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in 2002 china was labelled the next wave country by the national intelligence council in the us as china , together with four other countries ( ethiopia , india , nigeria and russia ) , was expected to have a serious hiv problem with a large population being at risk of hiv infection by 2010 ( intelligence community assessment , 2002 ) . it was projected that by the year 2010 ten to twenty million people could become infected with hiv in china ( intelligence community assessment , 2002 ; eberstadt , 2002 ; zhang , 2004 ; gu & renwick , 2008 ) . however , in contrast , according to the recent unaids estimates , 740,000 ( 540,0001,000,000 ) people ( adults and children ) lived with hiv / aids in china in 2009 ( unaids , 2010 ) . the estimated prevalence rate in china in 2009 was 0.05% ( 0.040.07% ) , and the estimated hiv prevalence among adults of reproductive age ( 1549 years ) was 0.1% ( unaids , 2010 ) . this large difference between projections and the real situation might suggest that projections were calculated inaccurately as they were based on unrealistic assumptions and potentially used poor quality data . alternatively , this difference could suggest that china is a success story that has managed to slow down and possibly control the hiv epidemic . despite the estimated low prevalence of hiv in china , the absolute number of people living with hiv / aids is large and growing and moving beyond high - risk groups to the general population , mainly through the heterosexual route of transmission ( ungass , 2008 ; merli & hertog , 2010 ) . china is characterized by demographic features unique in the world , such as oversupply of males for sexual partners and rapid fertility decline since the 1970s , which affect the chinese population age structure ( merli & hertog , 2010 ) . this oversupply of males and associated potential squeeze in the number of marriage and sexual partners will lead to an increase in occurrence of sex with commercial sex workers and will become an important contributor to the further spread of hiv through heterosexual transmission ( merli & hertog , 2010 ) . china 's early stage of the hiv epidemic , its large and diverse population , as well as its unique demographic features , make china an interesting case for studying changes in hiv awareness in a general population of women of reproductive age . this paper systematically examines the evolution of hiv awareness among women in china between 1997 and 2005 . the results of this analysis will feed directly into the evaluation of existing hiv prevention programmes in china and might suggest implications for both general policy as well as targeted interventions . these findings might also be helpful for designing and assessing the effectiveness of interventions for countries where the hiv epidemic is still at an early stage and where epidemics are still not stabilized ( for example , for countries in easter europe and central asia ) ( unaids , 2010 ) . in the situation where a vaccine against hiv is not available , the main force against the spread of hiv is prevention . hiv prevention efforts can only be effective if a combination of behavioural , structural and biomedical dimensions is considered when hiv prevention interventions and programmes are designed and implemented ( merson et al . to increase hiv awareness and knowledge is one of the main components of the behavioural dimension jigsaw ( snelling et al . , 2007 ) , and this is especially crucial in countries such as china where the hiv epidemic is still at an early stage . one of the main tasks of hiv prevention worldwide is the reduction of risky behaviours . information , i.e. hiv awareness and hiv knowledge , is a prerequisite of risk reduction behaviour as in order to change behaviours it is necessary to know that the behaviour is risky ( fisher & fisher , 1992 ; ross & kelaher , 1993 ; ingham , 1995 ; ambati et al . , 1997 ) . therefore , it is important to study the dynamics of hiv awareness in different cultural contexts . hiv awareness is typically measured by one question ( have you ever heard of hiv / aids ? ) in surveys . the more mature an hiv epidemic becomes , the higher is the level of hiv awareness observed in countries , and with time it approaches saturation among different groups of people . however , the early stage of the hiv epidemic in china allows the study of hiv awareness as it has not yet reached saturation point and still differs substantially by population strata . this paper acknowledges the importance of both hiv awareness and hiv knowledge but focuses specifically on hiv awareness as the first step in the investigation of hiv awareness and hiv knowledge in china . hiv awareness is consistently increasing all over the world ( snelling et al . , 2007 ) . the change in hiv awareness can be attributed to a number of reasons such as change in political commitment , interventions , individual change and change in population structure or population characteristics , as well as to the increased level of the hiv epidemic in the country , which could foster increased awareness , or the increased level of access to antiretroviral treatment for hiv - positive people . it is important to establish what drives the change in hiv awareness in different contexts , as well as what brings larger change in hiv awareness . political commitment plays a crucial role in the fight against hiv in every country of the world ( un , 2001 ) . in 2001 a crucial report hiv / aids : china 's titanic peril was produced by the un theme group on hiv / aids in china ( unaids , 2002 ) . this document provided an update of the hiv / aids situation in china and concluded that china was at the brink of a serious hiv epidemic and that the government should take the situation seriously , otherwise it could be too late ( unaids , 2002 ) . at that time the chinese government denied the existence of the problem in the country . a significant change in the government 's attitude towards the problem of hiv / aids was observed in december 2003 . wen jiabao , the chinese premier , brought hiv / aids to the governmental agenda and the problem of hiv / aids has been given the highest priority . following from this , many positive changes have happened in the field of hiv / aids in china ( zhang , 2004 ; he & rehnstorm , 2005 ; wu , 2005 ; gill & okie , 2007 ) . the priority to advance hiv awareness and hiv knowledge among all groups of people was already stressed in the 1994 cairo international conference on population and development ( icpd , 1994 ) . in response to this programme services in china have started providing education on hiv / aids for rural women , young people , as well as for migrants ( fang & kaufman , 2008 ) . this was later reiterated in the unfpa country programmes in china , which aimed to promote client - centred reproductive health and family planning services in china and to assess the state of reproductive health and family planning in the country , as well as to help design necessary interventions in order to achieve goals formulated at the icpd . as a part of the intervention efforts , two surveys were conducted in 2003 and 2005 in 30 selected counties within 30 provinces across china under the fifth country programme ( cp5 ) ( unfpa , 2003 ) . hiv prevention and improvement of the level of hiv awareness and knowledge among the general population were the core themes for the cp5 ( unfpa , 2003 ) . one of the recommendations from the cp5 baseline report was that intervention measures should place an additional emphasis on issues related to hiv / aids ( li et al . , the evaluation report concluded that there was an urgent need to increase the level of hiv awareness and hiv knowledge among different strata of the chinese population . young people , both males and females , as well as women ( especially those unmarried and currently married ) from the western region of the country ( which is the least developed part of china ) , as well as those from the central region , were identified as being least aware of hiv as 30% of them had never heard of hiv / aids ( li et al . , 2004a ) . these lagging - behind groups of people were then specifically targeted in the areas covered by the cp5 interventions . a major focus of the cp5 interventions was the dissemination of standard education materials on hiv / aids prevention , including wall posters , leaflets , pamphlets and video materials explicitly focusing on prevention of hiv transmission and reducing stigma and discrimination and providing information about care and treatment of people living with hiv / aids . health care providers , including doctors , nurses and community health workers , were provided with training sessions on hiv prevention and treatment compliance strategies , including partner referral and follow - up and counselling , as well as on emphasizing the relevance of reducing stigma and discrimination against those living with hiv / aids . however , the majority examined the hiv awareness of specific groups of the population such as university students ( li et al . , 2004b ; , 2007 ; zuo et al . , 2007 ) , health care workers ( wu et al . , 1999 ) , std clinic patients ( wang et al . , 2001 ) and migrants or the so - called floating population ( dong et al . , 2003 ; guo & xu 2006 ; zhu & tan , 2006 ; zhou et al . , 2007 ) . other studies investigated the hiv awareness of pregnant women ( wu et al . , 2007 ) , female sex workers ( lau et al . , 2002 ; huang et al . , 2004 ; hesketh et al . , 2005 ; qi et al . , 2007 ) , market vendors ( cao et al . , 2006 ) and entertainment industry workers ( pei & wang , 2007 ) . some of these studies did not use appropriate sample designs and , therefore , the generalization of the results , even within these groups , was questionable . a small number of studies examined the level of hiv knowledge in china in the general population at one point in time ( manchester , 2002 ; holtzman et al . , 2003 ; zhu & tan , 2006 ) . while data exist , to date there has not been any systematic analysis that has examined the changes in hiv awareness in the general population of women in china over time . therefore , the analysis presented in this paper will address this gap by conducting a thorough investigation of the evolution of hiv awareness in china among women of reproductive age . data sources that were available and which asked a standard hiv awareness question ( have you ever heard of hiv / aids ? ) were used for the analysis . four cross - sectional datasets for women of reproductive age ( 1549 years ) were analysed : the china national population and reproductive health survey 1997 , the china national family planning and reproductive health survey 2001 , as well as the china unfpa reproductive health and family planning surveys 2003 ( cp5 baseline survey ) and 2005 ( cp5 endline survey ) . the first two surveys are representative of the whole of china , whereas the other two surveys are representative of 30 counties that were purposefully selected from the 30 provinces in china ( jiang , 2000 ; pan et al . , 2003 ; li et al . , 2004c ; li et al . , 2008 ) . hiv prevention was not a governmental priority in china before 2003 but the situation changed and prevention efforts became more pronounced in the whole country after 2003 . the 1997 survey used a two - stage sample : first 1041 communities were selected , then 15,213 women were selected within those communities ( jiang , 2000 ) . the sample of women in this survey is self - weighted ( jiang , 2000 ) . the quality of the data collected met all objectives the survey team had ( jiang , 2000 ) . the 2001 survey was conducted in the same 1041 communities that were selected for the 1997 survey ( pan et al . , the quality of data was also satisfactory according to the survey team ( pan et al . , 2003 ) . the last two surveys ( cp5 baseline and endline surveys ) are not nationally representative . they were conducted in 30 purposely selected counties from the 30 provinces in china , and are representative of those 30 counties in china . in every province reproductive health and family planning knowledge , including hiv awareness and knowledge of individuals and knowledge about family planning services , were the primary interests of the survey . the 2003 survey used a stratified multi - stage sample of women aged 1549 years ( li et al . , 2004c ) . thirty counties were stratified into the three regions ( eastern , central and western ) and then regions were stratified by urban and rural areas . the sample size for each region was equal . within each of the three regions , systematic random sampling with probabilities proportional to population of women aged 1549 within each township was used for the selection of townships within each stratum in the baseline survey ( li et al . then within each township four communities were selected using systematic random sampling with probabilities proportional to the population of women aged 1549 years within each community . at the final stage of the sample selection a systematic random sample of 20 women was selected from a list ordered by age of all women aged 1549 years within each selected community ( li et al . , 2004c ) . in the 2005 survey a main change in sampling occurred : the sampling procedure was moved from the sample of individuals to the sample of households ( li et al . , 2008 ) . the 2005 survey used a stratified multi - stage sample of households ( li et al . thirty counties were first stratified into the three regions ( eastern , central and western ) and then regions were stratified by urban and rural areas . the sample size for each region was equal . within each of the three regions , systematic random sampling with probabilities proportional to the number of households within each township was used for the selection of townships in the endline survey ( li et al . , 2008 ) . then within each township four communities were selected using systematic random sampling with probabilities proportional to the number of households within each community . in the final stage , a systematic random sample of fifteen households was selected for a list ordered by local geography , and all women of reproductive age were considered eligible for the survey in these households ( li et al . , 2008 ) . women were asked similar question at two points of time ( 2003 and 2005 ) , which allows trends to be studied over time in the context of the 30 selected counties . however , the surveys are cross - sectional in nature and different samples of women were selected for the two surveys . due to the different representativeness levels of the two sets of surveys ( 19972001 and 20032005 ) and the similarity of the representation levels within these two sets , these two periods of time will be considered separately in this analysis . binary logistic regression analysis was used to model hiv awareness at different years in china and to initially estimate the effects of different factors on the probability of being aware of hiv in the chinese context among women of reproductive age at four points in time . 1 for women who reported being aware of hiv and 0 otherwise . in our case , as the comparability between surveys is more important than the model selection process , all variables that are comparable within the two sets of surveys ( age , residence , education , ethnicity and marital status within the first set of surveys ; and the same variables together with region , occupation and media exposure within the second set of surveys ) were included in the analysis . as comparability between surveys is of main importance , only the main effects discussed above were included in the models and interactions were not tested for significance or added to the final models . decomposition analysis is a widely used technique in different disciplines ( oaxaca , 1973 ; gomulka & stern , 1990 ; nielsen , 1998 ; canudas romo , 2003 ) that helps to separate the essential components and to explain the phenomena under study in more detail . researchers investigating different aspects of health have recently started employing the technique ( charasse - poul & fournier , 2006 ; jacobson et al . , 2007 ; gonzlez lvarez & clavero barranquero , 2009 ; stewart williams , 2009 ) . the main aim of this is to disentangle the two main components driving a total change : a change in a population structure and a change in relationships or effect sizes ( coefficients ) , i.e. do more people have a university degree this year than in a previous year versus does a higher proportion of people with only primary education know about hiv this year than in a previous year ? the total change contains both elements and decomposition analysis allows the separation of these two different components . decomposition analysis has been applied to different models : linear models ( oaxaca , 1973 ; blinder , 1973 ) , probit models ( gomulka & stern , 1990 ) , logit ( nielsen , 1998 ) and other models . in this study its use for the logistic regression model has advantages when compared with linear models as further decomposition analysis , i.e. decomposition of the second component into effects of specific characteristics of individuals , is possible . oaxaca & ransom ( 1999 ) in their note highlighted that detailed decomposition analysis is misleading for linear regressions due to the fact that the arbitrary choice of reference category in categorical variables will lead to differences in estimated intercepts and coefficients . however , this problem will not affect decomposition analysis using logistic regression as calculated predicted probabilities for different observations are not affected by the parameterization of the model as attention is not specifically on coefficients that might change because of changes in baseline categories . the main interest of this study was to decompose the total change in the proportions of women being aware of hiv at different points in time ( 1997 and 2001 ; 2003 and 2005 ) into the change due to difference in population structure and due to the change in hiv awareness that is influenced by the external or environmental factors such as interventions and political commitment . detailed decomposition analysis was conducted in order to establish which sub - groups experienced the larger changes in effect sizes , or in other words which variables contribute most to the total change in relationship or effect sizes between hiv awareness and characteristics of women . if no interventions are implemented or interventions are not effective , similar changes in probabilities are expected to be observed across different groups of women . however , if interventions are effective , larger changes are expected within the groups that were targeted by specific interventions . the bootstrap method ( efron & tibshirani , 1993 ) was used to estimate confidence intervals for the probabilities of being aware of hiv in order to determine if observed changes in probabilities of being aware of hiv at different points in time are statistically significant or if they are observed simply by chance . the literature suggests that the following factors are associated with hiv awareness in the chinese context : sex , age , education , income , place of residence , type of job or occupation and media exposure ( manchester , 2002 ; dong et al . , 2003 ; li et al . , 2004b ; tan et al . , 2007 variations in hiv awareness by the selected characteristics ( if available ) are presented in table 1 . since in the chinese context hiv awareness was initially promoted through family planning services , which tend to focus mostly on married women ( qian et al . , 2004 ) , it was considered to be important to include marital status as an explanatory variable when modelling hiv awareness and , therefore , hiv awareness by marital status is also presented in table 1 . table 1.hiv awareness by selected characteristics of women , china , 19972005percentage distribution by variablespercentage aware of hiv within groupsvariablecategory19972001200320051997200120032005total sample size15,21339,58684007356residencerural76.774.671.468.555.065.775.991.8urban23.325.428.631.591.493.493.795.5age group ( years)<2012.010.66.96.568.775.483.793.8202932.625.022.021.570.179.084.696.2303930.338.042.439.764.174.081.393.7405025.026.428.732.351.563.777.689.6ethnicitynon - han9.39.614.013.642.850.767.389.2han90.790.486.086.465.675.083.593.5educationno education21.416.69.24.328.436.545.870.6primary22.928.628.324.354.262.171.285.4junior secondary32.236.141.645.079.485.288.296.0senior secondary and above16.618.721.026.494.397.196.098.7marital statusnever married20.416.311.610.573.280.084.795.6married or remarried78.182.086.887.561.371.380.792.8divorced or widowed1.41.71.51.964.369.382.788.4regioneastern37.639.488.494.5central30.428.182.692.2western32.032.672.592.2occupationagricultural work44.140.072.789.6non - agricultural manual work18.516.285.195.5non - agricultural intellectual work9.612.297.998.8housework and others21.022.784.592.8 in school and out of work6.99.091.296.7radioregularly14.711.191.896.0occasionally32.526.988.295.4never52.862.074.191.4tvregularly78.185.486.194.4occasionally19.513.667.884.9never2.51.033.276.0newspaperregularly24.425.095.099.1occasionally34.136.290.795.9never41.638.881.286.7 hiv awareness by selected characteristics of women , china , 19972005 the results in table 1 suggest that , as expected , hiv awareness increased with time among the different groups of women . they also suggest that the percentage of women with no education decreased from 21.4% of women being illiterate in the 1997 dataset , to 16.6% in the 2001 dataset , and also from 9.2% in the 2003 data to only 4.3% in the 2005 data ( see table 1 ) . this change can partially be attributed to policies related to the eradication of illiteracy in china and partially to differences in the survey representativeness levels between the two sets of surveys . urban composition : the proportion of women reported being from rural areas also decreased with time ( see table 1 ) . therefore , differences in the level of hiv awareness can be attributed partially to variations in population structure . it is hypothesized that , due to the short time period between two sets of consecutive surveys , the difference in population structure is not expected to be large between 1997 and 2001 or between 2003 and 2005 and , therefore , changes in population structure may not be the main driver of the change in hiv awareness between two consecutive surveys . table 2 presents results of the binary logistic regression models for the four different surveys . hiv awareness was the outcome variable . for models 1 and 2 ( for the 1997 and 2001 surveys respectively ) the following variables were used as explanatory variables : residence , ethnicity , age , education and marital status of women ; for models 3 and 4 ( for the 2003 and 2005 surveys respectively ) the same five variables were used for the analysis , together with region , occupation and media exposure ( radio , tv and newspaper ) , as these variables were available in the 2003 and 2005 surveys and they were also reported as being important in the literature in determining hiv awareness ( manchester , 2002 ; dong et al . , 2003 ; wu et al . , 2007 ) . in both models 1 and 2 the following variables were found to be significantly associated with hiv awareness : respondent 's residence , age , ethnicity and education . marital status was not significant in either model . the fact that the same variables were found to be significantly associated with hiv awareness in 1997 and 2001 suggests that , between 1997 and 2001 , there were no specific interventions that were targeting specific groups of women , or if interventions were in place , they were not effective . if interventions were in place and were effective , hiv awareness would be expected to become more homogenous among groups of women and fewer variables would be expected to be associated with hiv awareness in 2001 . education , as expected , was found to be an important predictor of hiv awareness among women in both models . the higher the level of education , the higher the odds of being aware of hiv . women of han ethnicity ( which represents more than 90% of the population of china ) were found to be more likely to be aware of hiv in both models than woman who belong to ethnic minorities . the same was correct for women from urban areas when compared with women from rural areas . slightly different relations between age and hiv awareness are observed at different points in time : in 1997 women aged 2029 had a significantly higher probability of being aware of hiv than younger women , whereas in 2001 all women older than 20 had a higher probability of being aware of hiv than younger women . table 2.results of logistic regressionsmodel 1 ( 1997)model 2 ( 2001)model 3 ( 2003)model 4 ( 2005)variablecategory ( se) ( se) ( se) ( se)constant1.804 ( 0.092)1.928 ( 0.062)0.0145 ( 0.233)1.775 ( 0.442)residencerural ( ref.)0.0000.0000.0000.000urban1.250 ( 0.071)***0.980 ( 0.047)***0.590 ( 0.106)***0.218 ( 0.135)age group ( years)<20 ( ref.)0.0000.0000.0000.00020290.465 ( 0.101)***0.649 ( 0.077)***0.033 ( 0.210)1.061 ( 0.461)*30390.170 ( 0.111)0.534 ( 0.084)***0.031 ( 0.227)0.889 ( 0.479)40500.076 ( 0.114)0.373 ( 0.086)***0.033 ( 0.227)0.526 ( 0.479)ethnicitynon - han ( ref.)0.0000.0000.0000.000han0.727 ( 0.065)***0.921 ( 0.040)***0.843 ( 0.090)***0.593 ( 0.134)***educationno education ( ref.)0.0000.0000.0000.000primary0.983 ( 0.051)***0.998 ( 0.034)***0.720 ( 0.096)***0.644 ( 0.149)***junior secondary2.013 ( 0.058)***2.168 ( 0.039)***1.327 ( 0.109)***1.691 ( 0.173)***senior secondary and above3.002 ( 0.103)***3.578 ( 0.080)***1.835 ( 0.175)***2.399 ( 0.289)***marital statusnever married ( ref.)0.0000.0000.0000.000married or remarried0.015 ( 0.093)0.071 ( 0.073)0.768 ( 0.183)***0.139 ( 0.439)divorced or widowed0.333 ( 0.202)0.106 ( 0.126)0.827 ( 0.329)*0.421 ( 0.524)regioneastern0.391 ( 0.087)***0.019 ( 0.128)central0.220 ( 0.083)**0.098 ( 0.124)western ( ref.)0.0000.000occupationagricultural work ( ref.)0.0000.000non - agricultural manual work0.041 ( 0.098)0.162 ( 0.171)non - agricultural intellectual work1.054 ( 0.278)***0.424 ( 0.365)housework and others0.348 ( 0.088)***0.085 ( 0.125)in school and out of work0.201 ( 0.206)0.250 ( 0.315)radioregularly ( ref.)0.0000.000occasionally0.100 ( 0.132)0.028 ( 0.227)never0.789 ( 0.123)***0.122 ( 0.206)tvregularly ( ref.)0.0000.000occasionally0.545 ( 0.073)***0.711 ( 0.112)***never1.412 ( 0.168)***0.894 ( 0.299)**newspaperregularly ( ref.)0.0000.000occasionally0.004 ( 0.132)1.082 ( 0.290)***never0.872 ( 0.131)***1.542 ( 0.294)******p<0.001 ; * * p<0.01 ; * p<0.05 ; ref . : results of logistic regressions * * * p<0.001 ; * * p<0.01 ; * p<0.05 ; ref . : reference category . in model 3 it was found that hiv awareness was significantly associated with women 's residence , ethnicity , education , marital status , region , occupation and exposure to radio , tv and newspapers . in model 4 it was found that hiv awareness was significantly associated only with ethnicity , education and exposure to tv and newspapers . in 2005 , significant difference at the 5% level in hiv awareness is observed between women aged 2029 and younger women , with younger women having a lower probability of being aware of hiv than women aged 2029 . education , as expected , was found to be an important predictor of hiv awareness among women in both models , with higher education indicating higher odds of being aware of hiv . in both models women of han ethnicity have higher odds of being aware of hiv than their non - han counterparts . region , residence , marital status , occupation and exposure to radio effects were significant in 2003 but they were not significant in 2005 . the trend in reduction in the number of significant variables from model 3 to 4 suggests that hiv awareness becomes more homogeneous across the 30 counties in china , with the main differential factors being the level of education , ethnicity and exposure to tv and newspapers . these results might suggest that interventions and campaigns that targeted specific groups of women , including the unfpa interventions , which were introduced in these countries in 2003 , were effective . as there were other interventions in place in china and the design of the surveys does not allow controlling for them ( no controls are available ) , it is unfortunately impossible to assess the specific effect of the unfpa intervention in this study . the differences in the predicted probabilities of being aware of hiv between 1997 and 2001 , and between 2003 and 2005 , that were obtained using the four logistic regression models discussed earlier were decomposed . average predicted probabilities during each year of interest can be found along the diagonals of both parts of the table . the upper part of the table presents results for 1997 and 2001 and the lower part those for 2003 and 2005 . when moving down the column , average predicted probabilities of hiv awareness are shown , obtained by applying the same logistic regression equation coefficients to different samples of women ( first 1997 logistic regression equation coefficients were obtained for 1997 data and then they were applied to 2001 data , then 2001 logistic regression coefficients were obtained for 2001 data and then they were applied to 1997 ; the same process was conducted for 2003 and 2005 data ) . the difference between these two predicted probabilities is due to the change in population structure . when going across the row , average predicted probabilities of hiv awareness are shown , obtained by applying different logistic regression equation coefficients ( s ) to the given sample . the difference between these two predicted probabilities is due to the change in effect sizes . table 3.results of decomposition analysis1997 s2001 schange due to s1997 structure0.6350.7000.0652001 structure0.6600.7270.067change due to structure0.0250.0272003 s2005 s2003 structure0.8120.9120.1002005 structure0.8370.9290.092change due to structure0.0250.017 results of decomposition analysis table 3 shows that the average predicted probabilities of hiv awareness increase with time . the total absolute change in hiv awareness between 1997 and 2001 is 0.092 or 9.2 percentage points . the change that can be attributed to the change in population structure between 1997 and 2001 is 2.5 percentage points , and between 2003 and 2005 it is also 2.5 percentage points . as hypothesized , this change is not large due to the short period of time between the two consecutive surveys and not enough time for the population structure to experience substantial changes . the rest of the change can be attributed to the change in relationships or in the effect sizes between hiv awareness and explanatory variables over time . between 1997 and 2001 this change is 6.7 percentage points , and between 2003 and 2005 it is 9.2 percentage points . in order to identify which subgroups experienced the larger changes in effect sizes in relation to hiv awareness , a detailed decomposition analysis was conducted . if various interventions effectively target specific groups of women , larger changes in hiv awareness within these groups would be expected across time . on the other hand , if no interventions are implemented or if they are not effective , similar or no changes in probabilities of hiv awareness are expected to be observed across different groups of women . figures 1 , 2 and 3 present the results of the detailed decomposition analysis , which can help visualize the differences and similarities in hiv awareness of different groups of women . the first two figures show the average predicted probabilities of hiv awareness for different groups of women together with confidence intervals obtained using the bootstrap method . figure 1 presents predicted probabilities of being aware of hiv for specific groups of women that were obtained by applying the two different logistic regression equation coefficients ( 1997 and 2001 ) to the 2001 sample . it shows a change in relationships or effect sizes between 1997 and 2001 for different subpopulations defined by the five main demographic characteristics of chinese women . no groups experienced substantial changes in awareness between 1997 and 2001 and the changes are more or less evenly spread across different groups , with slightly bigger changes observed in groups that were lagging behind ( rural women , women with no education and only primary education and women between 30 and 49 years of age ) . these relatively similar and not substantial changes can be attributed to the fact that the government was denying the existence of the problem at that time and , therefore , not much was happening in the field of hiv prevention . 1a ) might be partially attributed to the information about dangerous blood donation in rural parts of china that existed at that time ( zaller et al . the average probability of being aware of hiv is greater for women who were married or remarried and widowed or divorced in 2001 than in 1997 when compared with women who were never married ( fig . this might be explained by the fact that information regarding hiv was available for women mainly through family planning services ( fang & kaufman , 2008 ) and never married women were not using them . this finding might also indicate that family planning agencies in china continue to play an important role in hiv prevention . 1.change in effect sizes for groups of women in china between 1997 and 2001 . 2.change in effect sizes for groups of women in china between 2003 and 2005 . 3.differences between changes in effect sizes at two points in time for specific groups of women in china for two sets of surveys . differences between changes in effect sizes at two points in time for specific groups of women in china for two sets of surveys . figure 2 presents the predicted probabilities obtained by applying the two different logistic regression equation coefficients ( 2003 and 2005 ) to the 2005 sample . it shows change in effect sizes between 2003 and 2005 for different subpopulations defined by ten main characteristics of chinese women in 30 purposefully selected counties in china . the figure shows that between 2003 and 2005 there was a substantial change in hiv awareness for rural women ( fig . 2b ) , those with no education ( large change of 22 percentage points ) and with only primary education ( fig . 2c ) , as well as for women residing in central and western regions ( fig . the larger change in effect sizes can be seen in the western region as well as in the central region , which might be attributed to the effectiveness of different prevention governmental and non - governmental programmes and campaigns , including the unfpa interventions , as special attention was directed to women from central and western parts of the country . with time , lower awareness groups are catching up and the gaps between the groups are closing . a substantial change in hiv awareness between these two years also happened to women who reported no exposure to radio , tv and newspapers ( fig . this suggests the effectiveness of interventions other than ones that were introduced at the local and country levels to reach women through mass media . confidence intervals for differences in mean predicted probabilities between two points in time , which were obtained using the bootstrap method , suggest that all differences in probabilities of being aware of hiv between 1997 and 2001 , as well as between 2003 and 2005 , are significant , with the exception of widowed or divorced women for both sets of surveys , women who were in the age group below 20 years for the years 1997 and 2001 and women who reported having a professional job for the years 2003 and 2005 . figure 3 shows the differences between the changes in size effects at two points in time for specific groups of women for two sets of surveys . when conducting this comparison it is important to bear in mind that different variables were used for the two decomposition analyses ( the region , occupation and media exposure variables were missing in the analysis of 1997 and 2001 surveys ) and the two sets of surveys represented different populations . however , the main purpose of this graph is to show the general pattern in the level of change of the size of the effects between the two sets of consecutive surveys . 3 shows substantial differences for specific sub - groups of women between the two sets of surveys . for all groups of women except women with senior secondary education and above and divorced or widowed women , the change between 2003 and 2005 was higher than between 1997 and 2001 . the change was almost the same for women with the highest educational level and for women from urban areas . the reason for this might be an already high level of hiv awareness among these two groups at 1997 and 2003 . this figure shows substantial difference in changes for women from non - han backgrounds and from rural areas , for women with none or with only primary education , and for women aged 1529 years and those who were never married . the findings suggest that greater changes happened to these specific groups between 2003 and 2005 than between 1997 and 2001 , which might in turn suggest effectiveness of interventions that were introduced in the country after 2003 . the results might also suggest that either never - married women started being included in reproductive health services provision or they were targeted specifically through some interventions . as mentioned earlier , young people were specifically targeted by the unfpa interventions as they were least aware of hiv . figure 3 shows that for women below the age of 20 the change is substantially larger between 2003 and 2005 than between previous years , which might suggest that some of the existing interventions , including the unfpa interventions , might have reached younger women and succeeded in increasing the hiv awareness among this group . figure 3 shows the effect of interventions in the whole country and in 30 counties selected for the cp5 . if interventions and campaigns were not effective , we would have expected more or less the same changes across all groups . if efforts and measures triggered by the political commitment to fight hiv were not effective , the level of change would have been expected to be less substantial between 2003 and 2005 and would have been more similar to the level between 1997 and 2001 . the analysis confirms that china has responded well to generating population - level awareness of hiv in a short period of time . the results of the study suggest that the situation with hiv awareness in china is similar to other parts of the world , with the level of hiv awareness increasing over time . the patterns of hiv awareness increase by intra - population groups are similar to the ones observed in the african context in the 1990s ( ingham , 1995 ) or obtained in earlier studies conducted in the chinese context ( wu et al . , 1999 ; , 2003 ; holtzman et al . , 2003 ; huang et al . , 2004 ; li et al . , 2004b ; hesketh et al . , 2005 ; guo & xu , 2006 ; wu , 2006 ; zhu & tan , 2006 ; pei & wang , 2007 ; qi et al . , 2007 ; tan et al . , 2007 ; wu et al . , 2007 ; zhou et al . , 2007 ; zuo et al . , 2007 ) . in 2005 , education remained one of the main factors associated with hiv awareness , the other main factors being ethnicity and exposure to tv and newspapers . a smaller part of the observed change in hiv awareness over time is attributed to change in population structure , in other words to demographic change , but a larger part is due to changes in environment such as in political commitment , successful interventions and health promotion campaigns . however , part of the change in hiv awareness between the two sets of consecutive surveys is also due to the differences in representativeness levels of surveys and designs , which are different for different surveys used for the analysis . with time , lower awareness groups are catching up and gaps between groups with initially different awareness levels are closing . the joint efforts of interventions and programmes introduced in the whole of china and in 30 counties , including the unfpa interventions , have been effective in closing the gaps between groups between 2003 and 2005 , as suggested by the larger increase in hiv awareness in specific groups of women between 2003 and 2005 than between 1997 and 2001 . the increases observed between 1997 and 2001 are more evenly spread between groups of women with different demographic characteristics , whereas between 2003 and 2005 increases are more pronounced among the groups that were targeted by different interventions and programmes , including the unfpa interventions . the main limitation of the results obtained through the decomposition analysis is the limited number of variables that were available for the analysis for models 1 and 2 . it is possible that there are other factors that could explain variation in the response variable but were not included in the logistic regression models because of lack of data . however , despite this limitation the results yielded a better understanding of evolution of hiv awareness in china . also , decomposition analysis is a good technique that allows the separation of different components that contribute to the total change in hiv awareness between different points in time . yet another limitation of the present analysis is that the results of the decomposition analysis between 2003 and 2005 can not be generalized to the whole country , but are indicative of the possibility that if the right interventions are in place , the level of hiv awareness can be substantially improved within a short period of time . the analysis also could not establish any causal associations because of the cross - sectional nature of the data available for the analysis . the analysis would have benefited from including a study that collected information about all hiv awareness and hiv knowledge - related interventions that took place in china before 2005 . it would also have helped to identify if there are specific interventions in place in china targeting groups such as msm ( men who have sex with men ) , which might be excluded from the provision performed through family planning services . the results of this type of study would help to isolate specific effects of the interventions , including unfpa interventions , which were implemented in china between 2003 and 2005 , and to assess the effectiveness of these interventions rather than just speculate about their potential effectiveness . finally , another limitation of the study is that it is not possible to isolate the specific effects of the unfpa interventions due to the limitations of data such as absence of control observations . as mentioned earlier , the main target groups for the unfpa interventions were women who were lagging behind in terms of hiv awareness and hiv knowledge specifically , unmarried young women , rural women , and women who live in the central and western regions of china . knowing the targets of interventions between 2003 and 2005 , it might be suggested that the interventions by unfpa , together with other interventions and programmes in place , were effective and helpful in closing the gaps between lower awareness groups and higher hiv awareness groups of women between 2003 and 2005 . the fact that the government changed its attitude towards hiv and many interventions and campaigns started in the country in the year 2003 might also have contributed to the change in effect sizes . this study 's analysis suggests the importance of political commitment and the importance of educational campaigns and interventions for improvements in hiv awareness . the paper presents an application of a regression decomposition analysis technique in the context of hiv awareness in china . decomposition analysis provided a useful tool for assessing the change in levels of hiv awareness in china using cross - sectional data when longitudinal data were not available . it also allowed the assessment of macro - level change in hiv awareness in china over time by isolating the change in hiv awareness that was not due to change in individual demographic characteristics of women . it could be speculated that increased hiv awareness among different groups of women in china might have contributed to the adoption of protective behaviours such as avoidance of illegal blood donation and increased rate of condom use , which in turn might have influenced the decrease in speed of spread of hiv infection across the country . however , in order to establish these links , further work needs to be conducted .

summaryhiv prevalence in china is less than one per cent , but the absolute number of people living with hiv / aids is large and growing . given the limited scope of any potential cure for hiv , prevention plays a crucial role in controlling the epidemic . this paper examines the evolution of hiv awareness among women in china between 1997 and 2005 . a regression decomposition analysis technique was used to disentangle the two main components driving a change in hiv awareness . the results show that hiv awareness has increased over time in china . the gaps between groups are narrowing over time and lower hiv awareness groups are catching up with the higher awareness groups . in 2005 education remained one of the main factors associated with hiv awareness , the other main factors being ethnicity , exposure to tv and newspapers . the increases in hiv awareness observed between 1997 and 2001 are similar between groups of women with different demographic characteristics , whereas between 2003 and 2005 increases are more pronounced among specific groups of women such as women from rural areas , women from western parts of the country , women who belong to ethnic minorities and those with no education or with only primary education . the results suggest that the main driver of the observed change in hiv awareness over time in china is change in the environment such as in political commitment , interventions and campaigns rather than change in population structure .

Introduction Background Methods Results Discussion

despite the estimated low prevalence of hiv in china , the absolute number of people living with hiv / aids is large and growing and moving beyond high - risk groups to the general population , mainly through the heterosexual route of transmission ( ungass , 2008 ; merli & hertog , 2010 ) . this paper systematically examines the evolution of hiv awareness among women in china between 1997 and 2005 . the change in hiv awareness can be attributed to a number of reasons such as change in political commitment , interventions , individual change and change in population structure or population characteristics , as well as to the increased level of the hiv epidemic in the country , which could foster increased awareness , or the increased level of access to antiretroviral treatment for hiv - positive people . the main aim of this is to disentangle the two main components driving a total change : a change in a population structure and a change in relationships or effect sizes ( coefficients ) , i.e. the main interest of this study was to decompose the total change in the proportions of women being aware of hiv at different points in time ( 1997 and 2001 ; 2003 and 2005 ) into the change due to difference in population structure and due to the change in hiv awareness that is influenced by the external or environmental factors such as interventions and political commitment . it is hypothesized that , due to the short time period between two sets of consecutive surveys , the difference in population structure is not expected to be large between 1997 and 2001 or between 2003 and 2005 and , therefore , changes in population structure may not be the main driver of the change in hiv awareness between two consecutive surveys . the fact that the same variables were found to be significantly associated with hiv awareness in 1997 and 2001 suggests that , between 1997 and 2001 , there were no specific interventions that were targeting specific groups of women , or if interventions were in place , they were not effective . the trend in reduction in the number of significant variables from model 3 to 4 suggests that hiv awareness becomes more homogeneous across the 30 counties in china , with the main differential factors being the level of education , ethnicity and exposure to tv and newspapers . no groups experienced substantial changes in awareness between 1997 and 2001 and the changes are more or less evenly spread across different groups , with slightly bigger changes observed in groups that were lagging behind ( rural women , women with no education and only primary education and women between 30 and 49 years of age ) . confidence intervals for differences in mean predicted probabilities between two points in time , which were obtained using the bootstrap method , suggest that all differences in probabilities of being aware of hiv between 1997 and 2001 , as well as between 2003 and 2005 , are significant , with the exception of widowed or divorced women for both sets of surveys , women who were in the age group below 20 years for the years 1997 and 2001 and women who reported having a professional job for the years 2003 and 2005 . for all groups of women except women with senior secondary education and above and divorced or widowed women , the change between 2003 and 2005 was higher than between 1997 and 2001 . this figure shows substantial difference in changes for women from non - han backgrounds and from rural areas , for women with none or with only primary education , and for women aged 1529 years and those who were never married . the findings suggest that greater changes happened to these specific groups between 2003 and 2005 than between 1997 and 2001 , which might in turn suggest effectiveness of interventions that were introduced in the country after 2003 . the results of the study suggest that the situation with hiv awareness in china is similar to other parts of the world , with the level of hiv awareness increasing over time . in 2005 , education remained one of the main factors associated with hiv awareness , the other main factors being ethnicity and exposure to tv and newspapers . a smaller part of the observed change in hiv awareness over time is attributed to change in population structure , in other words to demographic change , but a larger part is due to changes in environment such as in political commitment , successful interventions and health promotion campaigns . the joint efforts of interventions and programmes introduced in the whole of china and in 30 counties , including the unfpa interventions , have been effective in closing the gaps between groups between 2003 and 2005 , as suggested by the larger increase in hiv awareness in specific groups of women between 2003 and 2005 than between 1997 and 2001 . the increases observed between 1997 and 2001 are more evenly spread between groups of women with different demographic characteristics , whereas between 2003 and 2005 increases are more pronounced among the groups that were targeted by different interventions and programmes , including the unfpa interventions . yet another limitation of the present analysis is that the results of the decomposition analysis between 2003 and 2005 can not be generalized to the whole country , but are indicative of the possibility that if the right interventions are in place , the level of hiv awareness can be substantially improved within a short period of time . knowing the targets of interventions between 2003 and 2005 , it might be suggested that the interventions by unfpa , together with other interventions and programmes in place , were effective and helpful in closing the gaps between lower awareness groups and higher hiv awareness groups of women between 2003 and 2005 .

our institutional review board approved this research study , and waived the requirement for informed consent , as the study was retrospective . between february 2002 and february 2006 , 834 percutaneous us - vabs of consecutive breast lesions in patients were performed in our institution using the mammotome system ( biopsys / ethicon endo - surgery , cincinnati , oh ) . we reviewed the clinical and imaging findings of 837 consecutive lesions and 736 lesions from patients that underwent a vab for mass or architectural distortion were excluded from the study . after a review of follow - up imaging and the pathological findings of the remaining 101 lesions , we further excluded 39 lesions from patients who did not receive follow - up for at least two years nor underwent further pathological confirmation of the lesions . the remaining 62 lesions from patients who had undergone a vab due to the presence of microcalcifications as depicted on mammography and then had received at least two years follow - up or underwent excision for pathological confirmation of the lesions were included in this study . among the patients with the 62 lesions , three patients underwent a vab for each breast as they presented with microcalcifications in both breasts . the mean age of the 59 patients was 46.7 years ( age range , 27 to 70 years ) . three patients had a nipple discharge and another three patients complained of the presence of palpable lesions . one patient had an incidental finding of increased flurodeoxyglucose ( fdg ) uptake on the breast on a pet scan . mammography was performed with dedicated equipment ( dmr ; general electric medical systems , milwaukee , wi ) until april 2005 and a lorad / hologic selenia full field digital mammography system ( lorad / hologic , danbury , ct ) from may 2005 onwards . standard craniocaudal and mediolateral oblique views were routinely obtained and additional mammographic views were obtained as needed . digital mammography was performed for 29 lesions and film mammography was performed for 33 lesions . ultrasonography was performed using a high - resolution us unit ( hdi 5000 , philips - advanced technology laboratories , bothell , wa ) with 12-mhz linear array transducers . prior to a vab , lesions were assigned to the final assessment categories of the breast imaging reporting and data system ( bi - rads ) based on the findings of mammography and sonography ( 8) and data was entered prospectively into a database using a computerized spreadsheet ( excel , microsoft , redmond , wa ) . a us - guided automated core - needle is used for microcalcifications with an associated mass lesion . a us - vab an excisional biopsy is used after mammography - guided needle localization for microcalcifications that can not be delineated by us . except in cases where a patient or clinician requested a different procedure , after a meticulous us examination , a bb marker was placed on the skin above suspicious microcalcifications and the patient underwent mammography to assure that the calcification noted on us was consistent with the suspicious calcification depicted on mammography . the vab procedure was performed by one of two board - certificated radiologists with four and 10 years experience in breast imaging , respectively . after administration of local anesthesia , an 11-gauge probe was inserted into the breast through a small skin incision and was guided into the biopsy position under direct ultrasound visualization ( hdi 5000 ) . after the vab , specimen mammography was taken in every case to ensure that the aimed microcalcifications were retrieved . all of the specimens with calcification and without calcification were sent for a pathological examination separately . in cases where the pathological diagnosis was malignancy or borderline lesions such as atypical ductal hyperplasia ( adh ) , surgical resection was recommended . in the case of a benign finding where calcification was confirmed by specimen mammography with concordant imaging - pathological diagnosis , follow - up mammography in six months , 12 months and 24 months intervals were recommended . when the imaging - histological diagnosis was discordant or the histological diagnosis was benign but without calcification as depicted on specimen mammography , excision under mammographic localization was recommended . the clinical , pathological and imaging findings of the 62 lesions , including the results of subsequent excisions and follow - up imaging studies , were reviewed . for four lesions , with respect to the follow - up data , we evaluated whether there was histological underestimation or a false - negative result at follow - up . histological underestimation included a vab - diagnosed ductal carcinoma in situ ( dcis ) that was later revealed as an invasive carcinoma and a vab - diagnosed adh that was later revealed as an dcis or invasive cancer by surgery . the underestimation rate was determined from dividing the number of lesions with underestimation in the final diagnosis by the total number of lesions with adh or dcis as determined after a vab . a false negative was defined as the final diagnosis being malignancy with a benign diagnosis as determined by a vab . the false negative rate was determined by dividing the number of lesions with a false negative result in the final diagnosis by the total number of vabs performed . we reviewed the specimen mammography findings and assessed the average number of specimen samples with its range and standard deviation . we also estimated the number of specimens containing microcalcifications out of the total number of specimens and the number of microcalcifications to examine the efficacy of the use of us - vab to retrieve a targeted microcalcification . when the total number of microcalcifications was greater then 100 , it was referred to as ' innumerable ' . we analyzed the number of microcalcifications and the number of the core specimens containing microcalcifications to determine if there was any statistically significant difference between lesions identified as benign and malignant . the chi - squared or fisher 's exact tests for nonparametric variables and the t - test for parametric inference were performed . statistical analysis was performed with sas software ( sas system for windows , version 9.0 sas institute , cary , nc ) . between february 2002 and february 2006 , 834 percutaneous us - vabs of consecutive breast lesions in patients were performed in our institution using the mammotome system ( biopsys / ethicon endo - surgery , cincinnati , oh ) . we reviewed the clinical and imaging findings of 837 consecutive lesions and 736 lesions from patients that underwent a vab for mass or architectural distortion were excluded from the study . after a review of follow - up imaging and the pathological findings of the remaining 101 lesions , we further excluded 39 lesions from patients who did not receive follow - up for at least two years nor underwent further pathological confirmation of the lesions . the remaining 62 lesions from patients who had undergone a vab due to the presence of microcalcifications as depicted on mammography and then had received at least two years follow - up or underwent excision for pathological confirmation of the lesions were included in this study . among the patients with the 62 lesions , three patients underwent a vab for each breast as they presented with microcalcifications in both breasts . the mean age of the 59 patients was 46.7 years ( age range , 27 to 70 years ) . three patients had a nipple discharge and another three patients complained of the presence of palpable lesions . one patient had an incidental finding of increased flurodeoxyglucose ( fdg ) uptake on the breast on a pet scan . mammography was performed with dedicated equipment ( dmr ; general electric medical systems , milwaukee , wi ) until april 2005 and a lorad / hologic selenia full field digital mammography system ( lorad / hologic , danbury , ct ) from may 2005 onwards . standard craniocaudal and mediolateral oblique views were routinely obtained and additional mammographic views were obtained as needed . digital mammography was performed for 29 lesions and film mammography was performed for 33 lesions . ultrasonography was performed using a high - resolution us unit ( hdi 5000 , philips - advanced technology laboratories , bothell , wa ) with 12-mhz linear array transducers . prior to a vab , lesions were assigned to the final assessment categories of the breast imaging reporting and data system ( bi - rads ) based on the findings of mammography and sonography ( 8) and data was entered prospectively into a database using a computerized spreadsheet ( excel , microsoft , redmond , wa ) . a us - guided automated core - needle is used for microcalcifications with an associated mass lesion . a us - vab is used for microcalcifications visible by us but without an associated mass lesion . an excisional biopsy is used after mammography - guided needle localization for microcalcifications that can not be delineated by us . except in cases where a patient or clinician requested a different procedure , after a meticulous us examination , a bb marker was placed on the skin above suspicious microcalcifications and the patient underwent mammography to assure that the calcification noted on us was consistent with the suspicious calcification depicted on mammography . the vab procedure was performed by one of two board - certificated radiologists with four and 10 years experience in breast imaging , respectively . after administration of local anesthesia , an 11-gauge probe was inserted into the breast through a small skin incision and was guided into the biopsy position under direct ultrasound visualization ( hdi 5000 ) . after the vab , specimen mammography was taken in every case to ensure that the aimed microcalcifications were retrieved . all of the specimens with calcification and without calcification were sent for a pathological examination separately . in cases where the pathological diagnosis was malignancy or borderline lesions such as atypical ductal hyperplasia ( adh ) , surgical resection was recommended . in the case of a benign finding where calcification was confirmed by specimen mammography with concordant imaging - pathological diagnosis , follow - up mammography in six months , 12 months and 24 months intervals were recommended . when the imaging - histological diagnosis was discordant or the histological diagnosis was benign but without calcification as depicted on specimen mammography , excision under mammographic localization was recommended . the clinical , pathological and imaging findings of the 62 lesions , including the results of subsequent excisions and follow - up imaging studies , were reviewed . for four lesions , mammography films were not available and we reviewed the original mammography report instead . with respect to the follow - up data , we evaluated whether there was histological underestimation or a false - negative result at follow - up . histological underestimation included a vab - diagnosed ductal carcinoma in situ ( dcis ) that was later revealed as an invasive carcinoma and a vab - diagnosed adh that was later revealed as an dcis or invasive cancer by surgery . the underestimation rate was determined from dividing the number of lesions with underestimation in the final diagnosis by the total number of lesions with adh or dcis as determined after a vab . a false negative was defined as the final diagnosis being malignancy with a benign diagnosis as determined by a vab . the false negative rate was determined by dividing the number of lesions with a false negative result in the final diagnosis by the total number of vabs performed . we reviewed the specimen mammography findings and assessed the average number of specimen samples with its range and standard deviation . we also estimated the number of specimens containing microcalcifications out of the total number of specimens and the number of microcalcifications to examine the efficacy of the use of us - vab to retrieve a targeted microcalcification . when the total number of microcalcifications was greater then 100 , it was referred to as ' innumerable ' . we analyzed the number of microcalcifications and the number of the core specimens containing microcalcifications to determine if there was any statistically significant difference between lesions identified as benign and malignant . the chi - squared or fisher 's exact tests for nonparametric variables and the t - test for parametric inference were performed . statistical analysis was performed with sas software ( sas system for windows , version 9.0 sas institute , cary , nc ) . the extent of microcalcifications was assessed by measuring the longest diameter of the microcalcification distribution on routine mammography . the average diameter was 2.8 cm with a range from 0.5 cm to 8.6 cm and a standard deviation of 2.0 cm . twenty - nine out of 62 lesions were identified as a breast malignancy ( fig . twenty - seven lesions were identified as dcis by vab and two lesions were identified as invasive ductal carcinoma ( idc ) . the pathological results after surgery revealed the presence of an idc for two lesions of a vab - diagnosed dcis . one patient who was diagnosed with a dcis showed no residual cancer after surgery and all of the other patients had a residual malignancy as identified in the pathological specimens . five other lesions from patients diagnosed with benign disease by vab underwent excision . for two lesions , the patients wanted the lesions excised , as the lesions were palpable although they were imaging - histologically concordant . the follow - up period was 10 months , 23 months and 25 months , respectively with an average of 19.3 months . the lesions were excised after mammographically guided localization ; all of the lesions were finally identified with a fibrocystic change with microcalcifications . in addition to the five lesions that underwent excision with a pathological diagnosis of benign disease other than adh after a vab , the remaining 25 lesions had long - term follow - up results of more than two years . on follow - up mammography , there were no residual microcalcifications seen in four lesions and the remaining 21 lesions showed residual microcalcifications . there were no additional cases of malignancy after more than two years of follow - up . we were able to examine specimen mammography in all 58 lesions except for four patients where mammography films were not available . the average number of vab specimens per lesion was 9.6 specimens , ranging from three to 19 specimens with a standard deviation of 3.2 . the number of specimens with microcalcifications ranged from 0 to 16 , with an average of 4.5 specimens with microcalcifications seen on specimen mammography . the average number of microcalcifications was 43.8 countable microcalcification flecks ranging from 0 to over 100 . one lesion was identified with dcis after vab and though we could not validate the presence of microcalcifications on specimen mammography , pathology revealed a dcis with microcalcifications . the other lesion was from a patient who had a probably benign lesion with microcalcifications in the right breast and a lesion with low suspicious segmental microcalcifications in the left breast . us - vab was planned for the low suspicious lesion in the left breast but the patient also wanted confirmation of the probably benign lesion in the right breast . after vab of both lesions , specimen radiography showed microcalcifications in the left breast while there were no microcalcifications in the right breast . both lesions were identified as a fibrocystic change but there was no documentation of microcalcifications in the right breast on the pathology report . this patient has received follow - up without further pathological confirmation for more than four years and there has been no significant interval change . the number of underestimated lesions ( n = 3 ) was too small to statistically compare to the number of notunderestimated lesions ( n = 59 ) and the total number of specimens , the number of specimens containing microcalcifications and the number of microcalcifications are shown in table 3 . there was no significant statistical difference in the total number of specimens , the number of specimens containing microcalcifications and the number of microcalcifications between benign lesions and malignant lesions ( table 4 ) . the extent of microcalcifications was assessed by measuring the longest diameter of the microcalcification distribution on routine mammography . the average diameter was 2.8 cm with a range from 0.5 cm to 8.6 cm and a standard deviation of 2.0 cm . twenty - nine out of 62 lesions were identified as a breast malignancy ( fig . twenty - seven lesions were identified as dcis by vab and two lesions were identified as invasive ductal carcinoma ( idc ) . the pathological results after surgery revealed the presence of an idc for two lesions of a vab - diagnosed dcis . the underestimation rate was 7.4% . one patient who was diagnosed with a dcis showed no residual cancer after surgery and all of the other patients had a residual malignancy as identified in the pathological specimens . five other lesions from patients diagnosed with benign disease by vab underwent excision . for two lesions , the patients wanted the lesions excised , as the lesions were palpable although they were imaging - histologically concordant . the follow - up period was 10 months , 23 months and 25 months , respectively with an average of 19.3 months . the lesions were excised after mammographically guided localization ; all of the lesions were finally identified with a fibrocystic change with microcalcifications . in addition to the five lesions that underwent excision with a pathological diagnosis of benign disease other than adh after a vab , the remaining 25 lesions had long - term follow - up results of more than two years . on follow - up mammography , there were no residual microcalcifications seen in four lesions and the remaining 21 lesions showed residual microcalcifications . there were no additional cases of malignancy after more than two years of follow - up . we were able to examine specimen mammography in all 58 lesions except for four patients where mammography films were not available . the average number of vab specimens per lesion was 9.6 specimens , ranging from three to 19 specimens with a standard deviation of 3.2 . the number of specimens with microcalcifications ranged from 0 to 16 , with an average of 4.5 specimens with microcalcifications seen on specimen mammography . the average number of microcalcifications was 43.8 countable microcalcification flecks ranging from 0 to over 100 . one lesion was identified with dcis after vab and though we could not validate the presence of microcalcifications on specimen mammography , pathology revealed a dcis with microcalcifications . the other lesion was from a patient who had a probably benign lesion with microcalcifications in the right breast and a lesion with low suspicious segmental microcalcifications in the left breast . us - vab was planned for the low suspicious lesion in the left breast but the patient also wanted confirmation of the probably benign lesion in the right breast . after vab of both lesions , specimen radiography showed microcalcifications in the left breast while there were no microcalcifications in the right breast . both lesions were identified as a fibrocystic change but there was no documentation of microcalcifications in the right breast on the pathology report . this patient has received follow - up without further pathological confirmation for more than four years and there has been no significant interval change . the number of underestimated lesions ( n = 3 ) was too small to statistically compare to the number of notunderestimated lesions ( n = 59 ) and the total number of specimens , the number of specimens containing microcalcifications and the number of microcalcifications are shown in table 3 . there was no significant statistical difference in the total number of specimens , the number of specimens containing microcalcifications and the number of microcalcifications between benign lesions and malignant lesions ( table 4 ) . the increasing use of mammographic screening has led to the detection of smaller , earlier - stage malignancies , which most often present as microcalcifications ( 9 , 10 ) . microcalcifications detected on mammography represent a confounding situation as although the finding is the most common mammographic finding of dcis ( 10 ) , at the same time it is a very common finding in aging women . the majority of lesions of screening - provoked surgical biopsies that are ultimately identified as benign are due to calcifications ( 11 ) . for decades , the use of the needle - localized biopsy was accepted as the standard choice for a biopsy of calcification detected on mammography ( 12 , 13 ) . however , as the imaging equipment has evolved and the biopsy skills have accumulated , the use of the image - guided percutaneous biopsy became an another alternative ( 14 ) . moreover , when the patient condition is not adequate to undergo general anesthesia or when there is no stereotactic biopsy unit available , as in our institution , the use of us vab can be helpful if the microcalcifications can be delineated by us ( 7 ) . in addition , us guidance offers a number of advantages over the use of stereotactic guidance . a shortcoming of us is that microcalcifications are not depicted as clearly as in mammography . however , as the high - resolution us equipment has evolved , the chance of finding a microcalcification of question by careful us examination has also increased ( 15 ) . we were able to confirm retrieved microcalcifications by specimen mammography in 96.8% of cases , which is within the range of 95% to 100% that was previously reported by the use of successful stereotactic guided vab ( st - vab ) for microcalcifications ( 2 , 16 - 18 ) . though there were two lesions without microcalcifications seen on specimen mammography , there was no case of missed diagnosis of cancer in this study . the reported rate of a missed diagnosis of cancer after an st - vab is from 0% to 3% ( 19 ) . however , as microcalcifications are not well visualized on us as in mammography , to apply us - vab in cases of microcalcifications , a meticulous us examination by an experienced operator is mandatory and the insertion of a retrievable wire can be also helpful ( 20 ) to locate a microcalcification of question . the underestimation rate for a carcinoma was 7.4% in the current study with two of the dcis lesions identified as an idc and one of the dcis lesions identified as a dcis with microinvasion . in past studies , the underestimation rate of the use of an st - vab for a carcinoma was reported from 18% to 20% ( 16 , 21 ) . in the case of an adh identified after a vab , surgical excision was recommended , as over 50% of adh lesions are known to have a carcinoma at the time of excision ( 21 , 22 ) . the underestimation rate for an adh was 33.3% , one cancer out of three adh lesions in the current study , which is within the recently reported range of 7% to 35% after an st - vab . there was no statistical difference in the number of the specimen cores with microcalcifications and the total number of microcalcifications between the malignant lesions and benign lesions in this study . this result suggests that the efficacy of a us - vab for microcalcifications is independent of the nature of the lesion as being malignant or benign . in the case of a stereotactic - automated core - needle - biopsy , at least five or more flecks of calcium or three or more cores containing calcium are necessary to be present to ensure adequate sampling of microcalcifications with increasing retrieval of calcification that is associated with increasing sensitivity ( 23 ) . in this study , the average number of specimens with microcalcifications was 4.5 specimens , ranging from 0 to 16 specimens with a mean number of 43.8 calcification flecks and there was no statistically significant difference between benign lesions and the malignant lesions . this finding is concordant with a previous report with the use of automated core needle biopsies . the amount of microcalcification retrieval after a vab in this study was higher than that required for an automated core - needle - biopsy , which may explain why there was no statistically significant difference between the malignant lesions and benign lesions . to the best of our knowledge , there has been no previous report of an adequate amount of microcalcification retrieval after a us - vab . however , it has been reported that in the case of an st - vab , to yield 92% of diagnostic accuracy , 12 specimens were necessary and we obtained an average of 9.6 specimens in this study . this study only included patients with lesions that received follow - up for at least two years or patients that underwent surgery for further confirmation . a previous study on the use of an st - vab for breast microcalcifications has suggested that a 6-month follow - up might already provide evidence that a vab finding is representative ( 19 ) . in the case of us - vab for microcalcifications , given the limitations of the use of us for visualization of microcalcifications , long term follow up with mammography longer than six months should be necessary . in the case of a core needle biopsy , a two - year - follow - up is generally accepted to make a confident diagnosis of benign disease ( 19 ) and hence a follow - up period of more than two years can ensure a confident diagnosis for a us - vab of breast microcalcifications . first , only microcalcifications visible on linear probe us were included in this study and thus the study does not reflect all of the microcalcifications , including microcalcifications not delineated by us . second , patients without a follow - up period longer than two years were excluded in this study and there could be a possible missed diagnosis in these patients . also , we set a standard follow - up period of at least two years , which may be insufficient to diagnose a lesion with microcalcifications as benign and a further follow - up would be necessary . in conclusion , the use of a us - vab can be an effective alternative to the use of an st - vab in cases where microcalcifications are visible on high - resolution us .

objectiveto evaluate the diagnostic accuracy of the use of an ultrasonography ( us)-guided vacuum - assisted biopsy for microcalcifications of breast lesions and to evaluate the efficacy of the use of us - guided vacuum - assisted biopsy with long - term follow - up results.materials and methodsus - guided vacuum - assisted biopsy cases of breast lesions that were performed between 2002 and 2006 for microcalcifications were retrospectively reviewed . a total of 62 breast lesions were identified where further pathological confirmation was obtained or where at least two years of mammography follow - up was obtained . these lesions were divided into the benign and malignant lesions ( benign and malignant group ) and were divided into underestimated group and not - underestimated lesions ( underestimated and not - underestimated group ) according to the diagnosis after a vacuum - assisted biopsy . the total number of specimens that contained microcalcifications was analyzed and the total number of microcalcification flecks as depicted on specimen mammography was analyzed to determine if there was any statistical difference between the groups.resultsthere were no false negative cases after more than two years of follow - up . twenty - nine lesions were diagnosed as malignant ( two invasive carcinomas and 27 carcinoma in situ lesions ) . two of the 27 carcinoma in situ lesions were upgraded to invasive cancers after surgery . among three patients diagnosed with atypical ductal hyperplasia , the diagnosis was upgraded to a ductal carcinoma in situ after surgery in one patient . there was no statistically significant difference in the number of specimens with microcalcifications and the total number of microcalcification flecks between the benign group and malignant group of patients and between the underestimated group and not - underestimated group of patients.conclusionus-guided vacuum - assisted biopsy can be an effective alternative to stereotactic - guided vacuum - assisted biopsy in cases where microcalcifications are visible with the use of high - resolution us .

MATERIALS AND METHODS Study Population Imaging Evaluation US-Guided Vacuum-Assisted Biopsy and Management Follow-Up and Data Analysis RESULTS Mammography Pathological Diagnosis and Follow-Up Specimen Mammography DISCUSSION

after a review of follow - up imaging and the pathological findings of the remaining 101 lesions , we further excluded 39 lesions from patients who did not receive follow - up for at least two years nor underwent further pathological confirmation of the lesions . the remaining 62 lesions from patients who had undergone a vab due to the presence of microcalcifications as depicted on mammography and then had received at least two years follow - up or underwent excision for pathological confirmation of the lesions were included in this study . we also estimated the number of specimens containing microcalcifications out of the total number of specimens and the number of microcalcifications to examine the efficacy of the use of us - vab to retrieve a targeted microcalcification . we analyzed the number of microcalcifications and the number of the core specimens containing microcalcifications to determine if there was any statistically significant difference between lesions identified as benign and malignant . after a review of follow - up imaging and the pathological findings of the remaining 101 lesions , we further excluded 39 lesions from patients who did not receive follow - up for at least two years nor underwent further pathological confirmation of the lesions . the remaining 62 lesions from patients who had undergone a vab due to the presence of microcalcifications as depicted on mammography and then had received at least two years follow - up or underwent excision for pathological confirmation of the lesions were included in this study . we also estimated the number of specimens containing microcalcifications out of the total number of specimens and the number of microcalcifications to examine the efficacy of the use of us - vab to retrieve a targeted microcalcification . we analyzed the number of microcalcifications and the number of the core specimens containing microcalcifications to determine if there was any statistically significant difference between lesions identified as benign and malignant . in addition to the five lesions that underwent excision with a pathological diagnosis of benign disease other than adh after a vab , the remaining 25 lesions had long - term follow - up results of more than two years . there were no additional cases of malignancy after more than two years of follow - up . the number of underestimated lesions ( n = 3 ) was too small to statistically compare to the number of notunderestimated lesions ( n = 59 ) and the total number of specimens , the number of specimens containing microcalcifications and the number of microcalcifications are shown in table 3 . there was no significant statistical difference in the total number of specimens , the number of specimens containing microcalcifications and the number of microcalcifications between benign lesions and malignant lesions ( table 4 ) . in addition to the five lesions that underwent excision with a pathological diagnosis of benign disease other than adh after a vab , the remaining 25 lesions had long - term follow - up results of more than two years . there were no additional cases of malignancy after more than two years of follow - up . the number of specimens with microcalcifications ranged from 0 to 16 , with an average of 4.5 specimens with microcalcifications seen on specimen mammography . the number of underestimated lesions ( n = 3 ) was too small to statistically compare to the number of notunderestimated lesions ( n = 59 ) and the total number of specimens , the number of specimens containing microcalcifications and the number of microcalcifications are shown in table 3 . there was no significant statistical difference in the total number of specimens , the number of specimens containing microcalcifications and the number of microcalcifications between benign lesions and malignant lesions ( table 4 ) . there was no statistical difference in the number of the specimen cores with microcalcifications and the total number of microcalcifications between the malignant lesions and benign lesions in this study . in this study , the average number of specimens with microcalcifications was 4.5 specimens , ranging from 0 to 16 specimens with a mean number of 43.8 calcification flecks and there was no statistically significant difference between benign lesions and the malignant lesions . the amount of microcalcification retrieval after a vab in this study was higher than that required for an automated core - needle - biopsy , which may explain why there was no statistically significant difference between the malignant lesions and benign lesions . in the case of us - vab for microcalcifications , given the limitations of the use of us for visualization of microcalcifications , long term follow up with mammography longer than six months should be necessary . in the case of a core needle biopsy , a two - year - follow - up is generally accepted to make a confident diagnosis of benign disease ( 19 ) and hence a follow - up period of more than two years can ensure a confident diagnosis for a us - vab of breast microcalcifications . in conclusion , the use of a us - vab can be an effective alternative to the use of an st - vab in cases where microcalcifications are visible on high - resolution us .

the use of visible and near - infrared ( nir ) light , penetrable to deep tissue , is an attractive method of spatiotemporally controlling drug release from various drug delivery forms , such as prodrugs , liposomes , polymers , and other nano- and macrodelivery systems . however , because of its lower energy , it is difficult to directly cleave a chemical bond using such light . thus , novel mechanisms using lower energy light to trigger the release of biologically active compounds have been a major topic of interest . the photodynamic process and the unique chemistry of singlet oxygen ( so ) were adopted to mediate lower energy light release of drugs . so is formed during the photodynamic process and reacts with electron - rich olefins to form unstable dioxetanes . we named the cleavage of aminoacrylate by so photo - unclick chemistry , and demonstrated the release of the anticancer drug combretastatin a-4 ( ca4 ) from its prodrug using this method . we prepared cmp - l - ca4 , a ca4 prodrug that can be activated by far - red light ( 690 nm ) , by combining a so - labile aminoacrylate linker , a core - modified porphyrin ( cmp ) photosensitizer , and ca4 . the prodrug released ca4 and enhanced cytotoxicity upon illumination . the released ca4 damaged cancer cells , demonstrating our system s ability to generate bystander effects in vitro . we found that the antitumor effects of cmp - l - ca4 were superior to the effects of its pseudo - prodrug cmp - ncl - ca4 ( ncl = noncleavable linker ) . the ability to optically image the light - activatable prodrug is useful for determining an illumination time when the prodrug is at its maximum concentration at the target site . if the prodrug accumulates in the target sites , we can use optical imaging to detect the target areas , as well as to treat the disease . we expected that we could optically image our photounclickable prodrug in vivo using a fluorescent photosensitizer . thus , we could track the prodrugs and then treat the tumor with the combined effects of photodynamic therapy ( pdt ) and local chemotherapy ( figure 1a ) . the released drug could damage the cancer cells that survived the initial pdt damage through bystander effects ( figure 1b ) . ( a ) multifunctional prodrug for optical imaging and combined treatment with pdt and local chemotherapy . ( b ) bystander effects from the released drugs can kill cancer cells that survive pdt damage . [ the lifetime of so is short ( submicrosecond scale ) . thus , direct cell damage by so occurs only during illumination . the light dose used for ] we generated pc-(l - ca4)2 , an advanced multifunctioning ca4 prodrug , for both fluorescence optical imaging and combination therapy with pdt and released ca4 . we chose phthalocyanine ( pc ) because pc is a fluorescent photosensitizer that can generate both fluorescence and singlet oxygen . although fluorescence emission and so generation are competing processes , pc has uniquely balanced yields of both functionality ( i.e. , si - pc : o2 = 0.22 and f = 0.4 ) with a high molar extinction coefficient ( ) . its brightness ( bt ) is greater than that of cmp ( e.g. , = 150,000 m cm at 675 nm , bt = 6000 m cm for pc vs = 5000 at 690 nm , bt = 50 m cm for cmp ) . this pseudo - prodrug is similar to pc-(l - ca4)2 in structure , but can not release ca4 upon illumination . it will mimic the pdt effects of pc-(l - ca4)2 , but can not induce damage from released ca4 . we evaluated the cytotoxic effects of these two prodrugs with and without illumination , the inhibition of tubulin polymerization , the in vitro bystander effects , tumor localization using optical imaging , and the antitumor effects . we developed a synthetic scheme using high - yield reactions , such as esterification , nucleophilic substitution , and the yne - amine reaction , to make the process easily adaptable to other alcohol - containing drugs ( scheme 1 ) . a nucleophilic substitution reaction of silicon phthalocyanine dichloride ( pc - cl2 ) yielded compound 3 . pc-(l - ca4)2 was synthesized through a click ( yne - amine ) reaction of compounds 1 and 3 . under the basic condition , pc-(ncl - ca4)2 overall , the synthesis was straightforward and all steps gave high yields ( > 73% each step ) . reagents and conditions : ( i ) propynoic acid , dcc , dmap , ch2cl2 , room temp , 24 h ; ( ii ) 1,3-dibromopropane , anhydrous k2co3 , acetone , reflux , 12 h ; ( iii ) 2-(piperazin-1-yl)ethanol , pyridine , toluene , reflux , 4 h ; ( iv ) 1 , anhydrous thf , 30 min ; ( v ) 2 , anhydrous k2co3 , acetone , reflux , 12 h. we formulated the prodrugs using peg pla [ poly(ethylene glycol)-poly(d , l - lactide ) ] copolymer micelles to take advantage of the enhanced permeability and retention ( epr ) effect to enhance the delivery to tumor . the nanosized peg pla polymer micelle was expected to provide three advantages : ( 1 ) passive targeting to tumors via the epr effect , ( 2 ) prolonged circulation in the plasma , and ( 3 ) solubilization of the nonpolar prodrug . the zeta potentials and mean diameters of the micelles of pc-(l - ca4)2 and pc-(ncl - ca4)2 were determined by dynamic light scattering ( dls ) ( zeta potential = 11.64 1.38 mv , 16.81 1.67 mv and mean diameter = 71.96 1.34 nm , 75.07 1.45 nm , respectively ) . to visualize the formation of the polymeric micelles , we used transmission electron micrographs ( tem ) . tem images of the micelles showed consistent particle sizes ( 6178 nm for pc-(l - ca4)2 and 6580 nm for pc-(ncl - ca4)2 micelles ) . the stability of the micelles was monitored by their particle sizes and zeta potentials at 4 c under dark conditions . these values remained within 95% of the initial values for up to 21 days . ( a ) particle size distribution and tem images ( inset ) of micelles of ( a ) pc-(l - ca4)2 and ( b ) pc-(nlc - ca4)2 . ca4 is known to inhibit tubulin polymerization by binding to the colchicine binding pocket of tubulin . because the bulky groups ( pc - l and pc - ncl ) are attached to ca4 , we expected lower inhibitory activity of tubulin polymerization . we determined the effects of these prodrugs using the tubulin polymerization assay , in which fluorescence emission increases as tubulins polymerize ( figure 3a ) . consistent with our data on the previous ca4 prodrug cmp - l - ca4 , both pc-(l - ca4)2 and pc-(ncl - ca4)2 had significantly ( p < 0.02 ) lower inhibitory activity ( 23% and 17% , respectively ) than the parent drug ca4 ( 100% , figure 3b ) . effects of 3 m each of pcx , ca4 , pc-(l - ca4)2 , and pc-(ncl - ca4)2 on tubulin polymerization : ( a ) one data set of representative kinetic traces ( data from two more experiments are found in figure s1 , supporting information ) and ( b ) inhibition of tubulin polymerization by ca4 , pc-(l - ca4)2 , and pc-(ncl - ca4)2 after 1 h incubation ( sd of three experiments ) . due to the dramatic reduction of the inhibition of tubulin polymerization , it was expected that pc-(l - ca4)2 and pc-(ncl - ca4)2 would have lower dark toxicity ( cytotoxicity without illumination ) than ca4 . using mcf-7 cells , we found that the dark toxicity of the prodrugs decreased by 19- and 101-fold [ ic50d values = 9 , 173 , and 916 nm for ca4 , pc-(l - ca4)2 and pc-(ncl - ca4)2 , respectively ] . pc - l and pc - ncl reduced the cytotoxicity , presumably by interfering with its binding to tubulin ( figure 3 ) . however , illumination enhanced the toxicity of both the prodrug and the pseudo - prodrug [ ic50p = 6 nm and 34 nm for pc-(l - ca4)2 and pc-(ncl - ca4)2 , respectively ] . the difference in the magnitudes of phototoxicity and dark toxicity effects may be the result of each prodrug using a different mechanism . while pc-(ncl - ca4)2 can only kill cells via the pdt effects of so , pc-(l - ca4)2 can potentially kill cells by both pdt effects and released ca4 . to prove this mechanistic difference , we tested the bystander effects after the illumination . after treating the cells in wells of 24-well plates with the prodrug or pseudo - prodrug , one - half of each well was exposed to light . at 48 h post - illumination , live cells were stained with calcein am , and the center of each well was imaged using a fluorescence microscope . because the lifetime of so in aqueous medium / biological systems is very short ( 40 ns ) , so generated in the illuminated half of the so should decay before reaching the other half of the well ( diffusion distance of so = 20200 nm ) . however , the released ca4 can induce bystander effects because it can diffuse to the unilluminated half of the well . as we expected , bystander effects were found in the pc-(l - ca4)2-treated wells : cells in the nonilluminated side were damaged as much as cells in the illuminated side ( figure 5b ) . however , wells treated with pc-(ncl - ca4)2 had cell damage only in the illuminated halves of the wells ( figure 5c ) . this clearly supported our hypothesis that illuminated pc-(l - ca4)2 releases ca4 that can induce bystander effects , which is consistent with data from our previous study with cmp - l - ca4 . fluorescence live cell images of the center of each well treated with ( a ) vehicle ( diluting solution without a prodrug ) , ( b ) 25 nm pc-(l - ca4)2 , and ( c ) 25 nm pc-(ncl - ca4)2 . the left half of each well was illuminated with a 690 nm diode laser ( 11 mw / cm for 15 min ) . at these concentrations , pc-(l - ca4)2 and pc-(ncl - ca4)2 did not produce any significant dark toxicity . one of our major goals was to make pc-(l - ca4)2 detectable in tissues using fluorescence optical imaging . we expected that the fluorescent photosensitizer pc would provide sufficient fluorescence emission for this goal . pla polymer micelles of pc-(l - ca4)2 or pc-(ncl - ca4)2 . as a control formulation , we also made solutions of these prodrugs in 5% cremophor el , which does not produce epr effects . balb / c mice with sc tumors ( colon-26 cells , 46 mm in length ) were injected retro - orbitally with 2 mol / kg of the prodrug . then , the mice were imaged at various postinjection time points ( figure 6 and figure s4 ) . as anticipated , we could clearly see the fluorescence emissions from the two prodrugs in live mice . first , the images from the mice that received the polymer micelles of both prodrugs showed hot spots in tumors , with a peak at around 24 h postinjection , presumably due to the epr effects of the nanosized polymer micelles . these hot spots were more evident in the mice who received injections of pc-(l - ca4)2 . resulting from formulations with the polymer micelles persisted longer than those hot spots resulting from formulations with cremophor solutions . the polymer micelles appeared to delay the clearance of the prodrugs from the system . because the illumination for optical imaging could theoretically generate so and thus release ca4 from pc-(l - ca4)2 , we monitored the body weight change and tumor growth pattern of the mice imaged with pc-(l - ca4)2 . we did not see any significant impact on mouse body weight or tumor growth since the light dose used for imaging was negligible [ 675 filter ( 660690 nm ) at 1.5 w / cm for 2 s ( 3.0 10 j / cm ) ] . the treatment used a ( 1.2 10)-fold higher light dose than the dose used for imaging . fluorescence optical images of the mice after retro - orbital injection of 2 mol / kg of prodrug : ( a ) pc-(l - ca4)2 in polymer micelles , ( b ) pc-(ncl - ca4)2 in polymer micelles , ( c ) pc-(l - ca4)2 in 5% cremophor solution , and ( d ) pc-(ncl - ca4)2 in 5% cremophor solution . we expected that the new prodrug pc-(l - ca4)2 would show better antitumor effects than our previous prodrug cmp - l - ca4 , because pc has superior light absorption properties and the prodrug releases two ca4 instead of one . we used balb / c mice with sc tumors to evaluate the antitumor effects of the prodrugs with illumination . twenty - four hours post - retro - orbital injection of 1 or 2 mol / kg of the prodrug , the tumor was illuminated by a 690 nm diode laser for 30 min at 100 mw / cm ( 180 j / cm ) or 200 mw / cm ( 360 j / cm ) . six groups were used to assess the antitumor effects of the prodrug and pseudo - prodrug : g1 , negative control ; g2 , [ ca4 ( 1 mol / kg ) + no hv ] ; g3 , [ pc-(ncl - ca4)2 ( 1 mol / kg ) + hv ( 100 mw / cm ) ] ; g4 , [ pc-(l - ca4)2 ( 1 mol / kg ) + hv ( 100 mw / cm ) ] ; g5 , [ pc-(l - ca4)2 ( 2 mol / kg ) + hv ( 100 mw / cm ) ] ; and g6 , [ pc-(l - ca4)2 ( 2 mol / kg ) + hv ( 200 mw / cm ) ] . [ the tumor growth curves of g5 and g6 were nearly identical to g4 until day 15 . so , these curves were omitted from figure 7a for clarity ( figure s2 ) . ] we found outstanding antitumor effects in the mice treated with pc-(l - ca4)2 , g4g6 . after 24 h illumination , all tumors shrank to a nonmeasurable size and remained so for almost 15 days ( figure 7a ) . mice in group g4 experienced tumor growth only after day 16 ( figure s2 ) . all mice in g4g6 lived until day 30 , while tumor size of all 4 mice in the control group ( g1 ) reached > 800 mm in 12 days ( figure 7b ) . the pdt effects resulting from pc-(ncl - ca4)2 treatment ( g3 ) had a significant impact on tumor size until day 3 ( p < 0.05 ) . however , after day 3 , the tumors grew back at a rate similar to the tumors in the control group g1 . throughout the observation period , pc-(l - ca4)2 treatment yielded significantly better antitumor effects ( p < 0.01 ) , than pc-(ncl - ca4)2 treatment . thus , it seemed that the pdt effects alone might not be sufficient to produce such a robust antitumor effect as seen in group g4 . we hypothesized that our findings stemmed from the contribution of the released ca4 in addition to pdt effects . interestingly , no mice in g1g6 experienced a significant decrease in body weight ( figure s3 ) . the histological data were consistent with the antitumor effects ( figure 7c ) . after 24 h of treatment , tumors were collected and stained with h&e to visualize the tissue damage . while mice treated with pc-(ncl - ca4)2 experienced tissue damage only on the skin , mice receiving pc-(l - ca4)2 also experienced direct tumor damage . in fact , the volume of tumors treated with pc-(l - ca4)2 shrank to about 1/8 of the volume of the tumors in the control group . ( a ) tumor growth curves , drug iv administration : once a day on day 1 , illumination 24 h postdrug administration [ hv : 100 mw / cm for 30 min ( 180 j / cm ) or 200 mw / cm for 30 min ( 360 j / cm ) ] , 5 mice per group except the control group ( 4 mice ) . error bars represent se . in inset : the order of tumor size was g1 > g3 > g4 during day 1 to day 4 [ * * p < 0.01 ( g1 vs g3 , from day 1 to day 4 ) and # # p < 0.01 ( g3 vs g4 , from day 1 to day 4 ) ] . ( c ) h&e staining of tumors 24 h post - illumination from ( i ) control mice or mice treated with ( ii ) pc-(ncl - ca4)2 , or ( iii ) pc-(l - ca4)2 . prodrug administration and illumination conditions were same as for those in ( a ) . vt , viable tumor ; dt , damaged tumor ; and ds , damaged skin . we successfully demonstrated a multifunctional photounclickable prodrug that can be visualized by optical imaging , and ablates tumors with a combination of pdt and local chemotherapy . the prodrug pc-(l - ca4)2 and its pseudo - prodrug pc-(ncl - ca4)2 were prepared in high yields through a facile and flexible scheme . the cytotoxicity of these prodrugs was lower than that of the parent drug ca4 , but both prodrugs showed enhanced cytotoxicity upon illumination . the mechanisms of cell damage of pc-(l - ca4)2 and pc-(ncl - ca4)2 combined with illumination should be different . while the use of pc-(ncl - ca4)2 and illumination killed cancer cells through pdt effects , the use of pc-(l - ca4)2 and illumination combined these pdt effects with local chemotherapy through the released ca4 . this was supported by the bystander effects demonstrated in vitro . through the use of optical imaging , we found that both prodrugs were detected at the therapeutic dose within tumors . optical imaging also provided the information about the pk profiles of the prodrugs so that we could find the optimal time point for illumination . as expected , the antitumor effects in mice treated with pc-(l - ca4)2 were dramatically better than in mice treated with pc-(ncl - ca4)2 . treatment with either pc-(ncl - ca4)2 or ca4 produced minimal antitumor effects , suggesting that the outstanding antitumor effects of pc-(l - ca4)2 may have been a result of the synergistic effects of pdt and chemotherapy . in addition to confirming that our current so - activatable prodrug strategy provides improved antitumor efficacy , we demonstrated the innovative use of a fluorescent photosensitizer within the prodrug . using optical imaging , we were able to noninvasively generate pk information about the prodrug without causing any observable acute toxicity to mice . our multifunctional prodrug strategy includes ( 1 ) activation by the clinical translatable far - red light ( or nir ) , ( 2 ) the unique combination of pdt and local chemotherapy , and ( 3 ) the dual function of optical imaging and treatment with one prodrug . we anticipate that this strategy will be applicable to various drug delivery forms , clinically approved drugs , and advanced drug delivery systems targeted at tumors . ca4 , compound 1 , and compound 2(10 ) were synthesized as reported previously . the purity of the biologically evaluated compounds pc-(l - ca4)2 and pc-(ncl - ca4)2 was confirmed to be > 95% by high - performance liquid chromatography ( hplc ) ( figures s9 and s10 ) . 1-(2-hydroxyethyl)-piperazine ( 1.91 g , 14.70 mmol ) and pyridine ( 2.5 ml ) were added to a solution of silicon(iv ) phthalocyanine dichloride ( pc - cl2 , 1 g , 1.63 mmol ) in 50 ml toluene . the reaction mixture was refluxed for 12 h. the solvent was then removed under reduced pressure . the solvent of the combined organic layers was removed by evaporation , and the crude was recrystallized with chcl3/n - hexane ( 1:4 v / v ) to give a blue solid compound 3 ( 1.22 g , 94% ) . h nmr ( 300 mhz , cd2cl2 ) 1.19 ( t , j = 5.8 hz , 4h ) , 0.82 ( t , j = 5.8 hz , 1h ) , 0.28 ( m , 8h ) , 1.75 ( m , 8h ) , 8.308.40 ( m , 8h , pc - h ) , 9.559.69 ( m , 8h , pc - h ) ; c nmr ( 300 mhz , cd2cl2 ) 123.4 , 131.0 , 135.9 , 149.2 ; hrms esi ( m / z ) : [ m + h ] calculated for c44h43n12o2si , 799.3401 ; found , 799.3395 . compound 1 ( 46 mg , 0.13 mmol ) and compound 3 ( 50 mg , 0.06 mmol ) were dissolved in 20 ml dry thf , and the solution was stirred at room temperature for 15 min . the solvent was removed under reduced pressure to yield the crude product , which was then recrystallized from chcl3/n - hexane ( 1:5 v / v ) to give pc-(l - ca4)2 ( 83 mg , 87% ) . h nmr ( 300 mhz , cd2cl2 ) ) 1.96 ( t , j = 5.6 hz , 4h ) , 0.55 ( t , j = 5.0 hz , 4h ) , 0.29 ( br s , 8h ) , 2.20 ( br s , 8h ) , 3.67 ( s , 12h ) , 3.72 ( s , 6h ) , 3.77 ( s , 6h ) , 4.34 ( d , j = 12.8 hz , 2h ) , 6.45 ( d , j = 4.9 hz , 4h ) , 6.62 ( s , 4h ) , 6.84 ( d , j = 8.1 hz , 2h ) , 6.96 ( s , 2h ) , 7.00 ( s , 2h ) , 7.09 ( d , j = 12.8 hz , 2h ) , 8.388.43 ( m , 8h , pc - h ) , 9.659.70 ( m , 8h , pc - h ) ; c nmr ( 300 mhz , cd2cl2 ) 55.8 , 56.6 , 60.4 , 81.8 , 105.9 , 111.8 , 123.5 , 123.9 , 126.6 , 128.7 , 129.1 , 129.8 , 131.2 , 132.5 , 135.8 , 137.2 , 140.3 , 149.3 , 151.1 , 152.2 , 153.0 , 167.2 ; hrms esi ( m / z ) : [ m + h ] calculated for c86h83n12o14si , 1535.5921 ; found , 1535.5914 . anhydrous k2co3 ( 68 mg , 0.500 mmol ) and compound 3 ( 200 mg , 0.25 mmol ) were added to a solution of compound 2 ( 218 mg , 0.50 mmol ) in 10 ml dry dmf . the reaction mixture was stirred at room temperature for 12 h. the k2co3 was removed by suction filtration and the solvent was removed under reduced pressure . the crude product was then recrystallized from chcl3/n - hexane ( 1:5 v / v ) to give pc-(ncl - ca4)2 ( 302 mg , 80% ) . h nmr ( 300 mhz , cd2cl2 ) 1.96 ( t , j = 5.6 hz , 4h ) , 0.78 ( t , j = 5.8 hz , 4h ) , 0.41 ( br s , 8h ) , 1.50 ( m , 2h ) , 1.90 ( m , 2h ) , 2.18 ( m , 2h ) , 2.49 ( m , 2h ) , 3.32 ( s , 4h ) , 3.60 ( br s , 8h ) , 3.65 ( s , 12h ) , 3.75 ( s , 6h ) , 3.80 ( s , 6h ) , 6.46 ( m , 4h ) , 6.51 ( m , 4h ) , 6.74 ( m , 2h ) , 6.81 ( m , 2h ) , 6.92 ( m , 2h ) , 8.38 ( m , 8h , pc - h ) , 9.66 ( m , 8h , pc - h ) ; c nmr ( 300 mhz , cd2cl2 ) 55.8 , 60.4 , 105.1 , 111.4 , 113.6 , 115.2 , 117.5 , 123.4 , 126.7 , 126.8 , 126.9 , 130.9 , 132.8 , 135.9 , 149.1 ; hrms esi ( m / z ) : [ m + h ] calculated for c86h91n12o12si , 1511.6649 ; found , 1511.6612 . briefly , 3 mg of pc-(l - ca4)2 or pc-(ncl - ca4)2 was dissolved in 1.3 ml of thf . pla ( cat # ak09 , vendor : polyscitech ) was dissolved in 1 ml of thf . 600 l of the 3 mg of pc-(l - ca4)2 or pc-(ncl - ca4)2 dissolved in 1.3 ml thf was added to the mpeg - pla - thf mixture . the volume of the resulting mixture was reduced to 300 l under reduced pressure . the 300 l of the mixture of pc-(l - ca4)2 or pc-(ncl - ca4)2 and mpeg pla was added to 3 ml of distilled water dropwise while stirring . the mixture was stirred for 3 h , after which the organic solvent was evaporated under reduced pressure at 40 c . the concentration of pc-(l - ca4)2 or pc-(ncl - ca4)2 micelles was determined by diluting the micelles in thf : the absorbance was measured by absorbance of pc group . the concentration was calculated from the molar extinction coefficient ( ec ) of pc-(l - ca4)2 or pc-(ncl - ca4)2 at 675 nm in thf ( ec of pc-(l - ca4)2 = 205,000 ; pc-(ncl - ca4)2 = 206,110 m cm ) using the beer lambert law . the concentrations of pc-(l - ca4)2 or pc-(ncl - ca4)2 micelles were determined to be 211 and 210 m , respectively . freshly prepared pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles were used for all the experiments . 2.0 mm stock solutions of pc-(l - ca4)2 and pc-(ncl - ca4)2 prepared in thf were further diluted with 5% cremophor el in pbs to achieve appropriate concentrations . the pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in an aqueous solution were characterized by measuring their hydrodynamic diameter and zeta potential via dynamic light scattering ( dls ) . the size measurement was carried out at a concentration of 1.0 mg / ml of pc-(l - ca4)2 or pc-(ncl - ca4)2 . pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles were also imaged by transmission electron microscopy ( tem ) at 20,000 operating at 80 kv . tem samples were prepared by depositing 20 l of diluted pc-(l - ca4)2 and pc-(ncl - ca4)2 micelle solution on a 300 mesh copper tem grid with a carbon film . a fluorescence - based tubulin polymerization assay was conducted using a kit supplied by cytoskeleton , inc . the basic principle is that an increase in fluorescence will occur as a fluorescence reporter is incorporated into microtubules during the course of polymerization . the assay was performed following the experimental procedure described in version 2.1 of the tubulin polymerization assay kit manual . briefly , a drug in dmso stock solution was added to a mixture of tubulin and gtp in a buffer solution , to give a final concentration of 3 m . fluorescence was monitored ( excitation = 360 nm and emission = 450 nm ) every 2 min for 1 h. pcx and ca4 were included in the assay as positive controls , as well as a vehicle - only negative control . the cytotoxicity of pc-(l - ca4)2 and pc-(ncl - ca4)2 was determined with and without illumination . mcf-7 cells were maintained in minimum essential medium ( -mem ) supplemented with 10% bovine growth serum , 2 mm l - glutamine , 50 units / ml penicillin g , 50 g / ml streptomycin , and 1.0 g / ml fungizone . mcf-7 cells ( 5000 cells / well ) were seeded on 96-well plates in the medium and incubated for 24 h at 37 c in 5% co2 . stock pc-(l - ca4)2 and pc-(ncl - ca4)2 micelle solutions ( 200 m ) were prepared in distilled water . the stock solutions were further diluted with medium to obtain the necessary final concentrations . the diluted solution ( 10 l ) the plates were incubated for 24 h and then removed from the incubator . for the phototoxicity study : the uncovered plate was illuminated for 30 min using a diode laser ( 690 nm , 5.6 mw / cm ) . to ensure uniformity of the light during the illumination , each plate was shaken gently on an orbital shaker ( lab - line , barnstead international ) . for the dark toxicity study : plates were kept in the dark for 30 min and then returned to the incubator . briefly , a 10 l solution of mtt ( 10 mg in 1 ml pbs buffer ) was added to each well and the plate was incubated for 4 h at room temperature or 37 c . then , the mtt solution was removed and the cells were dissolved in 200 l of dmso . the absorbance of each well was measured at 570 nm with background subtraction at 650 nm . cell viability was quantified by measuring the absorbance of the treated wells compared to that of the untreated control wells , and expressed as a percentage . colon-26 cells were seeded at 5000 cells / well on 24-well plates and incubated for 24 h. stock pc-(l - ca4)2 and pc-(ncl - ca4)2 micelle solutions ( 200 m ) were prepared in distilled water . 200 m of the stock solutions were added to wells ( 1 ml ) to obtain appropriate final concentrations of both pc-(l - ca4)2 and pc-(ncl - ca4)2 at 25 nm . after 24 h incubation , the plates were illuminated from the bottom with a 690 nm diode laser at 11 mw / cm for 15 min . during illumination , half of each well was blocked with black masking tape ( cat # t743 - 1.0 , vendor : thorlabs ) . the illuminated plates were incubated for an additional 48 h. then , a calcein am live cell staining assay ( cat # 4892 - 010-k , vendor : molecular , probes tervigen ) was performed . the cells were washed once with 1 ml calcein am wash buffer , then 250 l fresh wash buffer and 250 l working reagent were added to the wells . fluorescent images were obtained with an olympus ix51 inverted microscope with a green fluorescence channel to visualize live cells . the concentrations of pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in aqueous solution were determined by diluting 10 l of the formulation stock in 1 ml of thf and measuring the absorbance of phthalocyanine . the concentration was calculated from the ec of pc-(l - ca4)2 at 672 nm in thf ( ec of pc-(l - ca4)2 = 205,000 ; pc-(ncl - ca4)2 = 206,110 m cm ) using the beer lambert law . we used four- to six - week - old balb / c mice to investigate the biodistribution and tumor targeting ability of the polymeric micelles . the mice were shaved before the imaging experiments , and were imaged using the ivis imaging system . the mice were injected with pc-(l - ca4)2 and pc-(ncl - ca4)2 in the micelle formulation ( 2 mol / kg , i.v . ) . as a comparison , fluorescence images were taken 0 , 3 , 6 , 12 , 24 , 48 , and 72 h after retro - orbital injection . before taking the images , the mice were anesthetized in an acrylic chamber with a 2.5% isoflurane / air mixture . the following parameters were used to acquire images with living image software : fluorescence mode , exposure time : 2 s , binning : medium , f / stop : 2 , excitation : 675 filter ( 660690 nm ) , and emission : 720 filter ( 710730 nm ) . during post processing , image counts were adjusted to 3 10 as minimum and 6.0 10 a.u . as maximum color scale . four- to six - week - old balb / c mice ( 1820 g ) were used for the murine tumor model . the mice were implanted sc with 2 10 colon 26 cells in pbs ( 100 l ) on the lower back of the neck . the longest axis of the tumor ( 1 ) and the axis perpendicular to l ( w ) were used to calculate tumor volume ( lw/2 ) . we used stock solutions of pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in aqueous solution and further dilutions to achieve final doses as follows : [ ca4 , ( 1 mol / kg each ) ] , [ pc-(ncl = ca4)2 , ( 1 mol / kg each ) ] , [ pc-(l - ca4)2 , ( 1 mol / kg each ) ] , and [ pc-(l - ca4)2 , ( 2 mol / kg each ) ] . to each mouse , 200 l of sample was injected via iv once on day 1 . twenty - four hours later mice were anesthetized by ip injection of 80 mg / kg ketamine and 6 mg / kg xylazine . tumors were illuminated with a 690 nm diode laser at 100 mw / cm or 200 mw / cm for 30 min 180 or 360 j / cm , respectively . tumor size was measured every day after the treatment . to evaluate antitumor effect , mice from various groups the specimens were fixed in 10% buffered formalin , embedded in paraffin , and 4 mm diameter tissue sections were stained with hematoxylin and eosin following a standard procedure at the tissue pathology core facility at ouhsc . the sections were viewed and photographed by bright - field microscopy at 4 magnification .

we recently developed photo - unclick chemistry , a novel chemical tool involving the cleavage of aminoacrylate by singlet oxygen , and demonstrated its application to visible light - activatable prodrugs . in this study , we prepared an advanced multifunctional prodrug , pc-(l - ca4)2 , composed of the fluorescent photosensitizer phthalocyanine ( pc ) , an so - labile aminoacrylate linker ( l ) , and a cytotoxic drug combretastatin a-4 ( ca4 ) . pc-(l - ca4)2 had reduced dark toxicity compared with ca4 . however , once illuminated , it showed improved toxicity similar to ca4 and displayed bystander effects in vitro . we monitored the time - dependent distribution of pc-(l - ca4)2 using optical imaging with live mice . we also effectively ablated tumors by the illumination with far - red light to the mice , presumably through the combined effects of photodynamic therapy ( pdt ) and released chemotherapy drug , without any sign of acute systemic toxicity .

Introduction Results and Discussion Conclusion Experimental Section

we named the cleavage of aminoacrylate by so photo - unclick chemistry , and demonstrated the release of the anticancer drug combretastatin a-4 ( ca4 ) from its prodrug using this method . we prepared cmp - l - ca4 , a ca4 prodrug that can be activated by far - red light ( 690 nm ) , by combining a so - labile aminoacrylate linker , a core - modified porphyrin ( cmp ) photosensitizer , and ca4 . thus , we could track the prodrugs and then treat the tumor with the combined effects of photodynamic therapy ( pdt ) and local chemotherapy ( figure 1a ) . the light dose used for ] we generated pc-(l - ca4)2 , an advanced multifunctioning ca4 prodrug , for both fluorescence optical imaging and combination therapy with pdt and released ca4 . we chose phthalocyanine ( pc ) because pc is a fluorescent photosensitizer that can generate both fluorescence and singlet oxygen . we evaluated the cytotoxic effects of these two prodrugs with and without illumination , the inhibition of tubulin polymerization , the in vitro bystander effects , tumor localization using optical imaging , and the antitumor effects . effects of 3 m each of pcx , ca4 , pc-(l - ca4)2 , and pc-(ncl - ca4)2 on tubulin polymerization : ( a ) one data set of representative kinetic traces ( data from two more experiments are found in figure s1 , supporting information ) and ( b ) inhibition of tubulin polymerization by ca4 , pc-(l - ca4)2 , and pc-(ncl - ca4)2 after 1 h incubation ( sd of three experiments ) . due to the dramatic reduction of the inhibition of tubulin polymerization , it was expected that pc-(l - ca4)2 and pc-(ncl - ca4)2 would have lower dark toxicity ( cytotoxicity without illumination ) than ca4 . using mcf-7 cells , we found that the dark toxicity of the prodrugs decreased by 19- and 101-fold [ ic50d values = 9 , 173 , and 916 nm for ca4 , pc-(l - ca4)2 and pc-(ncl - ca4)2 , respectively ] . fluorescence live cell images of the center of each well treated with ( a ) vehicle ( diluting solution without a prodrug ) , ( b ) 25 nm pc-(l - ca4)2 , and ( c ) 25 nm pc-(ncl - ca4)2 . because the illumination for optical imaging could theoretically generate so and thus release ca4 from pc-(l - ca4)2 , we monitored the body weight change and tumor growth pattern of the mice imaged with pc-(l - ca4)2 . fluorescence optical images of the mice after retro - orbital injection of 2 mol / kg of prodrug : ( a ) pc-(l - ca4)2 in polymer micelles , ( b ) pc-(ncl - ca4)2 in polymer micelles , ( c ) pc-(l - ca4)2 in 5% cremophor solution , and ( d ) pc-(ncl - ca4)2 in 5% cremophor solution . we found outstanding antitumor effects in the mice treated with pc-(l - ca4)2 , g4g6 . treatment with either pc-(ncl - ca4)2 or ca4 produced minimal antitumor effects , suggesting that the outstanding antitumor effects of pc-(l - ca4)2 may have been a result of the synergistic effects of pdt and chemotherapy . our multifunctional prodrug strategy includes ( 1 ) activation by the clinical translatable far - red light ( or nir ) , ( 2 ) the unique combination of pdt and local chemotherapy , and ( 3 ) the dual function of optical imaging and treatment with one prodrug . we used stock solutions of pc-(l - ca4)2 and pc-(ncl - ca4)2 micelles in aqueous solution and further dilutions to achieve final doses as follows : [ ca4 , ( 1 mol / kg each ) ] , [ pc-(ncl = ca4)2 , ( 1 mol / kg each ) ] , [ pc-(l - ca4)2 , ( 1 mol / kg each ) ] , and [ pc-(l - ca4)2 , ( 2 mol / kg each ) ] .

herbicide - resistant ( hr ) crops , particularly glyphosate - resistant ( gr ) crops , have transformed the way many growers manage weeds . however , after three decades and billions of dollars invested in research , only a few transgenic herbicide traits are commercially available . two transgenes code for a glyphosate - insensitive 5-enolpyruvylshikimate-3-phosphate synthase ( epsps ; ec 2.5.1.19 ) , the cp4 epsps gene from agrobacterium tumefaciens strain cp4 and the mutated zm-2mepsps from corn ( zea mays l. ) , and three transgenes code for metabolic inactivation . one gene from ochrobactrum anthropi strain lbaa encodes for glyphosate oxidoreductase ( gox ) , and two homologous genes , pat and bar from streptomyces viridochromogenes and streptomyces hygroscopicus , respectively , encode n - acetyltransferases that inactivate glufosinate . today , hr traits are used on > 80% of the estimated 134 million hectares of transgenic crops grown annually in 25 countries with a single trait , cp4 epsps , being by far the most utilized.(5 ) growers rapidly adopted the first gr crops because the technology enabled a new weed control practice with glyphosate that was effective , easy - to - use , economical , safe , and novel . the novel attribute of the gene technology was essential to get patents that protected the large investment needed to develop the technology , whereas growers touted the simplicity and convenience of the glyphosate - based crop systems . initially , glyphosate was exceedingly effective in gr crops , and many growers relied only on glyphosate to control weeds . some academic weed scientists were concerned about the sustainability of this approach and predicted the evolution of resistance . however , no cases of gr weeds had evolved after more than two decades of broad use in noncrop situations,(6 ) and some weed scientists and growers began to think that gr weeds would never be a problem . then the paradigm changed in 1996 with the discovery of gr rigid ryegrass ( lolium rigidum gaudin ) in australia . today , all accept the evolution of gr weeds is threatening the continued success of gr crops and the sustainability of glyphosate . nineteen weeds have evolved resistance to glyphosate ; about half evolved in gr crops.(9 ) the basis for resistance has been attributed to altered epsps target site,(10 ) reduced translocation or cellular transport to the plastid,(11 ) sequestration in the vacuole,(12 ) and gene amplification.(13 ) gr weeds increase the cost of weed control and diminish the benefits of glyphosate - based weed management systems . no matter how effective a herbicide is , weed management programs can not rely so heavily on one tactic or weeds will ultimately adapt and survive in large numbers . growers applied glyphosate alone over vast cropping areas to control genetically variable and prolific weeds year after year . many of these weeds had already evolved resistance to other herbicide modes of action , so there was no good herbicide alternative when these weeds subsequently evolved resistance to glyphosate.(14 ) of particular note is the case of the highly competitive and prolific palmer amaranth ( amaranthus palmeri s. wats . ) . the explosion of gr palmer amaranth populations in the southeastern united states became known as the pigweed disaster.(15 ) these gr populations are forcing growers to change their production practices and increase the costs for weed control , even to the extent of hand - weeding . because of these shortsighted use practices , glyphosate is not as effective as it used to be and growers must supplement glyphosate with other herbicides . growers now need to diversify the herbicides they use to mitigate the spread of gr weeds.(16 ) unfortunately , the chemical industry has not commercialized a herbicide with a new mode of action ( moa ) for over two decades.(17 ) this is partly because the number of chemicals that must be tested to discover a new herbicide has increased from fewer than 1000 in 1950 to more than 500,000 today and partly because companies are investing less money to discover new herbicides as the widespread use of gr crops has reduced the market opportunity . to address the gr weed problem , the industry is now developing new herbicide resistance traits that will expand the utility of currently available herbicides . however , it is critically important to recognize that these traits represent interim solutions for current weed problems and do not replace the long - term need to discover herbicides with new modes of action and to diversify weed management tactics . farm size was increasing , and the number of growers was declining ; thus , growers had to become more efficient . furthermore , weeds were rapidly evolving resistance to various herbicides , and growers perceived weed management as taking too much time . growers wanted new weed management tactics , and gr crops enabled an economical , efficient , and simple solution . the average rate and number of applications of glyphosate increased as its price declined , and the use of other herbicides decreased . competitors reacted by reducing the price of their herbicides , but those alternatives could not maintain their market presence.(20 ) in retrospect , gr crops could have helped to increase the diversity of herbicides that growers used ( table 1 ) . gr crops did not require that growers use only glyphosate and the added diversity of glyphosate combined with other herbicides would have mitigated the evolution of hr weeds . however , the use of tank mixtures and sequential application of different herbicides declined . in one year , from 1997 to 1998 , the use of glyphosate increased 81% in parallel with the increase of gr soybeans [ glycine max ( l. ) merr . ] from 13 to 36%.(21 ) the number of herbicide active ingredients used on at least 10% of the u.s . soybean area declined from 11 in 1995 to only 1 , glyphosate , in 2002.(22 ) even though the chance of weeds evolving resistance to glyphosate in a particular location is still predicted to be lower than that with other herbicides , weeds ultimately did evolve glyphosate resistance as a direct result of the lack of weed management diversification on incredibly large areas of gr crops . herbicides grouped according to the herbicide resistance action committee http://www.plantprotection.org/hrac . interestingly , hr weeds often do not decrease the amount of herbicide used because growers make herbicide decisions based on weed complexes , not individual species or biotypes . if a weed evolves resistance to a herbicide , that herbicide has not lost all of its value as it still controls other weeds , and growers often continue to use the herbicide in a program with another herbicide to control the resistant weed . furthermore , growers do not recognize the potential for weeds to evolve resistance to glyphosate until the biotypes appear in their fields.(25 ) unfortunately , this can lead to the practice of sequentially using herbicides until they are no longer effective , which is the fastest way to evolve multiple hr weeds.(16 ) a combination of herbicides , cultural , and mechanical tactics provides the greatest protection from hr weeds . some weed species are particularly troublesome to control and in their propensity to evolve resistance ( table 2 ) . problematic weeds in glyphosate - based production systems that have evolved genetic mutations that confer glyphosate resistance include palmer amaranth and waterhemp [ amaranthus tuberculatus ( moq . ) other weeds such as velvetleaf [ abutilon theophrasti ( l. ) medik . ] , morningglories ( ipomoea spp . ) , asiatic dayflower ( commelina communis l. ) , tropical spiderwort ( commelina benghalensis l. ) , and field bindweed ( convolvulus arvenis l. ) often survive because of naturally higher tolerance . populations of tolerant weed species increase when growers use less than full - labeled rates.(26 ) currently , at least seven gr weed species have evolved resistance to multiple herbicide moas , with one population of waterhemp in illinois being resistant to four.(27 ) the rapid expansion of multiple hr weed populations threatens the sustainability of current crop production systems.(16 ) weed selection determined by a market research survey of u.s . corn , soybean , and cotton growers by gfk kynetec , inc . , st . louis , mo ( used with permission ) . weed control ratings are summarized from u.s . extension guides with 0 being the lowest and 10 being the highest level of control . an r next to herbicide efficacy rating indicates that this weed has developed resistance to herbicide mode of action ( heap 2010 ) . accase trait currently under development and anticipated to be an issue in feral corn after commercialization . the best weed management strategy is to control weeds prior to the loss of crop yield potential and proactively delay the evolution of weed resistance . fortunately , most fields do not have gr weeds yet , and there is still time for many growers to implement diverse and proactive weed management practices ( table 3).(28 ) generally , the basic management tactics are the same for both the prevention and control of hr weeds , that is , diversification of tactics to reduce selection pressure imposed by specific herbicides . the challenge is to implement these practices under prevailing economic constraints when growers are not convinced resistance management tactics will be effective or they believe industry will continue to deliver new solutions to manage weeds.(29 ) many growers are reluctant to diversify weed management because they perceive alternative tactics as being less cost - effective despite growing evidence that such tactics can improve profitability as well as mitigate resistant weed issues.(30 ) more education will help overcome this perception as will the explosion of multiple hr weeds that emphatically persuades growers to diversify their weed management practices now or face serious long - term consequences . besides glyphosate , most current herbicides used for weed management in corn , soybean , and cotton are selective and typically used in mixtures to control a broad spectrum of weed species . the following section provides an overview of the utilities and limitations for various herbicide moas that have potential utility in hr crops . glyphosate is a nonselective , broad - spectrum foliar herbicide with no soil residual activity that has been used for > 30 years to manage annual , perennial , and biennial herbaceous grass , sedge , and broadleaf weeds as well as unwanted woody brush and trees . many glyphosate formulations and salts are commercially available ; the most common salts are the monopotassium and isopropylamine . the type and amount of adjuvant included in the various formulations differ greatly and strongly influence weed control . glyphosate strongly competes with the substrate phosphoenolpyruvate ( pep ) at the epsps enzyme - binding site in the chloroplast , resulting in the inhibition of the shikimate pathway . products of the shikimate pathway include the essential aromatic amino acids tryptophan , tyrosine , and phenylalanine and other important plant metabolic products.(31 ) the relatively slow moa and physicochemical characteristics result in glyphosate translocation throughout the plant and accumulation at the vital growing points before phytotoxicity occurs . favorable physicochemical characteristics , low cost , tight soil sorption , application flexibility , low mammalian toxicity , and availability of gr crops have helped make glyphosate the most widely used herbicide in the world.(32 ) a key advantage for glyphosate has been the consistent control of weeds almost without regard to size . however , the flexibility in glyphosate application timing and lack of soil residual have often resulted in growers delaying applications to help ensure that all of the weeds have emerged . unfortunately , such delay in application means that the weeds have begun to compete with the crop and thus reduced potential yield . the increased use of mixtures with herbicides that have soil residual activity will encourage growers to make earlier glyphosate applications and increase the likelihood that a single application gives season - long control . other commonly noted weaknesses with glyphosate are higher rates needed to control the more tolerant broadleaf weeds , antagonism by hard water and tank mixture partners , slow speed of action , and poor rainfastness . glufosinate is a nonselective , broad - spectrum foliar herbicide with no soil residual soil activity that inhibits glutamine synthetase [ gs ; ec 6.3.1.2 ] , an enzyme that catalyzes the conversion of glutamate plus ammonium to glutamine as part of nitrogen metabolism.(31 ) glufosinate is faster acting and controls key broadleaf weeds such as morningglories ( ipomoea spp . ) , hemp sesbania ( sesbania herbacea ( p. mill . ) mcvaugh ) , pennsylvania smartweed ( polygonum pensylvanicum l. ) , and yellow nutsedge ( cyperus esculentus l. ) better than glyphosate . however , glufosinate is used at higher rates and has historically been more expensive than glyphosate . cost and more restrictive application timing relative to weed size are probably its greatest disadvantages compared to glyphosate . because glufosinate behaves as a contact herbicide , it must be applied to smaller plants than glyphosate and is not as effective on perennials that require significant translocation for complete control . still , glufosinate is labeled to control > 120 broadleaf weeds and grasses including key gr weeds . no weeds have been formally reported as glufosinate - resistant yet.(9 ) synthetic auxin herbicides act as auxin agonists by mimicking the plant growth hormone indole-3-acetic acid ( iaa ) , disrupting growth and development processes , and eventually causing plant death , particularly in broadleaf species.(31 ) growers have used auxin herbicides widely for over 60 years as selective herbicides in monocotyledonous crops . auxins control a broad spectrum of broadleaf weeds , including key weeds that have evolved resistance to glyphosate . some synthetic auxins such as dicamba have fair soil residual activity with a half - life from 7 to 21 days . relatively few weeds have evolved resistance to auxin herbicides , which is noteworthy considering their long - term and widespread use . for example , only six weed species have evolved resistance to dicamba after 50 years of widespread use in cereal and noncrop environments.(9 ) the increased use of dicamba and other auxin herbicides in auxin - resistant crops has the potential of injuring other broadleaf crops and reducing biodiversity in field edges and nearby noncrop habitat if unmanaged.(33 ) off - target movement of auxin herbicides can occur via spray particle and vapor drift . particle drift is more problematic than vapor drift , but growers can manage with modified application techniques , drift control adjuvants , and correct decisions as to when , where , and how to apply . particularly troublesome for auxin herbicides would be any movement onto highly sensitive crops such as soybeans , cotton ( gossypium hirsutum l. ) , or grapes ( vitis vinifera l. ) . interestingly , 2,4-d is safer than dicamba on soybeans and dicamba is safer than 2,4-d on cotton.(34 ) as little as 0.01% of the labeled rate of dicamba can injure soybeans,(35 ) and 0.001% of the labeled rate of 2,4-d butyl ester formulation can injure tomatoes ( lycopersicon esculentum mill . ) and lettuce ( lactuca sativa l.).(36 ) some forms of dicamba and 2,4-d are highly volatile , especially at high temperatures . for example , the acid form of dicamba is more volatile than amine salt formulations , and some amine salts are more volatile than others . the manufacturer can also reduce potential off - target movement with application restrictions based on temperature , droplet size , humidity , and wind speed . because of their volatility and the sensitivity of nontarget crops , growers will probably not use auxin herbicides on vast areas during warm weather as is currently done with glyphosate . the enzyme 4-hydroxyphenyl pyruvate dioxygenase [ hppd ; ec 1.13.11.27 ] converts 4-hydroxyphenyl pyruvate to homogentisate , a key step in plastoquinone biosynthesis . this is the most recently discovered herbicide moa , and active analogue testing continues to generate new products.(37 ) inhibition of hppd causes bleaching symptoms on new growth by indirectly inhibiting carotenoid synthesis due to the requirement of plastoquinone as cofactor of phytoene desaturase [ pds ; ec 1.14.99].(38 ) visible injury depends on carotenoid turnover and thus is slower to appear on older tissues than young leaves.(31 ) hppd - inhibiting herbicides control a number of important weed species and may have soil residual activity , and no weeds have been formally reported to be resistant to this moa yet . corn is naturally tolerant to key hppd herbicides , but soybeans and cotton are generally sensitive . herbicides that inhibit acetolactate synthase ( als ; ec 2.2.1.6 ) , also known as acetohydroxyacid synthase ( ahas ) , were discovered in the mid-1970s and are still widely used . the als enzyme is a key step in the biosynthesis of the essential branched - chain amino acids valine , leucine , and isoleucine . als is a nuclear encoded enzyme that moves to the chloroplast via a transit peptide . more than 50 different als - inhibiting herbicides from five different chemical classes ( sulfonylureas , imidazolinones , triazolopyrimidines , pyrimidinylthiobenzoates , and sulfonylamino - carbonyl - triazolinones ) are commercially available . the characteristics of als herbicides vary in their soil residual properties , crop response , and types of weeds that are effectively controlled . als herbicides can provide foliar and soil residual activity on important grass and broadleaf weeds at low application rates . the tendency of weeds to evolve resistance to als herbicides limits their utility,(9 ) and their use is now mainly in mixtures with other types of herbicides . protoporphyrinogen oxidase ( ppo ; ec 1.3.3.4 ) is an essential enzyme that catalyzes the last common step in the biosynthesis of heme and ultimately chlorophyll by the oxidation of protoporphyrinogen ix to protoporphyrin ix . ppo - inhibiting herbicides cause the accumulation of protoporphyrinogen ix , which is photoactive , and exposure to light causes the formation of singlet oxygen and other oxidative chemicals that cause rapid burning and desiccation of leaf tissue . the soil residual and fast action characteristics of ppo herbicides complement the lack of soil residual and the slow activity of glyphosate . ppo enzyme mutations tend to reduce the enzymatic activity , which helps explain the relatively slow evolution of resistant weeds to this 40-year - old herbicide class.(41 ) companies continue to synthesize analogues and commercialize new ppo - inhibiting herbicides . for example , saflufenacil was introduced in 2010 and is labeled for use in a wide variety of crops , including corn , soybeans , and cotton.(42 ) its label describes burndown and residual control of 70 broadleaf weeds including key troublesome weeds in glyphosate - based systems such as common lambsquarters ( chenopodium album l. ) , horseweed [ conyza canadensis ( l. ) cronq . ] , waterhemp , and common ( ambrosia artemisiifolia l. ) and giant ( ambrosia trifida l. ) ragweeds . acetyl coenzyme a carboxylase [ accase ; ec 6.4.1.2 ] is the first step of fatty acid synthesis and catalyzes the adenosine triphosphate ( atp)-dependent carboxylation of malonyl - coa to form acetyl - coa in the cytoplasm , chloroplasts , mitochondria , and peroxisomes of cells.(43 ) accase - inhibiting herbicides generally inhibit the accase activity of monocot species and not dicots . the three chemical classes of accase inhibitors are cyclohexanediones ( dims ) ( e.g. , sethoxydim ) , aryloxyphenoxypropionates ( fops ) ( e.g. , quizalofop ) , and phenylpyrazolines ( dens ) ( e.g. , pinoxaden ) . the ability to use accase herbicides selectively in corn would be useful , but the tendency of weeds to evolve resistance to this herbicide class would limit its utility to being part of a weed management system.(9 ) currently used selective and burndown herbicides will continue to play important roles in weed management in hr crop systems ( table 1 ) . in addition to the herbicide types already discussed , photosystem ii ( psii ) inhibitors such as triazine and urea herbicides , lipid synthesis inhibitors such as s - metolachlor , and phytoene desaturase ( pds ) inhibitors such as clomazone will continue to be used as crop - selective herbicides to provide soil residual activity on key weeds . paraquat is a photosystem i ( psi ) inhibiting herbicide typically used in conservation and no - tillage production systems for nonselective burndown control of emerged weeds or as a directed spray with specialized application equipment in crop . paraquat controls a broad spectrum of weeds , and the lack of soil residual allows rotational crop flexibility similar to glyphosate and glufosinate . paraquat rapidly desiccates leaf tissue and thus does not translocate well enough to control perennial weeds . paraquat is relatively inexpensive , but its high mammalian toxicity imposes significant use and handling restrictions . a large number of transgenic and nontransgenic hr crops have been commercialized ( table 4 ) . these hr crops generally eliminated all crop injury concerns and allowed the grower to select new herbicide options with improved weed activity and environmental safety . before the advent of gr crops , most thought that the utility of hr crops would be limited to complementing selective herbicides . the full impact of hr crops really started in 1996 with the sales of gr soybeans . since then , the speed at which growers adopted gr crops has been unprecedented in corn , soybeans , and cotton.(4 ) success came despite an unpopular grower contract and strong objections by biotechnology opponents to potential unknown effects on the environment and human health and the ethical question of interfering with the natural order . with the exception of canada , nontransgenic hr traits are essentially unregulated . scientists have used a wide range of nontransgenic techniques to create crops with resistance to a number of herbicide moas ( table 5 ) . for example , the first commercial accase - resistant crop was a sethoxydim - resistant ( sr ) corn with an altered accase created using tissue culture selection.(49 ) a second accase trait is in the final stages of commercialization for use in sorghum . this trait was transferred with traditional breeding methods from feral sorghum ( shattercane , sorghum bicolor l. moench ) that had evolved accase herbicide resistance because of agronomic practices.(50 ) creating hr crops for als - inhibiting herbicides has been quite successful using tissue culture selection , pollen mutagenesis , microspore selection , seed mutagenesis , and gene transfer from close weedy relatives that had evolved herbicide resistance because of agronomic practices . today , at least seven different als - resistant crops are commercially available.(53 ) in all cases , resistance is due to an als mutation with three general crop phenotypes : broad resistance to als herbicides ; resistance only to imidazolinone and pyrimidinylthiobenzoate herbicides ; and resistance only to sulfonylurea and triazolopyrimidine herbicides . nontransgenic hr crops were only modestly successful ; the big success with hr crops began with transgenic gr soybeans in 1996 ( table 6 ) . growers perceived glyphosate resistance as the ideal herbicide trait because glyphosate controls over 300 annual and perennial weeds , has flexible application timings , and does not have any rotational crop restrictions.(56 ) gr crops allowed growers to use glyphosate as an in - crop selective herbicide and replace more expensive , selective herbicides that controlled a narrower weed spectrum and had other issues ( e.g. , crop tolerance ) . within a decade after glyphosate became commercially available , the search began to find crop resistance to glyphosate . nontransgenic approaches were not successful , and transgenic approaches were difficult and not initially successful.(57 ) initial attempts to find any natural enzymes in crops that could metabolically inactivate or were insensitive at the target site failed . eventually , a gene for a glyphosate insensitive epsps with enzymatic characteristics similar to plant epsps was isolated from a common soil bacterium , agrobacterium tumefaciens strain cp4 , which was surviving in a glyphosate manufacturing waste stream in luling , la.(57 ) this cp4 epsps gene has been used to develop gr soybeans , cotton , corn , canola , alfalfa ( medicago sativa l. ) , bentgrass ( agrostis stolonifera l. ) , and sugar beet ( beta vulgaris l.).(5 ) glyphosate resistance became the most rapidly adopted technology in the history of agriculture,(5 ) but the first gr crops were not perfect . the timing , rate , and number of glyphosate applications had to be restricted to ensure crop resistance,(5 ) and there were reports of a yield drag.(58 ) a new generation of herbicide traits currently in development will be combined with current and new glyphosate traits to help continue to improve this technology and extend the transgenic weed management revolution . glufosinate - resistant crops have been commercially available as long as gr crops ( table 6 ) , but have not been as successful for a number of reasons , particularly because of the higher cost of glufosinate and its more restrictive application timings . glufosinate resistance is widely available , not only because of its utility as a herbicide trait but also because it has been often used as a marker for other traits , particularly insect resistance traits . resistance to glufosinate is due to metabolic inactivation of the parent molecule by either of two homologous enzymes , phosphinothricin n - acetyltransferase ( pat ) or basta n - acetyltransferase ( bar ) , that catalyze the acetylation of glufosinate.(59 ) both genes were isolated from soil microorganisms , pat from streptomyces viridochromogenes and bar from streptomyces hygroscopicus . cotton and soybean growers who are troubled by difficult to control gr weeds such as palmer amaranth and waterhemp may rapidly adopt glufosinate - resistant crops and the use of glufosinate . crop cultivars that include resistance to both glufosinate and glyphosate are now commercially available in cotton , soybeans , and corn and provide growers a choice between two broad - spectrum herbicides as well as an array of naturally selective herbicides to diversify their weed management practices . whereas gr crops have been very successful , the evolution of gr weeds was faster and more widespread than many expected . this rapid evolution of gr weeds and the lack of any new selective herbicides with novel moas is encouraging hr crop technology to evolve again . the next wave of technologies will combine resistance to glyphosate and other herbicides to provide growers with more herbicide options with different moas as well as the possibility of using herbicides with both foliar and soil residual activity . scientists have discovered a plethora of herbicide traits that can be combined with glyphosate resistance to make multiple hr crops ( table 7 ) . if used correctly , multiple hr crops with these traits can sustain the usefulness of glyphosate . corn is relatively tolerant to most synthetic auxin herbicides , but soybeans and cotton are sensitive , and scientists have long sought a transgene to give these crops resistance and allow the use of auxin herbicides.(66 ) auxin herbicides control a broad spectrum of broadleaf weeds , including most known gr broadleaf weeds . because auxin herbicides act rapidly at multiple receptors and compete with an essential plant hormone pathway , making crops resistant by modifying the site of auxin action is difficult . in addition , these receptors respond differently to different auxin herbicide classes , for example , phenoxyacetates ( e.g. , 2,4-d ) , pyridinyloxyacetates ( e.g. , fluoroxypyr ) , benzoates ( e.g. , dicamba ) , picolinates ( e.g. , picloram ) , and quinolinecarboxylates ( e.g. , quinclorac).(67 ) so far , metabolic inactivation has proven to be a more successful strategy . a gene encoding for dicamba monooxygenase ( dmo ) , an enzyme that deactivates dicamba , was cloned from a soil bacterium , stenotrophomonas maltophilla , and used to make dicamba - resistant soybeans . the dmo enzyme encodes a rieske nonheme monooxygenase that metabolizes dicamba to 3,6-dichlorosalicylic acid ( dcsa ) . the complete bacterial dicamba o - demethylase complex consists of the monooxygenase , a reductase , and a ferredoxin . electrons are shuttled from reduced nicotinamide adenine dinucleotide ( nadh ) through the reductase to the ferredoxin and finally to the terminal component dmo . researchers can successfully express dmo in the cell nucleus with or without a transit peptide as well as in the chloroplasts where the monooxygenase would have a source of electrons produced by photosynthesis and where transgenic proteins can often be expressed at higher levels . commercialization of dicamba - resistant soybean and cotton is anticipated mid - decade . a family of aad genes that code for aryloxyalkanoate dioxygenase provides resistance to certain auxin herbicide . the aad-12 gene was isolated from delftia acidovorans and codes for a 2-ketoglutarate - dependent dioxygenase that inactivates phenoxyacetate auxins ( e.g. , 2,4-d ) and pyridinyloxyacetate auxins ( e.g. , triclopyr and fluoroxypyr).(62 ) this trait , dht2 , is being developed in soybeans . a second gene known as aad-1 was isolated from sphingomonas herbicideovarans and inactivates auxins and accase - inhibiting herbicides in the class known as fops ( e.g. , fluazifop).(62 ) this trait , dht1 , is being developed in corn . both traits are reported to provide resistance to high rates of 2,4-d with no adverse agronomic effects . the 2,4-d and dicamba resistance traits will always be used in stacks with at least one other herbicide - resistance trait . the expected increased use of auxin herbicides will increase the potential for off - target movement and injury to sensitive broadleaf plants . due to this potential environmental problem , the herbicide and trait providers will likely introduce improved herbicide formulations with better use directions before the traits are commercialized mid - decade . ironically , this risk of off - target movement could drive more rapid adoption of auxin traits because growers will want to protect their soybean and cotton crops from nearby applications of auxin herbicides . in some ways , many hppd herbicides have soil residual activity and control key broadleaf weeds that have already evolved resistance to glyphosate . increased resistance mechanisms for hppd herbicides include a less sensitive target site , overexpression of the enzyme , alternate pathway , increasing flux in the pathway , and metabolic inactivation . crops resistant to hppd herbicides have been in field development tests since 1999 , but there have been no technical disclosures of hppd resistance traits under developments thus far . technologies llc ( west point , ia ) and syngenta ( basel , switzerland ) have independently announced plans to develop hppd - resistant crops . bayer cropscience recently disclosed that they were developing soybeans resistant to three herbicide types : glyphosate , glufosinate , and hppd herbicides ( e.g. , isoxaflutole).(17 ) isoxaflutole can provide pre - emergence ( pre ) and postemergence ( post ) control of a relatively broad spectrum of annual weeds with soil residual activity . the triple stack offers the advantage of enabling the use of two herbicide moas to which weeds have not yet evolved resistance . for example , transgenic crops resistant to ppo - inhibiting herbicides have been developed , and the technology even received the trade name acuron.(41 ) the first ppo - resistant corn used a double mutant ppo , ppo-1 , from a. thaliana . similarly , ppo - resistant rice used overexpression of the naturally resistant bacillus subtilis ppo gene to confer resistance . other approaches including increasing gene copy number and tissue culture to select for overexpression of wild type ppo genes have also been successful.(41 ) the broad - spectrum weed control and soil residual activity of ppo herbicides could be useful in corn , soybeans , and cotton , but the existing widespread resistance to this class among some amaranthus species limits the value of the technology . a transgenic dht1 trait also gives resistance to accase - inhibiting herbicides by degrading the alkanoate side chains to a hydroxyl of the fop class of accase herbicides ( e.g. , quizalofop).(62 ) dht1 corn reportedly tolerates postemergence applications of quizalofop of up to 184 g / ha with no adverse agronomic effects . in addition , the specificity of its inactivation could allow the use of other accase herbicide types for hr volunteer corn management in rotational crops . metabolic inactivation systems based on cytochrome p450 monooxygenases ( p450 ) and glutathione transferase ( gst ) have the potential to inactivate a wide range of herbicide types ( table 7 ) . for example , native p450 enzymes can metabolically inactivate acetanilides , bentazon , dicamba , some als - inhibiting herbicides , isoxazoles , and urea herbicides . the chemical specificity of this metabolic system may offer the unique potential to allow growers to use herbicides in the same moa to control weeds in one season and still manage any feral volunteers with a herbicide in the same moa in the next year . no single herbicide resistance trait will be sustainable if the grower uses only the single herbicide type that the trait enables recurrently . multiple hr crops will help by allowing the use of new herbicide mixtures with multiple modes of action , but agriculture must manage this technology objectively and pragmatically , balancing short - term and long - term interests , so as not to create a transgenic treadmill.(18 ) the lack of soil residual activity has encouraged multiple in - crop applications glyphosate , as many as four or more applications per growing season . some of the new , multiple hr crop technologies will enable herbicide applications with soil residual activity and thus help growers to reduce selection pressure on the weed community by glyphosate.(74 ) for example , the glyphosate and als trait stack that has recently been deregulated in the united states can allow the use of als - inhibiting herbicides with soil residual that are too phytotoxic to use on conventional crop cultivars.(75 ) this stack consists of a metabolic system to inactivate glyphosate based on an enhanced glyphosate acetyltransferase enzyme from the soil bacterium bacillus licheniformis ( weigmann ) chester(76 ) and a highly resistant als allele ( hra ) with two mutations , tryp574leu and pro197ala.(75 ) a wide array of other combinations of current and new herbicide resistance traits is expected within the next decade . if used correctly , these multiple hr crops will provide new uses for existing herbicides to help growers better manage weeds and help sustain the utility of glyphosate and glyphosate resistance traits . weed management dramatically changed with the widespread adoption of gr crops . using glyphosate in gr crops made weed management too simple and convenient . importantly , the high initial efficacy of glyphosate declined with repeated use , and current glyphosate - based weed management systems are in jeopardy as evidenced by the speed at which weed populations are evolving resistance . still , glyphosate has not lost all utility ; it controls more weeds more effectively than other herbicides , but it can no longer be applied alone anytime on any weed anywhere . most growers still do not have any gr weeds in their fields and have time to implement proactive hr weed management practices to help sustain glyphosate . however , growers need to act now to diversify the herbicides and tactics they use , the crops they plant , their cultural practices , and field hygiene measures . the flexibility and range of alternative weed management practices will be narrow and require integration to replace glyphosate . these management practices will work better for the prevention rather than the control of gr weeds . once present , gr weeds can be managed but are difficult if not impossible to eradicate . current corn , soybean , and cotton cropping systems are based on a heavy reliance on glyphosate . given the changes in weed populations that are being reported , it is of paramount importance that other weed management alternatives be identified and implemented quickly . it is likely that no new herbicide or trait technology will match the impact of glyphosate and the first gr crops on agriculture . growers will use these new technologies in combinations to fill in efficacy gaps and diversify weed management practices . initially , it may look like an attempt to make glyphosate look as good as it used to be . some traits such as glufosinate resistance will enable a broad - spectrum alternative to glyphosate . others will enable options with soil residual activity or new moas to control key gr weeds . some hr crop technologies may benefit from incremental improvements in efficacy and properties of herbicides within long - standing herbicide moas that companies are still commercializing . growers must diversify their weed management practices now.(78 ) the more growers diversify , the less the risk that weeds will evolve herbicide resistance . diversification may make weed management more complex , but growers must not use new hr crop systems in the same way that some used initial gr crops , as a means to rely only on one herbicide until it is no longer effective and then switch herbicides . if growers use the new hr crops and the herbicides that they enable properly , hr crops will expand the utility of currently available herbicides and provide long - term solutions to manage gr weeds . hr crops will not replace the need for technical innovations , particularly the discovery of herbicides with new moas . diversification will be much easier if growers can chose from among multiple effective and economical weed management options . in areas of the world that have not yet adopted gr crops growers must not wait , but implement best management practices as soon as new trait and herbicide technologies are available . by using diverse weed management practices , growers will preserve the utility of herbicide resistance traits and herbicide technologies and help maintain profitable and environmentally sustainable crop production systems for future generations .

since 1996 , genetically modified herbicide - resistant ( hr ) crops , particularly glyphosate - resistant ( gr ) crops , have transformed the tactics that corn , soybean , and cotton growers use to manage weeds . the use of gr crops continues to grow , but weeds are adapting to the common practice of using only glyphosate to control weeds . growers using only a single mode of action to manage weeds need to change to a more diverse array of herbicidal , mechanical , and cultural practices to maintain the effectiveness of glyphosate . unfortunately , the introduction of gr crops and the high initial efficacy of glyphosate often lead to a decline in the use of other herbicide options and less investment by industry to discover new herbicide active ingredients . with some exceptions , most growers can still manage their weed problems with currently available selective and hr crop - enabled herbicides . however , current crop management systems are in jeopardy given the pace at which weed populations are evolving glyphosate resistance . new hr crop technologies will expand the utility of currently available herbicides and enable new interim solutions for growers to manage hr weeds , but will not replace the long - term need to diversify weed management tactics and discover herbicides with new modes of action . this paper reviews the strengths and weaknesses of anticipated weed management options and the best management practices that growers need to implement in hr crops to maximize the long - term benefits of current technologies and reduce weed shifts to difficult - to - control and hr weeds .

Introduction Utilities and Limitations of Current Herbicide Technologies Utilities and Limitations of Current and Future Herbicide-Resistant Crop Technologies Path Forward

herbicide - resistant ( hr ) crops , particularly glyphosate - resistant ( gr ) crops , have transformed the way many growers manage weeds . initially , glyphosate was exceedingly effective in gr crops , and many growers relied only on glyphosate to control weeds . (13 ) gr weeds increase the cost of weed control and diminish the benefits of glyphosate - based weed management systems . (17 ) this is partly because the number of chemicals that must be tested to discover a new herbicide has increased from fewer than 1000 in 1950 to more than 500,000 today and partly because companies are investing less money to discover new herbicides as the widespread use of gr crops has reduced the market opportunity . to address the gr weed problem , the industry is now developing new herbicide resistance traits that will expand the utility of currently available herbicides . however , it is critically important to recognize that these traits represent interim solutions for current weed problems and do not replace the long - term need to discover herbicides with new modes of action and to diversify weed management tactics . the average rate and number of applications of glyphosate increased as its price declined , and the use of other herbicides decreased . gr crops did not require that growers use only glyphosate and the added diversity of glyphosate combined with other herbicides would have mitigated the evolution of hr weeds . in one year , from 1997 to 1998 , the use of glyphosate increased 81% in parallel with the increase of gr soybeans [ glycine max ( l. ) merr . ] corn , soybean , and cotton growers by gfk kynetec , inc . the best weed management strategy is to control weeds prior to the loss of crop yield potential and proactively delay the evolution of weed resistance . fortunately , most fields do not have gr weeds yet , and there is still time for many growers to implement diverse and proactive weed management practices ( table 3). (30 ) more education will help overcome this perception as will the explosion of multiple hr weeds that emphatically persuades growers to diversify their weed management practices now or face serious long - term consequences . besides glyphosate , most current herbicides used for weed management in corn , soybean , and cotton are selective and typically used in mixtures to control a broad spectrum of weed species . before the advent of gr crops , most thought that the utility of hr crops would be limited to complementing selective herbicides . since then , the speed at which growers adopted gr crops has been unprecedented in corn , soybeans , and cotton. cotton and soybean growers who are troubled by difficult to control gr weeds such as palmer amaranth and waterhemp may rapidly adopt glufosinate - resistant crops and the use of glufosinate . crop cultivars that include resistance to both glufosinate and glyphosate are now commercially available in cotton , soybeans , and corn and provide growers a choice between two broad - spectrum herbicides as well as an array of naturally selective herbicides to diversify their weed management practices . corn is relatively tolerant to most synthetic auxin herbicides , but soybeans and cotton are sensitive , and scientists have long sought a transgene to give these crops resistance and allow the use of auxin herbicides. multiple hr crops will help by allowing the use of new herbicide mixtures with multiple modes of action , but agriculture must manage this technology objectively and pragmatically , balancing short - term and long - term interests , so as not to create a transgenic treadmill. (74 ) for example , the glyphosate and als trait stack that has recently been deregulated in the united states can allow the use of als - inhibiting herbicides with soil residual that are too phytotoxic to use on conventional crop cultivars. if used correctly , these multiple hr crops will provide new uses for existing herbicides to help growers better manage weeds and help sustain the utility of glyphosate and glyphosate resistance traits . importantly , the high initial efficacy of glyphosate declined with repeated use , and current glyphosate - based weed management systems are in jeopardy as evidenced by the speed at which weed populations are evolving resistance . most growers still do not have any gr weeds in their fields and have time to implement proactive hr weed management practices to help sustain glyphosate . however , growers need to act now to diversify the herbicides and tactics they use , the crops they plant , their cultural practices , and field hygiene measures . current corn , soybean , and cotton cropping systems are based on a heavy reliance on glyphosate . diversification may make weed management more complex , but growers must not use new hr crop systems in the same way that some used initial gr crops , as a means to rely only on one herbicide until it is no longer effective and then switch herbicides . if growers use the new hr crops and the herbicides that they enable properly , hr crops will expand the utility of currently available herbicides and provide long - term solutions to manage gr weeds . hr crops will not replace the need for technical innovations , particularly the discovery of herbicides with new moas . by using diverse weed management practices , growers will preserve the utility of herbicide resistance traits and herbicide technologies and help maintain profitable and environmentally sustainable crop production systems for future generations .

bacillus anthracis , the causative agent of the disease anthrax , has two major virulence factors , one in the form of exotoxins and the other in the form of unique capsule , which are encoded by the plasmids pxo1 and pxo2 , respectively [ 1 , 2 ] . the capsule is made up of poly - d--glutamic acid , a unique virulent structure that helps the bacterium to escape phagocytosis by macrophages inside the host . the exotoxins consist of three proteins , namely , protective antigen ( pa ) , lethal factor ( lf ) , and edema factor ( ef ) which together constitute the active anthrax toxins ( antx ) . basically , antx belongs to a - b toxin superfamily where the b moiety binds to the cell surface ( in this case , pa ) and assists in translocating the catalytic a moiety ( lf or ef ) inside the cell . individually , none of the proteins are toxic , but if pa combines with lf or ef , it results in the formation of lethal toxin ( letx ) or edema toxin ( edtx ) , respectively [ 5 , 6 ] . pa is a four - domain protein and can bind to either of the host cell receptors , capillary morphogenesis gene 2 ( cmg2 ) or tumor endothelial marker 8 ( tem8 ) , through its c - terminal domain [ 7 , 8 ] . once bound to the receptor , pa is cleaved at the sequence -rkkr- by host furin - like proteases to generate receptor bound 63 kda fragment ( pa63 ) and a free 20 kda fragment ( pa20 ) . the cleaved pa63 in its active state is oligomerized to form a pore like structure into which lf and ef can competitively bind with high affinity . once bound , lf / ef is translocated into the cell through receptor mediated endocytosis [ 10 , 11 ] . ef is calmodulin dependent adenylate cyclases which can increase the camp level inside the cell disturbing the water homeostasis resulting in characteristic edema observed during cutaneous anthrax . lf is a highly specific zinc metalloproteases that can cleave the members of map kinase kinases ( mapkk ) family at their n - terminus resulting in altered cell signalling , increased production of cytokines tumor necrosis factor- , interleukin-1 , and nitric oxide . lf is also known to activate inflammasome complex and caspases-1 mediated cellular apoptosis [ 1316 ] . in us , the fda approved anthrax vaccine adsorbed ( ava ) as the only vaccine commercially available for use against anthrax . it is prepared by adsorbing the cell free culture supernatant of acapsular strain of b. anthracis v770-np1-r on aluminium hydroxide gel [ 17 , 18 ] . in uk , anthrax vaccine precipitate ( avp ) is in use which is alum precipitated cell free culture supernatant of sterne strain 34f2 [ 19 , 20 ] . both ava and avp contain pa as major immunogen with trace amounts of lf and ef . although ava and avp are effective , their undefined composition , batch to batch variation , extensive dosing regimen , and adverse immunological side effects have made way for the search of second - generation anthrax vaccine containing recombinant pa that is under developmental stage [ 21 , 22 ] . these recombinant vaccines differ from their predecessors in that their composition will be defined , amenable to large scale production , and will be free from any adverse side effects . in case of large scale immunization of human population , huge quantities of biologically active recombinant pa will be required . for this purpose , a scalable purification process has been developed to generate recombinant pa in multigram quantities from recombinant e. coli . apart from e. coli , numerous other attempts to develop expression systems are based on variety of organisms including attenuated strains of b. anthracis , b. subtilis , b. brevis , and salmonella typhimurium [ 21 , 2426 ] . however , these approaches are not simpler and often require multiple harsh purification steps which may result in reduced yield and stability [ 27 , 28 ] . it is therefore always better to express a protein in a soluble form , as it can result in functionally active and biologically stable protein . moreover , soluble proteins are easy to purify and reduce the extra downstream processing that is involved in purifying the denatured proteins and refolding them to make functionally active form . in addition to this , most of the denatured proteins are susceptible to precipitation and protease degradation [ 29 , 30 ] during downstream processing . in the present study , we have expressed recombinant soluble pa in e. coli expression system using three different expression vectors , namely , pproexhta , pqe30 , and pet32c , bearing trc , t5 , and t7 promoters , respectively . further , each vector construct was transformed into different e. coli expression hosts and the growth conditions of the transformants were optimized so as to obtain the maximum yield in soluble form . purification was carried out using simple chromatographic techniques and the yield of protein was compared . further , the soluble pa from maximum producing clone pa - pet32c - de3-plyss was characterized by trypsin digestion and lf binding assay to confirm its functionality . the biological activity was confirmed through macrophage cell protection assay using the pa immunized sera . this study highlights the straightforward production of large quantity of recombinant pa in its biologically active soluble form using an optimized e. coli vector - host combination system . the expression vector pproexhta was purchased from invitogen ( usa ) , pqe30 from qiagen ( germany ) , and pet32c from novagen ( germany ) . luria - bertani broth / agar for maintaining the expression hosts was obtained from himedia ( india ) . all the primers ( table 2 ) used in this study were custom synthesized from sigma aldrich , india . the genomic dna was isolated from the b. anthracis clinical isolate by conventional method and was used as a template for pcr reaction . the pcr reaction was carried out in a 25 l reaction containing 50 ng of template dna , 0.1 mol l primers , 1x supertaq complete buffer ( 2 mmol l mgso4 ) , 0.2 mmol l dntps , and 2.5 units of super taq dna polymerase ( ambion , usa ) . the pcr condition was as follows : initial denaturation of 94c for 5 minutes , 30 rounds of 1 minute denaturation at 94c , 1 minute annealing at 50c , and 2 minutes of extension at 72c followed by 10 minutes of final extension at 72c . the pcr product was electrophoresed on 0.8% agarose gel and was extracted using qiagen gel extraction kit . the pcr product along with expression vectors pproexhta , pqe30 , and pet32c was restriction digested with bamhi / xhoi , bamhi / kpni , and bamhi / sali ( fermentas , usa ) combination , respectively , and was followed by ligation to obtain pa - pproexhta , pa - pqe30 , and pa - pet32c recombinant plasmids . the recombinant plasmid pa - pproexhta was transformed into e. coli dh5 , bl21-de3 , and de3-plyss , pa - pqe30 in e. coli m15 and xl-1 blue , and pa - pet32c plasmid in e. coli bl21-de3 and de3-plyss , respectively . after transformation , the positive clones were confirmed by restriction digestion of the isolated plasmid with the following enzymes : pa - pproexhta with bamhi / xhoi , pa - pqe30 with bamhi / kpni , and pa - pet32c with bamhi / sali combination . to determine the maximum expression of pa from individual clones , preliminary expression was carried out at different temperatures and time periods . all the e. coli cells carrying the recombinant plasmids were grown in 5 ml luria - bertani broth tubes at 37c till the od600 reached 0.6 . isopropyl -d-1-thiogalactopyranoside ( iptg ) was added at the final concentration of 1 mmol l and the clones were further grown at 37c and 22c temperature for 3 hours , for 5 hours , and overnight . the cells were harvested at 8000 g for 5 minutes and were analyzed on 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis ( sds - page ) . the temperature and time period at which particular clone showed maximum expression of pa were noted and the same conditions were applied for bulk production of pa . for purification , purification under native condition was performed by immobilized metal ion affinity chromatography ( imac ) using nickel trinitriloacetic acid ( ni - nta ) resin followed by anion exchange chromatography using diethylaminoethanol ( deae ) . briefly , the harvested cells were suspended in lysis buffer ( 300 mmol l sodium chloride , 50 mmol l sodium dihydrogen phosphate , and 1 mg ml lysozyme , ph 8.0 ) . following sonication on ice bath ( 300 watt , 50% burst/50% cooling ) , the lysate was centrifuged at 10,000 g for 10 minutes . the supernatant was collected and passed through a preequilibrated ni - nta agarose column . the matrix was washed with wash buffer ( 300 mmol l sodium chloride , 50 mmol l sodium dihydrogen phosphate , and 20 mmol l imidazole , ph 8.0 ) and finally protein was eluted with 250 mmol l imidazole . all the eluted fractions were pooled and dialyzed against 10 mmol l tris , ph 8 , and further loaded onto deae sepharose column . gradients of elutions from 10 mmol l to 500 mmol l sodium chloride were carried out and the fractions showing the maximum peak absorbance at 280 nm were pooled , dialyzed in pbs , concentrated to 1 ml volume , quantified by nanodrop ( thermo fisher 2000c ) , and stored at 20c until further use . for purification under denatured condition , the harvested cells were washed twice with lysis buffer before dissolving in urea buffer ( 8 mol l urea , 100 mmol l sodium dihydrogen phosphate , and 10 mmol l tris , ph 8) . the lysate was centrifuged at 10,000 g and the supernatant was loaded onto the urea buffer preequilibrated ni - nta agarose column . elution was carried out at ph 4.5 and the pooled elutes were dialyzed against two changes of pbs . the protein was concentrated to 1 ml volume , quantified by nanodrop , and stored at 20c until further use . to see the recombinant pa expression and purification profile of different clones , sds page was performed . briefly , all the seven clones after iptg induction were harvested and lysed in 2x sds sample lysis buffer . for purified proteins , 5 l of each sample was mixed with equal volume of 2x sds sample lysis buffer before loading onto 10% sds page . after electrophoresis , the gel was stained with coomassie brilliant blue and image was captured through gel doc xr+ imager system ( biorad , usa ) . for western blotting , the soluble cell lysate from all the clones was electrophoresed on 10% sds page and the gel was transferred to nitrocellulose membrane using te70xp semidry blot system ( hoefer inc , usa ) . following transfer , the membrane was blocked with 5% skim milk for 1 hour at room temperature and then probed with anti - pa monoclonal antibody at 1 : 1000 dilutions for 1 hour . after washing thrice with pbs - tween and pbs , the membrane was incubated with anti - mouse igg - hrp conjugate at 1 : 5000 dilutions and was finally developed with 3,3-diaminobenzidine tetrahydrochloride ( dab ) substrate . the soluble pa purified from pa - pet32c - de3plyss clones was tested for its proper conformation and functionality by incubating 5 g of pa with 5 ng of trypsin ( pa : trypsin at 1000 : 1 ratio ) for 30 minutes at room temperature following which the samples were electrophoresed on 12% sds - page and stained with coomassie brilliant blue . for the confirmation of functional activity of the expressed pa , 2 g of trypsin digested pa was incubated with 2 g of lf at room temperature for 30 minutes . the samples were run on 4% native polyacrylamide gel at 4c and visualized by staining the gel with coomassie brilliant blue . six female balb / c mice were procured from institutional animal care center , drde , and were provided with food and water ad libitum . each mouse was immunized subcutaneously with 10 g pa adsorbed on aluminium hydroxide adjuvant on days 0 , 14 , and 28 . all mice were bled prior to first immunization on day 0 and on 35th day . serum was separated and stored at 20c until further use . to test the reactivity of pa immunized sera , indirect elisa was performed by coating 1 g ml pa in elisa plate overnight at 4c . after blocking with 5% skim milk for one hour , serial twofold dilution of pa immunized sera starting from 1 : anti - mouse igg hrp was added and further incubated for one more hour and finally developed using orthophenylenediamine dihydrochloride ( opd ) as substrate . the reaction was stopped using 2 n h2so4 and the absorbance was recorded at 490 nm . the test was performed in triplicate and the data was represented as mean sd . cut - off value for the assays was calculated as the mean absorbance ( + 2 sd ) from sera of control group assayed at 1 : 100 dilutions . the endpoint igg titers were calculated as reciprocal of the highest serum dilution giving an absorbance more than the cut - off . mouse macrophage cell line j774.1 was procured from national centre for cell sciences , pune , india and was maintained in high glucose dulbecco 's modified eagle 's medium ( dmem ) containing 10% fetal bovine serum ( fbs ) and 100 u ml penicillin and streptomycin as antibiotics . one day before the experiment 4 10 cells were seeded in a 96 well plate and incubated at 37c in presence of 5% humidified co2 . the next day spent media was replaced by a fresh medium containing gradient concentration of soluble pa and native pa of b. anthracis from list biologicals ( 0.005 g ml to 5 g ml ) and was incubated at 37c along with 0.125 g ml lf . after 4 hours , 20 l of 5 mg ml mtt ( 3-(4,5 dimethylthiazol-2 yl)-2,5 diphenyl tetrazolium bromide ) was added to each well and further incubated for 30 minutes . the formazan crystals developed were dissolved in 100 l acidified isopropanol ( 25 l of concentrated hcl and 500 l 10% sds in 10 ml 90% isopropanol ) and the absorbance was measured using uv - vis microplate reader ( biotek , winooski , vt ) at 570 nm . all the experiments were performed in triplicate and repeated at least three times . for toxin neutralization assay , the next day , spent media were replaced with the medium containing 0.3 g ml of pa and 0.125 g ml of lf with twofold serial dilution of pa antiserum starting from 1 : 100 . after incubating the plate for 5 hours , 20 l of 5 mg ml mtt was added to each well and further incubated for 30 minutes . the formazan crystals developed were dissolved in 100 l acidified isopropanol and the absorbance was measured from uv - vis microplate reader ( biotek , winooski , vt ) at 540 nm with reference wavelength at 640 nm . wells containing only medium but no cells were taken as blank while the wells containing the cells with medium only were taken as positive control . percentage viability was calculated as follows:(1)asampleablankapcablank100,where asample is absorbance of test samples , ablank is absorbance of blank , and apc is absorbance of positive control . the neutralization titer was defined as the lowest serum dilution that kills 50% of the macrophage cells . a 2.23-kilo base pair structural gene for pa was pcr amplified ( figure 1 ) and was successfully inserted into the plasmids pproexhta , pqe30 , and pet32c by restriction digestion and ligation . the recombinant plasmid pa - pproexhta was transformed into e. coli hosts dh5 , bl21-de3 , and de3-plyss , pa - pqe30 into m15 and xl-1 blue e. coli host , and pa - pet32c into bl21-de3 and de3-plyss . the clones were confirmed by dna sequencing and restriction digestion analysis of the isolated plasmid which released a 2.2 kb insert from all the three expression vectors ( figure 2 ) . since no differences were observed by changing the iptg concentration , the expression studies were carried out with 1 mm iptg . after induction , the clones were incubated for 3 hours , for 5 hours , and overnight at 37c and 22c . for each vector construct and host combination , optimal expression conditions were selected at which recombinant pa production was maximum . for pa - pqe30 in m15 , pa - pproexhta in dh5 , and bl21-de3 , the growth condition was maintained at 37c for 5 hours after induction . the pa - pet32c construct in de3-plyss was grown at 22c for 5 hours while pa - pqe30 in xl-1 blue and pa - pet32c in bl21-de3 were grown at 22c overnight after iptg induction . the purification was carried out under both native and denaturing condition . among the seven clones , the highest yield of soluble pa , 15 mg l , was observed for pet32c construct in de3-plyss host . a pqe30 construct in xl-1 blue and m15 host produced 12 mg l and 7.5 mg the pproexhta harboring clone pa - pproexhta in bl21-de3 also yielded high amount of soluble recombinant pa at the range of 5.1 mg l. comparatively , all the other clones produced less amount of soluble recombinant pa . the pa - pproexhta construct in dh5 and de3-plyss produced 1.2 and 3 mg l of soluble protein while the pa - pet32c construct in bl21-de3 produced 1.5 mg apart from the soluble fractions , all the clones except pa - pqe30 in xl-1 blue yielded high amount of recombinant pa in denatured form . the purified proteins were dialyzed against pbs , concentrated to 1 ml volume , and stored in 20c until further use . the total yields of soluble and denatured recombinant pa from all the seven clones are outlined in table 3 . expression profile of the induced samples and purified recombinant pa was analyzed on 10% sds page . all the seven clones expressed at different growth parameters and time intervals were loaded onto the gel ( figure 3 ) . to determine the optimal growth conditions ( temperatures and time periods ) of each of the seven clones for maximum yield of soluble recombinant pa , western blotting with densitometric analysis was performed . following sds - page , the soluble cell lysate of each clone was transferred onto nitrocellulose membrane and incubated with anti - pa monoclonal antibody . all the pa producing clones reacted to anti - pa monoclonal antibody ( figure 4 ) . after purifying the proteins in soluble and denatured form , 5 l of the purified protein sample from each of the vector host combinations was electrophoresed on sds - page ( figure 5 ) . the relative molecular weights of pa expressed along with their respective tags were 83 kda in pqe30 , 86 kda in pproexhta , and 100 kda in pet32c . the total yield of recombinant pa in both soluble and denatured form has been depicted in graph ( figure 6 ) . since pa from pet32c - de3-plyss was produced in soluble form in large quantity , the same was considered for further characterization . trypsin digestion of pa resulted in the formation of two fragments of 20 kda and 63 kda ( figure 7(a ) ) . for binding assay , trypsin digested pa was incubated with lf for 30 minutes at room temperature and electrophoresed on 4% native - page . binding of lf to pa was confirmed by the shift in mobility as compared to pa and lf alone ( figure 7(b ) ) . the reciprocal serum dilution was found to be 1.35 10 for 35th day , explaining the high immunogenicity of immunized sera against recombinant pa ( figure 8) . to determine the biological activity of soluble pa , mtt based cytotoxicity and toxin neutralization assay was performed . recombinant soluble pa protein was found to be lethal for the growth of macrophage cell lines with 50% viability around 0.3 g ml ( figure 9(a ) ) . native pa also showed the similar cytotoxicity at the concentration 0.25 g ml , thereby confirming that the recombinant pa works equivalent to that of native pa . the toxin neutralization titer was found to be 6.4 10 ( figure 9(b ) ) . pa , the major component of b. anthracis tripartite toxin , is produced and secreted by the vegetative form of bacterium inside the host and recently it was reported to be present in trace amounts on its spore surfaces . eventually , the pa protein is the main component of commercial anthrax vaccine formulation and also the targeted molecule of various anthrax diagnostic assays . due to the risk associated with the handling of b. anthracis culture for production and purification of native pa from bacterial culture supernatant , various laboratories have opted for the production of recombinant pa expressed conveniently in e. coli host . although production of recombinant antigen as a heterologous protein in e. coli system is a well opted strategy , the suitable vector - host combination and the optimal growth condition to obtain maximal yield of functionally active protein are always empirical . in the present study , we have optimized the growth parameters for e. coli to obtain a maximum production of recombinant pa in soluble form from three different expression vectors . initially , the gene sequence coding for matured pa protein was amplified from b. anthracis and cloned into three different expression vectors pproexhta , pqe30 , and pet32c . these constructs were transformed to their respective hosts and were grown at optimized temperature and time period after iptg induction . owing to the hurdles experienced during the downstream processing of the inclusion bodies like solubilization and proper refolding of denatured proteins , we focused our interest solely on the soluble expression of pa . the pa - pet32c construct produced higher recombinant pa in soluble form ( 15 mg ml ) when expressed in e. coli de3-plyss . pa - pqe30 construct in m15 and xl-1 blue produced 7.5 and 12 mg ml of soluble recombinant pa . clones in pproexhta were initially expected to yield lower level of the recombinant protein owing to the trc promoters as compared to the stronger t7 and t5 promoters . however , when transformed with bl21-de3 e. coli strains as host , the expression of soluble pa was 5.1 mg ml . in comparison to the high expressing clones mentioned above , the pa - pet32c construct in bl21-de3 and pa - pproexhta construct in dh5 and bl21-de3 resulted in low level of soluble expression at 1.5 mg ml , 1.2 mg ml , and 3 mg ml , respectively . the low levels of soluble pa expression in these clones were accompanied by high expression of pa as inclusion bodies . although t7 promoter has been known as a very strong promoter occasionally , very high level of expression inside a bacterial host leads to the formation of cytoplasmic inclusion bodies which are insoluble and nonfunctional [ 33 , 34 ] . proper folding and biological activity of the recombinant proteins was confirmed by digestion with trypsin which produced a 63 kda active protein followed by binding assay with lf . pa from pet32c - bl21-de3-plyss clone resulted in two fragments after trypsin digestion and could bind to lf as determined by native - page . these functional activities were not observed with any of the resolubilized and refolded inclusion bodies from any of the constructs , as trypsin digestion resulted in the complete degradation which highlights the need for a functionally active pa protein ( data not provided ) . further trypsin digested pa could bind to lf in solution and resulted in anthrax lethal toxin complex as determined by the shift in mobility in native - page . these experiments proved that the expressed pa was functionally active . to determine the biological activity of purified recombinant pa , cytotoxicity and toxin cytotoxicity of the pa protein on macrophage cells was found to be 0.3 g ml which was almost equivalent to that of the native protein . toxin neutralization of pa immunized sera was robust with the neutralization titer of 6.4 10 . thus , the present study describes the production of biologically active soluble recombinant pa from e. coli expression system by simple purification steps without need for complicated downstream processing as reported by earlier studies [ 3537 ] . multigram quantities of soluble recombinant pa can be obtained by further employing scale - up facilities for the above procedure . therefore , these expression systems can be exploited for the production of cost - effective recombinant vaccine molecule against the deadly disease anthrax .

bacillus anthracis secretory protein protective antigen ( pa ) is primary candidate for subunit vaccine against anthrax . attempts to obtain large quantity of pa from escherichia coli expression system often result in the formation of insoluble inclusion bodies . therefore , it is always better to produce recombinant proteins in a soluble form . in the present study , we have obtained biologically active recombinant pa in small scale e. coli shake culture system using three different expression constructs . the pa gene was cloned in expression vectors bearing trc , t5 , and t7 promoters and transformed into their respective e. coli hosts . the growth conditions were optimized to obtain maximum expression of pa in soluble form . the expression construct pa - pet32c in de3-plyss e. coli host resulted in a maximum production of soluble pa ( 15 mg l1 ) compared to other combinations . purified pa was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein 's functionality . biological activity was confirmed by cytotoxicity assay on j774.1 cells . balb / c mice were immunized with pa and the immunogenicity was tested by elisa and toxin neutralization assay . this study highlights the expression of soluble and biologically active recombinant pa in larger quantity using simpler e. coli production platform .

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

the exotoxins consist of three proteins , namely , protective antigen ( pa ) , lethal factor ( lf ) , and edema factor ( ef ) which together constitute the active anthrax toxins ( antx ) . it is therefore always better to express a protein in a soluble form , as it can result in functionally active and biologically stable protein . in the present study , we have expressed recombinant soluble pa in e. coli expression system using three different expression vectors , namely , pproexhta , pqe30 , and pet32c , bearing trc , t5 , and t7 promoters , respectively . further , each vector construct was transformed into different e. coli expression hosts and the growth conditions of the transformants were optimized so as to obtain the maximum yield in soluble form . further , the soluble pa from maximum producing clone pa - pet32c - de3-plyss was characterized by trypsin digestion and lf binding assay to confirm its functionality . this study highlights the straightforward production of large quantity of recombinant pa in its biologically active soluble form using an optimized e. coli vector - host combination system . the recombinant plasmid pa - pproexhta was transformed into e. coli dh5 , bl21-de3 , and de3-plyss , pa - pqe30 in e. coli m15 and xl-1 blue , and pa - pet32c plasmid in e. coli bl21-de3 and de3-plyss , respectively . the temperature and time period at which particular clone showed maximum expression of pa were noted and the same conditions were applied for bulk production of pa . the soluble pa purified from pa - pet32c - de3plyss clones was tested for its proper conformation and functionality by incubating 5 g of pa with 5 ng of trypsin ( pa : trypsin at 1000 : 1 ratio ) for 30 minutes at room temperature following which the samples were electrophoresed on 12% sds - page and stained with coomassie brilliant blue . the recombinant plasmid pa - pproexhta was transformed into e. coli hosts dh5 , bl21-de3 , and de3-plyss , pa - pqe30 into m15 and xl-1 blue e. coli host , and pa - pet32c into bl21-de3 and de3-plyss . the pa - pet32c construct in de3-plyss was grown at 22c for 5 hours while pa - pqe30 in xl-1 blue and pa - pet32c in bl21-de3 were grown at 22c overnight after iptg induction . the pa - pproexhta construct in dh5 and de3-plyss produced 1.2 and 3 mg l of soluble protein while the pa - pet32c construct in bl21-de3 produced 1.5 mg apart from the soluble fractions , all the clones except pa - pqe30 in xl-1 blue yielded high amount of recombinant pa in denatured form . since pa from pet32c - de3-plyss was produced in soluble form in large quantity , the same was considered for further characterization . trypsin digestion of pa resulted in the formation of two fragments of 20 kda and 63 kda ( figure 7(a ) ) . binding of lf to pa was confirmed by the shift in mobility as compared to pa and lf alone ( figure 7(b ) ) . to determine the biological activity of soluble pa , mtt based cytotoxicity and toxin neutralization assay was performed . in the present study , we have optimized the growth parameters for e. coli to obtain a maximum production of recombinant pa in soluble form from three different expression vectors . the pa - pet32c construct produced higher recombinant pa in soluble form ( 15 mg ml ) when expressed in e. coli de3-plyss . however , when transformed with bl21-de3 e. coli strains as host , the expression of soluble pa was 5.1 mg ml . in comparison to the high expressing clones mentioned above , the pa - pet32c construct in bl21-de3 and pa - pproexhta construct in dh5 and bl21-de3 resulted in low level of soluble expression at 1.5 mg ml , 1.2 mg ml , and 3 mg ml , respectively . the low levels of soluble pa expression in these clones were accompanied by high expression of pa as inclusion bodies . proper folding and biological activity of the recombinant proteins was confirmed by digestion with trypsin which produced a 63 kda active protein followed by binding assay with lf . thus , the present study describes the production of biologically active soluble recombinant pa from e. coli expression system by simple purification steps without need for complicated downstream processing as reported by earlier studies [ 3537 ] .

early reports concerning myocyte size in the human left ventricle , relying heavily upon autopsy specimens [ 13 ] , often found that the individual contractile muscle cells are roughly cylindrical measuring 17 to 25 m in diameter and 60 to 140 m in length , giving length to diameter ratio of about 5 to 1 . recent studies , relying on enzymatic isolation of myocytes into suspension from surgical specimens [ 514 ] , commonly model myocytes as elliptic cylinders of variable eccentricities and length to width ratios under assorted conditions [ 57 ] . variations between these models , arising from disparate methodologies , call for further inquiry to resolve their causes . cross - sectional profiles of myocytes , whether isolated or in sections , display a daunting irregularity of outline , especially of major axis , although less so of the minor axis . measurements of minor axis are therefore emphasized in this report of findings with h&e - stained paraffin sections of forensic autopsy specimens . left ventricular mass increases in many hyperergopathic states , often culminating in systolic dysfunction [ 15 , 16 ] . behavior by the full range of myocyte sizes , and not just their average , merits attention . several authors [ 710 ] have reported unimodal distribution curves of myocyte sizes , consistently showing conspicuous upward skewness . these authors did not report statistical curve fitting ; however , graphic inspection raised the possibility of log normal fits , which could imply that the smallest and the largest myocytes may all respond alike [ 79 ] , a possibility seldom discussed . stated a rarely encountered suggestion that eccentric hypertrophy is analogous to normal growth , the process which converts a neonatal left ventricle into an adult chamber . it is therefore easy to envision that the process could become exaggerated by uncommon growth into the tallest statures , which could be viewed as a form of eccentric hypertrophy that is progressing toward pathological thresholds . myocyte breadth clearly increases in company with concentric hypertrophy irrespective of etiology [ 19 , 12 , 13 ] . however , the behavior of myocyte breadth as the chamber dilates is controversial [ 5 , 6 , 12 , 13 , 17 ] . this is done here for only one limited purpose , to relate myocyte sizes to body weight . this study in a series of 104 forensic autopsies included all encountered instances of cardiomegaly ( excluding coronary heart disease ) , instances that were often later attributed to hypertension , obesity , or idiopathic cardiomyopathy ( 46 cases ) , aortic stenosis ( 4 ) , mitral deformity ( 1 ) , or cor pulmonale ( 2 ) . between november 2007 and may 2009 , the orleans parish coroner 's office supplied 104 specimens . all cases with cardiomegaly ( whole heart weight > 399 gm in women and > 449 gm in men ) , other than coronary heart disease , were included . death was attributed to cardiovascular causes in 42 cases and to noncardiovascular causes in 62 cases ( with 46 instances of violence or drugs ) . left ventricular hypertrophy ( lvh ) occurred in 33 of the 42 cardiovascular cases and 20 of the 62 noncardiovascular cases ( many chosen because of the presence of incidental lvh ) . noncardiovascular cases were chosen to have < 12 hours postmortem interval , no immediately preceding hospitalization , and cause of death unrelated to cardiovascular disease . the 42 cases classed as cardiovascular deaths included chronic renal failure ( n = 1 ) , cerebrovascular disorders ( 3 ) , aortic stenosis ( 4 ) , mitral deformity ( 1 ) , pulmonary conditions ( 8 , cor pulmonale , pulmonary embolus , arrhythmogenic right ventricular dysplasia ) , dilated hypertrophy ( 23 ) , and lvh without dilatation ( 2 ) . clinical information , most notably on hypertension , was not available in these forensic cases , because review of clinical records was omitted ; therefore the irb declared this autopsy study exempt from their review . ventricles of the fresh heart were divided from the atria , separated , and the epicardial fat dissected away . the method of bove et al . was modified by using the fresh rather than the formalin - fixed heart and by placing the trabeculae carneae and papillary muscles from the right side of the interventricular septum with the right ventricle . data of deveraux et al . were used to calculate 219 grams in men and 199 grams in women as cutoff points for lvh . chamber diameter ( cd ) was measured with a ruler on the high chordal plane ( touching the edges of the opened mitral leaflets ) through the left ventricle . thickness of lateral and posterior walls was measured with a ruler and averaged to find lvt ; these exclude trabeculae carneae and epicardial fat . a slice of myocardium presenting the high chordal plane was formalin fixed for 16 days . samples were excised from lateral , posterior , and septal walls of the lv for sectioning both parallel and perpendicular to the plane in the first 34 cases , and thereafter only in the perpendicular plane . paraffin - embedded samples were sectioned at 6 m and stained with h&e . a subset of specimens measured before and after processing into slides showed mean shrinkage of 17.2% ( sem = 0.96% ) , agreeing with published values [ 21 , 22 ] . adjustment by 1.21 = 1/0.828 was done upon subsequent histological measurements , although this is not needed for the relative comparisons of myocyte breadths in specimens reported here . myocyte profiles in cross - section ( figure 1(a ) ) were evaluated by imposing an ellipse upon each profile and measuring the minor axis of the idealized outline . in longitudinal planes of cut , diameters were measured at positions exposing substantial lengths of uniform width ( figure 1(b ) ) . these measurements were made upon black - and - white photographic prints using a digital caliper accurate to 0.01 mm . thereafter , measurements were taken in 60 anucleate cross - sectioned profiles from each of 4 sites in the lv free wall . myocyte profiles with nucleus present were compared to profiles without visible nuclei as a ratio , and the mean ratio departed substantially from 1.0 ( mean = 1.195 , sem = 0.012 , n = 34 ) . in all following analyses , therefore , nucleated profiles were omitted from the samples for simplicity and consistently . the calibers measured in longitudinal profiles held a ratio to those in cross - sections of mean = 1.048 ( sem = 0 .012 ) ; since this is significantly different from 1.0 , longitudinal profiles were omitted from all but one of the following analyses . the two samples of lv free wall , posterior and lateral , yielded caliber ratios to each other of mean = 1.021 ( sem = 0.014 ) , which does not differ significantly from 1.0 ( p = .15 ) , but the ratio of caliber in the interventricular septum to that in the mean free wall did differ from 1.0 ( mean = 0.944 , sem = 0.012 , p < .001 ) ; hence the following analyses omit septal samples . between subendocardial and subepicardial sites the caliber ratio had mean = 1.002 ( sem = 0.009 , p = .84 ) , hence further analyses include sites at arbitrary depths into the ventricular wall . in the idealized model of a myocyte as an elliptic cylinder , its dimensions are major axis , a , minor axis , b , and length l , so that the volume is mv = abl/4 = rl . is often given to the quantity 2r in the imaginary circle of radius = r. however , the name width is also often used for the major axis or for the minor axis , making the name width ambiguous . to avoid this ambiguity , the name breadth , mb = myocyte breadth , is assigned to the quantity 2r calculated from the minor axis , as explained next . measurements on longitudinal planes of cut represent random diameters in all directions across the elliptic myocyte , and their average is a composite of these varying lengths . in the 34 specimens measured in both ways , the means of the two methods averaged a ratio of 1.048 ( sem = 0.012 ) . by a method of iteration it can be found that a / b = 11/10 yields a close approximation to the observed ratio of longitudinal / cross calibers = 1.048 given above . knowing that ab/4 = r and that a = 11b/10 , the area becomes ( b 11/10)/4 = r and 2b2((11/10)/4)=2r = mb=1.048b is the mean value for myocyte breadth . this is the function for determining mb from the measured minor axis , b , and conversion to mb allows comparisons with published data . in a subset of 59 specimens , z - bands were counted within 30 m long segments of myocytes ( figure 1(b ) ) . counts divided by 30 ( times 1.21 to correct for preparation shrinkage ) yields the sarcomere length . this value averaged 1.68 m ( sem = 0.044 m , n = 59 ) . in a study of 6 hearts from ordinary human autopsies , gerdes et al . found a comparable average sarcomere length of 1.68 m in isolated myocytes . hearts acquired for enzymatic isolation of myocytes typically undergo rapid fixation of fresh organs ; sarcomere lengths in those organs have generally ranged around 2.0 m [ 8 , 11 , 13 , 14 ] . the ratio 1.68/2.0 = 0.84 is needed later as an adjustment to allow transference of published diastolic data into formulas developed here for systolic data in this study . sarcomere lengths measured here did not correlate with lvw , renal evidence of hypertension , age , body length , body weight , or skewness of the log normal fits ( p = .15 to .98 ) . this study gives only limited and somewhat peripheral attention to myocyte volume ( mv ) . this quantity has three spacial dimensions and therefore seems appropriate for comparison with body weight ( a three - dimensional variable and the only one here calling for some passing attention ) . the necessary data require only mean values for each specimen and have no need for individual measurements . fortunately , some data offering a way to estimate these means are available in prominent publications , and this estimation is examined next : in a study of maturing spontaneously hypertensive rats from birth to 24 months , many with lvh , tamura et al . found that cell length should be directly reflected by proportional alterations in chamber circumference . in the rats , circumference of 20 mm corresponded to l = 135 m , indicating 148 myocytes in the circumference if they are envisioned in an imaginary arrangement end to end . a similar proportionality in the human can only be inferred indirectly from available sources . using the data of gerdes et al . , healthy nonhypertrophied hearts had l : mb = 7 : 1 , l = 134 m , and cd = 52 mm , hence a value of 1,313 can be estimated for the number of myocytes envisioned as circumferentially tandem , about 8.9 times those in the rat . : 1 , l = 203 m , and cd = 82 mm , yielding 1,269 as the estimate for number of circumferentially tandem myocytes , 8.6 times that in the rat . the ratio of cd to l is nearly identical in the human ventricles of both size ranges , in keeping with the result seen in rats of near proportionality of myocyte length to chamber diameter . in the data of this study , the hearts lacking hypertrophy with mean cd = 28.0 mm had mean mb = 17.4 m . hence a simple formula can yield approximate estimates of l for any particular cd ; l = 7 0.84 17.4 cd/28.0 = 3.654cd 10m , where the correction factor 0.84 adjusts for the systolic condition of the specimens examined here , and 17.4/28.0 is taken as the reference standard . the values for mv determined using this estimate of length correlates with mb with r = 0.59 ( p < .001 ) . total number of myocytes ( mn ) in a ventricle is of limited interest in this report . for that restricted purpose it can simplistically be calculated as mn = 0.75 lvw / mv , ignoring the negligible effect of specific gravity . the term 0.75 is a commonly reported estimate of myocytes as a proportion of total lvw ( myocyte volume fraction ) . mn calculated this way fails to remain constant across the range of ventricular weights in this data set , and it is possible to correct this defect by using an adjustment factor , j. although tamura et al . report their findings as favoring approximate proportionality of myocyte length to chamber circumference , the regression equation that they report actually contains a significant intercept term , showing departure from proportionality . with the help of that regression equation , ( 1 ) can be derived ( 1)j=1.2(cd28.0 ) . the adjusted myocyte length is ( 3.654 cd j ) 10 m . myocyte numbers , using mv calculated with this adjusted length , yields an adjusted quantity that does not correlate with lvw ( r = 0.11 , p = .29 ) and is therefore constant across the range of ventricular weights ; it also does not correlate with race ( p = .98 ) , sex ( p = .97 ) , height ( p = .35 ) , or body weight ( p = .60 ) , demonstrating the expected constancy of myocyte numbers between these groupings . this formula will be reconsidered in the later discussion . between mv and its adjusted value , r = 0.99 body mass index , bmi = weight / height ( kg / m ) , is often used as a proxy for body fat mass in proportionate comparisons between individuals . indexed left ventricular mass , lvw / height ( gm / m ) , is also often used in comparisons of individuals , implying that height is a proxy for lean body mass . in this data set , the 70 cases with complete data revealed a multiple correlation of these two proxies with total body weight of r = 0.997 , indicating that they jointly capture all of the variations between individuals in total body weight , so that their use as proxies passes this test of validity . the standardized regression equation is weight = 0.347height + 0.816bmi , implying that variation of fat mass between individuals is more than twice as important as variation of lean mass for determining total body weight . intimal thickness of interlobular arteries in the renal cortex was measured in 100 cases , and mean arterial pressure ( map ) calculated using a previously derived regression equation [ 24 , 25 ] ( map = ( s + 2d)/3 ) . these are all commonly used techniques available in the sas programs ( sas institute , cary nc ) . some of the linear regression equations yielded insignificant intercept terms so that the appropriate equations are forced through the origin ; the correlation coefficients reported with these equations are those retaining the intercept terms . for computing odds ratios in the logistic regressions , the variables were measured in units of standard deviation . for consistency and economy in testing of log figure 2 plots log normal distribution curves for mb as measured in the lv of a 6-week - old infant ( lvw = 15 gm ) , a pool of 6 cases of low ventricular weights , and a pool of 6 cases with high ventricular weights ( mean and sd were closely alike in these cases chosen for pooling ) . the low weight group was chosen to illustrate a distribution that fails to reject the log normal fit ( p > .05 by shapiro - wilk test ) , while the high weight group exemplifies the kind of misfit that is typical of curves that significantly depart from the log normal . the log normal was rejected as misfit in 33 of the 104 cases at p < .1 and 53 cases at p < .2 ( 32% and 51% resp . in table 1 , rather than the randomly expected 10% and 20% , resp . ) . the skewness had a mean of 0.140 ( p < .001 for difference from zero , sd = 0.211 ) , and was below zero in 73 specimens ( 70% ) . of the 33 seriously misfit cases , 22 displayed skewness < 0.140 ( correlation of skewness with p - values for the misfit was r = 0.38 , p < .001 in the 104 cases ) . the 56 cases with skewness > .139 had p - values for the log normal fits which appear acceptably random ( .1 > p > .2 by chi - square test in table 1 ) . the coefficient of variation ( cv ) of these logarithmic distributions ranged from 9.2 to 14.0% ( mean = 11.8% , sd = 1.2% ) ; the correlation of cv with the mean was r = 0.05 with p = .68 . with mean mb measured at 4 lv sites , the means can be compared in 6 pairings , testing the differences by the tukey test . of the 624 pairings in the pool of 104 cases , 174 ( 28% ) yielded p < .05 , showing members of the pairs to differ significantly from each other far more often than by chance ( 5% = 31 pairings ) . the numbers of pairs that differed significantly ( heterogeneity ) decreased with negative log normal skewness ( r = 0.25 with p = .011 ) indicating dissociation of significantly excessive heterogeneity from negative skewness . the p - value for fits of the log normal did not correlate with heterogeneity ( r = 0.05 with p = .59 ) . by components of variance analysis , the percentage of variance attributed to between sites variation averaged only 6.28% as much as error variance within sites ( sd = 1.00% , range = 1.6 to 32.6% ) in the 104 cases . heterogeneity did not correlate with between - site variance across cases ( r = 0.11 , p = .27 ) . in the total set of 104 subjects , mb ( a one dimensional quantity ) was found to correlate with body length ( bl , i.e. , height in meters ) . in figure 3 , the entire body of data revealed a correlation coefficient of 0.67 . however 46 subjects entered the series because of deaths related to violence or drugs , and these are thought to offer a reasonable look at the free - living population not entering the data set because of the incidental disease that happens also to be found . therefore , the regression equation fit only to these 46 subjects is plotted in figure 3 , r = 0.43 , p < .001 . this equation rejects an intercept term so that the appropriate equation is forced through the origin ( sloping line in figure 3 ) : ( 2)mb = 10.53bl . this equation rejects race ( p = .21 ) and sex ( p = .19 ) as offering no additionally significant correlation . a cutoff point for mb to define hypertrophy of myocytes was explored with logistic regression with lvh as response variable . the resulting equation gives mb = 19.7 m as the cutoff point ; above that value the mean of myocytes for that specimen can be declared hypertrophied . odds ratio in standard deviation units is 55.5 ( 10.3 to 299.3 as 95% ci ) . cd ( in mm ) correlated with mb ( m ) ( r = 0.63 ) ; the regression accepts the intercept term as significant , showing lack of proportionality ( sloping line in figure 4 ) : ( 3)mb=0.229cd+12.0 ( 0.229 has se=0.025 , 12.0 has se=0.91 ) . this equation is plotted in figure 4 as a dashed vertical line which optimally separates dots from circles ; when cd > 33.3 mm then lvh is judged to be present . in standard deviation units or = 25.4 ( ci = 7.6 to 85.1 ) . in the 53 hearts without dilatation , a cutoff point for recognizing wall thickening was explored with logistic regression , where myocyte hypertrophy ( mb > 19.7 m ) derived in section 3.4 was predicted using lv wall thickness , lvt . the resulting equation gives lvt = 16.5 m as the cutoff point ; above this value the left ventricular free wall can be declared thickened . using hypertrophy without dilatation to define concentric hypertrophy , table 2 shows this condition to display an appreciable increase in myocyte breadth compared with normal hearts , and this effect is enhanced in the presence of wall thickening . however , hypertrophied hearts with dilatation are far more numerous than those without dilatation ( 43 versus 10 cases ) , and these also express a greater degree of mb thickening , with the effect exceptionally strong when the ventricular free wall is thickened . the expected thinning of chamber wall as hypertrophied hearts dilate is not manifest in the group averages of table 2 ( derived from this forensic series of cases with only 4 instances of aortic stenosis contributing to the eccentric hypertrophy group ) . lvw ( gm , having three spacial dimensions ) has a linear relationship to mv ( m ) described by ( r = 0.86 , sloping line in figure 5 ) ( 5)lvw=3.90mv+72.0(r2=.748 , 3.90 has se=0.23 , 72.0 has se=11.2 ) . lvh is predicted to be present at values above the cutoff , and otherwise absent , hence that cutoff defines myocyte hypertrophy ( or = 931.0 with ci = 36.0 to > 999.9 in sd units ) . the cutoff point for using mv to recognize dilatation is found by logistic regression ( vertical dashed line in figure 5 ) : ( 7)37,927=mv . when mv > 37,927 m the chamber is predicted to be dilated ( or = 40.8 , ci = 8.8 to 187.5 sd units ) . in the 70 cases with data on body weight ( bw in kg , a quantity with 3 spatial dimensions ) mb = 0.573 bw + 14.3 ( r = 0.551 ) . the retention of an intercept term in this regression equation is inconvenient and results from comparing a three - dimensional quantity , body weight , to a one - dimensional quantity , mb . the three - dimensional quantity , mv , although limited by uncertainties in its indirect determination , correlates with bw ( r = 0.50 ) , and the regression equation rejects entry of an intercept term so that the appropriate equation is forced through the origin : ( 8)mv=435bw this equation rejects entry of height ( p = .92 ) , body mass index ( p = .21 ) , sex ( p = .61 ) , and race ( p = .43 ) . by logistic regression , the optimal classification of cases with myocyte hypertrophy from those without is 104.1 = bw ( or = 3.33 , ci = 1.72 to 6.45 in sd units ) . mv relates to these proxies with multiple correlation of r = 0.50 , thereby reproducing the value of r = 0.50 just found for total body weight ( 8) , as expected for the joint action of the two proxies together . the standardized regression equation for this multiple relationship is ( 9)mv=0.272height2.7 + 0.223bmi which implies that lean and fat mass proxies are of equal importance to the correlation with myocyte volume . map , derived from histological renal features in 100 of the cases , was found to correlate with the measure of myocyte volume , mv ( r = 0.21 , p = .04 ) . the intercept term is rejected so that the appropriate regression equation is forced through the origin : ( 10)mv=408map . by logistic regression the cutoff value for myocyte hypertrophy is map = 106.5 mm hg ; above this level myocyte hypertrophy becomes likely ( or = 1.68 , range 1.10 to 2.58 ) , and this cutoff point seems reasonable for diagnosing hypertension . mn , total myocytes in the left ventricle , is calculated in this paragraph using formulas explained with ( 1 ) , et seq . these numbers do not vary across groupings by lvw , race , sex , height , or body weight . ventricles with myocyte hypertrophy ( mv > 35,341 m ) revealed a small deficit in cell numbers compared with normal ventricles ( mean mn = 5.90 versus 6.69 10 cells , resp . , sem = 0.19 for both means , p < .001 ) . similarly , dilated ventricles ( cd > 33.3 mm ) also showed a small deficit compared to normal ( mean = 5.90 versus 6.89 10 cells resp . frequency distribution curves of mb in the present data set , in agreement with numerous published graphs [ 710 ] , showed unimodal curves with conspicuous upward skewness . fitting the log normal form in these data was seldom rejected by shapiro - wilk significance tests ( 32% of cases rejected at p < .1 ) , and the rejections appeared trivial to graphic inspection ( figure 2 , right frame ) . the means of the log normal curves were uncorrelated with the coefficients of variation across the range of ventricular sizes . this is the pattern expected if myocyte hypertrophy follows an allometric growth model , which would indicate that the enlargement of each myocyte remains proportional to that of all others ( in the absence of focal injury ) , a finding consistent with previous reports [ 79 ] . this inference might offer insights into the depressed contractility that affects myocytes as they elongate and approach failure in the dilated ventricle [ 15 , 16 ] . houser and margulies , for instance , imply that the depressed contractility in systolic heart failure , in the absence of coronary heart disease , is population wide over all myocytes , a suggestion consistent with figure 2 . , for instance , is that tissue oxygenation is faulty and increases the vulnerability of the hypertrophied myocardium . this mechanism would presumably act selectively upon the most hypertrophied myocytes while sparing the smallest ones , a mechanism hard to reconcile with the behavior of log normal curves during hypertrophy . the log normal distributions also seem inconsistent with the proposals of longitudinal splitting in the myocytes that are enlarged beyond the limit of hypertrophy , and of substantial cell loss to apoptosis or necrosis . those two effects might be expected to act selectively upon the most hypertrophied myocytes thereby truncating the upper range of the log normal distributions to generate a negative skewness coefficient . in the present data set , an infrequent and weak negative skewness happened as often in the smallest as in the largest ventricles thus showing no association with hypertrophy . the lengths of minor axes in cross - sectioned myocytes , as reflected in their proxy , mb , are not exactly random in their distribution among left ventricular samples . on average the variance between sites exhibits an excess of 6.28% above the expected value of 0% of the within - site random variance . these departures have only few and inconsistent associations with anatomic location throughout the left ventricular free wall , a finding in agreement with some authors , for example , campbell et al . there is no consistent pattern of regional myocyte size differences in normal mammalian hearts . these relatively small departures also do not contribute measurably to misfits of the widely ranging myocyte sizes to the log normal distributions or to their negative skewness . these departures may be of importance to cardiac function , but their significance seems negligible in the present analysis . more than 90% of human postnatal growth of the heart can be accounted for by hypertrophy of the fibers present at birth . it appears that this process may continue with growth into the upper ranges of stature ( figure 3 ) , and that the larger hearts in taller people are not manifesting physiological growth but instead are undergoing hypertrophy toward pathological boundaries . as stated by grossman et al . in our concept , the eccentric hypertrophy by which a child 's heart becomes that of an adult represents a physiologic volume overload , and may very well utilize the same mechanisms as seen with the pathologic volume overload of valvular insufficiency . equation ( 2 ) shows average myocyte breadth to increase in direct proportion to height , a result expected if tall persons did not receive any supplementary endowment of myocytes in infancy . these findings generate the conclusion that , in some special situations , it may be unwise to index left ventricular mass to height , a procedure that would artifactually discard a significant component of meaningful hypertrophy . as the left ventricular chamber diameter ( cd ) is found to be increasingly dilated from one specimen to another , the way that myocyte breadth ( mb ) follows or fails to follow this change is controversial . gerdes summarizes an often encountered suggestion that , volume overloading leads to proportionate growth in chamber diameter and wall thickness , which are reflected at the cellular level by proportionate growth of myocyte l ( length ) and csa ( cross - sectional area ) . however , gerdes in the same article later appears to confine the prior statement to compensated dilatation , because myocyte l / w ( length / width ) increased dramatically in humans with congestive heart failure due to ischemic and dilated cardiomyopathy . offer tabulations for normal and dilated failing hearts which indicate virtually no increase of myocyte breadth with dilated hypertrophy . the data in figure 3 are generally intermediate between these extremes . compared with the normal at mb / cd = 16/20 = 0.8 ( figure 4 ) , only one of the hypertrophied hearts fails to fall short of this value , so that a consistent proportionality of chamber diameter to myocyte breadth is unambiguously rejected in these data . the mean mb in ventricles without hypertrophy is 17.4 m , and all of the hypertrophied ventricles exceed this value , so that a consistent lack of increase in mb with dilatation is firmly rejected . the cube of myocyte breadth , mb , has a disappointing correlation with mv , r = 0.59 , so that this cubic term can substitute for mv only crudely for use , perhaps , in some exploratory settings . the methods section ( elliptic eccentricity ) gives a value of 1.048 for the ratio of major / minor axis in this data set . this outcome seems consistent with prior studies of autopsy materials , but is inconsistent with findings from enzymatically isolated myocytes wherein eccentricities are typically reported in the range of 2.0 . there appears to be an unexplained consistent discrepancy between autopsy studies of tissue sections as compared with myocytes enzymatically isolated from surgical specimens . some investigators have suggested that cardiac myocytes may assume a more flattened shape after dissociation compared with their shape in intact tissue specimens . this proposal may have some support from measurements of isolated myocyte thicknesses by confocal microscopy . an adjusted value of myocyte volume used here for some limited exploratory purposes adopts the axiom that , on average , myocyte numbers are alike across the range of ventricular sizes , which allows empirical construction of ( 1 ) , introducing an adjustment factor j. the origin of factor j began from the data of tamura et al . in which the myocyte length does not quite keep up as the chamber dilates with growth and hypertrophy . this outcome implies that the laggard myocyte length must be compensated by increased myocyte numbers , by a degree of slippage between adjacent parallel myocytes [ 2 , 12 , 13 ] , or by some other means . equation ( 8) reports the finding that average myocyte volume can be described as proportional to total body weight . this equation rejects entry of height , as expected if lean and fat body mass contribute equally . total body weight also suffices to statistically explain the sex difference in myocyte size , the cutoff point for myocyte hypertrophy found here to be 104.1 kg irrespective of sex , race , height , or fatness ( bmi ) . this surprising result derives from the little used methods of measuring myocytes as a way to diagnose hypertrophy . the conclusion is offered here as a first result of its kind and only to serve as hypothesis in need of testing .

cardiac myocytes are presumed to enlarge with left ventricular hypertrophy ( lvh ) . this study correlates histologically measured myocytes with lean and fat body mass . cases of lvh without coronary heart disease and normal controls came from forensic autopsies . the cross - sectional widths of myocytes in h&e - stained paraffin sections followed log normal distributions almost to perfection in all 104 specimens , with constant coefficient of variation across the full range of ventricular weight , as expected if myocytes of all sizes contribute proportionately to hypertrophy . myocyte sizes increased with height . by regression analysis , height2.7 as a proxy for lean body mass and body mass index ( bmi ) as a proxy for fat body mass , exerted equal effects in the multiple correlation with myocyte volume , and the equation rejected race and sex . in summary , myocyte sizes , as indexes of lvh , suggest that lean and fat body mass may contribute equally .

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

measurements of minor axis are therefore emphasized in this report of findings with h&e - stained paraffin sections of forensic autopsy specimens . behavior by the full range of myocyte sizes , and not just their average , merits attention . this study in a series of 104 forensic autopsies included all encountered instances of cardiomegaly ( excluding coronary heart disease ) , instances that were often later attributed to hypertension , obesity , or idiopathic cardiomyopathy ( 46 cases ) , aortic stenosis ( 4 ) , mitral deformity ( 1 ) , or cor pulmonale ( 2 ) . left ventricular hypertrophy ( lvh ) occurred in 33 of the 42 cardiovascular cases and 20 of the 62 noncardiovascular cases ( many chosen because of the presence of incidental lvh ) . the 42 cases classed as cardiovascular deaths included chronic renal failure ( n = 1 ) , cerebrovascular disorders ( 3 ) , aortic stenosis ( 4 ) , mitral deformity ( 1 ) , pulmonary conditions ( 8 , cor pulmonale , pulmonary embolus , arrhythmogenic right ventricular dysplasia ) , dilated hypertrophy ( 23 ) , and lvh without dilatation ( 2 ) . myocyte profiles with nucleus present were compared to profiles without visible nuclei as a ratio , and the mean ratio departed substantially from 1.0 ( mean = 1.195 , sem = 0.012 , n = 34 ) . the term 0.75 is a commonly reported estimate of myocytes as a proportion of total lvw ( myocyte volume fraction ) . mn calculated this way fails to remain constant across the range of ventricular weights in this data set , and it is possible to correct this defect by using an adjustment factor , j. although tamura et al . between mv and its adjusted value , r = 0.99 body mass index , bmi = weight / height ( kg / m ) , is often used as a proxy for body fat mass in proportionate comparisons between individuals . indexed left ventricular mass , lvw / height ( gm / m ) , is also often used in comparisons of individuals , implying that height is a proxy for lean body mass . for consistency and economy in testing of log figure 2 plots log normal distribution curves for mb as measured in the lv of a 6-week - old infant ( lvw = 15 gm ) , a pool of 6 cases of low ventricular weights , and a pool of 6 cases with high ventricular weights ( mean and sd were closely alike in these cases chosen for pooling ) . the three - dimensional quantity , mv , although limited by uncertainties in its indirect determination , correlates with bw ( r = 0.50 ) , and the regression equation rejects entry of an intercept term so that the appropriate equation is forced through the origin : ( 8)mv=435bw this equation rejects entry of height ( p = .92 ) , body mass index ( p = .21 ) , sex ( p = .61 ) , and race ( p = .43 ) . the standardized regression equation for this multiple relationship is ( 9)mv=0.272height2.7 + 0.223bmi which implies that lean and fat mass proxies are of equal importance to the correlation with myocyte volume . fitting the log normal form in these data was seldom rejected by shapiro - wilk significance tests ( 32% of cases rejected at p < .1 ) , and the rejections appeared trivial to graphic inspection ( figure 2 , right frame ) . the means of the log normal curves were uncorrelated with the coefficients of variation across the range of ventricular sizes . houser and margulies , for instance , imply that the depressed contractility in systolic heart failure , in the absence of coronary heart disease , is population wide over all myocytes , a suggestion consistent with figure 2 . the log normal distributions also seem inconsistent with the proposals of longitudinal splitting in the myocytes that are enlarged beyond the limit of hypertrophy , and of substantial cell loss to apoptosis or necrosis . those two effects might be expected to act selectively upon the most hypertrophied myocytes thereby truncating the upper range of the log normal distributions to generate a negative skewness coefficient . equation ( 2 ) shows average myocyte breadth to increase in direct proportion to height , a result expected if tall persons did not receive any supplementary endowment of myocytes in infancy . an adjusted value of myocyte volume used here for some limited exploratory purposes adopts the axiom that , on average , myocyte numbers are alike across the range of ventricular sizes , which allows empirical construction of ( 1 ) , introducing an adjustment factor j. the origin of factor j began from the data of tamura et al . this equation rejects entry of height , as expected if lean and fat body mass contribute equally .

androgens are a class of natural or synthetic steroid hormone compounds such as testosterone , dihydrotestosterone and androstenedione ( 1 ) . physiological androgens play vital roles in the regulation of anabolic metabolism and expression of male phenotype . they are actually eminent for regulation of male development particularly the male reproductive system ( 2 ) . the diverse effects of androgens are mediated by complex cell type - specific signaling pathways that always involve the androgen receptor ( ar ) ( 3 ) . the androgen receptor ( ar ) is a type of nuclear receptor that acts as a dna - binding transcription factor to regulate gene expression ( 4 ) . dysregulation of androgen / ar signaling perturbs normal reproductive development and accounts for a wide range of pathological conditions such as prostate cancer ( 2 ) . recent experimental findings conform the expression of ar in androgen - dependent , androgen - independent or hormone refractory prostate cancers , where ar expression is maintained throughout prostate cancer progression ( 5 ) . interestingly , a number of clinical studies suggest that prostate cancer initiation and progression is also uniquely dependent on ar expression ( 6 ) . this pathogenic role of ar has made it a major therapeutic target in aggressive prostate cancer ( 7 ) . phuti - kadam and has an abundant distribution in bangladesh and india . in traditional medicinal practice the bark of h. excelsumis used as an astringent and febrifuge and for treatment of fever and tumors , while the leaves are used to treat ulcers , sialitis , sore throat , tonsillitis and inflammatory conditions ( 8) . the stem bark of h. excelsum contains tannin , toxic alkaloid , hymenodictine , esculin , an apioglucoside of scopoletin andhymexelsin ( 9 ) . anthraquinones , rubiadin and its methyl ether , lucidin , nordamnacanthal , damnacanthal , 2-benzylzanthopurpurin , anthragallol , soranjidiol and morindone have also been isolated from roots ( 10 ) . the plant has been reported to have antioxidant and anti - inflammatory properties ( 11 ) . the present study aimed to investigate h. excelsum s phytochemicals with anti - prostate cancer activity and to predict their mechanisms of action using a computational molecular simulation study . the therapeutic effects of a medicinal plant are attributed to the phytochemical constituents of that plant . therefore , it is feasible to study individual phytochemicals present in plants when investigating the therapeutic effects of medicinal plants . from the literature search , eight phytochemicals were taken into consideration for the molecular simulation study including anthragallol , damnacanthal , esculin , lucidin , morindone , nordamnacanthal , rubiadin and soranjidiol . canonical smiles file of these selected ligand molecules were retrieved from the pubchem compound server ( https://pubchem.ncbi.nlm.nih.gov/ ) . canonical smiles files were entered in to the corina online demo to generate 3d protein data bank ( pdb ) files . protein data bank ( pdb ) file of human ar ligand binding domain ( pdb i d : 1e3 g ) was downloaded from rcsb pdb database ( http://www.rcsb.org/pdb/home/home.do ) . prior to molecular simulation study the drug likeness and safety of the chosen ligands were evolved by empirical computational tools . the molinspiration cheminformatics server was exploited to calculate the molecular properties associated with drug - likeness and to predict bioactivity of components including the nuclear receptor ligand and enzyme inhibitor ( 12 ) . the osiris property explorer was used to evolve the toxicity of the selected ligands in term of mutagenic , tumorigenic and irritant properties ( 13 ) . the site of the receptor that interacted with the ligands was predicted by the molegro virtual docker ( mdv ) inbuilt cavity detection algorithm and the pocket - finder server ( 14 ) . the outputs from both of the computational tools were synchronized to predict the ultimate ligand binding site and exploited in the subsequent docking study . molegro virtual docker ( mvd ) was used for the computer simulated docking study ( 15 ) . molegro virtual docker includes moldock and plants score for evaluating docking solutions followed by a rank for the best conformations . the solution to the function is the sum of intermolecular interaction energy between protein and ligand with the intra - molecular interaction energy of the ligand . the docking energy scoring function is based on a modified piecewise linear potential ( plp ) with new hydrogen bonding and electrostatics terms included . a lower score always indicates a higher affinity . before initiation of the docking operation , both protein and ligands were prepared by mvd through assigning bonds , bond orders , explicit hydrogens , charges and flexible torsions at the missing region only . potential binding sites ( cavities or active sites ) were identified using the built - in cavity detection algorithm of mvd with a grid resolution ( ) of 0.30 . internal electrostatic interactions , internal hydrogen bonds , and sp2-sp2 torsions were selected as ligand evaluation terms . the moldock se was set as the searching algorithm for ten runs using a maximum of 1500 iterations with a total population size of 50 . energy minimization and optimized h - bonds were enabled as post - docking steps for more accurate refinement of the docking results . multiple poses were clustered based on the rmsd threshold of 1.0 , ignoring similar poses with rmsd threshold of 1.0 . the therapeutic effects of a medicinal plant are attributed to the phytochemical constituents of that plant . therefore , it is feasible to study individual phytochemicals present in plants when investigating the therapeutic effects of medicinal plants . from the literature search , eight phytochemicals were taken into consideration for the molecular simulation study including anthragallol , damnacanthal , esculin , lucidin , morindone , nordamnacanthal , rubiadin and soranjidiol . canonical smiles file of these selected ligand molecules were retrieved from the pubchem compound server ( https://pubchem.ncbi.nlm.nih.gov/ ) . canonical smiles files were entered in to the corina online demo to generate 3d protein data bank ( pdb ) files . protein data bank ( pdb ) file of human ar ligand binding domain ( pdb i d : 1e3 g ) was downloaded from rcsb pdb database ( http://www.rcsb.org/pdb/home/home.do ) . prior to molecular simulation study the drug likeness and safety of the chosen ligands were evolved by empirical computational tools . the molinspiration cheminformatics server was exploited to calculate the molecular properties associated with drug - likeness and to predict bioactivity of components including the nuclear receptor ligand and enzyme inhibitor ( 12 ) . the osiris property explorer was used to evolve the toxicity of the selected ligands in term of mutagenic , tumorigenic and irritant properties ( 13 ) . the site of the receptor that interacted with the ligands was predicted by the molegro virtual docker ( mdv ) inbuilt cavity detection algorithm and the pocket - finder server ( 14 ) . the outputs from both of the computational tools were synchronized to predict the ultimate ligand binding site and exploited in the subsequent docking study . molegro virtual docker ( mvd ) was used for the computer simulated docking study ( 15 ) . molegro virtual docker includes moldock and plants score for evaluating docking solutions followed by a rank for the best conformations . the solution to the function is the sum of intermolecular interaction energy between protein and ligand with the intra - molecular interaction energy of the ligand . the docking energy scoring function is based on a modified piecewise linear potential ( plp ) with new hydrogen bonding and electrostatics terms included . a lower score always indicates a higher affinity . before initiation of the docking operation , both protein and ligands were prepared by mvd through assigning bonds , bond orders , explicit hydrogens , charges and flexible torsions at the missing region only . potential binding sites ( cavities or active sites ) were identified using the built - in cavity detection algorithm of mvd with a grid resolution ( ) of 0.30 . internal electrostatic interactions , internal hydrogen bonds , and sp2-sp2 torsions were selected as ligand evaluation terms . the moldock se was set as the searching algorithm for ten runs using a maximum of 1500 iterations with a total population size of 50 . energy minimization and optimized h - bonds were enabled as post - docking steps for more accurate refinement of the docking results . multiple poses were clustered based on the rmsd threshold of 1.0 , ignoring similar poses with rmsd threshold of 1.0 . the site of the receptor that interacted with the ligands was predicted by the molegro virtual docker ( mdv ) inbuilt cavity detection algorithm and the pocket - finder server ( 14 ) . the outputs from both of the computational tools were synchronized to predict the ultimate ligand binding site and exploited in the subsequent docking study . molegro virtual docker ( mvd ) was used for the computer simulated docking study ( 15 ) . molegro virtual docker includes moldock and plants score for evaluating docking solutions followed by a rank for the best conformations . the solution to the function is the sum of intermolecular interaction energy between protein and ligand with the intra - molecular interaction energy of the ligand . the docking energy scoring function is based on a modified piecewise linear potential ( plp ) with new hydrogen bonding and electrostatics terms included . before initiation of the docking operation , both protein and ligands were prepared by mvd through assigning bonds , bond orders , explicit hydrogens , charges and flexible torsions at the missing region only . potential binding sites ( cavities or active sites ) were identified using the built - in cavity detection algorithm of mvd with a grid resolution ( ) of 0.30 . internal electrostatic interactions , internal hydrogen bonds , and sp2-sp2 torsions were selected as ligand evaluation terms . the moldock se was set as the searching algorithm for ten runs using a maximum of 1500 iterations with a total population size of 50 . energy minimization and optimized h - bonds were enabled as post - docking steps for more accurate refinement of the docking results . multiple poses were clustered based on the rmsd threshold of 1.0 , ignoring similar poses with rmsd threshold of 1.0 . result of the drug likeness and toxicity evaluation of the ligand have been summarized in table 1 . the results from the molinspiration cheminformatics server showed that all the selected ligands met the lipinski 's rule of drug likeness without violation of any condition . additionally , all the ligands fitted more or less with nuclear receptor ligands and enzyme inhibitors . the output from the osiris property explorer suggested the esculin , lucidin , morindone and soranjidiol were safe ligands with no mutagenic or tumorigenic effects . thus , the probable phytochemicals were filtered , as they were more potent physiologically active molecules without any harmless effects . abbreviations : ei , enzyme inhibitor ; milogp , octanol - water partition coefficient developed at molinspiration ; n , no ; nv , number of violation of lipinski 's rule of drug likeness ; tpsa , total molecular polar surface area ; nrl , nuclear receptor ligand ; y , yes . initial prediction of the probable ligand - binding site was performed by the pocket - finder and mdv cavity search . the prediction showed an active site of 409 cubic angstroms with a maximum coordination of 8 42 12 from the pocket - finder output ( figure 1 ) . the cavity searching algorithm built in the mvd tool fetched a cavity of 86.02 cubic angstroms with a maximum coordination of 0.41 31.67 4.44 and surface of 204.8 square angstroms . output of the pocket - finder was synchronized with the mdv cavity search output for screening the prediction with higher degree of accuracy . as , these two outputs overlapped and the selected molecule had a surface of ~95 square angstroms at best , the cavity coordination of 0.41 31.67 4.44 was set as the desired ligand interaction site or binding site . the pocket - finder works by scanning the probe radius 1.6 angstroms along all gridlines of the grid resolution , 0.9 angstroms surrounding the protein . the output of the pocket - finder shows the highest precision ( 52.7 ) for site two , which was also energetically most favorable . the computational docking study of the ligand with the receptor ( coordinate : 0.41 31.67 4.44 ) by mvd was evolved in terms of moldock score for best pose . this observation is concordant with previous study with r 18 - 100 that has inhibitory effect on the ar . among the studied ligands , esculin exhibited the most favorable binding to the receptor followed by morindone , anthragallol , soranjidiol , lucidin and damnacanthal . the moldock score also revealed that esculin s interaction with the receptor is likely to be more energetically economic than that of the dihydrotestosterone ( table 2 ) . during dihydrotestosterone interaction with the receptor , the amino acid residues , including gln 711 , arg 752 and thr 877 , were involved in hydrogen bonding while gln 711 , met 745 and phn 764 were involved in steric interaction ( figure 2 a ) . the amino acid residues leu 704 , asn 705 , arg 752 and thr 877 were involved in hydrogen bonding in case of esculin s interaction receptor . esculin exploited leu 704 , gln 711 , leu 873 , phe 876 , met 787 and met 895 residues at the binding site to maintain a steric interaction with the receptor ( figure 2 b ) . thus , the amino acid residues arg 752 and thr 877 were the common residues involved in the hydrogen bonding of the receptor with esculin and dihydrotestosterone . , the residues of the receptor participating in hydrogen bonding with the corresponding ligand are shown in azure color . the interaction includes hydrogen bonding with gln 711 , arg 752 and thr 877 , steric interaction with gln 711 , met 745 and phn 764 . , the residues of the receptor participating in hydrogen bonding with corresponding ligands are shown in azure color . the interaction includes hydrogen bonding with leu 704 , asn 705 , arg 752 andthr 877steric interaction with leu 704 , gln 711 , leu 873 , phe 876 , met 787 and met 895 . result of the drug likeness and toxicity evaluation of the ligand have been summarized in table 1 . the results from the molinspiration cheminformatics server showed that all the selected ligands met the lipinski 's rule of drug likeness without violation of any condition . additionally , all the ligands fitted more or less with nuclear receptor ligands and enzyme inhibitors . the output from the osiris property explorer suggested the esculin , lucidin , morindone and soranjidiol were safe ligands with no mutagenic or tumorigenic effects . thus , the probable phytochemicals were filtered , as they were more potent physiologically active molecules without any harmless effects . abbreviations : ei , enzyme inhibitor ; milogp , octanol - water partition coefficient developed at molinspiration ; n , no ; nv , number of violation of lipinski 's rule of drug likeness ; tpsa , total molecular polar surface area ; nrl , nuclear receptor ligand ; y , yes . initial prediction of the probable ligand - binding site was performed by the pocket - finder and mdv cavity search . the prediction showed an active site of 409 cubic angstroms with a maximum coordination of 8 42 12 from the pocket - finder output ( figure 1 ) . the cavity searching algorithm built in the mvd tool fetched a cavity of 86.02 cubic angstroms with a maximum coordination of 0.41 31.67 4.44 and surface of 204.8 square angstroms . output of the pocket - finder was synchronized with the mdv cavity search output for screening the prediction with higher degree of accuracy . as , these two outputs overlapped and the selected molecule had a surface of ~95 square angstroms at best , the cavity coordination of 0.41 31.67 4.44 was set as the desired ligand interaction site or binding site . the pocket - finder works by scanning the probe radius 1.6 angstroms along all gridlines of the grid resolution , 0.9 angstroms surrounding the protein . the output of the pocket - finder shows the highest precision ( 52.7 ) for site two , which was also energetically most favorable . the computational docking study of the ligand with the receptor ( coordinate : 0.41 31.67 4.44 ) by mvd was evolved in terms of moldock score for best pose . this observation is concordant with previous study with r 18 - 100 that has inhibitory effect on the ar . among the studied ligands , esculin exhibited the most favorable binding to the receptor followed by morindone , anthragallol , soranjidiol , lucidin and damnacanthal . the moldock score also revealed that esculin s interaction with the receptor is likely to be more energetically economic than that of the dihydrotestosterone ( table 2 ) . during dihydrotestosterone interaction with the receptor , the amino acid residues , including gln 711 , arg 752 and thr 877 , were involved in hydrogen bonding while gln 711 , met 745 and phn 764 were involved in steric interaction ( figure 2 a ) . the amino acid residues leu 704 , asn 705 , arg 752 and thr 877 were involved in hydrogen bonding in case of esculin s interaction receptor . esculin exploited leu 704 , gln 711 , leu 873 , phe 876 , met 787 and met 895 residues at the binding site to maintain a steric interaction with the receptor ( figure 2 b ) . thus , the amino acid residues arg 752 and thr 877 were the common residues involved in the hydrogen bonding of the receptor with esculin and dihydrotestosterone . , the residues of the receptor participating in hydrogen bonding with the corresponding ligand are shown in azure color . the interaction includes hydrogen bonding with gln 711 , arg 752 and thr 877 , steric interaction with gln 711 , met 745 and phn 764 . , the residues of the receptor participating in hydrogen bonding with corresponding ligands are shown in azure color . the interaction includes hydrogen bonding with leu 704 , asn 705 , arg 752 andthr 877steric interaction with leu 704 , gln 711 , leu 873 , phe 876 , met 787 and met 895 . initial prediction of the probable ligand - binding site was performed by the pocket - finder and mdv cavity search . the prediction showed an active site of 409 cubic angstroms with a maximum coordination of 8 42 12 from the pocket - finder output ( figure 1 ) . the cavity searching algorithm built in the mvd tool fetched a cavity of 86.02 cubic angstroms with a maximum coordination of 0.41 31.67 4.44 and surface of 204.8 square angstroms . output of the pocket - finder was synchronized with the mdv cavity search output for screening the prediction with higher degree of accuracy . as , these two outputs overlapped and the selected molecule had a surface of ~95 square angstroms at best , the cavity coordination of 0.41 31.67 4.44 was set as the desired ligand interaction site or binding site . the pocket - finder works by scanning the probe radius 1.6 angstroms along all gridlines of the grid resolution , 0.9 angstroms surrounding the protein . the output of the pocket - finder shows the highest precision ( 52.7 ) for site two , which was also energetically most favorable . the computational docking study of the ligand with the receptor ( coordinate : 0.41 31.67 4.44 ) by mvd was evolved in terms of moldock score for best pose . this observation is concordant with previous study with r 18 - 100 that has inhibitory effect on the ar . among the studied ligands , esculin exhibited the most favorable binding to the receptor followed by morindone , anthragallol , soranjidiol , lucidin and damnacanthal . the moldock score also revealed that esculin s interaction with the receptor is likely to be more energetically economic than that of the dihydrotestosterone ( table 2 ) . during dihydrotestosterone interaction with the receptor , the amino acid residues , including gln 711 , arg 752 and thr 877 , were involved in hydrogen bonding while gln 711 , met 745 and phn 764 were involved in steric interaction ( figure 2 a ) . the amino acid residues leu 704 , asn 705 , arg 752 and thr 877 were involved in hydrogen bonding in case of esculin s interaction receptor . esculin exploited leu 704 , gln 711 , leu 873 , phe 876 , met 787 and met 895 residues at the binding site to maintain a steric interaction with the receptor ( figure 2 b ) . thus , the amino acid residues arg 752 and thr 877 were the common residues involved in the hydrogen bonding of the receptor with esculin and dihydrotestosterone . a ) dihydrotestosterone and androgen receptor interaction . here , the residues of the receptor participating in hydrogen bonding with the corresponding ligand are shown in azure color . the interaction includes hydrogen bonding with gln 711 , arg 752 and thr 877 , steric interaction with gln 711 , met 745 and phn 764 . , the residues of the receptor participating in hydrogen bonding with corresponding ligands are shown in azure color . the interaction includes hydrogen bonding with leu 704 , asn 705 , arg 752 andthr 877steric interaction with leu 704 , gln 711 , leu 873 , phe 876 , met 787 and met 895 . the purpose of the present study was to evaluate potential phytochemicals with anti - prostate cancer activity from h. excelsum with prediction of mechanism of action using a computational molecular simulation study . this study firstly investigated the anti - prostate cancer effect of h. excelsum phytochemicals with a mechanistic insight . from the literature search , eight phytochemicals were selected as the subject ligands including anthragallol , damnacanthal , esculin , lucidin , morindone , nordamnacanthal , rubiadin , and soranjidiol while dihydrotestosterone was considered as the control . besides , human androgen receptor ( ar ) ligand binding domain was selected as the receptor for the subsequent computational docking study . all the ligands were screened for drug - likeness rules , biological activity and probable toxicity . esculin , lucidin , morindone and soranjidiol were found as more potent physiologically active molecules without any harmless effects ( table 1 ) . upon the generation of the 3d pdb structure , the ligands were docked to the ar ligand binding residues by molegro virtual docker ( mvd ) tools . results of the docking study suggest a favorable binding of esculin to the receptor with respect to dihydrotestosterone ( table 2 ) . the coumarin glycoside esculin also scavenges superoxide radicals and decreases lipid peroxidation that is considered as a common strategy of a broad range on anti - cancer agents ( 16 ) . cichorium intybus , an esculin containing plant , has been reported to have a modest inhibitory effects on the proliferation of prostate , breast and colorectal cells ( 17 ) . analysis of amino acid residues involved in docking , hydrogen bonding energy contribution to docking score and steric interaction revealed a nearly similar ligand - receptor interaction for both of dihydrotestosterone and esculin . still , from the present molecular simulation study it was profound that the contribution of hydrogen bonding to moldock score was higher for esculin with respect to dihydrotestosterone ( table 2 ) . thus , hydrogen bonding and steric interaction patterns of esculin and dihydrotestosterone suggested a nearly similar ligand - receptor interaction pattern . therefore , esculin has the potency to act like an antagonist of dihydrotestosterone , while it also has a potential ar inhibitory effect .

background : hymenodictyon excelsum is a medicinal plant traditionally used for tumor treatment as it contains phytochemicals of anthraquinone and coumarin class.objectives:the aim of the present study was to unfold the therapeutic value of selected phytocompounds of hymenodictyon excelsum in prostate cancer.materials and methods : eight phytochemicals were selected based on the literature search including anthragallol , damnacanthal , esculin , lucidin , morindone , nordamnacanthal , rubiadin , and soranjidiol while dihydrotestosterone was considered as the control . human androgen receptor ( ar ) ligand binding domain ( pdb i d : 1e3 g ) was selected as the receptor for subsequent computational docking study . first , the selected phytocompounds were screened for their drug likeness and safety profile . molegro virtual docker ( mvd ) was subjected only to drug - like and safe phytocompounds for the computational docking study.results:except for anthragallol , nordamnacanthal and rubiadin , all the ligands were drug - like and safe . results of the docking study suggest a favorable binding of esculin to the receptor with respect to dihydrotestosterone . analysis of docking pattern revealed a nearly similar ligand - receptor interaction for both dihydrotestosterone and esculin.conclusions:the anthraquinone and coumarin principles of h. excelsum have an anti - prostate cancer effect that has been proposed to be exerted by antagonistic effects on ar .

