ) open reduction and fixation were performed at another hospital , and he fully recovered . patient 4 was a 35-year - old , 68.7 kg male who underwent right tibial graft surgery due to an odontogenic keratocyst of an anterior mandibular tooth and the premolar region . 2 ) noninvasive reduction and fixation were performed for 56 days ( figs . 3 , 4 ) , and the fracture was completely healed after six months.(fig . patient 3 was a 53-year - old , 55.6 kg male who underwent tibial graft surgery due to a maxillary dentigerous cyst.(fig . 10 ) fracture occurred when he slipped and hit a stair edge 42 days after surgery.(fig . 11 ) open reduction and fixation were performed at another hospital , and he fully recovered . patient 4 was a 35-year - old , 68.7 kg male who underwent right tibial graft surgery due to an odontogenic keratocyst of an anterior mandibular tooth and the premolar region . the ileum was initially recognized as the ideal region for cancellous bone collection and , until lately , has remained the favored donor site for auto - cancellous bone . o'keeffe et al.7 collected proximal - tibial cancellous bone and used it as graft preparation . they reported that tibial bone had easy accessibility for operation , appropriate qualitative and quantitative properties , and a rare complication rate , leading to its useful application . one of the possible complications of a tibial graft is that it can damage the articular surface and change the growth pattern for non - mature patients8,9 . additionally , a study by besly and ward booth10 comparing tibial and iliac grafts reported that complications such as pain , swelling , and gait disturbance might be present , in addition to the scarring at the operation site that is often considered acceptable by patients after tibia graft . one study5 about tibial grafts reported complications including swelling , gait disturbance for 3 - 4 weeks , and dehiscence of suture site . likewise , lezacano et al.11 explained that articular surface damage , tendon or nerve damage , and rare tibial fracture as potential complications . a comparison study10 with tibial and iliac grafts reported tibia graft advantages of low blood loss and fast gait recovery . in that study , all patients with tibial graft were able to walk within one day . moreover , a study by marchena et al.12 showed that , for outpatient procedures , complications such as infection , swelling , and fracture did not occur . on the other hand , o'keeffe et el.7 reported that tibial fracture occurred in one of 230 proximal tibial bone graft harvestings and suggested that patients should avoid external pressure on the region for at least six weeks after surgery . in two case series by van damme and merkx8,9 , two of nine patients experienced fracture at one week after the tibial bone graft surgery , while playing tennis in one patient and while running in the other . these authors insisted that patients should avoid exercise for at least 4 - 6 weeks after tibial bone graft surgery . hughes and revington13 reported that fracture occurred in two of 75 patients due to exercising at three months and falling at nine days after surgery . both patients were treated with immobilization using plaster of paris splints for tibial fractures . thor et al.14 reported that tibial fracture occurred for one patient who had 5 ml of compressed cancellous bone harvested for implantation after falling on the stairs at two weeks after surgery . they considered the fall a major factor in the fracture , but the design of the cortical bone window , the bone - harvesting technique , and immediate weight - bearing were suspected to be additional contributing factors . the conventional surgical procedure for medio - proximal tibial graft is performed through a vertical or oblique incision at the medial portion of the tibial tuberosity2,8,9,11 . in contrast , we created a horizontal incision in the wrinkle line just below the tibial tuberosity in order to reduce scar formation5,6 . accordingly , the bony window was located 1.0 cm lower than in the conventional procedure . however , no articles related to the frequency and risk of tibial fracture in accordance with the location of bony window were found in the studied literature . alt et al.15 conducted a study to evaluate the relation between volume of harvested cancellous bone and fracture risk during tibial bone graft . they collected cancellous bone from one cadaver tibia and did not collect from the other tibia . they made a 1 - 1.5 cm diameter bony window 1.5 cm inferior to the tibial tubercle and collected most of cancellous bone from each sites , which was then measured using a 10 ml syringe . subsequently , they pressed on the major axis of tibia in order to create a fracture . in five of eight cadavers , higher power was necessary to create fractures in the intact tibias , while the three other cadavers showed decancellated tibias that were more resistant to force than the intact tibias . furthermore , the magnitude of force for fracture showed no statistically significant difference between the two groups . as a result , they concluded that collecting the cancellous bone does not increase the risk of tibial fracture . n in a group of decancellated tibias and 9,491.13 n in a group of intact tibias . there was no significant difference between the two groups , although greater collection of bone tended to cause a decrease in bone resistance . accordingly , they recommended abstaining from exercise for 2 - 3 months after the surgery . a similar cadaver study by gerressen et al.17 showed that tibias from which cancellous bone was not collected were able to resist a greater amount of pressure in the major axis ( mean , 5,126.4 n ) than the group from which bone was collected ( mean , 3,766.9 n ) . regard , morrison18 suggested that pressure exerted on the tibia during walking reached 2 - 4 times the force of one 's weight regardless of sex . nisell19 described that isokinetic knee extension exerted 5 - 9 times the force of one 's weight on the tibia . gerressen et al.17 concluded that fractures could occur at lower pressures after tibial graft regardless of sex or age , and that tibial graft might greatly increase the possibility of tibial fracture . consequently , they recommended limiting activities to those involving a pressure equal to only half the body weight during the first week after surgery and resuming normal activities five weeks following surgery . in the present study , all patients were instructed to walk carefully starting the day after operation . additionally , we instructed patients not to perform exercise for at least three months after surgery and to be cautious about the risk of fracture due to external force . only four of 105 tibial graft patients experienced tibial fracture , and all fractures occurred between two and seven weeks after surgery . since tibial fractures occurred only during this certain period , we conclude that patient education is a very important factor to prevent tibial fractures . indirect vertical force caused by jumping in cases 1 and 2 and direct lateral force caused by bumping into objects in cases 3 and 4 were the causes of fracture in this study . from these results , we can assume that tibial fractures are vulnerable to direct or indirect external trauma rather than due to physical condition , such as body weight or age . in this study , tibial fractures occurred due to non - intended external force rather than the patient 's physical condition . in conclusion , we recommend that patients with tibial graft make an effort to avoid excessive external forces and exercise until 2 - 3 months after operation .

objectivesoral and maxillofacial defects often require bone grafts to restore missing tissues . well - recognized donor sites include the anterior and posterior iliac crest , rib , and intercalvarial diploic bone . the proximal tibia has also been explored as an alternative donor site . the use of the tibia for bone graft has many benefits , such as procedural ease , adequate volume of cancellous and cortical bone , and minimal complications . although patients rarely complain of pain , swelling , discomfort , or dysfunction , such as gait disturbance , both patients and surgeons should pay close attention to such after effects due to the possibility of tibial fracture . the purpose of this study is to analyze tibial fractures that occurring after osteotomy for a medioproximal tibial graft.materials and methodsan analysis was intended for patients who underwent medioproximal tibial graft between march 2004 and december 2011 in inha university hospital . a total of 105 subjects , 30 females and 75 males , ranged in age from 17 to 78 years . we investigated the age , weight , circumstance , and graft timing in relation to tibial fracture.resultstibial fractures occurred in four of 105 patients . there were no significant differences in graft region , shape , or scale between the fractured and non - fractured patients.conclusionpatients who undergo tibial grafts must be careful of excessive external force after the operation .

I. Introduction II. Materials and Methods III. Results 1. Case 1 2. Case 2 3. Case 3 4. Case 4 IV. Discussion V. Conclusion

PMC3282150

improper cooking is one of the main factors causing food borne illness [ 15 ] , and a large part , 4060% , of the cases of food borne illness are expected to originate from private households [ 69 ] . most consumers do not use a meat thermometer [ 10 , 11 ] but determine the doneness of meat most often by cutting the meat to evaluate changes in color and texture , or by other subjective techniques . especially for chicken breast fillet chicken breast fillet is , apart from minced meat , the most popular type of meat in the netherlands and a pathogen associated with it is campylobacter jejuni , a microorganism responsible for 50% of confirmed cases of bacterial gastroenteritis in western europe ( austria , belgium , finland , france , germany , ireland , italy , the netherlands , norway , portugal , sweden , switzerland , and the uk ) and the usa [ 1 , 1418 ] . furthermore , a predominant risk factor for c. jejuni infection is consumption of undercooked chicken meat [ 1922 ] . in a consumer study on food handling practices in the netherlands by our research group , focusing on the preparation of a chicken breast fillet salad artificially contaminated with nonmotile , nonpathogenic lactobacillus casei , it was shown that for consumers who apparently made no mistakes regarding cross - contamination , heating time of chicken meat ( boiled in chicken stock ) was negatively correlated with the bacterial contamination level of the prepared salad . although this correlation may be expected , it is surprising that the bacteria present were not immediately killed upon contact with boiling water , as the bacteria were only present on the surface of the meat and not , as with ground meat , on the inside as well . in addition , it was shown by bergsma et al . that consumer - style cooking ( pan frying ) of chicken breast inoculated with c. jejuni resulted in high bacterial heat resistance levels of this pathogen . ample research has been dedicated to heat inactivation of bacteria in meat , but most of these studies use thin - layered ground meat as a model system and normal cooking temperatures , that is , temperatures > 70c are hardly ever applied [ 2527 ] ( also see figure 3 ) . as these studies do not suffice to explain the observed heat resistance by bergsma et al . , the aim of this research was to further elaborate on this phenomenon . therefore , in the current study , the chicken fillets were boiled instead of pan fried to obtain better reproducible temperatures in the experiments and decimal reduction times of bacteria present on each sample were determined . heating profiles of chicken breast fillets during cooking were obtained both by measurement and by calculation . apart from c. jejuni , salmonella and escherichia coli were also studied as these bacteria are as well associated with food borne illness caused by consumption of raw or undercooked meat . in addition , l. casei was used , as this bacterium was used as indicator organism for c. jejuni to study real - life consumer practices by our research group . furthermore , the effect of food matrix , matrix size , and refrigerated storage on the survival of bacteria during cooking was studied as well as the effect of refrigerated storage on cell attachment . in addition , the obtained decimal reduction times were used to assess the relationship between the probability of illness ( campylobacteriosis ) due to consumption of chicken breast fillet and cooking time . five c. jejuni strains ( nctc 11168 , nctc 11828 , b258 , lb99hu , and 82/69 ) , a lactobacillus casei strain ( isolated from a food ) , and escherichia coli wg5 ( atcc 700078 ) were used as described by de jong et al . . c. jejuni , l. casei , and e. coli were cultured as described by de jong et al . . s. typhimurium dt104 was grown in brain heart infusion broth ( 37c ; bhi , oxoid , basingstoke , uk ) . the heat resistance of bacteria present on the surface of chicken breast fillets during cooking of the meat in boiling water was expressed as a decimal reduction time during boiling ( dboil ) : time needed to reduce the number of bacteria present on the outside of a large matrix exposed to boiling water by 10-fold ( min ) . this decimal reduction time is comparable to the formal , frequently applied d - value . however , to determine a dt - value of a bacterium on a matrix , the temperature of the matrix must be constant during the heating experiment . therefore , d - values of bacteria on food items are determined using small sized food samples , mainly with a 1 mm thickness . as these small - sized samples are not representative for sizes of meats prepared by consumers during cooking , the heating experiments in this study boil as index as the temperature could not be given explicitly , since the product temperature was dynamically changing ( see figure 2 ) . furthermore , the effect of food matrix and refrigerated storage on survival of c. jejuni during cooking was studied . in addition , cell attachments studies were conducted . to study the matrix - effect on heat resistance of c. jejuni , cold - stored ( 4c , 24 h ) inoculated chicken breast fillets and carrots were used as matrix and survival of c. jejuni on these matrices after a 5 minute cooking time was compared . the effect of refrigerated storage of chicken meat on heat resistance of c. jejuni present on the meat surface was studied by comparing survival of c. jejuni present after 5 min cooking using chicken breast fillets refrigerated ( 4c ) for 0 , 1 , or 24 h. the effect of refrigerated storage on cell attachment of c. jejuni to the matrix was studied for both chicken breast fillet and carrot . the inoculated matrices were stored in the refrigerator ( chicken meat : 0 , 1 , and 24 h at 4c ; carrots : 0 and 1 h at 4c ) and subsequently manually rinsed for 10 s under cold running water and the number of c. jejuni cells attached was determined . this was done by comparing the number of cells present on the food item after washing to the number of cells applied to the food item . a multiple - species cocktail of c. jejuni / l . casei was obtained by mixing a multiple - strain cocktail ( 5 strains mixed in equal volumes ) of c. jejuni with a single strain of l. casei in order to compare the behavior of these different bacterial species under equal test conditions , similar to what was done by the authors in a study on cross - contamination during consumer cooking practices . in addition , a multiple - species cocktail containing one strain of e. coli and one strain s. typhimurium was used . the cocktails were prepared by combining equal volumes of each single - species culture . an inoculum level of 10 cfu ml was used to inoculate the food items with . cell counts of bacterial suspensions were determined ( n0 ) by spread plating appropriate dilutions on appropriate media . whole chicken breast fillets ( 98218 g ; maximal thickness : 3.54 cm ) purchased in different batches at a local supermarket were used fresh or were stored frozen and defrosted before use . thawed or fresh fillets were dabbed with paper tissue to remove superficial moisture , after which each fillet was contaminated with 1 ml of inoculation culture ( each side 0.5 ml ) , which was evenly spread using a plastic , sterile rod . each contaminated fillet was stored overnight in a separate plastic bag at 4c to mimic retail storage . the ( large ) carrot was longitudinally sliced to obtain slices of similar weight ( 77198 g ) and similar thickness as the chicken breast fillets . both sides of these slices were allowed to dry in a laminar flow cabinet ( 30 min each side ) to remove moist , after which the carrot slices were similarly inoculated and stored as the chicken fillets . for the heating experiments , one single inoculated refrigerated food item was placed in a pan ( 24 cm ) with boiling water ( 4 - 5 l ) at a time ( each data point is one single heating experiment ) , thus limiting the decrease of the water temperature due to the addition of the food item . the water was constantly heated and boiling . at time zero the food item was placed in the boiling water and then heated for times ranging from 0 ( not heated ) to 15 min . after heat treatment , the food item was immediately transferred into a sterile waring commercial blender , weighed , and cooled with 200 ml peptone ( 1 g l ) physiological salt ( 9 g l ) solution ( pps ) of 4c and blended for 1 min resulting in blended chicken slurry or blended carrot slurry . the pan was washed up after cooking for every single food item and clean tap water was brought to the boil again . experiments were conducted at least in duplicate , on different days . for the heat - inactivation test , a cooking pan ( 24 cm ) with water ( 4 - 5 l ) was brought to the boil . the water was constantly heated and as the weight of the added matrices was small ( < 200 g ) compared to that of the water ( > 4000 g ) , the temperature profile of the water was considered to be constant at 100c . however , the temperature of refrigerated chicken meat has a come - up time and the temperature profile of the meat can be estimated using ( 1 ) : ( 1)uslab = uchicken fillet = tx , tt0tt0=1erf(x2at ) , in which u is the fraction of nontransferred heat ( 0 u 1 ) , tx , t is the temperature of the product at place coordinate x at time t , t0 is the temperature of the product at t = 0 , t is the temperature of the surroundings , erf is the standard error function from excel , x [ m ] is the place coordinate , and a [ m s ] is the product 's thermal diffusion coefficient , which is given by : ( 2)a=cp , where [ w m k ] is the product 's thermal conductivity , [ kg m ] is the product 's specific density , and cp [ ws kg k ] is the specific heat capacity of the product , and in which only conductive heat transfer effects via the determining dimension of the chicken fillet , the height , are taken into account ( the chicken fillet is thus considered to be an infinite slab , since the width and length of the fillet are more than 2.5 and 5 times as long as the height , resp . ) . in reality , the temperature of the chicken will increase more rapidly , so this is a fail safe assumption . equation ( 1 ) is applicable when the external heat transfer resistance is negligible ( boundary condition 1 ) and when the temperature of the centre of the product has not changed yet ( boundary condition 2 ) . to determine whether boundary condition 1 is met , the ratio between the transport resistance of the product and its surroundings , the biot number ( bi ) , can be calculated by : ( 3)bi = al=transport resistance producttransport resistance surroundings , where [ w m k ] is the convective - heat - transfer coefficient of the surroundings , l [ m ] is half height of the product , and [ w m k ] the product 's thermal conductivity . when the value of bi exceeds 10 , the transport resistance of the surroundings is considered to be negligible . with : ( 4)fo = atl2 , where fo ( fourier number ) is the dimensionless time , one can calculate until which heating time , the short times boundary , boundary condition 2 is met . for an infinite slab , the short times boundary is set by fo = 0.20 . the temperature profile of the chicken fillet ( at different distances from the surface of the meat ) in boiling water was estimated using ( 1 ) and the parameter values given in table 1 . a pt100 temperature probe attached to an applikon adi-1030 biocontroller ( applikon , schiedam , the netherlands ) the temperature probe was calibrated between 0c ( melting ice ) and 100c ( boiling water ) prior to use . according to the manufacturer , therefore , to measure the temperature at the surface of the meat , a 20 cm long stainless steel probe was bent in the shape of an ice - hockey stick , allowing the actual sensor ( last two cm ) to be pressed firmly to the surface of the meat . the temperature profiles of the water and meat surface were generated using noninoculated chicken fillets ( weight : 142172 g ) . the meat was placed on a plastic test tube rack so that only the last 7 cm of the temperature probe was under water . after adding the meat in the pan with boiling water , the temperature probe was immediately pressed on the meat surface or held in the water and temperature recording was started . culturability of the inoculated bacteria on the food items after heat - treatment was determined by use of the most probable number method ( mpn , see de man ) in combination with spread - plating suitable dilutions on agar plates . chicken breast fillets were sampled and analyzed as described by de jong et al . ; the carrots were similarly treated . the lower level of detection used was 1.4 cfu per food item . media used for the mpn method and plate counts , respectively , were as follows , as described by de jong et al . : preston broth and karmali agar for c. jejuni , mrs broth and agar for l. casei , and modified - tryptic soy broth and tryptic soy agar both supplemented with 1 g / l nalidixic acid for e. coli . for s. typhimurium buffered peptone water ( nvi ) and brilliant green agar ( oxoid ) were used . mpn samples were checked for growth of the respective organisms after incubation ( time / temperature details see ) by streak plating on abovementioned agar plates . suspected colonies of c. jejuni and l. casei were confirmed by phase contrast microscopy . c. jejuni media were microaerobically incubated ( broth : 48 h , agar : 72 h , at 37c ( to allow the recovery of any sublethally injured c. jejuni ) either in a three - gas incubator ( 5% co2 , 10% o2 , 85% n2 ) or in jars with bbl campypak ( becton dickinson , sparks , usa ) . for reasons of comparison , media for l. casei were aerobically incubated at 30c ( broth 48 h ; agar : 72 h ) , those for e. coli and s. typhimurium overnight at 37c . the contamination levels of the food items after heat treatment were calculated , taking into account the exact weights used for enumeration , using an excel spreadsheet based on the mpn method described by de man . data were represented as count data ( log cfu and mpn per fillet ) plotted versus heating time ( min ) , to which the linear and weibull inactivation models were fitted . levels below the detection limit were taken into account as censored data by using maximum likelihood estimation assuming poisson - distributed data . the best fitting model was determined by applying an f - test using the rss of the weibull and linear model . analyses were performed using mathematica 5.2 ( wolfram research inc , champaign , usa ) . according to the f - test , the linear model fitted best and therefore decimal reduction times during boiling ( dboil ) were calculated using the slope of the graphs , using : ( 5)lognt = logn0tdboil with log as the 10-based logarithm , nt as number of viable microorganisms at a given time , n0 as the initial number of microorganisms , t as time in min , and dboil as time needed to reduce the number of bacteria present on the meat surface by 10-fold when exposed to boiling water . the effect of different heat treatment scenarios was tested for its significance with anova on the log transformed data in spss ( spss , chicago , usa ) . for comparison of currently obtained data to literature data , d - values were collected from literature for different temperatures , strains , and products ( mainly meat ) or media following the approach of van asselt and zwietering . microorganisms studied were campylobacter jejuni ( n = 176 ) , lactobacilli ( n = 6 ) , escherichia coli ( n = 79 ) , and salmonella ( n = 287 ) . the relation between heating temperature and logd is linear : ( 6)log dref = intercept ( log d , t)trefz . logdref is the logarithm of the d - value ( log min ) at temperature tref , tref is the reference temperature ( c ) , and z is the temperature increase ( c ) needed to reduce the d - value with a factor of 10 . the logd versus temperature plot was used to compare our decimal reduction times with data ( d - values ) published in literature . decimal reduction times can be used to assess the human health risk of pathogen exposure consequential to undercooking . as an example , we studied campylobacter on chicken breast fillets using an empirical distribution of concentrations on fillets after cutting at the cutting plant , obtained from a dutch risk assessment model [ 21 , 33 ] . it showed that most contaminated fillets carried between 0 between 4 log cfu / fillet and less than 5% carried higher numbers , results which are similar to campylobacter contamination data reported for german retail products . after incorporation of the survival after storage [ 21 , 33 ] , yielding a decrease in concentration described by a betapert distribution with minimum 0.1 , most likely value 0.9 and maximum 2.1 log cfu / fillet , these results can be used as a distribution for input ( n0 ) of ( 5 ) . the exposure distribution after an inactivation time t is then given by the resulting distribution of nt . the associated health risk , expressed as the probability of illness per fillet , can be assessed by implementing this distribution of doses into the dose response relationship for campylobacter : ( 7)pill(nt)=0.33(1(+)(+nt)()(++nt ) ) with dose response parameters and . the effect of cooking on bacterial survival on whole chicken breast fillets was studied for a cocktail of c. jejuni . during cooking , cell numbers declined , following a straight line ( figure 1 ) . using ( 5 ) , we calculated that the decimal reduction time for c. jejuni when present on meat during cooking of the meat in boiling water was 1.90 min . in figure 2 , the estimated temperature profiles at the surface of a chicken fillet in boiling water and at 0.25 mm , 0.5 mm , and 0.75 mm depth are shown . to estimate the temperature profile of the chicken fillet in boiling water , the first condition states that the external heat transfer resistance is negligible , thus that bi > 10 ( 3 ) . to verify whether this boundary condition is met , for still water , bi ranges between 11.67 and 19.33 ; thus for boiling water bi will be higher and boundary condition 1 is met . to verify until which heating time boundary condition 2 was met , the short times boundary was calculated , using ( 4 ) . this implies that for heating times > 10.8 min the temperature according to the heating profile estimated by ( 1 ) is overestimated . after 30 sec , the average measured surface temperature of the chicken fillets approximated the estimated profile at 0.5 mm depth in the meat , and measured data ranged between the estimated profiles at 0.25 mm and 0.75 mm depth . within 1 min , the surface temperature of chicken fillets ( 4c at time zero ) reached a value of at least 85c ( figure 2 ) . the water temperature was only slightly affected by the addition of the refrigerated meat to the water and did not drop below 99.3c . ample research has been dedicated to heat resistance levels of food borne pathogens , and in figure 3 an overview is given from a selection of heat inactivation data not only of campylobacter but also of lactobacilli , e. coli , and salmonella . data are both from fluid and solid ( i.e. , meat ) matrices ( see also van asselt and zwietering . this graph shows the extremity of our obtained bacterial heat resistance levels ( encircled data ) . the heat resistance of bacteria depends on many factors . to investigate some specificities of the observed heat resistance , different heating experiments were conducted with c. jejuni . in addition , the heat resistance of c. jejuni was also determined when inoculated onto a slice of carrot of similar size as a chicken fillet to investigate whether the observed heat resistance was related to the matrix type . no bacteria were recovered from cold stored inoculated carrots ( 24 h , 4c ) after 5 min of boiling ( data not shown ) . the effect of refrigerated storage time on the heat resistance of c. jejuni on chicken meat was investigated . we observed that after a 24 h storage period , a 5 min cooking time reduced cell numbers by 3.0 ( stdev 0.5 ) log units , for 1 h stored fillets this was 4.3 ( stdev 0.8 ) log units , and for noncold stored fillets a 4.7 ( stdev 0.5 ) log reduction was observed . the heat resistances after 0 h and 1 h storage time were not significantly different ( p = 0.392 ) , but those observed after a 24 h storage time were significantly higher than the other two storage times ( 24 h versus 1 h storage time : p = 0.037 ; 24 h versus 0 h storage time : p = 0.002 ) . refrigerated storage for various periods of time did have no effect on the level of attachment of cells to chicken meat , as rinsing for 10 s under cold running water resulted in a 1.41.9 log cfu removal of campylobacter from chicken meat , independent of the storage time ( 0 , 1 , and 24 h ) . the heat resistance of l. casei , e. coli , and s. typhimurium was also tested under the same conditions as used for campylobacter . for all bacterial species tested cells could still be recovered from meat after a 10 minute heating time in boiling water . we calculated ( 5 ) decimal reduction times of 1.93 , 1.97 , and 2.20 min , respectively for l. casei , e. coli , and s. typhimurium . we finally determined the effect of the observed heat resistance levels on food safety health risks . for this purpose we applied a dutch risk assessment study on campylobacter in broiler meat , as explained in the methods section . equation ( 1 ) was used to calculate the expected ingested doses nt after boiling the meat for t minutes , given the value dboil = 1.90 min , as found in our experiments . after implementation of the dose response relationship , this resulted in figure 4 , showing the decreasing probability of illness per fillet bought at retail in the netherlands , as a function of cooking time . consumption of chicken breast fillets cooked for 10 minutes resulted in an estimated probability of illness of 5.5 10 . this research studied the effect of consumer cooking practices on survival of various bacterial species applied to chicken fillet meat . frying is the most frequently used method for the preparation of chicken meat by dutch consumers . the temperature at the surface of chicken meat during frying is on average 127c , but as the temperature is difficult to control ( stdev : 18c ) , we decided to study survival in boiling water . all tested species died off following a straight line from which a d - value of approximately 2 minutes was calculated . the advantage of the study design was that it immediately revealed the fate of bacteria present on meat during nonisothermal heating to high temperatures , mimicking a consumer 's style of cooking . thus , no extrapolation of data obtained from isothermal d- and z - value experiments with small - sized matrices at temperatures < 70c was needed . the disadvantage was the difficulty of determining the actual surface temperature of the meat and thus the exact temperature the bacteria were exposed to . we measured the surface temperature and we used a mathematical approach to determine the surface temperature during cooking . with a temperature probe , we measured that the surface temperature of chicken fillets reached 85c within 1 min . the mathematical approach can be applied when the external heat transfer resistance is negligible and when the temperature of the centre of the product has not changed . we calculated that during the first 10 minutes of cooking , both conditions are met . the calculated temperature at 0.5 mm depth in the meat approximated the average measured surface temperature of the chicken fillets . the shape of the calculated heating profile was similar to that of the measured profile , and the results of our calculations are comparable to those of houben and eckenhausen , who calculated the temperature at certain surface depths during pasteurization of a model product in a water bath of 96c . together this shows that both our measured and calculated temperature reliably reflect the temperature bacteria experience at the surface of a chicken breast fillet during cooking in boiling water . heat resistance studies are generally conducted in solid ( thin patties of mainly ground meat ) or in liquid matrices ( broth , milk , etc . ) , with higher heat resistance levels obtained in solid matrices [ 25 , 4046 ] . temperatures during such studies range from 55 to 72c , allowing accurate determination of d- and z - values . at higher temperatures , d - values the surface temperature of chicken fillets reached 85c within 1 minute . according to the literature ( after extrapolation ) the d - value of bacterial cells at 85c is less than one second [ 32 , 41 , 47 ] , yet in our experiments it took 2 minutes to obtain a 1 log reduction of bacteria . when inoculated on another solid matrix of approximately the same size ( slice of carrot ) bacteria showed different behaviour . the study of van asselt and zwietering revealed that heat resistance can be significantly increased for certain specific matrix ( high fat and low aw)/bacterium combinations . but as both carrot and chicken meat are low fat / high aw products , our results indicate that chicken meat itself ( e.g. , the presence of some specific component like iron , or an amino acid ) affects the heat resistance of bacteria . to determine the d - value of a bacterial species at a certain temperature , a small volume of a bacterial culture is transferred to a relatively large volume of a preheated menstruum . in this way , the temperature come - up time is as short as possible and bacteria experience a constant temperature . as the temperature of a large and solid matrix does not come up as fast as the temperature of a small or liquid matrix , the dimension of a product can affect the heating experiment . a weak size effect on heat resistance is indeed demonstrated in a similar study conducted in our laboratory by bergsma et al . , who pan fried chicken meat inoculated with c. jejuni using whole and diced fillets . in literature , purnell et al . observed that elevated heat resistance levels of naturally c. jejuni contaminated whole chicken carcasses during water immersion treatments at temperatures 70c ( 2030 s ) . the other campylobacter data points in figure 3 boxed by the dotted line were calculated using data from whyte et al . . these authors conducted hot water immersion treatments ( 7580c ; 0 , 10 , and 20 s ) with naturally and artificially contaminated chicken thighs . again high survival levels of campylobacter cells were obtained after heat treatments . in the study by purnell et al . not only was the size of the test products larger than that used in most other studies , but also the challenge temperatures in this studies were higher than normally used in heat resistance testing . interestingly , in small sized meat discs artificially inoculated with e. coli and salmonella ( see figure 3 in dotted box ) and pasteurized with hot steam ( 87c ) , also high survival levels of bacteria were measured after a 60 s heat treatment . although the calculated d - values or decimal reduction times for data from purnell , whyte , and mccann are based on very short heating times , 60 s , and may therefore be less accurate , these d - values or decimal reduction times and our current data show that bacteria on meat are not as easily killed by temperatures > 70c as predicted based on d - values obtained at temperatures < 70c . both our data and those discussed previously indicate that the combination of products size and challenge temperature might affect the level of heat resistance of bacteria . another difference of our study design compared to heating experiments described in literature is the overnight refrigerated storage of the contaminated fillets to mimic consumer / retail storage conditions . although overnight cold storage did not affect the number of bacteria attached to the meat , we observed that storing the meat in a refrigerator increased the number of surviving cells . at a low temperature ( no growth , stress ) physiological properties may have changed which may have affected the heat resistance of the bacteria , a phenomenon known as cross - protection [ 49 , 50 ] . although the pathogens used were all motile and thus can move from the surface to more inner parts of fillets , the observed high level of heat resistance can not be explained by such movement , as the heat survival of the non - motile l. casei was equal to that of the motile pathogens tested . so chicken meat , challenge temperature , or heating rate and cold storage have their effect on the heat resistance of c. jejuni , s. typhimurium , e. coli , and l. casei . because bacteria on the surface of poultry would be destroyed by limited cooking , the recurrent association of illness with undercooked poultry suggests either that the poultry is regularly re - contaminated after cooking or that campylobacter is somehow present deep in the tissues of a single poultry carcass , where it survives limited cooking , which is also in concordance with fao / who conclusions . however , data presented in our paper reveal that limited cooking does not necessarily eliminate all bacteria present on the surface of poultry meat . furthermore , our data add to the deeper understanding of the frequent association of food borne illness with consumption of undercooked poultry . as a consequence , our data show that research conclusions based on the assumption that cooked chicken meat does not substantially contribute to the risk of food borne illness [ 5355 ] must be interpreted with more consideration . consumption of chicken meat cooked for 10 min cooking still results in a probability of illness of 5.5 10 . the estimated probability of illness per meal containing chicken breast fillet and a salad cross - contaminated with c. jejuni from the chicken fillet to the salad , as assessed in the risk assessment of nauta et al . this not only confirms that the risk of acquiring campylobacteriosis consequential to undercooking is much smaller than that consequential to cross - contamination but also shows that the cooking time may be far more critical than previously assumed : with a cooking time of about 7.5 min the risk of undercooking is comparable to that calculated for cross - contamination ( see figure 4 ) . taking consumer behavior into account , using data of the observational study of fischer et al . , it becomes clear that undercooking of chicken meat by consumers is certainly not negligible , as 33% of the participants ( dutch consumers ) in their study applied heating times during cooking of chicken breast fillets of less than 7.5 min . when inoculated on chicken breast fillets , the heat resistance of bacteria increased to unexpected high levels . chicken meat , the challenge temperature , or heating rate and cold storage affected the level of resistance . it can be concluded from our study that , until now , the effect of cooking on the survival of bacteria present on the outside of chicken meat has been overestimated . it is therefore recommended to reconsider all statements made based on meat heating trials that do not use consumer - style meat types , sizes , and cooking techniques .

the aim of this research was to determine the decimal reduction times of bacteria present on chicken fillet in boiling water . the experiments were conducted with campylobacter jejuni , salmonella , and escherichia coli . whole chicken breast fillets were inoculated with the pathogens , stored overnight ( 4c ) , and subsequently cooked . the surface temperature reached 70c within 30 sec and 85c within one minute . extremely high decimal reduction times of 1.90 , 1.97 , and 2.20 min were obtained for c. jejuni , e. coli , and s. typhimurium , respectively . chicken meat and refrigerated storage before cooking enlarged the heat resistance of the food borne pathogens . additionally , a high challenge temperature or fast heating rate contributed to the level of heat resistance . the data were used to assess the probability of illness ( campylobacteriosis ) due to consumption of chicken fillet as a function of cooking time . the data revealed that cooking time may be far more critical than previously assumed .