1. Background 2. Objectives 3. Materials and Methods 3.1. Selection of Receptor and Ligands 3.2. Drug likeness and Toxicity Evaluation of the Ligands 3.3. Computational Simulation Study 3.3.1. Binding Site Prediction 3.3.2. Computational Simulation Tools and Algorithm 3.3.3. Molecular Docking Study 4. Results 4.1 Result of Drug Likeness and Toxicity Evaluation of the Ligands 4.2. Computational Simulation Study 4.2.1. Binding Site Prediction 4.2.2. Computational Molecular Docking 5. Discussion

anthraquinones , rubiadin and its methyl ether , lucidin , nordamnacanthal , damnacanthal , 2-benzylzanthopurpurin , anthragallol , soranjidiol and morindone have also been isolated from roots ( 10 ) . from the literature search , eight phytochemicals were taken into consideration for the molecular simulation study including anthragallol , damnacanthal , esculin , lucidin , morindone , nordamnacanthal , rubiadin and soranjidiol . protein data bank ( pdb ) file of human ar ligand binding domain ( pdb i d : 1e3 g ) was downloaded from rcsb pdb database ( http://www.rcsb.org/pdb/home/home.do ) . the site of the receptor that interacted with the ligands was predicted by the molegro virtual docker ( mdv ) inbuilt cavity detection algorithm and the pocket - finder server ( 14 ) . molegro virtual docker ( mvd ) was used for the computer simulated docking study ( 15 ) . from the literature search , eight phytochemicals were taken into consideration for the molecular simulation study including anthragallol , damnacanthal , esculin , lucidin , morindone , nordamnacanthal , rubiadin and soranjidiol . protein data bank ( pdb ) file of human ar ligand binding domain ( pdb i d : 1e3 g ) was downloaded from rcsb pdb database ( http://www.rcsb.org/pdb/home/home.do ) . the site of the receptor that interacted with the ligands was predicted by the molegro virtual docker ( mdv ) inbuilt cavity detection algorithm and the pocket - finder server ( 14 ) . molegro virtual docker ( mvd ) was used for the computer simulated docking study ( 15 ) . the site of the receptor that interacted with the ligands was predicted by the molegro virtual docker ( mdv ) inbuilt cavity detection algorithm and the pocket - finder server ( 14 ) . molegro virtual docker ( mvd ) was used for the computer simulated docking study ( 15 ) . the computational docking study of the ligand with the receptor ( coordinate : 0.41 31.67 4.44 ) by mvd was evolved in terms of moldock score for best pose . among the studied ligands , esculin exhibited the most favorable binding to the receptor followed by morindone , anthragallol , soranjidiol , lucidin and damnacanthal . the computational docking study of the ligand with the receptor ( coordinate : 0.41 31.67 4.44 ) by mvd was evolved in terms of moldock score for best pose . among the studied ligands , esculin exhibited the most favorable binding to the receptor followed by morindone , anthragallol , soranjidiol , lucidin and damnacanthal . the computational docking study of the ligand with the receptor ( coordinate : 0.41 31.67 4.44 ) by mvd was evolved in terms of moldock score for best pose . among the studied ligands , esculin exhibited the most favorable binding to the receptor followed by morindone , anthragallol , soranjidiol , lucidin and damnacanthal . the purpose of the present study was to evaluate potential phytochemicals with anti - prostate cancer activity from h. excelsum with prediction of mechanism of action using a computational molecular simulation study . this study firstly investigated the anti - prostate cancer effect of h. excelsum phytochemicals with a mechanistic insight . from the literature search , eight phytochemicals were selected as the subject ligands including anthragallol , damnacanthal , esculin , lucidin , morindone , nordamnacanthal , rubiadin , and soranjidiol while dihydrotestosterone was considered as the control . besides , human androgen receptor ( ar ) ligand binding domain was selected as the receptor for the subsequent computational docking study . all the ligands were screened for drug - likeness rules , biological activity and probable toxicity . upon the generation of the 3d pdb structure , the ligands were docked to the ar ligand binding residues by molegro virtual docker ( mvd ) tools . results of the docking study suggest a favorable binding of esculin to the receptor with respect to dihydrotestosterone ( table 2 ) . analysis of amino acid residues involved in docking , hydrogen bonding energy contribution to docking score and steric interaction revealed a nearly similar ligand - receptor interaction for both of dihydrotestosterone and esculin . thus , hydrogen bonding and steric interaction patterns of esculin and dihydrotestosterone suggested a nearly similar ligand - receptor interaction pattern .

all procedures were approved by the institutional animal care and use committee of the scripps research institute and were carried out on 3- to 4-month - old male c57bl/6j mice . animals were maintained on regular chow ( harlan teklad lm-485 diet 7012 ; carbohydrate 65% kcal , fat 13% , metabolizable energy 3.41 kcal / g ) . access to food and water was ad libitum , and the light : dark cycle was 12:12 h with lights on at 7:00 a.m. for telemetry studies , male mice were anesthetized with isoflorane ( induction 35% , maintenance 11.5% ) and surgically implanted with radio telemetry devices ( ta - f20 ; data sciences ) into the peritoneal cavity for cbt and locomotor activity measurement . mice were allowed to recover for 2 weeks and then were submitted for freely moving telemetry recording ( each group n = 46 ) for 7 days . mice were individually housed in a plexiglas cage in a room maintained at 25 0.5c on a 12:12 h light : dark cycle ( lights on at 6:00 a.m. ) with ad libitum access to food and water . the cages were positioned onto the receiver plates ( rpc-1 ; data sciences ) , and radio signals from the implanted transmitter were continuously monitored and recorded . cbt and locomotor activity ( number of horizontal movements ) were continuously monitored with a fully automated data acquisition system ( dataquest art ; data sciences ) . recordings were made for 72 h before treatment to ascertain that baseline levels of temperature were stable and that no ongoing febrile response confounds the results . louis , mo ) was predissolved in saline and subsequently diluted in artificial cerebrospinal fluid ( acsf ) . phosphatidylinositol 3kinase inhibitor ( pi3k - i ) , ly294002 ( 70920 ; cayman chemical , ann arbor , mi ) , was predissolved in dmso and subsequently diluted in acsf ( final concentration 5% dmso ) . injections of insulin ( or vehicle acsf ) , pi3k - i ( or vehicle acsf with 5% dmso ) , or insulin antibodies ( i8510 ; sigma , st . louis , mo ) ( or vehicle acsf ) were administered directly to the poa through the poa - implanted cannula ( anterior - posterior [ ap ] from bregma = 0.38 mm , lateral [ lat ] = midline , ventral [ v ] = 3.8 mm , cannula 26 ga , 10 mm length ) using an injector ( 33 ga , protruding 0.4 mm beyond the tip of the cannula , total length 10.4 mm ) connected to plastic tubing and a microsyringe ( 10 l ) in a volume of 0.5 l over a period of 5 min to allow diffusion ( n = 5 mice per group ) . fed mice using a computer - controlled open - circuit system ( oxymax system ) that is part of an integrated comprehensive lab animal monitoring system ( clams ; columbus instruments , columbus , oh ) . animals are tested in clear respiratory chambers ( 20 10 12.5 cm ) with a stainless steel elevated wire floor . room air is passed through chambers at a flow rate of 0.5 l / min . sample air is sequentially passed through o2 and co2 sensors ( columbus instruments ) for determination of o2 and co2 content , from which measures of oxygen consumption ( vo2 ) and carbon dioxide production ( vco2 ) are estimated . gas sensors are calibrated prior to the onset of experiments with primary gas standards containing known concentrations of o2 , co2 , and n2 ( airgas puritan medical , ontario , canada ) . respiratory exchange ratio ( rer ) is calculated as the ratio of vco2 to vo2 . energy expenditure measures ( vo2 , vco2 , and heat formation [ { 3.815 + 1.232 rer } vo2 { in liters } ] ) are corrected for estimated effective metabolic mass per kleiber power function . mice undergoing indirect calorimetry are acclimated to the respiratory chambers for 34 days before the onset of study . data are recorded under ambient room temperature clamped at 25c , beginning from the onset of the light cycle , 24 h / day for 3 days . positron emission tomography ( pet)/computed tomography ( ct ) imaging in this study was carried out with 2-[f]fluoro-2-deoxyglucose ( f - fdg ) on 12-week - old male sprague dawley rats ( mean body wt 250 g ) . rats are known to have a relatively large amount of bat in the interscapular region and thus are often used in research for bat activation ( 11 ) . each rat received 18.5 mbq of f - fdg via intraperitoneal injection , and 30 min later they were anesthetized with isoflorane ( 5% induction , 12% maintenance ) and imaged with a combined pet / ct scanner ( siemens medical solutions ) designed for small rodents at baseline and after microinjection of insulin into the preoptic area ( ap = 0.9 , lat = midline , v = 8.0 mmm from dura ) at 15 , 30 , 60 , and 180 min . in brief , ct for attenuation correction and 5-min emission pet were performed , followed by thin - slice ( 1.5 mm thick ) ct . pet and thin - slice ct images were reconstructed as 35-cm field - of - view images , and the pet and ct image sections at the same location were manually fused using image analysis software ( photoshop 6.0 ; adobe systems ) . the mild f - fdg uptake in skin and the contours of normal organs were used as landmarks . the pet and ct images were also intrinsically registered because of their acquisition on the dedicated pet / ct scanner . f - fdg uptake in interscapular bat was evaluated using the pet , ct , and fused pet / ct images . animal 's body temperature was maintained at 37c by a heat lamp ( temperature controller ret-3 temperature probe and hl-1 heat lamp ; physitemp instruments , clifton , nj ) in between periods of recording to avoid hypothermia during isoflorane anesthesia . pet data were analyzed by visual interpretation of coronal , sagittal , and transverse slices alone and in cross - referenced situations . when f - fdg pet uptake increase was observed , two levels were identified in comparison with normal activity : moderate ( more or less twice the activity in a reference region ) or intense ( markedly higher than the reference activity ) . pet and conventional imaging were interpreted separately , and the results were then compared with each other and , in certain cases , with the physiological information on hyperthermia . bone scans and ct images were read independently by two nuclear medicine physicians and by two radiologists , respectively . the brain was quickly removed from c57bl/6 mice at 2235 days old and submerged in ice - cold oxygenated ( 95% o2/5% co2 ) normal acsf ; composition was ( in mmol / l ) nacl 126 , kcl 3.5 , cacl2 2 , mgso4 1 , nah2po4 1.25 , nahco3 26 , and glucose 10 ( ph 7.4 ) . coronal slices ( 350 m thick ) containing the poa were cut by a microslicer ( vibratome ) . standard tight - seal recordings were performed in current clamp mode ( i - fast ) with an axopatch 200b amplifier to record spontaneous action potentials . the pipette solution used was ( in mmol / l ) 130 k - gluconate , 10 kcl , 10 hepes , 2 mgcl2 , 0.5 egta , 2 atp , and 1 gtp ( ph 7.4 ) . glass micropipettes were pulled with a horizontal puller ( p-87 ; sutter instruments , novato , ca ) using borosilicate glass . the electrode resistance after backfilling was 24 m. all voltage measurements were corrected for the liquid junction potential ( 9 mv ) . the temperature of the external solution was controlled with an hcc-100a heating / cooling bath temperature controller ( dagan corporation ) , a fast temperature controller equipped with a peltier element . to prevent changes induced in the electrode reference potential , the ground electrode was thermally isolated in a separate bath connected to the recording bath by a filter paper bridge . recordings were digitized using a digidata 1320a interface and analyzed using pclamp9 ( axon instruments , union city , ca ) software package and stored on the disk of a computer . after establishing whole - cell configuration ( or perforated whole cell ) , the spontaneous activity of the neuron was recorded for 24 min to determine its control behavior ( at 3637c ) , after which it was tested for temperature sensitivity with a temperature cycle of at least 3339c . the firing rate was determined for each 10-s interval and plotted against the temperature using sigmaplot software . the criteria for classifying a neuron as being warm sensitive were the same as those used by previous investigations of temperature - sensitive neurons ( 1 ) . briefly , the thermal coefficient is defined as the slope of the linear regression of the firing rate plotted as a function of temperature . this plot was determined over a temperature range of 3c in which a neuron was most sensitive . a warm - sensitive neuron is defined as having a thermal coefficient 0.8 impulses s c. neurons displaying nonreversible firing rate changes during changes in temperature were excluded . mice were anesthetized and poa tissues were obtained and homogenized in freshly prepared ice - cold radioimmunoprecipitation assay buffer ( thermo scientific , il ) including 1 mmol / l na3vo4 , 1 mmol / l naf , edta - free protease inhibitor , and phosstop ( roche , indianapolis , in ) 90 min after the injection of vehicle ( acsf ) or insulin ( 0.03 iu ) ( sigma ) into the poa . insoluble material was removed by centrifugation ( 15,000 g , 2 min ) at 4c . sample buffer ( 0.125 mol / l tris ph 6.8 , 0.04% glycerol , 0.4% sds , 0.01% -mercaptoethanol , 0.02% bromophenol blue ) was added and boiled for 5 min before separation in sds - page using 7.5% ready gel tris - hcl gels ( biorad , hercules , ca ) . electrotransfer of proteins from the gel to nitrocellulose membrane was performed for 60 min at 100 v. the nitrocellulose transfers were probed with anti insulin receptor- subunit , anti phospho akt ( ser473 ) antibodies from cell signaling technology as well as -actin ( millipore ) for the normalization . subsequently , the blots were incubated in supersignal west pico chemiluminescent substrates ( thermo scientific , hanover park , il ) for 5 min and visualized with autoradiography film ( denville scientific , metuchen , nj ) . factorial analyses of variance ( anova ) or student t tests were used for between - subject comparisons involving > 2 or exactly 2 levels , respectively . for analysis of in vivo studies on cbt one - way anova with a tukey post hoc test ( p < 0.05 ) was used to determine differences in the mean among multiple groups . area under the curve ( auc ) analysis was performed using graphpad prism 4 software . mice ( n = 4 ) were deeply anesthetized with isoflorane ( induction 35% ; maintenance 11.5% ) and placed in a kopf stereotactic frame ( kopf instruments ) . a 33 g injector ( sst-33/ft , plastics one , roanoak , va ) was connected to a hamilton syringe with a polyethylene tubing and filled with texas red conjugated dextran beads ( invitrogen , carlsbad , ca ; 1 mg / ml ) . the injector was inserted into the dorsomedial hypothalamus ( dmh ) ( ap = 1.58 mm , lat = 0.25 mm ; v = 4.6 mm from the surface of the brain ; n = 1 ) or raphe pallidus ( rpa ) ( ap = 6.12 mm , lat = 0.0 mm ; v = 5.8 mm from the surface of the brain ; n = 3 ) according to stereotactic coordinates ( 12 ) , and 1 l of tracer was injected over 1 min . animals were perfused via the ascending aorta with 10 ml of 0.9% nacl followed by 50 ml ice - cold 4% ( wt / vol ) paraformaldehyde in 0.16 mol / l pbs . brains were removed and postfixed for 2 h in the same fixative , then transferred to pbs containing 20% ( wt / vol ) sucrose and stored overnight at 4c . sections from the poa were cut on a cryostat ( leica ) at 40 m and transferred to individual wells containing pbs . for indirect immunohistochemistry free - floating sections were incubated overnight with a rabbit anti insulin receptor antibody ( 1:100 ; pfizer pp5 ) , rinsed in pbs , and incubated with a donkey anti - rabbit alexa488 ( 1:200 ; invitrogen ) for 1 h in room temperature . sections were rinsed in pbs , followed by incubation with 0.5 mol / l dapi ( invitrogen ) for 5 min , then rinsed in pbs and mounted onto superfrost plus sides ( vwr , ann arbor , mi ) and mounted with prolong gold . confocal images were captured using a zeiss laser confocal microscope using zen 2009 zeiss software suite ( carl zeiss , thornwood , ny ) . all serial optical image sections ( 0.3-m interval step slices ) were imported and spatially reassembled using imaris ( bitplane , saint paul , mn ) to generate a three - dimesional representation of the tissue and then maximum projected for two - dimensional images ( gray scale image panels ) in image pro plus ( media cybernetics , bethesda , md ) . positron emission tomography ( pet)/computed tomography ( ct ) imaging in this study was carried out with 2-[f]fluoro-2-deoxyglucose ( f - fdg ) on 12-week - old male sprague dawley rats ( mean body wt 250 g ) . rats are known to have a relatively large amount of bat in the interscapular region and thus are often used in research for bat activation ( 11 ) . each rat received 18.5 mbq of f - fdg via intraperitoneal injection , and 30 min later they were anesthetized with isoflorane ( 5% induction , 12% maintenance ) and imaged with a combined pet / ct scanner ( siemens medical solutions ) designed for small rodents at baseline and after microinjection of insulin into the preoptic area ( ap = 0.9 , lat = midline , v = 8.0 mmm from dura ) at 15 , 30 , 60 , and 180 min . in brief , ct for attenuation correction and 5-min emission pet were performed , followed by thin - slice ( 1.5 mm thick ) ct . pet and thin - slice ct images were reconstructed as 35-cm field - of - view images , and the pet and ct image sections at the same location were manually fused using image analysis software ( photoshop 6.0 ; adobe systems ) . the mild f - fdg uptake in skin and the contours of normal organs were used as landmarks . the pet and ct images were also intrinsically registered because of their acquisition on the dedicated pet / ct scanner . f - fdg uptake in interscapular bat was evaluated using the pet , ct , and fused pet / ct images . animal 's body temperature was maintained at 37c by a heat lamp ( temperature controller ret-3 temperature probe and hl-1 heat lamp ; physitemp instruments , clifton , nj ) in between periods of recording to avoid hypothermia during isoflorane anesthesia . pet data were analyzed by visual interpretation of coronal , sagittal , and transverse slices alone and in cross - referenced situations . when f - fdg pet uptake increase was observed , two levels were identified in comparison with normal activity : moderate ( more or less twice the activity in a reference region ) or intense ( markedly higher than the reference activity ) . pet and conventional imaging were interpreted separately , and the results were then compared with each other and , in certain cases , with the physiological information on hyperthermia . bone scans and ct images were read independently by two nuclear medicine physicians and by two radiologists , respectively . the brain was quickly removed from c57bl/6 mice at 2235 days old and submerged in ice - cold oxygenated ( 95% o2/5% co2 ) normal acsf ; composition was ( in mmol / l ) nacl 126 , kcl 3.5 , cacl2 2 , mgso4 1 , nah2po4 1.25 , nahco3 26 , and glucose 10 ( ph 7.4 ) . coronal slices ( 350 m thick ) containing the poa were cut by a microslicer ( vibratome ) . standard tight - seal recordings were performed in current clamp mode ( i - fast ) with an axopatch 200b amplifier to record spontaneous action potentials . the pipette solution used was ( in mmol / l ) 130 k - gluconate , 10 kcl , 10 hepes , 2 mgcl2 , 0.5 egta , 2 atp , and 1 gtp ( ph 7.4 ) . glass micropipettes were pulled with a horizontal puller ( p-87 ; sutter instruments , novato , ca ) using borosilicate glass . the electrode resistance after backfilling was 24 m. all voltage measurements were corrected for the liquid junction potential ( 9 mv ) . the temperature of the external solution was controlled with an hcc-100a heating / cooling bath temperature controller ( dagan corporation ) , a fast temperature controller equipped with a peltier element . to prevent changes induced in the electrode reference potential , the ground electrode was thermally isolated in a separate bath connected to the recording bath by a filter paper bridge . recordings were digitized using a digidata 1320a interface and analyzed using pclamp9 ( axon instruments , union city , ca ) software package and stored on the disk of a computer . after establishing whole - cell configuration ( or perforated whole cell ) , the spontaneous activity of the neuron was recorded for 24 min to determine its control behavior ( at 3637c ) , after which it was tested for temperature sensitivity with a temperature cycle of at least 3339c . the firing rate was determined for each 10-s interval and plotted against the temperature using sigmaplot software . the criteria for classifying a neuron as being warm sensitive were the same as those used by previous investigations of temperature - sensitive neurons ( 1 ) . briefly , the thermal coefficient is defined as the slope of the linear regression of the firing rate plotted as a function of temperature . this plot was determined over a temperature range of 3c in which a neuron was most sensitive . a warm - sensitive neuron is defined as having a thermal coefficient 0.8 impulses s c. neurons displaying nonreversible firing rate changes during changes in temperature were excluded . mice were anesthetized and poa tissues were obtained and homogenized in freshly prepared ice - cold radioimmunoprecipitation assay buffer ( thermo scientific , il ) including 1 mmol / l na3vo4 , 1 mmol / l naf , edta - free protease inhibitor , and phosstop ( roche , indianapolis , in ) 90 min after the injection of vehicle ( acsf ) or insulin ( 0.03 iu ) ( sigma ) into the poa . insoluble material was removed by centrifugation ( 15,000 g , 2 min ) at 4c . sample buffer ( 0.125 mol / l tris ph 6.8 , 0.04% glycerol , 0.4% sds , 0.01% -mercaptoethanol , 0.02% bromophenol blue ) was added and boiled for 5 min before separation in sds - page using 7.5% ready gel tris - hcl gels ( biorad , hercules , ca ) . electrotransfer of proteins from the gel to nitrocellulose membrane was performed for 60 min at 100 v. the nitrocellulose transfers were probed with anti phospho akt ( ser473 ) antibodies from cell signaling technology as well as -actin ( millipore ) for the normalization . subsequently , the blots were incubated in supersignal west pico chemiluminescent substrates ( thermo scientific , hanover park , il ) for 5 min and visualized with autoradiography film ( denville scientific , metuchen , nj ) . factorial analyses of variance ( anova ) or student t tests were used for between - subject comparisons involving > 2 or exactly 2 levels , respectively . for analysis of in vivo studies on cbt one - way anova with a tukey post hoc test ( p < 0.05 ) was used to determine differences in the mean among multiple groups . area under the curve ( auc ) analysis was performed using graphpad prism 4 software . mice ( n = 4 ) were deeply anesthetized with isoflorane ( induction 35% ; maintenance 11.5% ) and placed in a kopf stereotactic frame ( kopf instruments ) . a 33 g injector ( sst-33/ft , plastics one , roanoak , va ) was connected to a hamilton syringe with a polyethylene tubing and filled with texas red conjugated dextran beads ( invitrogen , carlsbad , ca ; 1 mg / ml ) . the injector was inserted into the dorsomedial hypothalamus ( dmh ) ( ap = 1.58 mm , lat = 0.25 mm ; v = 4.6 mm from the surface of the brain ; n = 1 ) or raphe pallidus ( rpa ) ( ap = 6.12 mm , lat = 0.0 mm ; v = 5.8 mm from the surface of the brain ; n = 3 ) according to stereotactic coordinates ( 12 ) , and 1 l of tracer was injected over 1 min . animals were perfused via the ascending aorta with 10 ml of 0.9% nacl followed by 50 ml ice - cold 4% ( wt / vol ) paraformaldehyde in 0.16 mol / l pbs . brains were removed and postfixed for 2 h in the same fixative , then transferred to pbs containing 20% ( wt / vol ) sucrose and stored overnight at 4c . sections from the poa were cut on a cryostat ( leica ) at 40 m and transferred to individual wells containing pbs . for indirect immunohistochemistry free - floating sections were incubated overnight with a rabbit anti insulin receptor antibody ( 1:100 ; pfizer pp5 ) , rinsed in pbs , and incubated with a donkey anti - rabbit alexa488 ( 1:200 ; invitrogen ) for 1 h in room temperature . sections were rinsed in pbs , followed by incubation with 0.5 mol / l dapi ( invitrogen ) for 5 min , then rinsed in pbs and mounted onto superfrost plus sides ( vwr , ann arbor , mi ) and mounted with prolong gold . confocal images were captured using a zeiss laser confocal microscope using zen 2009 zeiss software suite ( carl zeiss , thornwood , ny ) . all serial optical image sections ( 0.3-m interval step slices ) were imported and spatially reassembled using imaris ( bitplane , saint paul , mn ) to generate a three - dimesional representation of the tissue and then maximum projected for two - dimensional images ( gray scale image panels ) in image pro plus ( media cybernetics , bethesda , md ) . injection of insulin into the poa was followed by elevation of cbt lasting up to 6 h in a dose - dependent manner ( fig . similar to mice injected with a vehicle acsf , animals injected with insulin showed a stress - dependent elevation of cbt at time of treatment . however , cbt returned to baseline in vehicle - treated mice but not in animals that received insulin . the extent of cbt elevation was dose dependent : average cbt after injection ( after stress - induced peak , from 16 h after injection ) was 35.77 0.04c for vehicle and 36.21 0.07 , 36.98 0.03 , 37.11 0.04 , and 37.08 0.11c ( or + 0.44 , + 1.21 , + 1.34 , and + 1.31c , respectively ; p < 0.01 ) for 0.001 , 0.015 , 0.03 , and 0.06 iu of insulin , respectively . the hyperthermic effects of insulin were significantly larger in the poa ( 1.34c ) than in the dmh ( 0.78c ) ( p < 0.01 ) or the rpa ( 0.57c ; p < 0.01 ) , which are projection areas for the poa warm - sensitive neurons ( fig . 1c ) injected into the poa prior to injection of insulin prevented the insulin - evoked hyperthermic response . insulin did not display hyperthermic effects when injected peripherally ( intraperitoneally ) . the average temperature from 16 h after intraperitoneal injection was 36.28 0.07 and 36.22 0.10c for vehicle and insulin ( 0.03 iu ) , a : profile of the dose - dependent effects of local injection of four different doses of insulin ( 0.001 , ; 0.015 , ; 0.03 , ; and 0.06 iu , ) ( n = 8 animals per group ) . b : graph showing the differential effects on cbt of the injection of 0.03 iu of insulin in the poa , the dmh , and the rpa ( poa , and ; dmh , and ; rpa , and ; white = insulin and black = control ) . c : neutralizing antibody to insulin injected poa prior to insulin blocks the hyperthermic effects of insulin ( insulin antibody , and ; vehicle , and : white = insulin and black = vehicle ) . d : graph showing the effects on cbt of intraperitoneal injection of 0.03 iu of insulin or vehicle . arrow indicates time of injection ; the cbt increase observed in all conditions during the first hour is due to stress associated with injection . measurement of oxygen consumption , carbon dioxide production , and the rer demonstrated that poa injection of insulin was followed by increased oxygen consumption and lower rer compared with vehicle - treated mice , indicating a switch from glucose metabolism to elevated fatty acid utilization ( fig . . the lower rer upon poa insulin injection compared with that of vehicle - treated mice was prolonged and still present at 6 h postinjection . rer for vehicle and 0.03 iu insulin poa was 0.91 0.02 and 0.75 0.01 , respectively , ( p < 0.01 ) . locomotor activity did not contribute to cbt increase because it was not affected by either vehicle or insulin injection in the poa ( not shown ) . pet and ct imaging carried out on rats demonstrated that insulin injected into the poa activated bat thermogenesis because the insulin treatment induced up to fivefold ( p < 0.05 ) elevation in bat glucose f - fdg uptake ( fig . because in the arcuate nucleus , insulin was shown to activate the pi3k pathway ( 13 ) , the hyperthermic effects of insulin into the poa were also assessed in the presence of a pi3k - i . pretreatment with pi3k - i ly294002 ( 10 nmol in 0.5 l ) before insulin ( 0.03 iu ) , both administered to the poa 20 min apart , showed significant attenuation of insulin - induced hyperthermia ( p < 0.01 ) ( fig . comparison of the area under the curve ( auc ) for pretreatment with pi3k - i followed by either insulin or vehicle did not elicit statistically significant results . the peak hyperthermic response to insulin administered to the poa was observed 2 h after the pretreatment injection of vehicle . at this time point pretreatment with pi3k - i reduced the insulin - induced temperature elevation by 1.4c ( from 2.03 to 0.63c ) ( fig . poa injection of insulin increased fatty acid utilization and bat activity a : the 6-h profile of rer of mice treated with vehicle of 0.03 iu insulin in the poa , demonstrating that insulin injection decreased rer , indicating an elevation of fatty acid utilization . b : the 3-h profile of pet / ct of f - fdg uptake in rats injected with vehicle or 0.03 iu insulin in the poa . top panel shows the representative pet / ct ; squared in dotted lines is the area investigated after treatment with insulin or vehicle shown below . the anatomical position of bat c : the 3-h profile quantification of f - fdg uptake into bat after insulin treatment as indicated . the hyperthermic effects of poa injection of insulin are inhibited by the pi3k - i and induce an elevation of the activated phosphorylated form of akt . a : the 6-h profile of the effects of pi3k - i treatment on insulin - induced elevation of cbt . animals were pretreated with 10 nmoles of pi3k - i in 0.5 l 20 min before injection of 0.03 iu of insulin ; arrows indicate time of injections ( pi3k - i , and ; vehicle , and ; white = insulin and black = vehicle ) . b : western blot analysis of poa tissues confirmed the presence of the insulin receptor in the poa : the level of pakt increased 317 + 82.7% compared with vehicle 90 min after the injection of insulin to the poa . biochemical analysis of poa tissues confirmed the presence of the insulin receptor in the poa . comparative analysis demonstrated that insulin ( 0.03 iu ) treatment induced an elevation of the activated phosphorylated form of akt(ser 437 ) : the level of pakt increased 317 + 82.7% compared with vehicle 90 min after the injection ( fig . 3b ) , which is in line with the involvement of pi3k in the insulin signaling in this hypothalamic area . the hypothesis , suggested by the presence of the insulin receptor transcript found by single - neuron chipping of individual warm - sensitive neurons in the poa ( in preparation ) , that insulin might act directly on warm - sensitive neurons was also tested by electrophysiological recording of the effects of insulin on primary warm - sensitive po / anterior hypothalamus ( ah ) neurons ( wsn ) in hypothalamic slices . insulin was locally applied to warm - sensitive po / ah neurons using a microperfusion tip . wsns were identified by measuring the change in the firing rate during a temperature ramp ; the thermal coefficient of the wsns was 0.93 impulses s c ( fig . insulin ( 0.3 iu / ml ) decreased the firing rate of the wsn from 1.1 to 0.3 hz ( i.e. , by 73% ) ( fig . similar experiments revealed that the firing rates for 9 of 19 ( 43% ) warm - sensitive po / ah neurons tested were reversibly inhibited by insulin ( 0.3 iu / ml ) . the firing rate averaged 4.9 2.6 hz ( n = 9 ) in control and 2.6 2.4 hz ( n = 9 ) in the presence of insulin . this effect was accompanied by a hyperpolarization of 3.2 2.7 mv ( n = 9 ) . at 0.15 iu / ml insulin reduced the firing rate of warm - sensitive po / ah neurons by 10 7% ( n = 3 ) . these effects are consistent with the current models of central thermal regulation that state that the po / ah sends an inhibitory -aminobutyric acid ( gaba)ergic signal to the thermoregulatory centers dmh and/or rpa and controls thermogenesis induced by local or skin cooling or by binding of pyrogens to their receptors on warm - sensitive neurons ( 10 ) . thus , inhibition of neuronal activity in the po / ah results in hyperthermia , whereas a decrease in activity results in hypothermia ( reviewed in ) . as predicted by this model , insulin - induced inhibition of the activity of po / ah warm - sensitive neurons resulted in hyperthermia . effects of temperature and insulin on the firing - rate activity of a po / ah warm - sensitive neuron and the role of katp channels and pi3k in the inhibitory effects of insulin on warm - sensitive po / ah neurons . a : spontaneous firing activity of a po / ah neuron at three different temperatures . the slope of the linear regression , indicated by the solid line , is 0.93 impulses s c . c : application of insulin ( 0.3 iu / ml ) induced a small hyperpolarization ( 2mv ) and decreased the firing rate of the neuron from 1.1 to 0.3 hz . d : bath application of the katp channel blocker glybenclamide ( 20 mol / l ) reduced the firing rate of wsns by 43% . in the presence of the katp blocker , insulin ( 0.3 iu / ml ) did not affect the firing rate of wsns . e : bar chart summarizing the effect of insulin ( 0.3 iu / ml , n = 9 ; 0.1 iu / ml ; n = 5 ) , glybenclamide ( 20 mol / l ; n = 6 ) , and insulin ( 0.3 iu / ml , n = 6 ) in the presence of the blocker . f and g : bath application of the pi3k - i ly294002 ( 5 mol / l ) prevents the insulin- ( 0.3 iu / ml ; n = 6 ) caused inhibition of firing rate ( f ) . we then tested whether insulin caused hyperpolarization and whether reduction in firing rate involved the activation of atp - sensitive k channels ( katp channels ) as previously described on insulin effects in the arcuate neurons ( 5 ) . the katp channel blocker glybenclamide ( 20 mol / l ) was applied to the bath for 5 min prior to local insulin application . the channel blocker increased the firing rate of all neurons tested by 21 15% ( n = 6 ) ( fig . the presence of glybenclamide blocked the previously observed inhibitory effect of insulin ( 0.3 iu / ml ) on the firing rate in warm - sensitive poa / ah neurons ( fig . the pi3k - i ly249002 ( 5 /min bath ) inhibits the in vivo hyperthermic effects of insulin ( fig . 1c ) and also prevents the insulin - induced inhibition of the firing rate of the wsns in the poa . the inhibitory effect of ly249002 on insulin 's action ( fig . 4f and g ) suggests that at least some of the effects of insulin in the poa are exerted at the level of regulating the firing rate of wsns and , through this mechanism , the level of bat activation and thermogenesis . no effects of leptin ( 0.1 mol / l ) and hypoglycemia ( glucose 101 mmol / l ) on the activity of wsn were observed ( data not shown ) , although in other hypothalamic nuclei there are interactions among glucose , leptin , and insulin signaling . immunohistochemical detection of the insulin receptor shows colocalization with retrogradely traced neurons projecting to the rpa ( fig . texas red tracer ( a , red in d and e ) is colocalized with the insulin receptor ( b , green in d and e ) and the nucleus stained with dapi ( c , blue in d and e ) . e : a three - dimensional reconstruction of the same neuron as seen in a d showing an insulin receptor positive traced neuron ( arrow in d and e ) and an insulin receptor only positive neuron ( arrowhead ) . ( a high - quality color digital representation of this figure is available in the online issue . ) injection of insulin into the preoptic area of the hypothalamus induced a specific and dose - dependent elevation of cbt mediated by stimulation of bat thermogenesis as shown by imaging and rer measurement . these data show how insulin , primarily known for its peripheral action as a regulator of glucose uptake , may act as a central stimulator of fatty acid oxidation in bat . retrograde tracing indicates that insulin receptor expressing warm - sensitive neurons activate bat through projection via the rpa . these neurons are known to respond to local changes in temperature and to pyrogens during infection or diseases . insulin applied on hypothalamic slices acted directly on intrinsically warm - sensitive neurons by inducing hyperpolarization and reducing firing rates . it should be noted that the poa insulin doses used give rise to poa insulin concentrations that are in the range of circulating plasma concentrations of insulin and are unlikely to exert the bat effects via leakage to other brain areas or to the periphery . however , not all wsn tested responded to insulin , and none responded to leptin or changes in glucose concentrations ( not shown ) , indicating that wsns constitute a heterogeneous population of neurons and that insulin inhibits only a subset of these poa neurons ; however , this subset represents an input potent enough to the bat regulation to cause a hyperthermic response . the insulin - sensitive wsns may mediate actions important for the energy balance and may participate in the thermic effects of food , a phenomenon inversely correlated to the degree of insulin resistance and of body fat and proposed to be important in the development of obesity ( 16 ) . our data show how insulin can affect bat activity via central action on warm - sensitive neurons . this is important in light of recent findings on the role of bat activity in diabetes and in obesity ( 17,18 ) . the demonstration that wsns in the poa respond directly to insulin with increased thermogenesis provides an important link between thermoregulation and energy homeostasis .

objectivetemperature and nutrient homeostasis are two interdependent components of energy balance regulated by distinct sets of hypothalamic neurons . the objective is to examine the role of the metabolic signal insulin in the control of core body temperature ( cbt).research design and methodsthe effect of preoptic area administration of insulin on cbt in mice was measured by radiotelemetry and respiratory exchange ratio . in vivo 2-[18f]fluoro-2-deoxyglucose uptake into brown adipose tissue ( bat ) was measured in rats after insulin treatment by positron emission tomography combined with x - ray computed tomography imaging . insulin receptor positive neurons were identified by retrograde tracing from the raphe pallidus . insulin was locally applied on hypothalamic slices to determine the direct effects of insulin on intrinsically warm - sensitive neurons by inducing hyperpolarization and reducing firing rates.resultsinjection of insulin into the preoptic area of the hypothalamus induced a specific and dose - dependent elevation of cbt mediated by stimulation of bat thermogenesis as shown by imaging and respiratory ratio measurements . retrograde tracing indicates that insulin receptor expressing warm - sensitive neurons activate bat through projection via the raphe pallidus . insulin applied on hypothalamic slices acted directly on intrinsically warm - sensitive neurons by inducing hyperpolarization and reducing firing rates . the hyperthermic effects of insulin were blocked by pretreatment with antibodies to insulin or with a phosphatidylinositol 3kinase inhibitor.conclusionsour findings demonstrate that insulin can directly modulate hypothalamic neurons that regulate thermogenesis and cbt and indicate that insulin plays an important role in coupling metabolism and thermoregulation at the level of anterior hypothalamus .

RESEARCH DESIGN AND METHODS Positron emission tomography and computed tomography imaging. Slice preparation. Patch clamp recording. Temperature control. Data acquisition and analysis. Immunoblotting. Statistics. Immunohistochemistry. RESULTS DISCUSSION

each rat received 18.5 mbq of f - fdg via intraperitoneal injection , and 30 min later they were anesthetized with isoflorane ( 5% induction , 12% maintenance ) and imaged with a combined pet / ct scanner ( siemens medical solutions ) designed for small rodents at baseline and after microinjection of insulin into the preoptic area ( ap = 0.9 , lat = midline , v = 8.0 mmm from dura ) at 15 , 30 , 60 , and 180 min . for analysis of in vivo studies on cbt one - way anova with a tukey post hoc test ( p < 0.05 ) was used to determine differences in the mean among multiple groups . each rat received 18.5 mbq of f - fdg via intraperitoneal injection , and 30 min later they were anesthetized with isoflorane ( 5% induction , 12% maintenance ) and imaged with a combined pet / ct scanner ( siemens medical solutions ) designed for small rodents at baseline and after microinjection of insulin into the preoptic area ( ap = 0.9 , lat = midline , v = 8.0 mmm from dura ) at 15 , 30 , 60 , and 180 min . for analysis of in vivo studies on cbt one - way anova with a tukey post hoc test ( p < 0.05 ) was used to determine differences in the mean among multiple groups . injection of insulin into the poa was followed by elevation of cbt lasting up to 6 h in a dose - dependent manner ( fig . similar to mice injected with a vehicle acsf , animals injected with insulin showed a stress - dependent elevation of cbt at time of treatment . the hyperthermic effects of insulin were significantly larger in the poa ( 1.34c ) than in the dmh ( 0.78c ) ( p < 0.01 ) or the rpa ( 0.57c ; p < 0.01 ) , which are projection areas for the poa warm - sensitive neurons ( fig . c : neutralizing antibody to insulin injected poa prior to insulin blocks the hyperthermic effects of insulin ( insulin antibody , and ; vehicle , and : white = insulin and black = vehicle ) . because in the arcuate nucleus , insulin was shown to activate the pi3k pathway ( 13 ) , the hyperthermic effects of insulin into the poa were also assessed in the presence of a pi3k - i . poa injection of insulin increased fatty acid utilization and bat activity a : the 6-h profile of rer of mice treated with vehicle of 0.03 iu insulin in the poa , demonstrating that insulin injection decreased rer , indicating an elevation of fatty acid utilization . the hyperthermic effects of poa injection of insulin are inhibited by the pi3k - i and induce an elevation of the activated phosphorylated form of akt . b : western blot analysis of poa tissues confirmed the presence of the insulin receptor in the poa : the level of pakt increased 317 + 82.7% compared with vehicle 90 min after the injection of insulin to the poa . comparative analysis demonstrated that insulin ( 0.03 iu ) treatment induced an elevation of the activated phosphorylated form of akt(ser 437 ) : the level of pakt increased 317 + 82.7% compared with vehicle 90 min after the injection ( fig . the hypothesis , suggested by the presence of the insulin receptor transcript found by single - neuron chipping of individual warm - sensitive neurons in the poa ( in preparation ) , that insulin might act directly on warm - sensitive neurons was also tested by electrophysiological recording of the effects of insulin on primary warm - sensitive po / anterior hypothalamus ( ah ) neurons ( wsn ) in hypothalamic slices . insulin was locally applied to warm - sensitive po / ah neurons using a microperfusion tip . effects of temperature and insulin on the firing - rate activity of a po / ah warm - sensitive neuron and the role of katp channels and pi3k in the inhibitory effects of insulin on warm - sensitive po / ah neurons . 4f and g ) suggests that at least some of the effects of insulin in the poa are exerted at the level of regulating the firing rate of wsns and , through this mechanism , the level of bat activation and thermogenesis . injection of insulin into the preoptic area of the hypothalamus induced a specific and dose - dependent elevation of cbt mediated by stimulation of bat thermogenesis as shown by imaging and rer measurement . retrograde tracing indicates that insulin receptor expressing warm - sensitive neurons activate bat through projection via the rpa . insulin applied on hypothalamic slices acted directly on intrinsically warm - sensitive neurons by inducing hyperpolarization and reducing firing rates . the insulin - sensitive wsns may mediate actions important for the energy balance and may participate in the thermic effects of food , a phenomenon inversely correlated to the degree of insulin resistance and of body fat and proposed to be important in the development of obesity ( 16 ) .

the toronto charter for physical activity ( 2010 ) and several national physical activity plans ( usa , norway , scotland , and the u.k . ) advocate sports participation as an important part of population targeted physical activity for adolescents ( 2 , 3 ) . physical activity participation such as playing organized school sports has been promoted as a way to reduce the prevalence of overweight and obesity among adolescents , a public health challenge that continues to move in the wrong direction ( 25 ) . according to the world health organization ( 2012 ) , globally six percent of deaths are attributed to physical inactivity and physical inactivity is the 4 leading attributable cause of global deaths . numerous physical health benefits such as chronic disease prevention , healthy weight maintenance , stronger bones and muscles have been reported for adolescents who participate regularly in physical activity , however many adolescents continue to not meet physical activity recommendations ( 8 , 19 , 23 , 24 ) . emerging research evidence suggests that physical activity including organized sports participation is correlated positively to academic success and positive behaviors in middle school and high school students and that athletes perform better than non - athletes on cognitive performance tests ( 1 , 513 , 16 18 , 21 , 28 ) . in states like texas ( usa ) , students prior to 2011 were required to take and pass the texas assessment of knowledge and skills ( taks ) as part of state legislation ( 20 ) . texas students who were fitter based on fitnessgram scores , performed better on taks than low fit students ( 27 ) . while there are no published data that show athletes perform better than non - athletes on the fitnessgram , athletes in texas public schools are allowed to engage in practice eight hours per week ( plus competition time ) , which exceeds the one hour per day recommended u.s guidelines for daily physical activity in adolescents ( 22 , 23 ) . additionally , the vast majority of the scientific evidence indicates that being physically active ( like playing sports ) during the school day does not negatively affect academic success or progress ( 8 , 25 ) , although currently there is no consistent evidence that higher physical activity levels lead to increased intelligence scores ( 8 , 14 ) . unfortunately , the interpretation of previous studies that have shown positive academic outcomes associated with regular physical activity participation by adolescents in schools based on measures from organized sports , physical education , or physical fitness performance measures are inconclusive ( 8 , 25 ) . study limitations like differences in design , small sample sizes with limited ethnic diversity , lack of control for confounding variables , and self - reported versus individually measured performance have not allowed a clear understanding of how sports participation affects academic outcomes and adolescent behaviors . the purpose of this study was to further investigate and compare the academic and behavioral performance between male and female public school athletes and non - athletes in a large ethnically diverse population that included individual data reports . the hypotheses we tested were : 1 ) there are no significant differences in risk for dropping out of school between athletes and non - athletes . 2 ) there are no significant differences in disciplinary actions during the school year between athletes and non - athletes . 3 ) there are no significant differences in passing rates on academic tests like the taks between athletes and non - athletes . a total of 35,030 ( n=11,139 athletes and 23,891 non - athletes ; n = 16,673 girls and 18,357 boys ) in grades 712 were included in the study . all data were collected from a convenient sample of texas school districts ( n=7 ) that were recruited with the help of leaders from the texas high school coaches association ( thsca ) and the texas high school education foundation ( thsef ) . the grade levels and demographics of the athletes and non - athletes are presented in table 1 . data files from each school district were transferred to the researchers via password protected electronic media . individual informed consent was not necessary since the data is collected annually and mandated by the state of texas and is available through the freedom of information act . data for the study were analyzed retrospectively , and irb approval from texas state university ( # 2010r6869 ) was granted . in the spring of 2011 the investigators ( through the leadership at the thsca and the thsef recruited a convenient sample of school districts ( n=12 ) to participate in the study . individual student data ( student i d code , general demographics , taks scores , at risk for dropout status , and disciplinary actions ) organized by the institutional technologist ( it ) from each school district were submitted to the investigators . student taks scores included the achievement of the minimum standard by subject and grade ( meets standard ) as well as achievement of the commendable levels ( meets desirable standards for the majority of students set by the texas education agency at least the 90 percentile ) ( 20 ) . students were classified as athletes by each district s it , if they participated in at least one of the 20 plus university scholastic league ( uil , state governing body ) sponsored sports during the school year and as a non - athlete if they did not participate . once data quality analyses were finished , complete information from 7 districts was selected for use in the study for subsequent data analyses . to determine regular physical activity levels between athletes and non - athletes the following assumptions were made regarding observations that athletes ( adolescents ) are more physically active than their general population counterpart : it was estimated that athletes were physically active at moderate- to vigorous- intensities for a minimum of 8 hours per week outside the school day ( state practice rule limitations ) , which exceed the national guidelines for adolescents of 60 minutes of daily moderate to vigorous intensities ( 19 , 23 ) . many athletes also are active in texas as part of physical education classes that are taught by teacher / coaches as well . assumptions for non - athletes were based on the centers for disease control and prevention ( cdc ) youth behavior risk surveillance system ( yrbs ) 2011 data for high school youth in texas , which showed that 42.4% did not play on sports teams , 16.4 % did not participate in at least 60 minutes of physical activity on any day , and only 55.5% were active at least 60 minutes per day for less than 5 days ( 26 ) . since the data for the study were collected from a convenient sample , quasi - experimental design techniques were used to determine differences between group data . group comparisons to determine differences between athletes and non - athletes scores for the various study variables utilized chi - square or anova techniques found in spss version 15 depending on the variables value scale . data for the at - risk status of the student was scored nominally as either at risk or not at risk of dropping out of school therefore chi - square analyses were used to determine significant differences between athletes and non - athletes as well as differences among ethnicities between the two groups . scores for the taks test components were scored as either meeting or not meeting the mastery level of the minimum component therefore chi - square analyses were used to determine significant differences between athletes and non - athletes for the overall mastery of the test as well as its separate components and commendable achievement levels . finally , disciplinary actions were scored as the number of times within a year the student was disciplined by a faculty member or administrator ; therefore a oneway anova was used to compare groups ( athletes vs non - athletes ) by disciplinary actions to determine significant differences . for all analyses an a priori probability level was set at p < 0.05 to determine significant differences between the groups . a total of 35,030 ( n=11,139 athletes and 23,891 non - athletes ; n = 16,673 girls and 18,357 boys ) in grades 712 were included in the study . all data were collected from a convenient sample of texas school districts ( n=7 ) that were recruited with the help of leaders from the texas high school coaches association ( thsca ) and the texas high school education foundation ( thsef ) . the grade levels and demographics of the athletes and non - athletes are presented in table 1 . data files from each school district were transferred to the researchers via password protected electronic media . individual informed consent was not necessary since the data is collected annually and mandated by the state of texas and is available through the freedom of information act . data for the study were analyzed retrospectively , and irb approval from texas state university ( # 2010r6869 ) was granted . in the spring of 2011 the investigators ( through the leadership at the thsca and the thsef recruited a convenient sample of school districts ( n=12 ) to participate in the study . individual student data ( student i d code , general demographics , taks scores , at risk for dropout status , and disciplinary actions ) organized by the institutional technologist ( it ) from each school district were submitted to the investigators . student taks scores included the achievement of the minimum standard by subject and grade ( meets standard ) as well as achievement of the commendable levels ( meets desirable standards for the majority of students set by the texas education agency at least the 90 percentile ) ( 20 ) . students were classified as athletes by each district s it , if they participated in at least one of the 20 plus university scholastic league ( uil , state governing body ) sponsored sports during the school year and as a non - athlete if they did not participate . once data quality analyses were finished , complete information from 7 districts was selected for use in the study for subsequent data analyses . to determine regular physical activity levels between athletes and non - athletes the following assumptions were made regarding observations that athletes ( adolescents ) are more physically active than their general population counterpart : it was estimated that athletes were physically active at moderate- to vigorous- intensities for a minimum of 8 hours per week outside the school day ( state practice rule limitations ) , which exceed the national guidelines for adolescents of 60 minutes of daily moderate to vigorous intensities ( 19 , 23 ) . many athletes also are active in texas as part of physical education classes that are taught by teacher / coaches as well . assumptions for non - athletes were based on the centers for disease control and prevention ( cdc ) youth behavior risk surveillance system ( yrbs ) 2011 data for high school youth in texas , which showed that 42.4% did not play on sports teams , 16.4 % did not participate in at least 60 minutes of physical activity on any day , and only 55.5% were active at least 60 minutes per day for less than 5 days ( 26 ) . since the data for the study were collected from a convenient sample , quasi - experimental design techniques were used to determine differences between group data . group comparisons to determine differences between athletes and non - athletes scores for the various study variables utilized chi - square or anova techniques found in spss version 15 depending on the variables value scale . data for the at - risk status of the student was scored nominally as either at risk or not at risk of dropping out of school therefore chi - square analyses were used to determine significant differences between athletes and non - athletes as well as differences among ethnicities between the two groups . scores for the taks test components were scored as either meeting or not meeting the mastery level of the minimum component therefore chi - square analyses were used to determine significant differences between athletes and non - athletes for the overall mastery of the test as well as its separate components and commendable achievement levels . finally , disciplinary actions were scored as the number of times within a year the student was disciplined by a faculty member or administrator ; therefore a oneway anova was used to compare groups ( athletes vs non - athletes ) by disciplinary actions to determine significant differences . for all analyses an a priori probability level was set at p < 0.05 to determine significant differences between the groups . the results of the chi - square analyses comparing athletes and non - athletes for at - risk status , taks components , and taks commendable levels are presented in table 2 . chi - square analysis for the comparison of at - risk status between athletes ( n=11,139 ) and non - athletes ( n=23,891 ) resulted in a significant difference between the groups with a = 686.2 ; p<0.05 . representation of the differences is presented in figure 1 with fewer athletes ( 35.2% ) classified at - risk of dropping out of school compared to the non - athletes ( 52.3% ) . further examination of the at - risk data found significant decline in the at - risk factor among athletes from each ethnic group except for native americans . figure 2 is a graphical representation of these data . when compared to their non - athletic counterparts asian athletes were at a lower risk than asian non - athletes ( 24.2% vs 41.1% ; 2 = 32.18 ; p < 0.05 ) . black athletes were at a lower risk than black non - athletes ( 49.9% vs 56.7% ; 2 = 27.55 ; p < 0.05 ) . hispanic athletes were at a lower risk than hispanic non - athletes ( 43.1% vs 59.7% ; 2 = 147.17 ; p < 0.05 ) . white athletes were at a lower risk than white non - athletes ( 21.5% vs 39.8% ; 2 = 362.12 ; p < 0.05 ) . the only ethnic group that did not have a significantly lower at - risk factor for athletes compared to non - athletes were the native american group ( 39.6% vs 48.98% ; 2 = 1.93 ; p > 0.05 ) . comparison of the academic skills of the students associated with the taks pass rates for each of the taks components resulted in significantly higher pass rates for athletes compared to non - athletes . athletes had significantly higher passing rates compared to their non - athletic counterparts for the taks math component ( 2 = 1,390.62 ; p < 0.05 ) , the taks english language component ( 2 = 2,239.15 ; p < 0.05 ) , the taks reading component ( 2 = 1,732.63 ; p < 0.05 ) , the taks writing component ( 2 = 2,447.38 ; p < 0.05 ) , the taks science component ( 2 = 2,277.72 ; p < 0.05 ) , and the taks social studies component taksss ( 2 = 1,799.44 ; p < 0.05 ) . furthermore , when comparing the rates of students who achieved the commendable level ( highest achievement ) for the taks academic testing , a significantly greater number of athletes achieved the commendable level for each of the taks components compared to their non - athletic counterparts . figure 4 is a graphical representation of the commendable achievement for the taks component data . athletes had significantly higher commendable passing rates compared to their non - athletic counterparts for the taks math component ( 2 = 641.87 ; p < 0.05 ) , the taks english language component ( 2 = 657.23 ; p < 0.05 ) , the taks reading component ( 2 = 542.83 ; p < 0.05 ) , the taks writing component ( 2 = 730.84 ; p < 0.05 ) , the taks science component ( 2 = 765.90 ; p < 0.05 ) , and the taks social studies component taksss ( 2 = 573.64 ; p < 0.05 ) . in the final analysis , a oneway analysis of variance ( anova ) was used to compare the number of daily average disciplinary actions reported by faculty and administrators for the students by their group identification as an athlete or non - athlete . the anova revealed , as presented in figure 5 , that athletes had significantly fewer daily disciplinary actions during the school year compared to non - athletes ( f112.62 ; p < 0.05 ) . the present study is one of the larger studies ever conducted with a large ethnically diverse population of athletes and non - athletes that included individual data reports . an important finding of the study was that participation in athletics decreased the probability of students dropping out of school even when classified at higher risk for premature separation . this was true both generally and for each ethnic group , except the native americans . although the native american group was not significantly different for at - risk classification for athletes compared to non - athletes , it is likely due to the small sample size ( n=375 ) for native american students within this study . lumpkin and favor suggest that athletes drop out of high school less often than non - athletes ( 11 ) . they reported that in kansas , non - athletes were 15 times more likely to drop out of school than non - athletes in 20082009 . they also found that athletes across all ethnic groups in their study were less likely to drop out of school than non - athletes and suggested that participating in sports may help minorities matriculate and become acculturated when related to future academic success . our significant results indicate that non - athletes were at a 17% greater risk for dropping out than athletes for all ethnic groups except native americans in the study . these results are important because few previous related studies have included the ethnicity of their study populations ( 1 ) . our results show that participating in school sports was associated with between a 6.8% to 18.3% reduction of risk for dropping out based upon the school . howie and pate have noted that health disparities for minorities accompany the academic achievement gaps related to youth fitness levels ( 8) . pate and colleagues reported that low physical activity in adolescents was associated with many negative health behaviors ( 15 ) . although reasons for leaving school before graduating may vary among individuals , the positive effects of athletic participation such as improved self confidence , self esteem and self worth , coupled with the structured atmosphere and support system associated with athletics may improve the individual s decision to remain in school and graduate ( 11 ) . if participation in middle school and high school athletics can help reduce student drop out rates this would be important to school administrators with regards to improving academic outcomes based on 2001 , no child left behind legislation and as a way to promote positive public health messages . the athletes in our study had significantly higher passing rates on all taks components than non - athletes . the percentage passing rates percentages comparing athletes to non - athletes for each of the taks components were as follows : math 19.7% ; english language 27.6% ; reading - 31.6% , writing - 49.4% ; science 34.9% ; and social studies lumpkin and favor reported that athletes in their study also scored significantly higher than non - athletes for standardized state assessments in math , reading , history / government , writing , and science for 20062009 . others have found that physical activity through middle school and high school sports participation is associated with higher grade point averages and academic ability based on a variety of cognitive outcome measures ( 11 , 16 ) . our study data also supports the contention that participation in school athletics improves the probability of reaching the commendable level ( at least the 90 percentile ) of academic performance based on the taks ( 20 ) . the percentage passing rates percentages comparing athletes to non - athletes for each of the taks components at the commendable levels were as follows : math 11.8% ; english language 13% ; reading - 15.7% , writing 18.4% ; science 14.1% ; and social studies 18% . thus , athletes in our study not only were more likely to meet the minimal academic standards required , they also were more likely to achieve commendable levels , which are associated with post secondary educational success ( 20 ) . possible reasons for the athletes academic success may include increased interest in school , improved motivation to succeed in order to remain eligible , and the expectation of participating at the collegiate level . sports participation may also increase the athletes self esteem and self worth , while at the same time increase their parents interest in their performance both during and outside of the sport participation ( 29 ) . we found that the athletes in our study had significantly fewer disciplinary actions as compared to non - athletes . tomporowski and colleagues reported that regular participation in physical activity ( like school sports ) by adolescents can help improve cognitive control and attention and other measures of mental health that have been associated with enhanced academic achievement ( 21 ) . a student that is constantly facing disciplinary actions is more likely to perform poorly academically and at an increased risk for dropping out of school ( 6 , 21 ) . while a large body of evidence supports a positive effect regarding participation in physical activity and academic achievement , it is difficult to determine if the effects are causal ( 8) . this is also true with regards to school sports participation . other factors such as student identification with school related values could encourage students to perform better academically ( 7 ) . in texas , no pass , no play legislation has been credited with keeping students in school so that they remain eligible to participate in extracurricular activities ( 22 ) . the fact that coaches acting as mentors that strongly influence their athletes to maintain their eligibility and provide praise for success and negative feedback for failure can not be ignored . sports participation for school athletics has also been associated with higher levels of learned self - discipline and better time management skills ( 11 ) . our results have implications for policymakers , school administrators , and practitioners . according to the who , experience and scientific evidence show that regular participation in appropriate physical activity and sport provides people of both sexes and all ages and conditions , including persons with disabilities , with a wide range of physical , social and mental health benefits ( 30 ) . policy makers and school administrators should encourage opportunities for students to engage in athletics at the middle school and high school levels as one way to promote positive academic performance and behaviors ( 1 , 6 , 16 , 21 ) . practitioners should consider basic interventions that can help students become and remain physically active throughout the lifespan of education ( pre k to grade 12 ) that allows them to acquire the basic levels of fitness , self - esteem , and confidence to participate in school sports based on their motivational goals . a primary strength of this study is the large and diverse ethnic sample size based on individually reported taks score data . few previous related research studies have been able to evaluate individual athlete versus non - athlete academic or behavioral performance comparisons due to the reliance on aggregate data . we found that athletes versus non - athletes were at lower risk for dropping out of school , did better academically at the minimal and commendable levels for taks , and had fewer disciplinary actions . the main limitations of this study are its cross - sectional design and use of a convenient sample that may limit the generalizability of our study to other populations . while a strong case can be made that the athletes in our study were significantly more active than their non - athlete counterparts , we did not have actual physical activity measures for our subjects . our study also does not imply causal implications between participation in school sports and positive academic and behavioral outcomes . the results of this study support the research findings of others that participation in school sports is positively correlated with better academic and behavioral performances for athletes compared to non - athletes . the improved academic and behavioral performances of athletes may imply lifelong success through increased retention in high school , the increased possibility of matriculating to college , and successful college graduation . these implications may positively impact the individuals lifelong earning capacity and improve their quality of life and the quality of their community . further studies are warranted to determine if there are causal associations between academic and behavioral performances between athletes and non - athletes .

the toronto charter for physical activity ( 2010 ) and several national physical activity plans advocate sports participation as an important part of population targeted physical activity for youth . emerging research evidence also suggests that sports participation during adolescents is linked to significant positive correlations with academic and behavioral performance . the purpose of this study was to compare academic and behavioral performance between male and female public school athletes ( total n=11,139 ; 38% female ) and non - athletes ( total n=23,891 ; 52% female ) in a convenient , ethnicity diverse , sample ( grades 712 ) from the state of texas ( usa ) . we examined the passing rates of individual athletes and non - athletes on standardized tests ( texas assessment of knowledge and skills , taks ) for math , language arts , reading , writing , science , and social studies . we also examined the percentage of athletes and non - athletes for being at risk , for dropping out of school and for the total average number of disciplinary actions . chi - square statistical analyses comparing athletes to non - athletes showed that athletes scored significantly better ( p<0.05 ) on all standardized tests compared to non - athletes ( passing rate ranges ranged from 77.1% to 92.9% versus 27.7% to 66.5% respectively ) . athletes were at lower risk for dropout compared to non - athletes ( 35.6% versus 49.24% ; p<0.05 ) , and they had fewer disciplinary actions ( mean of 0.85 per athletes per year versus 1.23 for non - athletes ; anova , p<0.05 ) . our results support the research findings of others that participation in school sports is positively correlated to better academic and behavioral performances for athletes compared to non - athletes .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

the toronto charter for physical activity ( 2010 ) and several national physical activity plans ( usa , norway , scotland , and the u.k . ) advocate sports participation as an important part of population targeted physical activity for adolescents ( 2 , 3 ) . emerging research evidence suggests that physical activity including organized sports participation is correlated positively to academic success and positive behaviors in middle school and high school students and that athletes perform better than non - athletes on cognitive performance tests ( 1 , 513 , 16 18 , 21 , 28 ) . in states like texas ( usa ) , students prior to 2011 were required to take and pass the texas assessment of knowledge and skills ( taks ) as part of state legislation ( 20 ) . the purpose of this study was to further investigate and compare the academic and behavioral performance between male and female public school athletes and non - athletes in a large ethnically diverse population that included individual data reports . the hypotheses we tested were : 1 ) there are no significant differences in risk for dropping out of school between athletes and non - athletes . to determine regular physical activity levels between athletes and non - athletes the following assumptions were made regarding observations that athletes ( adolescents ) are more physically active than their general population counterpart : it was estimated that athletes were physically active at moderate- to vigorous- intensities for a minimum of 8 hours per week outside the school day ( state practice rule limitations ) , which exceed the national guidelines for adolescents of 60 minutes of daily moderate to vigorous intensities ( 19 , 23 ) . data for the at - risk status of the student was scored nominally as either at risk or not at risk of dropping out of school therefore chi - square analyses were used to determine significant differences between athletes and non - athletes as well as differences among ethnicities between the two groups . to determine regular physical activity levels between athletes and non - athletes the following assumptions were made regarding observations that athletes ( adolescents ) are more physically active than their general population counterpart : it was estimated that athletes were physically active at moderate- to vigorous- intensities for a minimum of 8 hours per week outside the school day ( state practice rule limitations ) , which exceed the national guidelines for adolescents of 60 minutes of daily moderate to vigorous intensities ( 19 , 23 ) . data for the at - risk status of the student was scored nominally as either at risk or not at risk of dropping out of school therefore chi - square analyses were used to determine significant differences between athletes and non - athletes as well as differences among ethnicities between the two groups . the results of the chi - square analyses comparing athletes and non - athletes for at - risk status , taks components , and taks commendable levels are presented in table 2 . chi - square analysis for the comparison of at - risk status between athletes ( n=11,139 ) and non - athletes ( n=23,891 ) resulted in a significant difference between the groups with a = 686.2 ; p<0.05 . representation of the differences is presented in figure 1 with fewer athletes ( 35.2% ) classified at - risk of dropping out of school compared to the non - athletes ( 52.3% ) . when compared to their non - athletic counterparts asian athletes were at a lower risk than asian non - athletes ( 24.2% vs 41.1% ; 2 = 32.18 ; p < 0.05 ) . the only ethnic group that did not have a significantly lower at - risk factor for athletes compared to non - athletes were the native american group ( 39.6% vs 48.98% ; 2 = 1.93 ; p > 0.05 ) . an important finding of the study was that participation in athletics decreased the probability of students dropping out of school even when classified at higher risk for premature separation . our significant results indicate that non - athletes were at a 17% greater risk for dropping out than athletes for all ethnic groups except native americans in the study . the percentage passing rates percentages comparing athletes to non - athletes for each of the taks components were as follows : math 19.7% ; english language 27.6% ; reading - 31.6% , writing - 49.4% ; science 34.9% ; and social studies lumpkin and favor reported that athletes in their study also scored significantly higher than non - athletes for standardized state assessments in math , reading , history / government , writing , and science for 20062009 . the percentage passing rates percentages comparing athletes to non - athletes for each of the taks components at the commendable levels were as follows : math 11.8% ; english language 13% ; reading - 15.7% , writing 18.4% ; science 14.1% ; and social studies 18% . we found that athletes versus non - athletes were at lower risk for dropping out of school , did better academically at the minimal and commendable levels for taks , and had fewer disciplinary actions . the results of this study support the research findings of others that participation in school sports is positively correlated with better academic and behavioral performances for athletes compared to non - athletes .

steroid hormones like progesterone have been extensively studied in the literature with controversies in early pregnancy usage with varied literature . the role in preventing abortions , recurrent pregnancy loss and preterm labor has been the aim of the review . adequate levels of circulating thyroid hormones are of primary importance for normal reproductive function progesterone is largely produced by the corpus luteum until about 10 weeks of gestation.1 a study in ovarian failure and assisted reproduction it was shown that one hundred mg of p were probably a supraphysiological dose to support pregnancy 6 to 8 weeks after conception . the fetoplacental unit was competent from 10 to 12 weeks gestation.2 when the pregnancy reaches term gestation , progesterone levels range from 100 - 200 ng / ml and the placenta produces about 250 mg / day . almost all of the progesterone produced by the placenta enters the placenta , contrast to oestrogen . progesterone production is independent of he precursor available , fetal status including the wellbeing . in early pregnancy , the maternal levels of 17 a - hydroxyprogesterone rise , marking the activity of the corpus luteum . by the tenth week of gestation , this compound has returned to baseline levels , indicating that the placenta has little 17a hydroxylase activity . however , beginning about the 32 week there is a second , more gradual rise in 17a - hydroxyprogesterone due to placental utilization of fetal precursors . progesterone is also important in suppressing the maternal immunologic response to fetal antigens , thereby preventing maternal rejection of the trophoblast . and , of course , progesterone prepares and maintains the endometrium to allow implantation earlier . studies have shown that the human corpus luteum makes significant amounts of estradiol , but it is progesterone and not oestrogen that is required for successful implantation.3 the placenta does not have all the necessary enzymes to make oestrogens from cholesterol , or even progesterone . human trophoblast lack 17-hydroxylase and therefore can not convert c21-steroids to c19-steroids , the immediate precursors of oestrogen . to bypass this deficit , dehydroisoandrosterone sulfate ( dha ) from the fetal adrenal is converted to estradiol-17 by trophoblasts . in its key location as a way station between mother and fetus , placenta can use precursors from either mother or fetus to circumvent its own deficiencies in enzyme activities . hormones act as catalysts for chemical changes at the cellular level that are necessary for growth , development and energy . fetus lacks 3 b hydroxysteroid dehydrogenase - hence unable to produce progesterone - borrows from placenta . in return , fetus give placenta what it lacks ( 19 carbon compounds)-precursor of oestrogen . protein hormones are : human placental lactogen ( hpl ) , human chorionic gonadotropin ( hcg ) , adrenocorticotropic ( acth ) , growth hormone variant ( hgh - v ) , parathyroid hormone - related protein ( pth - rp ) , calcitonin , relaxin , inhibins activins , atrial natriuretic peptide , hypothalamic - like releasing and inhibiting hormones , thyrotropin releasing hormone ( trh ) , gonadotropin releasing hormone ( gnrh ) , corticotropin - releasing hormone ( crh ) , somatostatin , growth hormone - releasing hormone ( ghrh ) , alpha fetoprotein , prolactin , relaxin and other decidual proteins . due to the comprehensiveness of the choices to describe hormones , clinical importance of hcg relevant for therapy is discussed in this chapter . this glycoprotein is critical since it rescues the corpus luteum from involution , and this maintains progesterone secretion by the ovarian granulosa cells . its usefulness as a diagnostic marker of pregnancy stems from the fact that it may be one of the earliest secreted products of the conceptus . in pregnancy , placental production of hcg is at its peak between the eighth to the tenth week of gestation , and tends to plateau at a lower level for the remainder of pregnancy . the only definitely known function for hcg is support of the corpus luteum ( cl ) , taking over for lh on about the eighth day after ovulation , 1 day after implantation , when b - hcg first can be detected in maternal blood . at 8 cell stage implantation occurs 5 - 6 days after ovulation and hcg must appear by 10 days of ovulation ( 4 days after ovulation ) to rescue corpus luteum . hence , blastocyst should implant in a narrow window of time . the hcg stimulation of cl has a daily secretion of 25 mg of p and 0.5 mg of e2 . hcg gene expression is present in both cytotrophoblast and syncytiotrophoblast , but it is synthesized mainly in the syncytiotrophoblast . the maternal circulating hcg concentration is approximately 100 iu / l at the time of the expected but missed menses . a maximal level of about 100,000 iu / l in the maternal circulation is reached at 8 - 10 weeks of gestation . there are two clinical conditions in which blood hcg titers are especially helpful : trophoblastic disease and ectopic pregnancies . trophoblastic disease is distinguished by very high b - hcg levels ( 3 - 100 times higher than normal pregnancy ) . ectopic production of a - and b - hcg by non - trophoblastic tumours is rare , but does occur . the human placental lactogen ( hpl ) is secreted primarily into the maternal circulation , most of its functions occur at sites of action in maternal tissues . human placental lactogen is thought to be responsible for the marked rise in maternal plasma insulin - like growth factor-1 ( igf-1 ) concentrations as the pregnancy approaches term . it is associated with insulin resistance , enhances insulin secretion which stimulates lipolysis , increases circulating free fatty acids , and inhibits gluconeogenesis ; in effect , it antagonizes insulin action , induces glucose intolerance , as well as lipolysis and proteolysis in the maternal system . hence the role of universal screening for abnormal blood sugar in the beginning of the third trimester is emphasized in clinical practice . in the fetus calcium concentrations , are regulated by the movement of calcium , across the placenta , from the maternal compartment . in order to maintain fetal bone growth , the maternal compartment undergoes adjustments that provide a net transfer of sufficient calcium to the fetus . maternal compartment changes that permit calcium accumulation include increases in maternal dietary intake , increases in maternal d3 levels , and increases in parathyroid hormone levels . progesterone supplement in pregnancy : an immunologic therapy there are several studies to understand the maintenance of pregnancy by progesterone . progesterone has been shown to increase the cytokines produced by th2 cells which predominate over those produced by th1 cells , resulting in the maintenance of pregnancy . the th2-derived cytokines , il-4 and il-6 , induce the release of hcg from trophoblasts and the hcg stimulates progesterone production from corpus luteum in pregnancy . progesterone has been shown to stimulate the secretion of th2 and reduces the secretion of th1 cytokines . this role in controlling the immune and endocrine system which promotes the function of the trophoblasts at the implantation site seems interesting.4 use of progestogen in threatened abortion is controversial.5 progesterone for recurrent miscarriage progestogen has been used for several years even before there was knowledge of the immunomodulatory properties of progesterone . since that time , studies of differing quality have been carried out to prove the benefits of progestogen supplementation in affected women . a study on 146 women who presented with mild or moderate vaginal bleeding during the first trimester of pregnancy was randomized to receive oral dydrogesterone ( 10 mg b.i.d . ) ( n=86 ) or no treatment ( n=60 ) . the incidence of miscarriage was significantly lower in the dydrogesterone group than in the untreated group ( 17.5% vs. 25% ; p<0.05).6 the majority of cited clinical trials revealed a trend to improved pregnancies and increased live birth rates in the progestogen treatment group , but unfortunately , many studies had poor designs and methodical weaknesses.7 several studies have shown that supportive care in early pregnancy is associated with a significant beneficial effect on pregnancy outcome . women with otherwise unexplained recurrent pregnancy loss should be counselled regarding the potential for successful pregnancy without any treatment except supportive therapy such as folic acid or vitamin supplementation.78 the route of progestogen administration are in various formulations , but it is generally recommend the exclusive use of progestogen without any ( anti- ) androgenic or ( anti- ) oestrogenic effect . progestogen supplementation is available as vaginal suppositories ( 0.4 g / day , preferably in the evening because natural progesterone can cause tiredness ) , intramuscular injection ( 250 mg hydroxyprogesterone weekly ) or oral intake ( e.g. 10 mg dydrogesterone , the stereo - isomer of natural progesterone.9 progesterone supplementation following assisted reproductive technology the use of the progesterone supplementation in art cycles has better clarity.10 the duration of progesterone supplementation following reproductive technology ( art ) has been studied in a retrospective cohort study . one group had progesterone supplementation through the first trimester of pregnancy ( first trimester protocol ) till 12 weeks and the second group had the progesterone discontinued after a positive beta hcg test 2 weeks after retrieval ( luteal protocol ) . a similar rate of clinical pregnancies occurred at 7 weeks ( 81.8% luteal protocol vs. 85.8% first trimester protocol ) and for live birth rates ( 76.8% luteal protocol vs. 75.0% first trimester protocol ) . there was a trend toward a higher rate of pregnancy loss after 7 weeks in the first trimester protocol group occurred ( 15.5% vs. 4.4% ) , indicating that first trimester progesterone supplementation may support early pregnancy through 7 weeks by delaying miscarriage but does not improve live birth rates . there are randomized trials supporting the routine use of luteal support in art cycles using gnrh agonists or antagonists . fifty - nine studies were included in a review to evaluate the luteal phase support with hcg compared to placebo or no treatment , in terms of increased ongoing pregnancy rates . luteal phase support with hcg or progesterone after assisted reproduction results in an increased pregnancy rate . hcg does not provide better results than progesterone , and is associated with a greater risk of ohss when used with gnrha . the optimal route of progesterone administration has not yet been established.11 a review showed a significant effect in favour of progesterone for luteal phase support , favouring synthetic progesterone over micronized progesterone.12 prevention of recurrent preterm delivery by 17 alpha - hydroxyprogesterone caproate preterm delivery should be anticipated and prevented to decrease perinatal morbidity and mortality . those women who have had a spontaneous preterm delivery earlier are at greatly increased risk for preterm delivery in subsequent pregnancies . the results of several small trials have suggested that 17 alpha - hydroxyprogesterone caproate ( 17p ) may reduce the risk of preterm delivery . a double - blind , placebo - controlled trial involving pregnant women with a documented history of spontaneous preterm delivery was done.13 a total of 19 clinical centers were taken for the study and pregnant women at 16 to 20 weeks of gestation were included and were randomly assigned by a central data center , in a 2:1 ratio , to receive either weekly injections of 250 mg of 17p or weekly injections of an inert oil placebo ; injections were continued until delivery or to 36 weeks of gestation . treatment with 17p significantly reduced the risk of delivery at less than 37 weeks of gestation which was 36.3 percent in the progesterone group vs. 54.9 percent in the placebo group ; relative risk , delivery at less than 35 weeks of gestation was 20.6 percent vs. 30.7 percent ; and delivery at less than 32 weeks of gestation was 11.4 percent vs. 19.6 percent . the incidence of necrotizing enterocolitis , intraventricular hemorrhage in infants of women treated with 17p had significantly lower rates of and need for supplemental oxygen . hence , the study concluded that weekly injections of 17p resulted in a substantial reduction in the rate of recurrent preterm delivery among women who were at particularly high risk for preterm delivery and reduced the likelihood of several complications in their infants . one double blind randomized placebo controlled trials reported lower preterm birth rate with the use of either intramuscular 17 alpha - hydroxyprogesterone caproate ( 17p ) or intravaginal micronized progesterone suppositories in women at risk for preterm delivery.14 the half - life of 17p was estimated to be approximately 7.8 days . the route of administration plays an important role in the drug 's safety and efficacy profile . oral progesterone has not been used for prevention of preterm labor because of its first - pass hepatic metabolism , and there is a lack of data on efficacy , high side - effect profile , and because of extreme variability in plasma concentrations . vaginal administration of progesterone avoids first - pass hepatic metabolism and is associated with rapid absorption , high bioavailability , and local endometrial effects.15 vaginal route offers no local pain and few side effects , it is associated with variable blood concentrations.16 to study the efficacy of progesterone for maintenance tocolytic therapy after threatened preterm labor was done in a randomized controlled trial.17 the study was on 70 women who presented with symptoms of threatened preterm labor , who after arrest of uterine activity were then randomized to progesterone therapy or no treatment and the purpose of this study was to determine whether supplementation of vaginal progesterone after inhibition of preterm labor is associated with an increased latency period and a decreased recurrent of preterm labor . treatment group received progesterone suppository ( 400 mg ) daily until delivery and control group received no treatment . the study concluded that the use of vaginal progesterone suppository after successful parenteral tocolysis associated with a longer latency preceding delivery but failed to reduce the incidence of readmission for preterm labor . dydrogesterone supplementation in women with threatened had preterm delivery the impact on cytokine profile , hormone profile , and progesterone - induced blocking factor.18 a study on eighty - three women with symptoms of threatened preterm birth were either randomized to study groups receiving tocolytic treatment combined with intravaginal micronized natural progesterone ( 200 mg daily ) or to a control group receiving only tocolysis . micronized natural progesterone treatment resulted in a prolonged latency period of 32.117.8 versus 21.216.3 days in the control group and heavier birth weights of 2,982.8697.8 g versus 2,585.3746.6 g.19 estradiol supplementation during the luteal phase of in vitro fertilization cycles a prospective randomized study was done to find the optimal dosage of estradiol ( e2 ) for luteal phase support through the addition of different doses of e2 to progesterone ( p ) luteal phase support in patients undergoing long gnrh agonist in vitro fertilization ( ivf ) treatments.20 two hundred and eighty - five women undergoing ivf treatment with a long gnrh agonist protocol were prospectively randomized into three groups . group 1 ( n=95 ) received p and 2 mg e2 , group 2 ( n=95 ) received p and 4 mg e2 and group 3 ( n=95 ) received p and 6 mg e2 as luteal phase support . the secondary variables of interest were the implantation rate ( ir ) , miscarriage rate and multiple pr . the clinical pr was 31.6% , 40% and 32% respectively in groups 1 , 2 and 3 and the differences between groups were not statistically significant . however , the miscarriage rate was significantly lower in group 2 ( 2.6% ) than in group 1 ( 20% ) but was not significantly lower than in group 3 ( 9.6% ) . the study concluded that the in luteal phase adding 2 , 4 or 6 mg of oral e2 to p creates no statistical difference in terms of pregnancy rates . however , a significantly higher miscarriage rate was found when 2 mg e2 was used . therefore , in the luteal phase support , 4 mg of oral estradiol in addition to progesterone can be considered to reduce the miscarriage rate . more research is still required on identification of at risk group , the optimal gestational age at initiation , mode of administration , dose of progesterone and long - term safety . it also changes prolactin , gonadotropin - releasing hormone , and sex steroid serum levels . it may also have a direct effect on oocytes , because it is known that specific binding sites for thyroxin are found on mouse and human oocytes . there is also an association between thyroid dysfunction in women and morbidity and outcome in pregnancy . in males it has been found that when euthyroidism is restored , both abnormalities improve or normalize . in women , hypothyroidism are the main thyroid diseases that have an adverse effect on female reproduction and cause menstrual disturbances - mainly hypomenorrhea and polymenorrhea in hyperthyroidism , and oligomenorrhea in hypothyroidism . adequate levels of circulating thyroid hormones are of primary importance for normal reproductive function.21 controlled ovarian hyperstimulation leads to increases in estradiol , which in turn may have an adverse effect on thyroid hormones and tsh . ovarian hyperstimulation may become severe when autoimmune thyroid disease is present , depending on preexisting thyroid abnormalities . isolated hypothyroxinemia has been described in approximately 2% of pregnancies , without serum tsh elevation and in the absence of thyroid auto antibodies . there is an association of increased rates of spontaneous abortion , premature delivery and/or low birth weight , fetal distress in labor , and perhaps gestation - induced hypertension and placental abruption in overt hypothyroidism . all antithyroid drugs cross the placenta and may potentially affect fetal thyroid function.22 thyroid disorders are common in women during pregnancy . if left untreated , both hypothyroidism and hyperthyroidism are associated with adverse effects on pregnancy and fetal outcomes . it is important to correctly identify these disorders and treat them appropriately to prevent pregnancy - related complications . indicated treatment is levothyroxine for hypothyroidism , and thioamides are the treatment of choice for hyperthyroidism ; thyroidectomy may be indicated in select cases.2324 cochrane review of three rcts involving 314 women showed in one trial of 115 women , levothyroxine therapy to treat pregnant euthyroid women with thyroid peroxidase antibodies was not shown to reduce pre - eclampsia but did significantly reduce preterm birth by 72% . one trial of 30 hypothyroid women compared levothyroxine doses , but only reported biochemical outcomes . a trial of 169 women compared the trace element selenomethionine ( selenium ) with placebo and no significant differences were seen for either pre - eclampsia or preterm birth . none of the three trials reported on childhood neurodevelopmental delay.25 progesterone is largely produced by the corpus luteum until about 10 weeks of gestation.1 a study in ovarian failure and assisted reproduction it was shown that one hundred mg of p were probably a supraphysiological dose to support pregnancy 6 to 8 weeks after conception . the fetoplacental unit was competent from 10 to 12 weeks gestation.2 when the pregnancy reaches term gestation , progesterone levels range from 100 - 200 ng / ml and the placenta produces about 250 mg / day . almost all of the progesterone produced by the placenta enters the placenta , contrast to oestrogen . progesterone production is independent of he precursor available , fetal status including the wellbeing . in early pregnancy , the maternal levels of 17 a - hydroxyprogesterone rise , marking the activity of the corpus luteum . by the tenth week of gestation , this compound has returned to baseline levels , indicating that the placenta has little 17a hydroxylase activity . however , beginning about the 32 week there is a second , more gradual rise in 17a - hydroxyprogesterone due to placental utilization of fetal precursors . progesterone is also important in suppressing the maternal immunologic response to fetal antigens , thereby preventing maternal rejection of the trophoblast . and , of course , progesterone prepares and maintains the endometrium to allow implantation earlier . studies have shown that the human corpus luteum makes significant amounts of estradiol , but it is progesterone and not oestrogen that is required for successful implantation.3 the placenta does not have all the necessary enzymes to make oestrogens from cholesterol , or even progesterone . human trophoblast lack 17-hydroxylase and therefore can not convert c21-steroids to c19-steroids , the immediate precursors of oestrogen . to bypass this deficit , dehydroisoandrosterone sulfate ( dha ) from the fetal adrenal is converted to estradiol-17 by trophoblasts . in its key location as a way station between mother and fetus , placenta can use precursors from either mother or fetus to circumvent its own deficiencies in enzyme activities . hormones act as catalysts for chemical changes at the cellular level that are necessary for growth , development and energy . fetus lacks 3 b hydroxysteroid dehydrogenase - hence unable to produce progesterone - borrows from placenta . in return , fetus give placenta what it lacks ( 19 carbon compounds)-precursor of oestrogen . protein hormones are : human placental lactogen ( hpl ) , human chorionic gonadotropin ( hcg ) , adrenocorticotropic ( acth ) , growth hormone variant ( hgh - v ) , parathyroid hormone - related protein ( pth - rp ) , calcitonin , relaxin , inhibins activins , atrial natriuretic peptide , hypothalamic - like releasing and inhibiting hormones , thyrotropin releasing hormone ( trh ) , gonadotropin releasing hormone ( gnrh ) , corticotropin - releasing hormone ( crh ) , somatostatin , growth hormone - releasing hormone ( ghrh ) , alpha fetoprotein , prolactin , relaxin and other decidual proteins . due to the comprehensiveness of the choices to describe hormones , clinical importance of hcg relevant for therapy this glycoprotein is critical since it rescues the corpus luteum from involution , and this maintains progesterone secretion by the ovarian granulosa cells . its usefulness as a diagnostic marker of pregnancy stems from the fact that it may be one of the earliest secreted products of the conceptus . in pregnancy , placental production of hcg is at its peak between the eighth to the tenth week of gestation , and tends to plateau at a lower level for the remainder of pregnancy . the only definitely known function for hcg is support of the corpus luteum ( cl ) , taking over for lh on about the eighth day after ovulation , 1 day after implantation , when b - hcg first can be detected in maternal blood . at 8 cell stage implantation occurs 5 - 6 days after ovulation and hcg must appear by 10 days of ovulation ( 4 days after ovulation ) to rescue corpus luteum . hence , blastocyst should implant in a narrow window of time . the hcg stimulation of cl has a daily secretion of 25 mg of p and 0.5 mg of e2 . hcg gene expression is present in both cytotrophoblast and syncytiotrophoblast , but it is synthesized mainly in the syncytiotrophoblast . the maternal circulating hcg concentration is approximately 100 iu / l at the time of the expected but missed menses . a maximal level of about 100,000 iu / l in the maternal circulation is reached at 8 - 10 weeks of gestation . there are two clinical conditions in which blood hcg titers are especially helpful : trophoblastic disease and ectopic pregnancies . trophoblastic disease is distinguished by very high b - hcg levels ( 3 - 100 times higher than normal pregnancy ) . ectopic production of a - and b - hcg by non - trophoblastic tumours is rare , but does occur . the human placental lactogen ( hpl ) is secreted primarily into the maternal circulation , most of its functions occur at sites of action in maternal tissues . human placental lactogen is thought to be responsible for the marked rise in maternal plasma insulin - like growth factor-1 ( igf-1 ) concentrations as the pregnancy approaches term . it is associated with insulin resistance , enhances insulin secretion which stimulates lipolysis , increases circulating free fatty acids , and inhibits gluconeogenesis ; in effect , it antagonizes insulin action , induces glucose intolerance , as well as lipolysis and proteolysis in the maternal system . hence the role of universal screening for abnormal blood sugar in the beginning of the third trimester is emphasized in clinical practice . in the fetus calcium concentrations , are regulated by the movement of calcium , across the placenta , from the maternal compartment . in order to maintain fetal bone growth , the maternal compartment undergoes adjustments that provide a net transfer of sufficient calcium to the fetus . maternal compartment changes that permit calcium accumulation include increases in maternal dietary intake , increases in maternal d3 levels , and increases in parathyroid hormone levels . progesterone supplement in pregnancy : an immunologic therapy there are several studies to understand the maintenance of pregnancy by progesterone . progesterone has been shown to increase the cytokines produced by th2 cells which predominate over those produced by th1 cells , resulting in the maintenance of pregnancy . the th2-derived cytokines , il-4 and il-6 , induce the release of hcg from trophoblasts and the hcg stimulates progesterone production from corpus luteum in pregnancy . progesterone has been shown to stimulate the secretion of th2 and reduces the secretion of th1 cytokines . this role in controlling the immune and endocrine system which promotes the function of the trophoblasts at the implantation site seems interesting.4 use of progestogen in threatened abortion is controversial.5 progesterone for recurrent miscarriage progestogen has been used for several years even before there was knowledge of the immunomodulatory properties of progesterone . since that time , studies of differing quality have been carried out to prove the benefits of progestogen supplementation in affected women . a study on 146 women who presented with mild or moderate vaginal bleeding during the first trimester of pregnancy was randomized to receive oral dydrogesterone ( 10 mg b.i.d . ) ( n=86 ) or no treatment ( n=60 ) . the incidence of miscarriage was significantly lower in the dydrogesterone group than in the untreated group ( 17.5% vs. 25% ; p<0.05).6 the majority of cited clinical trials revealed a trend to improved pregnancies and increased live birth rates in the progestogen treatment group , but unfortunately , many studies had poor designs and methodical weaknesses.7 several studies have shown that supportive care in early pregnancy is associated with a significant beneficial effect on pregnancy outcome . women with otherwise unexplained recurrent pregnancy loss should be counselled regarding the potential for successful pregnancy without any treatment except supportive therapy such as folic acid or vitamin supplementation.78 the route of progestogen administration are in various formulations , but it is generally recommend the exclusive use of progestogen without any ( anti- ) androgenic or ( anti- ) oestrogenic effect . progestogen supplementation is available as vaginal suppositories ( 0.4 g / day , preferably in the evening because natural progesterone can cause tiredness ) , intramuscular injection ( 250 mg hydroxyprogesterone weekly ) or oral intake ( e.g. 10 mg dydrogesterone , the stereo - isomer of natural progesterone.9 progesterone supplementation following assisted reproductive technology the use of the progesterone supplementation in art cycles has better clarity.10 the duration of progesterone supplementation following reproductive technology ( art ) has been studied in a retrospective cohort study . one group had progesterone supplementation through the first trimester of pregnancy ( first trimester protocol ) till 12 weeks and the second group had the progesterone discontinued after a positive beta hcg test 2 weeks after retrieval ( luteal protocol ) . a similar rate of clinical pregnancies occurred at 7 weeks ( 81.8% luteal protocol vs. 85.8% first trimester protocol ) and for live birth rates ( 76.8% luteal protocol vs. 75.0% first trimester protocol ) . there was a trend toward a higher rate of pregnancy loss after 7 weeks in the first trimester protocol group occurred ( 15.5% vs. 4.4% ) , indicating that first trimester progesterone supplementation may support early pregnancy through 7 weeks by delaying miscarriage but does not improve live birth rates . there are randomized trials supporting the routine use of luteal support in art cycles using gnrh agonists or antagonists . fifty - nine studies were included in a review to evaluate the luteal phase support with hcg compared to placebo or no treatment , in terms of increased ongoing pregnancy rates . luteal phase support with hcg or progesterone after assisted reproduction results in an increased pregnancy rate . hcg does not provide better results than progesterone , and is associated with a greater risk of ohss when used with gnrha . the optimal route of progesterone administration has not yet been established.11 a review showed a significant effect in favour of progesterone for luteal phase support , favouring synthetic progesterone over micronized progesterone.12 prevention of recurrent preterm delivery by 17 alpha - hydroxyprogesterone caproate preterm delivery should be anticipated and prevented to decrease perinatal morbidity and mortality . those women who have had a spontaneous preterm delivery earlier are at greatly increased risk for preterm delivery in subsequent pregnancies . the results of several small trials have suggested that 17 alpha - hydroxyprogesterone caproate ( 17p ) may reduce the risk of preterm delivery . a double - blind , placebo - controlled trial involving pregnant women with a documented history of spontaneous preterm delivery was done.13 a total of 19 clinical centers were taken for the study and pregnant women at 16 to 20 weeks of gestation were included and were randomly assigned by a central data center , in a 2:1 ratio , to receive either weekly injections of 250 mg of 17p or weekly injections of an inert oil placebo ; injections were continued until delivery or to 36 weeks of gestation . treatment with 17p significantly reduced the risk of delivery at less than 37 weeks of gestation which was 36.3 percent in the progesterone group vs. 54.9 percent in the placebo group ; relative risk , delivery at less than 35 weeks of gestation was 20.6 percent vs. 30.7 percent ; and delivery at less than 32 weeks of gestation was 11.4 percent vs. 19.6 percent . the incidence of necrotizing enterocolitis , intraventricular hemorrhage in infants of women treated with 17p had significantly lower rates of and need for supplemental oxygen . hence , the study concluded that weekly injections of 17p resulted in a substantial reduction in the rate of recurrent preterm delivery among women who were at particularly high risk for preterm delivery and reduced the likelihood of several complications in their infants . one double blind randomized placebo controlled trials reported lower preterm birth rate with the use of either intramuscular 17 alpha - hydroxyprogesterone caproate ( 17p ) or intravaginal micronized progesterone suppositories in women at risk for preterm delivery.14 the half - life of 17p was estimated to be approximately 7.8 days . the route of administration plays an important role in the drug 's safety and efficacy profile . oral progesterone has not been used for prevention of preterm labor because of its first - pass hepatic metabolism , and there is a lack of data on efficacy , high side - effect profile , and because of extreme variability in plasma concentrations . vaginal administration of progesterone avoids first - pass hepatic metabolism and is associated with rapid absorption , high bioavailability , and local endometrial effects.15 vaginal route offers no local pain and few side effects , it is associated with variable blood concentrations.16 to study the efficacy of progesterone for maintenance tocolytic therapy after threatened preterm labor was done in a randomized controlled trial.17 the study was on 70 women who presented with symptoms of threatened preterm labor , who after arrest of uterine activity were then randomized to progesterone therapy or no treatment and the purpose of this study was to determine whether supplementation of vaginal progesterone after inhibition of preterm labor is associated with an increased latency period and a decreased recurrent of preterm labor . treatment group received progesterone suppository ( 400 mg ) daily until delivery and control group received no treatment . the study concluded that the use of vaginal progesterone suppository after successful parenteral tocolysis associated with a longer latency preceding delivery but failed to reduce the incidence of readmission for preterm labor . dydrogesterone supplementation in women with threatened had preterm delivery the impact on cytokine profile , hormone profile , and progesterone - induced blocking factor.18 a study on eighty - three women with symptoms of threatened preterm birth were either randomized to study groups receiving tocolytic treatment combined with intravaginal micronized natural progesterone ( 200 mg daily ) or to a control group receiving only tocolysis . micronized natural progesterone treatment resulted in a prolonged latency period of 32.117.8 versus 21.216.3 days in the control group and heavier birth weights of 2,982.8697.8 g versus 2,585.3746.6 g.19 estradiol supplementation during the luteal phase of in vitro fertilization cycles a prospective randomized study was done to find the optimal dosage of estradiol ( e2 ) for luteal phase support through the addition of different doses of e2 to progesterone ( p ) luteal phase support in patients undergoing long gnrh agonist in vitro fertilization ( ivf ) treatments.20 two hundred and eighty - five women undergoing ivf treatment with a long gnrh agonist protocol were prospectively randomized into three groups . group 1 ( n=95 ) received p and 2 mg e2 , group 2 ( n=95 ) received p and 4 mg e2 and group 3 ( n=95 ) received p and 6 mg e2 as luteal phase support . the secondary variables of interest were the implantation rate ( ir ) , miscarriage rate and multiple pr . the clinical pr was 31.6% , 40% and 32% respectively in groups 1 , 2 and 3 and the differences between groups were not statistically significant . however , the miscarriage rate was significantly lower in group 2 ( 2.6% ) than in group 1 ( 20% ) but was not significantly lower than in group 3 ( 9.6% ) . the study concluded that the in luteal phase adding 2 , 4 or 6 mg of oral e2 to p creates no statistical difference in terms of pregnancy rates . however , a significantly higher miscarriage rate was found when 2 mg e2 was used . therefore , in the luteal phase support , 4 mg of oral estradiol in addition to progesterone can be considered to reduce the miscarriage rate . more research is still required on identification of at risk group , the optimal gestational age at initiation , mode of administration , dose of progesterone and long - term safety . it also changes prolactin , gonadotropin - releasing hormone , and sex steroid serum levels . it may also have a direct effect on oocytes , because it is known that specific binding sites for thyroxin are found on mouse and human oocytes . there is also an association between thyroid dysfunction in women and morbidity and outcome in pregnancy . in males it has been found that when euthyroidism is restored , both abnormalities improve or normalize . in women , hypothyroidism are the main thyroid diseases that have an adverse effect on female reproduction and cause menstrual disturbances - mainly hypomenorrhea and polymenorrhea in hyperthyroidism , and oligomenorrhea in hypothyroidism . adequate levels of circulating thyroid hormones are of primary importance for normal reproductive function.21 controlled ovarian hyperstimulation leads to increases in estradiol , which in turn may have an adverse effect on thyroid hormones and tsh . ovarian hyperstimulation may become severe when autoimmune thyroid disease is present , depending on preexisting thyroid abnormalities . isolated hypothyroxinemia has been described in approximately 2% of pregnancies , without serum tsh elevation and in the absence of thyroid auto antibodies . there is an association of increased rates of spontaneous abortion , premature delivery and/or low birth weight , fetal distress in labor , and perhaps gestation - induced hypertension and placental abruption in overt hypothyroidism . all antithyroid drugs cross the placenta and may potentially affect fetal thyroid function.22 thyroid disorders are common in women during pregnancy . if left untreated , both hypothyroidism and hyperthyroidism are associated with adverse effects on pregnancy and fetal outcomes . it is important to correctly identify these disorders and treat them appropriately to prevent pregnancy - related complications . indicated treatment is levothyroxine for hypothyroidism , and thioamides are the treatment of choice for hyperthyroidism ; thyroidectomy may be indicated in select cases.2324 cochrane review of three rcts involving 314 women showed in one trial of 115 women , levothyroxine therapy to treat pregnant euthyroid women with thyroid peroxidase antibodies was not shown to reduce pre - eclampsia but did significantly reduce preterm birth by 72% . one trial of 30 hypothyroid women compared levothyroxine doses , but only reported biochemical outcomes . a trial of 169 women compared the trace element selenomethionine ( selenium ) with placebo and no significant differences were seen for either pre - eclampsia or preterm birth . none of the three trials reported on childhood neurodevelopmental delay.25 sex steroids are the best known examples of hormones and hence the review is concentrating on these . progesterone is indispensable in creating a suitable endometrial environment for implantation , and also for the maintenance of pregnancy . progestogen supplementation should be used preferentially within the context of a clinical trial or , in selected cases , in a kind of pragmatic approach , because progestogen treatment seems to be without remarkable adverse effects and as no better treatment exists to date .

the endocrinology of human pregnancy involves endocrine and metabolic changes that result from physiological alterations at the boundary between mother and fetus . progesterone and oestrogen have a great role along with other hormones . the controversies of use of progestogen and others are discussed in this chapter . progesterone has been shown to stimulate the secretion of th2 and reduces the secretion of th1 cytokines which maintains pregnancy . supportive care in early pregnancy is associated with a significant beneficial effect on pregnancy outcome . prophylactic hormonal supplementation can be recommended for all assisted reproduction techniques cycles . preterm labor can be prevented by the use of progestogen . the route of administration plays an important role in the drug 's safety and efficacy profile in different trimesters of pregnancy . thyroid disorders have a great impact on pregnancy outcome and needs to be monitored and treated accordingly . method of locating review : pubmed , scopus

INTRODUCTION Steroid hormone production and uses Human chorionic gonadotropin CONCLUSION

the role in preventing abortions , recurrent pregnancy loss and preterm labor has been the aim of the review . progesterone has been shown to increase the cytokines produced by th2 cells which predominate over those produced by th1 cells , resulting in the maintenance of pregnancy . progesterone has been shown to stimulate the secretion of th2 and reduces the secretion of th1 cytokines . this role in controlling the immune and endocrine system which promotes the function of the trophoblasts at the implantation site seems interesting.4 use of progestogen in threatened abortion is controversial.5 progesterone for recurrent miscarriage progestogen has been used for several years even before there was knowledge of the immunomodulatory properties of progesterone . the incidence of miscarriage was significantly lower in the dydrogesterone group than in the untreated group ( 17.5% vs. 25% ; p<0.05).6 the majority of cited clinical trials revealed a trend to improved pregnancies and increased live birth rates in the progestogen treatment group , but unfortunately , many studies had poor designs and methodical weaknesses.7 several studies have shown that supportive care in early pregnancy is associated with a significant beneficial effect on pregnancy outcome . women with otherwise unexplained recurrent pregnancy loss should be counselled regarding the potential for successful pregnancy without any treatment except supportive therapy such as folic acid or vitamin supplementation.78 the route of progestogen administration are in various formulations , but it is generally recommend the exclusive use of progestogen without any ( anti- ) androgenic or ( anti- ) oestrogenic effect . there was a trend toward a higher rate of pregnancy loss after 7 weeks in the first trimester protocol group occurred ( 15.5% vs. 4.4% ) , indicating that first trimester progesterone supplementation may support early pregnancy through 7 weeks by delaying miscarriage but does not improve live birth rates . one double blind randomized placebo controlled trials reported lower preterm birth rate with the use of either intramuscular 17 alpha - hydroxyprogesterone caproate ( 17p ) or intravaginal micronized progesterone suppositories in women at risk for preterm delivery.14 the half - life of 17p was estimated to be approximately 7.8 days . the route of administration plays an important role in the drug 's safety and efficacy profile . the study concluded that the use of vaginal progesterone suppository after successful parenteral tocolysis associated with a longer latency preceding delivery but failed to reduce the incidence of readmission for preterm labor . in its key location as a way station between mother and fetus , placenta can use precursors from either mother or fetus to circumvent its own deficiencies in enzyme activities . progesterone has been shown to increase the cytokines produced by th2 cells which predominate over those produced by th1 cells , resulting in the maintenance of pregnancy . progesterone has been shown to stimulate the secretion of th2 and reduces the secretion of th1 cytokines . this role in controlling the immune and endocrine system which promotes the function of the trophoblasts at the implantation site seems interesting.4 use of progestogen in threatened abortion is controversial.5 progesterone for recurrent miscarriage progestogen has been used for several years even before there was knowledge of the immunomodulatory properties of progesterone . the incidence of miscarriage was significantly lower in the dydrogesterone group than in the untreated group ( 17.5% vs. 25% ; p<0.05).6 the majority of cited clinical trials revealed a trend to improved pregnancies and increased live birth rates in the progestogen treatment group , but unfortunately , many studies had poor designs and methodical weaknesses.7 several studies have shown that supportive care in early pregnancy is associated with a significant beneficial effect on pregnancy outcome . women with otherwise unexplained recurrent pregnancy loss should be counselled regarding the potential for successful pregnancy without any treatment except supportive therapy such as folic acid or vitamin supplementation.78 the route of progestogen administration are in various formulations , but it is generally recommend the exclusive use of progestogen without any ( anti- ) androgenic or ( anti- ) oestrogenic effect . there was a trend toward a higher rate of pregnancy loss after 7 weeks in the first trimester protocol group occurred ( 15.5% vs. 4.4% ) , indicating that first trimester progesterone supplementation may support early pregnancy through 7 weeks by delaying miscarriage but does not improve live birth rates . one double blind randomized placebo controlled trials reported lower preterm birth rate with the use of either intramuscular 17 alpha - hydroxyprogesterone caproate ( 17p ) or intravaginal micronized progesterone suppositories in women at risk for preterm delivery.14 the half - life of 17p was estimated to be approximately 7.8 days . the route of administration plays an important role in the drug 's safety and efficacy profile . the study concluded that the use of vaginal progesterone suppository after successful parenteral tocolysis associated with a longer latency preceding delivery but failed to reduce the incidence of readmission for preterm labor .

cachexia , an unintentional loss of lean body weight despite adequate nutrition , is a fatal complication of many diseases ( cancer , congestive heart failure , diabetes , kidney failure , chronic obstructive pulmonary disease , rheumatoid arthritis , and hiv / aids ) , while the primary causal molecular mechanisms still remain unknown [ 1 , 2 ] . actually , it seems obvious that all these complications have something in common despite distinct etiology . among several hypotheses , the insufficient anabolic action of insulin in response to rise in some cytokines during systemic inflammation attracted our attention . the loss of muscle tissue observed in elevated levels of proinflammatory cytokines seems to be linked to accelerated proteolysis rather than impaired protein synthesis . moreover , guttridge et al . demonstrated that inhibition of myogenesis is nf-b dependent , as cytokine activation is responsible for the reduction of myod protein that controls muscle development . , myod binding to myosin heavy chain iib promoter region is necessary for myosin expression in fast twitch muscles . interestingly , igf-1 could not stop the effect of proinflammatory cytokines in myotube atrophy , even though it was shown to repress atrogin1 gene . nowadays , efforts to fight cachexia are based on targeting genes prior to their effects evoked in target organs . it is believed that accelerated loss of skeletal muscle fibers and proteins which occur in muscle atrophy , muscle cachexia , and sarcopenia are driven by intrinsic mechanisms of autophagy , apoptosis , and decreased satellite cell activation . furthermore , the imbalance in regulation of skeletal muscle protein accretion leads to excessive activity of proteasome , cathepsins , calpains , and/or caspase proteolytic systems . a great deal of papers referring to muscle cachexia points to erroneous activity of signaling pathways triggered by certain cytokines , such as il-6 , il-1 , tnf- , ifn , and myostatin [ 1317 ] , respectively . tnf- acting through tnfr1 is known to trigger two functionally opposite and sequential signals : ( i ) first to support cell viability through nf-b - dependent gene regulation ; ( ii ) second to initiate intrinsic apoptosis with procaspase 8 activation . interestingly , nf-b transcription factor also plays a critical role in muscle cachexia [ 1921 ] . thus , deciphering the molecular mechanism of muscle cachexia would provide theoretical background for therapeutic use of some of the metabolic inhibitors . in this study murine c2c12 myotubes were subjected to short- and long term incubations with tnf- , ifn , ifn , and/or insulin as potent functional antagonists . the set - up of experiments was designed to verify the hypothesis that individually some cytokines could affect certain genes and proteins associated with muscle wasting , whereas together they add or synergize in their respective actions . it was assumed that they compete for the common transduction target ( stat-1 ) that blocks internal signal essential for accelerated protein loss ( nf-b ) . in this paper the results of both short ( minutes ) and long term ( hours ) studies performed on c2c12 myoblasts and myotubes are presented . we demonstrated that nf-b and jak / stat signaling pathways are intersected and that they control muscle growth and decay . ifn reduced the level of stat-1 protein linked to tradd protein to release nf-b from stat-1-dependent inhibition . in differentiating c2c12 myotubes , tnf- administration augmented cell growth , whereas it inhibited myhc iia protein expression in differentiated myotubes . to our surprise , tnf--induced effect was associated with both activation of myhc iia , atrogin1 ( also known as mafbx ) , and murf1 genes , but proteolysis took over protein accretion . media ( dulbecco 's modified eagles medium ( dmem ) with glutamax ) , pbs ( including ca and mg ) , antibiotics , and heat inactivated sera ( fetal bovine serum fbs and horse serum hs ) were purchased from gibco life technologies ( grand island , ny , usa ) . mouse tumor necrosis factor alpha ( tnf- ) , interferon alpha ( ifn ) , interferon gamma ( ifn ) , and insulin porcine ( sigma - aldrich chemical co. , st . louis , mo , usa ) were dissolved according to manufacturer 's recommendations and kept frozen at 20c . metabolic inhibitors : curcumin ( nf-b and proteasome inhibitor ) , ly294002 and pd98059 ( pi3-k and mek inhibitor , resp . , sigma - aldrich chemical co. , st . louis , mo , usa ) , and tyrphostin ( ag490 , jak inhibitor , merck , darmstadt , germany ) were dissolved in dmso and depending on the type were kept frozen at 20c or refrigerated at 48c . all other reagents were cell culture tested , of high purity , and unless otherwise stated they were purchased from sigma - aldrich chemical co. ( st . plastics were from becton dickinson ( bd biosciences , franklin lakes , nj , usa ) and tubes for deep freezing were from nunclon ( nunc , roskilde , denmark ) , while syringe filters were purchased from corning - costar inc . the mouse c2c12 cell line was obtained from the european collection of animal cell cultures ( ecaac ) . cells were initially suspended in growth media ( gm ) containing dmem with glutamax supplemented with 10% ( v / v ) fetal bovine serum ( fbs ) , pen : strep ( penicillin : streptomycin solution , 50 iu / ml/50 g / ml ) , gentamicin sulfate 20 g / ml , fungizone - amphotericin b 1 g / ml , and plated onto plastic noncoated culture flasks or petri dishes . they were cultured at 37c in a humidified 5% co2 and 95% air in incubator . after reaching 7080% confluence , myoblasts were subcultured by trypsynization and the same volume of cell suspension was seeded onto 100 mm petri dishes , 96-flatwell plates or multiwell 4 chamber culture slides ( becton dickinson , bd biosciences , franklin lakes , nj , usa ) depending on the experimental protocol . for differentiating and differentiation states when the myoblasts reached 80% confluence , growth media were switched to differentiation media ( dm ) containing dmem with glutamax supplemented with 2% ( v / v ) horse serum ( hs ) and the same antibiotic : antimycotic mixture . during acute and chronic study of myogenic differentiation , dm was replaced by freshly prepared media containing 2% bsa ( w / v ) with or without experimental factors . in the case of mitogenicity study , gm in nonconfluent cells was directly replaced by 2% bsa ( w / v)/dmem with or without experimental factors . when the experimental factors were dissolved in dmso , the equivalent volume of vehicle ( 0.1% v / v ) was added to the control cells . dmso - dissolved reagents were added exactly 30 min prior to the application of water - soluble reagents . preliminary experiments were carried out with increasing concentrations of cytokines for different time points in order to choose the best time / concentration combination to adopt in our short- and long term studies . differences in phosphorylation status and activities of particular proteins ( nf-b , stat-1 ) being fast and transient phenomena were carried out as short term studies . viability , mitogenicity , or changes in the protein expression were investigated in the long term study . there were dose- and time - dependent cell responses ( supplementary data 1 , see supplementary materials available online at http://dx.doi.org/10.1155/2013/171437 ) suggesting that the best concentration setting in our experimental model ( determined by the complete absence of cellular toxicity in mtt assay ) was obtained using 10 ng / ml of tnf- , ifn , ifn , and 10 nm of insulin . cells were removed from the culture plates using trypsin ( harvesting ) , centrifuged in gm at 1000 rpm for 5 min , media were aspirated off , and cell pellets were resuspended in gm . for the differentiating state , after 24 h in gm , cells were washed with pbs and then incubated in dm for 3 to 5 days . media were changed every day . for differentiation state , the cells were allowed to incubate in dm for 6 days prior to treatment with cytokines or inhibitors carried out for another 2 days ( 8 days altogether ) . myotubes were harvested on day 8 of differentiation ( i.e. , 6 days without treatment + 2 days with treatment ) . floating dead cells were removed during media change or washing with pbs and were not included in these experiments . cell viability was based on the ability of cells grown on 96-well plates to convert soluble mtt [ 3-(4,5-dimethylthiazol-2-yl)-2 - 5-diphenyltetrazolium bromide ] into an insoluble purple formazan reaction product with minor modifications to protocol described by jacobson et al . . briefly , cells were uniformly seeded in 96-well flat bottomed plates and grown in gm for 24 h. confluent cultures were washed with pbs and then exposed to dm including ( or not ) experimental factor(s ) for 5 successive days . media were changed every day and relative viability ( percent of control ) was evaluated at each day ( 1 , 2 , 3 , 4 , 5 ) . media were removed , cells were washed three times with pbs , and were further incubated with mtt for 1 h at 37c in a humidified 5% co2 and 95% air in incubator . next , mtt solution was removed and water insoluble formazan was immediately dissolved in dmso . cell mitogenicity was determined by crystal violet ( cv ) assay on identical 96-well flat bottomed multiwell plates . cells were uniformly seeded and grown in gm for 24 h. mitogenicity was measured on day 3 of culture . initially , cells were kept in gm for 24 h , followed by 24 h incubation with the experimental factor(s ) dissolved in serum - free 2% bsa / dmem , and finally recovered in gm for another 24 h. cells were exposed ( or not ) to experimental factors during 24 h. upon completing the experiment , cells were washed with pbs and fixed with two - step bath in ice - cold methanol ( 70% followed by 100% , v / v , 20 min , 4c ) . cells were immersed in 0.2% , w / v crystal violet solution in dd.h2o with ethanol 2% ( v / v ) for 10 min . subsequently , they were gently washed with dd.h2o , air dried , and incubated with sds solution ( 1% , w / v in dd.h2o ) . the absorbances for mtt and cv were measured at 490 and 570 nm , respectively , with elisa reader type infinite 200 ( tecan , austria ) . relative percentages ( versus nontreated control ) of viable or proliferating cells were measured by mtt conversion into purple formazan and quantity of cv bound to cellular dna , respectively . for whole - cell lysates the cells were cultured with or without experimental factors indicated in the legend of figure 7(a ) , harvested , washed , and lysed with ripa lysis buffer ( 1x pbs , 10 ml / l igepal ca-630 , 5 g / l sodium deoxycholate , 1 g / l sds ) supplemented with 0.4 mm pmsf ; 10 g / ml of aprotinin and 10 g / ml of sodium orthovanadate were added . to lyse the cell pellets , cells were broken up by repetitive triturating with the syringe with attached needle ( 21 g , 0.8 mm diameter ) . cell suspension was then left on ice ( 4c ) for 30 min and centrifuged for another 5 min ( 4c , 10 000 g ) . next , viscous solution was divided into smaller volumes and transferred to fresh eppendorf tubes and stored at 80c until used . for protein quantification in the whole - cell lysates , a protein - dye - binding method of bradford with commercial reagent was used ( bio - rad laboratories , hercules , ca , usa ) . to separate cytoplasmic and nuclear fractions , cells were washed , and after centrifugation cell pellets were resuspended in 400 l of ice - cold buffer ( 10 mm hepes ph 7.9 ; 10 mm kcl ; 0.1 mm edta ; 0.1 mm egta ; 1 mm dtt ; 0.5 mm pmsf ) and incubated on ice for 15 min . then 25 l of a 10% solution of igepal ca-630 was added . after centrifugation , supernatants containing cytoplasm were transferred to fresh tubes and were stored at 80c . nuclear pellets were resuspended in 200 l ripa buffer ( 1x pbs ; 1% igepal ca-630 ; 0.5% sodium deoxycholate ; 0.1% sds ; aprotinin ( available as a liquid from sigma - aldrich chemical co. ) ; 30 l added to 1 ml of buffer ; 1 mm sodium orthovanadate ) and were passed through a 21-gauge needle . pmsf ( 0.4 mm ) was added and cells were incubated 30 min on ice . cell lysates ( equal protein loads of 50 g ) were subjected to sds - page ( 1012.5% of gel , depending on the mw of protein ) at 150 v , next they were transferred at 100 v for 2 h to polyvinylidene difluoride ( pvdf ) membrane and immunoblotted with antibodies against phosphorylated and/or nonphosphorylated forms of proteins : actin , nf-b ( p65 ) , ib , stat1- , p(tyr701)-stat1- , myod , and myogenin ( santa cruz biotechnology , santa cruz , ca , usa ) or myhc iia ( sigma aldrich chemical co. , st . louis , mo , usa ) . working antibody concentrations ( from 1 : 200 to 1 : 1000 ) varied depending on the protein detected and were applied according to the manufacturer 's recommendations . after detection with antibodies which distinguished phosphorylated proteins , the same blot was reprobed with antibodies against the nonphosphorylated form of the same protein in order to verify that equal amounts of protein were always loaded and to identify the proteins . in order to reprobe a blot with a different antibody , the membrane was incubated in stripping buffer ( 100 mm -mercaptoethanol , 2% ( w / v ) sds , 62.3 mm tris , ph 6.7 ) for 30 min at 30c , and then reblocked . secondary polyclonal antibodies ( santa cruz biotechnology , santa cruz , ca , usa ) raised against respective species and conjugated to horseradish peroxidase were used for detection , followed by enhanced chemiluminescence assay ( amersham international , aylesbury , u.k . ) . after exposure and processing , the films were scanned and analyzed using kodak edas 290/kodak 1d 3.5 system . for immunoprecipitation at particular time points the cells were scraped from substratum in 0.5 ml ripa buffer , and after repetitive triturating with the 21 gauge needle , 0.4 mm pmsf was added and cells were incubated 30 min on ice . after centrifugation the protein concentration was determined by a protein - dye - binding method as previously described . cell lysates containing 900 g of protein were incubated overnight at 4c with 1.5 g rabbit polyclonal anti - stat-1 igg and for an additional 3 h were incubated with 30 l protein a / g bead slurry ( santa cruz , ca ) . beads were washed 4 times with ice - cold ripa buffer , boiled with sample buffer ( 2x laemmli buffer , sigma - aldrich chemical co. , st . after electrotransfer , the membranes were immunostained for tradd protein by standard western blot procedure . nf-b and stat-1 transcriptional activities were quantified with transam kits ( rixensart , belgium ) . these are sensitive , nonradioactive transcription factor elisa kits that facilitate the study of transcription factor activation in mammalian tissue and cell extracts . the active form of stat-1 contained in nuclear extracts was specifically bound to the immobilized oligonucleotide containing stat consensus binding site ( 5-ttcccggaa-3 ) . the primary antibody used to detect stat recognized only the alpha subunit of stat-1 , that is , accessible only when stat-1 was activated and bound to its target dna . similarly , transam nf-b kit contained a 96-well plate on which oligonucleotide containing the nf-b consensus site ( 5-gggactttcc-3 ) has been immobilized . the active form of nf-b contained in nuclear or whole - cell extract specifically binds to this oligonucleotide . the primary antibodies used to detect nf-b recognized an epitope on p65 or p50 , that is , accessible only when nf-b is activated and bound to its target dna . finally , an hrp - conjugated secondary antibody provided a sensitive colorimetric readout that was quantified by spectrophotometry ( 450 nm ) . for immunofluorescence , c2c12 myoblasts were propagated in multiwell 4 chamber culture slides and treated ( or untreated ) at appropriate times ( supplementary data 2 ) with experimental factors dissolved in 20 g / l bsa / dmem or 0.1% v / v dmso in 20 g / l bsa / dmem . after the experiment ended , the cells were fixed as follows : washed twice with pbs , fixed in 3.7% ( v / v ) formaldehyde for 15 minutes in room temperature , washed twice with pbs containing 10 g / l bsa / pbs , and incubated for 10 min in rt in triton x-100 solution ( 0.5% v / v in pbs ) . g / l bsa / pbs , and then incubated overnight at 4c with primary rabbit polyclonal anti - nf-b diluted 1 : 100 . g / l bsa / pbs and subsequently incubated for 1 hour in dark at rt with secondary chicken anti - rabbit antibody conjugated to alexa fluor 488 ( molecular probes inc . , eugene , or , usa ) diluted 1 : 500 . the cells were then washed three times with pbs and incubated with 7-aminoactinomycin d ( 7-aad ) water solution for 15 min at rt to visualize the cell nuclei . afterwards , the cells were washed five times with 10 g / l bsa / pbs , the chamber walls were removed , and coverslips were mounted on microscope slides using an antifade fluoromount ( sigma aldrich chemical co. , st . cells were visualized using confocal microscope fv-500 ( olympus optical co. , hamburg , germany ) . the fluorescence excitation was provided by 488 nm and 543 nm he - ne laser beams . fluorescence was measured using dichroic mirrors and filters for 505 , 525 nm , 560 , and 610 nm wavelengths . acquired data were stored in a series of 12 bit grey images separately and colored artificially by software . morphological changes and cell survival were monitored under an inverted phase - contrast microscope ( olympus ck40 , model : icd703wp ) . the formation of myotubes was monitored by obtaining photomicrographs using digital camera ( ccd color camera , hamburg , germany ) . total rna was extracted from myotubes with the total rna maxi kit ( a&a biotechnology , gdynia , poland ) , according to manufacturer 's protocol . rna was frozen at 76c before the performing the reverse transcription reaction ( rt - pcr ) . then , 500 g of total rna was purified on silica gel and reverse - transcribed with enhanced avian hs rt - pcr-100 kit ( sigma - aldrich , taufkirchen , germany ) . reaction mixture was based on anchored oligo(dt)23 ( 0.5 g/l in water for pcr ) . rt - pcr was carried out using mastercycler personal ( eppendorf , new york , ny , usa ) . first , the rna samples ( 200 ng / ml ) were incubated for 10 min at 70c . then water for pcr , 10xamv - rt buffer [ 500 mm tris - hcl , ph 8.3 , 400 mm kcl , 80 mm mgcl2 , 10 mm dtt , rnase inhibitor ( 20 u/l ) , and reverse transcriptase amv ( 20 u/l w 200 mm kh2po4 , ph 7.2 , 2 mm dtt , 0.2% ( v / v ) triton , 50% ( v / v ) glycerol ] were added at 4c . the samples were subjected to rt - pcr for 50 min at 48c . after completing the reaction the concentration of newly synthesized cdna was measured in the nanodrop 1000 ( nanodrop technologies , wilmington , nc , usa ) at = 230 nm . cdna was kept frozen at 76c until further analyses . to perform real time pcr reaction , cdna was combined with 25 m of each primer ( sense and antisense ) and sybr green ( lightcycler faststart dna master sybr green i and lightcycler control kit dna ; roche diagnostics , warsaw , poland ) . the qrt pcr measurements of individual cdnas were performed in triplicates using sybr green dye to measure duplex dna formation with the lightcycler ( roche diagnostics , warsaw , poland ) . the sequences of the primers sets used are shown in the attached table 1 ( genbank ) . the relative mrna levels of the target genes were determined using the relative standard curve . pcr conditions were as follows : denaturation for each cycle 95c ( 35 cycles , 10 sec for each cycle ) ; annealing both for myhc iia and atrogin1 60c ( 35 cycles , 010 sec for each cycle ) ; for murf1 58c ( 35 cycles , 010 sec for each cycle ) ; for 18s rrna 56c ( 35 cycles , 010 sec for each cycle ) ; and elongation 72c ( 35 cycles , 4 - 5 sec for each cycle ) . statistical analyses were performed using one - way analysis of variance ( anova ) followed by tukey 's , newman - keuls'a or benferroni multiple range test . if necessary , the selection of particular posthoc test ( newman - keuls , tukey , or benferroni ) was performed after the same critical difference for the first comparison was tested . regression analysis was carried out to draw appropriate dose - response or time - course curves . statistical differences from control cells were indicated by asterisks ( * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 ) , whereas statistical differences between the treatments and untreated control cells were ticked with different lower case letters ( bar charts ) . addition of tnf- ( 10 ng / ml ) to the medium stimulated viability ( by 88 2.82 to 140% 6.84 ) and mitogenicity ( by 30% 4.24 ) of differentiating c2c12 myoblasts ( figures 1(a ) and 1(c ) , p < 0.001 compared to initial control ) . ag490 ( 5 m ) used individually impaired cell viability at days 35 of differentiation ( figures 1(a ) and 1(b ) , p < 0.001 compared to initial control ) , but curcumin ( 1 m ) did not exert this effect . the effects of tnf- on cell viability and mitogenicity were abolished by curcumin ( nf-b inhibitor ) and ag490 ( jak inhibitor ) administration ( figures 1(b ) and 1(c ) ) . to verify the intracellular level of nf-b ( p65 ) in 3-day - old c2c12 myotubes , nuclear fractions were isolated and immunoblotted , whereas transcriptional activity was evaluated using transam method . nuclear expression of nf-b protein increased as early as 15 minutes after tnf- administration ( figure 1(f ) ) . there was a marked drop in cytoplasmic ib at the same time ( figure 1(f ) ) . as shown on figure 1(d ) , when c2c12 myotubes were exposed to tnf- ( 10 ng / ml ) , the transcriptional activity of nf-b was significantly elevated in 30th ( by 49% + 8.9 ) and 60th minute ( by 47% 7.15 ) of treatment ( p < 0.01 compared to control ) . furthermore , also after 24 hours of tnf- administration both nf-b expression and its activity were still significantly higher ( by 54% 2.89 , figures 1(e)-1(f ) , p < 0.01 compared to control ) . in contrast to short term treatment ( minutes ) , after 24 hours of tnf- administration the expression level of ib protein was increased in the cytoplasmic fraction ( figure 1(f ) ) . neither ifn nor ifn affected the activity of nf-b in c2c12 myotubes in the long term study ( figure 1(e ) , p > 0.05 compared to control ) . pretreatment with insulin ( 72 h , 10 nm ) prior to tnf- ( 10 ng / ml ) administration significantly increased tnf--dependent nf-b activity in 15th , 30th but 60th minute ( by 3-fold in the 15th and by 4.5-fold in the 30th minute of tnf- posttreatment , figure 1(h ) , p < 0.001 compared to control ) in 3-day old c2c12 myotubes . in contrast , insulin pretreatment neither changed nf-b activity ( figure 1(i ) , p > 0.05 ) nor expression when administered as pretreatment and cotreatment with ifn or ifn ( figure 1(g ) ) . preincubation with interferons ( ifn , ifn , 10 ng / ml ) sensitized cellular responses to tnf- administration in 3-day old c2c12 myotubes as evidenced by nf-b activity ( ifn pretreatment increased tnf- response by 2-fold in the 15th and by 3.5-fold in the 30th minute , while ifn by 45% 9.08 , 85% 9.18 , and 122% 15.16 at 15th , 30th , and 60th minute of treatment , respectively , figure 2(b ) , p < 0.001 compared to control ) . nf-b activity stimulated by tnf- and potentiated by ifn in 3-day - old c2c12 myotubes was additionally substantiated by nuclear nf-b setting observed in confocal microscopy ( supplementary data 2 ) . visualization confirmed that these reactions occurred , even though nf-b expression levels remained unchanged . when insulin was used together with interferons in one - day pretreatment , the effect of insulin on nf-b protein expression ( figure 2(c ) ) and activity in 3-day old c2c12 myotubes was also higher ( insulin + ifn pretreatment increased tnf- response by 52% 7.64 , 86% 22.63 , and 145% 11.33 at 15th , 30th , and 60th minute , respectively , ( figure 2(d ) , p < 0.01 ) , while insulin + ifn by 121% 25.19 and 225% 20.2 in the 15th and 30th minute of tnf- treatment ( figure 2(e ) , p < 0.001 compared to control ) . preincubation with cytokines ( tnf- or ifn , 10 ng / ml , 24 h ) increased stat-1-p - y - nuclear protein expression levels and activity in 3-day - old c2c12 myotubes by 39% 3.35 and 41% 2.78 , respectively , ( figure 2(g ) , p < 0.01 compared to control ) . insulin given together with ifn did not reduce elevated stat-1-p - y activity and nuclear protein expression level ( figures 2(g ) and 6(b ) , p > 0.05 compared to control ) . similarly to nf-b , myotubes responded to tnf- administration with considerable raise in nuclear protein expression levels and activity of stat-1-p - y ( increase by 28% 5.83 , 33% 1.98 , and 43.17 1.67 at 15th , 30th , and 60th minute of treatment , respectively , figure 3(a ) , p < 0.01 compared to control ) . insulin pretreatment ( 10 nm , 72 h ) hardly affected the cellular response to tnf- with respect to stat-1-p - y activity as it remained significantly higher than in control conditions during 15 ( 44% 6.24 , p < 0.001 ) , 30 ( 37.28% 4.54 , p < 0.001 ) , and 60 ( 17% 4.0 , p < 0.05 ) minutes of treatment ( figures 3(d ) and 3(f ) ) . when 3-day - old c2c12 myotubes were pretreated with ifn ( 10 ng / ml , 24 h ) followed by tnf- challenge , stat-1-p - y protein expression was similar to the conditions in which tnf- was given alone ( figure 3(c ) ) . additionally , ifn markedly ameliorated stat-1 activity by 52% 4.52 , 48.12 3.26 , and 23% 0.47 at 15th , 30th , and 60th minute of treatment , respectively , ( figure 3(d ) , p < 0.001 versus control ) . when ifn was replaced by ifn ( 10 ng / ml , 24 h ) , tnf--dependent rise in nuclear protein expression levels of stat-1-p - y was halted ( figure 3(c ) ) , whereas the activity of stat-1-p - y did not differ from tnf- administered alone ( increase by 28% 8.83 , 33% 1.98 , 43.17 1.67 at 15th , 30th , and 60th minute of tnf- treatment , resp . , additional preincubation with insulin ( 10 nm , 72 h ) neither affected the response to ifn nor to ifn pretreated cells in stat-1-p - y expression levels and transcriptional activities ( figures 3(e ) and 3(f ) ) . myhc iia protein expression level has been selected as a marker of protein gain / loss in c2c12 myotubes . its expression increased progressively during the consecutive days of differentiation ( figure 4(a ) ) . initially , the raise in myhc iia protein level was noted at the 3rd day of differentiation ( extensive fusion of myoblasts ) , and then it gradually increased to the 6th day with no additional elevation at day 7 ( figure 4(a ) ) . it was thought that at least 6 days of differentiation should be considered as the starting point for evaluation of changes in myhc iia protein expression . tnf- ( 10 ng / ml , 48 h ) was shown to inhibit the expression of myhc iia in c2c12 myotubes ( 8-day - old ) , while additional presence of ifn or ifn ( 10 ng / ml , 48 h ) amplified tnf--dependent drop in myhc iia protein expression ( figure 4(b ) ) . insulin pretreatment ( 10 nm , 24 h ) was apparently negligible to the tnf- effect induced in c2c12 myotubes ( figure 4(c ) ) . the administration of either curcumin ( 1 m , proteasome inhibitor ) or ag490 ( 5 m , jak inhibitor ) together with tnf- and/or ifn or ifn during 48 hours ( from 6th to 8th day of differentiation ) reversed the nuclear raise in nf-b protein expression to the control level ( figure 5(a ) ) . accordingly , as shown on figure 5(a ) , both inhibitors were equally potent in elevating cytosolic ib expression levels . as both nf-b activity and nuclear expression levels were associated with concomitant drop in myhc iia protein expression , the above - mentioned inhibitors were also used to determine whether they can protect c2c12 myotubes from the tnf--dependent loss of myhc iia . in agreement with our suggestion , both inhibitors ( curcumin and ag490 ) were capable to prevent the tnf--dependent loss of myhc iia expression levels in c2c12 myotubes , when compared with the control level ( figure 5(b ) ) . as mrfs are critical for muscle differentiation and maturation of myotubes , two different transcription factors were chosen for the study , namely , myod known as primary muscle regulatory factor and myogenin secondary to myod . as presented in figure 6 myod protein expression was reduced all along the treatment , irrespective to cytokine , whereas the level of myogenin protein was decreased by tnf- treatment or cotreatment only . additional administration of ifn or ifn did not alter the myogenin response to tnf- ( figure 6 ) . both the number and diameter of myotubes were reduced after individual treatment with tnf- or tnf- and ifn but not with ifn. they were restored to normal after simultaneous administration of curcumin or ag490 ( data not shown ) . in this study , it was assumed that stat-1 protein is activated either by ifn or ifn after binding to respective ifnar or ifngr . stat-1 was previously reported to be bound to tradd protein [ 4144 ] and inhibits nf-b - dependent death signal . as stat-1 protein is modulated by interferons , also nf-b signaling pathway can be released from inhibition when both tnf- and ifn or ifn are administered . to verify this assumption stat-1 protein complexes were isolated by immunoprecipitation , and tradd protein was immunoblotted after page . as it is shown in figure 7(a ) , the expression of tradd that was bound to stat-1 was the highest in untreated myotubes , and it was lowered by tnf- and further cotreatment with ifn and ifn. tnf- together with interferons attenuated the expression of myhc iia protein . on this evidence , we queried whether it is associated with lower activity of the respective gene ( myhc iia ) . surprisingly , tnf- , ifn , and ifn were shown to stimulate the activity of myhc iia by 3- , 1.8- , and 2.6-fold , respectively ( figure 7(b ) , p < 0.001 versus control ) . however , the myhc iia response to tnf- was controlled by nf-b and stat-1 transcription factors since the blockage of activation by curcumin ( nf-b ) or ag490 ( stat-1 ) diminished tnf--dependent rise in myhc iia activity ( figures 7(c ) and 7(d ) , p > 0.05 versus control ) . in contrast to tnf- , curcumin did not affect the activity of myhc iia upon cotreatment with ifn ( figure 7(c ) , p > 0.05 versus tnf- and ifn ) but it increased myhc iia activity when tnf- was added along with ifn ( figure 7(c ) , p < 0.01 versus tnf- and ifn ) . astonishingly , ag490 co - treatment with ifn elevated myhc iia activity by 2.5-fold ( figure 7(d ) , p < 0.001 versus control ) . when tnf- or ifn , or both ( 10 ng / ml ) were added to differentiating medium for the last 48 hours of differentiation ( days 68 ) , atrogin1 gene activity was stimulated by 1.8- and 1.6-fold at the 8th day of study ( figure 8(a ) , p < 0.01 compared to control ) . tnf- , but not ifn , dependent atrogin1 activation , was inhibited by curcumin ( 1 m , figure 8(a ) , p > 0.05 versus control ) . a more pronounced effect was induced by ag490 ( 5 m ) , which almost completely abolished tnf- and ifn effects during the last 48 hours of the 8-day incubation time ( figure 8(b ) , p < 0.001 compared to control ) . when tnf- , ifn , or ifn ( 10 ng / ml ) were added to differentiating medium for the last 48 hours murf1 gene activity was stimulated almost 4-fold at the 8th day of study ( figure 8(c ) , p < 0.001 compared to control ) . tnf- and ifn , but not the ifn-stimulated murf1 gene activity in nf-b - dependent manner , since curcumin ( 1 m ) was capable to reduce significantly the cellular response ( figure 8(d ) , p < 0.01 versus tnf- and ifn alone ) . no effect of curcumin was observed with respect to ifn ( figure 8(c ) , p > 0.05 versus ifn alone ) . ag490 ( 5 m ) abolished tnf--induced effect , but it potentiated the ifn effect on murf1 gene activity by almost 1.6-fold ( figure 8(d ) , p < 0.001 versus ifn alone ) , whereas it had no significant influence on ifn-dependent stimulation of murf1 gene activity ( figure 8(d ) , p > 0.05 versus ifn alone ) . several signaling pathways have been shown to participate in cancer cachexia [ 25 , 26 ] . it was reported that tgf- superfamily ( tgf- , activin , gdf-15 , and myostatin ) contributes to muscle wasting through smad pathway activation , whereas blockage of actriib partially reversed this effect . also jak / stat3 pathway inhibition was demonstrated to improve il-6-induced skeletal muscle cachexia in animal and c2c12 muscle cell models [ 13 , 15 ] . recently , we reported that leptin acts as mitogen but it markedly impairs muscle cell viability and myogenic differentiation through jak / stat3 signaling pathway . as leptin deficient obese mice ( ob / ob ) have also reduced lean body mass , apparently other than leptin - dependent signaling pathways must be engaged in muscle cachexia . in this study we showed that tnf- stimulated dna synthesis similarly to leptin ( figure 1(c ) , p < 0.001 versus control ) but it also increased c2c12 muscle cell viability ( figures 1(a)-1(b ) , p < 0.001 versus control ) . thus , both cytokines seem to actively participate in the activation of satellite cells ( myoblasts ) as was previously reported for leukemia inhibitory factor lif . this effect of cytokine does not necessarily mean that muscle tissue would grow in hyperplasia - associated hypertrophy conditions , as the key event in muscle growth is differentiation and fusion . moreover , mitogenicity has nothing to do with muscle fiber decay and resulting muscle cachexia . among inflammatory cytokines , tnf-/cachectin , il-1 , il-1 , ifn , and il-6 have been implicated in muscle wasting , and their elevated levels were detected in cachectic patients . in this experiment we sought to mimic the effects of increased concentrations of tnf- , ifn , and ifn during muscle differentiation using c2c12 mouse myoblasts . as tnf- improved viability and mitogenicity of differentiating c2c12 myoblasts , we suggest that tnf- might be capable to induce muscle dedifferentiation as demonstrated by buck and chojkier . tnf- improved cell respiration through nf-b - dependent mechanism , as curcumin ( 1 m ) administered with tnf- impaired muscle cell viability even stronger than curcumin given alone ( figure 1(a ) , p < 0.01 ) . inhibition of stat-1 kinase jak with tyrphostin ( ag490 ) also retarded tnf--induced mitogenicity and viability below control level ( figures 1(b)-1(c ) , p < 0.001 versus control ) . a similar effect of ag490 on c2c12 myoblast proliferation was observed by spangenburg and booth . moreover , nf-b and stat-1 responded to tnf- in elevated transcriptional activity , as shown in the 3-day myotubes in short- and long term experiments ( figures 1(d)-1(e ) , 3(b ) , p < 0.05 versus control ) . western blots confirmed transcriptional activation in the 3rd day of myogenesis as it was accompanied by higher nuclear nf-b and lower cytosolic ib protein levels after tnf- ( figure 1(f ) ) . insulin pretreatment amplified tnf--dependent activity of nf-b ( figure 1(h ) , p < 0.001 versus tnf- given alone ) , but it could hardly affect ifn or ifn effects ( figure 1(i ) , p > 0.05 ) . insulin however , after initial pretreatment , given in combined treatment with tnf- , inhibited nuclear translocation of nf-b ( figure 1(g ) ) . tnf- administration retarded muscle differentiation , as evidenced by lower mrfs ( myod and myogenin ) expression and reduced myotube formation ( figure 6(a ) and supplementary data 3 ) . it is well established that tnf- controls nf-b activity in muscle cells [ 5 , 32 ] and that transcriptional regulation through nf-b controls myod decay in c2c12 myotubes [ 5 , 33 ] . nf-b regulates the expression of a variety of muscle genes and proteins including those involved in control of cell proliferation , myogenesis , oxidative stress , and mitochondrial dysfunction . thus , higher nf-b activity observed after tnf- administration in this experiment suggests that differentiating myoblasts could be withdrawn from the myogenic program as shown recently in muscle - derived stem cells isolated from transgenic mice , or by enforced expression of c - flip in satellite cells . moreover , c - flip is a known intracellular modulator of death receptor mediated signaling ( inhibition of intrinsic apoptosis ) and nf-b activator . actually , this was not the case for ifn and ifn as neither cytokine ( 10 ng / ml ) could affect nf-b activity ( figure 1(e ) , p > 0.05 versus control ) . however , ifn and ifn used in one - day pretreatment sensitized c2c12 myotubes to tnf--induced nf-b activation on the 3rd day of myogenesis ( figure 2(b ) , p < 0.001 ) . higher nf-b transcriptional activity upon pretreatment with ifns was confirmed by higher and earlier nuclear expression of nf-b ( figure 2(a ) ) . indeed , systemic inflammation was often indicated as causal to skeletal muscle wasting [ 39 , 40 ] . in this study , we successfully verified that ifn and ifn affected tnf--dependent nf-b activation and showed that the latter occured through stat-1 activation . as shown in figure 2(f ) active stat-1 ( phosphorylated at tyrosine 701 , stat-1-p - y ) has been detected in the nucleus after tnf- , ifn , and ifn , whereas insulin pre- and cotreatment could not reduce the effect of ifns . thus , it is apparent that tnf- , ifn , and ifn cooperate in nf-b activation , and that nonactive stat-1 seems to inhibit this process . next , we assumed that stat-1 might inhibit nf-b in nonstimulated muscle cells as stat-1 was previously reported in other cell - types to be assembled with tnf - r1 through tradd protein partner [ 4143 ] . actually , during the immunoprecipitation study ifn and ifn caused reduction in the stat-1 protein quantity bound to tradd protein ( important component of tnf--induced signalosome , figure 7(a ) ) . thus , withdrawal of stat-1 from tnf - r1 assembly by ifn or ifn might explain their amplifying effect of tnf--induced nf-b activation . similarly , ifn mediated release from stat-1-mediated inhibition of nf-b activation in human colon adenocarcinoma colo 205 cells . raising evidence obtained from similar experiments performed in the past insulin , which is known to activate nf-b through pi3-k / akt pathway , was also capable to sensitize c2c12 myotubes to tnf--dependent nf-b activation ( figure 1(h ) , p < 0.001 versus control ) , but it did not change the response of nf-b or stat-1 to interferons ( figures 1(i ) and 2(g ) , p > 0.05 versus control ) . therefore , we assume that similarities between insulin and interferons in tnf--dependent nf-b activation must have different origins . insulin was capable to reduce nf-b activation in the presence of ifn- but not ifn ( figures 2(d)-2(e ) ) , although insulin did not influence nuclear nf-b and cytosolic ib protein levels , respectively , ( figure 2(c ) ) . presumably , the effects of insulin on tnf-- and ifns - mediated nf-b activation were indirect and not related to stat-1 activity ( figure 2(b ) , p > 0.05 versus tnf- or ifns ) . interestingly , tnf- raised nuclear stat-1-p - y protein levels , while insulin pre- and co - treatment additionally amplified this effect in the 3rd day of myogenic differentiation ( figure 3(a ) ) . at the same time , tnf- evoked higher transcriptional activity of stat-1 ( figure 3(b ) , p < 0.05 ) . these results are in concert with our previous report as ifn did not ( figure 3(b ) , p > 0.05 ) but ifn did increase tnf--dependent stat-1 activation ( figure 3(d ) , p < 0.05 ) . insulin pretreatment did not affect ifn-induced , tnf--dependent activation of stat-1 ( figure 3(d ) , p < 0.05 ) . the latter observation was corroborated by wb results obtained for stat-1-p - y protein levels ( figures 3(c ) and 3(e ) ) . one should keep in mind , that multiple signaling pathways are relevant to muscle cachexia ( nf-b , jak / stat , smad ) . from a pathophysiological point of view , muscle cachexia is an adaptation mechanism to chronic inflammation and long lasting anorexia ( cancer , anorexia nervosa , and aids ) . it has pushed through the demands for amino - acids needed to meet the requirements of acute phase protein synthesis . interestingly , it is clear now that skeletal muscles not only play a pivotal role in mobilization of amino - acids , but they are also the site of extensive production of acute phase proteins . overall , the goal is not to ultimately stop muscle cachexia by pharmacological or biotechnological intervention , but to make this task obsolete through the correction of associated symptoms of disease . the outcome of myhc iia protein expression in 6-day myotubes challenged with cytokines was chosen to examine their procachectic activity . initially , it should be stressed that muscle differentiation of c2c12 myoblasts was suppressed by tnf- but not ifn or ifn , except when they were administered together with tnf- ( figure 6(a ) ) . these effects were partially under the control of nf-b and/or jak / stat-1 , since either curcumin ( nf-b inhibitor ) or ag490 ( jak inhibitor ) reversed some of the tnf--induced effects . curcumin is a dominant nf-b inhibitor as it prevents proteasomal degradation of ib and subsequent nf-b activation . in 6-day myotubes , tnf- , given alone or together with ifn or ifn increased nuclear expression of nf-b with simultaneous drop in cytosolic expression of ib ( figure 5(a ) ) . at the same time , tnf- administered alone reduced the expression of the fast form of myhc iia protein ( figure 5(b ) ) . these data suggest that nf-b is a negative regulator of myogenesis , and that nf-b delays the rise in myhc iia protein expression . marked reduction was also observed after concomitant administration of ifn and ifn. however , neither cytokine administered separately nor in used combinations could induce nuclear expression of nf-b upon cotreatment with curcumin ( 1 m ) , and associated cytosolic ib expression level remained high ( figure 5(a ) ) . jak inhibitor ag490 ( 5 m ) was less potent than curcumin with respect to nf-b / ib expression levels ( figure 5(a ) ) . from this study it becomes clear that myogenesis is repressed by nf-b as reported by kumar et al . . the reduced expression of fast form of myhc iia was observed after tnf- , but not ifn or ifn given alone ( figure 5(b ) ) . combined treatment of tnf- and ifn and ifn and ifn was even more powerful to repress myhc iia expression in the cytosol . both inhibitors ( curcumin and ag490 ) almost totally abrogated the above - mentioned effects of cytokines ( figure 5(b ) ) . it should be underlined that tnf- markedly decreased the myod / myogenin protein levels ( figure 6 ) with resultant drop in myhc iia protein expression . neither ifn nor ifn could affect myogenin expression , but similarly to tnf- , both interferons considerably diminished myod protein expression levels ( figure 6 ) . summing up , combined treatment of tnf- with ifn or ifn led to a significant effect evoked by the former ( figure 6 ) . thus , in this experiment tnf- seems to play an important negative role in the control of myogenesis . to make the picture of cytokine - dependent muscle differentiation and muscle fiber decay more lucid , additional experiments followed by qrt pcr analysis observations obtained from this study suggest marked differences in the control of muscle differentiation and muscle wasting at genomic and translational levels . myhc iia gene expression was raised noticeably after tnf- administration ( figures 7(c)-7(d ) , p < 0.001 versus control ) , which was unexpected as myod level was diminished at the same time . interferons also stimulated myhc iia gene expression , but they were apparently weaker ( figures 7(c)-7(d ) , p < 0.001 versus control ) . astonishingly , curcumin ( 1 m ) was unable to cease the action of either cytokine ( figure 7(c ) , p > 0.05 versus cytokines alone ) , although it markedly reduced the effect of tnf- and ifn ( figure 7(c ) , p < 0.001 versus cytokines alone ) . ag490 significantly elevated myhc iia gene expression either alone or when it was given together with interferons ( figure 7(d ) , p < 0.01 versus control ) , but it abrogated tnf-- but not ifn-induced myhc iia gene activation . as myhc iia protein level decreased upon tnf- or tnf- and ifns administration , two genes atrogin1 and murf1 encoding respective ubiquitin ligases were examined . these genes were previously indicated as crucial for muscle fiber decay [ 48 , 49 ] . tnf- and ifn stimulated ( figure 8(a ) , p < 0.001 versus control ) , whereas ifn did not affect atrogin1 gene expression ( figure 8(a ) , p > 0.05 versus control ) . curcumin protected myotubes from tnf-- , tnf- and ifns - stimulated atrogin1 gene expression ( figure 8(a ) , p > 0.05 versus control ) . cytokine - induced atrogin1 gene activation was considerably diminished by ag490 ; gene expression even dropped below the control level ( figure 8(b ) , p < 0.05 versus control ) . surprisingly , ag490 given alone stimulated gene expression ( figure 8(b ) , p < 0.01 versus control ) . murf1 gene expression was activated by cytokines in a similar manner ( figures 8(c)-8(d ) , p < 0.001 versus control ) but curcumin and ag490 could repress only tnf--induced gene activation ( figures 8(c)-8(d ) , p < 0.001 versus tnf- alone ) . these results are in agreement with the well known effects of transcriptional activity of nf-b , as both genes are targets in muscle wasting [ 19 , 50 ] . much less is known about the control of these genes by stat-1. in this study , however , ag490 drastically reduced atrogin1 gene expression when added with cytokines . ag490 used individually elevated activity of atrogin1 gene ( figure 8(b ) , p < 0.01 versus control ) . monitoring of atrogin1 and murf1 genes activity showed dominant effect of tnf- ( also in combined treatment with ifns ) . we speculate that nf-b was a major player at the genomic level for tnf--induced effects . where tnf- was administered together with ifns , curcumin addition could not repress increased atrogin1 and murf1 genes activity ( figures 8(a ) and 8(c ) , p > 0.05 versus tnf- and ifns ) . a similar effect was noted in the case of treatment with ag490 . as mentioned above , this jak / stat inhibitor normalized atrogin1 gene activity which was raised by tnf- and ifns , but it did not affect murf1 gene activity except for the situation when tnf- was given alone ( figure 8(d ) , p < 0.001 versus control ) . our hypothesis addresses competition between cytokines ( tnf- , ifn , ifn ) to recruit stat-1. during this process release of nf-b allowed both nf-b and stat-1 to cooperate at the genomic level . as we showed , some of the cachectic effects were evoked differently for tnf- , ifns , and both , as tnf--induced activation of atrogin1 and murf1 genes was reduced by nf-b and stat-1 inhibitors ( curcumin and ag490 , resp . ) , while the presence of ifns deteriorated curcumin effect . astonishingly , tnf- was apparently mitogenic and stimulated viability of mononuclear muscle cells , whereas this cytokine efficiently inhibited muscle fiber formation . moreover , tnf- indirectly and directly diminished myhc iia protein level , even though it stimulated myhc iia gene expression . from qrt pcr study similarly to atrogin1 and murf1 , the activation of myhc iia gene by tnf- could be partially reversed by nf-b and stat-1 inhibitors . the effect of nf-b and stat-1 inhibitors was diminished upon ifns cotreatment pointing to the synergy between tnf- and ifns in action . insulin did not prevent combined tnf-- and ifn - s - dependent muscle decay , even though it is almost ultimate anabolic hormone . thus , where anyone thought to bring to an end the tnf--induced muscle cachexia , either the secretion of this cytokine or ifns seems to be essential .

tnf- was shown to stimulate mitogenicity in c2c12 myoblasts . selected cytokines tnf- , ifn , or ifn reduced the expression of myosin heavy chain ( myhc iia ) when given together . molecular mechanisms of cytokine activities were controlled by nf-b and jak / stat signaling pathways , as metabolic inhibitors , curcumin and ag490 , inhibited some of tnf- and ifn/ifn effects . insulin was hardly antagonistic to tnf-- and ifn/ifn-dependent decrease in myhc iia protein expression . cytokines used individually or together also repressed myogenesis of c2c12 cells . moreover , tnf-- and ifn/ifn-dependent effects on c2c12 myotubes were associated with increased activity of atrogin1 and murf1 genes , which code ubiquitin ligases . myhc iia gene activity was unaltered by cytokines . inhibition of nf-b or jak / stat with specific metabolic inhibitors decreased activity of atrogin1 and murf1 but not myhc iia gene . overall , these results suggest cooperation between cytokines in the reduction of myhc iia protein expression level via nf-b / jak / stat signaling pathways and activation of atrogin1 and murf1 genes as their molecular targets . insulin cotreatment or pretreatment does not protect against muscle decay induced by examined proinflammatory cytokines .

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

we demonstrated that nf-b and jak / stat signaling pathways are intersected and that they control muscle growth and decay . to our surprise , tnf--induced effect was associated with both activation of myhc iia , atrogin1 ( also known as mafbx ) , and murf1 genes , but proteolysis took over protein accretion . neither ifn nor ifn affected the activity of nf-b in c2c12 myotubes in the long term study ( figure 1(e ) , p > 0.05 compared to control ) . myhc iia protein expression level has been selected as a marker of protein gain / loss in c2c12 myotubes . tnf- ( 10 ng / ml , 48 h ) was shown to inhibit the expression of myhc iia in c2c12 myotubes ( 8-day - old ) , while additional presence of ifn or ifn ( 10 ng / ml , 48 h ) amplified tnf--dependent drop in myhc iia protein expression ( figure 4(b ) ) . as both nf-b activity and nuclear expression levels were associated with concomitant drop in myhc iia protein expression , the above - mentioned inhibitors were also used to determine whether they can protect c2c12 myotubes from the tnf--dependent loss of myhc iia . in agreement with our suggestion , both inhibitors ( curcumin and ag490 ) were capable to prevent the tnf--dependent loss of myhc iia expression levels in c2c12 myotubes , when compared with the control level ( figure 5(b ) ) . tnf- together with interferons attenuated the expression of myhc iia protein . on this evidence , we queried whether it is associated with lower activity of the respective gene ( myhc iia ) . surprisingly , tnf- , ifn , and ifn were shown to stimulate the activity of myhc iia by 3- , 1.8- , and 2.6-fold , respectively ( figure 7(b ) , p < 0.001 versus control ) . however , the myhc iia response to tnf- was controlled by nf-b and stat-1 transcription factors since the blockage of activation by curcumin ( nf-b ) or ag490 ( stat-1 ) diminished tnf--dependent rise in myhc iia activity ( figures 7(c ) and 7(d ) , p > 0.05 versus control ) . in contrast to tnf- , curcumin did not affect the activity of myhc iia upon cotreatment with ifn ( figure 7(c ) , p > 0.05 versus tnf- and ifn ) but it increased myhc iia activity when tnf- was added along with ifn ( figure 7(c ) , p < 0.01 versus tnf- and ifn ) . when tnf- , ifn , or ifn ( 10 ng / ml ) were added to differentiating medium for the last 48 hours murf1 gene activity was stimulated almost 4-fold at the 8th day of study ( figure 8(c ) , p < 0.001 compared to control ) . initially , it should be stressed that muscle differentiation of c2c12 myoblasts was suppressed by tnf- but not ifn or ifn , except when they were administered together with tnf- ( figure 6(a ) ) . at the same time , tnf- administered alone reduced the expression of the fast form of myhc iia protein ( figure 5(b ) ) . the reduced expression of fast form of myhc iia was observed after tnf- , but not ifn or ifn given alone ( figure 5(b ) ) . as myhc iia protein level decreased upon tnf- or tnf- and ifns administration , two genes atrogin1 and murf1 encoding respective ubiquitin ligases were examined . where tnf- was administered together with ifns , curcumin addition could not repress increased atrogin1 and murf1 genes activity ( figures 8(a ) and 8(c ) , p > 0.05 versus tnf- and ifns ) . as we showed , some of the cachectic effects were evoked differently for tnf- , ifns , and both , as tnf--induced activation of atrogin1 and murf1 genes was reduced by nf-b and stat-1 inhibitors ( curcumin and ag490 , resp . ) from qrt pcr study similarly to atrogin1 and murf1 , the activation of myhc iia gene by tnf- could be partially reversed by nf-b and stat-1 inhibitors .

nevertheless , when a bone defect can not heal through its normal wound healing response , surgical intervention is required to reconstruct the defect site with various forms of grafts and implants . currently , autografts , allografts and artificial biomaterials are commonly used to replace the missing tissue . autologous cancellous bone grafting is still the gold standard.1 however , donor site morbidity and limited graft availability push clinicians and researchers to use allograft bones and artificial biomaterials as alternatives.23 for allografts , possible pathogen transfer and degradation of the tissue 's material properties , bone mineral density and prevalence of microcracks is a major concern.45 biomaterials do not face similar problems and therefore , hold considerable promise . there are several kinds of materials , which have been designed into experimental and clinical uses including natural ingredients ( collagen , coral ) , synthetic polymers ( polylactic acid , polyglycolic acid and their copolymers ) , bioceramics ( hydroxyapatite , tricalciumphosphate ) . however , a single material may hardly cater to the increasing demands of biocompatibility , osteoconductivity , biodegradation and mechanical property that the implanted scaffold should have in practical cases . now - a - days , integrated hybrid biomaterials hold more promise than a single material . as reported in an earlier study6 we developed a three dimensional porous hybrid biomaterial consisting of plga , -tricalciumphosphate ( -tcp ) , collagen i and apatite , namely the plga/-tcp - collagen i / apatite scaffold . as the skeleton of this scaffold has been designed and produced by rapid prototyping ( rp ) technology , with low temperature deposition manufacturing ( ldm ) method , the scaffold was biodegradable , highly porous ( > 90% ) with interconnected macro pores of about 400 m and micropores less than 10 m , was mechanically strong and could be easily forged into desired shapes.7 however , there still remain problems to be solved not only relating to its hydrophobic surface , which is unfavorable for cell attachment and osteogenic development , but also relating to its decreased bone bonding property caused by the plga ingredient . to address those problems , earlier data demonstrated that the novel scaffold could improve cell proliferation and promote osteogenic differentiation with hydrophilic surfaces in vitro . the purpose of the current study was to evaluate whether the hybrid scaffolds combined with bone marrow mesenchymal stem cells ( bmscs ) can enhance bone defect repair in a rabbit model compared with the plga/-tcp skeleton . plga/-tcp ( 7:3 w / w ) skeleton was fabricated by a ldm system ( tissform 3 , the department of mechanical engineering of tsinghua university ) as we reported before.7 briefly , the hybridization procedure has been accomplished as follows : firstly , plga/-tcp was immersed in collagen i ( derived from fetal bovine corium ) acidic solutions ( ph 3.2 , 0.5 wt.% ) under vacuum for 4 h , then freeze - dried at 80c under a vacuum of 0.2 torr for an additional 72 h to form collagen microsponges in the skeleton pores . the new formed collagen microsponges were further crosslinked by treatment with glutaraldehyde vapor saturated with 25% glutaraldehyde aqueous solution at 37c for 4 h. after that , the scaffolds were incubated in a simulated body fluid ( sbf ) ( 50 ml in volume with ph 7.4 including 141 mm nacl , 4.0 mm kcl , 0.5 mm mgso4 , 1.0 mm mgcl2 , 4.2 mm nahco3 , 2.5 mm cacl2 and 1.0 mm kh2po4 , deionized water ) for up to 16 days at 37c to form mineral film . the sbf solution was changed every 24 h to ensure sufficient ion concentrations for mineral growth.6 the animal study was approved by the institutional animal review committee of the fourth military medical university . bmscs were obtained from the os longum of 1-day - old new zealand white rabbit and cultured under dulbecco 's modified eagle 's medium ( dmem ; gibco ) plus 10% fetal bovine serum ( fbs ) at 37c , 5% co2 . cell subcultures of third passages were osteogenically induced under dmem supplemented with 10% fbs , 10 mol / l dexamethasone , 10 mm -glycerolphosphate and 50 mg / ml l - ascorbic acid for 3 weeks.6 then the bmscs were trypsinized and suspended into each 15 3 3 mm sized hybrid scaffolds at a density of 7 10/ml to fabricate bmscs - plga/-tcp - collagen i / apatite composite and incubated for another 24 h waiting for implantation . in addition , the bmscs / plga/-tcp composite was prepared in the same way as control . 48 healthy new zealand white rabbits weighing between 2.5 and 3.5 kg were used in this study . rabbits were anesthetized with a cocktail of 50 mg / kg ketamine and 8 mg / kg xylazine administered intramuscularly . the operation sites over radii an approximately 2.5 cm long incision was made and the tissues overlying the distal diaphyseal radius were dissected . a 15 mm osteoperiosteal defect was created in the radius with a mini - oscillating saw bilaterally . the plga / btcp hybrid samples were inserted in the defect without external fixation . for experimental groups , the radial defects were implanted with bmscs - plga/-tcp - collagen i / apatite composites ( group a , n = 30 ) , bmscs / plga/-tcp composites ( group b , n = 30 ) , plga/-tcp - collagen i / apatite hybrid scaffolds ( group c , n = 15 ) and plga/-tcp scaffolds ( group d , n = 15 ) . six additional defects remained untreated as blank control ( group e , n = 6 ) . a balanced split - plot randomization scheme was used to locate the scaffolds so that the limb choice or formulation pairing would not influence the outcome . buprenorphine ( 0.05 mg / kg ) and enrofloxacin ( 50 mg / kg ) was given for pain relief and infection prophylaxis . water and food were supplied ad libitum . at 12 , 24 and 36 weeks postsurgery furthermore , each radiograph was examined by two independent observers and given a score depending on a standardized scoring system including an evaluation of bone formation ( 5 levels ) , persistence of the fracture line ( 3 levels ) and bone remodeling ( 3 levels).8 meanwhile , microcomputed tomography ( microct ) ( explore locus sp microct , ge company american ) was also done for three - dimensional ( 3d ) reconstruction of the original orthotopic defect sites in different groups postoperation . the specimens so obtained were preserved in 4% buffered formaldehyde , dehydrated and embedded in paraffin . serial sections were stained with hematine / eosine ( he ) and observed by light microscopy ( leica , rijswijk , the netherlands ) . as for morphometric analysis , five sequential sections per composite were selected for evaluation under low magnification , allowing coverage of the entire composite . using a leica - qwin 3.2 image analysis system ( leitz dmrd , leica microsystems , inc . , bannockburn , il , usa ) , all slides were seen by two independent observers to identify the type of bone tissue . the extent of bone formation was indicated by the percentage of the bone tissue area within the defect site and an average value was calculated for each composite . quantitative data were expressed as mean value standard deviation and student t - test was used to analyze the percentage of bone formation . data of radiographic scoring were processed as ordinal ranking and analyzed with mann - whitney u test . plga/-tcp ( 7:3 w / w ) skeleton was fabricated by a ldm system ( tissform 3 , the department of mechanical engineering of tsinghua university ) as we reported before.7 briefly , the hybridization procedure has been accomplished as follows : firstly , plga/-tcp was immersed in collagen i ( derived from fetal bovine corium ) acidic solutions ( ph 3.2 , 0.5 wt.% ) under vacuum for 4 h , then freeze - dried at 80c under a vacuum of 0.2 torr for an additional 72 h to form collagen microsponges in the skeleton pores . the new formed collagen microsponges were further crosslinked by treatment with glutaraldehyde vapor saturated with 25% glutaraldehyde aqueous solution at 37c for 4 h. after that , the scaffolds were incubated in a simulated body fluid ( sbf ) ( 50 ml in volume with ph 7.4 including 141 mm nacl , 4.0 mm kcl , 0.5 mm mgso4 , 1.0 mm mgcl2 , 4.2 mm nahco3 , 2.5 mm cacl2 and 1.0 mm kh2po4 , deionized water ) for up to 16 days at 37c to form mineral film . the sbf solution was changed every 24 h to ensure sufficient ion concentrations for mineral growth.6 the animal study was approved by the institutional animal review committee of the fourth military medical university . bmscs were obtained from the os longum of 1-day - old new zealand white rabbit and cultured under dulbecco 's modified eagle 's medium ( dmem ; gibco ) plus 10% fetal bovine serum ( fbs ) at 37c , 5% co2 . cell subcultures of third passages were osteogenically induced under dmem supplemented with 10% fbs , 10 mol / l dexamethasone , 10 mm -glycerolphosphate and 50 mg / ml l - ascorbic acid for 3 weeks.6 then the bmscs were trypsinized and suspended into each 15 3 3 mm sized hybrid scaffolds at a density of 7 10/ml to fabricate bmscs - plga/-tcp - collagen i / apatite composite and incubated for another 24 h waiting for implantation . in addition , the bmscs / plga/-tcp composite was prepared in the same way as control . 48 healthy new zealand white rabbits weighing between 2.5 and 3.5 kg were used in this study . rabbits were anesthetized with a cocktail of 50 mg / kg ketamine and 8 mg / kg xylazine administered intramuscularly . the operation sites over radii an approximately 2.5 cm long incision was made and the tissues overlying the distal diaphyseal radius were dissected . a 15 mm osteoperiosteal defect was created in the radius with a mini - oscillating saw bilaterally . the plga / btcp hybrid samples were inserted in the defect without external fixation . for experimental groups , the radial defects were implanted with bmscs - plga/-tcp - collagen i / apatite composites ( group a , n = 30 ) , bmscs / plga/-tcp composites ( group b , n = 30 ) , plga/-tcp - collagen i / apatite hybrid scaffolds ( group c , n = 15 ) and plga/-tcp scaffolds ( group d , n = 15 ) . six additional defects remained untreated as blank control ( group e , n = 6 ) . a balanced split - plot randomization scheme was used to locate the scaffolds so that the limb choice or formulation pairing would not influence the outcome . buprenorphine ( 0.05 mg / kg ) and enrofloxacin ( 50 mg / kg ) was given for pain relief and infection prophylaxis . at 12 , 24 and 36 weeks postsurgery , animals were euthanized with an over - dose of pentobarbital . furthermore , each radiograph was examined by two independent observers and given a score depending on a standardized scoring system including an evaluation of bone formation ( 5 levels ) , persistence of the fracture line ( 3 levels ) and bone remodeling ( 3 levels).8 meanwhile , microcomputed tomography ( microct ) ( explore locus sp microct , ge company american ) was also done for three - dimensional ( 3d ) reconstruction of the original orthotopic defect sites in different groups postoperation . the specimens so obtained were preserved in 4% buffered formaldehyde , dehydrated and embedded in paraffin . serial sections were stained with hematine / eosine ( he ) and observed by light microscopy ( leica , rijswijk , the netherlands ) . as for morphometric analysis , five sequential sections per composite were selected for evaluation under low magnification , allowing coverage of the entire composite . using a leica - qwin 3.2 image analysis system ( leitz dmrd , leica microsystems , inc . , bannockburn , il , usa ) , all slides were seen by two independent observers to identify the type of bone tissue . the extent of bone formation was indicated by the percentage of the bone tissue area within the defect site and an average value was calculated for each composite . quantitative data were expressed as mean value standard deviation and student t - test was used to analyze the percentage of bone formation . data of radiographic scoring were processed as ordinal ranking and analyzed with mann - whitney u test . x - rays of bone defects at operation day , 12 , 24 and 36 weeks after implantation were analysed [ figure 1a ] . immediately after the operation , the composites in different groups were nearly invisible because of the low x - ray attenuation coefficient [ figure 1b ] . when bone defect was treated with hybrid scaffolds ( group a ) , new bone tissue appeared at the proximal and distal ends of the radial defect sites 12 weeks postoperation [ figure 1c ] . in addition , the distance between both broken sides shortened and seemed almost united in the middle site [ figure 1c ] . another 12 weeks later , bone union was completely accomplished and bone medullary cavity was nearly recanalized . 36 weeks after the operation , the remodeling of bone contour ended [ figure 1e ] . no radiographic sign of bone formation was presented at the defect sites in group b during the whole experimental period and the bone remained un - united 36 weeks postoperation [ figure 1f - h ] . in groups c and d , which have not been combined with seed cells , the bone defect sites failed to repair 36 weeks after operation ( data not shown ) . the radial bone defect in blank control group e the x - ray image scores of group a were 5.12 at 12 weeks , 9.68 at 24 weeks and 11.86 at 36 weeks with full marks of 12 levels while groups b - d have no scores from the beginning to the end ( p > 0.05 ) . radiographic results : ( a ) bone defect caused by osteotomy ; ( b ) radiograph immediately after scaffolds implantation ; ( c - e ) 12 , 24 and 36 weeks postsurgery of the bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/-tricalciumphosphate ( -tcp)-collagen i / apatite composites ; ( f - h ) 12 , 24 and 36 weeks postsurgery of the bmscs - plga/-tcp composites micro ct was used to demonstrate 3d images of the bone defect area as well as to determine the quantity of newly formed bone . in group a , new bone along the lateral edge of the residual scaffolds was seen after 12 weeks [ figure 2a - c ] . bilateral bone cortices formed with nearly recanalized marrow cavity [ figure 2d - f ] after 24 weeks . after 36 weeks , the newly formed bone had a macroscopic structure and a ct value similar to that of the original radius with well remodeled bony outline [ figure 2g - i ] . meanwhile , no bone formation was detected after 12 , 24 and 36 weeks postoperation in group b except for the gradual degradation of implanted scaffolds until complete absorption [ figure 2j - r ] . microcomputed tomography results : 1 . bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/ -tricalciumphosphate ( -tcp)-collagen i / apatite composites ( a - i ) : 12 weeks postoperation showing new bone along lateral edge of residual scaffolds ( a - c ) ; 24 weeks postoperation showing nearly recanalized marrow cavity ( d - f ) ; 36 weeks postoperation showing remodelling ( g - i ) ; 2 . bmscs - plga/-tcp composites ( j - r ) : 12 weeks postoperation ( j - l ) ; 24 weeks postoperation ( m - o ) ; 36 weeks postoperation ( p - r ) . 3d reconstruction image ( a , d , g , j , m , p ) ; reconstruction image in the coronal plane ( b , e , h , k , n , q ) ; magnified view ( c , f , i , l , o , r ) of yellow cubic area ( a , d , g , j , m , p ) showing no new bone formation in the hybrid scaffolds group , the growing woven bone fully bridged the defect at 12 weeks after surgery . after 24 weeks , the woven bone remodeled into lamellar bone and bone marrow cavity began to form . solid union was documented by the transition from dense lamellar bone to dense cortical bone with normal marrow cavity in the defect site 36 weeks postsurgery [ figure 3a - c ] . in the original scaffolds group , no apparent bone tissue but remaining scaffolds after continual degradation finally , the scaffolds degraded completely and were replaced by connective tissues [ figure 3d - f ] . percent of bone forming area in group a was 48.56 3.12% at 12 weeks , 78.34 2.85% at 24 weeks and 97.27 2.65% at 36 weeks , which was significantly superior to no bone occupation of groups b - d ( p < 0.05 ) . bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/-tricalciumphosphate ( -tcp)-collagen i / apatite composites ( a - c ) : 12 weeks postoperation ( a ) ; 24 weeks postoperation ( b ) ; 36 weeks postoperation ( c ) ; 2 . bmscs - plga/ -tcp composites ( d - f ) : 12 weeks postoperation ( d ) ; 24 weeks postoperation ( e ) ; 36 weeks postoperation ( f ) . b = bone tissue , s = scaffold , mc = medullary cavity , ct = connective tissue , be = broken end of radius x - rays of bone defects at operation day , 12 , 24 and 36 weeks after implantation were analysed [ figure 1a ] . immediately after the operation , the composites in different groups were nearly invisible because of the low x - ray attenuation coefficient [ figure 1b ] . when bone defect was treated with hybrid scaffolds ( group a ) , new bone tissue appeared at the proximal and distal ends of the radial defect sites 12 weeks postoperation [ figure 1c ] . in addition , the distance between both broken sides shortened and seemed almost united in the middle site [ figure 1c ] . another 12 weeks later , bone union was completely accomplished and bone medullary cavity was nearly recanalized . 36 weeks after the operation , the remodeling of bone contour ended [ figure 1e ] . no radiographic sign of bone formation was presented at the defect sites in group b during the whole experimental period and the bone remained un - united 36 weeks postoperation [ figure 1f - h ] . in groups c and d , which have not been combined with seed cells , the bone defect sites failed to repair 36 weeks after operation ( data not shown ) . the radial bone defect in blank control group e the x - ray image scores of group a were 5.12 at 12 weeks , 9.68 at 24 weeks and 11.86 at 36 weeks with full marks of 12 levels while groups b - d have no scores from the beginning to the end ( p > 0.05 ) . radiographic results : ( a ) bone defect caused by osteotomy ; ( b ) radiograph immediately after scaffolds implantation ; ( c - e ) 12 , 24 and 36 weeks postsurgery of the bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/-tricalciumphosphate ( -tcp)-collagen i / apatite composites ; ( f - h ) 12 , 24 and 36 weeks postsurgery of the bmscs - plga/-tcp composites micro ct was used to demonstrate 3d images of the bone defect area as well as to determine the quantity of newly formed bone . in group a , new bone along the lateral edge of the residual scaffolds was seen after 12 weeks [ figure 2a - c ] . bilateral bone cortices formed with nearly recanalized marrow cavity [ figure 2d - f ] after 24 weeks . after 36 weeks , the newly formed bone had a macroscopic structure and a ct value similar to that of the original radius with well remodeled bony outline [ figure 2g - i ] . meanwhile , no bone formation was detected after 12 , 24 and 36 weeks postoperation in group b except for the gradual degradation of implanted scaffolds until complete absorption [ figure 2j - r ] . bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/ -tricalciumphosphate ( -tcp)-collagen i / apatite composites ( a - i ) : 12 weeks postoperation showing new bone along lateral edge of residual scaffolds ( a - c ) ; 24 weeks postoperation showing nearly recanalized marrow cavity ( d - f ) ; 36 weeks postoperation showing remodelling ( g - i ) ; 2 . bmscs - plga/-tcp composites ( j - r ) : 12 weeks postoperation ( j - l ) ; 24 weeks postoperation ( m - o ) ; 36 weeks postoperation ( p - r ) . 3d reconstruction image ( a , d , g , j , m , p ) ; reconstruction image in the coronal plane ( b , e , h , k , n , q ) ; magnified view ( c , f , i , l , o , r ) of yellow cubic area ( a , d , g , j , m , p ) showing no new bone formation in the hybrid scaffolds group , the growing woven bone fully bridged the defect at 12 weeks after surgery . after 24 weeks , the woven bone remodeled into lamellar bone and bone marrow cavity began to form . solid union was documented by the transition from dense lamellar bone to dense cortical bone with normal marrow cavity in the defect site 36 weeks postsurgery [ figure 3a - c ] . in the original scaffolds group , no apparent bone tissue but remaining scaffolds after continual degradation finally , the scaffolds degraded completely and were replaced by connective tissues [ figure 3d - f ] . percent of bone forming area in group a was 48.56 3.12% at 12 weeks , 78.34 2.85% at 24 weeks and 97.27 2.65% at 36 weeks , which was significantly superior to no bone occupation of groups b - d ( p < 0.05 ) . bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/-tricalciumphosphate ( -tcp)-collagen i / apatite composites ( a - c ) : 12 weeks postoperation ( a ) ; 24 weeks postoperation ( b ) ; 36 weeks postoperation ( c ) ; 2 . bmscs - plga/ -tcp composites ( d - f ) : 12 weeks postoperation ( d ) ; 24 weeks postoperation ( e ) ; 36 weeks postoperation ( f ) . b = bone tissue , s = scaffold , mc = medullary cavity , ct = connective tissue , be = broken end of radius in this study , we used a novel bioengineering composite by combining plga/-tcp - collagen i / apatite hybrid scaffold with bmscs and implanted these into critical - sized ( 15 mm ) segmental bone defects in a rabbit model . bone tissue engineering , temporary three - dimensional scaffolds play an important role in the manipulation of functions of bone forming cells and guidance of the formation of the new bones into desired shapes . generally , suitable scaffold should possess high porosity with a large surface - to - volume ratio and required hydrophilic surface properties that permit cell adhesion , differentiation and proliferation on one hand and adequate mechanical integrity to maintain the predesigned tissue structures on the other.910 nevertheless , very low number of seeded cells could remain on the surface of various kinds of biomaterials ( including autograft and allografts ) under standard cell culture conditions . most cells are lost due to the characteristics of surface free energy , namely the hydrophobicity of biomaterials . in order to enhance cell seeding efficiency and modulate cell - scaffold interactions , extracellular matrix ( ecm ) which critically affects cellular adhesion , proliferation and differentiation has been researched . previous studies have explored long chains of ecm proteins such as fibronectin ( fn ) , vitronectin ( vn ) for scaffold surface modification . biomaterials , which have been coated with these proteins have been proved to promote bmscs adhesion , proliferation and osteogenic potentiality to unite segmental bone defect.111213 mediated by cells integrins and ecm adhesion proteins , cells are continuously subjected to nanotopographical cues.14 as for bone tissue , the organic component type i collagen and inorganic component nanostructure hydroxyapatite constitute the main microenvironment . as far as biomimics theory is concerned therefore , the seeded cells may recognize the artificial nanotopography as natural milieu to a greater extent , leading to well integration with biomaterial and expectable bone formation in vitro and in vivo . it is expressed from the early stages of osteogenic differentiation and forms fibrils with a typical banding pattern of 68 nm width , a 3 - 5 nm banding depth and a 35 nm inter fibrillar spacing depth.14 by interacting with its natural nanoenvironment , type i collagen acts as a major regulator of cell osteogenic differentiation and can also facilitate bone deposition of in vitro cultured stromal cells.15 similar to fn and vn , collagen i also contains cell binding peptides and enhances cell attachment and osteogenesis in biomaterials by surface coating process.16 considering the superiority of type i collagen over fn and vn , we hybridized plga/-tcp with collagen i fibers to enhance the hydrophilic property of scaffold surface . compared to pure -tcp scaffolds , addition of plga achieved improved control of design parameters such as porosity , degradability , mechanical properties and varying degrees of pore size and shape for the composite scaffold . at the same time , the bioactive -tcp content is largely contained within the bulk of the plga/-tcp scaffold , rather than at the surface . since the interactions between biocomposite and bone tissue occur first at the pore surface , the non - exposed -tcp is in effect wasted . in order to enhance osteoconductivity of the scaffold , methods of surface modifications bioactive materials have been reported to bond to bone in vivo via formation of an apatite layer in the interface.17 furthermore , the calcium ions ( ca ) in the apatite layer are thought to play a key role . it is reported that the osteogenic proteins such as osteocalcin and osteopontin which are involved in the bone tissue bonding to materials surfaces are also ca binding proteins.18 similarly , to strengthen the bone - bonding ability of non - bioactive materials , biomimetic apatite coating methods involving the immersion of material substrates in sbf containing ca at physiological temperatures have been developed.19 the apatite coating can promote bone ingrowths , enhance direct bone contact,2021 and facilitate differentiation of bmscs along osteogenic lineage.22 in natural bone , hydroxyl apatite is deposited in a regular manner on the collagen matrix . in order to mimic this unique structure , we developed a hybrid scaffold of plga/-tcp skeleton , collagen i and apatite by depositing apatite particulates on the collagen i microsponge surfaces . the use of plga/-tcp skeleton as a mechanical skeleton advanced the formation of the hybrid scaffold with desired shapes and gave the hybrid scaffold good mechanical properties . as the primary components of bone ecm , collagen i and apatite should provide the novel hybrid scaffold with satisfactory cell interaction and osteoconductivity in vivo . histological and radiological results in our study clearly illustrated the statistical difference of osteogenic potency between bmscs - plga/-tcp - collagen i / apatite composites and bmscs - plga/-tcp composites . continuously active bone regeneration in the radial defects was found in the hybrid scaffolds group with the implantation time prolonged . the contact of bone to the material was intimate and direct without intervention of soft tissue . within 36 weeks postimplantation , new bone density was almost equal to that of the host bone , which indicated complete osteointegration of the defect area . while in bmscs - plga/-tcp composites group , connective tissues appeared between the native bone and implanted composites and no new bone tissue formed during the study . inconsistent development status concerning bone and connective tissues as demonstrated in two cellular composites groups should be attributed to the different scaffold handling process . to conclude , the results of reconstruction of rabbit 's radial defect has been very encouraging with composite constituted by plga/-tcp - collagen i / apatite scaffold and bmscs .

background : in bone tissue engineering , extracellular matrix exerts critical influence on cellular interaction with porous biomaterial and the apatite playing an important role in the bonding process of biomaterial to bone tissue . the aim of this study was to observe the therapeutic effects of hybrid rapid prototyping ( rp ) scaffolds comprising polylactic - co - glycolic acid ( plga ) , -tricalciumphosphate ( -tcp ) , collagen i and apatite ( plga/-tcp - collagen i / apatite ) on segmental bone defects in conjunction with combination with bone marrow mesenchymal stem cells ( bmscs).materials and methods : bmscs were seeded into the hybrid rp scaffolds to repair 15 mm defect in the radius of rabbits . radiograph , microcomputed tomography and histology were used to evaluate new bone formation.results:radiographic analysis done from 12 to 36 weeks postoperative period demonstrated that new bone formed at the radial defect site and continues to increase until the medullary cavity is recanalized and remodelling is complete . the bone defect remained unconnected in the original rp scaffolds ( plga/-tcp ) during the whole study . histological observations conformed to the radiographic images . in hybrid rp scaffold group , woven bone united the radial defect at 12 weeks and consecutively remodeled into lamellar bone 24 weeks postoperation and finally matured into cortical bone with normal marrow cavity after another 12 weeks . no bone formation but connective tissue has been detected in rp scaffold at the same time.conclusion:collagen i / apatite sponge composite coating could improve new bone formation in vivo . the hybrid rp scaffold of plga/-tcp skeleton with collagen i / apatite sponge composite coating is a promising candidate for bone tissue engineering .

I M Preparation of PLGA/-TCP skeleton and the PLGA/-TCP-collagen I/apatite hybrid scaffold Marrow Mesenchymal Stem Cells culture and combination with the hybrid scaffolds Operative procedure Evaluation of results Statistical evaluation R Macroscopic observations X-ray analyses Micro CT results Histology D

as reported in an earlier study6 we developed a three dimensional porous hybrid biomaterial consisting of plga , -tricalciumphosphate ( -tcp ) , collagen i and apatite , namely the plga/-tcp - collagen i / apatite scaffold . the purpose of the current study was to evaluate whether the hybrid scaffolds combined with bone marrow mesenchymal stem cells ( bmscs ) can enhance bone defect repair in a rabbit model compared with the plga/-tcp skeleton . radiographic results : ( a ) bone defect caused by osteotomy ; ( b ) radiograph immediately after scaffolds implantation ; ( c - e ) 12 , 24 and 36 weeks postsurgery of the bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/-tricalciumphosphate ( -tcp)-collagen i / apatite composites ; ( f - h ) 12 , 24 and 36 weeks postsurgery of the bmscs - plga/-tcp composites micro ct was used to demonstrate 3d images of the bone defect area as well as to determine the quantity of newly formed bone . bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/ -tricalciumphosphate ( -tcp)-collagen i / apatite composites ( a - i ) : 12 weeks postoperation showing new bone along lateral edge of residual scaffolds ( a - c ) ; 24 weeks postoperation showing nearly recanalized marrow cavity ( d - f ) ; 36 weeks postoperation showing remodelling ( g - i ) ; 2 . 3d reconstruction image ( a , d , g , j , m , p ) ; reconstruction image in the coronal plane ( b , e , h , k , n , q ) ; magnified view ( c , f , i , l , o , r ) of yellow cubic area ( a , d , g , j , m , p ) showing no new bone formation in the hybrid scaffolds group , the growing woven bone fully bridged the defect at 12 weeks after surgery . bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/-tricalciumphosphate ( -tcp)-collagen i / apatite composites ( a - c ) : 12 weeks postoperation ( a ) ; 24 weeks postoperation ( b ) ; 36 weeks postoperation ( c ) ; 2 . when bone defect was treated with hybrid scaffolds ( group a ) , new bone tissue appeared at the proximal and distal ends of the radial defect sites 12 weeks postoperation [ figure 1c ] . radiographic results : ( a ) bone defect caused by osteotomy ; ( b ) radiograph immediately after scaffolds implantation ; ( c - e ) 12 , 24 and 36 weeks postsurgery of the bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/-tricalciumphosphate ( -tcp)-collagen i / apatite composites ; ( f - h ) 12 , 24 and 36 weeks postsurgery of the bmscs - plga/-tcp composites micro ct was used to demonstrate 3d images of the bone defect area as well as to determine the quantity of newly formed bone . bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/ -tricalciumphosphate ( -tcp)-collagen i / apatite composites ( a - i ) : 12 weeks postoperation showing new bone along lateral edge of residual scaffolds ( a - c ) ; 24 weeks postoperation showing nearly recanalized marrow cavity ( d - f ) ; 36 weeks postoperation showing remodelling ( g - i ) ; 2 . 3d reconstruction image ( a , d , g , j , m , p ) ; reconstruction image in the coronal plane ( b , e , h , k , n , q ) ; magnified view ( c , f , i , l , o , r ) of yellow cubic area ( a , d , g , j , m , p ) showing no new bone formation in the hybrid scaffolds group , the growing woven bone fully bridged the defect at 12 weeks after surgery . bone marrow mesenchymal stem cells ( bmscs)-polylactic - co - glycolic acid ( plga)/-tricalciumphosphate ( -tcp)-collagen i / apatite composites ( a - c ) : 12 weeks postoperation ( a ) ; 24 weeks postoperation ( b ) ; 36 weeks postoperation ( c ) ; 2 . b = bone tissue , s = scaffold , mc = medullary cavity , ct = connective tissue , be = broken end of radius in this study , we used a novel bioengineering composite by combining plga/-tcp - collagen i / apatite hybrid scaffold with bmscs and implanted these into critical - sized ( 15 mm ) segmental bone defects in a rabbit model .

1 ) is the major active component of traditional herbal medicine peucedanum praeruptorum dunn and has been proved to be a novel ca influx blocker ( 1 ) . it has been reported that pd - ia could inhibit the expression of apoptosis related proteins , reduce the level of proinflammatory factors , and increase intermediate filament desmin and vimentin contents in ischemia / reperfusion myocardiocytes ( 24 ) . additionally , pd - ia has been reported to be an effective therapeutic drug for treatment of the contractile defects associated with cardiac hypertrophy ( 5 ) . therefore , pd - ia has drawn increasing attentions and displayed bright prospects in prevention and therapy of cardiac diseases . the pharmacokinetics , tissue distribution and excretion of pd - ia in rats following a single intravenous ( i.v . ) few intact form of pd - ia was excreted by bile and kidney , which might be resulted from liver first pass effects ( 6 ) . moreover , based on in vitro study , liver could be the main metabolizing organ and pd - ia is primarily metabolized via hepatic cytochrome p450 isozymes ( cyp ) 3a1 and 3a2 in rats ( not reported ) . the aim of this study was to investigate the influence of liver cirrhosis on the pharmacokinetics of pd - ia after i.v . drug metabolism by cyp isozymes has important clinical consequences because of the possibility of reduced drug metabolism and subsequent increased plasma drug concentrations in subjects with liver dysfunctions ( 7 ) . an experimental liver cirrhotic rat model induced by dimethylnitrosamine was used in this study , which simulates the clinical features of human liver cirrhosis such as mortality , ascites , hepatic parenchymal cell destruction , formation of connective tissue and nodular regeneration ( 8) . it has also been reported that dimethylnitrosamine - induced liver cirrhosis in rats is -reproducible ( 9 ) . diazepam(purity>98% , internal standard ) , dimethylnitrosamine and tris-buffer were obtained from sigma - aldrich(mo , usa).reduced form of -nicotinamide adenine dinucleotidephosphate ( nadph ) was purchased from roche ( basel , switzerland).detection kits for total proteins , albumin , glutamate oxaloacetate transaminase ( got ) , glutamate pyruvate transaminase ( gpt ) , total bilirubin and direct bilirubin were obtainedfrom nanjing jiancheng bioengineering institute(nanjing , china).other chemicals were of analytical or hplc grades . male sprague - dawley rats ( 45 weeks old , 180200 g ) were randomly divided into liver cirrhosis ( lc ) and control groups . freshly prepared dimethylnitrosamine ( solution in physiological saline ) at a dose of 0.01 mgkg was injected intraperitoneally to lc rats on three consecutive days of the week for 4 weeks ( 10 ) . for control rats , the serum of control and lc rats ( n=5 , respectively ) were collected for the measurement of total protein , albumin , got , gpt , total bilirubin and direct bilirubin . the whole kidney , spleen , and liver of each rat were excised , rinsed with physiological saline , blotted dry and weighed ( 10 ) . hepatic total rna was extracted using the trizol extraction method ( sigma - aldrich , usa ) according to the manufacturer 's instructions . reverse transcription of extracted total rna to cdna was performed using a reverse transcription kit ( takara , japan ) . then , the sybr green real - time pcr amplification and detection were performed using the abi 7500 system ( applied biosystems , usa ) and the selective primers for cyp3a1 ( sense primer : 5-gaggcccagctagagggacaaca-3 , antisense primer : 5-cgtagaggagcaccaggacgact-3 ) ( 125 bp ) , cyp3a2 ( sense primer : 5- ggattctaagcataagcaccgagtg-3 , antisense primer : 5-ggccaggaaatacaagacaaaggag-3 ) ( 187 bp ) , and -actin ( sense primer : 5-ccctgtgctgctcaccga-3 , antisense primer : 5-acagtgtgggtgaccccgtc-3 ) ( 170 bp ) genes . the cdna amplifications were carried out in a 20 l reaction volume containing 10 l of power sybr green master mix , 0.5 l each of forward and reverse primers ( 10 moll ) , 4 l of cdna and 5 l of purified water . thermal profile for real - time pcr was 95c for 10 min , and 45 cycles at 95c for 15 sec and 60c for 1 min , followed by the melting curve . the target mrna levels ( n=4 for both groups ) were normalized with the -actin mrna level . rat hepatic microsomes were prepared and the protein content was measured by the reported methods ( 11 , 12 ) . microsomal fractions ( equivalent to 0.5 mg of protein ) were mixed with 50 l of 0.1 moll tris-buffer , ph 7.4 , containing 1 mmoll nadph ; 5 l of pd - ia ( solutions in dimethylsulfoxide at concentrations of 5 , 10 , 25 , 50 , 100 , 250 and 500 moll ) and 0.1 moll tris-buffer , sufficient to a final volume of 0.5 ml . the reaction mixtures were incubated in a water - bath shaker at 37c for 3 min , and then terminated by placing them on ice . all above incubation conditions were linear and pd - ia was measured by lc - ms / ms ( 13 ) . the kinetic parameters ( km , vmax ) for the disappearance of pd - ia were calculated using the nonlinear regression method ( 14 ) . the intrinsic clearance ( clint ) for the disappearance of pd - ia per milligram of protein was calculated by dividing the vmax by the km . the cannulation of the jugular vein and carotid artery was performed for the pretreatment of rats ( 15 ) . pd - ia ( solution in peg400/ tween80/ physiological saline1:1:8 , v / v / v ) at a dose of 5mgkg was infused via the jugular vein to rats in both groups ( n=8 , respectively ) . following administration of pd - ia , a blood sample ( approximately 0.2 ml ) was collected via the carotid artery at 0 ( control ) , 1 ( at the end of the infusion ) , 5 , 15 , 30 , 60 , 90 , 120 , 180 , 240 , 360 and 480 min . each blood sample was immediately centrifuged and a 100 l of supernatant plasma layer was transferred into another tube and stored at 80c until analysis.the 8 hrs bile was collected via bile duct cannulation for each rat in both groups . at the end of the experiment ( 24 hrs ) , each metabolic cage was rinsed with 10 ml of distilled water and the rinsings were combined with 24 hrs urine . then each rat was sacrificed and the entire gastrointestinal tract was removed and homogenated . concentrations of pd - ia in the above samples were measured by lc - ms / ms ( 13 ) and pharmacokinetic parameters were calculated using winnonlin professional edition version 2.1 ( pharsight corporation , usa ) . diazepam(purity>98% , internal standard ) , dimethylnitrosamine and tris-buffer were obtained from sigma - aldrich(mo , usa).reduced form of -nicotinamide adenine dinucleotidephosphate ( nadph ) was purchased from roche ( basel , switzerland).detection kits for total proteins , albumin , glutamate oxaloacetate transaminase ( got ) , glutamate pyruvate transaminase ( gpt ) , total bilirubin and direct bilirubin were obtainedfrom nanjing jiancheng bioengineering institute(nanjing , china).other chemicals were of analytical or hplc grades . male sprague - dawley rats ( 45 weeks old , 180200 g ) were randomly divided into liver cirrhosis ( lc ) and control groups . freshly prepared dimethylnitrosamine ( solution in physiological saline ) at a dose of 0.01 mgkg was injected intraperitoneally to lc rats on three consecutive days of the week for 4 weeks ( 10 ) . for control rats , the serum of control and lc rats ( n=5 , respectively ) were collected for the measurement of total protein , albumin , got , gpt , total bilirubin and direct bilirubin . the whole kidney , spleen , and liver of each rat were excised , rinsed with physiological saline , blotted dry and weighed ( 10 ) . hepatic total rna was extracted using the trizol extraction method ( sigma - aldrich , usa ) according to the manufacturer 's instructions . reverse transcription of extracted total rna to cdna was performed using a reverse transcription kit ( takara , japan ) . then , the sybr green real - time pcr amplification and detection were performed using the abi 7500 system ( applied biosystems , usa ) and the selective primers for cyp3a1 ( sense primer : 5-gaggcccagctagagggacaaca-3 , antisense primer : 5-cgtagaggagcaccaggacgact-3 ) ( 125 bp ) , cyp3a2 ( sense primer : 5- ggattctaagcataagcaccgagtg-3 , antisense primer : 5-ggccaggaaatacaagacaaaggag-3 ) ( 187 bp ) , and -actin ( sense primer : 5-ccctgtgctgctcaccga-3 , antisense primer : 5-acagtgtgggtgaccccgtc-3 ) ( 170 bp ) genes . the cdna amplifications were carried out in a 20 l reaction volume containing 10 l of power sybr green master mix , 0.5 l each of forward and reverse primers ( 10 moll ) , 4 l of cdna and 5 l of purified water . thermal profile for real - time pcr was 95c for 10 min , and 45 cycles at 95c for 15 sec and 60c for 1 min , followed by the melting curve . the target mrna levels ( n=4 for both groups ) were normalized with the -actin mrna level . rat hepatic microsomes were prepared and the protein content was measured by the reported methods ( 11 , 12 ) . microsomal fractions ( equivalent to 0.5 mg of protein ) were mixed with 50 l of 0.1 moll tris-buffer , ph 7.4 , containing 1 mmoll nadph ; 5 l of pd - ia ( solutions in dimethylsulfoxide at concentrations of 5 , 10 , 25 , 50 , 100 , 250 and 500 moll ) and 0.1 moll tris-buffer , sufficient to a final volume of 0.5 ml . the reaction mixtures were incubated in a water - bath shaker at 37c for 3 min , and then terminated by placing them on ice . all above incubation conditions were linear and pd - ia was measured by lc - ms / ms ( 13 ) . the kinetic parameters ( km , vmax ) for the disappearance of pd - ia were calculated using the nonlinear regression method ( 14 ) . the intrinsic clearance ( clint ) for the disappearance of pd - ia per milligram of protein was calculated by dividing the vmax by the km . the cannulation of the jugular vein and carotid artery was performed for the pretreatment of rats ( 15 ) . pd - ia ( solution in peg400/ tween80/ physiological saline1:1:8 , v / v / v ) at a dose of 5mgkg was infused via the jugular vein to rats in both groups ( n=8 , respectively ) . following administration of pd - ia , a blood sample ( approximately 0.2 ml ) was collected via the carotid artery at 0 ( control ) , 1 ( at the end of the infusion ) , 5 , 15 , 30 , 60 , 90 , 120 , 180 , 240 , 360 and 480 min . each blood sample was immediately centrifuged and a 100 l of supernatant plasma layer was transferred into another tube and stored at 80c until analysis.the 8 hrs bile was collected via bile duct cannulation for each rat in both groups . at the end of the experiment ( 24 hrs ) , each metabolic cage was rinsed with 10 ml of distilled water and the rinsings were combined with 24 hrs urine . then each rat was sacrificed and the entire gastrointestinal tract was removed and homogenated . concentrations of pd - ia in the above samples were measured by lc - ms / ms ( 13 ) and pharmacokinetic parameters were calculated using winnonlin professional edition version 2.1 ( pharsight corporation , usa ) . diazepam(purity>98% , internal standard ) , dimethylnitrosamine and tris-buffer were obtained from sigma - aldrich(mo , usa).reduced form of -nicotinamide adenine dinucleotidephosphate ( nadph ) was purchased from roche ( basel , switzerland).detection kits for total proteins , albumin , glutamate oxaloacetate transaminase ( got ) , glutamate pyruvate transaminase ( gpt ) , total bilirubin and direct bilirubin were obtainedfrom nanjing jiancheng bioengineering institute(nanjing , china).other chemicals were of analytical or hplc grades . male sprague - dawley rats ( 45 weeks old , 180200 g ) were randomly divided into liver cirrhosis ( lc ) and control groups . freshly prepared dimethylnitrosamine ( solution in physiological saline ) at a dose of 0.01 mgkg was injected intraperitoneally to lc rats on three consecutive days of the week for 4 weeks ( 10 ) . for control rats , the serum of control and lc rats ( n=5 , respectively ) were collected for the measurement of total protein , albumin , got , gpt , total bilirubin and direct bilirubin . the whole kidney , spleen , and liver of each rat were excised , rinsed with physiological saline , blotted dry and weighed ( 10 ) . hepatic total rna was extracted using the trizol extraction method ( sigma - aldrich , usa ) according to the manufacturer 's instructions . reverse transcription of extracted total rna to cdna was performed using a reverse transcription kit ( takara , japan ) . then , the sybr green real - time pcr amplification and detection were performed using the abi 7500 system ( applied biosystems , usa ) and the selective primers for cyp3a1 ( sense primer : 5-gaggcccagctagagggacaaca-3 , antisense primer : 5-cgtagaggagcaccaggacgact-3 ) ( 125 bp ) , cyp3a2 ( sense primer : 5- ggattctaagcataagcaccgagtg-3 , antisense primer : 5-ggccaggaaatacaagacaaaggag-3 ) ( 187 bp ) , and -actin ( sense primer : 5-ccctgtgctgctcaccga-3 , antisense primer : 5-acagtgtgggtgaccccgtc-3 ) ( 170 bp ) genes . the cdna amplifications were carried out in a 20 l reaction volume containing 10 l of power sybr green master mix , 0.5 l each of forward and reverse primers ( 10 moll ) , 4 l of cdna and 5 l of purified water . thermal profile for real - time pcr was 95c for 10 min , and 45 cycles at 95c for 15 sec and 60c for 1 min , followed by the melting curve . the target mrna levels ( n=4 for both groups ) were normalized with the -actin mrna level . rat hepatic microsomes were prepared and the protein content was measured by the reported methods ( 11 , 12 ) . microsomal fractions ( equivalent to 0.5 mg of protein ) were mixed with 50 l of 0.1 moll tris-buffer , ph 7.4 , containing 1 mmoll nadph ; 5 l of pd - ia ( solutions in dimethylsulfoxide at concentrations of 5 , 10 , 25 , 50 , 100 , 250 and 500 moll ) and 0.1 moll tris-buffer , sufficient to a final volume of 0.5 ml . the reaction mixtures were incubated in a water - bath shaker at 37c for 3 min , and then terminated by placing them on ice . all above incubation conditions were linear and pd - ia was measured by lc - ms / ms ( 13 ) . the kinetic parameters ( km , vmax ) for the disappearance of pd - ia were calculated using the nonlinear regression method ( 14 ) . the intrinsic clearance ( clint ) for the disappearance of pd - ia per milligram of protein was calculated by dividing the vmax by the km . the cannulation of the jugular vein and carotid artery was performed for the pretreatment of rats ( 15 ) . pd - ia ( solution in peg400/ tween80/ physiological saline1:1:8 , v / v / v ) at a dose of 5mgkg was infused via the jugular vein to rats in both groups ( n=8 , respectively ) . following administration of pd - ia , a blood sample ( approximately 0.2 ml ) was collected via the carotid artery at 0 ( control ) , 1 ( at the end of the infusion ) , 5 , 15 , 30 , 60 , 90 , 120 , 180 , 240 , 360 and 480 min . each blood sample was immediately centrifuged and a 100 l of supernatant plasma layer was transferred into another tube and stored at 80c until analysis.the 8 hrs bile was collected via bile duct cannulation for each rat in both groups . at the end of the experiment ( 24 hrs ) , each metabolic cage was rinsed with 10 ml of distilled water and the rinsings were combined with 24 hrs urine . then each rat was sacrificed and the entire gastrointestinal tract was removed and homogenated . concentrations of pd - ia in the above samples were measured by lc - ms / ms ( 13 ) and pharmacokinetic parameters were calculated using winnonlin professional edition version 2.1 ( pharsight corporation , usa ) . body weight , organ weight and serum biochemical data in control and lc rats are listed in table 1 . body weight gain decreased significantly in lc rats ( from 192 to 274 g ) compared with that in control rats ( from 189 to 372 g ) . in lc rats , the whole liver and kidney weight were lower ( 55.1% and 17.9% decrease , respectively ) , but the whole spleen weight was significantly higher ( 52.4% increase ) than those in control rats . the liver weight based on percentage of body weight was significantly lower ( 39.9% decrease ) , however , the kidney and spleen weight based on percentage of body weight was higher ( 11.9% and 107.0% increase , respectively ) in lc rats . compared with the control rats , the lc ratshad significant decrease in seruml evels of total protein ( 37.9% ) and albumin ( 33.4% ) ; significant increased serum levels of got ( 84.5% increase ) , gpt ( 125.7% increase),total bilirubin ( 289.7% ) and direct bilirubin ( 447.8%).the data suggested that the presence of liver cirrhosis in lc rats was apparent . mean ( sd ) body weight , organ weight and serum biochemical data in control and lc rats ( n=5 , respectively ) . p<0.01 , significant difference compared to control cirrhosis may alter drug disposition by a variety of mechanisms including a reduction in absolute cell mass with function retained in surviving cells ; changes in the enzyme levels and/or activity in surviving hepatocytes ; impaired drug entry into hepatocytes ; and impaired uptake of oxygen ( 16).to further evaluate the potential mechanisms responsible for the metabolism of pd - ia in lc rats , changes in the hepatic mrna expression of cyp3a1 and 3a2 were investigated and are shown in figure 2 . the hepatic mrna expression of cyp3a1 and 3a2 in lc rats decreased significantly ( 73.1% and 64.1% , respectively ) , compared with that of control rats . hepatic mrna expression of cyp3a1 and cyp3a2 in control and lc rats was measured by real - time pcr . table 2 shows that the maximum velocity ( v max ) for the disappearance of pd - ia in lc rats was significantly slower ( 63.4% decrease ) than that of control rats , suggesting that the maximal ability to metabolize pd - ia in hepatic microsomes was significantly slower . however , the michaelis - menten constant ( k m ) was not significantly different between two groups suggesting that the affinity of pd - ia to the enzyme(s ) was not changed in lc rats . hence , the intrinsic clearance ( clint ) for the disappearance of pd - ia in lc rats was significantly slower ( 62.8% decrease ) than that of control rats suggesting that metabolism of pd - ia could be slower in lc rats . mean ( sd ) kinetic parameters for the disappearance of pd - ia in control and lc rats ( n=6 , respectively ) . vmax : maximum velocity ; k : michaelis - menten constant ; clint : intrinsic clearance p<0.01 , significant difference compared to control the mean arterial plasma concentration - time curves of pd - ia after i.v . administration at single dose of 5 mgkg to control and lc rats are shown in figure 3 , and the relevant pharmacokinetic parameters are listed in table 3 . administration , the mean arterial plasma concentrations of pd - ia were higher in lc rats , and this resulted in a significant greater auc0 - 8 ( 63.5% increase ) . the terminal half - life ( t 1/2 ) in lc rats was significantly longer ( 67.9% increase ) and the total body clearance ( cl ) was significantly lower ( 46.4% decrease ) . the total amount of unchanged pd ia excreted in 24 hrs urine ( ae0 - 24h , expressed in terms of percentage of i.v . these results indicated that the contribution of renal clearance ( clr ) to cl of pd - ia was almost negligible and the cl of pd - ia could represent non renal clearance ( clnr ) in rats . moreover , unchanged pd - ia recovered from the gastrointestinal tract at 24 hrs ( gi24 h ) was below detection limit for both groups ; the total amount of unchanged pd - ia in 8 hrs bile ( expressed in terms of percentage of i.v . dose ) were 0.092% and 0.126% for control and lc rats , suggesting that the contribution of gastrointestinal and biliary excretion to clnr of pd - ia was also negligible hence , the clnr could represent hepatic intrinsic clearance ( clint ) of pd - ia which was metabolized almost completely after i.v . mean ( sd ) plasma concentration - time curves of pd - ia after intravenous administration at a dose of 5 mgkg to control rats ( ; n=8 ) and lc rats ( ; n=8 ) . mean ( sd ) pharmacokinetic parameters of pd - ia after intravenous administration auc0 - 8 : area under the analyte concentrations versus time curve from time 0 to infinity ; t 1/2 : terminal half - life ; cl : total body clearance ; vz : terminal volume of distribution ; ae024 h : urine samples at 24 hrs ; gi24 h : gastrointestinal tract samples at 24 hrs bdl : below the detection limit . * * p<0.01 , significant difference compared to control . the auc0 - 8 after i.v . administration of pd - ia in lc rats was significantly higher than that in control rats , possibly as a result of the significant slower cl of pd - ia ( table 3 ) . since the clnr of pd - ia could represent its metabolic clearance and pd - ia was metabolized almost completely after i.v . administration in rats , the slower cl could be due to significant slower hepatic clint for the disappearance of pd - ia ( table 2 ) and slower hepatic blood flow rate ( 17 ) . the slower hepatic clint could be at least partly attributed to significant decrease in mrna expression of cyp3a1 and 3a2 compared with that in control rats ( fig . 2 ) since pd - ia is primarily metabolized via hepatic cyp3a1 and 3a2 in rats . moreover , it has been reported that the protein expression of hepatic cyp3a was decreased in lc rats ( 18 ) . the present results suggest that after i.v.administration of pd - ia at a single dose of 5 mgkg to lc rats , the auc0 - 8 was significantly greater than that of the control rats which could be due to slower hepatic blood flow rate and significant slower hepatic clint in rats . in addition , the decreased metabolic clearance of pd - ia in lc rats might be at least partly caused by the decreased levels of cyp3a1 and 3a2 responsible for pd - ia metabolism . these findings may provide new insights into the inter- and intra - individual pharmacokinetic variability of pd - ia .

background and the purpose of the study as a novel drug in the treatment of cardiac diseases , dl - praeruptorin a ( pd - ia ) is the major active component of traditional herbal medicine peucedanum praeruptorum dunn and is metabolized primarily via cytochrome p450 isozymes ( cyp ) 3a1 and 3a2 in rats . in the present study , the influence of liver cirrhosis on pharmacokinetics of pd - ia and hepatic mrna expression of cyp3a1 and 3a2 in rats with experimental liver cirrhosis ( lc rats ) were evaluated.methods pd - ia was given intravenously ( 5 mg kg1 ) to lc rats induced by dimethylnitrosamine and pharmacokinetic variables were measured . enzyme kinetic metabolism of pd - ia in rat hepatic microsomes was also investigated and hepatic mrna expression of cyp3a1 and 3a2 were measured by real - time pcr.results and major conclusion after intravenous administration in lc rats , the area under the plasma concentration - time curve from time zero to infinity ( auc0 - 8 ) was significantly greater than that in control rats , which might be due to slower rate of the hepatic blood flow and significant slower hepatic intrinsic clearance ( clint ) in rats . the decreased metabolic clearance of pd - ia in lc rats might be at least partly caused by the decreased levels of cyp3a1 and 3a2 responsible for pd - ia metabolism . these findings may provide new insights into the inter- and intra - individual pharmacokinetic variability of pd - ia .

INTRODUCTION MATERIALS AND METHODS None Materials Induction of liver cirrhosis Preliminary study and real-time PCR analysis of CYP3A1 and 3A2 Enzyme kinetic metabolism of Pd-Ia Pharmacokinetic study of Pd-Ia RESULTS AND DISCUSSION CONCLUSION

1 ) is the major active component of traditional herbal medicine peucedanum praeruptorum dunn and has been proved to be a novel ca influx blocker ( 1 ) . moreover , based on in vitro study , liver could be the main metabolizing organ and pd - ia is primarily metabolized via hepatic cytochrome p450 isozymes ( cyp ) 3a1 and 3a2 in rats ( not reported ) . the aim of this study was to investigate the influence of liver cirrhosis on the pharmacokinetics of pd - ia after i.v . concentrations of pd - ia in the above samples were measured by lc - ms / ms ( 13 ) and pharmacokinetic parameters were calculated using winnonlin professional edition version 2.1 ( pharsight corporation , usa ) . p<0.01 , significant difference compared to control cirrhosis may alter drug disposition by a variety of mechanisms including a reduction in absolute cell mass with function retained in surviving cells ; changes in the enzyme levels and/or activity in surviving hepatocytes ; impaired drug entry into hepatocytes ; and impaired uptake of oxygen ( 16).to further evaluate the potential mechanisms responsible for the metabolism of pd - ia in lc rats , changes in the hepatic mrna expression of cyp3a1 and 3a2 were investigated and are shown in figure 2 . the hepatic mrna expression of cyp3a1 and 3a2 in lc rats decreased significantly ( 73.1% and 64.1% , respectively ) , compared with that of control rats . hepatic mrna expression of cyp3a1 and cyp3a2 in control and lc rats was measured by real - time pcr . table 2 shows that the maximum velocity ( v max ) for the disappearance of pd - ia in lc rats was significantly slower ( 63.4% decrease ) than that of control rats , suggesting that the maximal ability to metabolize pd - ia in hepatic microsomes was significantly slower . hence , the intrinsic clearance ( clint ) for the disappearance of pd - ia in lc rats was significantly slower ( 62.8% decrease ) than that of control rats suggesting that metabolism of pd - ia could be slower in lc rats . administration , the mean arterial plasma concentrations of pd - ia were higher in lc rats , and this resulted in a significant greater auc0 - 8 ( 63.5% increase ) . these results indicated that the contribution of renal clearance ( clr ) to cl of pd - ia was almost negligible and the cl of pd - ia could represent non renal clearance ( clnr ) in rats . dose ) were 0.092% and 0.126% for control and lc rats , suggesting that the contribution of gastrointestinal and biliary excretion to clnr of pd - ia was also negligible hence , the clnr could represent hepatic intrinsic clearance ( clint ) of pd - ia which was metabolized almost completely after i.v . mean ( sd ) plasma concentration - time curves of pd - ia after intravenous administration at a dose of 5 mgkg to control rats ( ; n=8 ) and lc rats ( ; n=8 ) . mean ( sd ) pharmacokinetic parameters of pd - ia after intravenous administration auc0 - 8 : area under the analyte concentrations versus time curve from time 0 to infinity ; t 1/2 : terminal half - life ; cl : total body clearance ; vz : terminal volume of distribution ; ae024 h : urine samples at 24 hrs ; gi24 h : gastrointestinal tract samples at 24 hrs bdl : below the detection limit . administration of pd - ia in lc rats was significantly higher than that in control rats , possibly as a result of the significant slower cl of pd - ia ( table 3 ) . administration in rats , the slower cl could be due to significant slower hepatic clint for the disappearance of pd - ia ( table 2 ) and slower hepatic blood flow rate ( 17 ) . the slower hepatic clint could be at least partly attributed to significant decrease in mrna expression of cyp3a1 and 3a2 compared with that in control rats ( fig . the present results suggest that after i.v.administration of pd - ia at a single dose of 5 mgkg to lc rats , the auc0 - 8 was significantly greater than that of the control rats which could be due to slower hepatic blood flow rate and significant slower hepatic clint in rats . in addition , the decreased metabolic clearance of pd - ia in lc rats might be at least partly caused by the decreased levels of cyp3a1 and 3a2 responsible for pd - ia metabolism . these findings may provide new insights into the inter- and intra - individual pharmacokinetic variability of pd - ia .

anxiety is one of the most prevalent symptoms in pregnancy and has the largest share in labor . these effects include spontaneous abortion , low fetal weight , elevated levels of stress hormones , chronic increase in blood pressure , premature birth and infant mortality , change in endocrine secretion , change in hypothalamic - pituitary axis performance , immune system suppression , and changes in the number of lymphocytes and decrease in cd4/cd8 cells . the role of prenatal stress in the etiology of childhood illnesses and adulthood disorders and maternal postpartum depression is taken into consideration . however , pregnancy and the puerperium can be stressful enough to trigger mental disorders which may reflect the recurrence or exacerbation of ex - psychiatric disorders ( preexisting ) or may implicate the onset of a new disorder . chemical factors ( including hormones of progesterone - estrogen - cortisol - thyroid hormones ) and life stressors can have a significant impact on mental health . hence , logically it seems that pregnancy is supposed to have an impact on the number of concurrent psychiatric disorders . women in a variety of ways respond to the environmental stressors and ongoing concerns about fetus health , child care , lifestyle changes , or fear of labor pain . in a study , negative effects of high antenatal maternal anxiety related to impulsivity during a performance on cognitive tasks in 14 and 15 years old subjects were mentioned . 's study has shown that maternal trait anxiety , depression , and life event stress during pregnancy are associated with the incidence of complications in infant temperament at 64 months after birth . furthermore , the results of robertson 's study on 14,000 samples have shown that antenatal depression , anxiety , and life event stress in pregnancy can develop postpartum depression . in this respect , religion is supportive for people encountered with problems , and religious beliefs and activities such as prayer , worship , trust and appeal to god are defined as religious resources to increase tolerance . moreover , religious practices such as prayer , honesty and faith and also study of religious books in developing hope , encouragement and positive attitudes toward depressive conditions and helping the one to get out of the depressive crisis would create a kind of inner peace . some studies have shown that intrinsic religious orientation is among the organized psychological behavior of great importance in the treatment of diseases and plays basic roles in the treatment of pain , anxiety , depression , and stressors . since the late 20 century , the studies have revealed the effects of religious interventions for improving psychological suffering . furthermore , the effects of praying and reciting to control the psychological disorders are considerable . studies not only show the positive relationship between religion and mental health but also reflect the influence of religion on health status . the current study has been designed to investigate the religious teaching impact on the religious knowledge and attitudes of primiparous women referring to the selected prenatal clinics of tehran university of medical sciences in july 2013 up june 2014 . this study aimed at reducing antenatal maternal anxiety with religious education and use and improving mental health in pregnant women in pregnancy and postpartum periods . the present randomized clinical trial was conducted in the selected prenatal clinics of tehran university of medical sciences in 2013 . according to the statistical consultation , considering = 0.05 , = 0.2 , d = 2 , and power = 95% , and using the following formula , an 84-subject sample size ( 42 in each group ) was determined for the study [ figure 1 ] : the study research community protocol consort diagram after signing written informed consents , the individuals , based on the inclusion criteria , were enrolled in the study . sequence sampling was as follows . without the knowledge of researchers from mother 's attitude the final sample was selected in several stages : ( 1 ) 220 individuals were selected among those referring to the clinics affiliated with tehran university of medical sciences by purposive sampling . ( 2 ) they were randomly divided into a control and an intervention group using the table of random numbers . in doing so , the first qualified individual was allocated to the intervention group and the second one was allocated to the control group ( each group with 110 patients ) . ( 3 ) the final sample was obtained after the completion of the two scales ( knowledge assessment questionnaire and revised attitude scale - religious ( ras - r ) . ( 4 ) initially , 110 samples , 42 participants in each group , with average or weak religious knowledge and attitude were enrolled in the study , while those with high religious knowledge and attitude were excluded . both groups completed the demographic questionnaire , spielberger 's anxiety scale , knowledge questionnaire , and ras - r before and immediately and 2 months after the intervention . the state - trait anxiety inventory is a common and important construct in the study of the human experience of health and illness . various studies have shown that it is a sensitive scale for evaluation of the severity of anxiety . spielberger 's scale which is used for assessment of state and trait anxiety ( 20 items for each ) includes 40 items giving 80 scores . it is scored using a likert scale , and scores of 019 , 2040 , 4160 , and 6180 are assigned to normal , mild , average , and severe anxiety , respectively . the reliability of this scale was obtained as 0.97 . the reliability and validity indexes reported by aghamohammad ( 2007 ) are the basis of the present study . ras - r includes 25 items in 6 dimensions of pray , ethics and values , the effect of religion on life and behavior ( praying and fasting ) , social issues , world view and beliefs , and science and religion . this questionnaire is scored through a likert scale in such a way that scores of 45 , 12 , and 3 are given to the options with a positive attitude , options with a negative attitude , and those in between , respectively . scores > 100 , 50100 , and < 50 represent high , average , and low religious attitude , respectively . the correlation coefficient of the score of each item and the whole questionnaire score was reliable at 0.0001 level . the reliability of the questionnaire was obtained as 0.948 and 0.933 using spearman brown and guttman methods , respectively . the reliability and validity indexes reported in ebrahimi 's study were the basis of the current study . knowledge assessment questionnaire was a researcher - made instrument including 41 items in 6 dimensions of pray , ethics and values , the effect of religion on life and behavior ( praying and fasting ) , social issues , world view and beliefs , and science and religion . this questionnaire is scored through a likert scale in such a way that scores of 45 , 12 , and 3 are given to the options with a positive attitude , options with a negative attitude , and those in between , respectively . besides , scores > 164 , 82164 , and < 82 represent high , average , and low religious knowledge , respectively . to determine the validity of the questionnaire , it was given to 10 expert professors of schools of nursing and midwifery of shiraz and tehran universities of medical sciences . after application of the professors recommendations , it was given to 30 pregnant women in a pilot study and its reliability was assessed . in doing so , cronbach 's alpha coefficient was separately computed for different sections of the questionnaire revealing its internal consistency . in addition , the whole questionnaire 's reliability was confirmed by cronbach 's alpha = 81% . in the intervention group , instruction of religious doctrines the first three sessions were conducted by the researcher , whereas the last three ones were managed by a clergyman . after the end of the classes the content of the educational classes was as follows : first session : problems during the pregnancy period , importance of pregnancy period in islamic culture , and recommendations for the pregnancy period ( praying , dietary recommendations , and advices for after delivery ) , second session : breastfeeding , breastfeeding in quran , infant 's right for breastfeeding , and reward of breastfeeding , third session : individual ethics ( trust , sincerity , gratefulness ) , importance of mother 's characteristics such as good - temperedness , cheerfulness , kindness , modesty , forgiveness , and mental health in pregnancy , fourth session : collective ethics , good - temperedness , and showing empathy with others , fifth session : emotional feeling toward god including love of god , fear from god 's unhappiness , emotional feeling toward others , emotional feeling toward parents , positive emotion toward one - self , and positive emotion toward the world , and sixth session : doing one 's religious duties , praying ( individual praying , fasting ) , and importance of collective prayers . this research project was approved by the local ethics committee of shiraz university of medical sciences and written informed consents were obtained from all the participants . the research proposal no was 92 - 01 - 86 - 6947 and it was financially supported by endocrine and metabolism research center , shiraz university of medical sciences . the research in iranian registry of clinical trial was registered with registration number : irct : 2014030414041n3 . chicago , spss inc ) ; to evaluate the homogeneity of age ( independent sample t - test ) , education and level of income , chi - squared test was used . in addition to comparing the level of knowledge , attitude , and anxiety in the two groups , independent sample t - test were used . the present randomized clinical trial was conducted in the selected prenatal clinics of tehran university of medical sciences in 2013 . according to the statistical consultation , considering = 0.05 , = 0.2 , d = 2 , and power = 95% , and using the following formula , an 84-subject sample size ( 42 in each group ) was determined for the study [ figure 1 ] : the study research community protocol consort diagram after signing written informed consents , the individuals , based on the inclusion criteria , were enrolled in the study . sequence sampling was as follows . without the knowledge of researchers from mother 's attitude the final sample was selected in several stages : ( 1 ) 220 individuals were selected among those referring to the clinics affiliated with tehran university of medical sciences by purposive sampling . ( 2 ) they were randomly divided into a control and an intervention group using the table of random numbers . in doing so , the first qualified individual was allocated to the intervention group and the second one was allocated to the control group ( each group with 110 patients ) . ( 3 ) the final sample was obtained after the completion of the two scales ( knowledge assessment questionnaire and revised attitude scale - religious ( ras - r ) . ( 4 ) initially , 110 samples , 42 participants in each group , with average or weak religious knowledge and attitude were enrolled in the study , while those with high religious knowledge and attitude were excluded . both groups completed the demographic questionnaire , spielberger 's anxiety scale , knowledge questionnaire , and ras - r before and immediately and 2 months after the intervention . the state - trait anxiety inventory is a common and important construct in the study of the human experience of health and illness . various studies have shown that it is a sensitive scale for evaluation of the severity of anxiety . spielberger 's scale which is used for assessment of state and trait anxiety ( 20 items for each ) includes 40 items giving 80 scores . it is scored using a likert scale , and scores of 019 , 2040 , 4160 , and 6180 are assigned to normal , mild , average , and severe anxiety , respectively . the reliability of this scale was obtained as 0.97 . the reliability and validity indexes reported by aghamohammad ( 2007 ) are the basis of the present study . ras - r includes 25 items in 6 dimensions of pray , ethics and values , the effect of religion on life and behavior ( praying and fasting ) , social issues , world view and beliefs , and science and religion . this questionnaire is scored through a likert scale in such a way that scores of 45 , 12 , and 3 are given to the options with a positive attitude , options with a negative attitude , and those in between , respectively . scores > 100 , 50100 , and < 50 represent high , average , and low religious attitude , respectively . the correlation coefficient of the score of each item and the whole questionnaire score was reliable at 0.0001 level . the reliability of the questionnaire was obtained as 0.948 and 0.933 using spearman brown and guttman methods , respectively . the reliability and validity indexes reported in ebrahimi 's study were the basis of the current study . knowledge assessment questionnaire was a researcher - made instrument including 41 items in 6 dimensions of pray , ethics and values , the effect of religion on life and behavior ( praying and fasting ) , social issues , world view and beliefs , and science and religion . this questionnaire is scored through a likert scale in such a way that scores of 45 , 12 , and 3 are given to the options with a positive attitude , options with a negative attitude , and those in between , respectively . besides , scores > 164 , 82164 , and < 82 represent high , average , and low religious knowledge , respectively . to determine the validity of the questionnaire , it was given to 10 expert professors of schools of nursing and midwifery of shiraz and tehran universities of medical sciences . after application of the professors recommendations , it was given to 30 pregnant women in a pilot study and its reliability was assessed . in doing so , cronbach 's alpha coefficient was separately computed for different sections of the questionnaire revealing its internal consistency . in addition , the whole questionnaire 's reliability was confirmed by cronbach 's alpha = 81% . in the intervention group , instruction of religious doctrines the first three sessions were conducted by the researcher , whereas the last three ones were managed by a clergyman . after the end of the classes the content of the educational classes was as follows : first session : problems during the pregnancy period , importance of pregnancy period in islamic culture , and recommendations for the pregnancy period ( praying , dietary recommendations , and advices for after delivery ) , second session : breastfeeding , breastfeeding in quran , infant 's right for breastfeeding , and reward of breastfeeding , third session : individual ethics ( trust , sincerity , gratefulness ) , importance of mother 's characteristics such as good - temperedness , cheerfulness , kindness , modesty , forgiveness , and mental health in pregnancy , fourth session : collective ethics , good - temperedness , and showing empathy with others , fifth session : emotional feeling toward god including love of god , fear from god 's unhappiness , emotional feeling toward others , emotional feeling toward parents , positive emotion toward one - self , and positive emotion toward the world , and sixth session : doing one 's religious duties , praying ( individual praying , fasting ) , and importance of collective prayers . this research project was approved by the local ethics committee of shiraz university of medical sciences and written informed consents were obtained from all the participants . the research proposal no was 92 - 01 - 86 - 6947 and it was financially supported by endocrine and metabolism research center , shiraz university of medical sciences . the research in iranian registry of clinical trial was registered with registration number : irct : 2014030414041n3 . chicago , spss inc ) ; to evaluate the homogeneity of age ( independent sample t - test ) , education and level of income , chi - squared test was used . in addition to comparing the level of knowledge , attitude , and anxiety in the two groups , independent sample t - test were used . the results of the demographic data analysis in table 1 show that the subjects in the cohorts of intervention and control are homogeneous regarding demographic characteristics and some variables . demographic characteristics of primiparous women in intervention and control groups to evaluate the religious knowledge and attitudes in pregnant women , t - test results [ table 2 ] indicated that there was no difference in religious knowledge score between the intervention and control group subjects before the intervention p = 0.1 ; whereas there existed a significant difference between the intervention and control groups after the intervention and 2 months after the study ( p = 0.001 ) . independent t - test results , as shown in table 3 , indicated that there was no significant difference in the religious attitude score between the two groups of intervention and control before the intervention p = 0.9 , while between the intervention and control groups after the intervention ( p = 0.001 ) and 2 months after the study there was a significant difference ( p = 0.001 ) . comparison of religious knowledge scores before and after the intervention and 2 months after completion of the study in the intervention and control groups comparison of the religious attitude score before and after the intervention , and 2 months after completion of the study in the intervention and control groups in evaluating the anxiety level in the intervention group after training , the state anxiety in severe anxiety declined from 7.1% to 2.4% while the moderate anxiety remained unchanged ( 40.5% ) and the mild anxiety changed from 52.4% to 57.1% . thus , a slight increase in the state anxiety suggested that the volume of severe anxiety was reduced in the intervention group . in the control group , severe anxiety increased from 9.5% before the intervention to 11.9% after the intervention and to 24.4% within 2 months after the intervention . moderate state anxiety in the control group increased from 66.7% before the intervention to 64.3% after the intervention and to 71.4% within 2 months after the intervention . mild anxiety individuals reduced in size due to allocation of more individuals in the moderate and severe anxiety groups . the severe trait anxiety of individuals in the intervention group after religious education decreased from 7.1% to 0% and the reduction remained constant 2 months after completion of the intervention . however , the moderate trait anxiety of the individuals increased from 47.6% to 73.8% after the intervention and then remained fixed for up to 2 months after completion of the training . moreover also , mild personality anxiety slightly increased from 19% to 29% after the intervention and the volume reduced to 10% 2 months after the intervention . finally , the volume of more severe personality anxiety reduced in the intervention group . severe anxiety in the control group increased from 14.3% before the intervention to 16.7% after the intervention and to 23.8% within 2 months after the intervention . in the control group , moderate personality anxiety had increased from 59.5% before the intervention to 64.3% after the intervention and 73.8% after 2 months of completion of the intervention . furthermore , the number of individuals in the mild trait anxiety reduced and this was due to allocation of more people in the moderate and severe anxiety group . in evaluating the level of anxiety in pregnant women , the independent t - test results in table 4 show that there was a significant difference in the state anxiety score in primigravida women between the intervention and control groups before the intervention , ( p = 0.002 ) ; so , between the two cohorts of intervention and control after the intervention ( p = 0.001 ) and 2 months after the intervention there was a significant difference ( p = 0.001 ) . the independent t - test results in table 5 has shown that personality anxiety scores between the two cohorts have no difference preintervention ( p = 0.0197 ) so , between the intervention and control cohorts after the intervention ( p = 0.001 ) and 2 months later there existed a significant difference ( p = 0.001 ) . comparison of state anxiety scores before and after intervention and 2 months after completion of the intervention in the control and intervention groups comparison of personality anxiety scores before and after intervention and 2 months after completion of the intervention in the control and intervention groups based on the obtained results in this study , religious education has led to an increase in religious knowledge and attitudes among pregnant women . therefore , an increase in the knowledge and attitudes of pregnant women indicated the influence of religion on them . in the control group which had equal qualifications as the intervention group but instead of attending religious teachings was given only routine cares , no increase in the religious knowledge and attitudes was observed . the results indicated that by using religious education , the religious knowledge , and attitudes of participants in the training group in comparison with the controls went up and after 6 sessions of training , both state and trait anxiety have reduced . severe state anxiety in the first trimester of pregnancy in the intervention cohort ceased , and the individual stages of anxiety were in the range of mild and moderate , whereas those in the control group in the third trimester of pregnancy had 21.4% of severe state anxiety and 23.8% of severe trait anxiety . in a study on 4132 individuals aged 65 and older in canada , who regularly worshiped and participated in the religious gatherings , it was demonstrated that they suffered less from depression while there was no relationship between depression and worship . the depression was obvious among those who only listened to the religious radio and watched religious tv . involvement in religious rituals , social ties and social support from the religious gatherings may strengthen the beliefs and religious practices . in another study , it was indicated that belief or faith in god or reading or reciting bible and strong connection with the church could help the hospitalized medically ill patients . several groups of researchers by reviewing the studies have concluded that religious beliefs and religious practices have a positive impact on people 's physical and mental health status . a longitudinal study was conducted on 1091 secular individuals from east baltimore in canada from 1981 to 2004 . the study participants attended religious worship gatherings at least once a year . using a questionnaire , it was revealed that how often the individuals participated in the religious worship gatherings and to what extent the comfortable feelings and peace increased in them ( confidence interval : 0.310.99 adjusted odds ratios = 0.55 , 95% ) . the results of the mentioned study were consistent with those of this study . in a longitudinal study on 69 women in the 2840 weeks of gestation and older than 18 years in latin america , the relationship between spirituality and psychological , social , and maternal outcomes was investigated . in this study , 65% of the participants had high and moderate religious tendencies and 71.4% had spiritual tendencies and adhered to moral principles . the other results showed that : spiritual : religious values revealed a significant correlation with social protection and the average time of delivery , with perceived stress and depressive symptoms and the increased average birth weight . . spiritual : religious values revealed a significant correlation with social protection and the average time of delivery , with perceived stress and depressive symptoms and the increased average birth weight . the results of this study are consistent with ours in reducing the mothers anxiety . this cross - sectional study ( 2005 ) was carried out on 300 pregnant women referring to 6 health care centers of iran university of medical sciences in tehran , in the field of health knowledge of islam during pregnancy period . the results showed that knowledge of 20.6% of the participants was fine , and that of 64.7% and 14.7% was average and poor . health knowledge of islam during lactation was 3.6% good , 77% moderate , and 16.7% were poor . furthermore , it was shown that in 91.7% of the mothers who recited quran anxiety was reduced during pregnancy . the results of this study are consistent with our findings in reducing the anxiety of the mothers and the importance of education in religious teachings during pregnancy together with medical training . in a meta - analysis conducted in the field of mental health and religious studies , results showed a positive relationship between religion and mental health in 47% , in 23% of them there was a negative relationship , and in 30% no significant relationship was observed . evaluation studies show that religious concerns can have a protective effect on anxiety and depression during pregnancy . so , to raise knowledge and improve mental health during pregnancy and postpartum , training programs for prenatal care associated with islamic teachings and training of health staff are also recommended . in the end , it is recommended that pregnant women with spirituality can cope with the stress in pregnancy and spend pregnancy and postpartum period with more comfort . hence , medical teams can double the effectiveness by appealing to religious culture in addition to the gained science and knowledge . furthermore , women who were trained showed a good resistance against pregnancy and severe anxiety ; in general , the level of anxiety was reduced in comparison with the group who had received no training . this can act as a guide for planners and health authorities of the country to emphasize more on religious beliefs in the health education programs for pregnant women and use the strategic element of religion in counseling with mothers with mental health disorders . the study was financially supported by the research vice - chancellor of shiraz university of medical sciences , shiraz , iran . the study was financially supported by the research vice - chancellor of shiraz university of medical sciences , shiraz , iran .

background : anxiety is among the most common pregnancy complications . this study was conducted to examine the impact of religious teaching on anxiety in primiparous mothers referring to the selected perinatal clinics of tehran university of medical sciences in 2013.materials and methods : this randomized clinical trial was conducted on the pregnant women in 2028 weeks of gestation referring to the selected clinics of tehran university of medical sciences from july 2013 to june 2014 . the subjects were selected through simple random sampling and divided into religious education and control groups . to assess the individuals , a demographic questionnaire , an anxiety trait state - trait anxiety inventory and a religious knowledge and attitude trait ( pre- test and post - test and 1 or 2 months after the test ) were filled in by the two groups . training classes ( religious knowledge and attitude trait ) for the cases were held in 6 weeks , and the sessions lasted for 1 h.results:the knowledge and attitude scores showed significant differences in the controls and cases after the intervention ( p = 0.001 ) and 2 months after the study ( p = 0.001 ) . according to the results of independent t - test , a significant difference was found in the state anxiety score ( p = 0.002 ) and personal score ( p = 0.0197 ) between the two groups before the intervention ; however , the results were strongly significant different after the intervention and 2 months after the study ( p 0.001).conclusions : the improvement in the mothers knowledge and attitude in religious subjects will reduce anxiety in primiparas .