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

PMC4611897

paroxysmal dystonias are classified into paroxysmal kinesigenic dystonias ( pkds ) , paroxysmal nonkinesigenic dystonias , paroxysmal exertion induced dystonias ( peds ) , and paroxysmal hypnogenic dystonias . transient sudden movements triggered by exercise , which may or may not be associated with pain involving the lower limbs more than the upper limbs are designated as peds . mutation in glut-1 gene through slcza1 , which is a solute carrier for glucose transportation , causes energy deficiency on exertion . increase in cerebrospinal fluid homovanillic acid and 5-hydroxyindoleacetic acid with ped is reported in some patients with associated young - onset parkinson 's disease , epilepsy and migraine , and linkage to chromosome 16q11.2-q22.1 , as well as in 16p11.2-q11.2 has been reported . the role of mitochondria in movement disorders came to be known with reference to dystonia with the discovery of missense mutation in mitochondrial dna complex 1 , which is maternally inherited through mtnd6 gene . the oxidative phosphorylation ( oxphos ) components of the mitochondria are assembled from the genes distributed between mitochondrial dna and nuclear dna ( ndna ) . the components thus assembled are the inner membrane enzyme complexes i , ii , iii , iv , and v , protein kinases such as adenine dinucleotide translocators and mitochondrial biogenesis proteins which involves the apparatus to import cytoplasmically synthesized mitochondrial proteins needed for assembly and turnover . fifty - five percent of reduction in complex i occurs when alanine at position 72 is converted to valine by a mutation in mitochondrial dna complex i gene mtnd6 in leber 's hereditary optic atrophy causing early - onset hereditary dystonia . sixty - two percent reduction in platelet mitochondrial complex i is seen in patients with segmental and generalized dystonia and 37% reduction in focal dystonia . dfn-1 gene defect causes x - linked mohr - tranebjaerg syndrome with deafness and dystonia and skd3 protein is linked with dystonia and its gene product is functionally similar to dyt1 gene product raising strong possible role of ndna encoded mitochondrial genes in neurodegenerative disorders including movement disorders . leigh 's and leigh 's like syndromes due to complex v atpase 6 and complex i ndna subunits are reported to be associated with a wide variety of movement disorders but exertion induced dystonia due to mitochondrial disease has only a passing reference in the literature . paroxysmal dyskinesias are episodic childhood onset movement disorders usually precipitated by normal or sudden unexpected motor activity . the types of movements seen are sudden dystonia , chorea , athetosis , and ballism , or a combination of these . it can occur spontaneously or may be precipitated by sudden movements , prolonged exercise , caffeine and alcohol consumption , emotional stress , or fatigue . our patient is the second child of first degree consanguineous parents born naturally at full term . after 2 years , it was noticed that while running he was experiencing pain in the limbs which forced him to take brief periods of rest . he was poor in studies . from the age of 5 years , after walking some distance he used to experience abnormal twisting of the trunk to one side , most often to the right side with episodes of difficulty in speaking , and swallowing with abnormal posturing of the right upper and lower limbs . this was more in the morning hours and used to last for few minutes to few hours occasionally . however , unlike the typical kinesigenic dystonia , there was absolutely no response to diphenylhydantoin or carbamazepine . patient had an elder sibling who presented with similar symptoms and was treated in the similar fashion . later , at the age of 10 years , he developed poor vision , which was treated with steroids and vision improved . however , his exertion induced abnormal movements did not respond to diphenylhydantoin or carbamazepine and he was started on sodium valproate . with a single dose of sodium valproate child became drowsy followed by myoglobinuria , renal shutdown , and passed away within 24 h. the second child was brought to our center in february 2012 . at the time of admission , the child was found to be short statured , his head circumference was 48 cm . in general examination there was the mild prominence of the calf muscles and the paraspinal muscles with scoliosis to the left . he had no evidence of involvement of other systems . however , when made to walk about 20 m child was slowly tilting to the right with dysarthria . mother had cleft lip and father 's cousin had mental retardation [ figure 1 ] . in view of sudden deterioration and death in elder sibling with a single dose of 500 mgs sodium valproate , his blood routine , urine routine , renal functions , liver functions , calcium , and electrolytes were all normal . his creatine kinase was 3177 iu ; his lactate dehydrogenase was 2728 , lactate 12.55 , ammonia 46.6 , and uric acid 2.7 mg / dl . his urine ferric chloride test , benedict 's test , and cyanide nitroprusside tests were negative . his auditory brainstem response , visual evoked potential , somatosensory evoked potential , electroencephalography , and ultrasound abdomen were normal . magnetic resonance imaging brain showed mild hyperintensities in tectum and central tegmental tract and in the pontine tegmentum [ figure 2 ] . family history of mental retardation , deafness found in the paternal side and cleft lip in the mother ( a - c ) magnetic resonance imaging brain shows mild hyperintensities in tectum and central tegmental tract and in the pontine tegmentum left biceps biopsy stained with eosin , hematoxylin , modified gomori 's trichrome , and periodic acid - schiff was negative for ragged red or ragged blue fibers . mitochondrial genetics for t12706c , a13084 t , and g13513a in nd5 gene commonly associated with leigh 's syndrome was negative . his repetitive nerve stimulation showed decremental response elsewhere but when repeated in our center was normal . in view of the sudden death in the elder sibling , the child was clinically diagnosed as probable mitochondrial disease and started on a full mitochondrial cocktail . with however , his symptoms relapsed in november 2014 . therefore , the patient was again reassessed . his histopathology slide was reviewed with special stains and was reported as complex 1 deficiency . polg gene sequencing of all the exons revealed 2 mutations ( c. 2021 g > a and c. 3644 - 27 t > g ) . the c. 2021 g > a mutation was also found in the father suggesting a pathogenic mutation . the surf1 gene analysis revealed a novel and pathogenic mutation at c. 237 g > t , which is pathogenic and inherited from the father as an autosomal dominant trait . our patient is unique as both brothers showed the clinical phenotype of exertion induced dystonia and our only clue in the early phase of our management of this patient was the valproate induced death in his brother based on which he was put on mitochondrial cocktail . this was lifesaving for the patient , and he was asymptomatic for more than 2 years and had a minor relapse at present . reassessment genetically and histopathologically showed complex i deficiency with the novel , autosomal dominant , paternally inherited gene mutation . this patient is a unique case of ped involving the trunk , upper limbs in addition to dysarthria in two siblings born to first degree consanguineous parents . there was no response to treatment as pkd instead fatal complication occurred to the elder sibling with valproate which suggested the possibility of a mitochondrial disease and reinvestigation after 2 years showed a paternally inherited novel nuclear gene mutation causing complex i deficiency . well known syndromes of peds are also due to energy failure due to defective glucose transport . this constitutes the major entry point of electrons to oxphos system transferring it from nadh to ubiquinone , which is important for moving protons across the inner membrane essential for creating a negative membrane potential . premature electron leakage to oxygen in this process makes complex i an important source of superoxide production . complex i deficiency commonly presents with dystonia , failure to thrive , seizures , eye movement problems , lactic acidosis , and muscle weakness . exertion induced dystonia as a manifestation of mitochondrial disease is an uncommon presentation , and there is only one case reported in the literature .

paroxysmal dystonias are a group of relatively benign hyperkinetic childhood movement disorders of varied etiology . mitochondrial diseases are well known to produce persistent dystonias as sequelae , but paroxysmal exertion induced dystonia has been reported in only one case to the best of our knowledge . two siblings born to consanguineous parents presented with early - onset exertion induced dystonia , which was unresponsive to diphenylhydantoin and carbamazepine . a trial with valproate in one of the siblings turned fatal within 24 h. based on this clue , the second child was investigated and found to suffer from complex i deficiency with a paternally inherited dominant nuclear dna mutation , which is responsive to the mitochondrial cocktail . exertion induced dystonia can be a rare manifestation of complex i deficiency .

Introduction Case Report Discussion and Conclusion Financial support and sponsorship Conflicts of interest

PMC4485548

autism spectrum disorders ( asds ) describe a group of neurodevelopmental conditions in which the individuals face challenges with social engagement and age - appropriate play and fail to develop appropriate peer relationships according to their developmental level . although young people are frequently recommended to participate in leisure activities including play , sports , hobbies , and social activities , children with asd tend to spend time in passive play and maladaptive behaviors and they are less likely to spontaneously participate in organized leisure activities such as sports [ 2 , 3 ] . it could be attributed to their significant deficits in development of motor development and physical activity ( pa ) behavior [ 4 , 5 ] . social and behavioral impairments in asd can limit children opportunity to participate in physical activity and recreation programs that eventually end to their inactivity . physical inactivity predisposes children with asd to several comorbid conditions such as overweight and obesity [ 7 , 8 ] . to assess key correlates of physical activity , previous studies frequently addressed social variables as critical factors contribute to asd children physical activity . for example , pan showed that children with asd who had lower social engagement with adults displayed lower levels of physical activity than children had higher social involvement . indeed although children with asd receive rehabilitation services from an early age in order to improve daily performance and enhancement of active life , pa and leisure aspects of quality of life ( qol ) are underestimated in children with asd and their families [ 11 , 12 ] . to address the children needs , parents and caregivers have to spend many resources while making a balance between children needs and those of family or guardians is a difficult task . thus recently , studies examining qol in a wide range of individuals with asd indicated that adults with asd have lower scores in wellbeing measures [ 13 , 14 ] , and children also show a subideal outcome [ 15 , 16 ] . a recent study on asd demonstrated a positive connection between cheerfulness and participation in a quality leisure program ; authors also indicated that satisfaction is also correlated with leisure activities in individuals with asd . although some studies could not show any differences between the physical activity levels of children with and without asd , there is a general consensus that children with asd did not participate in enough pa necessary to meet the activity guidelines for wellbeing . most recent review of literature confirmed that children with asd fall short of the recommended physical activity levels and experience challenges in physical activity and physical education settings and thus recommended strategies to improve the physical activity statistics and quality of life among children with asd . limited research , to date , has looked at the barriers and facilitators of pa among children with asd . however , a number of barriers to physical activity from individual to social and environmental make pa participation of children with asd more difficult and may result in increasing their sedentary activities . a rare study examining parent - reported barriers to pa in children with asd reported significantly greater amount of barriers compared to td children . barriers reported by parents are too much supervision needed compared to td children , lack of skills , few friends , and exclusion by other children which are the most important barriers . however , children with asd themselves rated interpersonal ( such as screen activities ) , physical ( such as lack or unsafe equipment ) , and community ( such as lack of transportation to physical activity programs ) factors as the most frequent barriers . on the other hand , there are facilitators from personal ( individual versus team activities ) to collective ( such as social support ) that help children with asd to involve in a pa program . particularly the association between pa and social support has been established among children with asd . however a multiaspect approach is needed to assess the pa and leisure participation of children and adolescents with asd . of relevance to current study , it is important to examine whether the children with asd are provided with enough opportunities to participate in physical activities and what factors are playing a role in their physical activities . furthermore , identifying contributing factors into pa will be imperative to enhance the efficacy of interventions aimed to improve active life / wellbeing of children with asd . therefore , we aimed to assess the participation of a school based sample with asd in physical and daily activities . we further sought to examine individual ( e.g. , age and clinical symptoms ) and social ( e.g. , household structures ) factors contributing to the level of participation in leisure physical activities . a total sample of 83 children ( 53 boys and 31 girls ) with high functioning asd ( iq > 70 ) aged 6 to 15 years ( mean = 9.8 , sd = 1.8 ) were recruited from four autism specific schools in tehran . all of the subjects had received a clinical diagnosis of asd ( autism , asperger 's , or pervasive developmental disorder , not otherwise specified ) by a child psychiatrist or clinical psychologist and the diagnosis was confirmed using the revised autism diagnostic interview ( adi - r ) . this cross - sectional study was approved by the medical ethics committee of the tehran university of medical sciences . physical activity involvement during leisure time was examined using a modified checklist adapted from godin - shephard leisure time questionnaire ( glteq ) . we aimed to evaluate activities ( at least 15 minutes ) over a 7-day period asking two questions . the first question addresses the intensity of physical activities : strenuous ( e.g. , running , football ) , moderate ( e.g. , easy bicycling , easy swimming ) , and mild ( e.g. , yoga , easy walking ) . since children with asd were not acquainted to complete a self - report questionnaire , we modified the questions in order for parents / caregivers to reply . for instance how many times on the average do you do the following kinds of ? how many times on the average does your child do the following kinds of exercise ? we asked parents to consider physical activities all day long ( including school time ) when they answered the questions . also each child 's teacher was asked to help parent to include school time physical activities when asking the questions on how many times on the average does your child do the following kinds of exercise ? finally a composite score was calculated as activity score = ( 9 ( number of strenuous exercise episodes ) ) + ( 5 ( number of moderate exercise episodes ) ) + ( 3 ( number of mild exercise episodes ) ) . an additional question was presented as during a typical 7-day period ( a week ) how often does your child engage in any regular physical activity long enough to work up a sweat ( heart beats rapidly ) ? with three answer options as often , sometimes , and never / rarely . an overall total high score on both questions reflects a high level of physical activities . prior research showed an acceptable criterion validity and also reliability scores ( 0.74 and 0.80 ) ; our data on a subsample of participants ( 25 parents ) also showed a good test - retest reliability scores ( 0.79 and 0.81 ) . according to the activity guidelines , children were supposed to participate in exercise activities at least 60 minutes at a moderate to vigorous intensity on most ( five ) days of the week in order to be considered active ( glteq score = 5 5 days 4 ( 60/15 min ) 100 ) or they are considered inactive if their activity score was lower than the minimum recommendations ( glteq score < 100 ) . to assess the barriers to pa , parents were asked to specify the most frequent barriers to participation in leisure physical activity of their child . a list of barriers was provided including expense , lack of resources / opportunities , time limitation , motivation , and fear of injury and also an open item as other barriers . furthermore , parents were asked to complete a daily activity logbook for children , which was designed to obtain information about each child on their hourly engagement during a typical day . parents rated how much time , in average , children spent on solitary , social , home teaching , tv , feeding , school , and also sleep on daily basis . among those daily activities , social play ( i.e. , time spent on playing with peers ) and this questionnaire showed a good content validity and a satisfactory test - retest reliability ( intraclass correlation = 0.69 , p < 0.001 ) . moreover , we administered the autism treatment evaluation checklist ( atec ) to parents / caregivers to monitor the severity of autism symptoms . atec is a valid and helpful instrument to evaluate the severity asd symptoms in children with asd [ 25 , 26 ] . the checklist has four subscales , including language , sociability , sensory / cognitive awareness , health / physical / behavior , and the total score ( overall severity ) . finally , background demographic information of the participants was reviewed by the first author interviewing the families and using their medical profiles . in the next step , parental demographic variables including household structure ( single parent versus two parents ' family ) , household income , and the highest level of education obtained by parents were also recorded . parental education was examined by one question that asked participants to report the highest degree completed by either the father or the mother . for the current study , three education categories were created including low ( diploma and below ) , medium ( bachelor and below ) , and high ( master and above ) . participants were also asked to report total household income . for use in this paper , household income was categorized into four groups using the poverty income ratio ( based on poverty threshold from national central bank report ) . these categories ranged from below the poverty limit to incomes greater than three times the poverty limit . independent t - test was conducted to evaluate the statistical significance for the observed differences across gender ( boys and girls ) in outcome measurements ( physical activity score or daily activity measures ) . furthermore , in order to compare the time spent on solitary play and social play in total studied population , a paired t - test analysis was conducted . association between leisure scores or daily activities time and parental and child factors were assessed by correlation analysis . analyses were performed using the statistical package for the social sciences ( spss ) software version 17 for windows ( spss inc . , chicago , il , usa ) . however , it was very striking to find that only 10 ( 12% ) of children with asd were active and 73 ( 88% ) were inactive based upon activity guidelines and activity score measured by the glteq . addressing the frequency of activity participation , results showed that only 6% of children with asd often participated in physical activities , whereas 85.5% of them had never / rarely participated and 8.5% were sometimes involved in physical activities . furthermore , t - test analysis of glteq composite score showed that boys with asd participated in physical activities ( 58.8 22.1 ) more than girls with asd ( 35.5 14.5 ) ( t = 4.31 , 95% ci : 12.4833.13 , p < 0.001 ) . examining the correlates of children physical activities revealed that expectedly older children were less active than younger children ( r = 0.298 , p = 0.003 ) . there was no association between severity of disorder or parental education level and the activity score , but physical activity participation was positively correlated with the poverty income ratio ( r = 0.31 , p = 0.005 ) . t - test analysis showed that children in single - parent families had significantly lower activity scores than children in two parent 's families ( t = 3.91 , 95% ci : 9.3129.64 , p < 0.001 ) . based on the results obtained from independent t - test , girls were more involved in solitary play compared to boys ( t = 3.626 , 95% ci : 31.01106.22 , p < 0.001 ) . the results of paired t - test showed that children were predominantly more engaged in solitary play compared with social play activities ( t = 9.41 , 95% ci : 65.68100.80 , p < 0.001 ) . correlation analysis between daily activities and symptoms severity scores showed that social play participation was negatively correlated with language impairment ( r = 0.25 , p = 0.016 ) , sensory / cognitive awareness deficit ( r = 0.26 , p = 0.013 ) , and also overall severity score ( r = 0.29 , p = 0.005 ) . finally , parents reported that leading barriers of children adherence to physical activities were expense ( 31.7% ) and lack of resources and opportunities ( 30.1% ) followed by time ( 19.5% ) , motivation ( 17.1% ) , and fear of injury daily physical and play activities have an important role in the psychosocial development of children . in fact an appropriate activity profile prevents them from isolation in adulthood and significantly influences the wellbeing of children [ 17 , 28 ] . nevertheless , there was lack of studies assessing daily activities participation in children with asd and investigating the impact of individual and environmental factors on their physical activity parameters . results from the current study indicates that most of the children with asd do not have adequate physical activity participation since only few of our children met the minimum physical activity criteria . several studies have discovered that individuals with disabilities are more likely to be inactive and due to abundance of impediments they are less likely to participate in activities when they are compared with the general population [ 2932 ] . their findings show that asd and the severity of intellectual impairments place the people with disabilities at a higher risk for sedentariness [ 33 , 34 ] . a number of factors can limit the participation of children with asd in daily physical activities . those mainly include lack of positive experiences in exercises , frequent failures , emotional impairments , and low self - esteem . however , our data showed that such low participation was mostly due to the financial complaints and lack of resources or opportunities as reported by the parents . moreover , there were other factors ( e.g. , time constraints , lack of motivation , and fear of injury ) which can further limit the participation of autistic children in activities . interestingly , data from another developing country revealed similar barriers such as financial complaints , lack of knowledge , and perception of the situation in an asd sample . although there are differences in measurement of barriers across previous studies , almost similar patterns of barriers including time limits and financial constraints were reported as leading barriers to activity participation in children with disabilities particularly asd [ 15 , 36 , 37 ] . indeed this finding is not limited to asd and previous data from individuals with other disabilities revealed that disabled people face a number of barriers to pa participation even more than healthy population . expenditures of the child 's medical care impose a financial burden on families with an autistic child and therefore they require more financial resources . they also have to limit their productive working times in order to take care of their difficult children , which in turn will further challenge the possibility of secure a financial resource expansion . one of the important findings of the present study is that children from low - income families show lower level of pa than children from high - income families . children who grow up in a low - income family are more likely to live a sedentary lifestyle and experience more health problems related to physical inactivity compared to children from higher income families . there are a number of physical and social barriers to physical activity for low - income families including low access to parks and recreational services , traffic conditions and air pollution , lack of relevant alternatives of transportation , and lack of enough social support for physical activity . on the other hand , low - income families are often less able to overcome these barriers . due to financial constraints , there are fewer alternatives available for low - income people ; for example , they are not able to spend on a health club or recreation center membership . it can be expected that the problem is more complicated in families with an asd child . thus , the economically disadvantaged asd families may show a lower preference to participate in physical activities . furthermore , some parents have increasing concerns about their child 's health and possibility of an injury , which can explain their lack of interest toward activity participation of their autistic child . furthermore , they report lack of time and financial resources as the main impediments to participation in activities . our findings provide additional evidence regarding the effect of household structure on the leisure time activity involvement in children with asd . however , it is not clear if other variables such as presence of a sibling may influence the opportunities to engage in social play and daily social activities inside the family environment . expectedly children with asd showed a remarkably low social but high solitary play activity during a typical day . indeed a previous research has shown that the characteristics of asd as social , communicative , and motor impairments increase the likelihood of loneliness and decrease the opportunities for interactions in individuals with asd . previous studies suggested that a lower level of social play activities in addition to autistic character difficulties can have serious developmental and social consequences [ 4345 ] . examining the apparent role of autism symptom severity , we observed that the children with greater deficits ( e.g. , in communication ) had a lower engagement in social play activities . these results are in line with previous studies which indicated that there is an inverse correlation between severity of communication impairment and the level of life participation in individuals with disabilities . in fact , previous studies indicated that individuals with more severe motor or physical impairments or cognitive disabilities are at a higher risk of being excluded from daily activities [ 46 , 47 ] . our findings also indicated that there is a significant age and gender difference in level of physical activity and this is in line with studies of asd and general population . it can be explained that older children have low opportunities to participate in physical and recreation activities . furthermore , age may decrease the children motivation to participate in complex motor or physical activities . we also indicated that gender ( in favor of males ) influences the daily physical and play activities of children . gender differences in asd characteristics revealed that males with asd show more stereotypic and repetitive behaviors while there are more communication deficits in female counterparts . in addition , there is more achievements in motor skills and social competence in boys than girls with asd . one can argue that being a girl is associated with poor outcome in physical activity participation in asd . first , the cross - sectional design prevents an understanding of the exact nature of the daily activity participation , particularly with respect to its determinants . second , while a neurotypical control group was not included , it would be helpful to compare the scores of physical activity participation between neurotypical controls and children with asd . third , the measures solely relied on the parents ' ( or teachers ) self - report information ; thus , recall bias maybe a potential limitation . in conclusion , only a small part of children with asd are physically active according to activity guideline . financial concerns , lack of opportunities , and sociodemographic factors are indicated as major limitations of their physical activities .

autism spectrum disorder ( asd ) indicates several neurodevelopmental impairments which may end in impairments in motor or physical activities . daily physical activity involvement was investigated in a total of 83 children ( 52 boys and 31 girls ) with asd aged 615 years . results indicated that only 10 ( 12% ) of children with asd were physically active . children were predominantly engaged in solitary play rather than social play activities . gender , family income , and household structure were found to be associated with activity scores . financial burden and lack of opportunities were noted as the leading barriers to physical activities . in conclusion , findings indicated a low rate of physical activity participation in children with asd that is closely associated with sociodemographic variables .

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

PMC3577361

motivation is an innate feature affected by four factors , namely situation ( surroundings and the external stimuli ) , mood ( the organism 's internal state of mind and emotion ) , goal ( behavioral goal , purpose , tendency ) , and tool ( for goal achievement ) . humans acquire the necessary motivation for the fulfillment of their goals , needs , or instinct . as for university students , equipped with motivation , individual students are activated enough to complete an assignment successfully , achieve a goal , or accomplish some extent of mastery in their field , and thus are enabled to learn attain fruitfully and academic success . thus , motivation could be said to account for human behavior and explain why a particular behavior is demonstrated . motivated behavior is one with energy , and is purposeful and ongoing . in educational view educational motivation is viewed as a three - dimensional concept comprising the individual 's set of beliefs about his abilities , intention , and the relevant emotional response needed to display some particular activity . intrinsic motivation creates appeal for a person to act or react in a certain way , while extrinsic motivation accrues as a result of influence from outside sources driving a person to act purposefully . psychologists have emphasized the significance of motivation in learning new skills , strategies , and behavior , and have proposed academic achievement motivation as one of the main factors necessary for reaching a more accurate definition for motivation . academic achievement motivation refers to behavior resulting in learning and ( academic ) achievement . in other words , academic achievement motivation is the learner 's tendency to act in a certain way and evaluate his own performance spontaneously . behavior reflecting academic motivation mostly includes insisting on doing difficult assignments , working hard or making all the efforts to learn , and picking demanding assignments . therefore , academic achievement motivation , or the so - called intrinsic motivation , is a psychological state which accrues when an individual assures himself that he enjoys enough competence and autonomy to learn a particular subject . owing to the effect of academic achievement motivation on students success , in recent decades , psychologists have attempted to investigate and identify the factors involved . findings have shown factors pertaining to personality , family , place of study , and society are influential . as an example , one study showed that personality factors in general and self - esteem and academic achievement motivation in particular affected learning and academic achievement significantly . others have attempted to work on the integration of learning style , personality trait , intellectual capability , and academic achievement as prognostic factors for academic achievement . one study has reported that the most important motivation factors for medical students in choosing their field of study have been personality factors such as social status . in pennsylvania school of medicine , 90% of the students reportedly were satisfied with the educational programs and expressed their willingness to work in academic research centers after their graduation . in another study , dentistry students of different genders have mentioned different reasons for the selection of their field of study . in another study , for instance , male dentistry students were reported to be motivated by income more than females , while the former were more motivated by their family advice . on the other hand , researchers believe that low self - esteem , low educational quality , low family income , faint hope for future , and married life are the factors which diminish academic motivation . also , motivation loss causes pessimism , anxiety , and depression which , in turn , cause lower academic performance . in accordance with these findings , researchers believe that in order to develop motivation , learning is necessary to occur in an appropriate atmosphere , with the desired facility , and relevant to the learner 's needs . the issue has been studied to some extent ; however , little research has been done on academic motivation in the fields of medicine . considering physician 's role in health promotion and regarding the current research vacuum , this study investigated the relationship between academic motivation and academic achievement in medical students in clinical curses . the study in particular focused on academic motivation dimensions such as task , effort , complication , social power , affiliation , social concern , praise , and token . in this cross - sectional correlation study , 422 medical students ( from 4th to final year ) taking clinical courses in school of medicine of isfahan university of medical sciences completed the inventory of school motivation ( ism ) proposed by mc inerney and ali . , there are five choices ranging from strongly disagree to strongly agree ( rated 15 ) . the inventory investigates eight dimensions , namely task ( four items ) , effort ( seven items ) , competition ( six items ) , social power ( six items ) , affiliation ( three items ) , social concern ( five items ) , praise ( five items ) , and token ( seven items ) . for instance , considering competition , a student who had chosen strong agreement ( rated 5 ) for all the six questions was considered to be highly motivated for competition . the reliability of the tool has been calculated by a number of researches[1620 ] and cronbach 's estimates vary from 0.67 to 0.82 ( mean = 0.76 ) . in our study , cronbach 's turned out to be 0.93 when the tool was administered to 60 students . mentioning the confidentiality of the participant 's personal information , students average scores for the first 3 years of their studies ( a period of taking basic science courses ) and their average scores for the second 3 years of their studies ( a period of studying clinical courses ) were used as the criteria for academic achievement . both group and individual administration of the questionnaire were applied by the researcher in educational health care affiliated with isfahan university of medical sciences . students unwilling to participate and the incomplete questionnaires were excluded from the study ( 16.1% ) . data were analyzed using descriptive statistics indexes such as mean , standard deviation , and frequency percentile . analytical tests including pearson correlation coefficient and independent t - test were also applied . in order to investigate the relationship between the components of academic achievement motivation and the two average scores , we used pearson correlation coefficient . to compare the components in terms of gender , independent t - test response rate was 83.9% ( 344 students ) and most of the samples ( 63.4% ) were females . mean age was 24 1.67 years and most samples had an age ranging from 21 to 25 years . mean of the first 3-year average scores was 15.51 1.29 and mean of the second 3-year average scores was 15.81 1.10 . also , the mean and standard deviation for the eight components of academic motivation were as follows : task ( 4.25 0.52 ) , effort ( 3.77 0.67 ) , competition ( 3.35 0.82 ) , social power ( 3.16 0.85 ) , affiliation ( 3.61 0.91 ) , social concern ( 3.88 0.56 ) , praise ( 3.87 0.70 ) , token ( 3.28 0.87 ) , and motivation as a whole ( 3.64 0.48 ) . statistical analysis indicated that there was a direct significant relationship / correlation between total academic motivation and average scores on basic sciences ( r = 0.152 and p = 0.005 ) and average scores on clinical courses ( r = 0.160 and p = 0.003 ) . statistical analysis of the components showed a difference between girls ( n = 218 , mean of sd = 3.70 0.70 ) and boys ( n = 126 , mean of sd = 3.88 0.61 ) regarding the second component , namely effort ( t = 2.379 and p = 0.018 ) . there was also a difference between girls ( mean of sd = 3.25 0.8 ) and boys ( mean of sd = 3.52 0.84 ) regarding competition ( t = 2.94 and p = 0.004 ) . given the importance of academic motivation for academic achievement , the present study , trying to fill the current research gap , was designed to reveal the possible relationship between academic motivation and achievement . thus , the ism was administered to all medical students from 4th to last year of isfahan university of medical sciences . students average scores after finishing all basic courses and also their average scores in clinical courses were considered the criteria for academic achievement . high total motivation scores reflected the students quite high motivation and the task component appeared to gain the highest of the scores . there was also a relationship between motivation components and academic achievement , which was , to some extent , in accordance with other studies indicating a significant relationship between academic achievement and respect , power , praise , ability , competition , social status , goals , interest in education , and satisfying others . in our study , the low relationship between motivation components and academic achievement could be attributed to the students inaccurate reports of their average scores or the inaccuracy of the scores themselves . high scores on task component could be the result of the students greater involvement in cognitive processes and employing more cognitive strategies and supervisions which lead to higher academic achievements . in addition , a genuine belief that the learning content in valuable per se affects self - regulation and self - efficiency positively . provided that competition component , which showed to have a high correlation with the total average scores and basic sciences average scores , results in students more knowledge ability and expertise , it deserves admiration . however , if this motivation is merely for gaining a high score and outperforming others , this should be taken as an alarm by both the educational authorities and the whole society . as members of health teams , medical doctors need to be skilful enough to play their role in the team ; the study also showed male students had a higher motivation regarding competition and effort components . this could be accounted for by boys future responsibilities such as housing provision , life management , family supervision , or job achievements . since the research was conducted on medical students studying clinical courses , generalization of the findings for other academic levels or courses should be done with care . motivational factors affect academic achievement significantly and since medical students success is closely tied to public health , it is suggested that more attention be paid to motivation components by the authorities .

background : despite their ascribed intellectual ability and achieved academic pursuits , medical students academic achievement is influenced by motivation . this study is an endeavor to examine the role of motivation in the academic achievement of medical students.materials and methods : in this cross - sectional correlational study , out of the total 422 medical students , from 4th to final year during the academic year 20072008 , at school of medicine , isfahan university of medical sciences , 344 participated in completion of the inventory of school motivation ( ism ) , comprising 43 items and measuring eight aspects of motivation . the gold standard for academic achievement was their average academic marks at pre - clinical and clinical levels . data were computer analyzed by running a couple of descriptive and analytical tests including pearson correlation and student 's t-student.results:higher motivation scores in areas of competition , effort , social concern , and task were accompanied by higher average marks at pre - clinical as well as clinical levels . however , the latter ones showed greater motivation for social power as compared to the former group . task and competition motivation for boys was higher than for girls.conclusion:in view of our observations , students academic achievement requires coordination and interaction between different aspects of motivation .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC5282917

national external quality assurance programme pakistan ( neqapp ) is a system designed to objectively assess the quality results obtained by medical laboratories in pakistan . the primary aim of this proficiency testing ( pt ) program is to strengthen standards of clinical laboratories in pakistan by providing medical professionals with a comprehensive quality and cost effective external quality assessment ( eqa ) scheme at a national level and to reduce the risk of errors in laboratory results . this will help provide better patient care and quality results of clinical laboratories in pakistan along with fulfilling regulatory and accreditation requirements . erroneous lab results have great impact in delaying appropriate patient care along with increasing cost of diagnosis and management . results of a laboratory are judged against a comparator mean of instrument and method , or a pre - determined result as in the case of culture sensitivity for the microbiology program . the aim of this study is to highlight efforts taken to improve quality of laboratories by inculcating proficiency testing ( pt ) philosophy through neqapp , importance of laboratory pt in medical science and appraise current situation of quality reporting in our laboratories . as such , our study will help all those concerned with medical laboratories in pakistan and encourage a quality reporting culture according to international standards for better patient management . we conducted this cross section study at the department of chemical pathology and endocrinology , armed forces institute of pathology ( afip ) , rawalpindi , pakistan . results from january 2014 december 2015 were evaluated for clinical chemistry , hematology , microbiology and immunoassay programs . frequencies of unsatisfactory results of individual as well as all of the analytes were calculated for different laboratories . there were 88 laboratories enrolled with neqapp in 2011 , which increased to 140 laboratories in 2015 . ninety - six percent of the enrolled laboratories participated in the clinical chemistry programme followed by hematology ( 30% ) , microbiology ( 28% ) and immunoassay ( 14% ) . failure rates of newly enrolled laboratories were higher than those which were participating for the last two years . the percentage of unsatisfactory results of participating laboratories was 19% , 15% and 10% in the years 2014 and 2015 . this included ten parameters from clinical chemistry ( glucose , alkaline aminotransferase , bilirubin , creatinine , cholesterol , albumin , triglyceride , urea , alkaline phosphatase and amylase ) , three from hematology ( hemoglobin , red blood cell count and white blood cell count ) and two from immunoassay ( thyroid stimulating hormone and human chorionic gonadotropin ) . failure rates were highest for alkaline phosphatase ( 35% ) followed by creatinine ( 22% ) and urea ( 20% ) during the two years of the study period . in clinical chemistry , failure rates of the ten analytes for defence laboratories was 19% , 19.5% for public laboratories and 13.5% for private laboratories in the year 2014 . whereas failure rates declined to 8.5% in defence laboratories , 11% in public laboratories and 10.5% in private laboratories in the year 2015 . reasons for unsatisfactory results were evaluated and classified into five main categories : methodological ( 21% ) , clerical ( wrong entry of results or unit ) ( 42% ) , technical ( 20% ) , pt material stability ( 9% ) and random errors ( 8% ) . it has been observed that failure rates were low in laboratories which are supervised by technically qualified professionals in laboratory management and quality control , use of automation , standard methodologies and in those laboratories which are frequently participating in national and international proficiency testing programs for quite some time . good analytical agreement between laboratories is required as patients / clinicians move from one area / hospital to another . irrespective of the setting , i.e. , large reference laboratory with the latest equipment and professional staff or a small laboratory , a laboratory must report proficiency results of adequate quality to meet the stated guidelines . this can only be ensured by participating in external quality assessment ( eqa ) and taking appropriate actions when results do not meet acceptable performance . although once considered a theoretic entity , pt is now a regulatory requirement for laboratory licensing by health authorities . material used for proficiency testing are provided by external agencies , either mandated legislative bodies or voluntary organizations . these materials are used to check the quality performance of a laboratory relative to its peers in terms of standard of performance that is usually expressed as a total variation from a target value for each sample . erroneous proficiency results indicate that the laboratory is incapable of meeting the accepted standard of performance and can ultimately lead to the loss of the laboratory s license to perform that specific test or entire class of tests . external quality control program is usually selected by the laboratory and purchased from an external company . it is an external check of the analytical methods performance in an acceptable manner to produce clinically acceptable patient results within the stated criteria . qc samples provide us with data that represent the accuracy and precision of each method at the level of analyte present in each control . a laboratory must interpret that data in order to make daily decisions about the acceptability of each batch of patient samples , and ongoing decisions about the overall acceptability of method performance . in 1996 , at afip , rawalpindi , participation in the clinical chemistry survey of neqapp was commenced with the aim to expand it to other disciplines of pathology . the pakistan national accreditation council has launched the medical laboratory accreditation scheme in pakistan in compliance with international organization of standardization ( iso ) 15189 ( international standard for medical laboratories ) . it was at that time that neqapp program was expanded to hematology , microbiology and immunoassay to support the scientific and medical communities in pakistan . since then it has gained immense popularity and at present more than 140 clinical laboratories of pakistan including 40 defence hospital laboratories , 41 teaching institution laboratories ( medical colleges and postgraduate medical institutes ) , 59 public and private sector laboratories are voluntarily participating in this program ( fig . 1 ) . neqapp is a user friendly , cost effective program with immediate availability of customer service and technical support . laboratories have an option of submitting their test results electronically ( neqapp online ) or on paper ( results forms ) via post . acceptable performance in this national program is identified as falling within 2 standard deviation index ( sdi ) from ones comparator mean . this program should be adopted as mandatory requirement by regulatory authority for running and scrutinizing quality assurance of medical laboratories in pakistan . quality reporting can be ensured by selecting appropriate instrument with test method validation at instrument installment , improving transport and storage of reagents / calibrator / controls , scheduled equipment maintenance and ensuring quality checks . there is improvement in quality of pt results due to improvement in pre - analytic steps , laboratory automation , maintaining internal quality control and following standardize protocols for laboratory reporting . there are certain problems , as follows , which need to be highlighted for running the proficiency program effectively . laboratories not submitting results on due date , incomplete methodology details , failing to participate in all four quarters of a cycle , submitting results in units other than prescribed by neqapp , less use of electronic result submission and failing to update laboratory corresponding details especially in case of public laboratories . neqapp program plays a pivotal role in improving the quality of laboratory services in pakistan . in the last three years no health care facility can be totally self - reliant in terms of maintaining quality and this gap can be filled by participating in an external quality program , giving the true picture of level of quality reporting .