Introduction Materials and Methods Setting and design Ethics Statistical analysis Results Discussion Conclusions Financial support and sponsorship Conflicts of interest

the current study has been designed to investigate the religious teaching impact on the religious knowledge and attitudes of primiparous women referring to the selected prenatal clinics of tehran university of medical sciences in july 2013 up june 2014 . the present randomized clinical trial was conducted in the selected prenatal clinics of tehran university of medical sciences in 2013 . according to the statistical consultation , considering = 0.05 , = 0.2 , d = 2 , and power = 95% , and using the following formula , an 84-subject sample size ( 42 in each group ) was determined for the study [ figure 1 ] : the study research community protocol consort diagram after signing written informed consents , the individuals , based on the inclusion criteria , were enrolled in the study . both groups completed the demographic questionnaire , spielberger 's anxiety scale , knowledge questionnaire , and ras - r before and immediately and 2 months after the intervention . the present randomized clinical trial was conducted in the selected prenatal clinics of tehran university of medical sciences in 2013 . according to the statistical consultation , considering = 0.05 , = 0.2 , d = 2 , and power = 95% , and using the following formula , an 84-subject sample size ( 42 in each group ) was determined for the study [ figure 1 ] : the study research community protocol consort diagram after signing written informed consents , the individuals , based on the inclusion criteria , were enrolled in the study . both groups completed the demographic questionnaire , spielberger 's anxiety scale , knowledge questionnaire , and ras - r before and immediately and 2 months after the intervention . demographic characteristics of primiparous women in intervention and control groups to evaluate the religious knowledge and attitudes in pregnant women , t - test results [ table 2 ] indicated that there was no difference in religious knowledge score between the intervention and control group subjects before the intervention p = 0.1 ; whereas there existed a significant difference between the intervention and control groups after the intervention and 2 months after the study ( p = 0.001 ) . independent t - test results , as shown in table 3 , indicated that there was no significant difference in the religious attitude score between the two groups of intervention and control before the intervention p = 0.9 , while between the intervention and control groups after the intervention ( p = 0.001 ) and 2 months after the study there was a significant difference ( p = 0.001 ) . comparison of religious knowledge scores before and after the intervention and 2 months after completion of the study in the intervention and control groups comparison of the religious attitude score before and after the intervention , and 2 months after completion of the study in the intervention and control groups in evaluating the anxiety level in the intervention group after training , the state anxiety in severe anxiety declined from 7.1% to 2.4% while the moderate anxiety remained unchanged ( 40.5% ) and the mild anxiety changed from 52.4% to 57.1% . moderate state anxiety in the control group increased from 66.7% before the intervention to 64.3% after the intervention and to 71.4% within 2 months after the intervention . however , the moderate trait anxiety of the individuals increased from 47.6% to 73.8% after the intervention and then remained fixed for up to 2 months after completion of the training . severe anxiety in the control group increased from 14.3% before the intervention to 16.7% after the intervention and to 23.8% within 2 months after the intervention . in evaluating the level of anxiety in pregnant women , the independent t - test results in table 4 show that there was a significant difference in the state anxiety score in primigravida women between the intervention and control groups before the intervention , ( p = 0.002 ) ; so , between the two cohorts of intervention and control after the intervention ( p = 0.001 ) and 2 months after the intervention there was a significant difference ( p = 0.001 ) . the independent t - test results in table 5 has shown that personality anxiety scores between the two cohorts have no difference preintervention ( p = 0.0197 ) so , between the intervention and control cohorts after the intervention ( p = 0.001 ) and 2 months later there existed a significant difference ( p = 0.001 ) . comparison of state anxiety scores before and after intervention and 2 months after completion of the intervention in the control and intervention groups comparison of personality anxiety scores before and after intervention and 2 months after completion of the intervention in the control and intervention groups based on the obtained results in this study , religious education has led to an increase in religious knowledge and attitudes among pregnant women . the results indicated that by using religious education , the religious knowledge , and attitudes of participants in the training group in comparison with the controls went up and after 6 sessions of training , both state and trait anxiety have reduced . this cross - sectional study ( 2005 ) was carried out on 300 pregnant women referring to 6 health care centers of iran university of medical sciences in tehran , in the field of health knowledge of islam during pregnancy period .

species of the genus fusarium are among the most destructive fungal plant pathogens known and are responsible for major yield losses during cultivation of wheat , maize , barley , and soybeans . many species of fusarium produce mycotoxins as they grow parasitically within the plant , and ingestion of contaminated grain and food products processed from this grain causes acute and chronic health problems for both humans and animals . some species of fusarium produce trichothecenes , sesquiterpenoid mycotoxins that inhibit eukaryotic protein synthesis and other cellular functions in animals that ingest contaminated feed . type a trichothecenes ( e.g. , t-2 toxin , ht-2 toxin , diacetoxyscirpenol ) are of particular concern because they are considerably more toxic than the type b group ( e.g. , deoxynivalenol and nivalenol ) . as part of their defense response to xenobiotics , plants can modify the structure of several mycotoxins , including trichothecenes , by conjugation to sugars , organic acids , or sulfates , which reduce their phytotoxicity and may facilitate their sequestration . whereas conjugation to sugars may protect plants from the ill effects of the toxins , these so - called masked mycotoxins present a potential food safety concern because , although toxicological data are scarce , several studies highlight the potential threat to consumer safety from these substances . in particular , the possible hydrolysis of masked mycotoxins back to their toxic parents during mammalian digestion is of considerable concern . structurally t-2 toxin , 1 , r = ac ( figure 1 ) is ( 2,3,4,8)-4,15-bis(acetyloxy)-3-hydroxy-12,13-epoxytrichothec-9-en-8-yl 3-methylbutanoate , which in animals is known to be predominantly metabolized to the 4-o - deacetylated form known as ht-2 toxin , 1 , r = h ( figure 1 ) . glucoside conjugates of t-2 toxin have been reported in fusarium - infected grain and fusarium culture material , although to date the anomericity of the 3-o - linked glucosyl group is unknown . t-2 toxin--glucoside , 2 , and t-2 toxin--glucoside , 3 ( figure 1 ) , are anticipated to have very different physical properties . more importantly , the relative toxicities of the two anomers are as yet unknown , and the ability to test the naturally occurring form needs to be addressed . structures of t-2 toxin , 1 ( r = ac ) , ht-2 toxin , 1 ( r = h ) , t-2 toxin--glucoside , 2 , and t-2 toxin--glucoside , 3 . one of the most studied masked trichothecenes is deoxynivalenol-3-glucoside , which has been reported from contaminated cereal crops and in food products derived from these crops . nuclear magnetic resonance ( nmr ) studies have shown that the naturally occurring glucoside is deoxynivalenol-3--glucoside . plant glucosyltransferase genes that control the conversion of deoxynivalenol to deoxynivalenol-3--glucoside have been identified , and a barley glucosyltransferase gene has been engineered into wheat to improve resistance to fusarium head scab . in addition , a deoxynivalenol glucosyltransferase gene has been cloned and expressed in yeast . as a result , deoxynivalenol-3--glucoside has been prepared using yeast expression and has been available for studies on its stability during food processing and the digestive fate of this masked mycotoxin . although initial studies reported that deoxynivalenol-3--glucoside is relatively stable to gastric conditions , it was recently reported that masked mycotoxins can be deconjugated by human colon microbiota , thus releasing their parent forms . because parent toxins may be absorbed in the intestine , this cleavage should be considered of toxicological relevance depending on the colonic absorption of the target compound . we have recently shown that three species of blastobotrys are able to biotransform t-2 toxin to t-2 toxin--glucoside , 2 ( figure 1 ) , and these yeast species appeared to provide an efficient way to produce the material necessary to study the digestive fate or animal toxicity of t-2 toxin - glucoside and related compounds , as well as develop methods for their detection . it was first important to determine which anomeric form of t-2 toxin - glucoside was produced in fusarium - contaminated cereals before proceeding with larger scale studies using the yeast biotransformation product . for this reason , t-2 toxin--glucoside , 3 ( figure 1 ) , was chemically synthesized , and along with t-2 toxin--glucoside , 2 ( figure 1 ) , the yeast biotransformation product , compared to the t-2 toxin - glucoside found in contaminated grain . we present here a comparison of the chromatographic and spectroscopic properties of the two anomeric forms , their relative reactivities to antibodies prepared with t-2 toxin--glucoside or t-2 toxin , their relative phytotoxicities , the stabilities and bioavailabilities of these masked mycotoxins after ingestion , and the anomeric form of the naturally occurring t-2 toxin - glucoside . these results become particularly relevant if international standards are developed for the detection and quantitation of t-2 toxin and t-2 toxin - glucoside in cereal grains and products made from them . nmr experiments were performed with acetone - d6 as the solvent on a bruker avance amx 500 spectrometer ( bruker biospin corp . , billerica , ma , usa ) operating at 500.11 mhz using a standard 5 mm z - gradient bbi probe at 27 c . chemical shifts are reported as parts per million from tetramethylsilane calculated from the lock solvent . the deuterated solvents used were obtained from cambridge isotope laboratories ( andover , ma , usa ) . the pulse sequences used were those supplied by bruker , and processing was done with the bruker topspin software package ( v. 1.3 ) . additional nmr experiments with tetra - o - tips--glucosyl t-2 toxin , 6 , and t-2 toxin--glucoside , 3 , were performed on an agilent 600 mhz nmr spectrometer ( agilent , santa clara , ca , usa ) . one wheat and one oat sample were extracted for comparison with t-2 toxin - glucoside standards . samples were finely ground with a tecator cyclotec 1093 ( international pbi , milan , italy ) laboratory mill equipped with a 500 mm sieve . ten grams of each ground sample was extracted with 30 ml of acetonitrile / water ( 84:16 v / v ) by orbital shaking for 2 h. after filtration through filter paper , 10 ml of extract was cleaned with a mycosep 227 column ( romer laboratories , union , mo , usa ) . purified extract ( 6 ml , equivalent to 2 g of sample ) was dried under an air stream at 50 c . extracts of naturally contaminated cereals or t-2 toxin - glucoside standards were analyzed by lc coupled to tandem mass spectrometry ( ms / ms ) as previously described . lc - ms / ms analyses were performed by a qtrap ms / ms system , from applied biosystems ( foster city , ca , usa ) equipped with an electrospray ionization ( esi ) interface and an 1100 series micro - lc system comprising a binary pump and a microautosampler from agilent technologies ( waldbronn , germany ) . the column used was a 150 3 mm i.d . , 4 m , synergi hydro , with a 4 mm 2 mm i.d . , 10 m , aqua c18 guard column ( phenomenex , torrance , ca , usa ) . the flow rate of the mobile phase was 200 l / min , whereas the injection volume was 20 l . eluent a was water and eluent b was methanol , both containing 5 mm ammonium acetate . eluent b was increased from 20 to 40% in 3 min , then increased to 63% in 35 min , and kept constant for 9 min . for column re - equilibration , eluent b was decreased to 20% in 1 min and kept constant for 9 min . for ms analyses , the esi interface was used with the following settings : temperature , 350 c ; curtain gas , nitrogen , 30 psi ; nebulizer gas , air , 10 psi ; auxiliary gas , air , 30 psi ; ion spray voltage , + 4500 v , positive ion mode . the ms was operated in enhanced product ion ( epi ) mode , applying a collision energy ( ce ) for stimulation of ion fragmentation of 10 , or multiple reaction monitoring ( mrm ) mode to observe several distinctive ions produced upon fragmentation of the toxin with ammonium adduct [ m + nh4 ] ion . for t-2 toxin - glucoside , the parent ion ( m / z 646 ) and fragment ions were monitored to observe elution of the separated t-2 toxin conjugates . operation of the lc - ms / ms instrument and interpretation of the acquired data were done utilizing the analyst ( absciex ) software provided by the ms instrument manufacturer . t-2 toxin - glucosides were prepared using t-2 toxin that had been isolated and purified from liquid cultures of fusarium sporotrichioides strain 5493cos9 - 1#11 as described previously . t-2 toxin--glucoside , 2 ( optical rotation , [ ]d = + 79.74 , c 0.153 , ch3oh ) , was prepared by feeding t-2 toxin to blastobotrys muscicola cultures as described previously . t-2 toxin--glucoside , 3 , was synthesized as described below ( figure 2 ) . pentaacetyl--glucopyranose ( 1.0 g , 2.56 mmol ) was dissolved in dichloromethane ( 20 ml ) , and ethanethiol ( 220 l , 3.07 mmol ) was added followed by bf3oet ( 1.6 ml , 12.8 mmol ) at ambient temperature under an argon atmosphere . after 2 h , the reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate . the aqueous phase was extracted with dichloromethane , and the combined organic phase was washed with brine and dried over sodium sulfate . the filtrate was concentrated , and the residue was purified by column chromatography over silica gel ( hexane / ethyl acetate , 1:1 ) , giving 660 mg ( 67% ) of a colorless oil in the form of a 1:1 ,-mixture . to a solution of 4 ( 660 mg , 1.68 mmol ) in methanol ( 16.5 ml ) was added sodium methoxide ( 10 mg , 0.17 mmol ) at ambient temperature . after 1 h , the reaction mixture was neutralized with amberlite 120 . the resin was filtered off , and the filtrate was concentrated to give a colorless oil ( 340 mg ) , which was dissolved in 2,6-lutidine ( 15 ml ) ( sigma - aldrich , st . louis , mo , usa ) and treated with triisopropylsilyl trifluoromethanesulfonate ( 2 ml , 4.9 mmol ) ( sigma - aldrich ) . the reaction mixture was heated to 130 c , and after 1.5 h , further triisopropylsilyl trifluoromethanesulfonate ( 2 ml , 4.9 mmol ) was added . the reaction mixture was maintained at 130 c for 2 h , then further triisopropylsilyl trifluoromethanesulfonate ( 2 ml , 4.9 mmol ) was added . after 2 h , the reaction mixture was cooled to room temperature , diluted with hexane , and washed with 0.1 m hcl until the water phase remained acidic . the filtrate was concentrated and purified by column chromatography over silica gel eluting with hexane , a colorless oil ( 680 mg , 53% ) as a 1:1 mixture of ,-isomers . a solution of 5 ( 110 mg , 0.13 mmol ) and t-2 toxin ( 40 mg , 0.086 mmol ) was dissolved in dry dichloromethane ( 0.4 ml ) with 3 acid - washed molecular sieves ( alfa aesar , ward hill , ma , usa ) . the reaction mixture was cooled to 78 c under an argon atmosphere , and then n - iodosuccinimide ( 26 mg , 0.15 mmol ) ( chem - impex int . after 0.5 h , trifluoromethanesulfonic acid ( 2.3 l , 0.04 mmol ) ( sigma - aldrich ) was added to the cold solution . after 3 h , the reaction mixture was quenched with triethylamine ( emd , philadelphia , pa , usa ) , and the molecular sieves were filtered off . the filtrate was concentrated and purified by column chromatography over silica gel ( eluent , hexane / ethyl acetate 4:1 ) to give a colorless oil ( 100 mg , 95% ) as a 1:5 mixture of ,-isomers . h nmr ( 600 mhz in cdcl3 ) for -isomer : 5.66 ( d , j = 3.3 hz , 1h , h-4 ) , 5.63 ( d , j = 5.9 hz , 1h , h-10 ) , 5.27 ( d , j = 5.5 hz , 1h , h-8 ) , 4.91 ( d , j = 5.9 hz , 1h , h-26 ) , 4.31 ( d , j = 12.1 hz , 1h , h-15a ) , 4.27 ( dd , j = 4.8 , 3.67 hz , 1h , h-3 ) , 4.10 ( d , j = 5.5 hz , 1h , h-11 ) , 4.08 ( overlapped with h-15b , 1h , h-28 ) , 4.07 ( d , j = 12.5 hz , 1h , h-15b ) , 3.95 ( d , j = 2.9 hz , 1h , h-29 ) , 3.90 ( d , j = 5.9 hz , 1h , h-27 ) , 3.89 ( d , j = 5.8 hz , 1h , h-30 ) , 3.84 ( d , j = 5.1 hz , 2h , h-31a , b ) , 3.73 ( d , j = 4.8 hz , 1h , h-2 ) , 2.92 ( d , j = 4.0 hz , 1h , h-13a ) , 2.75 ( d , j = 4.0 hz , 1h , h-13b ) , 2.29 ( dd , j = 15.0 , 5.9 hz , 1h , h-7a ) , 2.09 ( m , 1h , h-18 ) , 2.07 ( s , 1h , h-23 ) , 2.05 ( m , 1h , h-19 ) , 2.00 ( s , h-25 ) , 1.99 ( overlapped with h-25 , 1h , h-7b ) , 1.72 ( s , h-16 ) , 0.95 ( d , j = 6.2 or 5.1 hz , 1h , h-20 ) , 0.94 ( d , j = 6.2 or 5.1 hz , 1h , h-21 ) , 0.67 ( s , h-14 ) ; c nmr ( 150 mhz in cd2cl3 ) 172.4 ( c-17 ) , 170.2 ( c-22 ) , 169.8 ( c-24 ) , 135.1 ( c-9 ) , 124.1 ( c-10 ) , 103.4 ( c-26 ) , 83.5 ( c-30 ) , 82.8 ( c-3 ) , 80.6 ( c-4 ) , 79.5 ( c-2 ) , 78.0 ( c-28 ) , 77.3 ( c-27 ) , 71.1 ( c-29 ) , 68.0 ( c-8 ) , 66.8 ( c-11 ) , 65.7 ( c-31 ) , 64.3 ( c-15 ) , 64.1 ( c-12 ) , 48.5 ( c-5 ) , 46.7 ( c-13 ) , 43.4 ( c-18 ) , 43.0 ( c-6 ) , 26.9 ( c-7 ) , 25.7 ( c-19 ) , 22.1 ( c-20 ) , 22.1 ( c-21 ) , 20.9 ( c-25 ) , 20.6 ( c-23 ) , 20.1 ( c-16 ) , 6.2 ( c-14 ) ; hrms - esi ( m / z ) [ m + na ] calcd for c66h124o14si4na 1275.7966 , found 1275.7947 . the above ,-mixture of 6 ( 100 mg , 0.08 mmol ) was dissolved in thf ( 1.0 ml ) and treated at 0 c with tetrabutylammonium fluoride ( 1.6 ml , 1.6 mmol ) ( acros - thermo fisher scientific inc . , the reaction mixture was allowed to warm to room temperature and stirred for 2.5 h , after which it was concentrated and purified by column chromatography over silica gel ( eluent , chloroform / methanol , 8:1 ) to give a colorless oil ( 42 mg , 84% ) of a 1:5 ,-mixture . further purification of this mixture by c18 hplc ( water acetonitrile 2540% ) and freeze - drying gave 21 mg ( 42% ) of the pure -isomer , 3 , as a white amorphous powder . h nmr ( 600 mhz in cd3od ) 5.96 ( d , j = 3.0 hz , 1h , h-4 ) , 5.77 ( d , j = 6.00 hz , 1h , h-10 ) , 5.31 ( d , j = 5.6 hz , 1h , h-8 ) , 4.46 ( dd , j = 4.9 , 3.2 hz , 1h , h-3 ) , 4.43 ( d , j = 7.7 hz , 1h , h-26 ) , 4.37 ( d , j = 6.2 hz , 1h , h-11 ) , 4.36 ( d , j = 12.7 hz , 1h , h-15a ) , 4.08 ( d , j = 12.5 hz , 1h , h-15b ) , 3.81 ( dd , j = 12.0 , 2.2 hz , 1h , h-31a ) , 3.70 ( d , j = 5.0 hz , 1h , h-2 ) , 3.63 ( dd , j = 12.0 , 5.8 hz , 1h , h-31b ) , 3.34 ( t , j = 9.0 , 8.9 hz , 1h , h-28 ) , 3.28 ( t , j = 9.0 , 9.0 hz , 1h , h-29 ) , 3.24 ( dd , j = 9.0 , 7.8 hz , 1h , h-27 ) , 3.19 ( ddd , j = 9.6 , 5.8 , 2.2 hz , 1h , h-30 ) , 3.03 ( d , j = 3.8 hz , 1h , h-13a ) , 2.85 ( d , j = 3.9 hz , 1h , h-13b ) , 2.36 ( dd , j = 15.2 , 6.0 hz , 1h , h-7a ) , 2.14 ( dd , j = 7.6 , 2.4 hz , 1h , h-18 ) , 2.07 ( s , h-23 ) , 2.05 ( s , 1h , h-25 ) , 2.05 ( m , h-19 ) , 1.92 ( d , j = 15.2 hz , 1h , h-7b ) , 1.73 ( s , h-16 ) , 0.95 ( d , j = 6.6 hz , 1h , h-20 ) , 0.94 ( d , j = 6.6 hz , 1h , h-21 ) , 0.72 ( s , 1h , h-14 ) ; c nmr ( 150 mhz in cd2cl3 ) 174.1 ( c-17 ) , 170.8 ( c-24 ) , 170.7 ( c-22 ) , 137.2 ( c-9 ) , 125.2 ( c-10 ) , 103.8 ( c-26 ) , 83.9 ( c-3 ) , 81.3 ( c-4 ) , 80.6 ( c-2 ) , 78.4 ( c-30 ) , 78.2 ( c-28 ) , 74.9 ( c-27 ) , 71.6 ( c-29 ) , 69.4 ( c-8 ) , 68.6 ( c-11 ) , 65.9 ( c-15 ) , 65.4 ( c-12 ) , 62.8 ( c-31 ) , 50.2 ( c-5 ) , 48.0 ( c-13 ) , 44.6 ( c-18 ) , 44.5 ( c-6 ) , 28.9 ( c-7 ) , 27.0 ( c-19 ) , 22.9 ( c-20 ) , 22.8 ( c-21 ) , 21.3 ( c-25 ) , 20.9 ( c-23 ) , 20.6 ( c-16 ) , 7.2 ( c-14 ) ; hrms - esi ( m / z ) [ m + na ] calcd for c30h44o14na 651.2629 , found 651.2618 ; optical rotation [ ]d = + 7.51 ( c 0.080 , ch3oh ) . the first of these was a competitive indirect enzyme - linked immunosorbent assay ( ci - elisa ) based upon an antibody ( mab 2 - 13 ) developed against t-2 toxin-3--glucoside , conjugated to ovalbumin ( e.g. , t-2g ova ) , as described previously . the second immunoassay format was a commercial test kit previously validated to detect t-2 toxin and ht-2 toxin ( veratox for t-2/ht-2 ) ( neogen corp . , lansing , mi , usa ) . the methanol content indicated in the test kit instructions , the t-2 toxin - glucoside standards were prepared in 35% ( v / v ) methanol / water . the single - celled alga chlamydomonas reinhardtii was used to assess the relative phytotoxicity of t-2 toxin , t-2 toxin--glucoside , and t-2 toxin--glucoside , as described previously . triplicate cultures ( 10 ml ) were initiated with 10 cells / ml containing a 100 m concentration of an individual trichothecene and grown for 6 days under fluorescent lights , with agitation of 200 rpm . culture doublings were calculated as follows : ( log of the final density log of the initial cell density)/log 2 . briefly , the main digestive juices were prepared by mixing salts and enzymes and were preheated at 37 c before use . a volume of 200 l of a water / methanol ( 25:75 , v / v ) solution containing the target compounds was transferred in a 4 ml septum vial , and the solvent was evaporated under nitrogen . the in vitro digestion was started by adding 75 l of artificial saliva to 200 l of the water / methanol ( 25:75 v / v ) solution containing the target analyte ( 500 g / l ) . after 5 min of incubation at 37 c , 150 l of artificial gastric juice was added and the mixture was incubated again for 2 h. at the end of the gastric step , 25 l of 1 m bicarbonate solution with 150 l of artificial duodenal juice and 75 l of artificial bile juice were added , and then a final incubation step of 2 h was performed . finally , 25 l of acetonitrile was added to stop the reactions , and the sample was centrifuged for 10 min at 10000 rpm , prior to direct lc - ms / ms analysis . human colonic fermentation of t-2 toxin--glucoside , t-2 toxin--glucoside , and t-2 toxin was carried out as described previously . these samples were immediately placed in an anaerobic environment and then mixed and weighed to obtain a 10% fecal solution in a phosphate saline buffer ( pbs ) . for each sample 1.8 ml of growth medium , prepared as described previously , and 1.8 ml of fecal solution were mixed and added to 0.4 ml of a toxin solution , at a final concentration of 500 g / l . each vial was then treated with a slight nitrogen flow to eliminate oxygen and perform fermentations under anaerobic condition . samples were incubated in a water bath at 37 c and shaken at a rate of 200 strokes / min . fermentations were stopped immediately to have controls and then after 30 min and 24 h , by cooling samples to room temperature and adding 0.4 ml of acetonitrile . samples were immediately centrifuged at 14000 rpm for 10 min and stored at 20 c . at the same time , samples without added mycotoxin were prepared . samples were diluted adding acetonitrile ( 1:2 v / v ) and directly analyzed for t-2 toxin - glucoside , t-2 toxin , and ht-2 toxin by lc - ms / ms using mrm as described above . nmr spectra were recorded for t-2 toxin--glucoside , obtained by yeast biotransformation ( figure 3a , c ) and for chemically synthesized t-2 toxin--glucoside ( figure 3b , d ) . both compounds are characterized by two spin systems due to a single glucopyranosyl ring and the trichothecene aglycone . the anomeric linkages are characterized by proton signals in the 4.55.5 ppm range and the c signals by proton signals between 95 and 105 ppm . t-2 toxin--glucoside gave a doublet at 4.98 ppm that integrates to a single proton ( figure 3a ) and correlates to an adjacent c signal at 98.18 ppm ( figure 3c ) . the chemically synthesized t-2 toxin--glucoside is similarly characterized by a larger h1ax h2ax bond angle , resulting in a definitive j1,2 coupling constant of 12 hz for the -anomeric proton at 4.46 ppm ( figure 3b ) . this h-1 proton correlates to the -c1 c nmr anomeric signal at 102.36 ppm ( figure 3d ) . hence , the t-2 toxin - glucoside anomers are defined by the observed h and c chemical shifts and by the characteristic j1,2 coupling constants . these assignments were confirmed by hsqc and , together with hmbc , dept , cosy , and noesy experiments , enable the complete nmr assignment of both epimers . proton nmr spectra of ( a ) t-2 toxin--glucoside , ( b ) t-2 toxin--glucoside with signals for the h1 anomeric proton indicated and c nmr anomeric carbon signals of ( c ) t-2 toxin--glucoside ( 98.18 ppm ) and ( d ) t-2 toxin--glucoside ( 102.36 ppm ) . the two epimeric standards , t-2 toxin--glucoside prepared with yeast and the synthetic t-2 toxin--glucoside , could be separated with lc ( figure 4 ) . in epi mode , t-2 toxin--glucoside and t-2 toxin--glucoside [ m + nh4 ] ions were subjected to collision - induced dissociation . under these conditions , t-2 toxin--glucoside more readily ionized to yield an [ m + h ] ion than the t-2 toxin -glucoside , as shown by the relative intensities of the residual [ m + nh4 ] and [ m + h ] ions ( figure 5 ) . other fragment ions were similar to those previously observed from fusarium - infected grain . lc - ms / ms chromatograms of ( a ) t-2 toxin--glucoside ( t-2glc ) and t-2 toxin--glucoside ( t-2glc ) standards , ( b ) t-2 toxin - glucoside extracted from wheat , and ( c ) t-2 toxin - glucoside extracted from oats . tandem mass spectrum of the [ m + nh4 ] ion of ( a ) t-2 toxin--glucoside , ( b ) t-2 toxin--glucoside , ( c ) t-2 toxin - glucoside from oats , and ( d ) t-2 toxin - glucoside from wheat . to determine the form of t-2 toxin - glucoside present in contaminated oats and wheat , extracts were analyzed with lc - ms / ms and compared to the standard t-2 toxin--glucoside and t-2 toxin--glucoside ( figure 4 ) . under the conditions used , t-2 toxin--glucoside eluted at 31.4 min and t-2 toxin--glucoside at 32.0 min . t-2 toxin - glucoside was detected at 31.5 min in oat and wheat samples , consistent with t-2 toxin--glucoside ( figure 4 ) . in addition , there were differences in the mass spectra of the two isomers , showing the same fragment ions but with different relative intensities . similar to t-2 toxin--glucoside , the yeast biotransformation product , the t-2 toxin - glucoside from oats and wheat , had the prominent [ m + h ] product ion from the [ m + nh4 ] ion ( figure 5 ) . in this study , the stability under in vitro gastrointestinal conditions and the catabolic fate of both t-2 toxin--glucoside and t-2 toxin--glucoside were tested in a human gut microbioma assay and compared to t-2 toxin . we found that the t-2 toxin - glucosides were unchanged after incubation with human artificial saliva . these results are similar to those reported for deoxynivalenol glucoside and zearalenone glucoside and further demonstrate the stability of some - and -glucosidic bonds upon digestion . in contrast to zearalenone glucoside , which was hydrolyzed in the first 30 min of colonic fermentation , t-2 toxin and the t-2 toxin - glucosides were relatively stable for the first 30 min ( figure 6 ) . t-2 toxin was mainly transformed into its derivative ht-2 toxin ( 83% at t = 24 h ) . t-2 toxin--glucoside and t-2 toxin--glucoside were both strongly degraded after 24 h , with < 30% of the starting material remaining . the latter was mainly cleaved to release its precursor t-2 toxin ( 58% ) with a smaller percentage of ht-2 toxin ( 12% ) , whereas the former is converted into t-2 toxin ( 13% ) , ht-2 toxin ( 30% ) , and other metabolites of unknown structure and toxicity ( 33% ) . degradation upon human colonic microbiota fermentation of ( a ) t-2 toxin--glucoside ( t-2glc ) , ( b ) t-2 toxin--glucoside ( t-2glc ) , and ( c ) t-2 toxin ( t-2 ) . ht-2 toxin ( ht-2 ) . because smaller amounts of t-2 toxin remained following colonic fermentation with t-2 toxin--glucoside , it may pose less of a risk than t-2 toxin--glucoside , which is not known to occur in crops . however , t-2 toxin--glucoside was also converted into other , as yet uncharacterized , compounds ( figure 6 ) . in addition to t-2 toxin and ht-2 toxin , other possible products of t-2 toxin - glucoside include ht-2 toxin - glucoside , t-2 triol , and t-2 tetraol . it is important to consider the toxicity of all of the products when the risk posed by t-2 toxin--glucoside in grain is assessed . the relative toxicity of t-2 toxin and the t-2 toxin - glucosides was tested with c. reinhardtii cultures . previous studies have shown that t-2 toxin is quite phytotoxic and t-2 toxin--glucoside is nontoxic . in this study , cultures grown in 100 m t-2 had only a slight increase in the number of cells , with 0.6 doubling after 4 days . in contrast , neither t-2 toxin--glucoside nor t-2 toxin--glucoside was phytotoxic , with 5.4 and 5.5 doublings , respectively , which are similar to the 5.3 doublings observed for cultures treated with acetone alone . the first immunoassay was a competitive indirect elisa format using mab 2 - 13 , an antibody developed with t-2 toxin--glucoside . mab 2 - 13 recognized t-2 toxin -glucoside slightly better than t-2 toxin ( cross reaction 108% ) , whereas t-2 toxin--glucoside was recognized more poorly , at about 57% relative to t-2 toxin ( table 1 ; figure 7a ) . this makes this antibody - based test suitable for the detection of both t-2 toxin and the naturally occurring t-2 toxin--glucoside . ( a ) ci - elisa responses of t-2 toxin--glucoside ( open circles ) and t-2 toxin--glucoside ( solid circles ) with mab 2 - 13 . data shown are the averages from four plates , with six replicates per toxin concentration per plate ( n = 24 ) , 1 standard deviation . response equation over the indicated concentration range ( 0.1200 ng / ml ) . ( b ) elisa responses of t-2 toxin ( open triangles ) , t-2 toxin--glucoside ( open circles ) , and t-2 toxin--glucoside ( solid circles ) to neogen veratox t-2/ht-2 antibody . data shown are the averages from three plates per toxin , with four replicates per toxin concentration per plate ( n = 12 ) , 1 standard deviation . data for t-2 toxin with this immunoassay are from maragos et al . the second immunoassay was a commercially available t-2/ht-2 kit . this kit was used to determine if an elisa , developed before the glucosides of t-2 and ht-2 toxins were even discovered , could detect these masked mycotoxins . the commercial elisa test kit for t-2 toxin and ht-2 toxin detection performed very well for detecting t-2 toxin with good sensitivity and good reproducibility . in addition , the kit cross - reacted fairly well with the t-2 toxin--glucoside , but poorly with the t-2 toxin--glucoside ( table 1 ; figure 7b ) . however , given that t-2 toxin--glucoside is the form found in naturally contaminated samples and that the cross - reaction with this anomer was only about 16% , this test kit would not be recommended for the simultaneous screening of t-2 toxin and t-2 toxin--glucoside . the ability of plants to form trichothecene glucosides can be seen as a detoxification mechanism that plants use when infected with trichothecene - producing fungi . because trichothecenes can be virulence factors in plant disease , expression of trichothecene ugt genes in plants is a potential way to increase disease resistance to trichothecene - producing fungi . however , both the trichothecenes and their masked forms need to be considered to accurately assess the risk to human and animal health posed by plant material contaminated with trichothecenes . sufficient amounts of t-2 toxin - glucoside are needed to determine the risk posed by the masked form of t-2 toxin in cereals , to develop reliable methods for their detection , and to study the stability , digestive fate , and toxicity of t-2 toxin - glucoside formed in plant materials . whereas the synthesis of deoxynivalenol - glucoside is relatively straightforward , this method was not amenable to the preparation of t-2 toxin - glucosides because the isovaleryl and acetyl groups are labile during the synthesis . a synthesis employing a superarmed tetra - o - triisopropylsilyl protected donor offered a way to selectively remove the protecting groups from the sugar portion of the glucoside without disturbing the isovaleryl and acetyl groups of t-2 toxin , thus providing a means to synthesize t-2 toxin--glucoside . microbial t-2 toxin--glucoside was prepared as a single anomer with b. muscicola yeast cultures , on the basis of lc and lc - ms chromatographic comparisons and ms / ms analyses , the t-2 toxin - glucoside occurring in contaminated wheat and oats was found to be identical to the yeast biotransformation product , t-2 toxin--glucoside . it is notable that naturally occurring t-2 glucoside has an -linked sugar , whereas naturally occurring deoxynivalenol glucoside has a -linked sugar . although -linked sugars are more commonly found with xenobiotics and natural products including flavonoids , there is a report of fusarium cultures producing an -linked glycoside , 15-monoacetoxyscirpenol-4--glucoside . the blastobotrys glucosyltransferase that converts t-2 toxin to t-2 toxin--glucoside has not been identified , but there may be substrate preferences between the ugt that lead to trichothecene - or -glucosides . although the focus of this study was the t-2 toxin - glucosides , ht-2 toxin - glucosides have also been reported in contaminated grain and in culture material . they may be formed either by hydrolysis of t-2 toxin--glucoside or by conversion of t-2 toxin to ht-2 toxin followed by glycosylation at c-3 or c-4 . it is possible that the latter route may utilize different glucosyltransferases that result in a orientation . to date , no plant ugt that converts t-2 toxin to t-2 toxin--glucoside has been identified . numerous glucosyltransferases were screened to identify one that had good substrate specificity for deoxynivalenol . although the t-2 toxin - glucoside found in naturally contaminated oats and wheat was identical to the t-2 toxin--glucoside prepared with yeast cultures , the anomericity of ht-2 toxin - glucosides has not been determined . the present study has demonstrated that naturally occurring t-2 toxin - glucoside has an -linked sugar and that t-2 toxin--glucoside can be prepared with b. muscicola in sufficient quantity to study its animal toxicity . the u.s . fda has not provided guidance levels to the agricultural industry for t-2 toxin or related trichothecenes . the european food safety authority ( efsa ) established a group tolerable daily intake of 100 ng / kg body weight for t-2 and ht-2 toxins . however , at present it is not possible to perform a proper risk assessment for masked mycotoxins in food , due to the lack of data on exposure and toxic properties . masked mycotoxins may elude analysis because of altered physical properties or because of modification of an epitope recognized by antibodies used for the detection , leading to an underestimation of the total mycotoxin load . possible release during food processing is also a concern , and efsa currently has a working group on the public health risk of masked mycotoxins in food and animal feed . research on masked mycotoxins is hampered by the nonavailability of analytical standards or calibrants , and only one compound , deoxynivalenol-3--glucoside , is commercially available . the occurrence of t-2 toxin--glucoside in wheat and oat samples , and its metabolism by microbes during digestion , suggests that there is a risk in underestimation of the total t-2 toxin load and a need to monitor both mycotoxins and their conjugated forms in cereals and food products . to promote research efforts in the area of masked mycotoxins , t-2 toxin--glucoside has now been made available to the scientific community by the authors .

t-2 toxin is a trichothecene mycotoxin produced when fusarium fungi infect grains , especially oats and wheat . ingestion of t-2 toxin contaminated grain can cause diarrhea , hemorrhaging , and feed refusal in livestock . cereal crops infected with mycotoxin - producing fungi form toxin glycosides , sometimes called masked mycotoxins , which are a potential food safety concern because they are not detectable by standard approaches and may be converted back to the parent toxin during digestion or food processing . the work reported here addresses four aspects of t-2 toxin - glucosides : phytotoxicity , stability after ingestion , antibody detection , and the anomericity of the naturally occurring t-2 toxin - glucoside found in cereal plants . t-2 toxin--glucoside was chemically synthesized and compared to t-2 toxin--glucoside prepared with blastobotrys muscicola cultures and the t-2 toxin - glucoside found in naturally contaminated oats and wheat . the anomeric forms were separated chromatographically and differ in both nmr and mass spectrometry . both anomers were significantly degraded to t-2 toxin and ht-2 toxin under conditions that mimic human digestion , but with different kinetics and metabolic end products . the naturally occurring t-2 toxin - glucoside from plants was found to be identical to t-2 toxin--glucoside prepared with b. muscicola . an antibody test for the detection of t-2 toxin was not effective for the detection of t-2 toxin--glucoside . this anomer was produced in sufficient quantity to assess its animal toxicity .

Introduction Materials and Methods Results and Discussion

whereas conjugation to sugars may protect plants from the ill effects of the toxins , these so - called masked mycotoxins present a potential food safety concern because , although toxicological data are scarce , several studies highlight the potential threat to consumer safety from these substances . we have recently shown that three species of blastobotrys are able to biotransform t-2 toxin to t-2 toxin--glucoside , 2 ( figure 1 ) , and these yeast species appeared to provide an efficient way to produce the material necessary to study the digestive fate or animal toxicity of t-2 toxin - glucoside and related compounds , as well as develop methods for their detection . for this reason , t-2 toxin--glucoside , 3 ( figure 1 ) , was chemically synthesized , and along with t-2 toxin--glucoside , 2 ( figure 1 ) , the yeast biotransformation product , compared to the t-2 toxin - glucoside found in contaminated grain . we present here a comparison of the chromatographic and spectroscopic properties of the two anomeric forms , their relative reactivities to antibodies prepared with t-2 toxin--glucoside or t-2 toxin , their relative phytotoxicities , the stabilities and bioavailabilities of these masked mycotoxins after ingestion , and the anomeric form of the naturally occurring t-2 toxin - glucoside . these results become particularly relevant if international standards are developed for the detection and quantitation of t-2 toxin and t-2 toxin - glucoside in cereal grains and products made from them . to determine the form of t-2 toxin - glucoside present in contaminated oats and wheat , extracts were analyzed with lc - ms / ms and compared to the standard t-2 toxin--glucoside and t-2 toxin--glucoside ( figure 4 ) . similar to t-2 toxin--glucoside , the yeast biotransformation product , the t-2 toxin - glucoside from oats and wheat , had the prominent [ m + h ] product ion from the [ m + nh4 ] ion ( figure 5 ) . in contrast to zearalenone glucoside , which was hydrolyzed in the first 30 min of colonic fermentation , t-2 toxin and the t-2 toxin - glucosides were relatively stable for the first 30 min ( figure 6 ) . in addition to t-2 toxin and ht-2 toxin , other possible products of t-2 toxin - glucoside include ht-2 toxin - glucoside , t-2 triol , and t-2 tetraol . this makes this antibody - based test suitable for the detection of both t-2 toxin and the naturally occurring t-2 toxin--glucoside . however , given that t-2 toxin--glucoside is the form found in naturally contaminated samples and that the cross - reaction with this anomer was only about 16% , this test kit would not be recommended for the simultaneous screening of t-2 toxin and t-2 toxin--glucoside . microbial t-2 toxin--glucoside was prepared as a single anomer with b. muscicola yeast cultures , on the basis of lc and lc - ms chromatographic comparisons and ms / ms analyses , the t-2 toxin - glucoside occurring in contaminated wheat and oats was found to be identical to the yeast biotransformation product , t-2 toxin--glucoside . although the t-2 toxin - glucoside found in naturally contaminated oats and wheat was identical to the t-2 toxin--glucoside prepared with yeast cultures , the anomericity of ht-2 toxin - glucosides has not been determined . the present study has demonstrated that naturally occurring t-2 toxin - glucoside has an -linked sugar and that t-2 toxin--glucoside can be prepared with b. muscicola in sufficient quantity to study its animal toxicity . the occurrence of t-2 toxin--glucoside in wheat and oat samples , and its metabolism by microbes during digestion , suggests that there is a risk in underestimation of the total t-2 toxin load and a need to monitor both mycotoxins and their conjugated forms in cereals and food products .

there is greater postprandial increase in circulating glucose with decrease in fasting glucose in late pregnancy with increased insulin level with progressive increase in maternal hormones such as progesterone , human chorionic somatomammotrophin ( hcs ) , and human placental lactogen ( hpl ) . these increase the responsiveness of the pancreatic islet cells in late pregnancy , thus increasing the insulin secretion and at the same time altering the insulin sensitivity in target organs . the rate of maternal to fetal glucose transfer is a function of transplacental concentration gradient . hpl increases the gradient by increasing maternal blood glucose concentration and decreasing the fetal glucose concentration . the glucose tolerance deteriorates in human pregnancy and is related to a pronounced peripheral resistance to insulin and is caused by postinsulin receptor events probably brought about by the cellular effects of the increased plasma levels of one or more of the pregnancy - associated hormones and free cortisol . the resistance is mainly confined to the muscle tissue , where significant reductions in the activities of phosphofructokinase and pyruvate kinase involved in glucose metabolism have been demonstrated , which along with increased muscle tissue in pregnancy , leads to a decrease in glycolysis in muscle . there was 65% increase in fasting insulin level in late gestation with 30% increase in basal endogenous glucose in 3436 weeks of gestation . the contribution of gluconeogenesis to total glucose was found to be 76% in late gestation ( at 34 weeks ) . the anti - insulinogenic and lipolytic activities of hcs , prolactin , cortisol , and glucagon , all of whose concentrations increased in the late gestation , contributed to the glucose intolerance , insulin resistance , and adipose tissue mobilization . the insulin resistance , as shown by insulin - glucose ratio , was higher at 37 weeks of gestation and less during postpartum , and this was consistent with the fact that there is persistent glucose production in fasting during pregnancy despite lower fasting glucose concentration . the change in carbohydrate and lipid metabolism in pregnancy is to ensure a continuous supply of nutrients to the fetus , even though mother takes food intermittently - these changes progress throughout pregnancy . there is fasting hypoglycemia due to fetal consumption , postprandial hyperglycemia , and hyperinsulinemia due to anti - insulin factors . this response is consistent with pregnancy - induced state of peripheral insulin resistance , the purpose of which is to ensure a sustained supply of glucose to fetus to meet its growth demand . certain factors secreted mainly by adipocytes called adipokines include leptin , adiponectin , tumor necrosis factor - alpha ( tnf - alpha ) , interleukin-6 , resistin , etc . , tnf - alpha is a cytokine produced by various cells such as monocytes , macrophages , neutrophils , adipocytes , and fibroblasts . tnf - alpha impairs insulin signaling by increasing serine phosphorylation of insulin receptor substrate-1 and decreasing insulin receptor tyrosine kinase activity and is definitely correlated with insulin resistance . the changes in insulin sensitivity from early gestation ( 2224 weeks ) to late gestation ( 3436 weeks ) , correlated with plasma tnf - alpha . pregnancy is a leptin - resistant state as it was seen that in spite of increased leptin levels , which would cause decreased appetite and energy intake , there was increased maternal energy intake with positive energy balance ; hence , leptin resistance exists in pregnancy . in addition , the variations in maternal insulin sensitivity are partly due to maternal adipocytokine and growth hormone / like growth factor axes . specific glucose transporter proteins help in transporting glucose across the placenta down the concentration gradient in normal non - diabetic pregnancy . macrosomia , hypoglycemia ( 18.4% ) , respiratory distress syndrome ( 5.2% ) , and hyperbilirubinemia ( 15.3% ) were found to be the most common perinatal problems in diabetic pregnancy . increased pregravid body mass index ( bmi ) and summed skinfold thickness ( triceps plus subscapular ) were associated with higher glucose concentrations at first visit and at 28 weeks . women whose plasma glucose concentrations were < 99 mg / dl with the others gravidas whose glucose concentrations were 99130 mg / dl and those with glucose concentrations of > 130 mg / dl . indicated that birth weights were approximately 50 g higher ( p < 0.05 ) for gravidas whose glucose concentrations were 99130 mg / dl and 200 g higher ( p < 0.005 ) for those with glucose concentrations of > 130 mg / dl . the difference in birth weight for women whose glucose concentrations were > 130 mg / dl versus above the median was 140 g ( p < 0.05 ) . increasing maternal plasma glucose concentration was associated with decreasing duration of gestation . high glucose concentrations are a risk factor or a risk marker for the subclinical infection that gives rise to chorioamnionitis . with increasing bmi , gravidas who are not diabetic , have higher glucose concentrations on the standard 50 g screening test . glucose challenge test is an acceptable screening test in which plasma glucose is measured 1 h after ingestion of 50 mg of pure glucose in 150 ml of fluid , to screen for gestational diabetes at 2428 weeks of pregnancy using a threshold of 140 mg / dl . glucose only is the glycosylating agent and that may be the reason for increase in the level of hba1c in diabetics and that the aldehyde of glucose and the amino group of valylhistidine can form a reversible schiff base linkage . the red blood cell is anucleated cell and hence it can not synthesize hemoglobin ( hb ) and all the proteins including hb is formed while it is in the bone marrow . the glycosylated hemoglobin ( hba1c ) levels increased in the lifespan of the cell and that the hba1c levels increased 2.8 times faster in diabetic than in normal mice . the formation of hba1c is a posttranslational modification and that since it is a very slow process , it is most likely to be nonenzymatic and an example of maillardreation ( nonenzymatic glycation ) . hba1c , the most abundant minor hb component in human erythrocytes , is formed by the condensation of glucose with the n - terminal amino groups of the beta - chains of hb a as both glucose and hb are found in large quantities in the red blood cells initially an unstable compound is formed , which is followed by the formation of a stable form and the second part of the reaction is irreversible . hba1c is slowly formed during the 120-day lifespan of the erythrocyte , and the concentration of hba1c would be higher in older red cells . the amount of hba1c formed is directly proportional to the amount of glucose concentration in the cell , and hence this reaction is faster in diabetics due to higher glucose concentration , and it may reveal information about adequate control of blood glucose over the period . glycosylation of hb is also a slow , nonenzymatic process reflecting the main hb - a fraction in diabetic patients , and the amount of sugar attached to hb correlated with average intracellular blood glucose level and with glycemic levels over the previous 610 weeks . statistically significant decrease in total hba1c including hba1c in healthy pregnant subjects occurred , at about twenty weeks and this decrease was maintained throughout pregnancy and hence its use to assess diabetic control in pregnancy . in a normal pregnancy , between 6 to 10 weeks , there is a decrease in the fasting blood glucose and this continues throughout pregnancy . this reduction in fasting glucose is smaller in subjects with higher bmi . in a study done by , hba1c values in diabetic pregnancy are lower than in nonpregnant diabetic subjects ( mean 7.8% 1.6% vs. 9.9% 1.9% ) and the mean value in pregnant nondiabetic was 4% 0.7% . a decrease in the upper reference limit of hba1c from 4.7% to 6.3% before pregnancy to 4.5% to 5.7% in early pregnancy and 4.4% to 5.6% in the third trimester occurs . average hba1c was found to be 5.1% 0.4% in early pregnancy ( p < 0.001 ) and 5.0% 0.3% ( p the changes in hba1c can be attributed to carbohydrate metabolism , changes in erythrocytes or both . new erythrocytes may be exposed to lower average concentration of glucose due to sustained fasting hypoglycemia , with decrease in glycosylation . the formation of hba1c is a posttranslational modification and that since it is a very slow process , it is most likely to be nonenzymatic and an example of maillardreation ( non - enzymatic glycation)and hba1c can be used as a reliable marker for the assessement of glycemic controlwith periodic monitoring being useful in diabetic patients with the upper range of hba1c increasing from 5% in first trimester to 5.9% in third trimeste . the glycosylated haemoglobin correlates best with the mean plasma glucose and urinary glucose levels over previous four to six weeks . an indian study has shown the mean hba1c in pregnant indian women with normal glucose tolerance to be 5.36+/-0.36% . the average plasma blood glucose corresponding to hba1c value of 5% is 101mg / dl and that corresponding to mean value of 5.29% is 111mg / dl ( range for 5.29%+/-0.514% in view of these maternal changes during a normal pregnancy in glucose levels , which increase as pregnancy progresses , and its effects and that hba1c correlates with the glycemic control over the past few weeks , the present study aims to estimate the levels of hba1c in pregnant nondiabetic women in the third trimester and its relation to adverse fetal outcomes . the study was conducted for dissertation purpose at lourdes hospital , ernakulam which is a 750 bedded multispecialty referral hospital catering to both urban and semi - urban population . the study is a unicentric , prospective , observational study and was conducted among the outpatient and inpatient pregnant women , in their third trimester that is from 24 weeks onward , attending the department of obstetrics and gynaecology in lourdes hospital , pachalam , kochi , kerala . data were collected with the help of a structured questionnaire by the researcher herself , from primigravida subjects in their third trimester at the nursing station near the outpatient department ( opd ) . the patients were followed up further in the opd and at the time of admission in the labor room . the collected data were analyzed to arrive at mean values standard deviation , for the hba1c value . the statistical techniques employed were mainly chi - square test - p < 0.05 was considered statistically significant . graphical representations in the form of tabulations , bar diagrams , and pie charts were also utilized . the exclusion criteria were abnormal gct value more than 140 mg , bmi > 25 kg / m , previous history of diabetes mellitus , on medications such as oral hypoglycemic agent , steroids , history of iron deficiency anemia , hemolytic anemia , hemoglobinopathies and other conditions such as uremia the gestational ages at the time of admission ranged from 37 weeks to term , and there were no preterm or postterm subjects . the prepregnant bmi [ figure 1 ] varied from 18.2 to 24.8 kg / m , the mean value being 21.56 1.37 kg / m . a significant association was found between the prepregnant bmi and hba1c ( chi - square value = 30.067 , p < 0.001 ) . pie diagram showing the distribution of body mass index the weight gain [ figure 2 ] of the subjects ranged from 8 to 16 kg with the mean value being 11 1.922 kg . hba1c was seen to correlate significantly with the weight gain ( chi - square value = 25.225 , p < 0.001 . pie diagram for distribution of weight gain in pregnancy the mean value of gct is 118.07 15.219 mg / dl , and the gct of the second trimester correlated significantly with hba1c of the third trimester ( p the random blood sugar in the third trimester ranged from 85 to 148 mg / dl . there was a significant association between the random blood sugar values and hba1c , both taken in the third trimester ( p < 0 05 ) . the hba1c values [ figure 3 ] in the third trimester of pregnancy in this study ranged from 4.5% to 6% . pie diagram showing the distribution for glycosylated hemoglobin values there were 54 normal deliveries and 45 cesarean sections with one ventouse delivery . there were in total 96 singleton deliveries , and 4 twin deliveries but one pair of twins had an intrauterine death . the indications for the cesarean sections varied from failed trial , fetal distress , meconium - stained liquor , premature rupture of membranes ( one case ) and placenta previa ( one case ) , corrected mitral stenosis ( elective ) . nineteen sections were for failed trial only , 4 were for failed trial and fetal distress . the birth weight ranged from 1930 to 3750 g with the mean birth weight being 3140 367 g. a significant association was found between the birth weight and hba1c value ( p < 0.05 ) . admission to neonatal intensive care unit ( icu ) was also found to correlate significantly with hba1c ( p < 0.05 ) . unfavorable outcomes were found the least in the 4.5%5% range ; hence , the upper cutoff of safe range can be taken as 5% , association with hba1c values and adverse outcomes being significant ( p < 0.05 ) . the various outcomes [ figure 4 ] in the newborn analyzed revealed that the majority were admitted for observation for transient tachypnea ( 49.5% ) and hyperbilirubinemia ( 16.5% ) requiring phototherapy , hypocalcemia requiring calcium supplements ( 12.6% ) , hypoglycemia requiring glucose ( 7.8% ) , and persistent tachypnea of newborn ( 5.8% ) . a few newborn required management such as intravenous antibiotics also . all the fetal outcomes correlated significantly with the hba1c value [ p < 0.05 , table 1 ] . bar diagram to show the distribution of adverse outcomes in the newborns correlation of fetal outcomes with glycosylated hemoglobin levels the study on primigravida in their third trimester who were nondiabetic was found to have a mean hba1c value of 5.29% 0.514% which was in fair agreement with other similar studies . hence , we may conclude that the upper cutoff of hba1c for a safe outcome is 5% for a nondiabetic pregnancy so that adverse outcomes can be minimized . the hba1c value was significantly associated with weight gain during pregnancy and prepregnant bmi , but no significant association was seen with prepregnant weight . for a female with normal bmi before pregnancy , weight gain of 1013 kg is recommended . as per a previous study , gain of > 13 kg is associated with 9% perinatal mortality in females with < 68 kg and 14% perinatal mortality in > 68 kg prepregnant weight and gestational age , pregravid weight , weight gain , and bmi all affect birth weight . there was association of hba1c in the third trimester with gct done in the thirdtrimester with gct done in the second trimester and random blood sugar in the thirdtrimester , which were both significant . similarly , a significant association was found betweenthe birth weight of the newborn , fetal outcomes such as observation in the icu for transienttachypnea , hyperbilirubinemia , hypocalcemia , and hypoglycemia . it is known that , inpregnant women , who are not overtly diabetic , association is found between increasingglucose levels and fetal outcomes such as birth weight , need for intensive care , hypoglycemia , and hyperbilirubinemia as also with obstetrical outcome like cesarean sections . in addition , neonatal hypoglycemia is increased in gestational diabetes patients due to fetal hyperinsulinemia , decreased hepatic glucose production , and decreased ability to use glycogen in the first few days of life . poor glycemic control in diabetic patient affects lung maturity and hence there is increased risk of respiratory distress syndrome . hyperbilirubinemia occurs probably due to decreased clearance of bilirubin and increased production of breakdown products as there is associated polycythemia in response to chronic fetal hypoxia . fetal hypocalcemia can occur due to decreased maternal parathyroid hormone and the severity of hypocalcemia correlates with degree of glycemic control . the average plasma blood glucose corresponding to hba1c value of 5% is 101 mg / dl and that corresponding to mean value of 5.29% is 111 mg / dl ( range for 5.29% 0.514% is 90129 mg / dl ) . hence , it may be concluded that hba1c can indeed be utilized for the monitoring of glycemic level , as a screening test , even in nondiabetic pregnancies in our clinical practice . the hba1c value should be kept between 4.5% and 5% for minimal adverse outcomes it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimesterthere is a need to keep the bmi also in the lower range since hba1c is found to increase with the bmi and weight gain , which will require lifestyle management in the preconception period itselfmore frequent measurements of hba1c beginning from the prepregnant state to the first , second , and third trimesters in our routine clinical practice is required so that the glycemic changes which occur throughout pregnancy , with follow - up into postnatal period , can be monitored . the hba1c value should be kept between 4.5% and 5% for minimal adverse outcomes it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimester there is a need to keep the bmi also in the lower range since hba1c is found to increase with the bmi and weight gain , which will require lifestyle management in the preconception period itself more frequent measurements of hba1c beginning from the prepregnant state to the first , second , and third trimesters in our routine clinical practice is required so that the glycemic changes which occur throughout pregnancy , with follow - up into postnatal period , can be monitored . the study is carried out during pregnancy , which is a very vulnerable period , but with minimum risks to the subjectsthis study being a hospital - based study and hba1c being a common test available in the hospital laboratory , the subjects could get it done with minimum effort . the study is carried out during pregnancy , which is a very vulnerable period , but with minimum risks to the subjects this study being a hospital - based study and hba1c being a common test available in the hospital laboratory , the subjects could get it done with minimum effort . sample size is smallconfounding factors such as family history and parity could not be avoided majority of subjects have history of diabetes mellitus in one or both parents , and primigravida were studied since the incidence of cesarean sections for an indication of previous cesarean sections is already quite highonly random blood sugars could be measured as and when the patient came to hospital in the third trimesterthe day of measurement of hba1c varied as and when the subjects came for their due date of checkup in the third trimestercounseling or any other advice was not provided as it is an observational study . confounding factors such as family history and parity could not be avoided majority of subjects have history of diabetes mellitus in one or both parents , and primigravida were studied since the incidence of cesarean sections for an indication of previous cesarean sections is already quite high only random blood sugars could be measured as and when the patient came to hospital in the third trimester the day of measurement of hba1c varied as and when the subjects came for their due date of checkup in the third trimester counseling or any other advice was not provided as it is an observational study . the hba1c value should be kept between 4.5% and 5% for minimal adverse outcomes it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimesterthere is a need to keep the bmi also in the lower range since hba1c is found to increase with the bmi and weight gain , which will require lifestyle management in the preconception period itselfmore frequent measurements of hba1c beginning from the prepregnant state to the first , second , and third trimesters in our routine clinical practice is required so that the glycemic changes which occur throughout pregnancy , with follow - up into postnatal period , can be monitored . the hba1c value should be kept between 4.5% and 5% for minimal adverse outcomes it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimester there is a need to keep the bmi also in the lower range since hba1c is found to increase with the bmi and weight gain , which will require lifestyle management in the preconception period itself more frequent measurements of hba1c beginning from the prepregnant state to the first , second , and third trimesters in our routine clinical practice is required so that the glycemic changes which occur throughout pregnancy , with follow - up into postnatal period , can be monitored . the study is carried out during pregnancy , which is a very vulnerable period , but with minimum risks to the subjectsthis study being a hospital - based study and hba1c being a common test available in the hospital laboratory , the subjects could get it done with minimum effort . the study is carried out during pregnancy , which is a very vulnerable period , but with minimum risks to the subjects this study being a hospital - based study and hba1c being a common test available in the hospital laboratory , the subjects could get it done with minimum effort . sample size is smallconfounding factors such as family history and parity could not be avoided majority of subjects have history of diabetes mellitus in one or both parents , and primigravida were studied since the incidence of cesarean sections for an indication of previous cesarean sections is already quite highonly random blood sugars could be measured as and when the patient came to hospital in the third trimesterthe day of measurement of hba1c varied as and when the subjects came for their due date of checkup in the third trimestercounseling or any other advice was not provided as it is an observational study . confounding factors such as family history and parity could not be avoided majority of subjects have history of diabetes mellitus in one or both parents , and primigravida were studied since the incidence of cesarean sections for an indication of previous cesarean sections is already quite high only random blood sugars could be measured as and when the patient came to hospital in the third trimester the day of measurement of hba1c varied as and when the subjects came for their due date of checkup in the third trimester counseling or any other advice was not provided as it is an observational study . thus , even though gct may be normal , blood sugars should be measured in the third trimester too , since dietary pattern also changes in the third trimester , due to sociocultural factors , in our society . the average blood plasma glucose corresponding to the mean hba1c value of 5.29% is 111 mg / dl ( range for 0.514% is 90129 mg / dl ) . the average plasma blood glucose corresponding to hba1c of 5% is 101 mg / dl . as hba1c value of 5% is considered to be the safe cutoff in this study , it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimester .

objective : the objective of the study is to estimate the level of glycosylated hemoglobin ( hba1c ) for a safe fetal outcome and to estimate the relation between this level and various adverse fetal outcomes . materials andmethodology : primigravidas who are diagnosed as not having gestational diabetes mellitus as per the glucose challenge test done at 24 weeks with a cutoff value up to 140 mg / dl are followed up at 3034 weeks for the estimation of hba1c in the blood and further till the time of delivery and postnatal period for the fetal outcomes . data were collected based on detailed patient interview , clinical examination , and laboratory investigations . data were analyzed to obtain the mean value of hba1c in the third trimester . fetal outcomes were analyzed with the hba1c value using chi - square test.results:the hba1c values in the third trimester of pregnancy in this study ranged from 4.5% to 6%.discussion : unfavorable outcomes were found the least in the 4.5%5% . the average plasma blood glucose corresponding to hba1c value of 5% is 101 mg / dl . the majority of the newborn were admitted for observation for transient tachypnea ( 49.5% ) and hyperbilirubinemia ( 16.5% ) requiring phototherapy , hypocalcemia requiring calcium supplements ( 12.6% ) , hypoglycemia requiring glucose ( 7.8% ) , and persistent tachypnea of newborn ( 5.8% ) and all the outcomes correlated significantly with hba1c values.conclusion:hence , hba1c can be utilized for the monitoring of glycemic level and as screening test .

Introduction Materials and Methodology Results Discussion Recommendations Strengths of the study Limitations of the study Conclusions Financial support and sponsorship Conflicts of interest

the average plasma blood glucose corresponding to hba1c value of 5% is 101mg / dl and that corresponding to mean value of 5.29% is 111mg / dl ( range for 5.29%+/-0.514% in view of these maternal changes during a normal pregnancy in glucose levels , which increase as pregnancy progresses , and its effects and that hba1c correlates with the glycemic control over the past few weeks , the present study aims to estimate the levels of hba1c in pregnant nondiabetic women in the third trimester and its relation to adverse fetal outcomes . pie diagram for distribution of weight gain in pregnancy the mean value of gct is 118.07 15.219 mg / dl , and the gct of the second trimester correlated significantly with hba1c of the third trimester ( p the random blood sugar in the third trimester ranged from 85 to 148 mg / dl . the hba1c values [ figure 3 ] in the third trimester of pregnancy in this study ranged from 4.5% to 6% . the various outcomes [ figure 4 ] in the newborn analyzed revealed that the majority were admitted for observation for transient tachypnea ( 49.5% ) and hyperbilirubinemia ( 16.5% ) requiring phototherapy , hypocalcemia requiring calcium supplements ( 12.6% ) , hypoglycemia requiring glucose ( 7.8% ) , and persistent tachypnea of newborn ( 5.8% ) . the average plasma blood glucose corresponding to hba1c value of 5% is 101 mg / dl and that corresponding to mean value of 5.29% is 111 mg / dl ( range for 5.29% 0.514% is 90129 mg / dl ) . the hba1c value should be kept between 4.5% and 5% for minimal adverse outcomes it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimesterthere is a need to keep the bmi also in the lower range since hba1c is found to increase with the bmi and weight gain , which will require lifestyle management in the preconception period itselfmore frequent measurements of hba1c beginning from the prepregnant state to the first , second , and third trimesters in our routine clinical practice is required so that the glycemic changes which occur throughout pregnancy , with follow - up into postnatal period , can be monitored . the hba1c value should be kept between 4.5% and 5% for minimal adverse outcomes it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimester there is a need to keep the bmi also in the lower range since hba1c is found to increase with the bmi and weight gain , which will require lifestyle management in the preconception period itself more frequent measurements of hba1c beginning from the prepregnant state to the first , second , and third trimesters in our routine clinical practice is required so that the glycemic changes which occur throughout pregnancy , with follow - up into postnatal period , can be monitored . the hba1c value should be kept between 4.5% and 5% for minimal adverse outcomes it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimesterthere is a need to keep the bmi also in the lower range since hba1c is found to increase with the bmi and weight gain , which will require lifestyle management in the preconception period itselfmore frequent measurements of hba1c beginning from the prepregnant state to the first , second , and third trimesters in our routine clinical practice is required so that the glycemic changes which occur throughout pregnancy , with follow - up into postnatal period , can be monitored . the hba1c value should be kept between 4.5% and 5% for minimal adverse outcomes it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimester there is a need to keep the bmi also in the lower range since hba1c is found to increase with the bmi and weight gain , which will require lifestyle management in the preconception period itself more frequent measurements of hba1c beginning from the prepregnant state to the first , second , and third trimesters in our routine clinical practice is required so that the glycemic changes which occur throughout pregnancy , with follow - up into postnatal period , can be monitored . as hba1c value of 5% is considered to be the safe cutoff in this study , it may well be useful to keep the blood sugar values , irrespective of fasting or postprandial levels , to below 101 mg / dl , in the third trimester .

tinnitus , the perception of sound in the absence of external acoustic stimulation , is a widespread pathology and affects around 10% of the adult population [ 14 ] . it is commonly the result of overexposure to noise or overconsumption of drugs such as salicylates . during the past decades , clinical studies , however , consistently reported noise overexposure as the main cause of tinnitus in human [ 1 , 2 , 5 , 6 ] . despite the strong alteration of the quality of life of patients suffering from tinnitus and its impact on public health systems , and despite increasing knowledge on the molecular mechanisms involved [ 710 ] , no efficient cure is currently available [ 4 , 6 , 9 ] . given the high prevalence and the suffering involved , the neurobiology of tinnitus is of great importance . what is it that alters a perceptual system to experience a phantom percept ? is this a form of runaway plasticity , and can it be blocked ? evidence has been accumulated to implicate neuroplasticity in tinnitus , including a role for cochlear n - methyl - d - aspartate ( nmda ) receptor [ 7 , 11 , 12 ] . most studies use salicylate - induced tinnitus as a model . this form accounts , however , for only a minor fraction of human tinnitus ; acoustic trauma is much more prevalent [ 2 , 5 ] . further , whereas salicylate - induced tinnitus is reversible , noise - induced tinnitus is frequently chronic . the mechanisms of noise - induced tinnitus are hardly understood . a particularly important question : once the noise insult took place , is there still an opportunity to abate tinnitus ? to achieve progress towards the aforementioned goals , an animal model is needed . the first behavioral model in the rat was introduced by jastreboff et al . . they used noise - controlled conditioned suppression of drinking , and showed that rats treated with salicylate are less likely to stop drinking when the noise is turned off . this has been taken to indicate that the salicylate - treated rats still hear the sound in its absence . this imaginative protocol , however , requires extensive training , utilizes footshock that may introduce confounding factors , and involves intense water deprivation . we have set out to develop a new behavioral test for tinnitus in the rat , based on navigation to a tone in a water t - maze ( wtm ) . based on the behavior of salicylate - treated rats , we define criteria for identification of tinnitus in the wtm , and use them to identify noise - induced tinnitus . here , we report that the induction of both salicylate - induced and noise - induced tinnitus can be blocked by the local cochlear application of ifenprodil , an antagonist of the 2b subunit of the nmda receptor ( nr2b ) , a molecule which is implicated in long - term potentiation and behavioral plasticity in the mammalian brain [ 1418 ] . we further report that this nr2b - dependent process undergoes consolidation , during which the development of long - term tinnitus can be prevented by an nr2b blocker . our data hence demonstrate a consolidation window in trauma - induced plasticity in a peripheral sensory system , and point to a potential method to abate the outcome of the sensory trauma . rats ( wistar males , ~60-day old , 250380 g , total n = 154 ) were caged individually at 22 2c in a 12-hour light / dark cycle . all experiments were approved by the weizmann institute of science animal care and use committee . the repartition of the animals in the different experimental groups is detailed in table 1 . we used a place - tone conditioning paradigm , in which the rat is conditioned in a water t - maze ( wtm , see figure 1 ) to associate a tone with the presence of a submerged escape platform in one of the arms ( tone arm ) , and the absence of that tone with the presence of the platform in the opposite arm ( no - tone arm ) . the arm - platform - tone permutations were counterbalanced between subjects in the experimental design to eliminate potential side preference . rats with tinnitus were expected to always behave as though the tone was present , even in its absence . the starting arm was 25-cm long and the two identical lateral arms 40 cm long each . all arms were 15-cm wide and 60-cm high , filled with water to a level of 24 cm . a sliding door was installed before the maze decision point . a submerged platform ( 12-cm diameter , 23 cm height ) the tones used were 10 khz or 6 khz ( as indicated under results ) continuous pure tones , 45 db spl , delivered from above by a speaker ( high performance 3 tweeter , best - star ) . in the conditioning phase each session consisted of 12 trials ( 3 no - tone + 3 tone alternating twice in that order ) . the platform was positioned at the end of one arm during the tone presentation and at the end of the opposite arm during the no - tone period . the rat was placed in the starting arm for 5 seconds before the opening of the door . the door was closed after the entrance of the rat to the lateral arm . when applicable , sound onset coincided with the placing of the rat in the starting arm and continued to overlap the first 5 seconds after the rat reached the platform . when the rat located the platform , it was allowed to stay on it for 30 seconds before being placed back in the starting arm ( beginning of the next trial ) or in the home cage ( after the last trial ) . the following two parameters were used to quantify conditioning in the course of training : time to reach the platform ( averaged over the 12 trials of each session ) , and correct decision ( percentage of correct responses over 12 decisions made in a session ) . the test protocol consisted of a single trial performed in the absence of the platform . except when otherwise specified , rats were tested in the absence of the tone . when rats were tested with the tone , the rats were placed in the starting arm 5 seconds before the opening of the door . the duration of the trial was 100 seconds from the entrance of the rat into the lateral arm . after this , the rat was placed back in its home cage . to quantify memory in the test session this is presented in section 3 as time spent in the indicated arm in the first 50 seconds and in the last 50 seconds , respectively . this breakdown into early and late test period was done to allow detection of possible alteration in behavior during the test itself . this was done to supplement at the group level the observations made of the behavior of the individual rats . these data are presented only when the size of the resulting subgroups was large enough to allow statistical analysis . this was performed in an acoustic chamber ( controlled acoustic environments , industrial acoustics company inc . rats were exposed to a 6 khz , 130 db spl pure - tone for 15 minutes . optimal tone detection in the rat , as assessed by electrophysiological recordings of auditory thresholds , is 10 khz . in previous morphological and electrophysiological studies , maximum damage was observed in the tonotopic cochlear area coding for 10 khz following an acoustic overexposure of a pure - tone of 6 khz . sound insult at 6 khz was therefore selected to maximize the occurrence of tinnitus of a frequency of 10 khz . the tone was delivered after an analog amplification ( ma 430 power amplifier , inkel , seoul , south korea ) via high - power speakers ( xd 120 , eighteen sound , reggio emilia , italy ) . rats were anesthetized before the noise overexposure by ip injection of 0.3 ml / kg sodium pentobarbital at 6% ( cts chemical industries ltd . all sound levels were measured and calibrated using a brel and kjaer microphone ( bk precision 732 , brel and kjaer , norcross , ga , usa ) . rats were anesthetized with ip injection of 0.3 ml / kg sodium pentobarbital at 6% and operated under aseptic conditions . the surgical protocol to place gelfoam ( gelita tampon ; b. braun medical , melsungen , germany ) on the round window of both cochleae was as described previously . after exposition of the cochlea via aposterior approach of the right bulla , gelfoam , soaked with 2.5 l of artificial perilymph containing the appropriate drug , was applied to the round window of the cochlea . the bulla was closed with dental cement , and the surgical wound was sutured . salicylate , ifenprodil , and metachlorophenylpiperazine ( mcpp ) were purchased from sigma ( sigma - aldrich , st . louis , miss , usa ) , 6,7-dinitroquinoxaline-2,3-dione ( dnqx ) was from tocris ( tocris bioscience , avonmouth , uk ) . the n - methyl - d - aspartate ( nmda ) receptor antagonist ifenprodil , specific to nr2b - subunit , was used at a concentration of 10 m . the -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa ) receptor antagonist dnqx was used at a concentration of 50 m . ifenprodil , dnqx , and mcpp were all dissolved in artificial perilymph ( ap , in mm : 140 nacl , 4 kcl , 2 cacl2 , 2 mgcl2 , 10 hepes , 10 glucose , ph 7.4 , and osmolarity , 300 10 mosm / kg h2o ) . neither nystagmus nor apparent dizziness was observed after local application of any of the pharmacological agents at the concentrations we used onto the round window , suggesting a lack of effect on the vestibular function . behavioral data were analyzed by one - way anova followed by post hoc comparisons ( tukey 's test ) . data are presented as mean sem . in experiments which involved time , comparisons were made using a two - way anova ( group time , with repeated measures on the last factor ) to test group effect , time effect , and group time interactions , followed by post hoc comparisons ( tukey 's test ) . proportions of rats experiencing tinnitus in various treatment groups were compared by chi - square test or mann - whitney test . in addition , the occurrence of tinnitus over time following pharmacological treatment was assessed using a wilcoxon test on the time spent in the tone arm . in the first phase of this study , we developed a novel behavioral paradigm that permits objective determination of tinnitus in the rat , and verified the power of this paradigm to detect tinnitus by the use of salicylate treatment under conditions that are well established to produce tinnitus . we used a place - tone conditioning paradigm , in which rats learn to associate the presence of a tone with the presence of an escape platform in one of the arms ( tone arm ) of a water t - maze ( wtm ) , and the absence of that tone with the presence of the escape platform in the opposite arm of the maze ( no - tone arm ) . akin to the reasoning of previous animal protocols intended to detect tinnitus [ 7 , 13 ] , we reasoned that rats with tinnitus , experiencing phantom tone , would always behave as though the tone is present , even in its absence . this frequency is optimally perceived by the intact rat and characterizes salicylate - induced tinnitus in the rat [ 7 , 20 ] . unconditioned rats displayed no arm preference in the wtm , as judged by the time spent in each arm ( see figure 1(b ) ) . this was regardless of whether they were tested in the presence of the tone , in its absence , or in the absence of the tone after being treated for 4 days with salicylate under conditions known to induce tinnitus [ 7 , 20 ] . thus , in the absence of the tone , the unconditioned rats spent 25.41.7 seconds and 23.72.7 seconds in the left arm during the first and the last 50 seconds of the test , respectively . when tested in presence of the 10-khz sound , finally , when tested in the absence of the tone , unconditioned salicylate - treated rats spent 24.91.1 seconds and 25.11.1 seconds in the left arm during the first and the last 50 seconds of the test , respectively . further , the first arm choice was made randomly : 4 of the 8 of the unconditioned rats selected the left arm as their first choice . in contrast , in conditioned rats , a clear preference became evident over training for the tone- /no tone - cued arm associations , as manifested in the time to reach the platform and in choice preference ( learning curve , figure 1(c ) ) . hence , on day 1 of training , the mean time to reach the platform was 9.80.1 seconds and accuracy was 43.30.8% , reaching 3.70.05 seconds and 74.40.7% , respectively , on day 3 ( see figure 1(c ) , n = 124 ) . the memory for the association remained robust two weeks after the end of training ( see figure 2 ) . when tested in the absence of the tone two weeks after the end of training , the conditioned rats spent only 13.41.2 seconds and 14.11.3 seconds during the first and the last 50 seconds of the test , respectively , in the tone arm ( see figure 2(a ) ) . in contrast , conditioned rats tested in presence of the tone displayed significant preference for the tone arm . they spent 35.81.1 seconds and 33.40.5 seconds in that arm during the first and last 50 seconds of the test , respectively ( different from the conditioned rats tested in the absence of the tone in both time windows , p < .001 , see figure 2(a ) ) . after salicylate treatment , the rats spent 40.31.4 seconds during the first 50 seconds and 30.80.8 during the last 50 seconds of the test in that arm ( different in both cases from the conditioned animals tested in the absence of the tone , p < .001 ) . in addition , despite being in both cases significantly different from the untreated conditioned animals tested in the absence of the tone , the time spent in the tone arm by salicylate - treated conditioned rats was significantly higher during the first 50 seconds of the test ( p < .01 ) . this might imply that the subjective perception of the cue in salicylate - treated rats is not exactly as in the untreated rats . an alternative hypothesis could be that animals behave differently in the test due to an elevation of their auditory thresholds . in the present protocol , given that animals are tested in the absence of the tone , deaf animals would be expected to have a greater probability to spend time in the no - tone arm . indeed , the physical absence of the tone would probably combine with the perceptive silence of deafness . this , however , is not the case : following salicylate treatment , animals spent more time in the tone arm . thus , a contamination of deafness in the interpretation of these data can be ruled out . rats treated with salicylate as above , but receiving a local cochlear application of ifenprodil , an nr2b antagonist , performed in the wtm similarly to untreated rats tested in the absence of the tone ( figure 2(a ) ) . they spent only 12.50.9 seconds during the first 50 first seconds and 14.30.9 seconds during the last 50 seconds of the test in the tone arm ( not significantly different from untreated rats ) . local application onto the cochleae of the vehicle only had no effect on salicylate - treated rats ( figure 2(a ) ) . the results obtained above allowed us to establish criteria to identify rats with tinnitus by their behavior in the wtm ( see figure 2(b ) ) . in the aforementioned experiments , two distinct behavioral patterns became apparent : one , tone , corresponding to the performance in the wtm of rats tested in presence of the tone , and the performance of rats treated with salicylate , or salicylate + vehicle , in the absence of the tone ; the other , no - tone , corresponding to the performance in the wtm of untreated rats and of salicylate + ifenprodil rats , tested in the absence of the tone . each rat could hence be designated as no - tone or tone on the basis of its individual performance . in addition , a complementary measure could also be recorded , that is , the number of rats within a given subgroup of rats assigned on the basis of the above individual criterion , which selected the arm associated with the tone as the first choice . rats displaying the tone behavior in the absence of exogenous tone were hence considered to suffer from tinnitus . rats were designated to the tone group if in the test they spent in the tone arm not more than 2 sd ( p > .05 ) below the mean time spent in the tone arm by untreated rats tested in the presence of the tone . in contrast , animals were designated to the no - tone group if in the test in the absence of the tone they spent in the tone arm not more than 2 sd above the mean time spent by untreated rats tested in the absence of the tone in the tone arm ( figure 2(b ) ) . in the following experiments , none of the animals displayed a behavior which did not fit into either the tone or the no - tone groups as defined above . having established the aforementioned criteria , we proceeded to test the effect of noise overexposure on the induction of long - term tinnitus . whereas salicylate is known to produce relatively short - term , reversible tinnitus , noise overexposure is known to be able to induce long - term , irreversible tinnitus . however , unlike salicylate treatment , the effectiveness of noise overexposure in inducing tinnitus is less predictable . in the noise overexposure experiments , animals were subjected to an intense overexposure of a 6 khz pure - tone . noise - induced tinnitus is often characterized by having rather high frequency [ 6 , 22 ] . furthermore , the frequency of noise - induced tinnitus is thought to be slightly higher than the frequency of the sound eliciting the tinnitus . hence , using a frequency of 6 khz to elicit tinnitus allowed us to analyze tinnitus with a frequency of around 10 khz . given that 10 khz is the best frequency in rat auditory perception , detection of tinnitus with this frequency should be more sensitive . however , to confirm this difference between the frequency of noise overexposure and the expected frequency of tinnitus , an additional group of animals was trained using a 6 khz pure - tone as the cs , before being subjected to noise overexposure . two weeks after noise overexposure , rats conditioned to a pure tone of 6 khz did not display behavioral evidence of tinnitus in the wtm test . rats in this group spent 14.30.8 seconds during the first 50 seconds and 15.50.6 seconds during the last 50 seconds of the test in the tone arm ( n = 8 , not significantly different from the conditioned animals tested in the absence of the tone ) . in contrast , two weeks after noise overexposure , rats conditioned to the 10 khz ( n = 26 ) tone did not represent a uniform and homogeneous population . rather , as demonstrated by the individual performance data portrayed in figure 3 , they segregated into two subgroups based on the aforementioned tinnitus - detection criteria . rats in the first subgroup ( n = 14 ) behaved like untreated rats tested in the absence of the tone ( see figure 3 ) . these rats spent 12.50.7 seconds during the first 50 seconds and 14.10.7 seconds during the last 50 seconds of the test in the tone arm ( not significantly different from untreated conditioned rats tested in the absence of the tone ) . rats in this group selected the arm associated with the tone as their first choice in only 3/14 of the cases . rats from the second subgroup ( n = 12 ) displayed a response similar to that expected in presence of the tone ( see figure 3 ) . these rats spent 33.30.8 seconds during the first 50 seconds and 34.60.9 seconds during the last 50 seconds of the test in the tone arm ( different from the conditioned animals tested in the absence of the tone , p < .001 ; not different from the conditioned animals tested in presence of the tone , see figure 3 ) . furthermore , they selected the tone arm in 8/12 of the cases , p < .01 compared to the first subgroup , mann - whitney test . all in all , our data demonstrate that noise overexposure induces long - term tinnitus , but that only about a half of the treated rats develop tinnitus . it is noteworthy that this partial effectiveness is similar to that observed in humans [ 2 , 5 ] . our previous findings on the role of nmda receptor , and particularly the present findings ( see above ) on the role of the 2b subunit of the nmda receptor ( nr2b ) in salicylate - induced tinnitus , have led us to investigate the role of the nr2b in noise - induced tinnitus . toward that end none of the rats receiving local application of ifenprodil just before sound overexposure ( n = 8) demonstrated behavioral evidence of tinnitus in the wtm ( see figure 4 ) . these rats spent 13.40.8 seconds and 13.80.8 seconds during the first and the last 50 seconds of the test in the tone arm ( not significantly different from the conditioned animals tested in the absence of the tone ) . similar results were observed when ifenprodil was applied to the cochlea 4 days after the noise overexposure ( see figure 4(a ) ) . two weeks after the noise overexposure , none of these rats ( n = 8) displayed behavioral evidence of tinnitus . they spent 12.9 1 seconds in the first 50 seconds and 13.10.6 seconds in the last 50 seconds of the test in the tone arm ( not significantly different from the conditioned rats tested in the absence of the tone ) . in contrast , when ifenprodil was applied 8 days after noise overexposure , 3 of the 8 rats in the group displayed behavioral evidence of tinnitus according to the individual criteria detailed above ( not statistically different from the noise - overexposed group , but statistically different from that observed when ifenprodil was applied just before or 4 days after sound overexposure , p < .05 ) . these rats spent 35.70.9 seconds and 33.70.9 seconds in the first and the last 50 seconds of the test in the tone arm , although the external tone was absent ( different from untreated conditioned animals tested in the absence of the tone , p < .001 ; not different from the untreated conditioned animals tested in presence of the tone ) . all the rats in this subgroup selected the tone arm as their first choice . the other 5 rats spent only 13.81.4 seconds during the first 50 seconds and 14.20.9 seconds during the last 50 seconds of the test in the tone arm , and four of them selected the no - tone arm as their first choice , in line with the absence of tinnitus in the group ( p < .05 compared to the group of animals receiving ifenprodil at day 0 , wilcoxon test ) . half of the rats ( 4/8 ) that received ifenprodil 12 days after the sound overexposure demonstrated evidence of tinnitus ( see figure 4(a ) ) . this proportion is not statistically different from the noise - overexposed group , but different from the proportion observed when ifenprodil was applied just before or 4 days after the sound overexposure ( p < .05 ) . the rats in this subgroup spent 13.51.3 seconds and 13.81.0 seconds in the tone arm in the first and the last 50 seconds of the test , respectively . this is not significantly different from conditioned intact rats tested in the absence of the tone . rats in the other subgroup ( i.e. , the other 4 ) spent 36.81.7 seconds and 34.81.0 seconds in the first and the last 50 seconds of the test , respectively , in the tone arm in the absence of the external tone . this is different from the conditioned animals tested in the absence of the tone ( p < .001 ) but not different from the conditioned animals tested in presence of the tone . none of the rats receiving the ampa receptor antagonist dnqx just before the sound overexposure on day 0 provided evidence of tinnitus ( n = 8) . in this case , the time spent in the tone arm was 12.81.0 seconds and 13.51.0 seconds , respectively , for the first and last 50 seconds of the test ( not significantly different from untreated conditioned rats tested in the absence of the tone ) . for rats that received the application of dnqx 4 days after the sound overexposure ( n = 8) , 50% demonstrated evidence of tinnitus ( see figure 4(b ) ; not statistically different from the noise - overexposed group , but different from the proportion observed when dnqx was applied just before the sound overexposure , p < .05 ) . four of these rats spent an average of 36.80.5 seconds in the first 50 seconds and 34.30.5 in the last 50 seconds of the test in the tone arm ( different from conditioned untreated rats tested in the absence of the tone , p < .001 , not different from conditioned untreated rats tested in presence of the tone ) . the other 4 rats in this 4-day postexposure dnqx group spent 10.30.9 seconds in the first 50 seconds and 13.01.4 in the last 50 seconds of the test in the tone arm ( not significantly different from the untreated conditioned rats tested in the absence of the tone ) . none of these animals preferred the tone arm as first choice ( p < .05 compared to the group of animals receiving ifenprodil at day 0 , and p < .05 compared to the group of animals receiving dnqx at day 0 , wilcoxon test ) . local application of the serotonergic agent mcpp into the cochlea just before noise overexposure did not prevent the occurrence of tinnitus ( n = 8 , see figure 4(b ) ) . two weeks after noise overexposure , 50% of these rats displayed tinnitus - like behavior ( not statistically different from the noise - overexposed group , but different from the proportion observed when ifenprodil was applied just before , or 4 days after the sound overexposure , p < .05 ) . rats in the subgroup that displayed behavioral evidence of tinnitus : four of these rats spent 37.30.9 seconds and 35.51.0 seconds in the first and the last 50 seconds of the test in the tone arm in the absence of the tone ( different from untreated conditioned rats tested in the absence of the tone , p < .001 ; not different from untreated conditioned rats tested in presence of the tone ) . in contrast , rats in the other subgroup spent 12.31.0 seconds and 14.30.9 seconds in the first and last 50 seconds of the test in the tone arm ( not significantly different from the untreated conditioned rats tested in the absence of the tone ) , and only one of these rats selected the no - tone arm as the first choice . it is estimated that about 10% of the adult population in industrialized societies suffer some form of chronic tinnitus [ 1 , 3 ] . in most cases , it is the consequence of noise trauma , posing a rather widespread and expanding occupational or clubbing hazard . drug toxicity , a side effect of medications such as salicylates and certain antibiotics , can also cause tinnitus . over the years , multiple hypotheses have been raised concerning the neuronal locale(s ) of the insult , including the peripheral auditory system , the central auditory system , or higher - order , limbic structures [ 4 , 8 , 9 ] . in this study , we set out to investigate neurobiological mechanisms of tinnitus , with the long - term objective of identifying targets for intervention to prevent or ameliorate the pathology . we first developed a new behavioral paradigm to measure tinnitus in the rat . using this paradigm , we demonstrated that local cochlear application of ifenprodil , an antagonist of the 2b subunit of the nmda receptor ( nr2b ) , prevents the long - term occurrence of noise - induced tinnitus , suggesting that the 2b subunit of the nmda receptor complex ( nr2b ) may be critically involved in the induction of tinnitus by salicylate . by analyzing the time - window of sensibility of noise - induced tinnitus to ifenprodil , we then discovered that long - term tinnitus undergoes a consolidation period of several days , during which tinnitus could be abated by blockade of nr2b in the cochlea . these results broaden our understanding of tinnitus and pave the way to the development of novel methods to prevent or ameliorate it . furthermore , these data also reflect on the notion of consolidation in neural plasticity in general . designing a behavioral paradigm to determine tinnitus in animals described here , the expectation was that animals with tinnitus would behave as though they hear a tone even in its absence . this was indeed proven to be the case : in a task involving conditioned tone arm association , treated animals expected to have tinnitus ( following either salicylate treatment or noise overexposure ) behaved in the absence of a tone as if they were hearing it . this new behavioral paradigm allowed us to define a criterion to decide whether freely moving rats are experiencing tinnitus , and validate it using salicylate treatment under conditions that are established to induce 100% tinnitus . the new protocol also provided us with the ability to dissect some perceptual attributes of tinnitus . by separating data obtained during the test interval into two 50-second segments , we were able to show difference in the behavior in the presence of the tone between salicylate- , noise - treated , and control rats . there was a decrease over the test in the time that the salicylate rats spent in the tone arm , which was not observed in the group of noise - exposed animals . this could be explained by assuming that salicylate rats underwent some perceptual depreciation during the test , realizing that salicylate - induced tinnitus does not really sound as the tone . an alternative interpretation of this difference could be that the tinnitus percept induced by salicylate changes over time . in the case of salicylate , tinnitus percept could have been modulated by other factors , such as the stress of not finding the platform . however , such a modulation is unlikely to have occurred , since animals presenting noise - induced tinnitus do not display such behavior . the fact that salicylate - induced tinnitus and noise - induced tinnitus differ in their perceptual characteristics is of major importance . given the fact that the vast majority of animal data in the field of tinnitus research was obtained using salicylate to induce tinnitus , caution should be practiced in generalizing the perceptual and mechanistic conclusions to noise - induced tinnitus . some authors indeed reported particular behavioral aspects related to noise - induced tinnitus , but a direct comparison of the validity of salicylate - induced tinnitus as a relevant model for noise - induced tinnitus was still lacking . our study reinforces the almost tautological conclusion that the best model of noise - induced tinnitus is noise - induced tinnitus . having said that , the molecular mechanisms of salicylate - induced tinnitus are of marked importance . both in vitro and in vivo data indicate that salicylate interferes in the cochlea with glutamatergic neurotransmission , particularly by selectively amplifying nmda - mediated responses [ 24 , 25 ] . furthermore , pharmacological experiments have shown that this pathway lies at the source of salicylate - induced tinnitus . we add to the elucidation of these mechanisms by suggesting here that the 2b subunit of the nmda receptor is particularly involved in this process . in contrast with noise - induced tinnitus , salicylate - induced tinnitus only lasts for a short period of time [ 7 , 20 ] . noise - induced tinnitus is therefore of a greater clinical significance . whereas under the conditions used in this study , salicylate treatment induced tinnitus in all treated animals ( as expected ) , the severe noise insult yielded tinnitus in only part of the treated group . this probabilistic characteristic of noise - induced tinnitus is well documented in humans , and has also been suggested in animals . in considering potential pharmacopeia for noise - induced tinnitus , the first synapse of the auditory pathways ( the synapse between inner hair cells and primary auditory neurons ) is an appealing target . indeed , in this study , when locally applied during the first 4 days following the noise overexposure , the nr2b - containing nmda receptor antagonist ifenprodil was able to completely abate long - term noise - induced tinnitus . taking into account the fact that the synapses between inner hair cells and primary auditory neurons are glutamatergic , the question arises whether the nmda receptor antagonist ifenprodil acts to repair the damage or protects against glutamate - induced excitotoxicity . glutamate - induced excitotoxicity is often associated with overactivity of ampa receptors , but nmda receptors have also been implicated [ 27 , 28 ] . whereas the local application of ifenprodil led to abolishment of tinnitus even when applied 4 days after the noise overexposure , local application of the ampa receptor antagonist dnqx had an effect only immediately before the noise overexposure . the fact that local application of dnqx failed to decrease the ratio of occurrence of tinnitus after exposure to an acoustic trauma when the application was done 4 days after the trauma shows that ampa involvement in noise - induced damage mainly occurs at the first stage after the insult , that is , during the period of the postulated response to excitotoxicity . the lack of effect of the serotonergic agent mcpp just before the acoustic trauma on the expression of tinnitus two weeks afterwards argues against nonspecific effects of the surgery or the dilution of endogenous perilymph with the vehicle . furthermore , to validate the specificity of the effect , we also locally applied a drug that is irrelevant to the genesis of salicylate - induced tinnitus : the serotonergic agent mcpp . incidentally , those results also ruled out a putative role of serotonin in the generation of noise - induced tinnitus . it is noteworthy that the postinsult time window during which the nmda receptor antagonist can abate tinnitus is limited to a few days only . a transient time - window of susceptibility to blockers of experience - dependent plasticity is a universal property of memory systems , and is referred to as consolidation [ 29 , 30 ] . furthermore , though the nmda receptor was considered to play a role in memory encoding only , evidence exists that it may play a key role in memory consolidation as well [ 32 , 33 ] . the exact mechanisms of the nmda receptor blockade in tinnitus notwithstanding ( see remark on repair or protection above ) the phenomenon of a transient sensitivity window during which an experience - dependent neuronal change can be abated may not be a hallmark of learning and memory systems only , and apply as well to experience - dependent modifications that are not construed as learning . indeed , tinnitus could be regarded as experience - dependent modification in a neuronal system , which may share mechanisms with memory systems even at the earliest station in which the insult leaves an imprint . this conclusion is not only of a conceptual nature , as it bears also upon the potential mechanisms of tinnitus . multiple hypotheses have been raised concerning the critical locale of the tinnitus - induced insult , including the peripheral auditory system , the central auditory system , or higher - order brain structures [ 4 , 9 ] . the possibility can not be excluded that tinnitus involves several or all of these locales , and that they are recruited over time , in a process that resembles memory systems consolidation , that is , the translocation of an engram from one location to another and its distribution over multiple loci [ 30 , 34 ] . if this is the case , clearly , the effectiveness of the treatment of long - term , noise - induced tinnitus is expected to decline over time after the insult . the finding that local application of a specific receptor antagonist into the first station of the auditory system is an effective treatment in the first days after the insult , under conditions that should minimize systemic side effects , is therefore of marked potential clinical implication .

tinnitus , the perception of sound in the absence of external acoustic stimulation , is a common and devastating pathology . it is often a consequence of acoustic trauma or drug toxicity . the neuronal mechanisms of tinnitus are neither yet fully understood nor are effective treatments available . using a novel behavioral paradigm for measuring tinnitus in the rat based on tone - guided navigation , we show here that the development of long - term noise - induced tinnitus , the most prevalent and clinically important form of human tinnitus , can be abated by local administration of the nmda antagonist ifenprodil into the cochlea in the first 4 days following the noise insult but not afterwards . this suggests that long - term tinnitus undergoes a consolidation - like process , resembling the ontogeny of items in long - term memory . furthermore , this finding paves the way to potential therapeutic strategies for the prevention of chronic tinnitus once the noise insult had taken place .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS 4. DISCUSSION

tinnitus , the perception of sound in the absence of external acoustic stimulation , is a widespread pathology and affects around 10% of the adult population [ 14 ] . based on the behavior of salicylate - treated rats , we define criteria for identification of tinnitus in the wtm , and use them to identify noise - induced tinnitus . here , we report that the induction of both salicylate - induced and noise - induced tinnitus can be blocked by the local cochlear application of ifenprodil , an antagonist of the 2b subunit of the nmda receptor ( nr2b ) , a molecule which is implicated in long - term potentiation and behavioral plasticity in the mammalian brain [ 1418 ] . we further report that this nr2b - dependent process undergoes consolidation , during which the development of long - term tinnitus can be prevented by an nr2b blocker . in the first phase of this study , we developed a novel behavioral paradigm that permits objective determination of tinnitus in the rat , and verified the power of this paradigm to detect tinnitus by the use of salicylate treatment under conditions that are well established to produce tinnitus . when tested in the absence of the tone two weeks after the end of training , the conditioned rats spent only 13.41.2 seconds and 14.11.3 seconds during the first and the last 50 seconds of the test , respectively , in the tone arm ( see figure 2(a ) ) . in addition , despite being in both cases significantly different from the untreated conditioned animals tested in the absence of the tone , the time spent in the tone arm by salicylate - treated conditioned rats was significantly higher during the first 50 seconds of the test ( p < .01 ) . the other 5 rats spent only 13.81.4 seconds during the first 50 seconds and 14.20.9 seconds during the last 50 seconds of the test in the tone arm , and four of them selected the no - tone arm as their first choice , in line with the absence of tinnitus in the group ( p < .05 compared to the group of animals receiving ifenprodil at day 0 , wilcoxon test ) . in this case , the time spent in the tone arm was 12.81.0 seconds and 13.51.0 seconds , respectively , for the first and last 50 seconds of the test ( not significantly different from untreated conditioned rats tested in the absence of the tone ) . using this paradigm , we demonstrated that local cochlear application of ifenprodil , an antagonist of the 2b subunit of the nmda receptor ( nr2b ) , prevents the long - term occurrence of noise - induced tinnitus , suggesting that the 2b subunit of the nmda receptor complex ( nr2b ) may be critically involved in the induction of tinnitus by salicylate . by analyzing the time - window of sensibility of noise - induced tinnitus to ifenprodil , we then discovered that long - term tinnitus undergoes a consolidation period of several days , during which tinnitus could be abated by blockade of nr2b in the cochlea . given the fact that the vast majority of animal data in the field of tinnitus research was obtained using salicylate to induce tinnitus , caution should be practiced in generalizing the perceptual and mechanistic conclusions to noise - induced tinnitus . in considering potential pharmacopeia for noise - induced tinnitus , the first synapse of the auditory pathways ( the synapse between inner hair cells and primary auditory neurons ) is an appealing target . indeed , in this study , when locally applied during the first 4 days following the noise overexposure , the nr2b - containing nmda receptor antagonist ifenprodil was able to completely abate long - term noise - induced tinnitus . the fact that local application of dnqx failed to decrease the ratio of occurrence of tinnitus after exposure to an acoustic trauma when the application was done 4 days after the trauma shows that ampa involvement in noise - induced damage mainly occurs at the first stage after the insult , that is , during the period of the postulated response to excitotoxicity . if this is the case , clearly , the effectiveness of the treatment of long - term , noise - induced tinnitus is expected to decline over time after the insult .

quantitative , valid , and inexpensive approaches to dietary assessment of large cohorts are needed to fulfill the promise of nutrigenomic research . recent findings suggest that 24-hour dietary recalls are likely to remain the most feasible method for gathering dietary intake information ( schatzkin et al . , 2003 ; schatzkin & kipnis , 2004 ) . advantages of the 24-hour dietary recall over other measures , such as food frequency questionnaires , include its reliance on short - term , rather than long - term memory , and its relatively low respondent burden . additionally , the 24-hour dietary recall is only moderately vulnerable to a telescoping effect , that is , the forward or backward displacement of the event in time ( janssen et al . , 2006 ) , is less likely to cause participants to modify their diets due to enhanced self - awareness ( wang et al . , 2002 ; kikunaga et al . , 2007 ) , and does not require tailoring to specific populations , or redesign and recalibration as the food supply changes . however , use of a single 24-hour dietary recall is likely not representative of an individual s habitual diet , and the number of days of assessment necessary to obtain a stable estimate is food and nutrient dependent ( beaton et al . , 1979 ) . further , recalls may be hindered by flawed short - term memory , resulting in unreported items ( omissions ) falsely - reported items ( intrusions ) ( smith , 1993 ; baxter et al . , 2006 ) , and inaccurate reporting of amounts consumed ( neuhouser et al . , web - based technology for acquiring multiple 24-hour dietary recalls is a demonstrated feasible approach towards improving dietary assessment ( arab et al . , 2010 ) even though a threshold effect is apparent ( schatzkin et al . , 2003 ) . further improvement may be afforded by coupling memory - enhancing technology with web - based recalls . the use of mobile phones with cameras for capturing food images as an alternative to the paper and pencil based dietary records has been tested by several groups , with most using self image - capture ( wang et al . , 2006 ; kikunaga et al . , 2007 ; six et al . , 2010 ; weiss et al . , 2010 ) , and others aimed toward automated image - capture and phone calling to trigger / remind participants to initiate recording ( sun et al . ) . challenges associated with the attempt to use camera phones to replace participant - based reporting have several difficulties , including 1 ) uncertainty regarding third party or automated identification of foods in the images , 2 ) flawed decision - making regarding whether foods imaged were consumed by the participant or not ( prepared for others , discarded ) , and 3 ) reduced reporting of consumption of socially undesirable items due to biased imaging . the current study examines the use of a novel , cost - effective image - assisted recall method that combines automatic image capture ( to reduce participant reactivity ) with a web - based 24-hour dietary recall . the goal was to test the feasibility of this approach ( pairing dietary images with the recall ) to enhance dietary assessment . mobile phone cameras equipped for automatic imaging were worn by participants during eating periods and images were uploaded to a secure website for use by participants during the reporting period the next day . since the images are used directly by the participant , this approach differs in both concept and substance from the use of non - automated camera phone - based dietary assessment that require decoding of images by automation or a third party ( wang et al . , 2006 ; kikunaga et al . nokia n80 mobile phones programmed with three mega pixel cameras , automatic flash , and a 4.7 mm lens ( 35 mm equivalent ) were used for image capture . the mobile phones were attached to a lanyard and worn around the neck of the participant as seen in figure 2 application - specific power management techniques ( kansal , 2003 ; raghunathan v , 2005 ; raghunathan v , 2006 ) were used to balance power savings and required performance ( kansal , 2006 . ) . software developed by engineers at the ucla center for embedded networked sensing , los angeles , ca ( cens ) was employed to automatically record , time stamp , and transmit ( via cellular service ) encrypted data from sensors integrated in the phone ( e.g. , camera , and microphone ) to the secure sensorbase server repository . images were captured every 10 seconds , allowing near - complete documentation of foods and beverages consumed throughout the target period covered in the 24-hour period and without minimal user intervention . industry - standard encrypted transmission was used for web access to the repository and transmission of images between mobile phones and data storage servers . images were filtered to remove blurry , poorly exposed , and unclear images ( bradski , 2000 ; kovesi , 2010 ) and filed chronologically in clusters , based on key the total number of images presented to the participant was limited to fewer than 100 . a customized web - based imageviewer was developed to allow participants private access to their own images and the ability to delete objectionable images when desired ( figure 4 ) . images flagged as private ( i.e. , not shared ) were immediately and permanently deleted from the repository . images marked as shared were made available to investigators via a separate private image browser ( reddy et al . , 2007 ) . to further protect the privacy of participants , the image repository did not store any other personal identifier information with the images . sensitivity to the risk of identification of study participants is needed in this area of research . participants can be identified if an individual passes a reflective surface when the camera takes an image . the ability to identify someone from a hand which enters the camera lens field of vision also presents a risk . this type of risk requires that images be stored in datasets that are independent and not cross - linked to those with personal data . for this we developed levels of protection that involve participation and approval of the informed participant at each level . the participants were asked whether they wish to share any , some , or all images with study investigators . any images marked as protected ( i.e. , not shared ) was permanently deleted from the data storage server when the participant logged out of imagedietday . image counts prior to deletion were maintained , along with the timestamps and sequence position of the deleted images to assist in understanding deletion patterns and intent . shared images were to be accessible to the investigators and their designees , who sign confidentiality agreements , via password - protected interfaces . images that were never viewed by the participant ( such as when the system determines them to be unclear and therefore unhelpful to display ) were deleted . to further protect the privacy of participants , the image repository did not store personally identifiable information . this study received irb approval for a three - step process : ( 1 ) automated image capture from the camera phone , ( 2 ) automatic , encrypted upload of images to a secure repository and removal from the device , ( 3 ) a private web portal for each participant that allowed them to review their images and permanently remove any that they did not wish to share with investigators . the general rule in the united states is that anyone may take photographs of whatever they want when they are in a public place . however , even on public property , photography can be prohibited if somebody has a reasonable expectation of privacy . two independent pilot studies of the approach were conducted in august 2007 : the first to test technical feasibility and the second to test subjective acceptability . ten young adult cens employees participated in a technical feasibility exercise designed to test the ability of the system to handle uploaded images collected by simultaneous users . participants wore and operated phones at all times including outside of the home , capturing six images per minute throughout the day . to test technical feasibility they work the phones for two to three days per person . participants in the acceptibility and feasibility study were a subset of the 261 generally healthy non - hispanic caucasian and african american adult men and women living in greater los angeles , california who had enrolled in the ucla energetics study , a biomarker - based validation study designed to test the feasibility and validity of a multi - media , computer - based nutrition questionnaire , dietday ( www.24hrrecall.com ) ( arab et al . , 2010 ) . to test feasibility of the automated image capture image - dietday , all participants scheduled to enter the energetics study in august and september of 2007 were invited to participate in a pilot study using the camera phone system to support 24-hour dietary recall reporting . participants in the pilot study wore phones on a lanyard around the neck with the camera facing outward for approximately one week- the period of time between their first and last visit , which ranged from 6 to 10 days . the camera was turned on prior to all eating occasions over a 24-hour period . a self - administered exit questionnaire containing structured and open - ended questions was administered at the end of the testing period . the participants were aware that their role was to provide objective feedback on the practicality of this new technology . during the consent process , technical aspects of the system were explained and participants were informed that they would have the ability to delete images they did not want to become part of the study record . the overall and pilot studies were approved by the ucla medical irb and the ucla general clinical research center . total energy expenditure ( tee ) was measured in these participants using the doubly - labeled water method ( subar et al . , ( schoeller , 1988 ) and coward and cole ( cole & coward , 1992 ) . doubly - labeled water was administered in a volume of one - fourth to one - half cup at a dose of approximately 2 g of 10 atom percent 18o labeled water and 0.12 g of 99.9 atom percent deuterium labeled water per kilogram of measured body weight ; subjects also consumed a 50-ml water rinse from the doubly - labeled water bottle . spot urine samples for determination of isotope enrichment were collected prior to dosing and at 1 , 2 , 3 , and 34 hours after dosing , and twice on the 15th day after dosing . deuterium and 18o levels in urine samples were quantified by mass spectroscopy and the values were used for calculation of total energy expenditure according to the plateau method ( schoeller , 1992 ) . all isotopic analyses were conducted at the university of wisconsin ( madison , wi ) . nokia n80 mobile phones programmed with three mega pixel cameras , automatic flash , and a 4.7 mm lens ( 35 mm equivalent ) were used for image capture . the mobile phones were attached to a lanyard and worn around the neck of the participant as seen in figure 2 application - specific power management techniques ( kansal , 2003 ; raghunathan v , 2005 ; raghunathan v , 2006 ) were used to balance power savings and required performance ( kansal , 2006 . ) . software developed by engineers at the ucla center for embedded networked sensing , los angeles , ca ( cens ) was employed to automatically record , time stamp , and transmit ( via cellular service ) encrypted data from sensors integrated in the phone ( e.g. , camera , and microphone ) to the secure sensorbase server repository . images were captured every 10 seconds , allowing near - complete documentation of foods and beverages consumed throughout the target period covered in the 24-hour period and without minimal user intervention . industry - standard encrypted transmission was used for web access to the repository and transmission of images between mobile phones and data storage servers . images were filtered to remove blurry , poorly exposed , and unclear images ( bradski , 2000 ; kovesi , 2010 ) and filed chronologically in clusters , based on key the total number of images presented to the participant was limited to fewer than 100 . a customized web - based imageviewer was developed to allow participants private access to their own images and the ability to delete objectionable images when desired ( figure 4 ) . images flagged as private ( i.e. , not shared ) were immediately and permanently deleted from the repository . images marked as shared were made available to investigators via a separate private image browser ( reddy et al . , 2007 ) . to further protect the privacy of participants , the image repository did not store any other personal identifier information with the images . sensitivity to the risk of identification of study participants is needed in this area of research . participants can be identified if an individual passes a reflective surface when the camera takes an image . the ability to identify someone from a hand which enters the camera lens field of vision also presents a risk . this type of risk requires that images be stored in datasets that are independent and not cross - linked to those with personal data . for this we developed levels of protection that involve participation and approval of the informed participant at each level . the participants were asked whether they wish to share any , some , or all images with study investigators . any images marked as protected ( i.e. , not shared ) was permanently deleted from the data storage server when the participant logged out of imagedietday . image counts prior to deletion were maintained , along with the timestamps and sequence position of the deleted images to assist in understanding deletion patterns and intent . shared images were to be accessible to the investigators and their designees , who sign confidentiality agreements , via password - protected interfaces . images that were never viewed by the participant ( such as when the system determines them to be unclear and therefore unhelpful to display ) were deleted . to further protect the privacy of participants , the image repository did not store personally identifiable information . this study received irb approval for a three - step process : ( 1 ) automated image capture from the camera phone , ( 2 ) automatic , encrypted upload of images to a secure repository and removal from the device , ( 3 ) a private web portal for each participant that allowed them to review their images and permanently remove any that they did not wish to share with investigators . the general rule in the united states is that anyone may take photographs of whatever they want when they are in a public place . however , even on public property , photography can be prohibited if somebody has a reasonable expectation of privacy . two independent pilot studies of the approach were conducted in august 2007 : the first to test technical feasibility and the second to test subjective acceptability . ten young adult cens employees participated in a technical feasibility exercise designed to test the ability of the system to handle uploaded images collected by simultaneous users . participants wore and operated phones at all times including outside of the home , capturing six images per minute throughout the day . to test technical feasibility they work the phones for two to three days per person . participants in the acceptibility and feasibility study were a subset of the 261 generally healthy non - hispanic caucasian and african american adult men and women living in greater los angeles , california who had enrolled in the ucla energetics study , a biomarker - based validation study designed to test the feasibility and validity of a multi - media , computer - based nutrition questionnaire , dietday ( www.24hrrecall.com ) ( arab et al . , 2010 ) . to test feasibility of the automated image capture image - dietday , all participants scheduled to enter the energetics study in august and september of 2007 were invited to participate in a pilot study using the camera phone system to support 24-hour dietary recall reporting . participants in the pilot study wore phones on a lanyard around the neck with the camera facing outward for approximately one week- the period of time between their first and last visit , which ranged from 6 to 10 days . the camera was turned on prior to all eating occasions over a 24-hour period . a self - administered exit questionnaire containing structured and open - ended questions the participants were aware that their role was to provide objective feedback on the practicality of this new technology . during the consent process , technical aspects of the system were explained and participants were informed that they would have the ability to delete images they did not want to become part of the study record . the overall and pilot studies were approved by the ucla medical irb and the ucla general clinical research center . total energy expenditure ( tee ) was measured in these participants using the doubly - labeled water method ( subar et al . , 2003 ) using isotope measurement methods described by schoeller et al . ( schoeller , 1988 ) and coward and cole ( cole & coward , 1992 ) . doubly - labeled water was administered in a volume of one - fourth to one - half cup at a dose of approximately 2 g of 10 atom percent 18o labeled water and 0.12 g of 99.9 atom percent deuterium labeled water per kilogram of measured body weight ; subjects also consumed a 50-ml water rinse from the doubly - labeled water bottle . spot urine samples for determination of isotope enrichment were collected prior to dosing and at 1 , 2 , 3 , and 34 hours after dosing , and twice on the 15th day after dosing . deuterium and 18o levels in urine samples were quantified by mass spectroscopy and the values were used for calculation of total energy expenditure according to the plateau method ( schoeller , 1992 ) . all isotopic analyses were conducted at the university of wisconsin ( madison , wi ) . ten young adult cens employees participated in a technical feasibility exercise designed to test the ability of the system to handle uploaded images collected by simultaneous users . participants wore and operated phones at all times including outside of the home , capturing six images per minute throughout the day . to test technical feasibility they work the phones for two to three days per person . participants in the acceptibility and feasibility study were a subset of the 261 generally healthy non - hispanic caucasian and african american adult men and women living in greater los angeles , california who had enrolled in the ucla energetics study , a biomarker - based validation study designed to test the feasibility and validity of a multi - media , computer - based nutrition questionnaire , dietday ( www.24hrrecall.com ) ( arab et al . , 2010 ) . to test feasibility of the automated image capture image - dietday , all participants scheduled to enter the energetics study in august and september of 2007 were invited to participate in a pilot study using the camera phone system to support 24-hour dietary recall reporting . participants in the pilot study wore phones on a lanyard around the neck with the camera facing outward for approximately one week- the period of time between their first and last visit , which ranged from 6 to 10 days . the camera was turned on prior to all eating occasions over a 24-hour period . a self - administered exit questionnaire containing structured and open - ended questions the participants were aware that their role was to provide objective feedback on the practicality of this new technology . during the consent process , technical aspects of the system were explained and participants were informed that they would have the ability to delete images they did not want to become part of the study record . the overall and pilot studies were approved by the ucla medical irb and the ucla general clinical research center . total energy expenditure ( tee ) was measured in these participants using the doubly - labeled water method ( subar et al . , 2003 ) using isotope measurement methods described by schoeller et al . ( schoeller , 1988 ) and coward and cole ( cole & coward , 1992 ) . doubly - labeled water was administered in a volume of one - fourth to one - half cup at a dose of approximately 2 g of 10 atom percent 18o labeled water and 0.12 g of 99.9 atom percent deuterium labeled water per kilogram of measured body weight ; subjects also consumed a 50-ml water rinse from the doubly - labeled water bottle . spot urine samples for determination of isotope enrichment were collected prior to dosing and at 1 , 2 , 3 , and 34 hours after dosing , and twice on the 15th day after dosing . deuterium and 18o levels in urine samples were quantified by mass spectroscopy and the values were used for calculation of total energy expenditure according to the plateau method ( schoeller , 1992 ) . all isotopic analyses were conducted at the university of wisconsin ( madison , wi ) . in the acceptability and feasibility study , 14 of 16 randomly recruited volunteers consented to the image - dietday add - on study and received phones . battery power was an issue , as one battery per day was not always adequate for the entire day . although most of the volunteers were technologically nave , 79% reported having no technical problems with the device ( table 2 ) . across all users , an average of 101 images were captured per eating episode , range ( 1 to 775 ) , reflecting the diversity of shorter and longer eating episodes across participants . when asked about the burden of wearing a camera phone throughout the day , 29% said it was easy , 50% said it was somewhat difficult , 14% said it was difficult , and 7% said it was very difficult . the racial breakdown of participants was fairly even ( 43% african - americans vs 57% caucasians ) , but the gender distribution was significantly different ( 79% female vs 21% male ) . the mean age and body mass index ( bmi ) of participants averaged 35 and 27 , respectively . upon analysis , a very close match between reported intake values aided by mobile camera phone images for reference and measured intake values from doubly labeled water data of total energy expenditure was found ( medians of 2359 reported vs. 2377 measured ) . these near identical values reveal the potential of the application of automated image - capture systems to supplement reporting of energy intake . in the current study , participants were offered the chance to provide open - ended comments anonymously at their exit interviews . the major themes noted in the open comment fields were related to the need to recharge the battery and self - consciousness about wearing the device in public , leading in some cases to changes in behavior such as eating out less often or more rapid eating . concern that the camera was missing foods not consumed at a dining table ( e.g. , snacks eaten on the run or couch consumption ) were also expressed . several participants noted that the device was cumbersome to wear and that they would have preferred a smaller , less conspicuous camera . future generations of phones that are lighter and smaller with a wide enough field of view to capture food under a variety of subject postures would enhance the system . an inexpensive camera phone fish - eye lens , which has been shown elsewhere to significantly increase the range of capture , might be a feasible alternative ( committee on networked systems of embedded computers , 2010 ) . in the technical feasibility study , all of the phones captured and transferred images as expected . both the secure image repository and image processing algorithms handled the full load of incoming images without failure . identification mechanisms for eating episodes and image deletion functioned , but warranted additional design refinement to enhance user - friendliness and maximal use of the images . this includes the development of more sophistication in the software that manages the number of images presented and the ability to access supporting images that are not initially presented . some phones required battery replacement during the trial , indicating need for more power than was provided by the batteries used if this imaging frequency were to be maintained or enhanced . analyses of the frequency of imaging suggests that if the goal is to capture total diet , more frequent imaging is necessary ( arab & winter , 2010 ) . image - dietday differs from several recently reported image - capture approaches ( wang et al . , ; weiss et al . , 2010 ) because the participants are using the autonomously collected images to help recall the foods that they ate during the target period . the image - assisted recall method tested in the current study should minimize confusion regarding identification of the food item , as the users in the current study did not perceive their own images as burdensome to view and nearly all ( 93% ) of participants indicated that images were beneficial ( helpful , 57% ; sort of helpful , 36% ) ; only 7% of participants indicated that the images were not helpful . additionally , the images captured using image - dietday may be distinct from those captured when there is intent to focus imaging on foods consumed ( wang et al . , 2006 ; kikunaga et al . , 2007 ; swanson , 2008 ; boushey et al . , 2009 ; six et al . ) . japanese investigators found that the self - consciousness of food intake caused by the process of intentional imaging may dramatically impact normal eating behavior ( wang et al . , 2006 ) and that enlisting people in actively imaging their consumption results in lower reporting of intakes ( kikunaga et al . , 2007 ) . however , the passive , automated image capture system used in the current study may reduce the likelihood that participants will alter their normal eating patterns in comparison to intentional imaging a method in which the participants set up each picture and are depended upon to capture their usual diet using a camera , without change in food selection , elimination of unfavorable foods , and forgetting to or deciding not to include certain eating situations . automated imaging offers the potential for improving documentation of all foods and beverages consumed if the application can be designed to reduce self - awareness of the times of observation . the availability of time - stamped images can be used to support participants memories and help prevent telescoping of the time a food was consumed and the reporting of phantom foods ( intrusions ) that were not actually consumed during the target period , and serve as reminders to report forgotten foods ( omissions ) ( baxter et al . , 2006 ) . the cost - effectiveness of mobile phones complements the accessibility of the internet as an interview and content - review tool , as they are widely available for data collection ( kovesi , 2010 ) . this approach would transform off - the - shelf mobile phones and web - based services into a system for monitoring individuals dietary intakes . the use of individuals cell phones also allows the option of prompted action ( e.g. , turning on the camera , repositioning the camera , locating connectivity for data upload ) either to trigger use or when data transfer issues is detected . depending on user preference this system can alert the investigator to subject use and compliance based on real - time statistics of uploaded data ( e.g. , the number , timing , and minimal quality threshold of images ) and provide immediate feedback to the subject if there are no images recorded at expected consumption times or if the image quality is poor ( blurry or dark ) , through an automated phone call or text message . these pilot studies demonstrate the feasibility of the image - assisted recall method for dietary recall in a select volunteer population . while the applicability of the findings to a randomly selected population are uncertain , the study nevertheless demonstrates that the technical requirements of adequate power , placement , technical stability , and download and image processing time , as well as concern regarding privacy issues , can be met . nearly all participants found the images helpful in reporting their dietary intakes from the previous day . this approach differs from other pilot studies using image capture in that the images are captured automatically after users initiate the camera and later the users themselves use the images to trigger memory of the foods that were consumed , eliminating the guesswork that would be required of third party reviewers . strategies to reduce self - awareness , such as having a run - in period of camera use , maintaining a random assignment of the 24-hour dietary recall , and miniaturizing and camouflaging the camera are important areas of development . a validation study will be required to determine how camera wear impacts eating behavior , whether the addition of the images improves objective validity of assessment and to allow closer assessment of the impact of imaging on omission and intrusion rates normally observed in recall based dietary assessment methods .

background / objectivesthe accuracy of dietary recalls might be enhanced by providing participants with photo images of foods they consumed during the test period.subjects/methodswe examined the feasibility of a system ( image - diet day ) that is a user - initiated camera - equipped mobile phone that is programmed to automatically capture and transmit images to a secure website in conjunction with computer - assisted , multi - pass , 24-hour dietary recalls in 14 participants during 2007 . participants used the device during eating periods on each of the three independent days . image processing filters successfully eliminated underexposed , over - exposed , and blurry images . captured images were accessed by participants using the imageviewer software while completing the 24-hour dietary recall on the following day.resultsnone of the participants reported difficulty using the imageviewer . images were deemed helpful or sort of helpful by 93% of participants . a majority ( 79% ) of users reported having no technical problems , but 71% rated the burden of wearing the device as somewhat to very difficult , owing to issues such as limited battery life , self - consciousness about wearing the device in public , and concerns about the camera s field of view.conclusionoverall , these findings suggest that automated imaging is a promising technology to facilitate dietary recall . the challenge of managing the thousands of images generated can be met . smaller devices with a broader field of view may aid in overcoming user s self - consciousness with using or wearing the device

INTRODUCTION METHODS System Components Pilot Feasibility Testing Technical Feasibility Study (n =10) Acceptability and Human Feasibility Study Energy Validation using Doubly Labeled Water RESULTS AND DISCUSSION CONCLUSIONS

advantages of the 24-hour dietary recall over other measures , such as food frequency questionnaires , include its reliance on short - term , rather than long - term memory , and its relatively low respondent burden . additionally , the 24-hour dietary recall is only moderately vulnerable to a telescoping effect , that is , the forward or backward displacement of the event in time ( janssen et al . the current study examines the use of a novel , cost - effective image - assisted recall method that combines automatic image capture ( to reduce participant reactivity ) with a web - based 24-hour dietary recall . mobile phone cameras equipped for automatic imaging were worn by participants during eating periods and images were uploaded to a secure website for use by participants during the reporting period the next day . this study received irb approval for a three - step process : ( 1 ) automated image capture from the camera phone , ( 2 ) automatic , encrypted upload of images to a secure repository and removal from the device , ( 3 ) a private web portal for each participant that allowed them to review their images and permanently remove any that they did not wish to share with investigators . participants in the acceptibility and feasibility study were a subset of the 261 generally healthy non - hispanic caucasian and african american adult men and women living in greater los angeles , california who had enrolled in the ucla energetics study , a biomarker - based validation study designed to test the feasibility and validity of a multi - media , computer - based nutrition questionnaire , dietday ( www.24hrrecall.com ) ( arab et al . to test feasibility of the automated image capture image - dietday , all participants scheduled to enter the energetics study in august and september of 2007 were invited to participate in a pilot study using the camera phone system to support 24-hour dietary recall reporting . this study received irb approval for a three - step process : ( 1 ) automated image capture from the camera phone , ( 2 ) automatic , encrypted upload of images to a secure repository and removal from the device , ( 3 ) a private web portal for each participant that allowed them to review their images and permanently remove any that they did not wish to share with investigators . participants in the acceptibility and feasibility study were a subset of the 261 generally healthy non - hispanic caucasian and african american adult men and women living in greater los angeles , california who had enrolled in the ucla energetics study , a biomarker - based validation study designed to test the feasibility and validity of a multi - media , computer - based nutrition questionnaire , dietday ( www.24hrrecall.com ) ( arab et al . to test feasibility of the automated image capture image - dietday , all participants scheduled to enter the energetics study in august and september of 2007 were invited to participate in a pilot study using the camera phone system to support 24-hour dietary recall reporting . to test feasibility of the automated image capture image - dietday , all participants scheduled to enter the energetics study in august and september of 2007 were invited to participate in a pilot study using the camera phone system to support 24-hour dietary recall reporting . although most of the volunteers were technologically nave , 79% reported having no technical problems with the device ( table 2 ) . when asked about the burden of wearing a camera phone throughout the day , 29% said it was easy , 50% said it was somewhat difficult , 14% said it was difficult , and 7% said it was very difficult . the major themes noted in the open comment fields were related to the need to recharge the battery and self - consciousness about wearing the device in public , leading in some cases to changes in behavior such as eating out less often or more rapid eating . , 2010 ) because the participants are using the autonomously collected images to help recall the foods that they ate during the target period . the image - assisted recall method tested in the current study should minimize confusion regarding identification of the food item , as the users in the current study did not perceive their own images as burdensome to view and nearly all ( 93% ) of participants indicated that images were beneficial ( helpful , 57% ; sort of helpful , 36% ) ; only 7% of participants indicated that the images were not helpful . the availability of time - stamped images can be used to support participants memories and help prevent telescoping of the time a food was consumed and the reporting of phantom foods ( intrusions ) that were not actually consumed during the target period , and serve as reminders to report forgotten foods ( omissions ) ( baxter et al . these pilot studies demonstrate the feasibility of the image - assisted recall method for dietary recall in a select volunteer population . while the applicability of the findings to a randomly selected population are uncertain , the study nevertheless demonstrates that the technical requirements of adequate power , placement , technical stability , and download and image processing time , as well as concern regarding privacy issues , can be met . strategies to reduce self - awareness , such as having a run - in period of camera use , maintaining a random assignment of the 24-hour dietary recall , and miniaturizing and camouflaging the camera are important areas of development .

all animal procedures were approved by the animal studies committee at washington university school of medicine , and all animals received humane care in compliance with the national institute of health s ( nih ) guide to care and use of laboratory animals.26 mice ( both sexes , 6 weeks to 5 months and 15 to 30 g in weight ) were anesthetized with 3% avertin ( 0.3 g of 2,2,2-tribromoethanol , 0.186 ml of 2-methyl2-butanol , and 9.814 ml of sterile water ) intraperitoneally , and rapid cardiectomy was performed . to isolate mitochondria , atria were excised and discarded whereas ventricular tissue was rapidly minced in cold buffer ( in mmol / l : 10 hepes ( n-[2-hydroxyethyl]1,35 piperazine - n-[4-butanesulfonic acid ] ) , 1 edta - potassium , 250 sucrose , adjusted to a ph of 7.1 with 20% koh and transferred to a 10-ml glass tube with a teflon pestle ( glas - col homogenizer , terre haute , in ) , and volume was adjusted to 7 ml with buffer . tissue was mechanically homogenized with a teflon pestle driven by a low - speed motor drive shaft set at 120 rpm . the homogenate was transferred to 6 microcentrifuge tubes and centrifuged at 900 g for 10 minutes at 4c . the supernatant was combined into a clean test tube and mixed to get a homogeneous solution that was divided equally between 6 clean microcentrifuge tubes and centrifuged at 5000 g for 15 minutes . one pellet was resuspended in 100 l of buffer , and 10 l was taken in duplicate for the bradford protein assay ( thermo scientific ; rockford , il ) to determine total protein . each pellet was maintained on ice and was resuspended in test media volume to equal 0.3 g/l in order to standardize protein content . the volume of isolated mitochondria was measured after suspension in test solution : ( 1 ) isolation buffer ( no atp ; n=13 ) ; ( 2 ) 200 mol / l of atp ( n=12 ) ; ( 3 ) 200 mol / l of atp and 100 mol / l of dzx ( sigma - aldrich , st . louis , mo ) ( n=12 ) ; ( 4 ) 200 mol / l of atp , 100 mol / l of dzx , and 500 nmol / l of tpn - q ( n=7 ) ; or ( 5 ) 200 mol / l of atp , 100 mol / l of dzx , and 100 nmol / l of tpn - q ( n=6 ) . isolation buffer ( 10 mmol / l of hepes , 200 mmol / l of mannitol , 50 mmol / l of sucrose , 1 mmol / l of egta ; ph 7.2 ) was used as a control solution . atp has been shown to close mitochondrial katp channels , so 200 mol / l of atp was used to slow the initial rate of mitochondrial swelling ( 0% mito katp activity).22 conversely , dzx activates mito katp channels , and 100 mol / l of dzx was added to achieve maximal activation of mitochondrial katp channels ( 100% mito katp activity ) . we were unable to reproduce the tpn - q dose - response relationships on mitochondrial volume demonstrated by other investigators using 4 tpn - q concentrations ( 0.5 , 10 , 90 , or 1000 pmol / l ) despite attempts utilizing 3 different vehicles for tpn - q : 20% acetonitrile , water , and hepes.24 in the present study , water was used as a vehicle for tpn - q because of its stability in the medium and because 20% acetonitrile alone resulted in myocyte swelling owing to its cyanide moiety . mitochondrial matrix volume measurements were obtained using a light - scattering technique,27 where the absorbance , at 520 nm , of a solution of isolated mitochondria was obtained every 14 seconds for the period of 3 minutes using uv probe 2.33 ( shimadzu scientific instruments , columbia , md ) and a spectrophotometer ( uv-1700 spectrophotometer ; shimadzu scientific instruments , columbia , md ) . ventricular myocytes were isolated from c57bl/6j mice of either sex ( age 6 weeks to 5 months and 15 to 30 g in weight ) , as previously described.15 mice were anesthetized with 2.5% avertin intraperitoneally . rapid cardiectomy was performed and solution a ( described below ) was perfused through the aorta for 5 minutes . the heart was then perfused at 37c for 12 to 20 minutes with solution b ( described below ) . ventricles were removed , minced , and placed into solution c ( described below ) and gently dispersed by glass pipette . cells were allowed to centrifuge by gravity , and serial washings were performed every 10 minutes for 15 to 20 minutes . solution a consisted of ( in mmol / l , except as noted ) 116 nacl ; 5.36 kcl ; 0.97 na2hpo4 ; 1.47 kh2po4 ; 21.10 hepes ( n-[2-hydroxyethyl ] piperazine - n-[4-butanesulfonic acid ] ) ; 11.65 glucose ; 26.50 mol / l of phenol red ( sigma - aldrich ) ; 3.72 mgcl2 ; 4.40 nahco3 ; essential vitamins ( 100 , 10 ml ; gibco , grand island , ny ) ; and amino acids ( 50 , 20 ml ; gibco ) . solution b consisted of solution a plus 10 mol / l of cacl2 ; 1.2 mg / ml of collagenase ( type 2 ; worthington biochemical corporation , freehold , nj ) . solution c consisted of solution a plus 5 mg / ml of bsa ( sigma - aldrich ) ; 1.25 mg / ml of taurine ; and 150 mol / l of cacl2 . myocytes were exposed to 37c control tyr for 5 minutes to obtain baseline volume . any changes in cell volume secondary to the isolation would be evident during this period . myocytes were then exposed to test solution ( 10 minutes ) followed by re - exposure to tyr solution ( 5 minutes ) . test solutions included the following groups ( n=12 for each ) : ( 1 ) tyr ; ( 2 ) cpg ; ( 3 ) cpg+100 mol / l of dzx ; ( 4 ) cpg+100 mol / l of dzx+tpn - q 200 nm / l ; ( 5 ) tyr+tpn - q ; and ( 6 ) cpg+tpn - q . cpg consisted of ( in mmol / l ) : nacl 110 , nahco3 10 , kcl 16 , mgcl2 16 , and cacl2 1.2 and was equilibrated with 95% o2 to 5% co2 and titrated to the ph of 7.3 with 10% nahco3 solution . diazoxide ( 7-chloro-3-methyl-1,2,4-benzothiadiazine-1,1-dioxide4 ; sigma - aldrich ) dose of 100 mol / l was utilized because it was effective in ameliorating cell swelling secondary to stress in previous studies.14 a stock solution of dzx was made by dissolving dzx in 0.1% dimethyl sulfoxide ( dmso ) , at which concentration dmso has no effect on cell volume.28 myocytes were used on the day of isolation and were not cultured . myocytes were visualized on an inverted microscope stage ( ix-51 ; olympus , tokyo , japan ) , as previously described.15 after 5 minutes , the chamber was perfused at a rate of 3 ml / min with tyr ( in mmol / l ) : nacl 130 , kcl 5 , cacl2 2.5 , mgso4 1.2 , nahco3 24 , na2hpo4 1.75 , and glucose 10 ( buffered to a ph of 7.4 using 95% o2 to 5% co2 ) . after viability was confirmed , myocyte images were captured using video - based edge detection software ( ionoptix , milton , ma ) and volume measured every 5 minutes , as previously described.5 myocyte contractility was measured using a video - based edge detection system ( ionoptix ) . cells were paced using a field stimulator ( myopacer ; ionoptix ) at a voltage of 105 above threshold at a frequency of 1 hz with a 5-ms duration to avoid occurrence of fusion beats . after 5 minutes of stimulation parameters of contractility included percentage of cell shortening , maximal velocity of shortening , and percentage of cell relengthening , as previously described.5 contractility was measured at baseline and after 5 minutes of re - exposure to tyr . data were analyzed using systat 13 ( systat software , inc , point richmond , ca ) . a repeated - measures anova was used for sequential time - based measurements for each test solution against its own baseline and control values . using fisher s least significant different test , post - hoc multiple comparisons were done between different test groups at different time points during test solution and re - exposure periods . a shapiro - wilk test was used to test for normality . if the data failed the normality test , a nonparametric ( friedman s nonparametric repeated - measures comparison ) mice ( both sexes , 6 weeks to 5 months and 15 to 30 g in weight ) were anesthetized with 3% avertin ( 0.3 g of 2,2,2-tribromoethanol , 0.186 ml of 2-methyl2-butanol , and 9.814 ml of sterile water ) intraperitoneally , and rapid cardiectomy was performed . to isolate mitochondria , atria were excised and discarded whereas ventricular tissue was rapidly minced in cold buffer ( in mmol / l : 10 hepes ( n-[2-hydroxyethyl]1,35 piperazine - n-[4-butanesulfonic acid ] ) , 1 edta - potassium , 250 sucrose , adjusted to a ph of 7.1 with 20% koh and transferred to a 10-ml glass tube with a teflon pestle ( glas - col homogenizer , terre haute , in ) , and volume was adjusted to 7 ml with buffer . tissue was mechanically homogenized with a teflon pestle driven by a low - speed motor drive shaft set at 120 rpm . the homogenate was transferred to 6 microcentrifuge tubes and centrifuged at 900 g for 10 minutes at 4c . the supernatant was combined into a clean test tube and mixed to get a homogeneous solution that was divided equally between 6 clean microcentrifuge tubes and centrifuged at 5000 g for 15 minutes . one pellet was resuspended in 100 l of buffer , and 10 l was taken in duplicate for the bradford protein assay ( thermo scientific ; rockford , il ) to determine total protein . each pellet was maintained on ice and was resuspended in test media volume to equal 0.3 g/l in order to standardize protein content . the volume of isolated mitochondria was measured after suspension in test solution : ( 1 ) isolation buffer ( no atp ; n=13 ) ; ( 2 ) 200 mol / l of atp ( n=12 ) ; ( 3 ) 200 mol / l of atp and 100 mol / l of dzx ( sigma - aldrich , st . louis , mo ) ( n=12 ) ; ( 4 ) 200 mol / l of atp , 100 mol / l of dzx , and 500 nmol / l of tpn - q ( n=7 ) ; or ( 5 ) 200 mol / l of atp , 100 mol / l of dzx , and 100 nmol / l of tpn - q ( n=6 ) . isolation buffer ( 10 mmol / l of hepes , 200 mmol / l of mannitol , 50 mmol / l of sucrose , 1 mmol / l of egta ; ph 7.2 ) was used as a control solution . atp has been shown to close mitochondrial katp channels , so 200 mol / l of atp was used to slow the initial rate of mitochondrial swelling ( 0% mito katp activity).22 conversely , dzx activates mito katp channels , and 100 mol / l of dzx was added to achieve maximal activation of mitochondrial katp channels ( 100% mito katp activity ) . we were unable to reproduce the tpn - q dose - response relationships on mitochondrial volume demonstrated by other investigators using 4 tpn - q concentrations ( 0.5 , 10 , 90 , or 1000 pmol / l ) despite attempts utilizing 3 different vehicles for tpn - q : 20% acetonitrile , water , and hepes.24 in the present study , water was used as a vehicle for tpn - q because of its stability in the medium and because 20% acetonitrile alone resulted in myocyte swelling owing to its cyanide moiety . mitochondrial matrix volume measurements were obtained using a light - scattering technique,27 where the absorbance , at 520 nm , of a solution of isolated mitochondria was obtained every 14 seconds for the period of 3 minutes using uv probe 2.33 ( shimadzu scientific instruments , columbia , md ) and a spectrophotometer ( uv-1700 spectrophotometer ; shimadzu scientific instruments , columbia , md ) . ventricular myocytes were isolated from c57bl/6j mice of either sex ( age 6 weeks to 5 months and 15 to 30 g in weight ) , as previously described.15 mice were anesthetized with 2.5% avertin intraperitoneally . rapid cardiectomy was performed and solution a ( described below ) was perfused through the aorta for 5 minutes . the heart was then perfused at 37c for 12 to 20 minutes with solution b ( described below ) . ventricles were removed , minced , and placed into solution c ( described below ) and gently dispersed by glass pipette . cells were allowed to centrifuge by gravity , and serial washings were performed every 10 minutes for 15 to 20 minutes . solution a consisted of ( in mmol / l , except as noted ) 116 nacl ; 5.36 kcl ; 0.97 na2hpo4 ; 1.47 kh2po4 ; 21.10 hepes ( n-[2-hydroxyethyl ] piperazine - n-[4-butanesulfonic acid ] ) ; 11.65 glucose ; 26.50 mol / l of phenol red ( sigma - aldrich ) ; 3.72 mgcl2 ; 4.40 nahco3 ; essential vitamins ( 100 , 10 ml ; gibco , grand island , ny ) ; and amino acids ( 50 , 20 ml ; gibco ) . solution b consisted of solution a plus 10 mol / l of cacl2 ; 1.2 mg / ml of collagenase ( type 2 ; worthington biochemical corporation , freehold , nj ) . solution c consisted of solution a plus 5 mg / ml of bsa ( sigma - aldrich ) ; 1.25 mg / ml of taurine ; and 150 mol / l of cacl2 . myocytes were exposed to 37c control tyr for 5 minutes to obtain baseline volume . any changes in cell volume secondary to the isolation would be evident during this period . myocytes were then exposed to test solution ( 10 minutes ) followed by re - exposure to tyr solution ( 5 minutes ) . test solutions included the following groups ( n=12 for each ) : ( 1 ) tyr ; ( 2 ) cpg ; ( 3 ) cpg+100 mol / l of dzx ; ( 4 ) cpg+100 mol / l of dzx+tpn - q 200 nm / l ; ( 5 ) tyr+tpn - q ; and ( 6 ) cpg+tpn - q . cpg consisted of ( in mmol / l ) : nacl 110 , nahco3 10 , kcl 16 , mgcl2 16 , and cacl2 1.2 and was equilibrated with 95% o2 to 5% co2 and titrated to the ph of 7.3 with 10% nahco3 solution . diazoxide ( 7-chloro-3-methyl-1,2,4-benzothiadiazine-1,1-dioxide4 ; sigma - aldrich ) dose of 100 mol / l was utilized because it was effective in ameliorating cell swelling secondary to stress in previous studies.14 a stock solution of dzx was made by dissolving dzx in 0.1% dimethyl sulfoxide ( dmso ) , at which concentration dmso has no effect on cell volume.28 myocytes were visualized on an inverted microscope stage ( ix-51 ; olympus , tokyo , japan ) , as previously described.15 after 5 minutes , the chamber was perfused at a rate of 3 ml / min with tyr ( in mmol / l ) : nacl 130 , kcl 5 , cacl2 2.5 , mgso4 1.2 , nahco3 24 , na2hpo4 1.75 , and glucose 10 ( buffered to a ph of 7.4 using 95% o2 to 5% co2 ) . after viability was confirmed , myocyte images were captured using video - based edge detection software ( ionoptix , milton , ma ) and volume measured every 5 minutes , as previously described.5 myocyte contractility was measured using a video - based edge detection system ( ionoptix ) . cells were paced using a field stimulator ( myopacer ; ionoptix ) at a voltage of 105 above threshold at a frequency of 1 hz with a 5-ms duration to avoid occurrence of fusion beats . after 5 minutes of stimulation , data were obtained from 12 to 30 consecutive beats and averaged . parameters of contractility included percentage of cell shortening , maximal velocity of shortening , and percentage of cell relengthening , as previously described.5 contractility was measured at baseline and after 5 minutes of re - exposure to tyr . data were analyzed using systat 13 ( systat software , inc , point richmond , ca ) . a repeated - measures anova was used for sequential time - based measurements for each test solution against its own baseline and control values . using fisher s least significant different test , post - hoc multiple comparisons were done between different test groups at different time points during test solution and re - exposure periods . if the data failed the normality test , a nonparametric ( friedman s nonparametric repeated - measures comparison ) was used . isolated mouse mitochondria were exposed to : ( 1 ) isolation buffer ( no atp ) ; ( 2 ) 200 mol / l of atp ; ( 3 ) 200 mol / l of atp and 100 mol / l of dzx ; ( 4 ) 200 mol / l of atp , 100 mol / l of dzx , and 500 nmol / l of tpn - q ; or ( 5 ) 200 mol / l of atp , 100 mol / l of dzx , and 100 nmol / l of tpn - q and volume measured using light scattering ( at 520 nm ) for 2 minutes . mitochondrial matrix volume is represented as 1/percent change in absorbance ( mitochondrial matrix swelling is inversely related to absorbance measured at 520 nm ) over time . dzx indicates diazoxide ; tpn - q , tertiapin q. myocyte volume over time is represented in figure 2 . myocytes demonstrated no significant volume change from baseline during exposure to tyr with or without tpn - q . the further addition of tpn - q prevented this benefit ( volume homeostasis ) of dzx ( cpg+dzx+tpn - q p<0.05 vs. cpg+dzx ) . as expected , the interaction between experimental groups and time was significant ( p<0.001 ) . isolated myocytes were exposed to control tyrode s physiologic solution ( tyr ) for 5 minutes ( time 0 to 5 ) , stress ( cpg , cpg+dzx , cpg+dzx+tpn - q , cpg+tpn - q , and tyr+tpn - q ) for 10 minutes ( time 5 to 15 ) , followed by tyr for 5 minutes ( time 15 to 20 ) . cpg indicates hypothermic hyperkalemic cardioplegia ; dzx , diazoxide ; tpn - q , tertiapin q. myocyte contractility is presented in figure 3 . myocytes demonstrated a significant decline in all 3 parameters of contractility after exposure to cpg ( p<0.05 vs. tyr ) that was worsened by the addition of tpn - q . the addition of dzx to cpg prevented the significant reduction in contractility observed in the cpg group . isolated mouse myocytes were exposed to control tyrode s physiologic solution ( tyr ) for 5 minutes ( time 0 to 5 ) , stress ( cpg , cpg+dzx , cpg+dzx+tpn - q , cpg+tpn - q , and tyr+tpn - q ) for 10 minutes ( time 5 to 15 ) , followed by tyr for 5 minutes ( time 15 to 20 ) . cpg indicates hypothermic hyperkalemic cardioplegia ; dzx , diazoxide ; tpn - q , tertiapin q isolated mouse mitochondria were exposed to : ( 1 ) isolation buffer ( no atp ) ; ( 2 ) 200 mol / l of atp ; ( 3 ) 200 mol / l of atp and 100 mol / l of dzx ; ( 4 ) 200 mol / l of atp , 100 mol / l of dzx , and 500 nmol / l of tpn - q ; or ( 5 ) 200 mol / l of atp , 100 mol / l of dzx , and 100 nmol / l of tpn - q and volume measured using light scattering ( at 520 nm ) for 2 minutes . mitochondrial matrix volume is represented as 1/percent change in absorbance ( mitochondrial matrix swelling is inversely related to absorbance measured at 520 nm ) over time . myocytes demonstrated no significant volume change from baseline during exposure to tyr with or without tpn - q . the further addition of tpn - q prevented this benefit ( volume homeostasis ) of dzx ( cpg+dzx+tpn - q p<0.05 vs. cpg+dzx ) . as expected , the interaction between experimental groups and time was significant ( p<0.001 ) . isolated myocytes were exposed to control tyrode s physiologic solution ( tyr ) for 5 minutes ( time 0 to 5 ) , stress ( cpg , cpg+dzx , cpg+dzx+tpn - q , cpg+tpn - q , and tyr+tpn - q ) for 10 minutes ( time 5 to 15 ) , followed by tyr for 5 minutes ( time 15 to 20 ) . . cpg indicates hypothermic hyperkalemic cardioplegia ; dzx , diazoxide ; tpn - q , tertiapin q. myocytes demonstrated a significant decline in all 3 parameters of contractility after exposure to cpg ( p<0.05 vs. tyr ) that was worsened by the addition of tpn - q . the addition of dzx to cpg prevented the significant reduction in contractility observed in the cpg group . isolated mouse myocytes were exposed to control tyrode s physiologic solution ( tyr ) for 5 minutes ( time 0 to 5 ) , stress ( cpg , cpg+dzx , cpg+dzx+tpn - q , cpg+tpn - q , and tyr+tpn - q ) for 10 minutes ( time 5 to 15 ) , followed by tyr for 5 minutes ( time 15 to 20 ) . cpg indicates hypothermic hyperkalemic cardioplegia ; dzx , diazoxide ; tpn - q , tertiapin q in previous studies , we have shown that myocyte swelling and reduced contractility in response to stress ( hyperkalemic cpg , hypoosmotic stress , and metabolic inhibition ) are ameliorated by addition of the katp channel opener , dzx.15 our efforts to localize the precise mechanism of cardioprotection afforded by dzx have implicated an involvement of the katp channel , sur1,16,19 but not the kir6.2 subunit.2 other investigators have provided evidence in support of the idea that kir1.1 ( the romk ) is a pore - forming subunit of the mkatp channel.24 in the current study , changes in mitochondrial and myocyte volume were observed in the presence of romk blocker tpn - q in the presence of dzx . a dose effect on mitochondrial volume was not detectable using tpn - q in the presence of dzx and multiple doses of tpn - q . the stability of tpn - q in the vehicle medium was thus evaluated in the media , in 20% acetonitrile , hepes , and in water . however , no significant change in mitochondrial volume was observed after the addition of tpn - q , even at high concentrations , in contrast to other investigators.24 thus , we conclude that tpn - q had no effect on mitochondrial volume in the presence of dzx , suggesting a location of action of dzx that was distinct from the channels targeted by tpn - q . myocytes demonstrated significant swelling in response to cpg that was prevented by the katp channel opener , dzx , which is consistent with previous results.2,3 similarly , a correlation between myocyte volume changes and contractility changes was consistent with previous work , supporting this myocyte model of stunning.4,5 tpn - q prevented this beneficial effect of dzx , thus implicating tpn - q targets other non kir tpn - q targets or undefined channel subunits sensitive to tpn - q may play a role ( girk1/4 , kach , and voltage - dependent ca - activated k channels ) in cardioprotection and will be the subject of future investigations.2931 the present study further characterizes the mechanism of cardioprotection provided by dzx . tpn - q did not alter dzx - induced mitochondrial swelling , but it did inhibit myocyte cardioprotection provided by dzx during stress . given that tpn - q inhibits kir1.1- , kir3.1- , and kir3.4-dependent k - channel activities , these data support that any of these subunits ( as well as undefined subunits sensitive to tpn - q ) could be involved in the cardioprotection afforded by dzx . however , these data also suggest that mitochondrial swelling by dzx does not involve kir1.1 , kir3.1 , or kir3.4 and likely results from a yet to be identified mechanism . future work to identify the site and mechanism of action of katp channel openers will involve indirect methods until genetic identification is accomplished . this study was supported by nih ro1 hl098182 - 01a1 ( lawton ) , nih 5t32hl007776 ( henn , janjua ) , and the barnes jewish hospital foundation ( lawton ) .

backgroundatp - sensitive potassium ( katp ) channel openers provide cardioprotection in multiple models . ion flux at an unidentified mitochondrial katp channel has been proposed as the mechanism . the renal outer medullary kidney potassium channel subunit , potassium inward rectifying ( kir)1.1 , has been implicated as a mitochondrial channel pore - forming subunit . we hypothesized that subunit kir1.1 is involved in cardioprotection ( maintenance of volume homeostasis and contractility ) of the katp channel opener diazoxide ( dzx ) during stress ( exposure to hyperkalemic cardioplegia [ cpg ] ) at the myocyte and mitochondrial levels.methods and resultskir subunit inhibitor tertiapin q ( tpn - q ) was utilized to evaluate response to stress . mouse ventricular mitochondrial volume was measured in the following groups : isolation buffer ; 200 mol / l of atp ; 100 mol / l of dzx+200 mol / l of atp ; or 100 mol / l of dzx+200 mol / l of atp+tpn - q ( 500 or 100 nmol / l ) . myocytes were exposed to tyrode s solution ( 5 minutes ) , test solution ( tyrode s , cardioplegia [ cpg ] , cpg+dzx , cpg+dzx+tpn - q , tyrodes+tpn - q , or cpg+tpn - q ) , n=12 for all ( 10 minutes ) ; followed by tyrode s ( 5 minutes ) . volumes were compared . tpn - q , with or without dzx , did not alter mitochondrial or myocyte volume . stress ( cpg ) resulted in myocyte swelling and reduced contractility that was prevented by dzx . tpn - q prevented the cardioprotection afforded by dzx ( volume homeostasis and maintenance of contractility).conclusionstpn - q inhibited myocyte cardioprotection provided by dzx during stress ; however , it did not alter mitochondrial volume . because tpn - q inhibits kir1.1 , kir3.1 , and kir3.4 , these data support that any of these kir subunits could be involved in the cardioprotection afforded by diazoxide . however , these data suggest that mitochondrial swelling by diazoxide does not involve kir1.1 , 3.1 , or 3.4 .

Methods Mitochondrial Isolation Mitochondrial Volume Measurement Myocyte Isolation Myocyte Volume Measurement Myocyte Contractility Statistical Analysis Results Mitochondrial Matrix Volume Myocyte Volume Myocyte Contractility Discussion Sources of Funding Disclosures

isolated mouse mitochondria were exposed to : ( 1 ) isolation buffer ( no atp ) ; ( 2 ) 200 mol / l of atp ; ( 3 ) 200 mol / l of atp and 100 mol / l of dzx ; ( 4 ) 200 mol / l of atp , 100 mol / l of dzx , and 500 nmol / l of tpn - q ; or ( 5 ) 200 mol / l of atp , 100 mol / l of dzx , and 100 nmol / l of tpn - q and volume measured using light scattering ( at 520 nm ) for 2 minutes . isolated myocytes were exposed to control tyrode s physiologic solution ( tyr ) for 5 minutes ( time 0 to 5 ) , stress ( cpg , cpg+dzx , cpg+dzx+tpn - q , cpg+tpn - q , and tyr+tpn - q ) for 10 minutes ( time 5 to 15 ) , followed by tyr for 5 minutes ( time 15 to 20 ) . isolated mouse myocytes were exposed to control tyrode s physiologic solution ( tyr ) for 5 minutes ( time 0 to 5 ) , stress ( cpg , cpg+dzx , cpg+dzx+tpn - q , cpg+tpn - q , and tyr+tpn - q ) for 10 minutes ( time 5 to 15 ) , followed by tyr for 5 minutes ( time 15 to 20 ) . cpg indicates hypothermic hyperkalemic cardioplegia ; dzx , diazoxide ; tpn - q , tertiapin q isolated mouse mitochondria were exposed to : ( 1 ) isolation buffer ( no atp ) ; ( 2 ) 200 mol / l of atp ; ( 3 ) 200 mol / l of atp and 100 mol / l of dzx ; ( 4 ) 200 mol / l of atp , 100 mol / l of dzx , and 500 nmol / l of tpn - q ; or ( 5 ) 200 mol / l of atp , 100 mol / l of dzx , and 100 nmol / l of tpn - q and volume measured using light scattering ( at 520 nm ) for 2 minutes . isolated myocytes were exposed to control tyrode s physiologic solution ( tyr ) for 5 minutes ( time 0 to 5 ) , stress ( cpg , cpg+dzx , cpg+dzx+tpn - q , cpg+tpn - q , and tyr+tpn - q ) for 10 minutes ( time 5 to 15 ) , followed by tyr for 5 minutes ( time 15 to 20 ) . isolated mouse myocytes were exposed to control tyrode s physiologic solution ( tyr ) for 5 minutes ( time 0 to 5 ) , stress ( cpg , cpg+dzx , cpg+dzx+tpn - q , cpg+tpn - q , and tyr+tpn - q ) for 10 minutes ( time 5 to 15 ) , followed by tyr for 5 minutes ( time 15 to 20 ) . cpg indicates hypothermic hyperkalemic cardioplegia ; dzx , diazoxide ; tpn - q , tertiapin q in previous studies , we have shown that myocyte swelling and reduced contractility in response to stress ( hyperkalemic cpg , hypoosmotic stress , and metabolic inhibition ) are ameliorated by addition of the katp channel opener , dzx.15 our efforts to localize the precise mechanism of cardioprotection afforded by dzx have implicated an involvement of the katp channel , sur1,16,19 but not the kir6.2 subunit.2 other investigators have provided evidence in support of the idea that kir1.1 ( the romk ) is a pore - forming subunit of the mkatp channel.24 in the current study , changes in mitochondrial and myocyte volume were observed in the presence of romk blocker tpn - q in the presence of dzx . myocytes demonstrated significant swelling in response to cpg that was prevented by the katp channel opener , dzx , which is consistent with previous results.2,3 similarly , a correlation between myocyte volume changes and contractility changes was consistent with previous work , supporting this myocyte model of stunning.4,5 tpn - q prevented this beneficial effect of dzx , thus implicating tpn - q targets other non kir tpn - q targets or undefined channel subunits sensitive to tpn - q may play a role ( girk1/4 , kach , and voltage - dependent ca - activated k channels ) in cardioprotection and will be the subject of future investigations.2931 the present study further characterizes the mechanism of cardioprotection provided by dzx . given that tpn - q inhibits kir1.1- , kir3.1- , and kir3.4-dependent k - channel activities , these data support that any of these subunits ( as well as undefined subunits sensitive to tpn - q ) could be involved in the cardioprotection afforded by dzx .

from the chemical view , the aetiology of dental erosion can be defined as the chronic exposure of the teeth to extrinsic or intrinsic acids under the condition that the oral fluids are undersaturated with respect to tooth mineral [ 23 , 26 ] . under in vitro conditions without physical impact , teeth demineralise centripetally ( fig . 1 ) , a feature of substance loss which is normally not observed in the mouth . in fact , the multitudes of physical and chemical assaults occurring during a lifetime result in a more or less characteristic pattern of tooth wear . the classification of wear is therefore made from morphological features which are frequently seen clinically . the tooth morphology as apparent after eruption is the idealised status , deviations of which , if not caries or trauma , are diagnosed as ( erosive ) tooth wear . various forms of wear including dental erosion are listed in the international classification of diseases thus defining them as a disease ( for critical discussion of this notion see ) . 1effect of the continuous exposure of a human third molar to 10% citric acid . the amorphous , centripetal tissue loss is obvious ( a unaffected tooth , b tissue loss after 4 , c 8 , and d 12 h immersion time ) effect of the continuous exposure of a human third molar to 10% citric acid . the amorphous , centripetal tissue loss is obvious ( a unaffected tooth , b tissue loss after 4 , c 8 , and d 12 h immersion time ) looking at what diagnosis is , one will find mostly definitions like identification of disease from signs or symptoms , implicating that the physician or scientist reads off from the patient thus detecting the disease . health and disease , however , is not a given condition , but is constituted by the theoretical concepts and the discursive practice the physician or scientist is subjected to . that is to say , rather than reading off the disease from the patient , a pattern of criteria is projected onto the diagnosed subject which determines the diagnostic procedure and outcome . following this approach , the diagnostic process , in a first step , is a theoretical concept ordering signs and symptoms to diseases which takes place within the configurative power of the discourses . in whatever discourse the physician acts , the conclusion reached through this process is called a diagnosis . from this background , it becomes obvious that the diagnostic process must be accompanied by a recurrent reconsideration of its theoretical concepts and the reevaluation of the criteria used . the diagnosis of tooth wear in general and erosion in particular is made from its lesion characteristics , from the results of nutritional , medical and occupational analysis , and from dietary records . the diagnostic process can be more differentiated with the individual patient , whereas in field trials it is restricted to the classification of lesion shape . throughout the literature , there is more or less consensus about the aetiology of the various forms of tooth wear , the clinical criteria for dental erosion and its differential diagnosis [ 12 , 14 ] . the early signs of erosive tooth wear appear as changes of the optical properties of enamel resulting in a smooth silky shining glazed surface . when the tissue loss continues , changes in the original morphology occur . on smooth surfaces , convex areas flatten or concavities develop , the width of which clearly exceeds the depth . on occlusal and incisal surfaces , rounding and cupping of the cusps and grooving of the incisal edges occur , and restorations may rise above the level of the adjacent tooth surfaces . in advanced cases , the validity of these clinical criteria for erosion , however , has not been under critical consideration . validity , in general terms , means the degree , to which a measurement measures what it purports to measure . for the issue addressed here , criterion validity is the relevant term , meaning the extent to which the measurement correlates with an external criterion under study . concurrent validity means to which extent the measurement and the criterion refer to the same time . in the case of erosion this would address the question if ( a ) the diagnostic criteria reflect lesions being an effect of an exposure to acids and ( b ) if the presence of characteristic signs is concurrent with an acid exposure . considering how the diagnostic criteria for erosion were established , the literature reveals that it was individual clinical experience and case reports rather than systematic research . one of the first publications on the characteristics of acid induced tissue loss was in 1946 from robinson and in 1947 from stafne and lovestedt . robinson , whilst being in doubt about the aetiology of dental erosion , described the lesions as located on smooth surfaces and as wedge shaped , cup shaped , disk shaped , irregular in form , l- or u - shaped , or simply as small round depressions or larger surface lesions . it is not clear how these descriptions were established , but robinson referred to a work from mcclure and ruzicka describing the morphology of lesions of rats teeth after being fed with lactate and citrate drinking fluid . stafne and lovestedt presented their observations in subjects with known acid exposure , amongst them 50 patients with frequent consumption of lemon juice . they did not give a concrete description of lesion shape , but attributed hypersensitivity , absence of stain and defects with rounded margins as effect of the action of the acids . their most important sign of diagnostic value was the presence of fillings projecting above the surface of the tooth . in their publication , a number of clinical images were included , presenting lesions clearly matching current erosion criteria . more than twenty years later , in 1970 , it was pindborg who gave the often cited definition of erosion as being superficial loss of dental hard tissue by a chemical process which does not involve bacteria . he described the clinical signs of chemically - induced tissue loss as usually located to the gingival third of the facial surfaces , possibly also located at proximal surfaces , lesions to appear shallow , disc - shaped , smooth , polished , or scooped out . in contrast to abrasion , he attributed erosive lesions to be located evenly on the left and right side . it appears noteworthy , that pindborg , as well as robinson , ascribed cupping of the cusps , loss of the occlusal morphology or loss of crown height , and incisal grooving to attrition which in their publications was defined as result of mastication . it is probably eccles and jenkins [ 8 , 10 ] who were the first to give a detailed and systematic description of the lesion characteristics and also suggested a system for classification . the basis of the development of the clinical criteria was a sample of 72 patients seen in the dental hospital over a period of 9 years . all cases were thoroughly documented with respect to their medical and dietary history , that is , it was a group of subjects with known exposure to intrinsic or dietary acids . the findings derived from this group of subjects ( table 1 ) have been retained nearly unchanged until now . the main aspects are in general terms loss of surface contour , shallow concavities on smooth surfaces , cupping and grooving on occlusal / incisal surfaces and restorations rising above the level of the adjacent tooth surface . table 1diagnostic criteria for dental erosion as outlined by eccles and jenkins and eccles diagnostic criteria for dental erosioninitialabsence of developmental ridges of the enamel , smooth glazed surfaceadvanced facial / oral surfacesconcavities whose breadth greatly exceeds their depthlesion ovoid or crescentic in outline , concave in cross section orlesion entirely in the crown , irregular in outline , punched out appearance occlusal / incisal surfacessurfaces appear flattened , depression of the cusps ( cupping ) and on the incisal edges ( grooving ) , edges of restorations raising above the level of the adjacent tooth surface diagnostic criteria for dental erosion as outlined by eccles and jenkins and eccles current criteria , derived from a relatively small sample , have been applied in case reports [ 8 , 9 , 16 , 32 , 39 , 50 ] and studies with risk groups [ 15 , 17 , 21 , 25 , 34 , 38 , 44 , 48 , 51 ] . indeed , when used in subjects with known or strongly assumed exposure to acids , most studies revealed a higher prevalence of lesions in the exposed groups compared to the control groups ( table 2 ) . table 2prevalence of lesions in risk groups deriving from the use of current diagnostic criteria for dental erosion indexgroup sizeprevalence risk groupprevalence control groupintrinsic acid exposuremeurman et al . reflux diseaseeccles and jenkins indexn = 11728/117 = 24%no control grouprytmaa et al . eating disorderseccles and jenkins indexn = 14022/35 = 63%12/105 = 11%hrn et al . eating disorderslussi indexn = 13379/81 = 98%minor , less severeincisal / occlusalincisal / occlusalgrade 1 : 93%grade 1 : 73%grade 2 : 52%grade 2 : 23%buccalbuccalgrade 1 : 30%grade 1 : 19%grade 2 : 9%grade 2 : 6%palatalpalatalgrade 1 : 21%grade 1 : 10%grade 2 : 5%grade 2 : 0%extrinsic acid exposurelinkosalo and markkanen vegetarianseccles and jenkins indexn wine tasterseccles and jenkins indexn = 1914/19 = 74%no control groupganss et al . raw food dietlussi indexn = 206127/130 = 98%66/76 = 87% prevalence of lesions in risk groups deriving from the use of current diagnostic criteria for dental erosion these observations support the finding that subjects with continuous exposure to acids have a higher rate of lesions with a specific characterisation . this is , however , not enough support for the assumption that , vice versa , subjects presenting with such defects must be exposed to acids . analytical epidemiological studies on random or cluster samples attempted to relate the occurrence of lesions with any of the known aetiological factors for erosion , but most of them include children or adolescents , but there is lack of studies on older population groups . furthermore , off the few studies , some used the tooth wear index ( twi ) which is not designed to assess dental erosion specifically . the overall findings ( table 3 ) are controversial since some authors found no or only a partial relation between aetiological factors and the occurrence or severity of lesions [ 2 , 3 , 5 , 7 , 20 , 30 , 35 , 37 , 49 ] , whereas others revealed strong relationships . in addition , a relation to the intake of yoghurt or other foodstuff which certainly has no erosive potential was mentioned [ 1 , 27 , 35 ] . table 3analytical epidemiological studies attempting to relate the occurrence of ( erosive ) wear to aetiological factors index , group size , age and prevalenceconclusionjrvinen et al . eccles & jenkins indexcitrus fruits : odds ratio ( or ) 2case - control , n = 100 eachsoft drinks : or 41383-year - oldslussi et al . lussi indexsignificant relation to the consumption of fruit , acidic drinks , yoghurt , vomitingn = 4172630- and 4650-year - olds at least 36 and 43% resp . with twi ( smith and knight)no significant relation to drinks or other acidic foodn = 210significant relation to heart burn1114-year - olds57% had wear in enamel on more than 10 teethjaeggi et al . lussi indexno relation to any aetiological factorn = 4171925-year - olds at least 82% with erosional - dlaigan et al . twi ( smith and knight)significant relation to drinks and fruit , but also to milk , yoghurt and beern = 41814-year - olds48% low , 51% moderate 1% severe lesionsal - majed at al . twi ( smith and knight ) modified for erosionno association to erosive drinks for the total samplen = 862significant association to frequency of drinks at night and duration of drinks retained in the mouth only in advanced cases ( n = 95)1214-year - olds95% with erosionmathew et al . lussi indexno relation to the intake of sport drinksn = 3041828-year - olds37% with erosionvan rijkom et al . modified lussi indexno relation to acidic drinks and fruitsn = 4001516-year - olds30% with visible smooth weararnadottir et al . modified lussi indexno significant association to risk factorsn = 27815-year - olds72% grade 124% twi ( smith and knight ) modified for erosionno significant association with dietary factorsn = 1726significant relationship with gastro - oesophageal symptoms418-year - olds36 , 56 and 34% with any erosion on buccal and palatal surfaces of the incisors , and first permanent molars resp.dugmore and rock twi ( smith and knight ) modified for erosiondrinking fizzy pop : odds ratio 1.592.52 depending on amount and frequencyn = 114912-year - oldsno relation to eating apples , citrus fruit56% with erosionmilosevic et al . twi ( smith and knight ) on labial and lingual surfaces in front teeth , occlusal surfaces of first molarsno association to apples , fresh orangesn = 2385weak association ( or 11.4 ) to yoghurt , grapefruit , salad dressing , vinegar , fruit juice , fizzy drinks14-year - oldsstrong association to herbal / lemon tea ( or 3.97)53.5% with exposed dentine analytical epidemiological studies attempting to relate the occurrence of ( erosive ) wear to aetiological factors there are many points of discussion for explanation . at first , ( erosive ) wear is the effect of various concurrent or past chemical exposures from different sources , the variety of which can hardly be covered by simple questionnaires , and especially in younger people the dietary intake and lifestyle might be open to variations . furthermore , questionnaires may reflect only in part the dietary habits due to interpretation by the responder or seasonal changes in daily life habits . when the young age of the groups studied is considered , the determinants for dental erosion may not have acted for long enough , and , in addition , there might be considerable differences in the individual susceptibility to erosive demineralisation . it must , however , also be taken into consideration that current diagnostic criteria might not be valid enough to really reflect the effect of a chronic acid exposure . taking into account that tooth wear is the result of various factors , the occlusal and incisal surfaces of teeth are not only exposed to acids but are particularly prone to physical impacts from mastication . from this background , the finding that many studies using erosion indices reveal that occlusal lesions are the most prevalent and that it is the first lower molar which is most affected , is of particular importance . occlusal surfaces might be the only location exhibiting a relation between lesion severity and prevalence , and age thus indicating a significant contribution of abrasion from mastication . furthermore , particularly the teeth from ancient remains exhibit defects being strikingly of the same shape as those attributed to erosion today ( fig . 2a occlusal aspect of a subject living on a raw food diet with multiple acid impacts , and a medieval subject b with an assumed abrasive diet ( images a , b , and c samples from [ 13 , 15 ] ) . occlusal / incisal defects in a subject with chronic vomiting d and in a medieval subject c. the shape of lesions from predominantly erosive and predominantly abrasive aetiology is strikingly similar a occlusal aspect of a subject living on a raw food diet with multiple acid impacts , and a medieval subject b with an assumed abrasive diet ( images a , b , and c samples from [ 13 , 15 ] ) . occlusal / incisal defects in a subject with chronic vomiting d and in a medieval subject c. the shape of lesions from predominantly erosive and predominantly abrasive aetiology is strikingly similar attempts have been made to characterise the shape of wear resulting from abrasion and erosion [ 6 , 13 ] . a comparison of subjects with known exposure to a non - acidic but coarse diet and subjects with an acidic , but refined diet using silicon impressions revealed that in the former , the scooped out dentine was significantly shallower than in the latter . furthermore , the deepest lesion region in teeth from subjects with abrasive diet was located at the functional cusps , whereas in teeth from subjects with an erosive diet , the deepest region of the defects was located more centrally . the authors concluded that in cases when the dentine is severely scooped out , the causative agent is most likely of erosive origin whereas scooping tends to be shallower when wear is caused by abrasion . the conclusion of the study was to suggest a quantitative diagnostic procedure by calculating the depth / breadth ratio for clinical differential diagnosis . another approach was to compare the shape of defects occurring in subjects with substantially different nutrition patterns . the study included three groups of age matched individuals randomly selected contemporary subjects , medieval remains and a group of subjects living on raw food diet . from the latter group , extensive information was available on their intake of acid food qualifying their nutrition as rather erosive . for the medieval group , an abrasive nutrition was assumed from knowledge about the general nature of the early medieval diet . as to the smooth surfaces , the most interesting finding was that in the medieval group , no lesions were observed ( table 4 ) . even in cases with severe wear , the lingual and buccal aspects of the teeth appeared undisturbed and , in many cases , even developmental ridges were present . the lack of cervical defects is also reported by aubry et al . and kaidonis ( in this issue ) and also inherently supported by the fact that the overwhelming number of anthropological studies on tooth wear only deal with occlusal and proximal wear and that the indices used there do not include criteria for cervical wear [ 11 , 36 ] . there are , however , few studies reporting wear on lingual surfaces in archaeological remains [ 18 , 41 ] and there is one publication discussing regurgitation as a possible aetiological factor . in this study indeed , buccal and lingual wear was observed to the same degree as in a contemporary comparison group , but cervical wear did not develop beyond criterion 0 in most individuals . in 20% of the skulls , however , a cervical wear to twi score of 23 occurred . no information was given about the shape of the lesions , and from the images included , it appears that their characteristics would not match the current criteria for dental erosion . table 4prevalence of lesions of defined shape in three groups ( n = 100 each ) of subjects with substantially different nutrition patterns abrasive diet ( medieval group)acidic diet ( raw food group)average western diet incisal / occlusal surfacesincisors / caninesgrooving93%96%90%n.s . molars / premolarsshallow cupping ( < 0.5 mm)87%59%47%p < 0.001deep cupping ( > 0.5 mm)78%45%4%p < 0.0001 smooth surfaces ( all teeth)concavity coronal to the cej0%63%8%p < 0.0001v - shaped defects0%38%10%p < 0.0001 prevalence of lesions of defined shape in three groups ( n = 100 each ) of subjects with substantially different nutrition patterns in contrast to the findings in the medieval group , shallow defects located coronal from the cej were considerably prevalent in the raw food group ( table 4 , fig . 3b ) , whereas in the western diet group , the prevalence of this kind of lesions was much lower and corresponds to its general prevalence in germany . fig . 3a buccal aspect of teeth 4447 with significant loss of crown height , but without any lesion in a medieval remain with severe generalised occlusal wear c. b occlusal defects in a subject living on a raw food diet with a high intake of acidic food . the shape of the occlusal lesions is similar to c , but combined with shallow lesions with intact cervical rim lesions . 4b with an initial buccal lesion a buccal aspect of teeth 4447 with significant loss of crown height , but without any lesion in a medieval remain with severe generalised occlusal wear c. b occlusal defects in a subject living on a raw food diet with a high intake of acidic food . the shape of the occlusal lesions is similar to c , but combined with shallow lesions with intact cervical rim lesions . 4b with an initial buccal lesion as to the occlusal / incisal surfaces , grooving and cupping was common in all groups even though most often in the medieval group , followed by the raw food group , and the western diet group . the conclusion from this study was that shallow defects on smooth surfaces might be a valid criterion for dental erosion , whereas cupping , and especially incisal grooving , was common in all groups and therefore not valid for a differential diagnosis . the similarity of occlusal / incisal defects in all groups might be explained from a tribological view . wear , as a result of abrasion , occurs as three body wear that means the intervention of an abrasive slurry or bolus . during mastication can occur when deeper enamel regions with lower microhardness are exposed or when the dentine is reached . in the case of erosion , tissue loss is on one hand caused by direct dissolution of mineral but also due to an increased susceptibility of acid softened surfaces to physical wear . in these cases , also a non- or less abrasive bolus could cause similar defects when acting on acid weakened surfaces . hence , the occlusal / incisal substance loss observed in individuals prone to dietary acids may be explained as pronounced abrasion / demastication of acid softened surfaces . therefore , it is questionable if the occlusal morphological criteria used for the diagnosis of occlusal erosion per se are valid . even though there are often striking similarities between lesions of predominantly abrasive or predominantly erosive origin , there is , on the other hand , often also considerable variation in lesion shape in cases of erosion . the morphology of occlusal lesions in subjects with known exposure to acids varies from deep hollowing out of the cusps to totally amorphous loss of the occlusal structure even in subjects with a similar dietary history ( fig . 4occlusal tissue loss from erosive aetiology can also be of strikingly different shape either presenting as deeply hollowed out lesions ( a subject with raw food diet , b subject with excessive consumption of orange juice ) or as amorphous generalised tissue loss affecting the entire surface ( c , d subjects with excessive consumption of erosive drinks ) . an interesting feature is seen in an adolescent with a history of severe anterior open bite with only the molars being in function e substance loss occurred from excessive consumption of a cola type drink . in the premolars , f hollowing out the entire occlusal surface with enamel remnants in the centre , aetiology is the excessive consumption of sport drinks occlusal tissue loss from erosive aetiology can also be of strikingly different shape either presenting as deeply hollowed out lesions ( a subject with raw food diet , b subject with excessive consumption of orange juice ) or as amorphous generalised tissue loss affecting the entire surface ( c , d subjects with excessive consumption of erosive drinks ) . an interesting feature is seen in an adolescent with a history of severe anterior open bite with only the molars being in function e substance loss occurred from excessive consumption of a cola type drink . in the premolars , f hollowing out the entire occlusal surface with enamel remnants in the centre , aetiology is the excessive consumption of sport drinks the validity of current diagnostic criteria for dental erosion has not been systematically studied , even though there is consensus about their definition . the following conclusions can be drawn : grooving of incisal surfaces is a common phenomenon and possibly the effect of any physical or chemical impact . it should be considered to abandon grooving of anterior teeth and canines as a clinical criterion for dental erosion.shallow defects located coronal from the cej may predominantly occur as effect of chronic acid exposure and might be pathogonomic for dental erosion . this assumption is supported by the finding that these types of lesions are not present in ancient remains even in cases of severe wear.cupping of cusps is the most uncertain criterion because it can be an effect of abrasion as well as of erosion . in industrialised countries , abrasion is not expected to be a significant factor in young people . cupping occurring at younger ages can therefore be an effect of erosion . at older ages , however , physical and chemical impacts add up increasingly and cupping will therefore be of little diagnostic value in adults . grooving of incisal surfaces is a common phenomenon and possibly the effect of any physical or chemical impact . it should be considered to abandon grooving of anterior teeth and canines as a clinical criterion for dental erosion . shallow defects located coronal from the cej may predominantly occur as effect of chronic acid exposure and might be pathogonomic for dental erosion . this assumption is supported by the finding that these types of lesions are not present in ancient remains even in cases of severe wear . cupping of cusps is the most uncertain criterion because it can be an effect of abrasion as well as of erosion . in industrialised countries cupping occurring at younger ages can therefore be an effect of erosion . at older ages , however , physical and chemical impacts add up increasingly and cupping will therefore be of little diagnostic value in adults . these conclusions are drawn from very few studies ; therefore systematic research on this issue is needed . nevertheless , there is enough support for a criticism of current diagnostic criteria particularly in the light of the development of a new index .

in principle , there is agreement about the clinical diagnostic criteria for dental erosion , basically defined as cupping and grooving of the occlusal / incisal surfaces , shallow defects on smooth surfaces located coronal from the enamel cementum junction with an intact cervical enamel rim and restorations rising above the adjacent tooth surface . this lesion characteristic was established from clinical experience and from observations in a small group of subjects with known exposure to acids rather than from systematic research . their prevalence is higher in risk groups for dental erosion compared to subjects not particularly exposed to acids , but analytical epidemiological studies on random or cluster samples often fail to find a relation between occurrence or severity of lesions and any aetiological factor . besides other aspects , this finding might be due to lack of validity with respect to diagnostic criteria . in particular , cupping and grooving might be an effect of abrasion as well as of erosion and their value for the specific diagnosis of erosion must be doubted . knowledge about the validity of current diagnostic criteria of different forms of tooth wear is incomplete , therefore further research is needed .

The process of diagnosis and current criteria for dental erosion The implementation of current diagnostic criteria Conclusions from epidemiological studies using current diagnostic criteria Comparative studies on lesion characteristics of wear Conclusion

throughout the literature , there is more or less consensus about the aetiology of the various forms of tooth wear , the clinical criteria for dental erosion and its differential diagnosis [ 12 , 14 ] . on occlusal and incisal surfaces , rounding and cupping of the cusps and grooving of the incisal edges occur , and restorations may rise above the level of the adjacent tooth surfaces . all cases were thoroughly documented with respect to their medical and dietary history , that is , it was a group of subjects with known exposure to intrinsic or dietary acids . the main aspects are in general terms loss of surface contour , shallow concavities on smooth surfaces , cupping and grooving on occlusal / incisal surfaces and restorations rising above the level of the adjacent tooth surface . table 1diagnostic criteria for dental erosion as outlined by eccles and jenkins and eccles diagnostic criteria for dental erosioninitialabsence of developmental ridges of the enamel , smooth glazed surfaceadvanced facial / oral surfacesconcavities whose breadth greatly exceeds their depthlesion ovoid or crescentic in outline , concave in cross section orlesion entirely in the crown , irregular in outline , punched out appearance occlusal / incisal surfacessurfaces appear flattened , depression of the cusps ( cupping ) and on the incisal edges ( grooving ) , edges of restorations raising above the level of the adjacent tooth surface diagnostic criteria for dental erosion as outlined by eccles and jenkins and eccles current criteria , derived from a relatively small sample , have been applied in case reports [ 8 , 9 , 16 , 32 , 39 , 50 ] and studies with risk groups [ 15 , 17 , 21 , 25 , 34 , 38 , 44 , 48 , 51 ] . table 2prevalence of lesions in risk groups deriving from the use of current diagnostic criteria for dental erosion indexgroup sizeprevalence risk groupprevalence control groupintrinsic acid exposuremeurman et al . raw food dietlussi indexn = 206127/130 = 98%66/76 = 87% prevalence of lesions in risk groups deriving from the use of current diagnostic criteria for dental erosion these observations support the finding that subjects with continuous exposure to acids have a higher rate of lesions with a specific characterisation . analytical epidemiological studies on random or cluster samples attempted to relate the occurrence of lesions with any of the known aetiological factors for erosion , but most of them include children or adolescents , but there is lack of studies on older population groups . molars / premolarsshallow cupping ( < 0.5 mm)87%59%47%p < 0.001deep cupping ( > 0.5 mm)78%45%4%p < 0.0001 smooth surfaces ( all teeth)concavity coronal to the cej0%63%8%p < 0.0001v - shaped defects0%38%10%p < 0.0001 prevalence of lesions of defined shape in three groups ( n = 100 each ) of subjects with substantially different nutrition patterns in contrast to the findings in the medieval group , shallow defects located coronal from the cej were considerably prevalent in the raw food group ( table 4 , fig . the conclusion from this study was that shallow defects on smooth surfaces might be a valid criterion for dental erosion , whereas cupping , and especially incisal grooving , was common in all groups and therefore not valid for a differential diagnosis . the morphology of occlusal lesions in subjects with known exposure to acids varies from deep hollowing out of the cusps to totally amorphous loss of the occlusal structure even in subjects with a similar dietary history ( fig . in the premolars , f hollowing out the entire occlusal surface with enamel remnants in the centre , aetiology is the excessive consumption of sport drinks the validity of current diagnostic criteria for dental erosion has not been systematically studied , even though there is consensus about their definition . it should be considered to abandon grooving of anterior teeth and canines as a clinical criterion for dental erosion.shallow defects located coronal from the cej may predominantly occur as effect of chronic acid exposure and might be pathogonomic for dental erosion . this assumption is supported by the finding that these types of lesions are not present in ancient remains even in cases of severe wear.cupping of cusps is the most uncertain criterion because it can be an effect of abrasion as well as of erosion . shallow defects located coronal from the cej may predominantly occur as effect of chronic acid exposure and might be pathogonomic for dental erosion . cupping of cusps is the most uncertain criterion because it can be an effect of abrasion as well as of erosion .

it is an important element to inspire thoughts out - of - the - box , or , more practically , to find new ways of organizing and correcting problems otherwise not considered . at a time when there is a debate of reforming the portuguese health - care system , the canadian system , though different , may provide answers to some of the questions that are being asked . this article is based on a report on a 1-month observership in family medicine that took place in march 2013 in edmonton , alberta , canada . every canadian citizen is assigned a personal health number ( phn ) which guarantees free access to health care at any health institution in canada . on the first approach , this may prove to be difficult regarding organizing healthcare resources ; however , patients usually choose one doctor as their family physician , thus guaranteeing the continuity of care . most physicians accept only patients without a family doctor and will not accept patients with physicians already . it rests on the physician to decide whether or not he is willing to accept new patients , giving him freedom of choice regarding the number of patients , he is willing to see and follow ( usually between 1500 and 2000 patients ) . this decision might not only be settled at individual level but also at organizational level as is it possible for a clinic to refuse new patients usually if their panel is full . furthermore , physicians are also allowed to refuse to treat a patient further in case the patient - physician relationship and interaction are compromised . besides regular patients , there are also the walk - ins , which are patients who require medical examination due to an acute situation . here , again , the physician may choose not to see walk - in patients or the clinic is allowed to state that it does not accept any walk - ins . for this matter , there are specified walk - in clinics that center their care on acute conditions and usually have easier accessibility due to longer opening hours . many clinics have specified walk - in times for their patients on top of booked appointments . as for the articulation of primary care and secondary care , it is quite well established in canada . usually , primary care physicians reference patients to a specialist by letter , which is registered in the computer system . the waiting time to get an appointment varies from 1 to 6 months ; however , in case it 's urgent , the primary care physician may also phone the specialist and book an appointment for the very same day . the whole reference process happens at individual level , that is , the primary care physician references a patient to a specialist , individually . therefore , it is also possible for the patients to choose the specialist they wish to go to . in case it is required , the primary care physician may also phone the specialist and discuss a patient 's case with him or her . in case it 's urgent , the specialist on call at the hospital can always be contacted by phone for advice . all physicians usually work at the hospital or at a clinic ( a building where physicians may rent an office to practice in ) . for the latter , it is required that physicians cover the rent and any expenses they may have with personnel , such as nurses and secretaries . medical services are always covered by the provincial government , so the patient is not required to pay for the visits . in case of an acute onset of symptoms , patients may go to their family physician , to the walk - in clinics , or directly to the hospital emergency room . at the hospital , patients are triaged by a nurse and if wait times are long and the patient is triaged low , they may choose to book an appointment with his family physician instead . describing the activity of the family physician , their approach is family - centered , rather than at individual level , as the patients physicians accept are members of a family . risk groups ( children , pregnant women , diabetic patients , etc . ) are seen in a special type of appointment throughout the day . other types of special appointments include the general physical examination , which consists in a yearly evaluation , from head to toe , of the patient 's overall health . during this visit , it is normal practice to review the patient 's personal history and habits to keep track of any change that may have occurred . in case , the patient suffers from a common chronic condition ( e.g. : hypertension , diabetes , chronic obstructive pulmonary disease , asthma , etc . ) it includes the problem list , lifestyle issues , the current medication , the involvement of other health care professionals , the advised scheduling of medical services throughout the year , end of life discussion , and the goals set by the patient and the physician . it is reviewed on a yearly basis and is expected to help the patient get a better grasp of his condition and manage medical visits accordingly . patients are advised to book their appointments according to their condition and their motive , always having the possibility of booking an appointment on the same day in case of an acute condition . home visits are optional , but most family doctors do not perform them . if the clinic has the necessary conditions , family physicians are also able to perform deliveries , small surgeries ( e.g. circumcisions , vasectomies , cyst removals , ingrown toenail incisions ) , biopsies ( includes endometrial biopsies ) , casts , use of liquid nitrogen , insert intrauterine devices , aspirations , and joint injections . with special training , family physicians are allowed other procedures , such as performing c - sections or anesthesia . family physicians also have the option to become involved in hospital care and stay responsible for a ward of 2025 patients . every major hospital has family medicine wards that admit patients with different types of pathologies such as hip fracture , delirium , sepsis , congestive heart failure , etc . , the family doctor is responsible for these patients , having to check on them on a daily basis and if required , ask for the collaboration from another specialist . , family physicians can do emergency work as well without extra training . in the larger centers , family physicians require 1 extra year of residency training in emergency room medicine and are almost considered equivalent to a specialist emergency room physician . regarding medication , it is sold in units and physicians may , in one prescription , prescribe chronic medication that lasts for a year . as patients are advised to pick up their medication at the same pharmacy , physicians may call up the pharmacy at any point and discuss the patient 's medication with the pharmacist . what 's more , pharmacists may also renovate prescriptions ( except for narcotics ) , which , consequently , make many patients not to go and see the doctor . nurses at the primary care clinics assist the doctors and have other duties , such as giving injections , ear wash , wart treatment , filling cognitive assessment forms , take away casts , review chronic disease management plans , collect the history in newborns , and obtain the ankle brachial index . the canadian health - care service is fundamentally public ; however , clinics are owned by a physician . physicians get their income according to a fee - for - service model , that is , the physician bills the government after seeing a patient . afterward , the physician has to pay about 30% of their income to the physician who owns the clinic , to cover staff salaries , rent , etc . , income taxes cover 38% of a physician 's income , and physicians have to pay their insurance fee . physicians are considered self - employed ; therefore , professionals do not have a right to pension or to sickness leave . there are also some salaried positions in the public system ( alternate relationship plan ) , but they are mainly reserved for academic physicians to promote research and teaching . as for working hours , this individual modeling could prove an obstacle for the management of the clinic ; however , there is a record of each doctor 's activity plan together with the number of patients he or she is currently seeing and the patients the physician should be seeing . in some provinces , there are initiatives to promote primary health creating structures such as the primary care network ( pcn ) . a pcn may include one clinic with several physicians and support staff or many physicians in various clinics across a health region . each network works toward developing programs and providing services to best meet the needs of local patients . in a pcn , patients have access to multiple services such as geriatrics long - term , women 's health clinic , shared care maternity , pediatric asthma , cast clinic , and after - hours clinic . overall , pcns aim to provide better services and to engage the community in activities to promote the role of physicians within primary care . regarding continued medical education , it is required that every doctor submits 250 mainpro ( maintenance of proficiency ) credits every 5 years - one credit is equal to 1 h of learning . these credits are authorized by the college of family physicians of canada , and they can be gained in any scientific activity participated besides practice , i.e. research , teaching , courses , reading journals , certified podcasts , group sessions , congress attendance , master 's degree , etc . every portuguese citizen has a health care number which allows them to have access to health care , after paying a token duty . the latter does not apply in certain situations , such as patients that demonstrate financial hardship ( it is mandatory to declare one 's income to the state ) as well as their dependents , pregnant women and women in labor , children until the age of twelve , patients with an inability level of 60% or higher , transplant patients , military and former military personnel who have been permanently incapacitated due to their military service , and certain unemployed people . blood , cell , tissue or organ donors , as well as firemen , are exempted of co - payment in case of primary care . demonstrating financial hardship proves a barrier to vulnerable groups such as homeless people as they have no means of proving they have a right to be exempted from the usual co - payment . furthermore , excepting emergency room visits , portuguese citizens may only use health - care resources that are available in their living area . when a family physician starts working at a health - care center , he / she is given a list of about 18002000 patients , which is made out of members of various families . the physician is expected to follow these patients throughout life , occasionally accepting a new family member by request from the family . thus , there is a very little variation in the patients the physician usually sees . however , this assures the continuity of care , aids the physician to get more familiar with the patients , and to establish a closer patient doctor relationship . in case doctor relationship is compromised , the physician may ask another physician , of the same health care unit , to see and follow a patient . because of the list system , there is a considerable amount of citizens that do not have access to primary care , let alone a family physician . as the portuguese health - care system is a mix between a public and a private system , citizens with higher incomes however , patients have to pay out - of - pocket for the full amount of that visit as they are not covered by the state . some public health care subsystems have an agreement with some doctors and partially cover for the visits , but only specific groups of the population ( such as police , firemen , military , and state workers ) have access to them . for those with fewer resources , access to health care equals , for the most part , paying a visit to the emergency room at the nearest hospital . outside the hospital , there are some centers that offer a public health care service ( sap ) , but they only cover matters of an acute onset . there has been an effort to cover the primary care health requirements of citizens that are not included in a family physician 's list . this is being accomplished by asking some family physicians to follow vulnerable risk groups , such as children and pregnant women , in addition to their usual clinical activity . although not enough , this measure slightly helps to alleviate the difficult access to family physicians . on the other hand , matters are quite different for citizens that have a family physician . in a health care practice , the physician 's schedule is organized by the type of visit and it includes allocated times for normal visits , urgencies , addressing risk groups ( children , pregnant women , family planning , diabetes , and hypertension ) , and home visits . a visit usually lasts for 1015 min and the patient may book a visit according to his current need . there are also appointments that do not require the presence of the patient and thus have a lower token duty than the one to pay for a regular visit . they include the renovation of a prescription , prescribing exams , a telephone contact , or filling out any other type of document that does not require the patient to be in the room . the longest a medicine prescription lasts is 6 months , and patients need to go to their physician to get a refill . when prescribing , physicians may only prescribe a whole package of pills , which causes patients to store many packages at home . it is not customary for pharmacies to keep track of a patient 's medication ; therefore , there is no dialog with the physician on the patient 's medication . when it comes to risk groups , all visits start with an evaluation by the nurse , followed by the visit to the physician . nurses play an important role in primary care as , besides giving injections and doing wound treatments , they are responsible for the teaching and giving advice to risk groups . they also perform home visits and in case they consider it to be necessary , may request the physician to reassess a patient . in some health - care centers , nurses may become a family nurse and work in partnership with one family physician . this means that a family nurse only follows the patients that are included in the physician 's list . family physicians do not usually perform any surgical or invasive activities unless they underwent a specific training in that area . this is the case for very few physicians hence when required , most physicians reference patients to the local hospital through a computer program . this application is the main means of referral between primary and secondary care as patients need to be referred through it , to have an appointment at a public hospital . when making a referral , the family physician chooses the required specialty instead of an individual doctor , and he may only refer to the local hospital . a patient is only transferred to a different hospital in case the local one considers it does not have the necessary means to provide the best care to the patient . apart from this application , the communication between primary and secondary care is scarce and difficult to establish . reports from the hospital specialists have to be requested by special notice and most of the time ; patients have to carry the notices themselves . work is being done to establish a national platform on the internet for this purpose , but it is not yet fully functional . a family physician 's schedule is made out of a weekly 40 h labor journey . family physicians only work on weekdays and do not have to make night shifts . like any other physician , family physicians are state workers and are paid a fixed salary at the end of each month , having the right for pension or sickness leave . the percentage of the salary that has to be paid to the state varies with the income of every physician . every canadian citizen is assigned a personal health number ( phn ) which guarantees free access to health care at any health institution in canada . on the first approach , this may prove to be difficult regarding organizing healthcare resources ; however , patients usually choose one doctor as their family physician , thus guaranteeing the continuity of care . most physicians accept only patients without a family doctor and will not accept patients with physicians already . it rests on the physician to decide whether or not he is willing to accept new patients , giving him freedom of choice regarding the number of patients , he is willing to see and follow ( usually between 1500 and 2000 patients ) . this decision might not only be settled at individual level but also at organizational level as is it possible for a clinic to refuse new patients usually if their panel is full . furthermore , physicians are also allowed to refuse to treat a patient further in case the patient - physician relationship and interaction are compromised . besides regular patients , there are also the walk - ins , which are patients who require medical examination due to an acute situation . here , again , the physician may choose not to see walk - in patients or the clinic is allowed to state that it does not accept any walk - ins . for this matter , there are specified walk - in clinics that center their care on acute conditions and usually have easier accessibility due to longer opening hours . many clinics have specified walk - in times for their patients on top of booked appointments . as for the articulation of primary care and secondary care , it is quite well established in canada . usually , primary care physicians reference patients to a specialist by letter , which is registered in the computer system . the waiting time to get an appointment varies from 1 to 6 months ; however , in case it 's urgent , the primary care physician may also phone the specialist and book an appointment for the very same day . the whole reference process happens at individual level , that is , the primary care physician references a patient to a specialist , individually . therefore , it is also possible for the patients to choose the specialist they wish to go to . in case it is required , the primary care physician may also phone the specialist and discuss a patient 's case with him or her . in case it 's urgent , the specialist on call at the hospital can always be contacted by phone for advice . all physicians usually work at the hospital or at a clinic ( a building where physicians may rent an office to practice in ) . for the latter , it is required that physicians cover the rent and any expenses they may have with personnel , such as nurses and secretaries . medical services are always covered by the provincial government , so the patient is not required to pay for the visits . in case of an acute onset of symptoms , patients may go to their family physician , to the walk - in clinics , or directly to the hospital emergency room . at the hospital , patients are triaged by a nurse and if wait times are long and the patient is triaged low , they may choose to book an appointment with his family physician instead . describing the activity of the family physician , visits their approach is family - centered , rather than at individual level , as the patients physicians accept are members of a family . risk groups ( children , pregnant women , diabetic patients , etc . ) are seen in a special type of appointment throughout the day . other types of special appointments include the general physical examination , which consists in a yearly evaluation , from head to toe , of the patient 's overall health . during this visit , it is normal practice to review the patient 's personal history and habits to keep track of any change that may have occurred . in case , the patient suffers from a common chronic condition ( e.g. : hypertension , diabetes , chronic obstructive pulmonary disease , asthma , etc . ) it includes the problem list , lifestyle issues , the current medication , the involvement of other health care professionals , the advised scheduling of medical services throughout the year , end of life discussion , and the goals set by the patient and the physician . it is reviewed on a yearly basis and is expected to help the patient get a better grasp of his condition and manage medical visits accordingly . patients are advised to book their appointments according to their condition and their motive , always having the possibility of booking an appointment on the same day in case of an acute condition . home visits are optional , but most family doctors do not perform them . if the clinic has the necessary conditions , family physicians are also able to perform deliveries , small surgeries ( e.g. circumcisions , vasectomies , cyst removals , ingrown toenail incisions ) , biopsies ( includes endometrial biopsies ) , casts , use of liquid nitrogen , insert intrauterine devices , aspirations , and joint injections . with special training , family physicians family physicians also have the option to become involved in hospital care and stay responsible for a ward of 2025 patients . every major hospital has family medicine wards that admit patients with different types of pathologies such as hip fracture , delirium , sepsis , congestive heart failure , etc . , the family doctor is responsible for these patients , having to check on them on a daily basis and if required , ask for the collaboration from another specialist . , family physicians can do emergency work as well without extra training . in the larger centers , family physicians require 1 extra year of residency training in emergency room medicine and are almost considered equivalent to a specialist emergency room physician . regarding medication , it is sold in units and physicians may , in one prescription , prescribe chronic medication that lasts for a year . as patients are advised to pick up their medication at the same pharmacy , physicians may call up the pharmacy at any point and discuss the patient 's medication with the pharmacist . what 's more , pharmacists may also renovate prescriptions ( except for narcotics ) , which , consequently , make many patients not to go and see the doctor . nurses at the primary care clinics assist the doctors and have other duties , such as giving injections , ear wash , wart treatment , filling cognitive assessment forms , take away casts , review chronic disease management plans , collect the history in newborns , and obtain the ankle brachial index . the canadian health - care service is fundamentally public ; however , clinics are owned by a physician . physicians get their income according to a fee - for - service model , that is , the physician bills the government after seeing a patient . afterward , the physician has to pay about 30% of their income to the physician who owns the clinic , to cover staff salaries , rent , etc . , income taxes cover 38% of a physician 's income , and physicians have to pay their insurance fee . physicians are considered self - employed ; therefore , professionals do not have a right to pension or to sickness leave . there are also some salaried positions in the public system ( alternate relationship plan ) , but they are mainly reserved for academic physicians to promote research and teaching . as for working hours , family physicians are free to plan their weekly schedule . this individual modeling could prove an obstacle for the management of the clinic ; however , there is a record of each doctor 's activity plan together with the number of patients he or she is currently seeing and the patients the physician should be seeing . in some provinces , there are initiatives to promote primary health creating structures such as the primary care network ( pcn ) . a pcn may include one clinic with several physicians and support staff or many physicians in various clinics across a health region . each network works toward developing programs and providing services to best meet the needs of local patients . in a pcn , patients have access to multiple services such as geriatrics long - term , women 's health clinic , shared care maternity , pediatric asthma , cast clinic , and after - hours clinic . overall , pcns aim to provide better services and to engage the community in activities to promote the role of physicians within primary care . regarding continued medical education , it is required that every doctor submits 250 mainpro ( maintenance of proficiency ) credits every 5 years - one credit is equal to 1 h of learning . these credits are authorized by the college of family physicians of canada , and they can be gained in any scientific activity participated besides practice , i.e. research , teaching , courses , reading journals , certified podcasts , group sessions , congress attendance , master 's degree , etc . every portuguese citizen has a health care number which allows them to have access to health care , after paying a token duty . the latter does not apply in certain situations , such as patients that demonstrate financial hardship ( it is mandatory to declare one 's income to the state ) as well as their dependents , pregnant women and women in labor , children until the age of twelve , patients with an inability level of 60% or higher , transplant patients , military and former military personnel who have been permanently incapacitated due to their military service , and certain unemployed people . blood , cell , tissue or organ donors , as well as firemen , are exempted of co - payment in case of primary care . demonstrating financial hardship proves a barrier to vulnerable groups such as homeless people as they have no means of proving they have a right to be exempted from the usual co - payment . furthermore , excepting emergency room visits , portuguese citizens may only use health - care resources that are available in their living area . when a family physician starts working at a health - care center , he / she is given a list of about 18002000 patients , which is made out of members of various families . the physician is expected to follow these patients throughout life , occasionally accepting a new family member by request from the family . thus , there is a very little variation in the patients the physician usually sees . however , this assures the continuity of care , aids the physician to get more familiar with the patients , and to establish a closer patient doctor relationship . in case doctor relationship is compromised , the physician may ask another physician , of the same health care unit , to see and follow a patient . because of the list system , there is a considerable amount of citizens that do not have access to primary care , let alone a family physician . as the portuguese health - care system is a mix between a public and a private system , citizens with higher incomes opt to see specialists at their offices directly . however , patients have to pay out - of - pocket for the full amount of that visit as they are not covered by the state . some public health care subsystems have an agreement with some doctors and partially cover for the visits , but only specific groups of the population ( such as police , firemen , military , and state workers ) have access to them . for those with fewer resources , access to health care equals , for the most part , paying a visit to the emergency room at the nearest hospital . outside the hospital , there are some centers that offer a public health care service ( sap ) , but they only cover matters of an acute onset . there has been an effort to cover the primary care health requirements of citizens that are not included in a family physician 's list . this is being accomplished by asking some family physicians to follow vulnerable risk groups , such as children and pregnant women , in addition to their usual clinical activity . although not enough , this measure slightly helps to alleviate the difficult access to family physicians . on the other hand , matters are quite different for citizens that have a family physician . in a health care practice , the physician 's schedule is organized by the type of visit and it includes allocated times for normal visits , urgencies , addressing risk groups ( children , pregnant women , family planning , diabetes , and hypertension ) , and home visits . a visit usually lasts for 1015 min and the patient may book a visit according to his current need . there are also appointments that do not require the presence of the patient and thus have a lower token duty than the one to pay for a regular visit . they include the renovation of a prescription , prescribing exams , a telephone contact , or filling out any other type of document that does not require the patient to be in the room . the longest a medicine prescription lasts is 6 months , and patients need to go to their physician to get a refill . when prescribing , physicians may only prescribe a whole package of pills , which causes patients to store many packages at home . it is not customary for pharmacies to keep track of a patient 's medication ; therefore , there is no dialog with the physician on the patient 's medication . when it comes to risk groups , all visits start with an evaluation by the nurse , followed by the visit to the physician . nurses play an important role in primary care as , besides giving injections and doing wound treatments , they are responsible for the teaching and giving advice to risk groups . they also perform home visits and in case they consider it to be necessary , may request the physician to reassess a patient . in some health - care centers , nurses may become a family nurse and work in partnership with one family physician . this means that a family nurse only follows the patients that are included in the physician 's list . family physicians do not usually perform any surgical or invasive activities unless they underwent a specific training in that area . this is the case for very few physicians hence when required , most physicians reference patients to the local hospital through a computer program . this application is the main means of referral between primary and secondary care as patients need to be referred through it , to have an appointment at a public hospital . when making a referral , the family physician chooses the required specialty instead of an individual doctor , and he may only refer to the local hospital . a patient is only transferred to a different hospital in case the local one considers it does not have the necessary means to provide the best care to the patient . apart from this application , the communication between primary and secondary care is scarce and difficult to establish . reports from the hospital specialists have to be requested by special notice and most of the time ; patients have to carry the notices themselves . work is being done to establish a national platform on the internet for this purpose , but it is not yet fully functional . a family physician 's schedule is made out of a weekly 40 h labor journey . family physicians only work on weekdays and do not have to make night shifts . like any other physician , family physicians are state workers and are paid a fixed salary at the end of each month , having the right for pension or sickness leave . the percentage of the salary that has to be paid to the state varies with the income of every physician . regarding the health - care system , accessing health care seems easier in canada than in portugal . thanks to the phn , citizens have free access to and choice of any health care institution or health professional in canada , even across provinces . on the other hand , accessibility is limited in portugal due to the geographic restriction and the co - payment that is requested to have access to any type of health care . when it comes to primary health care , all of these are aggravated by the static list of primary care physicians and the shortage of family doctors . this picture is very concerning as citizens with fewer resources , and thus , more vulnerable , are the ones who have the most difficulty to be granted access to health care . comparing the work of family physicians , the patients , a canadian doctor sees and follows may vary more than in portugal , which could pose a problem for continuity of care . in practice , however , patients tend to stay loyal to one doctor . as for their abilities , when they undergo a special training , they are allowed more complex interventions that could otherwise only be performed by a hospital specialist . besides that , they also have to be on call 24/7 and prepared to follow a patient that is hospitalized , especially if they work in a rural area . however , most family physicians in canada do not do any home visits , which is common practice in portugal . nurses play a more active role in portugal than in canada as they complement the work of family physician . on the other hand , the collaboration with other health professionals ( especially pharmacies ) is more established in canada than in portugal . overall , one may consider the canadian primary health - care system to be very good , and the way it is organized may inspire other countries that are experiencing difficulties in primary healthcare delivery , such as is happening in portugal . observing and experiencing other health - care services provides new insight on organization , planning , and structure of one owns health - care service . it is a highly valuable experience that enriches participants and actively contributes for the development and improvement of primary care . hereby , the author declares that she presents a conflict of interest as she has been involved in both the establishment and management of the family medicine 360 exchange program over the past years . hereby , the author declares that she presents a conflict of interest as she has been involved in both the establishment and management of the family medicine 360 exchange program over the past years .

background : the work of a family physician is quite different in each country , and if we consider different continents , differences are even more remarkable . social and cultural contexts justify a particular organization , not only of the health - care system but also its providers as well.objectives:by analyzing different health - care systems , new ideas may come about which may trigger positive changes in a health - care service to diminish healthcare disparities.methods:description and comparison of the primary healthcare service in canada and portugal.results:although both health - care systems are mainly public , organizational differences can be found that condition primary healthcare access.conclusion:exchanges in other health - care systems contribute for an active knowledge exchange that prompts participants to analyze options on how to improve healthcare access to citizens . this ultimately , leads to the development of primary care , the pillar of a well - functioning health - care system .

Introduction The work of a family physician in Canada The work of a family physician in Portugal Conclusion Financial support and sponsorship Conflicts of interest

at a time when there is a debate of reforming the portuguese health - care system , the canadian system , though different , may provide answers to some of the questions that are being asked . as for the articulation of primary care and secondary care , it is quite well established in canada . the waiting time to get an appointment varies from 1 to 6 months ; however , in case it 's urgent , the primary care physician may also phone the specialist and book an appointment for the very same day . the whole reference process happens at individual level , that is , the primary care physician references a patient to a specialist , individually . describing the activity of the family physician , their approach is family - centered , rather than at individual level , as the patients physicians accept are members of a family . every portuguese citizen has a health care number which allows them to have access to health care , after paying a token duty . blood , cell , tissue or organ donors , as well as firemen , are exempted of co - payment in case of primary care . when a family physician starts working at a health - care center , he / she is given a list of about 18002000 patients , which is made out of members of various families . because of the list system , there is a considerable amount of citizens that do not have access to primary care , let alone a family physician . there has been an effort to cover the primary care health requirements of citizens that are not included in a family physician 's list . on the other hand , matters are quite different for citizens that have a family physician . in a health care practice , the physician 's schedule is organized by the type of visit and it includes allocated times for normal visits , urgencies , addressing risk groups ( children , pregnant women , family planning , diabetes , and hypertension ) , and home visits . in some health - care centers , nurses may become a family nurse and work in partnership with one family physician . when making a referral , the family physician chooses the required specialty instead of an individual doctor , and he may only refer to the local hospital . as for the articulation of primary care and secondary care , it is quite well established in canada . in case it is required , the primary care physician may also phone the specialist and discuss a patient 's case with him or her . describing the activity of the family physician , visits their approach is family - centered , rather than at individual level , as the patients physicians accept are members of a family . nurses at the primary care clinics assist the doctors and have other duties , such as giving injections , ear wash , wart treatment , filling cognitive assessment forms , take away casts , review chronic disease management plans , collect the history in newborns , and obtain the ankle brachial index . in a pcn , patients have access to multiple services such as geriatrics long - term , women 's health clinic , shared care maternity , pediatric asthma , cast clinic , and after - hours clinic . every portuguese citizen has a health care number which allows them to have access to health care , after paying a token duty . when a family physician starts working at a health - care center , he / she is given a list of about 18002000 patients , which is made out of members of various families . because of the list system , there is a considerable amount of citizens that do not have access to primary care , let alone a family physician . there has been an effort to cover the primary care health requirements of citizens that are not included in a family physician 's list . on the other hand , matters are quite different for citizens that have a family physician . in a health care practice , the physician 's schedule is organized by the type of visit and it includes allocated times for normal visits , urgencies , addressing risk groups ( children , pregnant women , family planning , diabetes , and hypertension ) , and home visits . in some health - care centers , nurses may become a family nurse and work in partnership with one family physician . when making a referral , the family physician chooses the required specialty instead of an individual doctor , and he may only refer to the local hospital . regarding the health - care system , accessing health care seems easier in canada than in portugal . comparing the work of family physicians , the patients , a canadian doctor sees and follows may vary more than in portugal , which could pose a problem for continuity of care . nurses play a more active role in portugal than in canada as they complement the work of family physician . overall , one may consider the canadian primary health - care system to be very good , and the way it is organized may inspire other countries that are experiencing difficulties in primary healthcare delivery , such as is happening in portugal . observing and experiencing other health - care services provides new insight on organization , planning , and structure of one owns health - care service . it is a highly valuable experience that enriches participants and actively contributes for the development and improvement of primary care .

atrial septal defect ( asd ) is the second most common congenital heart defect ( chd ) and accounts for approximately 10% of all cardiac malformations.1 , 2 eighty percent of persistent foramen ovale and small asds close spontaneously during infancy or childhood , whereas large asds or those remaining open into adulthood may cause congestive heart failure , pneumonia , pulmonary vascular disease , atrial arrhythmias , and paradoxical embolism.3 , 4 , 5 , 6 , 7 also , cooccurrence with other cardiac malformations within the same individual is often observed.8 asd is correlated to mutations in the nkx25 gene , located on chromosome 5 ( 5q34).9 , 10 , 11 nkx25 is a cardiac transcription factor that plays a significant role in development of the atrioventricular node as well as maintaining function of the node throughout life.12 in recent years , nkx25 mutations have been reported in chd patients with nonfamilial as well as familial chd . familial atrioventricular block , observed as congenital or adultonset type , cooccur with asd.13 , 14 , 15 besides asd,9 , 10 , 11 congenital complete atrioventricular block cooccur with laterality defects such as levotransposition of the great arteries ( ltga ) or atrial isomerism.16 , 17 , 18 as opposed to the adultonset type , congenital complete atrioventricular block is diagnosed in utero or shortly after birth , and it is associated with mortality rates ranging from 33% to 80% if the heart rate is below 50 or it cooccurs with structural heart disease.17 , 18 conversely , the adultonset type of familial atrioventricular block is of a progressive nature , and there are several reports of patients with normal ecg or a harmless firstdegree atrioventricular block followed by sudden onset of second and thirddegree atrioventricular block or sudden death later in life.14 , 15 , 19 sudden cardiac death ( scd ) occur in patients with both types of atrioventricular block15 , 16 , 20 , 21 and in patients with nkx25 mutations.22 , 23 scd have been reported in pediatric as well as adult cases of atrioventricular block,15 , 16 , 20 , 21 and autopsy studies have shown fibrotic replacement of the avbundle,24 which explains the atrioventricular node malfunctioning in these patients . however , there has been an alarming number of scds in obligate carriers and relatives of patients with nkx25 mutations.22 , 23 , 25 , 26 this , and previous reports of patients with asd and/or atrioventricular block dying suddenly with a functioning pacemaker , suggest that the myocardium is also involved in the nkx25 phenotype.15 , 16 despite the large efforts in finding nkx25 mutations in chd patients , there have been no reviews of the existing literature to determine the frequency of the mutation or characterization of the phenotypic appearance of mutation carriers . we hypothesized , that mutations in nkx25 primarily occur in familial chd , and that the signature phenotype is asd with or without conduction disease or arrhythmia ( cd / a ) . , we report a novel truncating mutation in six members of a family with autosomal dominant transmission of asd ( n = 5 ) cooccurring with atrioventricular block and complex chd ( n = 1 ) . by reviewing the literature , we show that the majority of nkx25 mutation carriers are patients with familial asd and conduction disturbances , and we report an alarming large number of scds in such families . this finding has important implications for the management of patients with familial asd , because they could be carriers of a nkx25 mutation with an increased risk of scd . diagnoses of probands and their affected relatives were verified by a review of the patient file , and a diagnosis was considered confirmed if it was found during echocardiography , heart catheterization , surgery , or autopsy ( relations and diagnoses shown in supporting inforamtion ) . a total of 100 danish unaffected individuals , unrelated to the study subjects , were used as controls to investigate population frequency of the identified mutation . polymerase chain reaction products were sequenced bidirectionally with bigdye terminator v. 1.1 reagents ( applied biosystems , naerum , denmark ) and analyzed using an abi 3130xl genetic analyzer . also , a systematic search with the words nkx25/csx and congenital heart disease / asd / atrioventricular block / heart block was conducted in pubmed and omim . papers published in english peerreviewed journals investigating germline mutations were included , supplemented with literature cited in key papers . the study protocol was reviewed and approved by the local ethics committee and written informed consent was obtained from all participants or their legal guardians prior to investigation . we identified a single nucleotide deletion at position 112 in exon 1 of nkx25 in one family segregating autosomal dominant chd in three generations ( figure 1 ) . the mutation was not present in 100 danish controls or the exac database of variants in the exome ( http://exac.broadinstitute.org/ ) supporting that 112delg is a rare variant . the deletion causes a shift of the reading frame , leading to a deduced protein with abnormal amino acid sequence from amino acid 37 and a premature stop at amino acid 175 . i:2 ( 37 years old ) had surgical closure of a secundum atrial septal defect ( asd2 ) . ecg showed junctional rhythm with a heart rate of 49 beats per minute ( bpm ) . ii:5 ( 6 years old ) had surgical closure of an asd2 with intermittent 1 . and 2 . iii:1 ( 8 months old ) had complex chd [ double outlet right ventricle , fallot type ( dorvtof ) , coarctation of aorta ( coa ) , persistent left superior vena cava ( plsvc ) , and asd ] . iii:2 had an insignificant muscular ventricular septal defect ( vsd ) and a small asd2 at birth . iii:4 was a healthy carrier of the mutation , but an echocardiogram had never been done by wish of the parents . ( b ) section of the nucleotide sequence of nkx25 gene located on chromosome 5 ( 5q34 ) . the deletion of a single nucleotide at position 112 causes a frameshift , resulting in a truncated protein and a premature stop codon . in the danish family , the deletion segregated with chd and was observed in 5/5 affected individuals , where a blood sample was available , and in one apparently healthy individual , who , however , has not been thoroughly investigated for chd . asd was diagnosed in all six individuals affected with chd ( five live with documented chd , and one deceased with complex chd ) , three also had conduction disease , one a ventricular septal defect ( vsd ) and one individual ( iii:1 ) had asd in complex chd ( figure 1 ) . iii:1 was diagnosed with double outlet right ventricle , fallot type ( dorvtof ) , coarctation of aorta , asd , and persistent left superior vena cava . the patient died eight months old after complicated staged surgery and atrioventricular block or arrhythmia was not observed . fiftynine different nkx25 mutations were reported and confirmed in 202 patients in 31 papers.2 , 18 , 19 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 51 , 52 , 53 , 54 , 55 , 56 thus , including the mutation we identified in six danish individuals , a total of 60 nkx25 mutations have been identified in 208 patients . a total of 198 index patients from chd families ( asd = 117 ; atrioventricular block = 9 ; hlhs = 12 ; tof = 3 , mixed chd = 57 ) have been screened for mutations and in 18 ( 9.1% ) a nkx25 mutation was found cosegregating with the malformation . in comparison 1037 nonfamilial cases ( asd = 137 ; atrioventricular block = 4 ; hlhs = 7 ; tof = 290 ; mixed chd = 599 ) have been screened in which 17 ( 1.6% ) had a nkx25 mutation ( table 1 ) . a fisher 's exact test showed that this difference is significant ( p = 7.1 10 ) . studies by costa et al . , belvis et al . , and xie et al . were excluded ( table 1 ) , because the index patients did not have chd , but cardiomyopathy , stroke and atrial fibrillation , respectively . if these are included , the frequencies are 4.4% and 1.8% for familial and nonfamilial cases , respectively , and the difference is still significant ( p = .0032 ) . studies , in which an actual screening of affected individuals was undertaken and where the text stated the number of screened familial and sporadic cases , was included in the calculations . number of screened familial and sporadic cases published overview of the number of familial / sporadic index cases with chd screened for nkx25 mutations . only the number of index cases screened and the number of index cases ( ) with mutations are displayed . for example , in this study 39 index cases from 39 families were screened and one subject had a mutation . indicate that mutation carriers found had asd ( e.g. , in the study by costa et al . , where index patients had familial dilated cardiomyopathy , or xie et al . , where index patients had familial atrial fibrillation ) . the 208 patients were grouped according to diagnosis and presence / absence of cd / a ( figure 2 ) . patients with several malformations are part of several groups ( e.g. , a patient with asd and vsd were included in the asd and in the vsd column , respectively ) . asd was present in 145 ( 70% ) of the mutation carriers and 112 ( 54% ) also had cd / a . in addition , 17 patients had vsd and cd / a , however , 16 of these also had an asd . none of the three and thirteen patients with hlhs or tof , respectively , had cd / a . lastly , 11 patients had cardiomyopathy ( left ventricular noncompaction , left ventricular hypertrophy , dilated cardiomyopathy ) cooccurring with cd / a in six . the majority of patients with a confirmed mutation in nkx25 has asd with cd / a . asd , atrial septal defect ; tof , tetralogy of fallot ; vsd , ventricular septal defect ; hlhs , hypoplastic left heart syndrome ; cm , cardiomyopathy ( left ventricular noncompaction , dilated cardiomyopathy , left ventricle hypertrophy ) ; scd , sudden cardiac death ( sudden death in otherwise healthy individual before age 50 ) ; other : interrupted aortic arch = 1 ; truncus arteriosus = 1 ; levotransposition of the great arteries = 1 ; coarctation of aorta = 2 ; double outlet right ventricle = 1 ; tricuspid valve anomaly ( atresia , ebstein ) = 4 ; anomalous pulmonary venous return = 1 ; heterotaxy = 1 . in a total of 112 patients with asd and cd / a , 27 mutations were reported in 105 cases of familial chd , whereas only seven mutations were found in cases with nonfamilial chd . there were 19 scds in nine families with asd and cd / a , and cardiomyopathy was also present in four ( 44% ) of these nine families . a mutation ( p.512insglycys ) was documented in only one of these patients , however , additionally eight patients were obligate carriers ( supporting information figures ii the remaining 10 patients were all part of pedigrees with dominant traits of nkx25 mutations cosegregating with the malformations , and they all died suddenly before the age of 50 . assuming that these 10 were carriers of the mutations transmitted in their families , the total number of patients with familial asd with cd / a would be 130 ( 112 + 8 obligate + 10 possible carriers ) , corresponding to scds in 8% of mutation carriers with familial asd and cd / a and 15% if the possible carriers are included . we identified a single nucleotide deletion at position 112 in exon 1 of nkx25 in one family segregating autosomal dominant chd in three generations ( figure 1 ) . the mutation was not present in 100 danish controls or the exac database of variants in the exome ( http://exac.broadinstitute.org/ ) supporting that 112delg is a rare variant . the deletion causes a shift of the reading frame , leading to a deduced protein with abnormal amino acid sequence from amino acid 37 and a premature stop at amino acid 175 . i:2 ( 37 years old ) had surgical closure of a secundum atrial septal defect ( asd2 ) . ecg showed junctional rhythm with a heart rate of 49 beats per minute ( bpm ) . ii:5 ( 6 years old ) had surgical closure of an asd2 with intermittent 1 . and 2 . iii:1 ( 8 months old ) had complex chd [ double outlet right ventricle , fallot type ( dorvtof ) , coarctation of aorta ( coa ) , persistent left superior vena cava ( plsvc ) , and asd ] . iii:2 had an insignificant muscular ventricular septal defect ( vsd ) and a small asd2 at birth . iii:4 was a healthy carrier of the mutation , but an echocardiogram had never been done by wish of the parents . ( b ) section of the nucleotide sequence of nkx25 gene located on chromosome 5 ( 5q34 ) . the deletion of a single nucleotide at position 112 causes a frameshift , resulting in a truncated protein and a premature stop codon . in the danish family , the deletion segregated with chd and was observed in 5/5 affected individuals , where a blood sample was available , and in one apparently healthy individual , who , however , has not been thoroughly investigated for chd . asd was diagnosed in all six individuals affected with chd ( five live with documented chd , and one deceased with complex chd ) , three also had conduction disease , one a ventricular septal defect ( vsd ) and one individual ( iii:1 ) had asd in complex chd ( figure 1 ) . iii:1 was diagnosed with double outlet right ventricle , fallot type ( dorvtof ) , coarctation of aorta , asd , and persistent left superior vena cava . the patient died eight months old after complicated staged surgery and atrioventricular block or arrhythmia was not observed . fiftynine different nkx25 mutations were reported and confirmed in 202 patients in 31 papers.2 , 18 , 19 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 51 , 52 , 53 , 54 , 55 , 56 thus , including the mutation we identified in six danish individuals , a total of 60 nkx25 mutations have been identified in 208 patients . a total of 198 index patients from chd families ( asd = 117 ; atrioventricular block = 9 ; hlhs = 12 ; tof = 3 , mixed chd = 57 ) have been screened for mutations and in 18 ( 9.1% ) a nkx25 mutation was found cosegregating with the malformation . in comparison 1037 nonfamilial cases ( asd = 137 ; atrioventricular block = 4 ; hlhs = 7 ; tof = 290 ; mixed chd = 599 ) have been screened in which 17 ( 1.6% ) had a nkx25 mutation ( table 1 ) . a fisher 's exact test showed that this difference is significant ( p = 7.1 10 ) . studies by costa et al . , belvis et al . , and xie et al . were excluded ( table 1 ) , because the index patients did not have chd , but cardiomyopathy , stroke and atrial fibrillation , respectively . if these are included , the frequencies are 4.4% and 1.8% for familial and nonfamilial cases , respectively , and the difference is still significant ( p = .0032 ) . studies , in which an actual screening of affected individuals was undertaken and where the text stated the number of screened familial and sporadic cases , was included in the calculations . number of screened familial and sporadic cases published overview of the number of familial / sporadic index cases with chd screened for nkx25 mutations . only the number of index cases screened and the number of index cases ( ) with mutations are displayed . for example , in this study 39 index cases from 39 families were screened and one subject had a mutation . indicate that mutation carriers found had asd ( e.g. , in the study by costa et al . , where index patients had familial dilated cardiomyopathy , or xie et al . , where index patients had familial atrial fibrillation ) . the 208 patients were grouped according to diagnosis and presence / absence of cd / a ( figure 2 ) . patients with several malformations are part of several groups ( e.g. , a patient with asd and vsd were included in the asd and in the vsd column , respectively ) . asd was present in 145 ( 70% ) of the mutation carriers and 112 ( 54% ) also had cd / a . in addition , 17 patients had vsd and cd / a , however , 16 of these also had an asd . none of the three and thirteen patients with hlhs or tof , respectively , had cd / a . lastly , 11 patients had cardiomyopathy ( left ventricular noncompaction , left ventricular hypertrophy , dilated cardiomyopathy ) cooccurring with cd / a in six . the majority of patients with a confirmed mutation in nkx25 has asd with cd / a . asd , atrial septal defect ; tof , tetralogy of fallot ; vsd , ventricular septal defect ; hlhs , hypoplastic left heart syndrome ; cm , cardiomyopathy ( left ventricular noncompaction , dilated cardiomyopathy , left ventricle hypertrophy ) ; scd , sudden cardiac death ( sudden death in otherwise healthy individual before age 50 ) ; other : interrupted aortic arch = 1 ; truncus arteriosus = 1 ; levotransposition of the great arteries = 1 ; coarctation of aorta = 2 ; double outlet right ventricle = 1 ; tricuspid valve anomaly ( atresia , ebstein ) = 4 ; anomalous pulmonary venous return = 1 ; heterotaxy = 1 . in a total of 112 patients with asd and cd / a , 27 mutations were reported in 105 cases of familial chd , whereas only seven mutations were found in cases with nonfamilial chd . there were 19 scds in nine families with asd and cd / a , and cardiomyopathy was also present in four ( 44% ) of these nine families . a mutation ( p.512insglycys ) was documented in only one of these patients , however , additionally eight patients were obligate carriers ( supporting information figures ii the remaining 10 patients were all part of pedigrees with dominant traits of nkx25 mutations cosegregating with the malformations , and they all died suddenly before the age of 50 . assuming that these 10 were carriers of the mutations transmitted in their families , the total number of patients with familial asd with cd / a would be 130 ( 112 + 8 obligate + 10 possible carriers ) , corresponding to scds in 8% of mutation carriers with familial asd and cd / a and 15% if the possible carriers are included . a total of 198 index patients from chd families ( asd = 117 ; atrioventricular block = 9 ; hlhs = 12 ; tof = 3 , mixed chd = 57 ) have been screened for mutations and in 18 ( 9.1% ) a nkx25 mutation was found cosegregating with the malformation . in comparison 1037 nonfamilial cases ( asd = 137 ; atrioventricular block = 4 ; hlhs = 7 ; tof = 290 ; mixed chd = 599 ) have been screened in which 17 ( 1.6% ) had a nkx25 mutation ( table 1 ) . a fisher 's exact test showed that this difference is significant ( p = 7.1 10 ) . studies by costa et al . , belvis et al . , and xie et al . were excluded ( table 1 ) , because the index patients did not have chd , but cardiomyopathy , stroke and atrial fibrillation , respectively . if these are included , the frequencies are 4.4% and 1.8% for familial and nonfamilial cases , respectively , and the difference is still significant ( p = .0032 ) . studies , in which an actual screening of affected individuals was undertaken and where the text stated the number of screened familial and sporadic cases , was included in the calculations . number of screened familial and sporadic cases published overview of the number of familial / sporadic index cases with chd screened for nkx25 mutations . only the number of index cases screened and the number of index cases ( ) with mutations are displayed . for example , in this study 39 index cases from 39 families were screened and one subject had a mutation . indicate that mutation carriers found had asd ( e.g. , in the study by costa et al . , where index patients had familial dilated cardiomyopathy , or xie et al . , where index patients had familial atrial fibrillation ) . the 208 patients were grouped according to diagnosis and presence / absence of cd / a ( figure 2 ) . patients with several malformations are part of several groups ( e.g. , a patient with asd and vsd were included in the asd and in the vsd column , respectively ) . asd was present in 145 ( 70% ) of the mutation carriers and 112 ( 54% ) also had cd / a . in addition , 17 patients had vsd and cd / a , however , 16 of these also had an asd . none of the three and thirteen patients with hlhs or tof , respectively , had cd / a . lastly , 11 patients had cardiomyopathy ( left ventricular noncompaction , left ventricular hypertrophy , dilated cardiomyopathy ) cooccurring with cd / a in six . the majority of patients with a confirmed mutation in nkx25 has asd with cd / a . asd , atrial septal defect ; tof , tetralogy of fallot ; vsd , ventricular septal defect ; hlhs , hypoplastic left heart syndrome ; cm , cardiomyopathy ( left ventricular noncompaction , dilated cardiomyopathy , left ventricle hypertrophy ) ; scd , sudden cardiac death ( sudden death in otherwise healthy individual before age 50 ) ; other : interrupted aortic arch = 1 ; truncus arteriosus = 1 ; levotransposition of the great arteries = 1 ; coarctation of aorta = 2 ; double outlet right ventricle = 1 ; tricuspid valve anomaly ( atresia , ebstein ) = 4 ; anomalous pulmonary venous return = 1 ; heterotaxy = 1 . in a total of 112 patients with asd and cd / a , 27 mutations were reported in 105 cases of familial chd , whereas only seven mutations were found in cases with nonfamilial chd . there were 19 scds in nine families with asd and cd / a , and cardiomyopathy was also present in four ( 44% ) of these nine families . a mutation ( p.512insglycys ) was documented in only one of these patients , however , additionally eight patients were obligate carriers ( supporting information figures ii the remaining 10 patients were all part of pedigrees with dominant traits of nkx25 mutations cosegregating with the malformations , and they all died suddenly before the age of 50 . assuming that these 10 were carriers of the mutations transmitted in their families , the total number of patients with familial asd with cd / a would be 130 ( 112 + 8 obligate + 10 possible carriers ) , corresponding to scds in 8% of mutation carriers with familial asd and cd / a and 15% if the possible carriers are included . there were no sudden deaths reported in the nonfamilial cases . mutations in the nkx25 gene have been reported several times in chd patients , but a review of the phenotypic characteristics of the mutation carriers has not been presented . in this study , we identified a novel nkx25 mutation in a danish family and through a review of the literature , we found , that nkx25 mutations usually occur in familial cases , the signature phenotype is asd with cd / a and there is an alarming number of scds in the mutation carriers and their relatives . in a danish family with autosomal dominant transmission of asd , atrioventricular block and complex heart defect , we identified of a novel mutation in 5/5 affected individuals . the mutation causes a frameshift and is expected to cause haploinsufficiency , due to nonsense mediated mrna decay or production of a truncated version of the protein . the mutation cosegregated with chd in the family , and all but one of the healthy individuals was negative for this mutation . we can not exclude the possibility , that the unaffected carrier ( iii:4 ) had an insignificant asd that closed early , or that she later in life develops an adultonset atrioventricular block . nonpenetrance has been reported for a few mutations in nkx25 of which one ( arg25cys ) recently was suggested not to be causative due to the increasing number of unaffected relatives or controls carrying this mutation.27 , 51 the mutation reported in the present study has never been found in any healthy individuals and we strongly believe this mutation is causative . due to the lack of guidelines in this area , we decided to enrol the danish family in 5 yearly checkups to monitor their heart rhythm . including the danish family , 60 different mutations have been reported in 208 chd patients and 74% of the patients had asd , whereas only three documented cases of hlhs and 13 of tof was reported . we found a highly significant increased frequency of mutations in familial cases compared to nonfamilial cases ( p = 7.1 10 ) . lastly , we found that nine mutation carriers and 10 relatives died suddenly before the age of 50 , two of which had functional pacemakers at time of death . scd in individuals without progressive heart failure and with functional pacemakers can be assumed to be caused by tachyarrhythmias . however , the presence of cardiomyopathy in some of these individuals strongly suggests that this is a disease of the myocardium . we hypothesized , that nkx25 mutations are correlated to asd with cd / a and that the diseaserelated mutations predominantly occur in chd families , rather than in nonfamilial chd , and we confirmed this by review of the existing literature . nkx25 is necessary for cardiac development as well as maintaining proper function of the avnode and myocardium throughout adult life.12 , 47 nkx25 mutations in patients with asd and atrioventricular block has been reported sporadically , however , recent studies have also reported healthy mutation carriers exhibiting runs of nonsustained ventricular tachycardia , ventricular fibrillation , and paroxysmal atrial fibrillation during holtermonitoring or recordings from implantable cardioverterdefibrillators.22 , 35 , 45 during cardiogenesis nkx25 signal the heart to develop from primary slowly conducting into fast conducting working myocardium.48 , 49 however , in the areas of the developing conduction system timed repression of nkx25 is crucial for proper formation of the sinus node , the av node and the peripheral conduction system.49 our study is limited by the retrospective design and should be interpreted with certain precautions . first , we only included studies in which a clear screening procedure was reported , as well as studies in which the number of screened familial / nonfamilial cases was stated clearly . this could have biased our results ; however , when we included three screening studies of patients without chd ( cardiomyopathy , atrial fibrillation , and stroke ) the difference was still significant . second , we have proof of one mutation carrier and eight obligate carriers dying from scd , and the inclusion of the remaining 10 in the cohort as assumed mutation carriers could be considered as speculative . however , they were all part of pedigrees with confirmed mutations segregating with chd , which support our theory , that they were also mutation carriers . with this review , we have established a connection between scd , cardiomyopathy and familial asd , that necessitates clinicians not merely to see patients with familial asd as cured after successful asd repair , but as possible carriers of a mutation associated with increased risk of developing progressive arrhythmias , cardiomyopathy , and scd.23 , 25 , 29 , 41 we found that 44% of families with scd cases had nkx25 mutations combined with cardiomyopathy . due to the small number of reported cases , we can only speculate whether this combination of nkx25 mutation and cardiomyopathy increases the risk of scd . further studies are needed to confirm this theory , but in the meantime , we suggest , that patients with familial asds should be screened for mutations in nkx25 to assess the risk of malignant arrhythmias and sudden deaths . if they are mutation carriers , we suggest that a preventive implantable cardioverterdefibrillator should be considered in these patients , especially if there is also a family history of cardiomyopathy . all authors contributed to design of the study , data analysis as well as publication review . additional supporting information may be found in the online version of this article at the publisher 's website : table i. diagnoses of danish probands screened for nkx25 mutations and their relatives . ( 1999),26 ouyang et al . ( 2011),23 and perera et al . ( 2014).22 figure iv .

abstractobjectiveatrial septal defect ( asd ) is the second most common congenital heart defect ( chd ) and is observed in families as an autosomal dominant trait as well as in nonfamilial chd . mutations in the nkx25 gene , located on chromosome 5 , are associated with asd , often combined with conduction disturbances , cardiomyopathies , complex chd , and sudden cardiac death as well . here , we show that nkx25 mutations primarily occur in asd patients with conduction disturbances and heritable asd . furthermore , these families are at increased risk of sudden cardiac death.resultswe screened 39 probands with familial chd for mutations in nkx25 and discovered a novel mutation in one family ( 2.5% ) with asd and atrioventricular block . a review of the literature revealed 59 different nkx25 mutations in 202 patients . mutations were significantly more common in familial cases compared to nonfamilial cases ( p = 7.1 109 ) . the majority of patients ( 74% ) had asd with conduction disturbance . nineteen patients ( 15% ) of 120 with familial asd and conduction disturbance died from sudden cardiac death of which nine ( 8% ) were confirmed mutation carriers , and 10 were possible carriers.conclusions nkx25 mutations mainly occur in familial chd , the signature phenotype is asd with conduction disturbances and mutation carriers are at increased risk of sudden cardiac death . we suggest that familial asd patients should be screened for nkx25 mutations and , if they are mutation carriers , implantation of an implantable cardioverterdefibrillator should be considered in these patients .