objectivethe objective of this study was to highlight current status and importance of national external quality assurance program pakistan ( neqapp).study design : cross sectional studyplace and duration of studydepartment of chemical pathology and endocrinology , armed forces institute of pathology ( afip ) from august to october 2015.methodsthe study data was extracted from electronic neqapp database . results from 2014 - 2015 were evaluated for clinical chemistry , hematology , microbiology , and immunoassay programs . frequencies of unsatisfactory results of individual analytes as well as of all the participating laboratories were calculated.resultsfailure rate of newly enrolled laboratories were more as compared to those which were participating for the last two years . the percentages of unsatisfactory results of all laboratories were 19% and 15% in 2014 and 2015 , respectively . fifteen analytes were selected according to their increasing percentage of participation . failure rate was highest for alkaline phosphatase ( 35% ) followed by creatinine ( 22% ) and urea ( 20% ) in two years analysis . performance of laboratories in each quarter was evaluated depending upon number of analytes in which they fail to pass . the major failures were due to clerical and technical errors as determined during data compilation of results.conclusionthere is an increase in trend of participating in neqapp by health care laboratories which is a step towards laboratory quality management system in pakistan . nonetheless , there is a need for improving quality of laboratory results .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

PMC4095914

the question of how to effectively activate o2 for reaction is of great interest in biochemistry and organic and inorganic chemistry ( including materials science ) because the products and byproducts of o2 reactions can be variable and also because o2 often encounters kinetic barriers to the reaction , proton transfers , both sequential and concerted , are a major theme in the reaction chemistry of redox - active systems , including many biologically important metalloenzymes . proton - pumping redox enzymes are functionally essential for energy storage but diverse in their mechanisms and structures , comparing cytochrome c oxidases ( ccos , complex iv ) to cytochrome bc1 centers ( complex iii ) , for example . two main classes of mechanisms have been proposed for proton pumping in ccos : ( 1 ) a direct mechanism where the chemistry of o2 reduction is directly coupled to proton pumping within the o2 binding dinuclear complex ( dnc ) containing an fe - heme and cu sites ( fe - a3 and cub ) ; ( 2 ) an indirect mechanism where conformational or electrostatic interactions with the surrounding protein , in particular with the neighboring redox donor centers in the enzyme , are required . our current and past work has centered on potential direct mechanisms for both fundamental and practical reasons . the driving force for proton pumping resides in the metal redox and o2 binding / reduction chemistry of the dnc , so this is clearly a good place to start . by analyzing what the dnc can do with the other relevant sites as simple inputs , we want to identify the distinctive positive features as well as the potential limitations of our models . our viewpoint is modular , which simplifies analysis where separability is valid , and reveals its limitations , where predictions prove questionable or false . our work is an illustration of the theme that while the map is not the territory , to understand the territory , it is best to build and improve the map . following up on our earlier work , we explore how different metal and tyrosine redox and electronic states interact with molecular o2 binding to promote proton transfer and proton pumping in the dnc of cco . the theoretical / computational tools are spin - polarized broken - symmetry density functional theory ( bs - dft ) in conjunction with continuum solvation dielectric modeling for the surrounding protein solvent environment . mitochondrial and bacterial respiration are the major sources of functional energy via adenosine triphosphate ( atp ) synthesis in aerobic organisms . ccos are the terminal electron acceptors in the respiratory chain of mitochondria and many bacteria . essentially , o2 linked to the metal center is the final sink for electrons from the earlier electron - transfer complexes i iii and mobile carriers [ ubiquinone ( q ) and cytochrome c]1 there are a number of critical functional requirements for the operation of eq 1 within the dinuclear heme iron copper complex of cco . electrons and protons must be taken up at various steps of the catalytic cycle , o2 must be efficiently bound and activated for reaction without releasing oxygen - radical intermediates like superoxide anion o2 or other toxic products [ for example , hydrogen peroxide ( h2o2 ) ] , and protons must be transported to and react with metal - bound o2 and/or its subsequent intermediates . equation 1 summarizes the relevant net chemical reaction but not the mechanism or the essential coupling to proton pumping . proton pumps like cco are enzymes coupled to energy transduction machines . beyond the chemistry of eq 1 , the free energy of this reaction in cco has to be effectively utilized for proton pumping from the inside ( in ) to the outside ( out ) of the membrane against the electrochemical gradient ( figure 1 ) . figure 1 is a modified and annotated version of figure 1 in ref ( 2 ) representing the structure of the bacterial b - type enzyme from thermus thermophilus ( tt ) . cu reaction center ( dnc cu is cub and the corresponding fe is fe - a3 ) are shown . the dinuclear cu cu center called cua is shown by two small blue dots . heme - b , which is the immediate donor of electrons to the dnc fe cu center , is not shown ; heme - b lies behind heme - a3 in this orientation . many spectroscopic and kinetic studies show that electrons are donated from cua to heme - b fe and then to the dnc heme - a3 . further , o2 binding is ordered compared to electron ( e ) transfer , so that typically all four metal redox centers are 1e - reduced before o2 binds to the dnc reaction site between fe - a3 and cub . schematic ( left ) of cytochrome ba3 with noted functionalities and cartoon of the same ( right ) showing the o2 channel inside the lipid bilayer , the reaction chamber between cu and fe , and the electron- and proton - transfer pipes . n or negative side ( figure 1 , bottom , cytoplasm ) and the outside is the p or positive side of the membrane ( figure 1 , top , periplasm ) corresponding to the polarity of the membrane electrochemical gradient . the detailed mechanism of proton pumping is far from evident , but the overall reaction stoichiometry is2over a full reaction cycle . some language is useful for bookkeeping : of the ( 4 + n)hin entering the reaction chamber , 4h , called scalar protons , react with o2 to produce 2h2o , while the other nh , called vector protons , are pumped across the membrane . measurements of n have yielded values near n = 4 for a - type ccos , but lower values near n = 2 have been measured for b- and c - type ccos . ( for example , extrapolating the charge - transfer measurements for the oxidative half of the reaction cycle to the entire oxidative plus reductive reaction cycle for b - type tt ba3 yields n = 2.5 approximately . ) finding accurate experimental values for n is difficult , and significant uncertainties remain . in common with respiratory chain complexes i and iii and ordered sequentially by increasing redox potentials , complex iv links e transfer down the electron - transport chain to proton pumping across the inner membrane of the mitochondria and , analogously , across the plasma membrane in most aerobic bacteria . the resultant proton electrochemical gradient , or proton motive force , is utilized for atp synthesis from adenosine diphosphatase ( adp ) plus inorganic phosphate by complex v ( atp synthase ) , which also resides within the same membrane . the proton electrochemical gradient has a higher positive potential on the outside ( p ) of the membrane and a lower negative potential on the inside ( n ) of the membrane ( matrix side in mitochondria ) . is largely funneled through the atp synthase active site to generate atp from adp+pi . as expected from energy conservation , continued proton pumping is required to maintain the membrane potential against the leak and to provide the driving force for atp synthesis . alternatively , if the electrochemical gradient is depleted , proton pumping is required to restore the gradient . the electrochemical potential is the sum of an electrical potential ( voltage ) difference , , and a ph difference , ph , across the membrane3where f is the faraday constant . while the reaction inputs and outputs of the respiratory complexes are understood in broad stroke , in no case is the actual coupled electron redox / proton pumping mechanism understood in even reasonable detail . many features are known from spectroscopy and kinetics about the chemical mechanism of o2 reduction and subsequent mechanistic steps of the reaction cycle , but there is no current resolution about whether or how proton pumping is directly coupled to the chemistry of o2 reduction at the fe cu dnc or indirectly coupled through conformational or electrostatic changes in the surrounding protein , particularly involving the nearby redox centers ( the cua homodinuclear and mononuclear fe - heme donor centers ) . it is possible that the different enzyme classes a1 versus b could have significant differences in their mechanisms ( see the later discussion ) , including whether a direct or indirect mechanism ( or some mixture ) applies . x - ray crystallographic structures are now available for mitochondrial [ bovine heart ( mt ) ] and several bacterial ccos , including paracoccus denitrificans ( pd ) , rhodobacter spheroides ( rs ; all a1 type ) , and t. thermophilus ( tt ; b type ) the mt structure is by far the most complex , with 13 subunits ( 3 encoded by mitochondria and 10 by the nucleus ) . however , the three mitochondrial subunits ( i iii ) of mt closely resemble the corresponding subunits in the bacterial enzymes pd and rs , and all are of class aa3 . the tt enzyme is of a different class ( namely , ba3 ) but still has many similarities in sequence , structure , and mechanism to the other ccos . the bacterial enzymes are easier to work with than the more complex mt protein and have , therefore , been subjected to studies using systematic mutagenesis of different sites . our main focus will be on analysis of the tt enzyme , for which fee , stout , and co - workers have obtained high - resolution x - ray structures , including several different mutants , and measured kinetic spectroscopic parameters of many intermediates over the redox and catalytic cycle . kinetic analysis is enhanced in ba3-type enzymes including tt because the absorption spectrum of heme - b is well - separated from that of heme - a3 in contrast to aa3 ( a type ) enzymes , where the heme - a and -a3 bands overlap . also , importantly , the tt enzyme has a single input path ( kb path ) for protons into the dinuclear fe cu center , which simplifies analysis of the proton pathways compared to two paths for a1-type enzymes . while the kb path occupies a structurally analogous region compared to the k path in a1 enzymes , the kb path does not contain lysine , in contrast to the k path in a1 . the biological e - transfer sequence proceeds in one electron ( 1e ) steps from cytochrome c to the dinuclear mixed - valence cua site in subunit ii ( having a 2cu center in the oxidized state and 2cu in the reduced ) , to a mononuclear fe - heme redox center in subunit i ( which in tt is heme - b but in pd , rs , and mt is heme - a ) , and finally to the dinuclear heme - a3fe cub reaction center , which is also in subunit i and is where o2 binds . the proton entry pathway is simpler in the tt ba3 enzyme , having a single entry pathway called the kb path , compared to two potential entry pathways , called the k and d paths , in the class aa3 enzymes . o2 enters via a well - defined hydrophobic pocket near the dinuclear fe - a3-cub site . figure 1 ( left ) shows an overall structural model of cco from tt with the inputs and outputs superposed , and figure 1 ( right ) is a cartoon of the reaction chamber with reactants and products . in tt ba3 , the subunit i residue ile235 is present ( see figure 1 ) in place of a glutamate residue present in a1-type enzymes , so that the second proton - transfer pathway ( the d or q proton - transfer pathways seen in a1-type enzymes ) is absent in b - type enzymes . this also eliminates the corresponding path to the proton loading site as proposed in a - type enzymes . the significant implication is that the path into the dnc is common to both the scalar and vector protons . any subsequent bifurcation of proton flow between the scalar and vector pathways occurs at or after the entry point into the dnc . also , disruptions in o2 binding and electron- or proton - transfer reactions at cco can lead to an excess of reactive oxygen species including hydroxyl and superoxide radicals . these oxygen species can arise directly at cco or can be induced earlier in the electron - transport chain , for example at complexes i and iii , because of a blockage in electron or proton transport at cco . finally , disruptions in the normal proton - transport pathways may also have damaging effects on the membrane potential and acidity in the matrix or periplasmic space . understanding the structures , mechanisms , and functions of mitochondrial cco is therefore important for better analysis of genetic and metabolic diseases and cancer and is also relevant to pathologies in aging . mitochondrial dna is intrinsically subject to elevated mutation frequency possibly because of its proximity to leaky electron - transport chains that produce reactive oxygen species . the close functional and mechanistic relationship between mitochondrial cco and various bacterial ccos allows for the development of an overview of the catalytic proton - pumping cycle , while variations in the structures and energetics / kinetics can provide insight into the interactions between different redox centers and proton sources . the a - type enzymes in pd and rs reside in proteobacteria , some of which were probably ancestors of eukaryotic mitochondria . b - type enzymes , while simpler in some ways , also show significant variations in different species / genera . further , mutagenesis studies in bacteria provide a good way of examining blockage and inhibition of proton and electron transfer and o2 binding . there have also been very significant accomplishments in making synthetic analogues of the dnc , and testing the reaction , physical , and spectroscopic properties of these systems . electron flow in both a1- and b - type ccos has been studied extensively . electrons transfer from cytochrome c to cua , then on to heme - a ( or heme - b in a1 and b types , respectively ) , and from there to fe - a3cub . after o2 binding to the dnc , a number of oxygen - derived intermediates are formed . a good deal is known about the timing of o2 binding and e transfer by binding co to heme - a3 and then introducing o2 , followed by photolyzing the co in the fully reduced or half - reduced ( mixed - valent ) forms of the bovine and related bacterial enzymes . because of their considerable structural and functional similarities , a- and b - type enzymes probably have a common mechanism of o2 reduction . using optical spectroscopy to monitor transient kinetics , siletsky et al . and szundi et al . observed four intermediates for reduction and o2 binding and reactions in tt cytochrome ba3 at the dnc , as shown in figure 2 . to obtain proper timings for kinetics , the reaction of the fully reduced enzyme is started by photolysis of the reduced fe - a3co adduct of the enzyme in the presence of o2 , and then changes in a wide array of properties are followed from the nanosecond to second time scale . here the fully reduced enzyme includes the fully reduced state ( r ) of the dnc and the reduced states of heme - b and cua . concentration of o2 for r a is 90 m for 9 s , from szundi et al . compounds a , pi ( where pi is likely composed of both pm and pr ) , and o in tt ba3 are similar to their analogues in a1-type enzymes . more broadly , the optical absorption difference spectra of heme - a3 do not clearly report on the oxidation state ( or related chemical / electronic features ) of either cub or the unusual tyrosine called yoh . recently , szundi et al . found that the early oxidative steps of the reaction become unreliable with the co photodissociation method because co can then bind to cub , and this inhibits o2 binding to fe - a3 and cub . instead of using co bound to the reduced state r , they instead utilized a photolabile o2 carrier complex to deliver o2 to the dnc . the kinetics are then clarified by examining different concentrations of o2 delivered to the dnc . lifetimes ( = inverse of rate constants r ) from this study are assembled in figure 2 . these lifetimes differ from those we previously presented based on siletsky s kinetic studies . starting from state r , o2 binding occurs very rapidly ( even at the low effective [ o2 ] = 90 m ) and so does the following formation of ferryloxo states , spectroscopic state pi . state pi is a combination of electronic / protonation states pm and pr , and because the heme - b fe is also reduced , the very fast e transfer to reduce the tyrosine radical is not detectable in these experiments . ( the state pm is detected in separate experiments , where the o2 reaction is started from the mixed - valence state , with the dnc being in state r , while the heme - b and cua sites are oxidized . then the cycle stops at pm because of a lack of reducing electrons . ) the cycle depicted in figure 2 omits the next spectroscopically observed intermediate , pii , which corresponds to the timing for e transfer from reduced cua to oxidized heme - b fe , which was the redox center oxidized in the pm pr transition . after the heme - b fe is reduced , it is ready to deliver the final electron in the transition pr o , which is comparatively slow . end with state o. recent pulse radiolysis experiments have shown a comparatively slow 1e - transfer rate from state o ( reducing agent heme - b fe ) to heme - a3 fe ( iii ii ) , lifetime = 4800 s , compared to faster e transfer from cua to heme - b , = 260 s in tt ba3 . therefore , both pr o and o r have comparatively slow e transfers . we have argued in our published work that e transfer from heme - b ( reduced ) to the dnc may be determined by the nature of the oxidant center present on heme - a3 , which is likely to involve both reorganization energy terms and the driving force ( redox potentials ) for different reaction steps . for future reference , we comment briefly on the issue of the electrochemical potential ( load versus no or low load ) in kinetic studies and proton - pumping experiments . when experiments are conducted in detergents and/or buffers , there is no net directionality ( vectorial character ) to the orientation of the cco proteins in the media , and so there is no net electrochemical potential . for kinetic studies , these types of conditions are often used , and they lead to faster kinetics than those in the presence of an electrochemical potential ( experiments under load ) . even x - ray crystallographic experiments done in the lipidic cubic phase have no net directionality ( see tiefenbrunn et al . ) because the individual cco proteins are aligned antiparallel in the lipidic cubic phase . by contrast , experiments designed to measure the proton - pumping stoichiometry or net charge ( q ; e , h ) transfer across the membrane ( usually vesicle experiments ) are set up so that the cco proteins have specific directional alignment with respect to the inside versus outside of the vesicles . proton - pumping stoichiometry ( vector protons ) is typically determined by running a full or partial cycle and collapsing the electrical potential difference across the vesicle using an ionophore , which does not conduct protons but does conduct some other charged particles . ( for example , valinomycin allows countertransport of k ions when protons are pumped in the cco reaction cycle . ) in that case , ph is measured in the corresponding partial cycle . to measure full charge transfer , corresponding to electrons , scalar protons , and vector protons driven against the existing gradient , measurements of the electrical potential change over a partial cycle are obtained . both for proton pumping and net charge transfer , the standing electrochemical potential ( and its electrical component ) ( usually , the electrical term dominates eq 3 under physiological conditions in mitochodria and analogously in aerobic bacteria . so , in practice , most proton - pumping and charge - stoichiometry experiments are under low load . our dft calculations for the reaction cycle are performed under idealized ph gradient conditions , where we assume that each pumping of a vector proton across the dnc corresponds to transfer from a neutral ph = 7 cytoplasm ( n side ) to an acidic ph = 3 periplasm ( p side ) , and there is no electrical potential gradient , as in the vesicle experiments with an ionophore described above . this makes bookkeeping for the cost of proton pumping easy because only the vector protons have a cost , which is accounted for as the proton exits from the dnc . by contrast , a standing electrical potential across the membrane will lead to a pump cost for the vector protons and to a cost for charge transfer for the electrons and scalar protons because these combine in the reaction chamber of the dnc . the energy accounting here is more complex , and we will defer it to future work . ( for a simple analysis , see siletsky et al . ) however , there is one general observation that is very useful . if the same energy cost over an entire cycle ( say for pumping four vector protons from ph = 7 to 3 ) is instead distributed over both the vector proton transfers and the scalar proton plus electron transfers as occurs for an electrical potential difference , rather than a ph difference , the charge - transfer cost across the dnc over individual steps is considerably less ( roughly /2 or less because there are q = 8 charges transported across the membrane versus n = 4 vector protons ) . in our published work ( 2008 ) , we have introduced the first complete catalytic reaction cycle wheel for the cytochrome ba3 dnc , based on a detailed chemical model supported by large - scale dft calculations . our work is anchored in several known features of the experimental reaction scheme but goes well beyond what is presently established . very recently , we have further refined the dft model by examining alternative tautomeric and protonation states . we emphasize some important general points starting from figure 2.(1 ) electron and proton delivery . the points of electron entry into the cycle as well as o2 binding are well established , but only four protons are accounted for within this scheme . these are the scalar protons as in eq 1 , and the vector protons are not included ( see eq 2 ) . on the basis of eq 2 , the optimal proton - pumping ratio is 4h/4e , equivalent to 1h/1e . typically , experimental proton - pumping ratios are near 1 , but early published measurements with ba3 yielded values of about 0.40.5 . in any event , a pumping ratio of 1 requires a proton uptake ratio for hin/1e of ( 4 + n)/4 = 2 . moreover , under physiological conditions ( and in some experimental setups ) , the pumped protons must be moved against the electrochemical gradient at a cost in g , using the chemical driving force available from the redox / protonation reactions with o2 . there are critical and distinctive chemical questions raised by the catalytic reaction cycle and the structure / composition of the dnc . cu bridging peroxo intermediate is likely to form subsequent to the initial oxy complex ( a ) , which has been identified , but the peroxo intermediate(s ) has not been spectroscopically identified . the peroxo complex is likely to be very short - lived but is clearly essential to the energetics of o o bond cleavage and for avoidance of a substantial overpotential . both the energetics and properties of these intermediates are of great interest and are connected to binding modes and protonation states . ( 2b ) there is a unique tyrosine ( yoh in figure 2 ) that is formed from a covalent bond between the tyrosine phenol and a histidine imidazole ring ( residues y237 and h233 in tt ba3 ) . what is the physical mechanism connecting the redox and chemical bonding cycle to the proton pumping ? the lack of mobile proton carriers within cco precludes a redox loop mechanism , but cco pumps protons , nonetheless . we have already proposed a proton exit channel into the watery surface between subunits i and ii ( see figures 1 and 3 ) . specifically , this involves deprotonation of h376 ( from the positive imidazolium form ) . as an alternative , deprotonation may instead involve a nearby bound water ( probably a hydronium ion ) , which also looks reasonable . our current working hypothesis is that the dnc complex itself and the kb path conducting protons into the dnc are the major factors driving proton pumping . we also think that the redox potentials and timing of e transfer from heme - b fe to the dnc play a role , as will be described later . the central polar plot of the running sum , g , shows the energetic course of catalysis as computed at the level of pw91 . a combination of electrostatic and large - scale dft calculations with associated energy analysis has been used to examine the proton - pumping mechanism in class a1 ( aa3-type ) enzymes . in a number of papers , blomberg and siegbahn have proposed that 1e transfer from the cua center to the mononuclear heme - a drives protonation at a pump - loading site ( pls ) close to a propionate in heme - a3 , as well as protonation of the glutamate ( glu286 in bacterial rs and a homologous glutamate in mitochondria ) . the glu286 proton then activates proton exit from the pls after 1e transfer from heme - a to the dnc fe site in heme - a3 , with a coupled chemical proton transfer close to the reduced fe - a3 site ( the 1e - transfer step modeled is o e , the first 1e transfer in the o r 2e reduction ) . presumably , at other steps of the catalytic reduction o2 binding reaction cycle , some chemical protons originate from the k channel . in any event , in class b ( ba3-type ) enzymes , there is no d - type proton - transfer pathway , and in tt ba3 , there is an isoleucine ( ile235 ) in a position homologous to glu286 ( see figure 3 in ref ( 35 ) ) . nonetheless , ba3 enzymes do pump protons , although probably at lower efficiency than aa3.(4 ) proton entry and flow . therefore , we will focus here on the catalytic reaction cycle , including proton flow into the dnc from the kb path and particularly within the dnc itself . the vector and scalar protons almost certainly take different paths through the dnc . the currently available high - resolution x - ray structures of tt cytochrome ba3 from fee , stout and collaborators show a much richer array of waters bound to the dnc than that seen in earlier x - ray structures at lower resolution . ( a ) in our earlier paper ( 2008 ) , we constructed the entire catalytic reaction wheel for the cytochrome ba3 dnc , employing large - scale dft calculations . ( b ) on the basis of subsequent work , we have found an improved energy path by modifying the sequence of proton uptake and proton - transfer events and examining alternative proton tautomeric states . these two sets of models each contain 183189 atoms and are called s1 and s2 , for the original and for the newer model encompassing different tautomers for several states . both models are based on the hunsicker - wang and fee x - ray structure of ba3 . wang ( pw91 ) exchange - correlation potential for geometry optimization in a dielectric medium with dielectric constant = 18.5 for the surrounding environment . then single - point pw91 and b3lyp * calculations are compared over the reaction path . ( c ) for selected parts of the catalytic reaction path , we have now introduced a larger model ( l ) , incorporating additional waters and residues and with greater flexibility in the electronic states described . the larger model is built based on the high - resolution ( 1.8 ) x - ray structure of tiefenbrunn et al . in the lipidic cubic phase , which has a higher density of water in the protein interior . as discussed in a recent paper , the observed x - ray structure contains a bound dioxygen species that is probably hydroperoxide . as in the hunsicker - wang and fee ( 2.3 resolution ) x - ray structure , the dnc in the protein is probably radiolytically reduced , but within dft , we can control the oxidation state and the types of bound ligands that are introduced in the calculations . because of the increased cluster size , we found that the calculations could be performed with fewer geometric constraints . in particular , only the link atoms at the outside boundary of the quantum cluster were constrained to their positions in the tiefenbrunn x - ray structure , as specified in ref . the dielectric model is the same as models s1 and s2 , and all use the cosmo solvation model . the catalytic wheel diagram for the dnc from our new model s2 is presented in figure 3 . from geometry optimization and energy evaluation of 30 different tautomeric / net protonation states ( defined as model sall ) for the 14-step cycle , compared to model s1 , if a different tautomer is chosen in s2 , this is indicated by a subscript on the state number , for example , 72 versus 7 . where a proton has been added , this is indicated by an h , so ; for example , 2h compared to 2 and 8h2 compared to 8 indicate both a tautomeric shift and an additional proton added . figure 3 shows a detailed map of the catalytic reaction cycle for model s2 . we have augmented the figure by labeling intermediates o , r , a , pm , and pr , which have all been observed spectroscopically . all of these intermediates have approximate counterparts in figure 2 , and the theoretical scheme is supported by detailed dft calculations . most of the spectroscopic intermediates correspond to more than one detailed intermediate in the 14-state reaction cycle . for example , states 13 , 14 , and 1 correspond to experimental intermediate o ; all of these states are fe cu species , but they differ with respect to protonation state(s ) or h2o binding . states not seen spectroscopically , which are testable by energetic calculations and potentially by spectroscopic measurements when these ( or related states ) are isolated . structures 3 , 6 , 102 , and 13 are detailed 2d maps of the dinuclear fe - a3cub center , while the other states are represented by simple chemical formulas . a full 3d picture of the fe(o o)cu bridging peroxo state , intermediate 5 , is shown in figure 4 . by comparison with figure 3 , the coordination , covalent , and hydrogen bonds are quite clear in the 2d maps , but the 3d links are distorted ; for example , the heme proprionate side chains are attached to the fe - heme edge . active site structure of compound 5 of ba3 . left : smaller model used in the 2008 calculations . right : larger model used in recent calculations , which is taken from the dnc of the high - resolution ( 1.8 ) x - ray crystal structures ( pdb entry : 3s8 g ) of ba3 cco from tt . for ease of visualization , these two models are not on the same scale . the new 14-step cycle differs from the previous one in ( 1 ) having protons enter the reaction path earlier at two steps , 1 2h and 7 8h2 , ( 2 ) retaining water in state 112 compared to state 11 , and ( 3 ) utilizing different proton tautomers , for all of the states where there are subscripts in figure 3 . ( the original tautomers from model s1 are not subscripted . ) by convention , tautomers from model s1 are each tautomer 1 ; for example , 6 = 61 . in constructing the intermediates in figure 3 the one exception is state 6 , which is critical to the peroxide cleavage reaction . in our model s2 , this is 6 kcal mol higher ( in pw91 ) than the lowest tautomer , 62 , where the special tyrosine ( y237 ) is protonated in place of the peroxide . as we will show later the outcome of replacing our original reaction path ( model s1 ) with one constructed from the lowest - energy tautomers ( also alternate protonation states ) is shown in figure 5 , comparing parts a and b. the effect is striking . these changes to the previous mechanism result in a substantially smoother catalytic cycle . the largest positive free - energy difference g , accounting for successive uphill steps , is lowered to about 20 kcal mol for pw91 in model s2 ( 31 kcal mol in b3lyp * ) , while in the reaction pathway of model s1 for both pw91 and b3lyp * , this value is close to 30 kcal mol . it is easy to see that both exchange - correlation potentials run in a similar manner for energies over the reaction cycle and that figure 5b shows a more reasonable cycle for both pw91 and b3lyp*. the g values for individual steps are considerably less in all cases ( about 15 kcal mol maximum ) . accounting for transition states using standard transition state theory will also give barriers larger than those from g steps between intermediates , while concerted processes and/or proton tunneling may well lower the effective barriers . plots of g ( kcal mol ) along the trajectory from 1 to 14 . the top panel ( a ) is from the 2008 mechanism ( from table 4 for energies ) ; the bottom panel ( b ) represents new work presented here . solid black circles represent energies obtained from pw91 calculations , while reddish diamonds represent energies obtained from b3lyp * calculations . the experimental energies from 1 2 and 2 3 are obtained from refs ( 26 ) and ( 44). one protonation step that promotes the catalytic cycle is to add one additional proton to yo in the transition 1 2 to make the state 2h , so that the added proton transfer from the kb path to y237 is effectively concerted with 2e reduction of fe cu to give fe cu . our computed redox potentials for 1 2h are 0.24 v for pw91 and 0.02 v for b3lyp * , versus 0.275 v ( experimental average for cu cu , + 0.34 v , and fe fe , + 0.21 v , for mitochondria ) or 0.314 v using the experimental redox potential for fe fe for tt ba3 . the corresponding error in g is about 1.63.1 kcal mol for pw91 and about 13.515.5 kcal mol for b3lyp*. in contrast to state 1 2h ( o r ) , the calculated o2 binding affinity ( for standard state [ o2 ] ( aqueous ) = 1 m ) is much poorer for pw91 than b3lyp * , g = 17 and 10 kcal mol , respectively , versus 8.3 kcal mol experimental for tt ba3 from the reaction kinetics study of szundi et al . ( see also figure 2 ; state 2h 3 , r a ) . these few available experimental values are plotted in figure 5b . experimental kinetics work of hallen and nilsson shows that there is no proton uptake when o2 binds ( noting that these are experiments in mitochondria ) , which is consistent with state 2h 3 ( figure 5b ) rather than 2 3 ( figure 5a ) and is correctly represented in pw91 but not b3lyp*. the kb path into the dnc based on tiefenbrunn , fee , and stout s high - resolution x - ray structure is depicted in figure 6 . all residue side chains are constructed based on the x - ray density map , except for one added water ( center purple ) , which completes the hydrogen - bonding network from glu15(ii ) ( from subunit ii ) to y237 of the dnc and involves a3-roh from the lower heme edge . the orientation of figure 6 is 180 rotated about the vertical axis with respect to figures 3 and 4 so that the cu site is toward the left ( above y237 ) and fe is toward the right . figure 6 shows the structure of the kb path for protons into the dnc in tt ba3 . the glu15(ii ) anion is positioned to accept a proton from the solution ( n side ) and return to a hydrogen - bond alignment . then a proton can be readily transferred via a concerted grotthuss - type process ( in common language , a bucket brigade mechanism ) to y237 . usually we expect that the destination for the proton is y237 , but the geranylgeranyl a3-roh group provides an additional entry point into the complex where an alternative pathway is needed . structure of the k path in cytochrome ba3 as deduced from a combination of structural and mutational analyses . the top two residues are actually part of the active site structure per se . copyright 2009 national academy of sciences . to examine the physical / chemical forces that drive proton pumping across the dnc , we now look at the path from state 4 to 5 and include an additional tautomer 52 . compared to state 4 , state 52 has a proton shifted from h282 to h376 to form a state with h376h , h282 , and yoh . the proton - shift distance is much smaller than that directly from state 4 to 5 , and the predicted energy cost is small ( 7 kcal mol for state 4 52 , followed by 5 kcal mol for 52 5 , in place of 2 kcal mol for state 4 5 in pw91 ; in b3lyp * , both energy differences are near 2 kcal mol ) . state 5 is then generated by shifting a proton in 52 from neutral y237 ( yoh ) to h282 to form yo , h282h . in figure 7 , the structures of these states are presented with the dipole moment vectors superimposed in red and the magnitudes labeled [ in debye ( d ) units ] . because dipole moment vectors are oriented from negative to positive , it is clearly seen that the proton shifts strongly reorient the very large dipole moment vector from 4 52 5 , so that state 4 is almost oppositely aligned to state 5 . the corresponding electrostatic potential becomes more positive in the vicinity of h376h , or alternatively in the nearby water cluster ( top of figures 3 and 4 ) . the following proton entering via the kb path ends in state 6 ( see figure 3 ) , but the proton entry state is tautomer 62 ( fe o o cu , yoh ) . state 62 is 6 kcal mol below state 6 in pw91 , so the path 5 62 6 has energy differences ( 8.7 and + 6 kcal mol ) . after proton entry in state 62 , the proton shift to form the hydroperoxide in state 6 produces an increase in the dipole moment magnitude of about 13 d , in a direction similar to that in state 5 , again enhancing the electrostatic potential near h376 . as shown in figures 3 and 5b , the energy difference 6 72 is only mildly endergonic ( 11 kcal mol in pw91 ; pumping against an assumed proton gradient from the ph = 7 n side to the ph = 3 p side , equivalent to 5.5 kcal mol ) . these three structures are overall neutral . as expected on physical grounds , reducing cu cu in the preceding step 1 2 increases the proton affinity of the covalently linked y327h233 pair bonded to cu ( g = 11.4 kcal mol for pw91 for 2 2h , so g is lowered for 14 2h to 11.1 kcal mol compared to that for 14 2 , + 22.5 kcal mol ) . this is also consistent with the experimental observation that o2 binding is not ph - dependent because that proton enters earlier at state 2h . after o2 binding and formation of a peroxo - type bridge and higher fe and cu oxidation states , ( fe , fe ) and cu , we expect that cu would acidify y237h233 ( i.e. , the yoh site ) via through - bond interactions with cu . yo(h282h ) , where g = 8.7 2.7 and 6.5 3.7 kcal mol for pw91 and b3lyp * , respectively ( average , range ) . this average and range is based on our calculations for the tautomers of seven different states ( 5 , 6 , 9 , 10 , 11 , 13 , and 14 ; figure 3 ) , which are all cu species . from figures 5b and 3 , it is evident that the entire pathway from states 3 92 has only low - energy barriers , at least judging from the energy differences between these intermediates , and the overall pathway is highly exergonic , which is consistent with the rapid rate observed from states a pr ( observed as pi ) . the e transfer from heme - b ( fe ) to the tyrosine radical pm pr is clearly very fast , faster than 5 s from experiment ( figure 2 ) , and the earlier true peroxo intermediates are not observed ( figure 3 ) . the calculated g value for 8h2 92 is 23 kcal mol ( corresponding to a redox potential of about + 1.22 v ; we assume that the redox potential of the donor is near that of cytochrome c , about 0.22 v ) . if the proton coming in must go to y237 first , then g for tautomer 91 is more positive , 10.4 kcal mol ( redox potential of about + 0.67 versus she ) . however , this is still quite exergonic . for the next redox step , from state 112 to 12 , g is again very negative , 21 kcal mol ( redox potential of + 1.12 v versus she ) . there are two coupled protonations assumed here , and it is assumed that e transfer from heme - b fe is to cu cu . if state 12 is skipped in favor of state 13 , so that e transfer goes from heme - b fe to heme - a3 fe fe fe directly , the predicted g value is almost the same . the corresponding spectral state changes of the dnc are pr f and pr o for reduction at cu and fe , respectively . as is seen in figure 2 , the transition from pr o is observed to go fairly slowly , and pr f is not directly observed . cu is , however , not a good spectroscopic reporter using optical difference spectroscopy , and spectral analysis is focused on heme - a3 fe . typically , the state f is not seen in the normal reaction cycle of cco but can be generated by binding h2o2 to the reduced state r ( fe and cu ) in the dnc . this shortcircuits the normal catalytic cycle , and the intermediate state f slowly changes back to state o ( resting oxidized ) . in any event , the comparative slowness of the observed pr o transition may well be a consequence of the coupled proton transfers and associated proton rearrangement . ( all calculations above are with pw91 , unless noted otherwise . ) in tables 1 and 2 ( see also figure 4 , right ) , we present structures and energies for a number of states of the large model . we focus on two arcs with olyp potential , one encompassing the true peroxo states ( 4 5 6 ) , table 1 , and the second covering the last 1e reduction ending in the resting state o ( 112 12 13 14 1 ; pr f o ) , table 2 . the size of the quantum cluster is increased to 205207 atoms by adding structural waters observed in the tiefenbrunn x - ray structure and a glycine fragment that hydrogen bonds to internal waters near the reaction site . the larger size of the cluster allows us to model the reaction geometries with less constraints . cu distance is no longer constrained to x - ray geometries as in work by fee et al . also , the olyp potential used typically has a better balance in describing the relative energies of different site spin states for fe and typically yields more accurate spin coupling energies between transition - metal sites compared to pw91 . in work by fee ( 2008 ) , we employed parallel spin on ( ferromagnetic coupling , f ) fe and cu sites for ease of computation and kept the observed experimental site spins over the reaction cycle . by contrast , the newer calculations take account of both f and af ( antiferromagnetic ) alignment of the site spins , and different possible site spins as well ( see table 1 ) , and our recent paper . note that structure 4 of the larger model here does not have the water molecule above his376 . the energies given in the af - coupled states are broken - symmetry state energies . the calculated properties given here include energies ( e , offset by 28000 kcal mol ) , s expectation values , net charges of the cluster ( q ) , the mulliken net spin polarizations for the fe , cu , o1 , and o2 atoms , and the key geometric data ( distances in angstroms ) . f stands for ferromagnetic coupling and af for antiferromagnetic coupling . in the following notes , ls represents low spin and is represents intermediate spin . formally adding two protons to state 112 , one on the h282 side chain and another one on fe = o this is tautomer 1 ( t1 ) of 112_2h with lower energy ( see footnote d for tautomer 2 ) . h282h , y237 ) , and fe ohh2o cu(h376h , h282h , y237 ) . this is the second tautomer ( t2 ) of state 112_2h with higher energy . this is formal state 12 + h2o , where h2o is scalar water on cu . note that in this larger model no water molecule is added on the top of h376 . spin projection corrections are : 1.3 , 0.3 kcal mol for 14_h2o ( af ) and 1_h2o ( af ) . in tables 1 and 2 , simple redox potentials without coupled protonation events can be calculated with the equation4where the absolute standard hydrogen electrode energy value is taken as e(she ) = 4.34 ev = 100.1 kcal mol and eox and ered are the energies of the oxidized and reduced states . e(she ) corresponds to the best measured solvation free energy of a proton gsolv = 264.0 kcal mol , where the ionization threshold energy of a free electron is taken as 0 , with no account of electron entropy ( see the appendix and references therein ) . similarly , gdeprot can be computed from adf energies ; we take a typical e(zpe ) ( zpe = zero - point energy ) for deprotonation of hydroxyl oxygen as about 8.7 kcal mol , and this gives the protonation energy correction for single proton addition as 4.5 kcal mol at ph = 7 ( see the appendix , table a3 ) . this is a dft correction , 2.9 kcal mol plus an empirical correction ( 1.6 kcal mol ) , to the adf energy differences e(ha ) e(a ) , from table 1 . for different tautomers in tables 1 and 2 , energy differences are directly meaningful without correction , assuming only that e(zpe ) cancels for deprotonation / protonation . in table 1 , we include the small spin projection energy corrections from the broken symmetry ( af ) to the proper pure spin singlet states , in parentheses . from table 1 , state 4 , and ref ( 37 ) , 1e reduction of state 4 is very unfavorable , redox potential e = 0.73 v vs she , so cytochrome c ( or heme - b ) can not further reduce the fe(o protonation of state 6 at the tyrosine anion ( y237 ) is very unfavorable ( gprot = 7.2 kcal mol , about 5.3 ph units more acid than ph = 7 ) , which is important because the tyrosine anion is well adapted to become a radical after 1e transfer to fe(ooh)cu , which would be suppressed if the neutral tyrosine ( yoh ) were very stable . the reaction pathway 4 52 5 62 6 has g = + 5.1 , 6.8 , 4.1 , and + 6.5 kcal mol and is similar to the energy profile for the smaller model s2 . the protonation free energy for 5 62 4.1 kcal mol includes an empirical correction ( see the appendix , table a3 ) . structurally , it is clear that the hydroperoxide in state 6 is very important for breaking the o o bond because the activated af state has an o o distance of 1.49 . the calculated f - coupled state 6 has an even longer o o bond , 1.53 , and is only 4.3 kcal mol above af . even more importantly , the cu o2 ( bond to hydroperoxo o atom ) bond is significantly shorter in the f state , 2.35 compared to 2.66 in the af state , and is on the path to a cu o1 bond is also close to that expected for ferryl oxo in both f and af . also , the charged a3 rohoy hydrogen bond is very short , oo = 2.83 in state 5 , and still short in state 6 but much longer for all states with neutral tyr this is entirely consistent with what we saw in ref ( 37 ) , where the x - ray structure of tiefenbrunn was proposed to have a charged a3 roh bond . turning now to the reaction pathway for the final e transfer pr o , it is clear that obtaining fe oh would be facilitated by having a nearby proton , but in the earlier small model ( s2 ; figure 3 ) , we proposed that h2o exited the reaction pocket directly between fe and cu at this stage . instead , we now retain this scalar h2o in states 12_h2o , 13_h2o , 14_h2o , and 1_h2o ( f and o ) . in place of state 112 in figure 3 , we now add 2h to form two tautomeric states ( the first two lines in table 2 ) . we find that the addition of these two protons is very favorable and these two states are related by a proton shift from y237 ( yoh ) to replace the proton shifted from h2o cu to fe = o . our calculations indicate that to some extent an electron may accompany this proton transfer onto the fe , but this state is a resonance mixture of valence isomers . the proton - shifted tautomer 112_2h ( tautomer 1 ; table 2 , line 1 ) is lower in energy than 112_2h ( tautomer 2 ) by about 5 kcal mol , while upon 1e reduction , the cu state is predicted to be lower , by about 4 kcal mol ( 0.17 ev ) . the corresponding redox potential is about 0.57 v ( reduction of cu ) or 0.40 v ( reduction of fe ) . the actual reduction process may not follow the lowest energies because it is favorable to preserve the proton shift to form fe oh once this is formed . further , kinetic measurements of farver , fee , and co - workers implicate total reorganization energies in the range of 1.01.5 ev for e transfer between heme - b and heme - a3 fe . both the driving force and the reorganization energies then are important for the timing of e transfer . the fe ohh2o cu state ( 13_h2o ) will provide a focal point for the last part of the reaction cycle ( second arc ) extending from 112_2h 13_h2o 14_h2o 1_h2o . in figure 8 , we present the energy profile from the last 1e reduction to the last proton pumping as assumed within the model cycle to the final reset to the initial state 1 . we also compare the energetic pathway with one where the scalar h2o on cu has dissociated ( figure 9 ) . however , the presence of h2o on cu facilitates proton transfer between cu h2o and fe = o or fe oh . further , there is recent x - ray structural evidence for two waters or oh h2o between fe and cu in the resting state o. we do find that g(h2o dissociation ) = 9 kcal mol ( olyp ) , favoring dissociation , and less , 0.6 kcal mol ( pw91 ) and + 3.2 kcal mol ( b3lyp * ) , but based on the active site structure , kinetic trapping for the cu - bound h2o is likely . two waters are probably released after 2e reduction of state 1_h2o 2h ( o r ) with a lowering of the free energy . plots of g ( kcal mol ) relative to state 13_h2o along the trajectory from 112_2h ( lower energy tautomer 1 ) to 1_h2o in the cycle with the scalar water on cu . the pw91 and b3lyp * energies were obtained for the smaller model ( see table s2 in the supporting information , si ) . olyp calculations were performed on the larger models ( tables 2 and a3 ) . plots of g ( kcal mol ) relative to state 13 along the trajectory from 112_2h to 1 in the cycle without the scalar water on cu . the pw91 and b3lyp * energies were obtained for the smaller model ( see tables s2 and s3 in the si ) . olyp calculations were performed on the larger models ( tables 3 and a3 ) . the calculated properties given here include energies ( e in kcal mol ) , s expectation values , net charges of the cluster ( q ) , and the mulliken net spin polarizations for fe and cu sites . note that in this larger model no water molecule is added on the top of h376 . returning to the energy profiles in figure 8 , the calculated g values for 1_h2o relative to state 13_h2o are + 14.0 and + 15.6 kcal mol for the large olyp model ( without and with the empirical deprotonation correction ) and + 19.0 and + 18.3 kcal mol for pw91 and b3lyp*. in figure 9 , the corresponding energies are + 13.6 , + 15.2 , + 21.2 , and + 14.8 kcal mol for olyp , olypcorr , pw91 , and b3lyp*. these are not high energy differences for proton pumping considering that a + 5.5 kcal mol pumping cost ( for ph = 7 ph = 3 ) is included . in light of the experimental structural , kinetics , and mutagenesis data for ba3 from tt , the idea that there is a largely common pathway for scalar and vector protons and that this derives from the kb pathway should not be surprising . the first , mentioned earlier ( see figure 6 ) , is at a3-roh and the adjacent tyr237 . this bifurcation point is mainly utilized as needed to bypass tyr237 when it is in a radical state , and a proton needs to be passed on , especially a chemical proton entering the reaction chamber between fe and cu at that stage . the other bifurcation point , which we think is much more common in the reaction path , occurs at his282 . from this location , a proton on his282 ( neutral ) can move onto a cu - bound hydroxo or peroxo ( scalar proton ) , with the different tautomers being close in energy from our dft calculations . alternatively , a vector proton can move onto the exit position ( pls ) and proceed on the exit pathway . specifically , starting with his282 , a proton enters typically from tyr237 to generate his282 neutral . this will weaken the hydrogen bond to threonine ( t302 ) , allowing its side chain to rotate and deposit a proton on his376 or more probably in the nearby water pool ( see figure 4 , large model , right ) . ( this location is near propionate a , as previously proposed for the pls location . ) above his376 in figures 3 and 4 is an asp287arg225 salt bridge . indicate that asp287 is probably important for efficient proton pumping ; these results are still incomplete , and only one mutation has been examined for arg225 , which is so far inconclusive . we propose that the asp287arg225 salt bridge is potentially a simple electrostatic switch or gate , which could be opened by a strongly positive electrostatic potential nearby , for example , by having a proton in the adjacent water pool . arg salt bridge could be paid when the proton enters the water pool or during transit . also , the local electric field of this proton ( and from the surrounding charge distribution ) possibly acts in opposition to the long - range electric field from the membrane potential . on the basis of our earlier work on the catalytic cycle of the cytochrome ba3 dnc , we have found an improved reaction path by modifying the sequence of proton uptake and proton - transfer events and examining alternative tautomeric states . the new 14-step reaction cycle results in a much smoother energy pathway for the catalytic reaction cycle , comparing parts a and b of figure 5 with lower barriers between intermediates . the entire pathway from state 3 92 has low - energy barriers and proceeds downhill . we have also tracked the dft - calculated dipole moments of peroxo and hydroperoxo intermediates after o2 binding ( figure 7 ) . here we find that there are very substantial changes in the magnitude and direction of the dipole moment of the dnc with tautomeric shifts , and a progressive increase in the positive electrostatic potential near his376 ( in addition to that due to protonation of his376 itself ) . we then constructed large model structures and evaluated geometries and energies ( olyp potential ) for two arcs of the cycle , one for true peroxo states 4 5 6 and the other for the last 1e reduction from state 112 1 ( spectroscopic states pr o ) . important results from these dft calculations are presented in figures 8 and 9 and tables 2 and 3 . we have tested the effect of keeping the scalar water bound to cu in state 112 through the remainder of the catalytic cycle both for the large model ( olyp potential ) and for the smaller model ( pw91 and b3lyp * ) potentials . the energy profiles in both figures 8 and 9 are similar , but the water on cu facilitates protonation of fe - oxo . the energy barriers for the final proton - pumping step 13 1 are about 1516 kcal mol for olyp , large model , and somewhat higher for b3lyp * ( 1518 kcal mol for b3lyp * , small model ) and for pw91 ( 1921 kcal mol ) . these results include an assumed 5.5 kcal mol cost for proton pumping at one step . further , we discuss a potential proton exit pathway involving breaking of an asp the exiting proton would be carried or transferred via a water pool from inside to outside . earlier on the vector proton pathway , we have proposed that his282 and thr302 play an important role . there are many important issues still to be understood in the reaction cycle and proton - pumping mechanism of cco both in ba3 class enzymes and more broadly including also aa3 enzymes ( bacterial and mitochondrial ) . we have not yet addressed the kinetics in detail , including transition states , effects of sequence differences , and mutational effects . more needs to be done to integrate the kb path and the other redox centers into the models presented here . the scalar water exit pathway is also essential for proper analysis . in general , however , we expect that the structural and energetic maps of the reaction cycle in the dnc that we have generated will contribute to a better representation of the coupling of electron and proton transfer to proton pumping in cco .