Background Methods Results NKX25 Mutation in a Family with ASD, Complex Malformation, and Atrioventricular Block Review of Published NKX25 Mutations The Signature Phenotype of The Majority (94%) of Mutation Carriers with ASD and CD/A are Familial Cases SCD Occurred in 15% of Patients with Familial ASD and CD/A Discussion Author Contributions Supporting information

atrial septal defect ( asd ) is the second most common congenital heart defect ( chd ) and accounts for approximately 10% of all cardiac malformations.1 , 2 eighty percent of persistent foramen ovale and small asds close spontaneously during infancy or childhood , whereas large asds or those remaining open into adulthood may cause congestive heart failure , pneumonia , pulmonary vascular disease , atrial arrhythmias , and paradoxical embolism.3 , 4 , 5 , 6 , 7 also , cooccurrence with other cardiac malformations within the same individual is often observed.8 asd is correlated to mutations in the nkx25 gene , located on chromosome 5 ( 5q34).9 , 10 , 11 nkx25 is a cardiac transcription factor that plays a significant role in development of the atrioventricular node as well as maintaining function of the node throughout life.12 in recent years , nkx25 mutations have been reported in chd patients with nonfamilial as well as familial chd . familial atrioventricular block , observed as congenital or adultonset type , cooccur with asd.13 , 14 , 15 besides asd,9 , 10 , 11 congenital complete atrioventricular block cooccur with laterality defects such as levotransposition of the great arteries ( ltga ) or atrial isomerism.16 , 17 , 18 as opposed to the adultonset type , congenital complete atrioventricular block is diagnosed in utero or shortly after birth , and it is associated with mortality rates ranging from 33% to 80% if the heart rate is below 50 or it cooccurs with structural heart disease.17 , 18 conversely , the adultonset type of familial atrioventricular block is of a progressive nature , and there are several reports of patients with normal ecg or a harmless firstdegree atrioventricular block followed by sudden onset of second and thirddegree atrioventricular block or sudden death later in life.14 , 15 , 19 sudden cardiac death ( scd ) occur in patients with both types of atrioventricular block15 , 16 , 20 , 21 and in patients with nkx25 mutations.22 , 23 scd have been reported in pediatric as well as adult cases of atrioventricular block,15 , 16 , 20 , 21 and autopsy studies have shown fibrotic replacement of the avbundle,24 which explains the atrioventricular node malfunctioning in these patients . however , there has been an alarming number of scds in obligate carriers and relatives of patients with nkx25 mutations.22 , 23 , 25 , 26 this , and previous reports of patients with asd and/or atrioventricular block dying suddenly with a functioning pacemaker , suggest that the myocardium is also involved in the nkx25 phenotype.15 , 16 despite the large efforts in finding nkx25 mutations in chd patients , there have been no reviews of the existing literature to determine the frequency of the mutation or characterization of the phenotypic appearance of mutation carriers . we hypothesized , that mutations in nkx25 primarily occur in familial chd , and that the signature phenotype is asd with or without conduction disease or arrhythmia ( cd / a ) . by reviewing the literature , we show that the majority of nkx25 mutation carriers are patients with familial asd and conduction disturbances , and we report an alarming large number of scds in such families . in this study , we identified a novel nkx25 mutation in a danish family and through a review of the literature , we found , that nkx25 mutations usually occur in familial cases , the signature phenotype is asd with cd / a and there is an alarming number of scds in the mutation carriers and their relatives . nkx25 is necessary for cardiac development as well as maintaining proper function of the avnode and myocardium throughout adult life.12 , 47 nkx25 mutations in patients with asd and atrioventricular block has been reported sporadically , however , recent studies have also reported healthy mutation carriers exhibiting runs of nonsustained ventricular tachycardia , ventricular fibrillation , and paroxysmal atrial fibrillation during holtermonitoring or recordings from implantable cardioverterdefibrillators.22 , 35 , 45 during cardiogenesis nkx25 signal the heart to develop from primary slowly conducting into fast conducting working myocardium.48 , 49 however , in the areas of the developing conduction system timed repression of nkx25 is crucial for proper formation of the sinus node , the av node and the peripheral conduction system.49 our study is limited by the retrospective design and should be interpreted with certain precautions . further studies are needed to confirm this theory , but in the meantime , we suggest , that patients with familial asds should be screened for mutations in nkx25 to assess the risk of malignant arrhythmias and sudden deaths . if they are mutation carriers , we suggest that a preventive implantable cardioverterdefibrillator should be considered in these patients , especially if there is also a family history of cardiomyopathy .

data were derived from a cohort ( n = 4,933 ) of diabetic patients participating in the diabetes care system west - friesland ( dcs ) , the netherlands , in the period from 1998 to 2005 . the dcs coordinates regional diabetic care and provides additional care to diabetic care given by general practitioners . , this system provides for annual physical examinations , with assessment of glucose control , cardiovascular risk profile , and complications of diabetes , including fundus photography , and supports self - management . patient data of this annual visit and therapeutic advice are provided to the general practitioner of the patient to implement . in the study period between 1998 and 2005 , patients entered the dcs in different years and with different diabetic duration . for each patient , the year of entry was considered baseline measurement ( t0 ) . thus , patients who entered in the year 1998 could provide for up to eight visits . however , because only 349 of the 3,343 included patients had data at t7 , we excluded the t7 visit and used data from t0 to t6 in the analysis . for the present analysis of the developmental patterns of dr , we excluded patients with type 1 diabetes ( n = 49 ) and patients with only one or no visit with graded fundus photographs ( n = 1,541 ) . although statistically significant , small differences were seen between included ( n = 3,343 ) and excluded ( n = 1,590 ) patients at t0 ; the excluded patients were 5.6 years older , included 6.2% more women , had a 1.8-year longer duration of diabetes , entered the dcs 1.7 years later , had a 2 mmhg lower diastolic blood pressure , had a 4 mmhg higher systolic blood pressure , had a 0.6 lower bmi , had a slightly more favorable lipid profile , and had 10% more insulin use than patients who were included in the study . no significant differences were observed in fasting plasma glucose ( fpg ) , hba1c , retinopathy prevalence , and smoking behavior . systolic and diastolic blood pressures were measured on the right arm after 5 min of rest in a seated position using a random - zero sphygmomanometer ( hawksley - gelman , lancing , sussex , uk ) ; from 2003 on , an oscillometric device was used ( colin press - mate bp-8800 , komaki city , japan ) . fpg was measured by means of a hexokinase method ( roche diagnostics gmbh , mannheim , germany ) . levels of total cholesterol , hdl cholesterol , and triglycerides were measured using enzymatic techniques ( boehringer - mannheim , mannheim , germany ) . urinary albumin - creatinine ratio in milligrams per millimole was determined in an overnight first - voided urine sample . urinary albumin was measured by rate nephelometry ( array protein system ; beckman coulter , fullerton , ca ) , with an assay threshold of 2 mg / l . information on current medication use , smoking ( yes / no ) , and year of onset of diabetes was obtained by self - report . until 2000 , fundus photography of both eyes was performed with a kowa pro fundus camera with green filter ( kowa optical industry , torrance , ca ) . black and white 35-mm photographs were taken 30 min after mydriasis with 0.5% tropicamide and 2.5% phenylephrine eye drops . from the beginning of 2000 to 2004 , fundus photography of both eyes was performed with a nonmydriatic canon cr5 camera ( canon inc . , tokyo , japan ) . in 2004 , fundus photography of both eyes was performed with a nonmydriatic topcon trc nw 100 camera ( topcon , tokyo , japan ) . all fundus photographs were 45 degrees of two fields : one field centered on the macula and one nasal field with the optic disc positioned on a disc - diameter from the temporal edge of the field , according to the eurodiab protocol ( 14 ) . if fundus photography at the dcs had failed or was missing , data on dr were retrieved from the patient s medical file at the local hospital . all photographs were graded by an ophthalmologist according to the eurodiab study grading system , in which grade 0 is no retinopathy , grade 1 is minimal nonproliferative retinopathy , grade 2 is moderate nonproliferative retinopathy , grade 3 is severe nonproliferative or preproliferative retinopathy , grade 4 is photocoagulated retinopathy , and grade 5 is proliferative retinopathy . the eurodiab grading does not provide for more detailed distinctions of specific diabetic changes in the retina , such as macular edema . in the dcs , the eurodiab grades 4 and 5 have been combined in one grade , grade 4 . in case of photocoagulation at the local hospital , any existing grade in the data of the dcs was changed to eurodiab grade 4 at all time points after this therapy . if results of the grading of both eyes were available , the grading of the worst eye was used . the year of entry in the dcs of each individual was considered as the baseline measurement ( t0 ) . the prevalence of dr per grade was calculated at baseline , as well as the prevalence of any dr ( grades 14 ) at t0 to t6 . further analyses were conducted in two parts . in part one , latent class growth modeling ( lcgm ) was used to identify distinct developmental patterns of dr ( 15 ) . lcgm is a relatively new statistical technique used to analyze heterogeneity in the time course of , for example , dr . the underlying aim of the technique is to model this heterogeneity by identifying k number of distinct developmental patterns of dr called clusters . individuals are allocated to one cluster on the basis of a similar pattern of development of retinopathy at all measurements . the allocation is done only once for each individual . to determine the optimal number of clusters , a forward approach was taken starting with a model with one developmental pattern , implying that all individuals in the study had the same course of dr . subsequently , one cluster at a time was added , and the model fit was assessed by the bayesian information criterion ( bic ) , which is often used when conducting such analyses ( 16 ) . after each step of adding a new cluster , the model fit was considered better if the bic decreased . the final number of clusters was derived on the basis of the forward approach assessed by a low bic score and clinically relevant differences between clusters . if addition of a new cluster led to a model with two clinically indistinguishable developmental clusters , the model was not considered to be improved . individuals within each of the clusters have a probability of having each of the five eurodiab grades of retinopathy . identification of the retinopathy grade with the highest probability at each time point was used to derive a descriptive name of each cluster . the lcgm analyses were conducted with mplus 5.21 . in part two of our analyses , differences between clusters in baseline characteristics ( mean [ sd ] or proportion ) were tested with an anova with post hoc bonferroni or tests . baseline characteristics with a skewed distribution were calculated as median ( interquartile range ) within each cluster , and differences between clusters were analyzed by kruskal trends in mean levels of hba1c , systolic blood pressure , and total cholesterol were graphically compared between the clusters during follow - up ( spss 15.0.2 , spss inc . , data were derived from a cohort ( n = 4,933 ) of diabetic patients participating in the diabetes care system west - friesland ( dcs ) , the netherlands , in the period from 1998 to 2005 . the dcs coordinates regional diabetic care and provides additional care to diabetic care given by general practitioners . , this system provides for annual physical examinations , with assessment of glucose control , cardiovascular risk profile , and complications of diabetes , including fundus photography , and supports self - management . patient data of this annual visit and therapeutic advice are provided to the general practitioner of the patient to implement . in the study period between 1998 and 2005 , patients entered the dcs in different years and with different diabetic duration . for each patient , the year of entry was considered baseline measurement ( t0 ) . thus , patients who entered in the year 1998 could provide for up to eight visits . however , because only 349 of the 3,343 included patients had data at t7 , we excluded the t7 visit and used data from t0 to t6 in the analysis . for the present analysis of the developmental patterns of dr , we excluded patients with type 1 diabetes ( n = 49 ) and patients with only one or no visit with graded fundus photographs ( n = 1,541 ) . although statistically significant , small differences were seen between included ( n = 3,343 ) and excluded ( n = 1,590 ) patients at t0 ; the excluded patients were 5.6 years older , included 6.2% more women , had a 1.8-year longer duration of diabetes , entered the dcs 1.7 years later , had a 2 mmhg lower diastolic blood pressure , had a 4 mmhg higher systolic blood pressure , had a 0.6 lower bmi , had a slightly more favorable lipid profile , and had 10% more insulin use than patients who were included in the study . no significant differences were observed in fasting plasma glucose ( fpg ) , hba1c , retinopathy prevalence , and smoking behavior . systolic and diastolic blood pressures were measured on the right arm after 5 min of rest in a seated position using a random - zero sphygmomanometer ( hawksley - gelman , lancing , sussex , uk ) ; from 2003 on , an oscillometric device was used ( colin press - mate bp-8800 , komaki city , japan ) . fpg was measured by means of a hexokinase method ( roche diagnostics gmbh , mannheim , germany ) . levels of total cholesterol , hdl cholesterol , and triglycerides were measured using enzymatic techniques ( boehringer - mannheim , mannheim , germany ) . urinary albumin - creatinine ratio in milligrams per millimole was determined in an overnight first - voided urine sample . urinary albumin was measured by rate nephelometry ( array protein system ; beckman coulter , fullerton , ca ) , with an assay threshold of 2 information on current medication use , smoking ( yes / no ) , and year of onset of diabetes was obtained by self - report . until 2000 , fundus photography of both eyes was performed with a kowa pro fundus camera with green filter ( kowa optical industry , torrance , ca ) . black and white 35-mm photographs were taken 30 min after mydriasis with 0.5% tropicamide and 2.5% phenylephrine eye drops . from the beginning of 2000 to 2004 , fundus photography of both eyes was performed with a nonmydriatic canon cr5 camera ( canon inc . , tokyo , japan ) . in 2004 , fundus photography of both eyes was performed with a nonmydriatic topcon trc nw 100 camera ( topcon , tokyo , japan ) . all fundus photographs were 45 degrees of two fields : one field centered on the macula and one nasal field with the optic disc positioned on a disc - diameter from the temporal edge of the field , according to the eurodiab protocol ( 14 ) . if fundus photography at the dcs had failed or was missing , data on dr were retrieved from the patient s medical file at the local hospital . all photographs were graded by an ophthalmologist according to the eurodiab study grading system , in which grade 0 is no retinopathy , grade 1 is minimal nonproliferative retinopathy , grade 2 is moderate nonproliferative retinopathy , grade 3 is severe nonproliferative or preproliferative retinopathy , grade 4 is photocoagulated retinopathy , and grade 5 is proliferative retinopathy . the eurodiab grading does not provide for more detailed distinctions of specific diabetic changes in the retina , such as macular edema . in the dcs , the eurodiab grades 4 and 5 have been combined in one grade , grade 4 . in case of photocoagulation at the local hospital , any existing grade in the data of the dcs was changed to eurodiab grade 4 at all time points after this therapy . if results of the grading of both eyes were available , the grading of the worst eye was used . the year of entry in the dcs of each individual was considered as the baseline measurement ( t0 ) . the prevalence of dr per grade was calculated at baseline , as well as the prevalence of any dr ( grades 14 ) at t0 to t6 . further analyses were conducted in two parts . in part one , latent class growth modeling ( lcgm ) was used to identify distinct developmental patterns of dr ( 15 ) . lcgm is a relatively new statistical technique used to analyze heterogeneity in the time course of , for example , dr . the underlying aim of the technique is to model this heterogeneity by identifying k number of distinct developmental patterns of dr called clusters . individuals are allocated to one cluster on the basis of a similar pattern of development of retinopathy at all measurements . the allocation is done only once for each individual . to determine the optimal number of clusters , a forward approach was taken starting with a model with one developmental pattern , implying that all individuals in the study had the same course of dr . subsequently , one cluster at a time was added , and the model fit was assessed by the bayesian information criterion ( bic ) , which is often used when conducting such analyses ( 16 ) . after each step of adding a new cluster , the model fit was considered better if the bic decreased . the final number of clusters was derived on the basis of the forward approach assessed by a low bic score and clinically relevant differences between clusters . if addition of a new cluster led to a model with two clinically indistinguishable developmental clusters , the model was not considered to be improved . individuals within each of the clusters have a probability of having each of the five eurodiab grades of retinopathy . identification of the retinopathy grade with the highest probability at each time point was used to derive a descriptive name of each cluster . the lcgm analyses were conducted with mplus 5.21 . in part two of our analyses , differences between clusters in baseline characteristics ( mean [ sd ] or proportion ) were tested with an anova with post hoc bonferroni or tests . baseline characteristics with a skewed distribution were calculated as median ( interquartile range ) within each cluster , and differences between clusters were analyzed by kruskal trends in mean levels of hba1c , systolic blood pressure , and total cholesterol were graphically compared between the clusters during follow - up ( spss 15.0.2 , spss inc . , at baseline , our population with a mean age of 61.0 years ( sd 11.3 ) , 52.7% of whom were male , and a mean age at diagnosis of diabetes of 56.9 years ( sd 11.5 years ) ( table 1 ) had a prevalence of the separate eurodiab grades of 87.4% for grade 0 , 9.1% for grade 1 , 2.0% for grade 2 , 1.2% for grade 3 , and 0.4% for grade 4 . the prevalence of any dr ( grades 14 ) was 12.7% at baseline , 14.3% at t1 , 15.1% at t2 , 14.6% at t3 , 18.7% at t4 , 14.6% at t5 , and 16.9% at t6 ( p value for trend = 0.001 ) . differences in baseline characteristics between clusters of distinct developmental patterns of dr in the dcs population , the netherlands data represent mean sd , proportions , or median ( interquartile range ) . between - cluster differences were tested with anova and post hoc bonferroni for mean values , with tests for proportions and kruskal wallis test for median values . npdr , nonproliferative dr ; pdr , proliferative dr ; ns , nonsignificant ; oad , oral antidiabetic . on the basis of the lower bayes information criterion of a model with five clusters compared with a model with six clusters , the optimal number of clusters with distinct developmental patterns of dr was five ( clusters a this optimal number was also confirmed by clinical interpretation of the patterns of development of dr , which were distinct in the five cluster model . a model with six clusters persistent no retinopathy . within cluster a , the probability for patients having no retinopathy ( grade 0 ) was 0.92 or more at each of the 7 time points . within cluster b , the highest probability for these patients was 0.81 of having either grade 0 or grade 1 retinopathy at t0 , with a higher probability of having grade 1 ( 0.56 ) than grade 0 ( 0.25 ) . not only did the probability of having either grade 0 or 1 gradually increase over time to 0.99 at t6 , but after t2 , the probability of having grade 0 started being higher than the probability of having grade 1 . slow progression from minimal to moderate retinopathy , including patients with a slow and gradual progression . within cluster c , this probability decreased gradually over time , and after t4 the probability of having a higher grade ( grade 2 or 3 ) had increased to 0.22 , which further increased to 0.35 at t6 . another cluster of progression was cluster d ( n = 47 , 1.4% ) , characterized as fast progression from minimal or moderate to ( pre)proliferative or treated retinopathy . after 1 year the probability of having retinopathy grade 0 , 1 , or 2 already decreased for these patients from 0.81 to 0.62 , with a further decrease through t2 and t3 . after t4 , the probability of having retinopathy grade 3 or 4 was 0.91 or more in cluster d. the final cluster e ( n = 25 , 0.8% ) was characterized as the probability of having retinopathy grade 3 or 4 for these patients was 0.78 at t0 , which even increased after 1 year to 0.95 . pdr , proliferative dr . between cluster differences of baseline characteristics can be seen in table 1 . at t0 , patients in cluster a with persistent no retinopathy had a statistically significantly lower diabetes duration than patients in the other clusters . mean hba1c levels were lower in cluster a patients with at least 0.8% compared with patients in other clusters except for patients in cluster b slow regression was not statistically different from the hba1c of patients in cluster c slow progression from minimal to moderate retinopathy , but the hba1c in patients of cluster b was 1.3% lower than in patients of clusters d and e. mean fpg was lower in patients of both clusters a and b compared with patients in clusters c , d , and e. in cluster a , 67% of the patients used oral antidiabetic medication , which was higher than in the fast progressing cluster d but lower than in all other clusters . insulin use was highest in the progressive clusters c , d , and e , and lowest in cluster a. combination therapy of insulin and oral antidiabetic medication was also significantly lower in cluster a compared with all other clusters . persistent no retinopathy compared with patients in cluster c slow progression from minimal to moderate retinopathy at baseline . in both clusters a and c , a similar proportion of patients used antihypertensive medication ( 47.7 vs. 45.3% ) at baseline . hba1c , systolic blood pressure , and total cholesterol are shown per cluster as means at each time point in fig . patients in clusters a and b , the nonprogressive clusters , had lower mean values of hba1c and total cholesterol than patients in the progressive clusters c , d , and e over time . an increase over time for all clusters was seen in systolic blood pressure . for patients in cluster c , who started to show an increase in retinopathy severity after t4 , an increase of systolic blood pressure with 15 mmhg was seen between t3 and t5 . patients in cluster d , with an increase in retinopathy severity after t1 , showed an increase in systolic blood pressure of 10 mmhg after t1 . systolic blood pressure also increased in clusters a and b , but this increase never exceeded 5 mmhg in 1 year . mean hba1c ( a ) , systolic blood pressure ( b ) , and total cholesterol ( c ) by clusters of the development of dr . at baseline , our population with a mean age of 61.0 years ( sd 11.3 ) , 52.7% of whom were male , and a mean age at diagnosis of diabetes of 56.9 years ( sd 11.5 years ) ( table 1 ) had a prevalence of the separate eurodiab grades of 87.4% for grade 0 , 9.1% for grade 1 , 2.0% for grade 2 , 1.2% for grade 3 , and 0.4% for grade 4 . the prevalence of any dr ( grades 14 ) was 12.7% at baseline , 14.3% at t1 , 15.1% at t2 , 14.6% at t3 , 18.7% at t4 , 14.6% at t5 , and 16.9% at t6 ( p value for trend = 0.001 ) . differences in baseline characteristics between clusters of distinct developmental patterns of dr in the dcs population , the netherlands data represent mean sd , proportions , or median ( interquartile range ) . between - cluster differences were tested with anova and post hoc bonferroni for mean values , with tests for proportions and kruskal wallis test for median values . npdr , nonproliferative dr ; pdr , proliferative dr ; ns , nonsignificant ; oad , oral antidiabetic . on the basis of the lower bayes information criterion of a model with five clusters compared with a model with six clusters , the optimal number of clusters with distinct developmental patterns of dr was five ( clusters a this optimal number was also confirmed by clinical interpretation of the patterns of development of dr , which were distinct in the five cluster model . a model with six clusters 1 . the largest cluster a ( n = 2,971 , 88.9% ) was characterized by persistent no retinopathy . within cluster a , the probability for patients having no retinopathy ( grade 0 ) was 0.92 or more at each of the 7 time points . within cluster b , the highest probability for these patients was 0.81 of having either grade 0 or grade 1 retinopathy at t0 , with a higher probability of having grade 1 ( 0.56 ) than grade 0 ( 0.25 ) . not only did the probability of having either grade 0 or 1 gradually increase over time to 0.99 at t6 , but after t2 , the probability of having grade 0 started being higher than the probability of having grade 1 . slow progression from minimal to moderate retinopathy , including patients with a slow and gradual progression . within cluster c , patients had a probability of 0.92 of having grade 0 or 1 at t0 . this probability decreased gradually over time , and after t4 the probability of having a higher grade ( grade 2 or 3 ) had increased to 0.22 , which further increased to 0.35 at t6 . another cluster of progression was cluster d ( n = 47 , 1.4% ) , characterized as fast progression from minimal or moderate to ( pre)proliferative or treated retinopathy . after 1 year the probability of having retinopathy grade 0 , 1 , or 2 already decreased for these patients from 0.81 to 0.62 , with a further decrease through t2 and t3 . after t4 , the probability of having retinopathy grade 3 or 4 was 0.91 or more in cluster d. the final cluster e ( n = 25 , 0.8% ) was characterized as the probability of having retinopathy grade 3 or 4 for these patients was 0.78 at t0 , which even increased after 1 year to 0.95 . , cluster b slow regression . , cluster c slow progression from minimal to moderate nonproliferative dr . between cluster differences of baseline characteristics can be seen in table 1 . at t0 , patients in cluster a with persistent no retinopathy had a statistically significantly lower diabetes duration than patients in the other clusters . mean hba1c levels were lower in cluster a patients with at least 0.8% compared with patients in other clusters except for patients in cluster b slow regression . slow regression was not statistically different from the hba1c of patients in cluster c slow progression from minimal to moderate retinopathy , but the hba1c in patients of cluster b was 1.3% lower than in patients of clusters d and e. mean fpg was lower in patients of both clusters a and b compared with patients in clusters c , d , and e. in cluster a , 67% of the patients used oral antidiabetic medication , which was higher than in the fast progressing cluster d but lower than in all other clusters . insulin use was highest in the progressive clusters c , d , and e , and lowest in cluster a. combination therapy of insulin and oral antidiabetic medication was also significantly lower in cluster a compared with all other clusters . an 8 mmhg lower systolic blood pressure was seen in patients in cluster a persistent no retinopathy compared with patients in cluster c slow progression from minimal to moderate retinopathy at baseline . in both clusters a and c , a similar proportion of patients used antihypertensive medication ( 47.7 vs. 45.3% ) at baseline . hba1c , systolic blood pressure , and total cholesterol are shown per cluster as means at each time point in fig . patients in clusters a and b , the nonprogressive clusters , had lower mean values of hba1c and total cholesterol than patients in the progressive clusters c , d , and e over time . for patients in cluster c , who started to show an increase in retinopathy severity after t4 , an increase of systolic blood pressure with 15 mmhg was seen between t3 and t5 . patients in cluster d , with an increase in retinopathy severity after t1 , showed an increase in systolic blood pressure of 10 mmhg after t1 . systolic blood pressure also increased in clusters a and b , but this increase never exceeded 5 mmhg in 1 year . mean hba1c ( a ) , systolic blood pressure ( b ) , and total cholesterol ( c ) by clusters of the development of dr . in this study on the course of dr , we identified five clusters of developmental patterns of dr in patients with t2 dm and assessed the risk factor levels of these clusters . the largest cluster ( a ) represents diabetic patients who did not develop any dr over a time period of 6 years . one cluster showing regression ( b ) represented patients who had a high probability of having minimal dr , which disappeared over time . two progressive clusters ( c and d ) were identified that differed from each other in severity and in speed of developing dr . the smallest cluster ( e ) consisted of patients who had persistently ( pre)proliferative dr or photocoagulation scars . thus , in a large cohort of patients with t2 dm , the majority of patients did not develop dr . of the 372 patients in clusters b , c , d , and e who did develop dr , more than 44% ( n = 165 ) showed regression in cluster b. the two progressive clusters c and d had persistently higher levels of risk factors than clusters a and b , with no or little retinopathy . steep increases in systolic blood pressure in these progressive clusters were observed , which seemed to coincide with the point in time at which progression of dr occurred . to our knowledge these analyses were performed in a large population of patients with t2 dm , representative of 85% of the diabetic patients in the region with a maximum follow - up of 6 years . we accurately measured important risk factors within a standardized care system and at yearly time intervals . we used the eurodiab grading system to assess dr , a grading system recommended for large epidemiologic studies ( 14 ) . however , a limitation of our study is the use of one combined grade for eurodiab grades 4 and 5 ; thus , no distinction can be made between progression to proliferative dr ( pdr ) or treatment of dr with photocoagulation . furthermore , because the eurodiab was not designed to assess maculopathy , this study does not provide details on different causes of progression . nonetheless , to obtain an impression of clinically relevant progression of dr , the use of the eurodiab grading with 4/5 combined without more detailed grading seems to be sufficient . the current study was performed in a diabetic population of mainly caucasian origin , with a mean age of 61.0 years . therefore , these results may not be representative of a younger or non - caucasian population . furthermore , the study population consisted of patients with t2 dm with a variety of diabetic duration and relatively well treated hyperglycemia . however , the prevalence of an hba1c > 8.0% at some point between t0 and t6 was 47% , and the prevalence of an hba1c > 10.0% was 17% . nonetheless , less heterogeneous clusters or different clusters of developmental patterns might be found if analyses would be repeated in different populations , e.g. , with type 1 diabetes or new - onset t2 dm , with a longer follow - up than the time frame of 6 years in our study or with higher levels of blood glucose . several important longitudinal studies , which have studied risk factors influencing the onset or the progression of dr , differ from the current study . first , the aim of these studies was to describe the associations of onset or progression of dr with risk factors , but none of these studies were designed to study the developmental pattern of dr based on yearly measurements . second , some of these studies used more than two follow - up measures ( 10,17 ) , but most had only retinopathy data on baseline and one follow - up measurement ( 11,18 ) , and thus the outcome was incidence of new - onset dr or incidence of a certain degree of progression of dr . this hampers a sound analysis of progression and regression , because the possibility of regression of dr was not taken in account . we show that regression does occur in a small subgroup of patients , cluster b. although small , this cluster is the second largest cluster after cluster a with persistent no retinopathy . previous cross - sectional studies on dr prevalence have also shown that most diabetic patients have no dr or mild background dr . we also found that the majority of diabetic patients ( 88.9% ) do not develop dr in a 6-year period . this might have consequences for advice on differential possible screening intervals in selected patients . in the current study , clusters with progressive or stable high levels of dr have high levels of some risk factors compared with nonprogressive clusters . these results are in line with the aforementioned cohort studies , such as the uk prospective diabetes study , eurodiab , and wisconsin epidemiological study of diabetic retinopathy , which showed associations of incidence and progression of dr among others with hba1c , blood pressure , and diabetic duration . the steep increases of systolic blood pressure in the progressive clusters , coinciding with the time at which retinopathy progressed , suggest that progression of dr is influenced to a large extent by blood pressure and diabetes duration . nonetheless , these clusters also have high levels of blood glucose and other risk factors . thus , future research should focus on the influence of risk factors on both progression and regression of dr . in summary , we have described five clusters of distinct developmental patterns of dr that enable a more detailed examination of the influence of various risk factors on the course of retinopathy . the majority of patients with t2 dm in this population did not or only minimally developed retinopathy over a period of 6 years . these patients showed lower risk factor levels of diabetic duration , hba1c , fpg , and systolic blood pressure compared with patients in the two other small clusters of diabetic patients who showed progression of dr . patients in these two progressive clusters are at high risk of persistent pdr and are therefore in need of strict and regular screening . further research is needed to investigate how to distinguish patients in progressive developmental clusters from patients with no or minimal background dr .

objectiveto identify distinct developmental patterns of diabetic retinopathy ( dr ) and assess the risk factor levels of patients in these clusters.research design and methodsa cohort of 3,343 patients with type 2 diabetes mellitus ( t2 dm ) monitored and treated in the diabetes care system west - friesland , the netherlands , was followed from 2 to 6 years . risk factors were measured , and two - field fundus photographs were taken annually and graded according to the eurodiab study group . latent class growth modeling was used to identify distinct developmental patterns of dr over time.resultsfive clusters of patients with distinct developmental patterns of dr were identified : a , patients without any signs of dr ( 88.9% ) ; b , patients with a slow regression from minimal background to no dr ( 4.9% ) ; c , patients with a slow progression from minimal background to moderate nonproliferative dr ( 4.0% ) ; d , patients with a fast progression from minimal or moderate nonproliferative to ( pre)proliferative or treated dr ( 1.4% ) ; and e , patients with persistent proliferative dr ( 0.8% ) . patients in clusters a and b were characterized by lower risk factor levels , such as diabetes duration , hba1c , and systolic blood pressure compared with patients in progressive clusters ( c e).conclusionsclusters of patients with t2 dm with markedly different patterns of dr development were identified , including a cluster with regression of dr . these clusters enable a more detailed examination of the influence of various risk factors on dr .

RESEARCH DESIGN AND METHODS Study population Measurements Retinopathy Statistical analysis RESULTS Prevalence of retinopathy stages Course of retinopathy Characteristics CONCLUSIONS Supplementary Material

data were derived from a cohort ( n = 4,933 ) of diabetic patients participating in the diabetes care system west - friesland ( dcs ) , the netherlands , in the period from 1998 to 2005 . in part one , latent class growth modeling ( lcgm ) was used to identify distinct developmental patterns of dr ( 15 ) . , data were derived from a cohort ( n = 4,933 ) of diabetic patients participating in the diabetes care system west - friesland ( dcs ) , the netherlands , in the period from 1998 to 2005 . for the present analysis of the developmental patterns of dr , we excluded patients with type 1 diabetes ( n = 49 ) and patients with only one or no visit with graded fundus photographs ( n = 1,541 ) . in part one , latent class growth modeling ( lcgm ) was used to identify distinct developmental patterns of dr ( 15 ) . on the basis of the lower bayes information criterion of a model with five clusters compared with a model with six clusters , the optimal number of clusters with distinct developmental patterns of dr was five ( clusters a this optimal number was also confirmed by clinical interpretation of the patterns of development of dr , which were distinct in the five cluster model . slow progression from minimal to moderate retinopathy , including patients with a slow and gradual progression . another cluster of progression was cluster d ( n = 47 , 1.4% ) , characterized as fast progression from minimal or moderate to ( pre)proliferative or treated retinopathy . mean hba1c levels were lower in cluster a patients with at least 0.8% compared with patients in other clusters except for patients in cluster b slow regression was not statistically different from the hba1c of patients in cluster c slow progression from minimal to moderate retinopathy , but the hba1c in patients of cluster b was 1.3% lower than in patients of clusters d and e. mean fpg was lower in patients of both clusters a and b compared with patients in clusters c , d , and e. in cluster a , 67% of the patients used oral antidiabetic medication , which was higher than in the fast progressing cluster d but lower than in all other clusters . patients in clusters a and b , the nonprogressive clusters , had lower mean values of hba1c and total cholesterol than patients in the progressive clusters c , d , and e over time . on the basis of the lower bayes information criterion of a model with five clusters compared with a model with six clusters , the optimal number of clusters with distinct developmental patterns of dr was five ( clusters a this optimal number was also confirmed by clinical interpretation of the patterns of development of dr , which were distinct in the five cluster model . another cluster of progression was cluster d ( n = 47 , 1.4% ) , characterized as fast progression from minimal or moderate to ( pre)proliferative or treated retinopathy . slow regression was not statistically different from the hba1c of patients in cluster c slow progression from minimal to moderate retinopathy , but the hba1c in patients of cluster b was 1.3% lower than in patients of clusters d and e. mean fpg was lower in patients of both clusters a and b compared with patients in clusters c , d , and e. in cluster a , 67% of the patients used oral antidiabetic medication , which was higher than in the fast progressing cluster d but lower than in all other clusters . an 8 mmhg lower systolic blood pressure was seen in patients in cluster a persistent no retinopathy compared with patients in cluster c slow progression from minimal to moderate retinopathy at baseline . patients in clusters a and b , the nonprogressive clusters , had lower mean values of hba1c and total cholesterol than patients in the progressive clusters c , d , and e over time . in this study on the course of dr , we identified five clusters of developmental patterns of dr in patients with t2 dm and assessed the risk factor levels of these clusters . of the 372 patients in clusters b , c , d , and e who did develop dr , more than 44% ( n = 165 ) showed regression in cluster b. the two progressive clusters c and d had persistently higher levels of risk factors than clusters a and b , with no or little retinopathy . to our knowledge these analyses were performed in a large population of patients with t2 dm , representative of 85% of the diabetic patients in the region with a maximum follow - up of 6 years . in summary , we have described five clusters of distinct developmental patterns of dr that enable a more detailed examination of the influence of various risk factors on the course of retinopathy . these patients showed lower risk factor levels of diabetic duration , hba1c , fpg , and systolic blood pressure compared with patients in the two other small clusters of diabetic patients who showed progression of dr .

reactive oxygen and nitrogen species ( rons ) is an umbrella term that encompasses oxygen radicals , nitrogen radicals , and their nonradical derivatives.1 although rons can perform useful functions such as initiating cellular signaling and defending against harmful agents , an overexpression of this species can cause damage to lipids , protein , dna , and other molecules.2 to prevent this , organisms are equipped with antioxidant defense systems that scavenge rons . however , the balance between rons expression and antioxidant scavenging can become disturbed if rons production is increased and/or the antioxidant defense system is weakened.3 this undesirable phenomenon , which is referred to as oxidative stress , has been linked to over one hundred diseases as well as the aging process.2,4 several conditions have been demonstrated to induce oxidative stress , including physical exercise5 and exposure to various pollutants such as cigarette smoke,6 radiation,7 and ozone.8 another stressor that may be a more frequent cause of oxidative stress for many individuals is the metabolism of a high - fat and/or high - carbohydrate meal.9 such metabolism floods the circulatory and peripheral tissues with substrate , and the processing of these substrates overwhelms the mitochondrial electron transport chain . this accelerates electron leakage and subsequent superoxide formation , which in turn initiates a cascade of events that culminates in additional rons formation.10 the excessive rons formation and subsequent oxidative damage caused by this metabolism of foodstuff is termed postprandial oxidative stress . such dysmetabolism is thought to be involved in the pathogenesis of human disease , including insulin resistance11 and cardiovascular disease.12,13 antioxidant intake is thought to decrease oxidative stress , with foods and nutritional supplements containing antioxidants often recommended for this purpose . the reduction in oxidative stress following antioxidant intake has been shown to occur both in the fasted14 and postprandial state.15 coffee is a beverage that has been reported to increase blood antioxidant capacity following both acute16 and chronic intake.17 coffee is rich in antioxidants,18 including chlorogenic acid , melanoidins , and hydroxycinnamic acids.19 this may help explain the j - shaped relationship between daily coffee intake and cardiovascular risk observed in some studies ( reviewed in di castelnuovo et al20 ) and may indicate a potential for coffee to attenuate postprandial oxidative stress.2123 although all types of coffee contain relatively high concentrations of antioxidants,24,25 caffeinated coffee may possess even greater antioxidant activity . specifically , caffeine is noted to have antioxidant properties,26,27 and caffeinated coffee has been recently reported to attenuate lipid and protein oxidation in rat tibialis anterior muscle following exercise.28 that said , caffeine intake is also known to acutely elevate blood fatty acid concentrations,29 which may lead to increased lipid peroxidation in the presence of high concentrations of rons ( as observed following high - fat meal intake).30 hence , it is possible that any potential antioxidant effect of caffeine may be counteracted by the increased propensity for lipids to be oxidized following high - fat feeding . the purpose of this study was to determine the effect of coffee intake on postprandial oxidative stress . we hypothesized that intake of both caffeinated and decaffeinated coffee would attenuate oxidative stress . considering that individuals often consume coffee in conjunction with meals that are high in fat and/or simple sugar such as eggs , bacon , bagels , donuts , and other breakfast and dessert foods the potential ability of coffee intake to minimize postprandial oxidative stress may have implications for many people . subjects were recruited by word of mouth and recruitment flyers from the university of memphis campus and the local memphis community . subjects were required to be healthy at the time of enrollment , without a history of cardiovascular or metabolic disease . in addition , no subject smoked cigarettes , nor did any subject use medications or dietary supplements throughout the study period that may have impacted the outcome measures . no requirements were placed on subjects with regard to daily coffee consumption , as we were unaware of data indicating differing antioxidant effects of coffee intake in habitual versus nonhabitual coffee drinkers . a total of 16 healthy and physically active men ( n = 8) and women ( n = 8) participated in this study , 9 of whom were light coffee drinkers ( mean daily intake , 14 ounces ) and 7 of whom did not consume coffee . subjects height ( using a wall - mounted stadiometer ) , body weight ( using a medical - grade scale ) , body fat percentage ( using lange calipers and a 7-site skinfold test ) , waist and hip circumference ( using a tension - regulated tape measure ) , heart rate ( via 60 second palpation on the radial artery ) , and blood pressure ( via upper arm blood pressure cuff and auscultation with a stethoscope ) were measured and recorded . subjects also received diet logs and were provided with instructions on how to record food and beverage intake during the 2 days prior to each test day . during the initial visit to the lab , all experimental procedures were performed in accordance with the helsinki declaration , and the university of memphis human subjects committee approved all experimental procedures ( protocol number 062111772 ) . subjects reported to the lab on 3 different occasions separated by 5 to 8 days in a 10-hour fasted state and without having consumed caffeine during the prior 24 hours . following the measurement of resting heart rate ( 60-second palpation ) and blood pressure ( standard auscultation procedures using a cuff and stethoscope ) , a fasting blood sample was taken . the test meal consisted of a milk shake made with a combination of whole milk , ice cream ( breyers all natural vanilla ) , and heavy whipping cream . as we have done previously,31,32 the size ( dietary energy ) of the milk shake was determined based on subjects body weight , providing 0.8 grams of fat , 1.0 gram of carbohydrate , and 0.25 grams of protein per kilogram of body weight . this provided approximately 12.2 kilocalories / kilogram of body weight ( eg , a subject weighing 70 kilograms would consume a milk shake containing 854 kilocalories ) , yielding an energy content similar to milk shakes provided in many commercial establishments . the lipid content of the milk shake was similar to what we have used in 2 prior studies of postprandial oxidative stress31,32 and less than that used in some of our other work.33,34 subjects were allowed a maximum of 15 minutes to completely consume the milk shake . one subject who was mildly lactose intolerant was given a standard dose ( 9000 fcc lactase units ) of lactase enzyme . blood was again collected from subjects at 2 and 4 hours following intake of the milk shake ( time started as soon as subjects began consuming the milk shake ) . subjects remained in the lab during this 4-hour period and expended little energy ( ie , watched movies , worked on the computer , etc ) . no additional meals or calorie - containing beverages were allowed during this period . however , water was allowed ad libitum , in addition to the 16 ounces of water ( or coffee ) provided below . following milk shake consumption on each of the 3 visits , subjects consumed either 16 ounces of freshly brewed caffeinated or decaffeinated coffee ( black , unsweetened ) or 16 ounces of bottled water the order of assignment on the 3 days of testing was random , using a crossover design , that is , each subject received all 3 conditions ( caffeinated coffee , decaffeinated coffee , water ) over the course of the study period . the caffeinated coffee and decaffeinated coffee provided approximately 175 mg and 15 mg of caffeine , respectively . medium roast coffee beans ( eight o - clock coffee company , montvale , nj ) were ground ( approximate total of 30 grams of beans per 16 ounce serving of coffee ) and each serving of coffee was brewed fresh ( using bottled water ) and separately in an effort to maintain a similar brew - to - consume time for each subject . although results vary across studies,25 and all forms of coffee beans appear to be rich in antioxidants , it has been reported that medium roast coffee beans have the highest antioxidant content.35 this may be due in part to the fact that naturally occurring antioxidants are not significantly depleted in the roasting process and newly formed antioxidants are gained by roasting through the maillard reaction.36 subjects were provided with the freshly brewed coffee 15 minutes following brewing completion a time when the coffee s temperature did not provide discomfort to subjects . subjects were not told whether they were given caffeinated or decaffeinated coffee on a given day ; of course , many of them were able to infer which condition they received based on their bodies response to the caffeine intake . the coffee or water was completely consumed within 15 minutes following the consumption of the milk shake . venous blood samples were taken from an antecubital vein via needle and vacutainer before and at 2 and 4 hours following intake of the milk shake . blood triglyceride ( tag ) is highly responsive to high - fat feeding , and our prior work involving healthy adults indicates that the peak tag response to a high - fat milk shake occurs at 2 hours post ingestion , with oxidative stress biomarkers peaking between 2 and 4 hours post ingestion.34,37 blood samples that were collected in tubes containing edta were immediately centrifuged at 1500 g for 15 minutes at 4c for collection of plasma . blood samples that were collected in tubes containing no additives were allowed to clot at room temperature for 30 minutes and then separated by centrifugation at 1500 g for 15 minutes at 4c for collection of serum . plasma and serum samples were stored in multiple aliquots at 70c until analyzed for the following variables . triglycerides were analyzed in serum following standard enzymatic procedures as described by the reagent manufacturer ( thermo electron clinical chemistry ) . malondialdehyde ( mda ) was analyzed in plasma following the procedures of jentzsch et al38 using reagents purchased from northwest life science specialties ( vancouver , wa ) . hydrogen peroxide ( h2o2 ) was analyzed in plasma using the amplex red reagent method as described by the manufacturer ( molecular probes , invitrogen detection technologies , eugene , or ) and used as a gross surrogate measure of radical production . trolox equivalent antioxidant capacity ( teac ) was analyzed in serum according to the procedures outlined by the reagent provider ( sigma chemical , st . diet records reflecting the 48-hour periods that preceded each test day were analyzed ( food processor sql , version 9.9 , esha research , salem , or ) . subjects were asked to refrain from strenuous physical activity for the 48 hours prior to each test day , but they were instructed to otherwise maintain their usual physical activity during the entire course of the study . data were analyzed using a 3 ( condition ) 3 ( time ) analysis of variance . all analyses were performed using jmp statistical software ( version 4.0.3 , sas institute , cary , nc ) . statistical significance was set at p < 0.05 ( two tailed ) . subjects were recruited by word of mouth and recruitment flyers from the university of memphis campus and the local memphis community . subjects were required to be healthy at the time of enrollment , without a history of cardiovascular or metabolic disease . in addition , no subject smoked cigarettes , nor did any subject use medications or dietary supplements throughout the study period that may have impacted the outcome measures . no requirements were placed on subjects with regard to daily coffee consumption , as we were unaware of data indicating differing antioxidant effects of coffee intake in habitual versus nonhabitual coffee drinkers . a total of 16 healthy and physically active men ( n = 8) and women ( n = 8) participated in this study , 9 of whom were light coffee drinkers ( mean daily intake , 14 ounces ) and 7 of whom did not consume coffee . subjects height ( using a wall - mounted stadiometer ) , body weight ( using a medical - grade scale ) , body fat percentage ( using lange calipers and a 7-site skinfold test ) , waist and hip circumference ( using a tension - regulated tape measure ) , heart rate ( via 60 second palpation on the radial artery ) , and blood pressure ( via upper arm blood pressure cuff and auscultation with a stethoscope ) were measured and recorded . subjects also received diet logs and were provided with instructions on how to record food and beverage intake during the 2 days prior to each test day . during the initial visit to the lab , all experimental procedures were performed in accordance with the helsinki declaration , and the university of memphis human subjects committee approved all experimental procedures ( protocol number 062111772 ) . subjects reported to the lab on 3 different occasions separated by 5 to 8 days in a 10-hour fasted state and without having consumed caffeine during the prior 24 hours . following the measurement of resting heart rate ( 60-second palpation ) and blood pressure ( standard auscultation procedures using a cuff and stethoscope ) , a fasting blood sample was taken . the test meal consisted of a milk shake made with a combination of whole milk , ice cream ( breyers all natural vanilla ) , and heavy whipping cream . as we have done previously,31,32 the size ( dietary energy ) of the milk shake was determined based on subjects body weight , providing 0.8 grams of fat , 1.0 gram of carbohydrate , and 0.25 grams of protein per kilogram of body weight . this provided approximately 12.2 kilocalories / kilogram of body weight ( eg , a subject weighing 70 kilograms would consume a milk shake containing 854 kilocalories ) , yielding an energy content similar to milk shakes provided in many commercial establishments . the lipid content of the milk shake was similar to what we have used in 2 prior studies of postprandial oxidative stress31,32 and less than that used in some of our other work.33,34 subjects were allowed a maximum of 15 minutes to completely consume the milk shake . one subject who was mildly lactose intolerant was given a standard dose ( 9000 fcc lactase units ) of lactase enzyme . blood was again collected from subjects at 2 and 4 hours following intake of the milk shake ( time started as soon as subjects began consuming the milk shake ) . subjects remained in the lab during this 4-hour period and expended little energy ( ie , watched movies , worked on the computer , etc ) . no additional meals or calorie - containing beverages were allowed during this period . however , water was allowed ad libitum , in addition to the 16 ounces of water ( or coffee ) provided below . following milk shake consumption on each of the 3 visits , subjects consumed either 16 ounces of freshly brewed caffeinated or decaffeinated coffee ( black , unsweetened ) or 16 ounces of bottled water . the order of assignment on the 3 days of testing was random , using a crossover design , that is , each subject received all 3 conditions ( caffeinated coffee , decaffeinated coffee , water ) over the course of the study period . the caffeinated coffee and decaffeinated coffee provided approximately 175 mg and 15 mg of caffeine , respectively . medium roast coffee beans ( eight o - clock coffee company , montvale , nj ) were ground ( approximate total of 30 grams of beans per 16 ounce serving of coffee ) and each serving of coffee was brewed fresh ( using bottled water ) and separately in an effort to maintain a similar brew - to - consume time for each subject . although results vary across studies,25 and all forms of coffee beans appear to be rich in antioxidants , it has been reported that medium roast coffee beans have the highest antioxidant content.35 this may be due in part to the fact that naturally occurring antioxidants are not significantly depleted in the roasting process and newly formed antioxidants are gained by roasting through the maillard reaction.36 subjects were provided with the freshly brewed coffee 15 minutes following brewing completion a time when the coffee s temperature did not provide discomfort to subjects . subjects were not told whether they were given caffeinated or decaffeinated coffee on a given day ; of course , many of them were able to infer which condition they received based on their bodies response to the caffeine intake . the coffee or water was completely consumed within 15 minutes following the consumption of the milk shake . venous blood samples were taken from an antecubital vein via needle and vacutainer before and at 2 and 4 hours following intake of the milk shake . blood triglyceride ( tag ) is highly responsive to high - fat feeding , and our prior work involving healthy adults indicates that the peak tag response to a high - fat milk shake occurs at 2 hours post ingestion , with oxidative stress biomarkers peaking between 2 and 4 hours post ingestion.34,37 blood samples that were collected in tubes containing edta were immediately centrifuged at 1500 g for 15 minutes at 4c for collection of plasma . blood samples that were collected in tubes containing no additives were allowed to clot at room temperature for 30 minutes and then separated by centrifugation at 1500 g for 15 minutes at 4c for collection of serum . plasma and serum samples were stored in multiple aliquots at 70c until analyzed for the following variables . triglycerides were analyzed in serum following standard enzymatic procedures as described by the reagent manufacturer ( thermo electron clinical chemistry ) . malondialdehyde ( mda ) was analyzed in plasma following the procedures of jentzsch et al38 using reagents purchased from northwest life science specialties ( vancouver , wa ) . hydrogen peroxide ( h2o2 ) was analyzed in plasma using the amplex red reagent method as described by the manufacturer ( molecular probes , invitrogen detection technologies , eugene , or ) and used as a gross surrogate measure of radical production . trolox equivalent antioxidant capacity ( teac ) was analyzed in serum according to the procedures outlined by the reagent provider ( sigma chemical , st . diet records reflecting the 48-hour periods that preceded each test day were analyzed ( food processor sql , version 9.9 , esha research , salem , or ) . subjects were asked to refrain from strenuous physical activity for the 48 hours prior to each test day , but they were instructed to otherwise maintain their usual physical activity during the entire course of the study . data were analyzed using a 3 ( condition ) 3 ( time ) analysis of variance . all analyses were performed using jmp statistical software ( version 4.0.3 , sas institute , cary , nc ) . statistical significance was set at p < 0.05 ( two tailed ) . however , we did not include the data from one male subject in our analysis because this subject had a mean fasting tag concentration that was an outlier compared with the values of the remaining subjects ( 573 mg dl ) . no component of dietary intake was different for subjects across conditions ( p > 0.05 , table 2 ) . heart rate and blood pressure were not impacted by the milk shake and not different for any condition ( p > 0.05 , fig . tag values increased significantly over time in response to the milk shake ( p < 0.001 ) . values were higher at 2 and 4 hours post milk shake ingestion compared with pre and at 4 hours post ingestion compared with 2 hours ( p < 0.05 ) . no condition ( p = 0.70 ) or interaction effects ( p = 0.67 ) were noted for tag . data are presented in figure 2 panel a. mda values increased significantly over time in response to the milk shake ( p < 0.001 ) . values were higher at 2 and 4 hours post milk shake ingestion compared with pre ( p < 0.05 ) . no condition ( p = 0.22 ) or interaction effects ( p = 0.95 ) were noted for mda . data are presented in figure 2 panel b. h2o2 values increased significantly over time in response to the milk shake ( p < 0.001 ) . values were higher at 4 hours post milk shake ingestion compared with pre and 2 hours ( p < 0.05 ) . no condition ( p = 0.22 ) or interaction effects ( p = 0.94 ) were noted for h2o2 . data are presented in figure 3 panel a. teac was unaffected by the milk shake and not different across conditions . no time ( p = 0.36 ) , condition ( p = 0.86 ) , or interaction effects ( p = 0.94 ) were noted . our findings indicate that acute consumption of coffee has no effect on tag , mda , h2o2 , or teac for up to 4 hours following the consumption of a high - fat milk shake . whether a larger volume of coffee ( and hence , antioxidant content ) and/or a smaller volume of milk shake would yield different results remains unknown . likewise , coffee ingestion at a time prior to milk shake consumption may have yielded different findings . future study is needed to generate answers to these questions . moreover , the inclusion of additional antioxidant biomarkers ( eg , glutathione , superoxide dismutase , and catalase ) and oxidative stress biomarkers ( eg , isoprostanes and protein carbonyls ) may have strengthened the study . as we have reported in prior studies , intake of a high - fat milk shake causes an increase in the concentrations of both tag and oxidative stress biomarkers.31,33,34,37 surprisingly , the response of all variables for subjects in the present study was somewhat different than what we have noted in recent work,34,37 with values for tag , mda , and h2o2 continuing to increase 4 hour postmeal rather than peaking 2 hours postmeal . this delayed tag and oxidative stress response likely can not be explained by the addition of coffee to the study design , as we observed a similar response for the water condition ( with the exception of the tag data , for which water resulted in similar tag values at 2 and 4 hours postmeal ) . our failure to include a longer time course of measurement in the current design is a limitation of this work , as it is possible ( but not probable ) that we may have observed between - group differences in tag and oxidative stress responses at time points greater than 4 hours following milk shake ingestion . some prior studies have reported an attenuation in postprandial oxidative stress following acute anti - oxidant intake.3943 these findings led us to hypothesize that coffee consumption would also attenuate postprandial oxidative stress , as coffee has been reported in one study to provide 66% of dietary antioxidant intake,18 and is abundant in chlorogenic acid , melanoidins , and hydroxycinnamic acids.19 on the other hand , although we do not know the exact antioxidant potential of coffee compared with antioxidant supplements , it is possible that the antioxidant content of a 16-ounce serving of coffee was too low , relative to the content of a typical high - potency antioxidant supplement , to affect our outcome variables . we measured heart rate and blood pressure in the present study because we hypothesized that the rons production induced by the milk shake would promote an acute state of endothelial dysfunction44 and a concomitant increase in blood pressure , and we wanted to determine if the hypothesized attenuation in oxidative stress following coffee intake would prevent this rise . however , neither heart rate nor blood pressure was affected by the milk shake ; hence , testing the ability of coffee to alter this response was not possible . our line of questioning may have been more appropriately addressed if we were to include a sample of hypertensive individuals within the research design.45 as can be viewed in table 1 , the subjects baseline resting heart rate and blood pressure were both within healthy norms . we report for the first time that acute consumption of coffee does not affect tag , mda , h2o2 , or teac for up to 4 hours following the consumption of a high - fat milk shake . our findings suggest that coffee intake is not adequate to counteract the massive increase in rons generated by consumption of a high - fat meal . rather than attempting to counteract the increase in rons following high - fat consumption with antioxidant intake , it may be more effective to reduce the oxidative insult by reducing the meal s fat content.31,37 future work may ( 1 ) extend the time frame of measurement beyond the 4-hour postprandial period , ( 2 ) include a meal that is lower in fat content ( so as to not overwhelm the system with rons ) , ( 3 ) provide coffee at a time prior to intake of the high - fat meal , and/or ( 4 ) consider the inclusion of a sample of subjects with known chronic disease ( eg , diabetes or hypertension ) . such additional experiments may help to more fully elucidate the role of coffee on attenuating postprandial oxidative stress .

background : coffee has been reported to be rich in antioxidants , with both acute and chronic consumption leading to enhanced blood antioxidant capacity . high - fat feeding is known to result in excess production of reactive oxygen and nitrogen species , promoting a condition of postprandial oxidative stress.methods:we tested the hypothesis that coffee intake following a high - fat meal would attenuate the typical increase in blood oxidative stress during the acute postprandial period . on 3 different occasions , 16 men and women consumed a high - fat milk shake followed by either 16 ounces of caffeinated or decaffeinated coffee or bottled water . blood samples were collected before and at 2 and 4 hours following intake of the milk shake and analyzed for triglycerides ( tag ) , malondialdehyde ( mda ) , hydrogen peroxide ( h2o2 ) , and trolox equivalent antioxidant capacity ( teac).results : values for tag and mda ( p < 0.001 ) , as well as for h2o2 ( p < 0.001 ) , increased significantly following milk shake consumption , with values higher at 4 hours compared with 2 hours post consumption for tag and h2o2 ( p < 0.05 ) . teac was unaffected by the milk shake consumption . coffee had no impact on tag , mda , h2o2 , or teac , with no condition or interaction effects noted for any variable ( p > 0.05).conclusions : acute coffee consumption following a high - fat milk shake has no impact on postprandial oxidative stress .

Introduction Materials and Methods Subjects and screening (first laboratory visit) Testing (second, third, and fourth laboratory visits) Blood collection and biochemistry Dietary intake and physical activity Statistical analysis Results Discussion Conclusion

the reduction in oxidative stress following antioxidant intake has been shown to occur both in the fasted14 and postprandial state.15 coffee is a beverage that has been reported to increase blood antioxidant capacity following both acute16 and chronic intake.17 coffee is rich in antioxidants,18 including chlorogenic acid , melanoidins , and hydroxycinnamic acids.19 this may help explain the j - shaped relationship between daily coffee intake and cardiovascular risk observed in some studies ( reviewed in di castelnuovo et al20 ) and may indicate a potential for coffee to attenuate postprandial oxidative stress.2123 although all types of coffee contain relatively high concentrations of antioxidants,24,25 caffeinated coffee may possess even greater antioxidant activity . specifically , caffeine is noted to have antioxidant properties,26,27 and caffeinated coffee has been recently reported to attenuate lipid and protein oxidation in rat tibialis anterior muscle following exercise.28 that said , caffeine intake is also known to acutely elevate blood fatty acid concentrations,29 which may lead to increased lipid peroxidation in the presence of high concentrations of rons ( as observed following high - fat meal intake).30 hence , it is possible that any potential antioxidant effect of caffeine may be counteracted by the increased propensity for lipids to be oxidized following high - fat feeding . following milk shake consumption on each of the 3 visits , subjects consumed either 16 ounces of freshly brewed caffeinated or decaffeinated coffee ( black , unsweetened ) or 16 ounces of bottled water the order of assignment on the 3 days of testing was random , using a crossover design , that is , each subject received all 3 conditions ( caffeinated coffee , decaffeinated coffee , water ) over the course of the study period . venous blood samples were taken from an antecubital vein via needle and vacutainer before and at 2 and 4 hours following intake of the milk shake . blood triglyceride ( tag ) is highly responsive to high - fat feeding , and our prior work involving healthy adults indicates that the peak tag response to a high - fat milk shake occurs at 2 hours post ingestion , with oxidative stress biomarkers peaking between 2 and 4 hours post ingestion.34,37 blood samples that were collected in tubes containing edta were immediately centrifuged at 1500 g for 15 minutes at 4c for collection of plasma . blood was again collected from subjects at 2 and 4 hours following intake of the milk shake ( time started as soon as subjects began consuming the milk shake ) . following milk shake consumption on each of the 3 visits , subjects consumed either 16 ounces of freshly brewed caffeinated or decaffeinated coffee ( black , unsweetened ) or 16 ounces of bottled water . venous blood samples were taken from an antecubital vein via needle and vacutainer before and at 2 and 4 hours following intake of the milk shake . blood triglyceride ( tag ) is highly responsive to high - fat feeding , and our prior work involving healthy adults indicates that the peak tag response to a high - fat milk shake occurs at 2 hours post ingestion , with oxidative stress biomarkers peaking between 2 and 4 hours post ingestion.34,37 blood samples that were collected in tubes containing edta were immediately centrifuged at 1500 g for 15 minutes at 4c for collection of plasma . values were higher at 2 and 4 hours post milk shake ingestion compared with pre and at 4 hours post ingestion compared with 2 hours ( p < 0.05 ) . values were higher at 2 and 4 hours post milk shake ingestion compared with pre ( p < 0.05 ) . values were higher at 4 hours post milk shake ingestion compared with pre and 2 hours ( p < 0.05 ) . our findings indicate that acute consumption of coffee has no effect on tag , mda , h2o2 , or teac for up to 4 hours following the consumption of a high - fat milk shake . as we have reported in prior studies , intake of a high - fat milk shake causes an increase in the concentrations of both tag and oxidative stress biomarkers.31,33,34,37 surprisingly , the response of all variables for subjects in the present study was somewhat different than what we have noted in recent work,34,37 with values for tag , mda , and h2o2 continuing to increase 4 hour postmeal rather than peaking 2 hours postmeal . some prior studies have reported an attenuation in postprandial oxidative stress following acute anti - oxidant intake.3943 these findings led us to hypothesize that coffee consumption would also attenuate postprandial oxidative stress , as coffee has been reported in one study to provide 66% of dietary antioxidant intake,18 and is abundant in chlorogenic acid , melanoidins , and hydroxycinnamic acids.19 on the other hand , although we do not know the exact antioxidant potential of coffee compared with antioxidant supplements , it is possible that the antioxidant content of a 16-ounce serving of coffee was too low , relative to the content of a typical high - potency antioxidant supplement , to affect our outcome variables . we report for the first time that acute consumption of coffee does not affect tag , mda , h2o2 , or teac for up to 4 hours following the consumption of a high - fat milk shake .

a ca - sensitive k channel ( gardos channel , hik1 , or hsk4 ) is expressed in the plasma membrane of human red blood cells ( rbcs ) ( gardos , 1958 ; ishii et al . , 1997 ; vandorpe et al . , 1998 ; hoffman et al . , 2003 ) . at saturating intracellular ca concentrations , gardos channel activation increases the mean k flux through the plasma membrane by three orders of magnitude ( gardos , 1958 ; simons , 1976 ; lew and ferreira , 1978 ) , causing the loss of an alkaline , hypertonic effluent containing kcl and koh ( lew and bookchin , 1986 ; freeman et al . , 1987 ) . gardos channel activation plays an important role in the mechanism of sickle cell dehydration ( lew and bookchin , 2005a ) and may also participate in the physiological densification of aging rbcs ( clark , 1988 ; lutz , 1988 ) . early estimates of the number of gardos channels per cell described values ranging from 1 to 200 , with claims of wide differences among rbc subpopulations ( lew et al . , 1982 ; , 1984 ; alvarez and garca - sancho , 1987 ; wolff et al . , 1988 ; brugnara et al . , 1993 ; lew et al . , 2004 , 2005 ) . however , when ca ionophores were used to induce rapid and uniform ca loads ( simonsen et al . , 1982 ) , triggering instant and maximal gardos channel activation in all the cells ( fmax at saturating ca loads ) , the distribution of dehydration rates in the rbc population , as followed by flow cytometry , proceeded to lower mean volumes with no significant variation in standard deviation or appearance of multimodality ( lew et al . , 2005 ) . the discrepant estimates of the early experiments were attributed to unrecognized differences in intracellular rbc ca concentrations due to cell to cell variations in ca pump - leak balance ( lew et al . , 2003 , 2005 ) . we use fmax throughout to represent the experimental condition in which saturating ca loads maximally activate the k fluxes via gardos channels in each rbc in any given sample of rbcs . thus defined , fmax equals the npof product , where n represents the number of active gardos channels per cell , po is the open state probability condition of the channels in each cell , and f is the unit channel flux at saturating ca levels , as determined by single channel conductance . the apparent uniformity of gardos channel capacity among the rbcs was surprising because it did not reveal the expected decrease in driving force for dehydration among the aging , denser rbcs , which would be seen as a tail of slower dehydrating cells in the migrating gaussian curves . age - dependent changes in rbc metabolism , composition , and transport have been well documented ( beutler , 1985 ; lutz et al . , 1988 ; chasis et al . , 1989 ; mohandas and groner , 1989 ; , 1998 ; lew et al . , 2003 ; kuypers and de jong , 2004 ; arese et al . , 2005 ) . the unexpected finding that gardos channel activity appeared uniform , regardless of rbc age , was puzzling and prompted a reevaluation . to further investigate possible age variations in gardos channel fmax among the cells , we used glycated hemoglobin ( hb a1c ) as an age marker for rbcs from normal , nondiabetic subjects ( bookchin and gallop , 1968 ; bunn et al . to detect age - related variations in gardos channel activity among rbcs , we designed experimental conditions in which the rbc dehydration rates were determined by the gardos channel fmax and by the driving gradient for dehydration in each cell , and the relative ages of the fastest and slowest dehydrating rbcs were assessed by measuring their hb a1c contents . the rbcs were washed thrice in medium a containing ( in mm ) nacl , 142 ; kcl , 3 ; na - hepes , 10 , ph 7.5 at 37c ; mgcl2 , 0.15 , and suspended at 10% hematocrit ( hct ) in medium a containing 0.05 mm cacl2 and in which 10 mm nacl was replaced by nascn . this low concentration of scn was sufficient to bypass the rate - limiting effects of cl permeability on dehydration ( garca - sancho and lew , 1988 ; tiffert et al . , 2001 ) so that the rbcs ' dehydration rates were determined mainly by their gardos channel fmax . the suspension was incubated at 37c under continuous magnetic stirring and divided into equal aliquots that were processed every 15 , 20 , or 30 s in the different experiments . the ionophore a23187 was added at t = 0 to a final concentration of 10 m in each of the aliquots to generate an instant , uniform , and saturating [ ca]i load in all the rbcs . at the final set time for each aliquot , 1 mm egta was added to extract all rbc ca , which promptly inactivated the gardos channels and arrested further dehydration . the egta - treated aliquots were dispensed into microfuge tubes containing diethylphthalate oil ( dep oil , = 1.117 g / ml ) , and spun at 10,000 g for 1 min to separate the rbcs that exceeded that density at each time interval after gardos channel activation . the rbcs harvested from the pellets and from the top of the oil were analyzed for hemoglobin ( hb ) to estimate the proportion of rbcs recovered from each fraction , and for hb a1c by hplc as follows . the hb concentration in the rbc lysates was determined by light absorption at 415 nm ( the soret band ) using a plate reader ( spectramax 190 , molecular devices ) . the hb yields of the density - segregated cell fractions were expressed as a percent of the total hb in the unfractionated sample . hb a1c was measured on code - labeled samples by ion exchange hplc ( abraham et al . , 1984 ; bosch et al . , 1992 ) at the biochemistry laboratories , addenbrooke 's hospital , cambridge , uk ( project 719 ) and at jacobi medical center , bronx , ny , using variant ii instruments ( bio - rad laboratories ) . reproducibility of the measurements was estimated on 20 aliquots of a single blood sample , whose mean standard deviation was 5.71% 0.02 , giving a coefficient of variation of 0.35% . thus the intrinsic measurement error of the reported hb a1c values was within the size of the points in the figures below . in selected experiments the cells were washed three times in 10 volumes of isotonic mgcl2 , lysed , and diluted in distilled water to the required optimal reading range and measured against standards with similar na concentrations . 3 below were performed as previously described ( lew et al . , 1995 ; raftos et al . , 1996 ; raftos et al . , 1997 ) . in brief , pairs of 96-well plates were used , one of each pair having a u - shaped well bottom , and the other flat bottomed for optical density measurements . each row of the u - bottomed plate contained 250 l each of 12 lysis media solutions with different osmolarities , ranging from 1.0 to 0.01 relative tonicity ( rt ) units , prepared by mixing appropriate volumes of one solution containing 149 mm nacl and 2 mm na - hepes ( ph 7.5 at 20c ) , and a second solution containing only 2 mm of na- hepes ( ph 7.5 at 20c ) . 15 s before each sampling time , 0.5 ml of cell suspension was transferred to a groove in a plastic incubation tray ( accutran disposable incubation tray , schleicher & schuell ) at room temperature . at the exact sampling time , a 12-channel pipette ( finnpipette , lab systems ) was used to deliver 10-l samples of the suspension in the incubation tray to the row of 12 wells on a u - bottomed 96-microwell plate containing the 250 l of the lysis media , and rapidly mixed by repeated vigorous squirts with the multichannel dispenser . each timed sample generated a full osmotic fragility curve . to estimate the fraction of hemolysis in each well , the plate was centrifuged for 5 min at 1,500 g , 150-l samples of the supernatants were transferred with a 12-channel dispenser to the correspondingly labeled row on the flat - bottomed plate . the concentrations of hb were measured on the spectramax plate reader at the soret band ( 415 nm ) to calculate the percent hemolysis in each well . this was plotted as a function of relative tonicity for the osmotic fragility curves reported in fig . to detect age - related variations in gardos channel activity among rbcs , we designed experimental conditions in which the rbc dehydration rates were determined by the gardos channel fmax and by the driving gradient for dehydration in each cell , and the relative ages of the fastest and slowest dehydrating rbcs were assessed by measuring their hb a1c contents . the rbcs were washed thrice in medium a containing ( in mm ) nacl , 142 ; kcl , 3 ; na - hepes , 10 , ph 7.5 at 37c ; mgcl2 , 0.15 , and suspended at 10% hematocrit ( hct ) in medium a containing 0.05 mm cacl2 and in which 10 mm nacl was replaced by nascn . this low concentration of scn was sufficient to bypass the rate - limiting effects of cl permeability on dehydration ( garca - sancho and lew , 1988 ; tiffert et al . , 2001 ) so that the rbcs ' dehydration rates were determined mainly by their gardos channel fmax . the suspension was incubated at 37c under continuous magnetic stirring and divided into equal aliquots that were processed every 15 , 20 , or 30 s in the different experiments . the ionophore a23187 was added at t = 0 to a final concentration of 10 m in each of the aliquots to generate an instant , uniform , and saturating [ ca]i load in all the rbcs . at the final set time for each aliquot , 1 mm egta was added to extract all rbc ca , which promptly inactivated the gardos channels and arrested further dehydration . the egta - treated aliquots were dispensed into microfuge tubes containing diethylphthalate oil ( dep oil , = 1.117 g / ml ) , and spun at 10,000 g for 1 min to separate the rbcs that exceeded that density at each time interval after gardos channel activation . the rbcs harvested from the pellets and from the top of the oil were analyzed for hemoglobin ( hb ) to estimate the proportion of rbcs recovered from each fraction , and for hb a1c by hplc as follows . the hb concentration in the rbc lysates was determined by light absorption at 415 nm ( the soret band ) using a plate reader ( spectramax 190 , molecular devices ) . the hb yields of the density - segregated cell fractions were expressed as a percent of the total hb in the unfractionated sample . hb a1c was measured on code - labeled samples by ion exchange hplc ( abraham et al . , 1984 ; bosch et al . , 1992 ) at the biochemistry laboratories , addenbrooke 's hospital , cambridge , uk ( project 719 ) and at jacobi medical center , bronx , ny , using variant ii instruments ( bio - rad laboratories ) . reproducibility of the measurements was estimated on 20 aliquots of a single blood sample , whose mean standard deviation was 5.71% 0.02 , giving a coefficient of variation of 0.35% . thus the intrinsic measurement error of the reported hb a1c values was within the size of the points in the figures below . in selected experiments the cells were washed three times in 10 volumes of isotonic mgcl2 , lysed , and diluted in distilled water to the required optimal reading range and measured against standards with similar na concentrations . 3 below were performed as previously described ( lew et al . , 1995 ; raftos et al . , 1996 ; raftos et al . , 1997 ) . in brief , pairs of 96-well plates were used , one of each pair having a u - shaped well bottom , and the other flat bottomed for optical density measurements . each row of the u - bottomed plate contained 250 l each of 12 lysis media solutions with different osmolarities , ranging from 1.0 to 0.01 relative tonicity ( rt ) units , prepared by mixing appropriate volumes of one solution containing 149 mm nacl and 2 mm na - hepes ( ph 7.5 at 20c ) , and a second solution containing only 2 mm of na- hepes ( ph 7.5 at 20c ) . 15 s before each sampling time , 0.5 ml of cell suspension was transferred to a groove in a plastic incubation tray ( accutran disposable incubation tray , schleicher & schuell ) at room temperature . at the exact sampling time , a 12-channel pipette ( finnpipette , lab systems ) was used to deliver 10-l samples of the suspension in the incubation tray to the row of 12 wells on a u - bottomed 96-microwell plate containing the 250 l of the lysis media , and rapidly mixed by repeated vigorous squirts with the multichannel dispenser . each timed sample generated a full osmotic fragility curve . to estimate the fraction of hemolysis in each well , the plate was centrifuged for 5 min at 1,500 g , 150-l samples of the supernatants were transferred with a 12-channel dispenser to the correspondingly labeled row on the flat - bottomed plate . the concentrations of hb were measured on the spectramax plate reader at the soret band ( 415 nm ) to calculate the percent hemolysis in each well . this was plotted as a function of relative tonicity for the osmotic fragility curves reported in fig . fig . 1 , typical of three similar experiments , reports the time - dependent changes in rbc fraction and hb a1c content of the rbcs recovered above the dep oil after the onset of the ca load . as the fraction of rbcs remaining above the oil declined ( fig . 1 c shows that there was an inverse correlation between the percent hb a1c and the fraction of cells with density < 1.117 , indicating that those rbcs slowest to become dense had the highest hb a1c values . relation between hb a1c content and percent of cells harvested above diethylphthalate oil ( < 1.117 g / ml ) during gardos channel mediated dehydration . time = 0 in a and b corresponds to the onset of the ca load by addition of the ionophore a23187 . as the rbcs dehydrate , the fraction recovered above the oil decreases ( a ) , but its mean hb a1c increases ( b ) . using time as the parametric variable , the hb a1c content is plotted as a function of yield in c , which also gives the parameter values of the linear regression fit through the experimental points . the results are typical of three experiments performed with blood from three donors . the results in fig . 1 are in striking contrast with those expected from the known age density distribution of rbcs , and from the assumption that rbcs of all ages would have comparable gardos channel fmax activity , which would have been the simplest explanation of the observed conservation of the volume distribution profile during dehydration ( lew et al . , 2005 ) . if that explanation had been valid , then during gardos channel mediated dehydration , the rbcs expected to exceed the oil density first would have been the densest and oldest cells nearest to the density boundary ( fig . 1 ) was the opposite ; the youngest rbcs became dense first and the oldest rbcs last . these results suggest that rbc gardos channel activity is not uniform , but rather that it declines with cell age . diagram to illustrate the discrepancy between predicted and observed rbc age distribution during ca- induced dehydration . the gaussian curves surround rbc populations ( circles ) of differing age , represented by the grayscale progression from white ( youngest ) to black ( oldest ) . the x axis shows increasing density from left to right , and the vertical arrow shows the position of the 1.117 g / ml density boundary chosen for our experiments . prior to gardos channel activation , the initial rbc density distribution is represented as determined by age ( the lighter young cells on the left and the denser older cells on the right ) . after k permeabilization , the rbcs dehydrate and their increasing density is represented by the shift to the right of the gaussian curves . the standard deviation is shown conserved representing previous experimental results ( bookchin et al . , 2000 ; those findings led to the suggestion ( predicted ) that dehydration rates were similar in all the rbcs , with conservation of the original rbc age distribution within the migrating gaussians ( predicted ) . the present results showed that this pattern is valid only with valinomycin - induced k permeabilization . by contrast , for gardos channel mediated k permeabilization , the results suggest that conservation of the standard deviation conceals a substantial degree of age scrambling within the migrating gaussian curves ( observed ) , with the younger rbcs passing the older cells that started as most dense in the initial condition ; thus the rbcs that first pass the set density boundary are much younger than those reaching it last , reflecting a declining pattern of gardos fmax with rbc age . a close analysis of the factors influencing the age distribution of gardos - dehydrating rbcs suggests alternative possibilities . three main factors determine the speed at which k - permeabilized rbcs will traverse the high - density boundary in our experiments ( = 1.117 g / ml ) : ( 1 ) the initial density of each rbc determines its starting distance to the density boundary , ( 2 ) the strength of the k gradient provides the driving force for dehydration , which is progressively weaker the higher the individual cell 's na / k concentration ratio , which increases with cell age ( bookchin et al . , 2000 ) , and ( 3 ) the magnitude of the gardos channel fmax in each cell . therefore , the slower dehydration rate of the dense , aged rbcs could result from either a decreased driving force for dehydration , decay in gardos channel fmax , or both . to discriminate between the contributions of ion gradient dissipation and of gardos channel fmax to the observed pattern of rbc dehydration , we sought first to subject the rbcs to a uniform , high k permeability , bypassing gardos channel activation . under these conditions , any age - determined differences in dehydration rate could be attributed solely to the initial rbc densities and to cell - to - cell differences in driving gradient for dehydration , not to variations in induced k permeability among the rbcs . valinomycin , a k - selective ionophore ( harris and pressman , 1967 ; mueller and rudin , 1967 ) , has been extensively used to increase the k permeability of cell membranes , but for our purposes it was necessary to establish first whether the valinomycin - induced k permeability was uniform among the rbcs . if it could be shown that valinomycin rapidly equilibrated between treated and untreated rbcs , this would indicate that valinomycin distributes itself rapidly and in equal concentration per unit rbc membrane area , thus generating a uniform k permeability among the rbcs . to investigate this point , we mixed valinomycin - treated with untreated rbcs , and followed the dehydration response in the mixture using the profile migration method , in which the uniformity of the rbc dehydration is monitored by the extent of slope conservation during the left - shifted displacement of the osmotic fragility curves ( raftos et al . , 1996 ) . 3 a , the control condition , shows the fairly rapid and parallel displacement of the osmotic fragility curves generated by valinomycin addition to a suspension of rbcs in low - k media , as expected from uniformity of dehydration rates . whether this uniformity was indeed the result of uniform k 3 ( a and b ) shows that the 50% untreated rbcs in the mixed suspension dehydrated to the same final condition at a slower rate than that of the control rbcs but still sufficiently fast to secure a rapid redistribution of valinomycin between treated and untreated rbcs , thus ensuring uniform k permeabilization among valinomycin - exposed rbcs . ( a ) control condition : valinomycin was added at t = 0 to a 10% rbc suspension in medium a ( final concentration = 10 m ) . the suspension was incubated at 37c under continuous magnetic stirring , and samples for osmotic fragility curves were taken at the times indicated . the parallel displacement of the osmotic fragility curves to the left reflect uniformity of dehydration rates . ( b ) valinomycin redistribution rate : an aliquot of thoroughly washed , packed rbcs from the control suspension of valinomycin - treated , fully dehydrated rbcs was added at t=0 to a 5% hct suspension of untreated rbcs in medium a at 37c and under magnetic stirring , giving a final hct of 10% . the 50% untreated rbcs in the mixed suspension dehydrated to the same final condition as the control cells at a rate similar to that of the original population ( a ) , indicating a very rapid redistribution of valinomycin between treated and untreated rbcs . valinomycin could thus be applied to test the extent to which age - related differences in rbc dehydration rates result from their initial density distribution or from differences in their driving k gradients . 4 a shows that the first rbcs to enter the pellets ( filled circles , lower yields ) had relatively high hb a1c contents . these are the fastest dehydrating cells in the presence of valinomycin and must contain a considerable proportion of aged rbcs , a trend opposite to that observed with gardos - dehydrated rbcs ( fig . 4 a shows a much narrower variation among the valinomycin - treated cells than among the ca - loaded cells , a consistent observation in all the experiments of this series . this indicates a more substantial age mix in the fractions recovered from valinomycin - treated rbcs than from those dehydrated by gardos channel activation . hb a1c and na content of density - segregated rbcs during valinomycin - induced dehydration . a 10% rbc suspension in medium a at 37c was treated with 10 m valinomycin at t = 0 . samples were spun through diethylphthalate at frequent time intervals , and the rbcs harvested from the top and bottom of the oil were assayed for hb ( to determine yield ) , percent hb a1c , and na content ( [ na]c ) . ( a ) percent hb a1c is plotted as a function of the percent of cells recovered from pellets ( filled circles , > 1.117 ) or from above the oil ( open circles , < 1.117 ) in timed samples after the addition of valinomycin to the cell suspension . ( b ) the na content of the rbcs is plotted as a function of the percent of cells recovered above the dep oil ( < 1.117 ) . the coefficients of the best - fit curve are yo = 6.50 , a = 175 , and b = 7.84 . the hb a1c of the rbcs above the oil ( open circles ) did not differ significantly from the mean values , in any of the three experiments of this series , except for a tiny rbc fraction recovered after maximal dehydration ( fig . the latter rbcs also had the highest na content ( fig . 4 b ) , sufficient to lower the driving gradient to an extent that they would not dehydrate beyond the 1.117 g / ml density boundary . the yield of these high - na cells varied from 0.3 to 0.6% in the three experiments . in fig . 1 b , at the highest yields , the mean rbc na content was 710 mmol(340 g hb ) . at yields from 5 to 25% the mean na content was 10 to 16 mmol(340 g hb ) , indicating a minor but progressive increase in mean na content with rbc age , and consequent reduction in the driving gradient for dehydration . these results show that for uniformly k - permeabilized ( valinomycin - treated ) rbcs , the main determinant of dehydration rate was their initial density , i.e. , their initial distance from the set density boundary ( fig . there was also a progressive contribution of na / k gradient dissipation , slowing the older rbcs ' dehydration and thus generating a large age mix in the density - separated fractions , with reduced variations in hb a1c . gradient dissipation became extreme only in a minute , high - na fraction of cells . thus , following uniform k permeabilization with valinomycin , the oldest and densest cells pass the density boundary first , but because of their weaker driving force for dehydration they soon become admixed in the pellets with younger rbcs . thus in these conditions , dehydration rates are determined by the weak opposing effects of initial density distribution and progressively reduced driving gradient , whereas with ca - induced dehydration it is the variation of gardos channel fmax among the rbcs that dominates the dehydration rates , generating an inverse age density distribution ( fig . 2 , bottom ) . furthermore , the experimental need of relatively high hct ( 10% in our experiments ) to harvest sufficient rbcs for measurements of initial and final hb a1c and [ na ] also contributed to the larger age mix seen with valinomycin . in such concentrated cell suspensions , the net kcl loss generates a large rapid increase in the extracellular [ k ] , so that the faster dehydrating rbcs are driven by a higher outward k concentration gradient than those slower to dehydrate . with valinomycin , the two main contributors to the rate at which dehydrating rbcs approach the density boundary are the initial cell density distribution and the strength of the driving gradient . initial cell density places the older rbcs nearer the density boundary , but the driving gradient of the younger rbcs ( which start further from the boundary ) is stronger . the younger cells dehydrate faster and the k they release slows the rate at which the older cells approach the boundary , thus further mixing the ages of dehydrating , valinomycin - treated rbcs relative to their original age density distribution . with ca - induced dehydration , on the other hand , the rise in extracellular [ k ] during dehydration amplifies their age differences in gardos fmax , by further slowing the older rbcs ' dehydration rates . 1 can now be applied to analyze the distribution of gardos channel fmax among the rbcs . 1 . for a rough estimate of the gardos channel fmax distribution , we assume that the time it takes for any rbc to reach the boundary density is determined primarily by its gardos channel fmax , and that its initial density and the status of its driving gradient have negligible effects . with these assumptions , we can apply the lew - bookchin red cell model ( lew and bookchin , 1986 ) to simulate the present experimental conditions and compute the time needed for rbcs with different k permeabilities ( pk ) ( representing gardos channel fmax variations ) to attain a density of 1.117 g / ml . the simulation in fig . 5 b plots pk as a function of time . using time as the parametric variable , and the fit in fig . 5 b , we can now plot cell frequency as a function of pk from the data in fig . 5 a , and 5 c represents the integral of the distribution of gardos channel fmax among the rbcs . its derivative , representing the distribution of gardos channel fmax , is shown in fig . 5 d. the dashed line shows the best gaussian fit of the derivative curve , rendering a mean pk of 38 h and a coefficient of variation of 37% . fig . 5 e reports the variation in hb a1c in the rbcs recovered above the oil at the time intervals shown after the ca load . time as the parametric variable for fig . 5 ( b and e ) we can plot the percent hb a1c as a function of pk ( fig . 5 f ) ; this reveals an inverse relation between percent hb a1c and pk corresponding to an inverse relation between rbc age and gardos channel fmax . ( a ) the percent rbcs recovered from the top of the dep oil as a function of time after a saturating ca load . ( b ) the lew - bookchin red cell model ( lew and bookchin , 1986 ) was used to estimate the time needed for rbcs with different k permeabilities ( pk ) to exceed a density of 1.117 g / ml in conditions simulating the experiments in a. note that pk here corresponds to the gardos channel fmax . the coefficients of the best - fit curve are yo = 6.51 , a = 96.3 , and b = 1.08 . ( c ) using the best fit curve of b , and time as the parametric variable , the combined experimental points in a were redrawn as a function of pk from b. note the choice of axis reorientation : percent cells with > 1.117 instead of with < 1.117 as in a plotted as a function of increasing pk . the coefficients of the best - fit curve are yo = 2.68 , a = 105 , xo = 36.3 , and b = 11.4 . this curve represents the integral of the distribution of gardos channel activity in the rbc population . ( d ) the derivative of the best - fit curve of c is plotted as a function of pk , with a normalized scale for the cell fraction . superimposed on the derivative curve is a gaussian curve fit ( dashed line ) with the indicated statistical parameters . activity relation ; the percent hb a1c measured in the rbcs harvested from the top of dep ( a ) as a function of time . ( f ) using time as the parametric variable , the percent hb a1c in the rbcs from e is plotted as a function of pk , from b. with caution , given the simplifying assumptions inherent in this analysis and the unavoidable age mix in the samples , these results show that the gardos channel fmax declines monotonically with rbc age ( fig . 5 , e and f ) , and that the fmax values among the rbcs follows a fairly broad , approximate gaussian distribution ( fig . 5 it is important to note that even the lowest pk value in the oldest rbcs is 10 h , still three to four orders of magnitude above the ground k permeability of the human rbc ( beaug and lew , 1977 ; lew and beaug , 1979 ) . the present results show that gardos channel activity , measured at saturating ca loads , declines sharply throughout the lifespan of human rbcs . however , these results do not allow us to discriminate between the separate contributions of n or po to the age decline in gardos channel fmax . a reduction in n only is equivalent to a cumulative and progressive all - or - none inactivation of gardos channels within each cell . such an all - or - none type of inactivation was observed experimentally with the ca - mg - atpase of isolated rbc membranes exposed to high glucose concentrations ( gonzalez flecha et al . , 1999 ) . these authors showed that glycation of a single essential lys residue , probably located near the catalytic atp site of the atpase , caused full inhibition , whereas those molecules without glycation of the vulnerable lys residue remained functionally normal . the possible effects of glycation on gardos channel function have not yet been explored . a reduction in po with cell age may perhaps be inferred from the peculiar temperature response of the gardos channels observed in excised membrane patches from rbc membranes ( grygorczyk , 1987 ) . at saturating ca levels , po was shown to fall sharply , by > 90% , when the temperature was increased from 30c to 35c . on the other hand , when gardos channel mediated k fluxes were measured in intact rbcs , the fall in efflux rate constant between 27c and 37c was only about half ( hoffman et al . , 2003 ) . hoffman et al . ( 2003 ) considered the possibility that this discrepancy in the magnitude of the temperature effect may result from heterogeneity in the response of intact rbc populations . taken together with the present results , the discrepancy may be explained if what has been detected as a differential temperature effect reflects in fact a decline in po with rbc age . in this interpretation , the po of young rbcs would be high and show little change with temperature . as rbcs age , their channels undergo structural or regulatory changes that can be exposed experimentally as a progressive decline in po at temperatures > 30c . thus , age heterogeneity in the rbc population would generate a mean temperature differential that could mask the extreme high and low po values at 37c in young and old rbcs , respectively . this interpretation assumes that the reported patch clamp data were obtained preferentially from aged rbcs with low po responses at 35c . further research on excised patches from age - segregated rbcs is needed to investigate whether the temperature effect is related to rbc age and to assess the contribution of po to fmax decline . to the extent that the homeostatic changes in aging rbcs are driven by elevated [ ca]i levels , the effects of decline in gardos channel activity and of the minor reductions in driving force for dehydration , as quantified above , would have negligible effects on age - associated densification . 4 b ) suggests a slow and gradual na gain , with a sharp late increase leading to the formation of the light , high - na cells in a terminal homeostatic condition ( bookchin et al . , 2000 ) . the effect of age - related decline in gardos channel fmax may be relevant to the pattern of sickle cell dehydration ( lew and bookchin , 2005 ) . the interaction of the polymers of deoxygenated haemoglobin s with the membrane of sickle cells activates a poorly selective cation permeability pathway , psickle , with a stochastic distribution of ca permeabilities in the rbc population in each deoxygenation event ( lew et al . , 1997 ) . the variably elevated [ ca]i activates gardos channels , triggering rbc dehydration and acidification . in sickle reticulocytes and young sickle cells , which retain a high activity of acid - sensitive k : cl cotransporters ( brugnara et al . , 1986 ) , gardos - induced acidification stimulates further k : cl - mediated dehydration , segregating sickle cells into slow- and fast - track dehydrating cells , with a predominance of young cells in the densest fraction of sickle cells , rich in irreversible sickle cells ( bookchin et al . , 1991 ; lew et al . , 1991 the age distribution of gardos channel fmax documented here for normal rbcs reveals extraordinarily high fmax values in young rbcs . these features may contribute to the increased vulnerability of young ss cells to dehydration observed by us and others , and they provide an additional mechanism for enhancing the differences between slow- and fast - track dehydrating ss rbcs .

the ca2 + -sensitive k+ channel of human red blood cells ( rbcs ) ( gardos channel , hik1 , hsk4 ) was implicated in the progressive densification of rbcs during normal senescence and in the mechanism of sickle cell dehydration . saturating rbc ca2 + loads were shown before to induce rapid and homogeneous dehydration , suggesting that gardos channel capacity was uniform among the rbcs , regardless of age . using glycated hemoglobin as a reliable rbc age marker , we investigated the age activity relation of gardos channels by measuring the mean age of rbc subpopulations exceeding a set high density boundary during dehydration . when k+ permeabilization was induced with valinomycin , the oldest and densest cells , which started nearest to the set density boundary , crossed it first , reflecting conservation of the normal age density distribution pattern during dehydration . however , when ca2 + loads were used to induce maximal k+ fluxes via gardos channels in all rbcs ( fmax ) , the youngest rbcs passed the boundary first , ahead of the older rbcs , indicating that gardos channel fmax was highest in those young rbcs , and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process . further analysis of the gardos channel age activity relation revealed a monotonic decline in fmax with cell age , with a broad quasi - gaussian fmax distribution among the rbcs .

INTRODUCTION MATERIALS AND METHODS Experimental Design and Procedure RESULTS DISCUSSION

a ca - sensitive k channel ( gardos channel , hik1 , or hsk4 ) is expressed in the plasma membrane of human red blood cells ( rbcs ) ( gardos , 1958 ; ishii et al . gardos channel activation plays an important role in the mechanism of sickle cell dehydration ( lew and bookchin , 2005a ) and may also participate in the physiological densification of aging rbcs ( clark , 1988 ; lutz , 1988 ) . , 1982 ) , triggering instant and maximal gardos channel activation in all the cells ( fmax at saturating ca loads ) , the distribution of dehydration rates in the rbc population , as followed by flow cytometry , proceeded to lower mean volumes with no significant variation in standard deviation or appearance of multimodality ( lew et al . the apparent uniformity of gardos channel capacity among the rbcs was surprising because it did not reveal the expected decrease in driving force for dehydration among the aging , denser rbcs , which would be seen as a tail of slower dehydrating cells in the migrating gaussian curves . to detect age - related variations in gardos channel activity among rbcs , we designed experimental conditions in which the rbc dehydration rates were determined by the gardos channel fmax and by the driving gradient for dehydration in each cell , and the relative ages of the fastest and slowest dehydrating rbcs were assessed by measuring their hb a1c contents . to detect age - related variations in gardos channel activity among rbcs , we designed experimental conditions in which the rbc dehydration rates were determined by the gardos channel fmax and by the driving gradient for dehydration in each cell , and the relative ages of the fastest and slowest dehydrating rbcs were assessed by measuring their hb a1c contents . 1 are in striking contrast with those expected from the known age density distribution of rbcs , and from the assumption that rbcs of all ages would have comparable gardos channel fmax activity , which would have been the simplest explanation of the observed conservation of the volume distribution profile during dehydration ( lew et al . , 2000 ; those findings led to the suggestion ( predicted ) that dehydration rates were similar in all the rbcs , with conservation of the original rbc age distribution within the migrating gaussians ( predicted ) . by contrast , for gardos channel mediated k permeabilization , the results suggest that conservation of the standard deviation conceals a substantial degree of age scrambling within the migrating gaussian curves ( observed ) , with the younger rbcs passing the older cells that started as most dense in the initial condition ; thus the rbcs that first pass the set density boundary are much younger than those reaching it last , reflecting a declining pattern of gardos fmax with rbc age . three main factors determine the speed at which k - permeabilized rbcs will traverse the high - density boundary in our experiments ( = 1.117 g / ml ) : ( 1 ) the initial density of each rbc determines its starting distance to the density boundary , ( 2 ) the strength of the k gradient provides the driving force for dehydration , which is progressively weaker the higher the individual cell 's na / k concentration ratio , which increases with cell age ( bookchin et al . to discriminate between the contributions of ion gradient dissipation and of gardos channel fmax to the observed pattern of rbc dehydration , we sought first to subject the rbcs to a uniform , high k permeability , bypassing gardos channel activation . thus , following uniform k permeabilization with valinomycin , the oldest and densest cells pass the density boundary first , but because of their weaker driving force for dehydration they soon become admixed in the pellets with younger rbcs . thus in these conditions , dehydration rates are determined by the weak opposing effects of initial density distribution and progressively reduced driving gradient , whereas with ca - induced dehydration it is the variation of gardos channel fmax among the rbcs that dominates the dehydration rates , generating an inverse age density distribution ( fig . for a rough estimate of the gardos channel fmax distribution , we assume that the time it takes for any rbc to reach the boundary density is determined primarily by its gardos channel fmax , and that its initial density and the status of its driving gradient have negligible effects . ( f ) using time as the parametric variable , the percent hb a1c in the rbcs from e is plotted as a function of pk , from b. with caution , given the simplifying assumptions inherent in this analysis and the unavoidable age mix in the samples , these results show that the gardos channel fmax declines monotonically with rbc age ( fig . the effect of age - related decline in gardos channel fmax may be relevant to the pattern of sickle cell dehydration ( lew and bookchin , 2005 ) . , 1986 ) , gardos - induced acidification stimulates further k : cl - mediated dehydration , segregating sickle cells into slow- and fast - track dehydrating cells , with a predominance of young cells in the densest fraction of sickle cells , rich in irreversible sickle cells ( bookchin et al .

every year worldwide over 1.6 million people are diagnosed with lung cancer and over 1.3 million patients die from this disease.1 there is an urgent need to develop new therapeutic drugs and new drug delivery systems with higher activity against lung cancer and lower side effects than clinically used drugs . diferuloylmethane ( dfm ) , a natural polyphenolic extract of turmeric , definitely shows inhibitory activity against many cancer cells , including those of the lung , liver , stomach , ovaries , breast , bladder , head , and neck.2,3 meanwhile , dfm has shown almost no toxicity against normal cells and dose - limiting toxicity in all animal tests and human clinical trials.4 it seems very reasonable , then , that dfm is regarded as one of the most promising antitumor drugs , even though it has not been available clinically so far . the clinical translation and application of dfm may be severely confined , mainly by its very low solubility . molecular inclusion complexes often refer to special complex compounds in which the molecules of one component ( guest molecules ) are contained wholly or partially within the cavity structure of the other component ( host molecules ) . the cavity nature ( a hydrophilic opening and a hydrophobic interior ) of the molecules of cyclodextrin or their derivative ( the most frequently used host molecules ) defines the way in which these molecules form complexes with low - molecular - weight hydrophobic drugs . although molecular inclusion complex technology is already used to increase the solubility of different types of lipophilic drugs5,6 and the bioactivities ( such as ocular anti - inflammatory,7 anticervical cancer,8 antibreast cancer,9 antileukemia,10 and antiepileptic activities11 ) of a few drugs , so far only a few studies have employed it to improve the antilung cancer activity of lipophilic drugs . that is to say , further efforts need to be made before the potential for a molecular inclusion complex to be a good therapeutic antilung cancer drug delivery system can be judged . furthermore , considering that hydroxypropyl--cyclodextrin ( hbcd ) has better biocompatibility and higher water solubility than cyclodextrin or other cyclodextrin derivatives,12 a molecular inclusion complex consisting of dfm and hbcd ( micdh ) is worth exploring . micdh may have higher solubility than , and enhanced antilung cancer activity compared with , free dfm . in the experiments outlined in this article , an optimized formulation of micdh and the optimal process parameters for the preparation of micdh were investigated . the aim of this study was to explore and evaluate the enhanced physical properties and antitumor activity of micdh , including its solubility , in vitro release and model fitting , microscopic morphology , molecular structure simulation , antilung cancer activity , and action mechanisms . hbcd was purchased from xinxin pharmaceutical excipients co , ltd ( taixing , china ) . roswell park memorial institute 1640 medium containing 10% fetal bovine serum was purchased from life technologies ( carlsbad , ca ) . methylthiazol tetrazolium , propidium iodide , and ribonuclease a were purchased from sigma - aldrich ( st louis , mo ) . fluo-3 am , dcfh , and rhodamine 123 were purchased from beyotime institute of biotechnology ( shanghai , china ) . all other reagents and solvents used in this study were of analytical grade . weighted hbcd and dfm were mixed in a mortar . the mixture was kneaded , with an appropriate amount of distilled water intermittently added during this kneading process until the mixture formed a slurry . the mixture was then placed in a water bath , where the reaction temperature was controlled and the mixture became a paste . the paste was then placed in a vacuum drying oven at 35c for 3 hours and then in a desiccator chamber at 20c for another 24 hours . finally , the dried paste was crushed into a powder.13 results of the preliminary experiments revealed that the factors of the dfm - to - hbcd molar ratio ( mol : mol ) , preparation temperature ( in degrees celsius ) , and kneading time ( in hours ) had a relatively strong influence on the solubility . thus , a three - factor , three - level orthogonal experimental design was used to find the optimal levels of each factor ( table 1).14 in order to examine the influence of the complex formulation on the solubility , various formulations of micdh were prepared under different preparation conditions according to the orthogonal design . dfm concentrations were determined with an ultraviolet - visible spectrophotometer ( uv-3150 ; shimadzu corporation , kyoto , japan ) . as shown in figure 1 , hbcd served as a blank reference and did not interfere with the determination of dfm . the calibration curve was linear over the range of 1.15.5 g / ml ( a = 0.1493c + 0.0058 ; r = 0.9999 ; n = 7 ; a refers to the absorbance of dfm at 425 nm , and the intraday and interday relative standard derivation ranged from 0.35% to 1.23% . the mean recovery of dfm was 99.89% 0.41% the ultraviolet - visible method provided an assay that was both sensitive and specific for quantifying dfm . the morphology and the structure of all powder samples ( dfm , hbcd , physical mixture , and micdh ) were separately investigated using biomicroscopy ( xsp-35 - 1600x ; phoenix , shangrao , china ) , and photomicrographs were taken at suitable magnifications ( c-60 zoom ; olympus corporation , tokyo , japan ) . the schematic structure of micdh was further simulated according to the dfm - hpcd molecular inclusion complex with 1:1 stoichiometry , using stick and space - filling models . the studies were performed using a modified dialysis method.15 briefly , micdh containing 10 mg of dfm was loaded into dialysis bags and these were soaked in the release medium of either 0.1 mol / l hydrochloric acid ( hcl ) or ph 6.8 phosphate - buffered saline ( pbs ) containing 2% sodium dodecyl sulfate ( sds).13,16 this dissolution study was run at 100 rpm , and the dissolution vessel was maintained at a mean temperature of 37c 0.5c . aliquots ( 1.0 ml ) of the sample were withdrawn at designated time intervals . to maintain a constant volume , the quantitative determination of dfm was performed with a spectrophotometer and the cumulative release rate was then calculated . the procedures were applied to three batches of micdh and free dfm.16 the classic similarity factor ( f2 ) method was chosen to evaluate the similarity between two release profiles . the f2 value was calculated according to the following equation ( where f2 was the similarity factor , xii and xri were the cumulative amount of drug released at time t of two release profiles , n was the number of sampling points , and wt was the weight ) , equation ( 1 ) : the f2 value could be classified into one of the following levels : ( 1 ) equal to 100 , indicating two release curves were almost exactly the same ; ( 2 ) between 50 and 100 , indicating that the similarity between two release curves was statistically significant ; and ( 3 ) lower than 50 , indicating that the similarity was not statistically significant.17 human lung adenocarcinoma a549 cells were seeded at a density of 1 10 cells per well in 96-well plates for 24 hours before the culture media were replaced with fresh media containing 10 g / ml of micdh . after 24 hours , the media were exchanged with fresh media containing 5 mg / ml methylthiazol tetrazolium solution . the absorbance values were then measured at 570 nm with a microplate photometer ( sunrise ; tecan trading ag , salzburg , austria ) . furthermore , a549 cells cultured in 25 cm flasks were treated with 10 g / ml of micdh for 24 hours . cells were harvested by trypsinization and centrifugation and were then fixed with 70% ethanol at 4c overnight . after being rinsed twice with pbs , cells were resuspended in a dna - staining solution containing 40 g / ml propidium iodide and 0.1 mg / ml ribonuclease a at 25c for 30 minutes . the cell cycle was analyzed with a facsvantage flow cytometer ( becton , dickinson and company , san jose , ca ) equipped with cellquest software ( becton , dickinson and company ) . apoptotic cell quantification was determined using an annexin v - fitc / pi apoptosis detection kit ( tianjin sungene biotech co , ltd , tianjin , china ) . a549 cells treated with 10 g / ml of micdh for 24 hours were collected , centrifuged , and resuspended in a staining solution containing one kind of 5 mol / l fluorescent dye ( dcfh , fluo-3 am , or rhodamine 123 ) at 37c for 30 minutes . the reactive oxygen species ( ros ) and intracellular free calcium ion ( ca ) levels and the mitochondrial membrane potential unless otherwise specified , all data are shown as the mean plus or minus the standard deviation . one - way analysis of variance , scheff s f - test , and student s t - test were used for statistical analysis to determine significant differences . analyses were performed using the statview statistical package ( v 5.0 ; sas institute , inc , cary , nc).18 the mixture was kneaded , with an appropriate amount of distilled water intermittently added during this kneading process until the mixture formed a slurry . the mixture was then placed in a water bath , where the reaction temperature was controlled and the mixture became a paste . the paste was then placed in a vacuum drying oven at 35c for 3 hours and then in a desiccator chamber at 20c for another 24 hours . finally , the dried paste was crushed into a powder.13 results of the preliminary experiments revealed that the factors of the dfm - to - hbcd molar ratio ( mol : mol ) , preparation temperature ( in degrees celsius ) , and kneading time ( in hours ) had a relatively strong influence on the solubility . thus , a three - factor , three - level orthogonal experimental design was used to find the optimal levels of each factor ( table 1).14 in order to examine the influence of the complex formulation on the solubility , various formulations of micdh were prepared under different preparation conditions according to the orthogonal design . dfm concentrations were determined with an ultraviolet - visible spectrophotometer ( uv-3150 ; shimadzu corporation , kyoto , japan ) . as shown in figure 1 , hbcd served as a blank reference and did not interfere with the determination of dfm . the calibration curve was linear over the range of 1.15.5 g / ml ( a = 0.1493c + 0.0058 ; r = 0.9999 ; n = 7 ; a refers to the absorbance of dfm at 425 nm , and . the ultraviolet - visible method provided an assay that was both sensitive and specific for quantifying dfm . the morphology and the structure of all powder samples ( dfm , hbcd , physical mixture , and micdh ) were separately investigated using biomicroscopy ( xsp-35 - 1600x ; phoenix , shangrao , china ) , and photomicrographs were taken at suitable magnifications ( c-60 zoom ; olympus corporation , tokyo , japan ) . the schematic structure of micdh was further simulated according to the dfm - hpcd molecular inclusion complex with 1:1 stoichiometry , using stick and space - filling models . the studies were performed using a modified dialysis method.15 briefly , micdh containing 10 mg of dfm was loaded into dialysis bags and these were soaked in the release medium of either 0.1 mol / l hydrochloric acid ( hcl ) or ph 6.8 phosphate - buffered saline ( pbs ) containing 2% sodium dodecyl sulfate ( sds).13,16 this dissolution study was run at 100 rpm , and the dissolution vessel was maintained at a mean temperature of 37c 0.5c . aliquots ( 1.0 ml ) of the sample were withdrawn at designated time intervals . to maintain a constant volume , the quantitative determination of dfm was performed with a spectrophotometer and the cumulative release rate was then calculated . the procedures were applied to three batches of micdh and free dfm.16 the classic similarity factor ( f2 ) method was chosen to evaluate the similarity between two release profiles . the f2 value was calculated according to the following equation ( where f2 was the similarity factor , xii and xri were the cumulative amount of drug released at time t of two release profiles , n was the number of sampling points , and wt was the weight ) , equation ( 1 ) : the f2 value could be classified into one of the following levels : ( 1 ) equal to 100 , indicating two release curves were almost exactly the same ; ( 2 ) between 50 and 100 , indicating that the similarity between two release curves was statistically significant ; and ( 3 ) lower than 50 , indicating that the similarity was not statistically significant.17 human lung adenocarcinoma a549 cells were seeded at a density of 1 10 cells per well in 96-well plates for 24 hours before the culture media were replaced with fresh media containing 10 g / ml of micdh . after 24 hours , the media were exchanged with fresh media containing 5 mg / ml methylthiazol tetrazolium solution . the absorbance values were then measured at 570 nm with a microplate photometer ( sunrise ; tecan trading ag , salzburg , austria ) . furthermore , a549 cells cultured in 25 cm flasks were treated with 10 g / ml of micdh for 24 hours . cells were harvested by trypsinization and centrifugation and were then fixed with 70% ethanol at 4c overnight . after being rinsed twice with pbs , cells were resuspended in a dna - staining solution containing 40 g / ml propidium iodide and 0.1 mg / ml ribonuclease a at 25c for 30 minutes . the cell cycle was analyzed with a facsvantage flow cytometer ( becton , dickinson and company , san jose , ca ) equipped with cellquest software ( becton , dickinson and company ) . apoptotic cell quantification was determined using an annexin v - fitc / pi apoptosis detection kit ( tianjin sungene biotech co , ltd , tianjin , china ) . a549 cells treated with 10 g / ml of micdh for 24 hours were collected , centrifuged , and resuspended in a staining solution containing one kind of 5 mol / l fluorescent dye ( dcfh , fluo-3 am , or rhodamine 123 ) at 37c for 30 minutes . the reactive oxygen species ( ros ) and intracellular free calcium ion ( ca ) levels and the mitochondrial membrane potential were then analyzed using the facsvantage flow cytometer . unless otherwise specified , all data are shown as the mean plus or minus the standard deviation . one - way analysis of variance , scheff s f - test , and student s t - test were used for statistical analysis to determine significant differences . analyses were performed using the statview statistical package ( v 5.0 ; sas institute , inc , cary , nc).18 in this study , the optimal levels of each factor were found to be a1b2c3 ; that is , the optimal values for the dfm - to - hbcd molar ratio ( represented by a ) , the preparation temperature ( in degrees celsius and represented by b ) , and the kneading time ( in hours and represented by c ) were found to be 1:1 , 40c , and 1.5 hours , respectively ( see table 1 ) . the solubility of micdh prepared under the optimal protocol described was recorded as 1.76 0.03 mg / ml . micdh was much more soluble than free dfm ( 40 ng / ml ; n = 3 ) . as shown in figure 2 , under the optical microscope , the micdh powders ( ie , the micdh aggregates ) appeared irregular and amorphous , and they were much larger than the free dfm or hbcd aggregates , especially the former . the hbcd aggregates appeared spherical in shape , while the physical mixture showed simple additive effects of free dfm and hbcd . figure 3 shows the schematic structure of micdh simulated according to the dfm - hbcd complex with 1:1 stoichiometry , using stick and space - filling models . the drug release profiles of micdh and free dfm containing the same amount of dfm in two release media ( 0.1 mol / l hcl and ph 6.8 pbs containing 2% sds ) are shown in figure 4 . the release rates of dfm from micdh increased obviously compared with those from free dfm in every release medium . the cumulative release rates for micdh in the two release media over 10 hours were more than 35% , while the cumulative release rates for free dfm were zero . furthermore , rates of micdh and free dfm were ~55% versus ( vs ) ~10% in 48 hours , and ~80% vs < 30% in 120 hours , respectively . as shown in table 2 , several mathematical models were used to fit the micdh release data.17 the results suggest that both the first - order kinetic model and the higuchi model could be used to fit the release data well in the release media of 0.1 mol / l hcl and ph 6.8 pbs containing 2% sds . different f2 values indicated whether two release profiles shared statistically significant similarity . when the value was above 50 , the answer was yes ; otherwise , the answer was no . as shown in table 3 , the f2 values between two release curves in ph 6.8 pbs and 0.1 mol / l hcl of micdh or free dfm were above 50 ( ~59 and ~96 , respectively ) , while the f2 values between two release curves of micdh and free dfm in ph 6.8 pbs or 0.1 mol / l hcl were much lower than 50 ( ~21 and ~22 , respectively ) . as shown in figure 5 , both micdh and free dfm inhibited human lung adenocarcinoma a549 cell growth in a dose - dependent manner . the mean 50% inhibitory concentration ( the drug concentration causing a 50% decrease in cell survival ) value of micdh ( 8.50 g / ml ) decreased by almost a third , compared with that of free dfm ( 12.53 g / ml ) , suggesting that micdh exhibited a higher antitumor effect than free dfm . tumor cells treated with 10 g / ml of either micdh or free dfm for 24 hours were further analyzed to help better understand the mechanisms involved . as shown in figure 6 , more cells were arrested in the s / g2 phase of the cell cycle ( ~56% vs ~46% ) or were induced to undergo apoptosis ( ~99% vs ~55% ) when treated with micdh than when treated with free dfm . as shown in figure 7 , ros and intracellular ca levels in cells treated with micdh were much higher than in those treated with free dfm ( p < 0.05 ) , while the mitochondrial membrane potential in cells treated with micdh was only a little lower than in those treated with free dfm ( p > 0.05 ) . in this study , the optimal levels of each factor were found to be a1b2c3 ; that is , the optimal values for the dfm - to - hbcd molar ratio ( represented by a ) , the preparation temperature ( in degrees celsius and represented by b ) , and the kneading time ( in hours and represented by c ) were found to be 1:1 , 40c , and 1.5 hours , respectively ( see table 1 ) . the solubility of micdh prepared under the optimal protocol described was recorded as 1.76 0.03 mg / ml . micdh was much more soluble than free dfm ( 40 ng / ml ; n = 3 ) . as shown in figure 2 , under the optical microscope , the micdh powders ( ie , the micdh aggregates ) appeared irregular and amorphous , and they were much larger than the free dfm or hbcd aggregates , especially the former . the hbcd aggregates appeared spherical in shape , while the physical mixture showed simple additive effects of free dfm and hbcd . figure 3 shows the schematic structure of micdh simulated according to the dfm - hbcd complex with 1:1 stoichiometry , using stick and space - filling models . the drug release profiles of micdh and free dfm containing the same amount of dfm in two release media ( 0.1 mol / l hcl and ph 6.8 pbs containing 2% sds ) are shown in figure 4 . the release rates of dfm from micdh increased obviously compared with those from free dfm in every release medium . the cumulative release rates for micdh in the two release media over 10 hours were more than 35% , while the cumulative release rates for free dfm were zero . furthermore , rates of micdh and free dfm were ~55% versus ( vs ) ~10% in 48 hours , and ~80% vs < 30% in 120 hours , respectively . as shown in table 2 , several mathematical models were used to fit the micdh release data.17 the results suggest that both the first - order kinetic model and the higuchi model could be used to fit the release data well in the release media of 0.1 mol / l hcl and ph 6.8 pbs containing 2% sds . different f2 values indicated whether two release profiles shared statistically significant similarity . when the value was above 50 , the answer was yes ; otherwise , the answer was no . as shown in table 3 , the f2 values between two release curves in ph 6.8 pbs and 0.1 mol / l hcl of micdh or free dfm were above 50 ( ~59 and ~96 , respectively ) , while the f2 values between two release curves of micdh and free dfm in ph 6.8 pbs or 0.1 mol / l hcl were much lower than 50 ( ~21 and ~22 , respectively ) . as shown in figure 5 , both micdh and free dfm inhibited human lung adenocarcinoma a549 cell growth in a dose - dependent manner . the mean 50% inhibitory concentration ( the drug concentration causing a 50% decrease in cell survival ) value of micdh ( 8.50 g / ml ) decreased by almost a third , compared with that of free dfm ( 12.53 g / ml ) , suggesting that micdh exhibited a higher antitumor effect than free dfm . tumor cells treated with 10 g / ml of either micdh or free dfm for 24 hours were further analyzed to help better understand the mechanisms involved . as shown in figure 6 , more cells were arrested in the s / g2 phase of the cell cycle ( ~56% vs ~46% ) or were induced to undergo apoptosis ( ~99% vs ~55% ) when treated with micdh than when treated with free dfm . as shown in figure 7 , ros and intracellular ca levels in cells treated with micdh were much higher than in those treated with free dfm ( p < 0.05 ) , while the mitochondrial membrane potential in cells treated with micdh was only a little lower than in those treated with free dfm ( p > 0.05 ) . according to statistics , ~70% of new drug candidates and ~40% of oral drugs on the market are poorly soluble in water.19 the formulation of poorly soluble drugs for oral delivery has always presented a big challenge . poor aqueous solubility often leads to poor bioavailability and low bioactivity , so the first and most important point to be addressed for the practically insoluble drug dfm was the development of a suitable dfm formulation with better solubility than free dfm . as far as the methods to improve the solubility of low - solubility drugs are concerned , the molecular inclusion complexation technology can be as effective as other approaches such as crystal modification , self - emulsification , and nanogranulation or more so , while also being much simpler and easier for production scale - up.2022 in the preliminary experiments , several preparation methods ( eg , coprecipitation and sonication methods ) and the type of host molecules ( eg , - and -cyclodextrin ) were screened ( data not shown ) . following this , hbcd were chosen to prepare the micdh by kneading method . three critical factors ( the dfm - to - hbcd molar ratio , the preparation temperature , and the kneading time ) that had relatively strong influence on solubility were investigated . an orthogonal design was used to optimize the formulation and preparation process of micdh the orthogonal design , one of the most used designs in medical and pharmaceutical fields , can reflect the principal components associated with each experimental factor.23 the formulation with the highest solubility was then obtained . encouragingly , the solubility of micdh prepared under optimal conditions was 4400 times that of free dfh . it has been documented that the solubility of brominated noscapine ( antiprostate cancer drug ) could be improved approximately 11- and 21-fold upon complexation with -cyclodextrin and methyl--cyclodextrin , respectively,24 while the solubility of 9-nitrocamptothecin ( antiliver cancer drug ) could be improved 104-fold upon complexation with hbcd.25 the formation process of a molecular inclusion complex was a physical rather than a chemical process . a drug molecule was able to enter the host molecular cavity and was held there by intermolecular forces such as van der waals force and hydrogen bonding rather than covalent bonding . the formation of a molecular inclusion complex was mainly relative to three - dimensional structures and polarity.26 the drug molecule , having a suitable size and shape corresponding to that of the cavity , was usually easy to encapsulate within the host molecule . on the other hand , a guest molecule was able to enter and exit a host cavity in a dynamic process . the stability of a molecular inclusion complex mainly depended on the polarities of the guest and host molecules and on the strength of the intermolecular force . based on the information outlined here , it was easy to determine why the solubilizing effects of different molecular inclusion complexes were not the same . as expected , compared with free dfm , the release rates of dfm from micdh increased obviously in different release media ( 0.1 mol / l hcl or ph 6.8 pbs containing 2% sds ) . this f2 method , which is recommended by the us food and drug administration to evaluate the similarity between two dissolution profiles , was simple and reliable . as shown in table 3 , the f2 value calculated to be ~59 suggested that the similarity between two release curves for micdh in ph 6.8 pbs and 0.1 mol / l hcl was statistically significant . the fact that the release rate in ph 6.8 pbs was higher than the release rate in 0.1 mol / l hcl may be ascribed to the existence of the phenolic hydroxyl group in the dfm and the slow release of dfm from micdh . on the other hand , since there was a significant difference between two curves when the f2 value was below 50 , the difference between the curves of micdh and free dfm in each release medium was statistically significant . being an alternative system for the oral delivery of a water insoluble - drug , micdh could be expected to have better absorption and bioavailability than free dfm because of faster release . in clinical practice , this type of formulation may provide a faster , better , and longer duration of anticancer action than that provided by free dfm . the results from this study confirm that the release rates of dfm ( a practically water - insoluble drug ) could be controlled by molecular inclusion complexation with hbcd ( a biodegradable macromolecular excipient ) . micdh may be used as a carrier for the enhanced release of dfm in cancer therapy . apart from increasing solubility , micdh improved the antitumor activity against human lung adenocarcinoma a549 cells compared with free dfm . similar results have been reported in previous studies.11,12 for example , the anticancer activity of s1 ( a structure - specific b - cell lymphoma-2 inhibitor ) against human breast adenocarcinoma mcf-7 cells could be enhanced by complexation with -cyclodextrin;11 another example is the anticancer activity against leukemia cells of bis(tbutyl - s - acyl-2-thioethyl)-cytidine monophosophate , which could be enhanced by complexation with hbcd.12 the results of this study also showed that micdh had higher antitumor activity than free dfm . this may be because of the improved solubility and release of micdh , since micdh in aqueous solution favored the entrance of dfm into the cell and act . first , micdh induced s / g2 cell cycle modifications on human lung adenocarcinoma a549 cells ; an increased accumulation of cells at the s / g2 phase after micdh contact was found compared with that after free dfm contact.27 second , incubation of a549 cells with micdh for 24 hours caused an obvious increase in the proportion of apoptotic cells compared with incubation with free dfm.28 the increased induction of apoptosis by micdh in human lung adenocarcinoma a549 cells , compared with that by free dfm , may be due to the increases of ros29 and intracellular ca concentration30 and the decrease of mitochondrial membrane potential.31 a previous study has shown that dfm can inhibit tumor cell growth via different mechanisms other than those mentioned here.32 more efforts are needed in regard to understanding the mechanisms responsible for improvement of the antitumor activity of micdh . the results of this study indicate that micdh enhanced the physical properties and antitumor activity of dfm remarkably . an optimized molecular inclusion complex of dfm was successfully developed to greatly improve the physical properties and antitumor activity of dfm . the release rates of dfm from micdh were significantly higher than those from free dfm . micdh is a promising alternative to free drm as a formulation for dfm delivery in lung cancer treatment .

objectivethe purpose of this study was to explore and evaluate the enhanced physical properties and biological activity of a molecular inclusion complex ( micdh ) comprising diferuloylmethane ( dfm ) and hydroxypropyl--cyclodextrin.methodsthe preparation conditions of micdh were optimized using an orthogonal experimental design . the solubility , in vitro release and model fitting , microscopic morphology , molecular structure simulation , anti - lung cancer activity , and action mechanism of micdh were evaluated.resultsthe solubility of dfm was improved 4400-fold upon complexation with hydroxypropyl--cyclodextrin . the release rate of dfm was significantly higher from micdh than from free dfm . micdh exhibited higher antitumor activity against human lung adenocarcinoma a549 cells than free dfm . more cells were arrested in the s / g2 phase of the cell cycle or were induced to undergo apoptosis when treated with micdh than when treated with free dfm . furthermore , increased reactive oxygen species and intracellular calcium ion levels and decreased mitochondrial membrane potential were observed in cells treated with micdh.conclusionmicdh markedly improved the physical properties and antitumor activity of dfm . micdh may prove to be a preferred alternative to free dfm as a formulation for dfm delivery in lung cancer treatment .

Introduction Materials and methods Preparation and optimization of MICDH Morphology and structure In vitro release rate studies Antitumor activity of MICDH Statistical analysis Results Optimized formulation and preparation of MICDH Characterization of MICDH In vitro release studies Antitumor activities of MICDH Discussion Conclusion

the aim of this study was to explore and evaluate the enhanced physical properties and antitumor activity of micdh , including its solubility , in vitro release and model fitting , microscopic morphology , molecular structure simulation , antilung cancer activity , and action mechanisms . thus , a three - factor , three - level orthogonal experimental design was used to find the optimal levels of each factor ( table 1).14 in order to examine the influence of the complex formulation on the solubility , various formulations of micdh were prepared under different preparation conditions according to the orthogonal design . the f2 value was calculated according to the following equation ( where f2 was the similarity factor , xii and xri were the cumulative amount of drug released at time t of two release profiles , n was the number of sampling points , and wt was the weight ) , equation ( 1 ) : the f2 value could be classified into one of the following levels : ( 1 ) equal to 100 , indicating two release curves were almost exactly the same ; ( 2 ) between 50 and 100 , indicating that the similarity between two release curves was statistically significant ; and ( 3 ) lower than 50 , indicating that the similarity was not statistically significant.17 human lung adenocarcinoma a549 cells were seeded at a density of 1 10 cells per well in 96-well plates for 24 hours before the culture media were replaced with fresh media containing 10 g / ml of micdh . the reactive oxygen species ( ros ) and intracellular free calcium ion ( ca ) levels and the mitochondrial membrane potential were then analyzed using the facsvantage flow cytometer . as shown in figure 6 , more cells were arrested in the s / g2 phase of the cell cycle ( ~56% vs ~46% ) or were induced to undergo apoptosis ( ~99% vs ~55% ) when treated with micdh than when treated with free dfm . as shown in figure 7 , ros and intracellular ca levels in cells treated with micdh were much higher than in those treated with free dfm ( p < 0.05 ) , while the mitochondrial membrane potential in cells treated with micdh was only a little lower than in those treated with free dfm ( p > 0.05 ) . as shown in figure 6 , more cells were arrested in the s / g2 phase of the cell cycle ( ~56% vs ~46% ) or were induced to undergo apoptosis ( ~99% vs ~55% ) when treated with micdh than when treated with free dfm . as shown in figure 7 , ros and intracellular ca levels in cells treated with micdh were much higher than in those treated with free dfm ( p < 0.05 ) , while the mitochondrial membrane potential in cells treated with micdh was only a little lower than in those treated with free dfm ( p > 0.05 ) . apart from increasing solubility , micdh improved the antitumor activity against human lung adenocarcinoma a549 cells compared with free dfm . similar results have been reported in previous studies.11,12 for example , the anticancer activity of s1 ( a structure - specific b - cell lymphoma-2 inhibitor ) against human breast adenocarcinoma mcf-7 cells could be enhanced by complexation with -cyclodextrin;11 another example is the anticancer activity against leukemia cells of bis(tbutyl - s - acyl-2-thioethyl)-cytidine monophosophate , which could be enhanced by complexation with hbcd.12 the results of this study also showed that micdh had higher antitumor activity than free dfm . first , micdh induced s / g2 cell cycle modifications on human lung adenocarcinoma a549 cells ; an increased accumulation of cells at the s / g2 phase after micdh contact was found compared with that after free dfm contact.27 second , incubation of a549 cells with micdh for 24 hours caused an obvious increase in the proportion of apoptotic cells compared with incubation with free dfm.28 the increased induction of apoptosis by micdh in human lung adenocarcinoma a549 cells , compared with that by free dfm , may be due to the increases of ros29 and intracellular ca concentration30 and the decrease of mitochondrial membrane potential.31 a previous study has shown that dfm can inhibit tumor cell growth via different mechanisms other than those mentioned here.32 more efforts are needed in regard to understanding the mechanisms responsible for improvement of the antitumor activity of micdh . the results of this study indicate that micdh enhanced the physical properties and antitumor activity of dfm remarkably . an optimized molecular inclusion complex of dfm was successfully developed to greatly improve the physical properties and antitumor activity of dfm . micdh is a promising alternative to free drm as a formulation for dfm delivery in lung cancer treatment .

long - term health is profoundly influenced by environmental conditions experienced early in life . the ability to respond to environmental cues , or plasticity , is critical to survival but can result in adaptations that influence disease risk later in life . we previously demonstrated that a high protein diet consumed from weaning to four months of age predisposed rats to greater obesity risk in adulthood following a metabolic challenge with a high energy diet . body weight , fat mass and glycemia in adult males was higher following a high fat / sucrose challenge in rats that consumed a high protein versus a high prebiotic fiber diet from weaning . plasma concentrations of the anorexigenic hormone , glucagon - like peptide-1 ( glp-1 ) , were higher in the high prebiotic compared to high protein - fed rats , and leptin was elevated in high protein versus high prebiotic rats . whether recovery from the high fat / sucrose diet in terms of body fat and satiety hormones is better in rats consuming a long - term high protein versus high fiber diet is not known . consumption of a high fat diet is known to produce numerous detrimental metabolic effects including excess adiposity , insulin resistance and leptin resistance . what is less well understood is whether a transient high fat diet is less detrimental when introduced intermittently into a long - term dietary pattern high in protein or dietary fiber . a systematic evaluation of the metabolic response to a transient high fat diet is warranted . while it is common to measure a limited number of metabolites in serum , such as glucose , in response to dietary intervention or in disease states such as diabetes , it is now possible to measure global metabolic response using proton nuclear magnetic resonance - based metabolomic ( h nmr ) analysis . nmr has the advantage of providing the simultaneous quantitative measurement of many metabolites that could provide a more complete picture of metabolic response to diet and provide novel information that can be used to probe unrecognized mechanisms . therefore , our objective was to examine the metabolomic profile of adult wistar rats exposed to a temporary high fat / sucrose diet in the context of a long - term high protein or high fiber diet . body composition and satiety hormone response during an oral glucose tolerance test were also measured . ethical approval was obtained from the university of calgary health sciences animal care committee and was consistent with the national research council s guide for the care and use of laboratory animals . female wistar rats were obtained from charles river ( montreal , pq , canada ) and housed in a temperature and humidity controlled room with a 12-h light , 12-h dark cycle . after a period of acclimatization , females were mated with wistar males in wire - bottom cages . on the day a copulation plug was found , the females were isolated and given free access to control diet ( ain-93 g ) . one day following birth , the litters were culled to 10 pups ( 5 males and 5 females where possible ) to minimize differences in feeding between litters . at weaning ( 21 d ) , the male rats were randomized to one of 3 experimental diets : control ( c ) , high fiber ( hf , 21% wt / wt ) and high protein ( hp , 40% wt / wt ) . details of the composition of the diets have been previously published . a combination of the prebiotic fibers , inulin and oligofructose , at a ratio of 1:1 ( by weight ) were used in the hf diet . rats consumed these diets until 15 weeks of age when all rats were given a high fat / high sucrose ( hfs ) diet for 6 weeks . the hfs diet provided 40% of energy from fat and 45% from sucrose and was composed of ( g/100 g ) cornstarch ( 5 ) , casein ( 14 ) , sucrose ( 51 ) , soybean oil ( 10 ) , lard ( 10 ) , alphacel ( 5 ) , ain-93 m mineral mix ( 3.5 ) , ain-93 vitamin mix ( 1 ) , l - cystine ( 0.3 ) , and choline bitartrate ( 0.25 ) . after 6 weeks of hfs consumption , the rats were placed back on their respective weaning diet for an additional 4 weeks . diets met all nutritional requirements of growing rats based on ain-93 g recommendations , and those of adult rats for maintenance once the rats reached 10 weeks ( ain-93 m ) . rats were individually housed , and food and water provided ad libitum throughout the experiment . food intake was measured throughout the study by subtracting the weight of the feed cup from the previous days weight . energy intake was calculated by multiplying food intake by the energy density of each diet ( i.e. , 3.7 kcal / g for c and hp , and 3.3 kcal / g for hf ) . at the end of the study , body composition was measured ( fat mass , lean mass , bone mineral content ( bmc ) , and bone mineral density ( bmd ) ) using dual energy x - ray absorptiometry ( dxa ) with software for small animal analysis ( hologic qdr 4500 , hologic , inc . an oral glucose tolerance test ( ogtt ) was performed at the end of the study . after an overnight fast , rats were anesthetized with isoflurane , and a fasting cardiac blood sample was taken for metabolomics and glucose and satiety hormone analysis . rats were then given 50% glucose ( wt / vol ) by gavage at a dose of 2 g of glucose / kg . additional samples were taken at 15 , 30 , 60 , and 90 min postgavage according to our previous work . separate serum samples for metabolomics analysis were collected at fasting ( time = 0 ) and one postprandial time point ( t = 30 min ) . blood glucose concentrations were measured immediately using a blood glucose meter ( accu - chek blood glucose meter , laval , qc ) . blood for satiety hormone analysis was collected in tubes containing diprotinin - a ( 0.034 mg / ml of blood ; mp biomedicals , irvine , ca ) ; sigma protease inhibitor ( 1 mg / ml of blood ; sigma aldrich , oakville , on , canada ) and roche pefabloc ( 1 mg / ml of blood ; roche , mississauga , on , canada ) and then centrifuged at 1600 g for 15 min at 4 c. plasma was stored at 80 c until analysis . after the final blood sample , the rats were overanesthetized followed by an aortic cut . the liver , stomach , small intestine , cecum , and colon were weighed , and a sample was snap frozen in liquid nitrogen and then stored at 80 c . ghrelin ( active ) , amylin ( active ) , insulin , leptin , gip ( total ) , glp-1 ( active ) and pyy ( total ) concentrations were quantified using a rat gut hormone panel milliplex kit ( millipore , st . the sensitivity for the milliplex kit ( in pg / ml ) is 2 ( ghrelin ) , 20 ( amylin ) , 1 ( gip ) , 28 ( insulin ) , 27 ( leptin ) , 16 ( pyy ) and 28 ( glp-1 ) . total rna was extracted from the ileum and colon using trizol reagent ( invitrogen , carlsbad , usa ) . reverse transcription was performed with an input of 1 g of total rna using the first strand cdna synthesis kit for rt - pcr ( invitrogen , carlsbad , ca usa ) with oligo d(t)15 as a primer . real time pcr using primers for proglucagon and pyy was performed according to our previous work ( maurer et al . , 2009 ) . samples were thawed and filtered using 3-kda nanosep microcentrifuge filters , prewashed to reduce contamination . the filtrate was transferred to clean microfuge tubes ; the final sample volume ranged from 200 to 300 l . samples were brought to 650 l by addition of d2o , 130 l of phosphate buffer containing dimethyl - silapentane - sulfonate ( dss , final concentration 0.5 mm ) and 10 l of sodium azide . all nmr experiments were performed on a bruker advance 600 spectrometer ( bruker biospin , milton , canada ) operating at 600.22 mhz and equipped with a 5 mm txi probe at 298 k. all one - dimensional h nmr spectra of aqueous samples were acquired using a standard bruker noesygppr1d pulse sequence in which the residual water peak was irradiated during the relaxation delay of 1.0 s and during the mixing time of 100 ms . a total of 1024 scans were collected into 63 536 data points over a spectral width of 12 195 hz with a 90 pulse width of 10.4 s and a 5 s repetition time . a line broadening of 0.1 hz was applied to the spectra prior to fourier transformation , phasing , and baseline correction . additional two - dimensional nmr experiments were performed for the purpose of confirming chemical shift assignments , including total correlation spectroscopy ( 2d h processed spectra were imported into chenomx nmr suite software ( version 4.6 , edmonton , ab , canada ) for quantification . each compound concentration was then normalized to account for differences in sample filtration during preparation by dividing the measured concentration into the total concentration of all metabolites in that sample ( excluding glucose and lactate because of excessively large concentrations that otherwise dominate the normalization ) . differences between the diets were determined using a one - way anova with tukey s multiple comparison posthoc test . parameters with serial measurements were analyzed with a repeated measures anova [ with time as a within subject variable and diet as the between subject variable ] with bonferroni adjustment when applicable . statistical analyses were performed using spss v 17.0 software ( spss inc . , chicago , il ) . for metabolomics analysis , in addition to univariate tests , multivariate analysis was conducted using simca - p+ 12.0.1 software ( umetrics , sweden ) to better assess the concentration changes . a supervised partial least - squares discriminant analysis ( pls - da ) approach was chosen to compare the variance of metabolite concentrations between three sample classes ( three diet types : control diet , hf diet and hp diet ) . a supervised orthogonal partial least - squares analysis was used ( hf diet versus hp diet model ) for a direct comparison of the variance between diet type ( y variable ) and metabolite concentrations ( x variable ) . the results from the metabolomics analysis were also combined together with the plasma analysis of satiety hormones and other biological measurements and regressed to the diet type using o2-pls - da ( orthogonal pls - da ) . lasso regression , which is designed to handle the multivariate collinearity in high - dimensional omics this was accomplished using the gmlnet package in gnu r ( http://www.r-project.org ) . body weight over the course of the study was influenced by week ( p < 0.001 ) , diet ( p < 0.001 ) and their interaction ( p < 0.008 ) . in the first phase , rats consumed the c , hf , or hp diet from weaning until 15 weeks of age . at the end of this period the rats fed hf diet had significantly lower body weight than rats fed c and hp ( p < 0.001 , figure 1a ) . rats then consumed a hfs diet for 6 weeks as a metabolic challenge . throughout the entire 6 week period , rats fed the hf diet maintained a lower body weight than rats fed c and hp ( p < 0.01 ) . after the final 4 week period consuming their respective long - term diet , rats fed hf had lower body weight than hp but not c ( p < 0.01 ) . similar to body weight , there was a significant effect of week ( p < 0.001 ) , diet ( p < 0.03 ) and week diet ( p < 0.001 ) on energy intake . rats fed hp diet had consistently higher energy intake across all three dietary periods that was higher than c and hf at 12 weeks ( p < 0.04 , figure 1b ) and higher than hf at 21 weeks ( p < 0.02 ) . in the last 3 weeks of the study hp energy intake was higher than c ( p < 0.01 ) , which was in turn higher than hf ( p < 0.05 ) . body composition was measured at study termination , at which time rats fed hf diet had significantly lower percent body fat than hp rats ( p < 0.03 , table 1 ) . bone mineral density was lower in rats fed c and hp diet versus hf ( p < 0.05 ) . liver weight was lower ( p < 0.05 ) and cecum weight higher ( p < 0.001 ) in rats fed hf versus c and hp . body weight and energy consumption of rats rematched to control , high fiber , or high protein weaning diets following a high fat , sucrose diet challenge in adulthood . panel ( b ) represents energy intake measured throughout the 16 weeks of c , hf , or hp diet , followed by 6 weeks of high fat / sucrose diet , and 4 weeks of rematching to c , hf , or hp diet . in panel ( a ) , the * represents a difference ( p < 0.05 ) between hf versus c and hp . the represents a difference ( p < 0.05 ) among all 3 groups . in panel ( b ) , the * represents a difference ( p < 0.05 ) between hp versus c and hf . the represents a difference ( p < 0.05 ) between hf versus c and hp . the represents a difference ( p < 0.05 ) among all 3 groups . treatments with different superscript letters are significantly different ( p < 0.05 ) . at the end of the study the hf group showed significantly lower glucose auc than c ( p < 0.01 , figure 2b ) . insulin levels during the ogtt were higher in c versus hf rats at 15 and 30 min ( p < 0.02 , figure 2c ) and higher in hp rats at 90 min ( p < 0.03 ) . reflective of glucose , insulin auc was lower in rats fed hf versus c and hp ( p < 0.05 , figure 2d ) . fasting leptin was higher in rats fed c versus hf and higher in c versus hp at 30 min ( p < 0.05 , figure 2e ) . leptin auc was lower in hf versus c ( p < 0.01 , figure 2f ) . concentrations of blood glucose and plasma insulin and leptin in rats during an oral glucose tolerance test following the rematching period . panel ( a ) represents the serial values and panel ( b ) the tauc for blood glucose during the ogtt . panel ( c ) represents the serial values and panel ( d ) the tauc for plasma insulin during the ogtt . in panel ( c ) , the * represents a difference ( p < 0.05 ) between hf versus c and the a difference between hf and hp . in panel ( d ) , means with different superscripts are different ( p < 0.05 ) . panel ( e ) represents the serial values and panel ( d ) the tauc for plasma leptin during the ogtt . in panel ( e ) , the * represents a difference ( p < 0.05 ) between c versus hf and the a difference between c and hp . in panel ( f ) , means with different superscripts are different ( p < 0.05 ) . rats fed the c and hp diets had very similar profiles of glp-1 , pyy and gip secretion during the ogtt ( figure 3a f ) . rats fed the hf diet , however , had markedly higher glp-1 and pyy levels and lower gip levels compared to c and hp rats ( p < 0.01 , figure 3b , d , f ) . with the exception of the 30 min time point for glp-1 , levels of glp-1 and pyy were higher in rats fed hf versus c and hp for every time point during the ogtt ( figures 3a , c , p < 0.05 ) . rats fed the hf diet had lower levels of gip at 60 min compared to c and hp and lower than hp at 90 min ( p < 0.03 ) . concentrations of plasma glp-1 , pyy , gip , and ghrelin in rats during an oral glucose tolerance test following the rematching period . results are presented as mean sem , n = 89 per group . panel ( a ) represents the serial values and panel ( b ) the tauc for glp-1 during the ogtt . in panel ( a ) , the * represents a difference ( p < 0.05 ) between hf versus c and hp . in panel ( b ) , means with different superscripts are different ( p < 0.05 ) . panel ( c ) represents the serial values and panel ( d ) the tauc for pyy during the ogtt . in panel ( c ) , the * represents a difference ( p < 0.05 ) between hf versus c and hp . in panel ( d ) , means with different superscripts are different ( p < 0.05 ) . panel ( e ) represents the serial values and panel ( d ) the tauc for gip during the ogtt . in panel ( e ) , the * represents a difference ( p < 0.05 ) between c versus hf and hp and the a difference between hf and hp . in panel ( f ) , means with different superscripts are different ( p < 0.05 ) . panel ( g ) represents the serial values and panel ( h ) the tauc for ghrelin during the ogtt . there was an approximate 5-fold increase in pyy mrna levels in the colon of rats fed the hf diet ( supporting information , figure s1 ) , which was significantly higher than c and hp ( p < 0.05 ) . similarly , proglucagon mrna levels in the colon were 11-fold higher in rats fed the hf versus c and hp diet . in the ileum , pyy mrna levels were significantly higher in hf versus c and hp ( p = 0.002 ) , whereas the approximately 3-fold increase in proglucagon in the ileum with hf did not differ from the other groups ( p = 0.14 ) . a total of 50 metabolites were screened in serum at the end of the study . the pls - da scores plot of serum showed a significant separation of the c , hf and hp groups in the fasted state ( p < 0.05 , figure 4a ; coefficient plots shown in supporting information , figure s2 ) . this result suggests that the overall metabolic changes in metabolite levels are reflective of individual diets . the most important metabolites involved in the discrimination of the hp group in the fasted state were increases in leucine , isoleucine , isobutyrate , mannose and reductions in creatine , citrate , and serine ( figure 5a and table 2 ) . both groups were distinguished from the control group by a decrease in creatinine . in order to probe more precisely the metabolic shifts , an additional shared - and - unique structure ( sus ) analysis was conducted ( figure 6 ) . in this analysis metabolites that are altered in the same way in hp and hf ( interestingly , changes in levels of the short chain fatty acids ( scfa ) acetate and an unidentified scfa were elevated in the hf group ( positive coefficients on x - axis ) , while having coefficients close to zero in the hp group ( y - axis ) . scores plot and loadings plot for the ( a ) fasted and ( b ) postprandial state in rats at the end of the rematching period . one data point represents the combined metabolite measurement from one rat : control , high fiber , high protein . the t and t values represent the scores of each sample in principle components 1 and 2 , respectively . r is the explained variance ; q is the predictive ability of the model . the postprandial state represents blood collected at 30 min following an oral glucose load ( 2 g / kg bw ) . ( a ) fasted metabolomics measurements ( p = 4.2 10 , r = 0.857 , q = 0.795 ) , and ( b ) postprandial metabolomics measurements ( p = 4.0 10 , r = 0.833 , q = 0.74 ) . scores and loading biplot from the o2pls - da modeling are shown with scores as large points , and the loadings as small points labeled . only the variables with the largest influence on projection ( vip ) ( note that the reversal of axis between the two plots is a function of the mathematics and does not impact the relationships between the measured parameters and diets ) . shared and unique structures ( sus ) plots for the ( a ) fasted and ( b ) postprandial states . metabolites that have similar loadings in both the hf vs control ( x - axis ) and hp vs control ( y - axis ) opls - da models will fall along the diagonal with a positive slope . conversely , metabolites elevated in only one or the other will fall in the off - axis area . for example , citrate is elevated in the hf / control comparison under fasted and fed conditions ( positive loadings value ) and reduced in the hp / control comparison under both conditions ( negative loadings value ) . the most significant features ( either metabolomic or biological ) associated with each diet type using regularized general modeling techniques . blood was also collected at 30 min following an oral glucose load and 50 metabolites screened in the postprandial state . similar to the fasted state , the pls - da scores plot of postprandial serum showed a significant separation of the c , hf and hp groups ( p < 0.05 , figure 4b ) . similar to the fasted state , the hp group was characterized by an increase in mannose and decrease in serine , although interestingly no other metabolites were shown to be significantly important ( figure 5b and table 2 ) . both hp and hf groups were differentiated from the control group by decreases in creatine , creatinine , glutamate , and threonine . in order to better elucidate the metabolic relationships between the satiety hormone measurements , biological measurements , and metabolomics data , integrated o2pls - da models were generated in which all of the data was regressed to the diet type ( figure 5a , fasted ; figure 5b , postprandial ) . together , these analyses provide a detailed view of which metabolites are altered in response to dietary shifts . when examined together with an independent regularized modeling procedure ( lasso regression ) ( table 2 ) , the hf diet is associated with increases in plasma pyy and total auc for pyy in both states . similarly , the hf diet is associated with decreases in fat mass ( % ) and tauc for glucose in both states compared to the control group , and interestingly , the fasted measurements for hf are associated with increased bmd . organisms continually respond to cues in their environment , particularly nutritional cues . in humans , growth and development can be segregated into five overlapping periods including prenatal , infantile , childhood , juvenile , and pubertal growth . the transitions between these periods are times of enhanced plasticity or ability to adapt . we previously showed that introduction of a hp diet at weaning ( similar to infantile in humans ) predisposed rats to higher percent body fat and worse glucose control following a high fat and sucrose diet challenge in adulthood , in contrast to rats weaned onto a diet high in prebiotic fiber that were protected . from that study , however , we did not know if returning to the diet consumed throughout growth would mitigate any of the damage caused by the transient high energy diet intake phase . our objective in this study , therefore , was to determine if long - term consumption of these same high protein and fiber diets protects rats from the deleterious effects of a transient high fat , sucrose diet . our major findings include ( 1 ) a lower final body fat and glucose auc in hf rats and no difference between c and hp ; ( 2 ) markedly elevated plasma and intestinal mrna levels of the anorexigenic hormones , pyy and glp-1 ( proglucagon ) in hf compared to c and hp rats ; ( 3 ) significantly reduced secretion of gip in hf versus c and hp rats ; ( 4 ) metabolomic profiles that predicted class separation between the diets with > 90% accuracy ; and ( 5 ) a clearly distinguishable spectrum of metabolites for each diet that shows a meaningful relationship with biological measures such as satiety hormone secretion and body fat . overall , the hf rats had lower body weight , % body fat and glucose , leptin and gip auc and higher glp-1 and pyy auc and bmd compared to the hp rats . in addition , they had lower energy intake and insulin auc compared to hp and c rats . as predicted from our previous work , rats fed the hf diet from weaning to 15 weeks of age gained weight at a slower pace than rats consuming c or hp diets . rats fed the hp diet maintained the highest body weight throughout the study , and this was consistently higher than the rats fed the hf diet . from the longitudinal growth curves , it is clear that reduced weight gain by the rats fed hf during the first phase of the study provided them an advantage throughout the entire study . whereas the rats fed the hf diet gained an average of 405 g of body weight in the first 15 weeks , the rats fed the c and hp diets gained 520 and 533 g , respectively . weight gain during the hfs and final rematched periods were similar across groups , thereby resulting in a final body weight that was significantly lower in hf compared to hp rats . consistently higher body weight in the rats fed the hp diet appeared to be maintained in part because of higher energy intake , particularly in the final rematching period when their energy intake was markedly higher than hf rats throughout and significantly higher than c rats in the final 3 weeks . although this study is limited in having body fat measures only at termination due to restrictions on animal movement in and out of the facility , it is interesting that the rats fed hp diet had nearly identical body fat ( % ) to c rats . this is in contrast to our previous work in which body fat was measured following the hfs phase that showed significantly higher body fat in hp versus c and hf . two possibilities exist to explain this discrepancy . either the hp rats in the current study also had increased body fat ( % ) following the hfs phase and as a result of being rematched to their long - term diet had a beneficial reduction in fat mass by the end of the study , or the control rats were somehow different in this study . we have previously seen that some rats fed the ain-93 control diet gain more fat mass than expected ( unpublished results ) , and therefore it is possible the c group differs between studies even though they were all wistar rats from the same supplier . what remains consistent between studies , however , is that the hf group had significantly lower body fat ( % ) compared to the hp group . we previously showed that following a hfs diet challenge in adulthood , rats that consumed a hp diet during growth had higher blood glucose levels at 30 , 60 , and 90 min during an ogtt compared to rats fed hf during growth . this elevated glycemic response appears to have been partially corrected in the hp rats when placed back on their long - term diet given that there were no significant differences in glucose auc between hf and hp at the end of the study . nevertheless , taking into account the reduced insulin needed to manage the glucose load in the hf rats ( figure 2d ) , it would appear that hf as opposed to c or hp was associated with greater insulin sensitivity . glucagon - like peptide-1 ( glp-1 ) and peptide yy ( pyy ) are released from intestinal l cells and reduce food intake . the ability of prebiotic fibers to increase proglucagon mrna levels and glp-1 secretion is well supported . increased proglucagon expression typically takes place in the context of increased cecal weight due to the markedly increased bacterial fermentation that occurs with consumption of prebiotics . both glp-1 and pyy were markedly elevated in rats fed the hf diet , and the advanced regularized modeling confirmed a positive relationship between the metabolite profile of the hf diet and plasma pyy concentrations in the fasted and postprandial state . it is interesting to note that the prebiotic fiber diet appears to have acute and lasting effects on the ability of l cells to produce and secrete glp-1 and pyy . in the current study , measurements for glp-1 and pyy were made at the end of a 4 week period when fiber was physically in the diet , but we have also shown higher glp-1 secretion in hf rats at the end of the hfs diet period , a time when the diet is devoid of prebiotics . metabolomic profiling allows for the global assessment of endogenous metabolites within an organism . the use of h nmr in this study was able to clearly distinguish between the three diets examined . partial least - squares - discriminant analysis of serum samples showed clear separation of the c , hf and hp rats at the end of the study . essentially , the metabolite profile provided a distinct biochemical signature or snapshot of the combined gene function , enzyme activity and physiological response to the experimental diets . others have similarly shown clearly distinguished phylometabonomic patterns across five inbred strains of mice fed a high fat or normal carbohydrate diet . glucose was measured in two ways in the study , using h nmr analysis and using a blood glucose meter during the ogtt . glucose auc from the ogtt was significantly lower in rats fed hf versus c , and this was supported by o2pls - da modeling in which glucose was shown to be lower with hf diet . furthermore , we were able to show that the most important metabolites involved in the discrimination of the hp group in the fasted state were increases in isobutyrate , mannose and the branched chain amino acids ( bcaa ) leucine and isoleucine and reductions in creatine , citrate , and serine . the hf group was characterized by a decrease in arginine . while bcaa are recognized as key metabolites involved in protein synthesis and cell growth , there is controversial evidence surrounding their role in cellular metabolism linked to the development of obesity - associated insulin resistance . on the one hand , using principal component analysis , newgard et al identified a distinctive profile related to elevated bcaa in obese versus lean subjects . evidence suggests that altered metabolism , particularly a reduction in the catabolism of bcaa in liver and adipose tissue may contribute to higher plasma bcaa and potentially insulin resistance . on the other hand , oral supplementation with bcaa has been shown to improve insulin sensitivity and reduce diet - induced obesity . we detected elevated levels of the bcaa in our rats fed a long - term high protein diet ; however , given that the sole protein source in our diets was casein , a milk - derived protein that is rich in bcaa , it is not clear whether these levels simply reflect the diet or metabolic alterations per se . several metabolites are known to be of gut microbial origin including dimethylamine and trimethylamine . connor et al . identified a decrease in trimethylamine in db / db versus db/+ mice using urinary nmr - based metabolomic analysis . in our data set , trimethylamine was consistently shown to positively contribute to the separation between groups and may reflect the higher bacterial fermentation occurring in the rats fed hf . also consistent with increased bacterial fermentative action in the colon of the hf rats were the higher levels of the scfa , acetate and an unidentified scfa shown in the rats consuming the prebiotic fiber . scfa have been proposed as a chief mechanism responsible for increased expression and secretion of the anorexigenic hormones pyy and glp-1 . recently , we have also demonstrated that prebiotic fibers are associated with marked increases in bifidobacterium spp . in diet - induced and genetically lean and obese jcr : la cp rats.clostridium leptum , a member of the firmicutes phylum was also markedly reduced with prebiotic fiber intake in diet - induced obese rats . used h nmr - based metabolic profiling of fecal matter to probe the complex interaction between host and the gut microbiota . in that study the combination of a prebiotic ( galactosyl - oligosaccharide ) with a probiotic ( lactobacillus paracasei ) was associated with increased fecal acetate over time , which supports our observation of increased serum acetate in the hf group , assuming appropriate transport into the bloodstream . a point of interest is the ability of the independent o2pls - da models ( figure 5a , b ) and regularized modeling results ( table 2 ) to identify the significant relationship between the hf diet and increased bone mineral density . indeed , prebiotic fibers have been shown to significantly improve mineral absorption and metabolism and bone composition and architecture in animal models and increasingly in human studies . on a technical note , regularized modeling is an under - utilized tool in the study of metabolomics data . this approach is designed for analysis of data when multicollinearity is present among variables . this is clearly the situation when multiple metabolites belonging to similar pathways are being measured simultaneously . briefly , the principle for variable selection lies in the application of penalty terms to variable coefficients ( the lasso or ridge regression , or a combination , elastic net ) to shrink those coefficients not required for modeling accuracy . in contrast to the use of regularized modeling , the use of projection - based methods such as pca or pls is relatively prevalent in metabolomics analysis . rigorous statistical comparisons of variable selection using pls methods and regularized modeling have shown that each approach has individual strengths . in our own study , certain differences are noted between the two approaches , such as the association of mannose to the hp diet in the fasted state using regularized modeling , but not using o - pls - da . such differences are to be expected on the basis of the unique treatment of statistical variance in each approach and might provide interesting information about so - called edge - cases , which are are not clear - cut associations and differences . as a result , we suggest expanding the metabolomics statistical toolbox to include regularized modeling as a worthwhile pursuit . in conclusion , we demonstrate that despite higher energy intake , rats rematched to a high protein diet following a transient high fat , sucrose diet have similar percent body fat to those rematched to the control diet . this is in contrast to the increased fat mass that rats raised on a high protein diet display following a terminal high fat , sucrose diet . in contrast , a diet high in prebiotic fiber was protective against excess body fat and hyperglycemia throughout the study . in fact , rats reared on a high fiber diet were least affected by an intermittent high fat diet in adulthood when it was followed by a return to their long - term diet . finally , a clearly distinguishable spectrum of metabolites was associated with each diet . using h nmr it is possible to identify a unique metabolic phenotype for the experimental diets examined and their relationship to physiological and biochemical outcomes associated with consumption of those diets .

large differences in the composition of diet between early development and adulthood can have detrimental effects on obesity risk . we examined the effects of an intermittent high fat / sucrose diet ( hfs ) on satiety hormone and serum metabolite response in disparate diets . wistar rat pups were fed control ( c ) , high prebiotic fiber ( hf ) or high protein ( hp ) diets ( weaning to 16 weeks ) , hfs diet challenged ( 6 weeks ) , and finally reverted to their respective c , hf , or hp diet ( 4 weeks ) . at conclusion , measurement of body composition and satiety hormones was accompanied by 1h nmr metabolic profiles in fasted and postprandial states . metabolomic profiling predicted dietary source with > 90% accuracy . the hf group was characterized by lowest body weight and body fat ( p < 0.05 ) and increased satiety hormone levels ( glucagon - like peptide 1 and peptide - yy ) . regularized modeling confirmed that the hf diet is associated with higher gut hormone secretion that could reflect the known effects of prebiotics on gut microbiota and their fementative end products , the short chain fatty acids . rats reared on a hf diet appear to experience fewer adverse effects from an intermittent high fat diet in adulthood when rematched to their postnatal diet . metabolite profiles associated with the diets provide a distinct biochemical signature of their effects .

Introduction Materials and Methods Results Discussion Conclusions

body weight , fat mass and glycemia in adult males was higher following a high fat / sucrose challenge in rats that consumed a high protein versus a high prebiotic fiber diet from weaning . whether recovery from the high fat / sucrose diet in terms of body fat and satiety hormones is better in rats consuming a long - term high protein versus high fiber diet is not known . therefore , our objective was to examine the metabolomic profile of adult wistar rats exposed to a temporary high fat / sucrose diet in the context of a long - term high protein or high fiber diet . at weaning ( 21 d ) , the male rats were randomized to one of 3 experimental diets : control ( c ) , high fiber ( hf , 21% wt / wt ) and high protein ( hp , 40% wt / wt ) . body weight over the course of the study was influenced by week ( p < 0.001 ) , diet ( p < 0.001 ) and their interaction ( p < 0.008 ) . similar to body weight , there was a significant effect of week ( p < 0.001 ) , diet ( p < 0.03 ) and week diet ( p < 0.001 ) on energy intake . body weight and energy consumption of rats rematched to control , high fiber , or high protein weaning diets following a high fat , sucrose diet challenge in adulthood . panel ( b ) represents energy intake measured throughout the 16 weeks of c , hf , or hp diet , followed by 6 weeks of high fat / sucrose diet , and 4 weeks of rematching to c , hf , or hp diet . in panel ( c ) , the * represents a difference ( p < 0.05 ) between hf versus c and the a difference between hf and hp . in panel ( e ) , the * represents a difference ( p < 0.05 ) between c versus hf and the a difference between c and hp . in panel ( c ) , the * represents a difference ( p < 0.05 ) between hf versus c and hp . there was an approximate 5-fold increase in pyy mrna levels in the colon of rats fed the hf diet ( supporting information , figure s1 ) , which was significantly higher than c and hp ( p < 0.05 ) . in this analysis metabolites that are altered in the same way in hp and hf ( interestingly , changes in levels of the short chain fatty acids ( scfa ) acetate and an unidentified scfa were elevated in the hf group ( positive coefficients on x - axis ) , while having coefficients close to zero in the hp group ( y - axis ) . similar to the fasted state , the pls - da scores plot of postprandial serum showed a significant separation of the c , hf and hp groups ( p < 0.05 , figure 4b ) . when examined together with an independent regularized modeling procedure ( lasso regression ) ( table 2 ) , the hf diet is associated with increases in plasma pyy and total auc for pyy in both states . similarly , the hf diet is associated with decreases in fat mass ( % ) and tauc for glucose in both states compared to the control group , and interestingly , the fasted measurements for hf are associated with increased bmd . our major findings include ( 1 ) a lower final body fat and glucose auc in hf rats and no difference between c and hp ; ( 2 ) markedly elevated plasma and intestinal mrna levels of the anorexigenic hormones , pyy and glp-1 ( proglucagon ) in hf compared to c and hp rats ; ( 3 ) significantly reduced secretion of gip in hf versus c and hp rats ; ( 4 ) metabolomic profiles that predicted class separation between the diets with > 90% accuracy ; and ( 5 ) a clearly distinguishable spectrum of metabolites for each diet that shows a meaningful relationship with biological measures such as satiety hormone secretion and body fat . both glp-1 and pyy were markedly elevated in rats fed the hf diet , and the advanced regularized modeling confirmed a positive relationship between the metabolite profile of the hf diet and plasma pyy concentrations in the fasted and postprandial state . in fact , rats reared on a high fiber diet were least affected by an intermittent high fat diet in adulthood when it was followed by a return to their long - term diet .

in this individual patient data analysis , the first to be conducted in status epilepticus ( se ) , we did not find any variable associated with a likely response to topiramate ( tpm ) . however , considering the relatively small number of patients analyzed , the lack of a statistically significant difference may be due to statistical error type ii.the number of patients with super - refractory status epilepticus ( srse ) treated to date with tpm is probably much lower than commonly reported and is definitely too small to draw any conclusion about its definite role in the treatment of this condition.there is still an unmet need for high - quality observational and interventional studies to evaluate the role of tpm in se , including srse . status epilepticus ( se ) is defined as a condition resulting either from the failure of the mechanisms responsible for seizure termination or from the initiation of mechanisms , which lead to abnormally prolonged seizures . this condition represents a neurological and medical emergency with an estimated crude incidence of 1041/100,000 patients per year , an age - standardized incidence ranging from 4.61/100,000 to 18.3/100,000 , and an overall mortality of 20% [ 46 ] . se carries a high risk of long - term consequences including death and , as such , it needs to be promptly recognized and adequately treated . generalized convulsive ( tonic clonic ) se ( gcse ) is currently defined as a generalized tonic the treatment of gcse usually follows a stepwise approach , with initial administration of benzodiazepines followed by intravenous antiepileptic drugs ( aeds ) if seizure activity continues [ 5 , 6 ] . after failure of first- and second - line treatments , a more aggressive approach consisting of anesthetics ( propofol , pentobarbital , thiopentone , midazolam , or ketamine ) is required [ 5 , 6 ] . however , se may continue or recur 24 h or more after the onset of anesthetic therapy , or may recur on reduction or withdrawal of anesthesia ; this condition , which is associated with a mortality of 35% , has been termed super - refractory status epilepticus ( srse ) . the prognosis of se strongly depends on its duration , etiology , and intrinsic severity [ 9 , 10 ] . however , data on these prognostic factors are usually lacking in trials assessing the efficacy of treatments for se [ 11 , 12 ] . topiramate ( tpm ) is an aed that is effective against a broad spectrum of seizure types , possibly reflecting its multiple mechanisms of action . topiramate has good oral bioavailability , linear kinetics , low protein binding , and no active metabolites . in critically ill patients , tpm can be titrated relatively quickly and has been listed among possible therapeutic options for srse . the aim of this systematic review with individual patient data analysis was to evaluate the role of topiramate ( tpm ) in gcse , including srse . we systematically searched medline , central , lilacs , clinicaltrials.gov , google scholar , and opengrey.eu to identify studies assessing the role of tpm in the treatment of adult cases of gcse and reporting individual patient data . similarly , studies conducted in pediatric populations ( < 16 years of age ) were excluded . to minimize the risk of selective outcome reporting we also excluded single case reports . retrieved articles were independently evaluated for inclusion by two review authors ; any disagreement was resolved through discussion . the following individual patient data were collected : age , gender , weight , previous history of seizures , etiology ( acute symptomatic , remote symptomatic , progressive symptomatic , epilepsy , unknown / not specified ) , eeg features , degree of impairment of consciousness , status epilepticus severity score ( stess ) or epidemiology - based mortality score in se ( emse ) , number of aeds at admission , number of aeds prior to tpm administration , route of tpm administration , loading dose of tpm , maintenance dose of tpm , maximum daily dose of tpm , time from se to tpm administration , time from tpm administration to response , control of se ( yes / no ) , need for intubation ( yes / no ) , death ( yes / no ) , tpm used as last aed ( yes / no ) , name and order of aeds used to treat se . data were summarized using percentages , median , range , or mean and standard deviation , whichever was appropriate . because tpm was not the last aed administered to all patients prior to termination of se , we compared patients who received tpm as the last drug and those who did not . this comparison was performed to analyze whether there was any difference between the two groups that might help to identify some variables predicting a likely response to tpm . we also compared patients receiving tpm as the last drug and achieving se control to patients receiving tpm as the last drug but without termination of se . comparisons were conducted by analyzing variables such as : age , gender , previous history of seizures , etiology , number of aeds prior to tpm , maximum daily dose of tpm , patients with se controlled , number of deaths , and patients with tpm used as the last aed . differences between groups were calculated using the mann whitney u test , chi - squared test , and fisher s exact test . a p value of < 0.05 was considered statistically significant . a bonferroni correction was applied to correct for multiple testing . the literature search yielded 1164 articles ( medline : 119 ; clinicaltrials.gov : 1 ; central : 4 ; opengrey.eu : 0 ; lilacs : 0 ; google scholar : 1040 ) . after reading the full text , 15 articles initially considered for possible inclusion were eventually excluded ( fig . 1 ) . excluded articles with reasons for exclusion and number of patients receiving tpm for each excluded study are reported in the supplementary material . individual patient data were available for 35 patients with gcse ( six of whom with srse ) from four studies [ 1417 ] . the studies from which individual patient data were extracted adopted retrospective [ 14 , 15 , 17 ] and prospective designs.fig . details on clinical features , treatment , and outcomes of patients and results of statistical analyses are reported in tables 1 , 2 and 3.table 1clinical features , treatment details , and outcomes of patients receiving topiramate for generalized convulsive status epilepticuspatients with gcseno . ( % ) 16 ( 45.7)patients with previous history of seizures , no . ( % ) 7 ( 20)etiology , no . ( % ) acute symptomatic24 ( 68.6 ) epilepsy4 ( 11.4 ) remote symptomatic4 ( 11.4 ) unknown3 ( 8.6)aeds prior to tpm , no.median : 2 ( range 15)aeds prior to tpm , no . ( % ) 14 ( 11.4 ) 224 ( 68.6 ) 32 ( 5.7 ) 42 ( 5.7 ) 53 ( 8.6)tpm maximum daily dosage , mg median : 400 ( range 2001000)se controlled , no . ( % ) 6 ( 17.1 ) aeds antiepileptic drugs , gcse generalized convulsive status epilepticus , se status epilepticus , tpm topiramate patients from the study by hottinger et al . were excluded ( no data on maximum daily dosage was provided ) ; data reported in the table have been calculated from 32 patients we defined super - refractory status epilepticus as those cases where seizure activity ( assessed clinically and/or on eeg monitoring ) persisted after first - line and second - line treatments ( benzodiazepine followed by intravenous antiepileptic drugs ) and despite use of anesthetics ( propofol , pentobarbital , thiopentone , midazolam , or ketamine ) table 2clinical features , treatment details , and outcomes of patients receiving topiramate as the last drug for treatment of generalized convulsive status epilepticuspatients with controlled gcse ( tpm administered as last drug)patients with uncontrolled gcse ( tpm administered as last drug)statistical differenceno . of patients146age , yearsmedian : 47.5 ( range 1692)median : 41.5 ( range 2076 ) p = 0.562female , no . ( % ) 10 ( 71.4)3 ( 50 ) p = 0.613patients with previous history of seizures , no . ( % ) 2 ( 14.3)2 ( 33.3 ) p = 0.657etiology , no . ( % ) p = 0.666 acute symptomatic12 ( 85.7)5 ( 83.3 ) epilepsy1 ( 7.1)0 remote symptomatic1 ( 7.1)1 ( 16.7)aeds prior to tpm , no.median : 2 ( range 15)median : 2.5 ( range 15 ) p = 0.169aeds prior to tpm , no . ( % ) 13 ( 21.4)1 ( 16.7 ) 28 ( 57.1)2 ( 33.3 ) 302 ( 33.3 ) 42 ( 14.3)0 51 ( 7.1)1 ( 16.7)tpm maximum daily dosage , mg median : 400 ( range 3001000)median : 550 ( range 400800 ) p = 0.280death , no . ( % ) 7 ( 50)2 ( 33.3 ) p = 0.380 aeds antiepileptic drugs , gcse generalized convulsive status epilepticus , tpm topiramate patients from the study by hottinger et al . were excluded ( no data on maximum daily dosage was provided ) ; data reported in the table were calculated from 18 patients ( 12 with controlled and six with uncontrolled gcse ) table 3clinical features , treatment details , and outcomes of patients who did and did not receive topiramate as the last drug for treatment of generalized convulsive status epilepticuspatients with tpm used as last drugpatients with other aeds used as last drugstatistical differenceno . of patients2015age , yearsmedian : 47.5 ( range : 1692)median : 25 ( range : 1873 ) p = 0.929female , no . ( % ) 13 ( 65%)3 ( 20 ) p = 0.016 patients with previous history of seizures , no . ( % ) 4 ( 20%)3 ( 20 ) p = 1etiology , no . ( % ) p = 0.549acute symptomatic17 ( 85%)7 ( 46.7)epilepsy1 ( 5%)3 ( 20)remote symptomatic2 ( 10%)2 ( 13.3)unknown03 ( 20)aeds prior to tpm , no.median : 5 ( range 35)median : 2 ( range 25 ) p = 0.076aeds prior to tpm , no . ( % ) 14 ( 20%)0 210 ( 50%)14 ( 93.3 ) 32 ( 10%)0 42 ( 10%)0 52 ( 10%)1tpm maximum daily dosage , mg median : 425 ( range 4001000)median : 400 ( range 400400 ) p = 0.066death , no . ( % ) 9 ( 45%)5 ( 33.3 ) p = 0.728se controlled , no . ( % ) 14 ( 70%)10 ( 66.7 ) p = 1 aeds antiepileptic drugs , se status epilepticus , tpm topiramate patients from the study by hottinger et al . were excluded ( no data on maximum daily dosage was provided ) ; data reported in the table were calculated from 33 patients ( four with sr - gcse and 29 without sr - gcse ) the statistical significance was lost after applying the bonferroni correction for multiple comparisons ( p > 0.05 ) clinical features , treatment details , and outcomes of patients receiving topiramate for generalized convulsive status epilepticus aeds antiepileptic drugs , gcse generalized convulsive status epilepticus , se status epilepticus , tpm topiramate patients from the study by hottinger et al . were excluded ( no data on maximum daily dosage was provided ) ; data reported in the table have been calculated from 32 patients we defined super - refractory status epilepticus as those cases where seizure activity ( assessed clinically and/or on eeg monitoring ) persisted after first - line and second - line treatments ( benzodiazepine followed by intravenous antiepileptic drugs ) and despite use of anesthetics ( propofol , pentobarbital , thiopentone , midazolam , or ketamine ) clinical features , treatment details , and outcomes of patients receiving topiramate as the last drug for treatment of generalized convulsive status epilepticus aeds antiepileptic drugs , gcse generalized convulsive status epilepticus , tpm topiramate patients from the study by hottinger et al . were excluded ( no data on maximum daily dosage was provided ) ; data reported in the table were calculated from 18 patients ( 12 with controlled and six with uncontrolled gcse ) clinical features , treatment details , and outcomes of patients who did and did not receive topiramate as the last drug for treatment of generalized convulsive status epilepticus aeds antiepileptic drugs , se status epilepticus , tpm topiramate patients from the study by hottinger et al . were excluded ( no data on maximum daily dosage was provided ) ; data reported in the table were calculated from 33 patients ( four with sr - gcse and 29 without sr - gcse ) the statistical significance was lost after applying the bonferroni correction for multiple comparisons ( p > 0.05 ) despite our primary intention , it was impossible to analyze all variables initially considered because of lack of information for some of them . hence , only the following data were analyzed : age , gender , previous history of seizures , etiology ( acute symptomatic , remote symptomatic , progressive , epilepsy , unknown / not specified ) , number of aeds prior to tpm administration , maximum daily dosage of tpm , control of se ( yes / no ) , death ( yes / no ) , tpm used as last aed ( yes / no ) , order of aeds used to treat se . thirty - five patients with gcse ( median age : 40 years ; range : 1692 ; 46% women ) were included . in most cases ( 69% ) , the etiology underlying gcse was acute symptomatic . in the majority of cases ( 69% ) , tpm was used as third - line aed to control se . among the 35 patients receiving tpm either as the last drugs ( n = 20 ) or not ( n = 15 ) , se was controlled in 69% . in six out of 35 patients ( 17% ) , tpm was used to control se , which persisted after first - line and second - line treatments ( benzodiazepine followed by aed ) and despite the use of anesthetics ( srse ) ; in five out of these six cases , tpm was administered as the last drug , with a resolution of se in four cases . tpm was administered as the last drug in 57% of patients ( 20/35 ) , leading to se control in 14/20 ( 70% ) cases . no statistically significant difference was found for any of the variables considered when we compared patients who received tpm as the last drug to those who did not . the only difference was in the proportion of women , which was higher in those receiving tpm as the last drug ( 65 vs. 20% ; p = 0.016 ) ; however , the statistical significance was lost after applying the bonferroni correction for multiple comparisons ( p > 0.05 ) . similarly , no difference was found comparing patients receiving tpm as the last drug and achieving se control with those receiving tpm as the last drug but without termination of se . this is the first attempt in the literature to perform an individual patient data analysis in se . rather than extracting aggregate data from published studies , individual patient data analyses are carried out using the original research data reported in primary studies or provided by the researchers responsible for each study . individual patient data can then be re - analyzed and combined , if appropriate , into meta - analyses . individual patient data analyses have the potential to answer questions not posed by individual studies or conventional meta - analyses , as they can improve data quality and the type of analyses , producing more reliable results . as such , individual patient data analyses are considered the gold standard of review . however , as for conventional meta - analyses , their validity depends on the quality of the individual patient data obtained from primary studies . the use of individual patient data analyses has been particularly advocated for the assessment of the efficacy of treatments for se , a condition where several confounding factors are usually not adequately reported and analyzed with potential effect on the overall results [ 11 , 19 ] . in this scenario , individual patient data analyses may enable the conduction of subgroup analyses not conducted by the original researchers , taking into account relevant prognostic aspects such as etiology and seizure type . however , in order for these individual patient data analyses to be reliable , it is necessary that the data being analyzed are unbiased and complete for the variable(s ) of interest . results of case reports including data on tpm in se are likely to be affected by selective outcome reporting bias and positive - results publication bias , as favorable results have a higher chance of being reported and published . for this reason , individual patient data were extracted either from retrospective [ 14 , 15 , 17 ] or prospective trials . in all these studies the potential risk for selective outcome reporting seems low , at least according to the details provided in the methods section of the published reports . whether results obtained from these studies actually provide a reliable picture , however , remains unclear , as it is impossible to verify and quantify the existence and the amount of unpublished studies where tpm proved of limited utility in the treatment of se . however , despite searching multiple databases to identify bibliographical references for gray literature , we were not able to retrieve additional studies . hence , it is difficult to understand whether individual patient data included and analyzed in the present review reflect the prognosis of an unbiased patient population treated with tpm for se . in other terms , it is unclear whether the individual patient data collected and analyzed in our review are truly representative of patients treated with tpm for se in terms of clinical and prognostic features . however , even if the results had been obtained from unbiased sources , it would have been impossible to analyze the role of some prognostic factors such as duration of se and its intrinsic severity ( e.g. , assessed with stess , or emse ) , because of a lack of information on these variables . among the 35 patients receiving tpm either as the last drug or not , se was controlled in 69% . conversely , se was terminated in 14 of 20 patients receiving tpm as the last drug ( 70% ) . we considered these patients receiving tpm as the last drug and achieving se control as subjects in whom tpm was possibly / probably successful . however , it was impossible to adequately evaluate the definite efficacy of tpm because not all studies provided information on the time delay between tpm administration and se resolution . furthermore , not all studies specified whether modification of concomitant treatments were made in these patients . no statistically significant difference was found when comparing patients who received tpm as the last drug for any of the variables considered and those who did not . similarly , no difference was found comparing patients receiving tpm as the last drug and achieving se control with those receiving tpm as the last drug but without termination of se . the higher proportion of women found to have received tpm as the last drug was a false - positive result due to multiple comparisons , as the statistical significance was lost after applying the bonferroni correction . hence , we were unable to detect any variable associated with a likely response to tpm . however , considering the relatively small number of patients analyzed , the lack of a statistically significant difference may be likely due to statistical error type ii . interestingly , in most cases tpm was used as second- ( 11% ) or third - line ( 69% ) aed to control se . this finding is unexpected , as tpm is not recommended by any guidelines as treatment for se persisting despite failure of first- and second - line treatments [ 2024 ] . the use of tpm has been suggested as an option for srse , i.e. , se continuing or recurring 24 h or more after the onset of anesthetic therapy , or recurring on the reduction or withdrawal of anesthesia . however , in the present review , only six patients ( 17% ) received tpm for srse ; in five of these cases , tpm was administered as the last drug with resolution of se in four cases . in the literature , after excluding case reports , there is only one study ( not included in the present review as it did not provide individual patient data ) specifically assessing the role of tpm in srse in adults , whereas one study has evaluated tpm in se continuing despite two adequately dosed aeds ( i.e. refractory se ) . in the srse study , among the 28 patients receiving tpm as the last drug and discontinuing intravenous anesthetics with no additional aeds administered , cumulative cessation of rse in patients was 4/35 ( 11% ) at 1 day , 10/35 ( 29% ) at 2 days , and 14/35 ( 40% ) at 3 days . this retrospective study represents the largest reported group of patients with srse treated with tpm and suggests that tpm might be useful in the treatment of this serious condition in adult patients . in their review , ferlisi and shorvon reported that the published outcome of tpm in srse is restricted to 60 cases ( in 10 reports ) treated with topiramate however , a careful analysis of the data available in the literature indicates that the number of patients with srse treated to date with tpm is probably much lower than the reported figure , even after including pediatric patients . refractory se have high clinical heterogeneity , as they include both patients with srse and patients with se refractory to first- and second - line treatments but not receiving anesthetics [ 1517 , 26 , 28 ] . hence , the number of patients treated with tpm for srse is certainly too small to draw any conclusion about its definite role in the treatment of this condition . the lack of information on tpm in se can also be attributed to the fact that , even when tpm is widely used , single outcomes are usually not reported . for instance , in a recent retrospective study , tpm was found to be used in 47 out of 341 patients ( 14% ) , whereas in a 6-year cohort study tpm was administered as a third - line drug in 34 out of 171 patients ( 20% ) . however , the fact that none of them reported details on outcomes in subjects receiving tpm further highlights the need for studies reporting individual patient data . no statistically significant difference was found for any of the variables considered when we compared patients who received tpm as the last drug to those who did not . similarly , no difference was found comparing patients receiving tpm as the last drug and achieving se control with those receiving tpm as the last drug but without termination of se . as the present review demonstrates , despite their potential to answer questions not posed by individual studies or conventional meta - analyses , individual patient data analyses can do little if information has been obtained by biased studies or if data are incomplete . there is still an unmet need for high - quality observational and interventional studies to evaluate the role of tpm in se , including srse . several areas require attention in future research in the treatment of se : investigators should use uniform definitions of se and report results using clear and uniform methods to facilitate meta - analysis [ 11 , 12 ] . providing individual patient data is crucial , as they may be used to perform more detailed and informative analyses to assess the role of confounders . below is the link to the electronic supplementary material . supplementary material 1 ( pdf 195 kb ) supplementary material 2 ( pdf 318 kb ) supplementary material 3 ( pdf 172 kb ) supplementary material 1 ( pdf 195 kb ) supplementary material 2 ( pdf 318 kb ) supplementary material 3 ( pdf 172 kb ) eugen trinka has acted as a paid consultant to bial , biogen idec , eisai , ever neuropharma , medtronics , takeda , upsher - smith , and ucb ; has received speakers honoraria from bial , boehringer , eisai , gl lannacher , and ucb pharma ; and has received research funding from biogen idec , merck , novartis , red bull , ucb pharma , the european union , fwf ( sterreichischer fond zur wissenschaftsfrderung ) , and bundesministerium fr wissenschaft und forschung .

backgroundgeneralized convulsive status epilepticus ( gcse ) is a medical emergency associated with high morbidity and mortality that requires prompt medical intervention . topiramate ( tpm ) is an antiepileptic drug effective against a broad spectrum of seizure types , and has been proposed as a possible therapeutic option for super - refractory status epilepticus ( srse ) , the most severe form of gcse.aimthis review aimed to evaluate the role of tpm in gcse , including srse.methodsmedline , central , clinicaltrials.gov , lilacs , google scholar , and opengrey.eu were systematically searched . we compared : ( 1 ) patients who did and who did not receive tpm as their last drug ; ( 2 ) patients receiving tpm as the last drug and achieving se control and patients receiving tpm as the last drug but without termination of se.resultsthe literature search yielded 1164 results , with individual data available for 35 patients ( six with srse ) from four studies . se was controlled in 68.6% of patients receiving tpm either as the last drug ( 20 ) or not ( 15 ) , and in 14 of the 20 patients receiving tpm as the last drug ( 70% ) . only six patients received tpm for srse ; in five of them , tpm was administered as the last drug with resolution of se in four . when comparing patients who did and did not receive tpm as the last drug , no statistically significant difference was found for any of the variables considered ; similarly , no difference was found comparing patients receiving tpm as the last drug and achieving se control with those receiving tpm as the last drug but without termination of se.conclusionsthe lack of a statistically significant difference is likely to be due to the small sample size . in only a few patients was tpm used for srse . there is an unmet need for high - quality studies to evaluate the role of tpm in gcse.electronic supplementary materialthe online version of this article ( doi:10.1007/s40265 - 016 - 0672 - 2 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Key Points Introduction Methods Results Discussion Conclusions Electronic supplementary material Funding Conflict of interest

however , considering the relatively small number of patients analyzed , the lack of a statistically significant difference may be due to statistical error type ii.the number of patients with super - refractory status epilepticus ( srse ) treated to date with tpm is probably much lower than commonly reported and is definitely too small to draw any conclusion about its definite role in the treatment of this condition.there is still an unmet need for high - quality observational and interventional studies to evaluate the role of tpm in se , including srse . topiramate ( tpm ) is an aed that is effective against a broad spectrum of seizure types , possibly reflecting its multiple mechanisms of action . the aim of this systematic review with individual patient data analysis was to evaluate the role of topiramate ( tpm ) in gcse , including srse . we systematically searched medline , central , lilacs , clinicaltrials.gov , google scholar , and opengrey.eu to identify studies assessing the role of tpm in the treatment of adult cases of gcse and reporting individual patient data . because tpm was not the last aed administered to all patients prior to termination of se , we compared patients who received tpm as the last drug and those who did not . we also compared patients receiving tpm as the last drug and achieving se control to patients receiving tpm as the last drug but without termination of se . were excluded ( no data on maximum daily dosage was provided ) ; data reported in the table have been calculated from 32 patients we defined super - refractory status epilepticus as those cases where seizure activity ( assessed clinically and/or on eeg monitoring ) persisted after first - line and second - line treatments ( benzodiazepine followed by intravenous antiepileptic drugs ) and despite use of anesthetics ( propofol , pentobarbital , thiopentone , midazolam , or ketamine ) table 2clinical features , treatment details , and outcomes of patients receiving topiramate as the last drug for treatment of generalized convulsive status epilepticuspatients with controlled gcse ( tpm administered as last drug)patients with uncontrolled gcse ( tpm administered as last drug)statistical differenceno . were excluded ( no data on maximum daily dosage was provided ) ; data reported in the table were calculated from 18 patients ( 12 with controlled and six with uncontrolled gcse ) table 3clinical features , treatment details , and outcomes of patients who did and did not receive topiramate as the last drug for treatment of generalized convulsive status epilepticuspatients with tpm used as last drugpatients with other aeds used as last drugstatistical differenceno . were excluded ( no data on maximum daily dosage was provided ) ; data reported in the table were calculated from 18 patients ( 12 with controlled and six with uncontrolled gcse ) clinical features , treatment details , and outcomes of patients who did and did not receive topiramate as the last drug for treatment of generalized convulsive status epilepticus aeds antiepileptic drugs , se status epilepticus , tpm topiramate patients from the study by hottinger et al . among the 35 patients receiving tpm either as the last drugs ( n = 20 ) or not ( n = 15 ) , se was controlled in 69% . in six out of 35 patients ( 17% ) , tpm was used to control se , which persisted after first - line and second - line treatments ( benzodiazepine followed by aed ) and despite the use of anesthetics ( srse ) ; in five out of these six cases , tpm was administered as the last drug , with a resolution of se in four cases . no statistically significant difference was found for any of the variables considered when we compared patients who received tpm as the last drug to those who did not . similarly , no difference was found comparing patients receiving tpm as the last drug and achieving se control with those receiving tpm as the last drug but without termination of se . conversely , se was terminated in 14 of 20 patients receiving tpm as the last drug ( 70% ) . no statistically significant difference was found when comparing patients who received tpm as the last drug for any of the variables considered and those who did not . similarly , no difference was found comparing patients receiving tpm as the last drug and achieving se control with those receiving tpm as the last drug but without termination of se . however , in the present review , only six patients ( 17% ) received tpm for srse ; in five of these cases , tpm was administered as the last drug with resolution of se in four cases . no statistically significant difference was found for any of the variables considered when we compared patients who received tpm as the last drug to those who did not . similarly , no difference was found comparing patients receiving tpm as the last drug and achieving se control with those receiving tpm as the last drug but without termination of se . there is still an unmet need for high - quality observational and interventional studies to evaluate the role of tpm in se , including srse .

we used the oir model as a model for neovascular retinopathy as previously described . in brief , mice were exposed to hyperoxia ( 75% oxygen ) between p7 and p12 and subsequently transferred to room air . mice were euthanized by cervical translocation and eyes collected between p12 and p17 for analysis . quantification of the vaso - obliterated ( vo ) and neovascular ( nv ) area were performed according to established techniques . vaso - obliterated and nv rescue were determined per mouse , comparing the amount of vo and nv of the cntf - treated eye to the control - treated eye ( [ 1vo cntf - treated eye / vo control treated eye ] 100 ) . in all experiments , cntf ( rrcntf , # 557-nt-050 ; r&d systems , wiesbaden - nordenstadt , germany ) was administered intravitreally once using a 33-gauge needle . statistically significant changes in vo and nv were determined using unpaired two - tailed student 's t - tests with correction for multiple testing where appropriate . eyes were fixed in 4% paraformaldehyde and stained with commercial glygoprotein ( isolectin gs - ib4 , # i21412 ; life technologies , darmstadt , germany ) overnight at 4c . the following primary antibodies were used : goat anti - cntf - r ( # sc-1914 ; santa cruz biotechnology , inc . , dallas , tx , usa ) ; rabbit anti - collagen iv ( # ab19808 ; abcam , cambridge , ma , usa ) ; and pstat3 rabbit mab ( # 9145 ; cell signaling technology , inc . , danvers , ma , usa ) . for quantitative ( q)pcr analysis , we reverse transcribed rna into cdna for qpcr with the primers listed in the supplementary methods section . for western blot analysis , retinal lysates were blotted onto nitrocellulose membranes and incubated with the following antibodies : anti - bactin ab , mouse monoclonal ( # a1978 ; sigma - aldrich corp . , st . louis , mo , usa ) ; stat3 ( 79d7 ) , rabbit mab ( # 4904 ; cell signaling technology , inc . ) ; pstat3 ( tyr705 ) , and rabbit mab ( cell signaling technology , inc . ) . retinal cntf levels were determined using an elisa set ( rat cntf duoset elisa , # dy557 ; r&d systems ) . the endothelial spheroid sprouting assay was performed as previously published . in brief , human umbilical vein endothelial cells ( huvecs , # c-12203 ; promocell , heidelberg , germany ) were suspended in endothelial growth medium ( egm , # pb - mh-100 - 199 ; pelo biotech , planegg , germany ) containing 10% fbs and 0.25% ( wt / vol ) carboxy - methylcellulose ( # m0512 , sigma - aldrich corp . spheroids were harvested the next day and 30 spheroids seeded into 0.5 ml collagen i ( # 354236 ; corning , wiesbaden , germany ; final concentration of 1.5 mg / ml ) in 24-well plates . endothelial spheroids were photographed 24 hours after stimulation with 12.5 ng / ml hvegf 165 ( # 293-ve ; r&d systems ) , 833 ng / ml rrcntf , and/or 1667 ng / ml rrcntf - r diluted in 0.1 ml serum - free endothelial basal medium . endothelial cell sprouting was quantified using imagej ( http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . results are expressed in percent , normalized to the average sprout length of vegf - treated controls . huvecs , human retinal microvascular endothelial cells ( hrmvecs , # pb - ch-160 - 8511 , pelobiotech ) ; and human fetal astrocytes ( # pb-882 - 05f ; pelobiotech ) were seeded into six - well plates and grown to confluency . cells were stimulated with rrcntf ( 100 ng / ml ) , rrcntf - r ( # 558-cr , 200 ng / ml , r&d systems ) or cntf plus rrcntf - r for 6 hours before rna was collected as described above . we treated c57bl/6 mice in adherence to the arvo statement for the use of animals in ophthalmic and vision research and experiments were approved by the local animal welfare committee . we used the oir model as a model for neovascular retinopathy as previously described . in brief , mice were exposed to hyperoxia ( 75% oxygen ) between p7 and p12 and subsequently transferred to room air . mice were euthanized by cervical translocation and eyes collected between p12 and p17 for analysis . quantification of the vaso - obliterated ( vo ) and neovascular ( nv ) area were performed according to established techniques . vaso - obliterated and nv rescue were determined per mouse , comparing the amount of vo and nv of the cntf - treated eye to the control - treated eye ( [ 1vo cntf - treated eye / vo control treated eye ] 100 ) . in all experiments , cntf ( rrcntf , # 557-nt-050 ; r&d systems , wiesbaden - nordenstadt , germany ) was administered intravitreally once using a 33-gauge needle . statistically significant changes in vo and nv were determined using unpaired two - tailed student 's t - tests with correction for multiple testing where appropriate . for flatmount preparation , eyes were fixed in 4% paraformaldehyde and stained with commercial glygoprotein ( isolectin gs - ib4 , # i21412 ; life technologies , darmstadt , germany ) overnight at 4c . the following primary antibodies were used : goat anti - cntf - r ( # sc-1914 ; santa cruz biotechnology , inc . , dallas , tx , usa ) ; rabbit anti - collagen iv ( # ab19808 ; abcam , cambridge , ma , usa ) ; and pstat3 rabbit mab ( # 9145 ; cell signaling technology , inc . , danvers , ma , usa ) . for quantitative ( q)pcr analysis , we reverse transcribed rna into cdna for qpcr with the primers listed in the supplementary methods section . for western blot analysis , retinal lysates were blotted onto nitrocellulose membranes and incubated with the following antibodies : anti - bactin ab , mouse monoclonal ( # a1978 ; sigma - aldrich corp . , st . louis , mo , usa ) ; stat3 ( 79d7 ) , rabbit mab ( # 4904 ; cell signaling technology , inc . ) ; pstat3 ( tyr705 ) , and rabbit mab ( cell signaling technology , inc . ) . retinal cntf levels were determined using an elisa set ( rat cntf duoset elisa , # dy557 ; r&d systems ) . the endothelial spheroid sprouting assay was performed as previously published . in brief , human umbilical vein endothelial cells ( huvecs , # c-12203 ; promocell , heidelberg , germany ) were suspended in endothelial growth medium ( egm , # pb - mh-100 - 199 ; pelo biotech , planegg , germany ) containing 10% fbs and 0.25% ( wt / vol ) carboxy - methylcellulose ( # m0512 , sigma - aldrich corp . spheroids were harvested the next day and 30 spheroids seeded into 0.5 ml collagen i ( # 354236 ; corning , wiesbaden , germany ; final concentration of 1.5 mg / ml ) in 24-well plates . endothelial spheroids were photographed 24 hours after stimulation with 12.5 ng / ml hvegf 165 ( # 293-ve ; r&d systems ) , 833 ng / ml rrcntf , and/or 1667 ng / ml rrcntf - r diluted in 0.1 ml serum - free endothelial basal medium . endothelial cell sprouting was quantified using imagej ( http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . results are expressed in percent , normalized to the average sprout length of vegf - treated controls . huvecs , human retinal microvascular endothelial cells ( hrmvecs , # pb - ch-160 - 8511 , pelobiotech ) ; and human fetal astrocytes ( # pb-882 - 05f ; pelobiotech ) were seeded into six - well plates and grown to confluency . cells were stimulated with rrcntf ( 100 ng / ml ) , rrcntf - r ( # 558-cr , 200 ng / ml , r&d systems ) or cntf plus rrcntf - r for 6 hours before rna was collected as described above . recombinant rat cntf ( rrcntf ) was injected intravitreally at oir p12 to evaluate its effect on preretinal neovessel formation and capillary regrowth in a model of hypoxia - induced proliferative retinopathy . figure 2a shows the effect of three different doses of rrcntf ( 0.5 ng , 50 ng , and 500 ng ) on the vo and nv area at oir p17 . intravitreal injection of all doses resulted in a significant reduction in preretinal nv at oir p17 while only 50 ng and 0.5 ng rrcntf significantly decreased the vo area . overall , six different doses of rrcntf were tested to determine the maximum treatment effect of recombinant cntf on vo and nv in the oir model ( see supplementary fig . we determined vo and nv rescue by comparing cntf - injected eyes to control - treated eyes ( fig . intravitreal injection of 5 , 50 , or 250 ng of rrcntf yielded the strongest rescue effects with a maximum of 43% rescue in vo and 96% in nv area . recombinant rat cntf was injected intravitreally at different doses ( 0.05500 ng ) at oir p12 . treatment with rrcntf significantly decreased the area of vo and nv at oir p17 at almost all doses tested ( statistical significance is indicated by asterisk with * p < 0.05 ; error bars : sem ) . ( a ) shows vo and nv values for a selection of three out of six dosages tested . for bar graphs of additional doses in addition , vo area was also significantly improved by rrcntf treatment in all but the highest rrcntf dose group . ( b ) dose - response curve for the effect of rrcntf on retinal vo and nv . graphs represent the vo and nv rescue effect as a function of rrcntf dose : n = 0.05 ng , 10 mice ; n = 0.5 ng , 12 mice ; n = 5 ng , 11 mice ; n = 50 ng , 14 mice ; n = 250 ng , 12 mice ; n = 500 ng , 16 mice . ( c ) time - course analysis of retinal cntf levels measured 4 , 24 , and 48 hours after intravitreal injection of rrcntf at oir p12 ( rat cntf elisa ; n = 3 eyes per time and dose ) . intravitreal injection of 500 and 50 ng rrcntf increased retinal cntf levels for up to 48 hours . intravitreal injection of 0.5 ng rrcntf did not result in a significant increase in retinal rrcntf measurements . eyes injected with pbs / bsa served as control to determine baseline retinal cntf . since human recombinant cntf protein is known to have a relatively short half - life when administered systemically ( 120400 minutes following subcutaneous injection , 2.9 minutes following intravenous administration ) , we next analyzed retinal cntf levels following one single intravitreal injection of rrcntf ( fig . 2c ) . in analogy to the previous experiment , one of three different doses of cntf ( 0.5 , 50 , or 500 ng cntf ) was injected intravitreally at oir p12 and retinas were collected 4 , 24 , or 48 hours later . eyes injected with pbs / bsa served as controls to determine endogenous mouse cntf levels . endogenous baseline cntf levels were 0.28 to 0.33 ng cntf / mg protein for the three time points tested . intravitreal injection of 500 and 50 ng rrcntf strongly increased retinal cntf levels to 26.4 and 3.3 ng cntf / mg protein , respectively , 4 hours postinjection . twenty - four hours after intravitreal injection , retinal cntf levels were still increased to 1.0 and 0.8ng cntf / mg protein . at 48 hours , retinal cntf was at 0.45 ng cntf / mg protein for both treatment groups . intravitreal injection of 0.5 ng rrcntf , a dose that only resulted in 40% rescue of nv compared with a 95% rescue with higher cntf doses ( see fig . however , it has to be noted that cntf was injected intravitreally and cntf levels were measured in total retinal explants since the mouse vitreous does not yield enough material for analysis . taken together , these data suggest that intravitreal rrcntf has a high potency in preventing pathological nv development and improving capillary regrowth ( fig . furthermore , rrcntf showed a relatively broad therapeutic range yielding maximal rescue effects at treatment doses between 5 and 250 ng ( fig . an increase in retinal cntf levels was detectable for up to 48 hours after injection of doses above 50 ng . in order to better understand the role of endogenous cntf under hypoxic retinal stress , we measured cntf and cntf - r expression during oir . during the initial hyperoxic phase ( p10 and p12 , 0 hours ) , , however , mrna levels of cntf - r increased significantly ( p12 , 6 hours ) , followed by an upregulation of cntf expression at oir p14 . immunohistochemical stainings revealed a granular signal for cntf - r at the level of the outer photoreceptor segments and the ganglion cell layer in sections of oir p14 retinas ( fig . 3b ) . higher magnification images localized cntf - r expression in part to retinal endothelial cells . however , pcr analysis of cultured human endothelial cells and astrocytes showed that , in vitro , both cell types expressed lif - r and gp130 but not cntf - r ( fig . this difference in cntf - r expression between in vivo and in vitro might be explained by a change in gene expression patterns of both cell types in 2d - culture systems in vitro . alternatively , both cell types might also lack cntf - r expression in vivo and incorporate soluble cntf - r or cntf / cntf - r complexes provided by their surroundings . endogenous cntf and cntf - r levels are significantly upregulated during the hypoxic phase of oir . ( a ) retinal cntf and cntf - r levels remained unchanged during the hyperoxic phase ( p7-p12 ) of oir . however , after onset of retinal hypoxia , both cntf - r and cntf were significantly upregulated . this finding indicates that retinal tissue increases endogenous cntf and cntfr expression in response to hypoxic tissue injury ( normoxia / oir p10 , p14 , p15 : n = five mice per group and time point ; normoxia p12 : n = six mice ; oir p12 , 0 hours and oir p12 , 6 hours : n = four mice per time point ; t - test corrected for multiple testing ) . ( b ) in cross - sections of oir p14 retinas , expression of cntf - r was detected at the level of photoreceptor outer segments and the inner retina . in the inner retina , cntf - r signal localized to the superficial vascular plexus ( arrows ) as well as to surrounding cells . this staining pattern suggests that retinal endothelial cells are able to directly respond to cntf . ( c ) cultured human vascular endothelial cells and cerebral astrocytes were tested for the expression of components of the trimeric cntf receptor complex , lif - r , gp130 and cntf - r by pcr and gel electrophoresis . see text for more detail on the difference between in vivo and in vitro expression patterns . activation of the jak / stat3 signaling pathway represents a common downstream effector of cntf receptor activation . retinas injected intravitreally with 0.5 , 50 , or 500 ng rrcntf at oir p12 displayed significant increases in pstat3 levels at oir p13 , confirming the stat3 pathway as important mediator of cntf in ischemic retinas ( fig . retinas treated with 50 ng rrcntf displayed the strongest activation of the stat3 signaling pathway , matching the dose - response effect observed in cntf - treated eyes ( fig . importantly , low - dose rrcntf ( 0.5 ng ) also induced a significant 3-fold increase in pstat3 , explaining why low - dose rrcntf injections resulted in partial oir recovery , although no increase in overall retinal cntf - levels had been detected in retinal time - course experiments with this dose ( fig . 2c ) . ciliary neurotrophic factor activates stat3 signaling in the inner retina in a dose - dependent manner . ( a ) intravitreal rrcntf injections at oir p12 induced significant increases in phosphorylated stat3 levels in the retina at oir p13 compared with control - injected eyes ( n = 4 mice per dose ) . error bars : sem . a dose of 50 ng rrcntf most strongly activated stat3 signaling through both increased expression and phosphorylation of stat3 . ( b ) phosphorylation of stat3 was localized to the ganglion cell layer ( arrowheads ) and the superficial vascular plexus ( arrows ) , suggesting that multiple cell types , including vascular endothelial cells , respond to exogenous rrcntf . ( c ) intravitreal injection of rrcntf leads to upregulation of socs3 expression . while the increase in socs3 expression following 0.5 ng rrcntf injection was not statistically significant ( n = 4 per dose , p = 0.06 ) , doses of 50 and 500 ng rrcntf induced high socs3 mrna levels . from the doses investigated , 50 ng rrcntf had the strongest effect on socs3 upregulation , which is in line with the pstat3 results . no changes in vegf expression were observed at any dose , suggesting that the phenotypic changes induced by exogenous application of rrcntf in the oir model are independent of vegf . we next aimed to identify cntf responsive cells and stat3 downstream targets modulated by cntf treatment in ischemic retina . immunohistochemical staining in oir p13 retinas localized positive pstat3 signals to the level of the retinal ganglion cell layer and the superficial vascular plexus 24 hours after intravitreal cntf injections ( fig . 4b ) . recently , socs3 has been described to play an important role as endogenous inhibitor of pathologic angiogenesis in endothelial , neuronal , and glial cells . at the same time we hence hypothesized that the antiangiogenic effect of cntf in the retina may be mediated by jak / stat3/socs3 signaling . quantitative pcr analysis 24 hours post - cntf treatment ( oir p13 ) confirmed significant upregulation of retinal socs3 levels while vegf expression remained unchanged ( fig . 4c , supplementary fig . s2 ) , upregulation of socs3 was strongest in response to 50 ng rrcntf , which is in line with both the intensity of stat3 activation and oir rescue . in order to validate the proposed mechanism of socs3-mediated antiangiogenic effects of cntf , we performed in vitro experiments with endothelial and glial cells . as shown in figure 3c , endothelial cells as well as astrocytes did not express cntf - r in vitro . it is known , however , that cntf / cntf - r complexes can be incorporated into cell membranes of cells not intrinsically expressing cntf - r , thus allowing cntf signaling in these cells . therefore , all in vitro experiments were performed using four conditions : negative control , rrcntf alone , rcntf - r alone , and rrcntf + rcntf - r. we first tested if cntf exerts direct antiangiogenic effects on endothelial cells in a spheroid sprouting assay ( fig . recombinant rat cntf alone did not reduce vegf - induced endothelial cell sprouting in vitro . in contrast , when rrcntf was administered together with soluble rcntf - r , endothelial cell sprouting was significantly reduced , confirming the inhibitory effect of cntf on vegf - induced angiogenesis in the presence of cntf - r. ciliary neurotrophic factor has an anti - angiogenic effect on sprouting endothelial cells in the presence of cntf - r. ( a ) recombinant rat cntf decreases vegf - induced endothelial spheroid sprouting in the presence of cntf - r ( 1-way anova : * * * * p < 0.0001 ) . ( b ) in the presence of cntf - r , both huvecs and hrmvecs upregulate socs3 in response to rrcntf ( 1-way anova : * p < 0.05 ) . ( c ) in vitro , astrocytes also respond with socs3 upregulation to rrcntf stimulation in the presence of rcntf - r. however , this socs3 increase in astrocytes does not alter total vegf - a expression or expression of any of the vegf - a isoforms investigated . next , we tested whether exposure of endothelial cells to cntf , cntf - r , or a combination of rrcntf and rrcntf - r changes endothelial socs3 expression . in both huvecs and hrmvecs , combinatory treatment with rrcntf and rcntf - r indeed resulted in a significant upregulation of socs3 mrna in vitro ( fig . upregulation of socs3 expression was induced in astrocytes by combined stimulation with cntf plus cntf - r ( fig . these results confirm the observed retinal upregulation of socs3 upon cntf treatment in vivo ( fig . 4c ) . similarly in line with results from cntf - treated retina samples ( fig . 4c ) , expression of total vegf - a and vegf isoforms remained unaltered in human astrocytes upon stimulation with rrcntf plus rcntf - r ( fig . 5c ) . together , these results confirm upregulation of socs3 as one of the mechanisms by which the anti - angiogenic effect of cntf can be mediated . recombinant rat cntf ( rrcntf ) was injected intravitreally at oir p12 to evaluate its effect on preretinal neovessel formation and capillary regrowth in a model of hypoxia - induced proliferative retinopathy . figure 2a shows the effect of three different doses of rrcntf ( 0.5 ng , 50 ng , and 500 ng ) on the vo and nv area at oir p17 . intravitreal injection of all doses resulted in a significant reduction in preretinal nv at oir p17 while only 50 ng and 0.5 ng rrcntf significantly decreased the vo area . overall , six different doses of rrcntf were tested to determine the maximum treatment effect of recombinant cntf on vo and nv in the oir model ( see supplementary fig . we determined vo and nv rescue by comparing cntf - injected eyes to control - treated eyes ( fig . intravitreal injection of 5 , 50 , or 250 ng of rrcntf yielded the strongest rescue effects with a maximum of 43% rescue in vo and 96% in nv area . recombinant rat cntf was injected intravitreally at different doses ( 0.05500 ng ) at oir p12 . treatment with rrcntf significantly decreased the area of vo and nv at oir p17 at almost all doses tested ( statistical significance is indicated by asterisk with * p < 0.05 ; error bars : sem ) . ( a ) shows vo and nv values for a selection of three out of six dosages tested . for bar graphs of additional doses in addition , vo area was also significantly improved by rrcntf treatment in all but the highest rrcntf dose group . ( b ) dose - response curve for the effect of rrcntf on retinal vo and nv . graphs represent the vo and nv rescue effect as a function of rrcntf dose : n = 0.05 ng , 10 mice ; n = 0.5 ng , 12 mice ; n = 5 ng , 11 mice ; n = 50 ng , 14 mice ; n = 250 ng , 12 mice ; n = 500 ng , 16 mice . ( c ) time - course analysis of retinal cntf levels measured 4 , 24 , and 48 hours after intravitreal injection of rrcntf at oir p12 ( rat cntf elisa ; n = 3 eyes per time and dose ) . intravitreal injection of 500 and 50 ng rrcntf increased retinal cntf levels for up to 48 hours . intravitreal injection of 0.5 ng rrcntf did not result in a significant increase in retinal rrcntf measurements . eyes injected with pbs / bsa served as control to determine baseline retinal cntf . since human recombinant cntf protein is known to have a relatively short half - life when administered systemically ( 120400 minutes following subcutaneous injection , 2.9 minutes following intravenous administration ) , we next analyzed retinal cntf levels following one single intravitreal injection of rrcntf ( fig . 2c ) . in analogy to the previous experiment , one of three different doses of cntf ( 0.5 , 50 , or 500 ng cntf ) was injected intravitreally at oir p12 and retinas were collected 4 , 24 , or 48 hours later . eyes injected with pbs / bsa served as controls to determine endogenous mouse cntf levels . endogenous baseline cntf levels were 0.28 to 0.33 ng cntf / mg protein for the three time points tested . intravitreal injection of 500 and 50 ng rrcntf strongly increased retinal cntf levels to 26.4 and 3.3 ng cntf / mg protein , respectively , 4 hours postinjection . twenty - four hours after intravitreal injection , retinal cntf levels were still increased to 1.0 and 0.8ng cntf / mg protein . at 48 hours , retinal cntf was at 0.45 ng cntf / mg protein for both treatment groups . intravitreal injection of 0.5 ng rrcntf , a dose that only resulted in 40% rescue of nv compared with a 95% rescue with higher cntf doses ( see fig . however , it has to be noted that cntf was injected intravitreally and cntf levels were measured in total retinal explants since the mouse vitreous does not yield enough material for analysis . taken together , these data suggest that intravitreal rrcntf has a high potency in preventing pathological nv development and improving capillary regrowth ( fig . furthermore , rrcntf showed a relatively broad therapeutic range yielding maximal rescue effects at treatment doses between 5 and 250 ng ( fig . an increase in retinal cntf levels was detectable for up to 48 hours after injection of doses above 50 ng . in order to better understand the role of endogenous cntf under hypoxic retinal stress , we measured cntf and cntf - r expression during oir . during the initial hyperoxic phase ( p10 and p12 , 0 hours ) , cntf and cntf - r levels remained unchanged ( fig . 3a ) . within the first hours of hypoxia , however , mrna levels of cntf - r increased significantly ( p12 , 6 hours ) , followed by an upregulation of cntf expression at oir p14 . immunohistochemical stainings revealed a granular signal for cntf - r at the level of the outer photoreceptor segments and the ganglion cell layer in sections of oir p14 retinas ( fig . 3b ) . higher magnification images localized cntf - r expression in part to retinal endothelial cells . however , pcr analysis of cultured human endothelial cells and astrocytes showed that , in vitro , both cell types expressed lif - r and gp130 but not cntf - r ( fig . this difference in cntf - r expression between in vivo and in vitro might be explained by a change in gene expression patterns of both cell types in 2d - culture systems in vitro . alternatively , both cell types might also lack cntf - r expression in vivo and incorporate soluble cntf - r or cntf / cntf - r complexes provided by their surroundings . endogenous cntf and cntf - r levels are significantly upregulated during the hypoxic phase of oir . ( a ) retinal cntf and cntf - r levels remained unchanged during the hyperoxic phase ( p7-p12 ) of oir . however , after onset of retinal hypoxia , both cntf - r and cntf were significantly upregulated . this finding indicates that retinal tissue increases endogenous cntf and cntfr expression in response to hypoxic tissue injury ( normoxia / oir p10 , p14 , p15 : n = five mice per group and time point ; normoxia p12 : n = six mice ; oir p12 , 0 hours and oir p12 , 6 hours : n = four mice per time point ; t - test corrected for multiple testing ) . * ( b ) in cross - sections of oir p14 retinas , expression of cntf - r was detected at the level of photoreceptor outer segments and the inner retina . in the inner retina , cntf - r signal localized to the superficial vascular plexus ( arrows ) as well as to surrounding cells . this staining pattern suggests that retinal endothelial cells are able to directly respond to cntf . ( c ) cultured human vascular endothelial cells and cerebral astrocytes were tested for the expression of components of the trimeric cntf receptor complex , lif - r , gp130 and cntf - r by pcr and gel electrophoresis . see text for more detail on the difference between in vivo and in vitro expression patterns . activation of the jak / stat3 signaling pathway represents a common downstream effector of cntf receptor activation . retinas injected intravitreally with 0.5 , 50 , or 500 ng rrcntf at oir p12 displayed significant increases in pstat3 levels at oir p13 , confirming the stat3 pathway as important mediator of cntf in ischemic retinas ( fig . retinas treated with 50 ng rrcntf displayed the strongest activation of the stat3 signaling pathway , matching the dose - response effect observed in cntf - treated eyes ( fig . importantly , low - dose rrcntf ( 0.5 ng ) also induced a significant 3-fold increase in pstat3 , explaining why low - dose rrcntf injections resulted in partial oir recovery , although no increase in overall retinal cntf - levels had been detected in retinal time - course experiments with this dose ( fig . 2c ) . ciliary neurotrophic factor activates stat3 signaling in the inner retina in a dose - dependent manner . ( a ) intravitreal rrcntf injections at oir p12 induced significant increases in phosphorylated stat3 levels in the retina at oir p13 compared with control - injected eyes ( n = 4 mice per dose ) . error bars : sem . a dose of 50 ng rrcntf most strongly activated stat3 signaling through both increased expression and phosphorylation of stat3 . ( b ) phosphorylation of stat3 was localized to the ganglion cell layer ( arrowheads ) and the superficial vascular plexus ( arrows ) , suggesting that multiple cell types , including vascular endothelial cells , respond to exogenous rrcntf . while the increase in socs3 expression following 0.5 ng rrcntf injection was not statistically significant ( n = 4 per dose , p = 0.06 ) , doses of 50 and 500 ng rrcntf induced high socs3 mrna levels . from the doses investigated , 50 ng rrcntf had the strongest effect on socs3 upregulation , which is in line with the pstat3 results . no changes in vegf expression were observed at any dose , suggesting that the phenotypic changes induced by exogenous application of rrcntf in the oir model are independent of vegf . we next aimed to identify cntf responsive cells and stat3 downstream targets modulated by cntf treatment in ischemic retina . immunohistochemical staining in oir p13 retinas localized positive pstat3 signals to the level of the retinal ganglion cell layer and the superficial vascular plexus 24 hours after intravitreal cntf injections ( fig . 4b ) . recently , socs3 has been described to play an important role as endogenous inhibitor of pathologic angiogenesis in endothelial , neuronal , and glial cells . at the same time , socs3 represents an established target of the stat3 signaling pathway . we hence hypothesized that the antiangiogenic effect of cntf in the retina may be mediated by jak / stat3/socs3 signaling . quantitative pcr analysis 24 hours post - cntf treatment ( oir p13 ) confirmed significant upregulation of retinal socs3 levels while vegf expression remained unchanged ( fig . 4c , supplementary fig . s2 ) , upregulation of socs3 was strongest in response to 50 ng rrcntf , which is in line with both the intensity of stat3 activation and oir rescue . in order to validate the proposed mechanism of socs3-mediated antiangiogenic effects of cntf , we performed in vitro experiments with endothelial and glial cells . as shown in figure 3c , endothelial cells as well as astrocytes did not express cntf - r in vitro . it is known , however , that cntf / cntf - r complexes can be incorporated into cell membranes of cells not intrinsically expressing cntf - r , thus allowing cntf signaling in these cells . therefore , all in vitro experiments were performed using four conditions : negative control , rrcntf alone , rcntf - r alone , and rrcntf + rcntf - r. we first tested if cntf exerts direct antiangiogenic effects on endothelial cells in a spheroid sprouting assay ( fig . recombinant rat cntf alone did not reduce vegf - induced endothelial cell sprouting in vitro . in contrast , when rrcntf was administered together with soluble rcntf - r , endothelial cell sprouting was significantly reduced , confirming the inhibitory effect of cntf on vegf - induced angiogenesis in the presence of cntf - r. ciliary neurotrophic factor has an anti - angiogenic effect on sprouting endothelial cells in the presence of cntf - r. ( a ) recombinant rat cntf decreases vegf - induced endothelial spheroid sprouting in the presence of cntf - r ( 1-way anova : * * * * p < 0.0001 ) . ( b ) in the presence of cntf - r , both huvecs and hrmvecs upregulate socs3 in response to rrcntf ( 1-way anova : * p < 0.05 ) . ( c ) in vitro , astrocytes also respond with socs3 upregulation to rrcntf stimulation in the presence of rcntf - r. however , this socs3 increase in astrocytes does not alter total vegf - a expression or expression of any of the vegf - a isoforms investigated . next , we tested whether exposure of endothelial cells to cntf , cntf - r , or a combination of rrcntf and rrcntf - r changes endothelial socs3 expression . in both huvecs and hrmvecs , combinatory treatment with rrcntf and rcntf - r indeed resulted in a significant upregulation of socs3 mrna in vitro ( fig . upregulation of socs3 expression was induced in astrocytes by combined stimulation with cntf plus cntf - r ( fig . these results confirm the observed retinal upregulation of socs3 upon cntf treatment in vivo ( fig . 4c ) . similarly in line with results from cntf - treated retina samples ( fig . 4c ) , expression of total vegf - a and vegf isoforms remained unaltered in human astrocytes upon stimulation with rrcntf plus rcntf - r ( fig . 5c ) . together , these results confirm upregulation of socs3 as one of the mechanisms by which the anti - angiogenic effect of cntf can be mediated . this study demonstrates that intravitreal injections of recombinant cntf promote capillary regrowth and attenuate pre - retinal neovascularization in a mouse model of oxygen - induced retinopathy . with its pronounced vascular phenotype that includes vaso - obliteration and preretinal tuft formation , the oir model has developed into one of the most widely used vascular disease models . many studies have shown that neuronal and glial cells are strongly involved in the development of retinal vascular disease . the results from this study add a novel angioregulatory role for cntf to this emerging picture of complex neurovascular interplay in the retina . ciliary neurotrophic factor and cntf - r have been shown to be strongly expressed during rat retinal development , with mller cells being a major source for cntf expression and ganglion / amacrine cells the origin of cntf - r synthesis . in addition , cntf has been identified as an important mediator for photoreceptor differentiation . beyond development , multiple studies have shown that retinal cntf levels are also upregulated in response to injury . no studies have been performed to date on the role of cntf in the oir model . in this study , we observed significant upregulation of endogenous cntf - r and cntf at oir p12 ( 6 hours ) and oir p14 ( fig . the timing of cntf upregulation corresponds to the onset of intraretinal hypoxia unfolding in the central avascular area at oir p12 after transferring pups from hyperoxic atmosphere to normoxia . this finding indicates that the initial upregulation of cntf - r may represent a response of the inner retina to ischemic stress . however , activation of the downstream signaling pathway jak / stat3 of cntf was low at oir p13 in control - treated eyes , suggesting that endogenous activation of the cntf signaling pathway during the early hypoxic phase is minor , thus resulting in limited efficacy of this potential repair response ( fig . supplementation of recombinant cntf in the early hypoxic phase strongly upregulated the downstream signaling cascade , leading to a pronounced protective effect of cntf against ischemic vascular changes in the retina ( figs . 2 , 4 ) . the strong angiomodulatory effect of cntf observed in our study renders cntf a promising therapeutic approach for ischemic retinopathies . best oir rescue effects were achieved with cntf doses between 5 and 250 ng ( fig . both lower and higher doses of cntf resulted in lower stat3 phosphorylation , reduced socs3 upregulation and ultimately less protective effect . studies testing cntf in rodent models for treatment of retinitis pigmentosa have repeatedly discussed potential dose - dependent effects of cntf . in our model , high cntf doses from continuous delivery of cntf through adeno - associated virus ( aav ) vector transduction were reported to reduce electroretinogram ( erg ) amplitudes . a study by mcgill , however , found that while an intravitreal injection of high cntf doses ( up to 10 g ) as well as subretinal delivery of aav - vectored cntf in rat resulted in reduced erg amplitudes , this was not the case with low doses of cntf treatment ( 1100 ng ) . while cntf is a well - characterized neuroprotective agent capable of rescuing both photoreceptors and ganglion cells from apoptosis , the underlying mechanisms of the neuroprotective action of cntf are still incompletely understood . what is known , however , is that astroglia , microglia , and ganglion cells have all been reported to respond to cntf treatment . in our study , cntf is shown to have a therapeutic angiomodulatory effect in vivo that is partially mediated through a direct effect on endothelial cells . we confirmed the direct antiangiogenic effect on endothelial cells by using an endothelial spheroid sprouting model that resembles the pathologic neovascular tuft formation in the retina driven by high amounts of vegf . in this context it is important to note that our spheroidal sprouting model contains only endothelial cells . in this exclusive endothelial cell milieu , cntf - r has to be added exogenously in order to observe cntfs antiangiogenic effects since neither huvecs nor hrmvecs express cntf - r endogenously in vitro . it is well established that cntf signaling can occur in cell types that do not express the cntf - r by incorporation of soluble cntf - r into their cell membrane . further mechanistic workup in our study identified socs3 as one of the downstream mediators conveying the cntf effect in endothelial cells . while knockdown of socs3 in endothelial cells has been shown to increase proliferation and sprouting in these cells , its role in mediating cntf effects in endothelial cells has not yet been described . beside the direct anti - angiogenic effect of cntf on endothelial cells , it can not be discounted that indirect angiomodulatory effects of cntf via other cell types also play a role in the observed protective in vivo phenotype . similar to endothelial cells , human astrocytes do not express cntf - r in vitro ( fig . 3c ) . upon stimulation with cntf plus cntf - r , however , they upregulate socs3 similar to endothelial cells , suggesting that the beneficial effect of cntf may partially be mediated through glial cells . sun et al . recently reported that socs3 deficiency in glial and neuronal cells increases vegf expression , which in turn results in increased pathologic neovascularization in oir . however , cntf - mediated induction of socs3 did not alter vegf expression , suggesting that the cntf - induced protective effect in the oir model is independent of vegf . as a cautionary note , analysis of cntf effects in cell culture is reliable only if the cell cultures used are confirmed not to be contaminated with other cell types . taken together , our results demonstrate that cntf can have differential effects on retinal angiogenesis under hypoxic conditions , improving capillary regrowth while at the same time inhibiting the aberrant formation of new vessels . the magnitude of the effect of cntf on preventing pathologic nv is comparable with that of established anti - vegf agents in the oir model . we have identified both direct effects on endothelial cells as well as indirect effects of cntf on glial cells as important mechanistic pathways conveying these beneficial results . more work will be required to further elucidate the fine - tuned interplay of glial , neuronal , and endothelial cells in proliferative retinopathy . another open question lies in the duration of the treatment effect achieved by the use of recombinant cntf . in the oir model , the timeframe between intravitreal injection of cntf and analysis of the vascular phenotype spans only 5 days . while encapsulated cell implants provide a potential solution for continuous delivery of recombinant cntf in patients with degenerative retinal disease , it would be important to determine how long the angiomodulatory effect of a single intravitreal cntf injection lasts in patients with angioproliferative retinal disease .

purposeretinal vascular disease represents a major cause for vision loss in the western world . recent research has shown that neuronal and vascular damage are closely related in retinal disease . ciliary neurotrophic factor ( cntf ) is a well - studied neurotrophic factor that is currently being tested in clinical trials for the treatment of retinal degenerative diseases and macular telangiectasia . however , little is known about its effect on retinal vasculature . in this study , we investigate the effects of cntf in retinal neovascular disease using the mouse model of oxygen - induced retinopathy ( oir).methodsnewborn pups were exposed to 75% oxygen from postnatal day ( p)7 to p12 and subsequently returned to room air . ciliary neurotrophic factor was injected intravitreally at oir p12 and the vaso - obliterated and neovascular areas were quantified at oir p17 . immunohistochemistry , rna , and protein analysis were used to identify cntf - responsive cells . in vitro experiments were performed to analyze the effect of cntf on endothelial and astroglial cells.resultsin the oir model , cntf facilitated capillary regrowth and attenuated preretinal neovascularization in a dose - dependent manner . the protective effect of cntf was mediated via activation of the jak / stat3/socs3 signaling pathway . immunohistochemical studies identified endothelial cells among others as cntf - responsive cells in the retina . in vitro studies confirmed the anti - angiogenic effect of cntf on endothelial cell sprouting.conclusionsthis study provides evidence for a therapeutic potential of cntf beyond degenerative retinal disease . vasoproliferative retinopathies may benefit from a cntf - dependent and socs3-mediated angiomodulatory effect .

Materials and Methods Animal Models Immunohistochemistry, RNA, and Protein Analysis Endothelial Spheroid Sprouting Assay In Vitro CNTF Stimulation Experiments Results CNTF Promotes Capillary Regrowth and Attenuates Retinal Neovascularization in OIR Endogenous CNTF and CNTF-R Levels Are Significantly Upregulated in Response to Retinal Hypoxia CNTF Activates STAT3 Signaling and SOCS3 Negative Feedback Inhibition in the Inner Retina CNTF Inhibits Endothelial Sprouting and Induces Upregulation of SOCS3 in the Presence of CNTF-R Discussion Supplementary Material

in brief , mice were exposed to hyperoxia ( 75% oxygen ) between p7 and p12 and subsequently transferred to room air . recombinant rat cntf ( rrcntf ) was injected intravitreally at oir p12 to evaluate its effect on preretinal neovessel formation and capillary regrowth in a model of hypoxia - induced proliferative retinopathy . in analogy to the previous experiment , one of three different doses of cntf ( 0.5 , 50 , or 500 ng cntf ) was injected intravitreally at oir p12 and retinas were collected 4 , 24 , or 48 hours later . ciliary neurotrophic factor activates stat3 signaling in the inner retina in a dose - dependent manner . at the same time we hence hypothesized that the antiangiogenic effect of cntf in the retina may be mediated by jak / stat3/socs3 signaling . therefore , all in vitro experiments were performed using four conditions : negative control , rrcntf alone , rcntf - r alone , and rrcntf + rcntf - r. we first tested if cntf exerts direct antiangiogenic effects on endothelial cells in a spheroid sprouting assay ( fig . in contrast , when rrcntf was administered together with soluble rcntf - r , endothelial cell sprouting was significantly reduced , confirming the inhibitory effect of cntf on vegf - induced angiogenesis in the presence of cntf - r. ciliary neurotrophic factor has an anti - angiogenic effect on sprouting endothelial cells in the presence of cntf - r. ( a ) recombinant rat cntf decreases vegf - induced endothelial spheroid sprouting in the presence of cntf - r ( 1-way anova : * * * * p < 0.0001 ) . recombinant rat cntf ( rrcntf ) was injected intravitreally at oir p12 to evaluate its effect on preretinal neovessel formation and capillary regrowth in a model of hypoxia - induced proliferative retinopathy . in analogy to the previous experiment , one of three different doses of cntf ( 0.5 , 50 , or 500 ng cntf ) was injected intravitreally at oir p12 and retinas were collected 4 , 24 , or 48 hours later . ciliary neurotrophic factor activates stat3 signaling in the inner retina in a dose - dependent manner . we hence hypothesized that the antiangiogenic effect of cntf in the retina may be mediated by jak / stat3/socs3 signaling . therefore , all in vitro experiments were performed using four conditions : negative control , rrcntf alone , rcntf - r alone , and rrcntf + rcntf - r. we first tested if cntf exerts direct antiangiogenic effects on endothelial cells in a spheroid sprouting assay ( fig . in contrast , when rrcntf was administered together with soluble rcntf - r , endothelial cell sprouting was significantly reduced , confirming the inhibitory effect of cntf on vegf - induced angiogenesis in the presence of cntf - r. ciliary neurotrophic factor has an anti - angiogenic effect on sprouting endothelial cells in the presence of cntf - r. ( a ) recombinant rat cntf decreases vegf - induced endothelial spheroid sprouting in the presence of cntf - r ( 1-way anova : * * * * p < 0.0001 ) . this study demonstrates that intravitreal injections of recombinant cntf promote capillary regrowth and attenuate pre - retinal neovascularization in a mouse model of oxygen - induced retinopathy . however , activation of the downstream signaling pathway jak / stat3 of cntf was low at oir p13 in control - treated eyes , suggesting that endogenous activation of the cntf signaling pathway during the early hypoxic phase is minor , thus resulting in limited efficacy of this potential repair response ( fig . in our study , cntf is shown to have a therapeutic angiomodulatory effect in vivo that is partially mediated through a direct effect on endothelial cells . beside the direct anti - angiogenic effect of cntf on endothelial cells , it can not be discounted that indirect angiomodulatory effects of cntf via other cell types also play a role in the observed protective in vivo phenotype . however , cntf - mediated induction of socs3 did not alter vegf expression , suggesting that the cntf - induced protective effect in the oir model is independent of vegf . the magnitude of the effect of cntf on preventing pathologic nv is comparable with that of established anti - vegf agents in the oir model .

diarrhea is the fourth - leading cause of death among children under 5 y of age worldwide , killing an estimated 530,000 and hospitalizing millions in 2015 , mostly in developing countries . among the various causes of diarrheal diseases , etec and shigella recent impact studies indicate that immunizing against these two causes of diarrhea could benefit global public health significantly , particularly if they could be combined into a single vaccine for efficient immunization . such a vaccine would improve the health of infants and children in developing countries , and also could protect travelers and military personnel in areas where these bacteria are endemic . the oral route for administration of vaccines to infants is simpler , safer , and more acceptable than needle and syringe , and is particularly useful in developing countries that lack highly trained health care workers and face difficulty in disposing of sharps waste . the target tissue for oral vaccines is the mucosa of the small intestine ; however , the low ph of gastric fluid can degrade protein antigens and some adjuvant components of vaccine formulations before they reach the gut . the ability of antigens to resist degradation during transit through the stomach environment can have a profound effect on their ability to induce protective antibodies . because infants as young as 24 weeks gestational age are able to maintain the intragastric ph at the adult level below 4from the first day of life , even vaccines for young infants must survive this environment . thus , oral vaccines typically require a buffer , which can be given with the vaccine or incorporated into the formulation . oral enteric vaccine candidates in development include whole - cell inactivated bacterial and subunit vaccines most of which require a mucosal adjuvant to improve efficacy . although neutralizing stomach acid may not be as important for inactivated vaccines as for live ones , the stability of mucosal adjuvants in stomach acid must be evaluated , as some of these are sensitive to ph . the choice of buffer is important not only for maintaining vaccine efficacy , but also for avoiding unwanted side effects in infants . sodium bicarbonate , a commonly used antacid buffer , has been reported to cause discomfort from release of carbon dioxide gas after neutralization of stomach acid . this gas build - up in the stomach can create pressure that results in reflux of stomach contents into the esophagus , causing spitting , and may require re - administration of the vaccine . children can have reflux at any age , although it is more common in infants less than one year of age . other antacid buffers , such as bicarbonate - ascorbic acid , have been evaluated in clinical studies in infants and have been reported to cause less bloating than bicarbonate buffer alone . the phosphate - citrate buffer used in rotateq vaccine is another good candidate for the new enteric vaccines for infants . this buffer has sufficient stomach acid neutralizing capacity in a 2-ml dose volume , without unwanted side effects , and has shown good compatibility with different types of delivery containers . figure 1image of an infant receiving oral rotavirus vaccine from a prefilled polymer squeeze tube . image of an infant receiving oral rotavirus vaccine from a prefilled polymer squeeze tube . keeping the dose volume of vaccine formulation and buffer low is important for infants , since swallowing is slow and uncoordinated , and this can lead to spitting or vomiting of the vaccine . the total volume of the vaccine / adjuvant formulation and the buffer should not exceed 2 ml . the trivalent oral polio vaccine ( opv ) that requires 2 drops per dose is a gold standard formulation in terms of keeping the volume minimal ; however , opv is resistant to gastric acid and does not require an antacid buffer , allowing for this small dose volume . this will probably not be the case for new enteric vaccine candidates , such as a possible combination vaccine against etec and shigella , where the final vaccine presentation with multiple antigens , a mucosal adjuvant , and for vaccines formulated with antacid buffer , the largest contribution to overall dosing volume comes from the buffer component . the final formulation , comprising the vaccine and the adjuvant components , requires physiological ph and isotonicity ; thus , the buffer component must have low salt concentration to maintain viability of the antigens . at isotonic salt concentration , there is insufficient or low buffering capacity , which means large dose volumes of the formulation would be needed to effectively buffer the stomach acid . one way to maintain the low dose volume requirement for new combination vaccines is by separating the vaccine components . for example , antigens and adjuvant can be combined in one primary container , and the buffer concentrated in a low dose volume can have its own primary container . multiple administration steps are needed to fully vaccinate the infant , introducing greater risk of incomplete or improper administration of the full dose . requirements for containers and delivery devices for new oral vaccines include protection of and compatibility with the vaccine formulation , performance and ease of use in dose administration , safety , manufacturability , immunization program suitability , and cost . the stability of any new vaccine must be established for the specific material , design , and filling process for the intended container . glass vials are a common primary container for many injectable vaccines , and are also used for opv and some oral rotavirus vaccines . vials for oral administration are generally packaged with droppers to be attached at the point of use ; this approach is most suitable for very low - volume doses delivered by counting drops . polymer containers also are widely used for pharmaceuticals and for vaccines ; these include preformed containers that are sterilized and shipped to vaccine manufacturers for filling and sealing and blow - fill - seal containers that are formed , filled , and sealed in a single operation . polymer squeeze tubes are used as primary packaging for several opv and rotavirus vaccines and have the advantage of acting as the vaccine administration device , eliminating the need for an additional component for delivery . however , some adjuvants may adsorb to polymers , which would require modifying excipients to minimize this risk . polymer materials also are more permeable than glass to water vapor and oxygen transmission , and to leaching of label materials . protective secondary packaging , such as a foil pouch , may be used to prevent water and gas transfer through the container , and labeling can be applied to a tab rather than to the body of the container to minimize leaching of ink and adhesive components into the drug substance although both of these will increase the container 's cold chain volume . inclusion of a vaccine vial monitor ( vvm ) in the vaccine 's labeling is a requirement for vaccines assessed for world health organization ( who ) prequalification , in accordance with the programmatic suitability of vaccine candidates for who prequalification ( pspq ) , which provides guidelines on vaccine characteristics that will be considered during the prequalification process . for presentations requiring protective secondary packaging , placement of the vvm if multiple doses of vaccine are packaged together in the same pouch or overwrap , the vvm should be placed on the primary container so that it remains with the vaccine until each dose is delivered . however , in this case the vvm may be obscured by the secondary packaging , which could present challenges for inspection during storage . many of the features discussed here also provide safety and program suitability in low - resource settings , in addition to ease of use . for all new oral vaccine presentations , human factors and usability evaluations should be performed to assess ease of use , correct dosing , and any potential risks or errors resulting from the presentation . one important ergonomic factor for delivery of oral presentations is squeezability of the delivery device , which is determined by container shape , material stiffness , and viscosity of the vaccine . optimal usability and safety are achieved by minimizing the steps ( and subsequent risks ) associated with preparing and delivering the vaccine at the point of use , and by giving the vaccinator fine - tuned control over the dispensing speed . presentations requiring reconstitution or mixing of vaccine components at the point of use increase the time required for and the complexity of the delivery process , and have resulted in errors such as use of incorrect diluents or administration of the diluent alone by practitioners who mistake it for the complete vaccine . to this end , a critical characteristic of the pspq is that oral vaccine formulations be in a ready - to - use format ( no reconstitution needed ) for developing - country , public - sector immunization programs . compliance with this requirement is compulsory , but requests for exceptions are reviewed by the pspq standing committee , taking into account the public health need . if preparation steps are necessary , the mixing process and containers should be easy to use and should minimize the risk of errors , and all required components should be disposable and packaged with each dose of vaccine . this co - packaging requirement applies to water for reconstitution as well , as clean water may not be available at immunization sites in low - resource settings . integrated reconstitution technologies in which multiple liquid or dry vaccine components are packaged within the same primary container and mixed within the device prior to administration can simplify the preparation process and reduce the risk of errors , but can also be costlier . some features that impart ease of use to containers and delivery devices such as ready - to - use formulations also increase safety of use . an inherent safety feature of oral vaccines is that they are administered without needles , and thus needlestick injuries and sharps disposal can be avoided . another safety consideration is the use of packaging and delivery - device designs that reduce the risk of accidental injection of an oral vaccine , which can cause adverse events and render vaccination ineffective . this error has occurred with oral rotavirus vaccines , particularly those in packaging that appear similar to vaccines for injection . the risk of injection precludes use of oral vaccine presentations in glass vials intended for delivery with a syringe with the needle removed . manufacturing feasibility and cost are also important for selecting containers and delivery devices for oral vaccines . for vaccines that may be marketed to adult travelers as well as provided to infants in low - resource settings , different presentations and different final manufacturing processes may be needed . polymer containers may have lower per - unit costs than glass vials and be easier to use , but since manufacturers are likely to have glass - vial - filling facilities in place , using an alternative fill - finish process will entail start - up costs , could require building or repurposing facilities , and might increase a company 's technical burden and risks . for vaccines needing reconstitution , integrated reconstitution devices can simplify delivery but may increase manufacturing complexity and device costs . if a manufacturer is contracting with other companies for packaging or delivery components , having only a single source supplier is a risk , since a problem at this source could hold up the entire production process . immunization program suitability . for example , accurate dose preparation and measurement , as well as simple , controlled vaccine administration are desirable not only for ease of use but for program suitability in low - resource settings where health care workers may have minimal training . use of a polymer squeeze tube as the primary container and delivery device increases safety , addresses ease of use , and reduces packaging volume all of which contribute to program suitability . single - dose presentations offer advantages in ease of use , as they do not require measurement of doses ; however , they can increase manufacturing costs and cold chain storage volumes . multi - dose presentations reduce space needed in the cold chain , but they increase vaccine wastage , particularly if the vaccine is at risk of contamination or is not stable for more than one immunization session after opening . tradeoffs such as these must be assessed in determining the optimal primary container for a new vaccine . vaccine manufacturers as well as developers of packaging and administration components can get feedback on the suitability of proposed presentations of vaccines in development from the immunization practices advisory committee 's delivery technologies working group and from the who prequalification group . immunization program managers should ideally assess the overall program cost to deliver a vaccine in different presentations , in addition to vaccine price itself . some technologies may increase safety or ease of use , but likely will increase vaccine purchase costs and the burden on the cold chain , so these attributes must be balanced in selection of a vaccine presentation . developers will want to design primary containers and delivery devices with the needs of manufacturers and immunization programs in mind , in order to maximize suitability while minimizing costs . for example , for polymer tubes , a multi - mono - dose configuration in which multiple , conjoined , single - dose containers share a single label and vvm can reduce manufacturing costs and demands on storage space . however , this approach requires a design that prevents users from removing a single container without the label information . in summary , oral vaccines against bacterial pathogens such as etec and shigella that cause diarrhea in millions of children in low - resource countries could save lives and reduce the burden of serious illnesses . new oral enteric vaccine candidates must be buffered against stomach acid , formulated in minimal dose volumes , filled into containers that protect antigens and adjuvants but minimize the load on the cold chain , and can be safely administered in the correct dose by minimally trained vaccinators working in challenging environments all within the constraints of manufacturability and attention to cost . product designers who take these features into account in their development processes will be able to position new oral vaccines for implementation in routine immunization programs , and these programs in turn will achieve optimal vaccine impact . the oral route for administration of vaccines to infants is simpler , safer , and more acceptable than needle and syringe , and is particularly useful in developing countries that lack highly trained health care workers and face difficulty in disposing of sharps waste . the target tissue for oral vaccines is the mucosa of the small intestine ; however , the low ph of gastric fluid can degrade protein antigens and some adjuvant components of vaccine formulations before they reach the gut . the ability of antigens to resist degradation during transit through the stomach environment can have a profound effect on their ability to induce protective antibodies . because infants as young as 24 weeks gestational age are able to maintain the intragastric ph at the adult level below 4from the first day of life , even vaccines for young infants must survive this environment . thus , oral vaccines typically require a buffer , which can be given with the vaccine or incorporated into the formulation . oral enteric vaccine candidates in development include whole - cell inactivated bacterial and subunit vaccines most of which require a mucosal adjuvant to improve efficacy . although neutralizing stomach acid may not be as important for inactivated vaccines as for live ones , the stability of mucosal adjuvants in stomach acid must be evaluated , as some of these are sensitive to ph . the choice of buffer is important not only for maintaining vaccine efficacy , but also for avoiding unwanted side effects in infants . sodium bicarbonate , a commonly used antacid buffer , has been reported to cause discomfort from release of carbon dioxide gas after neutralization of stomach acid . this gas build - up in the stomach can create pressure that results in reflux of stomach contents into the esophagus , causing spitting , and may require re - administration of the vaccine . children can have reflux at any age , although it is more common in infants less than one year of age . other antacid buffers , such as bicarbonate - ascorbic acid , have been evaluated in clinical studies in infants and have been reported to cause less bloating than bicarbonate buffer alone . the phosphate - citrate buffer used in rotateq vaccine is another good candidate for the new enteric vaccines for infants . this buffer has sufficient stomach acid neutralizing capacity in a 2-ml dose volume , without unwanted side effects , and has shown good compatibility with different types of delivery containers . figure 1image of an infant receiving oral rotavirus vaccine from a prefilled polymer squeeze tube . keeping the dose volume of vaccine formulation and buffer low is important for infants , since swallowing is slow and uncoordinated , and this can lead to spitting or vomiting of the vaccine . the total volume of the vaccine / adjuvant formulation and the buffer should not exceed 2 ml . the trivalent oral polio vaccine ( opv ) that requires 2 drops per dose is a gold standard formulation in terms of keeping the volume minimal ; however , opv is resistant to gastric acid and does not require an antacid buffer , allowing for this small dose volume . this will probably not be the case for new enteric vaccine candidates , such as a possible combination vaccine against etec and shigella , where the final vaccine presentation with multiple antigens , a mucosal adjuvant , and a diluent / buffer may result in a large oral dose volume . for vaccines formulated with antacid buffer , the final formulation , comprising the vaccine and the adjuvant components , requires physiological ph and isotonicity ; thus , the buffer component must have low salt concentration to maintain viability of the antigens . at isotonic salt concentration , there is insufficient or low buffering capacity , which means large dose volumes of the formulation would be needed to effectively buffer the stomach acid . one way to maintain the low dose volume requirement for new combination vaccines is by separating the vaccine components . for example , antigens and adjuvant can be combined in one primary container , and the buffer concentrated in a low dose volume can have its own primary container . multiple administration steps are needed to fully vaccinate the infant , introducing greater risk of incomplete or improper administration of the full dose . requirements for containers and delivery devices for new oral vaccines include protection of and compatibility with the vaccine formulation , performance and ease of use in dose administration , safety , manufacturability , immunization program suitability , and cost . the stability of any new vaccine must be established for the specific material , design , and filling process for the intended container . glass vials are a common primary container for many injectable vaccines , and are also used for opv and some oral rotavirus vaccines . vials for oral administration are generally packaged with droppers to be attached at the point of use ; this approach is most suitable for very low - volume doses delivered by counting drops . polymer containers also are widely used for pharmaceuticals and for vaccines ; these include preformed containers that are sterilized and shipped to vaccine manufacturers for filling and sealing and blow - fill - seal containers that are formed , filled , and sealed in a single operation . polymer squeeze tubes are used as primary packaging for several opv and rotavirus vaccines and have the advantage of acting as the vaccine administration device , eliminating the need for an additional component for delivery . however , some adjuvants may adsorb to polymers , which would require modifying excipients to minimize this risk . polymer materials also are more permeable than glass to water vapor and oxygen transmission , and to leaching of label materials . protective secondary packaging , such as a foil pouch , may be used to prevent water and gas transfer through the container , and labeling can be applied to a tab rather than to the body of the container to minimize leaching of ink and adhesive components into the drug substance although both of these will increase the container 's cold chain volume . inclusion of a vaccine vial monitor ( vvm ) in the vaccine 's labeling is a requirement for vaccines assessed for world health organization ( who ) prequalification , in accordance with the programmatic suitability of vaccine candidates for who prequalification ( pspq ) , which provides guidelines on vaccine characteristics that will be considered during the prequalification process . for presentations requiring protective secondary packaging , placement of the vvm if multiple doses of vaccine are packaged together in the same pouch or overwrap , the vvm should be placed on the primary container so that it remains with the vaccine until each dose is delivered . however , in this case the vvm may be obscured by the secondary packaging , which could present challenges for inspection during storage . many of the features discussed here also provide safety and program suitability in low - resource settings , in addition to ease of use . for all new oral vaccine presentations , human factors and usability evaluations should be performed to assess ease of use , correct dosing , and any potential risks or errors resulting from the presentation . one important ergonomic factor for delivery of oral presentations is squeezability of the delivery device , which is determined by container shape , material stiffness , and viscosity of the vaccine . optimal usability and safety are achieved by minimizing the steps ( and subsequent risks ) associated with preparing and delivering the vaccine at the point of use , and by giving the vaccinator fine - tuned control over the dispensing speed . presentations requiring reconstitution or mixing of vaccine components at the point of use increase the time required for and the complexity of the delivery process , and have resulted in errors such as use of incorrect diluents or administration of the diluent alone by practitioners who mistake it for the complete vaccine . to this end , a critical characteristic of the pspq is that oral vaccine formulations be in a ready - to - use format ( no reconstitution needed ) for developing - country , public - sector immunization programs . compliance with this requirement is compulsory , but requests for exceptions are reviewed by the pspq standing committee , taking into account the public health need . if preparation steps are necessary , the mixing process and containers should be easy to use and should minimize the risk of errors , and all required components should be disposable and packaged with each dose of vaccine . this co - packaging requirement applies to water for reconstitution as well , as clean water may not be available at immunization sites in low - resource settings . integrated reconstitution technologies in which multiple liquid or dry vaccine components are packaged within the same primary container and mixed within the device prior to administration can simplify the preparation process and reduce the risk of errors , but can also be costlier . some features that impart ease of use to containers and delivery devices such as ready - to - use formulations also increase safety of use . an inherent safety feature of oral vaccines is that they are administered without needles , and thus needlestick injuries and sharps disposal can be avoided . another safety consideration is the use of packaging and delivery - device designs that reduce the risk of accidental injection of an oral vaccine , which can cause adverse events and render vaccination ineffective . this error has occurred with oral rotavirus vaccines , particularly those in packaging that appear similar to vaccines for injection . the risk of injection precludes use of oral vaccine presentations in glass vials intended for delivery with a syringe with the needle removed . manufacturing feasibility and cost are also important for selecting containers and delivery devices for oral vaccines . for vaccines that may be marketed to adult travelers as well as provided to infants in low - resource settings , different presentations and different final manufacturing processes may be needed . polymer containers may have lower per - unit costs than glass vials and be easier to use , but since manufacturers are likely to have glass - vial - filling facilities in place , using an alternative fill - finish process will entail start - up costs , could require building or repurposing facilities , and might increase a company 's technical burden and risks . for vaccines needing reconstitution , integrated reconstitution devices can simplify delivery but may increase manufacturing complexity and device costs . if a manufacturer is contracting with other companies for packaging or delivery components , having only a single source supplier is a risk , since a problem at this source could hold up the entire production process . immunization program suitability . for example , accurate dose preparation and measurement , as well as simple , controlled vaccine administration are desirable not only for ease of use but for program suitability in low - resource settings where health care workers may have minimal training . use of a polymer squeeze tube as the primary container and delivery device increases safety , addresses ease of use , and reduces packaging volume all of which contribute to program suitability single - dose presentations offer advantages in ease of use , as they do not require measurement of doses ; however , they can increase manufacturing costs and cold chain storage volumes . multi - dose presentations reduce space needed in the cold chain , but they increase vaccine wastage , particularly if the vaccine is at risk of contamination or is not stable for more than one immunization session after opening . tradeoffs such as these must be assessed in determining the optimal primary container for a new vaccine . vaccine manufacturers as well as developers of packaging and administration components can get feedback on the suitability of proposed presentations of vaccines in development from the immunization practices advisory committee 's delivery technologies working group and from the who prequalification group . cost containment . immunization program managers should ideally assess the overall program cost to deliver a vaccine in different presentations , in addition to vaccine price itself . some technologies may increase safety or ease of use , but likely will increase vaccine purchase costs and the burden on the cold chain , so these attributes must be balanced in selection of a vaccine presentation . developers will want to design primary containers and delivery devices with the needs of manufacturers and immunization programs in mind , in order to maximize suitability while minimizing costs . for example , for polymer tubes , a multi - mono - dose configuration in which multiple , conjoined , single - dose containers share a single label and vvm can reduce manufacturing costs and demands on storage space . however , this approach requires a design that prevents users from removing a single container without the label information . in summary , oral vaccines against bacterial pathogens such as etec and shigella that cause diarrhea in millions of children in low - resource countries could save lives and reduce the burden of serious illnesses . but new oral enteric vaccine candidates must be buffered against stomach acid , formulated in minimal dose volumes , filled into containers that protect antigens and adjuvants but minimize the load on the cold chain , and can be safely administered in the correct dose by minimally trained vaccinators working in challenging environments all within the constraints of manufacturability and attention to cost . product designers who take these features into account in their development processes will be able to position new oral vaccines for implementation in routine immunization programs , and these programs in turn will achieve optimal vaccine impact . this work was funded in whole by a grant from the bill & melinda gates foundation . the views expressed herein are solely those of the authors and do not necessarily reflect the views of the foundation .

abstractoral administration of vaccines is simpler and more acceptable than injection via needle and syringe , particularly for infants ( fig . 1 ) this route is promising for new vaccines in development against enterotoxigenic escherichia coli ( etec ) and shigella that cause childhood diarrhea with devastating consequences in low - resource countries . however , vaccine antigens and adjuvants given orally need buffering against the degradative effects of low stomach ph , and the type and volume of antacid buffer require special attention for infants . in addition , container / closure systems must be compatible with vaccine formulations , protect against water and gas transfer , and have minimal impact on the cold chain . health care workers in demanding low - resource settings need an administration device that is easy to use , yet will accurately measure and safely deliver the correct vaccine dose . developers must consider manufacturing capabilities , and immunization program managers want affordable vaccines . as new combination enteric vaccine candidates advance into clinical evaluation , features of the final vaccine presentation liquid or dry format , diluent , buffer , primary and secondary packaging , and administration device should be taken into account early in product development to achieve the greatest possible impact for the vaccine .