after a summary of the problem of coupling electron and proton transfer to proton pumping in cytochrome c oxidase , we present the results of our earlier and recent density functional theory calculations for the dinuclear fe - a3cub reaction center in this enzyme . a specific catalytic reaction wheel diagram is constructed from the calculations , based on the structures and relative energies of the intermediate states of the reaction cycle . a larger family of tautomers / protonation states is generated compared to our earlier work , and a new lowest - energy pathway is proposed . the entire reaction cycle is calculated for the new smaller model ( about 185190 atoms ) , and two selected arcs of the wheel are chosen for calculations using a larger model ( about 205 atoms ) . we compare the structural and redox energetics and protonation calculations with available experimental data . the reaction cycle map that we have built is positioned for further improvement and testing against experiment .

Introduction Biochemical Background Electron Flow within CcO Reaction Cycle of the DNC in CcO Objectives of the Theoretical/Computational Cycle Models and Methods: Catalytic Wheel Catalytic Reaction Cycle Results and Discussion Conclusions

PMC4845295

acute kidney injury ( aki ) occurs frequently after cardiac catheterization or percutaneous coronary intervention ( pci)1 with more than 5fold variability in incidence across hospitals.2 patients developing aki have an increased risk of short and longterm mortality3 , 4 and progression of renal disease.5 , 6 a number of preprocedural and periprocedural protocols have been developed to minimize the occurrence of aki in these patients.2 , 7 , 8 , 9 current risk models have been developed largely identifying patient risk factors at the time of the pci10 , 11 and others including procedural risk factors.12 , 13 , 14 however , current models have not evaluated the predictive ability of patient factors and clinical biomarkers from the year prior to the procedure , nor with external validation . as more healthcare systems participate in health information exchanges , such as the integrated veterans administration healthcare system , and provide increasing levels of integrated outpatient and inpatient management to address quality metrics and systemsbased care requirements , more clinical data are becoming available and should be considered in prediction rules . there are a number of postcatheterization aki risk prediction models published outside of the veterans health administration ( va).10 , 12 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 a national association study was done using the american college of cardiology 's national cardiovascular data registry ( accncdr ) for predicting aki , but these data have not yet been developed into a model and internally validated.13 additionally , no model for prospectively predicting risk of aki for patients undergoing either cardiac catheterization or pci in the va has been developed for routine clinical use . there is a growing body of literature that supports the need for each healthcare system to perform recalibration and remodeling on a regular basis within their own healthcare data in order to ensure sustained high performance for individual patient prediction . in addition , the comprehensive nature of the va electronic health record ( ehr ) data sources allows novel risk factors to be explored and tested for importance in risk prediction . therefore , we sought to develop an externally validated national prediction model for aki occurring after cardiac catheterization or pci for prospective automated surveillance . we hypothesized that risk factors from 1 year prior to , immediately before , and during the procedure would predict aki in the va . we developed and internally validated a set of risk prediction models for postprocedural aki following cardiac catheterization within a national va cohort . this cohort included 222 669 catheterization procedures among 70 centers between january 1 , 2009 and october 1 , 2013 . the clinical assessment , reporting and tracking program ( cart ) is a national clinical quality initiative for va cardiac catheterization laboratories that began in 2005 and was in use in all of these laboratories by the beginning of 2008.23 the cart program includes a clinical software application designed to collect standardized data on all coronary angiograms and pci . all data elements are mapped to the definitions and standards of the accncdr.24 , 25 the va is an integrated care network that includes acute inpatient hospitals , outpatient primary care and subspecialty clinics , outpatient pharmacies , rehabilitation facilities , and longterm care facilities and domiciliaries . all va clinical providers and allied health personnel are required to use the same ehr for documentation and execution of all clinical care . the va central irb and site va r&d committees at va tennessee valley healthcare system , va eastern colorado healthcare system , and white river junction , vermont va approved this study . the external validation cohort was provided by the northern new england cardiovascular disease study group ( nnecdsg ) , which includes participating medical centers in new hampshire , maine , and vermont . at total of 27 905 patients data were collected from a retrospective va cohort of adult patients with a cardiac catheterization , with or without intervention . all data for these patients were retrieved beginning 1 year prior to the first cardiac catheterization in the cohort ( january 1 , 2008 ) . each patient coronary angiogram or pci procedure that occurred within the same day as cardiac catheterization the data were collected from 2 national va sources : ( 1 ) the cart program , and ( 2 ) the ehr data available from the corporate data warehouse . ehr data from the corporate data warehouse was used for inpatient admission and administrative data , chemistry and hematology laboratory data , outpatient pharmacy fill records , and inpatient barcoded medication administration . both domains were merged for patient demographics . a complete description of data definitions is included in data s1 . estimated glomerular filtration rates were calculated for all serum creatinine measures using the readily available abbreviated modification of diet and renal disease equation . chronic kidney disease was defined as a baseline estimated glomerular filtration rate < 60 ml / min per 1.73 m.26 , 27 chronic comorbidities were defined using administrative current procedural terminology and international statistical classification of diseases , 9 revision ( icd9 ) diagnostic codes collected from data prior to hospital admission . substantial icd9 validation work has been performed previously in the veterans affairs and in general populations.28 , 29 additional related icd9 codes used were aimed at increasing sensitivity to these validated codes . procedures were included if they had at least 1 serum creatinine measurement within 1 day to 365 days prior to cardiac catheterization and at least 1 serum creatinine measurement from 0 to 7 days following the procedure . procedures were excluded if they had a renal transplant , or if they had endstage renal disease , which is defined as requiring chronic dialysis or having a baseline estimated glomerular filtration rate of < 15 ml / min per 1.73 m ( n=6998 ) at the time of the procedure . procedures with missing pre ( 21 014 ) or postprocedure serum creatinine ( 79 024 ) were excluded , which left a total of 115 633 patient procedures ( figure 1 ) . a comparison of procedures included in the cohort with procedures excluded due to missing serum creatinine is reported in table s1 . aki was defined using the kidney disease improving global outcomes guidelines definition : 0.3 mg / dl within 48 hours of the procedure or 50% increase in serum creatinine from baseline to the post cardiac catheterization peak serum creatinine at any time during the hospitalization or up to 7 days following the procedure.30 baseline creatinine was defined as the most recent serum creatinine between 365 and 7 days prior to the procedure , which is a method previously validated in the literature.31 acute kidney injury network stage 2 ( akin2 ) was defined as a doubling in serum creatinine from baseline or developing new dialysisdependent renal failure . contrastinduced nephropathy ( cin ) was defined by 0.5 mg / dl within 7 days following the procedure . dialysis was identified by any new dialysisdependent renal failure within 7 days following the procedure . the timing of the post cardiac catheterization creatinine values is + 1 to + 7 days with mean of 2 days and a sd of 5 days . dichotomous variables reflecting the presence of medical conditions were set to zero if they were coded as missing ( eg , absent ) . dummy variables were created for variables with greater proportions of missing values ( eg , intravenous fluids and laboratory values ) and modeled against dummy categories ( eg , missing values dummy modeled against known intravenous fluid status prior to the procedure dummy variables ) . predicted values were imputed for missing contrast volume using case complexity , number of vessels receiving direct intervention , and number of stents deployed . we developed prediction rules using logistic regression with variable selection by using the least absolute shrinkage and selection operator ( lasso ) . lasso obtains coefficient estimates by maximizing a likelihood with a l1 penalty on coefficient size , for a sequence of penalty parameters.32 lasso has the property that only a subset of variables in the model will have nonzero coefficients , which makes it a variable selection tool . we report the lasso models for which the penalty term resulted in a maximized crossvalidated likelihood . the predictive ability of the model is reported using the cstatistic ( area under the receiver operating characteristic ( roc ) curve , auc ) corrected for overfitting using bootstrap validation by sampling the dataset with 200 iterations , and by plotting calibration of the model using ( observed versus predicted rates of aki after binning of predicted values ) . the bootstrap distribution of cstatistic values was used to obtain 95% cis around the auc . sensitivity analyses were conducted rerunning the models based on a 48hour definition of aki end points and again on unique patients . we considered all pairwise interactions , but excluded all from the model because none increased the cstatistic by more than 0.001 . external validation was conducted as follows : first , as the nne cohort does not collect ehr variables from preceding outpatient encounters , each of the 4 models was reduced to only include variables also available in the nne external validation cohort . second , lasso was performed again on the common variables of the va and nne cohorts . third , the model development and bootstrapping methods were repeated in the va cohort for the reduced va models . last , the coefficients from the va reduced models were then used to calculate the nne external validation roc and 95% ci for each of the 4 models . we developed and internally validated a set of risk prediction models for postprocedural aki following cardiac catheterization within a national va cohort . this cohort included 222 669 catheterization procedures among 70 centers between january 1 , 2009 and october 1 , 2013 . the clinical assessment , reporting and tracking program ( cart ) is a national clinical quality initiative for va cardiac catheterization laboratories that began in 2005 and was in use in all of these laboratories by the beginning of 2008.23 the cart program includes a clinical software application designed to collect standardized data on all coronary angiograms and pci . all data elements are mapped to the definitions and standards of the accncdr.24 , 25 the va is an integrated care network that includes acute inpatient hospitals , outpatient primary care and subspecialty clinics , outpatient pharmacies , rehabilitation facilities , and longterm care facilities and domiciliaries . all va clinical providers and allied health personnel are required to use the same ehr for documentation and execution of all clinical care . the va central irb and site va r&d committees at va tennessee valley healthcare system , va eastern colorado healthcare system , and white river junction , vermont va approved this study . the external validation cohort was provided by the northern new england cardiovascular disease study group ( nnecdsg ) , which includes participating medical centers in new hampshire , maine , and vermont . at total of 27 905 patients data were collected from a retrospective va cohort of adult patients with a cardiac catheterization , with or without intervention . all data for these patients were retrieved beginning 1 year prior to the first cardiac catheterization in the cohort ( january 1 , 2008 ) . each patient coronary angiogram or pci procedure that occurred within the same day as cardiac catheterization the data were collected from 2 national va sources : ( 1 ) the cart program , and ( 2 ) the ehr data available from the corporate data warehouse . ehr data from the corporate data warehouse was used for inpatient admission and administrative data , chemistry and hematology laboratory data , outpatient pharmacy fill records , and inpatient barcoded medication administration . estimated glomerular filtration rates were calculated for all serum creatinine measures using the readily available abbreviated modification of diet and renal disease equation . chronic kidney disease was defined as a baseline estimated glomerular filtration rate < 60 ml / min per 1.73 m.26 , 27 chronic comorbidities were defined using administrative current procedural terminology and international statistical classification of diseases , 9 revision ( icd9 ) diagnostic codes collected from data prior to hospital admission . substantial icd9 validation work has been performed previously in the veterans affairs and in general populations.28 , 29 additional related icd9 codes used were aimed at increasing sensitivity to these validated codes . procedures were included if they had at least 1 serum creatinine measurement within 1 day to 365 days prior to cardiac catheterization and at least 1 serum creatinine measurement from 0 to 7 days following the procedure . procedures were excluded if they had a renal transplant , or if they had endstage renal disease , which is defined as requiring chronic dialysis or having a baseline estimated glomerular filtration rate of < 15 ml / min per 1.73 m ( n=6998 ) at the time of the procedure . procedures with missing pre ( 21 014 ) or postprocedure serum creatinine ( 79 024 ) were excluded , which left a total of 115 633 patient procedures ( figure 1 ) . a comparison of procedures included in the cohort with procedures excluded due to missing serum creatinine is reported in table s1 . aki was defined using the kidney disease improving global outcomes guidelines definition : 0.3 mg / dl within 48 hours of the procedure or 50% increase in serum creatinine from baseline to the post cardiac catheterization peak serum creatinine at any time during the hospitalization or up to 7 days following the procedure.30 baseline creatinine was defined as the most recent serum creatinine between 365 and 7 days prior to the procedure , which is a method previously validated in the literature.31 acute kidney injury network stage 2 ( akin2 ) was defined as a doubling in serum creatinine from baseline or developing new dialysisdependent renal failure . contrastinduced nephropathy ( cin ) was defined by 0.5 mg / dl within 7 days following the procedure . dialysis was identified by any new dialysisdependent renal failure within 7 days following the procedure . the timing of the post cardiac catheterization creatinine values is + 1 to + 7 days with mean of 2 days and a sd of 5 days . dichotomous variables reflecting the presence of medical conditions were set to zero if they were coded as missing ( eg , absent ) . dummy variables were created for variables with greater proportions of missing values ( eg , intravenous fluids and laboratory values ) and modeled against dummy categories ( eg , missing values dummy modeled against known intravenous fluid status prior to the procedure dummy variables ) . predicted values were imputed for missing contrast volume using case complexity , number of vessels receiving direct intervention , and number of stents deployed . we developed prediction rules using logistic regression with variable selection by using the least absolute shrinkage and selection operator ( lasso ) . lasso obtains coefficient estimates by maximizing a likelihood with a l1 penalty on coefficient size , for a sequence of penalty parameters.32 lasso has the property that only a subset of variables in the model will have nonzero coefficients , which makes it a variable selection tool . we report the lasso models for which the penalty term resulted in a maximized crossvalidated likelihood . the predictive ability of the model is reported using the cstatistic ( area under the receiver operating characteristic ( roc ) curve , auc ) corrected for overfitting using bootstrap validation by sampling the dataset with 200 iterations , and by plotting calibration of the model using ( observed versus predicted rates of aki after binning of predicted values ) . the bootstrap distribution of cstatistic values was used to obtain 95% cis around the auc . sensitivity analyses were conducted rerunning the models based on a 48hour definition of aki end points and again on unique patients . we considered all pairwise interactions , but excluded all from the model because none increased the cstatistic by more than 0.001 . external validation was conducted as follows : first , as the nne cohort does not collect ehr variables from preceding outpatient encounters , each of the 4 models was reduced to only include variables also available in the nne external validation cohort . second , lasso was performed again on the common variables of the va and nne cohorts . third , the model development and bootstrapping methods were repeated in the va cohort for the reduced va models . last , the coefficients from the va reduced models were then used to calculate the nne external validation roc and 95% ci for each of the 4 models . table 1 shows the characteristics of the cohort for the aki and nonaki groups , including demographics , prior comorbid events , comorbidities at presentation , and procedural aspects . the univariate association ( odds ratio ) of each characteristic with aki occurrence is also presented in table 1 . out of a total of 115 633 coronary angiography procedures , aki developed in 16 036 ( 13.9% ) , while the number with akin stage 2 was 2017 ( 1.7% ) , the number with cin was 13 763 ( 14.4% ) , and 476 ( 0.4% ) received dialysis treatment for aki . patient and procedural characteristics ace indicates angiotensinconverting enzyme ; aki , acute kidney injury ; akin , acute kidney injury network ; arb , angiotensin receptor blockers ; arf , acute renal failure ; cabg , coronary artery bypass grafting ; chf , congestive heart failure ; cin , contrastinduced nephropathy ; ckd , chronic kidney disease ; ckmb , creatinine kinase , muscle and brain subunits ; egfr , estimated glomerular filtration rate ; icd9 , current procedural terminology and international statistical classification of diseases , 9 revision ; iv , intravenous ; kdigo , kidney disease improving global outcomes ; mi , myocardial infarction ; nsaids , nonsteroidal antiinflammatory drugs ; or , odds ratio ; pci , percutaneous coronary intervention . using the variables included in table 1 table 2 contains the odds ratios we obtained from fitting this model to our data . the overfittingcorrected cstatistic for the aki prediction model was 0.742 ( 95% ci : 0.7380.747 ) . the second column contains the lassoderived model results for patients developing akin stage 2 , for which the biascorrected cstatistic was 0.826 ( 95% ci : 0.8160.836 ) . the third column contains our results from the model for patients developing 0.5 ( mg / dl ) increase in serum creatinine ( cin ) , with a biascorrected cstatistic of 0.741 ( 95% ci : 0.7370.746 ) . the last column shows the results from our lassoderived model for dialysis with a biascorrected cstatistic of 0.885 ( 95% ci : 0.8700.902 ) . each plot indicates strong agreement between the observed and predicted values , although for all 4 outcomes the observed odds are below our predictions for subjects with the lowest expected odds . we conducted sensitivity analyses for aki at 48 hours and by limiting the cohort to unique patient procedures as opposed to a unique procedure analysis . all rocs were robust for aki , akin2 , cin , and dialysis end points : 0.768 , 0.855 , 0.736 , and 0.895 , respectively . when limiting to unique patients ( n=90 105 ) , the rocs for aki , akin2 , cin , and dialysis end points were 0.738 , 0.826 , 0.739 , and 0.883 , respectively . both sensitivity analyses proved the model to perform well on a 48hour definition for aki end points and restricting to unique patient procedures . multivariable logistic regression models for predicting aki , akin stage 2 , cin , and dialysis selected using lasso with maximumized crossvalidation ace indicates angiotensinconverting enzyme ; aki , acute kidney injury ; akin , acute kidney injury network ; arb , angiotensin receptor blockers ; arf , acute renal failure ; cabg , coronary artery bypass grafting ; chf , congestive heart failure ; cin , contrastinduced nephropathy ; ckd , chronic kidney disease ; ckmb , creatinine kinase , muscle and brain subunits ; egfr , estimated glomerular filtration rate ; icd9 , current procedural terminology and international statistical classification of diseases , 9 revision ; iv , intravenous ; kdigo , kidney disease improving global outcomes ; lasso , least absolute shrinkage and selection operator ; mi , myocardial infarction ; nsaids , nonsteroidal antiinflammatory drugs ; or , odds ratio ; pci , percutaneous coronary intervention . observed vs expected calibration plots . this figure plots the observed vs expected calibration plots for aki , akin 2 , cin , and dialysis . aki indicates acute kidney injury ; akin 2 , acute kidney injury network 2 ; cin , contrastinduced nephropathy . a small number of candidate variables were statistically significant and associated consistently with occurrence of aki for each of our outcome definitions ( table 2 ) . history of diabetes was a risk factor , while prior coronary artery bypass grafting and pci were protective . prior instances of congestive heart failure and low albumin were also risk factors , as were prior aki by kidney disease improving global outcomes definition and chronic kidney disease by observed estimated glomerular filtration rates ( creatinine ) . exposure to either nacetylcysteine , angiotensin receptor blockers , or hydroxymethyl glutaryl coenzyme a reductase inhibitors at presentation was protective , whereas exposure to loop diuretics was a risk factor . clinical presentation risk factors were catheterization urgency status , shock , acute coronary syndrome , and anemia . external validation limited to variables included in the nnecdsg registry showed results consistent with our va aki population findings ( table 3 ) . the va reduced models performed with high agreement in roc statistics for all prediction end points , including any aki occurrence ( va : 0.70 , 95% ci 0.70 , 0.71 ; nnecdsg : 0.69 , 95% ci 0.68 , 0.71 ) , cin ( va : 0.68 , 95% ci 0.68 , 0.69 ; nnecdsg : 0.72 ; 95% ci 0.70 , 0.74 ) , akin stage 2 ( va : 0.80 , 95% ci 0.79 , 0.81 ; nnecdsg : 0.76 , 95% ci 0.73 , 0.79 ) , and dialysis ( va : 0.85 , 95% ci 0.82 , 0.87 ; nnecdsg : 0.86 , 95% ci 0.76 , 0.96 ) . northern new england cardiovascular disease study group external validation patient and disease characteristics aki indicates acute kidney injury ; cabg , coronary artery bypass grafting ; chf , congestive heart failure ; ckd , chronic kidney disease ; egfr , estimated glomerular filtration rate ; mi , myocardial infarction ; pci , percutaneous coronary intervention . va reduced multivariate models for external validation aki indicates acute kidney injury ; akin , acute kidney injury network ; cabg , coronary artery bypass grafting ; chf , congestive heart failure ; cin , contrastinduced nephropathy ; ckd , chronic kidney disease ; egfr , estimated glomerular filtration rate ; mi , myocardial infarction ; or , odds ratio ; pci , percutaneous coronary intervention ; va , veterans health administration . we developed a comprehensive and externally validated clinical prediction rule for aki among cardiac catheterization and/or pci procedures in the va . this is the first such tool developed from national va cart and ehr data , which explores novel outpatient and periprocedural risk factors for aki . all of the models developed for different severities of aki had good discrimination performance ( 0.7410.885 ) , and maintained adequate calibration across the full spectrum of risk in each model . reduced va models using routine ncdr variables and paired with the external validation cohort demonstrated the generalizability of our aki and dialysis prediction tool to nonva catheterization laboratories . there are a number of risk prediction models for aki following pci , but most were developed from data prior to 2005 with a variety of outcome definitions.34 the 2 most modern coronary angiography aki models were developed by tsai et al and gurm et al . both focused only on preprocedural risk estimation with a wider array of candidate variables than previously evaluated , including preprocedural inpatient medication exposures , clinical history and demographics , patient presentation characteristics , and laboratory assessments.11 , 13 this study extends prior research by exploring additional risk factors available within a comprehensive ehr for use within aki risk prediction models . only 1 study developed a model using both coronary angiography and pci procedures from 1218 patients at a single center and did not find a multivariate association between intervention status and aki.35 no prior modeling study has incorporated inpatient barcoded records of intravenous fluid administration , which have been shown in numerous studies to be protective of postprocedural aki . no prior study has included prevalent use of outpatient medications , such nonsteroidal antiinflammatory drugs , angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , or diuretics , all of which have been shown to be associated with aki . in this study , risk factors at the time of presentation as well as renal function or prior renal events were key variables for predicting aki and dialysis . within these categories , the strongest risk factors included urgency of the procedure , shock , chronic kidney disease stage , and development of aki prior to the procedure . we previously developed a singlecenter risk model to predict hospitalacquired aki for use in an ehr ; this effort extends that model to coronary angiography within a national va cohort for use in the va ehr.36 because we intend these models to be used in a clinical setting , the potential predictors are limited to those available in realtime from the ehr and the cart clinical applications . however , because these models provide an automated way to predict aki risk , they allow clinicians to account for a larger number of variables while reducing data entry time . integration of these models into ehrs has not yet been met with wide acceptance and success for several reasons . third , we need to expand our capacity to automate data collection in real time , and last , we need better ways to visualize the clinical decision process.37 the cart program and the office of analytics and business intelligence have prioritized the implementation of a realtime high risk aki clinical reminder using these models to be delivered during preprocedural assessment . since the development of the mehran cin risk score,12 new investigations have led to the creation of new cin and aki risk models with variable aki definitions and levels of discrimination.10 , 11 , 13 , 14 a recent study by the ncdr cathpci registry including 954 729 pci patients examined the association of common aki risk factors known at presentation , in addition to contrast use and length of hospitalization.13 unlike the ncdr investigators , who did not focus on the development of a risk model , the blue cross blue shield of michigan cardiovascular consortium constructed a model for cin ( 0.5 mg / dl increase in serum creatinine from baseline to hospital discharge ) using 48 001 pci patients and found an roc of 0.85 in the study cohort and an roc of 0.84 in the 20 572patient internal validation cohort.11 similarly to the ncdr study , the blue cross blue shield of michigan cardiovascular consortium model incorporated common aki risk factors at presentation , including patient characteristics ( age , weight , height , and heart failure ) , coronary disease , pci indication , and priority.11 , 13 nevertheless , all of these modeling approaches have been limited only to pci patients and have broadly excluded diagnostic cardiac catheterization patients . in addition , past studies have limited the definition of aki or cin to inhospital serum creatinine measurement alone , which falls short of meeting the current kidney disease improving global outcomes guidelines for defining aki.30 our models advance the state of aki clinical risk modeling in several ways . second , we use a sample of consecutive patients in a large , national set of va catheterization laboratories . third , we investigate novel risk factors by leveraging a robust electronic medical record rich in clinical patient characteristics including both outpatient and perioperative factors not available in the ncdr or blue cross blue shield of michigan cardiovascular consortium clinical registries . fourth , we used lasso to aid in variable selection in place of the conventional stepwise regression approach . fifth , implementation of our model in electronic medical recordkeeping would eliminate the need for data entry at the time of use and reduces the need for model parsimony . seventh , we incorporated the new kidney disease outcomes quality initiative aki guidelines to include serum creatinine measures up to 7 days postprocedure from inpatient or outpatient blood draws.30 we developed our prediction model based on a sample of va users , in which females are underrepresented and lowincome and complex patients are overrepresented . because some patients receive some of their care outside of the va system , we might have been unable to identify all prior events . despite searching multiple data sets in the va , postprocedure serum creatinine was missing for a large proportion of the population . however , this issue is common across all cardiac catheterization populations for coronary angiography because patients are released the same day of the procedure without creatinine measurement followup . even though we need to account for this limitation , we elected to retain angiography patients in order to be able to assess risk on a larger patient population . baseline serum creatinine was defined as the most recent serum creatinine measurement taken between 365 and 7 days prior to the procedure , a method previously validated by siew and colleagues.31 next , the observed odds were lower than the predicted odds among the lowest risk of aki and therefore the models we derived may slightly overestimate risk for those whose risk is in the lower tercile of risk . the prediction model coefficients ( and odds ratios ) corresponding to collinear ( correlated ) groups of variables should be interpreted cautiously , as they correspond to unit changes in the variable keeping the correlated variables constant , which may be unrealistic . last , because external validation of the va models was performed using the nnecdsg registry , we could only use variables present in the nnecdsg registry to build our model . the only variables excluded were those created by the va ehr from the year prior to the angiography procedure . although the inclusion of these variables in an aki risk model is one of the innovations of our study , the external validation of the va reduced models proves that the standard variables in registries such as the nnecdsg , which harmonizes variables with the ncdr data collection , are generalizable largely to ncdr participating centers . therefore , the external validation of the va aki risk models in our investigation is valid and generalizable to nonva cardiac catheterization laboratories in the united states . we must also be mindful about the uncertain implications of aki risk modeling for patients and providers . we propose our preprocedural predictive tool will be used by providers to assess patients at high risk of aki and therefore implement necessary prophylactic strategies to minimize the risk of aki.38 second , providers will need to utilize this tool along with other tools to determine a safety threshold of contrast use during the procedure,8 , 33 and therefore use judgment regarding duration of case , ad hoc pci , and number of interventions . the unintended consequences of identifying high risk patients for aki may be delayed or postponed procedures . providers will need to balance the tradeoffs of readiness for cardiac catheterization for patients at high risk of aki and potential delays in the procedure with the other clinical needs and timing of revascularization . we developed our prediction model based on a sample of va users , in which females are underrepresented and lowincome and complex patients are overrepresented . because some patients receive some of their care outside of the va system , we might have been unable to identify all prior events . despite searching multiple data sets in the va , postprocedure serum creatinine was missing for a large proportion of the population . however , this issue is common across all cardiac catheterization populations for coronary angiography because patients are released the same day of the procedure without creatinine measurement followup . even though we need to account for this limitation , we elected to retain angiography patients in order to be able to assess risk on a larger patient population . baseline serum creatinine was defined as the most recent serum creatinine measurement taken between 365 and 7 days prior to the procedure , a method previously validated by siew and colleagues.31 next , the observed odds were lower than the predicted odds among the lowest risk of aki and therefore the models we derived may slightly overestimate risk for those whose risk is in the lower tercile of risk . the prediction model coefficients ( and odds ratios ) corresponding to collinear ( correlated ) groups of variables should be interpreted cautiously , as they correspond to unit changes in the variable keeping the correlated variables constant , which may be unrealistic . last , because external validation of the va models was performed using the nnecdsg registry , we could only use variables present in the nnecdsg registry to build our model . the only variables excluded were those created by the va ehr from the year prior to the angiography procedure . although the inclusion of these variables in an aki risk model is one of the innovations of our study , the external validation of the va reduced models proves that the standard variables in registries such as the nnecdsg , which harmonizes variables with the ncdr data collection , are generalizable largely to ncdr participating centers . therefore , the external validation of the va aki risk models in our investigation is valid and generalizable to nonva cardiac catheterization laboratories in the united states . we must also be mindful about the uncertain implications of aki risk modeling for patients and providers . we propose our preprocedural predictive tool will be used by providers to assess patients at high risk of aki and therefore implement necessary prophylactic strategies to minimize the risk of aki.38 second , providers will need to utilize this tool along with other tools to determine a safety threshold of contrast use during the procedure,8 , 33 and therefore use judgment regarding duration of case , ad hoc pci , and number of interventions . the unintended consequences of identifying high risk patients for aki may be delayed or postponed procedures . providers will need to balance the tradeoffs of readiness for cardiac catheterization for patients at high risk of aki and potential delays in the procedure with the other clinical needs and timing of revascularization . we developed a robust clinical prediction model for aki following cardiac catheterization or pci to be used in a prospective automated surveillance program in the va . these models both explore novel variables that may be useful in future prediction modeling and risk adjustment , and we have confirmed external validity in a reduced model within the nnecdsg registry . this model has been operationally prioritized by the va to be incorporated into routine clinical practice , with the intention to automatically identify patients at risk of aki before and immediately following a cardiac catheterization or pci procedure . it is our hope that automated surveillance of aki will help clinicians reduce the incidence of aki and incorporate protocols to prevent aki urgently among those patients identified as high risk . this project was supported in part by veterans health administration health services research and development ( hsr&d ) cda08020 ( matheny ) , hsr&d iir11292 ( matheny , tsai , fly , plomondon , resnic , brown , mackenzie ) , and hs018443 ( brown ) from the agency for healthcare research and quality . dr siew was supported by k23 dk08896401a1 and k24 dk62849 grants from the national institute of diabetes and digestive and kidney diseases . this project was executed in collaboration with the national cart program in collaboration with john rumsfeld and the predictive analytics group of the va office of analytics and business intelligence in collaboration with christopher nielson , clifton baker , and stephan fihn .