Health impact of diarrhea Oral vaccines usually require buffering The final presentation should permit a small dose volume Key features of effective containers and delivery devices Summary Disclosure of potential conflicts of interest Funding

the oral route for administration of vaccines to infants is simpler , safer , and more acceptable than needle and syringe , and is particularly useful in developing countries that lack highly trained health care workers and face difficulty in disposing of sharps waste . this will probably not be the case for new enteric vaccine candidates , such as a possible combination vaccine against etec and shigella , where the final vaccine presentation with multiple antigens , a mucosal adjuvant , and for vaccines formulated with antacid buffer , the largest contribution to overall dosing volume comes from the buffer component . protective secondary packaging , such as a foil pouch , may be used to prevent water and gas transfer through the container , and labeling can be applied to a tab rather than to the body of the container to minimize leaching of ink and adhesive components into the drug substance although both of these will increase the container 's cold chain volume . for presentations requiring protective secondary packaging , placement of the vvm if multiple doses of vaccine are packaged together in the same pouch or overwrap , the vvm should be placed on the primary container so that it remains with the vaccine until each dose is delivered . presentations requiring reconstitution or mixing of vaccine components at the point of use increase the time required for and the complexity of the delivery process , and have resulted in errors such as use of incorrect diluents or administration of the diluent alone by practitioners who mistake it for the complete vaccine . for example , accurate dose preparation and measurement , as well as simple , controlled vaccine administration are desirable not only for ease of use but for program suitability in low - resource settings where health care workers may have minimal training . some technologies may increase safety or ease of use , but likely will increase vaccine purchase costs and the burden on the cold chain , so these attributes must be balanced in selection of a vaccine presentation . in summary , oral vaccines against bacterial pathogens such as etec and shigella that cause diarrhea in millions of children in low - resource countries could save lives and reduce the burden of serious illnesses . new oral enteric vaccine candidates must be buffered against stomach acid , formulated in minimal dose volumes , filled into containers that protect antigens and adjuvants but minimize the load on the cold chain , and can be safely administered in the correct dose by minimally trained vaccinators working in challenging environments all within the constraints of manufacturability and attention to cost . the oral route for administration of vaccines to infants is simpler , safer , and more acceptable than needle and syringe , and is particularly useful in developing countries that lack highly trained health care workers and face difficulty in disposing of sharps waste . this will probably not be the case for new enteric vaccine candidates , such as a possible combination vaccine against etec and shigella , where the final vaccine presentation with multiple antigens , a mucosal adjuvant , and a diluent / buffer may result in a large oral dose volume . for vaccines formulated with antacid buffer , the final formulation , comprising the vaccine and the adjuvant components , requires physiological ph and isotonicity ; thus , the buffer component must have low salt concentration to maintain viability of the antigens . protective secondary packaging , such as a foil pouch , may be used to prevent water and gas transfer through the container , and labeling can be applied to a tab rather than to the body of the container to minimize leaching of ink and adhesive components into the drug substance although both of these will increase the container 's cold chain volume . for presentations requiring protective secondary packaging , placement of the vvm if multiple doses of vaccine are packaged together in the same pouch or overwrap , the vvm should be placed on the primary container so that it remains with the vaccine until each dose is delivered . many of the features discussed here also provide safety and program suitability in low - resource settings , in addition to ease of use . presentations requiring reconstitution or mixing of vaccine components at the point of use increase the time required for and the complexity of the delivery process , and have resulted in errors such as use of incorrect diluents or administration of the diluent alone by practitioners who mistake it for the complete vaccine . for example , accurate dose preparation and measurement , as well as simple , controlled vaccine administration are desirable not only for ease of use but for program suitability in low - resource settings where health care workers may have minimal training . some technologies may increase safety or ease of use , but likely will increase vaccine purchase costs and the burden on the cold chain , so these attributes must be balanced in selection of a vaccine presentation . in summary , oral vaccines against bacterial pathogens such as etec and shigella that cause diarrhea in millions of children in low - resource countries could save lives and reduce the burden of serious illnesses . but new oral enteric vaccine candidates must be buffered against stomach acid , formulated in minimal dose volumes , filled into containers that protect antigens and adjuvants but minimize the load on the cold chain , and can be safely administered in the correct dose by minimally trained vaccinators working in challenging environments all within the constraints of manufacturability and attention to cost .

considerable effort has been made to understand mechanisms leading to the initiation of autoimmune diseases , and yet the reason for the loss of self - tolerance is still not fully clarified . one hypothesis is that infection triggers autoimmune disease in genetically predisposed individuals by cross - reactivity between self - antigen and foreign antigen due to similarity between foreign and self - antigenic epitopes . a second proposal is that infections may also trigger autoimmune disease through bystander activation of autoreactive t cells , in which self - antigen released during tissue damage is presented by activated innate immune cells or b cells , for instance , during mycobacterial infection [ 2 , 3 ] . uveitis or intraocular inflammation is a sight - threatening condition , which affects mostly people of working age . despite improved therapeutic possibilities 10% of patients many cases of intraocular inflammation are directly caused by infections such as toxoplasmosis , viral infections , and other pathogens . some cases may be associated with systemic diseases such as sarcoidosis , multiple sclerosis , and ankylosing spondylitis in which infections as triggers of autoimmunity are also implicated . specific agents such as chlamydophila , human herpes virus 6 , and epstein - barr virus are suggested in either the development or the progression of multiple sclerosis . however , both for idiopathic uveitis and for uveitis associated with systemic disease , the search for infectious causes is frequently fruitless . accordingly , animal models of autoimmune diseases have been developed most of which utilize immunization with an evolutionarily conserved autoantigen together with one or more adjuvants which usually comprise a component of an infectious agent such as heat - killed mycobacteria and pertussis toxin . adjuvants are required in order to activate innate immune cells via pathogen recognition receptors which include at least four classes of receptors , particularly toll - like receptors and c - type lectins . the need for adjuvants in generating experimental models implicates infectious agents in the induction of autoimmune disease and in this context , there has been considerable interest in the role of the microbiome in modulating susceptibility to autoimmune diseases . the explosion of research into the nature of the microbiome using molecular genetic techniques to type and classify microbiota has revealed that commensal organisms exist in specific sites or niches in the body and are specific for each site . initial studies on the gut microbiome in the context of inflammatory bowel disease revealed the role of the gut microbiome in maintaining immunological homeostasis in situ , thereby reducing the risk of inflammatory bowel disease . these studies have been extended to other autoimmune disease models and for this purpose the use of germ - free ( gf ) mice has been extremely valuable [ 913 ] . interestingly , the susceptibility to experimental autoimmune disease varied with the type of the disease : for instance , experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein was found to be less severe in gf mice , while diabetes mellitus in nonobese diabetic mice was more severe [ 1517 ] . have previously reported in a surrogate model of gf mice that alteration of the gut microbiota by using a combination of orally administered broad - spectrum antibiotics modulates the severity of eau . more recently work from the same laboratory has used an interphotoreceptor retinoid - binding protein- ( irbp- ) transgenic mouse model of spontaneous uveitis in antibiotic - treated and gf mice to demonstrate suppression of eau . although this work describes a spontaneous model of eau , which is considered by some to be more clinically relevant than the complete freund 's adjuvant- ( cfa- ) induced model , the mice contain an artificially high number of irbp - specific cd4 t cells ( > 20% in the irbp-161h clone ) . however , in cv mice and in humans , the precursor frequency of any particular antigen - specific t cell is known to be very low , if not rare . it is therefore possible that in the irbp - transgenic spontaneous model of uveitis , the sheer weight of numbers of antigen - specific t cells predicates a greatly increased risk of microbial antigen - induced t cell receptor cross - reactivity , which would then permit entry of activated t cells into tissue sites and further activation via cognate antigen . we have therefore investigated whether reduction in the gut microbiome , by either antibiotic use as previously reported or as found in gf mice , modifies eau induction in cv mice in which t cell activation is induced with a mycobacterial adjuvant ( cfa ) and is dependent on innate immune cell activation and signaling via the c - type lectin , dectin-1 . we report that the severity of eau is markedly reduced in gf mice and in mice which have been pretreated with antibiotics to reduce microbial burden but not when the microbial burden is reduced after activation of t cells has been induced . we used inbred male and female mice of the c57bl/6j strain ( 5 to 8 weeks old ) . mice were housed either at the conventional animal facility of department of pharmacology , first faculty of medicine , charles university in prague , where untreated cv mice were compared with cv mice treated with broad - spectrum antibiotics , or in the laboratory of gnotobiology at the institute of microbiology academy of sciences , czech republic , novy hradek , where experiments comparing gf and cv mice were performed . the mice were rederived into gf conditions using caesarean section and bred in sterile trexler - type plastic isolators for many generations . the bedding , food pellets , and water were sterilized by gamma irradiation ( 25 kgy ) or autoclaving . the long - term colonies of gf mice were supplied with sterile water and food pellets , hd2 extruded diet ( fitmin , czech republic ) , ad libitum . the gf status of colonies was evaluated weekly as fecal samples and cotton swabs from the isolator interior were tested for the presence of aerobic and anaerobic bacteria , mold , and yeast . the cv mice , which served as cv controls at this facility , were fed with the same diet and regularly tested for the absence of potential mouse pathogens , including strains of helicobacter muridarum and h. hepaticus , according to internationally recognized standards ( felasa ) . the use of animals for these experiments was approved by the commission for animal welfare of the first faculty of medicine of charles university in prague , czech republic , and the ministry of education , youth and sports and by the animal care and use committee of the institute of microbiology , academy of sciences of the czech republic , according to animal protection laws . eau was induced by subcutaneous inoculation of irbp peptide 500 g per mouse in complete freund 's adjuvant in conjunction with intraperitoneal application of pertussis toxin ( pt ) 0.6 g according to a standard protocol [ 23 , 24 ] . in brief , irbp peptide 120 ( interphotoreceptor retinoid - binding protein , also called retinol - binding protein 3-precursor fragment [ homo sapiens ] h2n - gpthlfqpslvldmakvlld - oh , new england peptide , gardner , usa ) dissolved in dmso ( dimethyl sulfoxide , sigma - aldrich , st . louis , usa ) was emulsified in ratio 1 : 1 with cfa ( difco , usa ) and the solution was applied subcutaneously . to reduce the microbial load , the mice were treated with broad - spectrum antibiotics ( mixture of 500 mg / l of metronidazole ( b. braun , czech republic ) and ciprofloxacin 100 mg / l ( ciprinol , krka , czech republic ) ) in the drinking water as previously described . metronidazole ( nitroimidazole ) has a limited spectrum of activity that encompasses various protozoans and most gram - negative and gram - positive anaerobic bacteria . ciprofloxacin is a second - generation fluoroquinolone , with a spectrum of activity , which includes gram - negative and gram - positive bacterial pathogens . to establish the importance of the microbiota with respect to disease induction , we initiated treatment either one week prior to eau induction or on the day of eau induction . in both experimental schedules , the antibiotic treatment continued until the end of the experiment . in vivo clinical examination ( fundus biomicroscopy ) an additional + 4.0 diopter lens between the camera and the otoscope was used . during the procedure , the mice were under general anesthesia ( ketamine 80 mg / kg and xylazine 5 mg / kg ( both bioveta , slovakia ) intraperitoneally ) . the otoscope was applied to the cornea using eye gel carbomerum ( vidisic gel , bausch and lomb , czech republic ) . a single image of the posterior central fundus from each eye was taken , transferred to a computer for analysis . retinal inflammatory changes were evaluated separately for the optic disc , retinal vessels , and retinal tissue changes from the central fundus ( table 1 ) . all samples were evaluated by two experienced ophthalmologists ( pss , ak ) and the discussed consensus of the two evaluations was used . the mice were sacrificed on day 35 and the eyes were enucleated and immediately immersed in tissue - tek o.c.t . torrance , ca , usa ) and frozen in 2-methylbutane ( sigma - aldrich , st . the samples were stored at 70c until sectioning to 7 m thick slices ( at 19 to 21c ) . the samples were cut with a cryostat ( leica cm 1850 ) and stained with hematoxylin and eosin . these samples were then evaluated by two experienced ophthalmologists and graded using a standardized scoring system as previously published [ 7 , 29 , 30 ] and modified by the authors ( table 2 ) . eyes with congenital defects , such as microphthalmia or cataract , have been excluded from evaluation , which led to odd numbers in some graphs . t - lymphocytes were detected using a three - step immunoperoxidase method with polyclonal rabbit anti - human cd3 ( dako denmark a / s , glostrup , denmark ) diluted 1 : 200 in pbs containing 1.5% normal goat serum visualization of primary antibody binding was performed using secondary biotinylated anti - rabbit antibody ( dako ) and the vectastain elite abc kit standard ( vector laboratories , usa ) . macrophages were detected using a three - step immunoperoxidase method with monoclonal rat anti - mouse f4/80 antibody ( clone bm8 , abcam , cambridge , uk ) diluted 1 : 100 in pbs containing 1.5% normal goat serum . visualization of primary antibody binding was performed using secondary biotinylated anti - rat antibody ( abcam ) and the vectastain elite abc kit standard ( vector laboratories , usa ) . positive cells were counted in two sections per eye , one from periphery and one from the centre , to obtain quantitative data . mouse mesenteric and cervical lymph nodes were separately harvested , mashed into cell suspension , washed in complete rpmi medium , and filtered through a 70 m cell strainer . for detection of regulatory t cells , the cell suspensions were washed , labeled with fixable viability dye ( ebioscience ) , blocked with anti - cd16/cd32 antibody , stained for surface cd4 and cd25 , fixed and permeabilized overnight with fixation / permeabilization buffer ( ebioscience ) , and stained for intracellular foxp3 . to analyze intracellular cytokine production , cells ( 2 10 cells / ml in complete rpmi ) were incubated for 5 hours with 50 ng / ml pma , 500 ng / ml ionomycin ( both from sigma - aldrich ) , and 2 m monensin ( ebioscience ) . after the incubation , the cells were washed , labeled with a viability dye , blocked , stained for surface cd4 , fixed , and permeabilized as described above . next , the cells were stained for intracellular cytokines with antibodies against ifn- , il-17 , and tnf-. the data were acquired on a facscalibur flow cytometer and analyzed with flowjo software . data were analyzed using graphpad prism version 6.04 for windows ( graphpad software , san diego , ca , usa , http://www.graphpad.com/ ) . kruskal - wallis and mann - whitney nonparametric tests were used to evaluate differences between the groups and p < 0.05 was considered significant . eau was induced with sterile reagents in either gf or cv mice and the level of inflammation at day 35 by fundoscopy and histology was compared . , no inflammation was observed in the gf mice at day 35 after induction , whereas in control cv mice , severe inflammation was observed as extensive signs of chorioretinal lesions , vascular sheathing ( vasculitis ) , and vitreous haze ( table 1 and figures 1(a ) and 1(b ) ; p < 0.001 ) . on histological evaluation , minimal to no signs of uveitis were observed in gf mice compared to severe uveitis in cv mice ( table 2 and figures 1(c ) and 1(d ) ; p < 0.001 ) . since gf mice appeared to develop less severe eau disease than cv housed mice , we speculated whether reduction in microbial load using antibiotic therapy would have the same effect as the gf state . we performed two experiments by administering antibiotics in cv mice either from the day of eau induction or from one week before . mice treated with metronidazole and ciprofloxacin ( see section 2 ) commencing one week prior to eau induction and continued for the course of the experiment ( a treatment which significantly reduces microbial burden ) had significantly lower levels of eau compared to controls both clinically ( figures 2(a ) and 2(b ) ; maximal difference observed at day 35 ; p < 0.05 ) and histologically ( figures 2(c ) and 2(d ) ; p < 0.05 ) . in contrast , mice treated with the same antibiotic regime but commencing on the day of immunization showed little difference in the level of eau compared to controls ( figures 3(a ) and 3(b ) ) . immunohistological studies of the eyes performed on gf mice and littermate controls at day 35 ( see section 2 ) showed that qualitatively there was no difference in the nature of the cell infiltrate in the retina and choroid , which was composed of t cells and macrophages , distributed as individual cells or small cell aggregates ( granulomas ) . however , quantitatively there was a significant reduction in cd3 t cells ( figure 4(a ) ; p < 0.05 ) and a similar but nonsignificant reduction in f4/80 macrophages ( figure 4(b ) ; p = 0.093 ) in the gf mice compared to the controls . immunohistology was also performed on eyes of mice treated with metronidazole and ciprofloxacin from one week before or on the day of eau induction ( see section 2 ; figures 4(c ) , 4(d ) , 4(e ) , and 4(f ) ) . our data show that there was no significant qualitative or quantitative difference in the numbers and distribution of cd3 t cells ( figures 4(c ) and 4(e ) ) or f4/80 macrophages ( figures 4(d ) and 4(f ) ) , when compared to controls . since in cfa immunized mice , t cell activation occurs extraocularly with clonal expansion in the skin - draining lymph nodes beginning as early as 6 days after immunization and subsequently in the eye - draining nodes as disease develops , we evaluated the phenotypes of lymph node cells by flow cytometry . in eye - draining cervical lymph nodes of cv mice , we observed an expansion of ifn--producing ( p < 0.01 ) and il-17-producing cd4 t cells ( p < 0.01 ) and a reduced percentage of foxp3 tregs ( p < 0.01 ) in cv mice at day 35 after immunization with cfa and irbp . however , the observed t cell expansion was considerably reduced in gf mice , which were similarly immunized . interestingly , the percentage of cd4tnf- t cells was similar in both cv and gf mice . cell populations in non - eye - draining mesenteric lymph nodes showed a small increase in the percentage of ifn--producing cd4 t cells in cv mice ( p < 0.05 ) which was significantly greater than ifn- t cells in gf mice but there was no difference in il-17-producing t cells in the mesenteric lymph nodes ( figure 5 ) . the gut microbiota plays a significant role in the development of many inflammatory diseases , both in the gut [ 33 , 34 ] and in distant organs [ 10 , 3538 ] . this is particularly important during the early stages of development , when the presence of microbiota is crucial for the maturation of the immune system in adult life . as mentioned in section 1 , the presence of microbiota usually enhances inflammation in most animal models of colitis , multiple sclerosis , arthritis , or ankylosing spondylitis [ 13 , 14 , 41 , 42 ] , but it decreases the inflammation in models of type 1 diabetes . here , we show that the severity of the ocular inflammation in a murine model of autoimmune uveoretinitis is significantly lowered if the bacterial load is reduced either by rearing the mice in gf conditions ( figure 1 ) or by prophylactic treatment with broad - spectrum antibiotics ( metronidazole and ciprofloxacin , figure 2 ) . similar results were recently reported in the irbp - transgenic mouse model of spontaneous uveitis . however , the mode of uveitis pathogenesis in this model is dependent on a high peripheral precursor frequency of antigen - specific t cells ( around 20% antigen - specific t cells are required in the periphery for expression of disease ) which is far in excess of antigen - specific t cell precursor frequency in normal mice and humans . our data now show that in the standard model of eau induced by irbp in cfa , which more closely resembles human uveitis , gf mice have markedly reduced but not completely suppressed disease . we also show as has been reported previously [ 18 , 19 ] that , by reducing the bacterial load by administration of broad - spectrum antibiotics , eau is significantly reduced in severity . importantly , in our study , when antibiotics were administered from the time of eau induction , there was no significant effect on eau severity . these experiments suggest that the preexisting microenvironment , particularly of the gut , has a significant role in determining the level of susceptibility to eau and in addition that broad - spectrum antibiotic treatment may at least partially modify this environment , although not as effectively as in germ - free mice . this might be relevant for human medicine as treatment of uveitis patients with antibiotics in the past has not been limited only to infectious forms of uveitis . the timing of the antibiotic treatment in patients , however , was always initiated after the development of immune response . in addition , some antibiotics , such as metronidazole , are known to suppress certain aspects of cell - mediated immunity , even when administered orally . these experiments do not exclude the role of microbiota in the establishment of the immune response , since commensal microbes can produce molecules that regulate the immune system and these microbes may also be influenced by oral antibiotics [ 4547 ] . in our experiments , when average water consumption and average mouse weight is calculated , the exposure to metronidazole was approximately 65 mg / kg for either gender of mice . although this amount may have some minor suppressive effect on cellular immunity , the resistance of gf mice to eau and the sensitivity of cv mice treated from the day of eau induction suggest that it is more the effect on the microbiota than the immunosuppressive effect of metronidazole . the surface of the eye , as for all mucosal surfaces , is colonized by microbes , and it is possible that the conjunctival microbiome may influence the development of ocular inflammation . however , such an event is unlikely unless there is a breach of the ocular surface barrier and any effect would likely be mediated through breakdown of the blood ocular barrier , in a similar manner that must apply to the gut microbiome . in this context , horai et al . have suggested that il-17-producing antigen - specific t cells in the gut lamina propria are activated through their t cell receptor via noncognate microbial antigen derived from commensal bacteria . these activated cells then have the possibility to cross the blood retinal barrier and induce disease . in our experiments , t cells are a prominent infiltrating subset in the retina / choroid and both ifn--producing ( th1 ) and il-17-producing t cells are found in the eye - draining lymph node . however only a small induction of th1 and no induction of th17 cells was observed in the mesenteric lymph nodes draining the gut ( figure 5 ) . in contrast , we have previously shown that t cell activation by cfa during eau induction is mediated via dectin-1 , a known activator of il-17-producing t cells , and that both th1 and th17 cells are involved . in addition , other innate receptors activated by mycobacterial proteins such as mincle have been identified all of which appear to act via the card9 signaling complex . we therefore propose , in the cfa model of eau described here , that antigen - specific th1 and th17 cells are activated locally in the skin - draining lymph node from the immunization site , which then traffic through many tissues and sites including the gut , where the gut microbiome amplifies their activation status in a bystander fashion , rather than by inducing antigen - specific t cells via commensal microbial antigen . inflammation is a strictly compartmentalized process , although there is often some systemic reflection of this event . therefore , we compared t cells in the eye , the local ( cervical ) and the distant ( mesenteric ) lymph nodes between gf and cv mice 35 days after the eau induction . using immunohistochemistry , we found that gf mice have significantly less cd3 t cells and a similar but not significant reduction in f4/80 macrophages in their eyes when compared with cv mice ( figure 4 ) . in both groups , the localization of cells was similar , with t cells either clustered in granulomas or scattered in inner and outer retinal layers and macrophages located in the inner retinal layers . these data suggest that the lower eye infiltration with t cells in gf mice is the consequence and not the cause of the reduced level of eau . the higher number or regulatory t cells in the cervical lymph nodes of gf mice , but not in mesenteric lymph nodes , also suggests that these cells are attracted to the local site of inflammation and may regulate the local immune response by bystander suppression . in the current study , we show that the absence of microbiota or decrease of bacterial load before induction of inflammation significantly decreases the susceptibility of mice to eau induced by irbp in cfa . we show that reduction in the microbial burden induces changes in the strength of the t cell response in gf mice , with reduced t cell infiltration in the retina and also reduced th1 and th17-type t cell numbers in the eye - draining lymph node . this effect was not reiterated in antibiotic - treated mice suggesting that the reduction in the microbiota in antibiotic - treated mice was incomplete . we propose that the presence of the microbiota promotes organ specific autoimmunity by amplifying the activation of antigen - specific t cells when these cells are induced in the secondary lymphoid organs as would occur in human disease . these results support the notion that the microbiota is important in pathogenesis of autoantigen - induced uveitis and that treatment with antibiotics may constitute an adjunct therapy for sight - threatening uveitis .

the microbiota is a crucial modulator of the immune system . here , we evaluated how its absence or reduction modifies the inflammatory response in the murine model of experimental autoimmune uveoretinitis ( eau ) . we induced eau in germ - free ( gf ) or conventionally housed ( cv ) mice and in cv mice treated with a combination of broad - spectrum antibiotics either from the day of eau induction or from one week prior to induction of disease . the severity of the inflammation was assessed by fundus biomicroscopy or by histology , including immunohistology . the immunophenotyping of t cells in local and distant lymph nodes was performed by flow cytometry . we found that gf mice and mice where the microbiota was reduced one week before eau induction were protected from severe autoimmune inflammation . gf mice had lower numbers of infiltrating macrophages and significantly less t cell infiltration in the retina than cv mice with eau . gf mice also had reduced numbers of ifn- and il-17-producing t cells and increased numbers of regulatory t cells in the eye - draining lymph nodes . these data suggest that the presence of microbiota during autoantigen recognition regulates the inflammatory response by influencing the adaptive immune response .

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

have previously reported in a surrogate model of gf mice that alteration of the gut microbiota by using a combination of orally administered broad - spectrum antibiotics modulates the severity of eau . we report that the severity of eau is markedly reduced in gf mice and in mice which have been pretreated with antibiotics to reduce microbial burden but not when the microbial burden is reduced after activation of t cells has been induced . to establish the importance of the microbiota with respect to disease induction , we initiated treatment either one week prior to eau induction or on the day of eau induction . we performed two experiments by administering antibiotics in cv mice either from the day of eau induction or from one week before . mice treated with metronidazole and ciprofloxacin ( see section 2 ) commencing one week prior to eau induction and continued for the course of the experiment ( a treatment which significantly reduces microbial burden ) had significantly lower levels of eau compared to controls both clinically ( figures 2(a ) and 2(b ) ; maximal difference observed at day 35 ; p < 0.05 ) and histologically ( figures 2(c ) and 2(d ) ; p < 0.05 ) . immunohistological studies of the eyes performed on gf mice and littermate controls at day 35 ( see section 2 ) showed that qualitatively there was no difference in the nature of the cell infiltrate in the retina and choroid , which was composed of t cells and macrophages , distributed as individual cells or small cell aggregates ( granulomas ) . immunohistology was also performed on eyes of mice treated with metronidazole and ciprofloxacin from one week before or on the day of eau induction ( see section 2 ; figures 4(c ) , 4(d ) , 4(e ) , and 4(f ) ) . since in cfa immunized mice , t cell activation occurs extraocularly with clonal expansion in the skin - draining lymph nodes beginning as early as 6 days after immunization and subsequently in the eye - draining nodes as disease develops , we evaluated the phenotypes of lymph node cells by flow cytometry . in eye - draining cervical lymph nodes of cv mice , we observed an expansion of ifn--producing ( p < 0.01 ) and il-17-producing cd4 t cells ( p < 0.01 ) and a reduced percentage of foxp3 tregs ( p < 0.01 ) in cv mice at day 35 after immunization with cfa and irbp . cell populations in non - eye - draining mesenteric lymph nodes showed a small increase in the percentage of ifn--producing cd4 t cells in cv mice ( p < 0.05 ) which was significantly greater than ifn- t cells in gf mice but there was no difference in il-17-producing t cells in the mesenteric lymph nodes ( figure 5 ) . here , we show that the severity of the ocular inflammation in a murine model of autoimmune uveoretinitis is significantly lowered if the bacterial load is reduced either by rearing the mice in gf conditions ( figure 1 ) or by prophylactic treatment with broad - spectrum antibiotics ( metronidazole and ciprofloxacin , figure 2 ) . these experiments suggest that the preexisting microenvironment , particularly of the gut , has a significant role in determining the level of susceptibility to eau and in addition that broad - spectrum antibiotic treatment may at least partially modify this environment , although not as effectively as in germ - free mice . although this amount may have some minor suppressive effect on cellular immunity , the resistance of gf mice to eau and the sensitivity of cv mice treated from the day of eau induction suggest that it is more the effect on the microbiota than the immunosuppressive effect of metronidazole . in our experiments , t cells are a prominent infiltrating subset in the retina / choroid and both ifn--producing ( th1 ) and il-17-producing t cells are found in the eye - draining lymph node . we therefore propose , in the cfa model of eau described here , that antigen - specific th1 and th17 cells are activated locally in the skin - draining lymph node from the immunization site , which then traffic through many tissues and sites including the gut , where the gut microbiome amplifies their activation status in a bystander fashion , rather than by inducing antigen - specific t cells via commensal microbial antigen . therefore , we compared t cells in the eye , the local ( cervical ) and the distant ( mesenteric ) lymph nodes between gf and cv mice 35 days after the eau induction . using immunohistochemistry , we found that gf mice have significantly less cd3 t cells and a similar but not significant reduction in f4/80 macrophages in their eyes when compared with cv mice ( figure 4 ) . these data suggest that the lower eye infiltration with t cells in gf mice is the consequence and not the cause of the reduced level of eau . we show that reduction in the microbial burden induces changes in the strength of the t cell response in gf mice , with reduced t cell infiltration in the retina and also reduced th1 and th17-type t cell numbers in the eye - draining lymph node .

since its introduction in 1973 , x - ray ct has revolutionized clinical imaging and become a cornerstone of radiology departments . closely correlated to the development of x - ray ct , the research for better image quality at lower dose has been pursued for important medical applications with cardiac ct being the most challenging example . the first dynamic ct system is the dynamic spatial reconstructor ( dsr ) built at the mayo clinic in 1979 [ 2 , 3 ] . in a 1991 spie conference , for the first time we presented a spiral cone - beam scanning mode to solve the long object problem [ 4 , 5 ] ( reconstruction of a long object from longitudinally truncated cone - beam data ) . in 1990s , single - slice spiral ct became the standard scanning mode of clinical ct . in 1998 , multislice spiral ct entered the market [ 7 , 8 ] . with the fast evolution of the technology , moreover , just as there have been strong needs for clinical imaging , there are equally strong demands for preclinical imaging , especially of genetically engineered mice [ 911 ] . to meet the biomedical needs and technical challenges , it is imperative that cone - beam ct methods and architectures must be developed in a systematic and innovative manner so that the momentum of the ct technical development as well as clinical and preclinical applications can be sustained and increased . for that purpose , our ct research has been for superior dynamic volumetric low - dose imaging capabilities . since the long object problem has been well studied by now , we recently started working on the quasi - short object problem ( reconstruction of a short portion of a long object from longitudinally truncated cone - beam data involving the short object ) . currently , the state - of - the - art cone - beam scanning for clinical cardiac imaging follows either circular or helical trajectories . the former only permits approximate cone - beam reconstruction because of the inherent data incompleteness . the latter allows theoretically exact reconstruction but due to the openness of helical scanning there is no satisfactory scheme to utilize cone - beam data collected near the two ends of the involved helical segment . recently , saddle - curve cone - beam scanning was studied for cardiac ct [ 12 , 13 ] , which can be directly implemented by compositing circular and linear motions : while the x - ray source is rotated in the vertical x - y plane , it is also driven back and forth along the z - axis . because the electromechanical needs are very challenging for converting a motor rotation to the linear oscillation and handling the acceleration of the x - ray source along the z - axis , it is rather difficult to implement directly the saddle - curve scanning mode in practice , and it has not been employed by any ct company . however , it does represent a very promising solution to the quasi - short object problem . early this year , we invented a composite - circling scanning principle to solve the quasi - short object problem . in section 2 , we will describe a backprojection filtration- ( bpf- ) based exact reconstruction algorithm . in section 4 , we will present representative simulation results . in section 5 , when an x - ray focal spot is in a 2d ( no , linear , circular , or other types ) motion on the plane , or more general in a 3d motion within a neighborhood , facing a short object to be reconstructed , and the x - ray source is at the same time rotated in a transverse plane of a patient , the synthesized 3d scanning trajectory can take various forms with respect to the short object . specifically , let r1a0 and r1b0 be the lengths of the two semiaxes of the scanning range in the focal spot plane facing the short object , and r2>0 the radius of the tube scanning circle on the x - y plane , we define a family of saddle - like composite trajectory as ( 1)={(s)|1(s)=r2cos(2s)r1bsin(1s)sin(2s)2(s)=r2sin(2s)+r1bsin(1s)cos(2s)3(s)=r1acos(1s),},where s represents time , 1 and 2 are the angular frequencies of the focal spot and tube rotations , respectively . when the ratio between 1 and 2 is an irrational number or a rational number with large numerator in its reduced form , the scanning curve covers a band of width 2r1a , allowing a uniform sampling pattern . with all the possible settings of r1a , r1b , r2 , 1 , and 2 , we have numerous cone - beam scanning trajectories including saddle curves and composite - circling loci that can be used to solve the quasi - short problem exactly . we are particularly interested in a rational ratio between 1 and 2 in this paper , which will result in a periodical scanning trajectory . without loss of generality , we reexpress ( 1 ) as ( 2)={(s)|1(s)=r2cos(s)r1bsin(ms)sin(s)2(s)=r2sin(s)+r1bsin(ms)cos(s)3(s)=r1acos(ms)},where m>1 is a rational number . when r1b=0 and m=2 , we obtain the standard saddle curve . some representative composite - circling curves are shown in figure 1 . as mentioned in the introduction , while the saddle curve cone - beam scanning does meet the requirement for exact cone - beam cardiac ct , it imposes quite hard mechanical constraints . in contrast to the saddle curve cone - beam scanning , our proposed composite - circling requires that the x - ray focal spot undergo a circular motion in a plane facing the short object to be reconstructed , while the x - ray source is rotated in the main gantry plane ( see figure 2 ) . preferably , we may let the patient sit or stand straight and make the gantry plane parallel to the earth surface . because of the symmetry of the proposed mechanical rotations and the compatibility with the physiological conditions , we believe that this approach to cone - beam ct of the short object has significant advantages over the existing cardiac ct methods and the standard saddle curve oriented systems from perspectives of both engineering implementation and clinical applications . assume an object function f(r ) is located at the origin of the natural coordinate system o. for any unit vector , let us define a cone - beam projection of f(r ) from a source point (s ) on a composite - circling trajectory by ( 3)df((s),):=0f((s)+t)dt.then , we define a unit vector as the one pointing to r from (s ) on the composite - circling trajectory ( 4)(r , s):=r(s)|r(s)|.as shown in figure 3 , a generalized pi - line can be defined as the line through a point and across the composite - circling trajectory at two points (sb(r ) ) and (st(r ) ) , where sb = sb(r ) and st = st(r ) are the rotation angles corresponding to these two points . at the same time , the pi - segment ( also referred to as a chord ) is defined as the part of the generalized pi - line between (sb(r ) ) and (st(r ) ) , the pi - arc as the part of the scanning trajectory between (sb(r ) ) and (st(r ) ) , and the pi - interval as ( sb , st ) . all the pi - segments form a convex hull h of the composite - circling curve where the exact reconstruction is achievable according to the generalized backprojection filtration ( bpf ) approach [ 15 , 16 ] . to perform the bpf reconstruction from data collected along a composite - circling trajectory , we define a unit vector along the chord ( 5)e(r):=(st(r))(sb(r))|(st(r))(sb(r))|,and set up a local coordinate system associated with the trajectory . initially , we only consider the circular scanning trajectory of the x - ray tube in the x - y plane which can be expressed as ( 6)={(s)1(s)=r2cos(s ) , 2(s)=r2sin(s ) , 3(s)=0}.for a given s , we define a local coordinate system for (s ) by three orthogonal unit vectors d1:=(sin(s),cos(s),0 ) , d2:=(0,0,1 ) , and d3:=(cos(s),sin(s),0 ) ( see figure 4 ) . equispatial cone - beam data are measured on a planar detector array parallel to d1 and d2 at a distance d from (s ) with d = r2+dc , where the constant dc is the distance between the z - axis and the detector plane . a detector position in the array is denoted by ( u , v ) , which are signed distances along d1 and d2 , respectively . let ( u , v)=(0,0 ) correspond to the orthogonal projection of (s ) onto the detector array . if s is given , ( u , v ) are determined by . thus , the cone - beam projection data along a direction from (s ) can be rewritten in the planar detector coordinate system as p(s , u , v):=df((s), ) with ( 7)u = dd1d3 , v = dd2d3.now , let us consider the circular rotation of the focal spot at the given time s. according to our definition ( 2 ) , the focal spot rotation plane is parallel to the local area detector , and the orthogonal projection of the circling focal spot position (s ) in the above - mentioned local area detector is ( r1b sin(ms),r1a cos(ms ) ) . thus , the cone - beam projection data along a direction from (s ) can be rewritten in the same local planar detector coordinate system as p(s , u , v):=df((s), ) with ( 8)u = dd1d3+r1bsin(ms ) , v = dd2d3+r1acos(ms ) . in 2002 , an exact and efficient helical cone - beam reconstruction method was developed by katsevich [ 17 , 18 ] , which is a breakthrough in the area of helical / spiral cone - beam ct . the katsevich formula is in a filtered backprojection ( fbp ) format using data from a pi - arc within a slightly enlarged tam - danielsson window . by interchanging the order of the hilbert filtering and backprojection , zou and pan proposed a backprojection filtration ( bpf ) formula in the standard helical scanning case . this bpf formula can reconstruct an object from the data within the tam - danielsson window . for important biomedical applications including bolus - chasing ct angiography and electron - beam ct / micro - ct , our group first proved the general validity of both the bpf and fbp formulae in the case of cone - beam scanning along a general smooth trajectory [ 15 , 16 , 22 , 23 ] . our group also formulated the generalized fbp and bpf algorithms in a unified framework , and applied them in the cases of generalized n - pi - window and saddle curve scanning . note that our generalized bpf and fbp formulae as well as others ' results on general cone - beam reconstruction are valid to any smooth scanning loci , and they can be certainly applied to the reconstruction problem with the proposed composite - circling trajectory . based on our experience with the cone - beam reconstruction from data along a saddle curve , the bpf algorithm is more computationally efficient than the pi - line - based fbp , and they have similar noise characteristics . therefore , here we will use the bpf method and describe its major steps as follows . step 1 ( cone - beam data differentiation)for every projection , compute the derivative data g(s , u , v ) from the projection data p(s , u , v ) : ( 9)g(s , u , v)sdf((s),)| fixed = ddsp(s , u , v)| fixed = ( s+usu+vsv)p(s , u , v),where ( 10)us=(ur1bsin(ms))2d+d+mr1bcos(ms),vs=(ur1bsin(ms))(vr1acos(ms))dmr1asin(ms).the detailed derivations of ( 10 ) are in appendix a. for every projection , compute the derivative data g(s , u , v ) from the projection data p(s , u , v ) : ( 9)g(s , u , v)sdf((s),)| fixed = ddsp(s , u , v)| fixed = ( s+usu+vsv)p(s , u , v),where ( 10)us=(ur1bsin(ms))2d+d+mr1bcos(ms),vs=(ur1bsin(ms))(vr1acos(ms))dmr1asin(ms).the detailed derivations of ( 10 ) are in appendix a. for every chord specified by sb and st and for every point r on the chord , compute the weighted backprojection data ( 11)b(r):=sb(r)st(r)g(s , u , v)ds|r(s)| with ( 12)u = d(r , s)d1d3+r1bsin(ms),v = d(r , s)d2d3+r1acos(ms ) . for every chord specified by sb and st , perform the inverse hilbert filtering along the 1d chord direction e(r ) to reconstruct f(r ) from b(r ) . the filtering formulation is essentially the same as in our previous papers [ 13 , 16 , 24 ] . rebin the reconstructed image into the natural coordinate system by determining the chord(s ) for each grid point in the natural coordinate system . the rebinning scheme is the same as what we used for the saddle curve . however , there are some differences in the method for determining a chord , which will be described in the next subsection . for our composite - circling mode , we assume that r1br2/(2 m ) . in this case , the projection of the trajectory in the x - y plane will be a convex single curve ( appendix b ) . among all the potential composite - circling trajectories , we now target the case m=2 which is similar to the popular saddle curve setting . that is , we will study how to determine a chord for a fixed point for m=2 in this subsection . as shown in figure 5 , to find a chord containing the fixed point r0=(x0,y0,z0 ) in the convex hull h , we first consider the projection curve of the trajectory in the x - y plane . due to the convexity of the projection curve , any line passing a point inside the curve in the x - y plane has two and only two intersections with the projection curve . then , we consider a special plane x = x0 . in this case , there are two intersection points between the plane and the projection curve . solving the equation r2cos(s)r1bsin(2s)sin(s)=x0 , that is , r2cos(s)2r1b(1cos2(s))cos(s)=x0 , we can obtain one and only one real root 1qcos1 for cos(s ) , and the view angles s1=cos1(qcos ) and s3=s1 that correspond to the two intersection points w1 and w3 . solving the equation r2sin(s)+r1bsin(2s)cos(s)=y0 , that is , r2sin(s)+2r1b(1sin2(s))sin(s)=y0 , we have the only real root 1qsin1 and the view angles s2=sin1(qsin ) and s4=s2 corresponding to the two intersection points w2 and w4 . clearly , the above four angles satisfy s1<s2<s3<s4 . now , we consider a chord l intersecting the line lz parallel to the z - axis through the point ( x0,y0,z0 ) . in the x - y plane , the projection of the line lz is the point ( x0,y0 ) and the projection of l passes through the point ( x0,y0 ) . according to the definition of a composite - circling curve , the line w1w3 intersects lz at ( x0,y0,r1acos(2s1 ) ) while w2w4 intersects lz at ( x0,y0,r1acos(2s2 ) ) . recall that we have assumed that r0 is inside the convex hull h , there will be r1acos(2s1)z0r1acos(2s2 ) , that is , r1a(2qcos21)z0r1a(12qsin2 ) . when the starting point wb of l moves from w1 to w2 smoothly , the corresponding end point wt will change from w3 to w4 smoothly , and the z - coordinate of its intersection with lz will vary from r1a(2qcos21 ) to r1a(12qsin2 ) continuously . therefore , there exists at least one chord l that intersects lz at r0 and satisfies sb1(s1,s2 ) , st1(s3,s4 ) . because the composite - circling trajectory is closed , we can immediately obtain another chord corresponding to the pi - interval ( st1,sb1 + 2 ) . similarly , we can find sb2(s2,s3 ) and st2(s4,s1 + 2 ) as well as the chord intervals ( sb2,st2 ) and ( st2,sb2 + 2 ) . hence , we can perform reconstruction at least four times for a given point inside the hull of a composite - circling trajectory . these properties are very similar to that of a saddle curve [ 12 , 13 ] . based on the above discussion , to illustrate the procedure for the chord determination , we list the following pseudocodes for numerically finding the chord corresponding to the pi - interval ( sb1,st1 ) : ( s1)set sbmin=s1 , sbmax=s2;(s2)set sb1=(sbmax+sbmin)/2 and find st1(s3,s4 ) so that (sb1)(st1) intersects lz : ( s2.1)compute the unit direction e in the x - y plane ( see figure 5);(s2.2)set stmin=s3 , stmax=s4 , and st1=(stmax+stmin)/2;(s2.3)compute the projection =((st1)r0)e;(s2.4)if =0 stop , else go to ( s2.2 ) and set stmax=st1 if >0 , and set stmin=st1 if <0 ; ( s3)compute z of the intersection point between (sb1)(st1) and lz;(s4)if z=z0 stop , else go to ( s2 ) and set sbmax=sb1 if z>z0 and set sbmin=sb1 z<z0 . note that e in s2.1 is the direction perpendicular to (sb1)(st1) and at the left side of (sb1)(st1). given the fact that implementation details of the above - described bpf method and chord determination scheme are similar to what we published in our previous papers [ 13 , 16 , 24 , 27 ] , we will not elaborate them further . set sbmin=s1 , sbmax=s2 ; set sb1=(sbmax+sbmin)/2 and find st1(s3,s4 ) so that (sb1)(st1) intersects lz : ( s2.1)compute the unit direction e in the x - y plane ( see figure 5);(s2.2)set stmin=s3 , stmax=s4 , and st1=(stmax+stmin)/2;(s2.3)compute the projection =((st1)r0)e;(s2.4)if =0 stop , else go to ( s2.2 ) and set stmax=st1 if >0 , and set stmin=st1 if <0 ; compute the unit direction e in the x - y plane ( see figure 5 ) ; set stmin=s3 , stmax=s4 , and st1=(stmax+stmin)/2 ; compute the projection =((st1)r0)e ; if =0 stop , else go to ( s2.2 ) and set stmax=st1 if >0 , and set stmin=st1 if <0 ; compute z of the intersection point between (sb1)(st1) and lz ; if z=z0 stop , else go to ( s2 ) and set sbmax=sb1 if z>z0 and set sbmin=sb1 z<z0 . to verify the correctness of the exact reconstruction method and demonstrate the merits of the composite - circling scanning mode , we implemented the reconstruction algorithm developed in section 3 in matlab on a pc ( 2.0 gagabyte memory , 2.8 ghz cpu ) , with all the computationally intensive parts coded in c. a composite - circling trajectory was made with r1a = r1b=10 cm , r2=57 cm , and m=2.0 , which is consistent with the specifications of available commercial ct scanners and satisfies the requirements for the exact reconstruction of a quasi - short object , such as the head and heart . in our simulation , we also assumed a virtual plane detector and set the distance from the detector array to the z - axis ( dc ) to zero . when the x - ray source was moved along a turn of the composite - circling trajectory , 1200 cone - beam projections were equiangularly acquired . similar to what we did for the reconstruction in the saddle curve case , 258 starting points sb were first uniformly selected from the interval [ 0.4492,0.0208 ] . from each (sb ) , 545 chords were made with the end - point parameter st uniformly in the interval [ sb+0.8883,sb+1.1150 ] . finally , the reconstructed images were rebinned into a 256256256 matrix in the natural coordinate system . beside , our method was also evaluated with noisy datasets . we assumed that n0 photons were emitted by the x - ray source but only n photons arrived at the detector element after being attenuated in the object , obeying a poisson distribution . the noise standard deviations in the reconstructed images were about 3.18103 and 10.05103 for n0=106 and 105 , respectively . figures 6 and 7 illustrate some typical image slices reconstructed from noise - free and noisy datasets collected along our composite - circling trajectory , as well as the counterparts from a saddle curve . while the composite - circling scanning is easier than a saddle curve in engineering implementation , there is no evident difference between the images reconstructed from the data collected along a composite - circling and a saddle curve because of their exactness . we remark that the stripe artifacts in figure 6 were introduced by the interpolation involving phantom edges . this type of artifacts disappeared when we used a modified differentiable shepp - logan head phantom . to solve the quasi - short object problem , we have proposed a family of saddle - like scanning trajectories but we have only numerically evaluated the composite - circling mode with m=2 . this does not mean that the case m=2 of the composite - circling mode is the optimal . we are actively working to investigate the properties of the saddle - like curves , and optimize the parameters and protocols . although the generalized bpf method has been developed for exact image reconstruction from data collected along a composite - circling trajectory , the method is not efficient because of its shift - variant property . recently , katsevich announced an important progress towards exact and efficient general cone - beam reconstruction for two classes of scanning loci . the second type covers generalizes circle - plus curves . inspired by his finding , we tend to believe that there exists an exact and efficient algorithm for exact cone - beam composite - circling reconstruction . we acknowledge that for cone - beam composite - circling , we would need to rotate an x - ray tube in a plane facing a short object or have a rotating focal spot in the tube , which is not a straightforward task . however , the situation with saddle curve cone - beam scanning is even more difficult , since an x - ray tube or focal spot must be moved back and forth rapidly along the z - axis for a high longitudinal sampling rate . given the paramount importance of exact cone - beam cardiac ct and the continued rapid development of the source and detector technology , our objective to solve the quasi - short object problem optimally with saddle - like cone - beam scanning curves is well justified . even if neither cone - beam saddle curve scanning nor composite - circling will be implemented in the near future , the use of a fixed focal spot in a rotating x - ray tube will be likely modified or replaced soon with the use of distributed sources . we believe that in the next decade , advances in distributed and other types of x - ray sources will define a new revolution in ct , which is the hardware foundation entirely consistent with our ongoing research on cone - beam saddle - like curve - based reconstruction algorithms . therefore , saddle - like curves , including saddle and composite - circling trajectories but not limited to them , will become increasingly important for cardiac cone - beam ct research and applications . regarding the engineering implementation of our composite - scanning mode because the x - ray source , detector array , and collimators are mounted on the same data acquisition system ( das ) , we can omit the rotation of the whole das . that is , the focal spot is circularly rotated in the plane parallel to the patient motion direction , and we need to have a collimation design to reject most of scattered photons for any focal spot position . during the scan , we can adjust the direction and position of the detector array and associated collimators to keep the line connecting the detector array center and the focal spot perpendicular to the detector plane and make all the collimators focus on the focal spot all the time . this can be mechanically done , synchronized by the rotation of the focal spot . in this case , the focal spot rotation plane and the detector plane are not parallel in general . other designs for the same purpose are possible in the same spirit of this invention . furthermore , our approach can also be adapted for inverse geometry based cone - beam ct . in conclusion , we have developed a novel composite - circling mode and method for solving the quasi - short object problem exactly , which has better mechanical rotation stability and physiological compatibility than saddle curve scanning . our generalized bpf method has been evaluated that reconstructs images from cone - beam data collected along a composite - circling trajectory for the case m=2 . the simulation results have demonstrated the correctness and merits of the proposed composite - circling mode and exact bpf reconstruction algorithm . for a given unit direction , its projection position in the local coordinate system can be expressed as ( a.1)u = dd1d3+r1bsin(ms ) , v = dd2d3+r1acos(ms).hence , we have ( a.2)us=(dd1d3)=dd1d3dd1d3(d3)2+mr1bcos(ms),vs=(dd2d3)=dd2d3dd2d3(d3)2mr1asin(ms).since d1=d3 , d2=0 and d3=d1 , we obtain ( a.3)us = dd3d3+d(d1)2(d3)2+mr1bcos(ms),vs = dd2d1(d3)2mr1asin(ms).by ( a.1 ) , it follows readily that ( a.4)us=(ur1bsin(ms))2d+d+mr1bcos(ms),vs=(ur1bsin(ms))(vr1acos(ms))dmr1asin(ms ) . the projection of our composite - circling trajectory on the x - y plane can be expressed as ( b.1)p={(s)1(s)=r2cos(s)r1bsin(ms)sin(s ) , 2(s)=r2sin(s)+r1bsin(ms)cos(s)}.according to liu and traas ( lemma 2.7 ) , a single close c2-continuous curve is globally convex if and only if the curvature at every point on the curve is nonpositive . hence , it is required that (s)(s)0 for any s. since ( b.2)1(s)=r2sin(s)r1bsin(ms)cos(s ) mr1bcos(ms)sin(s),2(s)=r2cos(s)r1bsin(ms)sin(s ) + mr1bcos(ms)cos(s),1(s)=r2cos(s)+r1b(m2 + 1)sin(ms)sin(s ) 2mr1bcos(ms)cos(s),2(s)=r2sin(s)r1b(m2 + 1)sin(ms)cos(s ) 2mr1bcos(ms)sin(s),we have ( b.3)(s)(s)=1(s)2(s)1(s)2(s)=(r2sin(s)+r1bsin(ms)cos(s ) + mr1bcos(ms)sin(s ) ) (r2sin(s)+r1b(m2 + 1)sin(ms)cos(s ) + 2mr1bcos(ms)sin(s ) ) + ( r2cos(s)r1b(m2 + 1)sin(ms)sin(s ) + 2mr1bcos(ms)cos(s ) ) (r2cos(s)r1bsin(ms)sin(s ) + mr1bcos(ms)cos(s))=(m21)r1b2cos2(ms)+3mr2r1bcos(ms ) + ( m2 + 1)r1b2+r22.letting z = tg2(ms/2 ) , we arrive at ( b.4)(s)(s)0 (m21)r1b2(1z1+z)2 + 3mr2r1b(1z1+z ) + ( m2 + 1)r1b2+r220 (r22 + 2m2r1b23mr2r1b)z2 + 2(r22 + 2r1b2)z + ( r22 + 2m2r1b2 + 3mr2r1b)0,where the relationship cos(ms)=(1z)/(1+z ) has been used . given that r2>0 , r1b0 , 2(r22 + 2r1b2)>0 , and ( r22 + 2m2r1b2 + 3mr2r1b)>0 , we obtain the following necessary and sufficient condition for (s)(s)0 at any s : ( b.5)r22 + 2m2r1b23mr2r1b0,which implies that r1br2/(2 m ) or r1br2/m . when r1br2/m , the curve p becomes a complex curve ( not single ) , and this case should be excluded . hence r1br2/(2 m ) is the necessary and sufficient condition for the convex projection of the composite - circling trajectory on the x - y plane .

here we propose a cone - beam composite - circling mode to solve the quasi - short object problem , which is to reconstruct a short portion of a long object from longitudinally truncated cone - beam data involving the short object . in contrast to the saddle curve cone - beam scanning , the proposed scanning mode requires that the x - ray focal spot undergoes a circular motion in a plane facing the short object , while the x - ray source is rotated in the gantry main plane . because of the symmetry of the proposed mechanical rotations and the compatibility with the physiological conditions , this new mode has significant advantages over the saddle curve from perspectives of both engineering implementation and clinical applications . as a feasibility study , a backprojection filtration ( bpf ) algorithm is developed to reconstruct images from data collected along a composite - circling trajectory . the initial simulation results demonstrate the correctness of the proposed exact reconstruction method and the merits of the proposed mode .

1. INTRODUCTION 2. COMPOSITE-CIRCLING SCANNING 3. EXACT RECONSTRUCTION 4. SIMULATION RESULTS 5. DISCUSSIONS AND CONCLUSIONS A. DERIVATIONS OF FORMULAE ( B. PROOF OF THE CONVEX PROJECTION CONDITION

in a 1991 spie conference , for the first time we presented a spiral cone - beam scanning mode to solve the long object problem [ 4 , 5 ] ( reconstruction of a long object from longitudinally truncated cone - beam data ) . since the long object problem has been well studied by now , we recently started working on the quasi - short object problem ( reconstruction of a short portion of a long object from longitudinally truncated cone - beam data involving the short object ) . recently , saddle - curve cone - beam scanning was studied for cardiac ct [ 12 , 13 ] , which can be directly implemented by compositing circular and linear motions : while the x - ray source is rotated in the vertical x - y plane , it is also driven back and forth along the z - axis . in section 5 , when an x - ray focal spot is in a 2d ( no , linear , circular , or other types ) motion on the plane , or more general in a 3d motion within a neighborhood , facing a short object to be reconstructed , and the x - ray source is at the same time rotated in a transverse plane of a patient , the synthesized 3d scanning trajectory can take various forms with respect to the short object . specifically , let r1a0 and r1b0 be the lengths of the two semiaxes of the scanning range in the focal spot plane facing the short object , and r2>0 the radius of the tube scanning circle on the x - y plane , we define a family of saddle - like composite trajectory as ( 1)={(s)|1(s)=r2cos(2s)r1bsin(1s)sin(2s)2(s)=r2sin(2s)+r1bsin(1s)cos(2s)3(s)=r1acos(1s),},where s represents time , 1 and 2 are the angular frequencies of the focal spot and tube rotations , respectively . with all the possible settings of r1a , r1b , r2 , 1 , and 2 , we have numerous cone - beam scanning trajectories including saddle curves and composite - circling loci that can be used to solve the quasi - short problem exactly . in contrast to the saddle curve cone - beam scanning , our proposed composite - circling requires that the x - ray focal spot undergo a circular motion in a plane facing the short object to be reconstructed , while the x - ray source is rotated in the main gantry plane ( see figure 2 ) . because of the symmetry of the proposed mechanical rotations and the compatibility with the physiological conditions , we believe that this approach to cone - beam ct of the short object has significant advantages over the existing cardiac ct methods and the standard saddle curve oriented systems from perspectives of both engineering implementation and clinical applications . to verify the correctness of the exact reconstruction method and demonstrate the merits of the composite - circling scanning mode , we implemented the reconstruction algorithm developed in section 3 in matlab on a pc ( 2.0 gagabyte memory , 2.8 ghz cpu ) , with all the computationally intensive parts coded in c. a composite - circling trajectory was made with r1a = r1b=10 cm , r2=57 cm , and m=2.0 , which is consistent with the specifications of available commercial ct scanners and satisfies the requirements for the exact reconstruction of a quasi - short object , such as the head and heart . when the x - ray source was moved along a turn of the composite - circling trajectory , 1200 cone - beam projections were equiangularly acquired . while the composite - circling scanning is easier than a saddle curve in engineering implementation , there is no evident difference between the images reconstructed from the data collected along a composite - circling and a saddle curve because of their exactness . to solve the quasi - short object problem , we have proposed a family of saddle - like scanning trajectories but we have only numerically evaluated the composite - circling mode with m=2 . although the generalized bpf method has been developed for exact image reconstruction from data collected along a composite - circling trajectory , the method is not efficient because of its shift - variant property . we acknowledge that for cone - beam composite - circling , we would need to rotate an x - ray tube in a plane facing a short object or have a rotating focal spot in the tube , which is not a straightforward task . given the paramount importance of exact cone - beam cardiac ct and the continued rapid development of the source and detector technology , our objective to solve the quasi - short object problem optimally with saddle - like cone - beam scanning curves is well justified . even if neither cone - beam saddle curve scanning nor composite - circling will be implemented in the near future , the use of a fixed focal spot in a rotating x - ray tube will be likely modified or replaced soon with the use of distributed sources . in conclusion , we have developed a novel composite - circling mode and method for solving the quasi - short object problem exactly , which has better mechanical rotation stability and physiological compatibility than saddle curve scanning . our generalized bpf method has been evaluated that reconstructs images from cone - beam data collected along a composite - circling trajectory for the case m=2 .

seed starch is the major storage compound in cereals providing as much as 80% of the calories consumed by mankind . this starch is also a major source of feed , fiber , biofuels , and biopolymers in many industrial applications . understanding the molecular basis of starch physicochemical properties and the control of its synthesis in the seed is a necessary step in improving and modifying starch properties tailored to an increasing variety of end - uses . it is composed of two glucose polymers , called amylose and amylopectin , which share the same basic glucan structure but differ in length and degree of branching . amylose is essentially a linear molecule of -1,4-linked glucose residues with a few -1,6-glycosidic linkages . the degree of polymerization of glucose in amylose molecules is species dependent and averages about 800 residues in wheat . amylopectin molecules , on the other hand , are much larger ( up to millions of residues ) and highly branched with a high frequency of -1,6-glycosidic linkages . the branching of the glucan chains of amylopectin occurs with regular periodicity and its length and pattern are critical for the proper formation of the starch granule . in wheat , starch granules exhibit a bimodal size distribution a characteristic unique to members of the grass triticeae family . the starch granules , designated a- , and b - starch granules , can be distinguished based on size , shape , relative proportion , and the timing of their initiation in the endosperm a process which , presumably , is under a defined genetic program . a - granules are lens - shaped , 1050 m in diameter , and make up to 70% of the volume and 10% of the total number of starch granules [ 5 , 6 ] . in contrast , b - granules are spherical , 59 m in diameter , and represent ~30% of the volume and 90% of the total number of granules . more recent evidence indicates the presence of c - type starch granules with diameter less than 5 m [ 4 , 7 ] . the small size of the c - granules makes them difficult to isolate and quantify which commonly leads to their being classified with b - granules . a - granules are formed around 414 days postanthesis ( dpa ) when the endosperm is still actively dividing [ 4 , 8 , 9 ] . b - granules are initiated at about 1016 dpa in stromules ( stroma containing tubules ) that are extruded from a - granule containing plastids [ 6 , 7 ] , and the small c - granules first appear about 21 dpa . the genetic basis of the multimodal size distribution of starch in wheat and barley is of great interest because the physiochemical properties of each type of starch granule vary and contribute to the food and industrial end - uses of triticeae starch [ 1012 ] . the posttranslational control of many enzymes involved in starch biosynthesis has been well documented [ 1315 ] . in contrast , the transcriptional regulation of the genes coding for these enzymes has not yet been fully explored . transcriptional regulation may be a more important mechanism for long - term control of genes expression especially during caryopsis development . several studies show that carbohydrate synthesis genes are strongly regulated by sugars especially sucrose and glucose and that sugars are important sensors and regulators of multiple pathways [ 16 , 1820 ] . in this report , results of a global gene expression profiling experiment were overlaid with the analysis of soluble sugar accumulation , starch content , and starch granule particle size distribution on the same batch of biological materials used for the microarray experiment . results showed multiple temporal expression patterns of key genes involved in starch synthesis , suggesting the relative importance of the different enzymes throughout caryopsis development . correlative analysis identified genes that showed similar patterns of expression to the accumulation profiles of starch and amylose and the distribution of starch granules suggesting these as possible candidate genes for further investigation for their roles in seed starch synthesis and potential targets for modulating carbohydrate metabolism in wheat by genetic engineering or molecular breeding efforts . hereward plants used for tissue sampling and rna extraction for cdna and oligoarrays experiments , respectively , were grown in the greenhouse under conditions as previously described [ 21 , 22 ] . caryopses from the head of the main stem of each bobwhite plant were harvested at different time - points for fresh - weight , dry - weight , total starch , and total protein content determinations . the total starch and total protein data obtained from our briefly , wheat caryopses from 10 to 25 heads per time - point were independently harvested , freeze - dried , and ground to flour using the udy mill ( udy corporation , fort collins , co , usa ) for measurement of total starch and protein . the megazyme total starch determination assay kit ( megazyme international , county wicklow , ireland ) was used for starch determination . amylose was determined for caryopses at 7 , 10 , 14 , 18 , 21 , 25 , 28 , 35 , and 50 dpa using the megazyme amylose / amylopectin determination kit . total protein content was determined by n combustion analysis with a flashea 1112 n / protein analyzer ( thermofisher scientific , waltham , ma usa ) . soluble sugars were determined at 7 , 14 , 21 , 28 , and 35 dpa by boiling each sample in 5 ml 80% ( v / v ) ethanol for 5 minutes . the samples were centrifuged at 4000 g for 15 minutes , the ethanol was decanted , and an additional 5 ml ethanol added to the pellet which was resuspended and boiled again for 5 minutes . the ethanol was removed by drying samples in a speed - vac and the residue reconstituted in 300 l of water . samples were filtered through a 0.45 m filter and injected onto a hamilton rx-10 anion exchange column ( hamilton company , reno , nv ) . sucrose , fructose , and glucose were measured by hplc on a dionex biolc system ( dionex , sunnyvale , ca ) with pulse amperometric detection . the gradient elution schedule consisted of 15 minutes of 15% of 200 mm naoh , then 30% over 7 minutes , and 15% naoh for 10 minutes . standard solutions of sucrose , fructose , and glucose ( fluka biochemika company ; steinheim , germany ) were mixed and 10 l injected on to the column at a flow rate of 2.0 ml / min . trehalose eluted at 2.7 minutes , glucose at 6.1 minutes , fructose at 7.6 minutes , and sucrose at 9.6 minutes . starch granules from caryopses at each time - point were isolated and purified using a established protocol . freeze dried whole caryopses were ground in a mortar and pestle and homogenized gently in 0.5 m nacl . the homogenate was filtered with a 90 m mesh filter , and the retentate was collected and gently ground further to release starch . this procedure was repeated until most of the starch was washed off from this fraction . no preferential loss of small - granules was detected as monitored by iodine - staining of the flow - thru in each wash . starch fractions collected were resuspended by vortexing in 5 volumes of 0.5 m nacl , and then centrifuged at 10,000 g for 10 minutes . debris at the starch - liquid interface was carefully removed and the pellet resuspended in 0.5 m nacl and then recentrifuged . the pellet was then washed in water ( three times ) , 2% ( v / v ) sds ( twice ) , in water ( three times ) , and once with 80% ( v / v ) acetone and then dried overnight . approximately 50 mg of purified starch was diluted in 50 ml of water and particle size analysis was processed using the horiba 900 laser scattering particle size distribution analyzer ( irvine , ca ) . for the 7 dpa sample , was adapted wherein all granules 5 m were considered spherical and those bigger than 5 m in diameter were considered oblate spheroid with thickness of 5 m and varying equatorial diameters . starch granules purified from the different developmental stages were dusted on the surface of a carbon adhesive tab and sputter coated with gold palladium particles using dentum vacuum desk ii . samples were viewed at 2.0 kv with the hitachi model s-4700 scanning electron microscope . two sets of microarray data were examined for expression of genes involved in starch metabolism in the developing wheat caryopsis . the first data set was obtained from our previous work which utilized an 8 k cdna array enriched for genes expressed in the endosperm . bobwhite was examined using rna from six time - points ( 3 , 7 , 14 , 21 , 28 , and 35 dpa ) which covered the critical stages in caryopsis development from coenocytic to desiccation stage . the 3 dpa data were omitted for correlative analyses since starch accumulation begins at about 5 dpa and at earlier times nonendosperm tissues predominate . data visualization and coordinate transcript expression analyses were accomplished using the built - in statistical modules in genespring gx software ( agilent technologies , santa clara , ca ) . the tigr website ( http://compbio.dfci.harvard.edu/tgi/ ) was used to determine if different ests belonged to the same contig , therefore belonging to the same tentatively unique gene . the second data set was derived from a time - series experiment in grain development using the affymetrix wheat genome short oligo dna expression arrays ( referred to hereafter as the oligoarray ) with 61,127 probes representing 55,052 potential genes . in this experiment , the expression of genes was examined in the developing caryopses of winter wheat t. aestivum cv . hereward caryopses at ten time - points ( 6 , 8 , 10 , 12 , 14 , 17 , 21 , 28 , 35 , and 42 dpa ) covering the onset of grain - filling stage to grain maturation . new analyses were carried out on 20 oligoarrays corresponding to the caryopses developmental series downloaded from the gene expression omnibus ( e_mexp-1193 , http://www.ncbi.nlm.nih.gov/geo/ ) to allow extraction of gene expression profiles relevant to the current report . probe set signal normalization and summarization was carried out using gcrma , the modified robust multiarray normalization algorithm that takes into consideration the gc content of each probe , as implemented in genespring software . a list of differentially expressed genes was generated using one - way anova using sas version 9.0 . the wheat oligoarray database at http://www.plexdb.org/ was used to verify the most recent annotations for the probesets . the ncbi est assembly nomenclature was used to name the oligoarray probes ; that is , ta.6869 is t. aestivum unigene 6869 . for the purpose of this work , a probe set is deemed to represent a potentially unique wheat gene and the accumulation of its transcript as measured by the signal intensities in each probe set represents the expression of the gene . in this study , it will be understood that transcript accumulation and gene expression will refer to gene transcript steady - state levels and will be considered to approximate the level of expression of the relevant gene . the mapman software was adapted to display the change in transcript expression during wheat caryopsis development using a color code , which were overlaid onto a custom - made sucrose - to - starch pathway . the soluble sugar content measured in developing caryopses showed that sucrose , fructose and glucose were at their highest levels at 7 dpa , then decreased to lower levels by about 21 dpa , and remained fairly constant through 35 dpa ( figure 1 ) . fructose was the predominant sugar at 7 dpa caryopses and was about 2.5 times higher than sucrose and glucose , and sucrose has lower levels than fructose at 7 and 14 dpa . sucrose levels became the most abundant sugar by 21 dpa with the relative ratio of sucrose to both glucose , and fructose rising through 35 dpa , which may indicate its active import into the endosperm . measurements of sucrose , glucose and fructose contents were in agreement with an earlier published work in desiccated whole kernel flours from hard red spring and durum varieties . there were 49 , 377 , and 409 genes which expression correlated with sucrose , glucose , and fructose pattern of accumulation , respectively ( see supplementary material 1 available online at doi:10.1155/2009/407426 ) . among the specific genes correlating with changes in sucrose levels include a putative transcription factor ( tf ) described as btf3 , as well as with another putative tf , some carbohydrate - metabolic genes , and an atp / adp carrier protein ( table 1 ) . glucose accumulation was tightly linked with the transcript accumulation profile of calmodulin , two putative tfs , a 14 - 3 - 3 protein , a mads - box protein 9 , a putative pgi , putative and probable protein kinases as well as other carbohydrate - metabolic genes ( table 1 and supplementary material 1 ) . the fructose accumulation profile shows very high similarity to the pattern of expression of a cdpk protein - like calmodulin gene from rice that may play a role in signal transduction pathways that involve calcium as a second messenger as well as other regulatory and carbohydrate - metabolism genes ( table 1 ) . when the data were further examined for genes correlating to more than one of the 3 sugars , only two showed shared genes in common , both involving glucose . of the 23 genes that correlated with glucose and sucrose , only the atp / adp carrier protein was known to be involved in the starch synthesis pathway ; there were no regulatory factors identified ( see supplementary material 2 ) . of the 249 genes that correlated well with the changes in the levels of both fructose and glucose were metabolic and transcriptional regulatory genes , for example , putative pyruvate kinase , a yabby protein , a probable kinase , 14 - 3 - 3 regulatory proteins , a pistillata - like mads - box protein , and other tfs . we observed 249 overlapping coexpressed genes with the levels of fructose and glucose in the grain , even though fructose levels were about three times higher than glucose levels at 7 and 14 dpa ( figure 1 ) . there were 23 genes coordinately expressed with both sucrose and glucose levels while there were none for sucrose and fructose . it is difficult to explain the high correlation between transcripts that have similar accumulation profile as both glucose and fructose levels and the difference in the number of transcripts that follow sucrose / glucose ( 23 ) versus sucrose / fructose levels ( 0 ) . what our results perhaps signify is that the metabolism of glucose , rather than fructose , more closely reflects changes in sucrose and starch metabolism . this is consistent with starch as the major sink for sucrose , and through its activated form adp - glucose , glucose is the precursor for starch biosynthesis . however this is not reconciled with the report that glucose and fructose appear to contribute equally to starch metabolism . there are very few studies that show the extent to which fructose is a regulator of global gene expression in cereal endosperm or any other plant organ , and the interrelationship of sugars to starch formation requires more research . the accumulation of storage reserves in the developing caryopsis showed that total protein , total starch , and the amylose content ( figure 2(a ) ) steadily accumulated from 10 dpa to a peak at 35 dpa followed by a slight reduction at 50 dpa . the amount of starch that accumulates as amylose increased from 7 dpa to 35 dpa of development . the change in amylose content in the caryopsis correlated with changes in granule distribution during grain maturation ( see below ) and transcripts of major seed storage proteins including - and -gliadins ( table 1 and table 2 ) . the rate at which amylose accumulated was highly similar to an adpg transporter ( bt1 ) , two sucrose synthase ( susy , ec 2.4.1.13 ) clones , and several regulatory proteins . changes in the proportion of starch as amylopectin ( total starch minus amylose ) correlated well with four clones for putative zn - finger proteins , a putative kinase interacting protein , a putative lrk1 ( receptor - type ) protein kinase , a probable protein kinase , as well as with carbohydrate - metabolic genes ( table 1 ) . when the rate of both amylose and amylopectin accumulation is expressed as mg / caryopsis / day , high correlative patterns with the expression of a number of storage proteins , as well as genes encoding two zn - finger , leucine - rich wd domain containing proteins was found ( see supplementary material 1 ) . detailed inspection of the rate of storage product accumulation revealed a biphasic pattern ( figure 2(b ) ) . the rate of total protein accumulation increased steadily from 10 dpa and reaching a maximum rate at 21 dpa , then dropped 2-fold at 25 dpa , and then increased 3-fold at 28 dpa before declining as the grain matures . similarly , the rate of total starch accumulation peaked at 18 dpa , dropped 3-fold at 25 dpa , and then increased 4-fold before declining through desiccation . the rate of amylose accumulation increased from the onset of the grain filling stage , peaking at 14 dpa , then leveling off until 21 dpa before declining 10-fold at 25 dpa . in contrast , the rate of total starch accumulation continued to increase from 10 dpa to 18 dpa then gradually declined until 25 dpa . the rate of total starch and amylose accumulation then increased 4- and 18-fold , respectively , to 28 dpa before the major decline through the end of grain development . to verify whether the biphasic pattern of storage reserve rate of accumulation we observed is not exclusive to the biological material we used , a set of external data independently obtained from another cultivar ( courtesy of dr . frances du pont , usda - ars ; personal communication ) were similarly analyzed for rates of starch accumulation . measurements of storage starch accumulation in the control ( untreated ) sample used in determining the effect of high temperature in developing caryopsis of t. aestivum var . butte 86 [ 30 , 31 ] showed the same biphasic pattern in the rate of starch accumulation ( see supplementary material 3 ) consistent with our observation . the observed biphasic pattern in the rates of protein , amylose and starch accumulation ( figure 2(b ) ) , coincides with the biphasic reprogramming of genes during caryopsis development . we previously showed that the number of genes that are differentially expressed only between two consecutive time - points peaked twice early to mid - development ( between 3 to 14 dpa ) and in later stages ( between 21 to 28 dpa ) of the caryopsis development . in a separate study with a different wheat germplasm , significant changes in transcript abundance during maximum grain filling at 1221 and at desiccation / maturation at 2842 days after anthesis were also reported . when looking at wheat storage protein expression , kawaura et al . found two distinct expression patterns , even within a single gene family , with peaks at 10 and 20 dpa . taken together , these reports suggest that this biphasic changes may be a fundamental biological phenomenon in wheat caryopsis development . the formation and size distribution of starch granules in developing caryopses were monitored by laser diffraction ( figure 3 ) and the morphology was examined by scanning electron microscopy ( figure 4 ) . the data are consistent with the presence of a small granule fraction detected at 14 dpa , which are the b - type granules , and as previously reported granules detected at 7 dpa are of the a - type . it is reasonable that some a - type granules that are still growing and are still < 10 m in size may contribute to total volume of b - type granules at 14 dpa . in fact , sem image of starch granules at 14 dpa shows the presence of oblate spheroid granules , a characteristic shape of growing a - type granules , at 10 m in diameter or less ( figure 4 ) . at 2128 dpa , the a - type granules continue to contribute most of the total starch granule volume ( ~58% ) while the b - type granules make up most of the granule number ( ~37% ) although the percentage is lower than at 14 dpa ( ~41% ) . this reduction could be due to the growth of a - type granules originally counted as b - granules at 14 dpa . we detected another increase in the number of granule < 10 m at 2835 dpa which may be due to the initiation of c - type granules . at 35 dpa the b- and c - type granules continued to contribute most of the total granule number ( ~98% ) . growth of the c - granules probably increased its contribution to the volume of the small - sized granules from ~37% at 28 dpa to ~56% at 35 dpa . the rate of the change in volume of the a - granule population correlated with the expression profiles of two unknown genes ( be438268 , r = 0.922 ; and be422634 , r = 0.832 ) , a putative dead box rna helicase ( r = 0.849 ) , and chitinase2 ( r = 0.811 ) . the profile of b - granule distribution ( number and volume ) and the ratio of b- to a - granules in terms of their volume and number correlated predominantly with the changes in transcriptional expression of storage protein genes ( see table 1 , table 2 ; and supplemental material 1 ) . the distribution of the a - granules in terms of their percent volume correlated well with the expression profiles of two genes encoding regulatory proteins . in terms of their percent number , a - granules correlated with the expression profiles of genes encoding several transcription factors and other carbohydrate metabolism proteins ( table 1 ) . these identified genes could serve as entry points to gain further insights into the molecular basis of multimodal size distribution of starch granules in wheat . the expression profiles of genes known to be involved in the sucrose to starch biosynthetic pathway were examined ; genes represented on the cdna array and oligoarray are shown in figures 5 and 6 , respectively . the selected genes are presented according to their expression pattern as detected on the cdna array that could be distinguished based on the age of the caryopsis at which transcript accumulation was maximal . the early expressers ( figures 5(a)5(h ) ) had transcript levels highest at 37 dpa , when dry matter and storage reserves ( starch and protein ) start to accumulate , and then generally decline through caryopsis development . the middle expressers ( figures 5(i)5(r ) ) had a bell - shaped pattern with a sharp increase and maximum level at about 14 dpa , when dry matter and storage reserves are rapidly accumulating , and then declining as the caryopsis matures . one gene has a profile ( sbei ; figure 5(s ) ) with bell - shaped pattern but peaks at 21 dpa and was categorized as a late expresser . finally , two genes ( figures 5(t ) and 5(u ) ) have similar modulated patterns of expression throughout caryopsis development with lowest levels of transcripts at about 7 and 28 dpa . figure 7 shows the expression profiles of other critical genes represented on the oligoarray but not on the cdna array . the first committed step to starch biosynthesis is catalyzed by adp - glucose pyrophosphorylase ( agpase)a tetrameric enzyme composed of two catalytic small subunits ( ssus ) and two regulatory large subunits ( lsus ) , both encoded by several genes in wheat . the cdna array detected expression of agpase ssu ( figure 5(k ) ) and lsu ( figure 5(l ) ) genes both showed similar bell - shape patterns peaking around 14 dpa . agpase ssu genes average expression changed about 8-fold , compared to 2.6-fold observed for lsu . for both ssu and lsu , in addition to multiple contigs showing the bell - shaped middle expresser pattern , a single sequence ( be590582 for ssu and be399537 for lsu ) showed less variation in expression . both of these variants were sequences relatively 5 in the coding regions of genes compared to the more 3 sequences of the other contigs . the profile variation associated with more 5 sequence is not known but may be related to the fact that target mrna preparations tend to be 3 biased . examination of the agpase ssu and lsu gene expression profiles using the oligoarray also detected different patterns of expression ( figures 6(k ) and 6(l ) ) . ta.6869 , which represents the plastidic isoforms ( ay727927 , ) , showed little change in expression throughout development . in contrast , ta.242 expression rose rapidly from 6 dpa to 8 dpa and remained high thereafter . ta.242 target sequence shows about 99% sequence identity to a cytosolic ssu gene ( ef405961 ) and 95% sequence identity to a plastidic gene ( eu586278 ) . it has been shown previously that the plastidic and cytosolic isoforms of ssu can be derived from the same gene by differential splicing with the difference in sequence primarily confined to the 5 end of the gene . ta.242 also matches the set of agpase ssu sequences on the cdna array which suggests that they are encoded by the same gene . ta.23665 showed a slight increase in expression from 6 dpa to 8 dpa and then gradually dropped from 17 dpa onward , whereas the two ta.2797 probesets , derived from different regions of the same sequence , showed peaked expression at 14 dpa and remained high thereafter . ta.23665 showed close similarity to a gene ( genbank i d x14348 ) that encodes the plastidic isoforms of the enzyme agpase lsu . ta.2797 matches all contigs from the cdna array ( figure 5(l ) ) , which presumptively are cytosolic isoforms . starch synthases ( sss ) catalyze the transfer of the glucose moiety from adp - glucose to the reducing end of a preexisting -1,4-linked glucan polymer . the starch synthases are grouped into five classes by dna sequence ; that is , ssi , ssii , ssiii , ssiv , and gbss , and each has unique but occasionally overlapping roles . ssi ( figure 5(n ) ) , ssiia ( figure 5(o ) ) , and gbssi ( figure 5(p ) ) all showed bell - shaped expression profiles , which agreed with previous observations [ 30 , 31 ] . gbssii ( figure 5(g ) ) was an early expresser and its expression decreased through caryopsis development , consistent with its role in transient starch accumulation in the pericarp . the gbssi contigs on the oligoarray ( figure 6(p ) ) were presumably two different homeologs , which the cdna array failed to discriminate . the ssi , ( figure 6(n ) ) , ssii ( figure 6(o ) ) , and ssiii ( figure 7(a ) ) genes detected via oligoarrays also showed bell - shaped patterns of expression . the oligoarray discriminated between ssiia ( figure 6(o ) , ta.55 ) and ssiib ( figure 6(o ) , ta.4204 ) and also showed some variation in expression level . the general expression pattern of gbssi ( figure 6(p ) , ta.2795 , and ta.24114 ) was similar to that on the cdna arrays ( figure 5(p ) ) . although the gbssii expression patterns ( figures 5(g ) and 6(g ) ) seem dissimilar , the cdna array experiment included a high mrna level at the 3 dpa point ( figure 5(g ) ) not included in the oligoarray . starch branching enzymes ( sbes ) catalyze the hydrolysis of an -1,4-glucan linkage and form an -1,6 glucosidic bond on c6 of a glucosyl moiety of an -1,4 glucan to form a branch . the cdna array expression profiles of sbeiia ( figure 5(h ) ) , sbeiib ( figure 5(q ) ) , and sbei ( figure 5(s ) ) categorize these genes as early , middle , and middle - late expressers , respectively . this result agrees with previous observations [ 36 , 37 ] , and points to different mechanisms for the temporal expression of the sbes . in members of the triticeae , a form of sbei , termed sbeic , is present exclusively in a - starch granules . it contains two sbei - like domains and is proposed to have originated by trans - splicing of a sbei - like mrna and a sbei transcript . it is not possible to determine whether the sbei clone on the cdna array is sbeic or sbei because the sbei - like domain sequence is present in both genes . one clone for sbei was present on the oligoarray ( figure 6(s ) , ta.39 ) , which showed a rapid increase from 6 dpa with a plateau from 14 to 42 dpa . the three clones for sbeiia on the cdna array ( figure 5(h ) ) showed similar expression profiles with high transcript levels from 3 to 14 dpa and then gradually declined . there were three clones for sbeiib on the cdna array which are two different expression profiles ( figure 5(q ) ) . be424382 and be424382.b , which showed a similar expression profile , are duplicates of the same clone further confirming array data reproducibility . it should be noted that the bf201559 sequence aligns with the 5-end of the t. aestivum sbeiib ( ay740401 ) , whereas the be424382 sequences align with the same gene at the 3-end . the sbeiib genes on the oligoarrays ( figure 6(q ) ; ta.4204 and ta.11127 ) have a similar expression pattern to sbeiib on the cdna array that showed a rapid increase from 6 dpa with a peak at 14 dpa and then declined thereafter . however , the expression of sbeiib genes as detected by the oligorray remained at higher levels after 14 dpa than did the cdna array samples . the clone for sbeiia ( figure 6(h ) , ta.28697 ) detected with the oligoarray had a similar general shape to those detected by the cdna arrays which started high at 68 dpa and remained high until 14 dpa after which transcript levels declined . the starch debranching enzymes ( dbes ) catalyze the hydrolysis -1,6 glycosidic linkages important for maintaining starch granule crystallinity [ 2 , 40 ] . there are two types of dbe present in plants , the pullulanase and the isoamylase . there were no dbe genes represented in the cdna array but both were on the oligoarrays . both pullulanase ( figure 7(b ) ) and isoamylase1 ( figure 7(c ) ) genes showed the middle expressor pattern with a peak at 14 dpa and gradually declined after 21 dpa . the pullulanase result is consistent with the rna expression profile of a pullulanase gene cloned from wheat . the expression of isoamylase2 ( figure 7(d ) ) is distinct and has not been previously reported . the phosphoglucose isomerase ( pgi ; ec 5.3.1.9 ) gene detected by the cdna array encodes a plastidic isoform and was an early expresser ( figure 5(b ) ) , whereas the pgi gene ta.894 detected by the oligoarrays ( figure 6(b ) ) , which encodes a cytoplasmic isoform , rose early in development and then remained fairly constant but with some variation in level . with triosephosphate isomerase ( tpi ; ec 5.3.1.1 ) , another glycolytic enzyme , the gene detected by the cdna array ( figure 5(r ) ) , encoded a cytoplasmic isoform and was a middle expresser , similar to most of the genes encoding for starch synthesis enzymes . the tpi genes detected by the oligoarray ( figure 6(f ) ) , included two genes for cytoplasmic isoforms , ta.13669 and ta.27753 , and a plastidic isoform , ta.4315 . the cytoplasmic ta.13669 was a middle expresser , whereas ta.4315 and ta.27753 show a pattern of expression that were closer to an early expresser profile . phosphoglucomutase ( pgm ; ec 5.4.2.2 ) expression was detected by both the cdna array ( figure 5(c ) , bf484585 ) and the oligoarray ( figure 6(c ) , ta.24519 ) . the pgm genes on both arrays encoded cytoplasmic isoforms of the enzyme and were early expressers . udp - glucose pyrophosphorylase ( ugpase ; ec 2.7.7.9 ; also known as utp - glucose-1-phosphate uridylyltransferase ) is a cytosolic enzyme involved in the synthesis of starch in the developing caryopsis as part of the main path of glucose into plastids . the expression of genes encoding for ugpase expression was similar to the early expresser pattern as detected by both cdna ( figure 5(a ) ) and oligoarrays ( figure 6(a ) ) . sucrose synthase ( susy ; udp - glucose : d - fructose 2-glucosyltransferase , ec 2.4.1.13 ) catalyzes the reversible conversion of sucrose and udp into udp - glucose and fructose . two classes of sucrose synthase genes were represented on the cdna array , with susy1 and susy2 both showing a bell - shape patterns of expression ( figures 5(i ) and 5(j ) ) . for the susy2 gene , two 3 sequences had almost identical patterns ( be398649 and be423294 ) , while two 5 sequences had either a similar , but depressed pattern compared to the 3 sequences ( be604913 ) , or an almost flat pattern ( be498731 ) . the expression of the susy2 gene detected by oligoarrays ( figure 6(a ) ) gave a similar pattern as the 3 sequences observed for the cdna arrays but represents a different gene ( by sequence analysis ) . the bell - shape expression pattern detected by the cdna arrays for the susy1 gene ( figure 5(i ) ) is similar to susy2 . the susy1 probesets on the oligoarray ( figure 6(i ) ) do not distinguish the susy isoforms and may hybridize with other related genes . sucrose phosphate synthase ( sps ; ec 2.4.1.14 ) catalyzes the conversion of fructose-6-phosphate and udp - glucose into sucrose-6-phosphate and udp . recent reports have shown that plants have multiple forms of sps and are encoded by five different families of genes in wheat . the two sps cdna clones on the cdna array ( figure 5(t ) ) represent the same sps gene and showed modulated pattern of expression . the three sps probesets on the oligoarrays ( figure 6(t ) ) showed three different patterns of expression compared to the sps gene represented on the cdna array . analysis of the sps target dna sequences from which the probesets were designed indicated that the three sps probes on the oligoarray and the one on the cdna array represent four distinct genes . when focusing on specific known pathways , the most striking example of correlations using the cdna arrays was for sugar accumulation and synchronous expression of glycolytic and oppp ( oxidative pentose phosphate pathway ) genes ( supplementary material 1 ) . these pathways provide reducing power ( nadh ) , intermediates , and atp for starch biosynthesis . most of these genes are early expressers ; for example , fbpa ( fructose-1,6-bisphosphate aldolase ; figure 5(e ) ) , enolase ( figure 5(f ) ) , and those not shown in figure 5 such as a putative genes for 6-phosphogluconolactonase , cytosolic 6-phosphogluconate dehydrogenase , and transketolase . our results showed that most of the genes involved in starch metabolism were middle expressers . genes shared between glycolysis and starch biosynthesis , however , were in general early expressers , suggesting that they may be at a key juncture in energy generation and utilization in a major metabolic pathway in developing endosperm . these results agree broadly with that published by sreenivasulu et al . in developing barley caryopsis where they showed that genes involved in energy production are expressed in general earlier than genes involved primarily in starch metabolism . posttranslational regulation including phosphorylation , interaction with 14 - 3 - 3 regulatory proteins and posttranslational red - ox activation , appear to be essential regulatory mechanisms controlling starch biosynthesis by providing a rapid response to short - term environmental changes . there are fewer examples of coarse control of starch biosynthesis , either because this is not as important or has not been as extensively studied [ 20 , 47 ] . we performed correlative analysis to find genes which show synchronous expression with genes encoding proteins that regulate starch accumulation ; that is , 14 - 3 - 3 , pdk , wrky tfs , and protein kinases to identify possible targets of their action . there are hundreds of closely related forms and at least one member , a 14 - 3 - 3 protein from the -group , may directly regulate the synthesis of starch by binding ssiii . in barley caryopses , as many as 16 proteins involved in carbohydrate metabolism were able to bind 14 - 3 - 3a proteins in vitro and are all putative candidates for regulation by phosphorylation in vivo . our data showed that the genes for 14 - 3 - 3b and 14 - 3 - 3c had a pattern of expression ( figure 8(a ) ) similar to some of the genes encoding glycolytic genes , as well as to unknown and hypothetical proteins , making it possible that they have a role in interacting with these regulatory proteins ( see supplementary material 4 ) . in plants , protein phosphorylation has been implicated in many aspects of cellular regulation and metabolism including the regulation of carbohydrate metabolism [ 51 , 52 ] . we observed coordinated transcript accumulation between genes encoding putative protein kinases , a gsk-3/shaggy - related protein kinase , and a putative atn1 kinase ( figure 8(b ) ) . expression pattern of these kinase genes correlated with the expression of genes coding for aatp and sps as detected by the cdna array and the rate of starch accumulation ( see supplementary material 4 ) . this similar pattern of expression may suggest that aatp and sps are possible targets for the action of these protein kinases . there is good biochemical evidence for the regulation of starch metabolism and especially of sps by protein kinases . there is now direct evidence that a gene homologue of a gsk3-like kinase called msk4 , from medicago sativa , regulates carbohydrate metabolism under environmental stress . a reduction in the expression of ppdk in rice is responsible in part for the floury-4 endosperm phenotype characterized by loosely packed starch granules and reduced starch biosynthesis in the immature endosperm . ppdk expression in ( figure 8(c ) ) was similar to two clones of susy2 , sbeiib , a probable starch synthase precursor , and glycosyltransferase , as well as with several unknown proteins ( see supplementary material 4 ) . there were two clones on the array which belong to group 3 of the wrky superfamily . the wrky 71 clone had maximal expression at 7 dpa ( figure 8(d ) ) , while the wrky19a clone on the array changed sharply in expression level during the caryopsis early grain - filling stage and was synchronously expressed with two clones similar to the oat storage protein avenin , a wheat monomeric alpha - amylase inhibitor , the wheat cm2 protein , and an unknown protein ( see supplementary material 4 ) . the expression profiles of the genes involved in sucrose to starch pathway as detected by cdna and oligoarrays showed good agreement , particularly for those genes whose isoforms were represented on both array platforms . the agreement was in spite of the fact that the two global profiling experiments use different array platforms and rna targets from plant materials of distinct genotypes , these results differ from the report of poole et al . [ 5 , 7 ] who concluded that measures of global expression with cdna and oligoarray experiments were not consistent after comparing relative expression levels between two cultivars at a single developmental time point . al . compared the expression of genes from the cdna and oligoarrays that are similar at the expectation value of e or less , which could include members of gene families that have different patterns of expression . our report focused on changes in the patterns of expression of single genes over caryopsis development within a single germplasm . the resulting profile patterns are consistent at least at a level indicating major changes in patterns . as mentioned , the set of genes represented in the cdna array complemented the set in the oligoarrays and vice versa . for example , the plastidic pgi gene was present in the cdna array , whereas the cytosolic pgi was present in the oligoarray . the cdna array probes were sensitive enough to detect the expression of the genes coding for the different isoforms of the same enzyme . these differences in expression pattern , however , were more clearly distinguished with the isoforms detected in the oligoarrays . although the oligoarray contains a better representation of genes , extra effort should be taken to verify the annotations for the probe sets used since a significant number of the ncbi unigene assemblies used in the original design of short oligomers for probes have been removed and the ests belonging to the original assembly may now be assigned to different gene assemblies . thus far , the probesets on all wheat dna arrays available contain an incomplete representation of the transcriptome . future arrays should take advantage of increasing sequence resources and prior array studies to develop more complete transcriptome representation . since a complete sequence of wheat will not be available soon , it may be possible to utilize the recently complete sequence of the monocot brachypodium , a closer relative of wheat than rice , to fill gaps in knowledge of wheat genes . in this study , we investigated the transcriptional control of storage starch synthesis in developing wheat caryopsis . we overlaid the results of a global gene expression profiling experiment with the analysis of soluble sugar accumulation , starch content , and starch granule particle size distribution on the same batch of biomaterials used for the microarray experiment . the 8 k cdna array used in this study was supplemented with data from an oligoarray report . this approach allowed us to gain an overview of the changes in the expression of genes coding for key enzymes involved in starch biosynthesis . genes previously not associated with starch metabolism , including those for transcription factors , were tentatively identified by which expression profile showed a positive correlation with changes in the physicochemical properties and composition of starch during caryopsis development . there are two major routes to make adp - glucose available for the biosynthesis of storage starch in the cereal amyloplast . one route is through the cytosolic synthesis of adp - glucose ( adpg ) and its transport into the plastid by the adpg transporter ; the second route involves the cytosolic synthesis of glucose-6-phosphate and its transport to the plastid by the glucose-6-phosphate / phosphate transporter ( gpt ) . it has been suggested that the cytosolic generation of adpg provides a more efficient way of committing carbon to starch biosynthesis , rather than importing hexose - phosphates , which can be used for other competing metabolic pathways in the plastid . as summarized in figure 9 , our data support the idea that the major route to starch synthesis is through the cytosolic agpase - generated adp - glucose . ugpase gene expression remained high from 7 dpa to 28 dpa when major starch synthesis occurs . the expression of cytosolic pgm gene peaked at 7 dpa and rapidly declined thereafter . this suggests that the pgm - mediated conversion of glucose-1-phosphate to glucose-6-phosphate for subsequent export to the amyloplast plays a lesser part during maximal starch synthesis . furthermore , the expression of the genes that encode for the cytosolic isoforms of agpase ssu and lsu is expressed at a much higher level than the genes that encode specifically for the plastidic isoforms . finally , the expression profile of the gene coding for the plastid transporter adpgt expression peaked at 14 dpa when grain filling is maximal , whereas aatp and gpt expressions were downregulated suggesting their reduced role during this stage . gene - to - metabolite correlations have been successfully used to provide new insights into the regulatory processes of metabolite accumulation ; that is , see [ 6163 ] . this approach was used to broaden the potential gene candidates that may regulate starch biosynthesis in wheat . however , one should be cautious when analyzing gene - metabolite correlations due to the time lag between gene expression and metabolite accumulation / phenotype observation . in our study , transcript levels were assessed at 7-day intervals , which most likely would have been long enough to compensate for the lag between transcript and metabolite accumulation / phenotype observation . it should be noted that we would have missed positive gene - to - metabolite correlations for genes that changed their expression pattern exactly at the time - points sampled . correlative analysis of transcript and biochemical data can be useful in identifying previously unrecognized relationships . we observed a tight correlation between the expression pattern of aatp , an sps , several kinases , including putative kinases , and the rate of starch accumulation . one of these kinases ( gsk-3 ) has been shown to be definitively involved in the regulation of starch metabolism . therefore the coexpression of the putative kinases with aatp , sps and the rate of starch accumulation may prove useful for identifying regulators of starch biosynthesis , which have not yet been implicated as having a role in this process . some of the starch biochemical determinants we assayed correlated very well with transcript levels of major seed storage proteins . for example , changes in starch amount , b - granule number , and volume throughout development correlated with the expression pattern of genes for low and high - molecular weight glutenins , as well as with different classes of gliadins ( see supplementary material 1 ) . several studies in maize [ 64 , 65 ] similarly reported a positive correlation between the abundance of the -zein transcript , which encodes for a maize storage protein , and mature kernel dry weight , suggesting that -zein synthesis might in some way be linked to carbohydrate formation . in wheat , genes for hmw - glutenin subunits and agpl shared common regulatory loci that mapped to different chromosome arms . several mutants selected for high grain protein often have pleiotropic effects on starch , for example , high lysine barley [ 67 , 68 ] and rice mutants . our data add to the long - held view that there is a global mechanism that controls the accumulation of the major storage reserves in storage organs and that sugars are the primary control elements in this process [ 65 , 7072 ] .

the expression of genes involved in starch synthesis in wheat was analyzed together with the accumulation profiles of soluble sugars , starch , protein , and starch granule distribution in developing caryopses obtained from the same biological materials used for profiling of gene expression using dna microarrays . multiple expression patterns were detected for the different starch biosynthetic gene isoforms , suggesting their relative importance through caryopsis development . members of the adp - glucose pyrophosphorylase , starch synthase , starch branching enzyme , and sucrose synthase gene families showed different expression profiles ; expression of some members of these gene families coincided with a period of high accumulation of starch while others did not . a biphasic pattern was observed in the rates of starch and protein accumulation which paralleled changes in global gene expression . metabolic and regulatory genes that show a pattern of expression similar to starch accumulation and granule size distribution were identified , suggesting their coinvolvement in these biological processes .

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

the genetic basis of the multimodal size distribution of starch in wheat and barley is of great interest because the physiochemical properties of each type of starch granule vary and contribute to the food and industrial end - uses of triticeae starch [ 1012 ] . in this report , results of a global gene expression profiling experiment were overlaid with the analysis of soluble sugar accumulation , starch content , and starch granule particle size distribution on the same batch of biological materials used for the microarray experiment . results showed multiple temporal expression patterns of key genes involved in starch synthesis , suggesting the relative importance of the different enzymes throughout caryopsis development . correlative analysis identified genes that showed similar patterns of expression to the accumulation profiles of starch and amylose and the distribution of starch granules suggesting these as possible candidate genes for further investigation for their roles in seed starch synthesis and potential targets for modulating carbohydrate metabolism in wheat by genetic engineering or molecular breeding efforts . two sets of microarray data were examined for expression of genes involved in starch metabolism in the developing wheat caryopsis . for the purpose of this work , a probe set is deemed to represent a potentially unique wheat gene and the accumulation of its transcript as measured by the signal intensities in each probe set represents the expression of the gene . of the 23 genes that correlated with glucose and sucrose , only the atp / adp carrier protein was known to be involved in the starch synthesis pathway ; there were no regulatory factors identified ( see supplementary material 2 ) . of the 249 genes that correlated well with the changes in the levels of both fructose and glucose were metabolic and transcriptional regulatory genes , for example , putative pyruvate kinase , a yabby protein , a probable kinase , 14 - 3 - 3 regulatory proteins , a pistillata - like mads - box protein , and other tfs . the accumulation of storage reserves in the developing caryopsis showed that total protein , total starch , and the amylose content ( figure 2(a ) ) steadily accumulated from 10 dpa to a peak at 35 dpa followed by a slight reduction at 50 dpa . the observed biphasic pattern in the rates of protein , amylose and starch accumulation ( figure 2(b ) ) , coincides with the biphasic reprogramming of genes during caryopsis development . the rate of the change in volume of the a - granule population correlated with the expression profiles of two unknown genes ( be438268 , r = 0.922 ; and be422634 , r = 0.832 ) , a putative dead box rna helicase ( r = 0.849 ) , and chitinase2 ( r = 0.811 ) . the distribution of the a - granules in terms of their percent volume correlated well with the expression profiles of two genes encoding regulatory proteins . in terms of their percent number , a - granules correlated with the expression profiles of genes encoding several transcription factors and other carbohydrate metabolism proteins ( table 1 ) . the expression profiles of genes known to be involved in the sucrose to starch biosynthetic pathway were examined ; genes represented on the cdna array and oligoarray are shown in figures 5 and 6 , respectively . the early expressers ( figures 5(a)5(h ) ) had transcript levels highest at 37 dpa , when dry matter and storage reserves ( starch and protein ) start to accumulate , and then generally decline through caryopsis development . udp - glucose pyrophosphorylase ( ugpase ; ec 2.7.7.9 ; also known as utp - glucose-1-phosphate uridylyltransferase ) is a cytosolic enzyme involved in the synthesis of starch in the developing caryopsis as part of the main path of glucose into plastids . expression pattern of these kinase genes correlated with the expression of genes coding for aatp and sps as detected by the cdna array and the rate of starch accumulation ( see supplementary material 4 ) . the expression profiles of the genes involved in sucrose to starch pathway as detected by cdna and oligoarrays showed good agreement , particularly for those genes whose isoforms were represented on both array platforms . compared the expression of genes from the cdna and oligoarrays that are similar at the expectation value of e or less , which could include members of gene families that have different patterns of expression . the cdna array probes were sensitive enough to detect the expression of the genes coding for the different isoforms of the same enzyme . we overlaid the results of a global gene expression profiling experiment with the analysis of soluble sugar accumulation , starch content , and starch granule particle size distribution on the same batch of biomaterials used for the microarray experiment . this approach allowed us to gain an overview of the changes in the expression of genes coding for key enzymes involved in starch biosynthesis . for example , changes in starch amount , b - granule number , and volume throughout development correlated with the expression pattern of genes for low and high - molecular weight glutenins , as well as with different classes of gliadins ( see supplementary material 1 ) .

in the province of ontario , invasive infections with gas ( isolation of gas from blood or an otherwise sterile site ) and cases of suspected tss and necrotizing fasciitis must be reported to local public health authorities ( 15 ) . reports of invasive gas infection originating from long - term care institutions in the city of hamilton resulted in an epidemiologic investigation by the city of hamilton social and public health services department . in the two outbreaks described below , investigations consisted of site visits with line - listings created to identify facility residents and staff ; reviews of recent admissions , discharges , and deaths among residents ; and reviews of reports of illness among residents and staff . attempts were made to identify facility residents and staff with close epidemiologic linkage to the index patients ( e.g. , caregivers , roommates , and close friends ) . although typically only persons with epidemiologic linkage to index patients are cultured for possible gas carriage , the circumstances described below resulted in more widespread collection of culture specimens . specimens were obtained from noses and pharynges of residents and staff , and attempts were made to obtain cultures of perirectal areas and wounds in facility residents . in the second outbreak described below , staff provided self - collected rectal and vaginal swabs . swabs from institution residents and staff were plated onto blood agar , with group a streptococcus identified by -hemolysis and lancefield typing using standard commercially available latex agglutination methods , as described elsewhere ( 16 ) . isolates were subsequently evaluated for relatedness by using pulsed - field gel electrophoresis ( pfge ) . isolates were suspended in buffered saline , and solutions were adjusted in volume until optical densities ( od ) were identical ( 1.6 od at 610 nm ) . to prepare genomic dna , 500 l of cell suspension from each strain was mixed with an equal volume of 2% low melting agarose solution . the agarose plug was prepared by using a commercially available plug mould ( bio - rad canada limited , mississauga , ontario , canada ) . all gas plugs were treated in lysis buffer containing 50 g / ml lysozyme at 37c overnight , extensively washed in washing buffer , and then treated in a solution containing 50 g / ml protease k at 55c overnight . pfge was run by using a 1% agarose gel at 6 volts / cm ; run time was 26 h with an initial switch time of 20 s and a final switch time of 60 s. following gel electrophoresis , the gel was stained in a solution containing 0.5 g / ml ethidium bromide and photographed by using the bio - rad multi - analysis ccd camera system . m and t serologic typing and opacity factor determination were performed on all isolates with standardized methods ( 17 ) . in addition , antiopacity factor ( aof ) typing was performed on all isolates that were opacity factor positive ( 17 ) . all antisera were prepared in - house . because of the difficulty of producing antisera for some m types , aof typing has been frequently used to predict the m type ( 18,19 ) . although aof typing does not always have the same type specificity as m typing , this approach is considered valid for most strains from developed countries ( 20 ) . aof typing was used to predict the following m types : 9 , 11 , 25 , 28 , 48 , 77 , 78 and 92 ( pt5118 ) . in the province of ontario , invasive infections with gas ( isolation of gas from blood or an otherwise sterile site ) and cases of suspected tss and necrotizing fasciitis must be reported to local public health authorities ( 15 ) . reports of invasive gas infection originating from long - term care institutions in the city of hamilton resulted in an epidemiologic investigation by the city of hamilton social and public health services department . in the two outbreaks described below , investigations consisted of site visits with line - listings created to identify facility residents and staff ; reviews of recent admissions , discharges , and deaths among residents ; and reviews of reports of illness among residents and staff . attempts were made to identify facility residents and staff with close epidemiologic linkage to the index patients ( e.g. , caregivers , roommates , and close friends ) . although typically only persons with epidemiologic linkage to index patients are cultured for possible gas carriage , the circumstances described below resulted in more widespread collection of culture specimens . specimens were obtained from noses and pharynges of residents and staff , and attempts were made to obtain cultures of perirectal areas and wounds in facility residents . in the second outbreak described below , staff provided self - collected rectal and vaginal swabs . swabs from institution residents and staff were plated onto blood agar , with group a streptococcus identified by -hemolysis and lancefield typing using standard commercially available latex agglutination methods , as described elsewhere ( 16 ) . isolates were subsequently evaluated for relatedness by using pulsed - field gel electrophoresis ( pfge ) . isolates were suspended in buffered saline , and solutions were adjusted in volume until optical densities ( od ) were identical ( 1.6 od at 610 nm ) . to prepare genomic dna , 500 l of cell suspension from each strain was mixed with an equal volume of 2% low melting agarose solution . the agarose plug was prepared by using a commercially available plug mould ( bio - rad canada limited , mississauga , ontario , canada ) . all gas plugs were treated in lysis buffer containing 50 g / ml lysozyme at 37c overnight , extensively washed in washing buffer , and then treated in a solution containing 50 g / ml protease k at 55c overnight . pfge was run by using a 1% agarose gel at 6 volts / cm ; run time was 26 h with an initial switch time of 20 s and a final switch time of 60 s. following gel electrophoresis , the gel was stained in a solution containing 0.5 g / ml ethidium bromide and photographed by using the bio - rad multi - analysis ccd camera system . m and t serologic typing and opacity factor determination were performed on all isolates with standardized methods ( 17 ) . in addition , antiopacity factor ( aof ) typing was performed on all isolates that were opacity factor positive ( 17 ) . all antisera were prepared in - house . because of the difficulty of producing antisera for some m types , aof typing has been frequently used to predict the m type ( 18,19 ) . although aof typing does not always have the same type specificity as m typing , this approach is considered valid for most strains from developed countries ( 20 ) . aof typing was used to predict the following m types : 9 , 11 , 25 , 28 , 48 , 77 , 78 and 92 ( pt5118 ) . in october 2000 , the hamilton public health unit was notified of a 97-year - old female nursing home resident ( index case - patient 1 ) admitted to an area hospital with cellulitis and group a streptococcal bacteremia . cultures were obtained as described from 87 residents and staff who might have had contact with the person with invasive disease . this isolate was genetically indistinguishable by pfge analysis from the invasive isolate obtained from index case - patient 1 , and both strains were typed as m77 t9/10 , and positive for serum opacity factor . intervention consisted of reinforcement of standard infection control practices with facility staff and treatment of the person with pharyngeal colonization with a 10-day course of cephalexin , 250 mg four times per day . follow - up cultures from this person taken 1 month after completion of treatment were negative . index case - patient 1 died during the course of the investigation of apparently unrelated cardiac disease . in january 2001 , an 87-year - old female resident of the same long - term care facility was transported to hospital with fever ( index case - patient 2 ) , and gas was isolated from blood cultures drawn at admission . several days later , gas was isolated from the wound of another facility resident . screening cultures performed on 195 residents and staff with a history of contact with one of these two infected persons indicated that seven residents and one staff member were colonized with gas . pfge showed that the strains from all resident carriers were identical to the wound and blood isolates , and to the isolate obtained from index case 1 . the bacterial isolate obtained from the staff member colonized residents and staff were treated with cephalexin as had been done with the previous patient . cultures obtained from treated persons 1 month after completion of therapy were again negative . in may 2001 , another resident of this nursing home was transported to hospital with gas bacteremia ( index case - patient 3 ) . at this time , all 386 residents and 135 staff of the facility were screened for gas carriage by culturing nasopharynx specimens . thirteen of these ( 11 residents and two staff members ) subsequently proved to be carrying strains identified by pfge analysis to be identical to the strain obtained from index case - patient 2 ( figure 1 ) ( prevalence of colonization or infection among residents was 2.8% vs. prevalence of colonization 1.5% among staff , p=0.38 by chi - square test ) . pulsed field gel electrophoresis , demonstrating relatedness of group a streptococcal isolates from an person with clinical illness from gas , a person with chronic colonization with gas , and asymptomatically colonized facility staff and residents . the isolate in lane 2 ( large solid arrow ) was obtained from index case - patient 2 in january 2001 . the isolate in lane 7 ( large hollow arrow ) was obtained from a person with chronic gas colonization ( resident a ) in may 2001 . small solid arrows denote electrophoretically identical gas strains from other persons with asymptomatic colonization with group a streptococcus in may 2001 . one of the residents colonized with gas ( resident a ) was a 69-year - old woman , immobilized by severe neurologic disease , who had a suprapubic bladder catheter and a gastrostomy tube . resident a was subsequently determined to have had urine and wound cultures positive for gas in july 2000 , 3 months before index case - patient 1 sought treatment ; these isolates were unavailable for evaluation . after consultation with local communicable disease control experts , the public health team recommended administering antibiotics to all facility residents and staff . all residents were treated with a 10-day course of cephalexin or penicillin vk , with erythromycin given to persons allergic to -lactam agents . no data are available with regard to the degree of compliance with antibiotic therapy among staff . however , all previously colonized staff members were culture - negative for group a streptococcus on repeat culturing 1 month later . ten of 11 treated residents had cultures negative for group a streptococcus 1 month after treatment . resident a remained persistently colonized at gastrostomy and catheter sites , despite a subsequent prolonged course of oral clindamycin . this resident was placed on contact isolation precautions , with staff using gowns and gloves when participating in her care . no subsequent cases of invasive group a streptococcal disease have been reported at this facility as of february 2003 . however , in august 2002 , gas was identified in cultures of eye drainage and wound drainage from three residents of a single wing of the facility ( figure 2 ) . pfge demonstrated two of the three isolates to be indistinguishable by pfge from isolates obtained from resident a. neither of these persons had been present in the facility at the time of invasive cases , and neither had been housed in the same part of the facility as resident a. an audit of infection control procedures at that time showed several violations of standard infection control measures , including the reuse of a single washcloth on multiple residents of the facility . clinical cases ( black bars ) of invasive gas infection occurred at intervals of 3 to 4 months . with the occurrence of cases , acquisition of culture specimens resulted in identification of asymptomatic colonization with the outbreak strain ( white bars ) or unrelated strains ( hatched bars ) in other residents and staff . no additional clinical cases occurred after mass antibiotic treatment ( m.a.t . ) ; resident a died ( ) in july 2002 ; colonization of two residents with the outbreak strain was recognized 1 month later . in november 2001 , the hamilton public health unit was notified of two deaths from invasive group a streptococcal disease at a 126-bed long - term care facility . the first case - patient was a 78-year - old man who had been admitted to the hospital with fever and possible pneumonia 2 days before . the second case - patient was a 61-year - old man with parkinson s disease and dementia who had been admitted to the hospital 1 day before with soft skin tissue infection and likely necrotizing fasciitis . both patients died on the day the cases were reported to the public health department . the two cases had originated on different floors of the facility ( 2nd and 4th ) . a large holiday party had been held in the communal dining hall 4 days previously , providing an opportunity for residents , staff , and community members to mingle . in addition , a healthcare worker reportedly had pharyngitis , a rash , and desquamation of the palms and had received a diagnosis of scarlet fever 3 weeks earlier . due to the lack of any epidemiologic link between case - patients and the population mixing that had occurred at the holiday party , swab cultures were collected on all facility residents and all available staff members . epidemic control measures were introduced and included restriction of new admissions to the facility and reinforcement of infection control practices . based on the rapidity of events , the apparent high case - fatality rate , and recent experience with the failure of targeted culturing and treatment ( as described in outbreak 1 ) , a decision was made to initiate mass antibiotic treatment for all facility residents and staff . after that decision , but before mass antibiotic treatment could be initiated , an 89-year - old male resident was admitted to the hospital with congestive heart failure . this man died 36 h after admission ; gas was isolated from blood cultures taken at hospital admission ( figure 3 ) . solid lines represent the time of onset and duration of illness among three cases with invasive gas infection in outbreak 2 , relative to the initiation of the outbreak investigation ( date=0 ) mass antibiotic treatment was initiated approximately 48 h after the start of the investigation , with azithromycin , 250 mg orally each day , administered for 5 days to residents and to staff who wished to be treated at the facility . staff who wished to receive antibiotic treatment from their personal physicians were permitted to do so . subsequently , gas was isolated from nasal and pharyngeal cultures of two staff members and five residents of the facility . all colonized residents were negative for gas on repeat cultures , 4 weeks after receiving antibiotics . one staff member remained culture positive 2 weeks after completing a course of cephalexin provided by her personal physician ; she received two additional courses of -lactam antibiotics and was documented to be culture negative for gas 4 weeks after she completed the second antibiotic course . pfge performed on the three gas strains from case - patients and the seven strains from colonized residents and staff showed that four colonized persons had isolates identical to those from the deceased case - patients ; all were serotype m1 t1 and were serum opacity factor negative ( figure 4 ) . the proportion of residents colonized or infected with the outbreak strain was significantly higher than the proportion of staff colonized ( 4.7% vs. 0.7% , p=0.03 by chi - square test ) . pulsed - field gel electrophoresis , demonstrating relatedness of group a streptococcal isolates from facility staff and residents . solid arrows denote identical strains from two of the three persons in whom fatal invasive group a streptococcal infection developed ; the third person with invasive disease had an electrophoretically identical strain ( not shown ) . in october 2000 , the hamilton public health unit was notified of a 97-year - old female nursing home resident ( index case - patient 1 ) admitted to an area hospital with cellulitis and group a streptococcal bacteremia . cultures were obtained as described from 87 residents and staff who might have had contact with the person with invasive disease . this isolate was genetically indistinguishable by pfge analysis from the invasive isolate obtained from index case - patient 1 , and both strains were typed as m77 t9/10 , and positive for serum opacity factor . intervention consisted of reinforcement of standard infection control practices with facility staff and treatment of the person with pharyngeal colonization with a 10-day course of cephalexin , 250 mg four times per day . follow - up cultures from this person taken 1 month after completion of treatment were negative . index case - patient 1 died during the course of the investigation of apparently unrelated cardiac disease . in january 2001 , an 87-year - old female resident of the same long - term care facility was transported to hospital with fever ( index case - patient 2 ) , and gas was isolated from blood cultures drawn at admission . several days later , gas was isolated from the wound of another facility resident . screening cultures performed on 195 residents and staff with a history of contact with one of these two infected persons indicated that seven residents and one staff member were colonized with gas . pfge showed that the strains from all resident carriers were identical to the wound and blood isolates , and to the isolate obtained from index case 1 . the bacterial isolate obtained from the staff member colonized residents and staff were treated with cephalexin as had been done with the previous patient . cultures obtained from treated persons 1 month after completion of therapy were again negative . in may 2001 , another resident of this nursing home was transported to hospital with gas bacteremia ( index case - patient 3 ) . at this time , all 386 residents and 135 staff of the facility were screened for gas carriage by culturing nasopharynx specimens . thirteen of these ( 11 residents and two staff members ) subsequently proved to be carrying strains identified by pfge analysis to be identical to the strain obtained from index case - patient 2 ( figure 1 ) ( prevalence of colonization or infection among residents was 2.8% vs. prevalence of colonization 1.5% among staff , p=0.38 by chi - square test ) . pulsed field gel electrophoresis , demonstrating relatedness of group a streptococcal isolates from an person with clinical illness from gas , a person with chronic colonization with gas , and asymptomatically colonized facility staff and residents . the isolate in lane 2 ( large solid arrow ) was obtained from index case - patient 2 in january 2001 . the isolate in lane 7 ( large hollow arrow ) was obtained from a person with chronic gas colonization ( resident a ) in may 2001 . small solid arrows denote electrophoretically identical gas strains from other persons with asymptomatic colonization with group a streptococcus in may 2001 . one of the residents colonized with gas ( resident a ) was a 69-year - old woman , immobilized by severe neurologic disease , who had a suprapubic bladder catheter and a gastrostomy tube . resident a was subsequently determined to have had urine and wound cultures positive for gas in july 2000 , 3 months before index case - patient 1 sought treatment ; these isolates were unavailable for evaluation . after consultation with local communicable disease control experts , the public health team recommended administering antibiotics to all facility residents and staff . all residents were treated with a 10-day course of cephalexin or penicillin vk , with erythromycin given to persons allergic to -lactam agents . no data are available with regard to the degree of compliance with antibiotic therapy among staff . however , all previously colonized staff members were culture - negative for group a streptococcus on repeat culturing 1 month later . ten of 11 treated residents had cultures negative for group a streptococcus 1 month after treatment . resident a remained persistently colonized at gastrostomy and catheter sites , despite a subsequent prolonged course of oral clindamycin . this resident was placed on contact isolation precautions , with staff using gowns and gloves when participating in her care . no subsequent cases of invasive group a streptococcal disease have been reported at this facility as of february 2003 . however , in august 2002 , gas was identified in cultures of eye drainage and wound drainage from three residents of a single wing of the facility ( figure 2 ) . pfge demonstrated two of the three isolates to be indistinguishable by pfge from isolates obtained from resident a. neither of these persons had been present in the facility at the time of invasive cases , and neither had been housed in the same part of the facility as resident a. an audit of infection control procedures at that time showed several violations of standard infection control measures , including the reuse of a single washcloth on multiple residents of the facility . clinical cases ( black bars ) of invasive gas infection occurred at intervals of 3 to 4 months . with the occurrence of cases , acquisition of culture specimens resulted in identification of asymptomatic colonization with the outbreak strain ( white bars ) or unrelated strains ( hatched bars ) in other residents and staff . no additional clinical cases occurred after mass antibiotic treatment ( m.a.t . ) ; resident a died ( ) in july 2002 ; colonization of two residents with the outbreak strain was recognized 1 month later . in november 2001 , the hamilton public health unit was notified of two deaths from invasive group a streptococcal disease at a 126-bed long - term care facility . the first case - patient was a 78-year - old man who had been admitted to the hospital with fever and possible pneumonia 2 days before . the second case - patient was a 61-year - old man with parkinson s disease and dementia who had been admitted to the hospital 1 day before with soft skin tissue infection and likely necrotizing fasciitis . both patients died on the day the cases were reported to the public health department . the two cases had originated on different floors of the facility ( 2nd and 4th ) . a large holiday party had been held in the communal dining hall 4 days previously , providing an opportunity for residents , staff , and community members to mingle . in addition , a healthcare worker reportedly had pharyngitis , a rash , and desquamation of the palms and had received a diagnosis of scarlet fever 3 weeks earlier . due to the lack of any epidemiologic link between case - patients and the population mixing that had occurred at the holiday party , swab cultures were collected on all facility residents and all available staff members . epidemic control measures were introduced and included restriction of new admissions to the facility and reinforcement of infection control practices . based on the rapidity of events , the apparent high case - fatality rate , and recent experience with the failure of targeted culturing and treatment ( as described in outbreak 1 ) , a decision was made to initiate mass antibiotic treatment for all facility residents and staff . after that decision , but before mass antibiotic treatment could be initiated , an 89-year - old male resident was admitted to the hospital with congestive heart failure . this man died 36 h after admission ; gas was isolated from blood cultures taken at hospital admission ( figure 3 ) . solid lines represent the time of onset and duration of illness among three cases with invasive gas infection in outbreak 2 , relative to the initiation of the outbreak investigation ( date=0 ) . mass antibiotic treatment was initiated approximately 48 h after the start of the investigation , with azithromycin , 250 mg orally each day , administered for 5 days to residents and to staff who wished to be treated at the facility . staff who wished to receive antibiotic treatment from their personal physicians were permitted to do so . subsequently , gas was isolated from nasal and pharyngeal cultures of two staff members and five residents of the facility . all colonized residents were negative for gas on repeat cultures , 4 weeks after receiving antibiotics . one staff member remained culture positive 2 weeks after completing a course of cephalexin provided by her personal physician ; she received two additional courses of -lactam antibiotics and was documented to be culture negative for gas 4 weeks after she completed the second antibiotic course . pfge performed on the three gas strains from case - patients and the seven strains from colonized residents and staff showed that four colonized persons had isolates identical to those from the deceased case - patients ; all were serotype m1 t1 and were serum opacity factor negative ( figure 4 ) . the proportion of residents colonized or infected with the outbreak strain was significantly higher than the proportion of staff colonized ( 4.7% vs. 0.7% , p=0.03 by chi - square test ) . pulsed - field gel electrophoresis , demonstrating relatedness of group a streptococcal isolates from facility staff and residents . solid arrows denote identical strains from two of the three persons in whom fatal invasive group a streptococcal infection developed ; the third person with invasive disease had an electrophoretically identical strain ( not shown ) . our description of outbreaks of multiple cases of invasive disease attributable to a single , identified strain of gas in long - term care institutions is consistent with prior reports ( 510 ) . outbreak 1 was caused by an m77 strain , an uncommon cause of invasive streptococcal disease in canada ( 21 ) . the second outbreak was caused by an m1-type strain , the most common isolate type in canada , which may be more virulent than other serotypes ( 21,22 ) . epidemiologic investigation of these two outbreaks showed a higher prevalence of colonization with the outbreak strains among residents than among staff , although this difference was not statistically significant in outbreak 1 . given the limited mobility of the residents of both facilities , such a difference would be consistent with disease transmission involving asymptomatically colonized healthcare workers . transmission may also have occurred as a result of transient carriage of the organism by healthcare workers , as may occur with nosocomial transmission of clostridium difficile and vancomycin - resistant enterococcus ( 23,24 ) , or as a result of contaminated fomites , such the washcloth described in outbreak 1 . other possible mechanisms of transmission include direct resident - to - resident transmission in the context of such social events as the holiday party described in outbreak 2 and foodborne transmission ( 25 ) . any of these mechanisms of spread could limit the expected effectiveness of screening and targeted antibiotic treatment , as culturing of only residents and staff with a clear epidemiologic link to cases would not be expected to identify all colonized persons . this situation was well demonstrated in outbreak 1 , which involved probable worker transmission of a strain from an immobile , chronically colonized person ( resident a ) to other residents in this institution . although likely important in the perpetuation of the outbreak , resident a was identified incidentally when cultures were performed on all facility residents 8 months after the initial case of invasive disease was reported . the importance of silent carriage of gas in the perpetuation of outbreaks has been well - described in both healthcare ( 26,27 ) and community ( 28 ) contexts . our use of mass antibiotic treatment as an epidemic control measure appears to have been successful , inasmuch as no further cases of invasive gas disease have been reported from either facility at the time of writing ( february 2003 ) . however , the documented persistence of the bacterial strain implicated in outbreak 1 after the death of resident a suggests that breaches in infection control practices ( as identified during site visits ) , combined with the presence of a persistently colonized resident or staff person , may limit the effectiveness of this control measure . such a control measure would also not prevent the repeated reimportation of a circulating community strain of gas into a facility by healthcare workers or visiting family members ( 29,30 ) . the impact of wide - scale antibiotic use on pathogenic microorganisms in long - term care institutions also remains an issue of concern . resistance to macrolides among gas isolates appears to be increasing in frequency ( 31,32 ) , and the overuse of -lactam antibiotics and macrolides may adversely affect the antibiotic susceptibility patterns of such common pathogens as streptococcus pneumoniae ( 3336 ) . these two outbreaks of invasive gas in the long - term care setting highlight the limitations of targeted culturing and antibiotic treatment as an outbreak control strategy . however , our experience also highlights the limitations of mass treatment as a control strategy , particularly when poor infection control practices persist and chronically colonized residents are present . further research into the epidemiology and transmission of gas in the long - term care setting will help refine the optimal approach to managing these challenging outbreaks .

outbreaks of invasive infections caused by group a -hemolytic streptococcus ( gas ) may occur in long - term care settings and are associated with a high case - fatality rate in debilitated adults . targeted antibiotic treatment only to residents and staff known to be at specific risk of gas may be an ineffective outbreak control measure . we describe two institutional outbreaks in which mass antibiotic treatment was used as a control measure . in the first instance , mass treatment was used after targeted antibiotic treatment was not successful . in the second instance , mass treatment was used to control a rapidly evolving outbreak with a high case - fatality rate . although no further clinical cases were seen after the introduction of mass antibiotic treatment , persistence of the outbreak strain was documented in one institution > 1 year after cases had ceased . strain persistence was associated with the presence of a chronically colonized resident and poor infection control practices .

Methods Epidemiologic Investigations Microbiologic Evaluation and Characterization of GAS Strains Results Outbreak 1 Outbreak 2 Discussion

reports of invasive gas infection originating from long - term care institutions in the city of hamilton resulted in an epidemiologic investigation by the city of hamilton social and public health services department . reports of invasive gas infection originating from long - term care institutions in the city of hamilton resulted in an epidemiologic investigation by the city of hamilton social and public health services department . in january 2001 , an 87-year - old female resident of the same long - term care facility was transported to hospital with fever ( index case - patient 2 ) , and gas was isolated from blood cultures drawn at admission . pfge demonstrated two of the three isolates to be indistinguishable by pfge from isolates obtained from resident a. neither of these persons had been present in the facility at the time of invasive cases , and neither had been housed in the same part of the facility as resident a. an audit of infection control procedures at that time showed several violations of standard infection control measures , including the reuse of a single washcloth on multiple residents of the facility . with the occurrence of cases , acquisition of culture specimens resulted in identification of asymptomatic colonization with the outbreak strain ( white bars ) or unrelated strains ( hatched bars ) in other residents and staff . based on the rapidity of events , the apparent high case - fatality rate , and recent experience with the failure of targeted culturing and treatment ( as described in outbreak 1 ) , a decision was made to initiate mass antibiotic treatment for all facility residents and staff . solid lines represent the time of onset and duration of illness among three cases with invasive gas infection in outbreak 2 , relative to the initiation of the outbreak investigation ( date=0 ) mass antibiotic treatment was initiated approximately 48 h after the start of the investigation , with azithromycin , 250 mg orally each day , administered for 5 days to residents and to staff who wished to be treated at the facility . in january 2001 , an 87-year - old female resident of the same long - term care facility was transported to hospital with fever ( index case - patient 2 ) , and gas was isolated from blood cultures drawn at admission . pfge demonstrated two of the three isolates to be indistinguishable by pfge from isolates obtained from resident a. neither of these persons had been present in the facility at the time of invasive cases , and neither had been housed in the same part of the facility as resident a. an audit of infection control procedures at that time showed several violations of standard infection control measures , including the reuse of a single washcloth on multiple residents of the facility . with the occurrence of cases , acquisition of culture specimens resulted in identification of asymptomatic colonization with the outbreak strain ( white bars ) or unrelated strains ( hatched bars ) in other residents and staff . ; resident a died ( ) in july 2002 ; colonization of two residents with the outbreak strain was recognized 1 month later . in november 2001 , the hamilton public health unit was notified of two deaths from invasive group a streptococcal disease at a 126-bed long - term care facility . based on the rapidity of events , the apparent high case - fatality rate , and recent experience with the failure of targeted culturing and treatment ( as described in outbreak 1 ) , a decision was made to initiate mass antibiotic treatment for all facility residents and staff . mass antibiotic treatment was initiated approximately 48 h after the start of the investigation , with azithromycin , 250 mg orally each day , administered for 5 days to residents and to staff who wished to be treated at the facility . our description of outbreaks of multiple cases of invasive disease attributable to a single , identified strain of gas in long - term care institutions is consistent with prior reports ( 510 ) . any of these mechanisms of spread could limit the expected effectiveness of screening and targeted antibiotic treatment , as culturing of only residents and staff with a clear epidemiologic link to cases would not be expected to identify all colonized persons . although likely important in the perpetuation of the outbreak , resident a was identified incidentally when cultures were performed on all facility residents 8 months after the initial case of invasive disease was reported . our use of mass antibiotic treatment as an epidemic control measure appears to have been successful , inasmuch as no further cases of invasive gas disease have been reported from either facility at the time of writing ( february 2003 ) . however , the documented persistence of the bacterial strain implicated in outbreak 1 after the death of resident a suggests that breaches in infection control practices ( as identified during site visits ) , combined with the presence of a persistently colonized resident or staff person , may limit the effectiveness of this control measure . these two outbreaks of invasive gas in the long - term care setting highlight the limitations of targeted culturing and antibiotic treatment as an outbreak control strategy . however , our experience also highlights the limitations of mass treatment as a control strategy , particularly when poor infection control practices persist and chronically colonized residents are present . further research into the epidemiology and transmission of gas in the long - term care setting will help refine the optimal approach to managing these challenging outbreaks .

specially designed screwshaped implants of titanium ( grade 4 ) were manufactured with tight tolerances to induce controlled bone strains . the designs of the implants were identical to the implants in the study by halldin et al . the test implants comprised of a condensation portion that had an increased diameter of 0.05 mm compared to the cutting portion . all implants had the same surface as the commercially available osseospeed implants ( dentsply implants , mlndal , sweden ) . to confirm that both implant groups had the same surface topography the implant surfaces were characterized by use of optical light interferometry ( microxam ; ade phase shift technology , inc . , tucson , az , usa ) . in brief , each implant was measured on three thread peaks , three valleys , and three flanks ( scan area of 264 200 m , vertical measurement range of 100 m ) . data evaluation was performed with the mountainmaps software ( digital surf , besanon , france ) . waviness and form were filtered with a 50 50 m gaussian filter . the surfaces were characterized with the surface roughness parameters s a ( m ) , s dr ( % ) , and s ds ( 1/m ) ( definition of them can be found elsewhere ( stout 2000 ; thomas 1999 ) ) . twentyfour mature female new zealand white rabbits ( weight 34 kg ) were used in this study . the study was approved by the french ministre de lenseignement suprieur et de la recherche and performed at the colenationalevtrinaired'alfort ( maisonsalfort , valdemarne , france , approval number , 0039101 ) . prior to surgery , 250 g / kg of medetomidine ( domitor , zoetis , france ) , 20 mg / kg ketamine ( imalgne 1000 , merial , sanofi , france ) , and 1 mg / kg of diazepam ( valium , roche , france ) was injected intramuscularly to provide general anesthesia . subsequently , analgesics 30 g / kg , buprenorphine ( buprcare , animalcare , york , uk ) was injected subcutaneously and 0.2 mg / kg meloxicam ( metacam ; boehringer ingelheim vetmedica , inc . , st . thereafter , incision of the skin was made with a # 15 blade and the muscle layers and periosteum were elevated and separated from the bone . osteotomy was prepared in the proximal tibia by drilling with a sequence of burs , starting from a round bur , 2.8 mm drill and finally with a 3.3 mm drill , corresponding to the core diameter of the implants . each rabbit received two test implants in one leg ( proximal and distal placement ) and two control implants on the contralateral side . the implants were inserted with a rotation speed set to 25 revolutions / min using the w&h implant unit ( elcomed , w&h sa310 , burmoos , austria ) . after implant insertion , the muscle layers were sutured with a resorbable suture ( vicryl3.0 ) , and the skin with a 40 nylon suture ( ethicon , auneau , france ) . postsurgically , a patch of fentanyl ( 25 g / h , duragesic ; janssen pharmaceutica , beerse , belgium ) was applied to each animal for 3 days . an antibiotic ( enrofloxacin , 200 mg / l , baytril ; bayer animal health , leverkusen , germany ) was administrated in the water supply for 5 days . the rabbits were kept in separate cages and were allowed to move and eat freely . at 7 , 28 , and 84 days after implant placement , eights rabbits at each time point were euthanized with an overdose injection of sodium pentobarbital ( euthasol , virbac , fort worth , tx , usa ) . each tibia was dissected and excessive soft and hard tissue around the implant square head was carefully removed . the bone samples were firmly secured with several individual bone pins and the removal torque of the implants was measured . the removal torque , time , and angle of rotation were recorded with a calibrated torque measurement device ( dr2112 lorenz messtechnik gmbh , alfdorf , germany ) using a constant rotational speed of 0.03 rpm . the torque device was calibrated in the range 502000 nmm with an accuracy of 1 nmm . the data were sampled with 100 hz and thereafter analyzed in matlab software 2013b ( mathworksinc . , the data were initially smoothed with a running average over a set of 50 data points and the angle of rotation was set to 0 rad when the torque reached 10 nmm . the fracture torque [ t f ] was identified as the maximum torque . in the present study , the magnitude of pressure at the threads was simulated by a radial displacement of the thread profile of 0.025 mm as described in the 2daxis symmetric model of halldin et al . the 2d simulations were performed using bilinear mature bone material properties without hardening ( table 1 ) . the reduction in prestress over time , due to relaxation and remodeling , of mature bone was simulated by use of the constitutive model , illustrated by the rheological model ( fig . 1 ) , ( halldin et al . 2014a ) with model parameter values according to table 6 . in brief : the parameters values obtained for simulation ( halldin et al . 2014a ) of the crowninshield & pope ( 1974 ) experiment were used but recalibrated to represent rabbit bone . 1a ) were adjusted to 5008 , 2166 and 776 mpa , respectively , to represent young 's modulus of mature rabbit cortical bone ( 7950 mpa ) ( isaksson et al . hence , by changing the spring stiffness values the viscosity was consequently adjusted to fit the relaxation behavior ( halldin et al . thereafter , the initial strain was increased to achieve a pressure similar to the theoretical thread pressure , simulated in the 2d finite element method , of the test implant . finally , the remodeling parameter was set to result in no pressure at 30 days which represents the expected theoretical time point when the initially induced bone strainshave vanished ( halldin et al . . the theoretical interfacial shear strength of the moderately rough surface was simulated using the finite element model described in halldin et al . a representative patch of the implant surface was selected and a finite element model was developed according to fig . the implant was moved until bone failure occurred which was assumed to represent the interfacial shear strength . in the current investigation the interfacial shear strength of a bone condensation implant was simulated with adding an external pressure ( fig . the pressure was reduced by use of the constitutive model ( halldin et al . the theoretical interfacial shear strength was simulated both with mechanical properties of healing bone and mature bone ( halldin et al . mechanical properties of bone during healing derived from mineralization level during healing bone and the relationship between mineralization level and mechanical properties halldin et al . ( 2014a , b ) ( a ) rheological model and the model parameters for the constitutive model developed by halldin et al . ( b ) fea model to simulate the interfacial shear strength of a rough surface exposed to a pressure . pairwise differences in removal torque measurements between test and control implants that were assumed to be normally distributed were analyzed with student 's ttest . pairwise differences in measurements between test and control that were not assumed to be normally distributed were analyzed using sign test for median . pairwise ratios ( test / control ) for different healing times were analyzed using mood median test to test the equality of medians from two populations . specially designed screwshaped implants of titanium ( grade 4 ) were manufactured with tight tolerances to induce controlled bone strains . the designs of the implants were identical to the implants in the study by halldin et al . the test implants comprised of a condensation portion that had an increased diameter of 0.05 mm compared to the cutting portion . all implants had the same surface as the commercially available osseospeed implants ( dentsply implants , mlndal , sweden ) . to confirm that both implant groups had the same surface topography the implant surfaces were characterized by use of optical light interferometry ( microxam ; ade phase shift technology , inc . , tucson , az , usa ) . in brief , six randomly selected implants ( three controls and three tests ) were selected . each implant was measured on three thread peaks , three valleys , and three flanks ( scan area of 264 200 m , vertical measurement range of 100 m ) . data evaluation was performed with the mountainmaps software ( digital surf , besanon , france ) . waviness and form were filtered with a 50 50 m gaussian filter . the surfaces were characterized with the surface roughness parameters s a ( m ) , s dr ( % ) , and s ds ( 1/m ) ( definition of them can be found elsewhere ( stout 2000 ; thomas 1999 ) ) . twentyfour mature female new zealand white rabbits ( weight 34 kg ) were used in this study . the study was approved by the french ministre de lenseignement suprieur et de la recherche and performed at the colenationalevtrinaired'alfort ( maisonsalfort , valdemarne , france , approval number , 0039101 ) . prior to surgery , 250 g / kg of medetomidine ( domitor , zoetis , france ) , 20 mg / kg ketamine ( imalgne 1000 , merial , sanofi , france ) , and 1 mg / kg of diazepam ( valium , roche , france ) subsequently , analgesics 30 g / kg , buprenorphine ( buprcare , animalcare , york , uk ) was injected subcutaneously and 0.2 mg / kg meloxicam ( metacam ; boehringer ingelheim vetmedica , inc . , st . thereafter , incision of the skin was made with a # 15 blade and the muscle layers and periosteum were elevated and separated from the bone . osteotomy was prepared in the proximal tibia by drilling with a sequence of burs , starting from a round bur , 2.8 mm drill and finally with a 3.3 mm drill , corresponding to the core diameter of the implants . each rabbit received two test implants in one leg ( proximal and distal placement ) and two control implants on the contralateral side . the implants were inserted with a rotation speed set to 25 revolutions / min using the w&h implant unit ( elcomed , w&h sa310 , burmoos , austria ) . after implant insertion , the muscle layers were sutured with a resorbable suture ( vicryl3.0 ) , and the skin with a 40 nylon suture ( ethicon , auneau , france ) . postsurgically , a patch of fentanyl ( 25 g / h , duragesic ; janssen pharmaceutica , beerse , belgium ) was applied to each animal for 3 days . an antibiotic ( enrofloxacin , 200 mg / l , baytril ; bayer animal health , leverkusen , germany ) was administrated in the water supply for 5 days . the rabbits were kept in separate cages and were allowed to move and eat freely . at 7 , 28 , and 84 days after implant placement , eights rabbits at each time point were euthanized with an overdose injection of sodium pentobarbital ( euthasol , virbac , fort worth , tx , usa ) . each tibia was dissected and excessive soft and hard tissue around the implant square head was carefully removed . the bone samples were firmly secured with several individual bone pins and the removal torque of the implants was measured . the removal torque , time , and angle of rotation were recorded with a calibrated torque measurement device ( dr2112 lorenz messtechnik gmbh , alfdorf , germany ) using a constant rotational speed of 0.03 rpm . the torque device was calibrated in the range 502000 nmm with an accuracy of 1 nmm . the data were sampled with 100 hz and thereafter analyzed in matlab software 2013b ( mathworksinc . , the data were initially smoothed with a running average over a set of 50 data points and the angle of rotation was set to 0 rad when the torque reached 10 nmm . in the present study , the test implant induced a pressure at the threads in the condensation region . the magnitude of pressure at the threads was simulated by a radial displacement of the thread profile of 0.025 mm as described in the 2daxis symmetric model of halldin et al . the 2d simulations were performed using bilinear mature bone material properties without hardening ( table 1 ) . the reduction in prestress over time , due to relaxation and remodeling , of mature bone was simulated by use of the constitutive model , illustrated by the rheological model ( fig . 1 ) , ( halldin et al . 2014a ) with model parameter values according to table 6 . in brief : the parameters values obtained for simulation ( halldin et al . 2014a ) of the crowninshield & pope ( 1974 ) experiment were used but recalibrated to represent rabbit bone . 1a ) were adjusted to 5008 , 2166 and 776 mpa , respectively , to represent young 's modulus of mature rabbit cortical bone ( 7950 mpa ) ( isaksson et al . hence , by changing the spring stiffness values the viscosity was consequently adjusted to fit the relaxation behavior ( halldin et al . thereafter , the initial strain was increased to achieve a pressure similar to the theoretical thread pressure , simulated in the 2d finite element method , of the test implant . finally , the remodeling parameter was set to result in no pressure at 30 days which represents the expected theoretical time point when the initially induced bone strainshave vanished ( halldin et al . the theoretical interfacial shear strength of the moderately rough surface was simulated using the finite element model described in halldin et al . ( 2015 ) . in brief , a representative patch of the implant surface was selected and a finite element model was developed according to fig . the implant was moved until bone failure occurred which was assumed to represent the interfacial shear strength . in the current investigation the interfacial shear strength of a bone condensation implant was simulated with adding an external pressure ( fig . the pressure was reduced by use of the constitutive model ( halldin et al . the theoretical interfacial shear strength was simulated both with mechanical properties of healing bone and mature bone ( halldin et al . mechanical properties of bone during healing derived from mineralization level during healing bone and the relationship between mineralization level and mechanical properties halldin et al . ( 2014a , b ) ( a ) rheological model and the model parameters for the constitutive model developed by halldin et al . ( b ) fea model to simulate the interfacial shear strength of a rough surface exposed to a pressure . pairwise differences in removal torque measurements between test and control implants that were assumed to be normally distributed were analyzed with student 's ttest . pairwise differences in measurements between test and control that were not assumed to be normally distributed were analyzed using sign test for median . pairwise ratios ( test / control ) for different healing times were analyzed using mood median test to test the equality of medians from two populations . there was no difference ( p = 0.287 ) in s a value of test implants ( mean 1.55 m ; std 0.21 ) compared to control implants ( mean 1.49 m ; std 0.19 ) . there was no difference ( p = 0.441 ) in s dr value of test implants ( mean 109% ; std44 ) compared to control ( mean 103% ; std 49 ) and no difference ( p = 0.337 ) in s ds value of test implants ( mean 0.061/m ; std 0.005 ) compared to control implants ( mean 0.061/m 0.007 ) . the results of the removal torque measurement ( nmm ) , for the three different implantation times ( 7 , 28 , and 84 days ) are presented in a boxplot ( fig . one sample ( tibia proximal 28 days of healing ) was unfortunately lost during carefully removing the excessive soft and hard tissue . the ratios between test and control , placed distally , of the present study ( at 7 , 28 , and 84 days ) . ( b ) removal torque ratios of the present study and the study by halldin et al . mean and median of removal torque values and the statistical analysis between the pairwise differences of test and control and the corresponding ratios subscription is commonly used to differentiate between different statistical methods . by use of the 2d axisymmetric model a theoretical average normal pressure on the thread profile of 39.5 parameter values the relaxation behavior of mature rabbit bone is captured quite well ( fig . 3a ) . using the constitutive model , with an initial strain to obtain an initial pressure of 39.5 mpa and a tuned remodeling parameter value that eliminated the pressure after 30 days , the decrease in pressure on the implant surface during healing was simulated ( fig . the parameter values used in the constitutive model to simulate the decrease in pressure over time are presented in table 3 . the theoretical interfacial shear strength , with mechanical properties of healing bone together with no pressure over time ( represents control implants , simulation c ) , is presented in fig . the theoretical interfacial shear strengths , with mechanical properties of healing bone together with decreased pressure over time ( represents test implants , simulation b ) and mature bone mechanical properties together with decreased pressure over time ( represents test implants , simulation a ) , are presented in fig 4a . the ratios between test and control of the in vivo study and the corresponding theoretical ratios are presented in fig . ( a ) simulated behavior of mature bone with calibrated model parameters to fit the relaxation behavior described in halldin et al . ( b ) simulated behavior of increased initial strain to generate a pressure of 39.5 mpa ( which represents the theoretical thread pressure of the test implant ) and the tuned remodeling parameter value that eliminates the pressure after 30 days . ( 2014a , b ) to simulate the change of pressure over time ( a ) simulation of the interfacial shear strength for the test implants with constant mechanical properties of mature bone during healing time and mechanical properties of bone during healing time . simulation of the interfacial shear strength for the control implants was performed with mechanical properties of bone during healing time . ( b ) the ratios of the present in vivo study and of the simulations . there was no difference ( p = 0.287 ) in s a value of test implants ( mean 1.55 m ; std 0.21 ) compared to control implants ( mean 1.49 m ; std 0.19 ) . there was no difference ( p = 0.441 ) in s dr value of test implants ( mean 109% ; std44 ) compared to control ( mean 103% ; std 49 ) and no difference ( p = 0.337 ) in s ds value of test implants ( mean 0.061/m ; std 0.005 ) compared to control implants ( mean 0.061/m 0.007 ) . the results of the removal torque measurement ( nmm ) , for the three different implantation times ( 7 , 28 , and 84 days ) are presented in a boxplot ( fig . one sample ( tibia proximal 28 days of healing ) was unfortunately lost during carefully removing the excessive soft and hard tissue . the ratios between test and control , placed distally , of the present study ( at 7 , 28 , and 84 days ) . ( b ) removal torque ratios of the present study and the study by halldin et al . mean and median of removal torque values and the statistical analysis between the pairwise differences of test and control and the corresponding ratios subscription is commonly used to differentiate between different statistical methods . by use of the 2d axisymmetric model a theoretical average normal pressure on the thread profile of 39.5 mpa was obtained . by applying appropriate parameter values the relaxation behavior of mature rabbit bone is captured quite well ( fig . 3a ) . using the constitutive model , with an initial strain to obtain an initial pressure of 39.5 mpa and a tuned remodeling parameter value that eliminated the pressure after 30 days , the decrease in pressure on the implant surface during healing was simulated ( fig . the parameter values used in the constitutive model to simulate the decrease in pressure over time are presented in table 3 . the theoretical interfacial shear strength , with mechanical properties of healing bone together with no pressure over time ( represents control implants , simulation c ) , is presented in fig . the theoretical interfacial shear strengths , with mechanical properties of healing bone together with decreased pressure over time ( represents test implants , simulation b ) and mature bone mechanical properties together with decreased pressure over time ( represents test implants , simulation a ) , are presented in fig 4a . the ratios between test and control of the in vivo study and the corresponding theoretical ratios are presented in fig ( a ) simulated behavior of mature bone with calibrated model parameters to fit the relaxation behavior described in halldin et al . ( b ) simulated behavior of increased initial strain to generate a pressure of 39.5 mpa ( which represents the theoretical thread pressure of the test implant ) and the tuned remodeling parameter value that eliminates the pressure after 30 days . ( 2014a , b ) to simulate the change of pressure over time ( a ) simulation of the interfacial shear strength for the test implants with constant mechanical properties of mature bone during healing time and mechanical properties of bone during healing time . simulation of the interfacial shear strength for the control implants was performed with mechanical properties of bone during healing time . ( b ) the ratios of the present in vivo study and of the simulations . an in vivo study was conducted to investigate how the implant stability was affected by a moderately rough implant that induced moderate bone strains of 0.015 at the time of implant installation . the implants were allowed to heal in the rabbit tibiae for 7 , 28 , and 84 days . it was hypothesized that bone condensation implants will enhance the mechanical stability initially and that the moderately rough surface will further contribute to the secondary stability by enhanced osseointegration . the bone condensation implants ( i.e. test implants ) presented an increased removal torque at 7 days of healing compared to the noncondensation implants ( i.e. control implant ) , whereas at 28 and 84 days of healing no difference in removal torque between bone condensation implants and nonbone condensation implants ( fig . 2b ) was observed . in the previous in vivo studies by halldin et al . ( 2011 , 2014b ) turned implants with the same moderate bone condensation level were used . the turned implants had a healing time of 3 , 13 , and 24 days and the removal torque values were measured with a removal torque device different to that of the one utilized in the current study . in the previous studies , the test ( moderate bone condensation ) implants presented significantly increased removal torque compared to control implants . the removal torque values of the two different removal torque devices can not directly be compared . however , if the removal torque ratios ( test / control ) are calculated respectively for study , they may be compared . interestingly , there was a similar trend of decreased ratio over time regardless of the surface micro roughness ( turned or moderately roughened ) ( fig . the ratios were considerably higher than 1 ( or 100% ) during the early healing times , which , in turn , means higher removal torque values for the tests implants compared to the control implants . this suggests that the moderate bone condensation implants ( test ) enhances implant stability during the initial critical phases of healing . when the osseointegration consolidates ( at 28 and 84 days ) the ratios between test and control tend to reach the value 1 ( fig . it is interesting to observe that the ratio was higher of turned implants at 3 days compared to the ratio of the moderately rough implants at 7 days . this might be related to the fact that the controlled condensation is particularly effective for the retention the first few days of healing ( 3 days vs. 7 days ) regardless of surface structure . the absolute removal torque values of moderately rough surface were increased during healing for both test and control implants , which attests to the favorable bone formation around all implants with a moderately rough surface structure . an interesting reflection from the previous studies of turned implants is that the removal torque decreased over time for the test implants . this might reflect that the decreased pressure , due to relaxation and remodeling , had a more pronounced effect on removal torque than the interlocking of a turned surface structure . in the present study therefore , it might be speculated that the increased interlocking capacity of the moderately rough surface had a more pronounced effect on the removal torque than the reduction in pressure , due to relaxation and remodeling , over time . it should be noted that the removal torque values , especially for the longer healing times , might be affected by bone growth into the cutting feature affecting the results . in addition , the potential grinding effect a structured surface has on the bone might influence the result . the present study used a rabbit model which comprises of a dense cortical bone layer of 34 mm and it does not incorporate the implant stability of a potential trabecular bone . the ratios of the present in vivo study were compared to the theoretical interfacial shear strength ratios simulated by the use of the 3d finite element model . interestingly , it was found that the simulated ratios are in line with the in vivo ratios . in the 3d simulation the strength of the interfacial bone was increased over time , reflecting healing bone and increased mineralization , that increased the interlocking capacity ( halldin et al . the interfacial shear strength was simulated both with constant mechanical properties of mature bone and mechanical properties representing healing bone . therefore the simulated shear strength of mature bone was assumed to provide the highest interlocking capacity that can be achieved during healing , thus reflecting only how a decreased pressure affects the interfacial shear strength . in the 3d finite element model the decreased pressure on the surface over time was obtained by use of the constitutive model simulating the relaxation and remodeling behavior of cortical bone ( halldin et al . the characteristics of the actual mechanical properties of the in vivo bone during healing are unknown . therefore , the simulation was based on the estimated mechanical properties of the interfacial bone obtained in the study by halldin et al . ( 2015 ) . in the present study , the parameters of the constitutive model described in halldin et al . ( 2014a ) were recalibrated to fit the viscoelastic relaxation and remodeling behavior of rabbit mature bone . it is assumed that when analyzing the ratios these assumptions might have a limited impact . despite these assumptions , regarding the healing bone mechanical properties and the relaxation and remodeling behavior of rabbit bone ( fig . 3a , b ) , the simulated ratios had the same trend as the ratios obtained the in vivo experiment ( fig . this implies that finite element analysis , with appropriate material models , may be used to evaluate differences in structural interlocking capacity between two surfaces . in addition , it seems to be a more rapid decrease in ratios during the initial healing which is in line with the findings of perren et al . the results of the study suggest that modification of the implant macro geometry in relationship to the osteotomy preparation , to induce moderate bone strain levels of 0.015 during implant insertion does not result in reduction in stability of moderately rough implants . it is interesting to note that the ratios using finite element method were in line with the in vivo ratios . this is an indication that when the condensation level is controlled in vivo , the biological outcomes may be predicted , which could optimize implant design .

abstractobjectiveswhen implants are inserted , the initial implant stability is dependent on the mechanical stability . to increase the initial stability , it was hypothesized that bone condensation implants will enhance the mechanical stability initially and that the moderately rough surface will further contribute to the secondary stability by enhanced osseointegration . it was further hypothesized that as the healing progresses the difference in removal torque will diminish . in addition , a 3d model was developed to simulate the interfacial shear strength . this was converted to a theoretical removal torque that was compared to the removal torque obtained in vivo.material and methodscondensation implants , inducing bone strains of 0.015 , were installed into the left tibia of 24 rabbits . noncondensation implants were installed into the right tibia . all implants had a moderately rough surface . the implants had an implantation time of 7 , 28 , or 84 days before the removal torque was measured . the interfacial shear strength at different healing time was estimated by the means of finite element method.resultsat 7 days of healing , the condensation implant had an increased removal torque compared to the nonbonecondensation implant . at 28 and 84 days of healing , there was no difference in removal torque . the simulated interfacial shear strength ratios of bone condensation implants at different implantation time were in line with the in vivo data.conclusionsmoderately rough implants that initially induce bone strain during installation have increased stability during the early healing period . in addition , the finite element method may be used to evaluate differences in interlocking capacity .

Material and methods Implant design Interferometry Animal study Removal torque measurement Theoretical interfacial shear strength Statistical analysis Results Interferometry Animal study Theoretical interfacial shear strength Discussion Conflicts of interests

at 7 , 28 , and 84 days after implant placement , eights rabbits at each time point were euthanized with an overdose injection of sodium pentobarbital ( euthasol , virbac , fort worth , tx , usa ) . the theoretical interfacial shear strength of the moderately rough surface was simulated using the finite element model described in halldin et al . ( b ) fea model to simulate the interfacial shear strength of a rough surface exposed to a pressure . thereafter , the initial strain was increased to achieve a pressure similar to the theoretical thread pressure , simulated in the 2d finite element method , of the test implant . the theoretical interfacial shear strength of the moderately rough surface was simulated using the finite element model described in halldin et al . in the current investigation the interfacial shear strength of a bone condensation implant was simulated with adding an external pressure ( fig . ( b ) fea model to simulate the interfacial shear strength of a rough surface exposed to a pressure . the results of the removal torque measurement ( nmm ) , for the three different implantation times ( 7 , 28 , and 84 days ) are presented in a boxplot ( fig . ( 2014a , b ) to simulate the change of pressure over time ( a ) simulation of the interfacial shear strength for the test implants with constant mechanical properties of mature bone during healing time and mechanical properties of bone during healing time . the results of the removal torque measurement ( nmm ) , for the three different implantation times ( 7 , 28 , and 84 days ) are presented in a boxplot ( fig . ( 2014a , b ) to simulate the change of pressure over time ( a ) simulation of the interfacial shear strength for the test implants with constant mechanical properties of mature bone during healing time and mechanical properties of bone during healing time . simulation of the interfacial shear strength for the control implants was performed with mechanical properties of bone during healing time . an in vivo study was conducted to investigate how the implant stability was affected by a moderately rough implant that induced moderate bone strains of 0.015 at the time of implant installation . the implants were allowed to heal in the rabbit tibiae for 7 , 28 , and 84 days . it was hypothesized that bone condensation implants will enhance the mechanical stability initially and that the moderately rough surface will further contribute to the secondary stability by enhanced osseointegration . test implants ) presented an increased removal torque at 7 days of healing compared to the noncondensation implants ( i.e. control implant ) , whereas at 28 and 84 days of healing no difference in removal torque between bone condensation implants and nonbone condensation implants ( fig . the turned implants had a healing time of 3 , 13 , and 24 days and the removal torque values were measured with a removal torque device different to that of the one utilized in the current study . in the previous studies , the test ( moderate bone condensation ) implants presented significantly increased removal torque compared to control implants . the ratios were considerably higher than 1 ( or 100% ) during the early healing times , which , in turn , means higher removal torque values for the tests implants compared to the control implants . this suggests that the moderate bone condensation implants ( test ) enhances implant stability during the initial critical phases of healing . it is interesting to observe that the ratio was higher of turned implants at 3 days compared to the ratio of the moderately rough implants at 7 days . the absolute removal torque values of moderately rough surface were increased during healing for both test and control implants , which attests to the favorable bone formation around all implants with a moderately rough surface structure . in the present study therefore , it might be speculated that the increased interlocking capacity of the moderately rough surface had a more pronounced effect on the removal torque than the reduction in pressure , due to relaxation and remodeling , over time . the ratios of the present in vivo study were compared to the theoretical interfacial shear strength ratios simulated by the use of the 3d finite element model . interestingly , it was found that the simulated ratios are in line with the in vivo ratios . therefore the simulated shear strength of mature bone was assumed to provide the highest interlocking capacity that can be achieved during healing , thus reflecting only how a decreased pressure affects the interfacial shear strength . 3a , b ) , the simulated ratios had the same trend as the ratios obtained the in vivo experiment ( fig . this implies that finite element analysis , with appropriate material models , may be used to evaluate differences in structural interlocking capacity between two surfaces . in addition , it seems to be a more rapid decrease in ratios during the initial healing which is in line with the findings of perren et al . the results of the study suggest that modification of the implant macro geometry in relationship to the osteotomy preparation , to induce moderate bone strain levels of 0.015 during implant insertion does not result in reduction in stability of moderately rough implants . it is interesting to note that the ratios using finite element method were in line with the in vivo ratios .

basal cell carcinoma ( bcc ) is the most common cancer in the caucasian population with an increasing incidence in recent years . it is a malignant epidermal tumor with a slow growth rate , limited local invasion , and a very low metastatic potential . basal cell carcinoma is related to chronic exposure to ultraviolet radiation and therefore occurs most commonly on the face . this can have a psychological impact on the patient in terms of both the disease and the possible sequelae of treatment . left untreated , local invasion results ( in very advanced cases ) in destruction of soft tissues involving muscle , bone , nerves , or sensory organs , such as the eyes . all these aspects contribute to the morbidity of the disease and the consequent impact on the healthcare system . the treatment goal for bcc is eradicate the tumor with the lowest possible functional and aesthetic impact and avoid relapses . treatment options include surgery , radiation therapy ( rt ) , photodynamic therapy , topical medications , and systemic medical therapy . although surgery is the first choice of treatment , rt is indicated in selected cases when surgery is not an option due either to the patient ( when surgery presents a high risk ) or for procedural ( cosmetic or functional ) reasons . the radiotherapeutic options , which have been used include superficial x - rays , electron beam , and low or high - dose - rate brachytherapy . it delivers low - energy radiation at a high - dose - rate through an applicator placed on the skin . as ebt uses an x - ray source rather than radioactive isotopes , it requires less room - shielding . the ability to switch the radiation source on and off reduces exposure of healthy tissues to unnecessary radiation . in this study , the esteya ( nucletron , an elekta company , elekta ab , stockholm , sweden ) electronic brachytherapy system was used . it has an articulated arm specifically designed for surface procedures that adapts to flat lesion locations . the skin applicator is constructed with tungsten shielding in such a way that radiation output is limited to the lesion of interest ; radiation leakage to healthy tissues is virtually zero . when compared to established hdr brachytherapy solutions with isotope based sources of radiation , a shorter treatment time is required in order to improve both the user and the patient experience . only a handful of studies have been reported to date [ 4 , 5 , 6 , 7 ] suggesting ebt as an effective treatment with few recurrences or side effects and excellent cosmetic results . however , aforementioned studies are often retrospective and not peer reviewed before publishing . higher level prospective research is needed on ebt for positioning this new technique and confirming improved clinical outcome when compared to existing technologies . this study aims to investigate the outcomes of ebt using the esteya ebt system for the treatment of superficial and nodular basal cell carcinoma using two different radiation dose regimens in two groups of patients . the fractionation schedules used in this study aimed to deliver the same biological effective dose ( bed ) as in the treatment with the valencia applicators ( nucletron , an elekta company , elekta ab , stockholm , sweden ) . as opposed to an ebt system , the latter are based on a ir radioactive source and a surface - specific applicator , which have been shown to provide excellent results in terms of control rate and cosmesis . the bed estimates the true biological dose delivered by a combination of dose per fraction and total dose to a given tissue characterized by a specific / ratio . it is calculated by the equation bed = nd [ 1 + d(/ ) ] , where n is the number of fractions , d is the dose / fraction , and / is a radiosensitivity coefficient . different histological classes of cancers have different / ratios and this can result in a different clinical response , despite the fact that the total dose has not changed . if the total dose is kept constant , the bed will increase if the dose per fraction is increased . in general , a value of / = 10 for the tumor is accepted [ 12 , 13 ] , although / = 8.5 has been suggested for skin cancers . in a previous study with the valencia applicators , the bed was 71.4 gy when considering / = 10 and 78.8 gy for / = 8 . to achieve this 6 fractions of 7 gy each prescribed at a given depth ( usually 3 or 4 mm ) , with 2 fractions per week with at least 48 h between consecutive fractions was used . in addition , the maximum skin dose ( at 0 mm depth ) per fraction was set to be lower than 10 gy in order to avoid skin injuries . in contrast to the valencia applicators , esteya is an ebt system based on a 69.5 kvp x - ray tube and a set of circular collimators that produce photon beams of 1 cm to 3 cm in diameter at a depth of 0 mm . thus , photons emitted in a treatment with esteya have considerably lower energy than photons emitted by a ir source . it has been reported that lower energy photons have a higher radiobiological effectiveness ( rbe ) . this implies that a lower physical dose should be prescribed with ebt sources in order to achieve the same clinical results ( i.e. the same bed ) as with the higher energy brachytherapy sources ( e.g. ir valencia applicators ) . the rbe depends on the photon spectrum and the dose per fraction applied . after a review of the literature [ 18 , 19 , 20 , 21 , 22 , 23 , 24 ] , it was estimated that the rbe for a 69.5 kvp x - ray source , such as the one used by esteya , is around 1.15 . based on this analysis , the same clinical results achieved with the valencia applicators could be expected by prescribing 7 gy/1.15 = 6.1 gy per fraction , during 6 fractions , with 2 fractions per week . because the recurrence rates obtained in early results for this group were not as low as with the valencia applicators , it was decided that the second group should be treated with the same fractionation as with the valencia applicators ( 7 gy per fraction ) , i.e. , no rbe correction was applied in comparison to group 1 . in both groups , because the tolerance in dose homogeneity for the esteya beam is within 5% , a 9.5 gy , threshold dose was established in order to be sure that the maximum skin dose per fraction was lower than 10 gy . the dose gradient for the esteya source is lower than that for the valencia applicators , which results in an even lower dose at the surface , and therefore this maximum skin dose per fraction was never reached either using 7 gy or 6.1 gy per fraction . two prospective , single - center , non - randomized , pilot studies to assess the outcome of electronic brachytherapy in superficial and nodular basal cell carcinoma treatment using esteya surface applicators were conducted sequentially . the second group studied received a differently calculated dose because similar results to the valencia applicator studies were not achieved with the dose used in the first group . the first group ( 1 ) included 20 patients with 20 lesions treated with 36.6 gy in 6 fractions of 6.1 gy , two times a week during three weeks , with at least 2 days between each consecutive fraction . the second group ( 2 ) included 20 patients with 20 lesions treated with 42 gy in 6 fractions of 7 gy , two times a week during three weeks , with at least 2 days between each consecutive fraction . thus , all fractionation and overall times were kept the same with the exception of the dose per fraction . in one arm , the 6.1 gy / fraction resulting from the theoretical rbe calculation was used , and in the second arm ( 7 gy / fraction ) , the same dose as in the valencia applicator study was used . it was approved by the ethics committee of clinical research of the la fe hospital . only adults with a primary superficial or nodular bcc with t1 and t2 clinical stage according to ajcc 2010 criteria t1 includes tumors 2 cm with less than 2 high risk features , and t2 includes tumors > 2 cm or any tumor with 2 or more high risk features . these high risk features are : > 2 mm thickness , clark level 4 , perineural invasion , tumor located on the ear or hair - bearing lip , and undifferentiated or poorly differentiated tumors . other forms of bcc or clinical stage more than t2 were excluded . due to applicator design , lesions bigger than 20 mm , deeper than 4 mm , or located on irregular surfaces were also excluded . tumor depth was assessed by high frequency ultrasonography ( hfus ) and a 3 mm punch - biopsy taken from the clinically most representative area in terms of depth . patients were seen after treatment at 2 weeks , 6 weeks , 3 months , 6 months , and 1 year . complete and partial response were defined by the absence or the presence of residual tumor clinically and aided by dermoscopy at each follow - up visit . when there was any doubt about tumor persistence or recurrence , a biopsy was performed for confirmation by histopathology . ctcae v4.0 ( common terminology criteria for adverse events ) toxicity scales were used to assess acute toxicity and rtog - eortc scales related to brachytherapy were used to assess cosmesis . mean standard deviation was reported for continuous data and percentage standard deviation for categorical data . to compare categorical data , we utilized a nonparametric test ( kendall tau b ) due to the presence of a percentage of < 5% in one group . statistical analysis was performed with the spss statistics 18 ( spss inc , chicago , usa ) program . the fractionation schedules used in this study aimed to deliver the same biological effective dose ( bed ) as in the treatment with the valencia applicators ( nucletron , an elekta company , elekta ab , stockholm , sweden ) . as opposed to an ebt system , the latter are based on a ir radioactive source and a surface - specific applicator , which have been shown to provide excellent results in terms of control rate and cosmesis . the bed estimates the true biological dose delivered by a combination of dose per fraction and total dose to a given tissue characterized by a specific / ratio . it is calculated by the equation bed = nd [ 1 + d(/ ) ] , where n is the number of fractions , d is the dose / fraction , and / is a radiosensitivity coefficient . different histological classes of cancers have different / ratios and this can result in a different clinical response , despite the fact that the total dose has not changed . if the total dose is kept constant , the bed will increase if the dose per fraction is increased . in general , a value of / = 10 for the tumor is accepted [ 12 , 13 ] , although / = 8.5 has been suggested for skin cancers . in a previous study with the valencia applicators , the bed was 71.4 gy when considering / = 10 and 78.8 gy for / = 8 . to achieve this 6 fractions of 7 gy each prescribed at a given depth ( usually 3 or 4 mm ) , with 2 fractions per week with at least 48 h between consecutive fractions was used . in addition , the maximum skin dose ( at 0 mm depth ) per fraction was set to be lower than 10 gy in order to avoid skin injuries . in contrast to the valencia applicators , esteya is an ebt system based on a 69.5 kvp x - ray tube and a set of circular collimators that produce photon beams of 1 cm to 3 cm in diameter at a depth of 0 mm . thus , photons emitted in a treatment with esteya have considerably lower energy than photons emitted by a ir source . it has been reported that lower energy photons have a higher radiobiological effectiveness ( rbe ) . this implies that a lower physical dose should be prescribed with ebt sources in order to achieve the same clinical results ( i.e. the same bed ) as with the higher energy brachytherapy sources ( e.g. ir valencia applicators ) . the rbe depends on the photon spectrum and the dose per fraction applied . after a review of the literature [ 18 , 19 , 20 , 21 , 22 , 23 , 24 ] , it was estimated that the rbe for a 69.5 kvp x - ray source , such as the one used by esteya , is around 1.15 . based on this analysis , the same clinical results achieved with the valencia applicators could be expected by prescribing 7 gy/1.15 = 6.1 gy per fraction , during 6 fractions , with 2 fractions per week . because the recurrence rates obtained in early results for this group were not as low as with the valencia applicators , it was decided that the second group should be treated with the same fractionation as with the valencia applicators ( 7 gy per fraction ) , i.e. , no rbe correction was applied in comparison to group 1 . in both groups , because the tolerance in dose homogeneity for the esteya beam is within 5% , a 9.5 gy , threshold dose was established in order to be sure that the maximum skin dose per fraction was lower than 10 gy . the dose gradient for the esteya source is lower than that for the valencia applicators , which results in an even lower dose at the surface , and therefore this maximum skin dose per fraction was never reached either using 7 gy or 6.1 gy per fraction . two prospective , single - center , non - randomized , pilot studies to assess the outcome of electronic brachytherapy in superficial and nodular basal cell carcinoma treatment using esteya surface applicators were conducted sequentially . the second group studied received a differently calculated dose because similar results to the valencia applicator studies were not achieved with the dose used in the first group . the first group ( 1 ) included 20 patients with 20 lesions treated with 36.6 gy in 6 fractions of 6.1 gy , two times a week during three weeks , with at least 2 days between each consecutive fraction . the second group ( 2 ) included 20 patients with 20 lesions treated with 42 gy in 6 fractions of 7 gy , two times a week during three weeks , with at least 2 days between each consecutive fraction . thus , all fractionation and overall times were kept the same with the exception of the dose per fraction . in one arm , the 6.1 gy / fraction resulting from the theoretical rbe calculation was used , and in the second arm ( 7 gy / fraction ) , the same dose as in the valencia applicator study was used . it was approved by the ethics committee of clinical research of the la fe hospital . only adults with a primary superficial or nodular bcc with t1 and t2 clinical stage according to ajcc 2010 criteria were included . t1 includes tumors 2 cm with less than 2 high risk features , and t2 includes tumors > 2 cm or any tumor with 2 or more high risk features . these high risk features are : > 2 mm thickness , clark level 4 , perineural invasion , tumor located on the ear or hair - bearing lip , and undifferentiated or poorly differentiated tumors . other forms of bcc or clinical stage more than t2 were excluded . due to applicator design , lesions bigger than 20 mm , deeper than 4 mm , or located on irregular surfaces were also excluded . tumor depth was assessed by high frequency ultrasonography ( hfus ) and a 3 mm punch - biopsy taken from the clinically most representative area in terms of depth . patients were seen after treatment at 2 weeks , 6 weeks , 3 months , 6 months , and 1 year . complete and partial response were defined by the absence or the presence of residual tumor clinically and aided by dermoscopy at each follow - up visit . when there was any doubt about tumor persistence or recurrence , a biopsy was performed for confirmation by histopathology . ctcae v4.0 ( common terminology criteria for adverse events ) toxicity scales were used to assess acute toxicity and rtog - eortc scales related to brachytherapy were used to assess cosmesis . mean standard deviation was reported for continuous data and percentage standard deviation for categorical data . to compare categorical data , we utilized a nonparametric test ( kendall tau b ) due to the presence of a percentage of < 5% in one group . statistical analysis was performed with the spss statistics 18 ( spss inc , chicago , usa ) program . patients treated with 36.6 gy are shown in the left column ( group 1 ) and patients treated with 42 gy are shown in the right column ( group 2 ) . both groups were comparable in all collected baseline characteristics except age ( p = 0.006 ) . baseline patients characteristics group 1 20 patients treated at 36.6 gy delivered in 6 fractions ; group 2 20 patients treated at 42 gy delivered in 6 fractions ; ns non - significant ( > 0.05 ) in group 1 , a complete response in 90% of cases was observed , whereas in group 2 the complete response was 95% ( figure 1 ) . example of complete response tumor persistence or recurrence was suspected clinically and dermoscopically in two patients in the first group at 3 and 6 months , respectively , and in one patient in the second group at 1 year follow - up . this was confirmed by histopathology after resection of the remaining tumor , which was a diagnostic as well as a curative procedure ( figure 2 ) . clinical and histopathological pictures of recurrent cases acute toxicity in the first group was g1 in 65% of cases due to erythema and g2 in 35% due to ulceration ( figure 3 ) . in the second group , 60% of patients presented with g1 toxicity and 40% with g2 . the cosmetic result was g0 ( no cutaneous alterations ) in 61% of patients in the first group and 55% in the second group . the rest of the patients only showed pigmentation alterations or alopecia , corresponding to a g1 cosmetic result ( figure 3 ) . these differences in acute toxicity and cosmetic results between the two treatment groups were not statistically significant ( p > 0.05 ) . examples of acute toxicity and cosmetic result group 1 20 patients treated at 36.6 gy delivered in 6 fractions ; group 2 20 patients treated at 42 gy delivered in 6 fractions ; ns non - significant ( > 0.05 ) comparison between different protocols of hdr - bt and ebt for bcc bcc basal cell carcinoma ; bed biological effective dose , nmsc non melanoma skin cancer ; ebt electronic brachytherapy ; hdr - bt high - dose - rate brachytherapy when treating bcc , dermatologists have a wide range of possibilities but surgery and rt are the treatments with the lowest recurrence rates . surgery is often the first choice of treatment due to its high efficacy and because it is a straightforward procedure . despite the high incidence of bcc , however , there is only one randomized study comparing surgery to rt , which was published in the late nineties . in this study , only primary facial bcc less than 4 cm were included . three hundred and forty - seven patients were treated , 174 with surgery and 173 with rt followed up over 4 years . it was concluded that surgery has a lower failure rate and better cosmesis than rt . firstly , the radiotherapy group was not homogeneous since patients were treated with interstitial brachytherapy , contact therapy , or conventional radiotherapy . further , doses and fractionation in each rt type were not the same . secondly , the use of flaps and grafts to close the wounds in the surgery group may have made the detection of persistence or recurrence more difficult . finally , only facial tumors were included , therefore we have no data about other locations . radiotherapy has been a part of the dermatologist 's treatment armamentarium for several decades but , since the eighties , this has been changing in favor of dermatologic surgery . this is basically due to the incorporation of surgery in dermatology and the difficulties of administering radiation in unshielded offices . in addition , in many countries dermatologists are not allowed anymore to administer rt themselves , thus they have to send patients to another department or clinic if they opt for rt . consequently , surgery has experienced a great surge in development in recent years in dermatology departments and offices , whereas rt has reduced noticeably in significance , being used only in cases when surgery is contraindicated . electronic brachytherapy has appeared as an alternative to more conventional rt techniques such as electron beam or high - dose - rate radionuclide - based brachytherapy . electron beams require a bolus and a more specific dosimetry , which makes it more complex in clinical practice . on the other hand , radionuclide - based brachytherapy uses a radioactive source , generally ir , which emits photons of higher energy than ebt sources . this results in ebt requiring less room shielding , and being safer , simpler and easier to apply . in radionuclide - based brachytherapy some applicators exist , which shield the radiation emitted by the ir source except for the region that needs to be irradiated . among them , the valencia applicators were designed specifically to produce a collimated and homogeneous dose distribution within the patient 's skin . compared to these applicators , ebt has the advantage of a shorter treatment time ( 2.5 minutes compared to 5 to 10 minutes ) , lower penumbra ( i.e. sharper lateral dose fall - off ) , less radiation leakage that implies lower peripheral dose , and a broader range of applicator sizes , resulting in a more conformal treatment . for these reasons , ebt is a promising technique for the treatment of skin lesions . so far , to the best of our knowledge , only four studies have been performed using ebt for bcc ( despite the fact that these authors treat other nmsc also ) . these four groups used the xoft axxent electronic ( ebx ) brachytherapy system ( xoft inc . these studies showed clinical cure rates higher than 98% with acceptable acute toxicities and very good cosmesis . all of them used a dose of 40 gy in 8 equal fractions , 5 gy per fraction , delivered twice weekly with at least 48 hours between each fraction . long - term follow - up has not yet been achieved because most patients have not reached their second year of follow - up . in our experience with the esteya system , the better dose to achieve the highest clinical cure rate is 42 gy in 6 equal fractions , i.e. 7 gy per fraction given at the prescription depth ( typically 3 mm ) . . also showed good toxicity results in patients treated in 6 or 7 fractions . although most brachytherapy treatment schemas in the literature use 8 to 12 fractions [ 35 , 36 , 37 , 38 , 39 , 40 ] , the fractionation used in this study does not result in a higher toxicity or a poorer cosmesis in comparison with a more fractionated treatment . thus , in an elderly population , a comfortable schema that facilitates compliance is preferred . for these reasons , a 6 fractions schema was chosen . in order to reduce the number of fractions to 5 , while keeping at the same time the same biological effective dose ( bed ) , 8 gy per fraction at the prescription depth ( typically 3 mm ) would be required . however , the latter would result in a skin dose ( i.e. at 0 mm depth ) of 9.9 gy per fraction , which , taking into account a 5% tolerance in dose homogeneity , would fail to guarantee compliance with the fda recommendations regarding the maximum skin dose to avoid toxicity . despite the solid radiobiological basis , three cases showed tumor persistence or recurrence . in group 1 , treated at 36.6 gy , this occurred early , one case at 3 and one at 6 months . the only failed case in group 2 , treated at 42 gy , occurred late , at 1 year follow - up . in this case , the lesion initially disappeared clinically but later reappeared . these cases were analyzed separately with regard to high frequency ultrasonography , previous biopsy , and histopathology from the persistent or recurrent tumor . medical records , clinical , and dermoscopic features of all visits were reviewed . despite this , we did not find any reason that could justify the failure of the treatment . the small sample size and the short - term follow - up being the main ones . the follow - up performed is probably insufficient to assess efficacy but these early results could be a trend in terms of clinical results . all patients will have further follow - up in order to assess long - term response and to rule out recurrences . as more studies are performed , we learn more about the biology and behavior of different cutaneous tumors . there are many patients with bcc and at the same time there are new treatments becoming available . all of this allows us to individualize treatment depending on patient and tumor characteristics . in the near future , ebt will probably be one more treatment option available for patients with bcc . dermatologists should know about this new technique in order to add it to their current treatment strategies . our initial experience with the esteya eb system to treat superficial and nodular bcc shows that a dose of 36.6 gy and 42 gy delivered in 6 fractions of 7 gy achieves a 90% and 95% clinical cure rate at 1 year , respectively . further investigation with respect to ebt for treating skin tumors is needed , ideally high - level evidence in the form of randomized clinical trials , to compare results with modern treatment protocols with those obtained with surgery . surgery remains the treatment of choice today , and ebt 's role and position is yet to be defined . this study was supported by nucletron ( nucletron , an elekta company , elekta ab , stockholm , sweden ) , by generalitat valenciana ( project prometeoii/2013/010 ) and by spanish government under project no .

purposebasal cell carcinoma ( bcc ) is a very common cancer in the caucasian population . treatment aims to eradicate the tumor with the lowest possible functional and aesthetic impact . electronic brachytherapy ( ebt ) is a treatment technique currently emerging . this study aims to show the outcomes of two consecutive prospective pilot clinical trials using different radiation doses of ebt with esteya eb system for the treatment of superficial and nodular basal cell carcinoma.material and methodstwo prospective , single - center , non - randomized , pilot studies were conducted . twenty patients were treated in each study with different doses . the first group ( 1 ) was treated with 36.6 gy in 6 fractions of 6.1 gy , and the second group ( 2 ) with 42 gy in 6 fractions of 7 gy . cure rate , acute toxicity , and late toxicity related to cosmesis were analyzed in the two treatment groups.results in group 1 , a complete response in 90% of cases was observed at the first year of follow - up , whereas in group 2 , the complete response was 95% . the differences with reference to acute toxicity and the cosmetic results between the two treatment groups were not statistically significant.conclusionsour initial experience with esteya eb system to treat superficial and nodular bcc shows that a dose of 36.6 gy and 42 gy delivered in 6 fraction of 7 gy achieves a 90% and 95% clinical cure rate at 1 year , respectively . both groups had a tolerable toxicity and a very good cosmesis . the role of ebt in the treatment of bcc is still to be defined . it will probably become an established option for selected patients in the near future .

Purpose Material and methods Rationale for the study fractionation schedules Study design Eligibility Procedure, monitoring, and follow up Statistics Results Discussion Conclusions Disclosure

basal cell carcinoma ( bcc ) is the most common cancer in the caucasian population with an increasing incidence in recent years . the treatment goal for bcc is eradicate the tumor with the lowest possible functional and aesthetic impact and avoid relapses . this study aims to investigate the outcomes of ebt using the esteya ebt system for the treatment of superficial and nodular basal cell carcinoma using two different radiation dose regimens in two groups of patients . the dose gradient for the esteya source is lower than that for the valencia applicators , which results in an even lower dose at the surface , and therefore this maximum skin dose per fraction was never reached either using 7 gy or 6.1 gy per fraction . two prospective , single - center , non - randomized , pilot studies to assess the outcome of electronic brachytherapy in superficial and nodular basal cell carcinoma treatment using esteya surface applicators were conducted sequentially . the second group studied received a differently calculated dose because similar results to the valencia applicator studies were not achieved with the dose used in the first group . the first group ( 1 ) included 20 patients with 20 lesions treated with 36.6 gy in 6 fractions of 6.1 gy , two times a week during three weeks , with at least 2 days between each consecutive fraction . the second group ( 2 ) included 20 patients with 20 lesions treated with 42 gy in 6 fractions of 7 gy , two times a week during three weeks , with at least 2 days between each consecutive fraction . the dose gradient for the esteya source is lower than that for the valencia applicators , which results in an even lower dose at the surface , and therefore this maximum skin dose per fraction was never reached either using 7 gy or 6.1 gy per fraction . two prospective , single - center , non - randomized , pilot studies to assess the outcome of electronic brachytherapy in superficial and nodular basal cell carcinoma treatment using esteya surface applicators were conducted sequentially . the second group studied received a differently calculated dose because similar results to the valencia applicator studies were not achieved with the dose used in the first group . the first group ( 1 ) included 20 patients with 20 lesions treated with 36.6 gy in 6 fractions of 6.1 gy , two times a week during three weeks , with at least 2 days between each consecutive fraction . the second group ( 2 ) included 20 patients with 20 lesions treated with 42 gy in 6 fractions of 7 gy , two times a week during three weeks , with at least 2 days between each consecutive fraction . in one arm , the 6.1 gy / fraction resulting from the theoretical rbe calculation was used , and in the second arm ( 7 gy / fraction ) , the same dose as in the valencia applicator study was used . patients treated with 36.6 gy are shown in the left column ( group 1 ) and patients treated with 42 gy are shown in the right column ( group 2 ) . baseline patients characteristics group 1 20 patients treated at 36.6 gy delivered in 6 fractions ; group 2 20 patients treated at 42 gy delivered in 6 fractions ; ns non - significant ( > 0.05 ) in group 1 , a complete response in 90% of cases was observed , whereas in group 2 the complete response was 95% ( figure 1 ) . example of complete response tumor persistence or recurrence was suspected clinically and dermoscopically in two patients in the first group at 3 and 6 months , respectively , and in one patient in the second group at 1 year follow - up . the cosmetic result was g0 ( no cutaneous alterations ) in 61% of patients in the first group and 55% in the second group . these differences in acute toxicity and cosmetic results between the two treatment groups were not statistically significant ( p > 0.05 ) . examples of acute toxicity and cosmetic result group 1 20 patients treated at 36.6 gy delivered in 6 fractions ; group 2 20 patients treated at 42 gy delivered in 6 fractions ; ns non - significant ( > 0.05 ) comparison between different protocols of hdr - bt and ebt for bcc bcc basal cell carcinoma ; bed biological effective dose , nmsc non melanoma skin cancer ; ebt electronic brachytherapy ; hdr - bt high - dose - rate brachytherapy when treating bcc , dermatologists have a wide range of possibilities but surgery and rt are the treatments with the lowest recurrence rates . in our experience with the esteya system , the better dose to achieve the highest clinical cure rate is 42 gy in 6 equal fractions , i.e. the only failed case in group 2 , treated at 42 gy , occurred late , at 1 year follow - up . our initial experience with the esteya eb system to treat superficial and nodular bcc shows that a dose of 36.6 gy and 42 gy delivered in 6 fractions of 7 gy achieves a 90% and 95% clinical cure rate at 1 year , respectively .

rare earth elements ( rees ) , referred to as industrial vitamins , are of particular importance and have extensive applications in various fields , especially in material science and industry , mainly due to their special chemical properties and luminous characters based on their 4f shell electrons . recently , along with large amounts of exploitation of rare earth resources and the discard of the rees contained materials , rees accessed inevitably environment , food chain , and then the human body . despite this , the positive or negative effect of rees on the biological body is completely unclear till now . clearly , a high - sensitive analysis method will promote the researches in this field and give an explicit pattern . similarly , the potential application fields of rees are also increasing in current society , and the demand of high - sensitivity analysis methods for rees is rising gradually . for instance , for biological samples , the sensitivity is general at the level of ng / g . in environmental analysis field , for soil , air , lake water , and so forth , the requirement detection level must at least achieve the magnitude of g / l , while for sea water and other high technique rees - containing materials , the according detection level should be the level of ng / l or pg / g . therefore , developing the sensitive analytical methods for rees is of vital significance and becomes one of the hot points in analytical science . because the chemical properties of the 17 rees are similar , the key point of rees analysis is the mutual separation of rees , thus the detection of rees is usually accompanied by extraction and preseparation . the development of the high - sensitive analytical methods also advances these new separation techniques . currently , numerous analytical techniques have been applied in the detection of rees with the aid of extraction and separation , including atomic absorption / fluorescence spectroscopy ( aas / afs ) , atomic emission spectroscopy ( aes ) , x - ray fluorescence spectroscopy ( xfs ) , neutron activation analysis ( naa ) , inductively coupled plasma optical emission spectrometer ( icp - oes ) , and inductively coupled plasma mass spectrometer ( icp - ms ) [ 9 , 10 ] . among them , icp - ms has become one of the most commonly used techniques because of its multiple advantages , including extremely high sensitivity , high analytical speed , broad dynamics range , and synchronous analysis of various elements . the sensitivity of icp - ms has reached the level of 0.01 ng / g for most rees and only 10 ng / g for icp - oes under the same conditions . however , restricted by its large and expensive equipment , the sample injection technique , matrix effect , and the difficulty in the direct analysis of solid sample , icp - ms is difficult to be applied in the fast field analysis of rees . ambient ionization techniques are a revolution in the mass spectroscopy , which enabled the ionization of samples in their native surrounding with minimum sample pretreatment and brought the breakthrough in the application of ms to high - throughput analysis [ 11 , 12 ] . there are more than 10 direct ionization techniques based on the atmospheric plasma that have been invented , including dielectric barrier discharge ionization ( dbdi ) , desorption atmospheric pressure chemical ionization ( dapci ) , desorption corona beam ionization ( dcbi ) , plasma - assisted desorption ionization ( padi ) , flowing atmospheric pressure afterglow ( fapa ) [ 18 , 19 ] , and microplasma discharge ionization ( mdi ) . usually , in these methods , the electronic field employed to produce plasma under atmospheric pressure is high discharge voltage ( usually several kilovolts ) and low frequency ( up to 850 hz ) . the temperature of plasma is low ( usually several hundred kelvin in excitation temperature ) , so that the plasma is weak and even invisible . the microwave plasma torch ( mpt ) is a kind of plasma generator at atmospheric pressure by means of the high - frequency ( 2450 mhz ) microwave field , which was developed initially at jilin university , and substantial improvements were made at indiana university . the mpt can be sustained in a variety of supporting gases , including ar , he , n2 , ne , and air and produce a stable flame - like plasma , similar to icp . based on its multiple merits , for instance , small size , low power consumption , low cost , easy operation , and high excitation efficiency , mpt has broad applications in aes portable spectrometer , supercritical fluid chromatography ( sfc ) , and liquid chromatography ( lc ) as an excitation light source for elemental analysis [ 24 , 25 ] . owing to the excellent ionization efficiency , mpt has also been employed as the ion source in mass spectrometry mainly for the elements analysis and the detection of halogenated hydrocarbons separated by a capillary gas chromatography . recently , zhang et al . studied the direct desorption / ionization approach of mpt on a linear ion trap ( ltq ) mass spectrometer to analyze a series of small organic molecules and showed that mpt is a useful alternative ambient ion source . however , mpt has been rarely used as the detection of metal elements in mass spectrometry . when the analyte is inorganic molecule , the desorption or ionization efficiency is bare due to the ultralow vapor pressure . moreover , in the usual direct desorption / ionization approach , plasma crossing sideling the sample surface restricts the efficient transformation of the energy from the plasma to the analytes due to the short interaction length . but this obstacle can be overcome by the approach of sample injection via the central tube of mpt . some previous researches had demonstrated that the mpt - ms is sensitive enough for the demand of field analysis of metal elements in aqueous liquid at the level of 0.021 ng / ml . in this study , an ar - assisted mpt coupled with the ltq mass spectrometer was used for the sensitive detection of trace levels of ree ions in water . the nebulized ree aqueous solution , without any pretreatment , flowed into the plasma through the central tube of the mpt to increase the interaction time of the plasma and the samples . for the slat solution of single rare earth elements , including yttrium , lanthanum , cerium , praseodymium , neodymium , samarium , and europium , the generated complex ions in plasma were detected in both positive and negative ion modes and further characterized in collision induced dissociated ( cid ) experiments . we further examined the semiquantitative capability of this method for the direct detection of rees in different aqueous liquids . the 2450 mhz microwave generator ( yy1 - 50 w-2450 ) and the coaxial line ( sfcj-50 - 9 ) were purchased from the nanjing electronic technology co. , ltd . lacl3 , cecl3 , prcl3 , eucl3 , and ndcl3 ( analytical grade ) were purchased from j&k chemical technology co. , ltd . yttrium , lanthanum , cerium , praseodymium , neodymium , samarium , and europium standard substance ( 10 mg / l in 1.0 mol / l the water used was deionized water provided by the chemistry facilities in the east china institute of technology ( ecit ) . we used pure water and rees standard substance to prepare a series of solutions with concentrations from 0 ng / ml to 500 ng / ml for the calibration curve measurements . for these rees , the maximum concentrations for the calibration curve measurements are different based on the practice signal intensities ; that is , the linear ranges are discriminating . for instance , for samarium , the maximum concentration is 500 ng / ml , but for yttrium and praseodymium the corresponding values are 100 ng / ml . the practical water sample was gathered in a well in ganzhou , jiangxi province , pr china . this practical aqueous was directly analyzed with any other pretreatment , except for the necessary dilution with deionized water . two mass spectrometers were employed in this study , including a commercial linear ion trap mass spectrometer ( thermo fisher scientific , san jose , ca , usa ) and a home - built quadrupole mass filter mass spectrometer . most of mpt - ms experiments were carried out by the ltq xl mass spectrometer ( see ) , and the quadrupole mass spectrometer is only used in some assisted experiments . to detect the metal ion in solution , a desolvation unit is combined with the mpt ion source and the ltq mass spectrometer . the desolvation unit . water samples , without any pretreatments , were pneumatically nebulized and generate the aerosols . the aerosols flow by carrier gas through a desolvation system to reduce the loss of the microwave power due to the absorption by water . the heated tube is 25 cm long and is wrapped with heating tape which is monitored with a thermocouple and a heater controller ( xmt61x , beijing huibang science technology co. ltd . , china ) . a 500 ml flask was filled with concentrated sulfuric acid ( 98% ) to further remove the h2o molecules from the aerosol . at last , the dry aerosols were introduced into the plasma through the central tube of the mpt ( see figure 1 ) . the mpt ion source . the mpt was described previously [ 29 , 30 ] , so only a brief summary of the main features is given here . the mpt consists of three tubes : ( 1 ) the outer tube , ( 2 ) the intermediate tube , and ( 3 ) the central tube . the central tube is made of quartz with an outer diameter of 3 mm and inner diameter of 2 mm . the intermediate tube ( 5.5 mm o.d and 4.5 mm i.d . ) and outer tube ( 26 mm o.d . and 22 mm i.d . ) are made of copper . the supporting gas flows in the intermediate tube . and the working gas is divided into two routes , one flows directly in the central tube and the other carries the dried salt in the argon aerosol into the central tube . this structure is called the dual - flow system and is beneficial to optimize the plasma jet shape . two adjustable rotameters ( 1001000 ml / min ) are controlled and optimized the two argon flow rates . a radio - frequency ( rf ) voltage is applied to the mpt . a 2450-mhz voltage with a maximum power of 100 w is applied to the intermediate tube by the center conductor of a coaxial cable . a simple tool from a paper clip , a rubber band , and a sheet of a4 paper is employed . the paper is wrapped around the paper clip and is secured with the rubber band . about 5 mm of the paper clip extends beyond the clip - paper junction . the tool was touching the torch between the intermediate tube and the inner tube to create a short circuit between the two tubes , then a cone - shaped plasma jet was generated on the top opening of the mpt . the horizontal distance between the opening end of mpt and the ion inlet of ltq was about 30 mm . the ltq mass spectrometer was set for the rees analysis with mainly parameters including the capillary voltage of (020 ) v and the tube lens voltage of 40 v. the temperature of the heated capillary was 150c . ions of interest were isolated with a mass - to - charge window width of 0.8 units for collision induced dissociation ( cid ) experiments by applying a collision energy ( ce ) of about 30% ( arbitrary unit defined by the ms manufacturer ) . in general , mpt possesses high excitation efficiency , because the excitation temperature of the plasma is relatively high , about 8002000 k , depending on the microwave power and the working gas . but the ionization degree of mpt is only 0.01% , far less than that of icp , 0.2% . thereby some groups with bright characterization can be as reagent ions and make the mass spectra easy assignment . these groups include no3 , nh2 , h2o , and oh , since the no3 is electrophilic , while metal ions are general electron donor ; the metal element m appears in the mpt mass spectra in the form of m(no3)n pulsing several h2o molecules or oh groups . depending on the number n , the result group can be cation or anion , so that the rees can be detected in positive and negative modes . in this study , the characteristic mpt mass spectra of some rees , including yttrium , lanthanum , cerium , praseodymium , neodymium , samarium , and europium , had been obtained in both positive and negative modes . figure 2(a ) showed a typical mpt mass spectrum collected from an experiment using 10 mg / l la standard solution in a full scan positive mode with background subtraction . lanthanum has two natural isotopes : la with a natural abundance of 99.91% and la with a natural abundance of 0.89% . combined with the empirical molecular formula mentioned above , the peak at m / z 317 and 334 can be assigned tentatively to [ la(no3)23h2o ] and [ la(no3)23h2ooh ] , respectively . to further confirm the assignment of m / z 317 , figure 2(b ) showed the mpt ms mass spectra , n up to 6 , in which the precursor ion m / z 317 lost successively three h2o groups to generate the peak at m / z 299 , 281 , and 263 in ms , ms , and ms spectra , respectively . the subprecursor should contain two no3 groups ; in further cid experiments , the main fragment was that of m / z 233 by the loss of no in ms spectrum . the ultimate fragment ion is that of m / z 155 , which was produced from the disaggregation of the ion of m / z 233 by throwing away a group of no4 and would not be dissociated further in cd experiments . using the lacl3 aqueous solution with the equal concentration , we can get the same results with the final fragment ions of m / z 155 . the results demonstrated again that the relevant anion groups no3 in the mpt mass spectra originated from the background in the plasma but not the sample solution . to further confirm this assignment , we repeated the same experiment on the quadrupole mass filter mass spectrometer with mpt as the ion source ; the straightforward ions of m / z 155 were gained , as shown in figure 2(c ) . it is worthy to pay serious attention to the precursor ion of [ la(no3)23h2o ] and [ la(no3)23h2ooh ] in which la element is still kept in its + 3 valence state , same as that in its original solution . this is a common character for rees in mpt - ms , because of the low atomization efficiency in the plasma produced by mpt . therefore , mpt - ms may gain the information on the original valence states of metallic ions and have a potential as a supplement of icp - ms . relative to the almost single isotope of lanthanum , neodymium is a representative multiple isotopic element , which has seven major natural isotopes : nd with a natural abundance of 27.2% , nd with a natural abundance of 12.2% , nd with a natural abundance of 23.8% , nd with a natural abundance of 8.3% , nd with a natural abundance of 17.2% , nd with a natural abundance of 5.7% , and nd with a natural abundance of 5.6% . figure 3 is a typical mpt mass spectrum collected from the experiments of 10 mg / l neodymium standard solution . as shown in figure 3(a ) , there were two parallel spectral - shape seven - peak bands with central positions at 320 and 338 . empirically , these two mass spectral bands can be assigned tentatively to [ nd(no3)23h2o ] and [ nd(no3)24h2o ] , respectively . here again , nd remains in original + 3 valence state . the inset in figure 3 showed the amplified view of the mass spectral region between m / z 310 and 350 . the intensity ratio of major seven peaks at m / z 320 , 321 , 322 , 323 , 324 , 326 , and 328 was 1 : 0.365 : 0.667 : 0.246 : 0.465 : 0.106 : 0.097 , which was integral lower than but yet approximately agreed with the neodymium natural isotopic ratio ( 1 : 0.448 : 0.875 : 0.305 : 0.632 : 0.209 : 0.206 ) . although further studies are necessary , one of the possible reasons is the complicated ionization mechanism for metal ions in mpt plasma . in fact , in mpt plasma , the phase transition for metal ions from liquid phase to gas phase may play an important role , as well as the electron collision and redox reactions in plasma . the ms spectra of these precursor ionic peaks exhibited analogous fragmentation patterns , that is , consecutively losing three h2o molecules , no and no4 to produce ( ndo ) , analogous to that of la(no3)2 3h2o . for simplicity , only ms ( n up to 5 ) spectra of m / z 320 , 322 , and 324 were shown in figure 3(b ) , which possesses vivid isotopic characterization . likewise , similar discussions on the mass spectral band of 338 can give the assignment of [ nd(no3)24h2o ] . table 1 summarized the dominated peak band in mpt mass spectra of rees in this study . rees can form the anion in the mpt plasma when the number of the attached no3 exceeds , so that rees can be detected in negative mode . in fact , the negative mode should be more suitable for the detection of rees since the negative mpt mass spectra are generally noisier than the positive mpt mass spectra . figure 4 is the typical mpt mass spectrum of cerium in negative mode collected in the range of m / z 320420 , where a 10 mg / l cerium standard solution was used . clearly , there are two pairs of peak , m / z 342 and 344 as well as m / z 388 and 390 , with intensity ratio of 7.65 and 8 . cerium has four natural isotopes , ce(0.19% ) , ce(0.25% ) , ce(88.48% ) , and ce(11.07% ) . ignoring the former two isotopes since their abundance is too small , the abundance ratio of the latter two isotopes is 7.99 , which nearly equals the intensity ratio of the two peaks in latter pair . based on the fine matching , combined with the empirical molecular formula , the ion of m / z 342 can be assigned to [ ceo(no3)3 ] and the ion of m / z 388 is [ ce(no3)4 ] . it is easy to see that the structure of [ ce(no3)4 ] is more symmetric so that the signals are more intense and the intensity ratio is much closer to the theoretical values . analogously , the mpt tandem mass spectrometry in negative mode also provides partial evidences about the structure of the rees anions . figures 5(a)5(c ) are the ms mass spectra of the precursor ions at m / z 388 , n = 2 , 3 , 4 , respectively . as shown , the precursor ions of m / z 388 produce first the fragment of m / z 342 by losing a no2 group . in ms , subprecursor of m / z 342 can produce the minor fragment of m / z 312 by the loss of no group and the major fragment of m / z 296 by throwing away a second no2 group , and the fragment of m / z 296 yields the peak at m / z 250 by the loss of a third no2 group and yields the peak at m / z 234 by losing a no3 group . the ions of m / z 234 would dissociate further under current experimental conditions possibly because the signal intensity in ms is low . however , sequential loss of 46-d group ( no2 ) hints that the precursor ion of m / z 388 is polynitrogen complex . figures 5(d)5(f ) depict the tandem mass spectra of the ions of m / z 390 showing parallel isotopic pattern . thus combined with the abundant ration , the fact that the ion of m / z 388 was assigned to [ ce(no3)4 ] is reasonable . the characteristic mpt anionic peaks of seven rees in this study were also summarized in table 1 . mpt - ltq ms in negative running mode is less noisier and more efficient than that in positive mode , and the quantitative performance of mpt - ltq negative ms is better than that of mpt - ltq positive ms in this study . therefore , we adopted the negative mode to measure semiquantitatively the concentration of single ree in water to verify the capacity of mpt - ms in the detection of rees . in addition , to reduce the possibility of a false signal originated from the isotopes of adjacent rees , the major fragments in the mpt - ms mass spectra were proposed as the signal for the qualification of rees in water samples . the linear relationships between the measured fragment signal intensities and the concentrations of each spiked ree in water were measured with the concentration range of 0 to several hundred ppb ( ng / ml ) , at least two orders of magnitude , with the determination coefficient r larger than 0.99 . to be noticed , for each data point , six measurements were repeated with the relative standard deviation ( rsd ) almost less than 15% . the lod of this method were estimated at the level of 0.1 ng / ml according to triple noise level at the lowest concentration . the lod for praseodymium is even as low as 0.04 ng / ml , almost close to the level of icp - ms [ 2 , 10 ] . the largest values of lod appear in yttrium , the only nonlanthanide rees in this study , arriving at 0.5 ng / ml . in addition , adding recovery experiments ( for almost all rees in this study , 5 ng / ml solutions were used , but for yttrium 7.5 ng / ml solution was used ) also give the recovery rates of these seven rees in a reasonable interval of 97.6%122.1% . totally , although these lod values are higher about one magnitude than those obtained in icp - ms , this mpt - ltq - ms is still valuable in particular applications as an alternative supplement of icp - ms for the field analysis of rees in water . as a primary application , according to these calibration curves , the two practical aqueous samples were directly analyzed to obtain the content of these seven rees . conclusively , a sensitive method based on mpt - ltq ms has been developed to determine individual content of some rees in water at trace levels , including yttrium , lanthanum , cerium , praseodymium , neodymium , samarium , and europium . by means of tandem mass spectrometry and isotopic abundant ratio , characteristic mpt mass spectra of these seven rees were identified both in positive and negative modes ; meanwhile the main fragment signals in ms mass spectra were also quantitative measurement . the results showed that this mpt - ms method has enough sensitivity to determine trace levels of these seven rees in water with the lod values at ~0.1 ng / ml level with reasonable semiquantitative performance . therefore , this method has potential applications in quality monitoring of single ree in water . nevertheless , in real applications , this mpt - ms ought to combine with some extraction and separation methods to isolate single ree for accurate analysis . however , as a low - cost and sensitive method , it is still promising and can be used as a supplement of icp applied in field analysis of single ree in water .

a sensitive mass spectrometric analysis method based on the microwave plasma technique is developed for the fast detection of trace rare earth elements ( rees ) in aqueous solution . the plasma was produced from a microwave plasma torch ( mpt ) under atmospheric pressure and was used as ambient ion source of a linear ion trap mass spectrometer ( ltq ) . water samples were directly pneumatically nebulized to flow into the plasma through the central tube of mpt . for some rees , the generated composite ions were detected in both positive and negative ion modes and further characterized in tandem mass spectrometry . under the optimized conditions , the limit of detection ( lod ) was at the level 0.1 ng / ml using ms2 procedure in negative mode . a single ree analysis can be completed within 2~3 minutes with the relative standard deviation ranging between 2.4% and 21.2% ( six repeated measurements ) for the 5 experimental runs . moreover , the recovery rates of these rees are between the range of 97.6%122.1% . two real samples have also been analyzed , including well and orange juice . these experimental data demonstrated that this method is a useful tool for the field analysis of rees in water and can be used as an alternative supplement of icp - ms .

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

currently , numerous analytical techniques have been applied in the detection of rees with the aid of extraction and separation , including atomic absorption / fluorescence spectroscopy ( aas / afs ) , atomic emission spectroscopy ( aes ) , x - ray fluorescence spectroscopy ( xfs ) , neutron activation analysis ( naa ) , inductively coupled plasma optical emission spectrometer ( icp - oes ) , and inductively coupled plasma mass spectrometer ( icp - ms ) [ 9 , 10 ] . however , restricted by its large and expensive equipment , the sample injection technique , matrix effect , and the difficulty in the direct analysis of solid sample , icp - ms is difficult to be applied in the fast field analysis of rees . the microwave plasma torch ( mpt ) is a kind of plasma generator at atmospheric pressure by means of the high - frequency ( 2450 mhz ) microwave field , which was developed initially at jilin university , and substantial improvements were made at indiana university . studied the direct desorption / ionization approach of mpt on a linear ion trap ( ltq ) mass spectrometer to analyze a series of small organic molecules and showed that mpt is a useful alternative ambient ion source . some previous researches had demonstrated that the mpt - ms is sensitive enough for the demand of field analysis of metal elements in aqueous liquid at the level of 0.021 ng / ml . in this study , an ar - assisted mpt coupled with the ltq mass spectrometer was used for the sensitive detection of trace levels of ree ions in water . the nebulized ree aqueous solution , without any pretreatment , flowed into the plasma through the central tube of the mpt to increase the interaction time of the plasma and the samples . for the slat solution of single rare earth elements , including yttrium , lanthanum , cerium , praseodymium , neodymium , samarium , and europium , the generated complex ions in plasma were detected in both positive and negative ion modes and further characterized in collision induced dissociated ( cid ) experiments . at last , the dry aerosols were introduced into the plasma through the central tube of the mpt ( see figure 1 ) . depending on the number n , the result group can be cation or anion , so that the rees can be detected in positive and negative modes . in this study , the characteristic mpt mass spectra of some rees , including yttrium , lanthanum , cerium , praseodymium , neodymium , samarium , and europium , had been obtained in both positive and negative modes . therefore , we adopted the negative mode to measure semiquantitatively the concentration of single ree in water to verify the capacity of mpt - ms in the detection of rees . in addition , to reduce the possibility of a false signal originated from the isotopes of adjacent rees , the major fragments in the mpt - ms mass spectra were proposed as the signal for the qualification of rees in water samples . the linear relationships between the measured fragment signal intensities and the concentrations of each spiked ree in water were measured with the concentration range of 0 to several hundred ppb ( ng / ml ) , at least two orders of magnitude , with the determination coefficient r larger than 0.99 . the lod of this method were estimated at the level of 0.1 ng / ml according to triple noise level at the lowest concentration . the lod for praseodymium is even as low as 0.04 ng / ml , almost close to the level of icp - ms [ 2 , 10 ] . in addition , adding recovery experiments ( for almost all rees in this study , 5 ng / ml solutions were used , but for yttrium 7.5 ng / ml solution was used ) also give the recovery rates of these seven rees in a reasonable interval of 97.6%122.1% . totally , although these lod values are higher about one magnitude than those obtained in icp - ms , this mpt - ltq - ms is still valuable in particular applications as an alternative supplement of icp - ms for the field analysis of rees in water . conclusively , a sensitive method based on mpt - ltq ms has been developed to determine individual content of some rees in water at trace levels , including yttrium , lanthanum , cerium , praseodymium , neodymium , samarium , and europium . the results showed that this mpt - ms method has enough sensitivity to determine trace levels of these seven rees in water with the lod values at ~0.1 ng / ml level with reasonable semiquantitative performance . however , as a low - cost and sensitive method , it is still promising and can be used as a supplement of icp applied in field analysis of single ree in water .

dendritic cells ( dcs ) and macrophages are a heterogeneous population of leukocytes that are critical in orchestrating immune responses ( steinman , 2007 ) . human tissues are populated by at least three dc subsets ; cd141 dcs ( haniffa et al . , 2012 ; 2014 ) , cd1c dcs ( lenz et al . , 1993 ; morelli et al . , 2005 ; angel et al . , 2006 ; zaba et al . , 2007 ) , and cd14 dcs ( nestle et al . , 1993 ; de gruijl et al . , 2006 ; klechevsky et al . , 2008 ; haniffa et al . , gene - expression studies suggest that human blood and tissue cd141 dcs are homologous to murine tissue cd103 and splenic cd8 dcs ( robbins et al . , 2008 ; bachem et al . , 2010 ; crozat et al . , 2010 ; 2010 ) and cd1c dcs are homologous to cd11bcd4 dcs in the spleen and cd11bcd24cd64 dcs in nonlymphoid tissues ( schlitzer et al . , 2013 ) . however , the precise relationship of human cd14 dcs to murine tissue populations remains unclear ( haniffa et al . , 2012 ) . excluding langerhans cells of the epidermis , the apparent paradox of three dc subsets in human interstitial tissues but only two in murine tissues remains unreconciled . human cd14 dcs were first identified as a spontaneously migrating population from dermal explants cultured ex vivo . these cells were classified as dcs based on major histocompatibility complex ( mhc ) class ii glycoprotein expression and their ex vivo migratory behavior . in vitro generated cd14 dcs from cd34 hematopoietic stem cells ( hscs ) have been used alongside primary cells to dissect their immunological properties ( caux et al . , 1996 ; klechevsky et al . , 2008 ; , 2009 ; haniffa et al . , 2012 ; matthews et al . , 2012 ; penel - sotirakis et al . , cd14 dcs secrete interleukin-10 ( il-10 ) and il-6 and have been shown to induce regulatory t cells ( tregs ) and helper follicular t cells ( tfh ) ( chu et al . a notable feature of cd14 dcs is their poor ability to stimulate allogeneic t cell proliferation ( klechevsky et al . , 2008 ; morelli et al . , 2005 ; de gruijl et al . , 2006 ) cd14 dcs also express cd141 , which is further upregulated during spontaneous migration from skin explant culture and initially presumed to be related to blood cd141 dcs ( chu et al . , 2012 ) . more recently , the true counterpart of blood cd141 dcs has been shown to be tissue cd14cd141 dcs ( haniffa et al . , 2012 ) . cd14 cells are related to human and mouse blood monocytes by gene expression and are rapidly reconstituted by donor - derived cells following hematopoietic stem cell transplantation ( hsct ) , unlike dermal macrophages , which turn over at a much slower rate ( haniffa et al . , 2009 ; haniffa et al . , steady - state dcs are derived from a lineage dependent on flt3 , in contrast to monocytes and macrophages , which are dependent on colony - stimulating factor-1 receptor ( csf-1r ) ( yoshida et al . , 1990 ; mckenna et al . , 2000 ; dai et al . , 2002 ) . circulating murine ly6c monocytes have been shown to extravasate into tissues existing as tissue monocytes ( jakubzick et al . , 2013 ; tamoutounour et al . , 2012 ) and also differentiate into dc - like and macrophage populations in the intestine and dermis ( bogunovic et al . , 2009 ; varol et al . , 2009 ; tamoutounour et al . , 2012 ; yona et al . , monocytes as a source of tissue inflammatory dcs are also well - documented ( zigmond et al . , 2012 ; plantinga et al . , 2013 ; human blood monocyte differentiation into dcs has been proposed in inflammation as the potential equivalent of in vitro cultured gm - csf and il-4 monocyte - derived dcs ( segura et al . , 2013 ) . however , the precise contribution of circulating monocytes to human tissue dcs and macrophages in steady state is unclear . altogether , these findings led us to question whether cd14 cells were bona fide dcs and which murine population was their homolog . in this study , we investigated the relationships between circulating blood cd14 monocytes and tissue macrophages with tissue mhc classiicd14 cells , currently defined as dcs . we defined the transcriptomic profile of the human monocyte - macrophage lineage distinct from the dc lineage and demonstrated the conserved gene transcripts defining these two lineages in humans and mice . our findings revealed that cd14 cells more closely resemble tissue resident , monocyte - derived macrophages than bone fide dcs . in addition , we showed that the murine dermal monocyte - derived macrophages are the homolog of human dermal cd14 cells . we previously showed that cd14 cells were distinct from dermal macrophages , which possessed dense cytoplasmic melanin granules by morphology , were autofluorescent by flow cytometry analysis , were adherent and nonmigratory , although both populations express cd14 ( haniffa et al . , 2009 ) . as the existence of tissue monocytes derived from ly6c classical monocytes was recently demonstrated in mice ( jakubzick et al . , 2013 ; tamoutounour et al . , 2013 ) , we therefore first set out to determine the phenotypic distinctions between dermal mhc classiiautofluorescent ( af)cd14 cells ( hereafter referred to as cd14 cells ) , afcd14 macrophages ( hereafter referred to as dermal macrophages ) , and blood cd14 monocytes , the homologs of murine ly6c monocytes . to control for the preparation conditions of freshly isolated cd14 cells by enzymatic digestion , we cultured cfse - labeled purified blood cd14 monocytes with enzymatically - digested skin overnight . gating on cfse - labeled cells allowed direct comparison of tissue cd14 with cd14 monocytes . overlay dot plot of cfse - labeled cd14 monocytes on digested skin cells showed that skin cd14 cells were phenotypically distinct from blood monocytes with higher side scatter ( ssc ) properties , expressing higher amounts of hla - dr and cd1c ( figure 1a ) . both skin cd14 cells and cfse - labeled cd14 monocytes spiked into the skin preparation had variable expression of cd141 ( figure 1a ) . we noted very few cd14 cells with an identical profile to the cfse - labeled monocytes cultured with digesting skin ( figure 1a ) , which might represent extravasated tissue monocytes in healthy human skin . we next compared the expression of selected antigens characterizing monocyte - macrophage cells in human skin and blood - antigen - presenting cell populations ( figure 1b ) . unlike cd1c dcs from blood and skin , cd14 cells had variable expression of cd163 similar to blood monocytes and macrophages ( figure 1b ) . we next compared the expression of skin antigen - presenting cell subsets and blood cd14 monocytes for the following transcripts ; dc transcription factor ( tf ) zbtb46 , cd209 , lymphatic vessel endothelial hyaluronan receptor ( lyve1 ) , and factor xiiia ( f13a1 ) . the latter three antigens were shown to identify dermal macrophages in situ ( wang et al . , 2014 ) skin cd1c and cd141 dcs expressed higher amounts of zbtb46 transcripts and low amounts of cd209 and lyve1 . in contrast , dermal cd14 cells expressed 40% and macrophages 15% of zbtb46 transcript amounts compared to cd1c dcs , consistent with previous reports on human inflammatory dcs and murine dermal macrophage populations ( segura et al . cd14 cells and macrophages expressed higher amounts of cd209 transcript compared to all other subsets , but lyve1 transcript expression was highest in macrophages ( figure 1c ) similar to f13a1 expression ( haniffa et al . , 2009 ) . in addition , dermal cd14 cells expressed high amounts of il1 and gamma - glutamyl transferase 5 ( ggt5 ) transcripts ( figure 1c ) , which were identified from our previous microarray analysis ( haniffa et al . immunostaining of whole - mount dermal sheet for lyve-1 , cd209 ( dc - sign ) , and fxiiia identified lyve-1fxiiiadc - sign cells corresponding to the cd14 cells in situ ( figure 1d ) . human tissue dcs are depleted in patients lacking circulating dcs and monocytes as shown by patients with genetic deficiency of monocytes and dcs due to gata2 or irf8 mutation , but the exact precursor - progeny relationships are difficult to demonstrate conclusively in humans ( bigley et al . , 2011 ; hambleton et al . , we previously showed that skin cd14 cells are derived from donor bone marrow within 40 days of hsct ( haniffa et al . , 2009 ) , but the kinetics of this relationship have not been resolved in detail . in this study , we followed the course of blood and skin monocytes , macrophages , and dcs during preparative cytotoxic therapy for transplantation and for up to 2 weeks after hsct ( clinical data in table s1 ) . cytotoxic therapy induced bone - marrow suppression and absolute monocytopenia by the day of transplantation ( day 0 ) ( figure 2a ) . this was mirrored by a rapid loss of cd14 cells from the skin within 6 days . after a delay of 6 days , macrophage numbers also declined to approximately 50% , where they remained stable . in the early recovery phase after hsct , there was a rapid rise in cd14 blood monocytes , which coincided with a rapid reconstitution of skin cd14 cells ( figure 2b ) . the kinetics of dc recovery were slower in the blood and skin and did not attain the same frequency as healthy controls ( figures 2a and 2b ) . the temporal relationship between blood cd14 monocytes and skin cd14 cells is consistent with a precursor - progeny relationship in which cd14 monocytes give rise to cd14 cells within a short timeframe . in support of the rapid differentiation step of cd14 monocytes into skin cd14 cells suggested by our in vivo findings , we showed that purified cd14 monocytes upregulated antigens and adopted morphological changes characteristic of cd14 tissue cells upon culture with primary endothelial cells over 3 days ( figure 2c ) . in order to evaluate the lineage identity of human skin cd14 cells , we performed microarray transcription profiling of human skin and blood dendritic cells , macrophages , and monocyte subsets . principal component analysis ( pca ) of all subsets analyzed showed separation of dcs from monocyte - macrophages in component one and further definition between monocyte - macrophage subsets and plasmacytoid dcs ( pdcs ) from myeloid dcs in component 2 ( figure 3a ) . we also performed a supervised connectivity map ( cmap ) gene set enrichment analysis and showed that the cd14 cell gene set was enriched in dermal macrophages and also weakly in cd14 blood monocytes but exhibited an inverse expression profile to blood and skin dcs ( figure 3b ) . this suggests that cd14 cells are more closely aligned to tissue macrophages and cd14 blood monocytes than to blood or tissue dcs . further examination of the relationship between cd14 cells , monocytes , macrophages , and dcs revealed a number of coregulated genes distinguishing monocytes , cd14 cells , and macrophages from blood and skin dcs ( figure 3c ) . functional pathways identified by the monocyte , cd14 cell , and macrophage gene sets include retinoid x receptor signaling , trem1 signaling , complement system , and communication between innate and adaptive cell regulation ( see figure s1 available online ) . in contrast , the human dc gene signature was enriched for cell - cycle control and amino acid , nucleic acid and cholesterol metabolism pathways ( figure s1 ) . we next performed cross - species analysis comparing human monocyte , macrophage , and cd14 cells with murine monocytes , macrophages , and dc subsets obtained from immgen ( gautier et al . , 2012 ) and gse49358 ( tamoutounour et al . , 2013 ) microarray data sets . this analysis identified a set of genes that are differentially expressed in a conserved manner , which include slc11a1 , mafb , cd14 , and fcgr2a ( figure 3d ; table s2 ) . similarly , human and mouse dc lineage also shared close homology of transcripts across species including flt3 , btla , hla - doa , and ciita ( figure 3d ; table s2 ) . a defining property of tissue - resident dcs is their migratory capacity to lymph node ( ln ) directed by ccl19 and ccl21 signaling through ccr7 . in vitro culture of explanted tissue mimics this process and resident dcs might be observed entering the lymphatic channels prior to emigrating from the tissue ( stoitzner et al . , 1999 ; ohl et al . , 2004 ; wang et al . , 2014 ) . in addition , this experiment showed that resident macrophages remain fixed in the tissue ( haniffa et al . . the ability of cd14 cells to leave tissue explants has been invoked as a dc credential ( nestle et al . , 1993 ) , but their route of migration in the ex vivo assay has not yet been established . if cd14 cells are not fixed in skin explants , then simple redistribution in ex vivo culture would result in apparent emigration from the tissue . like dermal macrophages , migrated and digested cd14 cells did not express ccr7 even upon stimulation ( figure 4a ) ( haniffa et al . , 2009 ) . time - course analysis of skin explants showed the presence of cd14 cells in the skin explant medium as early as 12 hr after culture ( figure 4b ) . however , at no stage were cd14 cells observed within lymphatics as assessed by whole - mount immunostaining of skin explants ( figure 4c ) . dc - sign expression is retained by spontaneously migrating cd14 cells ( figure s2 ) and would have permitted their localization within lymphatic channels if this had been the route of migration . the presence of spontaneously migrated cd14 cells , despite their absence within skin lymphatic lumen , suggests that cd14 cells exited from the skin without entering the lymphatic vasculature . dermal macrophages did not migrate spontaneously ( figure 4c ) , in keeping with previous observations ( haniffa et al . , 2009 ) . migratory cd14 cells have been much studied , but their relationship to cd14 cells isolated by enzymatic digestion has not been extensively evaluated . we compared cells isolated by digestion and migration for the expression of a custom selection of 96 genes reported in the literature to define dcs and macrophages by taqman low density array pcr . unsupervised clustering showed that migrated and digested cd1c dcs and cd14 cells clustered by subset rather than isolation method ( figure 4d ) . ccr7 , cyp27b1 , flt3 , fscn1 , indo , lamp3 , and ly75 were expressed at higher amounts by cd1c dcs compared to cd14 cells , which expressed higher amounts of ccl18 , ccl3 , ccr1 , cd163 , cd36 , clec10a , fcgr1a and 3a , il10 , marco , mmp12 , msr1 , siglec1 , and trem2 ( figure 4d ) , transcripts characteristic of monocyte - macrophage cells . a cardinal property of dcs as opposed to monocytes and macrophages is their superior ability to activate naive t cell proliferation . it has been previously shown that cd14 cells from the skin are inferior to cd1c dcs in inducing allogeneic naive t cell proliferation ( klechevsky et al . , 2008 ; angel et al . , after 5 days culture with cfse - labeled allogeneic naive cd4 t cells , cd14 cells induced 80% lower proliferation of naive cd4 t cell compared to cd1c dcs with negligible t cells proliferation observed with macrophages even at the highest apc : t cell ratio tested of 1:10 ( figure 5a ) . however , cd1c dcs , cd14 cells , and dermal macrophages were potent inducers of memory cd4 t cell proliferation and cytokine production upon stimulation with candida albicans ( figure 5b ) . both cd14 cells and dermal macrophages were comparable to cd1c in their ability to induce il-17 , il-22 , interferon- ( ifn- ) , and il-4 production by memory cd4 t cells ( figure 5b ) . recent studies have demonstrated heterogeneity within murine nonlymphoid tissues cd11b cells , which comprise dcs , monocytes , monocyte - derived dcs , and resident macrophages ( langlet et al . , 2012 ; tamoutounour et al . , 2012 ) . in order to precisely identify the murine equivalent of human cd14 cells , we performed comparative transcriptomics analysis by using microarray data of the recently described cd11b monocyte , dc , and macrophage populations in murine dermis ( tamoutounour et al . , 2013 ) . cmap analysis revealed highest enrichment of human cd1c dcs to their murine cd11b dc counterpart in both steady state and upon inflammation induced by the contact hypersensitivity allergen dnfb ( figure 6a ) . the reciprocal relationship was also observed that human dermal macrophages had low but positive cmap enrichment scores with murine macrophages and monocyte - derived cells ( figure 6a ) . blood cd14 monocytes had the highest enrichment score with mouse ly6c blood monocytes in the steady state ( figure 6a ) , in keeping with previous analysis ( haniffa et al . , 2012 ) . human cd14 cells were most enriched with murine macrophages ( p4 and p5 ) followed by ly6cmhc class ii monocyte - derived dc - like cells ( p2 and p3 ) ( figure 6a ) . these results suggest that human cd14 cells are related to monocytes but are not the equivalent of murine tissue monocytes ( p1 ) . the existence of murine monocyte - derived dermal macrophages was recently reported by tamatounour et al . as observed by a reduction in p4 and p5 dermal macrophages in ccr2 mice . we therefore hypothesized that human cd14 cells were the homolog of murine dermal monocyte - derived macrophages . in order to confirm the monocyte origin of some murine dermal macrophages , we used the s100a4-cre r26mice in which > 99% of hematopoietic stem cells ( hscs ) and this fate - mapping model previously demonstrated the independence of resident tissue macrophages from circulating monocytes and hsc progenitors ( hashimoto et al . our analysis of mouse dermal dc and macrophage fractions showed that > 90% of cd11b p1-p3 and 80%90% of p4 and p5 macrophages were indeed monocyte - derived ( figure 6b ) . collectively , these results provide further evidence of functional equivalence between monocyte - derived macrophages in the mouse dermis as the homologs of human cd14 cells . the results presented here suggest that cd14 dcs are not related to the human dc lineage but are monocyte - derived macrophages that are resident in healthy skin . their gene - expression program strongly overlaps with that of blood monocytes and resident tissue macrophages , but they are distinguishable from both of these by phenotypic and functional properties . although it is difficult to prove unequivocally that they have a monocyte origin , their absence in monocyte deficiency states , kinetics of renewal after hsct , and similarity to monocytes in short - term culture with endothelial cells are all consistent with a precursor - progeny relationship . their evident lack of ability to stimulate naive t cell proliferation is consistent with their status as a monocyte - derived macrophage . tissue monocytes coexpressing ly6c and mhc class ii were recently demonstrated as a distinct population in murine tissues ( jakubzick et al . , 2013 ; tamoutounour et al . , 2013 ) . these cells , derived from ly6cmhc class ii circulating monocytes , upregulate mhc class ii upon contact with endothelium ( jakubzick et al . , 2013 ) . in human , all blood and skin dcs , monocytes and macrophages express mhc class ii ( reviewed in haniffa et al . , ) . the linmhc class ii compartment in human blood does not contain any cd14-expressing cells and primarily comprises basophils ( autissier et al . , 2010 ) . spiking cfse - labeled blood cd14 monocytes into digesting skin allowed us to perform a direct comparison between dermal cd14 and blood monocytes . the identity of the circulating precursors of human tissue dcs and macrophages has been a subject of intense debate . despite the widespread use of in vitro culture protocols to generate monocyte - derived dcs and macrophages , in vivo evidence to support the contribution of such monocyte - derived cells in healthy tissue , we show the rapid decline and reconstitution of dermal cd14 cells that mirrors the kinetics of blood cd14 monocytes in patients undergoing hsct . in contrast , blood and skin cd1c dcs remained suppressed up to 14 days after hsct and are thus unlikely to be the precursors of dermal cd14 cells . we also show that blood cd14 monocytes acquire the morphology and phenotypic characteristics of dermal cd14 cells upon culture with endothelial cells in line with previous reports ( randolph et al . , 1998 ; chomarat et al . , 2000 the reduction of dermal macrophages after hsct by approximately 50% is in keeping with the reduction in dermal macrophages in patients with gata2 and irf8 mutation who are deficient in peripheral blood dcs and monocytes ( bigley et al . , 2011 ; hambleton et al . , longer - term follow up after hsct is required to evaluate macrophage recovery and whether proliferation of residual dermal macrophages or differentiation from cd14 cells is responsible for dermal macrophage reconstitution . the utility of transcriptomics analysis to map homologous subsets between humans and mice ( robbins et al . , 2008 ; haniffa et al . , 2012 ; schlitzer et al . , 2013 ; watchmaker et al . , 2014 ) and more recently to define dcs ( miller et al . , 2012 ) and macrophages ( gautier et al . , 2012 ) in mouse tissues is evident . extending our previous cmap analysis to human microarray data set incorporating dermal macrophages and epidermal lcs , we were able to precisely identify the monocyte - macrophage lineage of dermal cd14 cells . this identification enabled us to define the human dc and monocyte - macrophage transcriptomic signatures and to perform cross - species analysis with the murine dc , monocyte , and macrophage immgen data set . the extension of human dc , monocyte , and macrophage mapping further enhances the utility of the immgen data repository . our analysis confirms a high - degree of conservation for dc and macrophage transcription networks between humans and mice identifying shared genes such as mertk , cd14 , and slc11a1 ( nramp ) to define monocytes and macrophage and flt3 , btla , and kit to define dcs . comparative biology analysis of dc subsets has supported the relevance of murine models for both developmental and functional studies and the extension of a similar analysis to the macrophage lineage could present an additional perspective for future studies on these cells . the phenotypic and transcriptional assignment of dermal cd14 cells as monocyte - derived macrophages led us to reevaluate their ability to migrate spontaneously from skin explant cultures ex vivo which has been presumed to simulate lymphatic migration despite absent or very low expression of ccr7 even upon cytokine and lps stimulation . we observed that although 1 in 5 hla - dr cells after 12 hr and 48 hr culture of skin explant were cd14 cells , these cells were not observed within skin lymphatic lumen . the dichotomy between spontaneous migration and lymphatic migration was previously observed with murine lcs and dcs from ccr7 mice , which were capable of spontaneously migrating from skin explants but failed to form dermal cords indicative of lymphatic migration or enter skin draining lymph node ( ohl et al . it is well documented that cd14 cells are poor stimulators of naive t cells , a property that is expected of tissue - resident cells . however , cd14 cells and macrophages are on par with dcs in regulating memory cd4 t cell responses as shown here and in previous reports . dermal cd14 cells express high amounts of il-1 and ggt5 , a property not shared by any blood or skin dcs , monocytes , and macrophages , suggesting a role in maintaining epithelial integrity and regulation of skin inflammation including neutrophil migration ( han et al . murine tissue macrophages and epidermal langerhans cells ( lcs ) were recently shown to arise from embryonic yolk sac and fetal liver precursors challenging the traditional dogma of monocytes as precursors of all tissue macrophages ( van furth and cohn , 1968 ; schulz et al . , 2012 ; hoeffel et al . , , 2013 ; tamoutounour et al . , 2013 ) including this report suggests a contribution by circulating monocytes to the murine tissue macrophage pool . the contemporary view of the mononuclear phagocyte system encompasses several precursor origins including circulating monocytes as precursors of tissue macrophages . here , we report in humans , two populations of tissue resident cells , cd14 monocyte - derived macrophages and fixed macrophages that map to the cd11b pool of mouse macrophages . further work is required to evaluate the long - term contribution of monocytes to the resident macrophage pools . the functional classification of human tissue cd14 cells as monocyte - derived macrophages will redirect attention to functional regulation in the skin as opposed to lymph node . a greater understanding of the contribution of monocyte - derived cells in steady state , inflammation , wound healing , and pathology characterized by histiocytosis and granuloma formation will provide further insights into exploiting their origin and functional properties for clinical therapy . human samples were obtained in accordance with a favorable ethical opinion from newcastle , singapore singhealth , and national health care group research ethics committees . normal skin was obtained from mammoplasty and breast reconstruction surgery and digested whole ( 1 1 cm ) as previously described ( haniffa et al . , 2012 ) to obtain single - cell suspension . migrating cells were collected from whole skin pieces ( 1 1 cm ) cultured in rpmi with 10% fcs , and analyzed at serial time points between 0 and 72 hr . where stated , 100,000 cfse - labeled cd14 blood monocytes were cultured together with 1 1 cm skin piece during collagenase digestion . peripheral blood mononuclear cells were isolated by density centrifugation ( lymphoprep ; ge healthcare ) . blood and dermal dc subsets , naive and bulk cd4 t cells were isolated to > 91% purity by fluorescence - activated cell sorting ( facs ) using a facsariaii and facsfusion ( becton dickinson [ bd ] ) . flow cytometry was performed on a bdlsrii , bdfortessa , and facscanto , and data were analyzed with flowjo ( treestar ) . whole - mount immunofluorescence staining was performed with a previously described protocol ( wang et al . , 2014 ) . we fixed a 200 m skin sheet in pbs containing 2% paraformaldehyde and 30% sucrose overnight at 4c . skin was incubated overnight in pbs containing 0.5% bsa and 0.3% triton x-100 before staining with primary and secondary antibodies at 4c overnight at each stage . specimens were viewed using axio imager.z2 fluorescence microscope with axiovision software v4.8 and axiocam mr3 camera ( carl zeiss , inc . ) or leica leica tcs sp2 uv confocal microscope and lcs v 2.51 imaging software ( leica ) . we cultured 5,000 facs - sorted dermal dc subsets with 100,000 cfse - labeled allogeneic naive ( cd4cd25ccr7cd45ro ) t cells in u - bottomed 96-well plate . proliferation was measured by cfse dilution on day 6 . for memory t cell stimulation , autologous blood cd4 t cells were used to measure recall memory response to candida albicans . we pulsed 5,000 facs - sorted dermal dc subsets with candida albicans overnight , and cultured them with 100,000 cfse - labeled ( invitrogen ) autologous blood cd4 t cells on the following day . cytokine production was measured on day 10 , upon stimulation with 20 ng / ml pma ( sigma - aldrich ; sigma ) and 500 ng / ml ionomycin ( sigma ) for 5 hr in the presence of 10 g / ml brefeldin a ( sigma - aldrich ) for the last 2 hr . cells were fixed and permeabilized ( ebioscience fix / perm buffer set ) to allow intracellular cytokine staining . viaprobe staining ( becton dickinson ; bd ) was performed prior to cell fixation to distinguish viable cells . huvecs were cultured in endothelial cell basal medium 2 ( promo cell ) with the following supplements ( fcs , endothelial cell growth supplement , epidermal growth factor , insulin - like growth factor , vascular endothelial growth factor 165 , ascorbic acid , heparin , hydrocortisone [ promo cell ] ) . dermal fibroblasts were cultured in rpmi with 20% fcs , 1000 u / ml penicillin and streptomycin and 2 mm l - glutamine . huvecs or fibroblasts ( 30,000/well ) were seeded in a 24-well plate and cultured in their respective media ( 500 l ) for 18 hr at 37c , 5% co2 . facs - purified blood monocyte subsets were added to huvecs or fibroblast cell cultures ( 50,000 monocytes / well to a final volume 1 ml ) and analyzed on days 1 and 3 after coculture . absolute whole - blood leukocyte analysis was performed as described previously ( jardine et al . briefly , 100 l of whole blood was transferred directly to trucount tubes ( bd ) . antibodies were added directly and staining was performed at rt for 20 min , followed by red blood cell lysis by adding 900 l of bd pharmlyse reagent for 10 min at rt . absolute number of cells per microliters of blood was calculated according to the manufacturer s protocol . s100a4-cre mice were purchased from jackson laboratory and crossed to r26-stop - eyfp mice in house . animals positive for the r26-stop - eyfp - flox construct were used for the experiment . only sex ( female ) and aged ( 68 weeks ) matched mice were used . mouse skin cells were isolated as described previously ( ginhoux et al . , 2007 ) . briefly , mouse ears were split into dorsal and ventral halves and floated in rpmi-1640 medium ( sigma ) containing 1 mg / ml dispase ( invitrogen ) for 60 min to allow separation of epidermal and dermal sheets . dermal sheets were then cut into small pieces and incubated in rpmi containing 10% serum and 0.8 mg / ml collagenase type iv ( worthington - biochem ; 250 u / mg ) for 2 hr . cells were then passed through 19 g syringe and filtered through 100 um cell strainer ( bd falcon ) to obtain a homogenous cell suspension . total rna was extracted using the rneasy micro kit ( qiagen ) and treated with dnase i according to manufacturers instructions ( qiagen ) . rna was used as a template for complementary dna ( cdna ) synthesis using the revertaid h minus first strand cdna synthesis kit with the manufacturers protocol ( thermo scientific , fisher scientific uk ) . real - time pcr was performed with taqman gene - expression master mix and recommended taqman gene - expression assays according to manufacturers instructions ( life technologies ) . reactions were performed using an abi 7900ht fast real - time pcr system with the instrument s default settings for a standard run . relative quantification of the messenger rna ( mrna ) amounts was performed using the ct method and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) as the reference gene .

summarydendritic cells ( dcs ) , monocytes , and macrophages are leukocytes with critical roles in immunity and tolerance . the dc network is evolutionarily conserved ; the homologs of human tissue cd141hixcr1+clec9a+ dcs and cd1c+ dcs are murine cd103 + dcs and cd64cd11b+ dcs . in addition , human tissues also contain cd14 + cells , currently designated as dcs , with an as - yet unknown murine counterpart . here we have demonstrated that human dermal cd14 + cells are a tissue - resident population of monocyte - derived macrophages with a short half - life of < 6 days . the decline and reconstitution kinetics of human blood cd14 + monocytes and dermal cd14 + cells in vivo supported their precursor - progeny relationship . the murine homologs of human dermal cd14 + cells are cd11b+cd64 + monocyte - derived macrophages . human and mouse monocytes and macrophages were defined by highly conserved gene transcripts , which were distinct from dcs . the demonstration of monocyte - derived macrophages in the steady state in human tissue supports a conserved organization of human and mouse mononuclear phagocyte system .