backgroundacute kidney injury ( aki ) occurs frequently after cardiac catheterization and percutaneous coronary intervention . although a clinical risk model exists for percutaneous coronary intervention , no models exist for both procedures , nor do existing models account for risk factors prior to the index admission . we aimed to develop such a model for use in prospective automated surveillance programs in the veterans health administration.methods and resultswe collected data on all patients undergoing cardiac catheterization or percutaneous coronary intervention in the veterans health administration from january 01 , 2009 to september 30 , 2013 , excluding patients with chronic dialysis , endstage renal disease , renal transplant , and missing pre and postprocedural creatinine measurement . we used 4 aki definitions in model development and included risk factors from up to 1 year prior to the procedure and at presentation . we developed our prediction models for postprocedural aki using the least absolute shrinkage and selection operator ( lasso ) and internally validated using bootstrapping . we developed models using 115 633 angiogram procedures and externally validated using 27 905 procedures from a new england cohort . models had crossvalidated cstatistics of 0.74 ( 95% ci : 0.740.75 ) for aki , 0.83 ( 95% ci : 0.820.84 ) for akin2 , 0.74 ( 95% ci : 0.740.75 ) for contrastinduced nephropathy , and 0.89 ( 95% ci : 0.870.90 ) for dialysis.conclusionswe developed a robust , externally validated clinical prediction model for aki following cardiac catheterization or percutaneous coronary intervention to automatically identify highrisk patients before and immediately after a procedure in the veterans health administration . work is ongoing to incorporate these models into routine clinical practice .

Introduction Methods Study Setting and Design Data Collection Data Definitions Cohort Inclusion and Exclusion Criteria Outcome Definitions Statistical Analysis Results Discussion Limitations Conclusions Sources of Funding Disclosures Supporting information

PMC4552768

ambient temperature influences all living organisms , but none more than long - lived marine crustaceans . temperatures in the north atlantic ocean , where the lobster , homarus americanus and the crab , cancer borealis , live , routinely fluctuate from below 4 c to more than 25 c ( factor 1995 ; crossin et al . moreover , these animals can experience substantial temperature swings in very short times as a consequence of tides , currents , and storms . for those of us who use wild - caught marine crustaceans as experimental systems , it is important to understand the environmental challenges they face , as well as to appreciate that they also show temperature preferences that can contribute to where they choose to live ( krediet and donahue 2009 ; lewis and ayers 2014 ) . temperature is a global perturbation that influences all biological processes , to a greater or lesser degree . biologists often express the temperature dependence of a specific process by calculating the q10 , the factor by which a process increases for every 10 c increase in temperature . many biological processes , including most voltage - gated ion channels , have activation and inactivation rates with q10s in the 23 range , while the ion channels that are thought important for temperature sensing may have q10s as high as 50 or 100 ( garrity et al . importantly , if all of the component processes that govern a system s behavior have identical q10 s , the process will be temperature compensated ( robertson and money 2012 ) . for example , if all of the q10s that control the activation and inactivation kinetics of the ion channels contributing to action potential generation are the same , then the action potential waveform will be maintained as temperature is changed ( although the frequency may change ) . however , as the q10 describes an exponential function , a relatively modest difference in the q10s for two processes can nonetheless become quite significant in response to even a modest temperature perturbation . in this case , maintaining constant performance over a considerable temperature range becomes a non - trivial problem ( tang et al . 2014 ) , and there are relatively few combinations of conductance densities and q10s that allow a neuron to function over a range of temperatures ( wechselberger et al . , sensory and motor function often depends on relative temperature invariance ( tang et al . 2014 ) , so it becomes a challenge to understand how physiological performance can be preserved despite the fact that all the components of circuits in behavior are altered differentially by temperature . the effects of acute temperature change on the triphasic pyloric rhythm have been studied both in vivo ( soofi et al . 2014 ) and in vitro ( tang et al . 2010 , 2012 ) . in both cases , the frequency of the pyloric rhythm increases as the temperature increases over a permissive range , while the phase relationships , or the relative timing , among the component neurons are maintained ( tang et al . it is clear that phase maintenance is critical for appropriate movements , which result from muscle groups being activated in the correct order . the problem of maintaining phase despite changes in frequency has attracted a great deal of attention precisely because it requires understanding how biophysical processes which would tend to produce fixed delays can be combined instead to produce fixed phases ( harris - warrick et al . 2003 ; bucher et al . 2005 ; marder and bucher 2007 ; goaillard et al . the pacemaker kernel of the pyloric rhythm consists of the 2 pd neurons and a single ab neuron , which are tightly electrically coupled and are synchronously active ( marder and bucher 2007 ) . ( 2013 ) pharmacologically isolated the pacemaker kernel and then characterized the effects of temperature . as expected , as temperature unexpectedly , the duty cycle of the pacemaker kernel remained virtually constant over a large range of temperatures ( rinberg et al . this result goes a long way to explain the compensation of phase over temperature previously reported ( tang et al . 2010 ) , because the pacemaker kernel will provide rhythmic inhibition whose duration changes as the period changes . the response of the follower lp and py neurons to rhythmic inhibitory drive depends on the strength and time course of the inhibition they receive ( eisen and marder 1982 ; rabbah and nadim 2007 ) as well as the properties of ia and ih in the follower neurons ( hartline and gassie 1979 ; harris - warrick et al . all of these three processes are temperature dependent , with somewhat different q10s ( tang et al . 2010 ) . nonetheless , they balance well enough to maintain a constant phase ( tang et al . 2010 ) . as the temperature is increased beyond the permissive range , the preparations crash or lose their ability to maintain robust triphasic rhythms ( tang et al . figure 1 shows intracellular recordings of the pd and lp neurons and an extracellular recording of the lateral ventricular nerve ( lvn , which also shows activity of the py neurons ) , first at 11 c , then at 31 c , and then back at 11 c . note the normal alternation of the lp , py , and pd neurons at low temperatures , and then the disruption of the rhythm at 31 c , associated with loss of pd neuron activity , almost complete loss of lp neuron activity , but continued tonic firing in the py neuron.fig . example intracellular ( top lp and pd ) and extracellular ( bottom lvn ) traces from a preparation at 11 c ( stable rhythm ) , 31 c ( network crash ) and then back to 11 c ( recovery of a stable rhythm ) extreme temperature crashes the pyloric rhythm . example intracellular ( top lp and pd ) and extracellular ( bottom lvn ) traces from a preparation at 11 c ( stable rhythm ) , 31 c ( network crash ) and then back to 11 c ( recovery of a stable rhythm ) detailed analyses of high - temperature crashes using spectral analyses ( tang et al . 2012 ) show that individual preparations respond similarly to temperature changes in the permissive range , but crash differently at more extreme temperatures , as was predicted from studies that showed variability in network components ( golowasch et al . 1999 ; goldman et al . 2001 ; marder and goaillard 2006 ; schulz et al . 2006 , 2007 ; goaillard et al . in considering how the pyloric rhythm crashes at high temperatures , we were curious to determine how each component of the pyloric rhythm responds to extreme temperatures . at high temperatures , 2013 ) , but each preparation showed different crash dynamics . again , the behavior of the isolated pacemaker kernels was robust over a large permissive temperature range , but showed the effect of their underlying differences in conductance parameters as the oscillators made different transitions when they lost their ability to oscillate ( rinberg et al . when the circuit was intact , in many preparations it appeared that the lp neuron was often the first neuron to become silent or highly irregular at high temperature ( tang et al . consequently , we decided to pharmacologically isolate the lp neuron and study the effects of temperature on it . the top trace , labeled control , shows an intracellular recording of the lp neuron in the intact network . subsequently , the preparation was placed in picrotoxin to block the glutamatergic inputs to the lp neuron from the ab and py neurons ( marder and eisen 1984 ) . the second trace in fig . 2 shows the absence of large ipsps in comparison to the control , but that the lp neuron was still firing rhythmically , most likely because of a small inhibitory input remaining from the cholinergic pd neurons ( marder and eisen 1984 ) . as the temperature was further increased , the lp neuron started generating very long bursts and eventually stopped firing action potentials . figure 2b shows the pooled data from 15 lp neurons as a function of temperature . most neurons increased in firing frequency as temperature was raised from 11 to 23 c , but as the temperature was increased further many of them decreased or stopped firing . the effect of temperature on the coefficient of variation of the firing rate demonstrates clearly that as the temperature reaches 27 c , the neuronal activity patterns become highly variable , again as would be expected given the variable conductance densities of the lp neurons voltage - gated currents ( schulz et al . control , lp is active in the intact network . after applying picrotoxin ( ptx ) , glutamatergic connections to ab and py are blocked and some residual pd inhibition is left . with increasing temperature , lp spike frequency increases and pauses between lp bursts become larger . at 34 c lp crashes . b firing rate in isolated lp neurons increases with increasing temperature . at 23 c individual frequencies become more variable due to crashes . the coefficient of variation ( ratio between the standard deviation and the mean of the spike frequency ) increases at higher temperatures , due to variable spiking patterns in individual lp neurons . n = 15 lp neurons ; each color represents one lp neuron effect of temperature on the activity of isolated lp neurons . control , lp is active in the intact network . after applying picrotoxin ( ptx ) , glutamatergic connections to ab and py are blocked and some residual pd inhibition is left . with increasing temperature , lp spike frequency increases and pauses between lp bursts become larger . at 34 c lp crashes . b firing rate in isolated lp neurons increases with increasing temperature . at 23 c individual frequencies become more variable due to crashes . the coefficient of variation ( ratio between the standard deviation and the mean of the spike frequency ) increases at higher temperatures , due to variable spiking patterns in individual lp neurons . there is an enormous literature that demonstrates that animals acclimate to extended periods of time at different temperatures ( lehtikoivunen and kivivuori 1994 ; camacho et al . 2006 ; robertson and money 2012 ; tang et al . 2012 ) . we acclimated crabs for 34 weeks to low and high temperatures and found little change in the response to acute temperature ramps of in vitro preparations in the permissive temperature range , but found that preparations from warm - acclimated animals required higher temperatures to crash the pyloric rhythm ( tang et al . 2012 ) . the winter of 20112012 was unusually warm and ocean temperatures in the waters from which our animals were collected were 68 c warmer than normal , thus creating a natural acclimation period of many months . preparations from animals that had lived for extended periods of time in the ocean at much warmer temperatures than usual showed remarkable behavior in response to temperature . figure 3a shows recordings of the pyloric rhythm in response to acute temperature changes . but , as the temperature reached 31 c , the frequency was almost 5 hz , and at 35 c , it reached almost 7 hz ( fig . finally , at 3637 c , the pyloric rhythm crashed , but at a much higher temperature than we had ever seen in previous years . data from four preparations from this group of crabs that had over - wintered in warm waters are summarized in fig . 3b , c , and show the unusually high frequencies and crash points for these animals . thus , we conclude that an extended period of time at higher than usual winter water temperatures had shifted upwards the temperature tolerance of the animals . ( we should note that in the two subsequent years , the typical crash temperatures returned to lower values.)fig . the network reached a pyloric frequency of 7 hz at 35 c and then crashed at 36 c . c the temperatures at which the networks crashed from the same preparations unusually warm winter ocean temperatures alter the crash point of in vitro preparations . the network reached a pyloric frequency of 7 hz at 35 c and then crashed at 36 c . c the temperatures at which the networks crashed from the same preparations crustacean neuromuscular junctions are notoriously temperature sensitive ( stephens and atwood 1982 ) , with muscle tension and contraction compromised with temperature increase ( hamilton et al . 2007 ; thuma et al . 2013 ) . interestingly , circulating neuromodulators , such as dopamine and serotonin , may play a role in vivo to compensate for the potential loss of function at high temperature ( hamilton et al . therefore , we were curious to compare directly the effect of temperature on pyloric rhythms recorded both in vivo with implanted electrodes with results from dissected preparations in vitro ( soofi et al . 2014 ) . at the same temperature , the in vitro recorded motor patterns were faster than what was seen in vivo from the same animal . the steeper temperature dependence of the pyloric rhythm frequency in the in vitro preparations when compared to the in vivo preparations suggests that sensory feedback may reduce the overall effect of high temperature on pyloric rhythm frequency in vivo ( soofi et al . 2014 ) . because the pyloric rhythm frequencies recorded in vivo at high temperature appeared somewhat lower than those produced by the same animals after dissection and in vitro recordings , we were curious to examine the effects of temperature acclimation on the motor patterns recorded in vivo after the animals had been acclimated to higher temperatures for at least 1 month . figure 4a shows a comparison of the motor pattern recorded at 24 c from implanted electrodes in animals that had been acclimated for 4 weeks to 11 and 19 c , respectively . figure 4b shows that warm - acclimated animals became less sensitive to acute temperature increases in the permissive range . this may be physiologically advantageous , as muscle contraction is more likely to follow the motor pattern discharge when the pyloric rhythm frequency is less than 2 hz ( morris and hooper 1997 , 1998 , 2001 ) , although it would be interesting to see how long - term acclimation changes the properties of the neuromuscular junctions of the pyloric muscles.fig . a pyloric rhythms recorded at 24 c in animals that had been acclimated to 11 c ( top ) and 19 c ( bottom ) , recorded with an implanted extracellular electrode . b pooled and binned data from in vivo recordings in seven cold - acclimated and four warm - acclimated animals . warm - acclimated animals responded with a significantly shallower increase in frequencies as a function of temperature elevation ( two - way anova , p < 0.01 ) comparison of pyloric motor patterns recorded in vivo in animals acclimated to different temperatures . a pyloric rhythms recorded at 24 c in animals that had been acclimated to 11 c ( top ) and 19 c ( bottom ) , recorded with an implanted extracellular electrode . b pooled and binned data from in vivo recordings in seven cold - acclimated and four warm - acclimated animals . warm - acclimated animals responded with a significantly shallower increase in frequencies as a function of temperature elevation ( two - way anova , p < 0.01 ) maintaining a robust pyloric rhythm over a wide temperature range requires that many biophysical , biochemical , and molecular processes have temperature dependencies that collectively preserve the important features of circuit function . while this is difficult to achieve when building computational models ( caplan et al . 2014 ) , there must be many , yet to be discovered , biological rules that allow developing and growing animals to find sets of network parameters that allow them to retain stable circuit function in a temperature - fluctuating ocean .

marine invertebrates , such as lobsters and crabs , deal with a widely and wildly fluctuating temperature environment . here , we describe the effects of changing temperature on the motor patterns generated by the stomatogastric nervous system of the crab , cancer borealis . over a broad range of permissive temperatures , the pyloric rhythm increases in frequency but maintains its characteristic phase relationships . nonetheless , at more extreme high temperatures , the normal triphasic pyloric rhythm breaks down , or crashes . we present both experimental and computational approaches to understanding the stability of both single neurons and networks to temperature perturbations , and discuss data that shows that the crash temperatures themselves may be environmentally regulated . these approaches provide insight into how the nervous system can be stable to a global perturbation , such as temperature , in spite of the fact that all biological processes are temperature dependent .

Introduction The effects of temperature on the crab pyloric rhythm How do isolated neurons crash? Temperature acclimation of the pyloric rhythm Conclusions

PMC4017500

primary biliary cirrhosis ( pbc ) is an autoimmune liver disease characterized by the development of autoantibodies ( ama ) directed against mitochondrial components , antibodies against nuclear components , such as anti - sp100 and anti - gp 210 , and by progressive destruction of the intrahepatic bile ducts , resulting in fibrosis , cirrhosis , and finally liver failure ( 1 , 2 ) . tumor necrosis factor- ( tnf- ) might be related to both the severity and prognosis of patients with pbc ( 3 ) . crohn 's disease ( cd ) is frequently associated with a number of extraintestinal manifestations . many hepatobiliary disorders could complicate its course , with some of them , including autoimmune thyroiditis ( 4 ) , harboring immunological mechanisms in their pathogenesis . the treatment of cd with anti - tnf- agents has dramatically changed the outcome of the disease . on the other hand , the treatment of pbc is mainly symptomatic aiming to restore the increased levels of cholestatic liver enzymes , and reduce the severity of clinical symptoms . we describe herein the case of a male patient with long - standing cd who developed autoimmune hassimoto thyroiditis , pbc , and raynaud 's phenomenon , during the course of long - standing cd of the small bowel . in this patient , the underlying cd , and a male patient , aged 48 , was admitted in our department in 2009 , complaining mostly of diarrhoea , loss of weight , fatigue , and arthralgias . the family history revealed that his son , aged 17 , was suffering from cd of the large bowel accompanied by extraintestinal manifestations ( arthritis ) . his mother was suffering from systemic lupus erythematosus , while his sister from sjogren 's syndrome . from his past history , he mentioned cd of the small bowel accompanied by perineal fistula , diagnosed in 1994 . two years after the establishment of diagnosis of cd , he underwent total thyroidectomy because of hyperthyroidism were not responding to conservative treatment . the histology of the surgical specimen revealed that hyperthyroidism developed on the ground of thyroiditis hassimoto . in the subsequent years , cd was running with exacerbations of mild to moderate severity that promptly responded to oral administration of steroids and mesalamine . the patient did not receive azathioprine because of serious side - effects . in 2004 , because of a severe flare - up of the disease he was treated with infliximab 5mg / kg bw . although the biologic agent resulted in favorable clinical and laboratory effects , it was interrupted after 4 sessions , because of the appearance of high fever attributed to the drug itself . at that time , an abdominal ct scan revealed liver enlargement and a thickness of many loops of both jejunum and ileum . on admission in our department in 2009 , the laboratory investigation revealed esr 85 mm , hct 35.1% , hb 11.8g / dl , fibrinogen 517 mg / dl ( n.v . renal function tests , serum amylase , serum albumin , serum electrolytes , and tumor markers were normal . < 200 ) , hdl 56 mg / dl ( n.v . > 40 ) , ldl 178 mg / dl ( n.v . hepatitis b and c serum antibodies , p - anca , c - anca , anti - mpo , anti - pr3 , anti - endomysium , anti - gliadine ( g & a ) , and anti - ttg ( g & a ) were negative . however , anti - tg , anti - nuclear , anti - microsomial , antimitochondrial and anti - m2 serum autoantibodies revealed significantly higher values above the normal limits [ 2700 iu / ml ( n.v . < 200 ) , 1:640 ( n.v . 1:80 ) , 4100 iu / ml ( n.v . < 100 ) , 1:360 ( n.v . serum igg was 1560 mg / dl , iga 290 mg / dl ( n.v . serum inflammatory indices , including crp and a1-acid - glycoprotein , were increased [ 36 mg / l ( n.v <3 ) and 1.61 g / l ( n.v . abdominal mri revealed no abnormal findings from biliary tract , gallbladder , pancreas and liver . however , mri enterography showed the involvement of cd of multiple ileal loops , liver and spleen enlargement , and a small dilation of portal vein and of veins in the portal of spleen . the small bowel follow - through confirmed the involvement of many loops of jejunum and ileum . because of the previous intolerance of infliximab , the patient was treated with adalimumab 160 mg , then 80 mg and finally 40 mg every other week . anti - m2 antibodies remained positive . in 2011 , ursodeoxucholic acid ( 200mgx2/d ) was added , and was well tolerated . in 2012 , the latest ( 2012 ) laboratory investigation revealed no anemia with slightly elevated values of liver cholestatic enzymes . the titre of antimitochondrial antibodies further reduced to 1:80 . during the 3-year treatment with adalimumab we plan to continue the treatment with both the biologic agent and the ursodeoxycholic acid for the next few years . table 1 shows the values of cholestatic enzymes and serum ama in different time periods before and after the treatment with adalimumab . liver function tests in different time periods numbers in parentheses are normal values date of starting treatment with a dalimumab the described case presents some interesting clinical and epidemiological characteristics . the patient was a man with long - standing cd , having many members of his family suffering from a variety of autoimmune disorders . during the course of cd i ) he developed clinical symptoms of severe hyperthyroidism and as a consequence , he underwent total thyroidectomy the histology of which revealed that hyperthyroidism developed on the ground of hashimoto thyroiditis , ii ) for a long period of time he was suffering from raynaud 's phenomenon , iii ) the laboratory investigation revealed that he was also suffering from pbc . in this patient , the treatment with an anti - tnf- agent ( adalimumab ) resulted not only in a significant improvement of both cd and perineal disease , but also in a significant reduction in the values of cholestatic enzymes and serum titres of ama after a 4-month period . moreover , during the 3-year administration of adalimumab , reynaud 's phenomenon did not appear again . his clinical situation is now quite satisfactory and we plan to continue the administration of adalimumab at least for the next years . however , many questions should be addressed concerning the diagnosis and clinical outcome in this patient : i ) how strong is the diagnosis of pbc ? , ii ) are indeed the improvement of the titers of both serum ama and liver enzymes , due to the administration of the biologic agent ? , iii ) might the appearance of thyroid disease with other autoimmune disorders on the ground of cd be considered as an appearance by chance ? as far as the accuracy of the diagnosis of pbc is concerned , it is widely accepted that this diagnosis could be safely done if at least two of the following three criteria were fulfilled : i.e. serum ama titers > 1:40 , increased serum alkaline phosphatase levels for more than 6 months , and compatible liver histology in the absence of alternative explanation . however , liver biopsy at the time of diagnosis is currently not indicated except for patients participating in clinical trials and patients requiring accurate staging ( 6 ) . generally , high serum titers of ama are seen in more than 90% of patients with pbc ( 7 ) , while low - titers are considered to be not specific ( 8) . in our patient , the titers of ama and the levels of serum cholestatic enzymes were very high , thus establishing the diagnosis of pbc . today , the increased availability of serological tests have resulted in the establishment of diagnosis of pbc in a very early and almost asymptomatic clinical stage , as it was happened in the reported patient ( 9 ) . other immunological serum findings in patients with pbc include the presence of anti - nuclear ( ana ) and anti - smooth muscle antibodies in about 50% of them , a finding that has been linked to a worse prognosis ( 10 ) . the serum immunoglobulin m levels are also elevated without being correlated to the ama levels . in our patient , the serum titers of ana were very high , while the levels of serum immunoglobulin m were also above the upper normal limit . these antibodies have been correlated to elevated iga in patients with pbc suggesting that they might be an indirect sign of an enhanced mucosal immunity ( 11 ) . it has been long suggested that serum tnf- production is related to the severity and prognosis of pbc ( 12 ) . moreover , it has been shown that both the levels of serum tnf- and transforming growth factor- could be reduced in patients with pbc as a result of the treatment with ursodeoxycholic acid ( 13 ) . the patient responded well to the anti - tnf- treatment with etanercept but not with infliximab . arthritis , and serum levels of cholestatic liver enzymes , improved and this improvement persisted during a 30-month follow - up period ( 14 ) . to the best of our knowledge , our patient is the only case of concurrent pbc , cd and raynaud 's phenomenon described so far , in whom the biologic agent adalimumab was administered . adalimumab resulted in a great reduction in the serum titers of ama and cholestatic enzymes and a significant clinical improvement . although the combined results of recent metaanalysis regarding the role of ursodeoxycholic acid in pbc demonstrated no significant favourable effects concerning mortality , pruritus , fatigue , autoimmune conditions , liver histology , and portal pressure , we decided to add this agent in the therapeutic regime because it seems to improve some biochemical variables , like serum bilirubin and jaundice ( 15 ) . thyroid dysfunction is frequently associated with both pbc and cd sometimes predating the diagnosis of both diseases ( 16 ) . an imbalance of t cell regulation can initiate or propagate autoimmunity in both inflammatory bowel disease and pbc . in both disorders genetic , environmental and infectious agents it is concluded that the appearance of pbc during the course of cd , although quite rare , is a reality . extremely rare is also the combination of cd and pbc with autoimmune thyroiditis and raynaud 's phenomenon . it is not clear whether these co - morbidities appeared coincidentally or because they share some common immunopathogenetic elements .

we describe the case of a male patient suffering from long - lasting crohn 's disease of the small bowel who developed thyroiditis hassimoto , raynaud 's phenomenon , and primary biliary cirrhosis , during the course of the underlying bowel disease . it is not clear whether these co - morbidities appeared coincidentally , or because they share some common immunopathogenetic mechanisms . in this patient , crohn 's disease favorably responded to the treatment with an anti - tnf- agent ( adalimumab ) . the serum titers of antimitochondrial antibodyies and cholestatic enzymes considerably reduced during the 3-year treatment with the biologic agent . raynaud 's phenomenon , also , completely disappeared . bearing in mind the possible involvement of tnf- in the pathogenesis of primary biliary cirrhosis , it could be argued that the clinical and laboratory improvement of liver disease , as well as the reduction in serum titers of antimitochondrial antibodies , might be due to the anti - tnf- action of adalimumab . we suggest that it would be worth further investigating the role of biologic agents in the treatment of patients with primary biliary cirrhosis .