Introduction Results Discussion Experimental Procedures Author Contributions

dendritic cells ( dcs ) and macrophages are a heterogeneous population of leukocytes that are critical in orchestrating immune responses ( steinman , 2007 ) . cd14 cells are related to human and mouse blood monocytes by gene expression and are rapidly reconstituted by donor - derived cells following hematopoietic stem cell transplantation ( hsct ) , unlike dermal macrophages , which turn over at a much slower rate ( haniffa et al . in this study , we investigated the relationships between circulating blood cd14 monocytes and tissue macrophages with tissue mhc classiicd14 cells , currently defined as dcs . in addition , we showed that the murine dermal monocyte - derived macrophages are the homolog of human dermal cd14 cells . , 2013 ) , we therefore first set out to determine the phenotypic distinctions between dermal mhc classiiautofluorescent ( af)cd14 cells ( hereafter referred to as cd14 cells ) , afcd14 macrophages ( hereafter referred to as dermal macrophages ) , and blood cd14 monocytes , the homologs of murine ly6c monocytes . in addition , dermal cd14 cells expressed high amounts of il1 and gamma - glutamyl transferase 5 ( ggt5 ) transcripts ( figure 1c ) , which were identified from our previous microarray analysis ( haniffa et al . human tissue dcs are depleted in patients lacking circulating dcs and monocytes as shown by patients with genetic deficiency of monocytes and dcs due to gata2 or irf8 mutation , but the exact precursor - progeny relationships are difficult to demonstrate conclusively in humans ( bigley et al . the temporal relationship between blood cd14 monocytes and skin cd14 cells is consistent with a precursor - progeny relationship in which cd14 monocytes give rise to cd14 cells within a short timeframe . further examination of the relationship between cd14 cells , monocytes , macrophages , and dcs revealed a number of coregulated genes distinguishing monocytes , cd14 cells , and macrophages from blood and skin dcs ( figure 3c ) . ccr7 , cyp27b1 , flt3 , fscn1 , indo , lamp3 , and ly75 were expressed at higher amounts by cd1c dcs compared to cd14 cells , which expressed higher amounts of ccl18 , ccl3 , ccr1 , cd163 , cd36 , clec10a , fcgr1a and 3a , il10 , marco , mmp12 , msr1 , siglec1 , and trem2 ( figure 4d ) , transcripts characteristic of monocyte - macrophage cells . however , cd1c dcs , cd14 cells , and dermal macrophages were potent inducers of memory cd4 t cell proliferation and cytokine production upon stimulation with candida albicans ( figure 5b ) . both cd14 cells and dermal macrophages were comparable to cd1c in their ability to induce il-17 , il-22 , interferon- ( ifn- ) , and il-4 production by memory cd4 t cells ( figure 5b ) . recent studies have demonstrated heterogeneity within murine nonlymphoid tissues cd11b cells , which comprise dcs , monocytes , monocyte - derived dcs , and resident macrophages ( langlet et al . collectively , these results provide further evidence of functional equivalence between monocyte - derived macrophages in the mouse dermis as the homologs of human cd14 cells . although it is difficult to prove unequivocally that they have a monocyte origin , their absence in monocyte deficiency states , kinetics of renewal after hsct , and similarity to monocytes in short - term culture with endothelial cells are all consistent with a precursor - progeny relationship . despite the widespread use of in vitro culture protocols to generate monocyte - derived dcs and macrophages , in vivo evidence to support the contribution of such monocyte - derived cells in healthy tissue , we show the rapid decline and reconstitution of dermal cd14 cells that mirrors the kinetics of blood cd14 monocytes in patients undergoing hsct . dermal cd14 cells express high amounts of il-1 and ggt5 , a property not shared by any blood or skin dcs , monocytes , and macrophages , suggesting a role in maintaining epithelial integrity and regulation of skin inflammation including neutrophil migration ( han et al . the functional classification of human tissue cd14 cells as monocyte - derived macrophages will redirect attention to functional regulation in the skin as opposed to lymph node . a greater understanding of the contribution of monocyte - derived cells in steady state , inflammation , wound healing , and pathology characterized by histiocytosis and granuloma formation will provide further insights into exploiting their origin and functional properties for clinical therapy .

protein therapeutics ( including monoclonal antibodies [ mabs ] , peptides and recombinant proteins ) represent the largest group of new products in development by the biopharmaceutical industry ( durocher & butler , 2009 ; ho & chien , 2014 ) . these products are produced in a wide variety of platforms , including non - mammalian expression systems ( bacterial , yeast , plant and insect ) and mammalian expression systems ( including human cell lines ) ( ghaderi et al . , 2012 ) . importantly , the most appropriate expression system depends on the particular protein to be expressed . mammalian expression systems are generally the preferred platform for manufacturing biopharmaceuticals , as these cell lines are able to produce large , complex proteins with post - translational modifications ( ptms ; most notably glycosylation ) similar to those produced in humans ( durocher & butler , 2009 ; ghaderi et al . , 2012 ; swiech et al . , moreover , in the case of mammalian cell lines , and animal cell lines in general , most proteins can be secreted rather than requiring cell lysis to extract with subsequent protein refolding ( as is the case with bacteria / prokaryotes ) . the most common mammalian ( non - human ) cell lines used for therapeutic protein production include chinese hamster ovary ( cho ) cells , baby hamster kidney ( bhk21 ) cells and murine myeloma cells ( ns0 and sp2/0 ) ( estes & melville , 2014 ) . however , these non - human mammalian cell lines also produce ptms that are not expressed in humans , namely galactose-1,3-galactose ( -gal ) and n - glycolylneuraminic acid ( ngna ) . because humans possess circulating antibodies against both of these n - glycans , non - human cell lines are usually screened during their production to identify clones with acceptable glycan profiles ( ghaderi et al . , 2010 ) . human cell lines have the ability to produce proteins most similar to those synthesized naturally in humans , which may be an advantage compared with other mammalian expression systems ( ghaderi et al . . in particular , the structure , number and location of post - translational n - glycans can affect the biologic activity , protein stability , clearance rate and immunogenicity of biotherapeutic proteins ( arnold et al . , 2007 ; ghaderi et al . , 2010 ; swiech et al . , the first human cell line , hela , was established in 1951 from a cervical cancer ( scherer et al . , 1953 ) . human diploid cells were developed in the 1960s for vaccine manufacturing ; however , concerns for a latent oncogenic agent in these cell lines ( despite a lack of suggestive phenotypic characteristics ) delayed their acceptance . currently , human diploid cells are used in the manufacture of many viral vaccines ( petricciani & sheets , 2008 ) . however , due to their rapid growth , high protein yield , and the investment in system optimization , animal cells remained the substrate of choice for the production of recombinant proteins and mabs ( petricciani & sheets , 2008 ) . today , advances in technology have allowed for increased productivity with human cell lines , and there are now approved recombinant biotherapeutic products produced from the human embryonic kidney 293 ( hek293 ) and fibrosarcoma ht-1080 cell lines ( beck , 2009 ; casademunt et al . , 2012 ; additional biotherapeutic products produced in the per.c6 , hkb-11 , cap and huh-7 human cell lines are currently being evaluated ( enjolras et al . , 2012 ; estes & melville , 2014 ; jones et al . , 2003 ; mei et al . , 2006 ; swiech et al . , this article is a narrative review of the cell lines ( with a focus on human cell lines ) used for production of biotherapeutic proteins , both approved and in development . many non - human expression systems have been utilized in the production of currently approved biotherapeutic proteins ( table 1 ) . non - human expression systems used in the production of biotherapeutics approved in the united states and europe.expression systembiotherapeutic productfda approvalema approvalplant cellsenzymes taliglucerase alfaapprovednainsect cellsvaccines cervical cancer vaccineapprovedapprovedbacteriamonoclonal antibodies certolizumab pegolapprovedapproved cytokines tbo - filgrastimapprovedna romiplostimapprovedapproved enzymes asparaginase erwinia chrysanthemiapprovedna glucarpidaseapprovedna pegloticaseapprovedapproved collagenase clostridium histolyticumapprovedna peptidesapprovedna metreleptin therapeutic toxinsapprovedna incobotulinumtoxin a vaccines meningitis vaccineapprovedapproved pneumococcal vaccineapprovedapprovedyeastenzymes ocriplasminapprovedapproved peptides albiglutideapprovedapproved liraglutideapprovedapproved clotting factors catridecacogapprovedapprovedmammalian ( non - human ) cell lines chomonoclonal antibodies adalimumabapprovedapproved alemtuzumabapprovedna bevacizumabapprovedapproved brentuximab vedotinapprovedapproved denosumabapprovedapproved golimumabapprovedapproved ibritumomab tiuxetanapprovedapproved ipilimumabapprovedapproved obinutuzumabapprovedapproved omalizumabapprovedapproved panitumumabapprovedapproved pertuzumabapprovedapproved rituximabapprovedapproved siltuximabapprovedapproved tocilizumabapprovedapproved trastuzumabapprovedapproved vedolizumabapprovedapproved ado - trastuzumabemtansineapprovedapproved ustekinumabapprovedapproved cytokines darbepoetin alfaapprovedapproved interferon beta-1aapprovedapproved epoetin alfaapprovedapproved epoetin betanana epoetin thetanaapproved enzymes agalsidase betaapprovedapproved alglucosidase alfaapprovedapproved alteplaseapprovedapproved elosulfase alfaapprovedna galnac 4-sulfataseapprovedna human dnaseapprovedapproved hyaluronidaseapprovedna imigluceraseapprovedna laronidaseapprovedna tenecteplaseapprovedapproved fc - fusion proteins abataceptapprovedapproved afliberceptapprovedapproved alefaceptapprovedapproved belataceptapprovedapproved etanerceptapprovedna rilonaceptapprovedapproved ziv - afliberceptapprovedapproved hormones choriogonadotropin alfaapprovedna follitropin alfaapprovedapproved follitropin betaapprovedapproved luteinizing hormoneapprovedapproved osteogenic protein-1approvedapproved thyrotropin alfaapprovedapproved clotting factors factor viiiapprovedapproved factor ixapprovedapprovedns0monoclonal antibodies belimumabapprovedapproved natalizumabapprovedapproved ofatumumabapprovedapproved palivizumabapprovedapproved ramucirumabapprovednasp2/0monoclonal antibodies abciximabapprovedna basiliximabapprovedapproved canakinumabapprovedapproved cetuximabapprovedapproved infliximabapprovedapprovedbhkclotting factors factor viiaapprovedapproved factor viiiapprovedapprovedmurine c127hormones somatropinapprovedapprovedfda , us food and drug administration ; ema , european medicines agency ; na , not approved ; cho , chinese hamster ovary ; bhk , baby hamster kidney . data obtained from publically available resources ( october 2014 ) ; all approved products may not be included . references : ( abseamed , 2012 ; actemra , 2013 ; actilyse , 2014 ; activase , 2012 ; adcetris , 2013 ; advate , 2014 ; aldurazyme , 2008 ; aranesp , 2006 ; arcalyst , 2008 ; arzerra , 2014 ; avastin , 2010 ; avonex , 2007 ; benefix , 2012 ; benlysta , 2011 ; campath , 2014 ; cathflo activase , 2010 ; cerezyme , 2010 ; cervarix , 2012 ; cimzia , 2013 ; cimzia , 2014 ; cyramza , 2014 ; elelyso , 2014 ; enbrel , 2010 ; entyvio , 2014a , b ; eperzan , 2014 ; epoetin alfa hexal , 2012 ; eporatio , 2009 ; erbitux , 2009 ; erwinaze , 2011 ; eylea , 2012 ; eylea , 2013 ; fabrazyme , 2006 ; fertavid , 2009 ; follistim , 2011 ; gazyva , 2014 ; gazyvaro , 2014 ; ghaderi et al . , 2012 ; gonal - f , 2010 ; granix , 2014 ; helixate nexgen , 2010 ; herceptin , 2010 ; humira , 2008 ; hylenex , 2012 ; ilaris , 2014 ; jetrea , 2012 ; jetrea , 2013 ; kadcyla , 2013 , 2014 ; kogenate bayer , 2010 ; krystexxa , 2013 ; lumizyme , 2010 ; luveris , 2005 ; mabthera , 2008 ; menveo , 2010 ; metalyse , 2006 ; myalept , 2014 ; myozyme , 2011 ; naglazyme , 2005 ; novoseven , 2006 ; novothirteen , 2012 ; nplate , 2009 ; nulojix , 2011 ; obizur , 2014 ; office of device evaluation and center for devices and radiological health , 2001 ; opgenra , 2014 ; orencia , 2012 ; ovidrel , 2014 ; ovitrelle , 2006 ; perjeta , 2013a , b ; prevnar , 2009 ; procrit , 2000 ; prolia , 2010 ; pulmozyme , 2010 ; raxibacumab , 2014 ; rebif , 2008 ; refacto af , 2014 ; remicade , 2009 ; reopro , 2013 ; rituxan , 2014 ; roactemra , 2013 ; saizen , 1987 ; serostim , 1987 ; simponi , 2009 ; simulect , 2008 ; somatropin biopartners , 2013 ; stelara , 2013 ; swiech et al . , 2012 ; sylvant , 2015 ; sylvant , 2014 ; synagis , 2009 ; tanzeum , 2014 ; tbo - filgrastim , 2012 ; thyrogen , 2010 ; tnkase , 2011 ; tretten , 2014 ; tysabri , 2011 ; us food and drug administration , 2010 , 2011 , 2012 , 2013 , 2014 ; vectibix , 2014 ; victoza , 2009 ; vimizim , 2014a , b ; voraxaze , 2012 ; xeomin , 2014 ; xiaflex , 2014 ; xolair , 2010 ; xyntha , 2011 ; yervoy , 2011 ; zaltrap , 2013a , b ; zevalin , 2009 ) . fda , us food and drug administration ; ema , european medicines agency ; na , not approved ; cho , chinese hamster ovary ; bhk , baby hamster kidney . data obtained from publically available resources ( october 2014 ) ; all approved products may not be included . references : ( abseamed , 2012 ; actemra , 2013 ; actilyse , 2014 ; activase , 2012 ; adcetris , 2013 ; advate , 2014 ; aldurazyme , 2008 ; aranesp , 2006 ; arcalyst , 2008 ; arzerra , 2014 ; avastin , 2010 ; avonex , 2007 ; benefix , 2012 ; benlysta , 2011 ; campath , 2014 ; cathflo activase , 2010 ; cerezyme , 2010 ; cervarix , 2012 ; cimzia , 2013 ; cimzia , 2014 ; cyramza , 2014 ; elelyso , 2014 ; enbrel , 2010 ; entyvio , 2014a , b ; eperzan , 2014 ; epoetin alfa hexal , 2012 ; eporatio , 2009 ; erbitux , 2009 ; erwinaze , 2011 ; eylea , 2012 ; eylea , 2013 ; fabrazyme , 2006 ; fertavid , 2009 ; follistim , 2011 ; gazyva , 2014 ; gazyvaro , 2014 ; ghaderi et al . , 2012 ; gonal - f , 2010 ; granix , 2014 ; helixate nexgen , 2010 ; herceptin , 2010 ; humira , 2008 ; hylenex , 2012 ; ilaris , 2014 ; jetrea , 2012 ; jetrea , 2013 ; kadcyla , 2013 , 2014 ; kogenate bayer , 2010 ; krystexxa , 2013 ; lumizyme , 2010 ; luveris , 2005 ; mabthera , 2008 ; menveo , 2010 ; metalyse , 2006 ; myalept , 2014 ; myozyme , 2011 ; naglazyme , 2005 ; novoseven , 2006 ; novothirteen , 2012 ; nplate , 2009 ; nulojix , 2011 ; obizur , 2014 ; office of device evaluation and center for devices and radiological health , 2001 ; opgenra , 2014 ; orencia , 2012 ; ovidrel , 2014 ; ovitrelle , 2006 ; perjeta , 2013a , b ; prevnar , 2009 ; procrit , 2000 ; prolia , 2010 ; pulmozyme , 2010 ; raxibacumab , 2014 ; rebif , 2008 ; refacto af , 2014 ; remicade , 2009 ; reopro , 2013 ; rituxan , 2014 ; roactemra , 2013 ; saizen , 1987 ; serostim , 1987 ; simponi , 2009 ; simulect , 2008 ; somatropin biopartners , 2013 ; stelara , 2013 ; swiech et al . , 2012 ; sylvant , 2015 ; sylvant , 2014 ; synagis , 2009 ; tanzeum , 2014 ; tbo - filgrastim , 2012 ; thyrogen , 2010 ; tnkase , 2011 ; tretten , 2014 ; tysabri , 2011 ; us food and drug administration , 2010 , 2011 , 2012 , 2013 , 2014 ; vectibix , 2014 ; victoza , 2009 ; vimizim , 2014a , b ; voraxaze , 2012 ; xeomin , 2014 ; xiaflex , 2014 ; xolair , 2010 ; xyntha , 2011 ; yervoy , 2011 ; zaltrap , 2013a , b ; zevalin , 2009 ) . bacterial expression systems ( e.g. escherichia coli ) possess the advantages of being straightforward to culture , with rapid cell growth and high yields . in addition , protein expression can be initiated through promoter induction by addition of lactose or the lactose analogue isopropyl--d - thiogalactopyranoside ( iptg ; iptg induces the promoters lac , tac and trc ) . however , such systems are unable to produce complex , mammalian - like glycosylation due to the absence of the necessary enzymatic components and the intracellular compartmentalization required ( ghaderi et al . , 2012 ; graumann & premstaller , 2006 ) . in addition , mammalian proteins produced in these systems often aggregate , forming inclusion bodies , due to the low solubility of mammalian proteins in prokaryotic cells and absence of appropriate protein chaperone systems . proteins produced in bacterial expression systems must often be extracted from inclusion bodies and refolded . bacterial systems are therefore generally used for production of non - glycosylated proteins , including some mabs , hormones , cytokines and enzymes ( ghaderi et al . , 2012 ; graumann & premstaller , 2006 ) . similar to bacterial expression systems , yeast expression systems ( e.g. saccharomyces cerevisiae and pichia pastoris ) achieve rapid cell growth and high - protein yields with straightforward production scalability and without the need for animal - derived growth factors ( gerngross , 2004 ) . yeast cell lines may also be used to produce proteins that can not be obtained from e. coli due to the problems associated with folding and stereochemistry ( gerngross , 2004 ) . the key challenge associated with yeast expression systems is their production of high mannose residues within their expressed ptms ( 50200 vs three molecules in human cells , as part of either n- or o - linked glycan structures ) , which may confer a short half - life and render proteins less efficacious and even immunogenic in humans ( dean , 1999 ; gemmill & trimble , 1999 ; gerngross , 2004 ; lam et al . , 2007 ; the development of yeasts that have been genetically modified to address the issue of high mannose content has been reported ( chiba et al . , 1998 ; gerngross , 2004 ; ghaderi et al . , 2012 ; hamilton et al . , the expression of a fully humanized sialylated glycoprotein in glycoengineered yeast constitutes a major advance in the use of yeast expression systems for biopharmaceutical manufacturing ( hamilton & gerngross , 2007 ) . plant and insect cell expression systems are able to produce proteins with complex glycosylation patterns ; however , the glycan structures produced are significantly different from those produced in humans ( ghaderi et al . , 2012 ) . plants lack many of the key glycosylated residues present in humans , most notably sialic acids . in addition , they produce 1,3-fructose and 1,2-xylose , which are absent in humans and may be immunogenic ( ghaderi et al . , 2012 ) . notably , in 2012 , taliglucerase alfa ( elelyso ; pfizer , new york , ny ) was approved by the us food and drug administration ( fda ) for the treatment of type 1 gaucher disease . this therapy is produced using genetically modified carrot plant root cells that produce the enzyme with a human compatible glycan profile ( elelyso , 2014 ) . insect cells infected with the viral vector baculovirus ( baculovirus - insect cell expression system ) can also efficiently express recombinant proteins , and these systems are mostly used for the development of virus - like particles and , subsequently , vaccines ( kost et al . however , although they produce n - glycan precursors , these are trimmed , resulting in either high mannose or paucimannose residues that do not develop further into terminal galactose and/or sialic acid residues ( kost et al . , 2005 ) . this is evidenced by the lack of either galactosyltransferase or sialyltransferase activity . as in plants , insect systems however , in recent years , there have been developments in the use of transgenic insect cells , with humanized protein glycosylation mechanisms ( kost et al . , 2005 ) . the majority of currently licensed biotherapeutic products are produced in non - human mammalian expression systems ( table 1 ) , as these systems are able to produce ptms that ( outside of a human expression system ) most closely resemble those in humans ( ghaderi et al . these expression systems are used to produce mabs , hormones , cytokines , enzymes and clotting factors ( ghaderi et al . , 2012 ) . the most frequently used mammalian system is the cho cell line , which is used in the manufacture of > 70% of currently approved recombinant proteins ( butler & spearman , 2014 ) . first , cho cells are able to grow in suspension culture ( which enables large - scale production ; other cell lines , such as insect cells , also have this ability ) and serum - free chemically defined media ( enabling reproducibility across different batches of cultures with a better safety profile than in media that contain human- or animal - derived proteins ) ( kim et al . , 2012 ; lai et al . , 2013 historically , cho cells allowed gene amplification , resulting in a higher recombinant protein yield ( up to the gram per liter range for some proteins ) and specific productivity , which was previously an issue in other mammalian cell lines ( carlage et al . , 2012 ; kim et al . , 2012 ; yang et al other advances , such as the creation of stronger expression units and advanced hosts , better selection strategies ( e.g. through technologic advances in screening for high - productivity clones ) and targeting the transgene to transcriptional hotspots ( site - specific integration of transgenes ) , also contribute to the high protein yields attained from these cells ( kim et al . , 2012 ) . in addition , this expression system is highly tolerant to changes in ph , oxygen level , pressure or temperature during manufacturing ( ghaderi et al . , 2012 ; lai et al . , 2013 ) . furthermore , due to the long period of time that this cell line has been used , there is a degree of familiarity with the cho platform within development and manufacturing organizations , regulatory agencies , and suppliers ( e.g. cell culture media suppliers ) , which could potentially decrease overall timelines . this familiarity may also be beneficial when assessing contaminant profiles ( e.g. host cell proteins ) , which may be better characterized for cho cells compared with newer cell lines . the first recombinant biotherapeutic protein produced in cho cells was tissue plasminogen activator , approved in 1986 ( kim et al . , 2012 ) . therefore , the safety profile of cho cells has been established for more than 20 years ( butler & spearman , 2014 ; kim et al . , 2012 ) . cho cells have been shown to have reduced susceptibility to certain viral infections compared with other mammalian cell lines ( e.g. the bhk cell line ) , and routine screening systems for adventitious agents are effective in detecting cell line infections ( berting et al . , 2010 ) . this reduced susceptibility may be due to the fact that many viral entry genes are not expressed in cho cells ( xu et al . , 2011 ) . further , there is perceived species barrier protection with the use of hamster - derived cells , reducing the potential risk of transfer of contaminating adventitious agents to humans ( berting et al . , 2010 ; swiech et al . , however , many viruses have the ability to cross the species barrier and may still pose a risk ( pauwels et al . , 2007 ) . perhaps the most important advantage of cho cells is that they are able to produce proteins with complex bioactive ptms that are similar to those produced in humans . however , cho cells are unable to produce some types of human glycosylation ( cho cells lack [2 - 6 ] sialyltransferase [1 - 3/4 ] fucosyltransferases ) and they produce glycans that are not expressed in humans , namely -gal and ngna ( bosques et al . , 2010 ; dietmair et al . , 2012 ; ghaderi et al . , circulating antibodies against both of these n - glycans are present in humans , which may lead to increased immunogenicity and altered pharmacokinetics of these products when used in humans ( ghaderi et al . , 2010 ; padler - karavani et al . , additional screening in cho cells is required in order to isolate clones lacking the -gal and ngna glycans . this screening may result in otherwise productive clones needing to be discarded ( ghaderi et al . , 2010 ) . however , the attachment of non - human glycans may not be a concern for therapeutic proteins that do not require glycosylation , which illustrates the importance of considering the specific product molecule when choosing an appropriate cell line for production of a protein . other mammalian cell lines used for the production of biotherapeutic proteins include bhk-21 cells , used in the production of some coagulation factors such as factor viii ( wurm , 2004 ) . when murine myeloma cell lines ( ns0 and sp2/0 ) have been used historically , they have generally been used in the production of mabs , for example , palivizumab and ofatumumab ( barnes et al . , 2000 ; butler & spearman , 2014 ; ghaderi et al . , these myeloma cells were derived from immunoglobulin - producing tumor cells that no longer produced their original immunoglobulins ; these cells possess the appropriate machinery for producing and secreting these proteins ( barnes et al . , 2000 ) . for proteins produced in all of these non - human cell lines , as well as those produced in human cell lines , potential safety concerns arise from the possibility of process - related contaminants and immunogenicity ( world health organization , 2013 ) . process - related contaminants may include infectious agents ( viral , bacterial , fungal , etc . ) with the potential to result in host infection , nucleic acid contaminants with the potential to integrate into the host genome ( theoretical ) , and other contaminants from the manufacturing process , such as exogenous non - human epitopes ( e.g. from animal serum used during the manufacturing process ) that can be incorporated into human cells and the resultant biotherapeutic protein ( ghaderi et al . , 2012 ) . hek293 and ht-1080 are the two human cell lines most often used in the production of biotherapeutic proteins , which offer the advantage of producing fully human ptms ( tables 2 and 3 ) ( loignon et al . , 2008 ; swiech et al . , human cells lines and their therapeutic protein products.cell lineproductindicationfda approval statusema approval statushek293 drotrecogin alfasevere septicemia / septic shockapproved 2001 ; withdrawn 2011approved 2002 ; withdrawn 2011 rfviiifchemophilia aapproved 2014submitted 2014 rfixfchemophilia bapproved 2014na dulaglutidetype 2 diabetesapproved 2014submitted 2014 human - cl rhfviiihemophilia asubmitted to the fdaapproved 2014ht-1080 agalsidase alfafabry diseasenaapproved 2001 epoetin deltaanemia secondary to chronic renal failurenaapproved 2002 ; withdrawn 2009 ( europe ) idursulfasehunter syndromeapproved 2006approved 2007 velaglucerase alfatype 1 gaucher diseaseapproved 2010approved 2010per.c6 cl184rabies virus infectionsubmitted to the fdana mor103rheumatoid arthritis , multiple sclerosisphase 1 clinical developmentphase 1 clinical developmentfda , us food and drug administration ; ema , european medicines agency ; hek , human embryonic kidney ; na , not approved ; rfviiifc , recombinant factor viii fc fusion protein ; rfixfc , recombinant factor ix fc fusion protein ; rhfviii , recombinant human factor viii . data obtained from publically available resources ( october 2014 ) ; all approved products may not be included . 2005 ; behrens et al . , 2014 ; casademunt et al . , 2012 ; dynepo , 2007 ; elaprase , 2012 , 2013 ; eloctate , 2014 ; european medicines agency and committee for medicinal products for human use , 2014 ; glaesner et al . , 2010 ; octapharma , 2014 ; replagal , 2006 ; trulicity , 2014 ; vpriv , 2010a , b ; xigris , 2008 ) . comparison of human cell lines with other expression systems in the production of therapeutic proteins.advantagesdisadvantages absence of potentially immunogenic ptms due to human - compatible glycosylation easily grown in suspension serum - free culture amenable to a number of transfection methods clinical experience is not as extensive as for other cell lines , although experience is growing potential susceptibility to human viral contamination fda , us food and drug administration ; ema , european medicines agency ; hek , human embryonic kidney ; na , not approved ; rfviiifc , recombinant factor viii fc fusion protein ; rfixfc , recombinant factor ix fc fusion protein ; rhfviii , recombinant human factor viii . data obtained from publically available resources ( october 2014 ) ; all approved products may not be included . casademunt et al . , 2012 ; dynepo , 2007 ; elaprase , 2012 , 2013 ; eloctate , 2014 ; european medicines agency and committee for medicinal products for human use , 2014 ; glaesner et al . , 2010 ; octapharma , 2014 ; replagal , 2006 ; trulicity , 2014 ; vpriv , 2010a , b ; xigris , 2008 ) . hek293 cells are easily grown in suspension serum - free culture , reproduce rapidly , are amenable to a number of transfection methods , and are highly efficient at protein production ( swiech et al . , 2012 ; thomas & smart , 2005 ) . hek293-h ( berkner , 1993 ) and 293-f ( vink et al . , 2014 ) cell lines are clonal isolates of the hek293 cell line that were selected for fast growth in serum - free medium , superior transfection efficiency , and a high level of protein production ( gibco , 2014 ) . subclone 293-h also has improved adherence to monolayer culture ( when serum - supplemented media are used ) compared with other cell lines . the hek293-t ( 293-t ) cell line expresses the simian virus 40 large t antigen and is capable of expressing high titers of viral gene vectors for use in gene therapy ( yamaguchi et al . , 2003 ) . hek293-t cells are often used for the production of retroviral vectors ( yamaguchi et al . , 2003 ) . hek293-ebna1 cells stably express the epstein - barr virus ebna-1 gene , controlled by the cytomegalovirus promoter and demonstrate a greater growth rate and maximal cell density relative to parental hek293 cells ( schlaeger & christensen , 1999 ) . hek293 cells have been widely used to produce research - grade proteins for many years and , more recently , five therapeutic agents produced in hek293 cells have been approved by the fda or the european medicines agency ( ema ) for therapeutic use . these agents are drotrecogin alfa ( xigris ; eli lilly corporation , indianapolis , in ) , recombinant factor ix fc fusion protein ( rfixfc ; biogen , cambridge , ma ) , recombinant factor viii fc fusion protein ( rfviiifc ; biogen , cambridge , ma ) , human cell line recombinant factor viii ( human - cl rhfviii ; nuwiq ; octapharma , lachen , switzerland ) and dulaglutide ( trulicity ; eli lily , indianapolis , in ) . drotrecogin alfa is a recombinant activated protein c that was approved by the fda in 2001 and by the ema in 2002 for the treatment of patients with severe sepsis . hek293 cells were chosen by the manufacturer for production of drotrecogin alfa because its activity required two ptms , propeptide cleavage and -carboxylation of its glutamic acid residues , which cho cells can not produce with adequate efficiency ( berkner , 1993 ; durocher & butler , 2009 ) . the product was approved ( bernard et al . , 2001 ) , but was later voluntarily withdrawn from the market by its manufacturer ( eli lilly ) in 2011 following the randomized placebo - controlled prospective recombinant human activated protein c worldwide evaluation in severe sepsis and septic shock ( prowess - shock ) trial , which demonstrated no mortality benefit with drotrecogin alfa compared with placebo for patients experiencing septic shock ( green et al . , 2012 ; ranieri et al . , 2012 ) . rfviiifc and rfixfc are recombinant fusion proteins that were approved by the fda in 2014 for the control and prevention of bleeding episodes , perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in people with hemophilia a and b , respectively ( alprolix , 2014 ; eloctate , 2014 ; mahlangu et al . , 2014 ; powell et al . , rfviiifc consists of b domain deleted recombinant factor viii genetically fused to the fc portion of immunoglobulin g1 ( igg1 ) and is produced in hek293-h cells ( dumont et al . , 2012 ; eloctate , 2014 ; peters et al . , 2013 ) . the rfviiifc fed - batch culture process is robust at scales of 200 , 2000 and 15 000 liters , with the potential for a second - generation process to achieve even higher cell densities , on the order of 3.5 10 vc / ml ( huang et al . , 2014 ) . rfixfc was also produced using hek293-h cells , and consists of the factor ix sequence covalently linked to the fc domain of human igg1 ( alprolix , 2014 ; durocher & butler , 2009 ; mccue et al . , 2014 ; peters et al . , 2010 ) . an essential ptm for fix activity is -carboxylation of the first 12 glutamic acid residues in the gla domain by vitamin k cells have been reported to have a greater capacity for -carboxylation than cho cells ( berkner , 1993 ) . furthermore , fviii contains six potential tyrosine sulfation sites , which are vital for fviii functionality and binding to von willebrand factor . fviii expressed from human cell lines has been reported to be fully sulfated ( kannicht et al . , 2013 ; peters et al . , 2013 ) . the use of a human cell line for replacement coagulation factors , such as rfviiifc and rfixfc , may result in reduced immunogenicity relative to non - human mammalian cell lines , as -gal and ngna glycan moieties are absent from these manufactured protein products ( bosques et al . however , it should be noted that several recombinant clotting factor products produced in non - human mammalian cell lines have been used successfully for many years . the development of inhibitors ( neutralizing antibodies ) against replacement clotting factors occurs in 30% of people with severe hemophilia a and 5% of those with severe hemophilia b. the causative f8 or f9 gene mutation plays a pivotal role in inhibitor development in hemophilia a and b , respectively , with large or complete deletions , non - sense mutations or inversions ( e.g. intron 22 inversion in the f8 gene ) being the most commonly associated mutations ( franchini & mannucci , 2011 ) . the impact of ptms on inhibitor development is unknown , and will need further research . importantly , none of the previously treated people with hemophilia in the phase 1/2a or phase 3 clinical studies developed inhibitors to the rfviiifc and rfixfc fusion products ( mahlangu et al . , 2014 ; powell et al . , 2012 , 2013 ; shapiro et al . , human - cl rhfviii ( nuwiq ) , an additional factor viii replacement product for the management of hemophilia a , is being produced in the hek293-f cell line . like hek293-h cells , hek293-f cells are a derivation of hek293 cells that have been pre - adapted for growth in serum - free culture medium ( casademunt et al . , 2012 ) . human - cl rhfviii has been approved by the ema and submitted to the fda for approval ( octapharma , 2014 ) ; this product has been shown to exhibit a similar glycosylation profile to human plasma - derived factor viii , without -gal and ngna ( kannicht et al . , 2013 ) . glucagon-1-like peptide ( glp-1 ) fc fusion protein ( dulaglutide ) has been approved by the fda for the treatment of type 2 diabetes mellitus , and is produced using hek293-ebna cells ( glaesner et al . , 2010 ; trulicity , 2014 ) . large clinical trials have demonstrated its superiority over the dipeptidyl peptidase-4 inhibitor antagonist exenatide and its non - inferiority to liraglutide ( a glp-1 agonist ) , when added on to oral diabetic agents ( dungan et al . , 2014 ; wysham et al . , another human cell line , ht-1080 , was produced from a fibrosarcoma with an epithelial - like phenotype ( swiech et al . , 2012 ) . with the use of gene activation technology ( in which the endogenous dna promoter is replaced with a more potent type ) , four approved therapeutic proteins have been produced by shire ( swiech et al . , 2012 ) . 1 ) epoetin delta ( dynepo ) was approved by the ema in 2002 for the treatment of anemia secondary to chronic renal failure ( dynepo , 2007 ; elaprase , 2013 ; replagal , 2006 ; swiech et al . , 2012 ; vpriv , 2013 ) . 2 ) iduronate-2-sulfatase ( idursulfase ; elaprase ) is licensed as enzyme replacement therapy ( ema in 2007 and fda in 2006 ) for the treatment of hunter syndrome ( mucopolysaccharidosis ii ) , an x - linked lysosomal storage disorder ( elaprase , 2013 ) . 3 ) agalsidase alfa ( replagal ; shire human genetic therapies , danderyd , sweden ) was approved by the ema in 2001 for the treatment of fabry disease ( replagal , 2010 ) . compared with agalsidase beta ( fabrazyme ; genzyme therapeutics , cambridge , ma ) , which is produced using cho cells for a similar indication ( fabrazyme , 2010 , 2014 ) , agalsidase alfa has shown similar enzyme kinetics . however , agalsidase alfa demonstrates a lesser uptake into fibroblasts from patients with fabry disease and also lower concentrations in the kidney , heart and spleen of mice ( lee et al . , 2003 ) . a single clinical study has compared the two products ; this showed no significant differences for all efficacy outcomes , and there were no differences for the development of antibodies ( vedder et al . , 2007 ) . 4 ) the fourth agent produced in ht-1080 cells , velaglucerase alfa ( vpriv ; shire human genetic therapies , lexington , ma ) , was approved in 2010 ( fda and ema ) for the treatment of type 1 gaucher disease ( dynepo , 2007 ; elaprase , 2013 ; replagal , 2006 ; swiech et al . , 2012 ; vpriv , 2013 ) . velaglucerase alfa has been compared with two similar products : imiglucerase , produced using cho cells , and taliglucerase alfa , produced using carrot cells ( ben turkia et al . , 2013 ; tekoah et al . , these products have diverse glycan profiles and the studies have generally shown comparable uptake into macrophages , in vitro enzymatic activity , stability , organ distribution and efficacy ( ben turkia et al . , 2013 ; tekoah et al . , however , neutralizing antibodies to imiglucerase were noted in 24% of patients , which had an impact on enzyme activity . it was noted that various factors , such as the production cell line and glycosylation , may be responsible for the difference in immunogenicity , however , the specificity of the anti - imiglucerase antibodies was not stated ( ben turkia et al . , 2013 ) . notably , studies that evaluated epoetin delta produced in ht-1080 cells demonstrated differences in glycosylation compared with erythropoietin produced in cho cells , including a lack of ngna in the proteins ( butler & spearman , 2014 ; llop et al . , 2008 ; shahrokh et al . , 2011 however , there were additional overlapping isoforms present in endogenous human erythropoietin isolated from urine and serum relative to epoetin delta that could not be accounted for by sialic residues alone . human cell lines have been extensively utilized for the production of products that are currently in clinical development . in addition , human cell lines are a frequently used expression system for biomedical research due to their production of human ptms and high productivity . as productivity may vary across clonal isolates , it is important to screen for those clones with the highest yield of the therapeutic protein ( berkner , 1993 ) . the per.c6 cell line was created from human embryonic retinal cells , immortalized via transfection with the adenovirus e1 gene ( havenga et al . , 2008 ) . this system was originally developed for the production of human adenovirus vectors for use in vaccine development and gene therapy ( butler & spearman , 2014 ) . an investment was made in this cell line in order to develop a human expression system , and now an advantage of per.c6 is its ability to produce a high level of protein when used in the production of human igg ( jones et al . , 2003 ) . however , this does not require amplification of the incorporated gene ( jones et al . , 2003 ) . currently , a variety of products utilizing the per.c6 cell line are in phase 1 or 2 clinical trials ( durocher & butler , 2009 ) , including the mor103 mab , a human igg antibody against granulocyte macrophage colony - stimulating factor , and cl184 , an antibody against the rabies virus ( nagarajan et al . , 2014 ) . mor103 is in clinical development for the treatment of rheumatoid arthritis and multiple sclerosis . in a phase 1b/2a , randomized , placebo - controlled study , mor103 was active in patients with moderately severe rheumatoid arthritis ; a small number of patients developed anti - mor103 antibodies ( behrens et al . cl184 is a combination of two mabs , human igg1( ) and human igg1( ) ( bakker et al . , 2005 ) . in a phase 1 clinical study , it demonstrated a favorable safety profile and rapid development of rabies virus neutralizing activity , while there was no evidence to suggest the development of human anti - human antibodies ( bakker et al . , 2008 ) . the cap cell line is derived from human amniocytes obtained through amniocentesis ; these cells are immortalized through an adenovirus type 5 e1 gene ( schiedner et al . , 2008 ; swiech et al . , in addition to the ability to produce human ptms , the primary advantage of this cell is the potential for high protein yields ( schiedner et al . , 2008 ) . the hkb-11 cell line was created through polyethylene glycol fusion of hek293-s and a human b - cell line ( modified burkitt lymphoma cells ) ( cho et al . , 2003 ; durocher & butler , 2009 ; picanco - castro et al . , the advantages of this cell line include high - level protein production without the formation of aggregates , which can be a problem in other human cell lines ( picanco - castro et al . , 2013 ) . notably , hkb-11 has demonstrated increased expression of human fviii compared with expression in hek293 and bhk21 ( mei et al . , 2006 ) . similar to other human cell lines , it has been shown to produce human glycosylation patterns including ( 2,3 ) and ( 2,6 ) sialic acid linkages ( picanco - castro et al . , 2013 ) . hkb-11 has been used to produce a recombinant factor viii protein and tissue factor ( cho et al . , 2003 ) . a more recently developed cell line , huh-7 , originates from a human hepatocellular carcinoma ( enjolras et al . , 2012 ) . a recent study has shown that the huh-7-cd4 clone is capable of producing recombinant human factor ix with a human glycosylation profile . ptm profiles ( e.g. glycosylation , sialylation , phosphorylation and sulfation ) were similar to plasma - derived and recombinant factor ix ( rfix ) , and were improved relative to rfix produced in cho cells ( enjolras et al . , more recently , the huh-7 cell line has been used to produce mutant forms of rfix that have improved binding affinity for activated fviii , and also demonstrated enhanced clotting activity in mice ( perot et al . , 2015 ) . the human - specific glycosylation pattern of the ptms produced by human cell lines offer several advantages compared with those produced in animal cell lines . although other mammalian cells can produce similar ptms to human cells , most also produce -gal and ngna , ptms that are not present in the structure of human proteins ( ghaderi et al . , 2012 ) . patterns of post - translational glycosylation are known to affect protein yield , bioactivity , and clearance ( ghaderi et al . , 2010 ) . in addition , antibodies to ngna have been widely reported to occur in humans ( chung et al . , 2008 ; ghaderi et al . , one study utilizing an ngna knockout mouse model demonstrated increased immunogenicity of cetuximab due to anti - ngna antibodies ( ghaderi et al . , 2010 ) . in addition , in patients receiving the mab cetuximab for the treatment of colorectal or head and neck cancers , the majority of severe hypersensitivity reactions observed in clinical trials were associated with pre - existing ige antibodies against -gal ( chung et al . , 2008 ; ghaderi et al . , such antibodies may alter the efficacy or immunogenicity of proteins with the presence of non - human glycan structures . thus , human cell lines can serve as a valuable niche expression system for biotherapeutic proteins that require human ptms . a theoretical concern with the use of human cell lines is an increased risk of transfer of human adventitious agents , given the lack of a species barrier ( swiech et al . , however , current manufacturing technologies , typically inclusive of multiple viral inactivation or clearance steps , such as nanofiltration , have largely mitigated this concern and may provide more effective viral clearance than has been observed in cho cells ( kelley et al . production of biotherapeutic proteins in human cell lines is expanding , with several products currently approved for clinical use and others in clinical development in different therapeutic areas . advantages of human expression systems include achieving equal productivity to other mammalian cell lines and the production of proteins that lack potentially immunogenic , non - human ptms ( most notably -gal and ngna ) . in the future , with additional research investments and a continuation of the technologic advances that have already led to improvements in the use of human cell lines for protein manufacture , human cell lines will be further optimized , more sophisticated product collection strategies will be developed , and these cell lines may become one of the preferred platforms for protein biotherapeutic production . many non - human expression systems have been utilized in the production of currently approved biotherapeutic proteins ( table 1 ) . non - human expression systems used in the production of biotherapeutics approved in the united states and europe.expression systembiotherapeutic productfda approvalema approvalplant cellsenzymes taliglucerase alfaapprovednainsect cellsvaccines cervical cancer vaccineapprovedapprovedbacteriamonoclonal antibodies certolizumab pegolapprovedapproved cytokines tbo - filgrastimapprovedna romiplostimapprovedapproved enzymes asparaginase erwinia chrysanthemiapprovedna glucarpidaseapprovedna pegloticaseapprovedapproved collagenase clostridium histolyticumapprovedna peptidesapprovedna metreleptin therapeutic toxinsapprovedna incobotulinumtoxin a vaccines meningitis vaccineapprovedapproved pneumococcal vaccineapprovedapprovedyeastenzymes ocriplasminapprovedapproved peptides albiglutideapprovedapproved liraglutideapprovedapproved clotting factors catridecacogapprovedapprovedmammalian ( non - human ) cell lines chomonoclonal antibodies adalimumabapprovedapproved alemtuzumabapprovedna bevacizumabapprovedapproved brentuximab vedotinapprovedapproved denosumabapprovedapproved golimumabapprovedapproved ibritumomab tiuxetanapprovedapproved ipilimumabapprovedapproved obinutuzumabapprovedapproved omalizumabapprovedapproved panitumumabapprovedapproved pertuzumabapprovedapproved rituximabapprovedapproved siltuximabapprovedapproved tocilizumabapprovedapproved trastuzumabapprovedapproved vedolizumabapprovedapproved ado - trastuzumabemtansineapprovedapproved ustekinumabapprovedapproved cytokines darbepoetin alfaapprovedapproved interferon beta-1aapprovedapproved epoetin alfaapprovedapproved epoetin betanana epoetin thetanaapproved enzymes agalsidase betaapprovedapproved alglucosidase alfaapprovedapproved alteplaseapprovedapproved elosulfase alfaapprovedna galnac 4-sulfataseapprovedna human dnaseapprovedapproved hyaluronidaseapprovedna imigluceraseapprovedna laronidaseapprovedna tenecteplaseapprovedapproved fc - fusion proteins abataceptapprovedapproved afliberceptapprovedapproved alefaceptapprovedapproved belataceptapprovedapproved etanerceptapprovedna rilonaceptapprovedapproved ziv - afliberceptapprovedapproved hormones choriogonadotropin alfaapprovedna follitropin alfaapprovedapproved follitropin betaapprovedapproved luteinizing hormoneapprovedapproved osteogenic protein-1approvedapproved thyrotropin alfaapprovedapproved clotting factors factor viiiapprovedapproved factor ixapprovedapprovedns0monoclonal antibodies belimumabapprovedapproved natalizumabapprovedapproved ofatumumabapprovedapproved palivizumabapprovedapproved ramucirumabapprovednasp2/0monoclonal antibodies abciximabapprovedna basiliximabapprovedapproved canakinumabapprovedapproved cetuximabapprovedapproved infliximabapprovedapprovedbhkclotting factors factor viiaapprovedapproved factor viiiapprovedapprovedmurine c127hormones somatropinapprovedapprovedfda , us food and drug administration ; ema , european medicines agency ; na , not approved ; cho , chinese hamster ovary ; bhk , baby hamster kidney . data obtained from publically available resources ( october 2014 ) ; all approved products may not be included . references : ( abseamed , 2012 ; actemra , 2013 ; actilyse , 2014 ; activase , 2012 ; adcetris , 2013 ; advate , 2014 ; aldurazyme , 2008 ; aranesp , 2006 ; arcalyst , 2008 ; arzerra , 2014 ; avastin , 2010 ; avonex , 2007 ; benefix , 2012 ; benlysta , 2011 ; campath , 2014 ; cathflo activase , 2010 ; cerezyme , 2010 ; cervarix , 2012 ; cimzia , 2013 ; cimzia , 2014 ; cyramza , 2014 ; elelyso , 2014 ; enbrel , 2010 ; entyvio , 2014a , b ; eperzan , 2014 ; epoetin alfa hexal , 2012 ; eporatio , 2009 ; erbitux , 2009 ; erwinaze , 2011 ; eylea , 2012 ; eylea , 2013 ; fabrazyme , 2006 ; fertavid , 2009 ; follistim , 2011 ; gazyva , 2014 ; gazyvaro , 2014 ; ghaderi et al . , 2012 ; gonal - f , 2010 ; granix , 2014 ; helixate nexgen , 2010 ; herceptin , 2010 ; humira , 2008 ; hylenex , 2012 ; ilaris , 2014 ; jetrea , 2012 ; jetrea , 2013 ; kadcyla , 2013 , 2014 ; kogenate bayer , 2010 ; krystexxa , 2013 ; lumizyme , 2010 ; luveris , 2005 ; mabthera , 2008 ; menveo , 2010 ; metalyse , 2006 ; myalept , 2014 ; myozyme , 2011 ; naglazyme , 2005 ; novoseven , 2006 ; novothirteen , 2012 ; nplate , 2009 ; nulojix , 2011 ; obizur , 2014 ; office of device evaluation and center for devices and radiological health , 2001 ; opgenra , 2014 ; orencia , 2012 ; ovidrel , 2014 ; ovitrelle , 2006 ; perjeta , 2013a , b ; prevnar , 2009 ; procrit , 2000 ; prolia , 2010 ; pulmozyme , 2010 ; raxibacumab , 2014 ; rebif , 2008 ; refacto af , 2014 ; remicade , 2009 ; reopro , 2013 ; rituxan , 2014 ; roactemra , 2013 ; saizen , 1987 ; serostim , 1987 ; simponi , 2009 ; simulect , 2008 ; somatropin biopartners , 2013 ; stelara , 2013 ; swiech et al . , 2012 ; sylvant , 2015 ; sylvant , 2014 ; synagis , 2009 ; tanzeum , 2014 ; tbo - filgrastim , 2012 ; thyrogen , 2010 ; tnkase , 2011 ; tretten , 2014 ; tysabri , 2011 ; us food and drug administration , 2010 , 2011 , 2012 , 2013 , 2014 ; vectibix , 2014 ; victoza , 2009 ; vimizim , 2014a , b ; voraxaze , 2012 ; xeomin , 2014 ; xiaflex , 2014 ; xolair , 2010 ; xyntha , 2011 ; yervoy , 2011 ; zaltrap , 2013a , b ; zevalin , 2009 ) . fda , us food and drug administration ; ema , european medicines agency ; na , not approved ; cho , chinese hamster ovary ; bhk , baby hamster kidney . data obtained from publically available resources ( october 2014 ) ; all approved products may not be included . references : ( abseamed , 2012 ; actemra , 2013 ; actilyse , 2014 ; activase , 2012 ; adcetris , 2013 ; advate , 2014 ; aldurazyme , 2008 ; aranesp , 2006 ; arcalyst , 2008 ; arzerra , 2014 ; avastin , 2010 ; avonex , 2007 ; benefix , 2012 ; benlysta , 2011 ; campath , 2014 ; cathflo activase , 2010 ; cerezyme , 2010 ; cervarix , 2012 ; cimzia , 2013 ; cimzia , 2014 ; cyramza , 2014 ; elelyso , 2014 ; enbrel , 2010 ; entyvio , 2014a , b ; eperzan , 2014 ; epoetin alfa hexal , 2012 ; eporatio , 2009 ; erbitux , 2009 ; erwinaze , 2011 ; eylea , 2012 ; eylea , 2013 ; fabrazyme , 2006 ; fertavid , 2009 ; follistim , 2011 ; gazyva , 2014 ; gazyvaro , 2014 ; ghaderi et al . , 2012 ; gonal - f , 2010 ; granix , 2014 ; helixate nexgen , 2010 ; herceptin , 2010 ; humira , 2008 ; hylenex , 2012 ; ilaris , 2014 ; jetrea , 2012 ; jetrea , 2013 ; kadcyla , 2013 , 2014 ; kogenate bayer , 2010 ; krystexxa , 2013 ; lumizyme , 2010 ; luveris , 2005 ; mabthera , 2008 ; menveo , 2010 ; metalyse , 2006 ; myalept , 2014 ; myozyme , 2011 ; naglazyme , 2005 ; novoseven , 2006 ; novothirteen , 2012 ; nplate , 2009 ; nulojix , 2011 ; obizur , 2014 ; office of device evaluation and center for devices and radiological health , 2001 ; opgenra , 2014 ; orencia , 2012 ; ovidrel , 2014 ; ovitrelle , 2006 ; perjeta , 2013a , b ; prevnar , 2009 ; procrit , 2000 ; prolia , 2010 ; pulmozyme , 2010 ; raxibacumab , 2014 ; rebif , 2008 ; refacto af , 2014 ; remicade , 2009 ; reopro , 2013 ; rituxan , 2014 ; roactemra , 2013 ; saizen , 1987 ; serostim , 1987 ; simponi , 2009 ; simulect , 2008 ; somatropin biopartners , 2013 ; stelara , 2013 ; swiech et al . , 2012 ; sylvant , 2015 ; sylvant , 2014 ; synagis , 2009 ; tanzeum , 2014 ; tbo - filgrastim , 2012 ; thyrogen , 2010 ; tnkase , 2011 ; tretten , 2014 ; tysabri , 2011 ; us food and drug administration , 2010 , 2011 , 2012 , 2013 , 2014 ; vectibix , 2014 ; victoza , 2009 ; vimizim , 2014a , b ; voraxaze , 2012 ; xeomin , 2014 ; xiaflex , 2014 ; xolair , 2010 ; xyntha , 2011 ; yervoy , 2011 ; zaltrap , 2013a , b ; zevalin , 2009 ) . bacterial expression systems ( e.g. escherichia coli ) possess the advantages of being straightforward to culture , with rapid cell growth and high yields . in addition , protein expression can be initiated through promoter induction by addition of lactose or the lactose analogue isopropyl--d - thiogalactopyranoside ( iptg ; iptg induces the promoters lac , tac and trc ) . however , such systems are unable to produce complex , mammalian - like glycosylation due to the absence of the necessary enzymatic components and the intracellular compartmentalization required ( ghaderi et al . , 2012 ; graumann & premstaller , 2006 ) . in addition , mammalian proteins produced in these systems often aggregate , forming inclusion bodies , due to the low solubility of mammalian proteins in prokaryotic cells and absence of appropriate protein chaperone systems . proteins produced in bacterial expression systems must often be extracted from inclusion bodies and refolded . bacterial systems are therefore generally used for production of non - glycosylated proteins , including some mabs , hormones , cytokines and enzymes ( ghaderi et al . similar to bacterial expression systems , yeast expression systems ( e.g. saccharomyces cerevisiae and pichia pastoris ) achieve rapid cell growth and high - protein yields with straightforward production scalability and without the need for animal - derived growth factors ( gerngross , 2004 ) . yeast cell lines may also be used to produce proteins that can not be obtained from e. coli due to the problems associated with folding and stereochemistry ( gerngross , 2004 ) . the key challenge associated with yeast expression systems is their production of high mannose residues within their expressed ptms ( 50200 vs three molecules in human cells , as part of either n- or o - linked glycan structures ) , which may confer a short half - life and render proteins less efficacious and even immunogenic in humans ( dean , 1999 ; gemmill & trimble , 1999 ; gerngross , 2004 ; lam et al . the development of yeasts that have been genetically modified to address the issue of high mannose content has been reported ( chiba et al . , 1998 ; gerngross , 2004 ; ghaderi et al . the expression of a fully humanized sialylated glycoprotein in glycoengineered yeast constitutes a major advance in the use of yeast expression systems for biopharmaceutical manufacturing ( hamilton & gerngross , 2007 ) . plant and insect cell expression systems are able to produce proteins with complex glycosylation patterns ; however , the glycan structures produced are significantly different from those produced in humans ( ghaderi et al . , 2012 ) . plants lack many of the key glycosylated residues present in humans , most notably sialic acids . in addition , they produce 1,3-fructose and 1,2-xylose , which are absent in humans and may be immunogenic ( ghaderi et al . , 2012 ) . notably , in 2012 , taliglucerase alfa ( elelyso ; pfizer , new york , ny ) was approved by the us food and drug administration ( fda ) for the treatment of type 1 gaucher disease . this therapy is produced using genetically modified carrot plant root cells that produce the enzyme with a human compatible glycan profile ( elelyso , 2014 ) . insect cells infected with the viral vector baculovirus ( baculovirus - insect cell expression system ) can also efficiently express recombinant proteins , and these systems are mostly used for the development of virus - like particles and , subsequently , vaccines ( kost et al . , 2005 ; liu et al . , 2013 ) . however , although they produce n - glycan precursors , these are trimmed , resulting in either high mannose or paucimannose residues that do not develop further into terminal galactose and/or sialic acid residues ( kost et al . , 2005 ) . this is evidenced by the lack of either galactosyltransferase or sialyltransferase activity . as in plants . however , in recent years , there have been developments in the use of transgenic insect cells , with humanized protein glycosylation mechanisms ( kost et al . , the majority of currently licensed biotherapeutic products are produced in non - human mammalian expression systems ( table 1 ) , as these systems are able to produce ptms that ( outside of a human expression system ) most closely resemble those in humans ( ghaderi et al . , 2010 ) . these expression systems are used to produce mabs , hormones , cytokines , enzymes and clotting factors ( ghaderi et al . the most frequently used mammalian system is the cho cell line , which is used in the manufacture of > 70% of currently approved recombinant proteins ( butler & spearman , 2014 ) . first , cho cells are able to grow in suspension culture ( which enables large - scale production ; other cell lines , such as insect cells , also have this ability ) and serum - free chemically defined media ( enabling reproducibility across different batches of cultures with a better safety profile than in media that contain human- or animal - derived proteins ) ( kim et al . , 2012 ; lai et al . , 2013 ; rossi et al . , 2012 ) . historically , cho cells allowed gene amplification , resulting in a higher recombinant protein yield ( up to the gram per liter range for some proteins ) and specific productivity , which was previously an issue in other mammalian cell lines ( carlage et al . , 2012 ; kim et al . , 2012 ; yang et al . , 2014a , b ) . other advances , such as the creation of stronger expression units and advanced hosts , better selection strategies ( e.g. through technologic advances in screening for high - productivity clones ) and targeting the transgene to transcriptional hotspots ( site - specific integration of transgenes ) , also contribute to the high protein yields attained from these cells ( kim et al . , 2012 ) . in addition , this expression system is highly tolerant to changes in ph , oxygen level , pressure or temperature during manufacturing ( ghaderi et al . , 2012 ; lai et al . , 2013 ) . furthermore , due to the long period of time that this cell line has been used , there is a degree of familiarity with the cho platform within development and manufacturing organizations , regulatory agencies , and suppliers ( e.g. cell culture media suppliers ) , which could potentially decrease overall timelines . this familiarity may also be beneficial when assessing contaminant profiles ( e.g. host cell proteins ) , which may be better characterized for cho cells compared with newer cell lines . the first recombinant biotherapeutic protein produced in cho cells was tissue plasminogen activator , approved in 1986 ( kim et al . , 2012 ) . therefore , the safety profile of cho cells has been established for more than 20 years ( butler & spearman , 2014 ; kim et al . , 2012 ) . cho cells have been shown to have reduced susceptibility to certain viral infections compared with other mammalian cell lines ( e.g. the bhk cell line ) , and routine screening systems for adventitious agents are effective in detecting cell line infections ( berting et al . , 2010 ) . this reduced susceptibility may be due to the fact that many viral entry genes are not expressed in cho cells ( xu et al . , 2011 ) . further , there is perceived species barrier protection with the use of hamster - derived cells , reducing the potential risk of transfer of contaminating adventitious agents to humans ( berting et al . , 2010 ; swiech et al . , however , many viruses have the ability to cross the species barrier and may still pose a risk ( pauwels et al . , 2007 ) . perhaps the most important advantage of cho cells is that they are able to produce proteins with complex bioactive ptms that are similar to those produced in humans . however , cho cells are unable to produce some types of human glycosylation ( cho cells lack [2 - 6 ] sialyltransferase [1 - 3/4 ] fucosyltransferases ) and they produce glycans that are not expressed in humans , namely -gal and ngna ( bosques et al . , 2010 ; dietmair et al . , 2012 ; ghaderi et al . , circulating antibodies against both of these n - glycans are present in humans , which may lead to increased immunogenicity and altered pharmacokinetics of these products when used in humans ( ghaderi et al . , 2010 ; padler - karavani et al . , additional screening in cho cells is required in order to isolate clones lacking the -gal and ngna glycans . this screening may result in otherwise productive clones needing to be discarded ( ghaderi et al . , 2010 ) . however , the attachment of non - human glycans may not be a concern for therapeutic proteins that do not require glycosylation , which illustrates the importance of considering the specific product molecule when choosing an appropriate cell line for production of a protein . other mammalian cell lines used for the production of biotherapeutic proteins include bhk-21 cells , used in the production of some coagulation factors such as factor viii ( wurm , 2004 ) . when murine myeloma cell lines ( ns0 and sp2/0 ) have been used historically , they have generally been used in the production of mabs , for example , palivizumab and ofatumumab ( barnes et al . , 2000 ; butler & spearman , 2014 ; ghaderi et al . , these myeloma cells were derived from immunoglobulin - producing tumor cells that no longer produced their original immunoglobulins ; these cells possess the appropriate machinery for producing and secreting these proteins ( barnes et al . , 2000 ) . for proteins produced in all of these non - human cell lines , as well as those produced in human cell lines , potential safety concerns arise from the possibility of process - related contaminants and immunogenicity ( world health organization , 2013 ) . process - related contaminants may include infectious agents ( viral , bacterial , fungal , etc . ) with the potential to result in host infection , nucleic acid contaminants with the potential to integrate into the host genome ( theoretical ) , and other contaminants from the manufacturing process , such as exogenous non - human epitopes ( e.g. from animal serum used during the manufacturing process ) that can be incorporated into human cells and the resultant biotherapeutic protein ( ghaderi et al . , 2012 ) . hek293 and ht-1080 are the two human cell lines most often used in the production of biotherapeutic proteins , which offer the advantage of producing fully human ptms ( tables 2 and 3 ) ( loignon et al . , 2008 ; swiech et al . , human cells lines and their therapeutic protein products.cell lineproductindicationfda approval statusema approval statushek293 drotrecogin alfasevere septicemia / septic shockapproved 2001 ; withdrawn 2011approved 2002 ; withdrawn 2011 rfviiifchemophilia aapproved 2014submitted 2014 rfixfchemophilia bapproved 2014na dulaglutidetype 2 diabetesapproved 2014submitted 2014 human - cl rhfviiihemophilia asubmitted to the fdaapproved 2014ht-1080 agalsidase alfafabry diseasenaapproved 2001 epoetin deltaanemia secondary to chronic renal failurenaapproved 2002 ; withdrawn 2009 ( europe ) idursulfasehunter syndromeapproved 2006approved 2007 velaglucerase alfatype 1 gaucher diseaseapproved 2010approved 2010per.c6 cl184rabies virus infectionsubmitted to the fdana mor103rheumatoid arthritis , multiple sclerosisphase 1 clinical developmentphase 1 clinical developmentfda , us food and drug administration ; ema , european medicines agency ; hek , human embryonic kidney ; na , not approved ; rfviiifc , recombinant factor viii fc fusion protein ; rfixfc , recombinant factor ix fc fusion protein ; rhfviii , recombinant human factor viii . data obtained from publically available resources ( october 2014 ) ; all approved products may not be included . casademunt et al . , 2012 ; dynepo , 2007 ; elaprase , 2012 , 2013 ; eloctate , 2014 ; european medicines agency and committee for medicinal products for human use , 2014 ; glaesner et al . , 2010 ; octapharma , 2014 ; replagal , 2006 ; trulicity , 2014 ; vpriv , 2010a , b ; xigris , 2008 ) . comparison of human cell lines with other expression systems in the production of therapeutic proteins.advantagesdisadvantages absence of potentially immunogenic ptms due to human - compatible glycosylation easily grown in suspension serum - free culture amenable to a number of transfection methods clinical experience is not as extensive as for other cell lines , although experience is growing potential susceptibility to human viral contamination fda , us food and drug administration ; ema , european medicines agency ; hek , human embryonic kidney ; na , not approved ; rfviiifc , recombinant factor viii fc fusion protein ; rfixfc , recombinant factor ix fc fusion protein ; rhfviii , recombinant human factor viii . data obtained from publically available resources ( october 2014 ) ; all approved products may not be included . , 2012 ; dynepo , 2007 ; elaprase , 2012 , 2013 ; eloctate , 2014 ; european medicines agency and committee for medicinal products for human use , 2014 ; glaesner et al . , 2010 ; octapharma , 2014 ; replagal , 2006 ; trulicity , 2014 ; vpriv , 2010a , b ; xigris , 2008 ) . hek293 cells are easily grown in suspension serum - free culture , reproduce rapidly , are amenable to a number of transfection methods , and are highly efficient at protein production ( swiech et al . , 2012 ; thomas & smart , 2005 ) . hek293-h ( berkner , 1993 ) and 293-f ( vink et al . , 2014 ) cell lines are clonal isolates of the hek293 cell line that were selected for fast growth in serum - free medium , superior transfection efficiency , and a high level of protein production ( gibco , 2014 ) . subclone 293-h also has improved adherence to monolayer culture ( when serum - supplemented media are used ) compared with other cell lines . the hek293-t ( 293-t ) cell line expresses the simian virus 40 large t antigen and is capable of expressing high titers of viral gene vectors for use in gene therapy ( yamaguchi et al . , 2003 ) . hek293-t cells are often used for the production of retroviral vectors ( yamaguchi et al . , 2003 ) . hek293-ebna1 cells stably express the epstein - barr virus ebna-1 gene , controlled by the cytomegalovirus promoter and demonstrate a greater growth rate and maximal cell density relative to parental hek293 cells ( schlaeger & christensen , 1999 ) . hek293 cells have been widely used to produce research - grade proteins for many years and , more recently , five therapeutic agents produced in hek293 cells have been approved by the fda or the european medicines agency ( ema ) for therapeutic use . these agents are drotrecogin alfa ( xigris ; eli lilly corporation , indianapolis , in ) , recombinant factor ix fc fusion protein ( rfixfc ; biogen , cambridge , ma ) , recombinant factor viii fc fusion protein ( rfviiifc ; biogen , cambridge , ma ) , human cell line recombinant factor viii ( human - cl rhfviii ; nuwiq ; octapharma , lachen , switzerland ) and dulaglutide ( trulicity ; eli lily , indianapolis , in ) . drotrecogin alfa is a recombinant activated protein c that was approved by the fda in 2001 and by the ema in 2002 for the treatment of patients with severe sepsis . hek293 cells were chosen by the manufacturer for production of drotrecogin alfa because its activity required two ptms , propeptide cleavage and -carboxylation of its glutamic acid residues , which cho cells can not produce with adequate efficiency ( berkner , 1993 ; durocher & butler , 2009 ) . the product was approved ( bernard et al . , 2001 ) , but was later voluntarily withdrawn from the market by its manufacturer ( eli lilly ) in 2011 following the randomized placebo - controlled prospective recombinant human activated protein c worldwide evaluation in severe sepsis and septic shock ( prowess - shock ) trial , which demonstrated no mortality benefit with drotrecogin alfa compared with placebo for patients experiencing septic shock ( green et al . , 2012 ; ranieri et al . , 2012 ) . rfviiifc and rfixfc are recombinant fusion proteins that were approved by the fda in 2014 for the control and prevention of bleeding episodes , perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in people with hemophilia a and b , respectively ( alprolix , 2014 ; eloctate , 2014 ; mahlangu et al . , 2014 ; powell et al . , rfviiifc consists of b domain deleted recombinant factor viii genetically fused to the fc portion of immunoglobulin g1 ( igg1 ) and is produced in hek293-h cells ( dumont et al . the rfviiifc fed - batch culture process is robust at scales of 200 , 2000 and 15 000 liters , with the potential for a second - generation process to achieve even higher cell densities , on the order of 3.5 10 vc / ml ( huang et al . , 2014 ) . rfixfc was also produced using hek293-h cells , and consists of the factor ix sequence covalently linked to the fc domain of human igg1 ( alprolix , 2014 ; durocher & butler , 2009 ; mccue et al . , 2014 ; peters et al . , 2010 ) an essential ptm for fix activity is -carboxylation of the first 12 glutamic acid residues in the gla domain by vitamin k cells have been reported to have a greater capacity for -carboxylation than cho cells ( berkner , 1993 ) . furthermore , fviii contains six potential tyrosine sulfation sites , which are vital for fviii functionality and binding to von willebrand factor . fviii expressed from human cell lines has been reported to be fully sulfated ( kannicht et al . , 2013 ; peters et al . , 2013 ) . the use of a human cell line for replacement coagulation factors , such as rfviiifc and rfixfc , may result in reduced immunogenicity relative to non - human mammalian cell lines , as -gal and ngna glycan moieties are absent from these manufactured protein products ( bosques et al . , 2010 ; mccue et al . , 2014 , 2015 ; noguchi et al . , 1995 ) . however , it should be noted that several recombinant clotting factor products produced in non - human mammalian cell lines have been used successfully for many years . the development of inhibitors ( neutralizing antibodies ) against replacement clotting factors occurs in 30% of people with severe hemophilia a and 5% of those with severe hemophilia b. the causative f8 or f9 gene mutation plays a pivotal role in inhibitor development in hemophilia a and b , respectively , with large or complete deletions , non - sense mutations or inversions ( e.g. intron 22 inversion in the f8 gene ) being the most commonly associated mutations ( franchini & mannucci , 2011 ) . the impact of ptms on inhibitor development is unknown , and will need further research . importantly , none of the previously treated people with hemophilia in the phase 1/2a or phase 3 clinical studies developed inhibitors to the rfviiifc and rfixfc fusion products ( mahlangu et al . , 2014 ; powell et al . , 2012 , 2013 ; shapiro et al . , 2012 ) . human - cl rhfviii ( nuwiq ) , an additional factor viii replacement product for the management of hemophilia a , is being produced in the hek293-f cell line . like hek293-h cells , hek293-f cells are a derivation of hek293 cells that have been pre - adapted for growth in serum - free culture medium ( casademunt et al . , 2012 ) . human - cl rhfviii has been approved by the ema and submitted to the fda for approval ( octapharma , 2014 ) ; this product has been shown to exhibit a similar glycosylation profile to human plasma - derived factor viii , without -gal and ngna ( kannicht et al . , 2013 ) . glucagon-1-like peptide ( glp-1 ) fc fusion protein ( dulaglutide ) has been approved by the fda for the treatment of type 2 diabetes mellitus , and is produced using hek293-ebna cells ( glaesner et al . , 2010 ; trulicity , 2014 ) . large clinical trials have demonstrated its superiority over the dipeptidyl peptidase-4 inhibitor antagonist exenatide and its non - inferiority to liraglutide ( a glp-1 agonist ) , when added on to oral diabetic agents ( dungan et al . , 2014 ; wysham et al . , another human cell line , ht-1080 , was produced from a fibrosarcoma with an epithelial - like phenotype ( swiech et al . , 2012 ) . with the use of gene activation technology ( in which the endogenous dna promoter is replaced with a more potent type ) , four approved therapeutic proteins have been produced by shire ( swiech et al . , 2012 ) . 1 ) epoetin delta ( dynepo ) was approved by the ema in 2002 for the treatment of anemia secondary to chronic renal failure ( dynepo , 2007 ; elaprase , 2013 ; replagal , 2006 ; swiech et al . 2 ) iduronate-2-sulfatase ( idursulfase ; elaprase ) is licensed as enzyme replacement therapy ( ema in 2007 and fda in 2006 ) for the treatment of hunter syndrome ( mucopolysaccharidosis ii ) , an x - linked lysosomal storage disorder ( elaprase , 2013 ) . 3 ) agalsidase alfa ( replagal ; shire human genetic therapies , danderyd , sweden ) was approved by the ema in 2001 for the treatment of fabry disease ( replagal , 2010 ) . compared with agalsidase beta ( fabrazyme ; genzyme therapeutics , cambridge , ma ) , which is produced using cho cells for a similar indication ( fabrazyme , 2010 , 2014 ) , agalsidase alfa has shown similar enzyme kinetics . however , agalsidase alfa demonstrates a lesser uptake into fibroblasts from patients with fabry disease and also lower concentrations in the kidney , heart and spleen of mice ( lee et al . , 2003 ) . a single clinical study has compared the two products ; this showed no significant differences for all efficacy outcomes , and there were no differences for the development of antibodies ( vedder et al . , 2007 ) . 4 ) the fourth agent produced in ht-1080 cells , velaglucerase alfa ( vpriv ; shire human genetic therapies , lexington , ma ) , was approved in 2010 ( fda and ema ) for the treatment of type 1 gaucher disease ( dynepo , 2007 ; elaprase , 2013 ; replagal , 2006 ; swiech et al . velaglucerase alfa has been compared with two similar products : imiglucerase , produced using cho cells , and taliglucerase alfa , produced using carrot cells ( ben turkia et al . , 2013 ; tekoah et al . , these products have diverse glycan profiles and the studies have generally shown comparable uptake into macrophages , in vitro enzymatic activity , stability , organ distribution and efficacy ( ben turkia et al . however , neutralizing antibodies to imiglucerase were noted in 24% of patients , which had an impact on enzyme activity . it was noted that various factors , such as the production cell line and glycosylation , may be responsible for the difference in immunogenicity , however , the specificity of the anti - imiglucerase antibodies was not stated ( ben turkia et al . , 2013 ) . notably , studies that evaluated epoetin delta produced in ht-1080 cells demonstrated differences in glycosylation compared with erythropoietin produced in cho cells , including a lack of ngna in the proteins ( butler & spearman , 2014 ; llop et al . , 2008 ; shahrokh et al . , 2011 ) . however , there were additional overlapping isoforms present in endogenous human erythropoietin isolated from urine and serum relative to epoetin delta that could not be accounted for by sialic residues alone . human cell lines have been extensively utilized for the production of products that are currently in clinical development . in addition , human cell lines are a frequently used expression system for biomedical research due to their production of human ptms and high productivity . as productivity may vary across clonal isolates , it is important to screen for those clones with the highest yield of the therapeutic protein ( berkner , 1993 ) . the per.c6 cell line was created from human embryonic retinal cells , immortalized via transfection with the adenovirus e1 gene ( havenga et al . , 2008 ) . this system was originally developed for the production of human adenovirus vectors for use in vaccine development and gene therapy ( butler & spearman , 2014 ) . an investment was made in this cell line in order to develop a human expression system , and now an advantage of per.c6 is its ability to produce a high level of protein when used in the production of human igg ( jones et al . , 2003 ) . however , this does not require amplification of the incorporated gene ( jones et al . , 2003 ) . currently , a variety of products utilizing the per.c6 cell line are in phase 1 or 2 clinical trials ( durocher & butler , 2009 ) , including the mor103 mab , a human igg antibody against granulocyte macrophage colony - stimulating factor , and cl184 , an antibody against the rabies virus ( nagarajan et al . , 2014 ) . mor103 is in clinical development for the treatment of rheumatoid arthritis and multiple sclerosis . in a phase 1b/2a , randomized , placebo - controlled study , mor103 was active in patients with moderately severe rheumatoid arthritis ; a small number of patients developed anti - mor103 antibodies ( behrens et al . cl184 is a combination of two mabs , human igg1( ) and human igg1( ) ( bakker et al . , 2005 ) . in a phase 1 clinical study , it demonstrated a favorable safety profile and rapid development of rabies virus neutralizing activity , while there was no evidence to suggest the development of human anti - human antibodies ( bakker et al . , 2008 ) . the cap cell line is derived from human amniocytes obtained through amniocentesis ; these cells are immortalized through an adenovirus type 5 e1 gene ( schiedner et al . , 2008 ; in addition to the ability to produce human ptms , the primary advantage of this cell is the potential for high protein yields ( schiedner et al . , 2008 ) . the hkb-11 cell line was created through polyethylene glycol fusion of hek293-s and a human b - cell line ( modified burkitt lymphoma cells ) ( cho et al . , 2003 ; durocher & butler , 2009 ; picanco - castro et al . , 2013 ) . the advantages of this cell line include high - level protein production without the formation of aggregates , which can be a problem in other human cell lines ( picanco - castro et al . , 2013 ) . notably , hkb-11 has demonstrated increased expression of human fviii compared with expression in hek293 and bhk21 ( mei et al . , 2006 ) . similar to other human cell lines , it has been shown to produce human glycosylation patterns including ( 2,3 ) and ( 2,6 ) sialic acid linkages ( picanco - castro et al . , 2013 ) . hkb-11 has been used to produce a recombinant factor viii protein and tissue factor ( cho et al . , 2003 ) . a more recently developed cell line , huh-7 , originates from a human hepatocellular carcinoma ( enjolras et al . , 2012 ) . a recent study has shown that the huh-7-cd4 clone is capable of producing recombinant human factor ix with a human glycosylation profile . ptm profiles ( e.g. glycosylation , sialylation , phosphorylation and sulfation ) were similar to plasma - derived and recombinant factor ix ( rfix ) , and were improved relative to rfix produced in cho cells ( enjolras et al . , more recently , the huh-7 cell line has been used to produce mutant forms of rfix that have improved binding affinity for activated fviii , and also demonstrated enhanced clotting activity in mice ( perot et al . , 2015 ) . the human - specific glycosylation pattern of the ptms produced by human cell lines offer several advantages compared with those produced in animal cell lines . although other mammalian cells can produce similar ptms to human cells , most also produce -gal and ngna , ptms that are not present in the structure of human proteins ( ghaderi et al . , 2012 ) . patterns of post - translational glycosylation are known to affect protein yield , bioactivity , and clearance ( ghaderi et al . , 2010 ) . in addition , antibodies to ngna have been widely reported to occur in humans ( chung et al . , 2008 ; ghaderi et al . , one study utilizing an ngna knockout mouse model demonstrated increased immunogenicity of cetuximab due to anti - ngna antibodies ( ghaderi et al . , 2010 ) . in addition , in patients receiving the mab cetuximab for the treatment of colorectal or head and neck cancers , the majority of severe hypersensitivity reactions observed in clinical trials were associated with pre - existing ige antibodies against -gal ( chung et al . , 2008 ; such antibodies may alter the efficacy or immunogenicity of proteins with the presence of non - human glycan structures . thus , human cell lines can serve as a valuable niche expression system for biotherapeutic proteins that require human ptms . a theoretical concern with the use of human cell lines is an increased risk of transfer of human adventitious agents , given the lack of a species barrier ( swiech et al . , however , current manufacturing technologies , typically inclusive of multiple viral inactivation or clearance steps , such as nanofiltration , have largely mitigated this concern and may provide more effective viral clearance than has been observed in cho cells ( kelley et al . production of biotherapeutic proteins in human cell lines is expanding , with several products currently approved for clinical use and others in clinical development in different therapeutic areas . advantages of human expression systems include achieving equal productivity to other mammalian cell lines and the production of proteins that lack potentially immunogenic , non - human ptms ( most notably -gal and ngna ) . in the future , with additional research investments and a continuation of the technologic advances that have already led to improvements in the use of human cell lines for protein manufacture , human cell lines will be further optimized , more sophisticated product collection strategies will be developed , and these cell lines may become one of the preferred platforms for protein biotherapeutic production . editorial support for the writing of this manuscript was provided by melissa yuan , md , of medergy , and was funded by biogen .

abstractbiotherapeutic proteins represent a mainstay of treatment for a multitude of conditions , for example , autoimmune disorders , hematologic disorders , hormonal dysregulation , cancers , infectious diseases and genetic disorders . the technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s . although most biotherapeutic proteins developed to date have been produced using the mammalian chinese hamster ovary and murine myeloma ( ns0 , sp2/0 ) cell lines , there has been a recent shift toward the use of human cell lines . one of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post - translational modifications ( ptms ) that are consistent with those seen on endogenous human proteins . although other mammalian cell lines can produce ptms similar to human cells , they also produce non - human ptms , such as galactose-1,3-galactose and n - glycolylneuraminic acid , which are potentially immunogenic . in addition , human cell lines are grown easily in a serum - free suspension culture , reproduce rapidly and have efficient protein production . a possible disadvantage of using human cell lines is the potential for human - specific viral contamination , although this risk can be mitigated with multiple viral inactivation or clearance steps . in addition , while human cell lines are currently widely used for biopharmaceutical research , vaccine production and production of some licensed protein therapeutics , there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins ( compared with other types of cell lines ) . with additional research investment , human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins .

Introduction Non-human expression systems used to manufacture biotherapeutic products Human cell lines used to manufacture licensed products Human cell lines used in the expression of proteins in clinical and preclinical development Perceptions of risks versus benefits of using human cell lines Future perspectives Declaration of interest

the most common mammalian ( non - human ) cell lines used for therapeutic protein production include chinese hamster ovary ( cho ) cells , baby hamster kidney ( bhk21 ) cells and murine myeloma cells ( ns0 and sp2/0 ) ( estes & melville , 2014 ) . however , these non - human mammalian cell lines also produce ptms that are not expressed in humans , namely galactose-1,3-galactose ( -gal ) and n - glycolylneuraminic acid ( ngna ) . other mammalian cell lines used for the production of biotherapeutic proteins include bhk-21 cells , used in the production of some coagulation factors such as factor viii ( wurm , 2004 ) . when murine myeloma cell lines ( ns0 and sp2/0 ) have been used historically , they have generally been used in the production of mabs , for example , palivizumab and ofatumumab ( barnes et al . comparison of human cell lines with other expression systems in the production of therapeutic proteins.advantagesdisadvantages absence of potentially immunogenic ptms due to human - compatible glycosylation easily grown in suspension serum - free culture amenable to a number of transfection methods clinical experience is not as extensive as for other cell lines , although experience is growing potential susceptibility to human viral contamination fda , us food and drug administration ; ema , european medicines agency ; hek , human embryonic kidney ; na , not approved ; rfviiifc , recombinant factor viii fc fusion protein ; rfixfc , recombinant factor ix fc fusion protein ; rhfviii , recombinant human factor viii . the use of a human cell line for replacement coagulation factors , such as rfviiifc and rfixfc , may result in reduced immunogenicity relative to non - human mammalian cell lines , as -gal and ngna glycan moieties are absent from these manufactured protein products ( bosques et al . although other mammalian cells can produce similar ptms to human cells , most also produce -gal and ngna , ptms that are not present in the structure of human proteins ( ghaderi et al . advantages of human expression systems include achieving equal productivity to other mammalian cell lines and the production of proteins that lack potentially immunogenic , non - human ptms ( most notably -gal and ngna ) . in the future , with additional research investments and a continuation of the technologic advances that have already led to improvements in the use of human cell lines for protein manufacture , human cell lines will be further optimized , more sophisticated product collection strategies will be developed , and these cell lines may become one of the preferred platforms for protein biotherapeutic production . other mammalian cell lines used for the production of biotherapeutic proteins include bhk-21 cells , used in the production of some coagulation factors such as factor viii ( wurm , 2004 ) . when murine myeloma cell lines ( ns0 and sp2/0 ) have been used historically , they have generally been used in the production of mabs , for example , palivizumab and ofatumumab ( barnes et al . comparison of human cell lines with other expression systems in the production of therapeutic proteins.advantagesdisadvantages absence of potentially immunogenic ptms due to human - compatible glycosylation easily grown in suspension serum - free culture amenable to a number of transfection methods clinical experience is not as extensive as for other cell lines , although experience is growing potential susceptibility to human viral contamination fda , us food and drug administration ; ema , european medicines agency ; hek , human embryonic kidney ; na , not approved ; rfviiifc , recombinant factor viii fc fusion protein ; rfixfc , recombinant factor ix fc fusion protein ; rhfviii , recombinant human factor viii . the use of a human cell line for replacement coagulation factors , such as rfviiifc and rfixfc , may result in reduced immunogenicity relative to non - human mammalian cell lines , as -gal and ngna glycan moieties are absent from these manufactured protein products ( bosques et al . although other mammalian cells can produce similar ptms to human cells , most also produce -gal and ngna , ptms that are not present in the structure of human proteins ( ghaderi et al . advantages of human expression systems include achieving equal productivity to other mammalian cell lines and the production of proteins that lack potentially immunogenic , non - human ptms ( most notably -gal and ngna ) . in the future , with additional research investments and a continuation of the technologic advances that have already led to improvements in the use of human cell lines for protein manufacture , human cell lines will be further optimized , more sophisticated product collection strategies will be developed , and these cell lines may become one of the preferred platforms for protein biotherapeutic production .

an important feature of the color interaction in quantum chromodynamics ( qcd ) is that the outgoing partons produced in the hard interaction continue to interfere with each other during their fragmentation phase . this phenomenon , called color coherence , manifests itself by the relative abundance of soft radiation in the region between the color connected final - state partons and the suppression of soft radiation elsewhere . color coherence phenomena were initially observed in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathrm { e}^+\mathrm { e}^-$$\end{document}e+e- collisions by several experiments at petra , pep and lep [ 18 ] . these experiments showed the coherence effect in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } 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\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-\pi \le \vardelta \phi _ { 23}\le \pi $ $ \end{document}-23 ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \eta _ { 23 } = { { \mathrm{sign}}}(\eta _ 2 ) \cdot ( \eta _ 3 - \eta _ 2)$$\end{document}23=sign(2)(3-2 ) , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0 \le \beta \le \pi $ $ \end{document}0. the absolute value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \phi _ { 23}$$\end{document}23 in eq . 1 and the sign of the pseudorapidity of the second jet , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathrm{sign}}}(\eta _ 2)$$\end{document}sign(2 ) , in the definition of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \eta _ { 23}$$\end{document}23 are introduced to map symmetric configurations around \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \phi _ { 23 } = 0$$\end{document}23=0 or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta = 0$$\end{document}=0 onto the same \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} value . for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \phi _ { 23 } = 0$$\end{document}23=0 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} is defined to be zero or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi $ $ \end{document} depending on the sign of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \eta _ { 23}$$\end{document}23 being positive or negative . in the case of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \eta _ { 23 } = 0$$\end{document}23=0 , which can not happen simultaneously with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \phi _ { 23 } = 0$$\end{document}23=0 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} is defined to equal \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi /2$$\end{document}/2.fig . 1visualization of the observable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} in ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta , \phi $ $ \end{document}, ) space using a simulated three - jet event . the sizes of the rectangular boxes are proportional to the particle energies visualization of the observable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} in ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta , \phi $ $ \end{document}, ) space using a simulated three - jet event . the sizes of the rectangular boxes are proportional to the particle energies in a naive leading - order model the two partons are produced back - to - back in the transverse plane . one of the two partons may radiate a third parton . in the absence of color coherence effects there is no preferred direction of emission of this third parton around the radiating parton . in contrast , when color coherence effects are present , the third parton will tend to lie in the event plane defined by the emitting parton and the beam axis . therefore , in the presence of color coherence , the third jet population along the event plane ( in particular near \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta \approx 0$$\end{document}0 ) will be enhanced and out of the plane ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta \approx \pi /2$$\end{document}/2 ) will be suppressed . the color coherence effects are expected to become stronger in the region between the second jet and the remnant when the angle between them becomes smaller . therefore the study of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} variable is performed in two situations : when the second jet is rather central ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta _ 2 | \le 0.8$$\end{document}|2|0.8 ) and when the second jet is more forward ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.8 < |\eta _ 2 | \le 2.5$$\end{document}0.8<|2|2.5 ) . the aims of this paper areto measure the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distributions , normalized to the total number of events in each region , as 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}$$\beta $ $ \end{document} separately in the central ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta _ 2 | \le 0.8$$\end{document}|2|0.8 ) and forward region ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.8<|\eta _ 2 | \le 2.5$$\end{document}0.8<|2|2.5 ) : 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } f_{\eta _ 2 , i}(\beta ) = \frac{n_{\eta , i}}{n_{\eta } } , \end{aligned}$$\end{document}f2,i()=n,in , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n_{\eta } $ $ \end{document}n is the total number of events in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta _ 2$$\end{document}2 region , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n_{\eta , i}$$\end{document}n,i the number of events in the given \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i$$\end{document}ith \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} bin of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta _ 2$$\end{document}2 region . the choice of this normalization significantly reduces the impact of experimental systematic uncertainties such as the uncertainty in the luminosity.to gauge the sensitivity of the variable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} to color coherence effects.to compare our measurements to the predictions of mc event generators with various implementations of color coherence . to measure the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distributions , normalized to the total number of events in each region , as a function of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} separately in the central ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta _ 2 | \le 0.8$$\end{document}|2|0.8 ) and forward region ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.8<|\eta _ 2 | \le 2.5$$\end{document}0.8<|2|2.5 ) : 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } f_{\eta _ 2 , i}(\beta ) = \frac{n_{\eta , i}}{n_{\eta } } , \end{aligned}$$\end{document}f2,i()=n,in , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n_{\eta } $ $ \end{document}n is the total number of events in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta _ 2$$\end{document}2 region , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n_{\eta , i}$$\end{document}n,i the number of events in the given \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i$$\end{document}ith \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} bin of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta _ 2$$\end{document}2 region . the choice of this normalization significantly reduces the impact of experimental systematic uncertainties such as the uncertainty in the luminosity . to gauge the sensitivity of the variable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} to color coherence effects . to compare our measurements to the predictions of mc event generators with various implementations of color coherence a detailed description of the cms experiment can be found elsewhere ; so here we describe the detector systems most relevant to the present analysis . the central feature of the cms apparatus is a superconducting solenoid of 6 m internal diameter , providing a magnetic field of 3.8 t. within the field volume , a silicon pixel and strip tracker , a lead tungstate crystal electromagnetic calorimeter and a brass / scintillator hadron calorimeter ( hcal ) are installed . the central tracking system provides coverage up to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta |= 2.5$$\end{document}||=2.5 in pseudorapidity and the calorimeters up to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta |=3.0$$\end{document}||=3.0 . an iron and quartz - fiber cherenkov forward hadron calorimeter ( hf ) covers the pseudorapidity range \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$3.0<|\eta |<5.0$$\end{document}3.0<||<5.0 . the cms detector records events using a two - level trigger system consisting of a hardware - based level-1 ( l1 ) trigger and a software - based high - level trigger ( hlt ) . for this study , single jet triggers that reconstruct jets from calorimeter energy deposits at l1 and hlt are used to select events based on different \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t } } $ $ \end{document}pt jet thresholds . five different triggers with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t } } $ $ \end{document}pt thresholds of 30 , 50 , 70 , 100 , and 140\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { gev}$$\end{document}gev are used to select the events . the triggers were prescaled during the 2010 run when the associated rate exceeded the allocated band width except the highest - threshold one . therefore , the events are split into five different bins in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t1}}$$\end{document}pt1 with each bin containing the events collected during a period when the appropriate trigger was not prescaled . each bin starts at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t}\text { min}}$$\end{document}ptmin defined in such a way that the associated trigger efficiency exceeds 99 % . table 2 lists the binning in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t1}}$$\end{document}pt1 , and , for each bin , it gives the associated trigger , the number of selected events , and the integrated luminosity for the period during which the given trigger was not prescaled . jets are reconstructed with the anti-\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{\mathrm { t}}$$\end{document}kt algorithm , which is implemented in the fastjet package using a distance parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r = 0.5$$\end{document}r=0.5 , from a list of particle candidates reconstructed using the particle - flow ( pf ) algorithm . this pf algorithm reconstructs all particle candidates in each event using an optimized combination of information from all cms subdetector systems : muons , electrons ( with associated bremsstrahlung photons ) , photons ( unconverted and converted ) , and charged / neutral hadrons . the four - vectors of the neutral particles are computed by assuming that they come from the primary vertex , which is defined as the vertex with the highest sum of transverse momenta of all reconstructed tracks pointing to it . the reconstructed jet energy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e$$\end{document}e is defined as the scalar sum of the energies of the constituents , and the jet momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\varvec{p}}}$$\end{document}p is the vector sum of the momenta of the constituents . the jet transverse momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t}}$$\end{document}pt is the component of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\varvec{p}}}$$\end{document}p perpendicular to the beam . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e$$\end{document}e and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\varvec{p}}}$$\end{document}p values of a reconstructed jet are further corrected for the response of the detector , which is obtained from mc simulations , test beam results , and pp collision data [ 16 , 17 ] . the corrections account for the presence of multiple pp collisions in the same or adjacent bunch crossings ( pileup interactions ) using the jet area method . events are required to have a primary vertex reconstructed within 24 cm of the detector center along the beam line . additional selection criteria are applied to each event to remove any spurious jet - like features originating from isolated noise patterns in certain hcal regions . events having at least three jets with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t } } > 30$$\end{document}pt>30 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { gev}$$\end{document}gev are selected . the pseudorapidity of the two leading jets must be within \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta _ { 1 } |,|\eta _ { 2 } |\le 2.5$$\end{document}|1|,|2|2.5 , while for the third jet no constraints are applied in order to avoid a bias in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} measurement . to further reduce the background from misidentified jets , i.e. , jets resulting from noise in the electromagnetic , hadron and/or hadron forward calorimeters , a set of tight identification criteria are applied : each jet should contain at least two particles , one of which is a charged hadron , and the jet energy fraction carried by neutral hadrons , photons , muons , and electrons should be less than 90 % . with these criteria the contamination of the sample with misidentified jets is suppressed to a level less than 1 % . the dijet invariant mass of the two leading jets , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{12}$$\end{document}m12 , is required to exceed 220 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { gev}$$\end{document}gev to ensure a back - to - back configuration . with this requirement more than 98 % of the events have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\vardelta \phi _ { 12}-\pi |<1$$\end{document}|12-|<1 . finally the distance in the ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta , \phi $ $ \end{document}, ) space between the second and third jets is constrained to be \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.5<\vardelta r_{23 } = \sqrt{(\vardelta \eta _ { 23})^2 + ( \vardelta \phi _ { 23})^2}<1.5$$\end{document}0.5<r23=(23)2+(23)2<1.5 in order to ensure a three - jet topology where the third jet is closer to the second jet . the numbers of events passing the selection criteria in each \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t1}}$$\end{document}pt1 bin are summarized in table 2 . the measured \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \eta _ { 23}$$\end{document}23 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \phi _ { 23}$$\end{document}23 distributions are compared to various mc models in figs . 2 and 3 . a study of the amount of energy collected by the hf detector indicated that there is no diffractive component in the data sample.fig . 2observed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \eta _ { 23}$$\end{document}23 distributions , corrected for detector effects , compared to mc predictions by pythia 6 , pythia 8 , herwig++ , and madgraph + pythia 6 . 3observed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \phi _ { 23}$$\end{document}23 distributions , corrected for detector effects , compared to mc predictions by pythia 6 , pythia 8 , herwig++ , and madgraph + pythia 6 . the mc samples are normalized to the total number of events in datatable 1summary of the event selectionselection criteria \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t1}}>100$$\end{document}pt1>100 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { gev}$$\end{document}gev , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t3}}>30$$\end{document}pt3>30 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { gev}$$\end{document}gev \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta _ 1 |,|\eta _ 2 | \le 2.5$$\end{document}|1|,|2|2.5 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{12}>220$$\end{document}m12>220 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { gev}$$\end{document}gev \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } 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\usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${>}250$$\end{document}>250 1403631 99916 72815 271all193 13187 510105 621 observed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \eta _ { 23}$$\end{document}23 distributions , corrected for detector effects , compared to mc predictions by pythia 6 , pythia 8 , herwig++ , and madgraph + pythia 6 . the mc samples are normalized to the total number of events in data observed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \phi _ { 23}$$\end{document}23 distributions , corrected for detector effects , compared to mc predictions by pythia 6 , pythia 8 , herwig++ , and madgraph + pythia 6 . the mc samples are normalized to the total number of events in data summary of the event selection the binning in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t1}}$$\end{document}pt1 and , for each bin , the associated trigger , the integrated luminosity for the period during which the given trigger was not prescaled , and the number of selected events . the reconstructed jets are compared to the predictions of four different monte carlo generators that simulate jet production in pp collisions at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt{s } = 7\,\text { tev } $ $ \end{document}s=7tev . the numbers of events for all generator samples is much higher than the number of collected data events so the statistical uncertainties in the mc predictions are not visible in the figures . the pythia ( version 6.422 ) event generator uses leading - order ( lo ) matrix elements to generate the 2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rightarrow $ $ \end{document} 2 hard process in perturbative qcd ( pqcd ) and the parton shower ( ps ) model to simulate higher - order processes [ 2224 ] . the ps model gives a good description of parton emission when the emitted partons are close in phase space . , except that z2 uses the cteq6l1 parton distribution functions ( pdfs ) of the proton in which the parton showers are ordered in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t } } $ $ \end{document}pt . the older d6 t tune [ 2931 ] , where parton showers are ordered in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q^2$$\end{document}q2 , is considered for comparison . the d6 t tune was designed to describe the lower - energy results of ua5 and cdf . the color coherence effects are implemented in pythia 6 by means of an angular ordering algorithm where the effects can be switched on and off via the steering parameters mstp(67 ) and mstj(50 ) , which control the initial - state and the final - state showers , respectively . the pythia 8 ( version 8.145 ) event generator , used with tune 4c , orders the parton showers in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t } } $ $ \end{document}pt and models the underlying event using the multiple - parton interaction model from pythia 6 including initial- and final - state qcd radiation . the color coherence effects are implemented in a similar manner as for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t } } $ $ \end{document}pt - ordered showers in pythia 6 . the herwig++ [ 11 , 34 ] ( version 2.4.2 ) event generator takes lo matrix elements and simulates parton showers using the coherent branching algorithm with angular ordering of showers . the cluster hadronization model is used in the formation of hadrons from the quarks and gluons produced in the parton shower . the color coherence effects are implemented by the angular ordering of emissions in the parton shower using the coherent branching algorithm . the madgraph 4 ( version 2.24 ) event generator is interfaced with pythia 6 for the parton showering and the hadronization using the d6 t tune and uses fixed - order matrix element calculations for the multiparton topologies . from two to four partons the color coherence for the hard jets at leading order comes from the exact qcd color amplitudes in the model . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{\mathrm { t}}$$\end{document}kt mlm matching scheme applied with a matching parameter of 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { gev}$$\end{document}gev avoids double - counting between the partons from madgraph and the ps . the measurement of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution is performed in two regions defined by the pseudorapidity of the second jet : the central region \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta _ 2 | \le 0.8$$\end{document}|2|0.8 and the forward region \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.8<|\eta _ 2 | \le 2.5$$\end{document}0.8<|2|2.5 . the angular correlation effects considered in this analysis appear to have a reduced sensitivity to the transverse momentum of the leading jet \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t1}}$$\end{document}pt1 . consequently different \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t1}}$$\end{document}pt1 bins are merged into one single bin . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution in a given \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta _ 2$$\end{document}2 region is obtained as a sum of the events weighted by the luminosity collected by the trigger used in the associated \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_{\mathrm { t1}}$$\end{document}pt1 bin . in case of mc samples the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution is obtained by summing together the events weighted by their generation level weight in a given \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta _ 2$$\end{document}2 region . the normalized \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution is then obtained by dividing the weighted number of events in a given bin of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} by the total weighted number of events in the given \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta _ 2$$\end{document}2 region . in order to correct for the smearing effects induced by the detector resolution , an unfolding procedure is performed using the response matrices obtained from mc event generators . for this purpose the events generated with the mc programs ( pythia 6 , pythia 8 , madgraph + pythia 6 , and herwig++ ) are processed through a full cms detector simulation package based on geant 4 . particle - level jets are built from the four - vectors of the mc generated particles with hadronization , but without detector effects . these jets are obtained using the same jet algorithm as for the reconstructed events . the resolutions in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \eta _ { 23}$$\end{document}23 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vardelta \phi _ { 23}$$\end{document}23 are found to be of the order of 0.005 to 0.01 , depending on the transverse momentum and pseudorapidity of the jets . an iterative bayesian unfolding technique implemented in the roounfold package is used to derive the unfolding corrections to the measured \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distributions from the detector effects . the impact of the unfolding on the normalized distributions is typically of the order of 1 % . most of the systematic effects cancel out in the normalized \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution , but the residual influence of several sources of systematic uncertainty has been considered : the jet energy scale uncertainty is evaluated varying the jet response by 2.55 % , depending on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta $ $ \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}$$p_{\mathrm { t } } $ $ \end{document}pt of the jets . the impact of this source of systematic uncertainties is below \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$1~\%$$\end{document}1%.the jet energy and angular resolutions are accounted for by varying them by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\pm } 10~\%$$\end{document}10% and rebuilding the response matrices for the unfolding accordingly . .the uncertainty due to the unfolding procedure is estimated by the dependence of the response matrix on the choice of mc generator , alternative response matrices are built using alternative generators : pythia 6 , pythia 8 and madgraph + pythia 6 . the observed effect is of the order of 0.5 % .the measurement is found to be insensitive to the number of pileup interactions within statistical fluctuations . in the data corresponding to this analysis the total systematic uncertainties for each bin are about 2 % , and a list of the major uncertainties is summarized in table 3 . each systematic source was found to be fully correlated between \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \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}$$\eta _ 2$$\end{document}2 bins [ 43 , 44 ] . however , the various systematic sources are uncorrelated among themselves . table 3typical systematic and statistical uncertainties in the normalized \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} spectrum and the statistical errorsuncertainty sources \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta _ 2 | \le 0.8$$\end{document}|2|0.8 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.8<|\eta _ 2 | \le 2.5$$\end{document}0.8<|2|2.5 jet energy scale ( jes)1.0 % 1.0 % jet energy resolution ( jer)0.4 % 0.5 % jet angular resolution ( jar)0.5 % 0.6 % physics model ( pm ) used in unfolding0.6 % 0.7 % statistical uncertainty4.0 % 3.7 % the jet 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events compared to the pythia and madgraph generators near the event plane ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta = 0$$\end{document}=0 ) and a suppression in the transverse plane ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta = \pi /2$$\end{document}=/2 ) . the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi ^2$$\end{document}2 comparisons of data with mc simulation , taking into account the statistical and systematic correlations between different data points , are shown separately for the central and forward regions in table 6 . the number of degrees of freedom ( ndf ) is 17 , which is the number of bins minus one to account for the constraint imposed by the normalization.fig . 5the ratio of the various mc predictions to the measured \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution . the yellow band represents the systematic uncertainty , while the green band represents the total uncertaintytable 6values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi ^2$$\end{document}2 for comparisons of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution for the data with the predictions of various mc generators . the number of degrees of freedom for both regions is 17mc event generator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi ^2$$\end{document}2/ndf \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$|\eta _ 2 | \le 0.8$$\end{document}|2|0.8 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.8<|\eta _ 2 | \le 2.5$$\end{document}0.8<|2|2.5 pythia 6 z22.58.1 pythia 8 4c1.76.4 herwig++ 2.31.23.5 madgraph + pythia 61.63.3 the ratio of the various mc predictions to the measured \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution . the yellow band represents the systematic uncertainty , while the green band represents the total uncertainty values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi ^2$$\end{document}2 for comparisons of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution for the data with the predictions of various mc generators . the number of degrees of freedom for both regions is 17 none of the models used in the analysis describes the data satisfactorily . even though pythia 6 was adjusted with the tevatron data , it fails to describe the lhc data since the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi ^2$$\end{document}2/ndf is large . the pythia 8 tune 4c generator describes the data better than pythia 6 over the entire phase space , but the disagreement in the forward region is not negligible . the herwig++ event generator describes the data better than the other mc generators in the central region , but the agreement is poor in the forward region . finally , when madgraph is used with the exact \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2 \rightarrow 3$$\end{document}23 matrix element calculations at lo , the global description of the data is improved with respect to pythia 6 alone . the impact of the color coherence effects is studied by switching them on and off for the first emission in the initial- and final - state showers in pythia 6 . 6 that the agreement between the data and the simulation deteriorates when the color coherence effects in the mc events are suppressed . more quantitatively , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi ^2$$\end{document}2 divided by the number of degrees of freedom increases up to 7.7 in the central region and 11.5 in the forward region . the first emission in the initial- and final - state showers contributes roughly the same order . using pythia , it has been verified that the impact of the non - perturbative component of the qcd calculation ( hadronization and underlying event ) is negligible for this analysis . 6 , is that the data clearly support larger color coherence effects than in present mc implementations.fig . 6the mc predictions for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution from pythia 6 , with and without color coherence effects in the first branching of the initial- and final - state showers , compared to the measurement . the yellow band represents the systematic uncertainty , while the green band represents the total uncertainty the mc predictions for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} distribution from pythia 6 , with and without color coherence effects in the first branching of the initial- and final - state showers , compared to the measurement . the yellow band represents the systematic uncertainty , while the green band represents the total uncertainty color coherence effects in multijet events have been studied in a sample of pp collisions corresponding to an integrated luminosity of 36 pb\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{-1}$$\end{document}-1 , collected with the cms detector at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt{s } = 7\,\text { tev } $ $ \end{document}s=7tev . distributions of the variable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} , which was previously used in similar analyses at the tevatron , are used to measure the angular correlation between the second and third jets in transverse - momentum order , in the pseudorapidity and azimuthal angle space . the measurements , unfolded for detector effects , are compared to the predictions of the mc event generators pythia 6 , pythia 8 , herwig++ , and madgraph + pythia 6 in the central and forward rapidity regions . we have shown that the variable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} is sensitive to color coherence effects , and insensitive to the hadronization and underlying event . it is necessary to implement the color coherence effects in mc simulations to better describe the data . although the mc models in the analysis include this effect by default , none of them describes the data satisfactorily for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} values . the pythia 6 expectations predict weaker color coherence effects than those observed , while pythia 8 exhibits a better agreement with the data . the madgraph mc generator , which uses the exact \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2 \rightarrow 3$$\end{document}23 matrix element calculations at lo matched to pythia 6 for parton showering , improves the agreement with data with respect to pythia 6 alone , while herwig++ describes the data in the central region better than the other mc generators but shows discrepancies in the forward region .

a study of color coherence effects in pp collisions at a center - of - mass energy of 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { tev}$$\end{document}tev is presented . the data used in the analysis were collected in 2010 with the cms detector at the lhc and correspond to an integrated luminosity of 36 pb\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{-1}$$\end{document}-1 . events are selected that contain at least three jets and where the two jets with the largest transverse momentum exhibit a back - to - back topology . the measured angular correlation between the second- and third - leading jet is shown to be sensitive to color coherence effects , and is compared to the predictions of monte carlo models with various implementations of color coherence . none of the models describe the data satisfactorily .

Introduction The CMS detector Event selection Monte Carlo models Measurement of the normalized Results Summary

the technique originally developed by the tevatron experiments is used to study color coherence effects in pp collisions at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt{s}=7$$\end{document}s=7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { tev}$$\end{document}tev with the compact muon solenoid ( cms ) detector . the choice of this normalization significantly reduces the impact of experimental systematic uncertainties such as the uncertainty in the luminosity.to gauge the sensitivity of the variable \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta $ $ \end{document} to color coherence effects.to compare our measurements to the predictions of mc event generators with various implementations of color coherence . the dijet invariant mass of the two leading jets , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m_{12}$$\end{document}m12 , is required to exceed 220 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\,\text { gev}$$\end{document}gev to ensure a back - to - back configuration . the yellow band represents the systematic uncertainty , while the green band represents the total uncertainty color coherence effects in multijet events have been studied in a sample of pp collisions corresponding to an integrated luminosity of 36 pb\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{-1}$$\end{document}-1 , collected with the cms detector at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt{s } = 7\,\text { tev } $ $ \end{document}s=7tev .

tremor is the most common movement disorder which is defined as a compulsively , rhythmic , shaking movement of a body part . one of the most general reasons for tremor is idiopathic parkinson 's disease ( pd ) , a progressive neurodegenerative disease . the core clinical features of idiopathic pd include bradykinesia , followed by resting tremor , rigidity , and problems with stance stability . these symptoms will become evident after a loss of 5070% dopaminergic cells in the substantia . however , the differential diagnosis of idiopathic pd is difficult in the early stage due to the atypical and ambiguous symptoms and signs , especially when tremor is the solely obvious symptom . monosymptomatic resting tremor ( mrt ) is a descriptive term for resting tremor persisting for more than 2 years without other cardinal signs of pd . some reports demonstrated that mrt is characterized by a presynaptic dopaminergic deficit with the evidence of dopamine transporter positron emission tomography ( dat - pet ) and supported that mrt represents a phenotype of pd . most of these patients end up in the tremor - dominant variant of pd , but this may last more than a decade . however , in clinical practice , dopaminergic functional imaging revealed some patients with mrt to be scans without evidence of dopaminergic deficits ( swedds ) . few studies have been reported to reveal the prevalence of swedds in mrt patients and to illuminate the clinical and electrophysiological characteristics of mrt patients grouped by the dat - pet results . this study aimed to investigate the presynaptic dopamine function by dat - pet in a prospective mrt group and to evaluate the role of clinical features and electrophysiological characteristics in the differential diagnosis . we prospectively enrolled 43 consecutive patients with mrt who visited movement disorder clinics in beijing tiantan hospital from april 2011 to december 2013 . the diagnosis of mrt was confirmed by movement disorder specialists according to consensus statement of the movement disorder society on tremor . the mrt patients must meet the following criteria : ( 1 ) pure or predominant resting tremor ; ( 2 ) no sign of bradykinesia , rigidity , or abnormal gait and posture ; and ( 3 ) tremor 2 years . patients were excluded if they had : ( 1 ) specific etiologies for the asymmetric resting tremor ( e.g. , psychogeny , medication - induced , stroke , and multiple sclerosis ) ; ( 2 ) a clear history of pd or essential tremor ( et ) in the family ; ( 3 ) an obvious lesions of intracephalic structure on head computed tomography ( ct)/magnetic resonance imaging ( mri ) scans . information regarding demographics , medical and family history , disease course , and treatment were collected during a face - to - face interview in all patients . the unified parkinson 's disease rating scale ( updrs ) was tested by well - trained neurologists before dat - pet examination , thus there was a blindness between clinical data and dat - pet . the study was approved by the institutional review board ( committee for medical ethics ) of beijing tiantan hospital , capital medical university . surface electromyographic ( emg ) recordings of the most involved limbs in all positions were performed on all patients . the tremor frequency ( hz ) , mean amplitude ( mv ) , and pattern ( synchronous or alternating ) were assessed . the amplitude was measured peak to the valley for each burst , and the duration was measured peak to peak for each burst . the recording session included 5 min test and 5 min rest subsequently in each definite limb position . the examination was performed in a room where the patients had the opportunity to be familiar with the circumstances and felt relax before the test . c--cft , used as the dat radioligand for pet tracer , was synthesized at department of nuclear medicine , pla general hospital . c--cft was prepared by reaction of [ c ] methyl triflate with the n - desmethyl precursor , nor - p - cft ( rbi , natick , ma , usa ) . a high - resolution pet / ct scanner ( datscan ; gemini gxl-16 , philips , the netherlands ) was used for each patient at nuclear medicine center of the second hospital of armed police beijing office . anti - parkinsonism drugs must not be taken at least 12 h before pet , and dat agonists 72 h before the pet . the head of the caudate and putamen on the two consecutive planes with best articulation were selected to be the regions of interest ( rois ) on bilateral hemispheres . striatal asymmetry index ( sai ) was used to describe the asymmetry of the radioactivity uptake in the bilateral striatum . risp and rcon represent the uptake value of dat tracer in the striatum ipsilateral or contralateral to the clinically affected body side , respectively . continuous parameters and absolute counts were shown as mean standard deviation ( sd ) , and categorical parameters were shown as percentage . the differences in clinical and electrophysiological features between swedds and pd patients were calculated using the unpaired , two - tailed t - test for continuous variables and chi - square test or fisher exact test for categorical variables by spss 17.0 ( spss inc . , we prospectively enrolled 43 consecutive patients with mrt who visited movement disorder clinics in beijing tiantan hospital from april 2011 to december 2013 . the diagnosis of mrt was confirmed by movement disorder specialists according to consensus statement of the movement disorder society on tremor . the mrt patients must meet the following criteria : ( 1 ) pure or predominant resting tremor ; ( 2 ) no sign of bradykinesia , rigidity , or abnormal gait and posture ; and ( 3 ) tremor 2 years . patients were excluded if they had : ( 1 ) specific etiologies for the asymmetric resting tremor ( e.g. , psychogeny , medication - induced , stroke , and multiple sclerosis ) ; ( 2 ) a clear history of pd or essential tremor ( et ) in the family ; ( 3 ) an obvious lesions of intracephalic structure on head computed tomography ( ct)/magnetic resonance imaging ( mri ) scans . information regarding demographics , medical and family history , disease course , and treatment were collected during a face - to - face interview in all patients . the unified parkinson 's disease rating scale ( updrs ) was tested by well - trained neurologists before dat - pet examination , thus there was a blindness between clinical data and dat - pet . the study was approved by the institutional review board ( committee for medical ethics ) of beijing tiantan hospital , capital medical university . surface electromyographic ( emg ) recordings of the most involved limbs in all positions were performed on all patients . the tremor frequency ( hz ) , mean amplitude ( mv ) , and pattern ( synchronous or alternating ) were assessed . the amplitude was measured peak to the valley for each burst , and the duration was measured peak to peak for each burst . the recording session included 5 min test and 5 min rest subsequently in each definite limb position . the examination was performed in a room where the patients had the opportunity to be familiar with the circumstances and felt relax before the test . c--cft , used as the dat radioligand for pet tracer , was synthesized at department of nuclear medicine , pla general hospital . c--cft was prepared by reaction of [ c ] methyl triflate with the n - desmethyl precursor , nor - p - cft ( rbi , natick , ma , usa ) . a high - resolution pet / ct scanner ( datscan ; gemini gxl-16 , philips , the netherlands ) was used for each patient at nuclear medicine center of the second hospital of armed police beijing office . anti - parkinsonism drugs must not be taken at least 12 h before pet , and dat agonists 72 h before the pet . the head of the caudate and putamen on the two consecutive planes with best articulation were selected to be the regions of interest ( rois ) on bilateral hemispheres . striatal asymmetry index ( sai ) was used to describe the asymmetry of the radioactivity uptake in the bilateral striatum . risp and rcon represent the uptake value of dat tracer in the striatum ipsilateral or contralateral to the clinically affected body side , respectively . continuous parameters and absolute counts were shown as mean standard deviation ( sd ) , and categorical parameters were shown as percentage . the differences in clinical and electrophysiological features between swedds and pd patients were calculated using the unpaired , two - tailed t - test for continuous variables and chi - square test or fisher exact test for categorical variables by spss 17.0 ( spss inc . , eight patients were not involved in the present analyses for their symptoms of tremor lasting < 2 years and two patients were excluded due to incomplete documents of clinical features or electrophysiological measurements . a total of 33 patients with definite mrt ( mean age 55.3 11.6 years , 45.5% men ) constituted the final sample [ table 1 ] . pd were diagnosed in 84.8% ( n = 28 ) and sweeds in 15.2% ( n = 5 ) of cases , respectively . there were no significant differences in age , gender , and age at onset of tremor between swedds and pd groups . the duration of disease was longer in the sweeds group than in the pd group ( 13.0 years vs. 3.0 years , p < 0.05 ) . the percentage of hoehn - yahr score 1 was similar among two groups ( 20% vs. 25% , p > demographic and clinical characteristics of swedds and pd patients * these values were calculated by test ; others without star mark were calculated by t - test ; nonparametric test ; : not applicable , calculated by fisher exact test . sd : standard deviation ; swedds : scans without evidence of dopaminergic deficits ; updrs : unified parkinson s disease rating scale ; pd : parkinson s disease . according to the pet comparison with the norm values in the center , subjects were divided into two groups . a normal symmetrical uptake of c--cft in the bilateral striatum was seen in all five patients ( swedds group ) [ figures 1 and 2 ] . an asymmetrical reduced uptake in the striatum was seen in 28 patients ( pd group ) [ figures 1 and 3 ] . positron emission tomography ( pet ) to distinguish scans without evidence of dopaminergic deficits ( swedds ) from parkinson 's disease ( pd ) and tremor analysis between groups . transaxial slices of pet / tremor analysis of a patient with swedds ( left ) and another patient with right - sided pd ( right ) . dopamine transporter positron emission tomography help to distinguish scans without evidence of dopaminergic deficits from parkinson 's disease in monosymptomatic resting tremor patients . dopamine transporter positron emission tomography help to identify parkinson 's disease in the early age in monosymptomatic resting tremor patients . for nonmotor aspects of experiences of daily living ( part i of updrs ) , there were no significant differences in cognitive impairment and depression between two groups [ table 1 ] . for motor experiences of daily living ( part ii of updrs ) , pd patients were more likely to have difficulties in dressing and hygiene ( p < 0.05 ) . more patients in pd group tended to have difficulties in walking ( 67.9% in pd group and 20.0% in swedds group , p > 0.05 ) , although the difference was of no statistical significance . the two groups were similar in speech and freezing of gait . for motor examination ( part iii of updrs ) , the score was much lower in the sweeds group than in the pd patient group , and the difference was statistically significant ( 11.0 2.0 vs. 23.2 12.5 , p < 0.01 ) . eighteen ( 64.3% ) pd patients had a resting tremor , and 25 ( 89.3% ) had a postural tremor . a resting tremor was recorded in 3 ( 60.0% ) swedds patients and postural tremor was recorded in all 5 ( 100% ) swedds patients . the difference in tremor distribution ( resting tremor or postural tremor ) between two groups was not statistically significant . but the proportion of bilateral tremor was much higher in swedds group than in the pd group ( 100% vs. 32.1% , p < 0.05 ) [ table 2 ] . tremor analysis in patients with swedds and pd * these values were calculated by test ; others without star mark were calculated by t - test ; nonparametric test ; : not applicable , calculated by fisher exact test . swedds : scans without evidence of dopaminergic deficits ; pd : parkinson s disease ; sd : standard deviation . electrophysiological study the electrophysiological findings in 2 different conditions ( resting or postural tremor ) in studying group are shown in table 2 . no subject had a kinetic tremor in either of the two groups . there were no significant differences in the frequency and amplitude of resting or postural tremor between two groups . tremor analysis : frequency and amplitude table 2 shows the results of emg measurements in both resting and postural tremors . the frequency of resting tremor in swedds group tended to be higher than in the pd group , but without significant difference ( p = 0.08 ) . the amplitude of postural tremor tended to be higher in swedds group than in pd group , but the difference was of no significance ( p = 0.05 ) . tremor patterns analysis table 2 also shows the results of tremor patterns in resting or postural tremors . in resting tremor patterns analysis , the percentage of synchronous pattern was 60.0% in swedds group and 57.1% in pd group ( p > 0.05 ) , while the percentage of alternating pattern of resting tremor was 80.0% in swedds group and 78.6% in pd group ( p > 0.05 ) . in postural tremor patterns analysis , synchronous pattern was 80.0% in the swedds group and 56.7% in pd group , while alternating pattern was 60.0% in the swedds group and 75.0% in pd group ( p > 0.05 ) . eight patients were not involved in the present analyses for their symptoms of tremor lasting < 2 years and two patients were excluded due to incomplete documents of clinical features or electrophysiological measurements . a total of 33 patients with definite mrt ( mean age 55.3 11.6 years , 45.5% men ) constituted the final sample [ table 1 ] . pd were diagnosed in 84.8% ( n = 28 ) and sweeds in 15.2% ( n = 5 ) of cases , respectively . there were no significant differences in age , gender , and age at onset of tremor between swedds and pd groups . the duration of disease was longer in the sweeds group than in the pd group ( 13.0 years vs. 3.0 years , p < 0.05 ) . the percentage of hoehn - yahr score 1 was similar among two groups ( 20% vs. 25% , p > demographic and clinical characteristics of swedds and pd patients * these values were calculated by test ; others without star mark were calculated by t - test ; nonparametric test ; : not applicable , calculated by fisher exact test . sd : standard deviation ; swedds : scans without evidence of dopaminergic deficits ; updrs : unified parkinson s disease rating scale ; pd : parkinson s disease . according to the pet comparison with the norm values in the center , subjects were divided into two groups . a normal symmetrical uptake of c--cft in the bilateral striatum was seen in all five patients ( swedds group ) [ figures 1 and 2 ] . an asymmetrical reduced uptake in the striatum was seen in 28 patients ( pd group ) [ figures 1 and 3 ] . positron emission tomography ( pet ) to distinguish scans without evidence of dopaminergic deficits ( swedds ) from parkinson 's disease ( pd ) and tremor analysis between groups . transaxial slices of pet / tremor analysis of a patient with swedds ( left ) and another patient with right - sided pd ( right ) . dopamine transporter positron emission tomography help to distinguish scans without evidence of dopaminergic deficits from parkinson 's disease in monosymptomatic resting tremor patients . dopamine transporter positron emission tomography help to identify parkinson 's disease in the early age in monosymptomatic resting tremor patients . for nonmotor aspects of experiences of daily living ( part i of updrs ) , there were no significant differences in cognitive impairment and depression between two groups [ table 1 ] . for motor experiences of daily living ( part ii of updrs ) , pd patients were more likely to have difficulties in dressing and hygiene ( p < 0.05 ) . more patients in pd group tended to have difficulties in walking ( 67.9% in pd group and 20.0% in swedds group , p > 0.05 ) , although the difference was of no statistical significance . the two groups were similar in speech and freezing of gait . for motor examination ( part iii of updrs ) , the score was much lower in the sweeds group than in the pd patient group , and the difference was statistically significant ( 11.0 2.0 vs. 23.2 12.5 , p < 0.01 ) . eighteen ( 64.3% ) pd patients had a resting tremor , and 25 ( 89.3% ) had a postural tremor . a resting tremor was recorded in 3 ( 60.0% ) swedds patients and postural tremor was recorded in all 5 ( 100% ) swedds patients . the difference in tremor distribution ( resting tremor or postural tremor ) between two groups was not statistically significant . but the proportion of bilateral tremor was much higher in swedds group than in the pd group ( 100% vs. 32.1% , p < 0.05 ) [ table 2 ] . tremor analysis in patients with swedds and pd * these values were calculated by test ; others without star mark were calculated by t - test ; nonparametric test ; : not applicable , calculated by fisher exact test . swedds : scans without evidence of dopaminergic deficits ; pd : parkinson s disease ; sd : standard deviation . electrophysiological study the electrophysiological findings in 2 different conditions ( resting or postural tremor ) in studying group are shown in table 2 . there were no significant differences in the frequency and amplitude of resting or postural tremor between two groups . tremor analysis : frequency and amplitude table 2 shows the results of emg measurements in both resting and postural tremors . the frequency of resting tremor in swedds group tended to be higher than in the pd group , but without significant difference ( p = 0.08 ) . the amplitude of postural tremor tended to be higher in swedds group than in pd group , but the difference was of no significance ( p = 0.05 ) . tremor patterns analysis table 2 also shows the results of tremor patterns in resting or postural tremors . in resting tremor patterns analysis , the percentage of synchronous pattern was 60.0% in swedds group and 57.1% in pd group ( p > 0.05 ) , while the percentage of alternating pattern of resting tremor was 80.0% in swedds group and 78.6% in pd group ( p > 0.05 ) . in postural tremor patterns analysis , synchronous pattern was 80.0% in the swedds group and 56.7% in pd group , while alternating pattern was 60.0% in the swedds group and 75.0% in pd group ( p > 0.05 ) . there is still a debate on the etiology of mrt and its relation to pd . in this present study normal c - cft uptake in the striatum by dat - pet was found in 15.2% ( 5/33 ) of patients with mrt , who were diagnosed as the swedds , while reduced uptake in the striatum was found in 84.8% ( 28/33 ) of the patients , thus the corresponding diagnosis were pd . there were some differences in the clinical characteristics between pd and swedds groups in our study . we found that more patients in the pd group had difficulties in walking and balance . similar result was seen in a former study focusing on the gaits and posture of the patients . swedds patients were found to had a normal trunk and elbow posture , normal stride length variability , and normal bilateral step phase coordination , all of which were abnormal in pd patients . in addition , we found that dressing and hygiene were more likely to be difficult in pd group . we also found that the score in motor examination in updrs ( part iii ) was higher in pd group than in swedds group , which indicated dyskinesia was remarkably severe in the former group . thus , we concluded that the updrs might help to make a differential diagnosis between pd and sweeds . there are few studies on the difference in tremor patterns between pd and swedd patients . the basic tremor parameters overlapped between swedds , pd , segmental dystonia and et in previous electrophysiological studies . recent study found that pd feature re - emergent tremor coexisted with the highest amplitude in the resting tremor , while swedds , dystonia , and et subjects had the highest amplitude during action tremor . similarly , we found in our study that the action tremor amplitude tended to be higher in swedds group , although the difference was not significant . different tremor types have been separated in pd , including classic parkinsonian tremor at rest , postural and kinetic tremors of different frequencies , pure postural or kinetic tremor . the heterogeneity in swedds , which means dystonic tremors may be responsible for a number of swedds subjects as well as et , might explain partly that the tremor patterns were similar in the two groups . unfortunately , in the comprehensive analysis , when the tremor patterns were divided into different groups according to its features , we found there was no significant difference between pd and swedds groups . the diagnosis of mrt is made when the tremor syndrome persists for more than 2 years without bradykinesia , rigidity or postural dysfunction . the resting tremor in mrt differs obviously from the postural tremor or action tremor in et . the mrt patient has rarely family history and is nonresponsive to beta blockers when comparing to ets . ninety percent of the mrt patients can be thought to be subclinical pd with the evidence of presynaptic dopamine neuronal dysfunction by pet . besides careful clinical examination and electrophysiological test , functional imaging plays an important role in the differential diagnosis in patients with isolated tremor symptoms . recent studies found that when faced a diagnostic challenge in early pd , cardiac i - mibg scans alone or combined with dat - pet , may help to differentiate patients with swedds from patients with pd . in the present study , sweeds were diagnosed in 15.2% ( n = 5 ) of cases and the result was consistent with previous study , in which approximately 1115% of early pd patients showed normal dopaminergic functional imaging ( swedds ) . various ligands had been proved to provide identical data regardless of the kinetics and specificity for the dat . in light of i - fp - cit ( 123i - n - v - fluoropropyl-2b - carbomethoxy-3b-(4-iodophenyl ) nortropane ) , a widely used dat ligand in single photon emission computed tomography ( spect ) is difficult to prepare and expensive ; however , dat - pet is reasonable to detect presynaptic dopamine neuronal dysfunction . dat - pet is also an independent factor associated with the long - term motor and nonmotor outcomes when adjusted for age , gender , pd duration , baseline pd severity , and pd medications at the time of assessment . dat imaging has an important role in the differential diagnosis , management decision , and prognostic prediction in early pd and clinical uncertain parkinsonian syndrome . there are many other causes for tremor , such as physiological tremor , psychogenic tremor , et , and myoclonus - dystonia . differential diagnosis of tremor should base on history taking , physical examination , electrophysiological test , mri , and nuclear imaging of dat when the diagnosis remains uncertain . dat density in corpus striatum decreased 10% every year in the pd group , while 0.5% every year in the control group . we found that the patients with negative dat - pet results had no pd symptoms or signs in the next years while the patients with positive dat - pet results had some pd symptoms or signs in the following years . first , the findings of our study should be interpreted with caution considering the sample size . however , our prospective cohort is reasonable when compared with other researches focused on swedds , in which the sample size was generally small , varied from 8 to 33 . second , whether swedds patients had signs of dystonia was not recorded in our study . thus , we can not support the hypothesis that adult onset dystonia is the underlying diagnosis in this sub - group of patients with swedds . finally , transcranial sonography and susceptibility - weighted imaging can aid the differentiation in previous studies . these tests were not included in the analysis due to the integrity constraint and will be added in the further studies . in summary , dat - pet brain imaging can facilitate early and accurate diagnosis and help guide prognosis and treatment decisions . it can also help to avoid inappropriate dopaminergic drug therapy , as well as the long - term financial burden .

background : the relationship between monosymptomatic resting tremor ( mrt ) and parkinson 's disease ( pd ) remains controversial . in this study , we aimed to assess the function of presynaptic dopaminergic neurons in patients with mrt by dopamine transporter positron emission tomography ( dat - pet ) and to evaluate the utility of clinical features or electrophysiological studies in differential diagnosis.methods:thirty-three consecutive patients with mrt were enrolled prospectively . the unified parkinson 's disease rating scale and electromyography were tested before dat - pet . striatal asymmetry index ( sai ) was calculated , and a normal dat - pet was defined as a sai of < 15% . scans without evidence of dopaminergic deficits ( swedds ) were diagnosed in patients with a subsequent normal dat - pet and structural magnetic resonance imaging.results:twenty-eight mrt patients with a significant reduction in uptake of dat binding in the striatum were diagnosed with pd , while the remained 5 with a normal dat - pet scan were swedds . as for uprds , the dressing and hygiene score , walking in motor experiences of daily living ( part ii ) and motor examination ( part iii ) were significant different between two groups ( p < 0.05 and p < 0.01 , respectively ) . bilateral tremor was more frequent in the swedds group ( p < 0.05 ) . the frequency of resting tremor and the amplitude of postural tremor tend to be higher in the swedds group ( p = 0.08 and p = 0.05 , respectively).conclusions : mrt is heterogeneous in presynaptic nigrostriatal dopaminergic degeneration , which can be determined by dat - pet brain imaging . clinical and electrophysiological features may provide clues to distinguish pd from swedds .

I M Patients Electrophysiological test Dopamine transporter positron emission tomography Statistical analysis R Demographic data Positron emission tomography results Unified Parkinson's Disease Rating Scale Tremor analysis D

some reports demonstrated that mrt is characterized by a presynaptic dopaminergic deficit with the evidence of dopamine transporter positron emission tomography ( dat - pet ) and supported that mrt represents a phenotype of pd . however , in clinical practice , dopaminergic functional imaging revealed some patients with mrt to be scans without evidence of dopaminergic deficits ( swedds ) . this study aimed to investigate the presynaptic dopamine function by dat - pet in a prospective mrt group and to evaluate the role of clinical features and electrophysiological characteristics in the differential diagnosis . the unified parkinson 's disease rating scale ( updrs ) was tested by well - trained neurologists before dat - pet examination , thus there was a blindness between clinical data and dat - pet . the unified parkinson 's disease rating scale ( updrs ) was tested by well - trained neurologists before dat - pet examination , thus there was a blindness between clinical data and dat - pet . positron emission tomography ( pet ) to distinguish scans without evidence of dopaminergic deficits ( swedds ) from parkinson 's disease ( pd ) and tremor analysis between groups . dopamine transporter positron emission tomography help to distinguish scans without evidence of dopaminergic deficits from parkinson 's disease in monosymptomatic resting tremor patients . for motor experiences of daily living ( part ii of updrs ) , pd patients were more likely to have difficulties in dressing and hygiene ( p < 0.05 ) . for motor examination ( part iii of updrs ) , the score was much lower in the sweeds group than in the pd patient group , and the difference was statistically significant ( 11.0 2.0 vs. 23.2 12.5 , p < 0.01 ) . but the proportion of bilateral tremor was much higher in swedds group than in the pd group ( 100% vs. 32.1% , p < 0.05 ) [ table 2 ] . the frequency of resting tremor in swedds group tended to be higher than in the pd group , but without significant difference ( p = 0.08 ) . the amplitude of postural tremor tended to be higher in swedds group than in pd group , but the difference was of no significance ( p = 0.05 ) . in resting tremor patterns analysis , the percentage of synchronous pattern was 60.0% in swedds group and 57.1% in pd group ( p > 0.05 ) , while the percentage of alternating pattern of resting tremor was 80.0% in swedds group and 78.6% in pd group ( p > 0.05 ) . positron emission tomography ( pet ) to distinguish scans without evidence of dopaminergic deficits ( swedds ) from parkinson 's disease ( pd ) and tremor analysis between groups . dopamine transporter positron emission tomography help to distinguish scans without evidence of dopaminergic deficits from parkinson 's disease in monosymptomatic resting tremor patients . for motor experiences of daily living ( part ii of updrs ) , pd patients were more likely to have difficulties in dressing and hygiene ( p < 0.05 ) . for motor examination ( part iii of updrs ) , the score was much lower in the sweeds group than in the pd patient group , and the difference was statistically significant ( 11.0 2.0 vs. 23.2 12.5 , p < 0.01 ) . but the proportion of bilateral tremor was much higher in swedds group than in the pd group ( 100% vs. 32.1% , p < 0.05 ) [ table 2 ] . the frequency of resting tremor in swedds group tended to be higher than in the pd group , but without significant difference ( p = 0.08 ) . the amplitude of postural tremor tended to be higher in swedds group than in pd group , but the difference was of no significance ( p = 0.05 ) . in resting tremor patterns analysis , the percentage of synchronous pattern was 60.0% in swedds group and 57.1% in pd group ( p > 0.05 ) , while the percentage of alternating pattern of resting tremor was 80.0% in swedds group and 78.6% in pd group ( p > 0.05 ) . in this present study normal c - cft uptake in the striatum by dat - pet was found in 15.2% ( 5/33 ) of patients with mrt , who were diagnosed as the swedds , while reduced uptake in the striatum was found in 84.8% ( 28/33 ) of the patients , thus the corresponding diagnosis were pd . we also found that the score in motor examination in updrs ( part iii ) was higher in pd group than in swedds group , which indicated dyskinesia was remarkably severe in the former group .

eales disease ( ed ) is an idiopathic inflammatory venous occlusion that primarily affects the peripheral retina of adults . young adult males have been reported to have an increased prevalence , with the peak age of onset as 2035 years and a reported range of 1363 years [ 1 , 2 ] . however , a study of 55 patients by gieser et al . showed that men and women were affected equally . ed has been reported from the uk , usa , and canada in the later half of nineteenth and early twentieth centuries . however , the disease is more prevalent in india and portions of the middle east . the number of new cases per year in one of the referral hospital in india where this study was conducted ( sankara nethralaya , chennai , india ) for the last 10 years shows an average of 310 fresh patients per year . most patients present with symptoms of floaters , specks , cobwebs , blurring , or decreased vision associated with vitreous hemorrhage . often , patients complain of uniocular symptoms , but ophthalmic examination reveals early changes of ed in the other eye . the major manifestations of ed are inflammation , neovascularization , vitreous hemorrhage , and retinal detachment . stages of ed broadly include stage of retinal phlebitis , stage of peripheral nonperfusion , and stage of retinal neovascularization . a new classification system has been proposed for ed as stage 1 : periphlebitis of small ( 1a ) and large ( 1b ) caliber vessels with superficial retinal hemorrhages ; stage 2a : capillary nonperfusion , stage 2b : neovascularization elsewhere / of the disc ; stage 3a : fibrovascular proliferation , stage 3b : vitreous hemorrhage ; stage 4a : traction / combined rhegmatogenous retinal detachment , and stage 4b : rubeosis iridis , neovascular glaucoma , complicated cataract , and optic atrophy . association of ed with several systemic diseases , mainly tuberculosis , has been reported . however , several reports have shown association of a variety of neurological and hematological disorders with ed . the histopathological hallmark in ed is the adhesion of leukocytes to the endothelium and the infiltration of these cells into the retinal parenchyma . phagocyte - generated free radicals have been implicated in mediating tissue damage associated with various inflammatory vasculopathies . our earlier studies show elevated levels of reactive oxygen species and reactive nitrogen species products correlating with diminished antioxidant status in patients with ed , and based on these studies , increased oxidative stress has been strongly implicated in the etiopathogenesis of ed [ 2 , 68 ] . intraocular neovascularization occurs in numerous ischemic retinal disorders , including diabetic retinopathy , ischemic retinal - vein occlusion , and retinopathy of prematurity . this proliferation often results in vitreous hemorrhage , retinal detachment , and endothelial - cell - specific angiogenic and vasopermeable factor , namely , vegf , an ischemia - induced ocular angiogenic factor that has an important role in mediating the neovascular response of diabetic retinopathy and other ischemic retinal disorders . pigment epithelium - derived growth factor ( pedf ) is a glycoprotein and a potent inhibitor of ischemia induced neovascularization . decrease in pedf is reported in the vitreous of the proliferative diabetic retinopathy patients [ 10 , 11 ] . as neovascularization is prominent in ed this study attempts to look at these two predominant factors one acting as pro- and the other as anti - angiogenic , by estimating the vegf and pedf levels , respectively , in the vitreous specimen of ed patients and compare it with that of the pdr which also involves neovascularization and with mh which does not involve the same . pdr is characterized by presence of newly formed blood vessels arising from the optic disc ( nvd ) or elsewhere on the surface of retina ( nve ) . these new vessels may be flat along the surface or elevated into the vitreous cavity and may be with or without a component of glial cell proliferation , fibrovascular proliferation ( fvp ) . as the fibrous tissue contracts , the eyes develop recurrent preretinal and vitreous hemorrhages , tractional retinal detachment , and is occasionally combined with rhegmatogenous retinal detachment . a macular hole is a full - thickness defect of retinal tissue involving the anatomic fovea , thereby affecting central visual acuity . the effect of clinical intervention in terms of the steroid and laser therapy given on the levels of these factors has also been looked into . undiluted vitreous samples ( 0.30.5 ml ) were harvested from the midvitreous at the start of vitrectomy . vitreous fluid samples were obtained from 26 ed patients ( 25 m , 1 f , 33 8 years ) , 17 pdr ( 11 m , 6 f , 54 9 years ) , and seven patients with macular hole ( 3 m , 4 f ; 60 10 years ) . all the patients were proved clinically by ophthalmologists based on fundus examination by indirect ophthalmoscopy . as far as the diagnosis of ed is concerned , initially , the patient presents with retinal perivasculitis predominantly affecting the peripheral retina ( inflammatory stage ) , then sclerosis of retinal veins indicating retinal ischemia ( ischemic stage ) , and finally retinal or optic disk neovascularization , recurrent vitreous hemorrhage with or without retinal detachment ( proliferative stage ) . all patients with idiopathic , full - thickness retinal defect of more than 400 m and with posterior vitreous detachment ( stage 4 idiopathic macular hole ; ftmh ) were included in the study for the vitreous specimen as the disease control . careful slit lamp biomicroscopy is usually sufficient to establish the diagnosis in the majority of cases . optical coherence tomography is used to allow detailed cross - sectional examination of macular holes and may be effective in distinguishing them from other lesions where doubt exists . all cases of types 1 or 2 diabetes mellitus with ocular signs namely , nve , nvd , fvp , vitreous hemorrhage , tractional retinal detachment , or combined retinal detachment were included in the study for the vitreous specimen from the pdr cases . systemic steroid is given to the ed cases in the form of prednisolone , 1 mg / kg bodyweight given orally and is gradually tapered by 10 mg / week . tables 1 , 2 , and 3 show the clinical details of the patients in the study . table 1clinical details of the eales disease casess . no.age/sexod/osclassificationquadranglessteroidlasersystemicillness126/mod3b4nonot donenil230/mos3b4nosectoral phcnil319/mod3b4yessectoral phcnil435/mod3b4nosectoral phcnil525/mod3a4noprpnil639/mos3b4nosectoral phcnil733/mod3b4nonot donenil829/mos3b4nonot donenil936/mos4a4nonot doneht1030/mod3b4noprpnil1133/mod4a4yesprpnil1214/mod3b4yesprpnil1351/fod3b4noprpnil1436/mos3b4noprpnil1524/mos3b4noprpnil1628/mod4a4nonot donenil1745/mos3b4nonot donenil1835/mod3b4nonot donenil1934/mos3b4yesnot doneht2043/mos4a4nonot donenil2133/mos3b4noprpnil2238/mos4a4nonot donenil2323/mos3b4noprpnil2433/mos4a4noprpnil2536/mos3b4nonot donenil2636/mos3b4nonot doneniltable 2clinical details of the proliferative diabetic retinopathy casess . no.age/sexod/ostype of dmlasersystemic complicationsdiabetes duration152/mos1prpht , crf19260/mod2not donenil15358/mos2not donenil11450/mos2prpcrf6550/fos2prpht , crf5659/mos2prpht24742/fos1prpht , crf15861/mos2prpht15943/fod1prpht , crf , dyslipidemia31038/mod2not donenil101157/fos2not donedyslipidemia71252/mos2not donenil81367/fod2not doneht61442/mod1not donenil101565/mos2prpht71661/mos2prpht161755/fos2not doneht10table 3clinical details of macular hole casess . no.age/sexod/oslasersystemic complications168/modnot doneihd241/modnot donedm362/fosnot doneht458/fodnot donedm569/modnot donedm652/fodnot donenil762/fodnot doneht clinical details of the eales disease cases clinical details of the proliferative diabetic retinopathy cases clinical details of macular hole cases sample collection vitreous specimens were collected in a sterile tube , placed immediately on ice , centrifuged at 1,500 rpm for 5 min to separate the cell contents , then rapidly distributed in two vials , one for vegf and other for pedf immediately frozen at 80c until processed . during vitrectomy membrane peeling was done in the eyes with the ed / pdr , and these were transported in ice to the lab for the immunohistochemistry ( ihc ) . measurement of vegf and pedf vegf levels were measured in vitreous samples by enzyme - linked immunosorbent assay ( elisa ) using the quantikine vegf assay kit ( r&d systems ) and measurement of pedf by using chemicon international pedf sandwich elisa kit ( the elisa kit and pedf antibody for immunohistochemistry and western blot were the generous gift from dr . ihc for vegf and pedf in epiretinal membranes the ihc for vegf and pedf was done to confirm the presence of these factors in the epiretinal membrane ( erm ) of ed patients at the protein level . the epiretinal / fibrovascular membrane specimen obtained by surgical excision from ed and pdr cases , respectively , the deparaffinized sections were incubated with trypsin - edta for 30 min and washed with tris buffered saline ( ph 7.6 ) . further steps were carried out with novolink min polymer detection system ( kit from novacastra laboratories ltd , ne12 8ew , uk ) . the slides were blocked for peroxidase with hydrogen peroxide provided in the kit for 10 min , and tbs wash was subsequently given followed by incubation with protein block for 30 min . after this blocking step , slides were incubated for 2 h with rabbit polyclonal antibody directed against vegf ( santa cruz biotechnology , santa cruz , ca , usa ) and rabbit polyclonal pedf . these antibodies were characterized as described by the suppliers and in the literature and were used at a dilution of 1:200 for vegf and 1:150 for pedf . after washing with tbs , the slides were incubated for 45 min with post - primary block ( novolink min polymer detection system ) . the slides were washed again and incubated for 45 min with polymer link ( novolink min polymer detection system ) . finally , the slides were washed in tbs and rinsed with 0.1 m acetate buffer ph 5.05.2 and stained for 20 min with 0.8% amino ethyl carbazone in acetate buffer ( ph 5.0 ) ( sigma , st louis , mo , usa ) or dab as the chromogen ( novolink min polymer detection system ) . the sections were counterstained with mayer s haematoxylin for 15 s , rinsed in tap water and air - dried . western blot analysis for pedf fifty - microgram proteins from the vitreous of ed , pdr , and mh were loaded on 10% sds - page and transblotted to the pvdf membrane . pretreatment was done with 5% nonfat dried milk in 50 mm tris buffer ( ph 7.4 ) followed by incubation for 1 h with a rabbit polyclonal antibody prepared against human pedf diluted at 1:3,000 in tris buffer containing 0.5% milk . after this treatment , the membranes were washed extensively with tris buffer and subjected to further incubation for 30 min in an appropriate dilution of mouse anti - rabbit igg compared to the vitreous of macular hole which showed a mean vegf level of 14.9 9.25 pg / ml in the ed , there was a significant increase to a mean level of 850.5 1832 similarly , in pdr cases , the vitreal vegf level was significantly increased to 1001.8 1165.8 pg / ml ( p = 0.001 ) compared to disease control namely macular hole . the median shows that the vegf levels are similar in both pdr and ed groups compared to mh ( table 4 ) . but the levels of pedf in the vitreous of pdr patients showed a mean level of 3.74 3.8 g / ml which is lower than in ed that had a mean level of 4.8 3.9 g / ml compared to macular hole with a mean pedf level of 7.5 0.7 g / ml , ( pdr vs. mh : p = 0.027 and ed vs. mh : p = 0.008 ) . the median is lower in pdr patients compared to ed cases , though in both groups , it is lower than mh ( table 5 ) . the ratio of vegf to pedf was found to be significantly increased in ed ( ratio = 151 304 ; p = 0.01 ) and pdr ( ratio = 528 742 ; p = 0.000 ) compared to the macular hole ( ratio = 2.01 1.2 ) . however , the ratio was 3.5-fold higher in pdr than ed , and this ratio is significantly higher in pdr than in ed ( p = 0.009 ; fig . 1 ) . 1vegf / pedf ratio in ed ( n = 26)/mh ( n = 27)/pdr ( n = 17 ) . the p values are given as comparison between mh ( disease control ) vs. ed ( p = 0.01 ) ; mh vs. pdr ( p = 0.000 ) and pdr vs. ed ( p = 0.009)table 4vitreous levels of vegf in mh , ed , and pdrparticularsmacular holeeales diseasepdrno . of specimen72617mean age ( year)59 10 ( m3 , f4)32 8 ( m25 , f1)54 8 ( m11 , f6)mean vegf ( pg / ml)14.9 9.25850 1,832 p = 0.0001,001 1,165 p values are expressed based on the comparison of the individual disease with macular holetable 5pedf levels in the vitreous specimen of ed / pdr / mhparticularsmacular holeeales diseasepdrno . of specimen72617mean age59 1032 854 8mean pedf ( g / ml)7.5 0.74.8 3.9 , p = 0.0273.74 3.80 , p = 0.008median7.33.91.8pedf is expressed as mean sd . p values are expressed based on the comparison of the individual disease with macular hole vegf / pedf ratio in ed ( n = 26)/mh ( n = 27)/pdr ( n = 17 ) . the p values are given as comparison between mh ( disease control ) vs. ed ( p = 0.01 ) ; mh vs. pdr ( p = 0.000 ) and pdr vs. ed ( p = 0.009 ) vitreous levels of vegf in mh , ed , and pdr vegf is expressed as mean sd . p values are expressed based on the comparison of the individual disease with macular hole pedf levels in the vitreous specimen of ed / pdr / mh pedf is expressed as mean sd . p values are expressed based on the comparison of the individual disease with macular hole the high angiogenic potential is seen as the ratio of vegf / pedf correlates with the peak clinical onset of the ed in the age group 2130 years , and the diseases usually self - resolves above the age of 40 , which is reflected by the low ratio of vegf / pedf ( table 6 ) . table 6relation between age and vegf , pedf levels in the vitreous of ed patientsage group ( year)no . of patientsparametervegf pg / mlpedf g / mlratio202mean1,701 5675.7 892.2 13021308mean2,064 2,9754.86 3.5371 475median5983.9214314013mean157 1434.86 4.158.9 111median1023.519.4>403mean50.1 143.63 3.16.6 6median435.47.3p value2130 vs. 3140 years0.03ns0.032130 vs. > 40 years0.28ns0.23 relation between age and vegf , pedf levels in the vitreous of ed patients the relation between laser treatment and the levels of vegf and pedf in the vitreous of ed patients is shown in table 7 . the mean vegf levels were found to be significantly elevated in the sectoral laser treated group than the no laser group ( patients who did not warrant laser treatment ; p = 0.026 ) . the pedf levels and the vegf / pedf ratio too were elevated but were not statistically significant . owing to the small sample size , the distribution profile was made , which shows that the vegf levels are distinctly clustered at relatively lower levels in the no laser group compared to both sectoral and prp . but in the pdr cases , the no laser-treated group ( who did not warrant laser treatment ) had vegf level ( mean and the median ) higher than the laser - treated group . table 7relation between laser treatment and vegf , pedf levels in the vitreous of ed patientsgroupno . of patientsparametervegf pg / mlpedf g / mlrationo laser12mean265 2974.28 3.998 132median883.620sectoral phc4mean2304 30346.85 3.6444 693median12306.25143prp10mean971 21764.56 4.298.6 176median2174.733.4paired t testno laser vs. sectoral phcp value0.0260.2650.100no laser vs. prpp value0.2770.8730.994sectoral phc vs. prpp value0.3700.3590.148 relation between laser treatment and vegf , pedf levels in the vitreous of ed patients the ed patients under no steroid treatment have lower vegf / pedf ratio in terms of both mean and the median , though it was not statistically significant . however , the distribution shows that the steroid - treated group has relatively higher ratio and was maximum in the group treated with both oral and periocular steroids ( table 8) . this can possibly be explained by the fact that clinically , the treatment is guided by the severity of disease . table 8relation between steroid intake and the vegf , pedf levelsgroupno . of patientsparametervegfpedfrationo steroid9mean222 2793.98 3.671.7 92.7median10230812.8oral steroids11mean842 2,0736.15125median3255.536.1oral + periocular steroids6mean1,807 2,5013.48 4.3320 576median986.52.5108.6paired t testno steroids vs. oral steroidsp value0.380.210.44no steroid vs. oral + periocular steroidsp value0.070.810.21oral steroids vs. oral + periocular steroidsp value0.400.210.31 relation between steroid intake and the vegf , pedf levels both vegf and pedf were detected in the erm specimen from the ed cases as well as in the fibrovascular membrane from the pdr case ( fig . 2 ) . 2a c vegf immunostaining in epiretinal membrane of ed and pdr is visualized with a red reaction product . a erm from a patient with ed negative for secondary antibody ; b erm from ed patient showing vegf posivity ; c erm from pdr patient showing vegf positivity . f pedf immunostaining in epiretinal membrane of ed , and pdr is visualized with a red reaction product . d erm from ed patient showing pedf positivity ; e erm from pdr patient showing pedf positivity ; f erm from a patient with ed negative for secondary antibody a c vegf immunostaining in epiretinal membrane of ed and pdr is visualized with a red reaction product . a erm from a patient with ed negative for secondary antibody ; b erm from ed patient showing vegf posivity ; c erm from pdr patient showing vegf positivity . f pedf immunostaining in epiretinal membrane of ed , and pdr is visualized with a red reaction product . d erm from ed patient showing pedf positivity ; e erm from pdr patient showing pedf positivity ; f erm from a patient with ed negative for secondary antibody the western blot analysis of the vitreous specimen for pedf in ed compared to the pdr and macular hole is shown in fig . 3 . the densitometry analysis showed that the band intensity of the pdr is the lowest followed by ed and macular hole compared to the donor eye vitreous . 3western blot analysis of the vitreous specimen for pedf in eales disease compared to the pdr and mh . panel 1 : western blot analysis of vitreous pedf in ed , pdr , mh , and donor eye vitreous . lane 1 donor eye vitreous , lane 2 mh , lane 3 ed , lane 4 ed , lane 5 pdr , lane 6 pdr , lane 7 pdr western blot analysis of the vitreous specimen for pedf in eales disease compared to the pdr and mh . panel 1 : western blot analysis of vitreous pedf in ed , pdr , mh , and donor eye vitreous . lane 1 donor eye vitreous , lane 2 mh , lane 3 ed , lane 4 ed , lane 5 pdr , lane 6 pdr , lane 7 pdr ocular neovascularization is regulated by a diffusible factor , vegf that promotes angiogenesis in the retina which is known to play a role in the pathogenesis of early diabetic retinopathy . in addition , lower levels of pigment epithelium - derived factor have been related to the angiogenesis in diabetic retinopathy that results in active proliferative diabetic retinopathy . it is also well documented that advanced glycation end products ( age)advanced glycation end product receptor ( rage ) interaction elicited angiogenesis through the transcriptional activation of the vegf gene . interaction of ages with rage leads to leukostasis and blood - retinal barrier breakdown that are characteristic findings in diabetic retinopathy . while ages in the form of methylglyoxal detected in diabetic membrane and serum suggest the role of glycation . results from our previous studies suggest that ages formed through glycoxidation may play an important role in the development of retinal neovascularization seen in ed . the immunoreactivity of cml - ages ( carboxy methyl lysine ) in neovascular membrane and its increased levels in serum suggest that in spite of the normoglycemic status , the glycoxidation resulting in age and oxidative stress may trigger the retinal neovascularization in ed . therefore , this study was aimed at estimating the vegf and pedf levels in the vitreous specimen of ed and to see the ratio in comparison with pdr . present study shows that the mean vegf level in the vitreous is significantly increased in ed compared to mh and the distribution is similar to that seen in the pdr cases . ninety - eight percent of the ed patients and 96% of the pdr cases showed vegf levels over and above the median level of the mh group . the increased levels of vegf might explain the severity of the neovascularization and hemorrhage in the ed , similar to the report on pdr wherein studies have shown that high levels of vegf accumulate in the vitreous of patients with proliferative diabetic retinopathy , which results in the mitotic effect on the retinal capillary endothelial cells . the report by kumar and sinha on intravitreal bevacizumab in ed has been indicative of role of vegf in the neovascularization seen in ed . however , there has been no report on the vitreous levels of vegf in the ed cases . the main antiangiogenic factor is the pedf though there are also factors such as transforming growth factor beta , thrombospondin , and somatostatin . the study revealed that there is a significant decrease in the pedf levels in the vitreous of ed cases . seventy percent of ed cases show lowered pedf levels compared to the median level of mh , and in the pdr group , it is 82% . pedf has been proposed as a therapeutic target for oxidative stress - involved eye diseases , such as pdr . moreover , it is also earlier reported as an anti - inflammatory factor that can inhibit vegf - induced vascular permeability . in this study , the ihc shows the vegf and pedf at the protein level in the erm of the ed cases which is comparable to that of pdr . the balance between the angiogenic and anti - angiogenic factors rather than angiogenic factors themselves is said to be crucial in determining the progression of the angiogenic vitreoretinopathies such as pdr . therefore , the ratio of vegf / pedf was analyzed , and the study revealed that the pdr had the highest ratio followed by ed and the desirable levels were seen in mh . although vegf was raised in both diseases , pedf was found to be more lowered in the pdr than in ed . however , the ratio gives a clear picture of the angiogenic potential that was significantly higher in pdr compared to ed . clinically , ed is a self - limiting disease while pdr is a progressive disease . while looking at the effect of steroid on vegf , pedf levels , and vegf / pedf ratio in the ed cases , a subgroup analysis of the interval between last steroid intake and time of vitrectomy showed an observation that the vegf levels and the vegf / pedf ratio were lower in those receiving steroids between 1 and 6 months preceding surgery as compared to those receiving steroids in the month immediately preceding surgery . although multiple factors can influence the instantaneous levels of vegf and pedf in the vitreous , the treatment with effect to time lag can explain the above observation . similarly , the interval between last laser treatment and time of vitrectomy was looked into . however , no particular trend was noted here , and no statistical difference was noted in vegf , pedf , and vegf / pedf ratio between the groups ( data not shown ) . the vegf levels in the sectoral laser treatment were found to be significantly higher than the no laser group , and the ratio of vegf / pedf was also higher in spite of the increased pedf levels . the sectoral laser is done when the disease is relatively still active while in the prp the disease is in the involuntary stage . have shown a correlation between the vegf levels and the grade of photocoagulation . in the pdr patients , the laser - treated group showed lower angiogenic potential than the no laser group . vitreous vegf levels are reportedly higher in pdr patients with no laser treatment than the pdr patients treated with laser . how little the laser treatment is and the timely treatment can possibly influence the vegf and the pedf levels . the earlier study from this laboratory has shown the cml - age immunoreactivity detected in all cases of ed and 61% cases of diabetic retinopathy and none in idiopathic erm . age accumulation seen also in the serum of these patients inspite of normoglycemia as against diabetes was explained based on the glycoxidation process in ed triggered by the iron - induced oxidative stress . also , increasing evidence indicates that ages promote retinal alterations through oxidative stress [ 27 , 28 ] . though ageing and hyperglycemia as in armd and diabetic retinopathy , respectively , can result in age formation , in ed , it is very clear that oxidative stress and nitrosative stress precedes age accumulation [ 6 , 29 ] . low levels of pedf along with increased vegf has a net angiogenic effect that result in the neovascularization seen in ed . thus , in ed , increased oxidative stress leads to age formation owing to glycoxidation , which is followed by age rage - mediated vegf elaborated ( fig . 4 ) . inflammation , nv , and retinal damage in ed could be explained in terms of accumulation of lipid and oxygen - free radicals caused by neovascularization . intraocular neovascularization develops in numerous ischemic retinal vein disorders . in proliferative diabetic retinopathy , there is active and extensive proliferation of new vessels that leads to vitreous hemorrhage and retinal detachment . though ed is not exactly similar to diabetic retinopathy wherein the disease is continuous and ongoing process , in ed , there is an initial insult that may end up with the proliferative phase . the management depends on the stage of the disease and consists of ( a ) treatment with oral corticosteroids in the active inflammatory stage and ( b ) laser photocoagulation in the neovascularization state and vitrectomy when there is vitreous hemorrhage or retinal detachment .

eales disease ( ed ) is an idiopathic inflammatory venous occlusion of the peripheral retina . as neovascularization is prominent in ed , this study attempts to look at the ratio of vegf , the angiogenic factor , and pedf , an anti - angiogenic factor in the vitreous of ed patients in comparison with the macular hole ( mh ) and proliferative diabetic retinopathy ( pdr ) . vitreous levels of vegf and pedf were determined in the undiluted vitreous specimen obtained from 26 ed cases , 17 pdr , and seven patients with mh . the vitreous levels of vegf and pedf were estimated by elisa . the immunohistochemistry ( ihc ) for vegf and pedf were done in the epiretinal membrane of ed and pdr case . the vegf / pedf ratio was found to be significantly increased in ed ( p = 0.014 ) and pdr ( p = 0.000 ) compared to mh . however the ratio was 3.5-fold higher in pdr than ed ( p = 0.009 ) . the ihc data on the erm specimen from ed showed the presence of vegf and pedf similar to pdr . the high angiogenic potential seen as the ratio of vegf / pedf correlates with the peak clinical onset of the disease in the age group 2130 years and the diseases usually self - resolves above the age of 40 , which is reflected by the low ratio of vegf / pedf . the study shows that the vegf / pedf ratio is significantly increased in ed though the angiogenic potential is higher in pdr than in ed . clinically eales disease is known as a self - limiting disease , while pdr is a progressive disease .

Introduction Materials and methods Results Discussion

as neovascularization is prominent in ed this study attempts to look at these two predominant factors one acting as pro- and the other as anti - angiogenic , by estimating the vegf and pedf levels , respectively , in the vitreous specimen of ed patients and compare it with that of the pdr which also involves neovascularization and with mh which does not involve the same . vitreous fluid samples were obtained from 26 ed patients ( 25 m , 1 f , 33 8 years ) , 17 pdr ( 11 m , 6 f , 54 9 years ) , and seven patients with macular hole ( 3 m , 4 f ; 60 10 years ) . after this treatment , the membranes were washed extensively with tris buffer and subjected to further incubation for 30 min in an appropriate dilution of mouse anti - rabbit igg compared to the vitreous of macular hole which showed a mean vegf level of 14.9 9.25 pg / ml in the ed , there was a significant increase to a mean level of 850.5 1832 similarly , in pdr cases , the vitreal vegf level was significantly increased to 1001.8 1165.8 pg / ml ( p = 0.001 ) compared to disease control namely macular hole . the ratio of vegf to pedf was found to be significantly increased in ed ( ratio = 151 304 ; p = 0.01 ) and pdr ( ratio = 528 742 ; p = 0.000 ) compared to the macular hole ( ratio = 2.01 1.2 ) . however , the ratio was 3.5-fold higher in pdr than ed , and this ratio is significantly higher in pdr than in ed ( p = 0.009 ; fig . the p values are given as comparison between mh ( disease control ) vs. ed ( p = 0.01 ) ; mh vs. pdr ( p = 0.000 ) and pdr vs. ed ( p = 0.009)table 4vitreous levels of vegf in mh , ed , and pdrparticularsmacular holeeales diseasepdrno . the p values are given as comparison between mh ( disease control ) vs. ed ( p = 0.01 ) ; mh vs. pdr ( p = 0.000 ) and pdr vs. ed ( p = 0.009 ) vitreous levels of vegf in mh , ed , and pdr vegf is expressed as mean sd . p values are expressed based on the comparison of the individual disease with macular hole the high angiogenic potential is seen as the ratio of vegf / pedf correlates with the peak clinical onset of the ed in the age group 2130 years , and the diseases usually self - resolves above the age of 40 , which is reflected by the low ratio of vegf / pedf ( table 6 ) . of patientsparametervegfpedfrationo steroid9mean222 2793.98 3.671.7 92.7median10230812.8oral steroids11mean842 2,0736.15125median3255.536.1oral + periocular steroids6mean1,807 2,5013.48 4.3320 576median986.52.5108.6paired t testno steroids vs. oral steroidsp value0.380.210.44no steroid vs. oral + periocular steroidsp value0.070.810.21oral steroids vs. oral + periocular steroidsp value0.400.210.31 relation between steroid intake and the vegf , pedf levels both vegf and pedf were detected in the erm specimen from the ed cases as well as in the fibrovascular membrane from the pdr case ( fig . therefore , this study was aimed at estimating the vegf and pedf levels in the vitreous specimen of ed and to see the ratio in comparison with pdr . present study shows that the mean vegf level in the vitreous is significantly increased in ed compared to mh and the distribution is similar to that seen in the pdr cases . the increased levels of vegf might explain the severity of the neovascularization and hemorrhage in the ed , similar to the report on pdr wherein studies have shown that high levels of vegf accumulate in the vitreous of patients with proliferative diabetic retinopathy , which results in the mitotic effect on the retinal capillary endothelial cells . while looking at the effect of steroid on vegf , pedf levels , and vegf / pedf ratio in the ed cases , a subgroup analysis of the interval between last steroid intake and time of vitrectomy showed an observation that the vegf levels and the vegf / pedf ratio were lower in those receiving steroids between 1 and 6 months preceding surgery as compared to those receiving steroids in the month immediately preceding surgery . the vegf levels in the sectoral laser treatment were found to be significantly higher than the no laser group , and the ratio of vegf / pedf was also higher in spite of the increased pedf levels .

the processes of professionalisation and specialisation in health care have long been of interest to both historians and sociologists , among others . both can be seen as at least in part about laying claim to , developing and protecting knowledge and expertise that finally allows control over a particular domain . both are essentially political and social processes , depending on the acceptance or acquiescence of others for success , while also employing internal justifications ( for example , the development and elaboration of theory ) to vindicate their claims . professionalisation seeks to turn an occupation into a profession , with all that this implies in terms of power and autonomy within a given sphere of our social world specialisation within medicine and health care seeks to create specialisms of practice , to normalise categories of health and sickness , at least partly in order to allow individuals control and jurisdiction over particular categories . it seems sensible to propose , at least in some cases , a relationship between processes of professionalisation and specialisation . if a group of individuals collectively seek to professionalise , part of their justification for doing so must lie in the belief that they are specialists in a particular area and so warrant being given control over this domain . however , those involved in a newly emerging medical specialism would presumably see no need to try and professionalise this , in the sense of turning it into a profession , simply because they were already members of the medical profession . ( although of course they would be interested in describing , developing and otherwise laying claim to what they conceived of as the specialist area . ) equally , however , it is easy to imagine a group of individuals who were engaged in practices that they considered to be specialist , but who were lacking the structures of a profession within which these might be developed . such a group would presumably want to engage in processes of both specialisation and professionalisation . that is to say , they would wish both to lay claim to an area which they regarded as specialist , and to occupy that area as professionals with rights , responsibilities and privileges they had been instrumental in drawing up . in this sort of case , concerns with demarcation and division , the development of theoretical knowledge and practical expertise , appropriate training and so on would transfer between the projects of specialisation and professionalisation , with one reinforcing the other . for some involved ( but not necessarily for all ) , the projects might become indistinguishable from each other . this paper examines such a case , that of specialist health promotion in england during the final years of the twentieth century ( roughly between 1980 and 2000 ) . here , a relatively small group , who eventually came to be known as health promotion specialists , tried to lay particular claim to , and develop theoretical knowledge and practical expertise in , the then - emerging field of health promotion. this field had at least in part grown from that of health education , a set of loose and contingent practices broadly centred on the communication of health messages through teaching , propaganda and other means . after a brief period of relative development , specialist health promotion finally declined and those who were attempting to professionalise it failed in their project . for clarity s sake , i will refer throughout this paper to specialist health promotion ( shp ) and health promotion specialists ( hpss ) , although of course my central purpose here is to examine the difficulties these faced in becoming and being regarded as specialists in any sense at all . describing and understanding the failure of shp is important because the late twentieth century has been characterised as an era in which the ideological development of a new kind of public health ( which included health promotion ) took place . this development was supposedly quite distinct from the narrow clinical concerns that often seemed to preoccupy medicine and health care for much of the earlier part of that century . this ideological development is represented in part by a wave of policy documents , especially those coming from the world health organisation ( who ) . the development has been characterised as part of a broad recognition that momentous global issues such as inequality and sustainability require radical action , including novel ways of thinking about the problems of health and health care . clearly there may often be a gap between ideology and theoretical reconceptualisation on the one hand and policy and practice on the other . nevertheless , it seems odd that in an era during which the ideology of health promotion developed , shp in england declined and failed . the mismatch here between ideology and reality is a significant prompt for historical attention and the development of historical understanding . i argue in this paper that the failure of shp , both in its claim to specialisation and in the professionalisation project that was developed and supported by some hpss , was the result of three strongly connected difficulties . first , there was the difficulty of the specialists agreeing what they should be doing and how they should be doing it . second , there was the difficulty of them finding a place within the national health service ( nhs ) to do their work . third , there was the difficulty of another , much more powerful group- medicine and its ancillary of public health medicine staking claim to the domain . explaining and analysing these problems in detail starts to tell us much about the possibility or otherwise of reorienting and reshaping health services in the twenty - first century . according to the now moribund association that purported to represent hpss across the united kingdom ( uk ) , they were the uk s experts in health promotion. in 1985 there were 518 specialists ( or equivalents ) working across the four uk nations , rising to 859 by 1988 and roughly 1,000 by the mid-1990s , the great majority of them being employed by the nhs . the story being told here , mainly because of differences in policy and its effects within the four nations , is the english one . while figures for england alone are difficult to extrapolate , it is reasonable to assume that a large proportion of the roughly 1,000 specialists mentioned as working within the uk by the mid-1990s were doing so in the english context . what were they actually doing ? in order to address these questions , and to begin to understand the claims and interests of shp in the final years of the twentieth century , it is necessary to consider some aspects of earlier history . health promotion , as a concept and related set of practices , emerged during the late 1970s and early 1980s partly from a relatively lengthy tradition of health information and propaganda , which it is probably best to refer to as health education . the voluntary central council for health education ( cche ) had been established in 1927 , largely at the instigation of the society of medical officers of health . until 1950 the cche was formed from , among others , representatives of the ministries of health and education , the professional organisations of medical officers of health and the various associations of local authorities . the council s remit was to provide expert advice and resources in matters of health education . in 1950 it was reconstituted to bring it in line with the provisions of the nhs act 1946 , as its aims were now more closely identified with local authorities who largely had responsibility for this area through their public health departments . thus their representatives and associations at this point assumed full control of the cche . despite the council s remit and membership , however , commentators have noted that work in this field both before and after its inception had been sporadic , lacking in a sense of proper strategic planning and largely propagandist in approach . however , developments in the social and medical sciences in the second half of the century began to alter views about health education , what it might be capable of and what it should be doing . the 1950s saw the strengthening of the academic field of social psychology , particularly in the united states of america , a field that sought amongst other things to develop more sophisticated understandings of health behaviour , including how it could be changed . during the same decade , the power of the then new medium of television had gradually increased so that , by the 1960s , it had become a key media player. relatively sophisticated methods of advertising through this consumers through advertising led social psychologists towards attempting to understand these new methods and their impact on individual behaviour . at the same time , epidemiology was beginning to connect lifestyle behaviours such as diet and smoking with the onset of morbidity and premature mortality . the establishment of a connection between smoking and lung cancer at the beginning of the 1950s was especially important in drawing attention to the value of efforts aimed at lifestyle prevention , although , in the case of smoking , the caution of policymakers in tackling a widespread , culturally embedded behaviour meant that it took some time to establish reasonable anti - smoking programmes . these developments in the fields of psychology and epidemiology greater understanding of how people behaved , how they were influenced in their behaviour and how lifestyle was connected to ill health contributed to the belief that health education could play an increasingly important part in preventing illness and disease as an emerging specialist area . the joint committee of the central and scottish health services councils ( better known as the cohen committee on health education ) was formed in 1959 to consider how health education could be developed in order to play such a part and in 1963 it produced its report . perhaps unsurprisingly , given the lifestyle prevention context within which the cohen committee was originally established , the cohen report strongly recommended that health education should address issues such as cancer , diet and smoking . however , it also made recommendations relevant to understanding the development of health education as a putative specialism , two of which are especially important here . in the first place , the report recommended that a strong central board be set up to replace the voluntary cche . this board should co - ordinate activity at a national level and encourage the development of specialist health educators . second , it recommended that local authorities should more systematically recruit and train these educators , who were coming increasingly to be known as health education officers ( heos ) . strong central board was finally established in 1968 as the health education council ( hec ) . its formation and indeed most of its working life was difficult , as were the lives of its successor organisations . this was at least in part due to tensions over who should control the work of this body , which was attempting at the national level to co - ordinate and develop the embryonic specialism of health education . should it simply be an adjunct to medicine or should it be allowed somehow to develop at arm s length from medical influence ? the problematic question of medicine s influence over those working in the fields of health education and health promotion extends throughout the history being considered . the task of developing and embedding heos in local authority structures was possibly even more difficult than the creation of the hec . in 1968 , five years after the publication of the cohen report , one medical officer of health ( moh ) reported that it was generally unclear how many heos were actually employed by local authorities , what qualifications they possessed and what their background experience was . a small number of secondary sources start to cast some light on the identity and practices of heos working in local authorities during the late 1960s and early 1970s . in 1972 a survey was undertaken by keith tones , an academic who in 1974 would establish one of the first postgraduate diploma courses in health education ( at leeds polytechnic ) , and who would become one of the key drivers of the theorising component within attempts to develop the putative specialism of health education in the decade or so following . tones reported that over half the heos then in post in england , wales and northern ireland ( just under 200 ) had the remainder had come from teaching , dietetics , administration or another local authority public health - related role . in addition to his own survey work , tones , using data derived from the hec , identified that , while heos were based in units within local authorities ( of which at that point there were forty ) , about fifty per cent of these units comprised just one person . as to what heos actually did , tones described their involvement in programme planning , public relations , policy making , teaching , lecturing and routine administration . this picture of heos as involved in a range of diverse practices is supported by the single published account of life as a heo in the late 1960s and early 1970s that i have been able to identify . linda ewles worked during this period for bristol city council public health department : i cut my health education teeth working on campaigns such as one for the new measles immunisation exhibitions such as an annual three - day event at the local flower show where we promoted topics such as accident prevention and demonstrated the kiss of life ; running stop - smoking and slimming groups ( as we called them then ) ; and giving talks on nutrition , accident prevention and first aid to groups such as mothers unions and the brownies . and , of course , there was the audio - visual aid loan service of films , slides and film - strips , and the inevitable posters and leaflets . activities requiring more skills and experience , such as working with the lea [ local education authority ] on devising a health education syllabus for local schools , and in - service training for teachers and health visitors , were mainly undertaken by my senior heo colleagues . i cut my health education teeth working on campaigns such as one for the new measles immunisation exhibitions such as an annual three - day event at the local flower show where we promoted topics such as accident prevention and demonstrated the kiss of life ; running stop - smoking and slimming groups ( as we called them then ) ; and giving talks on nutrition , accident prevention and first aid to groups such as mothers unions and the brownies . and , of course , there was the audio - visual aid loan service of films , slides and film - strips , and the inevitable posters and leaflets . activities requiring more skills and experience , such as working with the lea [ local education authority ] on devising a health education syllabus for local schools , and in - service training for teachers and health visitors , were mainly undertaken by my senior heo colleagues . ewles picture of a loose set of activities and practices ( from running exhibitions at flower shows to planning a school s health education syllabus ) is made more vivid by the fact that she does not explain how they were decided on . they do not appear to have been based on particular normative conceptions of what a heo should be doing , or any established views of priority . in this sense , as is the case with tones survey account , the practices seem not only to be diverse but also contingent . yet at almost exactly the same time that linda ewles was involved as a health education officer in giving talks to local societies and lending film material , a striking global agenda for health improvement was emerging , which would ultimately affect the work of heos such as ewles . its initial impetus was the lalonde report , produced under the direction of the canadian minister of health , marc lalonde . as a politician , he was faced with the realisation that , despite the ever - increasing capacity of medical services to deal with illness and disease , these alone could now not offer the dramatic improvements in health required by advanced societies . the lalonde report was important not least because it emphasised the crucial place of social structures in determining and dealing with problems of ill health . to this extent , it opened up the possibilities of action for health improvement well beyond the lifestyle prevention activities and practices described by tones and ewles in the early 1970s . the lalonde report was quickly followed by a string of declarations from the who . these sought to establish the philosophical and practical basis of what was now being talked about as health promotion , defined a short time later by one theorist as the process of enabling individuals and communities to increase control over the determinants of health and thereby improve their health. as well as forming the basis of health promotion , the focus on the wider , structural determinants of health inspired by lalonde also prompted some to alter their conceptions about the nature of public health . public health as being largely constituted by concerns to do with the narrow mapping of disease and the planning of treatment services . now , for some , the idea of the so - called new public health began to emerge . as with health promotion , the new public health was largely defined by its altered focus on the structures creating health or causing illness . to this extent , it often seems hard to split a hair between health promotion and new conceptions of public health , a point that will become significant in explaining the failure of shp both properly to become a specialism and to professionalise . the expansive and possibly idealistic international agenda that i have described , and which gave rise both to health promotion and the new public health , was being developed at almost exactly the same time that in england heos were facing reorganisation . they were being forced to move from their local authority bases into the national health service ( nhs ) under the terms of the 1974 nhs reorganisation . they were not alone in this , being accompanied by their moh bosses and the much - diminished community health empire over which mohs had responsibility . ( this also included community midwifery and health visiting services . ) the nhs reorganisation that took heos into the health service followed the reshaping of local authority welfare and social work services in the wake of the 1968 seebohm report . mohs ( renamed specialists in community medicine ) , heos and their former local authority community health colleagues now found themselves in a service dominated by , and essentially only interested in , imperatives related to acute treatment and care . commentators have noted that the effects of the 1974 reorganisation on uk health services and on the broad health policy agenda have been relatively under - researched . what is clear , however , is that the influence of mohs on local community health was weakened . this was partly because of the rather nebulous role they assumed now that they were within the nhs , which was mainly to do with preventing and dealing with communicable and infectious diseases . it was also because , in moving to the health service , mohs lost their long - standing immediate organisational relationship with environmental health officers who , after some policy vacillation , remained in local authorities . the public health agenda after 1974 was consequently diffused , largely as a result of the fracturing of the public health work force that i have described . some local authorities maintained an interest in health education and the emerging idea of health promotion , although this was often difficult , given organisational barriers . heos struggled to maintain role and identity , as well as relationships with traditional allies such as health visitors who , while also now in the nhs , were located in a different organisational tier . ( heos worked at area level , while health visitors were part of the district work force . ) the impact of the 1974 reforms generally on local public health is complex but it certainly involved severed ties , disrupted relationships and still further re - balancing of the political economy of health in favour of treatment and care.43 45 nationally , a government consultative document on prevention was published in 1976 , shortly after the reorganisation that had adversely affected local health education and public health services to such a significant extent . prevention and health was firmly rooted in the idea of individual responsibility for disease prevention . it suffered significant criticism for failing to acknowledge wider structural influences on health and neglecting the gathering international agenda that emphasised the importance of dealing with structures in order to improve health . the contrast between uk government thinking about prevention on the one hand and global conceptions of there were sporadic challenges to the narrowing and medicalisation of public health that had occurred following the 1974 reorganisation . attempts at directing attention towards the impact of economic and social structures on health were actively thwarted by the conservative government that was elected in 1979 , one of the best known examples being suppression of the 1980 black report on inequalities in health . the suppression was deeply disappointing to many but it was hardly surprising given the incoming government s unilateral abandonment of the post - war economic and social welfare consensus . despite the organisational difficulties and conceptual tensions that i have described , the early 1980s saw some efforts towards providing greater shape to the work of heos . policy and other initiatives attempted to move them from the kind of embroilment in loosely or perhaps even unconnected practices described by linda ewles and towards more careful description of their domain , their putative expertise and the systematic application of this in practice . one of these was the so - called kirby report on the training and development of health education officers . kirby had started off by reviewing the grading , pay and conditions of clerical and administrative staff in the nhs . almost by accident , this review had uncovered heos among the labyrinthine administrative grading systems of the health service . recognising heos appeared to be doing quite different work from other administratively graded staff , the review sought to consider and report on this group separately . for the first time , kirby identified common job descriptions for heos that could be applied across health authorities , emphasising the health education programme planning , co - ordination , training and expert advice role of the officers . the report also established the nature of an induction programme for those who were in the early stages of their careers as heos . moreover , it provided official acceptance of the postgraduate diploma in health education ( which by now had been taught in leeds polytechnic and a small number of other higher education institutions for several years ) as the basic professional qualification for heos . however , it had at least provided a degree of tightness to the heo role . in effect , the role having been tightened , there was now the possibility of considering more exactly what kind of specialist knowledge and theory it might require . attempts to develop health education and health promotion theory constituted a further effort to better define and describe the putative specialism in which heos were working . george weisz argues that a community of scholars addressing a research problem is a condition for the development of specialism in the medical field . in england , a small and scattered community of scholars addressing the problem of the theoretical foundations of health education and health promotion emerged through their association with the founding of postgraduate courses related to the field . the first course was begun at leeds polytechnic in 1974 , followed shortly afterwards by further courses at bristol and south bank polytechnics and chelsea college london . the research problem faced by this small band of scholars was to describe health education and the emerging idea of health promotion carefully , draw the two together , make clear exactly where their theoretical roots lay or might be developed , and make a case for how this theory might support practice . it was a difficult problem , partly because the separate histories of health education and health promotion ( and the quite different contexts in which ideas relating to them had developed ) gave rise to a range of conceptions about what they actually were and what they should be trying to do . these included at one extreme the kind of individualistic approach represented by prevention and health and at the other the emphasis on structural change of lalonde and the who . some saw the research problem as partly involving an attempt at reconciliation , trying to convince health promoters ( as those working in the field were now being increasingly called ) that these and other approaches were equally valid . others saw theorising as at least to some degree about making explicit the values underlying the separate approaches or even possibly asserting that one particular approach was better than the rest . the different conceptions of health promotion that emerged from this early theorising contributed to later rifts between those working in shp and , ultimately , as i will argue , to the failure of specialisation and professionalsation . the early 1980s also saw a more careful , or at least clearer , description of the health promotion domain through the emergence , in the english context , of work related to the global who agenda . from its declarations , a number of significant figures within who , including the sociologist ilona kickbusch ( unusual for being both a woman and a prominent figure in global public health ) , renewed the idea that local authorities should have a central role in promoting the health of their communities , an idea that gradually turned into the healthy cities movement . the idea was taken up enthusiastically in some areas of england ( as well as other parts of the uk ) , notably liverpool , a declining port city with significant social problems . john ashton , a specialist in community medicine and an advocate of the new public health , appointed howard seymour as a health promotion specialist . both were employed by the nhs but worked to draw together the health service and liverpool city council in a programme of work aimed at creating the healthy city. the same kind of process took place in other areas and represented one way in which practitioners tried to overcome the fractures and divisions that had been caused by the 1974 reforms ; and to challenge , at least implicitly , the ideology of the libertarian conservative government . despite all this , however , numbers of heos ( or hpss , as some started to call themselves during this period of theoretical and practical development in health promotion ) remained low . however , a further and tragic development during this period provided the opportunity to expand the numbers of specialist staff . the pandemic , according to one historian , offered public health and its policy tool kit of epidemiology and health education [ the chance ] to define the parameters of policy - making in a way unique in post- war british public health history . hiv / aids could not yet be treated and its potential for catastrophic spread was deeply alarming . while programmes of research into causation developed , and thus whatever potential might exist for treatment was discovered , prevention was essentially the only strategy open to policy makers . realisation of this led to relatively large amounts of money going to health authorities for hiv / aids prevention work . rawson and grigg , in the only extensive study actually undertaken during this period of the development of specialist health education ( as these authors were still referring to it ) report a significant rise in heo posts . in england , wales and northern ireland the number of posts increased by over fifty per cent from 518 in 1985 to more than 850 in 1987 . another analysis , conducted for the health education authority ( which had replaced the hec in 1987 ) , suggested that a quarter of these new posts were related to hiv / aids and were fixed - term contracts connected to year - on - year ring - fenced hiv prevention monies . this suggests that the expansion of services came out of political necessity and short - term expediency rather than careful planning and development of the putative specialism . topics like hiv ( along with others such as illicit drugs and alcohol ) as driven by medicine and a relatively narrow conception of health ; as atomising health behaviour and thus limiting or even dismissing attention towards the social context on which health promotion ( or at least the version inspired by lalonde and promoted by the ottawa charter ) was supposed to be based . a sense emerges of practice being disputed , dichotomised and not necessarily grounded in coherent thoughts about what health promotion , if it was indeed a specialist field , should be doing . yet in other ways the advent of hiv suited the aspirations and interests of some involved in health promotion very well . in the early stages of the pandemic , one important kind of response to it was through grassroots activism . this connected neatly with the radical tendencies of some people involved in shp , which perhaps for those individuals was an almost necessary consequence of doing work that was at the margins of health policy . the 1980s saw the development of radical activism in health education and health promotion through , for example , groups such as the feminist health education officers group ( founded in 1980 ) and the radical health promotion collective ( 1985 ) . the radical voice also appeared in research and other activity that was funded or sponsored by the medical or other mainstream . while these were often small - scale activities , they had a relatively wide impact in terms of shaping the work philosophies of health education officers . rawson and grigg , for example , report a strong preference on the part of heos for moving away from addressing questions of individual behaviour change and towards the structural issues that formed the basis of the global rhetoric on health promotion . so was a consensus emerging in the mid-1980s about what hpss should be doing , and if this was the case does it suggest that a coherent specialism was beginning to emerge ? the radical activists and those sympathetic to a radical agenda were mainly working in the highly structured and largely medically focussed nhs . the capacity that they had to enact a radical agenda was virtually non - existent and their organisational power base extremely limited . at the same time as the radical agenda was being cherished and sympathised with , others considered that the best route to more power lay in attempts to professionalise shp ; to develop the kinds of structures that would give it greater autonomy and control over its affairs . professionalisation project was a reasonable one in the context of the dispute that i have described about what shp should be doing , together with the ambiguity that existed in its associated practices , is an important one that i will return to in later discussion . the general acceptance by the kirby report that heos should undergo postgraduate training had provided an impetus for the association that represented some of them known by the mid-1980s as the society of health education and health promotion officers to develop plans for a national training scheme . at about the same time , a code of conduct for hpss was being formulated , as well as plans for a national register of specialists . the society was thus attempting to drive , almost entirely alone , the implementation of the three key descriptive elements associated with an occupation becoming a profession : statutory training , registration and regulation . they saw professionalisation as no more than an attempt to mimic medicine s structures of power and control . set against this was the professionalisers beliefs that shifts within the nhs towards a more directive system of general management , recommended by the 1983 griffiths report , made it even more important that hpss should try to gain greater control over their work through professionalisation . in fact , griffiths turned out to be just a provisional test for hpss . further organisational reform of the nhs in the late 1980s and early 1990s presented them with much more dramatic problems . the introduction of the so - called internal market within the health service , dividing the nhs into a system of hpss awkwardly straddled the purchaser provider divide that was the central feature of the new internal market . some of what hpss did could be understood as purchasing activity ; for example , identifying areas of health promotion need and planning responses to this . some of it , however , was clearly provider work ; for example , organising health awareness campaigns or training on health issues . district health authorities ( the location of most hpss prior to the internal market reforms ) made some attempt to address this fundamental problem of organisational location , although it was seldom a priority . some placed health promotion departments within provider units ( most by now becoming nhs trusts ) ; others put them as purchasers within their own planning and public health functions . a further organisational arrangement was the allocation of some staff within a department to purchaser and others to provider . the health education authority analysed organisational options and tried to make national recommendations as to what might work best . however , regardless of whether departments were literally divided or not , the general effect was for the planning- provision continuum in shp to be broken . the organisation representing some hpss , now known as the society of health education and health promotion specialists ( sheps ) , reported multiple tales of service fragmentation and dislocation . ironically , the reforms that fragmented specialist health promotion were followed by the conservative government s first public - health strategy , the first attempt at national strategy in this area since prevention and health back in 1976 . this strategy , entitled the health of the nation , can in some ways be interpreted as a health promotion strategy , or at least as a strategy for a particular kind of health promotion based on principles of individual responsibility and lifestyle change . despite ideological opposition to this sort of approach on the part of some hpss , others welcomed the heath of the nation because at least now government policy was actively talking about health improvement rather than just disease treatment or service organisation . the dph role had been created as part of an attempt to re - invigorate public health medicine , which many saw as having lost its way since the 1974 reforms , the loss of the moh and the arrival of the specialist in community medicine . the health of the nation added to this impetus by giving dphs new responsibilities for such things as partnership working for health improvement between local and health authorities . as part of its renaissance , public health medicine had also quite cleverly capitalised on the international who agenda . public health medicine , a renaming that at least sometimes made easier an elision to the new public health , with its focus much like health promotion , as i have argued on structures and environment . there was still a firm belief that medics ( public health doctors ) should be leading public health work . however , there were also vague rhetorical commitments to the idea of public health as a multidisciplinary enterprise , a nod to the implications of the renaming that was taking place . these turned out to be enough to kick - start a movement that became known as multidisciplinary public health ( mdph ) . the contrast from this point on between the shp struggle to professionalise on the one hand , and the confident emergence of mdph on the other , is striking . the register of hpss that was supposed to form one of the cornerstones of professionalisation was finally published in 1991 . however , it stood alone : statutory training and registration remained as distant as ever . at the same time , those activists who were opposed to professionalisation continued in their efforts to represent health promotion in radical terms , as the antithesis of the power and control over a domain that was seemingly sought by sheps . yet there was also a sense on the activists part that their radical vision of health promotion had not moved forward much , at least partly because conservative government policy had comprehensively thwarted genuinely radical direction . as a commentator at one activist event , a symposium on health promotion theory involving practitioners and academics , noted : once the symposium got underway it soon became apparent that the key aim , as far as a significant number of the participants were concerned , was to find a way of regrouping in the face of hostile day - to - day working conditions . debates was : how , if at all , can health promotion specialists employed by the state put their radical principles into practice? or , as one participant succinctly put it : how can we hang on to once the symposium got underway it soon became apparent that the key aim , as far as a significant number of the participants were concerned , was to find a way of regrouping in the face of hostile day - to - day working conditions . debates was : how , if at all , can health promotion specialists employed by the state put their radical principles into practice? or , as one participant succinctly put it : how can we hang on to health of the nation on a day - to - day basis , many hpss had to find ways of working with the individual lifestyle agenda central to government strategy in the area , even if they were privately distasteful of it . specialisation in the broad field of medicine , as i have claimed , seeks in part to normalise categories of health in order to allow those with claims over the area concerned to gain control and jurisdiction . normalised category. if normalisation is part of the ambition of those who are attempting to specialise , and if such a goal is so deeply elusive as it seems to have been in the case of shp , it is unlikely that processes of professionalisation ( seeking and being granted power over the field concerned ) will be accepted by those who are able to grant that power . it is perhaps then hardly a surprise that the department of health made clear in 1996 it could not support the attempts being made by sheps to move shp towards becoming a profession . this lack of official sanction led the society almost immediately to suspend efforts to gain mandatory status for its registration scheme . i now want to move back to mdph and claim again the starkness of the contrast between its gathering pace during this period and what happened to shp . informal networks within the non - medical public health field finally led in 1997 to statements of intent between the embryonic mdph forum , the royal institute of public health and the faculty of public health ( fph ) with regard to the development of multidisciplinary public health education and accreditation . gatekeeper to the public health profession , opened its part 1 examinations to non - medics in 1998 . the scene was set for liberalisation of the public health workforce , potentially at all levels ; up to this point , at least in terms of the most influential positions , it had been completely the preserve of medicine . it is hard to avoid the conclusion , when comparing these relatively rapid and positive developments in mdph with what was happening at the same time in shp , that , for at least some hpss , moving into the arena of so - called multidisciplinary public health as reconstituted public health specialists , away from the disputes and doubts of specialist health promotion , must have seemed very attractive . the new labour government , elected in may 1997 , published its first public health strategy almost two years after the fph opened up its part 1 exams to non - medically qualified candidates . the saving lives strategy contained no reference to health promotion , either as a broad field or as a specialism . the health challenges of the new century were to be met by a rather vaguely described multidisciplinary public health workforce. of course , this would include some whose background was in shp . the hps workforce was diffused and dispersed to the extent that , by the beginning of the twenty - first century , a significant number of primary care trusts ( pcts ) , which were supposed to be the key vehicles for health improvement under the reorganisation of the nhs implemented by the new government , reported that they had no access at all to specialist health promotion staff . a succession of further health service reorganisations , particularly affecting pcts , led to more fragmentation of the hps workforce , which in any case had not really recovered from the introduction of the purchaser provider split a decade earlier . this time , however , the background policy rhetoric excluded health promotion and reinforced the concept of public health. the result was the further marginalisation of shp and a strong sense that it had lost control over that part of the health improvement agenda about which it was supposed to be expert . in studies , health promotion : of how this identified particular kinds of practice , and how these had been lost among the other activities of the public health departments that they now worked in . to the extent that the hps role was recognised at all , it was as some kind of loosely specified adjunct to public health. shp had become a specialised element within the larger public health workforce . professionalisation had already faltered and failed ; now too had the idea of health promotion as a specialism . the specialisation was public health , within which the knowledge , understanding and expertise that shp had struggled to describe and develop might or might not be found . i began this paper with the claim that , at least with regard to some cases in the field of medicine and health care , a relationship exists between processes of specialisation and professionalisation . a group seeking to professionalise , to become a profession , must believe that it has particular knowledge and expertise in relation to a given domain . that is to say , it must believe that it is , or has the potential to become , a specialism . the group must therefore develop or enhance its specialism in order to promote claims to becoming a profession ( claims to power and control over the domain concerned ) . in the most effective cases , presumably , the relationship between specialisation and professionalisation is a symbiotic one . so the easy , but not very helpful , answer to the questions above is that shp failed to professionalise because it failed to advance sufficiently as a specialism . the question then becomes : why did it fail to advance and develop as a specialism ? i argue that my narrative has demonstrated three key closely connected difficulties impeding the development of health promotion as a specialism , a field to which hpss could reasonably lay claim . first , there was the difficulty of the specialists agreeing what they should be doing and how they should be doing it . second , there was the problem of them finding a place within the nhs to do their work . third , there was the difficulty that another much more powerful group ( medicine and its ancillary public health medicine ) was staking claim to the domain . taking the first difficulty , throughout the period i have been considering , shp suffered a profound crisis of identity . it was almost constantly asking itself ( and having asked of it ) questions about what it actually was and what it should be doing . an apparently clear and important global rhetoric ( lalonde , the ottawa charter and so on ) seemed very hard to translate into local practice or even national recommendations for practice . if ottawa talked about the need to change structures , the experience of most hpss for most of the time was that they were engaged in the projects of individual behaviour change that government strategy during the period consistently identified as being the appropriate priority for health promotion . in effect one of these was officially sanctioned and the other provided a kind of rallying cry for the radical activists among their number . the consequences of this were that it divided shp into the separate camps of those who were pragmatic about practice on the one hand , and those who in the various ways that i have described rejected pragmatism on the other . authentic practice , the true nature of health promotion . under these conditions of division and dispute it is hard to see either how a specialism could be developed or , of course , a professionalisation project succeed . one thing that might possibly have moved hpss some way towards resolving the issue of what constituted authentic practice was a clear answer to the question of what actually worked in terms of improving health . an answer might have encouraged hpss to think in more unified ways about their practice . it might also have made policymakers consider ( or possibly reconsider ) the direction in which they were encouraging work to go ( either confirming that direction or possibly changing it ) . some answers were certainly found ; for example , the understanding that efforts at individual behaviour change in the primary care context were to some extent effective but were also extremely costly . ( it is difficult if not impossible to apply randomised controlled trials to health promotion simply because an intervention of this kind and its effects can not be isolated ; and therefore the relationship between intervention and effects is seldom if ever clear . ) moreover , simply because of the disputed nature of health promotion , research findings were often subject to ideological interpretation . moving on to the second problem , that of finding a place within the nhs for hpss to work , it seems reasonable to suppose that a specialism requires an acceptable and appropriate organisational place from which the putative specialist area can successfully develop . the removal of heos from local government to the nhs in 1974 exposed them to what turned out to be the first of a series of almost continuous attempts to reorganise the service according to the requirements of its various political controllers . historians of health and health care have pointed to the need for more analytical attention to be paid to the implications for the political economy of health that arose as a result of the 1974 reforms . health economy , where this is understood in much wider terms than simply treatment and care . the problem was that those controlling the health economy were largely concerned simply about treatment and care , allowing little opportunity for the careful development of a specialism that fell outside of these areas . the third and final difficulty was that of another , much more powerful group medicine and its ancillary of public health medicine staking claim to the domain of concern . the influence of medicine on health education and health promotion is quite clear within this narrative . the cohen committee , which essentially provided for the development of health education in the second half of the twentieth century , was formed of a medical majority . from the era of the moh the medical model of individual behaviour change in relation to specific disease or disease risk also dominated approaches to health promotion , often to the distaste of hpss . descriptions of medicine s influence on health promotion , however , only go some way towards explaining the impact that it had on attempts by shp to create a specialism . medical historians have argued that the emergence of specialisation in the broad field of medicine depends on a number of conditions , including confident technical development and an increasing public understanding of the area that is in the process of emerging as a specialism . rational state , which supports the development of medical knowledge on which specialisation depends . such support is founded on a belief that both knowledge and specialisation will be beneficial to the state s citizens . the long - standing dominance of medicine within the broad field of health is clear . if health education ( and then health promotion ) were to develop at all , they would need to do so in ways that somehow involved medical patronage . medicine would need to accept the legitimacy of the putative specialism , both on its own terms and also on behalf of the public and policy interests that it thought itself to be representing . for this to happen , at the very least the conditions described above would need to be present . health education ( and health promotion ) knowledge grew from ideas emerging in the relatively new field of the social sciences . technical developments ( in the form of , for example , the understanding and use of mass media ) . however , these were fragmentary and emanated from fields that were in themselves contested and viewed with suspicion by objective science. public understanding of health education and health promotion was essentially mediated by medicine , which cast the health education facts in a broad context of medical knowledge , suitably diluted for public consumption by medical diktat . rational policy making in the field has often been founded on beliefs about the value of objective medical knowledge , scepticism as to the social science foundations of health promotion and the consequent view that , if it is to exist at all , it should do so as an adjunct of medicine . essentially , medicine could see no reason why health promotion should exist as a specialism in the way that those working in shp conceived it . ( although , as i have emphasised , exactly what this conception might be was far from clear and highly disputed among hpss themselves . ) the professionalisation project of shp failed because health promotion failed to advance as a specialism . it failed to advance because of internal dispute , lack of an adequate organisational place from which to develop and the much more powerful interests of medicine staking claim to the putative specialism s area . these three reasons for the failure of specialisation are closely connected and , taken together , demonstrate the importance of the narrative presented here . it has been claimed with some reason that we are entering a new era in health policy and planning , an era in which traditional concern with treatment and care , while remaining important , will need to be seen in the context of much larger and ultimately significantly threatening problems . these include climate change , resource depletion and dealing with inequalities that exist both within and between states . in some ways , addressing these kinds of problems requires the sort of action proposed by the global strategies that have formed part of the narrative in this paper ; for example , the ottawa charter . it is unlikely that the problems will be effectively addressed through an individual , disease prevention orientation . yet it is this orientation that has persisted through the recent history of shp and eventually contributed to its decline and failure . a focus on the individual divided hpss : it failed to give them a proper organisational place , and it was fundamental to medicine staking a successful claim to health promotion . the failure of shp in effect demonstrates a failure in constructing alternative ways of thinking about health problems . of course , hpss were small in number , and even if successful in specialising and professionalising , would still have had to face enormous problems in conveying any kind of challenge to political and other orthodoxies . the point is that structures and contexts did not even allow them to begin . yet if reorientation is required , as has been claimed , the kinds of alternatives posed by shp have to be considered . thus historical understanding of the failure and how it is related to conceptual , organisational and ideological difficulties might help in determining how the reorientation in health policy and planning that some insist is now required can be better achieved in the future . with this in mind , what i began by claiming was an under - researched area is worthy of more extensive exploration than this paper by itself allows . global comparison , understanding how other states might have coped ( or otherwise ) with the challenge of finding a place for the putative specialism of health promotion in their structures and contexts is one interesting possibility . another is the deeper exploration of what has happened in the english context ; moving beyond the policy and other texts that have been instrumental in the construction of this narrative and towards fieldwork that actually listens to the voices of those who experienced the failure of shp . these are just two possible further research directions for an important and so - far neglected area , which if treated carefully has much to tell us about future possibilities for effective , appropriate and sustainable health policy and practice .

significant attention has been paid to the history of public health in england during the final part of the twentieth century . within this , however , the field that came to be known as specialist health promotion ( shp ) has been relatively neglected . between 1980 and 2000 those working in this field , generally known as health promotion specialists ( hpss ) , enjoyed a relative rise in policy and practice prominence before shp was effectively abandoned by government and others charged with developing and sustaining public - health structures . this paper seeks to explain why the fall of shp is important ; to move towards explaining its rise and decline ; and to argue for greater historical attention to be paid to an important but neglected field within health and health care . essentially , shp emerged from a set of loose and contingent practices known as health education . a range of important social , economic , organisational and political influences contributed to the slow construction of a putative specialism in health promotion , accompanied by the desire on the part of some ( but not all ) hpss to professionalise their role . finally the projects of both specialisation and professionalisation failed , again as a result of then prevailing organisational and political influences . the importance of such a failure in a so - called era of public health is discussed . in the light of this , the paper concludes by briefly setting out an agenda for further research related to the history of shp .

Introduction: Professionalisation, Specialisation and Health Promotion Who Were the Specialists and Where Did They Come From? Health Education and the Origins of Health Promotion, 196379 Disease Prevention and Radical Activism: Health Promotion and the Progress of Specialisation in the 1980s The Hopeless Pursuit of Professionalisation: Multidisciplinary Public Health and the Decline of Specialist Health Promotion in the 1990s Why did Specialisation fail? Why did the Professionalisation Project Collapse? Conclusion

this paper examines such a case , that of specialist health promotion in england during the final years of the twentieth century ( roughly between 1980 and 2000 ) . here , a relatively small group , who eventually came to be known as health promotion specialists , tried to lay particular claim to , and develop theoretical knowledge and practical expertise in , the then - emerging field of health promotion. this field had at least in part grown from that of health education , a set of loose and contingent practices broadly centred on the communication of health messages through teaching , propaganda and other means . for clarity s sake , i will refer throughout this paper to specialist health promotion ( shp ) and health promotion specialists ( hpss ) , although of course my central purpose here is to examine the difficulties these faced in becoming and being regarded as specialists in any sense at all . describing and understanding the failure of shp is important because the late twentieth century has been characterised as an era in which the ideological development of a new kind of public health ( which included health promotion ) took place . the development has been characterised as part of a broad recognition that momentous global issues such as inequality and sustainability require radical action , including novel ways of thinking about the problems of health and health care . in order to address these questions , and to begin to understand the claims and interests of shp in the final years of the twentieth century , it is necessary to consider some aspects of earlier history . health promotion , as a concept and related set of practices , emerged during the late 1970s and early 1980s partly from a relatively lengthy tradition of health information and propaganda , which it is probably best to refer to as health education . the establishment of a connection between smoking and lung cancer at the beginning of the 1950s was especially important in drawing attention to the value of efforts aimed at lifestyle prevention , although , in the case of smoking , the caution of policymakers in tackling a widespread , culturally embedded behaviour meant that it took some time to establish reasonable anti - smoking programmes . the problematic question of medicine s influence over those working in the fields of health education and health promotion extends throughout the history being considered . in 1972 a survey was undertaken by keith tones , an academic who in 1974 would establish one of the first postgraduate diploma courses in health education ( at leeds polytechnic ) , and who would become one of the key drivers of the theorising component within attempts to develop the putative specialism of health education in the decade or so following . the impact of the 1974 reforms generally on local public health is complex but it certainly involved severed ties , disrupted relationships and still further re - balancing of the political economy of health in favour of treatment and care.43 45 nationally , a government consultative document on prevention was published in 1976 , shortly after the reorganisation that had adversely affected local health education and public health services to such a significant extent . in england , a small and scattered community of scholars addressing the problem of the theoretical foundations of health education and health promotion emerged through their association with the founding of postgraduate courses related to the field . the different conceptions of health promotion that emerged from this early theorising contributed to later rifts between those working in shp and , ultimately , as i will argue , to the failure of specialisation and professionalsation . the organisation representing some hpss , now known as the society of health education and health promotion specialists ( sheps ) , reported multiple tales of service fragmentation and dislocation . ironically , the reforms that fragmented specialist health promotion were followed by the conservative government s first public - health strategy , the first attempt at national strategy in this area since prevention and health back in 1976 . the hps workforce was diffused and dispersed to the extent that , by the beginning of the twenty - first century , a significant number of primary care trusts ( pcts ) , which were supposed to be the key vehicles for health improvement under the reorganisation of the nhs implemented by the new government , reported that they had no access at all to specialist health promotion staff . i began this paper with the claim that , at least with regard to some cases in the field of medicine and health care , a relationship exists between processes of specialisation and professionalisation . if ottawa talked about the need to change structures , the experience of most hpss for most of the time was that they were engaged in the projects of individual behaviour change that government strategy during the period consistently identified as being the appropriate priority for health promotion . historians of health and health care have pointed to the need for more analytical attention to be paid to the implications for the political economy of health that arose as a result of the 1974 reforms . essentially , medicine could see no reason why health promotion should exist as a specialism in the way that those working in shp conceived it . thus historical understanding of the failure and how it is related to conceptual , organisational and ideological difficulties might help in determining how the reorientation in health policy and planning that some insist is now required can be better achieved in the future .

the maintenance of cardio- and cerebrovascular health is based on a complex relationship between the heart and the brain . while some responses to stress are vital for survival , mental stress has also been claimed to cause cardiovascular disease . the japanese observation from the early 1990s of a reversible stress - induced cardiomyopathy ( sic ) , the takotsubo , a peculiar type of left ventricular ( lv ) dysfunction triggered by an acute strong emotional or physical stressor , supports this notion . the syndrome , mostly affecting postmenopausal women , presents signs and symptoms of acute coronary syndrome without evidence of obstructive coronary artery disease . the cause for the evident female predisposition of sic is unknown , but may be related to gender differences in vulnerability to emotional stress and myocardial sensitivity to catecholamine toxicity . interestingly , a lower level of estrogen in absence of testosterone in postmenopausal women was recently suggested to explain their greater vulnerability to sic . though the definite pathophysiology of sic remains to be identified , exaggerated sympathetic activation is proposed to be a major contributor to the pathogenesis [ 8 , 9 ] and forms the basis for treatment of this medical entity . against this background , the aim of the present study was to directly record and evaluate efferent sympathetic nerve traffic in patients suffering from sic . twelve patients with sic and twelve healthy age , gender and bmi matched controls were recruited for this study . of the twelve sci patients , 5 patients were consecutively recruited 2448 h ( acute phase ) after sic onset , while 7 patients ( recovery phase , 16 months after onset ) were recruited from the scaar ( swedish coronary angiography and angioplasty register ) and riks - hia ( register of information and knowledge about swedish heart intensive care admission ) registries . scaar and riks hia registries contain data on consecutive patients from swedish hospitals that perform coronary interventions and provide intensive coronary care in sweden and are sponsored by the swedish health authorities . all patients showed pathologic ecg with evidence of either st - segment elevation or st - segment depression and had undergone coronary angiography based on the clinical suspicion of acute coronary syndrome . the following definition for sic was used : ( 1 ) transient hypokinesis , akinesis or dyskinesis in the left or right ventricular segments and frequently , but not always , a stressful trigger ( psychical or physical ) . ( 2 ) the absence of other pathological conditions ( e.g. ischemia , myocarditis , toxic damage , tachycardia etc . ) that may more credibly explain the regional dysfunction . ( 3 ) no or modest elevation in cardiac troponin ( i.e. disparity between troponin level and the amount of the dysfunctional myocardium present . standard pharmacologic therapy with acetylsalicylic acid , clopidogrel , simvastatin and ramipril was introduced during the first days of hospitalization . clopidogrel and simvastatin were discontinued after the sic diagnosis was established while the treatment with acetylsalicylic acid and ramipril was continued . metoprolol was first introduced after substantial recovery of left ventricular function , which usually occurs within 45 days . one of the 12 patients in this study was on beta blockade treatment due to a different illness at time of onset of sic . apart from one patient , none of the patients in this study were on antidepressant or anxiolytic medication . the body mass score ( bmi ) was calculated as body weight in kilograms divided by height in meters squared . systolic ( sbp ) and diastolic ( dbp ) blood pressure was measured using a calibrated sphygmomanometer to the nearest 5 mmhg during 15 min of supine rest . mean arterial pressure ( map ) was calculated by the formula map = dbp + ( sbp dbp)/3 . the patients in the recovery group were investigated without withdrawal of ongoing medication recommended for secondary prevention after myocardial infarction and for long - term treatment of heart failure ( beta - blockers , ace inhibitors , diuretics , nitrates , and digitalis).the study was approved by the local ethical committee . mixed peripheral nerves contain fascicles innervating muscle or skin areas selectively , surrounded by a barrier of connective tissue . c nerve fibres to muscle ( msna ) or skin vascular beds ( ssna ) in limb nerves of awake , unanaesthetized humans . direct recordings of multiunit postganglionic muscle sympathetic nerve activity ( msna ) were obtained with a tungsten microelectrode inserted into a muscle fascicle of the peroneal nerve posterior to the fibular head . details of the nerve recording technique and criteria for msna have been reported previously [ 12 , 13 ] . when a muscle nerve fascicle had been identified , small electrode adjustments were made until a site was found at which spontaneous , pulse - synchronous bursts of neural activity could be recorded . the original nerve signal was amplified with a gain of 50,000 and fed through a bandpass filter with a band width of 7002,000 hz and then through an integrating network with a time constant of 0.1 s , to obtain a mean voltage display of nerve activity . both the filtered and mean voltage neurograms were stored on a computer ( sampling frequency 200 hz ) , together with an electrocardiogram ( via standard chest leads ) and respiratory movements ( via a strain gauge attached to a rubber strap around the chest ) . bursts were identified by inspection of the mean voltage neurogram , aided by a computer program developed in the laboratory . msna was expressed as burst frequency ( bursts / min ) and burst incidence ( bursts/100 heartbeats ) . a relative burst amplitude spectrum was obtained and from it a median relative burst amplitude value was extracted and used for statistical analysis . during the microneurographic recording , finger arterial blood pressure was measured non - invasively by the volume - clamp method , the plethysmographic cuff being placed around the middle phalanx of the third finger ( finapres 2300 ; ohmeda , louisville , kentucky , usa ) and heart rate was monitored via ecg - chest electrodes . results between the study groups are presented as the mean standard error of the mean ( sem ) and range . results within the sic group are presented as the median and 25th and 75th percentiles due to the small number of subjects in each group . comparison between groups was performed with the students t - test for unpaired comparison and within the sci group comparison was performed with the mann twelve patients with sic and twelve healthy age , gender and bmi matched controls were recruited for this study . of the twelve sci patients , 5 patients were consecutively recruited 2448 h ( acute phase ) after sic onset , while 7 patients ( recovery phase , 16 months after onset ) were recruited from the scaar ( swedish coronary angiography and angioplasty register ) and riks - hia ( register of information and knowledge about swedish heart intensive care admission ) registries . scaar and riks hia registries contain data on consecutive patients from swedish hospitals that perform coronary interventions and provide intensive coronary care in sweden and are sponsored by the swedish health authorities . all patients showed pathologic ecg with evidence of either st - segment elevation or st - segment depression and had undergone coronary angiography based on the clinical suspicion of acute coronary syndrome . the following definition for sic was used : ( 1 ) transient hypokinesis , akinesis or dyskinesis in the left or right ventricular segments and frequently , but not always , a stressful trigger ( psychical or physical ) . ( 2 ) the absence of other pathological conditions ( e.g. ischemia , myocarditis , toxic damage , tachycardia etc . ) that may more credibly explain the regional dysfunction . ( 3 ) no or modest elevation in cardiac troponin ( i.e. disparity between troponin level and the amount of the dysfunctional myocardium present . standard pharmacologic therapy with acetylsalicylic acid , clopidogrel , simvastatin and ramipril was introduced during the first days of hospitalization . clopidogrel and simvastatin were discontinued after the sic diagnosis was established while the treatment with acetylsalicylic acid and ramipril was continued . metoprolol was first introduced after substantial recovery of left ventricular function , which usually occurs within 45 days . one of the 12 patients in this study was on beta blockade treatment due to a different illness at time of onset of sic . apart from one patient , none of the patients in this study were on antidepressant or anxiolytic medication . the body mass score ( bmi ) was calculated as body weight in kilograms divided by height in meters squared . systolic ( sbp ) and diastolic ( dbp ) blood pressure was measured using a calibrated sphygmomanometer to the nearest 5 mmhg during 15 min of supine rest . mean arterial pressure ( map ) was calculated by the formula map = dbp + ( sbp dbp)/3 . the patients in the recovery group were investigated without withdrawal of ongoing medication recommended for secondary prevention after myocardial infarction and for long - term treatment of heart failure ( beta - blockers , ace inhibitors , diuretics , nitrates , and digitalis).the study was approved by the local ethical committee . mixed peripheral nerves contain fascicles innervating muscle or skin areas selectively , surrounded by a barrier of connective tissue . c nerve fibres to muscle ( msna ) or skin vascular beds ( ssna ) in limb nerves of awake , unanaesthetized humans . direct recordings of multiunit postganglionic muscle sympathetic nerve activity ( msna ) were obtained with a tungsten microelectrode inserted into a muscle fascicle of the peroneal nerve posterior to the fibular head . details of the nerve recording technique and criteria for msna have been reported previously [ 12 , 13 ] . when a muscle nerve fascicle had been identified , small electrode adjustments were made until a site was found at which spontaneous , pulse - synchronous bursts of neural activity could be recorded . the original nerve signal was amplified with a gain of 50,000 and fed through a bandpass filter with a band width of 7002,000 hz and then through an integrating network with a time constant of 0.1 s , to obtain a mean voltage display of nerve activity . both the filtered and mean voltage neurograms were stored on a computer ( sampling frequency 200 hz ) , together with an electrocardiogram ( via standard chest leads ) and respiratory movements ( via a strain gauge attached to a rubber strap around the chest ) . bursts were identified by inspection of the mean voltage neurogram , aided by a computer program developed in the laboratory . msna was expressed as burst frequency ( bursts / min ) and burst incidence ( bursts/100 heartbeats ) . a relative burst amplitude spectrum was obtained and from it a median relative burst amplitude value was extracted and used for statistical analysis . during the microneurographic recording , finger arterial blood pressure was measured non - invasively by the volume - clamp method , the plethysmographic cuff being placed around the middle phalanx of the third finger ( finapres 2300 ; ohmeda , louisville , kentucky , usa ) and heart rate was monitored via ecg - chest electrodes . results between the study groups are presented as the mean standard error of the mean ( sem ) and range . results within the sic group are presented as the median and 25th and 75th percentiles due to the small number of subjects in each group . comparison between groups was performed with the students t - test for unpaired comparison and within the sci group comparison was performed with the mann besides being matched for age and bmi , the study groups did not differ in terms of heart rate and blood pressure level ( table 1).table 1basic characteristics of the study groupspatientsrangecontrolsrangep valuenumber / gender12 f12 fage ( years)66 3.3488562 2.746740.5bmi ( kg / m)25 1.9163424 1.222310.8hemodynamic data sbp ( mmhg)113 1592140126 15921500.6 dbp ( mmhg)64 14478073 1357950.2 map ( mmhg)77 10549290 13721100.4 hr ( beats / min)62 14459164 755760.6 r r interval ( sec)1.00 0.130.651.30.95 0.060.781.080.5microneurography msna ( burst / min)32 5.0136842 1224680.1 msna ( burst/100 heartbeats)49 17267765 1243900.03 msna ( median amp % ) 37 1.6304740 4.633460.1results are presented as mean sem and rangebmi body mass index , hr heart rate , map mean arterial pressure basic characteristics of the study groups results are presented as mean sem and range bmi body mass index , hr heart rate , map mean arterial pressure muscle sympathetic nerve activity ( msna ) expressed as burst incidence ( bi ) was significantly lower in patients with sic compared to matched healthy controls and though not reaching significance , tended to be lower also when expressed as burst frequency ( bf ) ( table 1 ) . the relative median burst amplitude did not deviate between the groups ( table 1 ) . within the sic group , there was no difference in msna bf , bi or relative median burst amplitude between the acute and recovery phase ( table 2).table 2muscle sympathetic nerve activity in patients with sic in the acute and recovery phasevariablesic acute phase ( n = 5)sic recovery phase ( n = 7)p valueage ( years)70 ( 6079)64 ( 5672)0.5bmi ( kg / m)29 ( 2434)22 ( 2224)0.05hemodynamic variables sbp ( mmhg)105 ( 104122)113 ( 98115)1.0 dbp ( mmhg)56 ( 4067)60 ( 5766)0.4 map ( mmhg)74 ( 6280)78 ( 7780)0.5 hr ( beats / min)77 ( 5183)60 ( 4961)0.4 r r interval ( sec)0.78 ( 0.701.16)1.00 ( 0.981.2)0.4 lvef ( % ) 50 ( 3560)60 ( 5760)0.2microneurography msna ( bursts / min)47 ( 2050)25 ( 1734)0.2 msna ( bursts/100 beats)57 ( 3965)51 ( 2956)0.3 msna ( median amp % ) 34 ( 3342)39 ( 3241)1.0measures are shown as the median and 25th and 75th percentilesbmi body mass index , hr heart rate , lvef ( % ) left ventricular ejection fraction , msna muscle sympathetic nerve activity muscle sympathetic nerve activity in patients with sic in the acute and recovery phase measures are shown as the median and 25th and 75th percentiles bmi body mass index , hr heart rate , lvef ( % ) left ventricular ejection fraction , msna muscle sympathetic nerve activity msna bf and bi did not differ between the control group and the sic patients in the acute phase ( 41 vs. 40 b / min , p = 0.8 and 63 vs. 54 b/100 hb , p = 0.2 , respectively ) , but was significantly lower in the sic patients in the recovery phase as compared to the control group ( 41 vs. 26 b / min , p = 0.02 and 63 vs. 46 b/100 hb , p = 0.03 , respectively ) , ( fig . 1 ) . there was no relation between time from onset of sic and degree of sympathetic activity.fig . 1a sympathetic nerve activity , expressed as burst incidence ( bi ) in 12 patients with sic ( 5 in the acute phase and 7 in the recovery phase ) and 12 matched controls ( * p = 0.03 ) . b examples of microneurographic recordings from a muscle ( msna ) and skin ( ssna ) fascicle in a sic patient in the recovery phase . the figures show a integrated neurogram of msna ( a ) and ssna ( b ) in a 64-year - old female patient with msna bf of 17 bursts / min and ssna bf of 27 bursts / min a sympathetic nerve activity , expressed as burst incidence ( bi ) in 12 patients with sic ( 5 in the acute phase and 7 in the recovery phase ) and 12 matched controls ( * p = 0.03 ) . b examples of microneurographic recordings from a muscle ( msna ) and skin ( ssna ) fascicle in a sic patient in the recovery phase . the figures show a integrated neurogram of msna ( a ) and ssna ( b ) in a 64-year - old female patient with msna bf of 17 bursts / min and ssna bf of 27 bursts / min systolic- , diastolic- and mean arterial blood pressure values did not differ between the groups ( tables 1 , 2 ) and showed no relation to msna bf and bi . heart rate and r r interval of the ecg was not significantly different between the groups ( tables 1 , 2 ) . ejection fraction ( ef % ) , as measured by ultrasonic echocardiography , did not deviate between the sic patients in the acute and recovery phase ( table 2 ) . an apparent inverse relationship between msna and ef % was lost when adjusted for age . muscle sympathetic nerve activity ( msna ) expressed as burst incidence ( bi ) was significantly lower in patients with sic compared to matched healthy controls and though not reaching significance , tended to be lower also when expressed as burst frequency ( bf ) ( table 1 ) . the relative median burst amplitude did not deviate between the groups ( table 1 ) . within the sic group , there was no difference in msna bf , bi or relative median burst amplitude between the acute and recovery phase ( table 2).table 2muscle sympathetic nerve activity in patients with sic in the acute and recovery phasevariablesic acute phase ( n = 5)sic recovery phase ( n = 7)p valueage ( years)70 ( 6079)64 ( 5672)0.5bmi ( kg / m)29 ( 2434)22 ( 2224)0.05hemodynamic variables sbp ( mmhg)105 ( 104122)113 ( 98115)1.0 dbp ( mmhg)56 ( 4067)60 ( 5766)0.4 map ( mmhg)74 ( 6280)78 ( 7780)0.5 hr ( beats / min)77 ( 5183)60 ( 4961)0.4 r r interval ( sec)0.78 ( 0.701.16)1.00 ( 0.981.2)0.4 lvef ( % ) 50 ( 3560)60 ( 5760)0.2microneurography msna ( bursts / min)47 ( 2050)25 ( 1734)0.2 msna ( bursts/100 beats)57 ( 3965)51 ( 2956)0.3 msna ( median amp % ) 34 ( 3342)39 ( 3241)1.0measures are shown as the median and 25th and 75th percentilesbmi body mass index , hr heart rate , lvef ( % ) left ventricular ejection fraction , msna muscle sympathetic nerve activity muscle sympathetic nerve activity in patients with sic in the acute and recovery phase measures are shown as the median and 25th and 75th percentiles bmi body mass index , hr heart rate , lvef ( % ) left ventricular ejection fraction , msna muscle sympathetic nerve activity msna bf and bi did not differ between the control group and the sic patients in the acute phase ( 41 vs. 40 b / min , p = 0.8 and 63 vs. 54 b/100 hb , p = 0.2 , respectively ) , but was significantly lower in the sic patients in the recovery phase as compared to the control group ( 41 vs. 26 b / min , p = 0.02 and 63 vs. 46 b/100 hb , p = 0.03 , respectively ) , ( fig . 1 ) there was no relation between time from onset of sic and degree of sympathetic activity.fig . 1a sympathetic nerve activity , expressed as burst incidence ( bi ) in 12 patients with sic ( 5 in the acute phase and 7 in the recovery phase ) and 12 matched controls ( * p = 0.03 ) . b examples of microneurographic recordings from a muscle ( msna ) and skin ( ssna ) fascicle in a sic patient in the recovery phase . the figures show a integrated neurogram of msna ( a ) and ssna ( b ) in a 64-year - old female patient with msna bf of 17 bursts / min and ssna bf of 27 bursts / min a sympathetic nerve activity , expressed as burst incidence ( bi ) in 12 patients with sic ( 5 in the acute phase and 7 in the recovery phase ) and 12 matched controls ( * p = 0.03 ) . b examples of microneurographic recordings from a muscle ( msna ) and skin ( ssna ) fascicle in a sic patient in the recovery phase . the figures show a integrated neurogram of msna ( a ) and ssna ( b ) in a 64-year - old female patient with msna bf of 17 bursts / min and ssna bf of 27 bursts / min systolic- , diastolic- and mean arterial blood pressure values did not differ between the groups ( tables 1 , 2 ) and showed no relation to msna bf and bi . heart rate and r r interval of the ecg was not significantly different between the groups ( tables 1 , 2 ) . ejection fraction ( ef % ) , as measured by ultrasonic echocardiography , did not deviate between the sic patients in the acute and recovery phase ( table 2 ) . an apparent inverse relationship between msna and ef % was lost when adjusted for age . the novel finding in this study is that sympathetic outflow to the muscle vascular bed is shown to be lower in patients suffering from stress induced cardiomyopathy as compared to healthy matched controls . this is in stark contrast to findings in both female and male patients following uncomplicated acute myocardial infarction ( ami ) , where msna was severely augmented as compared to controls , an activation shown to be sustained up to 6 - 9 months following ami , despite optimal pharmacological treatment including diuretics , ace - inhibitors and beta - blockers . in our study , msna did not significantly differ between controls and sic patients in the acute phase , indicating that sympathetic neuronal outflow is rapidly reduced following the initial acute phase of sic , while it was lower in patients in the recovery phase , which may in part be due to medication , though degree of nerve activity was not related to time from onset of sic . increased plasma catecholamine levels have been reported in sic and interpreted as evidence for generally increased sympathetic activity , related to the fact that the syndrome is elicited by an acute stressor and associated with an increase in stress - related neuropeptides . endomyocardial biopsies in some tc patients show contraction band necrosis , a finding consistent with catecholamine mediated cardiotoxicity . our finding of a reduced msna in sic compared to healthy controls may seem contradictory to previous findings of increased plasma catecholamine , but they may instead reflect different phases of the syndrome . an initial intense sympathetic activation in the acute phase of sic may cause excessive catecholamine release over the heart and distension of the ventricles . the left ventricular myocardium contains unmyelinated afferents with receptors that are excited both by mechanical and chemical stimuli . it has been proposed that in myocardial distress , these intra - cardiac receptors may function as protective nociceptors , which when activated , can inhibit cardiac contraction and decrease peripheral resistance . a distension of the ventricular myocardium , due to excessive catecholamine release over the heart in the acute phase , could increase the rate of discharge of these unmylellinated cardiac c - fibre afferents , resulting in reflex vagal bradycardia and widespread sympathetic inhibition . due to such a reflex mechanism , even though recorded in the acute phase , msna would not capture the early acute catecholamine release as the reflex would already have kicked in. the sympathetic nervous system is a highly differentiated system with a clear distinction in neuronal outflow between sympathetic subdivisions . msna and the sympathetic branch supplying the skin vascular bed ( ssna ) are the two subdivisions of the sympathetic nervous system accessible with microneurography . msna , is mainly involved in hemodynamic regulation and is under strong baroreflex control and correlates well with total body norepinephrine spillover as well as with regional ( heart , kidney and subcortical ) norepinephrine spillover [ 18 , 19 ] . in human heart failure though , cardiac adrenergic drive , thought to be of pathogenic importance , has been shown to precede the augmentation in sympathetic outflow to the kidney and muscle vascular beds . ssna , on the other hand , is mainly involved in thermoregulation and under limited baroreflex control , where only the sudomotor component of ssna exhibits some cardiac rhythmicity . in thermoneutral conditions , however and despite differences in baroreflex control , it has been speculated that under certain conditions , a common central pacemaker may influence sympathetic outflow to both the heart and the skin . in the present study , recordings of both msna and ssna were obtained in three sic patients in the recovery phase . interestingly , all three patients exhibited surprisingly high ssna , given that the recordings were performed in supine rest at a comfortable room temperature . the ssna showed clear cardiac rhythmicity suggesting a strong sudomotor contribution , and all three patients exhibited more pulse - synchronous ssna than msna bursts per minute ( fig . thus , while msna was low in our sic patients , our observations suggest that ssna , which is not involved in systemic hemodynamics , may be high , possibly due to enhanced responsiveness to emotional stimuli and may therefore reflect an aroused state . dissociation in the outflow of these two sympathetic branches has been shown previously in conditions such as aging , heart failure , obesity and essential hypertension , but contrary to sic , in these conditions msna is shown to be high while ssna is low or unchanged . the sympathetic nervous system is a highly differentiated system with a clear distinction in neuronal outflow between sympathetic subdivisions . msna and the sympathetic branch supplying the skin vascular bed ( ssna ) are the two subdivisions of the sympathetic nervous system accessible with microneurography . msna , is mainly involved in hemodynamic regulation and is under strong baroreflex control and correlates well with total body norepinephrine spillover as well as with regional ( heart , kidney and subcortical ) norepinephrine spillover [ 18 , 19 ] . in human heart failure though , cardiac adrenergic drive , thought to be of pathogenic importance , has been shown to precede the augmentation in sympathetic outflow to the kidney and muscle vascular beds . ssna , on the other hand , is mainly involved in thermoregulation and under limited baroreflex control , where only the sudomotor component of ssna exhibits some cardiac rhythmicity . in thermoneutral conditions , however and despite differences in baroreflex control , it has been speculated that under certain conditions , a common central pacemaker may influence sympathetic outflow to both the heart and the skin . in the present study , recordings of both msna and ssna were obtained in three sic patients in the recovery phase . interestingly , all three patients exhibited surprisingly high ssna , given that the recordings were performed in supine rest at a comfortable room temperature . the ssna showed clear cardiac rhythmicity suggesting a strong sudomotor contribution , and all three patients exhibited more pulse - synchronous ssna than msna bursts per minute ( fig . thus , while msna was low in our sic patients , our observations suggest that ssna , which is not involved in systemic hemodynamics , may be high , possibly due to enhanced responsiveness to emotional stimuli and may therefore reflect an aroused state . dissociation in the outflow of these two sympathetic branches has been shown previously in conditions such as aging , heart failure , obesity and essential hypertension , but contrary to sic , in these conditions msna is shown to be high while ssna is low or unchanged . though optimal pharmacological treatment for sic is not known , most sic patients are treated according to the general guidelines for treatment of acute myocardial infarction . this therapy includes diuretics , ace - inhibitors and beta - blockers , medication known in the long - term to inhibit the sympathetic nervous system . although based on a limited number of patients , our results clearly suggest that sympathetic neuronal outflow is rapidly reduced following the initial acute phase of sic . thus , a further understanding of the underlying mechanisms is needed for optimal treatment of the condition . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

purposeto evaluate directly recorded efferent sympathetic nerve traffic in patients with stress - induced cardiomyopathy ( sic).backgroundsic is a syndrome affecting mostly postmenopausal women following severe emotional stress . though the precise pathophysiology is not well understood , a catecholamine overstimulation of the myocardium is thought to underlie the pathogenesis.methodsdirect recordings of multiunit efferent postganglionic muscle sympathetic nerve activity ( msna ) were obtained from 12 female patients , 5 in the acute ( 2448 h ) and 7 in the recovery phase ( 16 months ) , with apical ballooning pattern and 12 healthy matched controls . msna was expressed as burst frequency ( bf ) , burst incidence ( bi ) and relative median burst amplitude ( rmba % ) . one of the twelve patients in this study was on beta blockade treatment due to a different illness , at time of onset of sic . all patients were investigated with ongoing medication.resultsmsna was lower in patients with sic as compared to matched controls , but did not differ between the acute and recovery phase of sic . rmba % , blood pressure and heart rate did not differ between the groups.conclusionmsna is shown to be lower in patients with sic compared to healthy controls , suggesting that sympathetic neuronal outflow is rapidly reduced following the initial phase of sic . a distension of the ventricular myocardium , due to excessive catecholamine release over the heart in the acute phase , may increase the firing rate of unmyelinated cardiac c - fibre afferents resulting in widespread sympathetic inhibition . such a mechanism may underlie the lower msna reported in our patients .

Introduction Methods Subjects Microneurography Statistics Results Muscle sympathetic nerve activity Hemodynamic variables Discussion Sympathetic differentiation Conclusion Open Access

comparison between groups was performed with the students t - test for unpaired comparison and within the sci group comparison was performed with the mann besides being matched for age and bmi , the study groups did not differ in terms of heart rate and blood pressure level ( table 1).table 1basic characteristics of the study groupspatientsrangecontrolsrangep valuenumber / gender12 f12 fage ( years)66 3.3488562 2.746740.5bmi ( kg / m)25 1.9163424 1.222310.8hemodynamic data sbp ( mmhg)113 1592140126 15921500.6 dbp ( mmhg)64 14478073 1357950.2 map ( mmhg)77 10549290 13721100.4 hr ( beats / min)62 14459164 755760.6 r r interval ( sec)1.00 0.130.651.30.95 0.060.781.080.5microneurography msna ( burst / min)32 5.0136842 1224680.1 msna ( burst/100 heartbeats)49 17267765 1243900.03 msna ( median amp % ) 37 1.6304740 4.633460.1results are presented as mean sem and rangebmi body mass index , hr heart rate , map mean arterial pressure basic characteristics of the study groups results are presented as mean sem and range bmi body mass index , hr heart rate , map mean arterial pressure muscle sympathetic nerve activity ( msna ) expressed as burst incidence ( bi ) was significantly lower in patients with sic compared to matched healthy controls and though not reaching significance , tended to be lower also when expressed as burst frequency ( bf ) ( table 1 ) . within the sic group , there was no difference in msna bf , bi or relative median burst amplitude between the acute and recovery phase ( table 2).table 2muscle sympathetic nerve activity in patients with sic in the acute and recovery phasevariablesic acute phase ( n = 5)sic recovery phase ( n = 7)p valueage ( years)70 ( 6079)64 ( 5672)0.5bmi ( kg / m)29 ( 2434)22 ( 2224)0.05hemodynamic variables sbp ( mmhg)105 ( 104122)113 ( 98115)1.0 dbp ( mmhg)56 ( 4067)60 ( 5766)0.4 map ( mmhg)74 ( 6280)78 ( 7780)0.5 hr ( beats / min)77 ( 5183)60 ( 4961)0.4 r r interval ( sec)0.78 ( 0.701.16)1.00 ( 0.981.2)0.4 lvef ( % ) 50 ( 3560)60 ( 5760)0.2microneurography msna ( bursts / min)47 ( 2050)25 ( 1734)0.2 msna ( bursts/100 beats)57 ( 3965)51 ( 2956)0.3 msna ( median amp % ) 34 ( 3342)39 ( 3241)1.0measures are shown as the median and 25th and 75th percentilesbmi body mass index , hr heart rate , lvef ( % ) left ventricular ejection fraction , msna muscle sympathetic nerve activity muscle sympathetic nerve activity in patients with sic in the acute and recovery phase measures are shown as the median and 25th and 75th percentiles bmi body mass index , hr heart rate , lvef ( % ) left ventricular ejection fraction , msna muscle sympathetic nerve activity msna bf and bi did not differ between the control group and the sic patients in the acute phase ( 41 vs. 40 b / min , p = 0.8 and 63 vs. 54 b/100 hb , p = 0.2 , respectively ) , but was significantly lower in the sic patients in the recovery phase as compared to the control group ( 41 vs. 26 b / min , p = 0.02 and 63 vs. 46 b/100 hb , p = 0.03 , respectively ) , ( fig . within the sic group , there was no difference in msna bf , bi or relative median burst amplitude between the acute and recovery phase ( table 2).table 2muscle sympathetic nerve activity in patients with sic in the acute and recovery phasevariablesic acute phase ( n = 5)sic recovery phase ( n = 7)p valueage ( years)70 ( 6079)64 ( 5672)0.5bmi ( kg / m)29 ( 2434)22 ( 2224)0.05hemodynamic variables sbp ( mmhg)105 ( 104122)113 ( 98115)1.0 dbp ( mmhg)56 ( 4067)60 ( 5766)0.4 map ( mmhg)74 ( 6280)78 ( 7780)0.5 hr ( beats / min)77 ( 5183)60 ( 4961)0.4 r r interval ( sec)0.78 ( 0.701.16)1.00 ( 0.981.2)0.4 lvef ( % ) 50 ( 3560)60 ( 5760)0.2microneurography msna ( bursts / min)47 ( 2050)25 ( 1734)0.2 msna ( bursts/100 beats)57 ( 3965)51 ( 2956)0.3 msna ( median amp % ) 34 ( 3342)39 ( 3241)1.0measures are shown as the median and 25th and 75th percentilesbmi body mass index , hr heart rate , lvef ( % ) left ventricular ejection fraction , msna muscle sympathetic nerve activity muscle sympathetic nerve activity in patients with sic in the acute and recovery phase measures are shown as the median and 25th and 75th percentiles bmi body mass index , hr heart rate , lvef ( % ) left ventricular ejection fraction , msna muscle sympathetic nerve activity msna bf and bi did not differ between the control group and the sic patients in the acute phase ( 41 vs. 40 b / min , p = 0.8 and 63 vs. 54 b/100 hb , p = 0.2 , respectively ) , but was significantly lower in the sic patients in the recovery phase as compared to the control group ( 41 vs. 26 b / min , p = 0.02 and 63 vs. 46 b/100 hb , p = 0.03 , respectively ) , ( fig .

laromustine ( also called 101 m , vnp40101 m , cloretazine , and onrigin ) , 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine ) , ks119 ( 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine ) , and ks119w ( 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-phospho-4-nitrophenyl)ethoxy]carbonyl]hydrazine ) are relatively new anticancer alkylating agents . laromustine , which has shown significant clinical activity in phase i and ii clinical trials in aml , generates 90ce upon base catalyzed decomposition ; whereas , ks119 and ks119w generate 90ce when reductively activated , with the aim of adding selectivity toward hypoxic solid tumor regions . the 90ce moiety has considerable potential in tumor targeted prodrugs because it is an excellent leaving group , and the rapid spontaneous decomposition of 90ce ( t1/2 30 s ) , which can be prevented by the substitution of the n-2 hydrogen with a cleavable chemical trigger , results in the delivery of alkylation stress close to the site of activation . the cytotoxic action of 90ce appears to result from the generation of chloroethylating intermediates that alkylate biomolecules , particularly the o-6 position of dna guanine . this latter alkylation accounts for the vast majority of its anticancer activity and leads to the eventual formation of a 1-(n - cytosinyl),-2-(n - guaninyl)ethane dna dna interstrand cross - link ( g - c ethane cross - link ) via an n , o - ethanoguanine intermediate ( figure 1 ) . selectivity for tumor cells arises from differentials between normal and tumor cells in their effective levels of mgmt , the resistance protein responsible for the repair of dna o-6 guanine lesions , and in their ability to repair the resultant cross - links generated from lesions that evade restoration by mgmt . each mgmt molecule can only repair a single o-6 guanine lesion ; therefore , the mgmt content correlates with sensitivity to agents of this type . few tumors are completely devoid of mgmt activity , but those that are exhibit high sensitivities to agents that generate guanine o-6 lesions among their repertoire of dna damage . summarizing scheme illustrating the generation of therapeutic guanine o-6 lesions and g - c ethane cross - links exploiting tumor cell dna repair deficiencies by 90ce and prodrugs thereof and bcnu . ( panel a ) the generation from 90ce and prodrugs thereof of primary and potential secondary chloroethylating species . ( panel b ) the generation from bcnu of aminoethylating , carbamoylating , hydroxyethylating , and vinylating species not strongly associated with therapeutic activity , and the formation of therapeutic anticancer chloroethylating species which are potentially in common with the secondary chloroethylating species generated by 90ce . ( panel c ) the overall stoichiometry for the generation of chloroethanol via the chloroethylation of water by 90ce via the primary or the secondary chloroethylating species . ( panel d ) scheme showing the major therapeutically relevant dna lesions generated by oxophilic chloroethylating electrophiles and the cellular processes involved in their repair . this lesion can be restored by mgmt or cyclize to form 1-(n - cytosinyl)-2-(n - guaninyl)ethane . this latter lesion can either react with the opposing cytosine to form a high cytotoxic g - c ethane cross - link or with mgmt preventing cross - link formation , but additional processes are required to complete the repair . cells have a limited capacity to repair the resultant g - c ethane cross - links via hdr . once formed , the g - c ethane cross - link , which does not contain an o-6 linkage , can not be repaired by mgmt . however , cells can repair a limited number of g - c ethane cross - links , probably using homology directed repair ( hdr ) . as expected , cells possessing defective hdr show additional sensitivity to laromustine , and cells lacking both hdr and mgmt are hypersensitive to this agent . a very small proportion of tumors are likely to possess both of these defects , but it is possible to screen for subsets of cancers that would likely exhibit exceptional sensitivity to 90ce prodrugs . compared to bcnu ( 1,3-bis(2-chloroethyl)-1-nitrosourea ) , which generates a wide array of electrophiles , including carbamoylating species ( figure 1 ) , a greater proportion of the overall cytotoxicity of 90ce appears to depend upon the o-6 chloroethylation of guanine . in support of this assertion , a 22-fold differential in clonogenic lc90 values was found between l1210 cell lines expressing and not expressing mgmt for 90ce , compared to only a 2-fold differential for bcnu in the same matched cell line pair . furthermore , laromustine produces 100% cures in many mgmt - deficient in vivo tumor models and has a therapeutic index ( ld50/ed50 ) against the l1210 leukemia of > 8 , more than double that of over 300 nitrosoureas tested . electrophilic and nucleophilic species can be described as hard or soft , and this concept can be used to predict their reaction site preference . hard electrophiles have a high positive charge density and tend to react via sn1 reaction mechanisms with hard nucleophiles which have a high negative charge density . in contrast , soft electrophiles have a low charge density or are easily polarized and tend to react via sn2 reaction mechanisms with soft nucleophiles that have a low negative charge density or are easily polarized . the relative hardness and softness of electrophiles determines the spectrum of nucleophilic positions available in dna for preferred reactions . the dna - backbone phosphate groups are the hardest nucleophilic sites in dna , and the o-6 position of guanine is the hardest base centered site , while the n-7 position of guanine is the softest . a single therapeutic alkylating agent can generate a wide array of lesions on sites of diverse hardness / softness in dna because complex decomposition pathways can result in the production of many alkylating species with a wide range of hardness / softness and hence different and/or overlapping alkylation site preferences . this is the case with bcnu , which generates several potential chloroethylating species together with species that hydroxyethylate , vinylate , aminoethylate , and carbamoylate . while o-6 chloroethylation of guanine still dominates in terms of the anticancer activity of bcnu , it is a relatively minor product compared to alkylations on the n-7 or n-1 positions of dna guanine . 90ce was designed to generate a unique primary chloroethylating species ( clch2ch2n = nso2ch3 ) , but this intermediate could potentially give rise to three other secondary chloroethylating species ( 2-chloroethyldiazohydroxide , 2-chloroethyldiazonium , and chloronium ions ) that are in common with those believed to be generated by bcnu ( figure 1 ) . nevertheless , the pattern of dna alkylation by 90ce appears to have a greater o-6 guanine to n-7 guanine alkylation bias under biologically relevant conditions and in in vitro and in vivo models than that of bcnu . we have previously studied the decomposition mechanism of 90ce in non - pi and low pi buffers ; under these conditions , 90ce appears to decompose in a relatively simplistic manner , as initially intended by design , to generate approximately two moles of methanesulfinate , one mole of nitrogen gas , and one mole of 2-chloroethanol , produced as a consequence of the chloroethylation of water ( figure 1 ) . it was found that the rate determining step during the decomposition of 90ce and 1,2-bis(sulfonyl)-1-alkylhydrazines was the elimination of the n-1 sulfonyl moiety from the 1,2-bis(sulfonyl)-1-alkylhydrazine anion , which in the case of 90ce results in the formation of the primary chloroethylating species clch2ch2n = nso2ch3 ( figure 1 ) . subsequently , we have found that in the presence of high concentrations of pi , the alkylation of 4-(4-nitrobenzyl)pyridine ( 4nbp ) by 90ce was greatly increased . 4nbp is the most commonly used colorimetric reagent for determining alkylating activity , and the pyridine nitrogen which serves as the nucleophilic target in this molecule is a moderately soft site . thus , 4nbp would normally be expected to be poorly alkylated by 90ce , as is found in the absence of pi . this pi dependent change in character was not observed with 1,2-bis(methylsulfonyl)-1-methylhydrazine ( ks90 ) , the methylating analogue of 90ce . in view of the ubiquitous cellular presence of pi , this interaction was further investigated , and a second decomposition pathway for 90ce has been elucidated . the implications of these findings in terms of future drug designs , tumor cell selectivity , agent toxicity , and possible novel resistance mechanisms are discussed . both 90ce and ks90 are potentially carcinogenic and mutagenic and should be handled carefully using personal protective equipment . all other chemicals were purchased from the sigma - aldrich chemical co. , st . eugene , or ; hpcd , ( 2-hydroxypropyl)--cyclodextrin was obtained from american maize products company ( 1100 indianapolis boulevard , hammond , in ) . murine l1210 leukemia cells were used as a source of dna for dna cross - linking assays . l1210 leukemia cell lines were grown in suspension culture in rpmi 1640 medium supplemented with 10% fbs in air/5% co2 at 37 c . dna was isolated using a puregene dna isolation kit ( gentra systems , minneapolis , mn ) using procedures recommended by the manufacturer . isolated l1210 dna was diluted to 400 g / ml with 5 mm tris , 1 mm edta , and 1 mm nan3 at ph 7.4 buffer , and stored at 4 c until required . the alkylation of 4nbp by 90ce in the presence of various concentrations of pi was studied using a method that is dependent upon the high ph aqueous stabilization of alkylated 4nbp using ( 2-hydroxypropyl)--cyclodextrin ( hpcd ) . all buffers contained 20 mm tris - hcl ( ph 7.4 ) and various concentrations of potassium phosphate 0200 mm ( ph 7.4 ) . 4nbp was added to give a final concentration of 1 mg / ml ( close to its solubility at 37 c ) from a 50 stock solution in dmso , and -thioglycerol ( tg ) was added as a competing nucleophile in some experiments to give final concentrations of either 1 or 20 mm from 100 aqueous stock solutions . the reactions were initiated by the addition of 200 m 90ce ( 100 mm stock solutions in dmso ) and incubated at 37 c for 15 min . some analogous experiments were performed using bcnu in place of 90ce with 20 h overnight incubations being utilized due to its more than 60-fold longer t1/2 time . the samples were assayed for 4nbp alkylation products by rapidly mixing equal volumes of the above reaction mixtures with 38% hpcd in 0.5 m koh at room temperature and recording the absorbance at 635 nm within 1 min . aging experiments to follow the loss of the ability of 90ce to alkylate 4nbp under high pi concentrations were performed in a very similar manner . the reaction was initiated by the addition of 200 m 90ce to 200 mm potassium phosphate buffer ( ph 7.4 ) at 37 c with very rapid mixing . one milliliter samples were then taken from this mixture and rapidly mixed with 20 l of 50 mg / ml of 4nbp in dmso , 0 , 30 , 60 , 90 , 120 , and 300 s after reaction initiation , and incubated for a further 15 min . mnp is a highly chromophoric thiol / thiolate at physiological ph values , absorbing with a max at 389 nm . in these experiments , observations were made at 440 nm because at this wavelength , the extinction coefficient is still substantial , and derivatization of the thiol / thiolate causes a complete rather than a partial bleaching of the absorbance . mnp was generated in situ from 2,2-dithiobis(5-nitropyridine ) by reduction with thioglycerol ( tg ) . to a 60 m dtbnp solution ( 10 mm stock in dmso ) in 200 mm potassium phosphate buffer ( ph 7.4 , 37 c ) , a limiting quantity of 100 m tg ( 10 mm stock in h2o ) was added ; this should convert 50 m of the dtbnp to 100 m of mnp , exhausting the tg and leaving mnp as essentially the only thiol present . this assumption was confirmed by observing an equivalent increase in absorption at 440 nm upon adding a 100 molar - fold excess of tg to 50 m dtbnp . the absorbance of 100 m mnp at 440 nm was 0.775 au and was attained within a few minutes of the addition of 100 m tg at 37 c and remained stable thereafter at this level . addition of excess 90ce ( 500 m ) fully bleached the mnp absorbance in < 1 min . this method for producing mnp was used to follow the extent and kinetics of alkylation / bleaching of mnp upon the addition of limiting aliquots of 90ce in 200 mm potassium phosphate buffer . to examine the effects of anions / solutes other than phosphate on the generation of soft electrophilic species from 90ce , identical experiments were performed in 20 mm tris - hcl buffer ( ph 7.4 , 37 c ) containing 100 m mnp ( generated in situ as described ) in which the buffer was supplemented with various solutes ( ph adjusted to 7.4 ) at 50 mm . the bleaching of mnp was followed after the addition of 100 m 90ce over 5 min ( 50 mm pi results in a submaximal effect ) . in the case of atp , some experiments were also performed in the presence of a 1.05-fold molar excess of mgcl2 . reaction mixtures had a final total volume of 40 l and were made up in 5 mm tris - hcl , 1 mm edta , and 1 mm nan3 buffer ( ph 7.4 ) and contained l1210 leukemia dna ( 120 g / ml ) , various final concentrations of pi ( 0100 mm ) , and 20 mm tg . the reactions were initiated by the addition of 90ce to give a final concentration of 100 m ( 4 l of 1.0 mm 90ce in 1.0 mm hcl ) . these samples were then incubated for 15 min at 37 c , then diluted by the addition of 560 l of 5 mm tris - hcl , 1 mm edta , and 1 mm nan3 ( ph 8.0 ) buffer to give a total volume of 600 l . at this point , these dnas then contained a monoadduct cross - link precursor ( o-(2-chloroethyl)guanine and n , o - ethanoguanine ) but insignificant cross - links . these samples were then incubated at 50 c for a further 3 h to allow the monoadducts sufficient time to fully react with the complementary strand and generate dna interstrand cross - links . from these samples , the level of dna cross - linking was then determined using a modification of a previously described assay . this assay is based upon the fact that upon rapid cooling thermally denatured dna containing one or more covalent interstrand cross - links rapidly renatures , yielding a highly fluorescent complex with h33258 dye . the 200 l aliquots of l1210 dna containing various levels of interstrand cross - links were added to 1.5 ml of 5 mm tris - hcl , 1.0 mm edta , and 1.0 mm nan3 buffer ( ph 8.0 ) containing 0.1 g / ml of h33258 , heated to 100 c for 3 min , then plunged into a water bath at room temperature for 3 min . fluorescence measurements were taken before the heating phase and after the 3 min chill using a hoefer scientific instruments tko 100 fluorometer , and the fraction of the dna molecules that were cross - linked ( i.e. , that contained at least one cross - link per dna molecule ) and the average number of cross - link moieties per dna molecule calculated assuming a poisson distribution as previously described . the decomposition / reaction of 90ce involves the formation of a strong acid ( methanesulfinic acid , pka 2 ) and thus can be followed using protonometric assays . we utilized a simple colorimetric assay that relies upon the measurement of the relatively linear change in absorption at 560 nm of phenol red that occurs in proportion to the generation / addition of small quantities of hydrogen ions when a weakly buffered solution of this ph indicator is subjected to incremental acidification over a narrow ph range ( ph < 0.1 unit ) close to the pka values of the buffer / indicator components . using a 20 g / ml solution of phenol red in 2 mm tris - hcl buffer , the absorbance at 560 nm was followed at an initial ph value of 7.4 at 20 and 37 c upon the addition of 50 m 90ce ( 5 l / ml of 10 mm 90ce in dmso ) . the assay mixtures were sealed with parafilm in 1 ml cuvettes to minimize changes in ph due to co2 exchange and brought to the appropriate temperature prior to the addition of agent via injection through the parafilm and rapid mixing . decomposition / reaction kinetics of 90ce ( 100 m ) were followed at both 20 and 37 c in 200 mm potassium phosphate ( ph 7.4 ) and 20 mm tris - hcl ( ph 7.4 ) by recording the appearance of uv absorbing material at 240 nm after the addition of 10 l / ml of a 10 mm 90ce solution in dmso . because of the temperature dependence of the dissociation constants of the buffer constituents , in particular that of tris - hcl , the buffer ph values must be set at the appropriate assay temperature . the loss of the ability of 90ce to chloroethylate the o-6 position of dna guanine was examined by following the exhaustion of dna cross - linking capability versus time at ph 7.4 and 37 c in 20 mm tris buffer . to 500 l of 20 mm tris buffer ( ph 7.4 at 37 c ) , 100 m 90ce was added ( 10 l of 10 mm 90ce in dmso ) to initiate electrophile generation ; at various times after initiation and mixing ( 0 , 30 , 60 , 90 , 120 , and 300 s ) , aliquots ( 12 l ) were withdrawn and mixed with an equal volume of l1210 dna 400 g / ml in 5 mm tris , 1 mm edta , and 1 mm nan3 ( ph 7.4 ) buffer and rapidly mixed . these mixtures were then incubated for 15 min at 37 c , diluted to a volume of 600 l with 5 mm tris , 1 mm edta , and 1 mm nan3 ( ph 8.0 ) buffer then incubated at 50 c for a further 3 h. the level of dna cross - linking and the number of cross - link moieties per dna molecule were then measured and calculated as described above . water is a particularly good trapping agent since it is present at a concentration of 55 m , which results in it sequestering the vast majority of the harder electrophiles generated . since 2-chloroethanol lacks any strong spectroscopic features and is relatively unreactive , it is necessary to transform it into a chromophoric derivative . therefore , the 2-chloroethanol was oxidized to its corresponding aldehyde using pichia pastoris alcohol oxidase ( ao ) , a relatively nonspecific enzyme which oxidizes short - chain , linear aliphatic alcohols to their respective aldehydes . the resultant aldehyde was then reacted with 2,4-dinitrophenylhydrazine ( 2,4-dnph ) to generate a hydrazone product which was quantified by hplc . to 0.5 ml samples of ph 7.4 buffers containing various concentrations of pi ( 0200 mm ) , 1.0 mm 90ce ( 5 l of 100 mm 90ce in dmso ) was added . the 0 mm pi buffer experiments contained 20 mm tris ( ph 7.4 ) to allow the decomposition to fully proceed , which would normally slow substantially as the ph fell , due to the liberation of methanesulfinic acid . these mixtures were incubated for 5 min at 40 c , then diluted 10-fold with distilled h2o . to 0.5 ml of this diluted mixture , 10 unit / ml of ao was added and the mixture incubated for 30 min at 40 c in sealed tubes with occasional shaking to maintain aeration . aliquots ( 0.5 ml ) were then mixed with an equal volume of a 0.4% solution of 2,4-dnph in ch3cn and 50 l of 1 m hclo4 , and the mixture incubated at 40 c for 5 min and then assayed directly by hplc . the hplc protocol utilized a 250 mm 4.6 mm varian microsorb 100 - 5 c-18 reverse phase column ( varian inc . , lake forest , california , usa ) and a constant composition buffer ( 52.5% ch3cn and 47.5% 30 mm potassium phosphate , ph 5.4 ) at a flow rate of 0.8 ml / min . all hplc measurements were performed using a beckman 127p solvent module and a beckman 168 uv / vis detector ( beckman , fullerton , ca , usa ) . this assay gave good linearity in the test range using authentic 2-chloroethanol samples and was not significantly affected by the initial buffer composition . the limit of 2-chloroethanol detection using this method was 1 m . assays were compared with identical reagent blanks containing all components except 90ce . acetaldehyde yields were determined using a protocol identical to that used for measuring 2-chloroethanol , except that the addition of and incubation with ao were not required . the assay was calibrated using authentic acetaldehyde standards , and the resultant hydrazone eluting at 14.9 min gave a linear auc ( area under the curve ) for the resultant hydrazone peak versus the initial concentration of acetaldehyde . the auc of the hydrazone product peak over that of dmso controls was used to calculate the acetaldehyde yields by comparison with authentic standards . ethylene glycol was measured in a manner similar to that used with 2-chloroethanol , except that a longer 60 min incubation with ao was utilized to oxidize the ethylene glycol to glyoxal since ethylene glycol is a poorer substrate for this enzyme . even with this longer incubation time , the oxidation was not complete , and the yield of glyoxal was approximately 50% at all concentrations ( based upon comparisons with authentic glyoxal samples ) . in addition , the hydrazone generated from glyoxal required the use of an increased ch3cn concentration ( 75% ch3cn and 25% 30 mm potassium phosphate , ph 5.4 ) in the hplc protocol compared to the previous method to avoid excessively long elution times . under these revised hplc conditions , the hydrazone product eluted at 9 min . ethylene glycol standards gave a linear auc for the resultant hydrazone peak versus the initial concentration of ethylene glycol . the auc of the glyoxal hydrazone product peak over that of dmso controls was used to calculate the ethylene glycol yield by comparison with authentic standards . the generation of free chloride during the decomposition of 90ce in buffers containing various concentrations of pi was assayed using a modification of the method of jrg and bertau . this is a highly sensitive colorimetric assay based on the formation and strong absorbance of [ fecl ] at max 340 nm . a 200 mm tris - acetate buffer ( ph 7.4 ) ( produced using tris free base and glacial acetic acid ) and a 200 mm potassium phosphate buffer ( ph 7.4 ) ( produced using kh2po4 and koh ) were combined in various proportions to generate chloride free buffers with pi concentrations between 0 and 200 mm . this was circumvented by formulating 0.5 m 90ce stock solutions in hydroxyacetone ( acetol ) for these experiments . two additional problems were encountered , one associated with the determination of chloride in the presence of high pi levels in an fe based assay and a second due to an additional background generated from nonchloride 90ce decomposition products ( discovered by utilizing ks90 as a control agent , with ks90 being a 90ce analog in which the chloroethyl moiety is replaced by a methyl group and therefore unable to liberate chloride ) . the first problem was resolved by precipitation of the pi using ca(no3)2 prior to the assay . the second problem was negated by splitting each sample into two , removing the chloride from one portion by utilizing silver acetate precipitation and using this chloride free counterpart as the reagent blank against which the second portion was measured . to 2 ml samples of buffers containing various levels of pi , formulated as described above , 40 l of 0.5 m 90ce dissolved in acetol was added to give a final 90ce concentration of 10 mm . this mixture was then incubated for 20 min at 37 c to allow for complete decomposition . these samples were then diluted 10-fold with distilled water and 0.1 ml of saturated 1.6 m ca(no3)2 solution added per ml and the precipitate removed by centrifugation . the supernatants were then split into two portions and one - half treated with 40 l / ml of 50 mm silver acetate and the other with 40 l / ml of distilled water . these samples were then centrifuged to remove any precipitate and 0.5 ml samples of supernatant mixed with an equal volume of 40 mm fe(no3)3 in 50% perchloric acid , and the absorbance of the experimental sample measured at 340 nm versus its chloride depleted counterpart . this assay was linear using chloride standards and chloride spiked experimental controls independent of the buffer pi concentration . methanesulfinic acid was assayed by a modification of the method of babbs and gale , which is based upon the reaction of an aromatic diazonium salt ( ar n = n ) , in this case , fast blue bb salt with methanesulfinic acid to produce a chromophoric diazosulfone derivative ( ar n = n - sooch3 ) , which is highly hydrophobic and thus can be selectively extracted into an organic solvent and determined spectrophotometrically . ten microliter aliquots of 100 mm 90ce in dmso were added to 1.0 ml samples of ph 7.4 buffers containing various pi contents ( 0200 mm ) to give a final 90ce concentration of 1 mm . the 0 mm pi buffer experiments contained 20 mm tris - hcl buffer ( ph 7.4 ) to allow the decomposition to fully proceed , which would normally slow substantially as the ph fell due to the liberation of methanesulfinic acid . these mixtures were incubated for 5 min at 37 c to allow for a complete decomposition / reaction , and then aliquots were diluted 40-fold with 500 mm nah2po4/h3po4 ( ph 2.2 ) containing 1 part in 80 of saturated fast blue bb salt solution in 500 mm nah2po4/h3po4 ( ph 2.2 ) . these mixtures were allowed to react at 40 c for 10 min in the dark . an equal volume ( 0.8 ml ) of n - octanol pre - equilibrated with 500 mm nah2po4/h3po4 was then added and the tubes vigorously shaken for 2 min en masse ; the samples were then centrifuged at 10,000 g for 120 s , and the upper octanol layer was separated and the absorbance measured at 460 nm versus a reagent blank within 5 min . calibration curves were generated using solutions of authentic sodium methanesulfinate at concentrations of 0.0 , 0.5 , 1.0 , 1.5 , 2 , and 3 mm in buffers with various pi contents ( 0200 mm ) , and a linear response ( absorbance 460 nm versus methanesulfinate concentration ) was observed that was independent of the pi concentration in the initial samples . the strongly absorbing thiol mnp produced by reducing dtbnp was used as a convenient electrophile trapping agent suitable for the separation by hplc . the hplc protocol utilized a 250 mm 4.6 mm varian microsorb 100 - 5 c-18 reverse phase column ( varian inc . , ) and elution with an unbuffered ch3cn / h2o solvent system , starting with 5% acetonitrile for 5 min followed by a 550% ch3cn linear gradient over the subsequent 30 min . after this point , the concentration of ch3cn was maintained at this level for 5 min , then returned to the starting concentration over an additional 5 min . absorbance was monitored at 340 nm using a beckman 168 uv / vis detector ; 340 nm was chosen because the strong absorption of the 5-nitropyridine moiety is largely independent of the derivatization or oxidation state of the attached thiol group at this wavelength . the reduced thiol ( mnp ) and oxidized disulfide dimer ( dtbnp ) elute at 5 and 38 min , respectively . the reaction protocol was as follows : 1 mm dtbnp in 200 mm k2hpo4 was reduced to mnp by the addition of 2 mm thioglycerol to generate 2 mm mnp in a high pi buffer environment . after a few minutes of gentle shaking at room temperature to ensure that the reduction was complete , an aliquot of 100 mm 90ce in dmso was added to give a final concentration of 2 mm 90ce . this mixture was incubated for 4 min at 37 c , centrifuged at 10,000 g for 1 min , then diluted 10-fold with distilled water and immediately analyzed by hplc . this reaction sequence is easily followed by the eye , as reduction to mnp caused the sample to develop a strong yellow color , which is then largely discharged over a few minutes upon alkylation by 90ce . under these reaction conditions , only one new mnp/90ce derived major product , eluting as a broad tailing peak ( 1013 min , peak 11.6 min ) , was observed . a similar experiment was performed at four times these concentrations of mnp and 90ce and the alkylated product peak collected for analysis by lcms . the alkylated product prepared , separated , and collected as described above was analyzed by mass spectroscopy using a chromatographic system consisting of an agilent 1200 series hplc system , including a binary pump ( model g1312b ) , a vacuum degasser ( model g1379b ) , an autosampler ( model g1367c ) , and a column oven ( model g1316b ) . the mass spectrometer was an applied biosystems sciex 4000 q - trap mass spectrometer ( applied biosystems sciex ; foster , ca ) . data acquisition was carried out by analyst 1.4.2 software on a dell computer . a declustering potential of 50 v , excitation energy of 100 v , and a collision energy of 10 v were utilized . previous studies , in the absence of high pi concentrations , demonstrated that the initial elimination of the n-1 sulfinate moiety was the rate determining step in the decomposition of 1,2-bis(sulfonyl)-1-alkylhydrazines ( figure 1 panel a ) as measured by hydrogen ion production . this time course also corresponded to the loss of biological activity upon agent aging . the instantaneous release of the first mole of protons represents the ionization of the acidic n-2 hydrogen and can not reflect the elimination of the n-1 sulfinic acid moiety from the un - ionized parental 1,2-bis(sulfonyl)-1-alkylhydrazine because the t1/2 for the first order release of the second mole of protons decreases with the increasing leaving group ability of the n-1 sulfinate moiety , and this could not occur if the n-1 sulfinate moiety had already left the molecule . the release of the second mole of protons occurs upon nucleophile alkylation by the primary oxophilic alkylating species rn = nso2ch3 ( e.g. , figure 1 panel d ) or secondary alkylating species derived from these . this biphasic release of two moles of hydrogen ions is observed during the decomposition of both methylating and chloroethylating analogues in a pi free or low pi buffers . in tris - hcl buffer , both ks90 and 90ce poorly alkylated 4nbp as would be expected for generators of relatively hard electrophiles . however , in 200 mm pi buffer , the behavior of 90ce changed markedly , and a > 10-fold increase in the alkylation of 4nbp was observed . we therefore investigated the pi concentration dependence of the alkylation of 4nbp ( 1 mg / ml , 4.7 mm ) by 200 m 90ce over a 0200 mm pi concentration range ( figure 2 ) . the presence of pi resulted in a marked , but saturatable , concentration dependent increase in 4nbp alkylation , with approximately 30 mm pi eliciting a half - maximal effect under these conditions . the inclusion of 20 mm tg , as a competing soft nucleophile , decreased the alkylation of 4nbp at the highest pi concentration by > 96% . the addition of 1 mm tg was almost as effective , resulting in an 90% reduction in 4nbp alkylation at 200 mm pi ( data not shown ) . thus , the generated electrophile must be extremely soft in nature since only 1 mm tg could out compete the alkylation of 5 mm 4nbp by 90% . this very strong thiol preference is more reminiscent of a michael addition reaction than the reaction of a nucleophile with a carbonium ion or highly polarized alkyl group . in comparison , bcnu at an equivalent concentration poorly alkylated 4nbp over a 0200 mm pi concentration range , and this nbp alkylation was marginally decreased rather than greatly increased at higher pi concentrations ( figure 2 ) , probably due to some alkylation of pi . in addition , no pi dependent increase in thiol preference was observed with bcnu ( data not shown ) . since 90ce can potentially generate three secondary chloroethylating species which are in common with the hard oxophilic chloroethylating species proposed for bcnu ( figure 1 , panel b ) , these findings imply that the pi dependent increase in soft nucleophile preference seen with 90ce does not involve these secondary chloroethylating species ( figure 1 , panel a ) but a component unique to 90ce . effect of pi concentration on the preference of 90ce and bcnu for 4nbp . reactions containing either 200 m 90ce ( 20 mm tg ) or 200 m bcnu and 1 mg / ml of 4nbp ( 4.7 mm ) in 20 mm tris - hcl buffer ( ph 7.4 ) containing various concentrations of pi ( ph 7.4 ) samples were reacted for either 15 min at 37 c for 90ce or overnight at 37 c for bcnu due to its > 60-fold longer t1/2 . the resultant level of 4nbp alkylation was then determined by measuring the absorbance at 635 nm versus a reagent blank after mixing samples with an equal volume of 38% hpcd dissolved in 0.5 m koh . ( ) 200 m 90ce in the absence of tg ; ( ) 200 m 90ce in the presence of 20 mm tg ; and ( ) 200 m bcnu in the absence of tg . all values are the result of at least 3 determinations se . in view of the pi concentration dependent increase in the generation of soft thiophilic alkylating species with increasing pi concentration from 90ce , we decided to examine the effects of pi concentration on the formation of dna interstrand cross - links which are dependent upon the generation of hard oxophilic chloroethylating electrophiles . dna interstrand cross - link formation is of particular importance since it is crucial to the mode of action of 90ce prodrugs . it can be seen ( figure 3 ) that increasing the pi concentration results in a progressive decrease in the yield of dna cross - linking moieties per dna molecule and that the electrophiles responsible for generating these lesions are hard oxophilic electrophiles that are resistant to thiol interception since 20 mm tg only decreased the yields 15% . only a very small proportion of the oxophilic chloroethylating electrophiles generated react with the o-6 position of dna guanine since the bulk chloroethylate water , which is present at 55 m , to generate 2-chloroethanol ( figure 1 , panel c ) . this is especially true for hard oxophilic chloroethylating species , which have a strong preference for water - like nucleophiles . however , it is expected that water in the absence of competing favored nucleophiles , due to its high concentration , would also trap the majority of any generated soft chloroethylating species as 2-chloroethanol . therefore , we looked at the production of 2-chloroethanol versus pi concentration to ascertain the changes occurring in the overall yields of chloroethylating electrophiles ( figure 4 , panel a ) . a large decrease in chloroethanol production from 83% this change approximately parallels the decrease seen in dna cross - link formation and is the inverse of the increase observed in soft thiophilic alkylators in the 4nbp alkylation experiments . the corresponding decrease in chloroethanol production suggested that the soft thiophilic alkylator was not a chloroethylating species . the reduction in 2-chloroethanol yields could mean that the chlorine was lost as chloride or remained as part of a different species . we therefore measured chloride release and found that 80% of the total chlorine was liberated as chloride under high pi conditions ( figure 4 , panel a ) . this finding confirmed that the chlorine was largely lost in the pi catalyzed pathway and was no longer part of the donated electrophile moiety . it was thought that the loss of chloride by elimination or by a hydrolytic mechanism could possibly lead to either vinylating or hydroxyethylating species , respectively . acetaldehyde and ethylene glycol are formed by the vinylation and hydroxyethylation of water , respectively , and can be generated in quite high yields by bcnu via decomposition pathways involving chloride loss ( figure 1 , panel b ) . for this reason , we assayed for the generation of these two chlorine - free two - carbon atom species during the decomposition of 90ce . at 200 mm pi , the highest level tested , the yields of acetaldehyde and ethylene glycol were approximately 5% and 2% , respectively ( figure 4 , panel a ) . these yields combined were insufficient in quantity to account for the large decrease in the yields of 2-chloroethanol ( 83% to 17% ) between 0 mm pi and 200 mm pi , respectively ( figure 4 panel a ) . in previous studies following the decomposition of gram quantities of ks90 , both the volume and composition of the gas liberated were determined ; and very close to a mole equivalent of nitrogen was found to be released during ks90 decomposition . while it is difficult to quantify and analyze microliter volumes of gas liberated during the decomposition of small quantities of dilute aqueous solutions of 90ce , at decomposition concentrations of 1 mm or greater gas bubbles can be observed nucleating on the vessel s walls . we therefore quantified the bubble formation number during the decomposition of 2 mm solutions of 90ce in both tris - hcl and 200 mm potassium phosphate and noted a > 80% reduction in bubble numbers under high pi conditions ( figure 4 , panel b ) . this observation implied that the volume of gas liberated under high pi conditions was also significantly reduced . the apparent failure of the bulk of the 90ce , under high pi conditions , to liberate either the two carbon alkylating moiety as chloroethanol , acetaldehyde , or ethylene glycol and the hydrazine derived nitrogen , suggested that the molecule failed to fully fragment under these conditions . consequently , we determined the effects of pi concentration on the liberation of methanesulfinate during the decomposition of 90ce ( figure 5 ) . the quantity of methanesulfinate was found to be reduced from 2 to 1.2 mols per mol of 90ce under high pi conditions . this finding is consistent with 80% of the 90ce failing to fully fragment after the initial elimination of the n-1 methanesulfinate moiety , thus liberating a single mole of methanesulfinate instead of two at the highest concentrations of pi . a small proportion ( 17% ) of the 90ce appeared to decompose , liberating chloride even in the absence of pi in the 200 mm tris - acetate buffer used in the chloride assay . if this proceeded by the 1 mol of methanesulfinate route , one would expect 1 mol of 90ce to liberate a maximum of 1.83 mols of methanesulfinate rather than 2 mols . this discrepancy could be due to a minor decomposition pathway(s ) where both the chloride and second methanesulfinate are lost in the absence of pi ( potential minor pathway a , see later ) , a small pi - like effect of the 200 mm tris - acetate buffer used in the chloride determinations ( see later ) , or an inaccuracy in the methanesulfinate standards due to hygroscopicity of the sodium salt used , or a combination of these possibilities . effects of pi concentration on the average number of cross - link moieties generated per dna molecule ( reaction concentration 120 g / ml of l1210 dna ) by 100 m 90ce at ph 7.4 in the presence and absence of 20 mm tg as a competing nucleophile . ( ) average number of cross - link moieties generated per dna molecule in the absence of tg ; and ( ) average number of cross - link moieties generated per dna molecule in the presence of 20 mm tg . effects of pi concentration on the yields of 2-chloroethanol , acetaldehyde , ethylene glycol , and chloride anions ( panel a ) , and nitrogen gas evolution from 90ce ( panel b ) at ph 7.4 and 37 c . panel a , ( ) 2-chloroethanol , ( ) acetaldehyde , ( ) ethylene glycol , and ( ) chloride anions . the experimental data indicate that the formation of ethylene glycol and acetaldehyde are insufficient to account for the decreases in chloroethanol and increases in chloride formation observed under high pi concentration conditions and that some other processes must account for these differences . ( panel b ) photographs taken just after the addition of 2 mm 90ce ( upper and lower left - hand images ) to microcentrifuge tubes containing either 20 mm tris - hcl ( upper row ) or 200 mm potassium phosphate ( lower row ) buffers at ph 7.4 , and after 5 min of incubation at 37 c ( upper and lower right - hand images ) , note the large decrease in nitrogen evolution under high phosphate conditions . effects of pi concentration on the yields of methanesulfinate formed during the decomposition of 90ce . one millimolar solutions of 90ce were decomposed at 37 c and ph 7.4 for 10 min in buffers containing various concentrations of pi . these samples were derivatized with the aromatic diazonium dye fast blue to give the octanol soluble highly chromophoric diazosulfone derivative . the diazosulfone was separated into octanol and the absorbance measured at 460 nm versus a reagent blank ( identical except for the absence of 90ce ) . ( insert ) decomposition scheme illustrating the generation of 2 mols of methanesulfinate per mol of 90ce in the absence of pi , with the downward arrow representing a potential decomposition pathway branch point in the presence of pi retaining a methanesulfinate moiety and both nitrogen atoms . differences in the overall kinetics of alkylation by 90ce reacting via hard oxophilic or soft thiophilic alkylating species could arise from the change in the decomposition pathway since this could be accompanied by a change in the rate determining step . furthermore , soft alkylating species could be expected to persist for longer time periods in aqueous solution and potentially extend the time course of alkylation in the absence of preferred nucleophiles or in the presence of rate limiting nucleophile concentrations . therefore , the kinetics of the following processes were measured and compared : the kinetics of the release of the second mole of hydrogen ions ( 0 mm pi ) , bleaching of mnp ( 200 mm pi ) , and the generation of uv absorbing species ( 200 mm pi ) , together with the loss of the ability of 90ce to produce dna cross - link moieties ( 0 mm pi ) and to alkylate 4nbp ( 200 mm pi ) upon aging ( figure 6 , panels a essentially identical half - reaction times of approximately 35 , 31 33 , 36 , and 30 s , respectively , were determined for all of these processes . the generation of uv absorbing product(s ) ( 16-fold greater under high pi conditions ) occurs with the inverse kinetics of the other processes ( exponential association rather than decay ) . the kinetics of all of these exponential decay processes and the exponential association process are compared by normalization in figure 6 , panel f ( 1 minus the normalized value has been plotted in the case of the exponential association ) . thus , the same rate determining step , i.e. , the previously identified elimination of the methylsufinate from n-1 , appears to control all of these processes . two of these processes , the kinetics of hydrogen ion generation at 0 mm pi and the kinetics of the production of uv absorbing material at 200 mm pi , were also examined at 20 c where the kinetics of both processes were equivalently slowed by a factor of 7-fold ( data not shown ) . these findings indicate that the rate determining steps for these two processes ( one at 0 mm pi and the other at 200 mm pi ) have both the same values and temperature dependencies , further implying the equivalency of their rate determining steps . because of inherent assay physical limitations , measurements of the kinetics of hydrogen ion generation are restricted to weakly buffered solutions . therefore , equivalent experiments could not be performed under high pi concentrations ; however , hydrogen ion liberation assays would be blind to a switch from methanesulfinate to chloride liberation since both would be accompanied by the liberation of a single hydrogen ion . the increase in the production of uv absorbing species under conditions of high pi concentration could imply the acquisition of double bonds , and this finding would be consistent with the elimination of hcl from the chloroethyl moiety . this would be favored after the elimination of the n-1 methanesulfinate moiety because it would then result in extended conjugation from the alkyl moiety all the way to the oxygen atoms on the remaining sulfonyl moiety . this would generate ch3so2n = nch = ch2 , a very thiophilic michael type acceptor , which would retain one of the methylsulfonyl moieties and the nitrogen atoms upon reaction with a thiol . kinetics of various 90ce dependent processes at 37 c and ph 7.4 under high and low pi conditions . ( panel a ) hydrogen ion generation versus time ( 0 mm pi ) ; ( panel b ) loss of dna cross - linking activity versus time ( 0 mm pi ) ; ( panel c ) loss of 4nbp alkylating activity versus time ( 200 mm pi ) ; ( panel d ) mnp bleaching versus time ( 200 mm pi ) ; ( panel e ) generation of uv absorbing material ( 240 nm ) versus time in the presence ( upper trace ) and absence ( lower trace ) of 200 mm pi ; and ( panel f ) comparison of the kinetics of the processes in panels a the calculated t1/2 and first order rate constant , determined by a nonlinear regression best curve fit analysis , for each reaction is given in the corresponding panel . the loss of activity upon aging of 90ce for dna cross - link formation ( 0 mm pi ) ( figure 6 , panel b ) and 4nbp alkylation ( 200 mm pi ) ( figure 6 , panel c ) indicate that neither the oxophilic nor thiophilic alkylating species persists in these reaction mixtures since the residual activity matches the kinetics for the rate determining elimination of the n-1 methanesulfinate moiety and thus is only equal to the remaining 90ce in both cases . therefore , the half - lives of both the oxophilic and thiophilic alkylating species must be considerably less than the 30 s rate determining elimination step at 37 c and ph 7.4 . the bleaching upon alkylation of highly chromophoric mnp at 440 nm is particularly useful since this can be used to follow real time alkylation by the thiophilic alkylating species as no workup is required . therefore , some additional studies were performed in which mnp ( 100 nmol / ml ) was progressively titrated by the repeated addition of initially limiting quantities of 90ce ( 20 nmol / ml ) until the mnp was completely exhausted ( figure 7 ) . during these successive additions , the same half - reaction time ( 30 s ) and extent of the reaction ( 20 nmol 90ce bleached 12.6 nmol of mnp ) was observed until the mnp was nearly completely consumed , despite the fact that initially there was a 5-fold molar ratio of mnp to 90ce , and when the sixth aliquot was added , this was reduced to only 1.2-fold . in the presence of 200 mm pi , 83% of the 90ce reacts via the chloride liberating pathways , which would largely comprise the pi catalyzed thiophilic electrophile pathway and the potential minor pathway a , while the remaining 17% results in chloroethanol formation . thus , 1416 nmol of the 20 nmol of 90ce would be expected to decompose via the phosphate catalyzed chloride liberating pathway , and 12.6 nmol ( 8090% ) of this was trapped by reacting with mnp . since this trapped fraction appeared to be independent of the mnp concentration until exhaustion , implying extremely fast and efficient scavenging ( figure 7 , panel a ) , it is possible that this small discrepancy reflects the presence of another minor reaction pathway that occurs in the presence of pi ( potential minor pathway b ) and not merely a failure of the mnp to fully scavenge the short - lived thiophilic electrophile generated . stoichiometry of mnp ( 100 m ) absorbance bleaching at 440 nm by 90ce at 37 c and ph 7.4 in 200 mm pi and 20 mm tris - hcl buffers . blue trace , bleaching of mnp ( 100 m ) by 100 m 90ce in 20 mm tris - hcl buffer ; red trace , bleaching of mnp ( 100 m ) by 100 m 90ce in 200 mm pi buffer ; and black trace , bleaching of mnp ( 100 m ) by successive 20 m additions of 90ce until mnp exhaustion in 200 mm pi buffer . ( insert a ) plot of the absorbance of a mnp ( 100 m ) solution at 440 nm , 300 s after the addition of various quantities ( 0200 m ) of 90ce at 37 c and ph 7.4 in 200 mm pi , indicating the stoichiometry of mnp titration . all values are the result of at least 3 determinations se . in situ generated mnp was chosen as a reagent to trap and identify the soft thiophilic electrophile because it efficiently scavenged the thiophilic species but would be expected to feebly trap any hard oxophilic electrophiles , resulting in few trapped products . furthermore , the strong uv absorbance at 340 nm of the nitropyridine moiety is largely independent of the state of reduction , oxidation , or alkylation of the thiol group , aiding the detection of any mnp derivatives formed . moreover , since we suspected that the thiophilic electrophile was a michael type acceptor , produced by the loss of hcl from the primary chloroethylating species , the resultant mnp adduct would be expected to be a neutral species suitable for lcms . schemes illustrating the in situ reduction of dtbnp by tg to yield 2 mols of mnp and its subsequent reaction with 90ce derived electrophiles in the presence of 200 mm pi are shown in figure 8 , panels a and b. hplc analysis of dtbnp gave a large 38 min peak for the oxidized material ( figure 8 , panel c and trace a ) , and reduction by a stoichiometric quantity of tg largely converted the dtbnp to mnp eluting at 5 min and a trace of the mixed disulfide eluting at 31.5 min ( figure 8 , panel c and trace b ) . treatment of this reduced material with 90ce in the presence of 200 mm pi resulted in an 80% decrease in the area of the 5 min mnp peak and the appearance of a single new broad tailing peak ( 1013 min , peak 11.6 min ) for the trapped product ( figure 9 , panel c and trace c ) . lcms analysis of collected material eluting between 11 and 12 min indicated a mass of 291 for the m+1 peak of the trapped mnp derivative ( figure 8 , insert d ) , which corresponded to a mass increase due to the electrophile moiety of 134 ( figure 8 , insert e ) . for a michael type acceptor , this would equal the mass of the attacking electrophile . this mass corresponds to the mass of our proposed thiophilic electrophile ch3so2n = nch = ch2 . reaction of this electrophile with a thiol adds the following moiety ch3so2nhn = chch2 to the sulfur atom . such adducts to both glutathione and n - acetylcysteine were previously postulated based on ms / nmr studies of the electrophilic metabolites of isotopically labeled laromustine and 90ce conducted at a pi concentration of 100 mm . it should be noted that the use of thiol traps would not be useful in trapping and identifying oxophilic or even thiophilic chloroethylating species because any adducts that were formed would rapidly eliminate the chloride to form a reactive cyclic sulfonium ion via an intramolecular nucleophilic substitution reaction and then react further with surrounding nucleophiles . trapping of ( panel a ) scheme illustrating the two stage reduction by tg of dtbnp to yield 2 mols of mnp . ( panel b ) scheme illustrating the trapping of the thiophilic electrophile derived from 90ce . ( panel c ) hplc traces recorded at 340 nm of 10-fold dilutions of reactions conducted in 200 mm pi buffer . trace ( a ) , 100 m dtbnp ( prereduction ) ; trace ( b ) , dtbnp postreduction by two molar equivalents of tg to yield mnp 200 m ; trace ( c ) , postreaction of in situ generated mnp with a molar equivalent of 90ce indicating the production of a trapped product eluting at 11 min . ( insert d ) lcms analysis of the trapped product ( eluting at 11 min ) , indicating a molecular plus hydrogen cation combined mass of 291 ( [ m + h ] = 291 ) . ( insert e ) calculation of the molecular weight of the donated alkyl moiety and postulated entity . effects of various solutes on the generation of thiophilic species from 90ce as measured by the bleaching of mnp . ( panel a ) bleaching of the absorbance of a 100 m mnp solution in various concentrations of tris - hcl buffer at ph 7.4 and 37 c at 440 nm , 300 s after the addition of 100 m 90ce . ( panel b ) the bleaching of the absorbance of a 100 m mnp solution in 20 mm tris - hcl at ph 7.4 and 37 c at 440 nm , 300 s after the addition of 100 m 90ce supplemented with various solutes at 50 mm ( adjusted to ph 7.4 ) . the horizontal line above the x - axis represents the magnitude of mnp bleaching due to the presence of 20 mm tris - hcl at ph 7.4 in these solute solutions ( the addition of 20 mm tris - hcl was required as many of these solutes lack buffering capacity at ph 7.4 ) . the mg atp sample contained 52.5 mm mgcl2 in addition to 50 mm atp . we examined 28 different anions / solutes including several phosphate esters to determine if other solutes behaved like pi and increased the yields of soft electrophilic species from 90ce ( figure 9 ) . nonphosphate containing anions of strong acids exhibited very little activity over the small effect due to the copresence of 20 mm tris - hcl , the addition of which was required to add buffering capacity . all reactions containing nonphosphate agents with pka values in the vicinity of the experimental ph ( edta , carbonate , citrate , imidazole , mops , triethanolamine - hcl , and tris - hcl with pka values of 6.20 , 6.35 , 6.40 , 6.95 , 7.20 , 7.76 , and 8.06 , respectively ) contain relatively high concentrations of potentially catalytic brnsted - lowry bases , and these all exhibited at least some to moderate activity . therefore , the mechanism by which these components catalyze the elimination of hydrogen chloride from the primary chloroethylating species probably involves a general acid / base catalyzed elimination reaction . however , factors other than the pka value appear to influence the catalytic efficiency since phosphoesters exhibited greater activity than other agents ( excluding arsenate ) with comparable pka values . we examined the activity of the three methyl esters of phosphate ( monomethylphosphate , dimethylphosphate , and trimethylphosphate ) since these successively replace the acidic protons of triprotic phosphoric acid and have relatively little steric bulk compared to other possible esters . compared to pi ( pka 7.21 ) , monomethylphosphate ( pka 6.31 ) was found to be approximately equiactive , while dimethylphosphate ( pka 1.29 ) surprisingly retained half the activity of pi despite being a relatively strong acid , and trimethylphosphate was inactive . this finding implies that at least one of the protic sites on pi must be unblocked for catalytic activity . glucose 6-phosphate ( pka 6.11 ) , a slightly more sterically hindered and acidic phosphomonoester , was only a little less active than monomethylphosphate . a number of polyphosphates were also examined ( atp , adp , and pyrophosphate ) , and these all proved to be approximately equiactive with pi ; thus , the addition of successive phosphate groups in a linear conformation does not significantly increase activity ( figure 9 ) . most ( 80% ) of the intracellular atp pool exists as a mg chelate , owing to the strong binding affinity of atp for mg . since this chelation involves the two terminal phosphates , we suspected that it may impact atp s ability to facilitate a switch in the decomposition pathway . therefore , the activity of atp - mg was assessed using a slight molar excess of mgcl2 ; this resulted in a 60% reduction in activity over that of atp alone . phosphocreatine ( creatine phosphate ) ( pka 4.5 ) , a guanidino phosphate in which a relatively acidic phosphate is involved in a n - phosphoguanidine linkage and a hydrogen bond with the guanidine moiety at physiological ph values , had 1/4th the activity of pi ( figure 9 ) . the only tested anion found to be superior to pi was arsenate , which can be viewed as a pi analogue . arsenic , like phosphorus , is a group 15 element and has very similar chemical properties ; moreover , arsenic acid ( h3aso4 ) and phosphoric acid ( h3po4 ) have analogous structures and near identical pka values of ( pka1 = 2.19 , pka2 = 6.94 , and pka3 = 11.5 ) and ( pka1 = 2.12 , pka2 = 7.21 , and pka3 = 12.67 ) for arsenic and phosphoric acids , respectively . two major decomposition pathways dominate , one resulting in the generation of the therapeutically important hard oxophilic chloroethylating species , which accounts for > 80% of the flux in tris - hcl buffers . the second pathway is facilitated by the presence of pi and some phosphoesters and generates a novel soft thiophilic electrophile ( ch3so2n = nch = ch2 ) , which is currently thought to have little therapeutic relevance . this pathway contributes 80% of the flux at pi concentrations of 200 mm or greater . a number of minor pathways undoubtedly occur ( potential minor pathways a and b , figure , 10 ) as evidenced by the generation of small yields of acetaldehyde / ethylene glycol and minor discrepancies in the anticipated yields of some of the major products . some of these discrepancies could be explained if the pathways that generated acetaldehyde / ethylene glycol resulted in the liberation of both sulfinate moieties in addition to chloride . scheme illustrating the proposed decomposition pathways for 90ce in the presence and absence of pi or catalytic brnsted - lowry base . the aqueous decomposition of 90ce ( pka 6.5 ) begins with the rate determining elimination of methanesulfinate ( t1/2 30 s ) from the 90ce anion to generate the primary oxophilic chloroethylating species . this intermediate can chloroethylate oxygen based nucleophiles ( roh ) directly or via the potential generation of secondary oxophilic chloroethylating species resulting in the liberation of a further mole of protons and methanesulfinate and a mole of nitrogen . in the presence of pi or suitable brnsted - lowry base , hcl is eliminated , generating a short - lived highly thiophilic conjungated soft electrophile which retains the remaining methylsufonyl moiety and both nitrogens on reaction with a thiol based nucleophile ( rsh ) . the positions of two potential minor pathways ( a and b ) are also indicated that could account for the generation of small yields of acetaldehyde / ethylene glycol and minor discrepancies in the anticipated yields of methanesulfinate and trapped mnp product . the generation of highly cytotoxic g - c ethane dna interstrand cross - links arising from dna guanine o-6 chloroethylation , caused by the oxophilic chloroethylating species , is critical to the antineoplastic activity of 90ce . these guanine o-6 lesions are specifically repaired by mgmt , and a 22-fold greater clonogenic lc90 value was observed in l1210 cell lines expressing mgmt . this result implies that the total cytotoxicity due to the alkylation of all other biomolecules by both the oxophilic and thiophilic alkylating species , in these mgmt deficient cells , is probably 5% of that due to guanine o-6 chloroethylations . moreover , it is expected that the vast majority of the thiophilic electrophiles would be scavenged by glutathione . in the presence of normal intracellular pi and phosphate ester concentrations , it is likely that 1025% of the 90ce reacts via the thiophilic pathway , and this can be regarded as a loss of potentially active agent . therefore , increases in the concentration of intracellular pi and active phosphoesters could contribute to the total tumor resistance by decreasing the yields of the therapeutically relevant oxophilic electrophiles . such a resistance mechanism would be expected to be limited to low level resistance ( 2-fold ) to 90ce prodrugs . the overall sensitivity of a tumor cell would be the product of several major and minor factors , including the dna repair activities of mgmt and hdr , protective glutathione - s - transferases able to intercept a portion of the oxophilic electrophiles prior to reaction with dna , and the levels of pi and phosphoesters able to divert the decomposition pathway away from oxophilic chloroethylating electrophile generation . aberrant microvascular systems in solid tumors result in hypoxic regions where the o2 concentrations can approach anoxia . under these conditions , the levels of phosphocreatine and atp / atp - mg fall markedly , while the levels of adp , amp , pi , and phosphomonoesters exhibit a commensurate rise . since atp / mg and phosphocreatine have modest activity , while the total activities of their hydrolysis products are much greater , this change would be expected to result in a decrease in the percentage yield of oxophilic chloroethylating electrophiles . this action could contribute toward a modest resistance to 90ce prodrugs in hypoxic regions . despite this , ks119 , a hypoxically targeted prodrug of 90ce synthesized in our laboratory , exhibits a remarkable 5 logs of differential cell kill in in vitro experiments between oxic and hypoxic environments , and selective targeting in in vivo models . this high degree of hypoxic selectivity is likely the result of the efficiency of the targeting system utilized by this agent . the development of 90ce analogues which eliminate or diminish the phosphate catalyzed decomposition route would be expected to remove this weakness , increase potency , and remove potential toxicities from the thiophilic electrophiles which likely do not significantly contribute to the anticancer activity . the rate determining step in the generation of reactive electrophiles from 90ce at ph 7.4 and 37 c involves the initial elimination of the n-1 methylsulfinate moiety ( t1/2 30 s ) from the 90ce anion . after this point , two major routes of further decomposition exist : ( i ) generating therapeutically relevant hard oxophilic chloroethylating electrophiles and ( ii ) a pathway producing a soft thiophilic electrophile ( ch3so2n = nch = ch2 ) currently of no known therapeutic importance ( figure 10 ) . at this branch point , the proportion taking pathway ( ii ) is likely stimulated by the presence of brnsted - lowry bases , with pi and its mono- and diesters appearing to be the most potent influencing molecules of biological significance . it is expected that 1025% of 90ce decomposition proceeds via this thiophilic route under normal cellular pi / active pi ester concentration conditions , and this percentage is likely to increase further under hypoxic conditions . this decomposition pathway could potentially lead to novel resistance mechanisms whereby intracellular levels of pi / active pi esters or other catalytically active brnsted - lowry bases are elevated in concentration , imparting cells with modest resistance to 90ce and its prodrugs . this study highlights the largely overlooked influence that seemingly inert reaction mixtures and/or buffer components can potentially have on reaction pathways . while the generation of electrophiles from 90ce probably represent an extreme case , it may be prudent to conduct initial studies in a range of buffers or in buffers resembling the cellular milieu to detect such influences , or to ensure that the data obtained are physiologically relevant . we are currently designing 90ce analogues which are expected to diminish this pi catalyzed route with the aim of increasing the yields of oxophilic chloroethylating species . these analogues are likely to be superior to 90ce when incorporated into hypoxia targeted prodrugs and would lack potential toxicities arising from the generation of thiophilic electophiles .

prodrugs of the short - lived chloroethylating agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine ( 90ce ) and its methylating analogue 1,2-bis(methylsulfonyl)-1-(methyl)hydrazine ( ks90 ) are potentially useful anticancer agents . this class of agents frequently yields higher ratios of therapeutically active oxophilic electrophiles responsible for dna o6-guanine alkylations to other electrophiles with lower therapeutic relevance than the nitrosoureas . this results in improved selectivity toward tumors with diminished levels of o6-alkylguanine - dna alkyltransferase ( mgmt ) , the resistance protein responsible for o6-alkylguanine repair . the formation of o6-(2-chloroethyl)guanine , which leads to the formation of a dna dna interstrand cross - link , accounts for the bulk of the anticancer activity of 90ce prodrugs . herein , we describe a new decomposition pathway that is available to 90ce but not to its methylating counterpart . this pathway appears to be subject to general / acid base catalysis with phosphate ( pi ) , phosphomonoesters , and phosphodiesters , being particularly effective . this pathway does not yield a chloroethylating species and results in a major change in nucleophile preference since thiophilic rather than oxophilic electrophiles are produced . thus , a pi concentration dependent decrease in dna dna interstand cross - link formation was observed . changes in 90ce decomposition products but not alkylation kinetics occurred in the presence of pi since the prebranch point elimination of the n-1 methanesulfinate moiety remained the rate - limiting step . the pi catalyzed route is expected to dominate at pi and phosphoester concentrations totaling > 2535 mm . in view of the abundance of pi and phosphoesters in cells , this pathway may have important effects on agent toxicity , tumor selectivity , and resistance to prodrugs of 90ce . furthermore , it may be possible to design analogues that diminish this thiophile - generating pathway , which is likely superfluous at best and potentially detrimental to the targeting of hypoxic regions where pi concentrations can be significantly elevated .

Introduction Materials and Methods Results and Discussion Conclusions

the 90ce moiety has considerable potential in tumor targeted prodrugs because it is an excellent leaving group , and the rapid spontaneous decomposition of 90ce ( t1/2 30 s ) , which can be prevented by the substitution of the n-2 hydrogen with a cleavable chemical trigger , results in the delivery of alkylation stress close to the site of activation . this latter alkylation accounts for the vast majority of its anticancer activity and leads to the eventual formation of a 1-(n - cytosinyl),-2-(n - guaninyl)ethane dna dna interstrand cross - link ( g - c ethane cross - link ) via an n , o - ethanoguanine intermediate ( figure 1 ) . selectivity for tumor cells arises from differentials between normal and tumor cells in their effective levels of mgmt , the resistance protein responsible for the repair of dna o-6 guanine lesions , and in their ability to repair the resultant cross - links generated from lesions that evade restoration by mgmt . it was found that the rate determining step during the decomposition of 90ce and 1,2-bis(sulfonyl)-1-alkylhydrazines was the elimination of the n-1 sulfonyl moiety from the 1,2-bis(sulfonyl)-1-alkylhydrazine anion , which in the case of 90ce results in the formation of the primary chloroethylating species clch2ch2n = nso2ch3 ( figure 1 ) . in view of the pi concentration dependent increase in the generation of soft thiophilic alkylating species with increasing pi concentration from 90ce , we decided to examine the effects of pi concentration on the formation of dna interstrand cross - links which are dependent upon the generation of hard oxophilic chloroethylating electrophiles . it can be seen ( figure 3 ) that increasing the pi concentration results in a progressive decrease in the yield of dna cross - linking moieties per dna molecule and that the electrophiles responsible for generating these lesions are hard oxophilic electrophiles that are resistant to thiol interception since 20 mm tg only decreased the yields 15% . therefore , the kinetics of the following processes were measured and compared : the kinetics of the release of the second mole of hydrogen ions ( 0 mm pi ) , bleaching of mnp ( 200 mm pi ) , and the generation of uv absorbing species ( 200 mm pi ) , together with the loss of the ability of 90ce to produce dna cross - link moieties ( 0 mm pi ) and to alkylate 4nbp ( 200 mm pi ) upon aging ( figure 6 , panels a essentially identical half - reaction times of approximately 35 , 31 33 , 36 , and 30 s , respectively , were determined for all of these processes . the loss of activity upon aging of 90ce for dna cross - link formation ( 0 mm pi ) ( figure 6 , panel b ) and 4nbp alkylation ( 200 mm pi ) ( figure 6 , panel c ) indicate that neither the oxophilic nor thiophilic alkylating species persists in these reaction mixtures since the residual activity matches the kinetics for the rate determining elimination of the n-1 methanesulfinate moiety and thus is only equal to the remaining 90ce in both cases . since this trapped fraction appeared to be independent of the mnp concentration until exhaustion , implying extremely fast and efficient scavenging ( figure 7 , panel a ) , it is possible that this small discrepancy reflects the presence of another minor reaction pathway that occurs in the presence of pi ( potential minor pathway b ) and not merely a failure of the mnp to fully scavenge the short - lived thiophilic electrophile generated . in the presence of normal intracellular pi and phosphate ester concentrations , it is likely that 1025% of the 90ce reacts via the thiophilic pathway , and this can be regarded as a loss of potentially active agent . the overall sensitivity of a tumor cell would be the product of several major and minor factors , including the dna repair activities of mgmt and hdr , protective glutathione - s - transferases able to intercept a portion of the oxophilic electrophiles prior to reaction with dna , and the levels of pi and phosphoesters able to divert the decomposition pathway away from oxophilic chloroethylating electrophile generation .

t cells are the prime movers of the endogenous immune response to cancer , although they may be aided ( or hindered ) in this process by other cells . although a number of antibodies to molecules expressed on tumors ( and normal cells ) are now used to treat cancers , they are used as pharmacological rather than immunological agents . all the immunological agents approved for treatment of human cancers activate the t cell responses to cancers [ 13 ] . this discussion will therefore focus purely on the t cell epitopes of cancers and the responses elicited by them . much of our initial understanding about t cell epitopes came from study of viral immunity . t cell epitopes of viruses can be identified and can be used to elicit immune responses and protective immunity against viruses . as it became possible to generate t cells against mouse and human tumors , it was expected that identification of epitopes of cancers could be similarly used to elicit immune responses and protective immunity against cancers . it has now been over 20 years since it became possible to identify the t cell epitopes of mouse and human cancers of non - viral origins , and a large number of t cell epitopes have now been defined and characterized . such epitopes have fallen into two categories , one where the epitopes seen by the antitumor t cells are identical between normal and tumor cells , and second , where such epitopes are specific to the tumor cells and not seen in normal cells , by virtue of a tumor - associated mutation or other genetic event . the former class of the t cell tumor epitopes , the shared tumor epitopes , so - called because they are shared by tumors and by tumors and normal cells , has generated much of the enthusiasm and activity over the last 20 years . to be clear , the shared epitopes themselves consist of two sub - classes of epitopes the differentiation antigens ( such as tyrosinase ) , which are shared between normal tissues and tumors , and the cancer testes or ct antigens ( such as mage and ny - eso1 ) , which although un - mutated are expressed on germinal tissues and cancers , but not on normal adult tissues . it is important to point this here because the ct antigens , although un - mutated , are tumor specific ( if one discounts the germinal tissues ) . the shared epitopes have been tested extensively for their ability to immunize and generate t cell responses and to protect mice and men against cancers . notwithstanding a lone voice or two [ 68 ] , the fact that these shared epitopes are not tumor specific , and hence may not be immunogenic or immune protective , has been mostly glossed over . ( it is useful to remember that the t cell epitopes of viruses were of course all virus specific . ) indeed , the evidence from mouse models lends credence to the idea that the lack of tumor specificity of these epitopes is a barrier to their ability to elicit immune - protective anti - tumor responses [ 9 , 10 ] . instead , the argument has been that since these epitopes are common between tumors and normal tissues , and since there must exist a degree of tolerance to the antigens , the goal should be to break tolerance against such self - antigens . the possibility that such breaking of tolerance , if achieved , would lead to unacceptable toxicities , has not been generally considered to be a major problem . since the studies carried out thus far have failed to elicit potent antitumor responses , or potent autoimmunity for that matter , the issue of toxicities remains moot . however , two large randomized multi - center clinical trials , actually the largest ever trials in the history of cancer vaccination , are currently testing whether immune response to one such shared tumor antigen , mage , elicits clinical benefit in cancer patients . the outcome of these trials will reveal if immunization with the shared , non - tumor - specific epitopes is tumor protective ; if the answer is in the affirmative , the results will also reveal , if such immunizations elicit pathological autoimmunity . the latter class of t cell epitopes , the ones where the epitopes are tumor specific by virtue of the fact that a mutation in a normal sequence has created a new epitope , has been problematic as well : an overwhelming proportion of these mutations is found in only a given tumor , that is , the epitopes are individually distinct for a tumor . although immunization with such tumor specific epitopes in mouse models of cancer has shown them to be highly tumor protective for the tumor that harbor them [ 1115 ] ( table 1 ) , and the indirect evidence in humans has been tantalizing , what does one do with an individually unique epitope even though it is tumor specific and perhaps even immune protective against a tumor ? the prospect of generating t cells from individual patients , characterizing the individually unique tumor - specific epitopes from these t cells for each patient , and immunizing each patient with such epitopes , is simply not practical for a variety of obvious reasons . for these good reasons , this latter class of epitopes has not elicited much enthusiasm.table 1t - cell - defined epitopes of mouse tumors and their characteristicsproteintumor(s)mhc allelepeptide sequenceunique or sharedelicits tumor rejection?referencesl9 ribosomal protein6132a squamous carcinomaiedfnhinvelshlgkuniqueyesp68 helicase8101 squamous carcinomaksnfvfagiuniqueyesp53meth a fibro - sarcomakkyicnsscmuniqueyeserk2cms5 fibro - sarcomalqihsanvluniqueyesl11 ribosomal proteinmeth a fibro - sarcomaieeyelrkhnfsdtguniqueyesp1amanyllpylgwlvfsharedno[9 , 10]ah1manylspsyvyhqfsharedno , un - publishedthe letter in italics denoted the altered residue created by a mis - sense mutationp1a has been shown to mediate tumor rejection if p1a and b7 - 1 expressing cells are used as vaccines t - cell - defined epitopes of mouse tumors and their characteristics the letter in italics denoted the altered residue created by a mis - sense mutation p1a has been shown to mediate tumor rejection if p1a and b7 - 1 expressing cells are used as vaccines to sum up the above , there are powerful scientific reasons and data against the idea that the shared tumor antigens may elicit protective tumor immunity ; however , the denouement for this line of thinking is not far off : the two randomized trials with the shared mage antigen expect to be un - blinded within the next two years . the idea that the individually unique tumor antigens may be tumor protective is more appealing theoretically and is supported by considerable mouse data and some human evidence ; however , it appears at first blush , to be logistically untenable . as a matter of fact , efforts to harness the individuality of immunogenicity of each cancer have a rather long and interesting history . this is discussed in the next section , followed by an overview of the extraordinary opportunities now available for this pursuit because of the availability of high throughput dna sequencing technologies . the first hint that t cells may be recognizing individually specific mutations in each individual cancer came long before we knew of t cells . and others noted over 50 years ago that inbred mice could be immunized against syngeneic tumors , even autochthonous tumors , and that such immunity was individually specific : tumors of the same histological type , induced by the same carcinogen in mice of the same haplotype , still showed individually distinct antigenicity . in a dramatic demonstration , globerson and feldman showed that two tumors induced on each flank of a single mouse by two independent injections of the same carcinogen were individually distinct antigenically . basombrio tested this individuality in a large panel of 25 tumors and observed the extreme rarity of either totally or partially shared antigenic components between methylcholanthrene - induced tumors , as demonstrated by rejection of tumor cell inocula . these observations starting well over 50 years ago suggested that each time there was a new transforming event , there was a new and unique pattern of immunogenicity . one of the possibilities considered at the time was that this uniqueness of immunogenicity was simply a reflection of pre - existing unique patterns of immunogenicity in the normal cells . clever experiments , which tested the patterns of immunogenicity of cells , transformed in vitro with the same carcinogen soon suggested otherwise : they showed that progeny of the same normal cell , transformed in vitro , had unique patterns of immunogenicity . these ideas have simply stayed in the literature for lack of avenues to explore them , until now , as discussed in the next section . and now for the modern history . a number of human studies have attempted to harness the individually distinct immunogenicity of individual human tumors . b cell lymphomas present a unique opportunity ( no pun intended ) because the tumors each have a unique idiotype ( antigen ) . building on the pioneering studies of ronald levy using anti - idiotypic antibodies to treat b cell lymphomas , patients were immunized with the idiotypes of their tumors and monitored for disease free survival . two randomized trials failed to show statistically significant clinical benefit in the idiotype - immunized arm [ 23 , 24 ] , while a third trial did show such difference . among solid tumors , a series of randomized trials in patients with colon cancers was performed where patients were immunized post - surgical resection , with whole irradiated autologous tumor cells mixed with bcg , or were not immunized . the last such randomized trial showed statistically significant benefit in the immunized patients with stage ii , but not stage iii colon cancer . the heat shock protein ( hsp)-based vaccine is yet another way to harness the antigenic individuality of each cancer . this approach is based on the demonstration that molecules of hsps of the hsp70 and hsp90 families are associated non - covalently ( 1:1 or 2:1 ) with a broad array of peptides generated in the cells during proteolytic degradation . these peptides consist overwhelmingly of self - peptides , but also contain any non - self peptides generated in the source from which the hsps are isolated . such non - self peptides include viral peptides ( if hsps were isolated from virus - infected or transformed cells ) or tumor antigenic peptides if the hsps were isolated from tumor tissues [ 29 , 30 ] . thus , the purified hsp preparations are actually hsp - peptide preparations . upon immunization , the hsp - peptide complexes are taken up by antigen presenting cells of the host through hsp receptors [ 31 , 32 ] , and the peptides are cross - presented by the mhc molecules of the antigen presenting cells , which then engage the t cells , and mediate anti - tumor responses . a phase 3 trial in patients with renal cell carcinoma in the adjuvant setting , where each patient was immunized with hsp - peptide complexes isolated from his / her own tumor , failed to show statistically significant clinical activity in the overall population , although significant activity was observed in post hoc sub - sets of early and intermediate stage disease . a large randomized trial using this approach is currently underway in patients with glioblastoma multiforme . there are two ways to look at this history of individually specific vaccination against cancers . at first look , none of these three approaches have succeeded : none is widely used in cancer therapy today . the idiotype vaccine for b cell lymphoma and the whole cell vaccine for colon cancer showed statistically significant clinical activity , but are encumbered by difficulties in vaccine production or regulatory concerns about vaccine quality . the hsp - based vaccine failed to show statistically significant activity except in post hoc sub - sets and , although approved for use in russia , has not been cleared for use in the us or europe . however , if one looks at these three vaccines in the larger universe of all cancer vaccines tested in phase 3 trials , a somewhat different pattern emerges . with a single exception , all of the vaccines based on the idea of common antigenicity of cancers have failed [ 1 , 33 ] , and the only one that was approved for use in the us is struggling to achieve acceptance , partly because of lack of confidence in its clinical activity . the results of ongoing randomized clinical trials in patients with melanoma and lung cancer vaccinated with the shared , un - mutated vaccine mage , as also those of improved versions of the three autologous vaccines discussed here , will bring some clarity to the picture or may muddy it further . regardless , scientific and clinical data to date provide strong evidence for the existence of an individually distinct antigenic repertoire for each individual cancer , and the feasibility of using this repertoire for successful cancer therapy . 20 years ago , i suggested that randomness of passenger mutations in individual tumors generates this repertoire . the argument can be unfolded thus : because the process of dna replication is not completely accurate , and each cell division in any cell leads to a small number of errors , even after the repair mechanisms have corrected most of the errors . this error rate can range anywhere between one error in a billion to one error in a hundred thousand base pairs replicated per cell division depending upon the cell type and the degree of genomic instability in it [ 35 , 36 ] . even at the lowest rate of errors , a tumor will accumulate thousands of mutations by the time it progresses from the first transformed cell to a clinically or radiologically detectable tumor . simply by statistical probability , a small proportion of these mutations will create new epitopes for some of the mhc i alleles of the tumor . i had suggested that ( a ) such neo - antigens will be created by the passenger mutations that have nothing to do with the transformed phenotype and that may or may not confer any survival advantage to the tumor , and ( b ) since these are random mutations , their repertoire for any particular tumor is likely to be unique . this mechanism would explain the unique antigenicity of tumors as observed by earlier workers as discussed above . at the time this mechanism was predicted ( 1993 ) , high throughput dna sequencing was still far away , and the possibility that this hypothesis could be experimentally tested did not really exist . fast forward to 2008 , when high throughput dna sequencing technologies began to be usable . using banked samples of breast and colon cancers , and based on partial sequences of tumor transcripts , segal et al . utilized the algorithms for prediction of hla binding sequences , and in the first study of this kind , predicted individual breast and colon cancers to have between 7 and 10 new and tumor - specific hla a201-restricted epitopes ! we made use of probability theory in estimating the number of tumor - specific neo - epitopes in a tumor . some of the results were entirely expected , but provided the benefit of quantitation , while others were novel . among the expected results , the analysis showed that the number of potential neo - epitopes ( a ) varies directly as a function of the mutation rate and ( b ) increases exponentially with increasing number of cell divisions ( i.e. , the older a tumor , the more neo - epitopes it has ) . further , as expected , it showed that the tumors become more antigenically heterogeneous as they grow . in a novel deduction , the analysis showed that the death rate within a tumor has a profound effect on its immunogenicity . a tumor with a higher death rate will require many more cell divisions to achieve a certain mass as compared to a tumor with a lower death rate . this result places tumor immunogenicity at the intersection of a number of non - immunological characteristics such as tumor vascularization , hypoxia , size and remains to be fully understood or exploited . predictions and theoretical considerations aside , the first actual effort at genomics - guided definition of tumor - specific epitopes was published by sahin and colleagues . using exome sequencing of a cell line derived from the spontaneous mouse melanoma b16 , castle et al . uncovered tens of neo - epitopes generated by mis - sense mutations and characterized them with respect to their immunogenicity ; they observed that a significant proportion of the predicted neo - epitopes was actually immunogenic in vivo . they also showed that immunization with two of such neo - epitopes modulated the course of tumor growth in tumor - bearing and prophylactically treated animals . these findings , important in and of themselves , were particularly interesting because they were made in a poorly immunogenic tumor line . schreiber and colleagues used high throughput dna sequencing to build on their work on immunoediting of cancers ; they identified a mutation - generated epitope in a tumor arising in an immunodeficient mouse , and showed that this neo - epitope becomes a tumor - rejection antigen upon transplantation into an immunocompetent mouse , and becomes the subject of immunoediting . our laboratory has carried out genomics - guided identification of several chemically induced and spontaneous mouse tumors . using methods broadly similar to those of castle et al . , but with significant differences , these studies have un - covered hundreds of epitopes in the chemically induced tumors and a much smaller number in the spontaneous tumors and have shown a proportion of them to be immunogenic in vivo . collectively , the genomics - driven approach to identification of tumor - specific neo - epitopes has just begun and is beginning to support the postulate that ( a ) tumors do harbor an individually distinct repertoire and ( b ) that this repertoire is created by randomness of passenger mutations . while the previous approaches to harnessing this individually specific repertoire [ 26 , 34 ] were handicapped by the inability to actually identify this repertoire for individual tumors , the new genomics technologies promise to help overcome that critical hurdle . the genomics - driven approach to harnessing the individually distinct repertoire of tumor - specific mutations requires significant enquiry and resolution in mouse models ; regardless , it may not be entirely out of place to begin to consider the challenges in translating this approach to the human situation . rapid and cheap high throughput sequencing of exomes or transcriptomes is not a challenge anymore and most core facilities at academic institutions as well as commercial facilities do this readily . bio - informatic analysis of such sequences is also becoming more widely accessible through pipelines already generated . it is clear that a pipeline of potentially immunogenic epitopes can be generated through analyses in silico [ 42 , 43 ] . the challenge is to trim this ( expected - to - be - quite - long ) list into a list that is small enough to be practical and contains epitopes that will be truly tumor protective . ( see also the issue of antigenic heterogeneity below . ) not all immunogenic epitopes will be tumor protective , and we can not reasonably immunize patients with all the putative epitopes identified in silico . a better understanding of this question is perhaps the single most significant challenge in translating genomics into true tumor immunomics . another issue greatly worthy of consideration is the possibility that immunization with a mutated epitope may elicit cross - reactive t cell response against the wild - type epitope as well . this raises the specter of at least some degree of autoimmunity , which may or may not be pathological . one may draw some lesson from the fact that immunizations of patients with un - mutated self - epitopes have seldom elicited pathological autoimmunity [ 1 , 3 ] . regardless , there is need for caution in this regard , and only further studies in mice and humans shall clarify this issue . how do we immunize ? do we use a collection of gmp - grade peptides or do we use rna encoding multiple epitopes [ 44 , 45 ] ? what adjuvants do we use ? how much immunogen should be used ? what should be the regimen of immunization ? these questions do not require a conceptual leap , but they do need considerable examination and experimentation . this issue is a large one , but arguably less significant than it may appear . the idea that tumor - specific neo - epitopes are generated by random mutations inherently harbors the idea of extensive antigenic heterogeneity : the mutations that occurred earlier in the clonal expansion of a tumor are likely to be imprinted on a larger proportion of tumor cells than those that occur later , assuming that both classes of mutations are neutral with respect to any survival advantage or disadvantage on cells . this scenario creates an image of a highly compartmentalized tumor - cell population , which contains a large number of epitopes presented by narrower and narrower segments of the tumor ( fig . 1 ) . the reality is actually likely to be different from that caricaturized in fig . 1 . the new mutations are as likely to occur in cells that harbor the older mutations as in cells that do not ( see [ 4649 ] for stimulating discussion ) . the net result would be not a compartmentalized population of tumor cells as in fig . 1 , but a tumor mass that is a hopelessly mixed chimera of cells that each contain different sets of overlapping epitopes . . it should be possible to identify this cocktail for any given tumor ( and its metastatic progeny ) by the use of a suitable combination of sequencing and bio - informatic methods . this is important work that needs to be done , but can be done . finally in this regard , it is worth remembering that one does not need to eliminate 100 % of the cells to obtain significant clinical benefit ; bystander killing of antigen - negative tumor cells is a robust reality [ 50 , 51].fig . a schematic showing the emergence of random passenger mutations ( that are not required for the transformed phenotype and that do not confer any survival advantage or disadvantage and assuming zero tumor cell death ) in a growing tumor mass . the mutation that occurs at the first division of the transformed cell is imprinted on 50 % of the population , while mutations occurring in subsequent cell cycles ( red , green , purple , turquoise , and orange , in that order ) are presented on increasingly narrower population segments , leading to a tumor with various sub - population of cells expressing different sets of mutations . the figure may appear to suggest ( incorrectly ) that tumors are actually compartmentalized in this manner : since newer mutations are as likely to occur in cells that harbor older mutations as in the cells that do not , the tumor mass will actually be a chimera of cells presenting large numbers of overlapping sets of neo - epitopes antigenic heterogeneity in tumor masses . a schematic showing the emergence of random passenger mutations ( that are not required for the transformed phenotype and that do not confer any survival advantage or disadvantage and assuming zero tumor cell death ) in a growing tumor mass . the mutation that occurs at the first division of the transformed cell is imprinted on 50 % of the population , while mutations occurring in subsequent cell cycles ( red , green , purple , turquoise , and orange , in that order ) are presented on increasingly narrower population segments , leading to a tumor with various sub - population of cells expressing different sets of mutations . the figure may appear to suggest ( incorrectly ) that tumors are actually compartmentalized in this manner : since newer mutations are as likely to occur in cells that harbor older mutations as in the cells that do not , the tumor mass will actually be a chimera of cells presenting large numbers of overlapping sets of neo - epitopes there are obvious regulatory challenges in the use of an individual - specific platform of immunotherapy . the reasonable requirements of quality controls for each lot of drug are far more complex in an individualized therapy ( where the drug made for each individual patient is a new drug lot ) than in a traditional therapy where a single lot caters to a large patient population . however , many if not most of these regulatory challenges have already been addressed to a significant degree since a large number of clinical trials ( including randomized multi - center phase 3 clinical trials ) have been conducted previously with individual - specific immunotherapies [ 2 , 26 ] . meeting these challenges is our immediate task . to quote the 16th president of the united states , as our case is new , it is clear that a pipeline of potentially immunogenic epitopes can be generated through analyses in silico [ 42 , 43 ] . the challenge is to trim this ( expected - to - be - quite - long ) list into a list that is small enough to be practical and contains epitopes that will be truly tumor protective . ( see also the issue of antigenic heterogeneity below . ) not all immunogenic epitopes will be tumor protective , and we can not reasonably immunize patients with all the putative epitopes identified in silico . a better understanding of this question is perhaps the single most significant challenge in translating genomics into true tumor immunomics . another issue greatly worthy of consideration is the possibility that immunization with a mutated epitope may elicit cross - reactive t cell response against the wild - type epitope as well . this raises the specter of at least some degree of autoimmunity , which may or may not be pathological . one may draw some lesson from the fact that immunizations of patients with un - mutated self - epitopes have seldom elicited pathological autoimmunity [ 1 , 3 ] . regardless , there is need for caution in this regard , and only further studies in mice and humans shall clarify this issue . how do we immunize ? do we use a collection of gmp - grade peptides or do we use rna encoding multiple epitopes [ 44 , 45 ] ? what adjuvants do we use ? how much immunogen should be used ? what should be the regimen of immunization ? these questions do not require a conceptual leap , but they do need considerable examination and experimentation . this issue is a large one , but arguably less significant than it may appear . the idea that tumor - specific neo - epitopes are generated by random mutations inherently harbors the idea of extensive antigenic heterogeneity : the mutations that occurred earlier in the clonal expansion of a tumor are likely to be imprinted on a larger proportion of tumor cells than those that occur later , assuming that both classes of mutations are neutral with respect to any survival advantage or disadvantage on cells . this scenario creates an image of a highly compartmentalized tumor - cell population , which contains a large number of epitopes presented by narrower and narrower segments of the tumor ( fig . 1 ) . the reality is actually likely to be different from that caricaturized in fig . 1 . the new mutations are as likely to occur in cells that harbor the older mutations as in cells that do not ( see [ 4649 ] for stimulating discussion ) . the net result would be not a compartmentalized population of tumor cells as in fig . 1 , but a tumor mass that is a hopelessly mixed chimera of cells that each contain different sets of overlapping epitopes . . it should be possible to identify this cocktail for any given tumor ( and its metastatic progeny ) by the use of a suitable combination of sequencing and bio - informatic methods . this is important work that needs to be done , but can be done . finally in this regard , it is worth remembering that one does not need to eliminate 100 % of the cells to obtain significant clinical benefit ; bystander killing of antigen - negative tumor cells is a robust reality [ 50 , 51].fig . a schematic showing the emergence of random passenger mutations ( that are not required for the transformed phenotype and that do not confer any survival advantage or disadvantage and assuming zero tumor cell death ) in a growing tumor mass . the mutation that occurs at the first division of the transformed cell is imprinted on 50 % of the population , while mutations occurring in subsequent cell cycles ( red , green , purple , turquoise , and orange , in that order ) are presented on increasingly narrower population segments , leading to a tumor with various sub - population of cells expressing different sets of mutations . the figure may appear to suggest ( incorrectly ) that tumors are actually compartmentalized in this manner : since newer mutations are as likely to occur in cells that harbor older mutations as in the cells that do not , the tumor mass will actually be a chimera of cells presenting large numbers of overlapping sets of neo - epitopes antigenic heterogeneity in tumor masses . a schematic showing the emergence of random passenger mutations ( that are not required for the transformed phenotype and that do not confer any survival advantage or disadvantage and assuming zero tumor cell death ) in a growing tumor mass . the mutation that occurs at the first division of the transformed cell is imprinted on 50 % of the population , while mutations occurring in subsequent cell cycles ( red , green , purple , turquoise , and orange , in that order ) are presented on increasingly narrower population segments , leading to a tumor with various sub - population of cells expressing different sets of mutations . the figure may appear to suggest ( incorrectly ) that tumors are actually compartmentalized in this manner : since newer mutations are as likely to occur in cells that harbor older mutations as in the cells that do not , the tumor mass will actually be a chimera of cells presenting large numbers of overlapping sets of neo - epitopes there are obvious regulatory challenges in the use of an individual - specific platform of immunotherapy . the reasonable requirements of quality controls for each lot of drug are far more complex in an individualized therapy ( where the drug made for each individual patient is a new drug lot ) than in a traditional therapy where a single lot caters to a large patient population . however , many if not most of these regulatory challenges have already been addressed to a significant degree since a large number of clinical trials ( including randomized multi - center phase 3 clinical trials ) have been conducted previously with individual - specific immunotherapies [ 2 , 26 ] . meeting these challenges is our immediate task . to quote the 16th president of the united states , as our case is new ,

the idea that individual tumors are antigenically unique has been around since the very dawn of our recognition of adaptive immune response to tumors . that idea has inspired a small number of attempts at individualized immunotherapy of human cancers . such previous attempts for solid tumors have been hobbled by an inability to define the individually unique antigenic repertoire of tumors because of technological difficulties . the new availability of rapid and cheap high throughput dna sequencing promises to overcome that hurdle . using this new ability , coupled with bio - informatic tools , it is now possible to define the immunogenic repertoire of any tumor to a high degree of granularity within a practical time frame and an acceptable cost . the development of these ideas , and a small number of such studies that underscore this promise , is discussed . this new way of characterizing the tumor immunome through characterization of the tumor genome has distinct challenges , including selection of the appropriate peptides , choosing methods of immunizations that can incorporate tens of epitopes , and addressing issues of antigenic heterogeneity of tumors . however , tools for meeting these challenges exist and are emergent .

Tumors as seen by T lymphocytes Harnessing the individually distinct immunome of each individual cancer: some medieval and modern history Harnessing the individually distinct immunome of each individual cancer: the genomic way Challenges in translation to the human setting Selection of candidates of immunization Technology of immunization Antigenic heterogeneity Regulatory challenges

it has now been over 20 years since it became possible to identify the t cell epitopes of mouse and human cancers of non - viral origins , and a large number of t cell epitopes have now been defined and characterized . the latter class of t cell epitopes , the ones where the epitopes are tumor specific by virtue of the fact that a mutation in a normal sequence has created a new epitope , has been problematic as well : an overwhelming proportion of these mutations is found in only a given tumor , that is , the epitopes are individually distinct for a tumor . although immunization with such tumor specific epitopes in mouse models of cancer has shown them to be highly tumor protective for the tumor that harbor them [ 1115 ] ( table 1 ) , and the indirect evidence in humans has been tantalizing , what does one do with an individually unique epitope even though it is tumor specific and perhaps even immune protective against a tumor ? the prospect of generating t cells from individual patients , characterizing the individually unique tumor - specific epitopes from these t cells for each patient , and immunizing each patient with such epitopes , is simply not practical for a variety of obvious reasons . for these good reasons , this latter class of epitopes has not elicited much enthusiasm.table 1t - cell - defined epitopes of mouse tumors and their characteristicsproteintumor(s)mhc allelepeptide sequenceunique or sharedelicits tumor rejection?referencesl9 ribosomal protein6132a squamous carcinomaiedfnhinvelshlgkuniqueyesp68 helicase8101 squamous carcinomaksnfvfagiuniqueyesp53meth a fibro - sarcomakkyicnsscmuniqueyeserk2cms5 fibro - sarcomalqihsanvluniqueyesl11 ribosomal proteinmeth a fibro - sarcomaieeyelrkhnfsdtguniqueyesp1amanyllpylgwlvfsharedno[9 , 10]ah1manylspsyvyhqfsharedno , un - publishedthe letter in italics denoted the altered residue created by a mis - sense mutationp1a has been shown to mediate tumor rejection if p1a and b7 - 1 expressing cells are used as vaccines t - cell - defined epitopes of mouse tumors and their characteristics the letter in italics denoted the altered residue created by a mis - sense mutation p1a has been shown to mediate tumor rejection if p1a and b7 - 1 expressing cells are used as vaccines to sum up the above , there are powerful scientific reasons and data against the idea that the shared tumor antigens may elicit protective tumor immunity ; however , the denouement for this line of thinking is not far off : the two randomized trials with the shared mage antigen expect to be un - blinded within the next two years . the idea that the individually unique tumor antigens may be tumor protective is more appealing theoretically and is supported by considerable mouse data and some human evidence ; however , it appears at first blush , to be logistically untenable . this is discussed in the next section , followed by an overview of the extraordinary opportunities now available for this pursuit because of the availability of high throughput dna sequencing technologies . with a single exception , all of the vaccines based on the idea of common antigenicity of cancers have failed [ 1 , 33 ] , and the only one that was approved for use in the us is struggling to achieve acceptance , partly because of lack of confidence in its clinical activity . the argument can be unfolded thus : because the process of dna replication is not completely accurate , and each cell division in any cell leads to a small number of errors , even after the repair mechanisms have corrected most of the errors . at the time this mechanism was predicted ( 1993 ) , high throughput dna sequencing was still far away , and the possibility that this hypothesis could be experimentally tested did not really exist . while the previous approaches to harnessing this individually specific repertoire [ 26 , 34 ] were handicapped by the inability to actually identify this repertoire for individual tumors , the new genomics technologies promise to help overcome that critical hurdle . rapid and cheap high throughput sequencing of exomes or transcriptomes is not a challenge anymore and most core facilities at academic institutions as well as commercial facilities do this readily . this scenario creates an image of a highly compartmentalized tumor - cell population , which contains a large number of epitopes presented by narrower and narrower segments of the tumor ( fig . however , many if not most of these regulatory challenges have already been addressed to a significant degree since a large number of clinical trials ( including randomized multi - center phase 3 clinical trials ) have been conducted previously with individual - specific immunotherapies [ 2 , 26 ] . however , many if not most of these regulatory challenges have already been addressed to a significant degree since a large number of clinical trials ( including randomized multi - center phase 3 clinical trials ) have been conducted previously with individual - specific immunotherapies [ 2 , 26 ] .

initially introduced as a component of complex interventions and later as part of a two - stage operation in high - risk patients , the procedure is now more common as one - stage operation and subject of avid scientific discussion . this article aims to trace the historical development of resection of the greater curvature with particular reference to its origin in ulcer and bariatric surgery . furthermore , the current value of sleeve gastrectomy within the spectrum of bariatric surgical procedures will be discussed . besides bariatric surgery modern sleeve gastrectomy has one more so far largely neglected origin : segmental and later longitudinal gastric resection used in ulcer surgery . experience and achievements from ulcer surgery simplified and facilitated development of sleeve gastrectomy which is not the desired universal procedure for bariatric surgery but certainly an attractive treatment option . it should be performed in a more standardized manner and with due regard to future long - term results . obesity is gradually turning into an epidemic condition throughout the world and has become a social , psychological , and economic burden of growing proportions.1,2 it is associated with a large number of concomitant diseases ( including type-2 diabetes , cardiovascular and respiratory diseases , dyslipidemia , and elevated risk of cancer ) and also markedly shortens the obese person s life expectancy.3,4 due to the limited options and especially the poor long - term results of conservative treatment , the surgical approach of bariatric surgery has been established in the last few decades.3 a bariatric procedure is considered to be indicated in adult patients with morbid obesity ( bmi 40 kg / m ) or a bmi 35 kg / m with additional comorbidities.5,6 long - term results of the surgical approach have been convincing in terms of reduced morbidity and mortality as well as enhanced quality of life.7,8 due to growing experience and the introduction of the endoscopic technique , the procedures have become increasingly safe and can be performed more easily by the use of modern stapling devices . therefore , bariatric surgery is even considered in adolescents with a high - risk profile or in patients with bmi < 35 kg / m.9,10 several surgical procedures have been developed over time and nearly all of them are currently performed by the laparoscopic approach . a distinction has been made between restrictive , malabsorptive , combined restrictive and malabsorptive , and electrical procedures for gastric stimulation . this diversity and the ongoing modifications of the procedures highlight the fact that there is no ideal procedure for widespread application . the quality of the respective procedures is no longer established by the previously used primary parameter of excess weight loss , but by the procedure s potential to maintain sufficient weight reduction on a long - term basis while ensuring minimal mortality and morbidity . in recent times , one procedure has become increasingly popular in obesity surgery , namely longitudinal gastric resection or sleeve gastrectomy . it was initially used as a part of complex interventions ( including biliopancreatic diversion with duodenal switch ) and later as a two - step bridging procedure in high - risk patients prior to final intervention , but was then established as a stand - alone procedure and is currently a subject of avid scientific discussion . the current concept of tube gastrectomy by resection of the greater curvature is not new in bariatric surgery . it is largely neglected that segmental and especially longitudinal gastric resection were developed and used in ulcer surgery.1113 following the introduction of adequate conservative drug therapies , ulcer surgery is now almost exclusively used as an emergency procedure . however , longitudinal gastric resection can be regarded as precursor of modern tube gastrectomy , which is now known as sleeve gastrectomy and is experiencing a revival in obesity surgery . the aim of the present review is to trace the historical development of the current longitudinal gastric resection on the basis of its origins in ulcer and bariatric surgery and to elucidate the subject with suitable illustrations . furthermore the contribution of historic ulcer surgery to modern bariatric surgery and in particular sleeve gastrectomy is demonstrated . finally , contemporary sleeve gastrectomy , its complications , and especially the current value of this procedure in the therapy spectrum of bariatric surgery will be discussed on the basis of major recent studies . development of longitudinal gastric resection in ulcer surgerythe advancing use of gastric surgery is a milestone in the evolution of abdominal surgery . the first gastric resection procedures were performed by j. pan and l. rydygier in 1879 and 1880 , but with lethal outcomes.14,15 t. billroth is known as the pioneer of gastric surgery and its scientific foundation . in 1881 , he performed the first successful gastric resection in a patient with a pyloric carcinoma.16 his work served as the starting point for classical gastric resection procedures such as billroth i and billroth ii ( first performed on a human patient in 1885 ) , depending on the manner of restoration of the gastrointestinal passage . these procedures became essential elements of every general surgeon s repertoire , particularly for ulcer treatment . development of longitudinal gastric resection in ulcer surgery the advancing use of gastric surgery is a milestone in the evolution of abdominal surgery . the first gastric resection procedures were performed by j. pan and l. rydygier in 1879 and 1880 , but with lethal outcomes.14,15 t. billroth is known as the pioneer of gastric surgery and its scientific foundation . in 1881 , he performed the first successful gastric resection in a patient with a pyloric carcinoma.16 his work served as the starting point for classical gastric resection procedures such as billroth i and billroth ii ( first performed on a human patient in 1885 ) , depending on the manner of restoration of the gastrointestinal passage . these procedures became essential elements of every general surgeon s repertoire , particularly for ulcer treatment . k. schwarz s discovery of the concept of no ulcers without acids in 1910 had a decisive impact on the development of gastric resection procedures.17 after this time , the aim of upcoming ulcer surgery was to reduce acid levels adequately in order to avoid recurrences . the purpose of the first segmental gastric resection procedures was to perform wedge- or v - shaped ulcer excision ; these were conducted as early as 1897 by j. mikulicz , 1904 by b. riedel , and 1929 by f.g . connell.1820 however , initially the outcome of these procedures was impeded due to considerable side effects like gastric emptying disorders.11 segmental gastric resection was greatly modified by o.h . wangensteen who , in 1952 , investigated the surgical procedures in great depth and resolved the problem of postoperative gastric emptying disorders by performing additional pyloroplasty.21 the technique was developed further by d.j . ferguson ( 1960 ) , f. largiadr ( 1971 ) , and t. sekine ( 1975).2224 the outstanding aspect of these advancements was preservation of antral innervation in order to prevent the post - gastrectomy syndrome . influenced by his experiences in segmental gastric resection with its undesirable side effects ( among others dumping syndrome ) , he searched for an acceptable operation for ulcer treatment . he was aware of the fact that gastric parietal cells , which are responsible for the production of hcl , are most dense in the corpus region lengthwise along the greater curvature.11,25 when performing the previous classical resection procedures ( including bi and bii resections and segmental gastric resection ) , portions of the stomach were removed at right angles to its conceived longitudinal axis . however , performing gastric resection along the longitudinal axis was considered even earlier by f. neugebauer , a.a . strauss , and v. schmieden in 1921 and 1924.2628 in contrast to wangensteen they performed resection along the lesser curvature to remove the ulcer itself , independent of acid reduction . however , wangensteen performed the first experiments of longitudinal resection along the greater gastric curvature to reduce acidity of gastric juice in 1940 . in some cases , he added a gastrojejunostomy at the antral gastric end ( fig . 1).25,29 based on promising results , he subsequently evolved his method of tubular gastric resection with additional transverse gastroplasty in order to accomplish a gastric reservoir function . wangensteen started applying this technique in 90 patients with duodenal ulcers and reported convincing results.11 however , after performing further animal experiments in 1957 , he revised his initially positive verdict about the operation . the acid response of the stomach after test meals was many - fold higher in animals subjected to tubular resection than in those that had undergone segmental gastric resection . wangensteen and colleagues attributed this phenomenon to the preservation of antral innervation and the resulting higher gastrin and acid secretion of the residual parietal cells . he concluded that tubular gastric resection should be viewed with caution and stopped using this technique.30 it is noteworthy that l. leger and l. deloyers made use of tubular or longitudinal resection without supplementary transverse gastroplasty.31,32fig . wangensteen performed longitudinal gastric resection to excise acid - producing regions of the stomach29 as early as in 1940 , o.h . wangensteen performed longitudinal gastric resection to excise acid - producing regions of the stomach29 in 1966 , m. saegesser introduced the theoretical construct of ideal gastric resection including resection of the corpus / fundus and the antrum , in combination with a selective post - branchial vagotomy and pylorotomy ( fig . 2).33 by performing longitudinal resection of the greater curvature , he intended to reduce acid secretion while preserving gastric reservoir function and the natural food passage . in 19881990 , j. hauss and h.u . spiegel focused on this construct and wangensteen s results , and made further developments.12,34 however , they dispensed with the vagotomy and pylorotomy demanded by saegesser because they believed that partial resection of the antrum and postoperative reduction of parietal cells would achieve sufficient acid reduction . in animal experiments , they achieved a 70% reduction of acid secretion in the presence of a normal serum gastrin response ( fig . 3).34 in 1993 , subsequent animal studies using this model showed a linear correlation between the reduction of parietal cells and acid reduction levels.35 spiegel s model was based on longitudinal gastric resection on the side of the greater curvature using a stapler and a gastric probe as guide rail . thus , he created a modern gastric tube ( fig . 4a , b).35 subsequently , the procedure was used in the clinical setting with promising results.13,36fig . 2 in 1966 , m. saegesser propagated his construct of ideal gastric resection with longitudinal resection of the fundus and the antrum , selective post - branchial vagotomy , and pylorotomy33fig . 3 in 1988 , j. hauss and h.u . spiegel presented a modified longitudinal resection model without vagotomy and pyolorotomy and reported significant acid reduction34fig . spiegel used longitudinal gastric resection in a large study focused on the treatment of ulcers . he utilized a gastric probe as guide rail ( a ) and a linear stapler ( b ) to create a modern gastric tube35 in 1966 , m. saegesser propagated his construct of ideal gastric resection with longitudinal resection of the fundus and the antrum , selective post - branchial vagotomy , and pylorotomy33 in 1988 , j. hauss and h.u . spiegel presented a modified longitudinal resection model without vagotomy and pyolorotomy and reported significant acid reduction34 in 1993 , h.u . spiegel used longitudinal gastric resection in a large study focused on the treatment of ulcers . he utilized a gastric probe as guide rail ( a ) and a linear stapler ( b ) to create a modern gastric tube35 the use of gastric resection procedures in gastroduodenal ulcer surgery entered a phase of stagnation and regression after this time . the decisive change which led to the renunciation of conventional resection procedures was the fact of advancing knowledge about the pathogenesis of ulcers , particularly the introduction of h2 receptor antagonists at the end of the 1970s , the introduction of proton pump inhibitors at the end of the 1980s , and the discovery of helicobacter pylori in 1982 . these developments had an equally strong impact on various vagotomy procedures for denervation , which were used less , and less in ulcer surgery . currently , the use of gastroduodenal ulcer surgery is confined to classical ulcer complications ( hemorrhage , perforation , penetration , pyloric stenosis ) and to exclude malignant tumors in cases of ulcers refractory to conservative treatment . the clinical use of longitudinal gastric resection was therefore becoming increasingly insignificant soon after being established as a treatment option . development of longitudinal gastric resection in bariatric surgerya review of the essential steps in the historical development of bariatric surgery is helpful in order to understand how longitudinal gastric resection appeared as sleeve gastrectomy within the modern therapy options . obesity surgery started with purely malabsorptive procedures , moved on to combined malabsorptive and restrictive procedures , and finally consisted of mainly restrictive procedures . kremen and co - workers in 1954.37 numerous modifications followed , particularly in respect of location and type of the anastomosis.38 a significant reduction in weight was achieved . however , many of these procedures were accompanied by serious side effects ( including diarrhea , hepatic cirrhosis , and electrolyte imbalance ) and did not prevail in the long term.2,39 development of longitudinal gastric resection in bariatric surgery a review of the essential steps in the historical development of bariatric surgery is helpful in order to understand how longitudinal gastric resection appeared as sleeve gastrectomy within the modern therapy options . obesity surgery started with purely malabsorptive procedures , moved on to combined malabsorptive and restrictive procedures , and finally consisted of mainly restrictive procedures . kremen and co - workers in 1954.37 numerous modifications followed , particularly in respect of location and type of the anastomosis.38 a significant reduction in weight was achieved . however , many of these procedures were accompanied by serious side effects ( including diarrhea , hepatic cirrhosis , and electrolyte imbalance ) and did not prevail in the long term.2,39 gradually , bariatric interventions were increasingly focused on the stomach . furthermore , a malabsorptive component was additionally employed to create a gastrointestinal bypass . in 1967 , mason submitted the first report of a gastric bypass after horizontal division of the stomach with re - anastomosis of its proximal portion by the use of a raised jejunal loop.40 again , numerous variations regarding pouch size or replacing division of the stomach by applying a horizontal row of clip sutures followed . griffen in 1977 , using a gastrojejunostomy , and y - roux reconstruction , while avoiding bile reflux , provided the advantage of a tension - free anastomosis.41 after further modifications ( particularly in respect of placement of the pouch and the length of the respective loops ) , this technique evolved into a standard procedure in bariatric surgery , especially in the usa , because of its very favorable ratio between weight reduction and side effects.42 a further noteworthy milestone in the development of bariatric surgery is biliopancreatic diversion which was developed by n. scopinaro in 1979 . scopinaro combined horizontal gastric resection with closure of the duodenal stump and a gastrojejunostomy while creating a common tract by jejunoileostomy to exclude large portions of the small bowel ( fig . 5).43 scopinaro initially varied the lengths of the three segments of the small bowel . subsequently a common tract about 50 cm in length and an alimentary tract about 250 cm length became established.2,44 the disadvantages of the procedure include malassimilation of fat and deficiency syndromes such as those of protein , iron , or vitamins.44,45fig . 5 in 1979 , n. scopinaro introduced his procedure of biliopancreatic diversion . he performed horizontal partial resection of the stomach with closure of the duodenal stump , gastrojejunostomy , and a jejunoileal anastomosis to create an alimentary tract he performed horizontal partial resection of the stomach with closure of the duodenal stump , gastrojejunostomy , and a jejunoileal anastomosis to create an alimentary tract printen reported the first purely restrictive procedure by incomplete horizontal division of the stomach while forming a conduit on the side of the greater curvature . however , the technique did not gain wide acceptance because of poorly sustained weight reduction.46 subsequent variations were used to achieve a reduction of gastric volume but were not successful due to dilatation of the gastric pouch.2,38 this problem was finally resolved in 1982 , again by e.e . mason , who introduced vertical gastroplasty with creation of a pouch on the side of the lesser curvature by placing a vertical clip suture and providing additional reinforcement with a distal polypropylene mesh ring.47 finally , restriction of the stomach by the use of a gastric band was developed in 1978 , initially without the option of being adjustable.48 the adjustable gastric band initially introduced by l.i . kuzmak in 1986 was modified further and is the second most commonly used procedure in obesity surgery these days.38,49 the modern procedure of longitudinal gastric resection or sleeve gastrectomy was incorporated quite late in the repertoire of obesity surgery . in 1993 , p. marceau and co - workers modified biliopancreatic diversion which had been introduced by n. scopinaro and replaced horizontal gastric resection with longitudinal gastric resection on the side of greater curvature , combined with preservation of the pylorus , and additionally doubled the length of the common tract to 100 cm.44 initially the small bowel was anastomosed to the proximal duodenum with additional placing of a distal row of clip sutures without transsection of the duodenum . however , this procedure was frequently associated with insufficiency of clip sutures , followed by a renewed increase in weight.44 the problem was resolved by the advancements made by d.w . and d.s . hess , based on t.r . demeester.50,51 in 1998 , they introduced biliopancreatic diversion with placement of a duodenal switch under postpyloric transsection of the duodenum and subsequent anastomosis with the alimentary loop . the biliopancreatic loop was anastomosed in the region of the distal ileum by creating a 50- to 100-cm - long common channel , 6).51 in conjunction with the development of minimally invasive surgery , the first laparoscopic tube gastrectomy was performed in the course of biliopancreatic diversion with a duodenal switch in 2000 ( fig . hess published biliopancreatic diversion with an additional duodenal switch . while preserving the pylorus they also created a biliopancreatic , an alimentary , and a common small bowel segment . using longitudinal gastric resection , they established a combined restrictive - malabsorptive procedure51fig resection is performed on the side of the greater curvature by the use of a linear stapler along a calibration probe100 in 1998 , d.w . and d.s . hess published biliopancreatic diversion with an additional duodenal switch . while preserving the pylorus they also created a biliopancreatic , an alimentary , and a common small bowel segment . using longitudinal gastric resection , they established a combined restrictive - malabsorptive procedure51 intraoperative view of contemporary laparoscopic sleeve gastrectomy . resection is performed on the side of the greater curvature by the use of a linear stapler along a calibration probe100 one of the milestones in the development of tube gastrectomy was the concept of the magenstrasse and mill operation . developed with the aim of devising a physiological bariatric procedure while avoiding implant - related complications ( such as those encountered with an adjustable gastric band or vertical banded gastroplasty ) and reducing long - term metabolic complications , this procedure was described by d. johnson in 1987.53 a circular stapler is used to create a hole in the antrum region , and a linear stapler is used to create a gastric tube on the side of the lesser curvature ( the magenstrasse ) while dividing the stomach longitudinally in cranial direction . because of the preserved antral mill of food , the method is known as the magenstrasse and mill operation . the procedure aroused a lot of interest because of its low side effects and marked weight reduction , particularly in the early postoperative phase.53 thus , modern tube gastrectomy may also be regarded as continuation of the magenstrasse in distal direction with subsequent resection ( including the portions of the stomach that produce ghrelin ; see the discussion section ) . the use of tube gastrectomy as a bridging step in a two - step surgical procedure is probably one of the most recent developments . tube gastrectomy is used as an initial intervention in high - risk patients in order to achieve a marked reduction of weight and risk factors and then perform the final intervention.5456 based on the positive experience gained from this concept and the technical simplicity of the procedure , gastric tube formation was eventually used as a stand - alone and single - step procedure . longitudinal gastric resection in ulcer and bariatric surgery was developed and established for different purposes and apparently , in a mutually independent manner . nevertheless , achievements in historical ulcer surgery benefited the development of sleeve gastrectomy . beyond this , resemblance in the development of longitudinal gastric resection and sleeve gastrectomy , respectively , can be demonstrated . significant experience with the procedures of segmental gastric resection contributed decisively to increase the understanding of antral innervation and pyloric function.11 side effects like malnutrition , gastric emptying disorders , dumping syndrome following segmental gastric resection , or classical billroth procedures led to further development of pylorus - preserving gastrectomies providing knowledge of great value about the physiological consequences of resection procedures and vagotomy.58,59 gradually , the complex role of the stomach as storage unit ( fundus and oral corpus ) and as a mill ( distal corpus and antrum ) was recognized and the interference with surgical procedures evaluated.6062 particularly the above - mentioned side effects encouraged o.h . wangensteen to search for an acceptable operation for ulcer treatment and to introduce tubular or longitudinal gastric resection.11 after wangensteen turned away from the procedure research focusing on longitudinal gastric resection was not abandoned . his successors established a clinically applicable procedure for ulcer treatment despite preserving antral innervation by resection of the greater curvature and thus performing a sleeve gastrectomy . however , after a brief period of clinical use this procedure faded into insignificance due to the upcoming and widespread conservative treatment options . on closer inspection of the circumstances during introduction of longitudinal gastric resection in bariatric surgery one is initially surprised about remarkable similarities to ulcer surgery . in his article focusing on the introduction of a new type of gastrectomy in 1993 , p. marceau described the ulcer genesis in biliopancreatic diversion due to absence of a buffer for gastric secretions . duodenum segment ( in contrast to the procedures used until this time ) , as well as innervation . thus , he developed the concept of acid reduction by longitudinal gastric resection.44 d.s . and d.w hess also emphasized the role of ulcer reduction by longitudinal gastric resection.51 looking at the presented historic development particularly of longitudinal gastric resection and its underlying pathophysiological concept in ulcer surgery , it becomes evident that the procedure is not an entirely new concept of gastrectomy . rather , it is a revival of an established method in a different context . in summary , it may be said that the contribution of ulcer surgery towards the understanding of the gastrointestinal system and particularly its innervation should not be underestimated . even in recent times we benefit from this knowledge.63 especially bariatric surgery which focuses increasingly on the stomach as target organ obtains valuable information based on already discovered relations . finally , technological achievements gained from the developing gastric surgery with its initially high mortality and serious complications should be taken into account . while open conventional suture procedures were used initially , surgeons were eager to acquire skills in performing stapler procedures which then became established and were eventually used on a routine basis . candidates for bariatric surgery are subject to a massively increased risk of mortality and morbidity due to the presence of several obesity - associated concomitant diseases.3 peri- and postoperative risks could be markedly reduced by the introduction of minimally invasive techniques and the fact that they became established standard procedures over time.5,52,64 a closer look on longitudinal resection of the greater curvature is indicated . in ulcer surgery and early bariatric surgery , the resection was performed in order to reduce active parietal cells and not primarily as a restrictive step . however , already wangensteen observed patients losing weight following tubular gastric resection even though he tried to create a gastric reservoir by performing transverse gastroplasty.11 gradually the value of restriction was identified and especially one further important function of the resected gastric tissue . resection of the fundus , which produces ghrelin , and additional reduction of gastric volume with dilatation of the antrum exert a marked positive impact on the sensation of satiety and reduction of calorie intake.54,65,66 ghrelin plays a central role in modulating appetite and the feeling of satiety , influencing gastrointestinal motility , particularly , body weight regulation . consequently , both ghrelin agonists as prokinetics to treat gastroparesis and postoperative ileus and ghrelin antagonists in order to suppress appetite and to improve glycemic control are subject of intensive research.67 the effects of longitudinal gastric resection on the gastrointestinal hormone interplay are far from being sufficiently discovered . obviously , the hormonal effects are regulated in a complex manner involving among others agrp , neuropeptide y , and leptin.68 the important role of these hormonal relations and their influence on metabolic disorders like diabetes is reflected by the increasing use of the term metabolic surgery . bariatric surgery is undisputedly one of the cornerstones of the treatment of morbid obesity and is subdivided into a number of different procedures.3 traditionally their success is measured in terms of excess weight loss . however , the anticipated weight loss should not be the sole or even principal criterion for selection of a procedure . complications related to the procedures of bariatric surgery are of substantial magnitude and must always be taken into account . the complexity of the surgical techniques and the potential surgical and metabolic complications of the various procedures are inversely related to the anticipated course of weight loss.5 especially malabsorptive and combined procedures are technically demanding and associated with increased rates of morbidity and mortality in high - risk patients . postoperatively they are frequently associated with deficiency syndromes that require supplementation.6971 gastro - gastric fistulas can now be largely avoided by complete division of the stomach . however , like leakage of the anastomosis , gastro - gastric fistulas still are a part of the spectrum of complications.72,73 depending on their severity and the time point of diagnosis , leakage of the anastomosis and strictures can be largely treated by the minimal - invasive approach and the use of stents . however , insufficiency of the duodenal stump after duodenal switch , relevant hemorrhage from clip sutures , or insufficiencies associated with concomitant cardiovascular reactions ( particularly tachycardia as a warning sign ) are serious complications that often require timely revision.52,57,74 by modifications such as combined biliopancreatic diversion and sleeve gastrectomy , side effects like dumping syndromes or ulcers could be largely avoided over time , but still do occur especially in cases of gastric bypass.51,75,76 furthermore , extensive procedures favor the occurrence of obstructions , hernia , or inappropriate bacterial colonization of the intestines.77,78 purely restrictive procedures such as laparoscopic insertion of an adjustable gastric band are convincing at first glance because of their low perioperative morbidity and mortality rates , but bear the risk of band dislocation ( slippage ) , band migration , port complications , and also compliance - related late complications.42,79 due to these numerous risks and complications , a procedure like sleeve gastrectomy which apparently can be performed easily and has a favorable risk benefit ratio would appear to have arrived at the right moment . the renaissance , and the enormously rapid and widespread application of this method as a single - step procedure , is quite understandable.80 introduced as a stepwise mode of treatment , the procedure reduced the previously high mortality rates in high - risk patients ( > 6% with a bmi > 60 , it was convincing because of its low complication ( about 9% ) and mortality rates ( < 1% ) , as well as its low rate of gastrointestinal long - term side effects.8183 some authors give preference to sleeve gastrectomy as opposed to a gastric balloon as part of a stepwise treatment regimen in high - risk patients.84 analogous to the concept of the magenstrasse and mill operation , gastric tube formation avoids malabsorption and implant - related complications while ensuring physiological gastric emptying.54,81 in contrast , sleeve gastrectomy involves irreversible resection of parts of the fundus and the corpus . the humoral aspect of the procedure seems to be important ( see above ) . in trials , sleeve gastrectomy was found to achieve a mean excess weight loss of 33% to 83% 1 year after surgery.54 despite this wide range , it may be assumed that , even in the mid - term , the procedure is associated with a similar marked reduction of weight as the usual procedures while reducing obesity - associated concomitant diseases.85,86 if additional weight reduction is required subsequently , the procedure can be performed in a two - step manner with a malabsorptive component ( gastric bypass or biliopancreatic diversion ) , either in a combined manner or a repeat sleeve gastrectomy can be conducted.87,88 therefore , one is easily inclined to regard tube gastrectomy as the desired all - round procedure . when assessing the procedures carefully , one should consider the fact that longitudinal gastric resection on the side of the greater curvature is an irreversible step and is associated with placement of a long row of stapler sutures along a gastric wall of varied structure.54,89 the most frequent surgical complications of the procedure are leaks ( about 0.9% ) , strictures ( about 0.7% ) , and postoperative bleeding ( about 0.4% ) . revision rates are reported to be around 4%.81,82 in addition to intraoperative inspection of the sutures , for instance by endoscopy or the use of methylene blue , several authors recommend oversewing the row of clip sutures or the use of clip reinforcement.56,57,90 however , procedures of suture reinforcement or oversewing are controversially discussed . some authors express apprehensions about suture weakening , do not necessarily attribute the reduction of insufficiency rates to suture reinforcement , or warn against strictures due to oversewing.89,91 other authors recommend laparoscopic greater curvature plication in order to avoid gastric resection and associated complications.92 two factors deserve attention : firstly , a growing number of studies have been focused on the use of sleeve gastrectomy as a single - step procedure and report convincing results , although adequate evaluable long - term results ( > 5 years ) are not yet available.56,83,93 secondly , sleeve gastrectomy is not performed in a standardized manner : various tube diameters and calibration probes ( 32 to 60 french ) are used.68,80,94 besides , the extent of resection , particularly in respect of preservation / resection of the antrum varies.57,95 intraoperative measurement of the volume of the resected stomach is of great importance . a removed volume < 500 cm is apparently associated with an early weight regain.57 thus , the results of various workgroups must be compared with caution . currently , the surgeon also is a substantial factor influencing the outcome of the procedure . any person involved in the treatment of obese patients should be aware of the fact that bariatric surgery is a domain of complex interventions in high - risk patients.3 the ideal procedure does not exist . rather , the key to successful treatment lies in a careful assessment of the individual risk jointly by the surgeon and the patient , as well as in providing intensive care and information before the operation and particularly in the long - term after a bariatric operation.5,96 eating habits , baseline weight , the anticipated weight loss , comorbidities , gender , age , and compliance are some of the numerous factors that must be taken into account.5,97 a team experienced in handling a wide spectrum of bariatric operations with confidence is indispensable to perform successful obesity surgery with sustained enhancement of quality of life and life expectancy.98 particularly sleeve gastrectomy should be viewed separately and not as a universal procedure . in view of the above - mentioned criteria , however , it should be performed in a more standardized manner and with due regard to future long - term results .

introductionsleeve gastrectomy is becoming increasingly popular within bariatric surgery . initially introduced as a component of complex interventions and later as part of a two - stage operation in high - risk patients , the procedure is now more common as one - stage operation and subject of avid scientific discussion . however , the concept of longitudinal gastric resection is not new . the procedure was already established in ulcer surgery but soon faded into insignificance . this article aims to trace the historical development of resection of the greater curvature with particular reference to its origin in ulcer and bariatric surgery . the contribution of ulcer surgery to modern sleeve gastrectomy is highlighted . furthermore , the current value of sleeve gastrectomy within the spectrum of bariatric surgical procedures will be discussed . relevant medical literature from pubmed to april 2010 was reviewed.discussionbesides bariatric surgery modern sleeve gastrectomy has one more so far largely neglected origin : segmental and later longitudinal gastric resection used in ulcer surgery . experience and achievements from ulcer surgery simplified and facilitated development of sleeve gastrectomy which is not the desired universal procedure for bariatric surgery but certainly an attractive treatment option . it should be performed in a more standardized manner and with due regard to future long - term results .

Introduction Discussion Introduction Discussion

initially introduced as a component of complex interventions and later as part of a two - stage operation in high - risk patients , the procedure is now more common as one - stage operation and subject of avid scientific discussion . this article aims to trace the historical development of resection of the greater curvature with particular reference to its origin in ulcer and bariatric surgery . furthermore , the current value of sleeve gastrectomy within the spectrum of bariatric surgical procedures will be discussed . besides bariatric surgery modern sleeve gastrectomy has one more so far largely neglected origin : segmental and later longitudinal gastric resection used in ulcer surgery . experience and achievements from ulcer surgery simplified and facilitated development of sleeve gastrectomy which is not the desired universal procedure for bariatric surgery but certainly an attractive treatment option . it should be performed in a more standardized manner and with due regard to future long - term results . it was initially used as a part of complex interventions ( including biliopancreatic diversion with duodenal switch ) and later as a two - step bridging procedure in high - risk patients prior to final intervention , but was then established as a stand - alone procedure and is currently a subject of avid scientific discussion . the aim of the present review is to trace the historical development of the current longitudinal gastric resection on the basis of its origins in ulcer and bariatric surgery and to elucidate the subject with suitable illustrations . development of longitudinal gastric resection in bariatric surgerya review of the essential steps in the historical development of bariatric surgery is helpful in order to understand how longitudinal gastric resection appeared as sleeve gastrectomy within the modern therapy options . however , many of these procedures were accompanied by serious side effects ( including diarrhea , hepatic cirrhosis , and electrolyte imbalance ) and did not prevail in the long term.2,39 development of longitudinal gastric resection in bariatric surgery a review of the essential steps in the historical development of bariatric surgery is helpful in order to understand how longitudinal gastric resection appeared as sleeve gastrectomy within the modern therapy options . the renaissance , and the enormously rapid and widespread application of this method as a single - step procedure , is quite understandable.80 introduced as a stepwise mode of treatment , the procedure reduced the previously high mortality rates in high - risk patients ( > 6% with a bmi > 60 , it was convincing because of its low complication ( about 9% ) and mortality rates ( < 1% ) , as well as its low rate of gastrointestinal long - term side effects.8183 some authors give preference to sleeve gastrectomy as opposed to a gastric balloon as part of a stepwise treatment regimen in high - risk patients.84 analogous to the concept of the magenstrasse and mill operation , gastric tube formation avoids malabsorption and implant - related complications while ensuring physiological gastric emptying.54,81 in contrast , sleeve gastrectomy involves irreversible resection of parts of the fundus and the corpus . in trials , sleeve gastrectomy was found to achieve a mean excess weight loss of 33% to 83% 1 year after surgery.54 despite this wide range , it may be assumed that , even in the mid - term , the procedure is associated with a similar marked reduction of weight as the usual procedures while reducing obesity - associated concomitant diseases.85,86 if additional weight reduction is required subsequently , the procedure can be performed in a two - step manner with a malabsorptive component ( gastric bypass or biliopancreatic diversion ) , either in a combined manner or a repeat sleeve gastrectomy can be conducted.87,88 therefore , one is easily inclined to regard tube gastrectomy as the desired all - round procedure . some authors express apprehensions about suture weakening , do not necessarily attribute the reduction of insufficiency rates to suture reinforcement , or warn against strictures due to oversewing.89,91 other authors recommend laparoscopic greater curvature plication in order to avoid gastric resection and associated complications.92 two factors deserve attention : firstly , a growing number of studies have been focused on the use of sleeve gastrectomy as a single - step procedure and report convincing results , although adequate evaluable long - term results ( > 5 years ) are not yet available.56,83,93 secondly , sleeve gastrectomy is not performed in a standardized manner : various tube diameters and calibration probes ( 32 to 60 french ) are used.68,80,94 besides , the extent of resection , particularly in respect of preservation / resection of the antrum varies.57,95 intraoperative measurement of the volume of the resected stomach is of great importance . rather , the key to successful treatment lies in a careful assessment of the individual risk jointly by the surgeon and the patient , as well as in providing intensive care and information before the operation and particularly in the long - term after a bariatric operation.5,96 eating habits , baseline weight , the anticipated weight loss , comorbidities , gender , age , and compliance are some of the numerous factors that must be taken into account.5,97 a team experienced in handling a wide spectrum of bariatric operations with confidence is indispensable to perform successful obesity surgery with sustained enhancement of quality of life and life expectancy.98 particularly sleeve gastrectomy should be viewed separately and not as a universal procedure . in view of the above - mentioned criteria , however , it should be performed in a more standardized manner and with due regard to future long - term results .

the impact of oral disturbances on individuals and communities are notable , particularly if the consequences on oral function and quality of life , as well as the societal costs , are taken into consideration.1 oral diseases affect systemic health and social functioning of the patients ; the pain , the negative esthetic impact , and the discomfort has important repercussions on public and private relationships.2,3 furthermore , recent studies have suggested that periodontitis could be considered as a risk factor for cognitive decline.4 several factors , such as nutritional habits , hygiene , tobacco and alcohol use , stress , and traumatic events exert a role in the occurrence of oral health problems.5 in light of the risk factors , the high incidence of oral disorders all over the world , particularly among disadvantaged populations , needs no explanation.6 oral health should be interpreted as an issue pertaining general health and its promotion should not be limited to the dentistry field . in everyday medical practice , while performing the general examination , proper attention must be given also to oral health ; this could facilitate the achievement of the ambitious goal proposed by the world health organization : the improvement of oral health status in the world.7 in addition , special attention should be dedicated to the most vulnerable patients , who tend to adopt bad habits and would benefit from simple oral hygiene advice . among the at - risk categories , patients suffering from mental illnesses are certainly in need of consideration . several studies have demonstrated the high incidence of periodontal problems among psychotic patients , probably because of self - care impairment , the side effects of many psychotropic medications , the difficult financial conditions , and the lack of motivation.8,9 the care of these patients should not be limited to the psychopathological aspects ; their unhealthy habits can not be passively accepted as a natural consequence of their psychiatric illness . moreover , the problem of poor oral hygiene is not limited to the patients suffering from severe mental disorders and presenting limited management skills , but pertains also to patients affected by mild psychiatric disturbances . although to date there are no systematic studies on the prevalence of depression in dental patients , an association between stress , depression , and chronic forms of periodontal disease has been documented.10 major depression in the general population has a lifetime prevalence ranging from 10% to 15%11 with an increased rate in females.12 patients with major depression ( dp ) are notoriously poorly dedicated to their oral hygiene and tend to follow a self - destructive lifestyle in terms of nutrition and voluptuary habits.13 this self - destructive behavior exposes dp to the occurrence of oral health problems that worsen their quality of life through the occurrence of psychological disturbances ; in fact , oral clefts , missing teeth , severe malocclusion , and severe caries are associated with feelings of embarrassment , withdrawal , anxiety , and insomnia3,14 moreover , depression is frequently accompanied by alexithymia , a personality trait involving difficulties in emotional regulation ( difficulties in identifying feelings , difficulties in describing feelings , and externally oriented thinking).15,16 alexithymia has been found to be related to a poor oral health - related quality of life and dental fear;17,18 the latter could lead to the avoidance of oral health services . the primary aim of our study was to evaluate the impact of depression and alexithymia on periodontal status . due to the influence of personality traits on behavior in general , the secondary aim of our study was to investigate the possible impact of personality disorders ( pds ) on dental status . dp ( outpatients ) referred to our psychiatry unit for consultation were consecutively enrolled in the study . healthy subjects ( controls ) with no neurological or psychiatric disorders were recruited from the general population ( relatives of university students , hospital employees , acquaintances of the research team ) during the period april 2012september 2012 . individuals suffering from cancer and acquired or congenital maxillofacial pathologies were excluded from the study . the study was approved by the local ethics committee of the university hospital policlinico - vittorio emanuele of catania and each individual signed a written informed consent . all the enrolled subjects filled in a questionnaire pertaining to demographic data , voluptuary habits ( tobacco smoking , coffee , alcohol , and drugs abuse ) , oral hygiene ( frequency of teeth - brushing , type of toothbrush , use of mouthwash , use of flossing , etc ) , use of partial dentures or being edentulous . smokers were divided into three groups according to the number of cigarettes smoked per day , according to previous studies : mild ( less than 15 cigarettes / day ) , moderate ( from 15 to 24 cigarettes / day ) , and heavy ( 25 or more cigarettes / day ) smokers.19 drinkers were divided into three groups according to the quantity of alcohol consumption : mild ( from 1 to 14.9 g / day ) , moderate ( from 15 to 29.9 g / day ) , and heavy drinkers ( 30 or more g / day).20 the presence of diabetes was recorded . as for dp , the presence of previous depressive episodes and suicidal ideation during the current episode were recorded . all the participants underwent a psychiatric evaluation through the following questionnaires : 1 ) the hamilton rating scale for depression : it is a reliable tool consisting of 21 items for the assessment of depression . the severity of depression is evaluated as follows : 25 severe depression ; 1824 moderate depression ; 817 mild depression ; 7 absence of depression.21,22 as to controls , the test was administered in order to exclude the presence of depressive symptoms . 2 ) the hamilton rating scale for anxiety : it is a validated and reliable tool consisting of 14 items for the assessment of anxiety . a total score 18 was considered pathological.23,24 3 ) the 20-item toronto alexithymia scale : it is a reliable instrument consisting of three subscales assessing alexithymia : subscale i assessing the difficulty in identifying feelings ; subscale ii assessing the difficulty in describing feelings ; subscale iii assessing externally oriented thinking and the lack of introspective capacities . scores between 51 and 60 indicate a condition of borderline alexithymia.25,26 4 ) the structured clinical interview for dsm - iv axis ii personality disorders : it is a validated scale investigating the presence of pds . it considers the eleven dsm - iv pds ( including pd not otherwise specified ) and the appendix categories depressive pd and passive - aggressive pd.27,28 5 ) the oral health impact profile ( ohip-14 ) : it is a validated scale 29 assessing the burden of oral health status on the quality of life through 14 items . the higher the ohip-14 total score , the worse the oral health - related quality of life is.30,31 then , all the participants ( except the fully edentulous ones ) underwent a dental examination performed by a dentist evaluating six selected teeth ( maxillary right first molar , maxillary right lateral incisor , maxillary left first bicuspid , mandibular left first molar , mandibular left lateral incisor , mandibular right first bicuspid ) . the following indexes have been calculated : 1 ) plaque index ( pi ) : it is a validated index designed to estimate the presence of plaque.32 the total score for each tooth ranges from 0 to 3 . the scores of each tooth are then added together and the value is divided by six in order to obtain the score of the patient . four ratings have been assigned to each patient : 0 excellent ; from 0.1 to 0.9 good ; from 1.0 to 1.9 fair , from 2.0 to 3.0 poor.33 2 ) gingival index ( gi ) : it is a validated and reliable index34 designed to estimate different degrees of inflammation in the marginal gingiva . the total score ranges from 0 to 3 , according to the severity of the inflammation . the scores of each tooth are added together and the value is divided by six in order to obtain the score of the patient . four ratings have been assigned to each patient : 0 excellent ; from 0.1 to 1.0 good ; from 1.1 to 2.0 fair ; from 2.1 to 3.0 poor.35 3 ) simplified oral hygiene index ( ohi - s ) : it is a good instrument that evaluates the level of oral hygiene . the total score is the sum of the debris index ( that ranges from 0 to 3 ) and the calculus index ( that ranges from 0 to 3 ) . the following ratings have been assigned to each patient : from 0 to 1.2 good , from 1.3 to 3.0 fair , from 3.1 to 6.0 poor.36 4 ) periodontal index ( pei ) : it is a good index intended to estimate deeper periodontal disease by investigating the presence of gingival inflammation and its severity , the pocket formation , and masticatory function . the total score ranges from 0 to 8 as follows : from 0 to 0.2 normal ( no inflammation ) , from 0.3 to 0.9 simple gingivitis ( inflammation , not circumscribing the tooth ) , from 1.0 to 1.9 periodontal disease at early stage ( inflammation circumscribing the tooth ) , from 2.0 to 4.9 mild periodontal disease ( gingivitis with pocket formation ) , from 5.0 to 8.0 severe periodontal disease ( tissue destruction with loss of function ) . five ratings have been assigned to each patient : excellent ( 00.2 ) , good ( 0.30.9 ) , fair ( 1.01.9 ) , poor ( 2.04.9 ) , very poor ( 5.08.0).37 all data analyses were generated using the statistical package for social sciences for windows ( version 13.0 for windows ; spss inc . , the difference between means and the difference between proportions was evaluated by the t - test and the chi - square test respectively . in case of not a normal distribution , appropriate nonparametric tests were performed . only values of p<0.05 were considered statistically significant . a multivariate approach was then adopted in order to investigate the independent effect of a risk or protective factor after adjustment for the parameters significantly associated with the outcome at the univariate analysis . unconditional logistic regression analysis was performed and we calculated odds ratio ( or ) , 95% confidence interval ( ci ) , and p - value ( two - tailed test , =0.05 ) for each variable . all data analyses were generated using the statistical package for social sciences for windows ( version 13.0 for windows ; spss inc . , chicago , il , usa ) . the difference between means and the difference between proportions was evaluated by the t - test and the chi - square test respectively . in case of not a normal distribution , appropriate nonparametric tests were performed . only values of p<0.05 were considered statistically significant . a multivariate approach was then adopted in order to investigate the independent effect of a risk or protective factor after adjustment for the parameters significantly associated with the outcome at the univariate analysis . unconditional logistic regression analysis was performed and we calculated odds ratio ( or ) , 95% confidence interval ( ci ) , and p - value ( two - tailed test , =0.05 ) for each variable . then , 50 dp ( 15 men and 35 women ; aged 56.715.2 years ) and 40 controls ( 16 men and 24 women ; aged 56.115.9 years ) were enrolled in the study . no significant differences in age , sex , and years of schooling ( 9.83.6 years among dp versus 9.44.4 years among controls ) were recorded when comparing dp and controls . no significant differences in terms of voluptuary habits and diabetes were found between the two groups . fourteen percent of the dp presented mild depression , 38% moderate depression , and 48% severe depression . seventy - two percent of the dp presented anxiety versus 17.5% of the controls ( or 12.12 , 95% ci 4.3533.71 ; p<0.001 ) . in addition , 58% of the dp were alexithymic versus 7.5% of the controls ( or 35.04 , 95% ci 8.4145.44 ; p<0.001 ) . twenty - six percent of dp presented a condition of borderline alexithymia versus 22.5% of the controls ( or 5.89 , 95% ci 1.8119.13 ; p<0.001 ) . out of the 50 interviewed dp , 43 presented at least one pd at the structured clinical interview for dsm - iv axis ii personality disorders , while only 14 controls were affected by at least one pd . the frequency of pds among dp was as follows : absence of pd , seven ( 14% ) ; borderline pd , seven ( 14% ) ; histrionic pd , four ( 8% ) ; narcissistic pd , one ( 2% ) ; obsessive - compulsive pd , 12 ( 24% ) ; paranoid pd , three ( 6% ) ; avoidant pd , four ( 8% ) ; depressive pd , two ( 4% ) ; dependent pd , two ( 4% ) ; passive - aggressive pd , zero ( 0% ) , not otherwise specified ( intending more than one pd ) pd , eight ( 16% ) . the frequency of pds among controls was as follows : absence of pd , 26 ( 65% ) ; borderline pd , zero ( 0% ) ; histrionic pd , two ( 5% ) ; narcissistic pd , two ( 5% ) ; obsessive - compulsive pd , three ( 7.5% ) ; paranoid pd , three ( 7.5% ) ; avoidant pd , one ( 2.5% ) ; depressive pd , zero ( 0% ) ; dependent pd , zero ( 0% ) ; passive - aggressive pd , one ( 2.5% ) ; not otherwise specified pd , two ( 5% ) . the only pd that presented a statistically significant difference between dp and controls was the obsessive - compulsive pd , recorded in 12 ( 24% ) dp and in three ( 7.5% ) controls ( or 14.85 , 95% ci 3.2667.63 , p<0.001 ) . for more details no significant differences in terms of frequency of teeth - brushing , type of toothbrush , use of mouthwash , flossing , use of partial dentures , being edentulous or not , were recorded when comparing dp and controls . on the contrary , significant differences were found when considering pi : 22% of dp versus 47.5% of controls presented a fair pi ( or 13.93 , 95% ci 2.7769.88 , p=0.001 ) ; 38% of dp versus 5% of controls had a poor pi ( or 36.1 , 95% ci 6.21209.5 , p=0.001 ) . in addition , 44% of dp versus 12.5% of controls presented a poor ohi - s ( or 4.4 , 95% ci 1.3114.7 , p=0.01 ) . thirty - four percent of dp presented a fair / poor gi versus 22.5% of controls ( or 3.1 , 95% ci 1.019.33 , p=0.04 ) . also , the pei score was significantly different between the two groups : 42% of dp versus 20% of controls presented a fair / poor pei ( or 4.43 , 95% ci 1.4213.75 , p=0.009 ) . as to the ohip-14 , the dp presented a mean score of 17.313.6 versus the 3.95.3 registered among the controls ( or 1.16 , 95% ci 1.081.25 , p<0.001 ) . for more details , see table 1 . a multivariate approach was adopted in order to investigate the independent effect of depression on periodontal status after adjustment for the parameters significantly associated with the outcome at the univariate analysis . since few individuals were heavy smokers , a single category was created ( moderate / heavy ) . the unconditional logistic regression performed for each dental index ( ohip-14 , pi , gi , ohi - s , and pei ) adjusting for age , sex , smoking , alexithymia , anxiety , and pd confirmed only the strength of the association between depression and ohip-14 found at the univariate analysis ( adjusted or 1.19 , 95% ci 1.061.34 , p=0.002 ) . when considering the severity of depression , no significant differences in terms of ohip-14 were found comparing the three levels ( mild , moderate , and severe ) . on the contrary , there were significant differences pertaining to the frequency of excellent pi ( 57.1% in dp suffering from mild depression versus 0% in dp with moderate depression versus 4.2% in dp with severe depression ; p<0.05 ) , excellent gi ( 85.7% in mild depression versus 21.1% in moderate depression versus 20.8% in severe depression ; p<0.05 ) , good ohi - s ( 85.7% in mild depression versus 26% in moderate depression versus 16.7% in severe depression ; p<0.05 ) and excellent pei ( 85.7% in mild depression versus 15.8% in moderate depression and 25% in severe depression ; p<0.05 ) . for more details , see table 2 . with regards to the correlations between periodontal status and alexithymia , 55.2% of dp with alexithymia presented a poor pi score versus 12.5% of dp without alexithymia and 15.4% of dp with borderline alexithymia ( p<0.05 ) . a significant difference was also found when considering pei : only 17.2% of dp with alexithymia presented an excellent pei , versus 53.8% of dp with borderline alexithymia and 37.5% of dp without alexithymia ( p<0.05 ) . no other significant differences were found when comparing the three groups ( dp with / without alexithymia , dp with borderline alexithymia ) . for more details , see table 3 . no correlations between status and the other variables evaluated in this study ( eg , anxiety and personality ) were found . then , 50 dp ( 15 men and 35 women ; aged 56.715.2 years ) and 40 controls ( 16 men and 24 women ; aged 56.115.9 years ) were enrolled in the study . no significant differences in age , sex , and years of schooling ( 9.83.6 years among dp versus 9.44.4 years among controls ) were recorded when comparing dp and controls . no significant differences in terms of voluptuary habits and diabetes were found between the two groups . fourteen percent of the dp presented mild depression , 38% moderate depression , and 48% severe depression . seventy - two percent of the dp presented anxiety versus 17.5% of the controls ( or 12.12 , 95% ci 4.3533.71 ; p<0.001 ) . in addition , 58% of the dp were alexithymic versus 7.5% of the controls ( or 35.04 , 95% ci 8.4145.44 ; p<0.001 ) . twenty - six percent of dp presented a condition of borderline alexithymia versus 22.5% of the controls ( or 5.89 , 95% ci 1.8119.13 ; p<0.001 ) . out of the 50 interviewed dp , 43 presented at least one pd at the structured clinical interview for dsm - iv axis ii personality disorders , while only 14 controls were affected by at least one pd . the frequency of pds among dp was as follows : absence of pd , seven ( 14% ) ; borderline pd , seven ( 14% ) ; histrionic pd , four ( 8% ) ; narcissistic pd , one ( 2% ) ; obsessive - compulsive pd , 12 ( 24% ) ; paranoid pd , three ( 6% ) ; avoidant pd , four ( 8% ) ; depressive pd , two ( 4% ) ; dependent pd , two ( 4% ) ; passive - aggressive pd , zero ( 0% ) , not otherwise specified ( intending more than one pd ) pd , eight ( 16% ) . the frequency of pds among controls was as follows : absence of pd , 26 ( 65% ) ; borderline pd , zero ( 0% ) ; histrionic pd , two ( 5% ) ; narcissistic pd , two ( 5% ) ; obsessive - compulsive pd , three ( 7.5% ) ; paranoid pd , three ( 7.5% ) ; avoidant pd , one ( 2.5% ) ; depressive pd , zero ( 0% ) ; dependent pd , zero ( 0% ) ; passive - aggressive pd , one ( 2.5% ) ; not otherwise specified pd , two ( 5% ) . the only pd that presented a statistically significant difference between dp and controls was the obsessive - compulsive pd , recorded in 12 ( 24% ) dp and in three ( 7.5% ) controls ( or 14.85 , 95% ci 3.2667.63 , p<0.001 ) . for more details as to periodontal status , no significant differences in terms of frequency of teeth - brushing , type of toothbrush , use of mouthwash , flossing , use of partial dentures , being edentulous or not , were recorded when comparing dp and controls . on the contrary , significant differences were found when considering pi : 22% of dp versus 47.5% of controls presented a fair pi ( or 13.93 , 95% ci 2.7769.88 , p=0.001 ) ; 38% of dp versus 5% of controls had a poor pi ( or 36.1 , 95% ci 6.21209.5 , p=0.001 ) . in addition , 44% of dp versus 12.5% of controls presented a poor ohi - s ( or 4.4 , 95% ci 1.3114.7 , p=0.01 ) . thirty - four percent of dp presented a fair / poor gi versus 22.5% of controls ( or 3.1 , 95% ci 1.019.33 , p=0.04 ) . also , the pei score was significantly different between the two groups : 42% of dp versus 20% of controls presented a fair / poor pei ( or 4.43 , 95% ci 1.4213.75 , p=0.009 ) . as to the ohip-14 , the dp presented a mean score of 17.313.6 versus the 3.95.3 registered among the controls ( or 1.16 , 95% ci 1.081.25 , p<0.001 ) . for more details , see table 1 . a multivariate approach was adopted in order to investigate the independent effect of depression on periodontal status after adjustment for the parameters significantly associated with the outcome at the univariate analysis . since few individuals were heavy smokers , a single category was created ( moderate / heavy ) . the unconditional logistic regression performed for each dental index ( ohip-14 , pi , gi , ohi - s , and pei ) adjusting for age , sex , smoking , alexithymia , anxiety , and pd confirmed only the strength of the association between depression and ohip-14 found at the univariate analysis ( adjusted or 1.19 , 95% ci 1.061.34 , p=0.002 ) . when considering the severity of depression , no significant differences in terms of ohip-14 were found comparing the three levels ( mild , moderate , and severe ) . on the contrary , there were significant differences pertaining to the frequency of excellent pi ( 57.1% in dp suffering from mild depression versus 0% in dp with moderate depression versus 4.2% in dp with severe depression ; p<0.05 ) , excellent gi ( 85.7% in mild depression versus 21.1% in moderate depression versus 20.8% in severe depression ; p<0.05 ) , good ohi - s ( 85.7% in mild depression versus 26% in moderate depression versus 16.7% in severe depression ; p<0.05 ) and excellent pei ( 85.7% in mild depression versus 15.8% in moderate depression and 25% in severe depression ; p<0.05 ) . for more details , see table 2 . with regards to the correlations between periodontal status and alexithymia , 55.2% of dp with alexithymia presented a poor pi score versus 12.5% of dp without alexithymia and 15.4% of dp with borderline alexithymia ( p<0.05 ) . a significant difference was also found when considering pei : only 17.2% of dp with alexithymia presented an excellent pei , versus 53.8% of dp with borderline alexithymia and 37.5% of dp without alexithymia ( p<0.05 ) . no other significant differences were found when comparing the three groups ( dp with / without alexithymia , dp with borderline alexithymia ) . for more details , see table 3 . no correlations between status and the other variables evaluated in this study ( eg , anxiety and personality ) were found . poor oral health is a silent epidemic with considerable consequences on the quality of life.38 previous studies demonstrated poorer oral health in patients with mental disorders.39,40 our results support these literature data . in our study there were no significant differences in terms of age , voluptuary habits , and hygiene between dp and controls ; however , when comparing the two groups , statistically significant differences pertaining to oral health were found . in particular , dp presented a statistically significant worse oral condition in terms of : ohip-14 , pi , gi , pei , and ohi - s . some studies reported that depression was significantly associated with periodontal disease , as measured by clinical attachment loss or alveolar bone loss and with toothless and , in general , with a nonuse of oral health services.11,41,42 psychiatrists and dentists must strongly fight against this maladaptive habit . from an interdisciplinary point of view , patients should be referred to the centers for mental / dental health . the elevated frequency of periodontal problems among dp may be due , in part , to antidepressant therapy , apathy or , more generally , to the fact that people with mental illness are more likely to cancel or not attend appointments with dentists and/or oral hygienists.43 moreover , it is well - known that psychopharmacological treatments reduce the salivary flow rate that could contribute to the development of periodontal diseases.44 in addition , the severe impairment in self - care described in depression and anxiety can lead to oral health problems;13 however , in our sample , other variables than behavioral issues seem to determine a worse periodontal status among dp , since the two examined groups ( dp and controls ) presented similar hygienic habits . apart from hygiene , the association between depressive symptoms and higher lactobacillus counts or the association between depression and inflammation could offer an explanation of our findings.45,46 inflammation plays a crucial role in the development of oral pathology too , so that polymorphisms of the genes involved in the immune response can lead to a higher susceptibility to periodontitis.47 in particular , the cyclooxygenase , that converts arachidonic acid to prostaglandins , exerts a fundamental role in the inflammatory process involved in the tissue destruction occurring in periodontitis.4850 the same enzymatic way seems to be over - expressed in major depression . dp without other medical illnesses , in fact , present an increase of inflammatory markers both in serum and in the central nervous system.51 hence , beyond the psychological explanation of the association between poor oral health and depression , there are biological factors that may contribute to the development of oral health problems among dp . the association between oral and mental health should not be underestimated , also considering that poor oral hygiene represents a risk factor for cardiovascular and kidney diseases , pulmonary infections , rheumatoid arthritis , and diabetes.5256 generally speaking , the link between oral and general health is well - known in literature.57 another interesting finding of this study pertains to the association between the severity of depression and periodontal health : dp with mild depression were more likely to present better dental index scores . more specifically , only few dp with severe depression presented an excellent pi , gi , pei score , and a good ohi - s score . compared to other studies , this paper highlights not only the link between affective symptoms and periodontal pathologies in general but also the importance of the severity of depression in terms of oral health outcomes . in addition , the analysis of our sample demonstrated a link between alexithymia and periodontal health : a considerable number of alexithymic patients presented , in fact , a poor pi and a fair pei . even in this case , inflammation could offer an explanation of the finding : the altered immune response found in depression has been demonstrated also in alexithymia . in particular , inflammatory markers such as high - sensitivity c reactive protein and interleukin-6 were found to be significantly higher among alexithymic patients than in non - alexithymic ones.58 along with major depression , alexithymia and personality disturbances seem to exert a role on the patient s periodontal health : in our sample , the strength of the association between depression and dental indexes was reduced by adjusting for these psychiatric variables . in fact , only the association between oral - health related quality of life ( ohip-14 ) and depression was confirmed and was not influenced by the presence of alexithymia and pds . to the best of our knowledge , this is the first study examining the relationship between the severity of depression , alexithymia , and periodontal health . firstly , we did not use x - ray imaging , but a physical ( dental ) examination for the detection of oral health problems . secondly , we did not consider the antidepressant therapy as a variable when evaluating the patient s periodontal status . the relationship between depression and poor periodontal health may , in fact , be bidirectional : depression may affect oral health , behaviors , and quality of life but , conversely , people with dental problems may have a lower self - esteem and self - confidence and these factors may lead to depression or worsen a current depressive episode . although the present study can not ascertain causal association , it provides substantial evidence that oral and mental health are strictly linked . in particular , our study demonstrated a correlation between psychiatric variables and poor periodontal health . longitudinal studies are necessary to clarify , in particular , the impact of depression and alexithymia on periodontal status . as a matter of fact , due to the high frequency of dental problems , patients suffering from mental illnesses should be referred to the oral health services for evaluation . hence , in everyday clinical practice , the evaluation of the patient should imply special attention to general health .

backgroundseveral studies have demonstrated the high incidence of periodontal disorders among patients suffering from mental illnesses , probably because of self - care impairment , the difficult financial conditions , and the lack of motivation . the primary aim of this study was to evaluate the impact of depression and alexithymia on periodontal status . due to the influence of personality traits on behavior in general , the secondary aim of our study was to investigate the possible impact of personality disorders on dental status.methodspatients with major depression ( dp ) referred to our psychiatry unit and healthy individuals ( controls ) were consecutively enrolled during the period april 2012september 2012 . all the participants to the study underwent a psychiatric evaluation ( through questionnaires investigating the presence of depression , anxiety , personality disorders , and alexithymia ) and a dental examination through the following indexes : plaque index , gingival index , simplified oral hygiene index , periodontal index.resultsfifty dp ( aged 56.715.2 years ) and 40 controls ( aged 56.115.9 years ) were enrolled in the study . dp showed a worse oral hygiene status . in particular , statistically significant differences were found when comparing dp and controls in terms of plaque index , simplified oral hygiene index , gingival index , periodontal index , and oral health impact profile . in addition , periodontal health was found to be negatively related to the severity of depression and the presence of alexithymia . the strength of association between depression and dental indexes was reduced after adjusting for the other psychiatric variables ( alexithymia and personality disorders ) and was confirmed only for oral health impact profile.conclusionpsychiatric variables seem to affect the patients periodontal status ; due to the high frequency of dental problems , patients suffering from mental illnesses should be referred to the oral health services for evaluation .

Background Methods Statistical analysis Results General characteristics of the sample Periodontal status: DP versus controls Multivariate approach Periodontal status and severity of depression Periodontal status and alexithymia Discussion Conclusion

the impact of oral disturbances on individuals and communities are notable , particularly if the consequences on oral function and quality of life , as well as the societal costs , are taken into consideration.1 oral diseases affect systemic health and social functioning of the patients ; the pain , the negative esthetic impact , and the discomfort has important repercussions on public and private relationships.2,3 furthermore , recent studies have suggested that periodontitis could be considered as a risk factor for cognitive decline.4 several factors , such as nutritional habits , hygiene , tobacco and alcohol use , stress , and traumatic events exert a role in the occurrence of oral health problems.5 in light of the risk factors , the high incidence of oral disorders all over the world , particularly among disadvantaged populations , needs no explanation.6 oral health should be interpreted as an issue pertaining general health and its promotion should not be limited to the dentistry field . several studies have demonstrated the high incidence of periodontal problems among psychotic patients , probably because of self - care impairment , the side effects of many psychotropic medications , the difficult financial conditions , and the lack of motivation.8,9 the care of these patients should not be limited to the psychopathological aspects ; their unhealthy habits can not be passively accepted as a natural consequence of their psychiatric illness . although to date there are no systematic studies on the prevalence of depression in dental patients , an association between stress , depression , and chronic forms of periodontal disease has been documented.10 major depression in the general population has a lifetime prevalence ranging from 10% to 15%11 with an increased rate in females.12 patients with major depression ( dp ) are notoriously poorly dedicated to their oral hygiene and tend to follow a self - destructive lifestyle in terms of nutrition and voluptuary habits.13 this self - destructive behavior exposes dp to the occurrence of oral health problems that worsen their quality of life through the occurrence of psychological disturbances ; in fact , oral clefts , missing teeth , severe malocclusion , and severe caries are associated with feelings of embarrassment , withdrawal , anxiety , and insomnia3,14 moreover , depression is frequently accompanied by alexithymia , a personality trait involving difficulties in emotional regulation ( difficulties in identifying feelings , difficulties in describing feelings , and externally oriented thinking).15,16 alexithymia has been found to be related to a poor oral health - related quality of life and dental fear;17,18 the latter could lead to the avoidance of oral health services . the primary aim of our study was to evaluate the impact of depression and alexithymia on periodontal status . due to the influence of personality traits on behavior in general , the secondary aim of our study was to investigate the possible impact of personality disorders ( pds ) on dental status . then , 50 dp ( 15 men and 35 women ; aged 56.715.2 years ) and 40 controls ( 16 men and 24 women ; aged 56.115.9 years ) were enrolled in the study . then , 50 dp ( 15 men and 35 women ; aged 56.715.2 years ) and 40 controls ( 16 men and 24 women ; aged 56.115.9 years ) were enrolled in the study . the elevated frequency of periodontal problems among dp may be due , in part , to antidepressant therapy , apathy or , more generally , to the fact that people with mental illness are more likely to cancel or not attend appointments with dentists and/or oral hygienists.43 moreover , it is well - known that psychopharmacological treatments reduce the salivary flow rate that could contribute to the development of periodontal diseases.44 in addition , the severe impairment in self - care described in depression and anxiety can lead to oral health problems;13 however , in our sample , other variables than behavioral issues seem to determine a worse periodontal status among dp , since the two examined groups ( dp and controls ) presented similar hygienic habits . the association between oral and mental health should not be underestimated , also considering that poor oral hygiene represents a risk factor for cardiovascular and kidney diseases , pulmonary infections , rheumatoid arthritis , and diabetes.5256 generally speaking , the link between oral and general health is well - known in literature.57 another interesting finding of this study pertains to the association between the severity of depression and periodontal health : dp with mild depression were more likely to present better dental index scores . in particular , inflammatory markers such as high - sensitivity c reactive protein and interleukin-6 were found to be significantly higher among alexithymic patients than in non - alexithymic ones.58 along with major depression , alexithymia and personality disturbances seem to exert a role on the patient s periodontal health : in our sample , the strength of the association between depression and dental indexes was reduced by adjusting for these psychiatric variables . as a matter of fact , due to the high frequency of dental problems , patients suffering from mental illnesses should be referred to the oral health services for evaluation .

human papillomavirus ( hpv ) is the most common sexually transmitted virus , infecting 75% of the sexually active canadian population [ 1 , 2 ] . it can cause cancer of the cervix , vulva , oropharynx , penis , and anus , as well as genital warts [ 35 ] . until recently , there were no medical interventions against hpv infections except monitoring the virus 's progress through the papanicolaou test ( or pap test ) and treating the ailments that developed [ 6 , 7 ] . one ( cervarix ) protects against types 16 and 18 while the other ( gardasil ) also protects against hpv types 6 and 11 . these vaccines offer protection for at least eight years . in 2007 , the canadian federal government provided the provinces and territories with $ 300 million to spend over 3 years for hpv immunization programs . by 2009 , every province and territory in canada had an hpv vaccination program in place . the creation and implementation of the programs are the responsibility of the individual provinces and territories . this results in eight distinct strategies throughout canada , which can be seen in table 1 . these strategies vary in the number of doses of the quadrivalent vaccine given ( only two doses in british columbia and quebec , with three doses administered elsewhere ) , the grade of the girls who are given the vaccine ( ranging from 4 to 10 ) , and the coverage rate ( 4986% within the first two years of the program ) . although the implementation of these programs differs between provinces , they all share a common goal of reducing the impact of hpv on the canadian population . this is measured through the morbidity and mortality rates of hpv . since the vaccine 's introduction , there have been a number of mathematical models assessing its impact on several populations . in terms of the cost - effectiveness of the vaccine , brown and white used an optimal control model for vaccinating adolescent females and males in the united kingdom . brisson et al . used a cohort model to estimate how many girls need to be vaccinated to prevent cervical cancer and genital warts in canada . in terms of the disease impact on a population , barnabas et al . developed a transmission model to measure the impact of vaccinating against hpv-16 in finland for both women and men . although it does consider the effects caused by hpv later in life , this model neglects to consider herd immunity . there are current clinical trials in british columbia looking at the effect of taking two or three doses of the quadrivalent vaccine . this will provide valuable information about the levels of protection provided by two or three doses . llamazares and smith ? developed an epidemiological model that includes a widespread childhood vaccination program with voluntary adult vaccination . the model is used as a preliminary investigation to determine whether provincial healthcare programs in canada should fund adult hpv vaccination . the authors determined a threshold of eradication for the targeted types of hpv and critical efficacy and immunogenicity levels such that eradication is not possible at any coverage rate . here we develop a mathematical model to explore the current vaccination strategies across canada as well as potential alternative strategies . these strategies are defined by the number of doses given , the grade of the girls the vaccine is given to , and the coverage rate achieved . our model will provide similar insight into the effectiveness of two and three doses of the quadrivalent vaccine . this model also provides insight into what the programs should look like in order to achieve a desired outcome . this model provides a unique perspective in its evaluation of the provincial programs based on the grade of vaccination , number of doses given , and the necessary coverage rate . we address the following research questions : ( 1 ) does the grade at which the girls are vaccinated significantly affect the outcome of the program ? ( 2 ) what coverage rate must the provinces reach in order to reduce the impact of hpv on the canadian population ? ( 3 ) what are the implications of vaccinating with two versus three doses ? this paper is organized as follows . section 4 gives the expressions for critical thresholds of efficacy and probability of protection . section 5 explores how the parameters were estimated , the results on the infection when varying the grade , dosage and coverage rate , and the sensitivity analysis of the model . our model is composed of 19 equations , 13 that describe the childhood vaccination strategy and six that describe the disease propagation through adults . the female children are broken up into seven grades ( 4 through 10 ) . within each appropriate grade class , there are unvaccinated ( c iu , where 4 i 10 ) and vaccinated female children ( c iv , where 4 i 10 ) . in the adult portion of the model , women are broken up into unvaccinated uninfected susceptible women ( a u ) , vaccinated uninfected susceptible women ( a v ) , unvaccinated and infected women ( i u ) , or vaccinated infected women ( i v ) . girls in grade 4 ( approximately 9 years old ) are described as ( 2)dc4dt=w1+cc4.for girls in grade i , where 5 i 10 , we have ( 3)dc(i+1)udt=1piciu1+cci+1u , dc(i+1)vdt=piciu+civ1+cc(i+1)v.uninfected adult women are described as ( 4)daudt=(1u)c10u+uiuf(wpw)au waunaau+av , davdt=(1v)c10v+viv+f(wpw)au (1)wavnaavav.infected adult women are described as ( 5)diudt=uc10u+waunuiuaiu+iv , divdt=vc10v+(1)wavnvivaiviv.uninfected men are described as ( 6)dmdt=mmiummivm+mnam.infected men are described as ( 7)dndt=mium+mivmmnan . a girl enters the model unvaccinated in grade 4 at a constant rate w. at some grade between 4 and 10 , a proportion of the girls become vaccinated at rate p i ( where p i is the proportion of girls given the vaccine in grade i and represents the vaccine efficacy for girls ) . when the girls enter grade 11 , a proportion of unvaccinated girls ( 1 u ) grow up to become uninfected unvaccinated women ( a u ) , while the remaining unvaccinated girls ( u ) are categorized as infected unvaccinated women ( i u ) to account for early childhood sexual activity . a proportion of vaccinated girls ( 1 v ) grow up to become uninfected vaccinated women ( a v ) , while the rest of the vaccinated girls ( v ) are categorized as infected vaccinated women ( i v ) to account for a proportion of girls who have contracted at least one of the targeted types before receiving the vaccine . once in the adult stage ( grade 11 to around the age of 26 ) unvaccinated adult women ( a u ) can become vaccinated at rate f( w p w ) , where w is the efficacy of the vaccine for adult women and p w is the proportion of women who get the vaccine . the vaccine wanes at rate . unvaccinated adult women become infected ( i u ) at rate w when coming into contact with an infected man ( n ) . vaccinated adult women ( a v ) can also become infected ( i v ) at rate ( 1 ) w , where represents the ability of the vaccine to protect against all targeted types . men enter the model as susceptible ( m ) through a constant rate m and stay in the model for approximately 10 years . unvaccinated susceptible men ( m ) can become infected after sexual activity with an infected woman ( i u or i v ) with transmission rate m. infection clears at rate u for unvaccinated women , at rate v for vaccinated women , and m for men . each compartment also includes a natural leaving rate , c or a , depending on child or adult status . parameter descriptions , ranges , and sample values can be found in table 2 . for this model , it is assumed that hpv is only heterosexually transmitted . female children are in grades 4 through 10 , which are the years childhood vaccination can take place . it is assumed that vaccination only occurs in one year , that male vaccination is negligible , and that the proportion of female children vaccinated during other years is negligible . the rate at which the hpv infection is cleared is independent of previous infection status . the transmission from women to men is higher than the transmission from men to women . both women and men are active in the adult model for approximately 10 years because the vaccine is not recommended for women over the age of 26 . we keep men in the model for the same length of time as women , on the grounds that , while there may be an age difference between young women and older men , such men are aging out with their sexual cohorts . that is , they may choose new partners within this cohort ( e.g. , friends of existing partners ) , but we assume they do not revert to an even younger cohort after such a cohort ages out . ( however , we have explored this assumption in more detail elsewhere and shown that relaxing it has a negligible effect on the epidemic , so it is only included here for analytical convenience . ) the assumptions made about the vaccine are that the vaccine does not wane in children , that it may not protect 100% ( this is based on the probability of protection and the efficacy ) , and that the vaccine does not protect someone who is already infected with the virus [ 32 , 33 ] . the difference between a two- and three - dose schedule is the efficacy of the vaccine . lastly , we do not consider disease - induced death since it does not play a role in removing sexually active individuals from the pool that we are considering ( complications due to hpv , such as cervical cancer , are generally seen much later in life ) [ 7 , 34 , 35 ] . the disease - free equilibrium is(8)c4,c5u,c5v,c6u,c6v,c7u,c7v,c8u,c8v,c9u,c9v , c10u,c10v,au,av,iu,iv,m,n,where ( 9)c4=w1+c , and we define c4u=c4 , c4v=0.then , for 5 i 10 , we have ( 10)ciu=1pi1c(i1)u1+c , civ=p(i1)c(i1)u+c(i1)v1+c , au=1uc10ufw pw+a , av=fw pwau+1vc10va , iu=0 , iv=0,m=ma , n=0 . the jacobian matrix for this model evaluated at the disease - free equilibrium is j dfe = [ j dfe | j dfe | j dfe ] , where(11)the characteristic polynomial of the jacobian is ( 12)detji=1c13adethdetl , where ( 13)h=fafa,l=uawau0va(1)wavmmmmma. solving det(h ) = 0 , we get(14)2+f+2a++af+a+=0,which has only eigenvalues with negative real part . solving det(l ) = 0 , we get ( 15)3+2++=0,where ( noting that m= ) ( 16)=3a+u+v+m+,=3a2+uv++um+v+m + 2au+v+m+1wm av wmau,=a3+a2u+v+m+ + auv++um+v+m + u(v+)m 1wm ava+u+ wmaua+v+. in order to determine the stability of the disease - free equilibrium in the linearized system , we use the routh hurwitz stability criterion . for our cubic characteristic polynomial ( 15 ) , we have four routh hurwitz conditions that must all be satisfied in order for the disease - free equilibrium to be locally stable:(1 ) > 0;(2 ) > 0;(3 ) > 0;(4 ) > . the first condition is satisfied , since all of the parameters are positive . the second condition is our threshold . if < 0 , then we are guaranteed to have at least one positive real root of the characteristic polynomial . if = 0 , then we have a nonhyperbolic fixed point and must use stability theory to determine the stability of the disease - free equilibrium . we determine stability for the case when = 0 = > 0 ( where is a small positive perturbation ) . we will show that > 0 and > 0 in order to satisfy the routh hurwitz criterion . we find ( 18)(1)wmav = 1a+u+a3+a2+a = 1a+u+wmaua+v+ = 1a+u++ uv+ma3 . substituting this into , we have ( 19)=3a2 + 2a+ 1a+u+ a3+a2+ awmaua+v+ + u(v+)mwmauwmau. to require > 0 , we need ( 20)(a+u+)3a2 + 2a+ + wmaua+v++ > a3+a2+ a+wmaua+u+ + uv+m,3a3 + 2a2+ a+3a2u+2au+ u+3a2 + 2a+ +(a+v+)wmau+ > a3+a2+ a+(a+u+)wmau + uv+m,2a3+a2(+3u+3)+2au+ uu(v+)m + 2a+ +(vu)wmau+>0.note that ( 21)uuv+m = uuv++um+v+m uvmum>0.it follows that > 0 if ( 22)vu , that is , if the recovery rate from infection is faster for vaccinated individuals ( or at least not worse ) , which we expect to be the case . when = 0 , the routh hurwitz conditions are satisfied , all roots have a negative real part , and our system is stable at the disease - free equilibrium . since > 0 and > 0 when = 0 , then = 0 is our threshold of stability . we used the product = w m to numerically examine the routh and . figure 2(a ) shows the scenario before vaccination while figure 2(b ) shows the scenario with 100% children and 100% adult vaccination . the region of stability does not significantly change depending on the efficacies ( c and w ) or the probability of protection ( ) . in figure 2 , is represented by the solid red line while is represented by the dashed blue line . when > 0 , > 0 . the disease - free equilibrium is stable only if > 0 and > . the value of indicated on the inset graph of figure 2 shows the threshold of transmission between a stable and unstable disease - free equilibrium . if < , then > 0 and > . this shows that we can use as a ( local ) threshold of stability the basic reproductive number r 0 is a threshold that represents the average number of secondary infections caused by one infectious person in a completely susceptible population . from the analysis in section 3.1 , the r 0 threshold is ( 23)r0=wm((1)(a+u+)av+(a+v+)au)[a3+a2+ a+u(v+)],where au and av are the values at the disease - free equilibrium . knowing this threshold is significant , since it will tell us which parameters are involved in shifting the disease from persistence to eradication . this threshold will be used to determine which parameters have the greatest influence on r 0 , which in turn will give us insight into the most effective intervention strategies ( section 5.5 ) . there are critical vaccine efficacies for both children ( ) and women ( w ) and there is a critical probability of protection ( ) that serves as a threshold where , even with 100% vaccination , the targeted types of hpv can not be eradicated in the population . these values are determined by first setting r 0 = 1 and rearranging for the desired parameter . in order to simplify the expression for r 0 , we rewrite the equilibrium values for our population in a general form . note here that k represents the grade of childhood vaccination . for 4 k 10 , we find ( 24)cku=w1+ck3 for kk,cku=w1pk11+ck3 for k > k,ckv=0 for kk,ckv=w1+ck3 for k > k,au=wfpww+a1c7,av=wffpww+a1c7 . to determine the expression for , we look at only childhood vaccination ( i.e. , no adult vaccination ) , which we get by setting p w = 0 . since childhood vaccination only occurs during one year , then(25)pk=1,when k = k,0,when kk. rearranging the expression r 0 = 1 and solving for , we find(26)=1+c7a2wm(a+u+)(1u)wa noticing that 0 ( i.e. , is not constant ) , we construct the following inequality from the definition of . we know that(27)wamc,w=wcwa=g , where w is the size of the female population when there are only women and g is the size of the female population when there are only girls . < ( w ) , then 100% coverage rate of girls will not be sufficient for eradication , since r 0 > 1 . if > ( g ) , then with a sufficient coverage rate it is possible for female - only vaccination to eradicate the targeted types of hpv . if is between the bounds , we can not accurately predict the outcome of the vaccine intervention . the critical adult efficacy occurs when there is no childhood vaccination but there is 100% adult vaccination . using a similar approach to the critical childhood efficacy , we find(29)w=(1+)wmw(a+u+)(1u)a ad = ld(ca)wmw(a+u+)=lllll1u1ca1,where d = ( 1 + c) a( a + a + a + u( v + ) ) . w is bounded as in ( 30)www()wg.the interpretation of w is similar to that of . if the transmission in canada is below = 1.02 , then the disease would be eradicated without any intervention . since hpv is endemic throughout the world and has not become eradicated , we conclude that the actual transmission parameters must be greater than 1.02 ( assuming all other parameters used in the simulation are correct ) . figure 2(b ) shows the region of stability for 100% childhood and 100% adult vaccination . as long as the transmission is lower than = 2.95 however , if the transmission is above 2.95 , then even 100% vaccination can not eradicate hpv . these values allow us to estimate a range of likely transmission values , which are currently unknown . the probability of protection , , was estimated by including the transmission of the targeted hpv types and an estimate for the proportion of girls who become sexually active before grade 11 ( 30% ) . assuming all of the sexually active girls came into contact with someone able to transmit one of the targeted hpv types , the infection for this grade class would spread at a rate of 30% , giving a range of 0 0.3 . the recovery rate was found by determining the average infectious period for both high- and low - risk types of hpv ( 1/ ) and solving for [ 26 , 27 ] . we looked at the significance of vaccinating girls at different ages by constructing a box plot as seen in figure 3 . for a specific grade , the coverage rate was varied between 0 and 100% . in figures 3(a ) and 3(b ) , the vaccine efficacy in adults was used to represent two ( 5096% ) and three ( 7188% ) doses , respectively . latin hypercube sampling was used to sample parameter values from the given ranges in table 2 using a uniform distribution . latin hypercube sampling is a statistical sampling method in which parameters are assigned a range of values , and a distribution of plausible collections of these parameter values are created . note that only one grade ( or adult ) is considered during each run ; for example , a box plot representing grade 4 only takes into account vaccinating girls in grade 4 and does not include vaccinating any other grade or adults . the thick red horizontal lines represent the median value of r 0 , while the box indicates the location of the upper and lower quartiles . comparing the median values in figures 3(a ) and 3(b ) , we notice that when girls are between grades 4 and 10 , the values are all around 1 , whereas the median r 0 is always above one for adult vaccination regardless of the number of doses given . the upper quartile values and lower quartile values also follow this trend , where the childhood vaccination values all have a similar range that is noticeably smaller than when adult vaccination occurs . figure 4 shows the mean r 0 values , rather than the median . while there is more variation between grades , vaccinating with 3 doses is always superior to vaccinating with 2 doses . due to transmission rates ( see figure 5 ) , neither form of vaccination will lead to eradication . however , childhood vaccination is always superior to adult vaccination . comparing figures 3(a ) and 3(b ) , we notice that the r 0 values are generally smaller in the case of three doses , which is expected since the vaccine is less effective on a two - dose schedule . this can also be validated by figure 5 , which shows a low correlation between or w and r 0 . figure 4 shows the difference in the mean r 0 when using a two - dose versus a three - dose regime . once again , having three doses is clearly superior to two , but the difference is not significant . since the true value of each parameter may fluctuate , we explore the sensitivity of r 0 to the parameter values in table 2 using latin hypercube sampling . this in turn allows us to use partial rank correlation coefficients to rank the parameters in terms of their influence on r 0 , be it a positive or negative influence . here r 0 is most sensitive to the transmission of hpv from men to women , w , and from women to men , m. the next influential parameter is the probability of protection , . notice that the coverage rates ( p i , where 4 i 10 , and p w ) and vaccine efficacy ( and w ) do not significantly affect the value of r 0 . figure 6 shows the output of the latin hypercube sampling for the three most influential parameters as well as some other parameters of interest : the recovery rates and the waning rate . note the extremely weak correlation between the latter parameters and r 0 . the coverage rate required to eliminate the targeted high - risk hpv types can be seen in figure 7 . above the curves is the region where r 0 < 1 and the targeted types are theoretically eradicated , whereas below the curves is the region where the disease persists in the population . the curves indicate when r 0 = 1 for either two doses ( lower red curves ) or three doses ( upper black curves ) for different values of the waning rate . as expected , the curve for two doses is higher than three , since the efficacy for two doses is slightly lower . the targeted types can be eradicated if childhood vaccination is supplemented with significant adult vaccination . however , as the waning rate of the vaccine increases , the window for eradication shrinks , requiring a significant amount of both childhood and adult vaccination . a childhood - only vaccination program can theoretically eradicate the targeted hpv types with either two or three doses , as long as the appropriate coverage rate is achieved ( figure 7 ) . for example , if 80% of children are vaccinated with a 3-dose vaccine that does not wane ( the lowest of the curves ) , then at least 40% of adults must be vaccinated to achieve eradication of targeted types . these requirements become harsher as the number of doses decreases or as the vaccine wanes . based on the results of the model , we conclude that , with sufficient childhood and adult vaccination , it is theoretically possible to eradicate targeted hpv types . we also determine that the grade of vaccination before sexual debut does not significantly affect the prevalence of the targeted hpv types for the canadian population . comparing figures 3(a ) and 3(b ) , we observe that the median value of r 0 is always close to one when children are vaccinated , while the median value of r 0 is above one when only adult women are vaccinated , independent of the number of doses given . this suggests that the targeted types are candidates for eradication if significant childhood vaccination can be achieved , whereas eradication is less likely from adult - only vaccination . looking only at childhood - only vaccination , there does not seem to be a large difference between the grades . from a mathematical standpoint , it is easy to understand why the grade of vaccination does not matter in the model , since there is no impact in terms of the disease dynamics , whether girls are vaccinated in grade 4 or 10 . the large jump is due to the change in vaccine effectiveness for those previously exposed to the considered hpv types , as well as the possibility of overvaccinating women , since they remain within the vaccination cohort for 10 years , whereas children are only vaccinated within a single year . this is important to determine because it shows there are likely no underlying grade - dependent trends . however , from an epidemiological standpoint , we know that the vaccine has little to no impact on those individuals who have a previous infection with a targeted type [ 32 , 33 ] . therefore public vaccination strategies should choose the grade of vaccination based on epidemiological and vaccination program limitations . assuming that the number of doses only changes the efficacy of the vaccine , the evidence from this model suggests that the number of doses does not significantly change the outcome of the vaccine strategy . figure 7 shows that the possibility of eradication of targeted hpv types exists with either two or three doses , independent of the grade the childhood vaccine is given in . this evidence follows the findings by several clinical studies that suggest that two doses of the bivalent vaccine may protect just as well as three doses [ 5 , 15 , 22 ] . the higher the coverage rate , the higher the success of the vaccine program ( figure 7 ) . based on this evidence , we suggest each province chooses the number of doses based on cost or ease of program implementation , as long as an appropriate coverage rate is matched to achieve the desired level of infection in the community . the value of r 0 is mainly affected by the ability of the targeted hpv types to transmit and the ability of the vaccine to prevent the infection . thus the most effective way to decrease r 0 is to decrease the transmission of the targeted types of hpv . this could be done through increasing condom use , reducing the number of sexual partners , or increasing the heterogeneity of the sexual contact network of the population [ 16 , 38 ] . since hpv has been infecting humans for millions of years and has not been eradicated , the real transmission rate in the canadian population must be greater than 1.02 . by studying the critical transmission value for 100% childhood and adult vaccination , we know that if the actual transmission rate is greater than 2.95 , then , even with 100% vaccine coverage , the targeted hpv types could not be eradicated . looking at the stability of the disease - free equilibrium based on the transmission is reasonable since the transmission parameters are the only two parameters that significantly affect the value of the basic reproductive number , as shown by the sensitivity analysis . we chose the estimates for the efficacies ( and w ) based on clinical evidence of both the bivalent and the quadrivalent vaccine . obviously , a single vaccine program will only use either the bivalent or the quadrivalent vaccine . since this model does not differentiate between different hpv types and the efficacies for either vaccine do not differ greatly , the numerical results hold for either vaccine . through sensitivity analysis , using larger ranges of the efficacy than observed for either vaccine , we see in figure 5 that it is not important to narrow down the range of efficacies since neither nor w affects the value of r 0 significantly . in order to estimate the efficacy of a two - dose schedule of the quadrivalent vaccine , we used the efficacy of the bivalent vaccine from the costa rica clinical trial . this model should be updated once better data is available for the efficacy and effectiveness of a two - dose quadrivalent hpv vaccine . it is important to note that our model has several limitations . in terms of vaccination programs , the model does not include catch - up programs unless included in the initial population conditions . each person receiving the vaccine with either two or three doses must complete the regimen within one year . the possibility of male vaccination is not included , although it is now approved by health canada [ 1 , 5 , 11 ] . the model does not differentiate in hpv 6 , 11 , 16 , or 18 or include any other hpv types . we have also not ruled out the possibility of a backward bifurcation , meaning the disease may persist even for r 0 < 1 , complicating eradication efforts . from the base adult model , we assume that men who have sexual relations with women in the sexually active cohort ( i.e. , women who are eligible for adult vaccination ) do not continue to find new partners in this age group as time goes on . the sexually active cohorts of men and women are linked only for the time ( approximately 10 years ) that adult women are sexually active and eligible for vaccination . note , however , that we have previously shown that this cohort assumption is negligible . since the provincial vaccination programs are already in place and each province has chosen their dosing schedule ( although it is possible they may change ) , we recommend that all of the provinces focus on increasing their coverage rate for both the public vaccination of girls and private vaccination of women to at least 80% . this number is realistic for many provinces , since several have already reached this goal ( quebec , nova scotia , newfoundland , and pei ) . for the other provinces , increasing their coverage rate may be accomplished by creating public - education campaigns or may involve making protocols similar to vaccines necessary to attend school such as those for hepatitis b [ 17 , 39 ] . our model suggests that the grade of vaccination does not affect the outcome of the vaccination program . therefore we suggest provinces vaccinate girls as early as possible to avoid vaccine failure due to previous infection . the main focus should be on obtaining large enough coverage rates for children and/or adults in order to achieve the desired outcome : using the vaccine to reduce the prevalence of hpv types 6 , 11 , 16 , and 18 in the population .

human papillomavirus ( hpv ) infection is the most common sexually transmitted infection , which is linked to several cancers and genital warts . depending on the canadian province , the quadrivalent vaccine is given to girls in grades 4 through 10 with either a two- or three - dose schedule . we use a mathematical model to address the following research questions : ( 1 ) does the grade at which the girls are vaccinated significantly affect the outcome of the program ? ( 2 ) what coverage rate must the provinces reach in order to reduce the impact of hpv on the canadian population ? ( 3 ) what are the implications of vaccinating with two versus three doses ? the model suggests the grade of vaccination and the number of doses do not make a significant difference to the outcome of the public vaccination program . the most significant factor is the coverage rate of children and adults . we recommend that provinces vaccinate as early as possible to avoid vaccine failure due to previous infection . we also recommend that the main focus of the program should be on obtaining a large enough coverage rate for children and/or adults in order to achieve the desired outcome with either two or three doses of the vaccine .

1. Introduction 2. The Model 3. Analysis 4. Critical Thresholds 5. Numerical Simulations 6. Discussion

human papillomavirus ( hpv ) is the most common sexually transmitted virus , infecting 75% of the sexually active canadian population [ 1 , 2 ] . these strategies vary in the number of doses of the quadrivalent vaccine given ( only two doses in british columbia and quebec , with three doses administered elsewhere ) , the grade of the girls who are given the vaccine ( ranging from 4 to 10 ) , and the coverage rate ( 4986% within the first two years of the program ) . although the implementation of these programs differs between provinces , they all share a common goal of reducing the impact of hpv on the canadian population . there are current clinical trials in british columbia looking at the effect of taking two or three doses of the quadrivalent vaccine . these strategies are defined by the number of doses given , the grade of the girls the vaccine is given to , and the coverage rate achieved . our model will provide similar insight into the effectiveness of two and three doses of the quadrivalent vaccine . this model provides a unique perspective in its evaluation of the provincial programs based on the grade of vaccination , number of doses given , and the necessary coverage rate . we address the following research questions : ( 1 ) does the grade at which the girls are vaccinated significantly affect the outcome of the program ? ( 2 ) what coverage rate must the provinces reach in order to reduce the impact of hpv on the canadian population ? ( 3 ) what are the implications of vaccinating with two versus three doses ? section 5 explores how the parameters were estimated , the results on the infection when varying the grade , dosage and coverage rate , and the sensitivity analysis of the model . female children are in grades 4 through 10 , which are the years childhood vaccination can take place . the difference between a two- and three - dose schedule is the efficacy of the vaccine . comparing the median values in figures 3(a ) and 3(b ) , we notice that when girls are between grades 4 and 10 , the values are all around 1 , whereas the median r 0 is always above one for adult vaccination regardless of the number of doses given . comparing figures 3(a ) and 3(b ) , we notice that the r 0 values are generally smaller in the case of three doses , which is expected since the vaccine is less effective on a two - dose schedule . notice that the coverage rates ( p i , where 4 i 10 , and p w ) and vaccine efficacy ( and w ) do not significantly affect the value of r 0 . a childhood - only vaccination program can theoretically eradicate the targeted hpv types with either two or three doses , as long as the appropriate coverage rate is achieved ( figure 7 ) . we also determine that the grade of vaccination before sexual debut does not significantly affect the prevalence of the targeted hpv types for the canadian population . comparing figures 3(a ) and 3(b ) , we observe that the median value of r 0 is always close to one when children are vaccinated , while the median value of r 0 is above one when only adult women are vaccinated , independent of the number of doses given . from a mathematical standpoint , it is easy to understand why the grade of vaccination does not matter in the model , since there is no impact in terms of the disease dynamics , whether girls are vaccinated in grade 4 or 10 . assuming that the number of doses only changes the efficacy of the vaccine , the evidence from this model suggests that the number of doses does not significantly change the outcome of the vaccine strategy . figure 7 shows that the possibility of eradication of targeted hpv types exists with either two or three doses , independent of the grade the childhood vaccine is given in . based on this evidence , we suggest each province chooses the number of doses based on cost or ease of program implementation , as long as an appropriate coverage rate is matched to achieve the desired level of infection in the community . in order to estimate the efficacy of a two - dose schedule of the quadrivalent vaccine , we used the efficacy of the bivalent vaccine from the costa rica clinical trial . each person receiving the vaccine with either two or three doses must complete the regimen within one year . since the provincial vaccination programs are already in place and each province has chosen their dosing schedule ( although it is possible they may change ) , we recommend that all of the provinces focus on increasing their coverage rate for both the public vaccination of girls and private vaccination of women to at least 80% . our model suggests that the grade of vaccination does not affect the outcome of the vaccination program . therefore we suggest provinces vaccinate girls as early as possible to avoid vaccine failure due to previous infection . the main focus should be on obtaining large enough coverage rates for children and/or adults in order to achieve the desired outcome : using the vaccine to reduce the prevalence of hpv types 6 , 11 , 16 , and 18 in the population .

pancreatic ductal adenocarcinoma is a significant health concern worldwide . according to the international agency for research on cancer ( iarc ) , it is the 15th cancer in overall incidence in the world , with an estimated 277,000 new cases diagnosed per year . it is one of the few diseases in which the mortality rate equals the incidence rate ; as a result the five - year survival rate for this disease remains a dismal 5% , and this has remained constant over many years . in the us , pancreatic cancer mortality is projected to exceed that of breast cancer in the coming decade . pancreatic ductal adenocarcinoma is the most common form of cancer affecting the pancreas , and this is the form that we discuss here . first , there are currently no screening methods for identifying it at stages when it could be cured ; it remains largely asymptomatic and thus undetected until it reaches an advanced stage , when surgical resection , the only potentially curative treatment , is not possible [ 5 - 7 ] . the search for sensitive and specific biomarkers of early stage disease is therefore of utmost importance . the standard of care for patients with advanced stage disease has been gemcitabine , even though this drug confers only modest survival advantages on its own . when used in combination with other agents gemcitabine has shown increased effectiveness ; for example , the combination of gemcitabine with the epidermal growth factor receptor ( egfr ) inhibitor erlotinib has been shown to provide a survival advantage in pancreatic cancer patients compared with gemcitabine alone , although the overall response rate is still low . gemcitabine in combination with agents to target desmoplastic ( fibrosis - causing ) stroma , such as nab - paclitaxel ( albumin - bound paclitaxel ) , has also shown promise , presumably because they deplete tumor stroma , which leads to better delivery of gemcitabine to the tumor cells . beyond the use of gemcitabine alone or in combination with other agents , preliminary success has been achieved with chemotherapeutic combination regimen folfirinox ( folinic acid , fluorouracil , irinotecan , and oxaliplatin ) , although toxicities associated with these treatments limit their utility in many patients . finally , studies are also ongoing to investigate the effectiveness of hedgehog inhibitors in pancreatic cancer . inhibition of this pathway has been proposed to target both the tumor stroma and the cancer stem cell population , although success has not yet been achieved in the clinic . current and potential future chemotherapeutic options for pancreatic ductal adenocarcinoma these regimens have shown promise based on preliminary data in pancreatic cancer or in clinical trials in other cancer types . if more than one trial has been reported the range of median overall survivals is listed . in recent years , advances in sequencing technologies have enabled the genetic and genomic events that underlie pancreatic carcinogenesis and progression to be deciphered in great detail . these efforts have greatly advanced our understanding of the key molecular events and mechanisms - for example , the driver genes characteristic of this tumor type and the core signaling pathways to which they correspond . we now also understand the timing of occurrence of these genetic events in pancreatic carcinogenesis and progression , and the implications of this information for targeted therapies in the setting of personalized medicine . herein , we summarize these discoveries and their potential for improved clinical management of pancreatic cancer . there are four genes that are mutated at high frequency in pancreatic cancer : kras , cdkn2a , tp53 , and smad4 ( table 2 ) ; these are referred to as ' driver ' genes . the most common of these are genetic aberrations in kras ( v - ki - ras2 kirsten rat sarcoma viral oncogene homolog ) at codons 12 , 13 , and occasionally 61 . kras encodes a gtpase that activates downstream effectors of receptor tyrosine kinases , such as the mitogen - activated protein kinase ( mapk ) cascades . inactivation of cdkn2a ( p16 , cyclin - dependent kinase inhibitor 2a gene ) is also a common event in pancreatic cancer , occurring by intragenic mutation in association with allelic loss , homozygous deletion , or hypermethylation . cdkn2a encodes a cyclin - dependent kinase inhibitor that controls the g1-s transition of the cell cycle ; loss of cdkn2a removes this important cellular brake mechanism . alterations in both kras and cdkn2a have been detected at the earliest stages , in pancreatic cancer precursor lesions ( called pancreatic intraepithelial neoplasia or panin ) . however , mutations in kras are predicted to precede those of cdkn2a because of a higher prevalence of kras mutations in early stage precursor lesions and the observation that most panin lesions containing cdkn2a inactivation also harbor a kras mutation ( figure 1 ) . tp53 ( encoding the tumor protein p53 ) , a master regulator of cell stress responses , is a frequent mutational target in many solid tumors [ 22 - 24 ] , and pancreatic cancer is no exception ; mutations in tp53 occur in up to 75% of pancreatic cancers , most often by point mutation or small intragenic deletion . finally , smad4 ( dpc4 , smad family member 4 gene ) , encoding a transcription factor that mediates transforming growth factor- ( tgf- ) and bone morphogenetic protein ( bmp ) signaling , is affected by homozygous deletion or inactivating mutations and allelic loss in up to 55% of pancreatic cancer patients . unlike kras and cdkn2a , the tp53 and smad4 genes are mutated in late stage panins , typically panin-3 lesions ( figure 1 ) . sporadic and inherited genetic alterations in pancreatic ductal adenocarcinoma effect of mutation : act , activating ; in , inactivating . commonly sporadic ( s ) or inherited ( i ) . pancreatic intraepithelial neoplasia ( panin ) is an established precursor lesion of infiltrating pancreatic ductal adenocarcinoma that involves the normal ductal epithelium of the pancreas . panin lesions develop from normal acinar cells in the pancreas , probably as the result of an activating kras mutation , leading to the formation of a panin-1 lesion characterized by a tall columnar epithelium lining the duct system but with little nuclear atypia . the development of inactivating mutations in cdkn2a coincides with the progression of a panin-1 to a panin-2 lesion , characterized by loss of polarity , pseudostratification , papillary formations , and nuclear atypia . inactivating mutations of tp53 and smad4 are late events and most often detected in panin-3 stage lesions . panin-3 lesions are recognized by their complete lack of polarity , marked nuclear atypia , high nuclear / cytoplasmic ratio , and pseudopapillary formation . mutations in additional genes may also occur during panin formation that are not illustrated in this example . a variety of genes are also mutated at low frequency in sporadic pancreatic cancer , such as tgfbr1 , tgfbr2 , and acvr1b , which encode ligand receptors in the tgf-/activin signaling pathway , and the protein kinase mkk4 . additional low - frequency targets are germline variants associated with the familial aggregation of pancreatic cancer . for example , germline mutations in the liver kinase b1 gene ( lkb1 ) are associated with the development of hamartomatous polyps in association with peutz - jeghers syndrome , and patients with this syndrome have a > 100-fold increased risk of developing pancreatic cancer in the context of intraductal papillary mucinous neoplasms . inherited mutations in the brca2 gene ( encoding breast cancer type 2 susceptibility protein , which is involved in dna damage repair ) are perhaps the best characterized of the germline variants . in addition to the increased risk of developing breast or ovarian cancer , brca2 mutations are associated with a 3.5- to 10-fold increased risk of developing pancreatic cancer . following the identification of brca2 mutations in familial pancreatic cancer , germline mutations in the fanconi anemia genes fancc , fancg , and palb2 ( fancn ) , whose protein product interacts with that of brca2 , have also been implicated in familial pancreatic cancer [ 36 - 39 ] . most recently , germline mutations in atm ( encoding the protein kinase ataxia telangiectasia mutated ) have been described in subsets of familial pancreatic cancer families . table 2 summarizes the somatic and germline alterations in known pancreatic cancer genes , their functions , and the relative risks that they confer . although the hallmark genetic changes contributing to pancreatic cancer have been well established , only recently has the pancreatic cancer genome been analyzed on a larger scale by whole - exome sequencing . in analyzing the exomes of 17 primary tumors and 7 metastases , jones et al . reaffirmed the known common genetic alterations in pancreatic cancer and revealed previously unrecognized alterations in genes that have a role in chromatin remodeling ( arid1a and mll3 ) . moreover , although they demonstrated that there are several core pathways that are recurrently targeted in most pancreatic cancers , such as those that control cell division , cell death , adhesion , and various signaling pathways , the pathway components that were altered in any individual tumor varied widely ( figure 2 ) . performed whole - exome sequencing in combination with copy number analysis of 99 early stage ( clinical stage i and ii ) infiltrating pancreatic cancers that yielded sequencing data with a high depth of coverage . a pathway - based analysis of this new comprehensive set of mutations confirmed the pancreatic cancer core pathways previously described by jones et al . . in addition , because of the larger sample size , biankin et al . could identify novel genetic targets in each core signaling pathway and also many alterations in genes encoding axon guidance factors that are typically expressed during embryogenesis . was also confirmed in a murine pancreatic cancer model based on transposon - mediated mutagenesis . the pathways and processes whose component genes are genetically altered in most pancreatic cancers based on whole - exome sequencing are shown . although some genes may correspond to a single pathway ( for example , kras2 mutations and the kras signaling core pathway ) others may have a role in more than one pathway ( for example , tp53 mutations and the apoptosis , dna damage , and jnk core signaling pathways ) . therapeutic targeting of one or more of these pathways , rather than specific gene alterations that occur within a pathway , provides a new way of treating pancreatic cancer . tgf- , transforming growth factor . the clinical significance of these findings is that there is extensive genetic heterogeneity among different patients ' pancreatic cancers , partly explaining why many gene - based therapies will prove ineffective at targeting a genetic alteration that are present in only a small subset of carcinomas . by contrast , therapies that target core pathways , rather than the diverse genetic alterations that can occur within that pathway , may prove more effective , as such therapies would focus on the convergent phenotypes and not the diverse genotypes observed . despite these sobering implications for therapy , the genetic heterogeneity observed in pancreatic cancer provides important additional information : the diverse somatic alterations can be used as evolutionary markers to reveal the life history of pancreatic cancer . such studies are essential for understanding the contribution of genetic mutations to pancreatic carcinogenesis , progression , and metastasis . it has been over 150 years since charles darwin first described natural selection as a force in evolutionary change . in 1976 , peter nowell implicated evolutionary change in cancer when he hypothesized that variant subclones undergo stepwise selection in tumor progression . we now consider cancer to be a genetic disease in which mutations accumulate over time leading to the eventual acquisition of advantageous ' hallmarks ' or traits . moreover , large scale sequencing studies have revealed the various intragenic alterations , copy number variants and chromosomal rearrangements that characterize the many distinct types of cancer . although darwin and nowell provided the overall framework , the current challenge is to translate these concepts of genetic change and clonal evolution in cancer to our ability to diagnose and treat this disease . metastasis is a key feature of many aggressive cancers , including pancreatic cancer , and is caused by a variety of factors such as changes in gene expression of the tumor cell , the microenvironment , and angiogenesis . however , although the genetic events associated with carcinogenesis are well delineated , the relevance of genetic events to these steps of tumor progression is unknown . to address this lack of knowledge for pancreatic cancer , yachida et al . reported the combinatorial effects of the four most commonly mutated genes in pancreatic cancer ( kras , cdkn2a , tp53 , and smad4 ) on patient outcome , with the hypothesis that the combination of somatic alterations in these four genes may be the predominant biological features of that neoplasm . patients whose carcinomas had at least three of these driver genes mutated showed a worse prognosis than those patients who had only one or two of these genes mutated in their carcinoma ; patients whose carcinomas harbored at least three mutated genes also had high metastatic burden at autopsy . although no relationships were found for kras or cdkn2a , they noted that widely metastatic pancreatic cancer ( characterized by hundreds to thousands of metastases present at rapid autopsy ) typically arose from carcinomas with tp53 or smad4 mutations . moreover , there were non - random patterns in which genetic alterations in tp53 and smad4 coexisted . for example , smad4 loss of function almost always occurred in association with genetic inactivation of tp53 , whereas the converse was not true . this indicates that smad4 alterations are selected for in association with tp53 genetic alterations during panin progression . this also suggests that smad4 inactivation occurs later than tp53 inactivation in the genetic progression model of pancreatic carcinogenesis ( figure 1 ) . in contrast , tp53 inactivation in association with wild - type smad4 was highly enriched for nonsense , deletion , or frameshift mutations that abolish p53 's dna binding activity , suggesting that loss of smad4 during panin progression is selected for because of its cytostatic and apoptotic functions conferred by its own ability to bind dna in association with smad2 or smad3 . collectively , these findings indicate that the genetic features of a primary carcinoma that accumulate during carcinogenesis strongly influence its metastatic propensity . thus , the genetic features of a carcinoma that can be exploited for the purposes of early detection , for example tp53 , might also provide information about the metastatic potential of that carcinoma . the genetic alterations that underlie pancreatic cancer metastasis have also been explored on a more global level . used whole - exome sequencing data of seven metastases to explore the timing and dynamics of genetic events in metastatic dissemination of pancreatic cancer . this approach revealed that mutations in a carcinoma could be classified into founders and progressors ( figure 3 ) . founder mutations are those that are present in all samples analyzed for a patient ; from an evolutionary perspective , founder mutations are those clonal events that characterize the ' most recent common ancestor ' of all cells in the neoplasm , or the parental clone that gave rise to it . these mutations were acquired during panin progression culminating in formation of the parental clone . by contrast , progressor mutations were those that occurred in only a subset of the samples analyzed for a patient . these sets of alterations were acquired later than founder mutations and thus highlight subclonal lineages that arose from the parental clone after the infiltrating carcinoma formed . importantly , campbell and colleagues observed a similar pattern while exploring genomic instability and rearrangements in a set of 13 pancreatic cancer patients . many genomic rearrangements were shared among all samples analyzed for a given patient , yet subsets of rearrangements were found in only a subset of samples or uniquely to a single metastasis . this study also described several phylogenetic relationships between the primary and metastatic neoplasms within a patient and also evidence of organ - specific signatures indicating selection for and adaptation of subclones in the new environment . carcinogenesis begins with an initiating alteration in a normal epithelial cell progenitor that provides a selective advantage . over time , waves of clonal expansion take place in association with the acquisition of mutations in genes such as cdkn2a , tp53 , or smad4 , corresponding to the genetic progression model of pancreatic intraepithelial neoplasia ( panin ) . this clonal expansion is expected to generate more than one subclone within a panin , one of which will give rise to the founder cell that will eventually become the parental clone ( p ) of cells that initiate the infiltrating carcinoma . the time taken for a cell with an initiating alteration to accumulate all mutations eventually present in the founder cell that forms the parental clone of the neoplasm additional waves of clonal expansion and accumulation of mutations continue to occur in cell lineages derived from the parental clone leading to the formation of numerous subclones and a genetically heterogeneous primary carcinoma . there are at least two conclusions that can be made from genomic studies of pancreatic cancer progression . first , the time required for accumulation of all genetic alterations in a parental clone suggests a relatively long interval for potentially curative screening methods in this disease , in the order of a decade or longer ( figure 3 ) . second , therapies that target subclonal genetic alterations ( progressor events ) will eventually lead to tumor recurrence , as subclones without those alterations will in theory be selected for by such therapies . therefore , successful approaches will ideally target genetic alterations in the parental clone of that pancreatic cancer ( founder events ) . a recent study by haeno and colleagues using computational modeling further supports this by indicating that pancreatic cancer is commonly in an exponential growth phase at diagnosis . thus , although it may take many years for a tumor to develop , very little time is needed for the cancer to have significant consequences and aggressive behavior once it is clinically evident . this exponential growth phase probably occurs after the formation of the parental clone , as subclones are continuously created and selected for and as they adapt to the dynamic microenvironment . only by identifying and targeting the alterations representing the parental clone approaches for targeting the tumor stroma or stem cell populations of pancreatic cancer also remain viable targets , as evidenced by prolonged survival in patients treated with agents that target them . the ultimate goal of deciphering the genomic changes that occur in pancreatic cancer is to use the information gleaned from each individual patient to guide their treatment . research in this area is still in progress , but there have been advances in both pancreatic and other cancer models that suggest promising directions . some success has been achieved in familial pancreatic cancer patients . as discussed earlier , subsets of familial cases of pancreatic cancer arise through inherited mutations in brca2 , fancc , fancg , and palb2 [ 34,36 - 39 ] . cancers deficient in these genes are missing a key component of the double - strand break repair pathways necessary for error - free dna repair , and treatment of cell lines or xenografts harboring defects in these genes with dna crosslinking agents has proven a potent therapeutic option by exploiting these deficiencies [ 59 - 62 ] . it was also hypothesized that targeting an alternative dna repair pathway - such as base - excision repair , in which parp-1 ( poly ( adp - ribose ) polymerase 1 ) is a central player - would lead to the accumulation of enough dna damage to result in growth arrest or apoptosis of tumor cells . this was first shown to be true in cell lines ( embryonic stem cells , chinese hamster ovary cells , and mcf7 and mda - mb-231 cell lines ) that were isogenic for loss of either brca1 or brca2 [ 64 - 66 ] . a subsequent experiment revealed high efficacy of parp inhibition in capan-1 pancreatic cancer cells , which have a somatic mutation in brca2 . although these data are preliminary in pancreatic cancer so far , it is exciting to consider parp inhibitors as treatment options for patients with germline or somatic brca2 or related pathway member mutations . currently , olaparib , a parp inhibitor , is undergoing clinical trials in breast and ovarian cancers for patients with brca1/2 mutations , with 40% response rates reported . it remains to be seen whether parp inhibition will also be a viable option for treating pancreatic cancer patients with germline or somatic mutations in these genes , and clinical trials to explore this possibility are currently underway . however , because patients with mutations in familial pancreatic cancer genes , such as those described earlier , are relatively uncommon among patients newly diagnosed with this disease , agents that target pancreatic cancer based on more common somatic driver mutations outlined in table 2 would be applicable to a greater proportion of patients . one strategy for genomics - based therapeutics has been to induce synthetic lethality in cancer cells , in which certain cellular events , if present simultaneously , result in the death of the cell . in cancer , the goal is to discover agents that , in the presence of specific mutations , induce this phenomenon . if possible , it seems that the ultimate target for pancreatic cancer treatment would be the kras protein itself , given that it is oncogenic , it is nearly ubiquitously mutated in pancreatic cancer , and it is a mutation found in the parental clone ( and thus all cells ) of the cancer . kras itself does not appear to be druggable , and a recent screen for synthetic lethality with kras activation proved ineffective in pancreatic cancer cell lines . nevertheless , in other model systems there have been genes identified whose inhibition is synthetically lethal with mutant kras , including tbk1 ( involved in activation of nf-b ) , stk33 ( a serine / threonine kinase that activates s6k1 ) , plk1 ( a serine / threonine kinase involved in mitosis ) , and members of the apc / c complex ( anaphase - promoting complex ) [ 74 - 76 ] . a proposed alternative would be a synthetic lethal screen of cells with cdkn2a inactivation , as this gene is also altered during carcinogenesis in the majority of pancreatic cancers , and is thus also harbored in the parental clone of the cancer . although limited success has been achieved in targeting kras directly , recent studies suggest that targeting its downstream effectors may prove more effective . a recent report from collisson et al . reported success in treating a model of pancreatic cancer with a combination of pathway inhibitors . inhibition of the mek - erk mapk signaling pathway was effective in pancreatic cancer cell lines and orthotopic tumors , but activation of akt ( protein kinase b ) signaling was induced by this treatment . however , when the authors treated cell lines with a combination of mek and akt inhibitors , a synergistic effect in killing the tumor cells was observed . given that both of these pathways are activated by oncogenic kras , it is intriguing to consider targeting the downstream effectors of kras signaling as a bypass to the inability to target mutant kras itself . diep and colleagues found synergistic effects of simultaneous egfr and mek inhibition in pancreatic cancer . this was especially evident in cells with wild - type kras , but not in those with kras mutations . however , certain cell lines with kras mutations were sensitive to mek inhibition alone . these data suggest that downstream inhibition of kras may be a promising option for pancreatic cancer patients . synthetic lethality screens in cell lines isogenic for smad4 expression identified two novel compounds , ua62001 and ua62784 , which selectively targeted smad4-negative cells . of particular interest , the effectiveness of ua62001 was shown to correlate well with overall tgf- pathway mutational status in a panel of 11 commonly available pancreatic cancer cell lines . more recently , cui et al . expanded the synthetic lethality concept by examining the efficacies of broadly acting drugs on 18 cell lines studied by jones et al . cui and colleagues were able to identify a decreased sensitivity of cells with cdkn2a mutations to gemcitabine and mitomycin c , an increased sensitivity of cells with tp53 mutations to triptolide , a decreased sensitivity of cells with smad4 mutations to gemcitabine , and an increased sensitivity of cells with smad4 mutations to irinotecan and cisplatin . although the differences in half maximal inhibitory concentration that were observed with these drugs were significant across these genotypes , the fold changes themselves were relatively small . in an alternative approach demonstrating the utility of unbiased molecular profiling for identifying therapeutic vulnerabilities , von hoff et al . used immunohistochemistry , fluorescent in situ hybridization , and gene expression microarray analyses of a large series of refractory patient tumors , including pancreatic cancer , to identify differentially expressed genes that interact with a known therapeutic agent . of 86 patients for which molecular profiling was performed , a molecular target was identified in 84 ( 98% ) patients , 66 of whom were treated according to the results of the molecular profile . of these 66 patients , 27% had a progression - free survival that was longer than expected from their times to disease progression while on previous failed regimens . this supports the use of therapies targeting specific molecular profiles identified in a patient 's tumor tissues . more work will be required to validate these types of studies , but they represent important first steps in identifying genetic - based susceptibilities to therapies used in patients with pancreatic cancer . the complexity of pancreatic cancer from its inception to metastatic colonization is now firmly established , leading to new insights into options for therapeutic targeting based on the genetic features of the neoplasm . for example , it is now evident that the genetic heterogeneity of a pancreatic cancer can be combined into a small number of pathways whose downstream effects can be targeted . moreover , genetic alterations in cancer can also be categorized by the timing of their development , and those that occur during carcinogenesis may be better targets for therapeutic development as they are contained within every cell of the neoplasm . related to this , patients with inherited germline mutations in brca2 and related genes now have options for chemotherapeutic management that exploit their cancers ' defects in dna damage repair , for example mitomycin c or parp inhibitors . the elucidation of the pancreatic cancer genome has implications beyond that of treatment , such as for risk assessment or early detection . for example , sequencing the germline of any individual with a strong family history of pancreatic cancer could identify those with a genetic predisposition to the disease , thus prioritizing them for careful screening of their pancreas or other at - risk organs . moreover , given that most patients do not have a family history of pancreatic cancer , the development of technologies and biomarkers to detect pancreatic cancer while still in the curative stage is of utmost importance . for example , up to 3% of individuals have a pancreatic cyst that is detectable by computerized tomography , some of which are precursors to pancreatic cancer . the distinction of precancerous cysts from those that do not require clinical intervention remains a challenge , but recent studies indicate this could be done simply by sequencing endoscopically obtained cyst fluid , thereby identifying patients who can be potentially cured by surgical management . although there is potential for the use of genetic and genomic information to guide pancreatic cancer diagnosis and treatment , how this will be done on a population level remains to be seen . for example , there are still no reliably sensitive or specific markers to diagnose pancreatic cancer in the curative stage . however , considering exciting new data showing the ability to detect cancer dna in circulating blood- or cell - based assays of patients with colorectal , breast , and gynecological malignancies , similar strategies could probably be applied to patients with pancreatic cancer . for example , in theory sequencing of circulating genomic dna may be better than sequencing a single sample of a neoplasm , as the latter does not indicate the extent of its genetic heterogeneity following subclonal evolution . however , the extent to which circulating dna or shed cells reflect the heterogeneous nature of a pancreatic cancer remains to be determined . nonetheless , genome sequencing of pancreatic cancers and their metastases has provided invaluable insight into the biological features of this disease , and in the future it will no doubt help in identifying potential therapies . brca2 : breast cancer 2 ; cdkn2a : cyclin - dependent kinase inhibitor 2a ; folfirinox : folinic acid , fluorouracil , irinotecan , and oxaliplatin ; kras : v - ki - ras2 kirsten rat sarcoma viral oncogene homolog ; mapk : mitogen - activated protein kinase ; mek : mapk kinase ; panin : pancreatic intraepithelial neoplasia ; parp : poly ( adp - ribose ) polymerase ; smad4 : smad family member 4 ; tp53 : tumor protein p53 ; tgf- : transforming growth factor . this work was supported by nih / nci grants 140599 , ca101955 , ca62624 and ca121113 , the skip viragh pancreatic cancer center and the sol goldman pancreatic cancer research center .

pancreatic cancer is a highly lethal tumor type for which there are few viable therapeutic options . it is also caused by the accumulation of mutations in a variety of genes . these genetic alterations can be grouped into those that accumulate during pancreatic intraepithelial neoplasia ( precursor lesions ) and thus are present in all cells of the infiltrating carcinoma , and those that accumulate specifically within the infiltrating carcinoma during subclonal evolution , resulting in genetic heterogeneity . despite this heterogeneity there are nonetheless commonly altered cellular functions , such as pathways controlling the cell cycle , dna damage repair , intracellular signaling and development , which could provide for a variety of drug targets . this review aims to summarize current knowledge of the genetics and genomics of pancreatic cancer from its inception to metastatic colonization , and to provide examples of how this information can be translated into the clinical setting for therapeutic benefit and personalized medicine .