Introduction Case Report Discussion

PMC5196023

to reconstruct the ancestral habitat , we first built the phylogeny for our sample using 14 molecular markers ( figures 1a and s1a ) . we then assigned each species to one of four niches based on previous descriptions and our own field observations : terrestrial , marginal aquatic with preference for solid substrates , marginal aquatic with preference for water surface , and open water surface [ 5 , 11 , 12 , 13 , 14 ] . our ancestral character state reconstruction suggests a gradual transition from the ancestral terrestrial to the derived open water habitat ( figure 1a ) , consistent with a previous reconstruction by andersen [ 5 , 11 ] . to test whether adaptation to locomotion on water has favored high speed in the gerromorpha , we first measured maximum speed in 16 species with clear differences in niche preference ( figure 1 ) . we used body length per second ( bl / s ) as a speed unit to account for differences in body size found among species . the terrestrial outgroups and the gerromorpha that specialize in marginal aquatic niches with preference for solid substrates [ 5 , 11 , 12 , 14 ] produce maximum speeds ranging between 6 and 9 bl / s ( figure 1 ; table s1 ) . in contrast , gerromorpha with preference for water surface ( marginal aquatic and open water ; [ 5 , 11 ] ) produce higher maximum speeds ranging between 27 and 113 bl / s ( figure 1 ; table s1 ) . formal correlation tests , after taking phylogeny into account , revealed a strong correlation between maximum speed and niche preference such that species that occupy solid substrates are slow , whereas those that occupy the water surface are remarkably faster and their maximum speeds were invariably higher on water than on ground ( figures 1 , s1b , and s4a ; table s1 ; spearman correlation test with phylogenetic independent contrast [ pic ] rho = 0.88 , adjusted p = 6.5e5 ; similar results were obtained with pearson correlations ) [ 15 , 16 ] . the increase in locomotion speed can not be explained merely by the plastic nature of animal behavior [ 2 , 17 , 18 ] , as lineages with preference for solid substrates were not able to deliver high maximum speeds when tested on water . altogether , these findings corroborate the hypothesis that water surface lifestyle has selected in favor of increased locomotion speed . because speed depends on leg length , we tested whether there is a correlation between leg length , speed , and niche preference in the gerromorpha . the terrestrial outgroups and the species that prefer solid substrates [ 5 , 11 , 12 , 14 ] exhibit low values of leg length to body length ( figures 2 and s2 ; table s2 ) . conversely , species that prefer water surface , including the basally branching mesovelia and the derived veliidae and gerridae [ 5 , 11 ] , exhibit higher values of leg length to body length ( figures 2 and s2 ; table s2 ) . pairwise correlation tests detected a strong and significant correlation between the length of the midlegs and both speed and niche preference , but not between the forelegs or hindlegs and these two variables ( figures 2 and s4a ; spearman correlation tests with pic ; midleg and speed : rho = 0.82 , adjusted p = 5.3e4 ; midleg and habitat : rho = 0.88 , adjusted p = 6.5e5 ) . increased leg length is known to contribute to generating faster movement through increasing the amplitude of strokes [ 20 , 21 ] . when we analyzed how the legs act during locomotion on fluid compared to solid substrates , we failed to detect any difference in the amplitude of leg strokes between water and ground locomotion in species with short midlegs , preference for solid substrates , and that use the tripod gait ( hebrus ) ( figures 3a and 3b ; table s3 ) . however , in species with elongated midlegs , with preference for water surface , and that also use the tripod gait ( microvelia and mesovelia ; movie s1 ) , we detected a significant increase in the amplitude of midleg strokes when these species moved on water compared to when they moved on solid substrates ( figures 3c , 3d , 3f , and 3 g ; table s3 ) . finally we tested gerris , a species that employs rowing ( movie s1 ) and that generates movement through distortion of the water surface and generation and shedding of vortices [ 8 , 22 ] . in this species , we found that the midlegs stretched in a straight shape and executed considerably large amplitudes of stroke , whereas the amplitudes of the forelegs and hindlegs were minimal if any ( figures 3e3h ; table s3 ) . altogether , these results suggest that the preference for various water surface niches have favored increased speed through increasing the length and the amplitude of stroke of the midlegs . leg motion pattern includes two timescales representing a stance phase ( leg pushing against substrate ) and a swing phase ( leg loses contact with substrate ; [ 23 , 24 ] ) . we therefore compared stroke frequency ( number of steps per second ) and pattern of leg motion ( stance and swing phases separately ) between 16 extant species ( figures 4a , 4b , and s3 ) . exclusively terrestrial species in addition to all gerromorpha that occupy solid substrates move with a highly similar stroke frequency ( ranging between 14 and 24 strokes per second [ st / s ] ) , and their pattern of motion is characterized by a longer stance phase ( figures 4a , 4b , and s3 ) . some of these species ( hebrus and hydrometra ) employed the same pattern of leg motion on water as the pattern they used on ground ( figures 4a , 4b , and s3 ) . the gerromorpha that have preference for water surface habitats and that also use the tripod gait move both on water and on ground with a much higher stroke frequency ( varying between 42 and 71 st / s ; figures 4a , 4b , and s3a ) . their motion pattern on ground resembled that of species that prefer solid substrates , except for mesovelia furcata ( figure s3b ) . interestingly , when they moved on water , these species were able to change their motion pattern such that the stance and the swing phases were executed with the same duration ( figure s3c ) . in addition , we detected a strong correlation between stroke frequency , speed , and preference for water surface in the gerromorpha that retain the ancestral tripod gait ( figures 4a and s4b ; spearman correlation tests with pic ; stroke frequency and speed : rho = 0.92 , adjusted p = 3.6e3 ; stroke frequency and habitat : rho = 0.83 , adjusted p = 0.013 ) . these findings are surprising because there is a trade - off between leg length and stroke frequency such that stroke frequency decreases with increasing leg length . the gerromorpha that move on water using the ancestral tripod gait show a significant increase in both leg length and stroke frequency , thus indicating that this trade - off was overcome during the transition to water surface - dwelling lifestyle . in species that employ rowing as a mode of water surface locomotion ( three gerridae and three veliidae ) , stroke frequency was dramatically low ( varying between 6 and 16 st / s ) ( figures 4b and s3a ; table s1 ) despite the high speed that they were able to generate ( varying between 27 and 113 bl / s ; figures 1b and 4b ; table s1 ) . in addition , the pattern of leg motion was reversed in most rowing species compared to species that specialize in solid substrates , such that the stance phase is now shorter than the swing phase ( figure s3c ) . therefore , niche specialization across the gerromorpha was accompanied by changes in the mode of locomotion , leg length , patterns of leg motion , and stroke frequency . species that use the derived rowing gait show a substantially lower stroke frequency when compared to those using the ancestral tripod gait . therefore , we tested whether the evolution of rowing could be associated with increased efficiency during water surface locomotion . first , we inferred ( see supplemental experimental procedures ) the amount of energy spent in the two veliids stridulivelia strigosa and paravelia bullialata , which have a similar body mass ( table s1 ) , generate comparable speeds , but differ in their mode of locomotion ( figure 1 ) . stridulivelia strigosa stroked 9 times to generate a speed of 64 body lengths ( 28 cm ) in 1 s , whereas paravelia bullialata stroked 61 times to generate a speed of 47 body lengths ( 18 cm ) in 1 s ( figures 1b and 4d ) . we calculated that stridulivelia strigosa spent 2.21e5 mj / mg / st ( millijoule per milligram per stroke ) , and paravelia bullialata spent 1.17e5 mj / mg / st ( figure 4c ; table s1 ) . therefore , by taking into account the number of strokes per unit of time , stridulivelia strigosa ( rowing ) spent 2.9e4 mj / mg / s ( millijoule per milligram per second ) , and paravelia bullialata ( tripod ) spent 2.35e2 mj / mg / s , which is over 80-fold higher in the latter ( figure 4d ; table s1 ) . when we extended this analysis to the entire sample of the gerromorpha that transited to the derived water surface habitat , we found that the rowing species consistently spent less energy to generate movement than species that employ the ancestral tripod gait ( figure 4d ; table s1 ; student s t test ; p = 4.6e4 ) . species using the ancestral tripod gait on water spend much of the time on aquatic plants and would only execute bursts of fast movement when crossing free water patches , presumably , to minimize the risk of capture by bottom - striking predators such as fish [ 5 , 10 ] . therefore , the maintenance of the tripod gait may have been advantageous , despite the high - energy demand , as it allows these animals to be versatile . rowing species , however , spend much more time on the open water where they forage , mate , and interact with predators [ 5 , 10 ] . this lifestyle may have increased demands on frequent , fast , and energy - efficient locomotion . these results indicate that the derived mode of water surface locomotion through rowing gait , characteristic of derived species that specialize in open water , is more energy efficient when compared to the ancestral mode using the tripod gait . another important trade - off in walking systems , including arthropods , exists between body size and stroke frequency , such that stroke frequency decreases with increasing body size [ 21 , 26 ] . because the evolution of rowing in derived lineages is associated with a dramatic decrease in stroke frequency , we hypothesized that this decrease may have removed the constraint on body size . to test this hypothesis , we plotted body size by stroke frequency in a total of 25 species that exhibit large variation in these two traits ( figure 4e ) . we found that species with small body size ( < 5,000 m ) may exhibit either low or high stroke frequency ( figure 4e ) . conversely , all species with larger body size ( > 5,000 m ) showed a significantly low stroke frequency ( figure 4e ) . in our dataset , we could not find any species with both high stroke frequency and large body size ( figure 4e ) . litterbugs ( dipsocoromorpha ; figure 1a ; ) are characteristically small , and similarly the ancestral state of body size in the gerromorpha has been undoubtedly small to very small . it is possible that because of their small body size , the ancestors of the gerromorpha were free from the constraint imposed on larger bugs , such that stroke frequency increased and facilitated the transition to water surface life . subsequently , the evolution of rowing combined with the significant increase in the length of the driving midlegs [ 10 , 30 , 31 , 32 , 33 ] , and most likely changes in the associated innervation and musculature [ 22 , 34 , 35 ] , may have contributed to reducing stroke frequency while maintaining high speeds . these structural and behavioral changes may have contributed to the evolution of larger bodies including the largest semi - aquatic bug known , the gerrid gigantometra gigas , whose body length is over 3 cm and leg span over 25 cm . understanding how the evolution of behavior and morphology can be associated with niche expansion and species diversification is a major challenge in evolutionary biology [ 1 , 3 , 17 , 18 ] . we have shown that lineages that remained in the ancestral habitat , composed of solid substrates , produce low maximum speeds and that lineages that specialize in open water surface niches generate significantly higher maximum speeds . this increase in locomotion speed is associated with the evolution of increased midleg length , changes in leg motion patterns , and increased frequency of leg strokes . the subsequent evolution of rowing , as a novel mode of locomotion on the water , removed the requirement for high stroke frequency , reduced energy expenditure , and enabled derived lineages to specialize in open water zones . finally , we have shown how the evolution of rowing , by removing the requirement for high stroke frequency , may have led to the evolution of larger gerromorpha . by uncovering the link between the ecology of the semi - aquatic bugs and the variety of phenotypes they exhibit , this work draws a comprehensive picture of how a combination of behavioral and structural changes can impact the evolutionary trajectory of groups of animals and can be associated with niche expansion and lineage diversification . all species were kept in water tanks at 25c , 50% humidity , and 14 hr of daylight and fed on live crickets . phylogeny reconstruction was conducted using 14 molecular markers with the software geneious version 7.1.9 using plugins mrbayes version 3.2.6 ( one million generations ; 25% burnin ) and phyml version 3.0 , using gtr model with 100 bootstraps . more details can be found in the supplemental experimental procedures . to enable reconstruction , we consolidated andersen s habitat classes [ 5 , 11 ] into the following four : terrestrial , marginal aquatic with preference for solid substrates , marginal aquatic with preference for water surface , and open water surface . each species was assigned to one of these four classes based on previous descriptions [ 5 , 11 , 12 , 13 , 14 ] and on the environment where we caught them . a set of adult individuals for each species were filmed at 2,000 frames per second , both on water surface and on a solid substrate with a grid paper in the background as a calibration scale . to calculate speed , a mean value was extracted from a defined interval plateau phase from velocity curve along each video . stroke frequency was determined as the number of steps performed by the individuals during a given locomotion duration and converted into number of strokes per second . reconstruction of ancestral character states was performed in rstudio version 0.99.486 using a maximum likelihood method adapted to discrete characters ( ace , package ape ; ) and represented using phytools . details about methods of reconstruction and scripts used can be found in the supplemental experimental procedures . kinetic energy ( ek in joules ) used during a stroke is determined using the following expression : ek = 0.5mv , where m is the mass of the insect in grams , and v is the velocity generated during one stroke in meters per second . details and r script used for statistical analyses are provided in the supplemental experimental procedures . ; investigation , a.j.j.c . ; formal analysis , a.j.j.c . , m.e.s . , and m.s .

summaryacquisition of new ecological opportunities is a major driver of adaptation and species diversification [ 1 , 2 , 3 , 4 ] . however , how groups of organisms expand their habitat range is often unclear [ 3 ] . we study the gerromorpha , a monophyletic group of heteropteran insects that occupy a large variety of water surface - associated niches , from small puddles to open oceans [ 5 , 6 ] . due to constraints related to fluid dynamics [ 7 , 8 , 9 ] and exposure to predation [ 5 , 10 ] , we hypothesize that selection will favor high speed of locomotion in the gerromorpha that occupy water - air interface niches relative to the ancestral terrestrial life style . through biomechanical assays and phylogenetic reconstruction , we show that only species that occupy water surface niches can generate high maximum speeds . basally branching lineages with ancestral mode of locomotion , consisting of tripod gait , achieved increased speed on the water through increasing midleg length , stroke amplitude , and stroke frequency . derived lineages evolved rowing as a novel mode of locomotion through simultaneous sculling motion almost exclusively of the midlegs . we demonstrate that this change in locomotory behavior significantly reduced the requirement for high stroke frequency and energy expenditure . furthermore , we show how the evolution of rowing , by reducing stroke frequency , may have eliminated the constraint on body size , which may explain the evolution of larger gerromorpha . this correlation between the diversity in locomotion behaviors and niche specialization suggests that changes in morphology and behavior may facilitate the invasion and diversification in novel environments .

Results and Discussion Experimental Procedures Author Contributions

PMC4540218

according to results of the pre - census house count conducted in 2011 , it is among the fastest growing megacities in the world and perhaps the fastest growing if measured by the annual increment in the population . its estimated 2010 population of 15.4 million is projected to reach 18.04 million by 2015 . 9 per cent of pakistan s total population and 24 per cent of the country s urban population live in karachi . the city also generates 15 per cent of national gdp , 25 per cent of the revenues and 62 per cent of income tax . there are also powerful federal land - owning interests in the city in the form of the karachi port trust , port qasim , customs , civil aviation authority , railways and the armed forces and their various industrial and real estate activities . the city contains 62 per cent of sindh s urban population and 30 per cent of its total population . by contrast , the country s second largest city , lahore , capital of punjab province , contains only 7 per cent of punjab s total population . karachi s large - scale industrial sector employs 71.6 per cent of the total industrial labour force in sindh ; the city produces 74.8 per cent of the province s total industrial output and contains 78 per cent of its formal private sector jobs . before the partition of india that accompanied the creation of pakistan , 61 per cent of karachi s population was sindhi speaking and only 6 per cent was urdu / hindi speaking . however , because of the migration of 600,000 urdu speakers from india between 1947 and 1951 , all this changed . pushto speakers from the north - west and other ethnic groups from other parts of pakistan and india have also continuously migrated in since the 1950s . by 1998 , the census estimated the number of urdu speakers in the city at 48.5 per cent and sindhi speakers had decreased to 7.2 per cent . this makes karachi the non - sindhi speaking capital of a predominantly sindhi speaking province . this is a cause of a major political conflict between the mutahida quomi movement ( mqm ) , which represents the urdu speaking population , and the pakistan peoples party ( ppp ) , which represents the sindhi speaking majority in the province . this conflict expresses itself in disagreement on the form of local government for sindh and on the control of the city s immense resources . as a result of this conflict , the local governance system has been altered four times in the last four years and there is still no consensus in sight . the pushto speaking population , estimated to make up 13 per cent of karachi s population , also has powerful political interests since its members control intra - city , inter - city and cargo - related transport activity . consequently , there is a battle for turf among the mqm , the ppp and the political parties and groups representing the pushto speaking population . this has led to ethnic violence and targeted killings of real estate developers and political workers . the city has remained the centre for supplying arms , ammunitions and food , first to the anti - soviet jihad in the 1980s , then to the jihadists in the war of attrition in afghanistan in the 1990s , and now to north atlantic treaty organization ( nato ) troops in their fight against the taliban . as a result of this involvement in addition , a war economy , supported by drugs , arms and the provision of supplies to the nato troops in afghanistan , has developed in the city . the anti - taliban army action in the north - west has been responsible for the migration of many more pushto speakers , who have migrated for safety to their relations in karachi . taliban fighters have also migrated and established their bases in the city s peripheral low - income settlements . this has led to further targeted killings of pro - liberal anti - taliban activists and political workers . according to the national aliens registration authority ( nara ) of the government of pakistan , karachi has over 1.7 million illegal immigrants . most of these are bangladeshis ( economic migrants ) , muslims from burma ( political refugees ) and afghans displaced by the war in their country . as a result of the security problems created by this situation , investment in karachi has diminished and a large number of industries have relocated to other areas of pakistan and also to other countries such as bangladesh . meanwhile , the absence of an agreed local government structure for the city compounds many of its security - related issues in 1947 , at the time of partition , the built - up area of karachi was just above 100 square kilometres . today its metropolitan area covers 3,527 square kilometres . before partition , land ownership and control all land belonged to the government of sindh , which had given large parcels of land to the karachi port trust , the civil aviation authority , the railways and the army cantonments exclusively for use of their functions . the pre - british properties had freehold ownership ; all other urban properties had a 99-year lease . the rest of the land in the district was vested with the provincial board of revenue ( bor ) and was used as pasture land by the 4,000-plus small goths or villages that were scattered all over it . the agricultural areas of the karachi district consisted of the malir oasis , irrigated by wells from the seasonal malir river , as well as lands along the lyari river , a seasonal stream with adjacent wells for irrigation . karachi s expansion , both formal and informal , has taken place on the board of revenue lands and on a major part of the irrigated farms . as a result , the rural economy of the karachi district has been devastated and much of the pasture land has become a desert . over 60 billion cubic feet of sand and gravel have been illegally removed from the seasonal rivers and streams since 1947 to be used as aggregate for concrete for the building industry . this means that rainwater run - off can not be controlled and aquifers can not be recharged . the social indicators and physical conditions of the rural population and its settlements are far below those of the low - income urban informal settlements in the karachi region . owing to the expansion of the city and the rules and regulations developed over time for converting rural land to urban , the land ownership and control patterns have changed significantly . the city district government karachi ( cdgk ) , created in 2001 , directly controls 30.9 per cent of the land in the city , as well as the 1.9 per cent of land allocated to civilian cooperative housing societies . the rest of the land is controlled by federal agencies , including the military cantonment boards and the defence housing authority , all of which have their own development programmes , building byelaws and zoning regulations , while the city government has its own plans and regulatory institutions . there is no coordination among these different agencies for planning purposes except for dealing with issues related to utilities . the city government , by virtue of being governed by an elected council , is more influenced by community and citizen concerns than the federal agencies . many of them occupied the houses vacated by departing hindus and sikhs , most of whom came from the merchant and trading communities . they had lived in lavish homes and beautiful neighbourhoods in what is today karachi s inner city . houses formerly inhabited by a single rich hindu family became occupied by numerous poor muslim families . to accommodate this increase in population , many community buildings were also occupied and converted into homes . also located in the old city were cargo terminals , the port and the inter - city railway network . wholesale markets and small manufacturing units were also within the old city but formed no more than 3 per cent of its area . as karachi grew , these markets and manufacturing units and their related warehousing also expanded . in the process , a growing population of male - only working class migrants moved in to serve these facilities . most of the two - and three - storey homes were pulled down and replaced by buildings with warehousing and commercial and industrial activities on the ground floor , with workers accommodation on the five to six floors above . these developments meant a large increase in the number of heavy vehicles moving through the narrow lanes of the inner city , resulting in further degradation and traffic congestion . as a result of this degradation , much of the better - off population of the old city relocated to new housing schemes developed by the cantonment boards and the karachi development authority ( kda ) . very few old neighbourhoods survive in the old city and where they do , it is in in a very hostile environment . another result of the migration of 1947 was the unorganized invasions of board of revenue land on the then periphery of the city . as the state reasserted itself , these invasions were halted , and some of the invaded areas were vacated . but inhabitants with political power stayed on and the land they occupied has been regularized , with the result that they have become owners of valuable properties , where high - rise apartments are now being built . the demand for land for shelter remained when the invasions were stopped , and in the 1950s a new phenomenon emerged . this was a joint venture among government officials , police and middlemen to informally occupy bor land and convert it into plots , which were then sold at affordable prices to the homeless residents of the city . the majority of karachiites live in the settlements , known as katchi abadis , that were developed through this process . the state responded to the crisis presented by large numbers of migrants by creating the karachi improvement trust in 1951 , which was responsible for providing basic services and infrastructure in colonies ( settlements ) and neighbourhoods developed for the refugees under the overall supervision of the ministry of rehabilitation and resettlement . the trust was upgraded in 1957 into the karachi development authority , which had a mandate to plan , develop and allocate ( not only for the refugees but for the expansion of the city as a whole ) various types and sizes of plots in the city from land acquired from bor . criteria for allotting such plots were also developed , but a sizeable number were earmarked for the defence establishment and others were left to the discretion of bureaucrats and politicians . the kda process is described in figure 1 ( kda requests bor ; bor notifies for conversion , removes encumbrances and hands over to kda ; kda plans , ballots and transfers to individuals ) . transformation of rural land to urban land source : hasan , a , a noman , m raza , a sadiq , s ahmed and m b sarwar ( 2013 ) , land ownership , control and contestation in karachi and its implications for low income housing , international institute for environment and development , uk . the costs of these plots were kept at far below market prices and as a result many of them , in the absence of a non - utilization fee , were held for speculation and remained vacant for considerable periods of time . in spite of the low cost , lower - income groups found the schemes unaffordable compared to the katchi abadis ( informal settlements ) developed by middlemen . the process of acquiring them was also long and cumbersome , and the lower - income groups felt they were viewed by government officials with suspicion and hostility . one of the earliest attempts at re - housing the migrant population was through the creation of cooperative housing societies in the 1950s . however , the housing societies over time decided to relax their rules and open their properties to the market so as to benefit their members . one of the important developments in karachi was the creation of the defence housing authority ( dha ) , which began as the pakistan defence officers housing society in 1960 . the dha consists of 3,530 hectares of land and is the most elite area of the city . it contains luxury apartments and homes , schools , colleges , clubs , posh shopping centres with designer boutiques , and five- and six - star hotels . the heads of the governing body and the executive board of the authority are pakistan army officers . the dha controls more than 5 per cent of the metropolitan area of the city , and is the most secure and safe area in the city , attracting rich and middle class families from other parts of the city , and further dividing the city into rich and poor areas . the dha also holds about 18 kilometres of coastline and creeks , the nearest coastal areas to the city . the government also made a number of attempts at providing housing through its many master plans . two satellite towns , north karachi and landhi - korangi , were developed at 25 to 30 kilometres from the city centre and the plan was to resettle the refugees in these towns . the planned industrial areas in these satellites were expected to provide the refugees with jobs . however , the industries did not materialize for many years and after 10,000 houses were constructed , the plan was terminated . the doxiadis plan turned a high density , multi - ethnic and multi - class city into a low density sprawl , potentially divided by ethnicity and class . it also created immense transport problems , with regard to both time and cost , for those who worked in the city centre while living in the satellite towns , as a result of which they became poorer . katchi abadis developed over time on the junctions of the roads linking the city to the satellites and the natural drainage channels . another attempt was made through the karachi development plan ( 197585 ) , when 110,000 sites and service plots were developed , ostensibly for low - income groups . in addition , 1,040 hectares were developed as sites and service plots through cooperative societies . these too were of little benefit to the urban poor owing to the extremely slow pace of development , encroachments onto the site ( often by armed gangs ) , opportunities for speculation and the fraudulent relocation of land parcels . the karachi development plan 2000 ( prepared in the late 1980s ) recommended that no new residential development take place until the 300,000-plus empty plots developed under the earlier plans were occupied . the plan never became law and its provisions were violated by both the formal and informal sectors . by 1978 , the katchi abadis of karachi had a population of two million or 55 per cent of the city s population . it aimed at regularizing all katchi abadis existing before the date of enactment , provided they had a minimum of 40 housing units and were not on ecologically unsafe land . the beneficiaries were to pay a small lease charge that was well below the market value of the land . to oversee the process , the sindh katchi abadi authority ( skaa ) 89 per cent of katchi abadis were marked for regularization and the cut - off date for regularization was extended to 20 december 1997 . the state responded to the crisis presented by large numbers of migrants by creating the karachi improvement trust in 1951 , which was responsible for providing basic services and infrastructure in colonies ( settlements ) and neighbourhoods developed for the refugees under the overall supervision of the ministry of rehabilitation and resettlement . the trust was upgraded in 1957 into the karachi development authority , which had a mandate to plan , develop and allocate ( not only for the refugees but for the expansion of the city as a whole ) various types and sizes of plots in the city from land acquired from bor . criteria for allotting such plots were also developed , but a sizeable number were earmarked for the defence establishment and others were left to the discretion of bureaucrats and politicians . the kda process is described in figure 1 ( kda requests bor ; bor notifies for conversion , removes encumbrances and hands over to kda ; kda plans , ballots and transfers to individuals ) . transformation of rural land to urban land source : hasan , a , a noman , m raza , a sadiq , s ahmed and m b sarwar ( 2013 ) , land ownership , control and contestation in karachi and its implications for low income housing , international institute for environment and development , uk . the costs of these plots were kept at far below market prices and as a result many of them , in the absence of a non - utilization fee , were held for speculation and remained vacant for considerable periods of time . in spite of the low cost , lower - income groups found the schemes unaffordable compared to the katchi abadis ( informal settlements ) developed by middlemen . the process of acquiring them was also long and cumbersome , and the lower - income groups felt they were viewed by government officials with suspicion and hostility . one of the earliest attempts at re - housing the migrant population was through the creation of cooperative housing societies in the 1950s . however , the housing societies over time decided to relax their rules and open their properties to the market so as to benefit their members . one of the important developments in karachi was the creation of the defence housing authority ( dha ) , which began as the pakistan defence officers housing society in 1960 . the dha consists of 3,530 hectares of land and is the most elite area of the city . it contains luxury apartments and homes , schools , colleges , clubs , posh shopping centres with designer boutiques , and five- and six - star hotels . the heads of the governing body and the executive board of the authority are pakistan army officers . the dha controls more than 5 per cent of the metropolitan area of the city , and is the most secure and safe area in the city , attracting rich and middle class families from other parts of the city , and further dividing the city into rich and poor areas . the dha also holds about 18 kilometres of coastline and creeks , the nearest coastal areas to the city . the government also made a number of attempts at providing housing through its many master plans . two satellite towns , north karachi and landhi - korangi , were developed at 25 to 30 kilometres from the city centre and the plan was to resettle the refugees in these towns . the planned industrial areas in these satellites were expected to provide the refugees with jobs . however , the industries did not materialize for many years and after 10,000 houses were constructed , the plan was terminated . the doxiadis plan turned a high density , multi - ethnic and multi - class city into a low density sprawl , potentially divided by ethnicity and class . it also created immense transport problems , with regard to both time and cost , for those who worked in the city centre while living in the satellite towns , as a result of which they became poorer . katchi abadis developed over time on the junctions of the roads linking the city to the satellites and the natural drainage channels . another attempt was made through the karachi development plan ( 197585 ) , when 110,000 sites and service plots were developed , ostensibly for low - income groups . in addition , 1,040 hectares were developed as sites and service plots through cooperative societies . these too were of little benefit to the urban poor owing to the extremely slow pace of development , encroachments onto the site ( often by armed gangs ) , opportunities for speculation and the fraudulent relocation of land parcels . the karachi development plan 2000 ( prepared in the late 1980s ) recommended that no new residential development take place until the 300,000-plus empty plots developed under the earlier plans were occupied . the plan never became law and its provisions were violated by both the formal and informal sectors . by 1978 , the katchi abadis of karachi had a population of two million or 55 per cent of the city s population . it aimed at regularizing all katchi abadis existing before the date of enactment , provided they had a minimum of 40 housing units and were not on ecologically unsafe land . the beneficiaries were to pay a small lease charge that was well below the market value of the land . to oversee the process , the sindh katchi abadi authority ( skaa ) 89 per cent of katchi abadis were marked for regularization and the cut - off date for regularization was extended to 20 december 1997 . as a result of the sindh local government ordinance 2001 , the old colonial system of local government was replaced by a three - tier decentralized system in the province , and an elected city council was created and headed by an indirectly elected mayor ( elected by city councillors ) . the karachi development authority , along with all its departments , was absorbed into the cdgk . work on the karachi strategic development plan ( ksdp ) 2020 was commenced and the plan was approved by the city council in 2006 . it aimed at creating residential and commercial development through urban renewal and high- and medium - rise development . new financial districts were also proposed on the northern bypass and along a road that cuts through mangrove marshes . the lands opened by the construction of the northern bypass soon became a battle for turf among the mqm , the ppp and the awami national party ( anp ) , representing the pushto speaking population . objections have also been raised by environmentalists ( for ecological reasons ) to the development on the mangrove marshes . owing to these conflicts and to an unclear and constantly changing local government structure , none of the provisions of the plan have been implemented . however , large - scale investments have been made for construction of signal - free roads , flyovers and underpasses , which are considered to be investment - friendly infrastructure . these investments have not improved traffic conditions in rush hour and have considerably increased the number of fatal accidents , especially of pedestrians and motorcyclists . the world class city vision for karachi coincided with the emergence of a strong neo - liberal lobby in the federal and provincial cabinets . as a result , a massive coastal development programme consisting of high - end apartments , hotels , marinas and commercial plazas was initiated . it was resisted by fishermen s organizations , environmentalists , important citizens from the elite of the city and civil society groups , which argued that the beaches would be lost to the people of karachi as places of recreation and entertainment . the proposals have been shelved , at least for the time being . while this opposition was being voiced , however , the provisions of the ksdp apply only to those coastal areas that come under the jurisdiction of the cdgk and not those coastal areas that come under the control of the karachi port trust and the cantonment boards . since little progress has been made on the provisions of the ksdp 2020 , requirements for the expanding commercial activities of the city are not being met . the cdgk has responded by commercializing the residential areas along the 17 major arteries of the city , thereby increasing their floor to area ratios . in the process , a number of court cases by residents who opposed this move have also been filed in the sindh high court and there have been objections to it from professional bodies as well . with globalization and liberalization , to respond to global demand , looms have been set up to produce yarn that can be processed further in formal sector factories . to save on labour and related transport costs , these looms have not been set up in industrial zones but in low - income settlements , where local people are employed . the factories lack proper light along with ventilation and safety safeguards , and do not follow the building regulations of the sindh building control authority . in violation of court judgements , the armed forces , politicians , city government officials , and the national and international corporate sector are all involved in this illegal activity . the formal sector supplies about 32,000 housing units and an additional 32,000 are built in katchi abadis . meanwhile , 75.5 per cent of karachiites are classified as poor and as such they constitute the majority of the unmet demand . the result has been the continuous expansion of katchi abadis . the absence of provision for the poor is also reflected in residential land usage . karachi s total residential area is 36 per cent of the total area of the district . 74 per cent of it is developed formally for 38 per cent of the population while 22 per cent is developed informally for 62.2 per cent of the population . 88 per cent of the housing stock is located on plots of 120 square yards or less , while houses on 400 to 2,000 square yards constitute only 2 per cent of the housing stock . the government policy on housing is all about accessing the market . to make that possible , the government has transformed the former housing building finance corporation into a private company and has liberalized the loan process in terms of both procedures and better packages . as a result of these reforms , housing finance availability increased by 400 per cent to pakistani rupees ( rs ) 3.5 billion ( us$ 33 million ) in 2003 . other housing - related provisions of the ksdp 2020 include strengthening the katchi abadi improvement and regularisation programme ( kairp ) ; converting sites for single - storey unit houses into apartment complexes ; and , as mentioned earlier , commercializing the main arteries of the city . except for kairp , none of the proposals will provide housing to the urban poor . in spite of these changes , housing finance does not reach the poor because they have no collateral and/or formal sector jobs ( 72 per cent of karachiites work in the informal sector ) to be able to fulfil loan conditions or afford the high rates of interest . meanwhile , the cdgk is continuing to develop residential and commercial schemes that are purchased by the formal sector developers from the sindh building control authority . developers claim that to get building and utility connection approvals and bridge financing from the house building finance company , they have to pay large sums as bribes to officials . this increases costs by about 25 per cent and this cost is passed on to the purchasers . there are a lot of hidden costs as well that the purchasers discover after entering into agreement with the developers . because of the informal procedures involved in the formal building sector , the old developers , who were professional engineers and managers , have been replaced by operators whose origins and sources of money are dubious . they have close relations with political parties and the disputes over land - related transactions among the builders , estate agents , political patrons and government officials are settled through money , connections , police patronage , and even killing and making an example of the opponent . karachi s conflicts and the informalization of the formal sector in housing and development have created a number of problems for citizens who wish to buy , sell or rent accommodation . they are not sure whether or not they are being defrauded and whether or not the schemes they are investing in are legal . they are cautious about renting their apartments to people who are not of their own ethnicity and/or religion and increasingly prefer to live in the neighbourhoods of their own ethnic or religious community . this has created a sense of xenophobia that is dividing the city along class and ethnic lines . owing to these security - related concerns , those who can afford it prefer to live in the cantonment areas , where land records and transactions are properly maintained and the related laws are strictly followed . as a result of pressure from civil society and community organizations , the sindh government has made a decision that there will be no evictions without compensation or relocation except to settlements which contain both social and physical infrastructure ( a policy that applies only to the areas governed by the cdgk ) . the relocation projects developed so far are far from the city centre and important work areas . studies show that as a result of relocation , communities become poorer in both economic and social terms . in addition to these land - related socioeconomic issues , the disposal of sewage is an important concern . karachi generates over 500 million gallons of sewage per day , of which 400 million go untreated into the sea , adversely affecting marine life and the livelihoods of fishermen communities . fishermen s associations are constantly pointing this out and agitating for appropriate development projects and rehabilitation of marine habitats . karachi has also started to flood more frequently since the mid-1980s and especially during the last decade . there is no evidence to date to suggest that this is because of a change in rainfall patterns . research has established that it is related to drainage encroachments of three kinds : encroachments on outfalls to the sea by elite housing schemes ; encroachments by informal settlements on the natural drainage channels , considerably reducing their width ; and encroachments by recent development schemes , formal and informal , upon the natural drainage channels on the periphery of karachi . as a result , water drains very slowly to the sea and also floods those areas where the drainage channels have disappeared . the formal sector supplies about 32,000 housing units and an additional 32,000 are built in katchi abadis . meanwhile , 75.5 per cent of karachiites are classified as poor and as such they constitute the majority of the unmet demand . the result has been the continuous expansion of katchi abadis . the absence of provision for the poor is also reflected in residential land usage . karachi s total residential area is 36 per cent of the total area of the district . 74 per cent of it is developed formally for 38 per cent of the population while 22 per cent is developed informally for 62.2 per cent of the population . 88 per cent of the housing stock is located on plots of 120 square yards or less , while houses on 400 to 2,000 square yards constitute only 2 per cent of the housing stock . the government policy on housing is all about accessing the market . to make that possible , the government has transformed the former housing building finance corporation into a private company and has liberalized the loan process in terms of both procedures and better packages . as a result of these reforms , housing finance availability increased by 400 per cent to pakistani rupees ( rs ) 3.5 billion ( us$ 33 million ) in 2003 . other housing - related provisions of the ksdp 2020 include strengthening the katchi abadi improvement and regularisation programme ( kairp ) ; converting sites for single - storey unit houses into apartment complexes ; and , as mentioned earlier , commercializing the main arteries of the city . except for kairp , none of the proposals will provide housing to the urban poor . in spite of these changes , housing finance does not reach the poor because they have no collateral and/or formal sector jobs ( 72 per cent of karachiites work in the informal sector ) to be able to fulfil loan conditions or afford the high rates of interest . meanwhile , the cdgk is continuing to develop residential and commercial schemes that are purchased by the formal sector developers from the sindh building control authority . developers claim that to get building and utility connection approvals and bridge financing from the house building finance company , they have to pay large sums as bribes to officials . this increases costs by about 25 per cent and this cost is passed on to the purchasers . there are a lot of hidden costs as well that the purchasers discover after entering into agreement with the developers . because of the informal procedures involved in the formal building sector , the old developers , who were professional engineers and managers , have been replaced by operators whose origins and sources of money are dubious . they have close relations with political parties and the disputes over land - related transactions among the builders , estate agents , political patrons and government officials are settled through money , connections , police patronage , and even killing and making an example of the opponent . karachi s conflicts and the informalization of the formal sector in housing and development have created a number of problems for citizens who wish to buy , sell or rent accommodation . they are not sure whether or not they are being defrauded and whether or not the schemes they are investing in are legal . they are cautious about renting their apartments to people who are not of their own ethnicity and/or religion and increasingly prefer to live in the neighbourhoods of their own ethnic or religious community . this has created a sense of xenophobia that is dividing the city along class and ethnic lines . owing to these security - related concerns , those who can afford it prefer to live in the cantonment areas , where land records and transactions are properly maintained and the related laws are strictly followed . as a result of pressure from civil society and community organizations , the sindh government has made a decision that there will be no evictions without compensation or relocation except to settlements which contain both social and physical infrastructure ( a policy that applies only to the areas governed by the cdgk ) . the relocation projects developed so far are far from the city centre and important work areas . studies show that as a result of relocation , communities become poorer in both economic and social terms . in addition to these land - related socioeconomic issues , the disposal of sewage is an important concern . karachi generates over 500 million gallons of sewage per day , of which 400 million go untreated into the sea , adversely affecting marine life and the livelihoods of fishermen communities . fishermen s associations are constantly pointing this out and agitating for appropriate development projects and rehabilitation of marine habitats . karachi has also started to flood more frequently since the mid-1980s and especially during the last decade . there is no evidence to date to suggest that this is because of a change in rainfall patterns . research has established that it is related to drainage encroachments of three kinds : encroachments on outfalls to the sea by elite housing schemes ; encroachments by informal settlements on the natural drainage channels , considerably reducing their width ; and encroachments by recent development schemes , formal and informal , upon the natural drainage channels on the periphery of karachi . as a result , water drains very slowly to the sea and also floods those areas where the drainage channels have disappeared . until 1971 , karachi s transport system consisted of a tramway and large buses operated both by private sector companies and by the karachi transport corporation ( ktc ) , a public sector organization . together these transport systems were unable to cater to the needs of karachi s expanding population . in addition , the ktc was running at a loss and so it was privatized in 1996 . to address the transport issue , the government permitted the introduction of individually owned minibuses , purchased through loans from investors . the investors and the operators ( who eventually become owners after paying off heavy interest loans ) are referred to as the transport mafia by karachiites . at present , over 15,000 minibuses operate in the city . karachi s transport issues have been intensively studied and various schemes involving all forms of transport have been proposed . it is claimed that this is due to lack of political will and opposition from the transport mafia . because of the rising cost of fuel , transport can no longer be a profitable business unless fares are increased . the government can not permit such an increase because it would be unaffordable to commuters . as a result , there has been a decrease in the number of vehicles over the last decade while the commuting public has increased . as a result , travelling has become uncomfortable , time - consuming and expensive ( costs have more than doubled in the last seven years ) . living on the periphery ( where the only land affordable to the poor is located ) now means additional costs in transportation , time and discomfort . it also means that women who look after families can not work , children can not go to proper schools and fathers leave early in the morning and return late at night . it has become cheaper and more convenient to rent in the city rather than owning a home on the periphery . also , land and construction prices have increased relative to incomes since the early 1990s . the first involves families building upward incrementally , often the result of the wish to keep at least one son and his family with them . a study of an apartment complex shows that between 1974 ( when the apartments were built ) and 2010 , the number of persons per room increased from 56 to 1015 due to the growth in the number of family members and the absence of affordable housing alternatives . the third process involves the conversion of small single- or double - storey houses into 4- to 10-storey apartment blocks . here , a developer buys the plot of land on which the house stands , builds an apartment block and , in addition to paying for the land , gives the owner one or two apartments free of cost . most of these apartments are two - room affairs and are rented out through the pugri system . a pugri of rs 700,000 ( us$ 775 ) to rs 750,000 ( us$ 834 ) is demanded , after which the rent for the apartment is reduced to rs 200 ( us$ 2.3 ) per month . without pugri , the rent is rs 3,000 ( us$ 34 ) per month . in either case , an increase of 5 per cent per year in rent is agreed upon . in this way , the developer recovers more than 100 per cent of the costs and at the same time establishes an income . this sort of development is carried out in most cases without formal approval from the building control authorities and it follows no building byelaws and zoning regulations . case studies of settlements in the inner city areas of karachi and previously peripheral areas show that their densities increased from 600 persons per hectare to 4,000 persons per hectare and from 200 persons per hectare to 1,195 persons per hectare respectively . in the case of the inner city settlements , there is no private space for newlywed couples . where high - rises are being developed children can not be supervised , so they form gangs and take drugs . as a result of affordability issues and the processes described above , the housing units being developed are becoming smaller and smaller . the high - rise buildings have no lifts , which has an adverse effect on children , old persons and family relations . such high densities can be achieved without adverse physical and social conditions if properly planned . as a result of the densification through the investor supported process in the inner city areas and the high cost of acquiring land and building a house , the number of rentals is increasing sharply and will probably continue to increase in the future . research establishes that tenants are the most vulnerable of all groups in the housing drama . many live in extremely cramped conditions with five to seven family members in a room . surveys show that they can not feed themselves and their families if they have to pay for both rent and transport . with their low incomes they have no prospect of owning a house at any time in their lives . some tenants surveyed could afford a home in an informal settlement but were not willing to because of the insecurity of tenure or because living in an informal settlement would damage their dignity . rent laws in pakistan are very pro - tenant , but do not apply in low - income settlements where musclemen and dons are the owners and arrangements are informal . rickshaws that carried three persons each . in the last four years over 40,000 three - wheelers , carrying six passengers each and known as qingqis , have been added to the city . their annual turnover is estimated at rs 8.64 billion ( us$ 81.5 million ) per year . the transporters are against the qingqis because they take away business from the transporters ; the police are opposed to them because they create congestion . in october 2013 , the government banned the plying of qingqis but this ban was overturned by the sindh high court . the public has also responded to the crisis by purchasing motorbikes and the market has responded by providing them on hire purchase . in 1990 , there were 450,000 motorbikes in karachi ; in 2004 , there were 500,000 ; and in 2010 , there were over one million . studies show that karachiites , both male and female , have a preference for motorbikes since , apart from the initial capital investment , they are cheaper , faster and more flexible than public transport . one is the relationship among transport , its cost and nature , and location for housing . the second is that , in the absence of subsidies , means of transportation that are alternatives to the conventional transport system are available . as noted , the reported pre - census preliminary results based on house counts show karachi to be the fastest growing large city in the world . between 1998 and 2011 , it grew from 9.8 million to 21.2 million ( 115 per cent ) . no metropolitan region in the world has ever grown so much in so short a time . the results indicate that karachi s urban area has a population of approximately 19.5 million . the city has an average density of approximately 24,000 per square kilometre , making it among the world s most dense megacities . average household size is decreasing in the rest of pakistan , but in karachi there are indications that it rose from 6.7 to 7.3 between 1998 and 2011 , which means that 10 per cent of the recent density increase is within housing units . this is corroborated by the discussion on housing and transport in the previous section of this paper . migration is also a factor , however , and there is every indication that migration to karachi will not only continue but increase . this is because traditional governance systems in the rural areas have collapsed and the rural population increasingly depends on urban produced goods . for this , cash is required and with mechanization and corporate farming methods , the landless labour has no possibilities of earning a livelihood . housing , therefore , is an important priority for both the migrant and longer - term populations , and its appropriate location is related to the availability , cost and nature of transport . the city has considerable land near the centre and places of work , in both the areas controlled by the cdgk and especially the cantonments . except for the cantonments , this land has not been properly demarcated and much of its ownership is unclear . therefore , a land settlement leading to a proper documentation of ownership and categories is required . the main problem here is not the cost of the land ( which communities could pay for over a 15-year period ) , but a strong anti - poor bias in planning and policy that can only be resolved through civil society pressure and pro - poor political movements . it is eating into karachi s potential green areas , whose aquifers also need to be recharged by the building of small check dams and the prevention of sand and gravel removal from seasonal rivers and streams . this ad hoc expansion has to be arrested and replaced by high density housing that will save land for national parks and agriculture and make mass transit systems economically viable . meanwhile , the investor - induced densification of the katchi abadis into apartments needs to be understood , and processes need to evolve to make it safe and environmentally healthy . but none of this will work unless credit on affordable terms is available to the poor for the purchase or building of a home . the security situation in karachi is related to an absence of governance , criminalization of sections of the police , slow legal processes , and an absence of consensus between the different political parties on how to deal with this issue . security concerns , as described in the previous sections , are responsible not only for investment not coming into karachi but also for the departure of industry to other locations in pakistan and to bangladesh and the uae . an important issue is the absence of affordable , fast and comfortable transport that could perhaps make the periphery more acceptable to low - income groups ; the market and the better - off people in the lower and lower middle income groups have tried to overcome this problem . the city today has more than 1.3 million motorbikes ( which means one for every nine adults ) , mostly purchased on hire purchase from the market . apart from capital costs motorbikes are cheaper and faster than karachi s public transport and are flexible ; the number of motorbikes is increasingly rapidly . a decision has to be taken as to whether motorbikes should be promoted by reducing duties on them or not . apart from being affordable they require no subsidy and no loans for purchase , unlike buses . however , if motorbikes and qingqis are to be promoted , then the nature of the current mass transit proposals for the city will have to change and be limited to only a few major corridors of movement . beyond these corridors , qingqis could be a mode of para - transit . to accommodate motorbikes , zoning and environmental regulations especially related to parking , noise pollution and safety local engineering projects to accommodate both of these modes of transport will have to be developed , segregating them from other modes . but before this an understanding of the physical needs of these forms of transport will have to be established , along with an understanding of how they could be utilized in planning for access to appropriately located and affordable land for housing . the issues related to housing , transport , environment and the constraints the land market faces can best be dealt with through the establishment of an effective governance system and an appropriate vision for the city as a commuter and pedestrian friendly city as opposed to a world class city . the security issue , on the other hand , has a regional context although it could improve with the establishment of an effective local government supported by the provincial politicians and establishment . however , for the establishment of a viable governance system , the political parties of sindh will have to rise above their narrow constituency - related issues and politicians will have to think as statesmen for the larger benefit of the city and the province , and through this bridge the ethnic divide to begin with . with such a consensus it may also be possible to make the afghan war - related economy subservient to the provincial administration . karachi has a very active civil society that has developed various appropriate models for the provision of social and physical infrastructure at neighbourhood and sector levels . it has also fought , often successfully , against insensitive projects and for the rights of the katchi abadi dwellers . the involvement of this civil society , because of its knowledge and connections , is essential in the reform process . for this , civil society organizations will have to come together and function as a network rather simply than guarding their turf .

karachi is one of the world s fastest growing large cities . this paper describes the complex processes by which land is ( formally and informally ) made available for housing ( and for commercial development ) , as well as who benefits and how the low - income majority of karachi citizens lose out . it also describes what underpins this especially the political complications in a city that has grown so rapidly , has had fundamental changes in its ethnic composition ( and thus also in its politics ) and has attracted so many illegal immigrants . the paper describes the changes in formal and informal land markets over the last 50 years and the changing responses by government agencies to housing ( and land for housing ) issues . also explored are the connections among land , housing and transport ( which include different processes of densification ) and the complex politics involved . the paper ends with recommendations for land titling , for changes in transport policies , for better use of land already owned by government agencies , for cross - political party agreement on how to address serious security issues ( that are leading to loss of investment ) and for increased political effectiveness of karachi s active civil society organizations .

I. Background II. Changing Land Ownership and Control Since 1947 III. Evolution Over Time a. The response of the state IV. The Situation Today a. Housing V. The DensificationTransport Link VI. Issues

PMC4983308

the resin - to - enamel bond following etching of the enamel surface with phosphoric acid was first described by buonocore in 1955 . it has been suggested that etching duration is a factor that can influence the bond to enamel [ 25 ] , but the optimal enamel etching time varies depending on several parameters namely content and concentration of acid used , physical state of the etchant , duration of etching and rinsing , method of activation , enamel surface instrumentation , patient s age , chemical composition and condition of enamel , primary or permanent tooth , prism structure , previous exposure to fluoride and demineralization . at first , the recommended time of acid etching was 30 seconds for 85% phosphoric acid . in the 1960s , it was extended to 60 seconds . in the 1980s , the recommended etching time was reduced to 30 seconds . extending the etching time to 60 seconds has been shown to produce rougher enamel surfaces with no enhancement of bond strength . meanwhile , some authors recommended reducing the etching time to 15 seconds when 32% to 40% phosphoric acid gel was used , as it did not significantly reduce the bond strength of composite resin or orthodontic brackets to enamel [ 810 ] . uncut enamel on the surface of permanent teeth is more acid resistant to cut enamel because of higher fluoride content , and aged enamel may require an extended etching time up to 60 seconds . most of the manufacturers of adhesive systems have recommended 15 seconds because it saves chair side time without compromising the adhesive performance . this time duration is considered adequate for creating a retentive enamel surface with no difference in the enamel etching pattern or decrease of bond strength to instrumented enamel [ 9,13 , 14 ] . adversely , it is generally believed that shorter etching times may compromise bond strength and durability , but some previous studies have suggested that shorter enamel etching time may be efficient . a laboratory study by beech and jalaly in 1980 showed no significant differences in sbs among five- , 15- and 60-second etching times for bonding orthodontic adhesive resin to enamel . more recently , self - etching adhesives were introduced ; these adhesive systems have an acidic ph that creates some etching effects on the enamel and dentin . while a separate etching step for dentin is widely believed to be unnecessary , there is a general consensus that selective etching of enamel can still have benefits for self - etching adhesives and the latest multi - mode universal adhesives . clinicians are interested in approaches and techniques that can help reduce chair time without compromising the result . in this manner , a reduced etching time of enamel appears to be an interesting topic to explore . in theory , an accelerated interaction of phosphoric acid with enamel should be able to reduce the etching time . from the chemical point of view , etching capability of a reagent can be increased either by increasing the temperature of the etching solution or by changing the concentration of the reactants . absorption of photons at specific wavelengths has been considered as an effective approach for local temperature rise . in this manner , several previous works in the dental field have focused on methods to raise temperature of professionally applied bleaching gels . it has been suggested that the use of light curing units commonly available in dental practices can also be effective in this regard . among the different types available today , leds have the advantage of narrower spectrum which promoting photo - chemical reactions at their respective wavelength ( typically around 470 nm ) without generating excessive heat . there is no report in the literature on the effect of temperature rise induced by light curing units on enamel etching . therefore , the aim of this in - vitro study was to assess the thermal effect of led light curing unit on the enamel etching time . the null hypotheses were that reducing the enamel etching time from 15 seconds to five seconds would not affect the sbs of composite resin to enamel , and that the application of led light simultaneously with five seconds of etching would not affect the sbs of composite resin to enamel . this study was approved by the ethical committee of shahid beheshti dental school ( approval i d : 91512 ) . extracted human permanent molar teeth stored in 0.5% chloramine t solution were cut approximately 1 mm below the cementoenamel junction . after the teeth were mesiodistally sectioned , buccal and lingual coronal enamel surfaces were flattened by gentle polishing using 600-grit silicon carbide papers under running water . group 1 ( g1 ) received 15 seconds of etching . in group 2 ( g 2 ) , the gel was left undisturbed for only five seconds and group 3 ( g 3 ) had a five - second acid - etching time with led light irradiation ( 1200mw / cm ) simultaneously ( le demetron ii , kerr , orange , ca , usa ) . in g1 , 37% phosphoric acid etchant gel ( super etch , sdi , bayswater , australia ) was applied with a small brush using a continuous brushing method , while in g2 and g3 the etchant gel was left undisturbed on the enamel surface . after that , the etchant was thoroughly rinsed with air and water spray , and then the surface was air - dried . on each enamel surface , a thin layer of light cured unfilled resin ( margin bond , coltene , altstatten , switzerland ) was applied with a micro brush . bonding of composite cylinder to the pretreated enamel surface was achieved by packing light cured composite resin ( filtek z 250 3 m espe , st . paul , mn , usa ) with a2 shade into a cylindrical plastic tube ( tygon tubes , norton performance plastic co. , cleveland , oh , usa ) , which was placed on the bonding substrate . the internal diameter of the tube was 0.7 mm and the height was 1 mm . the composite was light cured for 20 seconds . after one hour at room temperature ( 23c ) , the tygon tubes were cut with a scalpel and removed from the substrates ; the specimens were finally stored in distilled water in an incubator ( 37c ) for 24 hours . the sbs test was carried out with a testing machine ( microtensile tester , bisco , schaumburg , il , usa ) . in order to modify the design of this testing apparatus for sbs test , a flat metal plate was used with three perpendicular metal cylinders 0.7 mm in diameter and 8 , 9 or 10 mm in height along a straight line . this plate was attached with cyanoacrylate glue to the microtensile jig in order to enable wire loop sbs test . the force was applied to each composite cylinder at a crosshead speed of 1mm / min using an orthodontic wire ( 0.2 mm in diameter ) looped around the composite cylinder and a metal cylinder until the failure occurred . the load at fracture was recorded and sbs in mpa was calculated by dividing the fracture load ( n ) by the boding surface area ( mm ) . the statistical analysis was performed by comparing the mean sbs values among the groups using one - way anova followed by tukey s hsd post - hoc test in spss 19 at a significance level of 0.05 . the results of sbs test are presented in fig . 1 and table 1 . the mean sbs valuesstandard deviation were 51.282.35 , 40.472.75 and 50.002.59 mpa in groups 1 , 2 and 3 , respectively . the statistical analysis suggested that there was a significant difference among the groups in this regard ( p=0.009 ) ; post - hoc analysis showed that there was a significant difference between groups 1 and 2 ( p=0.013 ) and between groups 2 and 3 ( p=0.032 ) , while there was no difference between groups 1 and 3 ( p=0.932 ) . the error bar of means and 95% confidence intervals of means bond strengths value in the study group . microshear bond strength ( mpa ) of study groups statistical analysis of microshear bond strength ( mpa ) the null hypotheses of the study were rejected as the reduced etching time resulted in lower sbs to enamel , and led light irradiation during five seconds of etching improved sbs to a level similar to that of 15 seconds of etching . these results confirm that application of led light can accelerate and enhance the efficacy of enamel etching during the shortened etching period . the idea of using additional light or heat to activate the bleaching gel dates back to 1918 , when abbot reported the use of high intensity light to increase the temperature of a bleaching agent applied to the tooth surface . when light is projected onto a bleaching gel , it is absorbed and its energy is converted to heat . thus , irradiation of light can have thermal catalytic and photolytic effects . wavelengths with a high absorption coefficient in water and in tooth minerals are readily absorbed by the tooth surface , where heat conversion takes place . asmussen and peutzfeld showed that temperature rise induced by light curing unit was correlated with power density of light . it was concluded that temperature rise for a led device with a power density of 780 mw / cm was around 9c after 20 seconds of irradiation , which was not significantly different from that of a quartz tungsten device with power density of 650 mw / cm . gomes showed that the temperature increase at the light guide tip in units with power density of more than 1000 mw / cm was around 35c after five seconds of irradiation . it should be noted that excessive heat on the tooth surface might damage the pulp in vital teeth . it was shown that that there was a wide range of differences in temperature rise among light curing units ranging from 4.10c to 14.60c over 60 seconds of application . however , the temperature rise after five seconds of application of led light is unlikely to affect dental pulp , since the duration and temperature rise are limited and the absorbance of light by the etching gel will minimize the heat transferred to the underlying tooth structure . this speculation on rapid and superficial temperature rise gains strength from the fact that led irradiation was effective in improving bond strength . reducing the etching time from 15 seconds to five seconds appears to be a valuable approach , but from a clinical and practical point of view , this procedure will require an assistant to readily apply the led light concomitantly with the application of etching gel on the tooth surface . in conclusion , this study showed for the first time that application of phosphoric acid gel to the enamel surface along with led light irradiation decreased the enamel etching time to five seconds without compromising the bond strength .

objectives : the aim of this in - vitro study was to assess the thermal effect of light emitting diode ( led ) light curing unit on the enamel etching time.materials and methods : three treatment groups with 15 enamel specimens each were used in this study : g1 : fifteen seconds of etching , g2 : five seconds of etching , g3 : five seconds of etching plus led light irradiation ( simultaneously ) . the micro shear bond strength ( sbs ) of composite resin to enamel was measured.results:the mean sbs values standard deviation were 51.282.35 , 40.472.75 and 50.002.59 mpa in groups 1 , 2 and 3 , respectively . there was a significant difference between groups 1 and 2 ( p=0.013 ) and between groups 2 and 3 ( p=0.032 ) in this respect , while there was no difference between groups 1 and 3 ( p=0.932).conclusion : simultaneous application of phosphoric acid gel over enamel surface and light irradiation using a led light curing unit decreased enamel etching time to five seconds without compromising the sbs .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

PMC3274684

the laparoscopic roux - en - y gastric bypass ( lrgb ) is currently the preferred procedure in the management of severely obese patients . with increasing numbers of operated patients , late complications internal hernias are the main cause of late postoperative morbidity , causing abdominal discomfort and sometimes leading to bowel obstruction . we present a large series from a single team of surgeons with a high internal follow - up rate . the closure of mesenteric defects involves the suturing or other manipulation of the small bowel mesentery with the risk of haematomas and disturbed anatomy of the enteroanastomosis . against the background of our previous experience , we wanted to test whether a new , simple approach to closing the mesenteric defects could be introduced without a high cost in short - term complications . aleris hospital in oslo , norway , and aleris obesitas skne in kristianstad , sweden , are private clinics with dedicated bariatric units , using the same rotating team of surgeons . indications for surgery were the same as generally accepted as a good standard for bariatric surgery ( european guidelines).table 1patient demographics 2005may 2010june 2010june 2011total number of patients2,4721,630age at operation42 ( 1774)41 ( 1769)bmi at operation43 ( 2968)42 ( 2962)fu time ( months)23 ( 764)follow - up at the time of writing insufficient for analysis follow - up at the time of writing insufficient for analysis previous policy during the period from september 2005 until june 2010 , we performed , in total , 2,472 lrgb without closing the mesenteric defects . new policy from 1 june 2010 to 1 june 2011 , we performed 1,630 lrgb and closed the mesenteric defect in all our patients with our new technique . five ports were used : two 5-mm and two 12-mm ports for instruments and one 10-mm for the camera ( fig . 1 ) . 1position of trocars for gastric bypass position of trocars for gastric bypass the first part of the operation was the same in all cases . a very small gastric pouch was created and the bowel brought up , first as an omega loop in an antecolic and antegastric fashion . following linear stapling of the anastomoses and division of the omega loop , the last step was testing the integrity of the gastrojejunostomy by inflating it with methylene - blue - dyed saline via a nasogastric tube . the closing procedure was started after dividing the omega loop , with the patient still in the reverse trendelenburg position . petersens defect the endo universal 4.8 mm stapler ( autosuture ) was inserted via trocar # 4 ( 12 mm ) . with gentle manoeuvres , a pair of graspers was used to expose the petersen 's defect by lifting the transverse mesocolon ( fig . 2 ) . we closed the petersen 's defect from the root of the mesentery of the roux limb and transverse colon up to the transverse colon itself . to avoid vascular injury , 2closure of petersen s space closure of petersen s space jejunal mesenteric defect the assistant pulled up the end of the duodenal limb in order to expose properly this mesenteric defect as shown in fig . we used the same type of laparoscopic stapler via trocar # 4 also for the closing of this defect . we took care to avoid kinking of the roux - limb at level of the jejuno-jejunostomy.fig . 3closure of jejunal mesenteric defect closure of jejunal mesenteric defect data from all patients during the study period were prospectively collected in a proprietary database . the closing procedure was started after dividing the omega loop , with the patient still in the reverse trendelenburg position . petersens defect the endo universal 4.8 mm stapler ( autosuture ) was inserted via trocar # 4 ( 12 mm ) . with gentle manoeuvres , a pair of graspers was used to expose the petersen 's defect by lifting the transverse mesocolon ( fig . 2 ) . we closed the petersen 's defect from the root of the mesentery of the roux limb and transverse colon up to the transverse colon itself . to avoid vascular injury , 2closure of petersen s space closure of petersen s space jejunal mesenteric defect the assistant pulled up the end of the duodenal limb in order to expose properly this mesenteric defect as shown in fig . we used the same type of laparoscopic stapler via trocar # 4 also for the closing of this defect . we took care to avoid kinking of the roux - limb at level of the jejuno-jejunostomy.fig . data from all patients during the study period were prospectively collected in a proprietary database . we performed 2,472 lrgb in our two hospitals between september 2005 and june 2010 ( table 1 ) . one hundred seventeen patients ( 100 women and 17 men ) with a mean age of 41 years ( range , 2267 years ) developed an internal hernia ( 4.7% ) at a mean interval after lrgb of 13 ( range , 443 months ) . the mean bmi the time of internal hernia was 27 ( range , 1838 kg / m ) . in 46 ( 39% ) cases , the internal hernia was at the jejuno - jejunostomy ; 28 ( 24% ) cases had an internal hernia at petersens defect . forty - three patients were found only to have a mesenteric defect without overt herniation of bowel . the diagnosis of internal herniation was confirmed laparoscopically and the reduction of the hernia and closure of the mesenteric defects undertaken with a non - absorbable running suture . seven were operated on by open surgery ; four patients needed bowel resections ( 70300 cm ) because of severe ischemia . a life - table analysis was performed , which estimates an 11.1% 5-year incidence of clinically significant internal hernia requiring operation ( fig . 4a survival function diagram drawn using the life - table method demonstrates well the relation of internal hernia occurrence to post - operative time . the analysis is based on 1,671 patients operated in oslo with lrgb from 2006 to mid 2010 . one hundred four ( 6.2% overall ) cases had been reoperated for confirmed , clinically significant internal hernia . this diagram illustrates how the internal hernia cases start to occur 3 months post - operatively and seem to be uniformly frequent through the first 3 years . internal hernia operation - free survival by this method is estimated to be 88.9% at the end of a 5-year follow - up a survival function diagram drawn using the life - table method demonstrates well the relation of internal hernia occurrence to post - operative time . the analysis is based on 1,671 patients operated in oslo with lrgb from 2006 to mid 2010 . one hundred four ( 6.2% overall ) cases had been reoperated for confirmed , clinically significant internal hernia . this diagram illustrates how the internal hernia cases start to occur 3 months post - operatively and seem to be uniformly frequent through the first 3 years . internal hernia operation - free survival by this method is estimated to be 88.9% at the end of a 5-year follow - up between june 2010 and june 2011 , we operated 1,630 consecutive lrgb patients where the mesenteric defects were closed using the endohernia stapling device ( endo universal 4.8 mm , autosuture ) . stopwatch timing in 20 cases showed an average time used for closing both mesenteric defects of 1 min and 49 s ( 1:20 to 2:50 ) . four patients ( 0.2% ) in the group with mesenteric closure have been re - operated on because of bowel obstruction at the entero - anastomosis directly related to the closure of this mesenteric defect . all were early in the series and operated within 4 days of the primary operation . twenty - two patients operated by this method have been re - operated on later for other reasons , and in all cases , the two mesenteric defects were examined and complete closure ascertained and documented ( fig . 5).fig . 5jejunal mesenteric defect ( left ) and petersen`s defect ( right ) 11 months after closure with ms endohernia stapling device jejunal mesenteric defect ( left ) and petersen`s defect ( right ) 11 months after closure with ms endohernia stapling device we performed 2,472 lrgb in our two hospitals between september 2005 and june 2010 ( table 1 ) . one hundred seventeen patients ( 100 women and 17 men ) with a mean age of 41 years ( range , 2267 years ) developed an internal hernia ( 4.7% ) at a mean interval after lrgb of 13 ( range , 443 months ) . the mean bmi the time of internal hernia was 27 ( range , 1838 kg / m ) . in 46 ( 39% ) cases , the internal hernia was at the jejuno - jejunostomy ; 28 ( 24% ) cases had an internal hernia at petersens defect . forty - three patients were found only to have a mesenteric defect without overt herniation of bowel . the diagnosis of internal herniation was confirmed laparoscopically and the reduction of the hernia and closure of the mesenteric defects undertaken with a non - absorbable running suture . seven were operated on by open surgery ; four patients needed bowel resections ( 70300 cm ) because of severe ischemia . a life - table analysis was performed , which estimates an 11.1% 5-year incidence of clinically significant internal hernia requiring operation ( fig . 4a survival function diagram drawn using the life - table method demonstrates well the relation of internal hernia occurrence to post - operative time . the analysis is based on 1,671 patients operated in oslo with lrgb from 2006 to mid 2010 . one hundred four ( 6.2% overall ) cases had been reoperated for confirmed , clinically significant internal hernia . this diagram illustrates how the internal hernia cases start to occur 3 months post - operatively and seem to be uniformly frequent through the first 3 years . internal hernia operation - free survival by this method is estimated to be 88.9% at the end of a 5-year follow - up a survival function diagram drawn using the life - table method demonstrates well the relation of internal hernia occurrence to post - operative time . the analysis is based on 1,671 patients operated in oslo with lrgb from 2006 to mid 2010 . one hundred four ( 6.2% overall ) cases had been reoperated for confirmed , clinically significant internal hernia . this diagram illustrates how the internal hernia cases start to occur 3 months post - operatively and seem to be uniformly frequent through the first 3 years . internal hernia operation - free survival by this method is estimated to be 88.9% at the end of a 5-year follow - up between june 2010 and june 2011 , we operated 1,630 consecutive lrgb patients where the mesenteric defects were closed using the endohernia stapling device ( endo universal 4.8 mm , autosuture ) . stopwatch timing in 20 cases showed an average time used for closing both mesenteric defects of 1 min and 49 s ( 1:20 to 2:50 ) . four patients ( 0.2% ) in the group with mesenteric closure have been re - operated on because of bowel obstruction at the entero - anastomosis directly related to the closure of this mesenteric defect . all were early in the series and operated within 4 days of the primary operation . twenty - two patients operated by this method have been re - operated on later for other reasons , and in all cases , the two mesenteric defects were examined and complete closure ascertained and documented ( fig . 5jejunal mesenteric defect ( left ) and petersen`s defect ( right ) 11 months after closure with ms endohernia stapling device jejunal mesenteric defect ( left ) and petersen`s defect ( right ) 11 months after closure with ms endohernia stapling device the clinical presentation of internal hernia ranges from intermittent pain , often in the left upper abdomen through more constant abdominal pain , with or without nausea and vomiting to severe , acute abdominal pain.2 a certain diagnosis can most often only be made by laparoscopy . in case of uncertain abdominal pain in patients with lrgb , our policy is to perform gastroscopy to exclude pouch ulcers and ultrasound examination , revealing cholecystolithiasis . whenever a laparoscopy is indicated in these patients , internal hernia may often co - exist with ulcer or gallstones , which can lead to diagnostic error . a high suspicion of internal hernia is in order , even in the presence of other possible causes of pain such as pouch - ulcer . it seems that the laparoscopic approach is associated with a higher risk of internal hernias than that of open gastric bypass , due to less postoperative adhesions . the reported incidence of internal hernia after lrgb ranges widely , from 0.2% to 9%.27 the incidence also depends on the surgical method , with the retrocolic route of the alimentary - limb causing more internal hernias than the antecolic route.4,8 primary closure of the mesenteric defects seems to reduce the incidence of internal hernia5 , but can also be associated with complications . by suturing the mesentery , closure of the mesenteric defects can also result in increased rate of obstruction at the entero - anastomosis caused by adhesion or rotation of the anastomosis.9 our present cumulative incidence of internal herniation of 4.7% lies within the published limits.7,10 however , we are convinced this incidence is underestimated because the duration of the follow - up is still limited . a lifetime risk in excess of the estimated 5-year incidence of 11.1% is what can be expected by a longer follow - up ( fig . 4 ) . internal hernia is a potentially dangerous complication , causing mortality in one of our patients . therefore , when extracting this significant fact from our database in june 2010 , we decided to commence primary closure of both mesenteric defects in all our patients . our team is currently performing 1,700 lrgb per year ( aleris hospital oslo and aleris obesitas skne ) , thus secondary closure of the defects in 11.1% of patients will demand a great deal of resources . by using the endohernia device , we did not have to change the placement of our trocar ports , and our operation time was only slightly increased ( 23 min ) . mean time from primary operation without mesenteric closure to reoperation for internal hernia was 13 months . the hernia - free survival diagram indicates that the incidence of clinically significant herniations continues from about 3 months post - op through at least the first 3 years . in our view , it is very important at this stage to present our early observations and experience with the methodology as it is already gaining wide popularity . we will certainly report the results when follow - up duration has become appropriate for analysis . however , the primary closures with our method have hitherto only resulted in four reoperations due to kinking of the enteroanastomosis and no mesenteric haematomas . our experience is in line with increasing evidence from several centres that the mesenteric defects should be closed primarily . we can not , however , at this moment , present long - term results on the efficacy of this closure method , but the results so far are promising .

backgroundbowel obstruction due to internal hernia is a well - known complication of laparoscopic roux - en - y gastric bypass ( lrgb ) . increasing evidence supports primary closing of the mesenteric defects , but controversy continues about surgical technique of systematic closure . this paper reviews our experience with internal hernia after lrgb and describes a new method of preemptive closure of the mesenteric defects.material and methodstwo thousand four hundred seventy - two consecutive patients undergoing lrgb from september 2005 to june 2010 were entered into our prospective longitudinal database . the mesenteric defects were not closed . patients entered a 5-year follow - up program , and all who subsequently presented with internal hernia were analyzed . a further 1,630 patients operated on in the last 12 months were subjected to our new technique of closing the defects ; data were entered in our own database as well as in the scandinavian quality registry . follow - up time for these patients is limited.resultsin the first group , 117 patients developed an internal hernia ( 4.7% ) at a mean interval after lrgb of 13 ( range , 443 months ) . four patients needed bowel resections because of severe ischemia . there was one death associated with complication of the internal hernia . in the primary closure group , four patients early in the series had reoperations for kinking of the enteroanastomosis . there have been no mesenteric haematomas encountered.conclusionsinternal hernia should be ruled out in patients with previous lrgb and abdominal pain . our technique for primary closing of the mesenteric defects seems to be safe and is so far promising .

Introduction Patient Material Operative Methods Technique for Primary Laparoscopic Closing of Mesenteric Defects Statistics Results Non-closure of Mesenteric Defects Closure of Mesenteric Defects by Our New Technique Discussion Conclusion

PMC4534688

the proteasome inhibitor ( pi ) bortezomib is used as a first- and second - line treatment of multiple myeloma ( mm ) ( anderson et al . , 2011 ) . 2012 ) main function is to degrade ubiquitinated proteins in a controlled manner ( bedford et al . proteasomes are comprised of a cylindrical core particle ( cp ) capped at each end by a regulatory particle ( rp ) ( lander et al . , 2012 , he et al . , 2012 ) . the rp captures and denatures ubiquitin - marked protein substrates , and translocates their unfolded polypeptide chains towards proteolytic active - sites in the cp 's lumen ( finley , 2009 ) . cp contains three types of active - sites , each of which comprises a peptide - docking area and an exposed catalytic threonine . pis including bortezomib prevent protein hydrolysis by forming covalent adducts with the catalytic threonines of the active - sites ( groll et al . , 2009 , some proteasome activity is necessary for any cell to live ( heinemeyer et al . , 1997 ) , not just mm cells ( craxton et al . , 2012 , although the bortezomib concentrations at which cells of different cancers die differ widely , from low nanomolar to high micromolar ic concentrations , cells of the ( incurable ) b - cell malignancy mm are exquisitely sensitive ( shabaneh et al . , 2013 ) , hence bortezomib 's success in treatment of mm ( anderson et al . , 2011 ) . intriguingly , bortezomib at its low ic concentration causes only a small reduction in proteasomes ' ability to degrade proteins ( kisselev et al . the reduction is small because bortezomib preferentially inhibits the chymotrypsin - like ( ct - like ) active - site , but at ic does not inhibit the caspase - like and trypsin - like active - sites ( fig . s2a ) ( kisselev et al . , 2006 ) ; however , protein substrates can be hydrolysed by any of the three types of active - sites ( kessler et al . , 2001 , kisselev et al . , 2006 ) . thus , the question arises why minimal inhibition of proteasome function suffices to induce apoptosis in mm but not in bortezomib - insensitive cells . several explanations have been proposed , including high proteasome workload in mm cells ( bianchi et al . , 2009 , meister et al . we report that , surprisingly , a ( low ) ic bortezomib challenge which in - vitro minimally inhibits proteasomes in living mm cells severely inhibits proteasomes ' hydrolytic activity . our data suggest that , in living mm cells , a bortezomib - induced structural change in the proteasome ( pitcher et al . , 2014 ) is responsible for this severe degree of proteasome inhibition . antibodies from enzo life sciences : -ubiquitin ( fk2 , pw8810 ) , -cleaved caspase 3 ( pab , adi - aas-103 ) , -20s 7 ( moab ln43 , bml - pw8110 ) , -rpn12/s14 ( pab , bml - pw8815 ) , -rpn10/s5a ( moab s5a-18 , bml - pw9250 ) , -rpt5/s6a ( moab tbp1 - 19 , pw8770 ) , -rpt4/s10b ( moab p42 - 23 , pw8830 ) , -rpt2/s4 ( pab , bml - pw8305 ) , and -5i / lmp7 ( pab , pw8355 ) . antibodies from other sources : -streptag ( qiagen , moab 34850 ) , -parp ( cellsignalling , pab , # 9542 ) and -procaspase3 ( cellsignalling , pab , # 9661 ) . additional reagents : ada - k(biotin)-ahx3-l3-vs , epoxomicin ( enzo lifesciences ) , bortezomib ( millennium / takeda ) , streptactin resin ( qiagen , 30004 ) , ninta resin ( sigma - aldrich , his select hf nickel affinity gel , h0537 ( fig . annexinv/7aad apoptosis staining ( ebioscience 88 - 8007 - 74 ) was used to assess cell viability . enzymes : dnase1 , micrococcal nuclease , rnasea / t1 , rnaseh , s1 nuclease and rnase1 ( fermentas / thermo ) , pde1 from crotalus adamanteus venom ( sigma , p3243 - 1vl ) . caspase inhibitor set iii ( enzo life sciences , alx-850 - 227-ki01 ) , used at 1:500 dilution = 4 m . antibodies were diluted in pbs + 0.5% tween20 for western blotting , pvdf membranes were re - probed multiple times ( yeung and stanley , 2009 ) . myeloma cell lines nci - h929 , kms12-bm , rpmi-8226 , opm2 , and the t - lymphocyte jurkat cell line , were grown in rpmi ( sigma ) , supplemented with 10% v / v fbs , and 1% v / v pen / strep . for fractionation , a cytosol extraction ( ce ) buffer ( 25 mm tris hcl [ ph 7.8 ] , 5 mm mgcl2/edta , 1 mm atp / adp , 2 mm dtt , 150 mm nacl , 0.1 or 0.5% np-40/igepal ) and nuclear extraction ( ne ) buffer ( bakondi et al . , 2011 ) ( 20 mm hepes [ ph 7.4 ] , 420 mm nacl , 0.5 mm edta , 0.5 mm egta , 1 mm dtt , 1 tablet of inhibitor cocktail ( roche , 11 - 873 - 580 - 001 ) per 50 ml ) were used . when using the ne for subsequent affinity - purification , 2 volumes of h2o were added to 1 volume ne to reduce the nacl concentration to physiological levels . for non - fractionated lysate , cell pellets snap - frozen in ln and stored at 80 c were resuspended and combined in 10 volume of ce buffer and passed through a nanodebee ( bee international ) homogeniser at 19,000 psi . proteasome activity was measured basically as described ( kisselev and goldberg , 2005 , vilchez et al . , 2012 ) , using fluorogenic proteasome substrates suc - llvy - amc , ac - rlr - amc , ac - gpld - amc ( enzo life sciences ) for the three types of active - site . hcl , ph 7.5 , 250 mm sucrose , 5 mm mgcl2 , 0.5 mm edta , 2 mm atp and 1 mm dtt ) by passing cells through a 29 g needle ten times . fluorescence ( 380 nm excitation , 460 nm emission ) was monitored continuously on a microplate fluorometer for 1 h at 37 c . to measure for the presence of free 20s proteasomes , a second was set up with the addition of 40 mm bortezomib ; any activity in the bortezomib wells was subtracted from corresponding wells to compensate for unspecific protease activity . the ubiquitinated substrate ( g3p ) ( matyskiela et al . , 2013 ) was kindly provided by dr andreas martin , and used as described . in - vitro degradation with purified yeast ( fig . 3c , d ) proteasomes was done in pbs supplemented with 2.5 mm atp , 2.5 mm mgcl2 , 2.5 mm dtt , and 10% dmso in a total volume of 20 l . reaction was performed for 1 h either at 30 c or 37 c ( fig . 3c , d ) , after which the reaction was stopped by addition of 20 l 2 sds sample buffer and boiled . data plotted were mean of replicates , with error bars plotted of the standard error of mean ( sem ) . research was supported by leukaemia lymphoma research uk ( llr grant10016 to mfk , llr gordon piller studentship award grant11043 to mfk , ak , dp ) , by an mrc - imperial confidence - in - concept ( icic ) grant to mfk , and by the nihr biomedical research centre at imperial college nhs trust , london . nci - h929 mm cells were challenged with a lethal ( 10 nm ) bortezomib concentration for varying lengths of time . cells were then lysed and an artificial fluorogenic peptide substrate was used to measure proteasomes ' hydrolytic activity ( fig . only when it was nearly absent ( < 10% ) , after 68 hour incubation , was an increase in ubiquitin conjugate levels observed ; intracellular accumulation of ubiquitinated proteins indicates that proteolytic workload exceeds proteasome capacity , accumulation being a phenotype which integrates other regulatory mechanisms in the cell including deubiquitinating enzyme activity ( fig . s1a ) . however , when cells were first lysed and then challenged with 10 nm bortezomib , only a 40% reduction in proteasomal ( ct - like ) activity was observed ( fig . s2a : 61.5% ct - like activity left , and as previously reported ( kisselev et al . , 2006 ) ) . a much stronger - than - expected in - vivo inhibitory effect was also observed for the other types of proteasomal active - sites ( figs . predictable explanations for bortezomib 's severe inhibition of proteasomes ' activity in mm cells did not apply : ( 1 ) severe inhibition was not due to prolonged incubation , because exposing purified proteasomes in the test tube to 10 nm bortezomib for longer periods of time still showed modest inhibition in line with fig . furthermore , we identified a mm cell line , rpmi-8226 , in which ct - like proteasome activity dropped very rapidly , within 2 h , to < 10% ( fig . 1e ) ; this time - frame approaches that of the in - vitro test using cell lysate ( fig . ( 2 ) although 10 nm bortezomib induced apoptosis , and apoptosis is known to inhibit proteasomes via caspase activation ( sun et al . , 2004 , adrain et al . , 2004 ) , caspase activation was not responsible for the observed proteasome inhibition : inhibition preceded caspase activation ( fig . 1d ) , cells were alive until 10 h post - challenge as measured by annexin - v staining ( fig . s2b ) , and there was a non - myeloma , t - lymphocyte cell line that did not die with 10 nm bortezomib but in which proteasome activity still dropped well beyond expectation to ~ 20% ( figs . 1f , s2d ; see also lung carcinoma a549 cells : fig . ( 3 ) severe inhibition was not because mm cells actively pumped in and/or retained bortezomib to establish a 10100 fold higher ( fig . we used the irreversible biotinylated inhibitor ada - k(biotin)-ahx3-l3-vs ( kessler et al . , 2001 ) to simultaneously measure inhibition of activity and the degree to which active - sites were inhibited , i.e. biotinylated ( kessler et al . , 2001 ) . we observed in - vivo a discrepancy between active - site inhibition and reduction in hydrolytic activity of those active - sites ( fig . we now report a structural change in the proteasome which does correlate with bortezomib - triggered early shutdown of proteasomal activity : bortezomib - triggered early changes in posttranslational modifications on proteasomal subunits . we recently reported that human nuclear proteasomes carry a constitutive , ctab - page - detectable , posttranslational modification that , in some respects , resembles poly(adp)-ribose ( pitcher et al . , 2014 ) . we reported that exposing mm cells to bortezomib induced changes in these ctab - page - detectable proteasome modifications , exactly at bortezomib 's ic concentration and above ( pitcher et al . , 2014 ) . 2b ctab - page analysis of total cell lysate shows that changes in these modifications of the proteasomal rpn12 subunit occur between 1 h ( rpmi-8226 ) and 4 h ( h929 ) of pi challenge . to examine these bortezomib - triggered changes in finer detail , we combined cell fractionation with affinity - purification of human proteasomes . ( purification is a technical trick that enables modified subunits to also become compatible with , and visible on , sds - page ( pitcher et al . , 2014 ) . ) we generated retrovirally - transduced opm2 mm cells , which expressed a rpn11 subunit tagged n - terminally with a his6strepii strepii tev - affinity cassette . cell fractionation and then affinity - purification ( using streptactin or ninta ) showed that subunits from specifically nuclear proteasomes are extensively modified , as we reported previously ( pitcher et al . , 2014 ) ( fig . 2c : rpn11 subunit , detected with anti - streptag antibody ) . repeating this fractionation - then - affinity - purification procedure , but this time after opm2 cells had been treated with 20 nm ( lethal ) bortezomib for up to 57 h , showed that patterns of modified subunits changed ( fig . generalising , these changes involved ( a ) a reduction in complexity for nuclear proteasomes ( e.g. rpt5 , rpn12 ) , and/or ( b ) the appearance of modified subunit species in the cytosol fraction ( e.g. 5i , rpn12 ) . ( a ) and ( b ) raise the possibility that severe inhibition of proteasome activity in total lysate ( fig . 1 ) may result from changes in proteasome structure which happen both in the cytosolic and nuclear compartments of the cell after cells are challenged with bortezomib . we are currently investigating the exact nature of the proteasome modifications . in order to test directly if changes in these proteasome modifications affect proteasome function , we first searched for commercial enzyme preparations that can digest the modifications of nuclear proteasomes . we discovered that a combination of venom phosphodiesterase-1 and s1 nuclease was efficient in trimming these modifications and collapsing modified rpt2 species into its correct subunit - size species ( fig . next , we combined this enzyme protocol with an in - vitro proteasome - mediated proteolysis assay , which uses a ubiquitinated model protein as substrate ( matyskiela et al . , 2013 ) we scaled up cell growth and affinity - purified proteasomes from total ( i.e. including nuclear ) lysate . proteasomes bound to affinity - resin were mock - treated or treated with pde1/s1 at a sub - optimal ( 20 c ) temperature , after which resin was washed to remove enzymes . most subunits of pde1/s1-treated versus untreated proteasomes were very similar , but for example rpn12 showed pronounced differences ( fig . we then incubated the proteasome preparations with a ubiquitinated model protein ( matyskiela et al . , s4 ) in order to assess the ability of these proteasome preparations to process a ubiquitinated substrate . at low proteasome / substrate ratio at 30 c , however , increasing the ratio overcame this defect , with both preparations degrading substrate equivalently . at even higher proteasome / substrate ratio , and at 37 c , proteasomes processed the substrate towards deubiquitination rather than degradation , and again both preparations behaved equivalently ( fig . in addition , we found that changes in the redox state of the reaction conditions uncovered qualitative differences between enzyme - treated and untreated proteasomes ( fig . 3d ) : in the oxidizing conditions minus dtt , enzyme - treated proteasomes removed streptag epitope ( i.e. substrate ) more efficiently than untreated proteasomes but did not shift down the ubiquitin signal correspondingly , indicating that these enzyme - treated proteasomes only partially digested the substrate protein starting from the tagged carboxyterminus before premature release . in contrast , under reducing conditions , untreated proteasomes were more efficient in degrading substrate than enzyme - treated proteasomes ( see also fig . our data indicate that , under certain experimental conditions , changes in proteasome modifications affect proteasome function , thereby strengthening the case that bortezomib - triggered changes in proteasome modifications within cells also affect proteasome function . in summary , our data reveal a dramatic inhibition of proteasome activity in mm cells after a ( low ) ic bortezomib challenge , and suggest that this inhibition is the compound result of , first , inhibition of a subset of active - sites , and , second , structural changes in the proteasome which further impair hydrolytic activity ( fig . engagement of pis with active - sites changes proteasome conformation and stabilizes the ( distant ) cp rp ( kleijnen et al . , 2007 , park et al . , nov 28 2008 ) and rp - hplic / ubiquilin ( kleijnen et al . , 2000 ) interactions , thus providing a possible signalling mechanism into the cell that may enable active - site inhibition to directly trigger activation of the cellular machinery that then changes the posttranslational modifications of the proteasomes . whereas it has been very difficult to explain why mm cells die from a nanomolar ic bortezomib challenge when assuming that the modest level of proteasome inhibition observed in - vitro holds true in - vivo ( bianchi et al . , 2009 , kisselev et al . , 2006 , shabaneh et al . , 2013 ) , it is not surprising that a myeloma cell with over 95% ct - inhibition and observable proteasome stress ( i.e. accumulation of ubiquitin conjugates , fig . in addition , our data show that bortezomib , in cancer cells which are bortezomib - resistant , does not achieve the same degree of proteasome inhibition as in ( bortezomib - sensitive ) mm cells ( figs . 1f , s2d ) , thus providing a molecular mechanism explaining what differentiates mm from most other cancers which bortezomib can not treat . please note that our data indicate that a high proteasome workload in mm cells ( bianchi et al . , 2009 , meister et al . , 2007 , shabaneh et al . , 2013 ) can not be the primary reason for mm cells ' sensitivity to bortezomib : for this explanation to work , all proteasomes in a cell would need to be fully engaged with no spare capacity left in order for a minimal inhibition of proteasomes to produce proteasome stress ; instead , we observed that mm cells have much spare proteasome capacity , and that reducing capacity even to 20% still did not yield proteasome stress ( fig . its effect on proteasome function may enable future intervention to re - sensitize bortezomib - resistant cells to treatment .

the proteasome inhibitor bortezomib is used to treat multiple myeloma ( mm ) . bortezomib inhibits protein degradation by inactivating proteasomes ' active - sites . mm cells are exquisitely sensitive to bortezomib exhibiting a low - nanomolar ic50 suggesting that minimal inhibition of degradation suffices to kill mm cells . instead , we report , a low bortezomib concentration , contrary to expectation , achieves severe inhibition of proteasome activity in mm cells : the degree of inhibition exceeds what one would expect from the small proportion of active - sites that bortezomib inhibits . our data indicate that bortezomib achieves this severe inhibition by triggering secondary changes in proteasome structure that further inhibit proteasome activity . comparing mm cells to other , bortezomib - resistant , cancer cells shows that the degree of proteasome inhibition is the greatest in mm cells and only there leads to proteasome stress , providing an explanation for why bortezomib is effective against mm but not other cancers .

Introduction Experimental procedures Results Discussion Authorship contributions Disclosure of conflicts of interest Supplementary data

PMC2948936

modified bases in dna pose a severe threat for genome integrity , ( figure 1 ) . dna could be directly damaged by environmental factors such as ionizing radiation , chemical mutagens or endogenous factors , such as oxidative stress and inflammation [ 24 ] . many of these factors also damage nucleotides in dna precursor pools [ 57 ] . additionally , cellular metabolism per se also contributes to the contamination of pools by nucleobase - analogs . base - analogs in the deoxyribonucleosidetriphosphate form are incorporated into dna by dna polymerases and are the source of genetic changes . potent repair systems remove not only the lesions from dna , but also the harmful triphosphates from the dna precursor pools ( figure 1 , [ 1 , 8 ] ) . defects of these protection mechanisms lead to hypermutagenesis or hyperrecombination [ 1012 ] and result in genome instability , which predisposes individuals to diseases like cancer [ 13 , 14 ] . base - analogs are clinically important and are widely used as immunosuppressants as well , antiviral and anticancer agents . the 8-oxoguanine in deoxyribonucleoside triphosphate form can be incorporated into dna by replicative as well as specialized dna polymerases [ 1618 ] . it can form base pairs with cytosine and adenine and , therefore , can lead to transversion mutations . the mutt protein of e. coli hydrolyzes 8-oxodgtp to 8-oxodgmp , preventing incorporation into dna . mutational inactivation of this gene leads to a 10.000-fold increase in the rates of transversions , but no elevation of dna fragmentation is detected in mutt strains . deamination of normal purines , as well as disregulation of the purine biosynthesis , leads to contamination of nucleotide pools with deoxyinosine triphosphate and deoxyxanthine triphosphate . the latter can generate mutagenic derivatives of adenine and guanine , hap and its 2-amino derivative in vitro . hap also can be generated by adenylosuccinate synthase , an enzyme of the de novo purine biosynthesis pathway wherein hydroxylamine is provided instead of aspartate in the reaction with imp . thus , hap can be a natural contaminant of dntp pools [ 23 , 26 ] , however the literature does not report direct measurements of hap derivatives in nucleotide pools . the dhaptp is incorporated into dna by various dna polymerases in vitro [ 28 , 29 ] . overwhelming indirect genetic evidence in microorganisms suggests that the mutagenic effect is mediated by dhaptp incorporation into dna . there is only fragmentary information about the effects of endogenous and exogenous base - analogs , such as hap , in multicellular eukaryotes . there are a number of evolutionarily conserved enzymatic systems that sanitize the nucleotide pool by selectively breaking deoxynucleoside triphosphate forms of base - analog dna precursors , either to monophosphate [ 17 , 30 ] or to nucleoside [ 31 , 32 ] or diphosphate . polymorphisms in the genes encoding protective enzymes are associated with an increased risk of cancer , a predisposition to base - analog - associated adverse drug reactions or a modulation of response to therapy in hepatitis c patients . itpa is a prominent enzyme protecting from base - analogs [ 27 , 39 ] . itpa orthologs from humans , yeast ( encoded by the ham1 gene ) , and bacteria ( encoded by the rdgb gene ) control levels of itp , ditp and dhaptp by hydrolyzing itp / ditp to ppi and imp / dimp [ 27 , 30 , 40 ] . itpa is highly conserved among species [ 26 , 40 , 42 ] . in e. coli , the rdgb mutation is synthetically lethal with the reca mutation abolishing homologous recombination [ 27 , 39 ] . the rdgb mutation sensitizes to the mutagenic and recombinogenic effects of hap in molybdenum - cofactor defective strain background ( another system protecting from hap [ 4345 ] ) because of a massive accumulation of breaks in dna [ 27 , 39 , 46 ] . dna damage is caused by intermediates in the repair of base - analogs in dna by endo v encoded by the nfi gene . a defect in the yeast homolog of itpa , ham1 , leads to elevated hapmutagenesis , but it does not affect spontaneous mutagenesis . the natural substrate , ditp , seems to be non - mutagenic when it is incorporated in dna , because hypoxanthine base pairs properly with cytosine , and yeast apparently do not possess an enzyme able to recognize hypoxanthine , xanthine and hap in dna similar to endo v. therefore , dna with these bases is not nicked and no recombination and dna breakage are seen . inosine and xanthine in dna are recognized in most organisms by a specialized repair system initiated by the orthologs of endonuclease v and elicit dna repair reactions that lead to dna fragmentation and genomic instability when the level of analogues is high [ 3 , 39 , 46 ] . mutations in the human itpa gene lead to the accumulation of itp in erythrocytes but do not show a clear disease phenotype , perhaps due to compensation by other cleansing enzymes [ 52 , 53 ] . human itpa p32 t variant , abolishing the itpa activity in erythrocytes , has been associated in most publications with adverse reactions to purine analogues used in the treatment of blood cancers , transplant , and inflammatory bowel diseases [ 37 , 5459 ] . the itpa p32 t variant causes sensitivity to mercaptopurine used for the treatment of acute lymphoblastic leukemia . knockout of the itpa gene in mice is lethal primarily because of heart failure . primary embryonic fibroblasts exhibit moderate chromosome instability phenotype , and the inviability of the itpa knockout mice suggests that the enzyme performs essential functions in addition to the prevention of the misincorporation of purine base - analogs in dna . if we assume that the role of itpa is the same in humans , the itpa p32 t variant should result in an incomplete loss of activity , at least in the heart . indeed , itpa with the change possesses enzymatic activity but is thermally unstable [ 62 , 63 ] , suggesting a possibility that the levels of the protein and its activity could vary among tissues . in this study , we demonstrate by comet assay that the model purine base - analog , hap , induces dna strand breaks in three different human cell lines . this suggests that there is active recognition of incorporated base - analogs and nicking of dna . the inducibility of the system of hap repair was indicated by the shape of hap dose response on the frequency of dna strand breaks . we suggest and provide the evidence that one component of the repair system is itpa . consistent with this , the level of spontaneous and base - analog - induced dna damage was elevated in cell line p32 t with compromised itpa activity . the levels and distribution of itpa p32 t as determined by immunostaining have been changed in the p32 t cell line . the results suggest that patients with the 94c->a polymorphism in the itpa in addition to drug intolerance might possess increased predisposition to diseases resulting from dna repair defects . the normal , diploid , human lung fibroblast cell line , wi-38 , ( atcc ccl-75 ) was kindly provided by dr . the human fibroblast cell line ( coriell institute biorepository ( gmo1617 ) , called here p32 t , is homozygous for a c > a transversion of nucleotide 94 ( 94c > a ) in exon 2 of the itpa gene . this leads to a proline to threonine substitution at codon 32 ( p32 t ) . these untransformed cell lines were cultivated as a monolayer in mem ( invitrogen , usa ) supplemented with 10% fetal calf serum ( gibco ) and 1 mm sodium pyruvate ( invitrogen , usa ) at 37c in a 5% co2 atmosphere . for the fibroblasts , cells at early passages ( < 25 passages ) were used in all experiments to avoid complications of replicative senescence because wi-38 cells have a mean lifespan of approximately 45 to 60 population doublings . the epithelial colorectal cancer cell line hct116 ( atcc , ccl247 , kindly provided by dr . robert e. lewis , unmc ) , was cultivated in dmem ( invitrogen , usa ) containing 10% fetal calf serum at 37c in a 5% co2 atmosphere . the colorectal cancer hct116 cells are defective in mismatch repair due to a nonsense mutation in the mlh1 gene . for the experiment addressing the specificity of our antibodies against itpa , we transfected colorectal carcinoma 293 cells by pegfp plasmid ( obtained from dr . a. rizzino , unmc ) with itpa cloned into bamh1-ecori sites in frame with egfp . we verified the presence of the 94c->a change in the itpa gene by sequencing of exon 2 amplified from genomic dna ( as exon 24 fragment ) or from rna . for genomic dna isolation , 1 10 cells were harvested for fibroblasts bearing wild - type ( wi-38 ) or mutant ( p32 t ) itpa , and dna was isolated according to the fermentas inc . briefly , cell pellets were lysed with sds and proteinase k. after incubation with nacl , dna was phenol : chloroform extracted and precipitated with ethanol . the cdna synthesis was performed using the qscript dna synthesis kit ( quanta biosciences # 95047 - 025 ) . for amplification of the specific itpa region encompassing the site of the 94c->a change , either genomic dna or cdna was diluted 100-fold and exons 2 through 4 were amplified using exons 2 , 3 , 4 forward and reverse primers and conditions . sequencing of the genomic fragment was performed by the same primers and the sequence of the cdna fragment was performed using itpa - sn 5tcattggtggggaagaagatc and itpa - sc 5aagctgccaaactgccaaa . the sequencing confirmed that the p32 t cell line possesses 94c->a transversions ( figure 2 ) . we also detected the hallmark accumulation of itp in the p32 t cell line by hplc , confirming that itpa activity is compromised in this cell line ( figure 3 ) . the comet assay was carried out under alkaline conditions , as described in the attached trevigen instructions . cells ( wi-38 , hct116 and p32 t ) with or without h2o2 or hap treatment were suspended in 1% low melting point agarose in 1xpbs , ph 7.4 , at 37c and immediately pipetted onto a cometslide . the agarose was allowed to set at 4c for 1030 min and the slide was immersed in a lysis solution at 4c for 50 min to remove cellular proteins . slides were then placed at 0.3 m naoh and 1 mm edta for 45 min at room temperature before electrophoresis at 300 ma for 60 min at 4c . the slides were then washed two times for 10 min each with water and then dehydrated in 70% ethanol for 5 min before staining with 1xsybr green i staining solution . to prevent background dna damage , handling samples and all the steps included in the preparation of the slides for the comet assay were conducted under yellow light or in the dark . comets were randomly captured at a constant depth of the gel , avoiding the edges of the gel , occasional dead cells , and superimposed comets . the percent ( % ) of dna in the comet tail was used in this study as the measure of dna damage . application guide , the average content of dna in the comet tail of untreated normal cells is less than ten percent . the amount of dna in the comet tail was estimated by computerized image analysis of selected comets using cometscore software . the statistical significance of differences was estimated by student 's t - criterion . for metaphase chromosome spreads , wi-38 , hct116 , and p32 t cells ( treated for 23 h with dmso or hap in dmso ) were arrested in metaphase by a 1 h treatment with 0.5 g / ml colcemide ( gibco url , usa ) , treated hypotonically with 0.075 m kcl , fixed three times in a 3 : 1 methanol - acetic acid mixture , spread on glass slides , and air dried . twenty spreads for each culture were analyzed by cytovision software ( genetix corp . , ca ) ; only the total number and size of the chromosomes were determined . hct116 , wi-38 , and p32 t cells were cultivated until subconfluence ( 5 10 cells per plate ) . the cells were harvested and resuspended in the lysis buffer ( 1xpbs containing a protease inhibitor cocktail ( roche biochemicals , in , usa ) , ph = 7.4 ) . the lysate was cleared by centrifugation and protein content determined by bradford reagent from biorad . the lysate equivalent to 100 g of protein was boiled in laemli 's buffer ( invitrogen , usa ) . the protein samples were resolved on a 10%20% tris - glycine gel ( invitrogen , usa ) by sds - page and transferred to nitrocellulose membranes . membranes were blocked overnight in commercially available blocking buffer ( thermo scientific , usa ) . membranes were then incubated in 1 : 500 dilutions of primary antibody against itpa ( the in - house polyclonal antibodies against itpa are described elsewhere ) and gapdh ( cell signaling # 2118 ) for one hour at room temperature . the membranes were then washed with commercially available washing buffer ( thermoscientific , usa ) five times for 10 min each . this was followed by incubation with secondary antibody ( 1 : 1500 dilution ) ( cell signaling # 7074 ) for 40 min at room temperature . this was followed by washing ( 5 times for 10 min each ) and detection by the ecl system ( thermoscientific , usa ) according to the manufacturer 's instructions . the results are presented in figure 4 . a major visible band , corresponding to itpa ( compare with lane with pure itpa ) , is prominent in wi-38 fibroblasts , and cancer hct116 cell extracts ( gray arrow ) but is less pronounced in p32 t cells , as we described before . the intensity of the non - specific 30 kda band is similar in all three cell lines . the position of the immunoreactive band was shifted up , closer to the 30 kda marker , in lane with 6 his - tagged pure itpa - p32 t ( predicted molecular mass 23.6 kda ) . we also analyzed the extract of 293 cell lines transfected with a plasmid expressing the gene for itpa - egfp fusion protein ( 51.4 kda ) and have found that a major band was detected at a position corresponding to 50 kda . for the analysis of itpa induction by hap by western blots we cultivated hct116 cells with or without hap until subconfluence ( 5 10 cells per plate ) . the cells were harvested and resuspended in the lysis buffer ( 50 mm tris , ph = 8.0 , 1 mm pmsf , 10% ( v / v ) glycerol , 0.5% triton x-100 ) and disrupted and processed as before with the following modifications to improve the quality and resolution . the lysate equivalent to 30 g of protein was boiled in laemli 's buffer containing 100 mm dtt and loaded on a 10%20% tris - glycine gel by sds - page . after trial run and coomassie staining the amount of extracts was further adjusted to produce equal amount of loaded protein in the control and treatment and run in the new gel . membranes were incubated in 1 : 2000 dilutions of primary antibody against itpa described above for one hour at room temperature . the membranes were washed with 1x pbst three times for 15 min each , and , incubated with secondary hrp - linked antibody ( 1 : 100000 dilution ) ( cell signaling # 7074 ) for 1 hour at room temperature . membrane was washed three times for 15 min each and signals were detected by supersignal west femto maximum sensitivity substrate ( thermoscientific , usa ) according to the manufacturer 's instructions . cells ( wi-38 , hct116 and p32 t ) with or without hap treatment were fixed with methanol acetic acid mixture ( 3 : 1 ) . all procedures were performed at room temperature . to prevent non - specific binding in the consequent antibody detection , we have used the primary antibodies against itpa described in the previous section and a goat - antirabbit antibody alexa fluor 488 nm conjugated ( thermoscientific , usa ) . both antibodies were diluted 1 : 1000 in 1xpbs containing 5% bsa and 0.05% triton x-100 . slides were washed three times in 1xpbst buffer between incubations with primary and secondary antibodies and after incubations . after washing , the cells were counterstained with dapi , mounted in antifade medium and analyzed by fluorescent microscopy . no fluorescence was detected when primary antibodies were omitted from the protocol , and very low signal was detected when preimmune rabbit serum was used in place of primary antibody , suggesting that fluorescence signals were absolutely dependent on antiitpa antibodies . after the comet assay or immunocytochemistry procedures , cells were examined on a nikon eclipse 80i microscope . images were recorded separately by a ccd device photometrics coolsnap cf and merged using adobe photoshop software . it has been reported previously that treatment of human epidermoid carcinoma cells with 1 mm hap lead to massive chromosome fragmentation ( figure 2 in ) . we examined hap effects on chromosomes in wi-38 , hct116 , and p32 t lines . we studied chromosome spreads of untreated wi-38 , hct116 , and p32 t cells versus the same cells treated with 3.3 mm hap . there were no more chromosomal abnormalities after treatment with 3.3 mm hap than in the untreated hct116 chromosome spreads ( figure 5 ) . we did not observe any striking differences in the rates of chromosomal abnormalities in wi-38 and p32 t chromosome spreads as well ( data not shown ) . it is possible that the cell line used in earlier studies was hypersensitive to hap . we investigated the effect of 24 h treatment by different hap concentrations on the frequency of single - stranded breaks in wi-38 , hct116 and p32 t cell cultures by single cell electrophoresis at ph > 13 . under our experimental conditions and with doses of mutagens used , no substantial cell killing occurred . the percent of dna in the comet tail ( thereafter named tail dna ) of the total dna was used in the study as the estimate of the amount of single - stranded breaks . the mean tail dna was statistically significantly different in the three lines : 8.42% in untreated normal wi-38 fibroblasts , 12.4% in colorectal cancer cell culture hct116 , and the highest 17.6% , in p32 t fibroblast cell culture , a large change for this type of assay two - fold increase over normal fibroblasts ( figure 6(a ) ) . the increased level of tail dna in untreated cells likely indicates persistent unrepaired endogenous damage in hct116 and p32 t cells . wi-38 normal fibroblasts were quite resistant to hap : the highest concentration of 3.3 mm hap increased tail dna two - fold to 20.5% , much less than the positive control hydrogen peroxide ( 42.8% tail dna ) ( figure 6(a ) ) . the induction curve had quite a gentle slope with a small hump / plateau of resistance when the dose increased from 0.66 to 1.32 ( seen clearly in the insert in figure 6(a ) ) . the comet tails in wi-38 cells after treatment with 0.66 mm3.3 mm of hap were the shortest among the variables studied ( figure 6(b ) ) . the response of the hct116 cells to the hydrogen peroxide treatment was similar to that of the wi-38 cells ( tail dna was 42.9% ) . an initial eight percent increase at 0.66 mm was followed by the plateau of around 25% tail dna at doses 1.32 mm2.64 mm ( figure 6(a ) , insert ) . this apparent resistance to the induction of breaks was finally concurred by 3.3 mm of hap and tail dna reached 30.3% ( figure 6(a ) ) . p32 t cells were most sensitive to hap , while the sensitivity to hydrogen peroxide was similar to other cell lines ( 43.4% ) . the lowest dose of hap induced as much tail dna in p32 t as the highest dose in normal fibroblasts ( 21% ) . the 1.98 mm hap produced 30.7% tail dna in p32 t , exceeding the maximum of tail dna induced by hap in other cell cultures studied at the same dose . after 3.3 mm hap treatment of these cells , the level of tail dna reached 52.7% and the tail was much longer than in wi-38 and hct116 under the same conditions ( figures 6(a ) and 6(b ) ) . in summary , hap was moderately active in normal wi-38 fibroblasts , hct116 cancer cells were more sensitive than wi-38 cells and p32 t itpa - deficient fibroblasts were the most susceptible . the presence of the plateau in the dose - response curves for hap indicated that wild - type cells might possess an inducible protection system , which is activated at 0.661.32 mm hap . one possible candidate is itpa and we studied itpa distribution after hap treatment in wi-38 , hct116 and p32 t cells by whole - cell immunostaining with specific antibodies against itpa ( specificity of antibodies has been verified by western blot , because of the shift of detected gfp - tagged itpa to higher position , figure 4 ) . itpa was not readily detected in untreated wi-38 fibroblasts , and cells were stained very weakly ( figure 7(a ) ) . the detected itpa amount increased after the treatment with 0.66 mm and 1.32 mm hap . the untreated hct116 cancer cells were stained with antibody against itpa somewhat more efficiently than wi-38 cells ( figure 7(a ) ) . the itpa amount increased after 0.66 mm and 1.32 mm hap treatment , the latter dose producing strikingly bright cytoplasm ( figure 7(a ) ) . some itpa was detected in untreated itpa - deficient p32 t fibroblasts , similar to hct116 cells ( figure 7(a ) , lower row ) . after treatment with 0.66 mm hap , the itpa amount increased , so induction occurred at lower dose . another type was small granules localized in nuclei ( figure 7(b ) , upper row ) . the third was one big bright granule , typically at the border of nuclei and cytoplasm or localized in the nuclei close to its edge . in hct116 , the heterogeneity was less pronounced and the net structure was not observed ( compare to figure 7(a ) ) . it is possible that it is undetectable due to a small cytoplasmic part of the cell and very bright fluorescence . in p32 t some brighter regions adjacent to nuclei were present but they were unstructured ( figure 7(b ) , lower row ) . we also have used western blot analysis in hct116 cells to confirm and quantify induction of itpa by hap ( figure 8) . clearly , hap treatment leads to up to 2.6-fold elevation of the amount of itpa . it is known that hap is mutagenic , clastogenic and carcinogenic in hamster cells [ 67 , 68 ] . using comet assay the most logical explanation of this and other currently available data is that the base - analog was incorporated into dna and either human endo v homolog ( encoded by the loc284131 , see description of closely related mouse homolog in ) or other an unknown glycosylase / endonuclease recognized incorporated hap and incised dna strands ( figure 1 ) . analysis of the dose - response curves revealed a hump / plateau region indicative of the induction of a cellular protective response / repair system . the haptreated wi-38 fibroblasts with wild - type itpa had the lowest rates of dna breaks , and untreated wi-38 cells had the lowest levels of spontaneous breaks among cell lines studied . the hump in the dose - response curve indicated that there is an induction of the repair activity at 0.661.32 mm hap . it remains to be determined whether this effect is related to the defect of a mismatch - repair system . the levels of dna breaks by hap in hct116 cells were higher than in wi-38 fibroblasts and the analysis of the dose - response curve indicated that the repair system most likely was activated completely only by 1.32 mm hap . the p32 t itpa - deficient fibroblasts were the most sensitive to the induction of dna breaks by hap treatment . we propose that deoxyribonucleoside triphosphates of hap , usually removed by itpa in wild - type cell lines , remain in the detectable amount in the dna precursor pools of itpa p32 t fibroblasts . this observation correlates well with the accumulation of itp only in this cell line ( figure 3 ) . it is known that itpa p32 t produced ectopically protects bacteria and yeast from hap to the same extent as wild - type itpa , but the level of itpa p32 t in human cell extracts is much lower than in normal fibroblasts . apparently , the human repair system does not work properly in p32 t cells alleviating dna damage by downstream repair enzymes . the spontaneous level of dna breaks in this cell line was the highest , raising the possibility that there is persistent endogenous dna damage in these cells . it is tempting to speculate that the damage is caused by endogeous ditp / dxtp . we have found that itpa levels increased significantly upon treatment with hap in all the investigated cells , although it was significantly lower in the itpa p32 t fibroblasts than in wi-38 and hct116 cells . p32 t itpa - deficient fibroblasts responded by the production of itpa to much lower doses of hap , because the destruction of haptp is less efficient in these cells and effective concentrations of substrates for the itpa are higher . the antibody staining revealed a net - like structure adjacent to nuclei and other , granular structures in the wi-38 normal fibroblasts , when itpa is induced by hap . this is consistent with previous analyses of the localization of itpa to purine biosynthetic complexes . it has been shown previously that human enzymes involved in de novo purine biosynthesis ( for example , htrifgart protein , and formylglycinamidine ribonucleotide synthase , prpp amidotransferase , hpaics , adenylosuccinate lyase and hatic ) , colocalize and cluster in human cell cytoplasm . usually these clusters localized throughout the whole cytoplasm volume but there were one or two big clusters on the border of nuclei and cytoplasm . one possible interpretation of our results is that itpa , the enzyme of purine salvage pathway , constitutes a part of this purinosome . the candidate structure is a big fluorescent granule . hypothetically , the net - like structure described here may represent a factory checking quality of dntp pools . it is possible that this abnormal itpa distribution is one of the reasons for a lack of itpa function in p32 t individuals , despite almost normal activity of the enzyme . taken together , our results suggest that human cells possess a repair system for purine base - analogs similar , in part , to bacteria . some dhaptp is presumably incorporated into dna , and subsequent repair of hap leads to dna strand breaks .

base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress , inflammation , and aberrant nucleotide biosynthesis . human inosine triphosphate pyrophosphatase ( itpa ) hydrolyzes triphosphates of noncanonical purine bases ( i.e. , itp , ditp , xtp , dxtp , or their mimic : 6-hydroxyaminopurine ( hap ) deoxynucleoside triphosphate ) and thus regulates nucleotide pools and protects cells from dna damage . we demonstrate that the model purine base analog hap induces dna breaks in human cells and leads to elevation of levels of itpa . a human polymorphic allele of the itpa , 94c->a encodes for the enzyme with a p32 t amino - acid change and leads to accumulation of nonhydrolyzed itp . the polymorphism has been associated with adverse reaction to purine base - analog drugs . the level of both spontaneous and hap - induced dna breaks is elevated in the cell line with the itpa p32 t variant . the results suggested that human itpa plays a pivotal role in the protection of dna from noncanonical purine base analogs .

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