The promise of genetics and genomics for targeted therapies of pancreatic cancer Pancreatic cancer genetics and genomics The role of genetics and genomics in subclonal evolution Using genomic information to guide treatment of pancreatic cancer Concluding remarks and future directions Abbreviations Competing interests Acknowledgements

we now also understand the timing of occurrence of these genetic events in pancreatic carcinogenesis and progression , and the implications of this information for targeted therapies in the setting of personalized medicine . alterations in both kras and cdkn2a have been detected at the earliest stages , in pancreatic cancer precursor lesions ( called pancreatic intraepithelial neoplasia or panin ) . tp53 ( encoding the tumor protein p53 ) , a master regulator of cell stress responses , is a frequent mutational target in many solid tumors [ 22 - 24 ] , and pancreatic cancer is no exception ; mutations in tp53 occur in up to 75% of pancreatic cancers , most often by point mutation or small intragenic deletion . a variety of genes are also mutated at low frequency in sporadic pancreatic cancer , such as tgfbr1 , tgfbr2 , and acvr1b , which encode ligand receptors in the tgf-/activin signaling pathway , and the protein kinase mkk4 . inherited mutations in the brca2 gene ( encoding breast cancer type 2 susceptibility protein , which is involved in dna damage repair ) are perhaps the best characterized of the germline variants . moreover , although they demonstrated that there are several core pathways that are recurrently targeted in most pancreatic cancers , such as those that control cell division , cell death , adhesion , and various signaling pathways , the pathway components that were altered in any individual tumor varied widely ( figure 2 ) . the clinical significance of these findings is that there is extensive genetic heterogeneity among different patients ' pancreatic cancers , partly explaining why many gene - based therapies will prove ineffective at targeting a genetic alteration that are present in only a small subset of carcinomas . despite these sobering implications for therapy , the genetic heterogeneity observed in pancreatic cancer provides important additional information : the diverse somatic alterations can be used as evolutionary markers to reveal the life history of pancreatic cancer . metastasis is a key feature of many aggressive cancers , including pancreatic cancer , and is caused by a variety of factors such as changes in gene expression of the tumor cell , the microenvironment , and angiogenesis . founder mutations are those that are present in all samples analyzed for a patient ; from an evolutionary perspective , founder mutations are those clonal events that characterize the ' most recent common ancestor ' of all cells in the neoplasm , or the parental clone that gave rise to it . over time , waves of clonal expansion take place in association with the acquisition of mutations in genes such as cdkn2a , tp53 , or smad4 , corresponding to the genetic progression model of pancreatic intraepithelial neoplasia ( panin ) . the time taken for a cell with an initiating alteration to accumulate all mutations eventually present in the founder cell that forms the parental clone of the neoplasm additional waves of clonal expansion and accumulation of mutations continue to occur in cell lineages derived from the parental clone leading to the formation of numerous subclones and a genetically heterogeneous primary carcinoma . there are at least two conclusions that can be made from genomic studies of pancreatic cancer progression . as discussed earlier , subsets of familial cases of pancreatic cancer arise through inherited mutations in brca2 , fancc , fancg , and palb2 [ 34,36 - 39 ] . it was also hypothesized that targeting an alternative dna repair pathway - such as base - excision repair , in which parp-1 ( poly ( adp - ribose ) polymerase 1 ) is a central player - would lead to the accumulation of enough dna damage to result in growth arrest or apoptosis of tumor cells . if possible , it seems that the ultimate target for pancreatic cancer treatment would be the kras protein itself , given that it is oncogenic , it is nearly ubiquitously mutated in pancreatic cancer , and it is a mutation found in the parental clone ( and thus all cells ) of the cancer . a proposed alternative would be a synthetic lethal screen of cells with cdkn2a inactivation , as this gene is also altered during carcinogenesis in the majority of pancreatic cancers , and is thus also harbored in the parental clone of the cancer . the complexity of pancreatic cancer from its inception to metastatic colonization is now firmly established , leading to new insights into options for therapeutic targeting based on the genetic features of the neoplasm . moreover , genetic alterations in cancer can also be categorized by the timing of their development , and those that occur during carcinogenesis may be better targets for therapeutic development as they are contained within every cell of the neoplasm . related to this , patients with inherited germline mutations in brca2 and related genes now have options for chemotherapeutic management that exploit their cancers ' defects in dna damage repair , for example mitomycin c or parp inhibitors . the elucidation of the pancreatic cancer genome has implications beyond that of treatment , such as for risk assessment or early detection .

the world in which we live has been under the influence of an extensive territory of goal - oriented human efforts . we are moving from destiny - based societies toward the societies where evolution is made by human factors . dealing with this ever - changing world requires a kind of trust in the people who make more contributions to the formation of social life . although the idea of trust is a century - long thought process , almost over the past decade , it has gained a significant position within the sociological ideas . moreover , there is such a strong relationship between trust and risk that luhmann defines trust as the solution to the specific risk - related problems . a risk which , in its most general term , means being subject to a particular danger or hazard has undergone a lot of changes in meaning and function , in a way that it has become more inclusive and has been used under different conditions . at the conceptual level , risk has been distinguished from danger and hazard ; at the phenomenon level , also , risks of contemporary world are claimed to be effects and consequences of human actions and decisions . in other words , risks are constructed adventures ; while danger is a harm , which is suffered from outside . explaining the contemporary world , the sociologists of risk refer to the concepts such as reflexivity , insecurity and uncertainty , individualization and reflective biography and consider different arenas of life as a constructed and not assigned affair . such evolutions have totally influenced the functions of the social sciences , in general , and sociology , in particular . accordingly , riskology has been considered as the essential element and the pioneer of the sociological modern reformation . meanwhile , the medicine and health , as one of the most important fields of research in sociology , is closely related to risk . the importance of the study of risks within the field of body and health is due to the fact that body and beauty are now prone to manipulation and personal motivation more than ever and discourse of beauty has been replaced by discourse of attractiveness . this means that attractiveness in appearance , thanks to the change in view of beauty and advances in the field of cosmetic surgery , has imparted an acquired aspect to beauty . the use of cosmetic surgery suggests that the beauty is more a social - cultural notion than a natural and god - given phenomenon . advances in the field of medicine have also contributed to the emergence of the idea that body can be a function of medical technologies . accordingly , notions of uncertainty of body and risk taking make sense . a glance at the previous research shows that these studies have mainly been conducted by the quantitative method in which the cosmetic surgery and the body management were considered as dependent variables . in these studies , attempt has been made to measure the effect of variables such as age , gender , education , marriage , socioeconomic status , the status of different resources ( economic , social , cultural ) , social - normative pressure , consumerism , and media consumption on the domain of body . what makes the present study different from others is that it aims to reconstruct the experience of the cosmetic surgery as a risky enterprise under a qualitative approach and describe and interpret different forms of the confidence experienced by those who apply for this surgery . the present study has been conducted within the interpretive paradigm , employing phenomenological method , which is of the qualitative design . this method , which is applied with the purpose of description , understanding and interpretation of the meanings in human 's living experiences , is very creative and flexible and does not follow a particular or preplanned procedure . in phenomenological studies , purposive sampling or criterion - based sampling is used . this experience , which is called lived experience , is in contrast to second - hand experience . in this study , mixed purposive sampling technique was applied . that is to say , based on extreme case sampling , snowball or chain sampling and opportunistic sampling were simultaneously used . according to extreme case sampling , namely , looking for outstanding and not necessarily very unusual cases from the phenomenon under investigation , it was tried to select the ones who met the characteristics including experience of the surgery , wide and extensive communication with different physicians , ability in stating the experience exactly and in details and being in a context of those who had the experience of the surgery in their relatives , and to include all groups with different demographic information in the sample and to examine , as much as possible , all aspects of the individual cases . then participants were asked to introduce the ones with the same characteristics in order to have the similar interview . also , with regard to the requirements of the study and emerging the new themes and concepts in analysis of data , participants in accordance with the given requirements were selected . totally , 26 participants were sampled among those who had the cosmetic surgery in isfahan . it should be mentioned that , medical information was not of importance in this study and solely the demographic information was collected . in accordance with the criterion of data saturation , sampling procedure continued until the researcher found out that , with regard to the content and the notions being created , no further new or appropriate piece of information was elicited . the variation among participants in terms of the gender , age , level of education , marital status , and type of the surgery the research data was collected in an extensive phenomenological interview as developed by dolbear and schuman . in this three - phase set , the first interview forms the context of the participants experiences . the second interview allows the participants to reconstruct the details of their experiences within the context in which the experiences have occurred . and the third interview encourages the participants to contemplate over the meaning , which is attributed to their experiences . data were gathered within the one - year period of the study , from february 20 , 2012 to february 20 , 2013 . the participants taking part in the research was based on their informed consent and guarantees of confidentiality and anonymity of data . indeed , participants were taken under consideration in clinics and offices of cosmetic surgeries in isfahan . next , in order to have their consent for cooperation in the study , in an informal and friendly environment , aims and methodology of the study were implicitly discussed with them . after initial consent , they were asked to give their final approval or disapproval for taking part in the interview in the following phone call . the interviews were recorded by two interviewers using a checklist of all the factors to be asked , guided by all the body movements , facial expressions , diction , and emotions . in the first 1520 minutes of the interview , participants life histories were discussed . in this phase , participants were invited to talk about their personal experiences regarding cosmetic surgeries as much as possible . in the next 2030 minutes , the focus was on the concrete details of the lived experience of the participants with regard to the risks of cosmetic surgeries and how to trust to this process . in the last 2030 minutes , they were asked to share their conception of risk and trust , based on their sense from their experiences . all the conversations were tape recorded and then , were transcribed word by word . before transcribing the interviews , researchers listened to each conversation several times so that a better understanding was achieved of the data . seven - stage colaizzi method of data analysis was used for analyzing the data and extracting different descriptive , interpretive , and explanatory codes . since the participants reflection as a persistent process in data analysis contributes to the higher reliability in the qualitative research , according to the colaizzi method , the researcher gave the interview transcription to the participants and asked them to examine the findings and control their conformity with their own experiences . furthermore , since devoting enough time for collecting the data enhances the profundity , hence , actuality of the data , in addition to the application of the intense three - phase interviewing technique and devoting enough time for each phase of the interview , the research data were collected in a 9-month period being supported by a group of skillful colleagues who had expertise in using qualitative method . the present study has been conducted within the interpretive paradigm , employing phenomenological method , which is of the qualitative design . this method , which is applied with the purpose of description , understanding and interpretation of the meanings in human 's living experiences , is very creative and flexible and does not follow a particular or preplanned procedure . in phenomenological studies , purposive sampling or criterion - based sampling is used . this experience , which is called lived experience , is in contrast to second - hand experience . in this study , mixed purposive sampling technique was applied . that is to say , based on extreme case sampling , snowball or chain sampling and opportunistic sampling were simultaneously used . according to extreme case sampling , namely , looking for outstanding and not necessarily very unusual cases from the phenomenon under investigation , it was tried to select the ones who met the characteristics including experience of the surgery , wide and extensive communication with different physicians , ability in stating the experience exactly and in details and being in a context of those who had the experience of the surgery in their relatives , and to include all groups with different demographic information in the sample and to examine , as much as possible , all aspects of the individual cases . then participants were asked to introduce the ones with the same characteristics in order to have the similar interview . also , with regard to the requirements of the study and emerging the new themes and concepts in analysis of data , participants in accordance with the given requirements were selected . totally , 26 participants were sampled among those who had the cosmetic surgery in isfahan . it should be mentioned that , medical information was not of importance in this study and solely the demographic information was collected . in accordance with the criterion of data saturation , sampling procedure continued until the researcher found out that , with regard to the content and the notions being created , no further new or appropriate piece of information was elicited . the variation among participants in terms of the gender , age , level of education , marital status , and type of the surgery the research data was collected in an extensive phenomenological interview as developed by dolbear and schuman . in this three - phase set , the first interview forms the context of the participants experiences . the second interview allows the participants to reconstruct the details of their experiences within the context in which the experiences have occurred . and the third interview encourages the participants to contemplate over the meaning , which is attributed to their experiences . data were gathered within the one - year period of the study , from february 20 , 2012 to february 20 , 2013 . the participants taking part in the research was based on their informed consent and guarantees of confidentiality and anonymity of data . indeed , participants were taken under consideration in clinics and offices of cosmetic surgeries in isfahan . next , in order to have their consent for cooperation in the study , in an informal and friendly environment , aims and methodology of the study were implicitly discussed with them . after initial consent , they were asked to give their final approval or disapproval for taking part in the interview in the following phone call . the interviews were recorded by two interviewers using a checklist of all the factors to be asked , guided by all the body movements , facial expressions , diction , and emotions . in the first 1520 minutes of the interview , participants life histories were discussed . in this phase , participants were invited to talk about their personal experiences regarding cosmetic surgeries as much as possible . in the next 2030 minutes , the focus was on the concrete details of the lived experience of the participants with regard to the risks of cosmetic surgeries and how to trust to this process . in the last 2030 minutes , they were asked to share their conception of risk and trust , based on their sense from their experiences . all the conversations were tape recorded and then , were transcribed word by word . before transcribing the interviews , researchers listened to each conversation several times so that a better understanding was achieved of the data seven - stage colaizzi method of data analysis was used for analyzing the data and extracting different descriptive , interpretive , and explanatory codes . since the participants reflection as a persistent process in data analysis contributes to the higher reliability in the qualitative research , according to the colaizzi method , the researcher gave the interview transcription to the participants and asked them to examine the findings and control their conformity with their own experiences . furthermore , since devoting enough time for collecting the data enhances the profundity , hence , actuality of the data , in addition to the application of the intense three - phase interviewing technique and devoting enough time for each phase of the interview , the research data were collected in a 9-month period being supported by a group of skillful colleagues who had expertise in using qualitative method . in fact , for making a decision and burdening the risk associated with that decision , the cases need to overcome the negative experiences ( here means fear and stress ) and gain some positive experiences ( trust ) . the major trusting procedure here occurs between the doctor and the patient and is considered a bilateral act ; gaining trust on the part of the patient and drawing trust on the part of the doctor . below , different types of the participants experienced trust have been classified . in this type of trust , little energy is used and the participants do not have accurate information about the surgery ; expressions such as maybe , i do nt know , and the words that show their uncertainty have been frequently used in their speech and they only visit one special doctor ( the doctor that is the target in vicarious trust ) . s / he said : such and such a thing or s / he asked : what do you want to do ? and in other words , not only does this person have little preplanned ideas about his / her wants but also s / he acts passively in his / her visits to the doctor and get subdued by the doctor 's ideas . i said : first of all , treat my nasal polyp ; then , s / he examined my nose and said : your left septum is deviated . i said : okay ! then , s / she said : do nt you want to change your nose shape ? s / he told me that the men 's nose should be long , should not be humpbacked or hooked , or in any special shape ; i mean , it is possible to give it a special shape , if you want , but after a while , you will regret your decision . on the other hand , since these participants , as those who passively put their trust on others , do not make any effort to pass trusting procedure , and limit themselves to the friends experiences and their trust on doctor , no changes occur in their decision upon different experiences with the doctor . the sub - codes of the vicarious trust , therefore , can be as follows : inaccurate data , gaining trust by using the least amount of energy , the first hand and the unique experience with the doctor . in the criterion trust , the person who builds the trust , having considered a criterion or criteria , put the trust on a doctor who is the surgeon of those who have those criteria instead of putting trust directly on the person or the object of the trust . this criterion can be the treatment process , the shape and form of the nose , the type of the relationship , etc . describing his / her experiences , one of the participants states : as to my brother who was also one of my encouragers in this case , some of his friends had had surgery ; then , he said that one of his friends had had surgery and he was satisfied by his surgery and his doctor was such and such a person and the check - up procedure is in this manner . the persons with the criterion trust are polarizing the doctors largely based on the concerned criterion and making a dichotomy of good and bad like this : before i became determined , among the addresses i had of the cosmetic surgeons , one of them was a doctor who was also in isfahan . it is always said that s / he performs awful surgeries and a few friends of mine who had undergone surgery with him / her looks really bad . but , when he introduced the doctor , my brother and specially those who had already visited him i talked with them ; i felt that the fear of { under whose care you are going to be } is going away . accordingly , in this group of the participants , trusting the doctor is partly due to the mistrust in the doctor who is located at the bad pole of the dichotomy . upon the appearance of such a dichotomy , most of the participants become determined quickly and use the least amount of energy for data collection and trust building processes . based on the participants experiences , the sub - codes of the criterion trust are : relatively accurate information , gaining trust through using a lot of energy , and little polarized experience with doctors ( limited to the extensions of good and bad dichotomy ) . in this type of trust , the required trust for the surgery will be wrapped and presented to the doctor after a long , careful , and empirical endeavor . this type of trust is in a way that the doctor : at first told me : do nt have a high expectation of your nose ! but , since i believed in his work , i said : no ! i waited for 2 years to see how my friend 's nose looks after the surgery . of course , the required ingredients for this type of trust are picked up from the specialized showcase of the doctor in a way that the participants , explaining the reasons for putting trust on the doctor , refer to doctor 's professional competencies outweighing over the financial benefits , the doctor 's fame in doing natural and specialized surgeries , and the use of certain techniques such as the application of smaller tampons or local anesthesia . in this type of the trust , participants , while presenting their wrapped trust to their selected doctor ; do not necessarily observe all the doctor 's prescriptions , stating that they have a comprehensive knowledge of the cosmetic surgeries : i knew all of these points completely , for example , my own research , my own information about the nose surgery was much more than [ stated in a higher tone of voice and with more stress ] what others are saying . i was saying : no , what doctor is saying works only for himself . according to the above mentioned experiences , the wrapped trust can be described as constructed upon the following sub - codes : accurate and detailed information , gaining trust by using the maximum amount of energy , person 's utmost capacity , making definite decisions , and selecting the doctor as a case study . in this type of trust , participants have chosen their options within a predetermined framework or structural limitations and referred to the only possible case within reach . this framework may be an organizational option like this : my father works for the oil company ! for this one of the participants , stating : i did nt go after visiting a doctor , acknowledges his / her lack of sense of selection and effort to choose his / her treating physician : i was in such a hurry that i did nt go after finding a doctor ! i went to the office of this doctor ; they said : he is good . i m telling you i wanted to travel to germany ; then , i traveled to germany right away , [ laughter ] to show it to my sister ! [ laughter ] . if s / he would be in shahinshahr , we ll visit him / her . i rarely commuted to isfahan ; honestly , because i had to take care of my children . according to the results , the sub - codes of the trust within reach can be described as follows : being satisfied with the available information , gaining trust by using little energy , structural limitations , making haste in decision making , and limitations in selecting the doctor . in this type of the trust , the participants show their emphasis on the specialized role of the doctor and their awareness of such a role and describe their first visit to the doctor , saying : that doctor is a cosmetic super - specialist or because he is a cosmetic and nose prosthesis super - specialist , i said : i want to have a nose surgery. it is where their trust arises . one of the participants , pointing out that s / he had been very careful in selecting the doctor and trusting him / her , asks : first of all , what is his / her specialty ? believing in the distinction between the specialties , s / he claims : the cosmetic surgeries are , now , performed by ent specialists with a lower fee . it is not their specialty ; just as we see there were some patients who died in such surgeries . most of the people who experience this type of the trust , consider the experience of the surgery as a simple experience to overcome , and this simplification is carried out by the reliance on the doctor 's specialty . in fact , surgery , as a rerun experience of the doctors , is conceptualized as a simple and nonthreatening procedure . one of the participants says : specially my daddy ! he saw the whole issue as a very simple matter : { honey , take it easy ! you just go ! this issue made me calm ; s / she knows his / her work! . some of the works are within my specialty and i can perform them with my eyes shut . it has been for several years that such a thing has happened to him ; with about thousands of individuals ! only in this way , i got myself together . in this type of the trust , the participants trust in doctor is to the extent that they do not state anything as to existing dichotomies such as private / public hospitals , snub / sharp nasal septum , open / closed surgery , using suture / burning the surgical site , and do not challenge and question the doctor for his decision about the selection of one of these alternatives : just as , i told him : { doctor , i put all the things under your care . based on what is said , the institutionalized trust can be described as constructed based on the following sub - codes : the quantitative and general information , gaining trust by using a relatively large amount of energy , total self - submission to the doctor 's specialty , simplification of the surgery , and selection of the doctor pending on the specialty . in this type of trust , little energy is used and the participants do not have accurate information about the surgery ; expressions such as maybe , i do nt know , and the words that show their uncertainty have been frequently used in their speech and they only visit one special doctor ( the doctor that is the target in vicarious trust ) . in other words , not only does this person have little preplanned ideas about his / her wants but also s / he acts passively in his / her visits to the doctor and get subdued by the doctor 's ideas . i said : first of all , treat my nasal polyp ; then , s / he examined my nose and said : your left septum is deviated . s / he told me that the men 's nose should be long , should not be humpbacked or hooked , or in any special shape ; i mean , it is possible to give it a special shape , if you want , but after a while , you will regret your decision . on the other hand , since these participants , as those who passively put their trust on others , do not make any effort to pass trusting procedure , and limit themselves to the friends experiences and their trust on doctor , no changes occur in their decision upon different experiences with the doctor . the sub - codes of the vicarious trust , therefore , can be as follows : inaccurate data , gaining trust by using the least amount of energy , the first hand and the unique experience with the doctor . in the criterion trust , the person who builds the trust , having considered a criterion or criteria , put the trust on a doctor who is the surgeon of those who have those criteria instead of putting trust directly on the person or the object of the trust . this criterion can be the treatment process , the shape and form of the nose , the type of the relationship , etc . describing his / her experiences , one of the participants states : as to my brother who was also one of my encouragers in this case , some of his friends had had surgery ; then , he said that one of his friends had had surgery and he was satisfied by his surgery and his doctor was such and such a person and the check - up procedure is in this manner . the persons with the criterion trust are polarizing the doctors largely based on the concerned criterion and making a dichotomy of good and bad like this : before i became determined , among the addresses i had of the cosmetic surgeons , one of them was a doctor who was also in isfahan . it is always said that s / he performs awful surgeries and a few friends of mine who had undergone surgery with him / her looks really bad . but , when he introduced the doctor , my brother and specially those who had already visited him i talked with them ; i felt that the fear of { under whose care you are going to be } is going away . accordingly , in this group of the participants , trusting the doctor is partly due to the mistrust in the doctor who is located at the bad pole of the dichotomy . upon the appearance of such a dichotomy , most of the participants become determined quickly and use the least amount of energy for data collection and trust building processes . based on the participants experiences , the sub - codes of the criterion trust are : relatively accurate information , gaining trust through using a lot of energy , and little polarized experience with doctors ( limited to the extensions of good and bad dichotomy ) . in this type of trust , the required trust for the surgery will be wrapped and presented to the doctor after a long , careful , and empirical endeavor . this type of trust is in a way that the doctor : at first told me : do nt have a high expectation of your nose ! but , since i believed in his work , i said : no ! i m sure it will get well . i waited for 2 years to see how my friend 's nose looks after the surgery . of course , the required ingredients for this type of trust are picked up from the specialized showcase of the doctor in a way that the participants , explaining the reasons for putting trust on the doctor , refer to doctor 's professional competencies outweighing over the financial benefits , the doctor 's fame in doing natural and specialized surgeries , and the use of certain techniques such as the application of smaller tampons or local anesthesia . in this type of the trust , participants , while presenting their wrapped trust to their selected doctor ; do not necessarily observe all the doctor 's prescriptions , stating that they have a comprehensive knowledge of the cosmetic surgeries : i knew all of these points completely , for example , my own research , my own information about the nose surgery was much more than [ stated in a higher tone of voice and with more stress ] what others are saying . i was saying : no , what doctor is saying works only for himself . according to the above mentioned experiences , the wrapped trust can be described as constructed upon the following sub - codes : accurate and detailed information , gaining trust by using the maximum amount of energy , person 's utmost capacity , making definite decisions , and selecting the doctor as a case study . in this type of trust , participants have chosen their options within a predetermined framework or structural limitations and referred to the only possible case within reach . this framework may be an organizational option like this : my father works for the oil company ! for this , we went there ( the oil company ) ; they recommended one of the participants , stating : i did nt go after visiting a doctor , acknowledges his / her lack of sense of selection and effort to choose his / her treating physician : i was in such a hurry that i did nt go after finding a doctor ! i went to the office of this doctor ; they said : he is good . i m telling you i wanted to travel to germany ; then , i traveled to germany right away , [ laughter ] to show it to my sister ! [ laughter ] . if s / he would be in shahinshahr , we ll visit him / her . i rarely commuted to isfahan ; honestly , because i had to take care of my children . according to the results , the sub - codes of the trust within reach can be described as follows : being satisfied with the available information , gaining trust by using little energy , structural limitations , making haste in decision making , and limitations in selecting the doctor . in this type of the trust , the participants show their emphasis on the specialized role of the doctor and their awareness of such a role and describe their first visit to the doctor , saying : that doctor is a cosmetic super - specialist or well , what is your problem ? because he is a cosmetic and nose prosthesis super - specialist , i said : i want to have a nose surgery. it is where their trust arises . one of the participants , pointing out that s / he had been very careful in selecting the doctor and trusting him / her , asks : first of all , what is his / her specialty ? believing in the distinction between the specialties , s / he claims : the cosmetic surgeries are , now , performed by ent specialists with a lower fee . it is not their specialty ; just as we see there were some patients who died in such surgeries . most of the people who experience this type of the trust , consider the experience of the surgery as a simple experience to overcome , and this simplification is carried out by the reliance on the doctor 's specialty . in fact , surgery , as a rerun experience of the doctors , is conceptualized as a simple and nonthreatening procedure . ! he saw the whole issue as a very simple matter : { honey , take it easy ! you just go ! this issue made me calm ; s / she knows his / her work! . some of the works are within my specialty and i can perform them with my eyes shut . it has been for several years that such a thing has happened to him ; with about thousands of individuals ! only in this way , i got myself together . in this type of the trust , the participants trust in doctor is to the extent that they do not state anything as to existing dichotomies such as private / public hospitals , snub / sharp nasal septum , open / closed surgery , using suture / burning the surgical site , and do not challenge and question the doctor for his decision about the selection of one of these alternatives : just as , i told him : { doctor , i put all the things under your care . based on what is said , the institutionalized trust can be described as constructed based on the following sub - codes : the quantitative and general information , gaining trust by using a relatively large amount of energy , total self - submission to the doctor 's specialty , simplification of the surgery , and selection of the doctor pending on the specialty . the present study was to describe , understand and interpret different types of the experienced trust in one risky medical enterprise . in other words , based on the participants experience , the effort of the cases to have surgery is a kind of risky enterprise . for the cases under study , making decision for the surgery ( or putting their decisions for the surgery into effect ) is not easy . the cases are faced with the propelling and encouraging factors ( justifications ) on the one hand , and the dissuading factors ( doubts ) , on the other . the conflict between justifications and doubts , change the decision making process for the surgery into a challenging experience . in this challenge , the phenomenon of trust and its different types gain significance based on the participants experiences . as the results of the present study indicate , five types of trust were experienced by the participants . the construct of these five types of the trust has been based on the common sub - codes that have had different levels . for example , different types of the trust can be scaled on a spectrum based on the amount of energy used or the amount of information . the spectrum for different types of the experienced trust based on the common sub - codes to sum up , what seems to be common among all the forms of the trust is that the trust is an event , which has occurred as a result of the diligent endeavor of the cases ( participants ) against that there is no remarkable act on the part of the doctors for building or drawing this trust . if , in terms of the participants degree of subjectivity , vicarious trust can be placed at the bottom and the wrapped trust can be placed at the top , in all the experiences , participants , having considered the necessity of gaining trust as a presupposition , have made a unilateral effort to create this phenomenon . to be more exact , the doctors , having presented a showcase of the premade frameworks for drawing trust , have dealt with all the patients in a same manner and according to a unilateral premade plan , and have made no effort to give some special meaning to each dual relationship and cases , here as patients , are left alone in interpreting these premade frameworks of drawing trust ( e.g. reference to the specialty , offering the catalogue of the work sample , the effect of the doctor 's office as the meeting place ) . based on the habermas classification of the instrumental and communicative rationality , it can be said that none of the participants have reached the level of communicative rationality . to be more exact , this choice has not been changed by those who trust into a relationship between those who trust and those who are trusted . meanwhile , doctors do not make any effort to create such a relationship and it seems that they do not even feel the need to do that . they , as the specialists , in one unequal relationship , do not feel the need to exchange information with the public and they make no distinction between the optional and compulsory surgeries . while , according to the communicative rationality expected by the participants , in each new visit , based on the existing exigencies on the part of the patient ( e.g. age , gender , type of the concerns , fears , and the previous experiences of the patient ) , a new doctor as to this issue , it should be said that one of the major concerns of the participants is that there is no room for the conversation in which they can convey their demands and preferences , while , doctors , having referred to their technical - specialized competence , do not acknowledge the general knowledge of the participants and the conversation atmosphere is not created . this is while the presupposition for the communicative rationality is the extrapolation of knowledge and the possibility for establishing the relationship and expressing the ideas about the field under discussion . this finding is supported by a few similar studies within the same area in iran in the world of medicine . in a study titled : the cultural plays of the death and dying , utilizing the field theory , the researchers investigated the cultural patterns of the death and dying within people who live with cancer . in one part of the results of the study , it has been pointed out that the patient finds the opportunity for talking with the specialists and advisers more effective than the any other treatment and medicine . doctor , however , focuses his attention on the clinical signs , body and the injured member and do not set a time to talk with the patient . meanwhile , for patients , talking with a doctor who is well informed of their disease is very important . the most important consequences of lack of conversation with therapists is that the patient is changed into a solitary , defenseless , unaware , and submissive person ; the right for selection and making decision in the crisis is reduced ; and patient experiences a kind of mental breakdown . according to the results of this study , further research should focus on doctors offices as a meeting place for cases and the role of such a meeting in stimulating , encouraging , and facilitating the process of making decision or giving up , as well . more studies can describe and interpret the mechanism used by doctors to impress others and the process of negotiation with patients . it is worth mentioning that this study was based on the conceptual framework of giddens and beck in which cultural differences among societies are not of importance .

background : in all areas of life including health , choices have widely increased and concerns over getting hold of further choices have made trust a necessary element . this study , taking into consideration the interconnection of three concepts of trust , risk , and body , aims at describing and interpreting different types of trust experienced in a risky medical operation ( cosmetic surgery).materials and methods : to achieve the given purpose , within interpretative paradigm and employing qualitative method , in - depth phenomenological interviews were conducted with 26 people who volunteered to have a cosmetic surgery . participants , who have been selected through purposive sampling techniques , were fully aware of their participation in the study and were insured that the data would be confidential and would be used only for the purpose of the study . data were gathered within a one - year period of the study , from february 20 , 2012 to february 20 , 2013 . results of three - phase interviews were validated against participatory feedback and researchers triangulation and were further analyzed by means of seven - stage colaizzi method.findings:consequently , five main themes , namely , vicarious trust , trust within the reach , institutionalized trust , criterion trust , and wrapped trust were extracted.conclusion:apart from existing differences among these five themes ( e.g. degree of the subjectivity and objectivity in the patient ) , they can be regarded comparable in terms of being single - sided ( from the patient 's side ) . in other words , in all experiences , participants , having considered the necessity of gaining trust as a presupposition , have made a unilateral effort in creating the aforementioned phenomenon .

INTRODUCTION MATERIALS AND METHODS Participants and research design Data collection procedure Data analysis RESULTS Vicarious trust Criterion trust Wrapped trust Trust within reach Trust in specialized role (institutionalized) DISCUSSION AND CONCLUSION

according to extreme case sampling , namely , looking for outstanding and not necessarily very unusual cases from the phenomenon under investigation , it was tried to select the ones who met the characteristics including experience of the surgery , wide and extensive communication with different physicians , ability in stating the experience exactly and in details and being in a context of those who had the experience of the surgery in their relatives , and to include all groups with different demographic information in the sample and to examine , as much as possible , all aspects of the individual cases . data were gathered within the one - year period of the study , from february 20 , 2012 to february 20 , 2013 . next , in order to have their consent for cooperation in the study , in an informal and friendly environment , aims and methodology of the study were implicitly discussed with them . furthermore , since devoting enough time for collecting the data enhances the profundity , hence , actuality of the data , in addition to the application of the intense three - phase interviewing technique and devoting enough time for each phase of the interview , the research data were collected in a 9-month period being supported by a group of skillful colleagues who had expertise in using qualitative method . according to extreme case sampling , namely , looking for outstanding and not necessarily very unusual cases from the phenomenon under investigation , it was tried to select the ones who met the characteristics including experience of the surgery , wide and extensive communication with different physicians , ability in stating the experience exactly and in details and being in a context of those who had the experience of the surgery in their relatives , and to include all groups with different demographic information in the sample and to examine , as much as possible , all aspects of the individual cases . data were gathered within the one - year period of the study , from february 20 , 2012 to february 20 , 2013 . next , in order to have their consent for cooperation in the study , in an informal and friendly environment , aims and methodology of the study were implicitly discussed with them . before transcribing the interviews , researchers listened to each conversation several times so that a better understanding was achieved of the data seven - stage colaizzi method of data analysis was used for analyzing the data and extracting different descriptive , interpretive , and explanatory codes . furthermore , since devoting enough time for collecting the data enhances the profundity , hence , actuality of the data , in addition to the application of the intense three - phase interviewing technique and devoting enough time for each phase of the interview , the research data were collected in a 9-month period being supported by a group of skillful colleagues who had expertise in using qualitative method . in the criterion trust , the person who builds the trust , having considered a criterion or criteria , put the trust on a doctor who is the surgeon of those who have those criteria instead of putting trust directly on the person or the object of the trust . in this type of the trust , participants , while presenting their wrapped trust to their selected doctor ; do not necessarily observe all the doctor 's prescriptions , stating that they have a comprehensive knowledge of the cosmetic surgeries : i knew all of these points completely , for example , my own research , my own information about the nose surgery was much more than [ stated in a higher tone of voice and with more stress ] what others are saying . of course , the required ingredients for this type of trust are picked up from the specialized showcase of the doctor in a way that the participants , explaining the reasons for putting trust on the doctor , refer to doctor 's professional competencies outweighing over the financial benefits , the doctor 's fame in doing natural and specialized surgeries , and the use of certain techniques such as the application of smaller tampons or local anesthesia . in this type of the trust , participants , while presenting their wrapped trust to their selected doctor ; do not necessarily observe all the doctor 's prescriptions , stating that they have a comprehensive knowledge of the cosmetic surgeries : i knew all of these points completely , for example , my own research , my own information about the nose surgery was much more than [ stated in a higher tone of voice and with more stress ] what others are saying . if , in terms of the participants degree of subjectivity , vicarious trust can be placed at the bottom and the wrapped trust can be placed at the top , in all the experiences , participants , having considered the necessity of gaining trust as a presupposition , have made a unilateral effort to create this phenomenon . to be more exact , the doctors , having presented a showcase of the premade frameworks for drawing trust , have dealt with all the patients in a same manner and according to a unilateral premade plan , and have made no effort to give some special meaning to each dual relationship and cases , here as patients , are left alone in interpreting these premade frameworks of drawing trust ( e.g. age , gender , type of the concerns , fears , and the previous experiences of the patient ) , a new doctor as to this issue , it should be said that one of the major concerns of the participants is that there is no room for the conversation in which they can convey their demands and preferences , while , doctors , having referred to their technical - specialized competence , do not acknowledge the general knowledge of the participants and the conversation atmosphere is not created .

whole - body magnetic resonance imaging ( wbmri ) is now recognized as an important tool in both diagnosis and follow - up of various oncologic and non - oncologic conditions in children . there are a few studies comparing these two field strengths in adults for whole - body mr angiography , oxygen - enhanced mri of lungs , cardiac and coronary imaging , and abdominal diffusion - weighted imaging ( dwi ) . comparison of image quality and artifacts of wbmri performed at 1.5 and 3 t has also been performed in adults . however , to the best of our knowledge , there has been no study comparing the image quality of wbmri at these two magnetic strengths in children . it would be ideal to compare studies performed on the same patient analyzing the same pathology simultaneously at these two magnetic field strengths in order to avoid bias secondary to the temporal variation in imaged pathology . however , this would not be feasible , especially in children . we attempted to compare the technical quality at the two magnetic field strengths in the same patients performed at different times but at a time interval . the aim of our study was to retrospectively perform intra - subject comparison of technical quality , artifacts , and the visibility of selected fixed structures between wbmri coronal short tau inversion recovery ( stir ) sequences performed at 1.5 and 3 t. institutional research ethics board approval and waiver for consent were obtained for this study . a list of children who had undergone mri studies on both 1.5 and 3 t at our institution , a tertiary pediatric referral center , for various indications was obtained from a database that keeps record of all mri exams . children who had wbmri at least once each on our 1.5 and 3 t philips ( achieva ; philips medical system , best , the netherlands ) mri scanners from january 2008 to april 2011 were included in this study . selection of 1.5 t versus 3 t scanner for wbmri is done clinically based on 3 t compatibility ( based on certain devices and prosthesis in the body ) and availability of the scanner . of 22 children who fulfilled these criteria , one child with a gap of 22 months between two wbmri exams was excluded , as this was deemed to be a long time interval which could potentially cause significant changes in the body physique , physiology , and ability of the child to stay still during the exam . remaining 21 ( 6 boys , 15 girls ; age between 4.6 and 17 years with average age of 12.03 years at the time of first wbmri ) children who formed the final study group had a maximum gap of 13 months between the wbmri exams . the minimum interval between wbmri exams was 3 months , with an average interval of 8.6 months . this interval was thought to be not long enough to change significantly physical characteristics of the body in a child , thus allowing fair comparison of wbmri technical quality at two field strengths . the indications in 21 children included 4 cases of li - fraumeni syndrome , 5 neuroblastoma , 2 retinoblastoma , 3 rhabdomyosarcoma , and 1 each of acinic cell carcinoma , epithelioid hemangioendothelioma , tuberous sclerosis with epithelioid angiomyolipoma , nasopharyngeal undifferentiated sarcoma , ewing sarcoma , desmoplastic round cell tumor , and metastatic adenocarcinoma of colon . nineteen children had wbmri on both 1.5 and 3 t without sedation or anesthesia ; remaining two were under general anesthesia for both 1.5 and 3 t scans . wbmri was performed using 1.5 and 3 t philips ( achieva ; philips medical system ) mri scanners equipped with high - performance gradient systems , sliding table platform , and quadrature body coil integrated in the magnet bore obviating the need to reposition the patient with each station . the exam included single stir coronal sequence at multiple stations covering the entire body from vertex to toes at multiple stations . the images from multiple stations were retrospectively stitched using software ( mobiview ) provided by the vendor to obtain whole - body images . there was some overlap between the stations and stitching was done based on table position . the coronal stir sequence at 1.5 t was performed with the following parameters : time to repeat ( tr ) 3000 ms , echo time ( te ) 60 ms , inversion time ( ti ) 165 ms , field of view ( fov ) 400500 mm , matrix 380 290 , slice thickness of 5 mm with 1 mm gap , number of signal averages 6 , echo train length ( etl ) 26 , and pixel bandwidth 446 hz . stir parameters at 3 t included tr 9126 ms , te 70 ms , ti 230 ms , fov 400500 mm , matrix 460 360 , slice thickness of 6 mm with 1 mm gap , number of signal averages 2 , etl 27 , and pixel bandwidth 256 hz . approximate time for each station at 1.5 t was 5 min and at 3 t was 4 min . the total scan time varied from 20 to 35 min depending on the height of the child . the acquisition was free breathing , no sagittal images of the spine were acquired , and no contrast or bowel paralysis was used . wbmri images were reviewed by a pediatric radiologist ( 8 years of experience in reading pediatric body mri ) and a pediatric radiology fellow ( a year of experience in reading pediatric body mri ) independently on picture archival and communication system ( pacs ) . images at 1.5 and 3 t were reviewed by both at separate occasions with a gap of at least 4 weeks . individual station images as well as whole - body stitched images of each wbmri exam were reviewed to assess five aspects / categories : vertebral column visibility , liver visibility , visibility of distal tibia / fibula , artifact grading , and overall image quality . these five aspects were analyzed using the grading system summarized in tables 1 and 2 and some are illustrated in figures 1 and 2 . one that may be affected by breathing movement ( vertebral column ) and another that is unlikely to be affected by movement ( distal tibia / fibula ) . for soft tissue visibility , this scoring system was developed by us . before reviewing the actual images of the study group , both reviewers together reviewed 10 wbmri exams ( 5 at 1.5 t and 5 at 3 t ) outside the study group to ensure consensus regarding the scoring system used in this study . grading for fixed structure visibility grading for severity of artifacts and overall image quality ( a - d ) overall image quality comparison . ( a ) wbmri image at 1.5 t shows excellent image quality rated as grade 4 . ( b ) wbmri image at 3 t shows good image quality rated as grade 3 that shows some movement artifacts affecting the lower legs and feet ( arrows ) and prominent chemical shift artifacts ( arrowheads ) . ( c ) wbmri image at 3 t shows fair image quality rated as grade 2 because of artifacts like ghosting ( broken arrows ) , chemical shift ( arrowheads ) , moire fringe - like artifacts ( arrows ) , and reduced visibility of liver and marrow in legs . ( d ) wbmri image at 3 t shows poor image quality rated as grade 1/non - diagnostic because of extensive movement artifacts ( a - e ) artifacts ( arrows ) . ( d ) chemical shift at the interface of subcutaneous and muscle compartments in thighs . ( e ) moire fringe - like artifacts at the body surface inter - observer agreement between the two reviewers was calculated for all five categories of image analysis using kendall 's coefficient of concordance ( w ) . w - values less than 0.20 were considered as poor agreement , 0.210.40 as fair agreement , 0.410.60 as moderate agreement , 0.610.80 as substantial agreement , and greater than 0.81 as almost perfect agreement . average of the mean scores given by the two readers for each category was used to compare 1.5 and 3 t using non - parametric signed rank test . a list of children who had undergone mri studies on both 1.5 and 3 t at our institution , a tertiary pediatric referral center , for various indications was obtained from a database that keeps record of all mri exams . children who had wbmri at least once each on our 1.5 and 3 t philips ( achieva ; philips medical system , best , the netherlands ) mri scanners from january 2008 to april 2011 were included in this study . selection of 1.5 t versus 3 t scanner for wbmri is done clinically based on 3 t compatibility ( based on certain devices and prosthesis in the body ) and availability of the scanner . of 22 children who fulfilled these criteria , one child with a gap of 22 months between two wbmri exams was excluded , as this was deemed to be a long time interval which could potentially cause significant changes in the body physique , physiology , and ability of the child to stay still during the exam . remaining 21 ( 6 boys , 15 girls ; age between 4.6 and 17 years with average age of 12.03 years at the time of first wbmri ) children who formed the final study group had a maximum gap of 13 months between the wbmri exams . the minimum interval between wbmri exams was 3 months , with an average interval of 8.6 months . this interval was thought to be not long enough to change significantly physical characteristics of the body in a child , thus allowing fair comparison of wbmri technical quality at two field strengths . the indications in 21 children included 4 cases of li - fraumeni syndrome , 5 neuroblastoma , 2 retinoblastoma , 3 rhabdomyosarcoma , and 1 each of acinic cell carcinoma , epithelioid hemangioendothelioma , tuberous sclerosis with epithelioid angiomyolipoma , nasopharyngeal undifferentiated sarcoma , ewing sarcoma , desmoplastic round cell tumor , and metastatic adenocarcinoma of colon . nineteen children had wbmri on both 1.5 and 3 t without sedation or anesthesia ; remaining two were under general anesthesia for both 1.5 and 3 t scans . wbmri was performed using 1.5 and 3 t philips ( achieva ; philips medical system ) mri scanners equipped with high - performance gradient systems , sliding table platform , and quadrature body coil integrated in the magnet bore obviating the need to reposition the patient with each station . the exam included single stir coronal sequence at multiple stations covering the entire body from vertex to toes at multiple stations . the images from multiple stations were retrospectively stitched using software ( mobiview ) provided by the vendor to obtain whole - body images . there was some overlap between the stations and stitching was done based on table position . the coronal stir sequence at 1.5 t was performed with the following parameters : time to repeat ( tr ) 3000 ms , echo time ( te ) 60 ms , inversion time ( ti ) 165 ms , field of view ( fov ) 400500 mm , matrix 380 290 , slice thickness of 5 mm with 1 mm gap , number of signal averages 6 , echo train length ( etl ) 26 , and pixel bandwidth 446 hz . stir parameters at 3 t included tr 9126 ms , te 70 ms , ti 230 ms , fov 400500 mm , matrix 460 360 , slice thickness of 6 mm with 1 mm gap , number of signal averages 2 , etl 27 , and pixel bandwidth 256 hz . approximate time for each station at 1.5 t was 5 min and at 3 t was 4 min . the total scan time varied from 20 to 35 min depending on the height of the child . the acquisition was free breathing , no sagittal images of the spine were acquired , and no contrast or bowel paralysis was used . wbmri images were reviewed by a pediatric radiologist ( 8 years of experience in reading pediatric body mri ) and a pediatric radiology fellow ( a year of experience in reading pediatric body mri ) independently on picture archival and communication system ( pacs ) . images at 1.5 and 3 t were reviewed by both at separate occasions with a gap of at least 4 weeks . individual station images as well as whole - body stitched images of each wbmri exam were reviewed to assess five aspects / categories : vertebral column visibility , liver visibility , visibility of distal tibia / fibula , artifact grading , and overall image quality . these five aspects were analyzed using the grading system summarized in tables 1 and 2 and some are illustrated in figures 1 and 2 . one that may be affected by breathing movement ( vertebral column ) and another that is unlikely to be affected by movement ( distal tibia / fibula ) . for soft tissue visibility , this scoring system was developed by us . before reviewing the actual images of the study group , both reviewers together reviewed 10 wbmri exams ( 5 at 1.5 t and 5 at 3 t ) outside the study group to ensure consensus regarding the scoring system used in this study . grading for fixed structure visibility grading for severity of artifacts and overall image quality ( a - d ) overall image quality comparison . ( a ) wbmri image at 1.5 t shows excellent image quality rated as grade 4 . ( b ) wbmri image at 3 t shows good image quality rated as grade 3 that shows some movement artifacts affecting the lower legs and feet ( arrows ) and prominent chemical shift artifacts ( arrowheads ) . ( c ) wbmri image at 3 t shows fair image quality rated as grade 2 because of artifacts like ghosting ( broken arrows ) , chemical shift ( arrowheads ) , moire fringe - like artifacts ( arrows ) , and reduced visibility of liver and marrow in legs . ( d ) wbmri image at 3 t shows poor image quality rated as grade 1/non - diagnostic because of extensive movement artifacts ( a - e ) artifacts ( arrows ) . ( d ) chemical shift at the interface of subcutaneous and muscle compartments in thighs . inter - observer agreement between the two reviewers was calculated for all five categories of image analysis using kendall 's coefficient of concordance ( w ) . w - values less than 0.20 were considered as poor agreement , 0.210.40 as fair agreement , 0.410.60 as moderate agreement , 0.610.80 as substantial agreement , and greater than 0.81 as almost perfect agreement . average of the mean scores given by the two readers for each category was used to compare 1.5 and 3 t using non - parametric signed rank test . there was substantial agreement between the two reviewers for all five categories and both field strengths . w - values of marrow visibility , liver visibility , and overall image quality were higher for 3 t as compared to 1.5 t. w - values are summarized in table 3 . inter - observer agreement the mean scores for all five categories by both readers were higher for 1.5 t images [ table 4 ] . comparison was done between the mean scores at 1.5 and 3 t averaged between the two readers . the difference between the averages of mean scores of the two field strengths was statistically significant [ table 5 ] , indicating less artifact , better fixed structure visibility and overall image quality at 1.5 t as compared to 3 t. mean scores for all categories comparison of average of mean scores by two readers between 1.5 and 3 t artifacts listed by both readers were summarized and compared for two field strengths . motion - related artifacts were seen to be the most common on both sets of images , including patient movement , motion due to pulsation , and bowel peristalsis . one form or the other of motion - related artifacts was seen in all the examinations ( 100% ) on both 1.5 and 3 t. ghosting was present in six cases on 1.5 t ( 28% ) and nine cases on 3 t ( 42% ) . pulsation artifacts from vessels were seen in three cases ( 14% ) on 1.5 t images as compared to those seen in nine cases ( 42% ) on 3 t images . chemical shift artifacts , predominantly affecting the interface between subcutaneous tissue and muscles in the extremities , were seen in 1 case ( 5% ) on 1.5 t and in all 21 cases on 3 t ( 100% ) . interference pattern seen as alternate curved bands of bright and dark signal at the periphery of the images , similar to moire fringe artifacts resulting from field inhomogeneity , were seen in nine cases on 3 t ( 42% ) , but on none of the 1.5 t images . susceptibility artifacts related to bowel and marrow were seen in six cases on 3 t ( 28% ) , but on none of the 1.5 t images . wbmri is being used with increasing frequency in children for various oncologic and non - oncologic indications . it has been shown to be useful in the assessment of lymphoma . even though this application , even in combination with dwi , is limited by its inability to differentiate between normal and abnormal lymph nodes , wbmri has been shown to have high sensitivity and specificity for detection of malignant lymph nodes based on size criteria ( short - axis diameter > 1 cm ) . it is used for screening of children with cancer predisposition syndromes like li - fraumeni syndrome and in children with retinoblastoma for detection of metastases and osteosarcoma . some non - oncologic applications of wbmri in children include chronic recurrent multifocal osteomyelitis ( crmo ) , langerhans cell histiocytosis , generalized osteonecrosis after cancer treatment , generalized vascular malformations like hemangiomatosis , lymphangiomatosis , and klippel trenaunay syndrome , and fever of unknown origin . wbmri in combination with dwi has the potential to replace pet / ct in the future . pet / mri is likely to replace pet / ct in children because of lack of ionizing radiation from the ct component . coronal stir sequence alone or in combination with other sequences is the most commonly used one for wbmri . most pathologic tissues are proton rich and have prolonged t1 and t2 relaxation times resulting in high signal intensity on stir images . robust and homogeneous fat suppression is another advantage of stir sequence that is useful for evaluating bone marrow in children , most of whom have hypercellular marrow . it is performed with a body coil integrated within the magnet bore , which in combination with sliding table platform obviates repositioning of patients at each station . imaging with body coil has lower signal - to - noise ratio ( snr ) as compared to phased - array coil . despite this and other limitations like suboptimal depiction of sternum , ribs , scapula , and skull , and the lower sensitivity of coronal plane for detection of lymphadenopathy , coronal stir serves the purpose well as a quick screening or search sequence for marrow and soft tissue abnormalities . with newer state - of - the - art systems with the capability to cover entire body with surface coil , mr imaging at 3 t provides high snr , spatial resolution , and temporal resolution . improved diagnostic accuracy and image quality has been reported with 3 t for imaging of brain , heart , vessels , musculoskeletal structures , and mr cholangiopancreatography . wbmri at 3 t is expected to provide good - quality images in shorter time as compared to 1.5 t. however , these advantages are associated with increased motion - and pulsation - related artifacts , field inhomogeneity - related artifacts , increased susceptibility , increased chemical shift , and reduced t1 contrast due to longer t1 relaxation time at 3 t [ figure 3 ] . increased chemical shift , increased susceptibility , and difficulty in achieving homogeneous magnetic field at 3 t as compared to 1.5 t result in greater artifacts at 3 t. artifacts related to motion and susceptibility at 3 t can be minimized by use of parallel imaging where high signal at 3 t and parallel imaging act complementary to each other . ( a and b ) overall comparison of images at 1.5 and 3 t. wbmri images at 1.5 t ( a ) and at 3 t ( b ) in a 10-year - old boy do not show any abnormality . the overall image quality was rated as 3 for both images . however , there are some inherent differences between the two images , including better lung visibility at 1.5 t , darker bones at 3 t ( seen in proximal humeri , pelvic bones , and upper lumbar vertebrae ) , better signal in arms at 3 t , and greater chemical shift artifact at 3 t ( arrows ) the previous comparison study between 1.5 and 3 t wbmri in adults by schmidt et al . even though overall image quality at 1.5 t was significantly better than at 3 t , it was rated as good on both field strengths in their study , suggesting feasibility of 3 t for wbmri . the difference in the image quality of stir between 1.5 and 3 t was due to significantly more artifacts affecting stir at 3 t , including dielectric effects , pulsation artifacts , and image inhomogeneity . similar to that study , our results showed significantly better overall image quality at 1.5 t and also suggested feasibility of 3 t for performance of wbmri , with overall image quality rated at 2.74 for 3 t versus 3.10 for 1.5 t , both of which are within a good range . most artifacts were seen with greater frequency at 3 t in our study , with some like chemical shift , susceptibility , and moire fringe - like artifacts seen almost exclusively at 3 t. abdominal dwi performed with free breathing has been shown to be of better quality with less artifacts at 1.5 t as compared to 3 t. however , in the same study , image quality of dwi with breath hold and use of parallel imaging was better at 3 t as compared to 1.5 t , with similar artifacts scores . our wbmri coronal stir sequence is performed with free breathing even in stations involving the chest and abdomen . moreover , we could not use parallel imaging because our wbmri is performed with the body coil . since we could not compare assessment of pathology in this study , we tried to evaluate fixed structure visibility , which in our opinion indirectly reflects diagnostic ability of wbmri at 1.5 and 3 t. two regions of bone marrow space were selected one that may be affected by breathing movement ( vertebral column ) and another that is unlikely to be affected by movement ( distal tibia / fibula ) . for soft tissue visibility , visibility of all three regions was significantly better at 1.5 t than at 3 t. however , similar to overall image quality , fixed structure visibility for both field strengths was within a good range [ table 5 ] . higher visibility scores at 1.5 t for liver and vertebral column were mainly due to fewer artifacts related to breathing and other motion - related artifacts , while higher scores for marrow visibility in distal tibia and fibula at 1.5 t were due to less inhomogeneity and darkening as compared to 3 t. higher inter - observer agreement ( w - values ) for scores at 3 t may be related to more severe degree of artifacts and relatively less visibility of fixed structures that are usually more obvious . the study period falls between 2008 and 2011 . in the last few years since then , mri technology has improved and some of the artifacts may have been reduced and image quality has improved . it does not directly compare assessment of pathology on two field strengths or impact of image quality and artifacts on lesion detection . nonetheless , it provides comparative data on image quality and artifacts on the most commonly used sequence for wbmri at two most commonly used field strengths in children . similar to previous studies in adults , wbmri performed with coronal stir sequence at 1.5 t has significantly better image quality , fixed structure visibility and fewer artifacts , as compared to wbmri at 3 t in children . this difference is unlikely to significantly affect detection of pathology on 3 t wbmri , as the image quality score at 3 t was also within a good range .

purpose : to compare whole - body magnetic resonance imaging ( wbmri ) performed at 1.5 and 3 t for technical quality , artifacts , and visibility of selected fixed structures.patients and methods:21 children who had wbmri at both 1.5 and 3 t scanners within a relatively short interval ( 3 - 13 months ; average-8.6 months ) were included . the images were objectively compared with scores from 4 to 1 for five parameters including severity of artifacts ; visibility of liver , vertebral column , and marrow in legs ; and overall image quality . inter - observer agreement was calculated using kendall 's coefficient of concordance ( w ) and scores were compared using signed rank test.results:there was substantial inter - observer agreement for all five categories at both field strengths . the difference between averages of mean scores of all five parameters for two field strengths was statistically significant ( p < 0.05 ) , indicating less artifact , better fixed structure visibility , and overall image quality at 1.5 t as compared to 3 t. however , scores at 3 t were also rated within a good range ( around 3 ) indicating its feasibility for wbmri in children.conclusion:wbmri at 1.5 t has significantly better image quality , fixed structure visibility , and fewer artifacts , as compared to wbmri at 3 t in children . this difference is unlikely to significantly affect detection of pathology on 3 t wbmri as the image quality score at 3 t was also within good range .

Introduction Patients and Methods Patients Magnetic resonance imaging technique Image analysis Statistical analysis Results Discussion Conclusion Financial support and sponsorship Conflicts of interest

the aim of our study was to retrospectively perform intra - subject comparison of technical quality , artifacts , and the visibility of selected fixed structures between wbmri coronal short tau inversion recovery ( stir ) sequences performed at 1.5 and 3 t. institutional research ethics board approval and waiver for consent were obtained for this study . ( e ) moire fringe - like artifacts at the body surface inter - observer agreement between the two reviewers was calculated for all five categories of image analysis using kendall 's coefficient of concordance ( w ) . ( c ) wbmri image at 3 t shows fair image quality rated as grade 2 because of artifacts like ghosting ( broken arrows ) , chemical shift ( arrowheads ) , moire fringe - like artifacts ( arrows ) , and reduced visibility of liver and marrow in legs . inter - observer agreement between the two reviewers was calculated for all five categories of image analysis using kendall 's coefficient of concordance ( w ) . w - values of marrow visibility , liver visibility , and overall image quality were higher for 3 t as compared to 1.5 t. w - values are summarized in table 3 . the difference between the averages of mean scores of the two field strengths was statistically significant [ table 5 ] , indicating less artifact , better fixed structure visibility and overall image quality at 1.5 t as compared to 3 t. mean scores for all categories comparison of average of mean scores by two readers between 1.5 and 3 t artifacts listed by both readers were summarized and compared for two field strengths . wbmri at 3 t is expected to provide good - quality images in shorter time as compared to 1.5 t. however , these advantages are associated with increased motion - and pulsation - related artifacts , field inhomogeneity - related artifacts , increased susceptibility , increased chemical shift , and reduced t1 contrast due to longer t1 relaxation time at 3 t [ figure 3 ] . however , there are some inherent differences between the two images , including better lung visibility at 1.5 t , darker bones at 3 t ( seen in proximal humeri , pelvic bones , and upper lumbar vertebrae ) , better signal in arms at 3 t , and greater chemical shift artifact at 3 t ( arrows ) the previous comparison study between 1.5 and 3 t wbmri in adults by schmidt et al . even though overall image quality at 1.5 t was significantly better than at 3 t , it was rated as good on both field strengths in their study , suggesting feasibility of 3 t for wbmri . the difference in the image quality of stir between 1.5 and 3 t was due to significantly more artifacts affecting stir at 3 t , including dielectric effects , pulsation artifacts , and image inhomogeneity . similar to that study , our results showed significantly better overall image quality at 1.5 t and also suggested feasibility of 3 t for performance of wbmri , with overall image quality rated at 2.74 for 3 t versus 3.10 for 1.5 t , both of which are within a good range . most artifacts were seen with greater frequency at 3 t in our study , with some like chemical shift , susceptibility , and moire fringe - like artifacts seen almost exclusively at 3 t. abdominal dwi performed with free breathing has been shown to be of better quality with less artifacts at 1.5 t as compared to 3 t. however , in the same study , image quality of dwi with breath hold and use of parallel imaging was better at 3 t as compared to 1.5 t , with similar artifacts scores . since we could not compare assessment of pathology in this study , we tried to evaluate fixed structure visibility , which in our opinion indirectly reflects diagnostic ability of wbmri at 1.5 and 3 t. two regions of bone marrow space were selected one that may be affected by breathing movement ( vertebral column ) and another that is unlikely to be affected by movement ( distal tibia / fibula ) . for soft tissue visibility , visibility of all three regions was significantly better at 1.5 t than at 3 t. however , similar to overall image quality , fixed structure visibility for both field strengths was within a good range [ table 5 ] . higher visibility scores at 1.5 t for liver and vertebral column were mainly due to fewer artifacts related to breathing and other motion - related artifacts , while higher scores for marrow visibility in distal tibia and fibula at 1.5 t were due to less inhomogeneity and darkening as compared to 3 t. higher inter - observer agreement ( w - values ) for scores at 3 t may be related to more severe degree of artifacts and relatively less visibility of fixed structures that are usually more obvious . similar to previous studies in adults , wbmri performed with coronal stir sequence at 1.5 t has significantly better image quality , fixed structure visibility and fewer artifacts , as compared to wbmri at 3 t in children . this difference is unlikely to significantly affect detection of pathology on 3 t wbmri , as the image quality score at 3 t was also within a good range .

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

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 . a defect in the yeast homolog of itpa , ham1 , leads to elevated hapmutagenesis , but it does not affect spontaneous mutagenesis . 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 . 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 . 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 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 . 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 ) . 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 . 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 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 . 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 . 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 . 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 . 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 . 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 . 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 .

the dermal healing response is a multistep process ( inflammation , granulation tissue proliferation , epithelialization , and remodeling of the wound site ) which may result in a number of different outcomes : complete healing , scarred healing , or a chronic nonhealing wound [ 1 , 2 ] . in nonhealing wounds , such as pressure and diabetic ulcers , neutrophils accumulate in the wound site and leave the wound stuck in a state of chronic inflammation . while inflammation normally resolves within 1 - 2 days as neutrophil number decreases , the prolonged presence of these cells contributes to a disordered network of regulatory cytokines . this aberrant set of regulatory signals has far - reaching effects on all the cells involved in dermal healing ( macrophages , fibroblasts , etc . ) and results in increased proteolytic activity and improper extracellular matrix ( ecm ) deposition . in order for these wounds to heal current clinical treatments often center around surgical debridement , exudate management , and minimization of bacterial adherence ( biofilm ) to remove inflammatory stimuli . other treatments involve the utilization of recombinant growth factors , synthetic protease inhibitors , or ph modifying ointments [ 1 , 2 ] . however , to date , there has been no single treatment that has proven to be optimal at stimulating the resolution of chronic wounds , and the future may lie in regenerative medicine 's ability to modify cellular behavior within the wound site . honey had been used medicinally for centuries , due to its inherent wound healing capacity . recently , with the emergence of antibiotic - resistant bacteria and a better scientific understanding of how honey influences healing , honey ( specifically active leptospermum honey from new zealand , known as manuka ) has once again become an acceptable product in the treatment of wounds . honey has a high osmolarity and a high sugar content , the combination of which has been shown to inhibit microbial growth [ 5 , 9 , 10 ] . manuka honey is also known to have a relatively low ph ( 3.54.5 ) , which , in addition to inhibiting microbial growth , will stimulate the bactericidal actions of macrophages , and in chronic wounds reduce protease activity , increase fibroblast activity , and increase oxygenation [ 5 , 1012 ] . hydrogen peroxide is slowly released from honey placed on a wound through the interaction of wound exudates with the honey 's inherent glucose oxidase . this hydrogen peroxide is in sufficient concentration to be antibacterial , yet dilute enough to be nontoxic while promoting fibroblast proliferation and angiogenesis [ 3 , 5 , 9 , 10 , 12 ] . manuka honey also possesses nonperoxide antibacterial activity in what is called the unique manuka factor ( umf ) due to the presence of methylglyoxal [ 6 , 12 ] . honey has been shown to contain a number of phenolic compounds , which are known to scavenge and remove reactive oxygen species ( ros ) released by neutrophils . . demonstrated that honey can suppress oedema and leukocyte infiltration in a mouse model of neutrophilic inflammation . tonks et al . demonstrated that monocytes cultured in the presence of honey were stimulated to produce a number of pro and anti - inflammatory cytokines ( tumor necrosis factor alpha ( tnf- ) , interleukin-1 beta ( il-1 ) , and interleukin-6 ( il-6 ) ) and may indicate modulation towards resolution in nonhealing wounds . platelet - rich plasma ( prp ) therapy has been gaining momentum as a bedside regenerative medicine procedure and has been used to stimulate regeneration of osteochondral defects [ 1315 ] , tendon / ligament injuries [ 1319 ] , and chronic dermal wounds ( diabetic and pressure ulcers ) [ 14 , 15 , 20 , 21 ] in clinical studies . prp is a simple and cost - effective method for collecting and concentrating autologous platelets ( some clinical studies have published on the use of pooled - banked prp in order to overcome donor variability with no evidence of immunoreactions [ 2225 ] ) for the purpose of activating and releasing their growth factor - rich alpha and dense granules . these granules releases a number of growth factors and cytokines , including : platelet - derived growth factor ( pdgf ) , transforming growth factor beta ( tgf- ) , vascular endothelial growth factor ( vegf ) , fibroblast growth factor ( fgf ) , epidermal growth factor ( egf ) , and others [ 13 , 15 , 16 , 20 , 2628 ] . these listed factors , in conjunction with the numerous factors contained in prp not listed , are known to accelerate cell migration and proliferation , promote ecm production , as well as play a role in macrophage phenotype and inflammation resolution [ 13 , 26 , 27 , 2933 ] . there is currently no consensus as to how prp should be most effectively utilized in the treatment of wounds . there have been a number of methods reported on delivering prp ; most involve the creation of an activated - platelet gel with thrombin [ 1315 , 34 ] or cacl2 [ 1315 , 28 ] . however , studies have shown that the use of these prp gels are inefficient due to the rapid release and diffusion of the factors . several techniques have been evaluated for sustained release , including gelatin gel microspheres , lyophilized prp [ 3639 ] , and alginate beads . collectively , these studies demonstrated the importance of keeping preparations rich in growth factors ( prgf ) in the wound site and slowly activating / releasing them as the wound site becomes infiltrated with reparative cells . the purpose of this study was to determine the in vitro response of three cell types critical to wound healing ( fibroblasts , endothelial cells , and macrophages ) when subjected to culture media supplemented with manuka honey , a powdered prgf ( a lyophilized version of prp ) , or a combination of manuka honey and prgf . the hypothesis being that manuka honey and prgf will increase cellular activity over control media , with a corollary that the combination of honey and prgf will provide the greatest increase due to increased growth factor and cytokine activity through acid activation . manuka honey has been documented to have an acidic ph , and factors such as tgf- are known to become physiologically active when subjected to an acid treatment . briefly , fresh human whole blood from 3 donors was purchased ( biological specialty corp . , colmar , pa , usa ) , pooled , and used in a smartprep 2 ( harvest technologies corp . , prp was then subjected to a freeze - thaw - freeze ( ftf ) cycle in a 70c freezer for cell lysis ( centrifuge tubes containing prp were placed in a 70c freezer for 24 hrs followed by a 37c water bath for 1 hr , and then returned to the 70c freezer for 24 hrs ) . frozen prp was then lyophilized for 24 hrs to create a dry prgf powder which was finely ground in a mortar and pestle prior to use . this dry prgf powder was added to the test media used in this study as a weight percentage ( w / w % ) . the honeys used in this study were a pure manuka honey ( wedderspoon organic , umf 16 + , vancouver island , bc , canada ) and medihoney ( mh , dermasciences inc . , princeton , nj , usa ) , which is a medical grade , sterile , manuka honey that has been filtered to remove any potential residual pollen or bee byproducts . honeys were added to test medias as a volume percentage ( v / v % ) . it has been previously documented that tgf- can be transformed from a nonactive state to a physiologically active form through ph modification ( tgf- elisa instructions ) . to determine what , if any , activation potential the honey ( ph known to be 3.54.5 ) afforded , prp was mixed in varying ratios ( 1 : 1 , 1 : 5 , 1 : 10 , 5 : 1 , and 10 : 1 ) with mh and subjected to a modified western blot to determine physiologically active tgf- through fluorescence . briefly , the prp : mh solutions were blotted on a pvdf membrane , along with prp that was acid activated using a modified tgf- elisa protocol ( promega , madison , wi , usa ) . the membrane was blocked in odyssey blocking buffer for one hour at room temperature . after blocking , samples were incubated in anti - human tgf- antibody ( promega , madison , wi , usa ) at room temperature for 1.5 hours . all samples were then washed four times with 0.1% tween-20 in pbs , after which the signal from anti - human tgf- antibody was detected with goat anti - mouse igg secondary antibody tagged with a fluorescent 800 nm marker ( thermo scientific , waltham , ma , usa ) . to account for antibody background fluorescence , each sample samples were incubated in the secondary antibody for 1 hour at room temperature without exposure to light . after washing , the samples were scanned using the 800 nm channel of the odyssey infrared imaging system ( li - cor biosciences , lincoln , ne , usa ) at an intensity of 3.5 . background fluorescence that was obtained from samples incubated with secondary antibody only was subtracted from the signal intensities of the samples incubated with both primary and secondary antibodies . human dermal fibroblasts ( hdf ) were seeded subconfluently in a 48-well plate at 50,000 cells / well in 500 l of control media ( dmem / f12 supplemented with 10% fetal bovine serum ( fbs ) , 1% penicillin / streptomycin ( p / s ) ) . following adhesion of cells to the well - plate ( ~2 hrs ) control media was replaced with test media ( control media supplemented with manuka honey in 0.1 , 1 , 5 , 10 , or 20% v / v , mh in 0.1 , 1 , 5 , 10 , or 20% v / v , prgf in 0.1 , 1 , 5 , or 10 mg / ml or a combination of 0.1% mh and 1 mg / ml prgf ) . test media was changed on days 1 , and 4 , with an mts assay ( celltiter 96 aqueous non - radioactive cell proliferation assay , promega , madison , wi , usa ) performed on days 1 , 4 , and 7 to determine mean cell count . using a protocol nearly identical to that of the hdf proliferation study , immortalized human pulmonary microvascular endothelial cells ( hpmec , donated generously from dr . c. j. kirkpatrick ) were seeded subconfluently at 25,000 cells / well in a 48-well plate in 500 l of control media ( m199 media supplemented with 10% fbs , 1% p / s , glutamax 100x , g418 , heparin sodium salt ( 50 g / ml ) , and endothelial cell growth supplement ( ecgs , 50 g / ml ) ) . following adhesion of cells to the well - plate ( ~2 hrs ) control media was replaced with test media ( control media supplemented with manuka honey in 0.1 , 1 , 5 , 10 , or 20% v / v , mh in 0.1 , 1 , 5 , 10 , or 20% v / v , prgf in 0.1 , 1 , 5 , or 10 mg / ml or a combination of 0.1% mh and 1 mg / ml prgf ) . test media was changed on days 1 , and 4 , with an mts assay performed on days 1 , 4 , and 7 to determine mean cell count . human peripheral blood macrophages ( atcc , crl9855 ) were seeded subconfluently in a 48-well plate at 50,000 cells / well in 500 l of control media ( rpmi 1640 , supplemented with 10% fbs , 1% p / s ) . following adhesion of cells to the well - plate ( ~2 hrs ) control media was replaced with test media ( control media supplemented with either manuka honey or mh in 0.1 , 1 , 5 , 10 , or 20% v / v ) . test media were changed on days 1 , 4 , and 7 , and an mts assay was performed on the same time points to determine a mean cell count . changed media was retained and used for subsequent elisa analysis to determine what role the honey may play in stimulating macrophage inflammation . prgf supplemented media was not investigated with this cell type as previous work has demonstrated that the presence of prgf had little impact on macrophage proliferation . using the retained media from the proliferation study , elisas were run per manufacturer 's protocol to determine the inflammatory response of macrophages to either pure manuka honey or mh . elisas were conducted to determine levels of tnf- ( antigenix america inc . , huntington station , ny , usa ) , an indicator of inflammation and m1 phenotype , interleukin-10 ( il-10 , peprotech , rocky hill , nj , usa ) , an interleukin commonly associated with inflammation resolution and a regulatory phenotype , and vegf ( antigenix america inc . ) , which is critical to angiogenesis and is expressed by m2 macrophages . similar to the aforementioned cell proliferation studies , cell chemotaxis studies were conducted on both macrophages and hpmecs ( hdf chemotaxis was evaluated with an in vitro wound healing assay ) using a transwell plate and an mts assay . human macrophages were seeded in the top insert of a transwell plate ( 8 m diameter pores , corning inc . , lowell , ma , usa ) with 50,000 cells / well in 200 l control media , while the bottom insert was filled with 600 l of test media ( control media supplemented with manuka honey in 0.1 , 1 , 5 , 10 , or 20% v / v , mh in 0.1 , 1 , 5 , 10 , or 20% v / v , prgf in 0.1 , 1 , 5 , or 10 mg / ml or a combination of 0.1% mh and 1 mg / ml prgf ) . media from both the top insert and the bottom well were changed on days 1 , and 2 , and the cells were counted using an mts assay on day 3 . the mts assay was performed on both inserts and the bottom wells to distinguish between cell chemotaxis and proliferation . an in vitro wound healing assay was performed to determine the rates of migration of hdfs into a wound site , simulating hdf migration into dermal wounds in vivo . a 48-well plate was coated with fibronectin ( 10 g / ml ) and blocked with bovine serum albumin ( 2 mg / ml ) prior to cell seeding . the exterior underside of each well was marked with a permanent marker to serve as a reference line . each well was then seeded to confluency with 100,000 hdfs in a standard dmem / f12 control media ( containing 1% p / s , and 2% fbs to minimize cellular proliferation ) . the cells were allowed to attach and spread overnight , creating a completely uninterrupted layer of cells . after 18 hrs , a 200 l pipette tip was scraped across the well , perpendicular to the reference line to create an intersection point , to create the in vitro wound . the wells were gently washed with control media to remove any dislodged cells , and test media was added . test media consisted of control media supplemented with 1% v / v mh , 1 mg / ml prp , and a combination of 1% v / v mh and 1 mg / ml prp ( chosen based upon results of hdf proliferation study ) . images were taken at time 0 , 6 , 12 , 18 , and 30 hr using an inverted nikon microscope with a 4x objective and color camera . using the reference intersection point , images were taken at the same location at each successive time point . using image analysis software ( imagetool ) , the area of each wound was measured , and a rate of healing was determined . to determine the amount of ecm produced by the hdfs , a hydroxyproline assay was performed based upon previously published protocols [ 4446 ] . briefly , hdfs were seeded near confluency at 100,000 cells / well in a 48-well plate in 500 l of control media . following adhesion of cells to the well - plate ( ~2 hrs ) control media were replaced with test media ( control media supplemented with 1% mh v / v , 1 mg / ml prgf , or a combination of 1% mh and 1 mg / ml prgf ) . these specific test media were chosen for evaluation based upon results of previous hdf experiments conducted in this study . media was changed every other day and retained for assessment with the assay on days 1 , 7 , 14 , 21 , and 28 . retained media was hydrolyzed in 100 l of 6 m hydrochloric acid in a boiling water bath and subsequently lyophilized until dry . dry samples were diluted in 50 l of distilled water , and 450 l of chloramine t reagent were added and allowed to oxidize in the dark at room temperature for 25 minutes . 500 l ehrlich 's reagent added to each sample , mixed gently , and incubated for 20 minutes at 60c . 100 l of each sample were then transferred to a 96-well plate and read at 570 nm on a spectrophotometer ( spectramax plus , molecular devices , sunnyvale , ca , usa ) . a preliminary in vitro angiogenesis bead assay was performed , similar to that of chen , et al . . louis , mo , usa ) were autoclaved and hydrated overnight , 10 l of bead solution was placed in a 15 ml tube and rinsed 3 times with complete hpmec medium before being mixed with approximately 1 million hpmec . the tube was incubated in standard conditions ( 37c , 5% co2 ) , with inversion of the tube occurring every half hour over 3 hours to ensure cells attached to the beads . the media was then removed from the centrifuge tube , placed in a flask , and incubated overnight . after incubation , beads were rinsed in pbs and transferred back to a 15 ml tube . once the beads had settled to the bottom , any remaining media were removed , and beads were washed once more with pbs . after allowing the beads to settle to the bottom of the tube a second time , the beads with adherent cells were resuspended in a collagen gel solution ( 80% liquid purecol with 10% 0.1 m naoh and 10% 10x pbs ) containing 1000 kiu / ml aprotinin ( sigma - aldrich , st . simultaneously , 250 l of collagen gel solution with aprotinin was placed in the bottom of a 24 well plate and incubated at standard conditions until gelation occurred . once the first layer was gelled , 250 l of cell suspension was placed on the first collagen gel layer and was incubated for 2 hours to again allow for gelation . supplemented media was then created as described previously ( manuka honey in 0.1 , 1 , 5 , 10 , or 20% v / v , mh in 0.1 , 1 , 5 , 10 , or 20% v / v , prgf in 0.1 , 1 , 5 , or 10 mg / ml or a combination of 0.1% mh and 1 mg / ml prgf ) and 500 l was placed on top of the gel solution containing the cell covered beads and incubated under standard conditions for 6 days . at days 1 , 4 , and 6 , images were captured using a nikon eclipse te200 microscope with a dage - mts digital camera . three images were taken for each test media at each time point . to quantify the percentage of hpmec sprouts per bead , a circular grid was overlayed onto each bead , dividing it into 36 sections . all sprouts extending from the surface of the beads were counted , summed , and divided by 36 to determine the percentage of sprouts for each bead . in addition , the length of each sprout extending from the surface of the beads was quantified using imagetool 3.0 software ( shareware provided by uthscsa ) . all statistical analysis was based on a kruskal - wallis one - way anova on ranks and a tukey - kramer pairwise multiple comparison procedure ( = 0.05 ) performed with the jmp in 8.0 statistical software package ( sas institute , inc . ) . graphical depictions of mean data were constructed with microsoft excel 2007 , with error bars representing standard deviations from the mean . the western blot analysis of honey - induced prp activation is shown in figure 1 . while the modified western blot analysis used in this study does not provide an exact quantification of physiologically active tgf- , the differences in sample fluorescence can be used to provide a general indication about the presence of tgf- activated through either acid or honey treatment . statistical analysis revealed there to be no significant differences in activated tgf- regardless of whether prp was activated through acid treatment or mixing with mh . additionally , both the acid and mh - treated prp was significantly more fluorescent than the unactivated prp . results of the hdf , hpmec , and macrophage proliferation studies are shown in figure 2 . hdf proliferation was observed over 7 days in response to test medias containing varying concentrations of manuka honey , mh , prgf , and a combination of prgf+mh , and cell numbers ranged from near 0 for the higher manuka / mh concentrations to greater than 224,000 for the prgf+mh on day 7 . statistical analysis revealed the combination of prgf+mh to significantly enhance cell proliferation over all other groups at days 4 and 7 . additionally , the presence of prgf alone had a greater impact on cell proliferation than mh alone , as 0.1 , 1 , and 5 mg / ml prgf had significantly higher cell numbers than the 0.1 or 1% mh . there were no significant differences between manuka honey and mh - supplemented media of the same concentration at any time point . the results of the hpmec proliferation study were similar to those of the hdf study , with cell numbers ranging from near 0 for the higher manuka / mh concentrations to over 101,000 for the prgf+mh test media . however , the differences between the prgf+mh and the other test media were not as pronounced as with the hdf study . while the prgf+mh had the highest average cell number at day 7 , it was not statistically different from the 1 mg / ml prgf test media at day 7 ( p = 0.1343 ) . additionally , there were few differences seen between test medias on day 4 as well ; with the prgf+mh , 1 and 5 mg / ml prgf , 0.1 and 1% mh , and control all being statistically the same . as before , there were no significant differences between the manuka honey and mh supplemented medias . the macrophage proliferation study was conducted without the addition of prgf , as a previous study revealed that macrophage proliferation was not significantly influenced by the presence of prgf . therefore , macrophage proliferation was compared between test medias containing pure manuka honey and mh . macrophage cell numbers ranged from near 0 for higher honey concentrations to over 286,000 for the 0.1% manuka honey at day 4 . statistical analysis revealed there to be no significant differences between test medias of manuka honey and mh at the same concentration . macrophage proliferation peaked at day 4 for the 0.1 and 1% manuka and mh test medias , at which point cells became contact inhibited and cell number decreased for day 7 . media used in the macrophage proliferation study was retained and analyzed through elisa for the presence of tnf- , il-10 , and vegf ; three cytokines that can provide insight into macrophage function and phenotype . for the tnf- elisa , the 1% manuka test media yielded the only significant result , with a peak tnf- concentration of 0.48 ng / ml on day 1 and significant decreases in tnf- at days 4 and 7 . the il-10 elisa results ranged from 0 for 5% manuka on day 7 to 0.13 ng / ml for the control at day 7 . statistically , the control media on days 4 and 7 were significantly different from all other groups , with the exception of the 0.1% manuka on day 7 , 0.1% mh on day 7 , and the 20% manuka on day 1 . the 20% manuka day 1 results are most likely outliers as the 20% manuka proved to be cytotoxic . while there appears to be a trend of increasing il-10 release from the lower honey concentrations ( 0.1 and 1% ) , these increases were not statistically significant . values of vegf release ranged from 0.06 to 0.19 ng / ml for 5% manuka on day 1 , and 0.1% manuka on day 4 , respectively . test media were not statistically different from one another , with the exception of the 0.1% manuka on day 4 and the 10% mh on day 1 , and these were most likely outliers as there is no logical explanation for such a random spike in vegf production . while there does appear to be a trend of increasing vegf over time for all samples , the differences were not significant . the presence of manuka / mh did not induce significant macrophage chemotaxis , but did increase cell proliferation in the insert at the lower mh concentrations . similar to the results of the cell proliferation studies , there were no significant differences between the pure manuka honey and the mh medias . the higher honey concentrations ( 5 , 10 , and 20% v / v ) resulted in cell death and are therefore not included in figure 4 . the 10 mg / ml prgf resulted in a statistically significant increase in chemotaxis to the bottom well over the control media , but did not induce a statistically significant increase in cell proliferation in the insert . the combination of prgf+mh contributed mainly to the proliferation of macrophages , with the prgf+mh insert experiencing the largest increase in cell number ( significant over all other groups ) with chemotaxis that was not significantly different from those of the control media . the 10 mg / ml prgf formed a clot when added to the media , making it difficult to ensure accurate cell counting with the mts . while reasonable numbers were achieved , their accuracy can not be assured due to the clot formation and potential loss of cells . hpmec chemotaxis was also minimal , with only the 5 and 10 mg / ml prgf inducing a statistically significant amount of cells to travel from the insert to the bottom well . these test media actually formed a semisolid clot , making it difficult to accurately assess cell number in the bottom well as cells seemed to readily migrate into the clot . the 1% mh , prgf+mh , and 0.1 mg / ml prgf all stimulated significant cellular proliferation over control media in the insert , but did not induce chemotaxis . future studies will utilize a longer time course for chemotaxis studies , as the 72 hrs in this study may not have been long enough to see cellular chemotaxis on a quantifiable scale . results of the in vitro wound healing assay are shown in figure 5 . the combination of prgf+mh demonstrated more rapid hdf infiltration into the denuded area compared to all other groups at time points 12 and 18 hrs . by the 30 hr time point , the wounded area in the prgf+mh group was indistinguishable from surrounding areas , whereas the other groups still contained small areas of exposed culture plastic . however , while there was a trend towards increased healing with the prgf+mh over all other medias , these differences were not all statistically significant due to a limited sample population . the prgf+mh exhibited significantly more healing than the controls at 30 hr , but was not significantly different from the prgf or the mh medias at the same time point . at the 18 hr time point both the prgf+mh and prgf were significantly different from control media , while not statistically different from one another . the largest jump in area healed occurred with the prgf+mh at the 12 hr time point ( 69% area healed compared to 33% for control ) , which was significantly different from all other medias at that time . mean hydroxyproline concentrations ranged from 3.5 g / ml for the prgf+mh with no cells to 7.9 g / ml for the prgf+mh on day 7 . results indicate that hydroxyproline , and therefore collagen , content peaked on day 7 for the prgf and prgf+mh and then decreased to an almost steady concentration , potentially due to cell proliferation resulting in contact inhibition and apoptosis or loss of aggregated collagen through subsequent media changes . statistical analysis revealed the prgf and prgf+mh to be statistically the same at the day 7 time point , while superior to the mh and control medias at the same time point . mean sprout densities ranged from 2.7% for controls on day 1 to 90.7% for the 1 mg / ml prgf on day 6 . statistical analysis revealed the 0.1 mg / ml prgf , 1 mg / ml prgf , and prgf+mh to be significantly different from the control at day 6 , with the 1 mg / ml prgf and prgf+mh not being different from each other . at day 4 , only the 1 mg / ml prgf was significantly different from the control , while none of the test medias were significantly different from the control at day 1 . mean sprout lengths ranged from 12.6 m for control on day 1 to 100.5 m for 1 mg / ml prgf on day 6 . statistical analysis of the mean sprout length data was fairly inconclusive due to the large deviations in both sprout number and length from bead to bead , however ; it was determined that the 1 mg / ml prgf was significantly different from the control on day 6 . trends in sprout length mean tend to indicate that both sprout length and density increase with prgf concentration . throughout this study , the 5 and 10 mg / ml prgf test medias suffered from gel formation which made their analysis extremely difficult and reported results unreliable . surprisingly , the manuka and mh supplemented medias proved to be rather unsuccessful at promoting sprout formation , as the higher concentrations ( 5 , 10 , and 20% v / v ) resulted in hpmec death and the lower concentrations failed to demonstrate increases in sprout density or length over control media . prp , and the powdered prgf used in this study , is an attempt to harness the healing potential of the platelets and their inherent growth factors to initiate and accelerate the body 's normal healing response . while prp and prgf have been investigated previously , this study was the first attempt at enhancing their bioactivity through the addition of manuka honey , which in and of itself has been shown to be effective in treating chronic dermal wounds in a number of clinical settings . this preliminary in vitro work investigated the potential for prgf and manuka honey / mh to stimulate the activity of hdf , hpmecs , and macrophages in ways that would provide insight into their specific roles in the healing process . cell proliferation , chemotaxis , cytokine release , and ecm production were all tested in the presence of prgf and manuka honey media supplements in order to determine what response , if any , each cell type had to the additives and which additive / concentration proved to be most efficacious . prp , in its liquid form has been used previously as a media supplement in the culture of a number of cell lines ( hdfs , bone marrow and adipose derived mesenchymal stem cells , patellar tendon fibroblasts , anterior cruciate ligament fibroblasts ) [ 34 , 4856 ] . these studies have all documented that the addition of prp to culture media resulted in increased rates of cell proliferation or was at minimum effective in maintaining rates of cell proliferation in the place of a traditional serum supplement . in this study , rather than utilize prp , a dry prgf powder was created by subjecting prp to a freeze - thaw - freeze process and subsequent lyophilization . the creation of this powder allows for easier handling of the material , while further concentrating its contents through the removal of the liquid plasma portion . despite this processing , it has been previously demonstrated that prgf retains its bioactivity and can successfully impact cellular activity [ 41 , 42 ] . the use of the prgf in this study clearly demonstrates effectiveness in stimulating cell proliferation , chemotaxis , matrix production , and angiogenesis . the presence of prgf - supplemented media promoted significant increases in the proliferation of hdfs and hpmecs at lower concentrations , while higher concentrations resulted in the formation of a clot that impeded accurate cell quantification . while this clotting action was definitely a hindrance to obtaining accurate cell proliferation / chemotaxis results in this study , it also demonstrated the retained inherent activity of the prgf powder . while determining the clotting potential of the prgf was not the intent of this study , this result was seen as an added benefit and may prove beneficial as a hemostatic product . overall , the results of the use of prgf are similar to what has been previously published with prp . namely , prp can significantly increase the activity of a number of different cell types . the statistically significant increases in hdf proliferation and migration , as well as the increases in hpmec proliferation and chemotaxis clearly demonstrate that the cytokines present in the prgf retain their functionality after processing . as noted , manuka honey has been re - gaining popularity among the wound healing community [ 3 , 5 , 911 , 57 ] . the rather unique properties possessed by the active leptospermum honeys are all highly beneficial in tissue regeneration . to evaluate the hypothesis that the low ph of the manuka honey would effectively activate prgf - derived growth factors , the impact of manuka honey supplemented media on three distinct cell lines was evaluated . the modified western blot performed demonstrated the presence of activated tgf- derived from the combination of mh and prp . while this test did not provide a quantitative assessment of how much active tgf- was present , it offered a side by side comparison of the two treatment methods and showed them to be nearly identical . the presence of this activated tgf- , and potentially other acid activated cytokines , may have played a major role in the impact that prgf+mh had in accelerating cellular activity . interestingly , throughout the study , there were no significant differences between the two honey products tested : the pure manuka honey and the medical grade mh . this would indicate that the filtration and sterilization process used to make the mh has no ill effects on its efficacy . the only significant difference experienced between the two products was seen in the macrophage inflammation response , where tnf- release was significantly increased in the 1% manuka honey on days 1 and 4 . this makes sense as the release of tnf- commonly indicates an inflammatory response and an m1 macrophage phenotype [ 2932 , 58 , 59 ] . it is completely possible , and highly likely , that the pure manuka honey may contain some amount of allergens or bee byproducts that could induce an inflammatory response . it would then make sense that the sterilized mh product would be allergen and bee byproduct free . as small amounts of il-10 were produced by the macrophages during this study , that may indicate that the macrophages expressed an anti - inflammatory phenotype ( regulatory macrophage ) . looking at macrophage phenotype as a highly plastic , continuous spectrum , the regulatory macrophage serves as a transitional bridge through the wound healing cascade between the m1 and the wound healing macrophages ( m2 ) . the presence of quantifiable il-10 , and a lack of quantifiable vegf , would indicate that the macrophages resided within this regulatory phenotype and had not progressed to the m2 phenotype [ 31 , 32 ] . it may be possible that longer duration culture would have initiated the expression of the m2 phenotype and possible vegf production , and this may be investigated in the future . another interesting result provided by these in vitro cell - honey interaction studies was the potential for the manuka honey products to become cytotoxic . in nearly all the studies performed , with all three cell types utilized , concentrations of manuka honey or mh of 5% v / v and above resulted in the death of the cells to be studied . it is hypothesized that this cytotoxicity was due to the acidic ph and the closed nature of the in vitro studies performed . to the authors ' knowledge , there have been no reports of any manuka products being cytotoxic when used clinically . in a clinical setting , there are far better mechanisms for diffusion , nutrient exchange , and waste removal than there are in an in vitro study , where cells are essentially sequestered and highly responsive to changes in local ph . for this reason , it is highly likely that this preliminary in vitro study may not demonstrate the true potential of using a manuka honey product in a wound healing capacity and further in vivo and in situ studies will be required . it may be possible that the promising results achieved here with low concentrations of honey may be surpassed with higher honey concentrations in an in vivo setting , where the effects of localized ph changes will be less inhibitory . while the presence of prgf and manuka / mh supplemented media proved effective in stimulating a positive cellular response individually , the use of prgf+mh media demonstrated a further increase in cellular activity . this impact was seen most clearly in the hdf - based studies , and the in vitro wound healing assay study , where the prgf+mh media supplements resulted in statistically significant increases in cell proliferation and migration . as previously stated , this increase in cellular response may be due to the presence of physiologically activated growth factors such as tgf-. however , this may only be a small part of the overall potential for the prgf+mh combination to increase cellular activity . it is entirely possible that since the individual prgf and mh components each provide necessary pieces of the tissue regeneration puzzle , the combination of prgf+mh may provide an ideal combination to stimulate wound healing . however , determining the exact mechanism of cell stimulation is beyond the scope of this preliminary study and will need to be evaluated in future work . while the prgf+mh yielded results that exceeded the authors ' expectations in the hdf - specific studies , the angiogenesis studies proved to be less conclusive this would signify that the impact on the initiation of angiogenesis was due to the presence of prgf alone and was not influenced by the addition of the manuka / mh . this may indicate that the rate of sprout formation and angiogenesis are highly dependent upon the presence of available vegf , readily found in prgf , in which case the presence of honey would not offer much benefit . however , it was anticipated that the ph of the honey products alone may be enough to induce sprout formation , as it has been documented that an acidic environment may promote angiogenesis [ 2 , 4 ] . therefore , as previously mentioned , these results may be indicative of the need for an in vivo angiogenesis model . in summary , prp / prgf capitalizes on the body 's own initial healing component , the platelet , and concentrating them to thereby concentrate their inherent growth factors and jump start the healing process . in the lyophilized powdered form , the prgf powder becomes more versatile ( i.e. , able to be stored in a vacuum sealed package ) and less messy than the liquid prp while retaining its potent bioactive properties . additionally , it may be possible that this powdered prgf , further enhanced through the addition of a mh product , may be carried by first responders or military personnel to be used at the time of injury . it is anticipated that not only will the application of a prgf and manuka honey product accelerate the healing process , as demonstrated with the in vitro results described here , but may also promote coagulation and provide antibacterial properties to a wound in the field . it may also be possible that such a combination product could prove to be highly effective in treating chronic wounds , where cellular senescence , low tissue friability , and biofilm formation are all concerns . having an essentially all natural product known to be highly antibacterial , yet capable of stimulating a number of different cell types , at a patient 's bedside capable of advancing the wound from a state of chronic inflammation to a more conducive healing state would be of great benefit to the treatment of chronic wounds . in this study , it was demonstrated that a number of cell types critical to dermal regeneration ( hdf , hpmec , and macrophages ) are capable of being positively influenced by the presence of prgf and manuka honey media supplements . based upon these results , it is apparent that prgf has the capacity to enhance cellular chemotaxis , mitogenesis , ecm production , and angiogenesis . this bioactivity can be further augmented by combining prgf with mh , potentially due to the increased presence of growth factors that have become physiologically activated . the precise nature of this enhancement is not currently understood and may stem from a number of attributes unique to the two materials acting concomitantly : the presence of necessary sugars , growth factors physiologically activated through manuka honey ph , direct ph effects on cells , the presence of vitamin c , and others . while further research is needed to definitively determine the method of action in which the prgf and mh permutation promote cellular activity , the use of this combination of materials has great potential from a regenerative medicine perspective ; being able to apply a lyophilized prgf powder , with or without a manuka honey product , to the site of a dermal wound at the time of injury may not only assist with clotting but may also accelerate closure and healing of the wound . ongoing work includes in vivo evaluations of wound healing and angiogenesis in small animal models , in vitro antibacterial investigation , as well as in vitro work with a human keratinocyte cell line .

aim . the purpose of this study was to determine the in vitro response of cells critical to the wound healing process in culture media supplemented with a lyophilized preparation rich in growth factors ( prgf ) and manuka honey . materials and methods . this study utilized cell culture media supplemented with prgf , as well as whole manuka honey and the medical - grade medihoney ( mh ) , a manuka honey product . the response of human fibroblasts ( hdf ) , macrophages , and endothelial cells ( hpmec ) was evaluated , with respect to cell proliferation , chemotaxis , collagen matrix production , and angiogenic potential , when subjected to culture with media containing prgf , mh , manuka honey , and a combination of prgf and mh . results . all three cell types demonstrated increases in cellular activity in the presence of prgf , with further increases in activity seen in the presence of prgf+mh . hdfs proved to be the most positively responsive cells , as they experienced enhanced proliferation , collagen matrix production , and migration into an in vitro wound healing model with the prgf+mh - supplemented media . conclusion . this preliminary in vitro study is the first to evaluate the combination of prgf and manuka honey , two products with the potential to increase regeneration individually , as a combined product to enhance dermal regeneration .

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

collectively , these studies demonstrated the importance of keeping preparations rich in growth factors ( prgf ) in the wound site and slowly activating / releasing them as the wound site becomes infiltrated with reparative cells . the purpose of this study was to determine the in vitro response of three cell types critical to wound healing ( fibroblasts , endothelial cells , and macrophages ) when subjected to culture media supplemented with manuka honey , a powdered prgf ( a lyophilized version of prp ) , or a combination of manuka honey and prgf . following adhesion of cells to the well - plate ( ~2 hrs ) control media was replaced with test media ( control media supplemented with manuka honey in 0.1 , 1 , 5 , 10 , or 20% v / v , mh in 0.1 , 1 , 5 , 10 , or 20% v / v , prgf in 0.1 , 1 , 5 , or 10 mg / ml or a combination of 0.1% mh and 1 mg / ml prgf ) . following adhesion of cells to the well - plate ( ~2 hrs ) control media was replaced with test media ( control media supplemented with manuka honey in 0.1 , 1 , 5 , 10 , or 20% v / v , mh in 0.1 , 1 , 5 , 10 , or 20% v / v , prgf in 0.1 , 1 , 5 , or 10 mg / ml or a combination of 0.1% mh and 1 mg / ml prgf ) . similar to the aforementioned cell proliferation studies , cell chemotaxis studies were conducted on both macrophages and hpmecs ( hdf chemotaxis was evaluated with an in vitro wound healing assay ) using a transwell plate and an mts assay . hdf proliferation was observed over 7 days in response to test medias containing varying concentrations of manuka honey , mh , prgf , and a combination of prgf+mh , and cell numbers ranged from near 0 for the higher manuka / mh concentrations to greater than 224,000 for the prgf+mh on day 7 . the combination of prgf+mh contributed mainly to the proliferation of macrophages , with the prgf+mh insert experiencing the largest increase in cell number ( significant over all other groups ) with chemotaxis that was not significantly different from those of the control media . this preliminary in vitro work investigated the potential for prgf and manuka honey / mh to stimulate the activity of hdf , hpmecs , and macrophages in ways that would provide insight into their specific roles in the healing process . cell proliferation , chemotaxis , cytokine release , and ecm production were all tested in the presence of prgf and manuka honey media supplements in order to determine what response , if any , each cell type had to the additives and which additive / concentration proved to be most efficacious . for this reason , it is highly likely that this preliminary in vitro study may not demonstrate the true potential of using a manuka honey product in a wound healing capacity and further in vivo and in situ studies will be required . while the presence of prgf and manuka / mh supplemented media proved effective in stimulating a positive cellular response individually , the use of prgf+mh media demonstrated a further increase in cellular activity . this impact was seen most clearly in the hdf - based studies , and the in vitro wound healing assay study , where the prgf+mh media supplements resulted in statistically significant increases in cell proliferation and migration . while the prgf+mh yielded results that exceeded the authors ' expectations in the hdf - specific studies , the angiogenesis studies proved to be less conclusive this would signify that the impact on the initiation of angiogenesis was due to the presence of prgf alone and was not influenced by the addition of the manuka / mh . it is anticipated that not only will the application of a prgf and manuka honey product accelerate the healing process , as demonstrated with the in vitro results described here , but may also promote coagulation and provide antibacterial properties to a wound in the field . in this study , it was demonstrated that a number of cell types critical to dermal regeneration ( hdf , hpmec , and macrophages ) are capable of being positively influenced by the presence of prgf and manuka honey media supplements . while further research is needed to definitively determine the method of action in which the prgf and mh permutation promote cellular activity , the use of this combination of materials has great potential from a regenerative medicine perspective ; being able to apply a lyophilized prgf powder , with or without a manuka honey product , to the site of a dermal wound at the time of injury may not only assist with clotting but may also accelerate closure and healing of the wound .

the parasites of the genus eimeria can be found in almost all vertebrates and some species are of high economical relevance as they can cause coccidiosis in livestock . eimeria parasites are known to have a stringent stage progression in their life cycles in the host animals . they develop mainly in the intestinal tract , but some species are also found in the liver ( rabbit ) or kidney ( goose ) . to study all stages of the parasites , it is helpful to have the complete development in vitro . in primary cells , only a few eimeria species are able to form gametocytes or complete their life cycles in vitro . often , this is only possible if merozoites are used for infection [ 24 ] . for rodent eimeria species , which are frequently used as model organisms , a gametocyte or oocyst development was not achieved thus far . in previous studies with the rat parasite eimeria nieschulzi , the stage development in vitro did not exceed the second merozoite stage [ 57 ] . whereas , up to four schizont generations are found in the host before an in vivo gamont development occurs [ 8 , 9 ] . in a study by tilley and upton , the development of four schizont generations of eimeria nieschulzi in a permanent cell line under reducing conditions was described . , the progress of the in vitro development of eimeria nieschulzi was investigated using primary tissue cells from fetal rats and compared to permanent cell lines . for better documentation , a transgenic ( yellow fluorescent protein ; yfp expressing ) eimeria nieschulzi strain with an additional gamogony specific reporter ( tandem dimeric tomato ( tdt ) ) gene expression was used . the tdt expression served as an indicator for successful development up to the gametocyte stage and beyond . furthermore , we confirmed the localization of wild - type e. nieschulzi parasites in the crypts of the small intestine by immunohistology and compared our in vitro data in detail with the data in the literature . pregnant rats ( crl : cd ( sd ) ) were euthanized at day 1618 of gestation . the decapitated fetuses were washed in cold ( 4c ) dulbecco 's modified eagle 's medium ( dmem ) to remove blood . liver , kidney , and intestine were separately collected and one assay was performed with all inner organs together as fetal mix ( fm ) . all explanted tissues were washed twice with 37c preheated dmem , minced into small pieces , and ground through a sieve ( mesh size 0,5 mm ) with a plunger . the cell / cell conglomerate suspension was transferred to a centrifuge tube and washed once with completed dmem ( 37c ) and transferred into completed preheated dmem ( with 10% fbs , 2% l - glutamine , 2% penicillin / streptomycin , 1% hepes , and 1% sodium pyruvate ) . the fm - suspension was seeded in ibidi -slides with fibronectin ( ibidi ) , gelatin , matrigel - coating ( 60 l / well according to the manufacturer 's recommendations ) as well as in uncoated slides . for primary liver and kidney cells , fibronectin coated ibidi -slides were used . primary intestinal cells were seeded into ibidi -slides , coated with fibronectin or gelatine , as well as uncoated slides . quality evaluation of the parasite development in vitro was performed by comparing the following permanent cell lines : iec6 ( rat intestinal ) , vero ( green monkey kidney ) , st ( swine testicular ) , and ipec - j2 ( porcine small intestinal epithelial cell ) which were routinely cultivated in completed dmem . for infection with parasites , 100.000150,000 of these cells were seeded in ibidi -slides per well . prior to inoculation with parasites , permanent cells were incubated about 3 - 4 h and primary cell about 24 h at 37c and 5% co2 atmosphere . oocysts of the passages p2 and p3 of the previously described transgenic eimeria nieschulzi were used in this study . sporozoites were excysted and purified as previously described and resuspended in cell culture medium ( dmem , completed ) . for infection of the cells , 20.00050.000 sporozoites were incubated together with the cells in the -slide at 37c and 5% co2 atmosphere . every day , the cell culture medium ( dmem ) was partially ( 50% ) removed and replaced with fresh medium twice . rats ( cd ) were infected orally by gavage with 500.0001.000.000 sporulated oocysts of e. nieschulzi and euthanized at different time points after inoculation with the oocysts . the exact time points can be found in the description of figure 3 . living parasite stages , the small intestine was cut into pieces of 0.5 cm length and fixed in 4% paraformaldehyde/1xpbs ( ph 7.4 ) for 24 h at 4c . after washing in 1xpbs and dehydration in a graded series of ethanol , sections of 5 m were cut with a rotary microtome ( leica rm2125rt ) . the sections were transferred to slides and the paraffin was removed by roti - histol ( co. roth ) . the antigen retrieval was performed with 0.1 m sodium citrate buffer ph 6.0 for 30 min at 90c . if hrp - linked secondary antibodies were used , endogenous peroxidase was blocked by incubation with 1% h2o2 in 1xpbs about 15 min . after blocking of unspecific antigens with 20% normal goat serum ( ngs ) and 0.05% tween in 1xpbs about 1 h at room temperature , the sections were incubated with hybridoma supernatant of the b1c4 clone for about 2 h at room temperature ( 1 : 10 dilution in 20% ngs/0.05% tween/1xpbs ) . after the removal of the primary antibody , the slides were incubated for 1 h at room temperature with the secondary antibody diluted in 20% ngs/0.05% tween/1xpbs ( fitc conjugated anti - mouse igg , co sigma aldrich f6257 1 : 100 , or hrp conjugated anti - mouse igg 1 : 200 , co sigma aldrich a9044 ) . fluorescence probed sections were washed , counterstained with dapi ( 1 g / ml ) about 10 min , and washed three times about 10 min prior to mounting . the sections labelled with peroxidase ( hrp ) probes were equilibrated in 0.05 m tris buffer ph 7.6 about 5 min and further incubated in dab / h2o2 staining solution ( 5 mg 3,3-diaminobenzidine and 5 l h2o2 in 10 ml h2o ) . images were taken using the zeiss axiovert 100 microscope combined with the olympus f - viewii monochrome ccd - camera and the image processing software cell f ( olympus ) from 24 h270 h , inoculation ( p.i . ) . the fluorescence of intracellular as well as extracellular parasitic stages was observed directly in the -slides with fitc filter set ( ex . d480/30 ) for the yellow fluorescent protein ( yfp ) and with a rhodamine filter set ( ex . 560/40 ) for tandem dimer tomato ( tdt ) . for the visualization of the autofluorescence of the oocyst wall , the dapi filter set ( excitation 365 nm , emission long pass 420 nm ) was used . the reproduction of images was performed comparable to the microscopic view according to the filter set dependent emission wavelength . capturing of the peroxidase probed sections was performed with the olympus c7070 camera combined with the olympus c5060-adu ocular adapter or with the monochrome fviewii and cellf software . in the permanent cell lines iec6 , vero , ipec - j2 , and st , as well in primary liver , kidney , and intestinal cells the development stopped after formation of the second schizonts , respectively , merozoite generation ( not shown ) . we could not observe any further development until 268 h p.i . in these mentioned cells , and thereby , we confirm the results of previous studies . developmental stages beyond the second - generation merozoites were observed in the fetal cell mix ( fm ) derived from the inner organs on the coated slides , but not in the uncoated control slide . we have to remark that only in half of the performed experiments with coated slides , crypt - like organoid structures were observed . the success of development of macrogamonts and oocysts was directly connected with occurrence of these crypt - like structures . the particular experiments and the results of parasite development are listed in table 1 . in the fetal cell mix , we could identify schizonts of the first- ( not shown ) , second- ( figure 1(a ) ) , and fourth - generation ( figures 1(e ) and 1(f ) ) , as well as free merozoites of the second ( figure 1(d ) ) and fourth generation ( figure 1(g ) ) , and also gametocytes and unsporulated oocysts ( figures 1(h)1(j ) , and 2 ) . the third - generation schizonts and merozoites are difficult to distinguish from the second generation . considering the literature , the large schizont in figures 1(b ) and 1(c ) may belong to the second or third generation . despite the size , the 22 m long merozoite in figure 1(d ) schizonts of the fourth generation were found clustered with up to 15 schizonts ( picture details figures 1(e ) and 1(f ) ) from 164 h p.i . free merozoites iv were observed ( figure 1(g ) ) separately or close to the gametocytes ( figures 2(a)2(e ) , 2(j)2(m ) ) . the tdt - protein is expressed under the microgametocyte - specific promoter of the gam56 gene found in eimeria tenella [ 11 , 15 ] . the gametocytes often occurred in clusters with up to 100 cells and grew in cells which were situated in a crypt - like formation ( figures 1(h ) , 1(i ) , and 1(j ) ) . however , gametocytes were not found in all crypt - like structures ( figure 1(k ) ) . if crypt - like organoids had developed up to four clusters of macrogamonts in 2550% of the -slide , wells could be identified . the best results were obtained in one experiment with fm - cells on a fibronectin coated -slide . notwithstanding , unsporulated oocysts with autofluorescent oocyst walls could be observed , but these oocysts did not sporulate in the cell culture slide or outside under air supply ( figures 2(n)2(r ) ) . in summary , the fm - cells provide the qualitatively potential to develop eimeria nieschulzi sporozoites over four asexual generations and the gametocyte stage to the point of oocyst development in vitro . the presence of macrogametocytes in organoid crypt - like structure indicates that these cells have similar characteristics to the native host cells in the rat . in a selected number of performed experiments we localized the wild type e. nieschulzi in situ in the crypt of the small intestine by immunohistology at 72 , 120 , and 144 h p.i . the antibody b1c4 originally produced against e. tenella sporozoites and described 1995 by greif and entzeroth was used for immunohistology . the antibody showed cross - reactivity to e. nieschulzi schizonts and gamonts . at 72 h p.i . , schizonts were detected in the middle parts of the crypts ( figures 3(a ) and 3(b ) ) . at higher magnifications , it was not possible to distinguish which schizont generation is labelled ; only the visible multiple nuclei indicated schizonts . at 117 h p.i . , long merozoites with a length of more than 25 m and straight cell shape in the intestine ( figure 3(c ) ) were identified . , parasite stages were labelled in the middle and lower part of the crypts ( figure 3(d ) ) . the schizonts shown in figure 3(e ) containing long merozoites belong to the third generation and were found in middle parts of the crypts . figure 3(f ) shows schizonts of different sizes in the lower part of the crypts . marquardt described large schizonts with a maximum of 12 merozoites as second or third generation . however , corresponding to the observed in vitro stages , we would assign them to the second - schizont - generation . at 144 h p.i . , the parasite stages were found predominantly in the middle and upper part of the crypts , as well as at the base of the villi ( figure 3(g ) ) . at higher magnifications , it was possible to identify mono- and multinuclear stages . the mononuclear stages are probably young macrogamonts and the multinuclear stages fourth generation schizonts ( figure 3(h ) ) . the control experiments without the primary antibodies did not show any specific signal derived by the secondary antibodies ( not shown ) . in primary fetal cells , the in vitro development of a rodent eimeria species up to the oocyst stage could be shown . as affirmed by various cell lines in this study and in previous studies , the in vitro development of eimeria nieschulzi is limited to the second - generation merozoite [ 57 ] . tilley and upton reported a development until the fourth generation in vitro under reducing conditions . however , eimeria nieschulzi can have in vitro variations concerning the morphology of the schizont stages . there are large schizonts with more than 20 merozoites but also small schizonts less than 10 merozoites ( figure 1(a ) ) . thus , it is difficult to distinguish the second schizont stage from the third schizont stage with certainty . because in vivo , first- and second - generation schizonts can have delayed development up to 96 h p.i . [ 8 , 9 ] , the duration of the development can not lead to concrete conclusions . only the size of explicitly visible merozoites in or out of schizonts should be consulted for determination between second ( mzii ) and third - generation merozoite ( mziii ) , but there are further challenges concerning the interpretation of literature data . marquardt described the occurrence of mziii beginning from 48 h p.i . with an average length of 20.8 m , 25.5 m at 72 h p.i . , and 26.9 m at 120 h p.i .. at 120 h p.i . , the merozoites showed a high motility while the previous smaller merozoites were nearly inactive . the schizont ii , shown by marquardt 1966 ( compare figure 8 in marquardt 1966 ) , is similar to in vitro stages at 48 h p.i . [ 6 , 11 ] and we would assign them to the first - generation schizont stage . the schizont iii in figure 11 shown in the publication by marquardt 1966 is similar to that we assign to the schizont ii stages ( figure 1(a ) ) which are visible in permanent cell lines beginning from 68 h p.i . ( figure 1(a ) ) . a large schizont ii shown by tilley and upton ( compare figure 7 in tilley and upton 1988 ) a schizont iii shown by tilley and upton 1988 ( compare with figure 8 in tilley and upton ) we would assign to the schizont ii stage . rick et al . also described large schizonts and long merozoites ( 21 m ) at 73 h p.i . , and assigned them to the second schizont generation . the residual tandem dimeric tomato signal , described by hanig et al . , additionally supported our interpretation of the first- and second - generation stages occurring in the permanent cell culture . our observations around 117 h p.i . and 120 h p.i . . we could also identify large merozoites about 25 m and longer with a high motility ( figure 3(c ) , supplementary figure 3 ) . the merozoites moved forward and backward as previously described by marquardt . additionally , we observed movements of merozoites iii with distances of more than their own cell length ( supplementary figure 3 ) . the model of gliding motility would prefer the forward gliding , and in most apicomplexan parasites this is observed in connection with banana or arch - shaped parasite cells . backward moving we think that the e. nieschulzi merozoites perform this by rotating on their length axis during the gliding . the straight cell shape would support a rotation hypothesis , and we observed that the merozoites almost used the same trails during their dislocation . it is conceivable that through the high motility of this stage , a further expansion of the parasite can be accomplished . in vitro , we could not observe such high motile stages with more than 25 m length in vitro , and we are not sure if very large schizonts in figures 1(b ) and 1(c ) belong to the third - generation . third generation schizonts probably occur in very low numbers in our cell culture , which is discussed below . marquardt observed the fourth generation only at day six and described a very fast transition from merozoite iii to merozoite iv . in combination with its description of merozoites iii at 48 and 72 h p.i . hence , we have additional doubts concerning the early merozoites iii which are probably merozoites ii . to shed light on that issue , further studies with modern molecular methods should be performed . in size and shape , the schizonts of the fourth - generation are comparable to the first - generation schizonts originated by single sporozoites . in contrast to the isolated appearance of the first schizont stage , the fourth - generation schizonts occurred in a cluster observed from 164 h p.i . the assumed fourth - generation schizont shown by tilley and upton is probably a first or second - generation schizont in a delaminated host cell . our experiments suggest that a reduced environment is not necessary for the development of eimeria nieschulzi in vitro . it is assumed that the development of the third - generation schizont stage along with the invasion of the second - generation merozoites is a crucial point for the in vitro development . in order to invade and further develop into schizonts of the third - generation , in permanent cell lines and primary liver and kidney cells , no further development beyond the second - generation merozoite was observed . the second - generation schizont forms clusters too ( figure 1(a ) ) , and this would also be expected for the third generation . in the nonhomogenous fetal cell mix used in this study , only a few of the merozoites ii seem to find a proper host cell for invasion . thus , parasite expansion within the third - generation schizont in cell culture would be repressed . this interpretation is supported by the occurrence of macrogamonts in only a part of the cell culture slide wells . however , once in a proper cellular environment ( crypt - like organoid ) , the majority of the developed merozoites iii ( derived from a single schizont iii ) can infect a new host cell . an expansion of parasitic stages occurred once more , and merozoites iii developed into large clusters of schizonts iv ( figures 1(e ) and 1(f ) ) . if merozoites iv are infecting new proper host cells , they would form even larger clusters of macrogamonts and this is shown in this study . in the natural host eimeria nieschulzi directly infects crypt cells of the small intestine and can be also found in cells at the basis of the villi . in other parts of the epithelium they are absent . they are so - called transit - amplifying cells ( ta - cells ) , proliferative progenitor cells derived from stem cells at the base of the crypt . inside the villi , these cells are not infected by eimeria nieschulzi , but the ta - cells obviously harbor an attractor for the parasite . in the in vitro experiments shown here , the majority of the developed macrogamonts could be observed in crypt - like structures . this suggests that these cells have similar properties to the intestinal crypt cells in the host . these crypt - like structures were only observed in the fm - cell assay and may be based on the necessity of epithelial - mesenchymal interactions for the development and proliferation of crypt cells . these mesenchymal cells were found in the fm - cell assay but not in the assay with only primary intestinal cells . this is probably the reason why intestinal cells solely do not form crypt - like organoids . cell culture slides coated with matrigel , fibronectin , or gelatine may be necessary for the growth of the fm - cells and the formation of crypt - like structures , while the cell culture medium seems to be less important . nevertheless , the formation of crypt - like organoid occurred not absolutely reliable ( table 1 ) . the fm cells are a variable undefined cell mixture with different cell content in every preparation and this can influence the cell growth . furthermore , a high content of red blood cells seems to negatively affect the growth of the primary cells and this content alternates also in the cell preparations . thus , in further experiments the growth of the crypt - like organoids should be aspired in a more defined manner . the possibility to build crypt - villous structures in vitro from single lgr5 + stem cells without a mesenchymal niche by stimulating the cell proliferation with external factors their results could give further perspectives for the in vitro cultivation of potentially crypt cell affine eimeria species . the molecules involved in invasion of eimeria parasites , more precisely eimeria tenella , are relatively well known [ 21 , 22 ] . however , there is a little characterization of the surface molecules and gene expression of the natural host cells . eimeria species can infect multiple cell lines , but stop their development finally after one or two schizont generations . established cell lines seem to be only partly sufficient to explore the involved molecules for infection of host cells because they only harbour a minimum of qualities for infection and development of the parasites . it is striking that in vivo e. nieschulzi directly infects the cell of the crypts and passes the villous cells , whereas in vitro they infect several types of epithelial cells . evidently , there are factors of the villous cells which prevent an infection and factors of the crypt cells which trigger an infection in vivo . sialylated glycans on the host cell surface and mic3 protein on the parasites side interact and may play an important role in host cell tropism of eimeria tenella . however , a colocalization of e. tenella stages and the lectin maaii ( a lectin binding to 26 or 2 - 3 sialyl linked glycan ) positive cells as well as the existence of such sialylated glycans in primary chicken kidney cells , where a complete development of e. tenella in vitro is possible , was not shown . therefore , it is still unclear if sialylated glycans are the most important mediators for host cell recognition . thus , the characterization and isolation of natural host cells have to be improved to designate the host cell ligands needed for infection and development . genetically modified eimeria species are helpful for these studies . furthermore , it was remarkable that no microgamonts or microgametes in fm - cell culture were definitively observed , whereas macrogametocytes and oocysts were clearly developed . second , microgamonts developed , but microgametogenesis of the microgametes was disturbed , for example , by serum . it would be also possible that through the three - dimensional character of the crypt - like organoid , a detection of microgamonts could not be performed by transmitted light microscopy . only the macrogamonts have a specific marker , and microgamonts are supposed to express only the yfp fluorochrome . our observation in small bowel smears showed only weak fluorescence signals in microgametes ( not shown ) and no micrographs could be taken through the rapid movement of the microgametes . shi et al . described the development of yfp - expressing microgametes of eimeria tenella in vitro , but did not show any transmitted light micrographs . we could also see only yfp expressing stages between tdt - expressing macrogamonts with no distinct nuclear signals ( supplementary figure 1 ) which were regularly observed in schizont stages ( figures 1(e ) and 1(f ) ) . also , the macrogamont typical central nuclear ( figure 1(f ) ) signal was absent . since we had not observed free microgametes and the oocysts did not sporulate , we assume no fertilization of the macrogamete took place . as the oocyst walls were formed in vitro , the wall formation process seems to be independent from the fertilization process . ferguson assumed that even fertilization is not obligatory for sporulation in toxoplasma gondii and other apicomplexa . , the genus eimeria could even acts as a model for toxoplasma gondii because such experiments are more difficult to accomplish in cats than in rats or chicken . the transfection technology is already established for eimeria species infecting these host animals [ 7 , 11 , 26 , 27 ] and can give the opportunity to deepen the knowledge of sexual processes in oocyst - forming apicomplexa .

the in vitro production of gametocytes and oocysts of the apicomplexan parasite genus eimeria is still a challenge in coccidiosis research . until today , an in vitro development of gametocytes or oocysts had only been shown in some eimeria species . for several mammalian eimeria species , partial developments could be achieved in different cell types , but a development up to gametocytes or oocysts is still lacking . this study compares several permanent cell lines with primary fetal cells of the black rat ( rattus norvegicus ) concerning the qualitative in vitro development of the rat parasite eimeria nieschulzi . with the help of transgenic parasites , the developmental progress was documented . the selected eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato . in the majority of all investigated host cells the development stopped at the second merozoite stage . in a mixed culture of cells derived from inner fetal organs the development of schizont generations i - iv , macrogamonts , and oocysts were observed in crypt - like organoid structures . microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply . by immunohistology , we could confirm that wild - type e. nieschulzi stages can be found in the crypts of the small intestine . the results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for eimeria species .

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

the parasites of the genus eimeria can be found in almost all vertebrates and some species are of high economical relevance as they can cause coccidiosis in livestock . in previous studies with the rat parasite eimeria nieschulzi , the stage development in vitro did not exceed the second merozoite stage [ 57 ] . in a study by tilley and upton , the development of four schizont generations of eimeria nieschulzi in a permanent cell line under reducing conditions was described . , the progress of the in vitro development of eimeria nieschulzi was investigated using primary tissue cells from fetal rats and compared to permanent cell lines . furthermore , we confirmed the localization of wild - type e. nieschulzi parasites in the crypts of the small intestine by immunohistology and compared our in vitro data in detail with the data in the literature . quality evaluation of the parasite development in vitro was performed by comparing the following permanent cell lines : iec6 ( rat intestinal ) , vero ( green monkey kidney ) , st ( swine testicular ) , and ipec - j2 ( porcine small intestinal epithelial cell ) which were routinely cultivated in completed dmem . in the permanent cell lines iec6 , vero , ipec - j2 , and st , as well in primary liver , kidney , and intestinal cells the development stopped after formation of the second schizonts , respectively , merozoite generation ( not shown ) . developmental stages beyond the second - generation merozoites were observed in the fetal cell mix ( fm ) derived from the inner organs on the coated slides , but not in the uncoated control slide . we have to remark that only in half of the performed experiments with coated slides , crypt - like organoid structures were observed . in the fetal cell mix , we could identify schizonts of the first- ( not shown ) , second- ( figure 1(a ) ) , and fourth - generation ( figures 1(e ) and 1(f ) ) , as well as free merozoites of the second ( figure 1(d ) ) and fourth generation ( figure 1(g ) ) , and also gametocytes and unsporulated oocysts ( figures 1(h)1(j ) , and 2 ) . the gametocytes often occurred in clusters with up to 100 cells and grew in cells which were situated in a crypt - like formation ( figures 1(h ) , 1(i ) , and 1(j ) ) . if crypt - like organoids had developed up to four clusters of macrogamonts in 2550% of the -slide , wells could be identified . notwithstanding , unsporulated oocysts with autofluorescent oocyst walls could be observed , but these oocysts did not sporulate in the cell culture slide or outside under air supply ( figures 2(n)2(r ) ) . the presence of macrogametocytes in organoid crypt - like structure indicates that these cells have similar characteristics to the native host cells in the rat . in a selected number of performed experiments we localized the wild type e. nieschulzi in situ in the crypt of the small intestine by immunohistology at 72 , 120 , and 144 h p.i . in primary fetal cells , the in vitro development of a rodent eimeria species up to the oocyst stage could be shown . as affirmed by various cell lines in this study and in previous studies , the in vitro development of eimeria nieschulzi is limited to the second - generation merozoite [ 57 ] . it is assumed that the development of the third - generation schizont stage along with the invasion of the second - generation merozoites is a crucial point for the in vitro development . however , once in a proper cellular environment ( crypt - like organoid ) , the majority of the developed merozoites iii ( derived from a single schizont iii ) can infect a new host cell . in the natural host eimeria nieschulzi directly infects crypt cells of the small intestine and can be also found in cells at the basis of the villi . in the in vitro experiments shown here , the majority of the developed macrogamonts could be observed in crypt - like structures . these crypt - like structures were only observed in the fm - cell assay and may be based on the necessity of epithelial - mesenchymal interactions for the development and proliferation of crypt cells . the possibility to build crypt - villous structures in vitro from single lgr5 + stem cells without a mesenchymal niche by stimulating the cell proliferation with external factors their results could give further perspectives for the in vitro cultivation of potentially crypt cell affine eimeria species . it would be also possible that through the three - dimensional character of the crypt - like organoid , a detection of microgamonts could not be performed by transmitted light microscopy .

the gprd provided a reliable source of longitudinal , anonymous medical data from general practices across the u.k . , with links to other healthcare databases with their database of 5 million patients representing 8.5% of the population . our study cohort was drawn from practices linked by the office of national statistics to the gprd , all located in england . we obtained data on people with diabetes 4065 years of age on 1 january 2004 ( baseline date ) , diagnosed after 40 years of age . for each patient with diabetes , we matched up to three people without diabetes for age ( within 1 year ) , sex , and general practice . we analyzed data over 7 years , from 1 january 2004 to 31 december 2010 . the gprd provided data in accordance with our data specification document , which defined people with diabetes as those with a diagnostic code and/or a treatment code for diabetes , and excluded patients with secondary diabetes , e.g. , gestational or corticosteroid - induced diabetes . from the dataset received from the gprd , we excluded those who died before 1 january 2004 , < 40 years of age at baseline , with sex unrecorded , with diabetes but not matched to people without diabetes , with diabetes but not identified as having type 2 diabetes , and without diabetes matched to people with diabetes who had been excluded from the cohort . we identified people with type 2 diabetes using an algorithm , slightly modified from that published by the royal college of general practitioners and national health service ( nhs ) diabetes ( 24 ) , and based on the date of diagnosis and the diagnostic and treatment codes for diabetes ( fig . we aimed to compare the populations with and without diabetes in terms of the following : 1 ) baseline characteristics , including smoking , bmi , systolic and diastolic blood pressures , plasma total cholesterol , ldl , hdl , and triglyceride concentrations , and use , by proportions , of aspirin , statins , and antihypertensive drugs ; 2 ) rates of all - cause mortality and cardiovascular mortality ; and 3 ) average annual rates of blood pressure readings , cholesterol monitoring , and prescriptions for aspirin , statins , and antihypertensive drugs . we also reported the average annual rates of prescriptions for glucose - lowering treatments in the people with diabetes . all statistical analyses were carried out using stata v.12 ( statacorp , college station , tx ) . we defined the percentage of people on medication at baseline as having had a prescription issued within the 3 months before the baseline date . baseline values of all other variables were means in the 2 years before the baseline date , except bmi and smoking status , which we based on the most recent reading in the 5 years before the baseline date . we used bmi if given or we calculated it from the closest value of weight and height . for some patients , we calculated total cholesterol using the friedewald equation ( 25 ) . we tested only variables for which at least 80% of patients had ( nonmissing ) data , as we could not rule out the possibility that the absence of data correlated with the variable would bias the results . we measured the frequencies of blood pressure readings and cholesterol monitoring as the average number of tests per person per year , and the average annual prescription rates for aspirin , statins , antihypertensive drugs , and glucose - lowering treatments were measured by the average number of months with prescriptions for these drugs per person per year . we used conditional logistic regression to test for differences between those with and without diabetes . cox proportional hazards models were constructed to estimate hazard ratios ( hrs ) with 95% cis of all - cause mortality and cvd mortality in those with diabetes compared with those without diabetes . given the extent of missing data , in the analysis of all patients , we were able to adjust only for baseline smoking status , having previously matched for age , sex , and general practice . analyzing the subcohort of patients with complete data for the most recent values of bmi , blood pressure , and cholesterol , we also adjusted for the baseline values of these factors . in patients with type 2 diabetes we stratified the analyses by sex and age - group ( < 55 and > 55 years of age ) and calculated p values by the log - rank test . we plotted log cumulative hazard against time to test the proportional hazards assumption of the cox models ( 26 ) . we categorized smoking status as current smokers , nonsmokers ( not having smoked in the previous 5 years ) , ex - smokers , and unknown . bmi , measured as a continuous variable in kg / m , was divided into categories 918.5 , 18.525 , 2530 , 3035 , 3540 , 4050 , 5070 , > 70 , and unknown , incorporating categorization from the world health organization ( 27 ) . we considered that a value for bmi on a threshold between categories would fall into the lower category , and values < 9 kg / m were implausible ( and therefore categorized them as unknown ) . we defined systolic blood pressure categories as low ( < 120 mmhg ) , high ( > 120 mmhg ) , and unknown . we defined diastolic blood pressure categories as low ( < 80 mmhg ) , high ( > 80 mmhg ) , and unknown ( 28 ) and total cholesterol categories as low ( up to 5 mmol / l ) , high ( > 5 the duration of diabetes was categorized into < 5 , 510 , and > 10 years . we excluded values related to plasma ldl , hdl , and triglycerides in the cox models , given the extent of missing data ( 70% or over ) . we defined cvd mortality within the icd-10 codes listed as the primary cause of death , i20i25 ( ischemic heart disease ) , i26i28 ( pulmonary heart disease and diseases of pulmonary circulation ) , and i60i69 ( cerebrovascular diseases ) . for analysis of the risks of all - cause mortality , all survivors at 31 december 2010 were censored at that date . for analysis of the risks of cvd deaths , we censored survivors at 31 december 2010 and people died of causes other than cvd deaths at the date of death , reflecting our assumption that deaths from non - cvd causes were independent of the risk of cvd death . we conducted three sensitivity analyses on all patients to test the robustness of the estimated hrs . in the first analysis , we classified smoking status into three categories , by grouping nonsmokers and those with unknown smoking status . in the second analysis , we censored all patients who left the practice or died before 31 december 2010 at the date they were recorded as having left the practice or the date of death , or the earlier of the two dates if both events occurred . in the third analysis , we censored controls who developed diabetes before 31 december 2010 at the date 5 years before diabetes was diagnosed . we chose 5 years as an estimate of the time lag that occurs between the onset of diabetes and diagnosis . for comparability with the results of the national diabetes audit in england ( 24 ) , we calculated standardized mortality ratios for the same follow - up period ( 1 november 2008 to 31 october 2009 ) . as our study compared a clinically defined exposure ( diabetes ) against a hard outcome measure ( mortality ) , we did not need to consult patient or user groups . the gprd provided a reliable source of longitudinal , anonymous medical data from general practices across the u.k . , with links to other healthcare databases with their database of 5 million patients representing 8.5% of the population . our study cohort was drawn from practices linked by the office of national statistics to the gprd , all located in england . we obtained data on people with diabetes 4065 years of age on 1 january 2004 ( baseline date ) , diagnosed after 40 years of age . for each patient with diabetes , we matched up to three people without diabetes for age ( within 1 year ) , sex , and general practice . we analyzed data over 7 years , from 1 january 2004 to 31 december 2010 . the gprd provided data in accordance with our data specification document , which defined people with diabetes as those with a diagnostic code and/or a treatment code for diabetes , and excluded patients with secondary diabetes , e.g. , gestational or corticosteroid - induced diabetes . from the dataset received from the gprd , we excluded those who died before 1 january 2004 , < 40 years of age at baseline , with sex unrecorded , with diabetes but not matched to people without diabetes , with diabetes but not identified as having type 2 diabetes , and without diabetes matched to people with diabetes who had been excluded from the cohort . we identified people with type 2 diabetes using an algorithm , slightly modified from that published by the royal college of general practitioners and national health service ( nhs ) diabetes ( 24 ) , and based on the date of diagnosis and the diagnostic and treatment codes for diabetes ( fig . we aimed to compare the populations with and without diabetes in terms of the following : 1 ) baseline characteristics , including smoking , bmi , systolic and diastolic blood pressures , plasma total cholesterol , ldl , hdl , and triglyceride concentrations , and use , by proportions , of aspirin , statins , and antihypertensive drugs ; 2 ) rates of all - cause mortality and cardiovascular mortality ; and 3 ) average annual rates of blood pressure readings , cholesterol monitoring , and prescriptions for aspirin , statins , and antihypertensive drugs . we also reported the average annual rates of prescriptions for glucose - lowering treatments in the people with diabetes . all statistical analyses were carried out using stata v.12 ( statacorp , college station , tx ) . we defined the percentage of people on medication at baseline as having had a prescription issued within the 3 months before the baseline date . baseline values of all other variables were means in the 2 years before the baseline date , except bmi and smoking status , which we based on the most recent reading in the 5 years before the baseline date . we used bmi if given or we calculated it from the closest value of weight and height . for some patients , we calculated total cholesterol using the friedewald equation ( 25 ) . we tested only variables for which at least 80% of patients had ( nonmissing ) data , as we could not rule out the possibility that the absence of data correlated with the variable would bias the results . we measured the frequencies of blood pressure readings and cholesterol monitoring as the average number of tests per person per year , and the average annual prescription rates for aspirin , statins , antihypertensive drugs , and glucose - lowering treatments were measured by the average number of months with prescriptions for these drugs per person per year . we used conditional logistic regression to test for differences between those with and without diabetes . cox proportional hazards models were constructed to estimate hazard ratios ( hrs ) with 95% cis of all - cause mortality and cvd mortality in those with diabetes compared with those without diabetes . given the extent of missing data , in the analysis of all patients , we were able to adjust only for baseline smoking status , having previously matched for age , sex , and general practice . analyzing the subcohort of patients with complete data for the most recent values of bmi , blood pressure , and cholesterol , we also adjusted for the baseline values of these factors . in patients with type 2 diabetes we stratified the analyses by sex and age - group ( < 55 and > 55 years of age ) and calculated p values by the log - rank test . we plotted log cumulative hazard against time to test the proportional hazards assumption of the cox models ( 26 ) . we categorized smoking status as current smokers , nonsmokers ( not having smoked in the previous 5 years ) , ex - smokers , and unknown . bmi , measured as a continuous variable in kg / m , was divided into categories 918.5 , 18.525 , 2530 , 3035 , 3540 , 4050 , 5070 , > 70 , and unknown , incorporating categorization from the world health organization ( 27 ) . we considered that a value for bmi on a threshold between categories would fall into the lower category , and values < 9 kg / m were implausible ( and therefore categorized them as unknown ) . we defined systolic blood pressure categories as low ( < 120 mmhg ) , high ( > 120 mmhg ) , and unknown . we defined diastolic blood pressure categories as low ( < 80 mmhg ) , high ( > 80 mmhg ) , and unknown ( 28 ) and total cholesterol categories as low ( up to 5 mmol / l ) , high ( > 5 the duration of diabetes was categorized into < 5 , 510 , and > 10 years . we excluded values related to plasma ldl , hdl , and triglycerides in the cox models , given the extent of missing data ( 70% or over ) . we defined cvd mortality within the icd-10 codes listed as the primary cause of death , i20i25 ( ischemic heart disease ) , i26i28 ( pulmonary heart disease and diseases of pulmonary circulation ) , and i60i69 ( cerebrovascular diseases ) . for analysis of the risks of all - cause mortality , all survivors at 31 december 2010 were censored at that date . for analysis of the risks of cvd deaths , we censored survivors at 31 december 2010 and people died of causes other than cvd deaths at the date of death , reflecting our assumption that deaths from non - cvd causes were independent of the risk of cvd death . we conducted three sensitivity analyses on all patients to test the robustness of the estimated hrs . in the first analysis , we classified smoking status into three categories , by grouping nonsmokers and those with unknown smoking status . in the second analysis , we censored all patients who left the practice or died before 31 december 2010 at the date they were recorded as having left the practice or the date of death , or the earlier of the two dates if both events occurred . in the third analysis , we censored controls who developed diabetes before 31 december 2010 at the date 5 years before diabetes was diagnosed . we chose 5 years as an estimate of the time lag that occurs between the onset of diabetes and diagnosis . for comparability with the results of the national diabetes audit in england ( 24 ) , we calculated standardized mortality ratios for the same follow - up period ( 1 november 2008 to 31 october 2009 ) . as our study compared a clinically defined exposure ( diabetes ) against a hard outcome measure ( mortality ) , we did not need to consult patient or user groups . the gprd provided data on 99,151 people . after excluding 12,053 for the reasons given above , we analyzed data on 87,098 , including 21,798 with type 2 diabetes and 65,300 matched people without diabetes . the average duration of diabetes in the diabetes group was a median 3.9 years , with an interquartile range 1.88.0 years . the high levels of missing data for ldl , hdl , and triglycerides are apparent in table 1 . the percentage of people taking medications was significantly greater in those with diabetes for all drugs ( p < 0.001 ) . of the patients with diabetes , 0.3% used short - acting insulin , 15.1% used long - acting insulin , and 68.6% used only noninsulin diabetes drugs . baseline characteristics in the 7 years after baseline , among the 21,789 people with diabetes , there were 2,146 deaths from all causes ( 9.8% ) and 658 cvd deaths ( 3.0% ) . in the 65,300 without diabetes , there were 2,969 deaths from all causes ( 4.6% ) and 574 cvd deaths ( 0.9% ) . the hr for all - cause mortality for people with diabetes compared with people without diabetes , adjusted for smoking status ( model ii ) was 2.12 ( 95% ci 2.002.25 ) for all patients . people with diabetes were at a significantly higher risk of mortality than people without diabetes across all the subgroups . the adjusted relative risk of mortality was higher in women than in men ( men , 1.93 [ 1.792.07 ] ; women , 2.47 [ 2.232.72 ] ) and higher in the < 55 than in the > 55 years of age - group ( < 55 years of age , 2.72 [ 2.423.06 ] ; > 55 years of age , 2.03 [ 1.892.17 ] ) . hrs of all - cause mortality and cardiovascular mortality the hr for cvd mortality for people with diabetes compared with people without diabetes , adjusted for smoking status ( model ii ) , was 3.28 ( 95% ci 2.913.70 ) for all patients . people with diabetes were consistently at a higher risk of cvd mortality than people without diabetes across all subgroups . sensitivity analyses showed that hrs were not changed measurably by classifying smoking into three categories , nor by censoring all patients who left the practice ( 16,047 additional patients were censored before 31 december 2010 and 588 who died were censored before their death ) , nor by also censoring the people without diabetes at baseline who developed diabetes before 31 december 2010 ( involving 59 patients , of whom 5 died ) . when analyzing the relationship between mortality and duration of diabetes ( supplementary table 1 ) and comparing these with people diagnosed with diabetes 510 years before baseline , we observed a significantly reduced risk of all - cause mortality in those diagnosed < 5 years before baseline ( hr 0.77 [ 95% ci 0.700.86 ] ) and a significantly increased risk in those diagnosed > 10 years before baseline ( 1.69 [ 1.511.90 ] ) . the risk of cvd mortality also increased with duration of diabetes ( < 5 years , 0.75 [ 0.620.91 ] ; > 10 years , 2.21 [ 1.802.72 ] ) . increases in relative risk were observed in all subgroups and reached statistical significance in men , women , and the under 55 years of age - group considering all - cause mortality but only in women for cvd mortality . to estimate how much adjusting for major cvd risk factors would attenuate the hrs , we conducted a secondary analysis restricted to 29,361 people with complete data for bmi , blood pressure , and total cholesterol ( 18,591 people with type 2 diabetes and 10,146 without diabetes ) . in this subgroup , adjusting for smoking only did not alter the hr for all - cause mortality , which was 1.63 ( 95% ci 1.491.79 ) with or without adjusting for smoking , but adjusting for smoking , bmi , blood pressure , and cholesterol reduced the hr to 1.51 ( 1.371.67 ) . the corresponding hrs for cvd mortality were 2.28 ( 1.902.75 ) unadjusted , 2.26 ( 1.882.73 ) with adjustment for smoking , and 2.03 ( 1.662.47 ) with adjustment for smoking , bmi , blood pressure , and total cholesterol . the frequencies of blood pressure testing and cholesterol monitoring were significantly higher in those with diabetes than those without ( all p < 0.001 ) ( table 3 ) , as was the average annual use of aspirin , statins , and antihypertensive drugs ( all p < 0.001 ) ( table 3 ) . average annual monitoring and medication per person standardized mortality ratios for the same 1-year follow - up period as for the national diabetes audit were 2.91 ( 95% ci 2.543.14 ) in men and 3.75 ( 3.114.35 ) in women 4065 years of age . standardized mortality ratios reported by the national diabetes audit were 2.55 in men and 3.38 in women 3564 years of age . in this cohort , people with type 2 diabetes had twice the risk of dying from any cause and three times the risk of cvd death compared with people without diabetes . men were at a greater absolute risk of mortality than women , but the relative risk associated with diabetes in men was lower than in women . younger middle - aged people with type 2 diabetes ( < 55 years of age ) were at a greater relative risk than older middle - aged people without diabetes ( > 55 years of age ) . the association between diabetes and the risk of death was largely independent of smoking status . a secondary analysis , in a subcohort for which the relevant data were nonmissing , suggested that the relative risk was also largely independent of bmi , blood pressure , and cholesterol . among people with diabetes the strengths of our study included the data sources , with high - quality data on a large representative sample of the english population , complete and accurate data on date of death , and access to longitudinal data on the monitoring and treating of cardiovascular risk factors ( 30,31 ) . however , our study was limited by the extent of missing data for some covariates of interest . in our view , the assumptions necessary for multiple imputation ( 32 ) would not be justified . hence , we explored how far adjusting for bmi , blood pressure , and cholesterol might modify the results in a subcohort who had complete data for these variables . to identify patients with type 2 diabetes , it was necessary to modify the algorithm published by the royal college of general practitioners and nhs diabetes ( 24 ) , as the date of diagnosis of diabetes may be unreliable in people whose diagnosis predated entry to the gprd . a recent study showed that data on the duration of diabetes is 90% accurate for a duration < 5 years , but only 67% accurate for a duration > 15 years ( 33 ) . our modified algorithm was intended to produce a cohort that was representative of type 2 diabetes as far as possible . as we could not adjust for factors such as renal function , or ethnicity , some residual our findings are consistent with those of both older and more recent studies of middle - aged people with type 2 diabetes , in providing evidence of risk of mortality lower than before , although elevated with respect to the nondiabetic population , with a greater relative risk in women than men ( 1113,1518,21,23,34 ) , and with a lower relative risk at older ages ( 11,16,20,23,34 ) . our study is directly comparable with one of these studies , which also used gprd data to examine the risk of mortality associated with type 2 diabetes ( 13 ) . we produced hrs that were lower than the standardized mortality ratios reported by the national diabetes audit ( 23 ) . when we calculated standardized mortality ratios for the same 1-year period and compared with national diabetes audit results for a similar age - group , we obtained consistent results , but our standardized mortality ratios were higher than our hrs . this suggests that the differences between our hrs and the national diabetes audit results are attributable to methodological differences ( weakness of one - sample design and standardized mortality ratios ) rather than underlying differences in the mortality rates between these two large samples from the english population . although there is some evidence suggesting falling rates of mortality in people with incident ( 35,36 ) and prevalent type 2 diabetes ( 37 ) , our study shows that middle - aged patients with type 2 diabetes remain at a significantly increased risk of mortality , compared with people without diabetes , despite more active management of cardiovascular risk factors . therefore , mortality rates do not appear to have fallen more quickly in people with type 2 diabetes than in the general population . our study highlights the important need to continue efforts to improve life expectancy in people with type 2 diabetes . this appears to be particularly important for women and for younger middle - aged people .

objectivemiddle - aged people with diabetes have been reported to have significantly higher risks of cardiovascular events than people without diabetes . however , recent falls in cardiovascular disease rates and more active management of risk factors may have abolished the increased risk . we aimed to provide an up - to - date assessment of the relative risks associated with type 2 diabetes of all - cause and cardiovascular mortality in middle - aged people in the u.k.research design and methodsusing data from the general practice research database , from 2004 to 2010 , we conducted a cohort study of 87,098 people , 4065 years of age at baseline , comparing 21,798 with type 2 diabetes and 65,300 without diabetes , matched on age , sex , and general practice . we produced hazard ratios ( hrs ) for mortality and compared rates of blood pressure testing , cholesterol monitoring , and use of aspirin , statins , and antihypertensive drugs.resultspeople with type 2 diabetes , compared with people without diabetes , had a twofold increased risk of all - cause mortality ( hr 2.07 [ 95% ci 1.952.20 ] , adjusted for smoking ) and a threefold increased risk of cardiovascular mortality ( 3.25 [ 2.873.68 ] , adjusted for smoking ) . women had a higher relative risk than men , and people < 55 years of age had a higher relative risk than those > 55 years of age . monitoring and medication rates were higher in those with diabetes ( all p < 0.001).conclusionsdespite efforts to manage risk factors , administer effective treatments , and develop new therapies , middle - aged people with type 2 diabetes remain at significantly increased risk of death .

RESEARCH DESIGN AND METHODS Data sources and aims Statistical analysis RESULTS CONCLUSIONS

from the dataset received from the gprd , we excluded those who died before 1 january 2004 , < 40 years of age at baseline , with sex unrecorded , with diabetes but not matched to people without diabetes , with diabetes but not identified as having type 2 diabetes , and without diabetes matched to people with diabetes who had been excluded from the cohort . we aimed to compare the populations with and without diabetes in terms of the following : 1 ) baseline characteristics , including smoking , bmi , systolic and diastolic blood pressures , plasma total cholesterol , ldl , hdl , and triglyceride concentrations , and use , by proportions , of aspirin , statins , and antihypertensive drugs ; 2 ) rates of all - cause mortality and cardiovascular mortality ; and 3 ) average annual rates of blood pressure readings , cholesterol monitoring , and prescriptions for aspirin , statins , and antihypertensive drugs . cox proportional hazards models were constructed to estimate hazard ratios ( hrs ) with 95% cis of all - cause mortality and cvd mortality in those with diabetes compared with those without diabetes . from the dataset received from the gprd , we excluded those who died before 1 january 2004 , < 40 years of age at baseline , with sex unrecorded , with diabetes but not matched to people without diabetes , with diabetes but not identified as having type 2 diabetes , and without diabetes matched to people with diabetes who had been excluded from the cohort . we aimed to compare the populations with and without diabetes in terms of the following : 1 ) baseline characteristics , including smoking , bmi , systolic and diastolic blood pressures , plasma total cholesterol , ldl , hdl , and triglyceride concentrations , and use , by proportions , of aspirin , statins , and antihypertensive drugs ; 2 ) rates of all - cause mortality and cardiovascular mortality ; and 3 ) average annual rates of blood pressure readings , cholesterol monitoring , and prescriptions for aspirin , statins , and antihypertensive drugs . cox proportional hazards models were constructed to estimate hazard ratios ( hrs ) with 95% cis of all - cause mortality and cvd mortality in those with diabetes compared with those without diabetes . the hr for all - cause mortality for people with diabetes compared with people without diabetes , adjusted for smoking status ( model ii ) was 2.12 ( 95% ci 2.002.25 ) for all patients . the adjusted relative risk of mortality was higher in women than in men ( men , 1.93 [ 1.792.07 ] ; women , 2.47 [ 2.232.72 ] ) and higher in the < 55 than in the > 55 years of age - group ( < 55 years of age , 2.72 [ 2.423.06 ] ; > 55 years of age , 2.03 [ 1.892.17 ] ) . hrs of all - cause mortality and cardiovascular mortality the hr for cvd mortality for people with diabetes compared with people without diabetes , adjusted for smoking status ( model ii ) , was 3.28 ( 95% ci 2.913.70 ) for all patients . when analyzing the relationship between mortality and duration of diabetes ( supplementary table 1 ) and comparing these with people diagnosed with diabetes 510 years before baseline , we observed a significantly reduced risk of all - cause mortality in those diagnosed < 5 years before baseline ( hr 0.77 [ 95% ci 0.700.86 ] ) and a significantly increased risk in those diagnosed > 10 years before baseline ( 1.69 [ 1.511.90 ] ) . to estimate how much adjusting for major cvd risk factors would attenuate the hrs , we conducted a secondary analysis restricted to 29,361 people with complete data for bmi , blood pressure , and total cholesterol ( 18,591 people with type 2 diabetes and 10,146 without diabetes ) . the frequencies of blood pressure testing and cholesterol monitoring were significantly higher in those with diabetes than those without ( all p < 0.001 ) ( table 3 ) , as was the average annual use of aspirin , statins , and antihypertensive drugs ( all p < 0.001 ) ( table 3 ) . younger middle - aged people with type 2 diabetes ( < 55 years of age ) were at a greater relative risk than older middle - aged people without diabetes ( > 55 years of age ) . as we could not adjust for factors such as renal function , or ethnicity , some residual our findings are consistent with those of both older and more recent studies of middle - aged people with type 2 diabetes , in providing evidence of risk of mortality lower than before , although elevated with respect to the nondiabetic population , with a greater relative risk in women than men ( 1113,1518,21,23,34 ) , and with a lower relative risk at older ages ( 11,16,20,23,34 ) . although there is some evidence suggesting falling rates of mortality in people with incident ( 35,36 ) and prevalent type 2 diabetes ( 37 ) , our study shows that middle - aged patients with type 2 diabetes remain at a significantly increased risk of mortality , compared with people without diabetes , despite more active management of cardiovascular risk factors .

at the time of the birth of general relativity ( gr ) , experimental confirmation was almost a side issue . einstein did calculate observable effects of general relativity , such as the perihelion advance of mercury , which he knew to be an unsolved problem , and the deflection of light , which was subsequently verified . but compared to the inner consistency and elegance of the theory , he regarded such empirical questions as almost peripheral . today , experimental gravitation is a major component of the field , characterized by continuing efforts to test the theory s predictions , to search for gravitational imprints of high - energy particle interactions , and to detect gravitational waves from astronomical sources . the modern history of experimental relativity can be divided roughly into four periods : genesis , hibernation , a golden era , and the quest for strong gravity . the genesis ( 18871919 ) comprises the period of the two great experiments which were the foundation of relativistic physics the michelson - morley experiment and the etvs experiment and the two immediate confirmations of gr the deflection of light and the perihelion advance of mercury . following this was a period of hibernation ( 19201960 ) during which theoretical work temporarily outstripped technology and experimental possibilities , and , as a consequence , the field stagnated and was relegated to the backwaters of physics and astronomy . but beginning around 1960 , astronomical discoveries ( quasars , pulsars , cosmic background radiation ) and new experiments pushed gr to the forefront . experimental gravitation experienced a golden era ( 19601980 ) during which a systematic , world - wide effort took place to understand the observable predictions of gr , to compare and contrast them with the predictions of alternative theories of gravity , and to perform new experiments to test them . the period began with an experiment to confirm the gravitational frequency shift of light ( 1960 ) and ended with the reported decrease in the orbital period of the hulse - taylor binary pulsar at a rate consistent with the gr prediction of gravity wave energy loss ( 1979 ) . the results all supported gr , and most alternative theories of gravity fell by the wayside ( for a popular review , see ) . since 1980 , the field has entered what might be termed a quest for strong gravity . many of the remaining interesting weak - field predictions of the theory are extremely small and difficult to check , in some cases requiring further technological development to bring them into detectable range . the sense of a systematic assault on the weak - field predictions of gr has been supplanted to some extent by an opportunistic approach in which novel and unexpected ( and sometimes inexpensive ) tests of gravity have arisen from new theoretical ideas or experimental techniques , often from unlikely sources . examples include the use of laser - cooled atom and ion traps to perform ultra - precise tests of special relativity ; the proposal of a fifth force , which led to a host of new tests of the weak equivalence principle ; and recent ideas of large extra dimensions , which have motived new tests of the inverse square law of gravity at sub - millimeter scales . instead , much of the focus has shifted to experiments which can probe the effects of strong gravitational fields . the principal figure of merit that distinguishes strong from weak gravity is the quantity gm / ( rc ) , where g is the newtonian gravitational constant , m is the characteristic mass scale of the phenomenon , r is the characteristic distance scale , and c is the speed of light . near the event horizon of a non - rotating black hole , or for the expanding observable universe , 0.5 ; for neutron stars , 0.2 . these are the regimes of strong gravity . for the solar system , < 10 ; this is the regime of weak gravity . at one extreme . will unification of the forces , or quantization of gravity at this scale leave observable effects accessible by experiment ? dramatically improved tests of the equivalence principle , of the inverse square law , or of local lorentz invariance are being mounted , to search for or bound the imprinted effects of planck - scale phenomena . at the other extreme are the strong fields associated with compact objects such as black holes or neutron stars . astrophysical observations and gravitational wave detectors are being planned to explore and test gr in the strong - field , highly - dynamical regime associated with the formation and dynamics of these objects . in this living review , we shall survey the theoretical frameworks for studying experimental gravitation , summarize the current status of experiments , and attempt to chart the future of the subject . we shall not provide complete references to early work done in this field but instead will refer the reader to the appropriate review articles and monographs , specifically to theory and experiment in gravitational physics , hereafter referred to as tegp . additional recent reviews in this subject are [ 276 , 284 , 286 , 71 , 98 , 239 ] . references to tegp will be by chapter or section , e.g. , tegp 8.9 . the principle of equivalence has historically played an important role in the development of gravitation theory . newton regarded this principle as such a cornerstone of mechanics that he devoted the opening paragraph of the principia to it . in 1907 , einstein used the principle as a basic element in his development of general relativity . we now regard the principle of equivalence as the foundation , not of newtonian gravity or of gr , but of the broader idea that spacetime is curved . much of this viewpoint can be traced back to robert dicke , who contributed crucial ideas about the foundations of gravitation theory between 1960 and 1965 . these ideas were summarized in his influential les houches lectures of 1964 , and resulted in what has come to be called the einstein equivalence principle ( eep ) . one elementary equivalence principle is the kind newton had in mind when he stated that the property of a body called mass is proportional to the weight , and is known as the weak equivalence principle ( wep ) . an alternative statement of wep is that the trajectory of a freely falling test body ( one not acted upon by such forces as electromagnetism and too small to be affected by tidal gravitational forces ) is independent of its internal structure and composition . in the simplest case of dropping two different bodies in a gravitational field , wep states that the bodies fall with the same acceleration ( this is often termed the universality of free fall , or uff ) . the einstein equivalence principle ( eep ) is a more powerful and far - reaching concept ; it states that : wep is valid.the outcome of any local non - gravitational experiment is independent of the velocity of the freely - falling reference frame in which it is performed.the outcome of any local non - gravitational experiment is independent of where and when in the universe it is performed . the second piece of eep is called local lorentz invariance ( lli ) , and the third piece is called local position invariance ( lpi ) . the outcome of any local non - gravitational experiment is independent of the velocity of the freely - falling reference frame in which it is performed . the outcome of any local non - gravitational experiment is independent of where and when in the universe it is performed . for example , a measurement of the electric force between two charged bodies is a local non - gravitational experiment ; a measurement of the gravitational force between two bodies ( cavendish experiment ) is not . the einstein equivalence principle is the heart and soul of gravitational theory , for it is possible to argue convincingly that if eep is valid , then gravitation must be a curved spacetime phenomenon , in other words , the effects of gravity must be equivalent to the effects of living in a curved spacetime . as a consequence of this argument , the only theories of gravity that can fully embody eep are those that satisfy the postulates of metric theories of gravity , which are : spacetime is endowed with a symmetric metric.the trajectories of freely falling test bodies are geodesics of that metric.in local freely falling reference frames , the non - gravitational laws of physics are those written in the language of special relativity . local freely falling reference frames , the non - gravitational laws of physics are those written in the language of special relativity . the argument that leads to this conclusion simply notes that , if eep is valid , then in local freely falling frames , the laws governing experiments must be independent of the velocity of the frame ( local lorentz invariance ) , with constant values for the various atomic constants ( in order to be independent of location ) . the only laws we know of that fulfill this are those that are compatible with special relativity , such as maxwell s equations of electromagnetism . furthermore , in local freely falling frames , test bodies appear to be unaccelerated , in other words they move on straight lines ; but such locally straight lines simply correspond to geodesics in a curved spacetime ( tegp 2.3 ) . general relativity is a metric theory of gravity , but then so are many others , including the brans - dicke theory and its generalizations . theories in which varying non - gravitational constants are associated with dynamical fields that couple to matter directly are not metric theories . neither , in this narrow sense , is superstring theory ( see section 2.3 ) , which , while based fundamentally on a spacetime metric , introduces additional fields ( dilatons , moduli ) that can couple to material stress - energy in a way that can lead to violations , say , of wep . it is important to point out , however , that there is some ambiguity in whether one treats such fields as eep - violating gravitational fields , or simply as additional matter fields , like those that carry electromagnetism or the weak interactions . still , the notion of curved spacetime is a very general and fundamental one , and therefore it is important to test the various aspects of the einstein equivalence principle thoroughly . we first survey the experimental tests , and describe some of the theoretical formalisms that have been developed to interpret them . for other reviews of eep and its experimental and theoretical significance , see [ 126 , 162 ] . a direct test of wep is the comparison of the acceleration of two laboratory - sized bodies of different composition in an external gravitational field . the simplest way to quantify such possible violations of wep in a form suitable for comparison with experiment is to suppose that for a body with inertial mass mi , the passive gravitational mass mp is no longer equal to mi , so that in a gravitational field g , the acceleration is given by mia = mpg . now the inertial mass of a typical laboratory body is made up of several types of mass - energy : rest energy , electromagnetic energy , weak - interaction energy , and so on . if one of these forms of energy contributes to mp differently than it does to mi , a violation of wep would result . one could then write 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{\rm{p } } } = { m_{\rm{i } } } + \sum\limits_a { { { { \eta ^a}{e^a } } \over { { c^2}}},}$$\end{document } where e is the internal energy of the body generated by interaction a , is a dimensionless parameter that measures the strength of the violation of wep induced by that interaction , and c is the speed of light . a measurement or limit on the fractional difference in acceleration between two bodies then yields a quantity called the etvs ratio given by 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta \equiv 2{{\vert{a_1 } - { a_2}\vert } \over { \vert{a_1 } + { a_2}\vert } } = \sum\limits_a { { \eta ^a}\left({{{e_1^a } \over { { m_1}{c^2 } } } - { { e_2^a } \over { { m_2}{c^2 } } } } thus , experimental limits on place limits on the wep - violation parameters . many high - precision etvs - type experiments have been performed , from the pendulum experiments of newton , bessel , and potter to the classic torsion - balance measurements of etvs , dicke , braginsky , and their collaborators . in the modern torsion - balance experiments , two objects of different composition are connected by a rod or placed on a tray and suspended in a horizontal orientation by a fine wire . if the gravitational acceleration of the bodies differs , and this difference has a component perpendicular to the suspension wire , there will be a torque induced on the wire , related to the angle between the wire and the direction of the gravitational acceleration g. if the entire apparatus is rotated about some direction with angular velocity , the torque will be modulated with period 2/. in the experiments of etvs and his collaborators , the wire and g were not quite parallel because of the centripetal acceleration on the apparatus due to the earth s rotation ; the apparatus was rotated about the direction of the wire . in the dicke and braginsky experiments , g was that of the sun , and the rotation of the earth provided the modulation of the torque at a period of 24 hr ( tegp 2.4 ( a ) ) . beginning in the late 1980s , numerous experiments were carried out primarily to search for a fifth force ( see section 2.3.1 ) , but their null results also constituted tests of wep . in the free - fall galileo experiment performed at the university of colorado , the relative free - fall acceleration of two bodies made of uranium and copper was measured using a laser interferometric technique . et - wash experiments carried out at the university of washington used a sophisticated torsion balance tray to compare the accelerations of various materials toward local topography on earth , movable laboratory masses , the sun and the galaxy [ 249 , 19 ] , and have reached levels of 3 10 . the resulting upper limits on are summarized in figure 1 ( tegp 14.1 ; for a bibliography of experiments up to 1991 , see ) . figure 1selected tests of the weak equivalence principle , showing bounds on , which measures fractional difference in acceleration of different materials or bodies . the free - fall and et - wash experiments were originally performed to search for a fifth force ( green region , representing many experiments ) . the blue band shows evolving bounds on for gravitating bodies from lunar laser ranging ( llr ) . selected tests of the weak equivalence principle , showing bounds on , which measures fractional difference in acceleration of different materials or bodies . the free - fall and et - wash experiments were originally performed to search for a fifth force ( green region , representing many experiments ) . the blue band shows evolving bounds on for gravitating bodies from lunar laser ranging ( llr ) . a number of projects are in the development or planning stage to push the bounds on even lower . the project microscope ( micro - satellite traine compense pour lobservation du principe dquivalence ) is designed to test wep to 10 . it is being developed by the french space agency cnes for a possible launch in march , 2008 , for a one - year mission . the drag - compensated satellite will be in a sun - synchronous polar orbit at 700 km altitude , with a payload consisting of two differential accelerometers , one with elements made of the same material ( platinum ) , and another with elements made of different materials ( platinum and titanium ) . another , known as satellite test of the equivalence principle ( step ) , is under consideration as a possible joint effort of nasa and the european space agency ( esa ) , with the goal of a 10 test . step would improve upon microscope by using cryogenic techniques to reduce thermal noise , among other effects . at present , step ( along with a number of variants , called ministep and quickstep ) has not been approved by any agency beyond the level of basic design studies or supporting research and development . an alternative concept for a space test of wep is galileo - galilei , which uses a rapidly rotating differential accelerometer as its basic element . its goal is a bound on at the 10 level on the ground and 10 in space . although special relativity itself never benefited from the kind of crucial experiments , such as the perihelion advance of mercury and the deflection of light , that contributed so much to the initial acceptance of gr and to the fame of einstein , the steady accumulation of experimental support , together with the successful merger of special relativity with quantum mechanics , led to its being accepted by mainstream physicists by the late 1920s , ultimately to become part of the standard toolkit of every working physicist . this accumulation included the classic michelson - morley experiment and its descendents [ 186 , 237 , 141 , 46],the ives - stillwell , rossi - hall , and other tests of time - dilation [ 136 , 229 , 103],tests of the independence of the speed of light of the velocity of the source , using both binary x - ray stellar sources and high - energy pions [ 44 , 5],tests of the isotropy of the speed of light [ 50 , 227 , 159 ] . the classic michelson - morley experiment and its descendents [ 186 , 237 , 141 , 46 ] , the ives - stillwell , rossi - hall , and other tests of time - dilation [ 136 , 229 , 103 ] , tests of the independence of the speed of light of the velocity of the source , using both binary x - ray stellar sources and high - energy pions [ 44 , 5 ] , tests of the isotropy of the speed of light [ 50 , 227 , 159 ] . in addition to these direct experiments , there was the dirac equation of quantum mechanics and its prediction of anti - particles and spin ; later would come the stunningly successful relativistic theory of quantum electrodynamics . in 2005 , on the 100th anniversary of the introduction of special relativity , one might ask what is there to test ? . special relativity has been so thoroughly integrated into the fabric of modern physics that its validity is rarely challenged , except by cranks and crackpots . it is ironic then , that during the past several years , a vigorous theoretical and experimental effort has been launched , on an international scale , to find violations of special relativity . the motivation for this effort is not a desire to repudiate einstein , but to look for evidence of new physics beyond einstein , such as apparent violations of lorentz invariance that might result from certain models of quantum gravity . quantum gravity asserts that there is a fundamental length scale given by the planck length , lpl = ( g / c ) = 1.6 10 cm , but since length is not an invariant quantity ( lorentz - fitzgerald contraction ) , then there could be a violation of lorentz invariance at some level in quantum gravity . in brane world scenarios , while physics may be locally lorentz invariant in the higher dimensional world , the confinement of the interactions of normal physics to our four - dimensional brane could induce apparent lorentz violating effects . and in models such as string theory , the presence of additional scalar , vector , and tensor long - range fields that couple to matter of the standard model could induce effective violations of lorentz symmetry . these and other ideas have motivated a serious reconsideration of how to test lorentz invariance with better precision and in new ways . a simple and useful way of interpreting some of these modern experiments , called the c - formalism , is to suppose that the electromagnetic interactions suffer a slight violation of lorentz invariance , through a change in the speed of electromagnetic radiation c relative to the limiting speed of material test particles ( c0 , made to take the value unity via a choice of units ) , in other words , c 1 ( see section 2.2.3 ) . such a violation necessarily selects a preferred universal rest frame , presumably that of the cosmic background radiation , through which we are moving at about 370 km s . such a lorentz - non - invariant electromagnetic interaction would cause shifts in the energy levels of atoms and nuclei that depend on the orientation of the quantization axis of the state relative to our universal velocity vector , and on the quantum numbers of the state . the presence or absence of such energy shifts can be examined by measuring the energy of one such state relative to another state that is either unaffected or is affected differently by the supposed violation . one way is to look for a shifting of the energy levels of states that are ordinarily equally spaced , such as the zeeman - split 2j + 1 ground states of a nucleus of total spin j in a magnetic field ; another is to compare the levels of a complex nucleus with the atomic hyperfine levels of a hydrogen maser clock . the earliest clock anisotropy experiments were the hughes - drever experiments , performed in the period 195960 independently by hughes and collaborators at yale university , and by drever at glasgow university , although their original motivation was somewhat different [ 131 , 96 ] . the hughes - drever experiments yielded extremely accurate results , quoted as limits on the parameter c 1 in figure 2 . dramatic improvements were made in the 1980s using laser - cooled trapped atoms and ions [ 215 , 163 , 53 ] . this technique made it possible to reduce the broading of resonance lines caused by collisions , leading to improved bounds on shown in figure 2 ( experiments labelled nist , u. washington and harvard , respectively ) . figure 2selected tests of local lorentz invariance showing the bounds on the parameter , which measures the degree of violation of lorentz invariance in electromagnetism . the michelson - morley , joos , brillet - hall and cavity experiments test the isotropy of the round - trip speed of light . the centrifuge , two - photon absorption ( tpa ) and jpl experiments test the isotropy of light speed using one - way propagation . the limits assume a speed of earth of 370 km s relative to the mean rest frame of the universe . selected tests of local lorentz invariance showing the bounds on the parameter , which measures the degree of violation of lorentz invariance in electromagnetism . the michelson - morley , joos , brillet - hall and cavity experiments test the isotropy of the round - trip speed of light . the centrifuge , two - photon absorption ( tpa ) and jpl experiments test the isotropy of light speed using one - way propagation . the limits assume a speed of earth of 370 km s relative to the mean rest frame of the universe . also included for comparison is the corresponding limit obtained from michelson - morley type experiments ( for a review , see ) . in those experiments , when viewed from the preferred frame , the speed of light down the two arms of the moving interferometer is c , while it can be shown using the electrodynamics of the c formalism , that the compensating lorentz - fitzgerald contraction of the parallel arm is governed by the speed c0 = 1 . thus the michelson - morley experiment and its descendants also measure the coefficient c 1 . one of these is the brillet - hall experiment , which used a fabry - perot laser interferometer . in a recent series of experiments , the frequencies of electromagnetic cavity oscillators in various orientations were compared with each other or with atomic clocks as a function of the orientation of the laboratory [ 297 , 168 , 190 , 12 , 248 ] . these placed bounds on c 1 at the level of better than a part in 10 . haugan and lmmerzahl have considered the bounds that michelson - morley type experiments could place on a modified electrodynamics involving a vector - valued effective photon mass . it has recently been extended to the entire standard model of particle physics by kosteleck and colleagues [ 63 , 64 , 155 ] . ( sme ) has a large number of lorentz - violating parameters , opening up many new opportunities for experimental tests ( see section 2.2.4 ) . a variety of clock anisotropy experiments have been carried out to bound the electromagnetic parameters of the sme framework . for example , the cavity experiments described above [ 297 , 168 , 190 ] placed bounds on the coefficients of the tensors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde k}_{{\rm{e } } -}}$\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}${{\tilde k}_{{\rm{o + } } } } $ \end{document } ( see section 2.2.4 for definitions ) at the levels of 10 and 10 , respectively . direct comparisons between atomic clocks based on different nuclear species place bounds on sme parameters in the neutron and proton sectors , depending on the nature of the transitions involved . the bounds achieved range from 10 to 10 gev . for example , if photons satisfy the lorentz violating dispersion relation 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^2 } = { p^2}{c^2 } + { e_{{\rm{pl}}}}{f^{(1)}}\vert p\vert c + { f^{(2)}}{p^2}{c^2 } + { { { f^{(3 ) } } } \over { { e_{{\rm{pl}}}}}}\vert p\vert^{3}{c^3 } + \ldots,$$\end{document } where epl = ( c / g ) is the planck energy , then the speed of light = e/p would be given , to linear order in the f by 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{\upsilon_\gamma } } \over c } \approx 1 + \sum\limits_{n \geq 1 } { { { ( n - 1)f_\gamma ^{(n)}{e^{n - 2 } } } \over { 2e_{{\rm{pl}}}^{n - 2}}}.}$$\end{document } such a lorentz - violating dispersion relation could be a relic of quantum gravity , for instance . by bounding the difference in arrival time of high - energy photons from a burst source at large distances , one could bound contributions to the dispersion for n > 2 . one limit , |f| < 128 comes from observations of 1 and 2 tev gamma rays from the blazar markarian 421 . another limit comes from birefringence in photon propagation : in many lorentz violating models , different photon polarizations may propagate with different speeds , causing the plane of polarization of a wave to rotate . if the frequency dependence of this rotation has a dispersion relation similar to equation ( 3 ) , then by studying polarization diffusion of light from a polarized source in a given bandwidth , one can effectively place a bound |f| < 10 . other testable effects of lorentz invariance violation include threshold effects in particle reactions , gravitational cerenkov radiation , and neutrino oscillations . mattingly gives a thorough and up - to - date review of both the theoretical frameworks and the experimental results for tests of lli . the principle of local position invariance , the third part of eep , can be tested by the gravitational redshift experiment , the first experimental test of gravitation proposed by einstein . despite the fact that einstein regarded this as a crucial test of gr , we now realize that it does not distinguish between gr and any other metric theory of gravity , but is only a test of eep . / = / between two identical frequency standards ( clocks ) placed at rest at different heights in a static gravitational field . if the frequency of a given type of atomic clock is the same when measured in a local , momentarily comoving freely falling frame ( lorentz frame ) , independent of the location or velocity of that frame , then the comparison of frequencies of two clocks at rest at different locations boils down to a comparison of the velocities of two local lorentz frames , one at rest with respect to one clock at the moment of emission of its signal , the other at rest with respect to the other clock at the moment of reception of the signal . the frequency shift is then a consequence of the first - order doppler shift between the frames . the result is a shift 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z = { { \delta u } \over { { c^2}}},$$\end{document } where u is the difference in the newtonian gravitational potential between the receiver and the emitter . if lpi is not valid , then it turns out that the shift can be written 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z = ( 1 + \alpha){{\delta u } \over { { c^2}}},$$\end{document } where the parameter may depend upon the nature of the clock whose shift is being measured ( see tegp 2.4 ( c ) for details ) . the first successful , high - precision redshift measurement was the series of pound - rebka - snider experiments of 19601965 that measured the frequency shift of gamma - ray photons from fe as they ascended or descended the jefferson physical laboratory tower at harvard university . the high accuracy achieved one percent was obtained by making use of the mossbauer effect to produce a narrow resonance line whose shift could be accurately determined . other experiments since 1960 measured the shift of spectral lines in the sun s gravitational field and the change in rate of atomic clocks transported aloft on aircraft , rockets and satellites . figure 3 summarizes the important redshift experiments that have been performed since 1960 ( tegp 2.4 ( c ) ) . figure 3selected tests of local position invariance via gravitational redshift experiments , showing bounds on , which measures degree of deviation of redshift from the formula / = u / c . in null redshift experiments , selected tests of local position invariance via gravitational redshift experiments , showing bounds on , which measures degree of deviation of redshift from the formula / = u / c . in null redshift experiments , after almost 50 years of inconclusive or contradictory measurements , the gravitational redshift of solar spectral lines was finally measured reliably . during the early years of gr , the failure to measure this effect in solar lines was siezed upon by some as reason to doubt the theory . solar spectral lines are subject to the limb effect , a variation of spectral line wavelengths between the center of the solar disk and its edge or limb ; this effect is actually a doppler shift caused by complex convective and turbulent motions in the photosphere and lower chromosphere , and is expected to be minimized by observing at the solar limb , where the motions are predominantly transverse . the secret is to use strong , symmetrical lines , leading to unambiguous wavelength measurements . successful measurements were finally made in 1962 and 1972 ( tegp 2.4 ( c ) ) . in 1991 , lopresto et al . measured the solar shift in agreement with lpi to about 2 percent by observing the oxygen triplet lines both in absorption in the limb and in emission just off the limb . the most precise standard redshift test to date was the vessot - levine rocket experiment that took place in june 1976 . a hydrogen - maser clock was flown on a rocket to an altitude of about 10,000 km and its frequency compared to a similar clock on the ground . the experiment took advantage of the masers frequency stability by monitoring the frequency shift as a function of altitude . a sophisticated data acquisition scheme accurately eliminated all effects of the first - order doppler shift due to the rocket s motion , while tracking data were used to determine the payload s location and the velocity ( to evaluate the potential difference u , and the special relativistic time dilation ) redshift experiment performed in 1978 tested whether the relative rates of two different clocks depended upon position . two hydrogen maser clocks and an ensemble of three superconducting - cavity stabilized oscillator ( scso ) clocks were compared over a 10-day period . during the period of the experiment , the solar potential u / c changed sinusoidally with a 24-hour period by 3 10 because of the earth s rotation , and changed linearly at 3 10 per day because the earth is 90 degrees from perihelion in april . however , analysis of the data revealed no variations of either type within experimental errors , leading to a limit on the lpi violation parameter | this bound has been improved using more stable frequency standards , such as atomic fountain clocks [ 120 , 216 , 23 ] . the current bound , from comparing a cesium atomic fountain with a hydrogen maser for a year , is | | < 2.1 10 . the varying gravitational redshift of earth - bound clocks relative to the highly stable millisecond pulsar psr 1937 + 21 , caused by the earth s motion in the solar gravitational field around the earth - moon center of mass ( amplitude 4000 km ) , was measured to about 10 percent . two measurements of the redshift using stable oscillator clocks on spacecraft were made at the one percent level : one used the voyager spacecraft in saturn s gravitational field , while another used the galileo spacecraft in the sun s field . the gravitational redshift could be improved to the 10 level using an array of laser cooled atomic clocks on board a spacecraft which would travel to within four solar radii of the sun . modern advances in navigation using earth - orbiting atomic clocks and accurate time - transfer must routinely take gravitational redshift and time - dilation effects into account . for example , the global positioning system ( gps ) provides absolute positional accuracies of around 15 m ( even better in its military mode ) , and 50 nanoseconds in time transfer accuracy , anywhere on earth . yet the difference in rate between satellite and ground clocks as a result of relativistic effects is a whopping 39 microseconds per day ( 46 s from the gravitational redshift , and 7 s from time dilation ) . if these effects were not accurately accounted for , gps would fail to function at its stated accuracy . this represents a welcome practical application of gr ! ( for the role of gr in gps , see [ 15 , 16 ] ; for a popular essay , see . ) if lpi is satisfied , the fundamental constants of non - gravitational physics should be constants in time . table 1 shows current bounds on cosmological variations in selected dimensionless constants . for discussion and references to early work , see tegp 2.4 ( c ) or . for a comprehensive recent review both of experiments and of theoretical ideas that underly proposals for varying constants , see . table 1bounds on cosmological variation of fundamental constants of non - gravitational physics . for an in - depth review , see .constant klimit on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot k / k$\end{document } ( yr)redshiftmethod(yr)fine structure constant ( em = e / c ) < 30 100clock comparisons [ 181 , 31 , 111 , 209 ] < 0.5 100.15oklo natural reactor [ 72 , 116 , 210 ] < 3.4 100.45re decay in meteorites ( 6.4 1.4 ) 100.23.7spectra in distant quasars [ 269 , 193 ] < 1.2 100.42.3spectra in distant quasars [ 242 , 51]weak interaction constant \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\alpha _ { \rm{w } } } = { g_{\rm{f}}}m_{\rm{p}}^2c/{\hbar ^3})$\end{document } < 1 100.15oklo natural reactor < 5 1010big bang nucleosynthesis [ 179 , 223]e - p mass ratio < 3 102.63.0spectra in distant quasars bounds on cosmological variation of fundamental constants of non - gravitational physics . for an in - depth review , see . experimental bounds on varying constants come in two types : bounds on the present rate of variation , and bounds on the difference between today s value and a value in the distant past . the main example of the former type is the clock comparison test , in which highly stable atomic clocks of different fundamental type are intercompared over periods ranging from months to years ( variants of the null redshift experiment ) . if the frequencies of the clocks depend differently on the electromagnetic fine structure constant em , the electron - proton mass ratio me / mp , or the gyromagnetic ratio of the proton gp , for example , then a limit on a drift of the fractional frequency difference translates into a limit on a drift of the constant(s ) . the dependence of the frequencies on the constants may be quite complex , depending on the atomic species involved . the most recent experiments have exploited the techniques of laser cooling and trapping , and of atom fountains , in order to achieve extreme clock stability , and compared the rubidium-87 hyperfine transition , the mercury-199 ion electric quadrupole transition , the atomic hydrogen 1s2s transition , or an optical transition in ytterbium-171 , against the ground - state hyperfine transition in cesium-133 . these experiments show that , today , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\dot \alpha}_{{\rm{em}}}}/{\alpha _ { { \rm{em } } } } < 3 \times { 10^{- 15}}{\rm{y}}{{\rm{r}}^{- 1}}$\end{document}. the second type of bound involves measuring the relics of or signal from a process that occurred in the distant past and comparing the inferred value of the constant with the value measured in the laboratory today . one sub - type uses astronomical measurements of spectral lines at large redshift , while the other uses fossils of nuclear processes on earth to infer values of constants early in geological history . earlier comparisons of spectral lines of different atoms or transitions in distant galaxies and quasars produced bounds em or gp(me / mp ) on the order of a part in 10 per hubble time . dramatic improvements in the precision of astronomical and laboratory spectroscopy , in the ability to model the complex astronomical environments where emission and absorption lines are produced , and in the ability to reach large redshift have made it possible to improve the bounds significantly . in fact , in 1999 , webb et al . [ 269 , 193 ] announced that measurements of absorption lines in mg , al , si , cr , fe , ni , and zn in quasars in the redshift range 0.5 < z < 3.5 indicated a smaller value of em in earlier epochs , namely em/em = ( 0.72 0.18 ) 10 , corresponding to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\dot \alpha}_{{\rm{em}}}}/{\alpha _ { { \rm{em } } } } = ( 6.4 \pm 1.4 ) \times { 10^{- 16}}{\rm{y}}{{\rm{r}}^{{\rm{- 1}}}}$\end{document } ( assuming a linear drift with time ) . measurements by other groups have so far failed to confirm this non - zero effect [ 242 , 51 , 219 ] ; a recent analysis of mg absorption systems in quasars at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0.4 < z < 2.3\,{\rm{gave}}\,{{\dot \alpha}_{{\rm{em}}}}/{\alpha _ { { \rm{em } } } } = ( - 0.6 \pm 0.6 ) \times { 10^{- 16}}{\rm{y}}{{\rm{r}}^{- 1}}$\end{document } . another important set of bounds arises from studies of the oklo phenomenon , a group of natural , sustained u fission reactors that occurred in the oklo region of gabon , africa , around 1.8 billion years ago . measurements of ore samples yielded an abnormally low value for the ratio of two isotopes of samarium , sm / sm . neither of these isotopes is a fission product , but sm can be depleted by a flux of neutrons . estimates of the neutron fluence ( integrated dose ) during the reactors on phase , combined with the measured abundance anomaly , yield a value for the neutron cross - section for sm 1.8 billion years ago that agrees with the modern value . however , the capture cross - section is extremely sensitive to the energy of a low - lying level ( e 0.1 ev ) , so that a variation in the energy of this level of only 20 mev over a billion years would change the capture cross - section from its present value by more than the observed amount . recent reanalyses of the oklo data [ 72 , 116 , 210 ] lead to a bound on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\dot \alpha}_{{\rm{em}}}}$\end{document } at the level of around 5 10 yr . in a similar manner , recent reanalyses of decay rates of re in ancient meteorites ( 4.5 billion years old ) gave the bound \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\dot \alpha}_{{\rm{em}}}}/{\alpha _ { { \rm{em } } } } < 3.4 \times { 10^{- 16}}{\rm{y}}{{\rm{r}}^{{\rm{- 1}}}}$\end{document } . because the three parts of the einstein equivalence principle discussed above are so very different in their empirical consequences , it is tempting to regard them as independent theoretical principles . on the other hand , any complete and self - consistent gravitation theory must possess sufficient mathematical machinery to make predictions for the outcomes of experiments that test each principle , and because there are limits to the number of ways that gravitation can be meshed with the special relativistic laws of physics , one might not be surprised if there were theoretical connections between the three sub - principles . for instance , the same mathematical formalism that produces equations describing the free fall of a hydrogen atom must also produce equations that determine the energy levels of hydrogen in a gravitational field , and thereby the ticking rate of a hydrogen maser clock . hence a violation of eep in the fundamental machinery of a theory that manifests itself as a violation of wep might also be expected to show up as a violation of local position invariance . around 1960 , schiff conjectured that this kind of connection was a necessary feature of any self - consistent theory of gravity . more precisely , schiff s conjecture states that any complete , self - consistent theory of gravity that embodies wep necessarily embodies eep . in other words , the validity of wep alone guarantees the validity of local lorentz and position invariance , and thereby of eep . if schiff s conjecture is correct , then etvs experiments may be seen as the direct empirical foundation for eep , hence for the interpretation of gravity as a curved - spacetime phenomenon . of course , a rigorous proof of such a conjecture is impossible ( indeed , some special counter - examples are known [ 204 , 194 , 62 ] ) , yet a number of powerful plausibility arguments can be formulated . the most general and elegant of these arguments is based upon the assumption of energy conservation . this assumption allows one to perform very simple cyclic gedanken experiments in which the energy at the end of the cycle must equal that at the beginning of the cycle . this approach was pioneered by dicke , nordtvedt , and haugan ( see , e.g. , ) . a system in a quantum state a decays to state b , emitting a quantum of frequency . the quantum falls a height h in an external gravitational field and is shifted to frequency , while the system in state b falls with acceleration gb . at the bottom , state a is rebuilt out of state b , the quantum of frequency , and the kinetic energy mbgbh that state b has gained during its fall . the energy left over must be exactly enough , magah , to raise state a to its original location . ( here an assumption of local lorentz invariance permits the inertial masses ma and mb to be identified with the total energies of the bodies . ) if ga and gb depend on that portion of the internal energy of the states that was involved in the quantum transition from a to b according to 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_a } = g\left({1 + { { \alpha { e_a } } \over { { m_a}{c^2 } } } } \right),\ , \quad { g_b } = g\left({1 + { { \alpha { e_b } } \over { { m_b}{c^2 } } } } \right),\quad { e_a } - { e_b } \equiv h\nu$$\end{document } ( violation of wep ) , then by conservation of energy , there must be a corresponding violation of lpi in the frequency shift of the form ( to lowest order in h/mc ) 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$z = { { \nu{\prime } - \nu } \over { \nu{\prime } } } = ( 1 + \alpha){{gh } \over { { c^2 } } } = ( 1 + \alpha){{\delta u } \over { { c^2}}}.$$\end{document } haugan generalized this approach to include violations of lli ( tegp 2.5 ) . coordinate system and conventions : x = t : time coordinate associated with the static nature of the static spherically symmetric ( sss ) gravitational field ; x = ( x , y , z ) : isotropic quasi - cartesian spatial coordinates ; spatial vector and gradient operations as in cartesian space.matter and field variables : m0a : rest mass of particle a.ea : charge of particle a.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$x_a^\mu ( t)$\end{document } : world line of particle a.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\upsilon _ a^\mu = dx_a^\mu / dt$\end{document } : coordinate velocity of particle a.a = : electromagnetic vector potential ; e = a0 a/t , b = a.gravitational potential : u(x).arbitrary functions : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t(u),h(u),\epsilon ( u),\mu ( u);\,{\rm{eep}}\,{\rm{is}}\,{\rm{satisfied}}\,{\rm{if}}\,\epsilon = \mu = { ( h / t)^{1/2}}{\rm{for}}\,{\rm{all}}\,u.$$\end{document } action : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = - \sum\limits_a { { m_{{0a}}}\int { { { ( t - h\upsilon_a^2)}^{1/2}}dt } } + \sum\limits_a { { e_a}\int { { a_\mu}(x_a^\nu)\upsilon_a^\mu dt + { { ( 8\pi)}^{- 1}}\int { ( \epsilon { e^2 } - { \mu ^{- 1}}{b^2 } ) } { d^4}x.}}$$\end{document } non - metric parameters:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ 0 } = - c_0 ^ 2{\partial \over { \partial u}}\ln { [ \epsilon { ( t / h)^{1/2}}]_0},\quad { \lambda _ 0 } = - c_0 ^ 2{\partial \over { \partial u}}\ln { [ \mu { ( t / h)^{1/2}}]_0},\quad { \upsilon _ 0 } = 1 - { ( t{h^{- 1}}\epsilon \mu)_0},$$\end{document } where c0 = ( t0/h0 ) and subscript 0 refers to a chosen point in space . if eep is satisfied , 0 0 0 0 . coordinate system and conventions : x = t : time coordinate associated with the static nature of the static spherically symmetric ( sss ) gravitational field ; x = ( x , y , z ) : isotropic quasi - cartesian spatial coordinates ; spatial vector and gradient operations as in cartesian space . matter and field variables : m0a : rest mass of particle a.ea : charge of particle a.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$x_a^\mu ( t)$\end{document } : world line of particle a.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\upsilon _ a^\mu = dx_a^\mu / dt$\end{document } : coordinate velocity of particle a.a = : electromagnetic vector potential ; e = a0 a/t , b = a. m0a : rest mass of particle a. ea : charge of particle a. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$x_a^\mu ( t)$\end{document } : world line of particle a. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\upsilon _ a^\mu = dx_a^\mu / dt$\end{document } : coordinate velocity of particle a. a = : electromagnetic vector potential ; e = a0 a/t , b = a. gravitational potential : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t(u),h(u),\epsilon ( u),\mu ( u);\,{\rm{eep}}\,{\rm{is}}\,{\rm{satisfied}}\,{\rm{if}}\,\epsilon = \mu = { ( h / t)^{1/2}}{\rm{for}}\,{\rm{all}}\,u.$$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = - \sum\limits_a { { m_{{0a}}}\int { { { ( t - h\upsilon_a^2)}^{1/2}}dt } } + \sum\limits_a { { e_a}\int { { a_\mu}(x_a^\nu)\upsilon_a^\mu dt + { { ( 8\pi)}^{- 1}}\int { ( \epsilon { e^2 } - { \mu ^{- 1}}{b^2 } ) } { d^4}x.}}$$\end{document } non - metric parameters : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ 0 } = - c_0 ^ 2{\partial \over { \partial u}}\ln { [ \epsilon { ( t / h)^{1/2}}]_0},\quad { \lambda _ 0 } = - c_0 ^ 2{\partial \over { \partial u}}\ln { [ \mu { ( t / h)^{1/2}}]_0},\quad { \upsilon _ 0 } = 1 - { ( t{h^{- 1}}\epsilon \mu)_0},$$\end{document } where c0 = ( t0/h0 ) and subscript 0 refers to a chosen point in space . if eep is satisfied , 0 0 0 0 . the first successful attempt to prove schiff s conjecture more formally was made by lightman and lee . they developed a framework called the th formalism that encompasses all metric theories of gravity and many non - metric theories ( see box 1 ) . it restricts attention to the behavior of charged particles ( electromagnetic interactions only ) in an external static spherically symmetric ( sss ) gravitational field , described by a potential u. it characterizes the motion of the charged particles in the external potential by two arbitrary functions t(u ) and h(u ) , and characterizes the response of electromagnetic fields to the external potential ( gravitationally modified maxwell equations ) by two functions (u ) and (u ) . the forms of t , h , , and vary from theory to theory , but every metric theory satisfies 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\epsilon = \mu = { \left({{h \over t } } \right)^{1/2}},$$\end{document } for all u. this consequence follows from the action of electrodynamics with a minimal or metric coupling : 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = - \sum\limits_a { { m_{0a}}{{\int { ( - { g_{\mu \nu}}\upsilon _ \upsilon _ a^\nu)}}^{1/2 } } } \;dt + \sum\limits_a { { e_a}\int { { a_\mu}(x_a^\nu)\upsilon _ a^\mu } } \;dt - { 1 \over { 16\pi}}\int { \sqrt { - g } { g^{\mu \alpha}}{g^{\nu \beta}}{f_{\mu \nu}}{f_{\alpha \beta } } } \;{d^4}x,$$\end{document } where the variables are defined in box 1 , and where f a, a,. by identifying g00 = t and gij = hij in a sss field , fi0 = ei and fij = ijkbk , one obtains equation ( 9 ) . conversely , every theory within this class that satisfies equation ( 9 ) can have its electrodynamic equations cast into metric form . in a given non - metric theory , the functions t , h , , and will depend in general on the full gravitational environment , including the potential of the earth , sun , and galaxy , as well as on cosmological boundary conditions . which of these factors has the most influence on a given experiment will depend on the nature of the experiment . body made up of interacting charged particles , and found that the rate was independent of the internal electromagnetic structure of the body ( wep ) if and only if equation ( 9 ) was satisfied . in other words , wep eep and schiff s conjecture was verified , at least within the restrictions built into the formalism . certain combinations of the functions t , h , , and reflect different aspects of eep . for instance , position or u - dependence of either of the combinations (t / h ) and (t / h ) signals violations of lpi , the first combination playing the role of the locally measured electric charge or fine structure constant . the non - metric parameters 0 and 0 ( see box 1 ) are measures of such violations of eep . similarly , if the parameter 0 1 ( th)0 is non - zero anywhere , then violations of lli will occur . this parameter is related to the difference between the speed of light c , and the limiting speed of material test particles c0 , given by 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c = { ( { \epsilon _ 0}{\mu _ 0})^{- 1/2}},{c_0 } = { \left({{{{t_0 } } \over { { h_0 } } } } \right)^{1/2}}.$$\end{document } in many applications , by suitable definition of units , c0 can be set equal to unity . if eep is valid , 0 0 0 = 0 everywhere . the rate of fall of a composite spherical test body of electromagnetically interacting particles then has the form 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bf{a } } = { { { m_{\rm{p } } } } \over m}\nabla u,$$\end{document } 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{m_{\rm{p } } } } \over m } = 1 + { { e_{\rm{b}}^{{\rm{es } } } } \over { mc_0 ^ 2}}\left [ { 2{\gamma _ 0 } - { 8 \over 3}{\upsilon _ 0 } } \right ] + { { e_{\rm{b}}^{{\rm{ms } } } } \over { mc_0 ^ 2}}\left [ { 2{\lambda _ 0 } - { 4 \over 3}{\upsilon _ 0 } } \right ] + \cdots,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{\rm{b}}^{{\rm{es}}}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{\rm{b}}^{{\rm{ms}}}$\end{document } are the electrostatic and magnetostatic binding energies of the body , given by 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e_{\rm{b}}^{{\rm{es } } } = - { 1 \over 4}t_0^{1/2}h_0^{- 1}\epsilon _ 0^{- 1}\left\langle { \sum\limits_{ab } { { { { e_a}{e_b } } \over { { r_{ab } } } } } } \right\rangle,$$\end{document } 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e_{\rm{b}}^{{\rm{ms } } } = - { 1 \over 8}t_0^{1/2}h_0^{- 1}{\mu _ 0}\left\langle { \sum\limits_{ab } { { { { e_a}{e_b } } \over { { r_{ab}}}}[{{\bf{v}}_a } \cdot { { \bf{v}}_b } + ( { { \bf{v}}_a } \cdot { { \bf{n}}_{ab}})({{\bf{v}}_b } \cdot { { \bf{n}}_{ab } } ) ] } } \right\rangle,$$\end{document } where rab = |xa xb| , nab = ( xa xb)/rab , and the angle brackets denote an expectation value of the enclosed operator for the system s internal state . etvs experiments place limits on the wep - violating terms in equation ( 13 ) , and ultimately place limits on the non - metric parameters |0| < 2 10 and |0| < 3 10 . ( we set 0 = 0 because of very tight constraints on it from tests of lli ; see figure 2 , where = . ) these limits are sufficiently tight to rule out a number of non - metric theories of gravity thought previously to be viable ( tegp 2.6 ( f ) ) . the th formalism also yields a gravitationally modified dirac equation that can be used to determine the gravitational redshift experienced by a variety of atomic clocks . for the redshift parameter ( see equation ( 6 ) ) , the results are ( tegp 2.6 ( c ) ): 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha = \left\{{\begin{array}{*{20}c } { - 3{\gamma _ 0 } + { \lambda _ { 0\quad}}{\rm{hydrogen}}\;{\rm{hyperfine}}\;{\rm{transition,}}\;{\rm{h - maser}}\;{\rm{clock}},}\\ { - { 1 \over 2}(3{\gamma _ 0 } + { \lambda _ 0}){\rm{electromagnetic}}\;{\rm{mode}}\;{\rm{in}}\;{\rm{cavity,}}\;{\rm{scso}}\;{\rm{clock}},}\\ { - 2{\gamma _ 0}\quad \quad \quad \;{\rm{phonon}}\;{\rm{mode}}\;{\rm{in}}\;{\rm{solid,}}\;{\rm{principal}}\;{\rm{transition}}\;{\rm{in}}\;{\rm{hydrogen}}.}\\ \end{array } } \right.$$\end{document } the redshift is the standard one ( = 0 ) , independently of the nature of the clock if and only if 0 thus the vessot - levine rocket redshift experiment sets a limit on the parameter combination |30 0| ( see figure 3 ) ; the null - redshift experiment comparing hydrogen - maser and scso clocks sets a limit on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vert{\alpha _ { \rm{h } } } - { \alpha _ { { \rm{scso}}}}\vert = { 3 \over 2}\vert{\gamma _ 0 } - { \lambda _ 0}\vert$\end{document}. alvarez and mann [ 7 , 6 , 8 , 9 , 10 ] extended the th formalism to permit analysis of such effects as the lamb shift , anomalous magnetic moments and non - baryonic effects , and placed interesting bounds on eep violations . the th formalism can also be applied to tests of local lorentz invariance , but in this context it can be simplified . since most such tests do not concern themselves with the spatial variation of the functions t , h , , and , but rather with observations made in moving frames , we can treat them as spatial constants . then by rescaling the time and space coordinates , the charges and the electromagnetic fields , we can put the action in box 1 into the form ( tegp 2.6 ( a ) ) 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = - \sum\limits_a { { m_{{0a}}}\;{{\int { ( 1 - \upsilon_a^2)}}^{1/2 } } } \;dt + \sum\limits_a { { e_a}\int { { a_\mu}(x_a^\nu)\upsilon_a^\mu}}\ ; dt + { ( 8\pi)^{- 1}}\int { ( { e^2 } - { c^2}{b^2})}\ , { d^4}x,$$\end{document } where c h0/(t000 ) = ( 1 0 ) . this amounts to using units in which the limiting speed c0 of massive test particles is unity , and the speed of light is c. if c 1 , lli is violated ; furthermore , the form of the action above must be assumed to be valid only in some preferred universal rest frame . the natural candidate for such a frame is the rest frame of the microwave background . the electrodynamical equations which follow from equation ( 17 ) yield the behavior of rods and clocks , just as in the full th formalism . for example , the length of a rod which moves with velocity v relative to the rest frame in a direction parallel to its length will be observed by a rest observer to be contracted relative to an identical rod perpendicular to the motion by a factor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1 - { v^2}/2 + { \mathcal o}({v^4})$\end{document}. notice that c does not appear in this expression , because only electrostatic interactions are involved , and c appears only in the magnetic sector of the action ( 17 ) . the energy and momentum of an electromagnetically bound body moving with velocity v relative to the rest frame are given by 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { e = { m_{\rm{r } } } + { 1 \over 2}{m_{\rm{r}}}{v^2 } + { 1 \over 2}\delta m_{\rm{i}}^{ij}{v^i}{v^j } + { \mathcal o}(m{v^4}),}\\ { { p^i } = { m_{\rm{r}}}{v^i } + \delta m_{\rm{i}}^{ij}{v^j } + { \mathcal o}(m{v^3}),}\\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_r } = { m_0 } - e_{\rm{b}}^{{\rm{es}}}$\end{document } , m0 is the sum of the particle rest masses , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{\rm{b}}^{{\rm{es}}}$\end{document } is the electrostatic binding energy of the system ( see equation ( 14 ) with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_0^{1/2}{h_{{0^\epsilon}_0}}^{- 1 } = 1$\end{document } ) , and 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta m_{\rm{i}}^{ij } = - 2\left({{1 \over { { c^2 } } } - 1 } \right)\left [ { { 4 \over 3}e_{\rm{b}}^{{\rm{es}}}{\delta ^{ij } } + \tilde e_{\rm{b}}^{{\rm{es}}ij } } \right],$$\end{document } where 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde{e}_{\rm{b}}^{{\rm{es}}ij } = - { 1 \over 4}\left\langle { \sum\limits_{ab } { { { { e_a}{e_b } } \over { { r_{ab}}}}\left({n_{ab}^in_{ab}^j - { 1 \over 3}{\delta ^{ij } } } \right ) } } \right\rangle.$$\end{document } note that ( c 1 ) corresponds to the parameter plotted in figure 2 . the electrodynamics given by equation ( 17 ) can also be quantized , so that we may treat the interaction of photons with atoms via perturbation theory . the energy of a photon is times its frequency , while its momentum is /c . using this approach , one finds that the difference in round trip travel times of light along the two arms of the interferometer in the michelson - morley experiment is given by l0(/c)(c 1 ) . the experimental null result then leads to the bound on ( c 1 ) shown on figure 2 . similarly the anisotropy in energy levels is clearly illustrated by the tensorial terms in equations ( 18 , 20 ) ; by evaluating \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde e_{\rm{b}}^{{\rm{es}}ij}$\end{document } for each nucleus in the various hughes - drever - type experiments and comparing with the experimental limits on energy differences , one obtains the extremely tight bounds also shown on figure 2 . the behavior of moving atomic clocks can also be analyzed in detail , and bounds on ( c 1 ) can be placed using results from tests of time dilation and of the propagation of light . in some cases , it is advantageous to combine the c framework with a kinematical viewpoint that treats a general class of boost transformations between moving frames . such kinematical approaches have been discussed by robertson , mansouri and sexl , and will ( see ) . for example , in the jpl experiment , in which the phases of two hydrogen masers connected by a fiberoptic link were compared as a function of the earth s orientation , the predicted phase difference as a function of direction is , to first order in v , the velocity of the earth through the cosmic background , 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \phi } \over { \tilde \phi } } \approx - { 4 \over 3}(1 - { c^2})({\bf{v } } \cdot { \bf{n } } - { \bf{v } } \cdot { { \bf{n}}_0}),$$\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 \phi = 2\pi \nu l$\end{document } , is the maser frequency , l = 21 km is the baseline , and where n and n0 are unit vectors along the direction of propagation of the light at a given time and at the initial time of the experiment , respectively . the observed limit on a diurnal variation in the relative phase resulted in the bound |c tighter bounds were obtained from a two - photon absorption ( tpa ) experiment , and a 1960s series of mssbauer - rotor experiments , which tested the isotropy of time dilation between a gamma ray emitter on the rim of a rotating disk and an absorber placed at the center . kosteleck and collaborators developed a useful and elegant framework for discussing violations of lorentz symmetry in the context of the standard model of particle physics [ 63 , 64 , 155 ] . called the standard model extension ( sme ) , it takes the standard su(3 ) su(2 ) u(1 ) field theory of particle physics , and modifies the terms in the action by inserting a variety of tensorial quantities in the quark , lepton , higgs , and gauge boson sectors that could explicitly violate lli . sme extends the earlier classical th and c frameworks , and the g framework of ni to quantum field theory and particle physics . the modified terms split naturally into those that are odd under cpt ( i.e. that violate cpt ) and terms that are even under cpt . the result is a rich and complex framework , with many parameters to be analyzed and tested by experiment . such details are beyond the scope of this review ; for a review of sme and other frameworks , the reader is referred to the living review by mattingly . here we confine our attention to the electromagnetic sector , in order to link the sme with the c framework discussed above . in the sme , the lagrangian for a scalar particle charge e interacting with electrodynamics takes the form 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal l } = [ { \eta ^{\mu \nu } } + { ( { k_\phi})^{\mu \nu}}]{({d_\mu}\phi)^\dagger}{d_\nu}\phi - { m^2}{\phi ^\dagger}\phi - { 1 \over 4}\ , [ { \eta ^{\mu \alpha}}{\eta ^{\nu \beta } } + { ( { k_f})^{\mu \nu \alpha \beta}}]\ , { f_{\mu \nu}}{f_{\alpha \beta}},$$\end{document } where d = + iea , where ( k ) is a real symmetric trace - free tensor , and where ( kf ) is a tensor with the symmetries of the riemann tensor , and with vanishing double trace . there could also be a cpt - odd term in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal l}$\end{document } of the form ( ka)af , but because of a variety of pre - existing theoretical and experimental constraints , it is generally set to zero . components , by defining 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { ( { \kappa _ { de}})}^{jk } } = - 2{{({k_f})}^{0j0k}},}\\ { { { ( { \kappa _ { hb}})}^{jk } } = { 1 \over 2}{\epsilon ^{jpq}}{\epsilon ^{krs}}{{({k_f})}^{pqrs}},}\\ { { { ( { \kappa _ { db}})}^{kj } } = - { { ( { k_{he}})}^{jk } } = { \epsilon ^{jpq}}{{({k_f})}^{0kpq}}.}\\ \end{array}$$\end{document } in many applications it is useful to use the further decomposition 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \quad \;\;{{\tilde \kappa}_{{\rm{tr } } } } = { 1 \over 3}{{({\kappa _ { de}})}^{jj}},}\\ { { { ( { { \tilde \kappa}_{{\rm{e + } } } } ) } ^{jk } } = { 1 \over 2}{{({\kappa _ { de } } + { \kappa _ { hb}})}^{jk}},}\\ { { { ( { { \tilde \kappa}_{{\rm{e -}}}})}^{jk } } = { 1 \over 2}{{({\kappa _ { de } } - { \kappa _ { hb}})}^{jk } } - { 1 \over 3}{\delta ^{jk}}{{({\kappa _ { de}})}^{ii}},}\\ { { { ( { { \tilde \kappa}_{{\rm{o + } } } } ) } ^{jk } } = { 1 \over 2}{{({\kappa _ { db } } - { \kappa _ { he}})}^{jk}},}\\ { { { ( { { \tilde \kappa}_{{\rm{o -}}}})}^{jk } } = { 1 \over 2}{{({\kappa _ { db } } - { \kappa _ { he}})}^{jk}}.}\\ \end{array}$$\end{document } the first expression is a single number , the next three are symmetric trace - free matrices , and the final is an antisymmetric matrix , accounting thereby for the 19 components of the original tensor ( kf ) . in the rest frame of the universe , these tensors have some form that is established by the global nature of the solutions of the overarching theory being used . in a frame that is moving relative to the universe , the tensors will have components that depend on the velocity of the frame , and on the orientation of the frame relative to that velocity . in the case where the theory is rotationally symmetric in the preferred frame , the tensors ( k ) and ( kf ) can be expressed in the form 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${({k_\phi})^{\mu \nu } } = { \tilde \kappa _ \phi}\left({{u^\mu}{\mu ^\nu } + { 1 \over 4}{\eta ^{\mu \nu } } } \right),$$\end{document } 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${({k_f})^{\mu \nu \alpha \beta } } = { \tilde \kappa _ { { \rm{tr}}}}\left({4{u^{\left [ \mu \right.}}{\eta ^{\left . \beta \right]\nu } } } \right),$$\end{document } where [ ] around indices denote antisymmetrization , and where u is the four - velocity of an observer at rest in the preferred frame . with this assumption , all the tensorial quantities in equation ( 24 ) vanish in the preferred frame , and , after suitable rescalings of coordinates and fields , the action ( 22 ) can be put into the form of the c framework , with 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c = { \left({{{1 - { 3 \over 4}{{\tilde \kappa}_\phi } } \over { 1 + { 1 \over 4}{{\tilde \kappa}_\phi } } } } \right)^{1/2}}{\left({{{1 - { { \tilde \kappa}_{{\rm{tr } } } } } \over { 1 + { { \tilde \kappa}_{{\rm{tr } } } } } } } \right)^{1/2}}.$$\end{document } thus far , we have discussed eep as a principle that strictly divides the world into metric and non - metric theories , and have implied that a failure of eep might invalidate metric theories ( and thus general relativity ) . on the other hand , there is mounting theoretical evidence to suggest that eep is likely to be violated at some level , whether by quantum gravity effects , by effects arising from string theory , or by hitherto undetected interactions . roughly speaking , in addition to the pure einsteinian gravitational interaction , which respects eep , theories such as string theory predict other interactions which do not . in string theory , for example , the existence of such eep - violating fields is assured , but the theory is not yet mature enough to enable a robust calculation of their strength relative to gravity , or a determination of whether they are long range , like gravity , or short range , like the nuclear and weak interactions , and thus too short range to be detectable . in one simple example , one can write the lagrangian for the low - energy limit of a string - inspired theory in the so - called einstein frame , in which the gravitational lagrangian is purely general relativistic : 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \tilde{\mathcal l } = \sqrt { - \tilde g } { { \left({\tilde g } \right.}^{\mu \nu}}\left [ { { 1 \over { 2\kappa}}{{\tilde r}_{\mu \nu } } - { 1 \over 2}\tilde g(\varphi){\partial _ \mu}\varphi { \partial _ \nu}\varphi } \right ] - u(\varphi){{\tilde g}^{\mu \nu}}{{\tilde g}^{\alpha \beta}}{f_{\mu \alpha}}{f_{\nu \beta}}}\\ { \left . { \quad \quad \quad \quad + \overline { \tilde \psi } \left [ { i\tilde e_a^\mu { \gamma ^a}\left({{\partial _ \mu } + { { \tilde \omega}_\mu } + q{a_\mu } } \right ) - \tilde m(\varphi ) } \right]\tilde \psi } \right),}\\ \end{array}$$\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 non - 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 r}_{\mu \nu}}$\end{document } is the ricci tensor derived from it , is a dilaton field , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde g}$\end{document } , u and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde m}$\end{document } are functions of . the lagrangian includes that for the electromagnetic field f , and that for particles , written in terms of dirac spinors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \psi}$\end{document}. this is not a metric representation because of the coupling of to matter via \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde m(\varphi)$\end{document } and u( ) . a 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 } } = f(\varphi){g_{\mu \nu}},\tilde \psi = f{(\varphi)^{- 3/4}}\psi$\end{document } , puts the lagrangian in the form ( jordan frame ) 29\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 l } = \sqrt { - g } { { \left(g \right.}^{\mu \nu}}\left [ { { 1 \over { 2\kappa}}f(\varphi){r_{\mu \nu } } - { 1 \over 2}f(\varphi)\tilde g(\varphi){\partial _ \alpha}}{f_{\nu \beta } } + \overline \psi \left [ { ie_a^\mu { \gamma ^a}\left({{\partial _ \mu } + { \omega _ \mu } + q{a_\mu } } \right ) - \tilde m(\varphi){f^{1/2 } } } \right]\psi } \right).}\\ \end{array}$$\end{document } one may choose \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f(\varphi ) = { \rm{const}}./\tilde m{(\varphi)^2}$\end{document } so that the particle lagrangian takes the metric form ( no explicit coupling to ) , but the electromagnetic lagrangian will still couple non - metrically to u( ) . the gravitational lagrangian here takes the form of a scalar - tensor theory ( see section 3.3.2 ) . but the non - metric electromagnetic term will , in general , produce violations of eep . for examples of specific models , see [ 254 , 85 ] . another class of non - metric theories are included in the varying speed of light ( vsl ) theories ; for a detailed review , see . on the other hand , whether one views such effects as a violation of eep or as effects arising from additional matter fields whose interactions , like those of the electromagnetic field , do not fully embody eep , is to some degree a matter of semantics . unlike the fields of the standard model of electromagnetic , weak and strong interactions , which couple to properties other than mass - energy and are either short range or are strongly screened , the fields inspired by string theory could be long range ( if they remain massless by virtue of a symmetry , or at best , acquire a very small mass ) , and can couple to mass - energy , and thus can mimic gravitational fields . still , there appears to be no way to make this precise . as a result , eep and related tests are now viewed as ways to discover or place constraints on new physical interactions , or as a branch of non - accelerator particle physics , searching for the possible imprints of high - energy particle effects in the low - energy realm of gravity . whether current or proposed experiments can actually probe these phenomena meaningfully is an open question at the moment , largely because of a dearth of firm theoretical predictions . on the phenomenological side , the idea of using eep tests in this way may have originated in the middle 1980s , with the search for a fifth force . in 1986 , as a result of a detailed reanalysis of etvs original data , fischbach et al . suggested the existence of a fifth force of nature , with a strength of about a percent that of gravity , but with a range ( as defined by the range of a yukawa potential , e / r ) of a few hundred meters . this proposal dovetailed with earlier hints of a deviation from the inverse - square law of newtonian gravitation derived from measurements of the gravity profile down deep mines in australia , and with emerging ideas from particle physics suggesting the possible presence of very low - mass particles with gravitational - strength couplings . during the next four years numerous experiments looked for evidence of the fifth force by searching for composition - dependent differences in acceleration , with variants of the etvs experiment or with free - fall galileo - type experiments . although two early experiments reported positive evidence , the others all yielded null results . over the range between one and 10 meters , the null experiments produced upper limits on the strength of a postulated fifth force between 10 and 10 of the strength of gravity . interpreted as tests of wep ( corresponding to the limit of infinite - range forces ) , the results of two representative experiments from this period , the free - fall galileo experiment and the early et - wash experiment , are shown in figure 1 . at the same time , tests of the inverse - square law of gravity were carried out by comparing variations in gravity measurements up tall towers or down mines or boreholes with gravity variations predicted using the inverse square law together with earth models and surface gravity data mathematically continued up the tower or down the hole . despite early reports of anomalies , independent tower , borehole , and analyses of orbital data from planetary range measurements , lunar laser ranging ( llr ) , and laser tracking of the lageos satellite verified the inverse - square law to parts in 10 over scales of 10 to 10 km , and to parts in 10 over planetary scales of several astronomical units . a consensus emerged that there was no credible experimental evidence for a fifth force of nature , of a type and range proposed by fischbach et al . for reviews and bibliographies of this episode , see [ 107 , 109 , 110 , 4 , 278 ] . although the idea of an intermediate - range violation of newton s gravitational law was dropped , new ideas emerged to suggest the possibility that the inverse - square law could be violated at very short ranges , below the centimeter range of existing laboratory verifications of the 1/r behavior . one set of ideas [ 13 , 11 , 221 , 220 ] posited that some of the extra spatial dimensions that come with string theory could extend over macroscopic scales , rather than being rolled up at the planck scale of 10 cm , which was then the conventional viewpoint . on laboratory distances large compared to the relevant scale of the extra dimension , gravity would fall off as the inverse square , whereas on short scales , gravity would fall off as 1/r , where n is the number of large extra dimensions . other possibilities for effective modifications of gravity at short range involved the exchange of light scalar particles . following these proposals , many of the high - precision , low - noise methods that were developed for tests of wep were adapted to carry out laboratory tests of the inverse square law of newtonian gravitation at millimeter scales and below . the challenge of these experiments has been to distinguish gravitation - like interactions from electromagnetic and quantum mechanical ( casimir ) effects . no deviations from the inverse square law have been found to date at distances between 10 m and 10 mm[171 , 130 , 129 , 52 , 170 ] . for a comprehensive review of both the theory and the experiments , see . the empirical evidence supporting the einstein equivalence principle , discussed in the previous section 2 , supports the conclusion that the only theories of gravity that have a hope of being viable are metric theories , or possibly theories that are metric apart from very weak or short - range non - metric couplings ( as in string theory ) . therefore for the remainder of this review , we shall turn our attention exclusively to metric theories of gravity , which assume that there exists a symmetric metric , test bodies follow geodesics of the metric , andin local lorentz frames , the non - gravitational laws of physics are those of special relativity . there exists a symmetric metric , test bodies follow geodesics of the metric , and in local lorentz frames , the non - gravitational laws of physics are those of special relativity . the property that all non - gravitational fields should couple in the same manner to a single gravitational field is sometimes called universal coupling . because of it , one can discuss the metric as a property of spacetime itself rather than as a field over spacetime . this is because its properties may be measured and studied using a variety of different experimental devices , composed of different non - gravitational fields and particles , and , because of universal coupling , the results will be independent of the device . thus , for instance , the proper time between two events is a characteristic of spacetime and of the location of the events , not of the clocks used to measure it . consequently , if eep is valid , the non - gravitational laws of physics may be formulated by taking their special relativistic forms in terms of the minkowski metric and simply going over to new forms in terms of the curved spacetime metric g , using the mathematics of differential geometry . the details of this going over can be found in standard textbooks ( see [ 189 , 270 ] , tegp 3.2 . ) . in any metric theory of gravity , matter and non - gravitational fields respond only to the spacetime metric g. in principle , however , there could exist other gravitational fields besides the metric , such as scalar fields , vector fields , and so on . if , by our strict definition of metric theory , matter does not couple to these fields , what can their role in gravitation theory be ? their role must be that of mediating the manner in which matter and non - gravitational fields generate gravitational fields and produce the metric ; once determined , however , the metric alone acts back on the matter in the manner prescribed by eep . what distinguishes one metric theory from another , therefore , is the number and kind of gravitational fields it contains in addition to the metric , and the equations that determine the structure and evolution of these fields . from this viewpoint , one can divide all metric theories of gravity into two fundamental classes : purely dynamical and prior - geometric . by purely dynamical metric theory we mean any metric theory whose gravitational fields have their structure and evolution determined by coupled partial differential field equations . in other words , the behavior of each field is influenced to some extent by a coupling to at least one of the other fields in the theory . by prior geometric theory , we mean any metric theory that contains absolute elements , fields or equations whose structure and evolution are given a priori , and are independent of the structure and evolution of the other fields of the theory . these absolute elements typically include flat background metrics or cosmic time coordinates t. general relativity is a purely dynamical theory since it contains only one gravitational field , the metric itself , and its structure and evolution are governed by partial differential equations ( einstein s equations ) . brans - dicke theory and its generalizations are purely dynamical theories ; the field equation for the metric involves the scalar field ( as well as the matter as source ) , and that for the scalar field involves the metric . rosen s bimetric theory is a prior - geometric theory : it has a non - dynamical , riemann - flat background metric , and the field equations for the physical metric g involve . by discussing metric theories of gravity from this broad point of view , it is possible to draw some general conclusions about the nature of gravity in different metric theories , conclusions that are reminiscent of the einstein equivalence principle , but that are subsumed under the name strong equivalence principle . let this frame be small enough that inhomogeneities in the external gravitational fields can be neglected throughout its volume . on the other hand , let the frame be large enough to encompass a system of gravitating matter and its associated gravitational fields . the system could be a star , a black hole , the solar system , or a cavendish experiment . call this frame a quasi - local lorentz frame . to determine the behavior of the system we must calculate the metric . first we determine the external behavior of the metric and gravitational fields , thereby establishing boundary values for the fields generated by the local system , at a boundary of the quasi - local frame far from the local system . but because the metric is coupled directly or indirectly to the other fields of the theory , its structure and evolution will be influenced by those fields , and in particular by the boundary values taken on by those fields far from the local system . this will be true even if we work in a coordinate system in which the asymptotic form of g in the boundary region between the local system and the external world is that of the minkowski metric . thus the gravitational environment in which the local gravitating system resides can influence the metric generated by the local system via the boundary values of the auxiliary fields . consequently , the results of local gravitational experiments may depend on the location and velocity of the frame relative to the external environment . of course , local non - gravitational experiments are unaffected since the gravitational fields they generate are assumed to be negligible , and since those experiments couple only to the metric , whose form can always be made locally minkowskian at a given spacetime event . local gravitational experiments might include cavendish experiments , measurement of the acceleration of massive self - gravitating bodies , studies of the structure of stars and planets , or analyses of the periods of gravitational clocks . a theory which contains only the metric g yields local gravitational physics which is independent of the location and velocity of the local system . this follows from the fact that the only field coupling the local system to the environment is g , and it is always possible to find a coordinate system in which g takes the minkowski form at the boundary between the local system and the external environment ( neglecting inhomogeneities in the external gravitational field ) . thus the asymptotic values of g are constants independent of location , and are asymptotically lorentz invariant , thus independent of velocity . general relativity is an example of such a theory.a theory which contains the metric g and dynamical scalar fields a yields local gravitational physics which may depend on the location of the frame but which is independent of the velocity of the frame . this follows from the asymptotic lorentz invariance of the minkowski metric and of the scalar fields , but now the asymptotic values of the scalar fields may depend on the location of the frame . an example is brans - dicke theory , where the asymptotic scalar field determines the effective value of the gravitational constant , which can thus vary as varies . on the other hand , a form of velocity dependence in local physics can enter indirectly if the asymptotic values of the scalar field vary with time cosmologically . then the rate of variation of the gravitational constant could depend on the velocity of the frame.a theory which contains the metric g and additional dynamical vector or tensor fields or prior - geometric fields yields local gravitational physics which may have both location and velocity - dependent effects . a theory which contains only the metric g yields local gravitational physics which is independent of the location and velocity of the local system . this follows from the fact that the only field coupling the local system to the environment is g , and it is always possible to find a coordinate system in which g takes the minkowski form at the boundary between the local system and the external environment ( neglecting inhomogeneities in the external gravitational field ) . thus the asymptotic values of g are constants independent of location , and are asymptotically lorentz invariant , thus independent of velocity . a theory which contains the metric g and dynamical scalar fields a yields local gravitational physics which may depend on the location of the frame but which is independent of the velocity of the frame . this follows from the asymptotic lorentz invariance of the minkowski metric and of the scalar fields , but now the asymptotic values of the scalar fields may depend on the location of the frame . an example is brans - dicke theory , where the asymptotic scalar field determines the effective value of the gravitational constant , which can thus vary as varies . on the other hand , a form of velocity dependence in local physics can enter indirectly if the asymptotic values of the scalar field vary with time cosmologically . then the rate of variation of the gravitational constant could depend on the velocity of the frame . a theory which contains the metric g and additional dynamical vector or tensor fields or prior - geometric fields yields local gravitational physics which may have both location and velocity - dependent effects . these ideas can be summarized in the strong equivalence principle ( sep ) , which states that : wep is valid for self - gravitating bodies as well as for test bodies.the outcome of any local test experiment is independent of the velocity of the ( freely falling ) apparatus.the outcome of any local test experiment is independent of where and when in the universe it is performed . the distinction between sep and eep is the inclusion of bodies with self - gravitational interactions ( planets , stars ) and of experiments involving gravitational forces ( cavendish experiments , gravimeter measurements ) . note that sep contains eep as the special case in which local gravitational forces are ignored . the outcome of any local test experiment is independent of the velocity of the ( freely falling ) apparatus . the outcome of any local test experiment is independent of where and when in the universe it is performed . the above discussion of the coupling of auxiliary fields to local gravitating systems indicates that if sep is strictly valid , there must be one and only one gravitational field in the universe , the metric g. these arguments are only suggestive however , and no rigorous proof of this statement is available at present . empirically it has been found that almost every metric theory other than gr introduces auxiliary gravitational fields , either dynamical or prior geometric , and thus predicts violations of sep at some level ( here we ignore quantum - theory inspired modifications to gr involving the one exception is nordstrm s 1913 conformally - flat scalar theory , which can be written purely in terms of the metric ; the theory satisfies sep , but unfortunately violates experiment by predicting no deflection of light . general relativity seems to be the only viable metric theory that embodies sep completely . in section 3.6 , we shall discuss experimental evidence for the validity of sep . despite the possible existence of long - range gravitational fields in addition to the metric in various metric theories of gravity , the postulates of those theories demand that matter and non - gravitational fields be completely oblivious to them . the only gravitational field that enters the equations of motion is the metric g. the role of the other fields that a theory may contain can only be that of helping to generate the spacetime curvature associated with the metric . matter may create these fields , and they plus the matter may generate the metric , but they can not act back directly on the matter . thus the metric and the equations of motion for matter become the primary entities for calculating observable effects , and all that distinguishes one metric theory from another is the particular way in which matter and possibly other gravitational fields generate the metric . the comparison of metric theories of gravity with each other and with experiment becomes particularly simple when one takes the slow - motion , weak - field limit . this approximation , known as the post - newtonian limit , is sufficiently accurate to encompass most solar - system tests that can be performed in the foreseeable future . it turns out that , in this limit , the spacetime metric g predicted by nearly every metric theory of gravity has the same structure . it can be written as an expansion about the minkowski metric ( = diag(1,1,1,1 ) ) in terms of dimensionless gravitational potentials of varying degrees of smallness . these potentials are constructed from the matter variables ( see box 2 ) in imitation of the newtonian gravitational potential 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$u({\bf{x}},t ) \equiv \int \rho ( { \bf{x}}{\prime},t)\vert { \bf{x } } - t{\prime}\vert^{{- 1}}{d^3}x{\prime}.$$\end{document } the order of smallness is determined according to the rules u p/ , |d / dt|/|d|dx| , and so on ( we use units in which g = c = 1 ; see box 2 ) . a consistent post - newtonian limit requires determination of g00 correct through \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^2})$\end{document } , g0i through \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^{3/2}})$\end{document } , and gij through \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}(\epsilon)$\end{document } ( for details see tegp 4.1 ) . the only way that one metric theory differs from another is in the numerical values of the coefficients that appear in front of the metric potentials . the parametrized post - newtonian ( ppn ) formalism inserts parameters in place of these coefficients , parameters whose values depend on the theory under study . in the current version of the ppn formalism , summarized in box 2 , ten parameters are used , chosen in such a manner that they measure or indicate general properties of metric theories of gravity ( see table 2 ) . under reasonable assumptions about the kinds of potentials that can be present at post - newtonian order ( basically only poisson - like potentials ) , one finds that ten ppn parameters exhaust the possibilities . table 2the ppn parameters and their significance ( note that 3 has been shown twice to indicate that it is a measure of two effects).parameterwhat it measures relative to grvalue in grvalue in semi - conservative theoriesvalue in fully conservative theories how much space - curvature produced by unit rest mass?1 how much nonlinearity in the superposition law for gravity?1 preferred - location effects?0 1 preferred - frame effects?0 1 0 2 0 2 0 3 000 3 violation of conservation of total momentum?000 1 000 2 000 3 000 4 000 the ppn parameters and their significance ( note that 3 has been shown twice to indicate that it is a measure of two effects ) . the parameters and are the usual eddington - robertson - schiff parameters used to describe the classical tests of gr , and are in some sense the most important ; they are the only non - zero parameters in gr and scalar - tensor gravity . the parameter is non - zero in any theory of gravity that predicts preferred - location effects such as a galaxy - induced anisotropy in the local gravitational constant gl ( also called effects ) ; 1 , 2 , 3 measure whether or not the theory predicts post - newtonian preferred - frame effects ; 3 1 , 2 , 3 , 4 measure whether or not the theory predicts violations of global conservation laws for total momentum . in table 2 we show the values these parameters take in gr , in any theory of gravity that possesses conservation laws for total momentum , called semi - conservative ( any theory that is based on an invariant action principle is semi - conservative ) , andin any theory that in addition possesses six global conservation laws for angular momentum , called fully conservative ( such theories automatically predict no post - newtonian preferred - frame effects ) . semi - conservative theories have five free ppn parameters ( , , , 1 , 2 ) while fully conservative theories have three ( , , ) . in any theory of gravity that possesses conservation laws for total momentum , called semi - conservative ( any theory that is based on an invariant action principle is semi - conservative ) , and in any theory that in addition possesses six global conservation laws for angular momentum , called fully conservative ( such theories automatically predict no post - newtonian preferred - frame effects ) . the ppn formalism was pioneered by kenneth nordtvedt , who studied the post - newtonian metric of a system of gravitating point masses , extending earlier work by eddington , robertson and schiff ( tegp 4.2 ) . a general and unified version of the ppn formalism was developed by will and nordtvedt . the canonical version , with conventions altered to be more in accord with standard textbooks such as , is discussed in detail in tegp 4 . other versions of the ppn formalism have been developed to deal with point masses with charge , fluid with anisotropic stresses , bodies with strong internal gravity , and post - post - newtonian effects ( tegp 4.2 , 14.2 ) . coordinate system : the framework uses a nearly globally lorentz coordinate system in which the coordinates are ( t , x , x , x ) . three - dimensional , euclidean vector notation is used throughout . all coordinate arbitrariness ( gauge freedom ) has been removed by specialization of the coordinates to the standard ppn gauge ( tegp 4.2 ) . units are chosen so that g = c = 1 , where g is the physically measured newtonian constant far from the solar system.matter variables : : density of rest mass as measured in a local freely falling frame momentarily comoving with the gravitating matter.= ( dx / dt ) : coordinate velocity of the matter.w : coordinate velocity of the ppn coordinate system relative to the mean rest - frame of the universe.p : pressure as measured in a local freely falling frame momentarily comoving with the matter. : internal energy per unit rest mass ( it includes all forms of non - rest - mass , non - gravitational energy , e.g. , energy of compression and thermal energy).ppn parameters : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma , \,\beta , \,\xi , \,{\alpha _ 1},\,{\alpha _ 2},\,{\alpha _ 3},\,{\varsigma _ 1},\,{\varsigma _ 2},\,{\varsigma _ 3},\,{\varsigma _ 4}.$$\end{document } metric : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { g_{00 } } = - 1 + 2u - 2\beta { u^2 } - 2\xi { \phi _ w } + ( 2\gamma + 2 + { \alpha _ 3 } + { \zeta _ 1 } - 2\xi){\phi _ 1 } + 2(3\gamma - 2\beta + 1 + { \zeta _ 2 } + \xi){\phi _ 2}}\\ { \quad \quad + 2(1 + { \zeta _ 3}){\phi _ 3 } + 2(3\gamma + 3{\zeta _ 4 } - 2\xi){\phi _ 4 } - ( { \zeta _ 1 } - 2\xi){\mathcal a } - ( { \alpha _ 1 } - { \alpha _ 2 } - { \alpha _ 3}){w^2}u - { \alpha _ 2}{w^i}{w^j}{u_{ij}}}\\ { \quad \quad + ( 2{\alpha _ 3 } - { \alpha _ 1}){w^i}{v_i } + { \mathcal o}({\epsilon ^3}),}\\ { { g_{0i } } = - { 1 \over 2}(4\gamma + 3 + { \alpha _ 1 } - { \alpha _ 2 } + { \zeta _ 1 } - 2\xi){v_i } - { 1 \over 2}(1 + { \alpha _ 2 } - { \zeta _ 1 } + 2\xi){w_i } - { 1 \over 2}({\alpha _ 1 } - 2{\alpha _ 2}){w^i}u}\\ { \quad o}({\epsilon ^{5/2}}),}\\ { { g_{ij } } = ( 1 + 2\gamma u){\delta _ { ij } } + { \mathcal o}({\epsilon ^2}).}\\ \end{array}$$\end{document } metric potentials : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{u = \int { { { \rho \prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad \quad } } \\ { { u_{ij } } = \int { { \rho \prime { { ( x - x\prime)}_i}{{(x - x\prime)}_j } } \over { \vert { \rm{x } } - { \rm{x}}\prime { \vert ^3}}}{d^3}x\prime , \quad \quad \quad } \\ { \quad \quad \quad \quad \quad { \phi _ w } = \int { { \rho \prime \rho \prime \prime ( { \rm{x } } - { \rm{x}}\prime ) } \over { \vert { \rm{x } } - { \rm{x}}\prime { \vert ^3}}}\cdot\left({{{{\rm{x}}\prime - { \rm{x}}\prime \prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \prime \vert } } - { { { \rm{x } } - { \rm{x}}\prime \prime } \over { \vert { \rm{x}}\prime - { \rm{x}}\prime \prime \vert } } } \right){d^3}x\prime { d^3}x\prime \prime , } \\ { a = \int { { \rho \prime { { [ { \rm{v}}\prime \cdot({\rm{x } } - { \rm{x}}\prime)]}^2 } } \over { \vert { \rm{x } } - { \rm{x}}\prime { \vert ^3}}}{d^3}x\prime , \quad \quad \quad \quad } \\ { { \phi _ 1 } = \int { { { \rho \prime \upsilon { \prime ^2 } } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad } } \\ { { \phi _ 2 } = \int { { { \rho \prime u\prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad } } \\ { { \phi _ 3 } = \int { { { \rho \prime \pi \prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert } } } { d^3}x\prime , \quad \quad \quad \quad \quad \quad } \\ { { \phi _ 4 } = \int { { { p\prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad } } \\ { { v_i } = \int { { { \rho \prime \upsilon { \prime _ i } } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad } } \\ { \,\,\,\,{w_i } = \int { { \rho \prime [ { \rm{v}}\prime \cdot({\rm{x } } - { \rm{x}}\prime)]{{(x - x\prime)}_i } } \over { \vert { \rm{x } } - { \rm{x}}\prime { \vert ^3}}}{d^3}x\prime .\quad \quad } \\ \end{array}$$\end{document } stress - energy tensor ( perfect fluid ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { t^{00 } } = \rho ( 1 + \prod + { v^2 } + 2u)}\\ { { t^{0i } } = \rho { v^i}\left({1 + \prod + { v^2 } + 2u + { p \over p } } \right),}\\ { { t^{ij } } = \rho { v^i}{v^j}\left({1 + \prod + { v^2 } + 2u + { p \over p } } \right ) + p{\delta ^{ij}}(1 - 2\gamma u).}\\ \end{array}$$\end{document } equations of motion : stressed matter : t = 0.test bodies : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{{d^2}{x^\mu } } \over { d{\lambda ^2 } } } + { \gamma ^\mu}\nu \lambda { { d{x^\nu } } \over { d\lambda}}{{d{x^\lambda } } \over { d\lambda } } = 0$\end{document}.maxwell s equations : f = 4j , f = a; a;. the framework uses a nearly globally lorentz coordinate system in which the coordinates are ( t , x , x , x ) . three - dimensional , euclidean vector notation is used throughout . all coordinate arbitrariness ( gauge freedom ) has been removed by specialization of the coordinates to the standard ppn gauge ( tegp 4.2 ) . units are chosen so that g = c = 1 , where g is the physically measured newtonian constant far from the solar system . matter variables : : density of rest mass as measured in a local freely falling frame momentarily comoving with the gravitating matter.= ( dx / dt ) : coordinate velocity of the matter.w : coordinate velocity of the ppn coordinate system relative to the mean rest - frame of the universe.p : pressure as measured in a local freely falling frame momentarily comoving with the matter. : internal energy per unit rest mass ( it includes all forms of non - rest - mass , non - gravitational energy , e.g. , energy of compression and thermal energy ) . : density of rest mass as measured in a local freely falling frame momentarily comoving with the gravitating matter . = ( dx / dt ) : coordinate velocity of the matter . w : coordinate velocity of the ppn coordinate system relative to the mean rest - frame of the universe . p : pressure as measured in a local freely falling frame momentarily comoving with the matter . : internal energy per unit rest mass ( it includes all forms of non - rest - mass , non - gravitational energy , e.g. , energy of compression and thermal energy ) . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma , \,\beta , \,\xi , \,{\alpha _ 1},\,{\alpha _ 2},\,{\alpha _ 3},\,{\varsigma _ 1},\,{\varsigma _ 2},\,{\varsigma _ 3},\,{\varsigma _ 4}.$$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { g_{00 } } = - 1 + 2u - 2\beta { u^2 } - 2\xi { \phi _ w } + ( 2\gamma + 2 + { \alpha _ 3 } + { \zeta _ 1 } - 2\xi){\phi _ 1 } + 2(3\gamma - 2\beta + 1 + { \zeta _ 2 } + \xi){\phi _ 2}}\\ { \quad \quad + 2(1 + { \zeta _ 3}){\phi _ 3 } + 2(3\gamma + 3{\zeta _ 4 } - 2\xi){\phi _ 4 } - ( { \zeta _ 1 } - 2\xi){\mathcal a } - ( { \alpha _ 1 } - { \alpha _ 2 } - { \alpha _ 3}){w^2}u - { \alpha _ 2}{w^i}{w^j}{u_{ij}}}\\ { \quad \quad + ( 2{\alpha _ 3 } - { \alpha _ 1}){w^i}{v_i } + { \mathcal o}({\epsilon ^3}),}\\ { { g_{0i } } = - { 1 \over 2}(4\gamma + 3 + { \alpha _ 1 } - { \alpha _ 2 } + { \zeta _ 1 } - 2\xi){v_i } - { 1 \over 2}(1 + { \alpha _ 2 } - { \zeta _ 1 } + 2\xi){w_i } - { 1 \over 2}({\alpha _ 1 } - 2{\alpha _ 2}){w^i}u}\\ { \quad \quad - { \alpha _ 2}{w^j}{u_{ij } } + { \mathcal o}({\epsilon ^{5/2}}),}\\ { { g_{ij } } = ( 1 + 2\gamma u){\delta _ { ij } } + { \mathcal o}({\epsilon ^2}).}\\ \end{array}$$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c}{u = \int { { { \rho \prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad \quad } } \\ { { u_{ij } } = \int { { \rho \prime { { ( x - x\prime)}_i}{{(x - x\prime)}_j } } \over { \vert { \rm{x } } - { \rm{x}}\prime { \vert ^3}}}{d^3}x\prime , \quad \quad \quad } \\ { \quad \quad \quad \quad \quad { \phi _ w } = \int { { \rho \prime \rho \prime \prime ( { \rm{x } } - { \rm{x}}\prime ) } \over { \vert { \rm{x } } - { \rm{x}}\prime { \vert ^3}}}\cdot\left({{{{\rm{x}}\prime - { \rm{x}}\prime \prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \prime \vert } } - { { { \rm{x } } - { \rm{x}}\prime \prime } \over { \vert { \rm{x}}\prime - { \rm{x}}\prime \prime \vert } } } \right){d^3}x\prime { d^3}x\prime \prime , } \\ { a = \int { { \rho \prime { { [ { \rm{v}}\prime \cdot({\rm{x } } - { \rm{x}}\prime)]}^2 } } \over { \vert { \rm{x } } - { \rm{x}}\prime { \vert ^3}}}{d^3}x\prime , \quad \quad \quad \quad } \\ { { \phi _ 1 } = \int { { { \rho \prime \upsilon { \prime ^2 } } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad } } \\ { { \phi _ 2 } = \int { { { \rho \prime u\prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad } } \\ { { \phi _ 3 } = \int { { { \rho \prime \pi \prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert } } } { d^3}x\prime , \quad \quad \quad \quad \quad \quad } \\ { { \phi _ 4 } = \int { { { p\prime } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad } } \\ { { v_i } = \int { { { \rho \prime \upsilon { \prime _ i } } \over { \vert { \rm{x } } - { \rm{x}}\prime \vert}}{d^3}x\prime , \quad \quad \quad \quad \quad \quad } } \\ { \,\,\,\,{w_i } = \int { { \rho \prime [ { \rm{v}}\prime \cdot({\rm{x } } - { \rm{x}}\prime)]{{(x - x\prime)}_i } } \over { \vert { \rm{x } } - { \rm{x}}\prime { \vert ^3}}}{d^3}x\prime .\quad \quad } \\ \end{array}$$\end{document } stress - energy tensor ( perfect fluid ) : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { t^{00 } } = \rho ( 1 + \prod + { v^2 } + 2u)}\\ { { t^{0i } } = \rho { v^i}\left({1 + \prod + { v^2 } + 2u + { p \over p } } \right),}\\ { { t^{ij } } = \rho { v^i}{v^j}\left({1 + \prod + { v^2 } + 2u + { p \over p } } \right ) + p{\delta ^{ij}}(1 - 2\gamma u).}\\ \end{array}$$\end{document } equations of motion : stressed matter : t = 0.test bodies : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{{d^2}{x^\mu } } \over { d{\lambda ^2 } } } + { \gamma ^\mu}\nu \lambda { { d{x^\nu } } \over { d\lambda}}{{d{x^\lambda } } \over { d\lambda } } = 0$\end{document}.maxwell s equations : f = 4j , f = a; a;. stressed matter : t = 0 . test bodies : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{{d^2}{x^\mu } } \over { d{\lambda ^2 } } } + { \gamma ^\mu}\nu \lambda { { d{x^\nu } } \over { d\lambda}}{{d{x^\lambda } } \over { d\lambda } } = 0$\end{document}. maxwell s equations : f = 4j , f = a; a;. one of the important applications of the ppn formalism is the comparison and classification of alternative metric theories of gravity . the population of viable theories has fluctuated over the years as new effects and tests have been discovered , largely through the use of the ppn framework , which eliminated many theories thought previously to be viable . the theory population has also fluctuated as new , potentially viable theories have been invented . in this review , we shall focus on gr , the general class of scalar - tensor modifications of it , of which the jordan - fierz - brans - dicke theory ( brans - dicke , for short ) is the classic example , and vector - tensor theories . the reasons are several - fold : a full compendium of alternative theories circa 1981 is given in tegp 5 .many alternative metric theories developed during the 1970s and 1980s could be viewed as straw - man theories , invented to prove that such theories exist or to illustrate particular properties . few of these could be regarded as well - motivated theories from the point of view , say , of field theory or particle physics.a number of theories fall into the class of prior - geometric theories , with absolute elements such as a flat background metric in addition to the physical metric . most of these theories predict preferred - frame effects , that have been tightly constrained by observations ( see section 3.6.2 ) . an example is rosen s bimetric theory.a large number of alternative theories of gravity predict gravitational wave emission substantially different from that of general relativity , in strong disagreement with observations of the binary pulsar ( see section 7).scalar - tensor modifications of gr have become very popular in unification schemes such as string theory , and in cosmological model building . because the scalar fields could be massive , the potentials in the post - newtonian limit could be modified by yukawa - like terms.vector-tensor theories have attracted recent attention , in the spirit of the sme ( see section 2.2.4 ) , as models for violations of lorentz invariance in the gravitational sector . many alternative metric theories developed during the 1970s and 1980s could be viewed as straw - man few of these could be regarded as well - motivated theories from the point of view , say , of field theory or particle physics . a number of theories fall into the class of prior - geometric theories , with absolute elements such as a flat background metric in addition to the physical metric . most of these theories predict preferred - frame effects , that have been tightly constrained by observations ( see section 3.6.2 ) . a large number of alternative theories of gravity predict gravitational wave emission substantially different from that of general relativity , in strong disagreement with observations of the binary pulsar ( see section 7 ) . scalar - tensor modifications of gr have become very popular in unification schemes such as string theory , and in cosmological model building . because the scalar fields could be massive , the potentials in the post - newtonian limit could be modified by yukawa - like terms . vector - tensor theories have attracted recent attention , in the spirit of the sme ( see section 2.2.4 ) , as models for violations of lorentz invariance in the gravitational sector . the metric g is the sole dynamical field , and the theory contains no arbitrary functions or parameters , apart from the value of the newtonian coupling constant g , which is measurable in laboratory experiments . throughout this article we do this despite recent evidence , from supernova data , of an accelerating universe , which would indicate either a non - zero cosmological constant or a dynamical dark energy contributing about 70 percent of the critical density . although c has significance for quantum field theory , quantum gravity , and cosmology , on the scale of the solar - system or of stellar systems its effects are negligible , for the values of ac inferred from supernova observations . the field equations of gr are derivable from an invariant action principle i = 0 , where 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = { ( 16\pi g)^{- 1}}\int r { ( - g)^{1/2}}{d^4}x + { i_{\rm{m}}}({\psi _ { \rm{m}}},{g_{\mu \nu}}),$$\end{document } where r is the ricci scalar , and i m is the matter action , which depends on matter fields m universally coupled to the metric g. by varying the action with respect to g , we obtain the field equations 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } \equiv { r_{\mu \nu } } - { 1 \over 2}{g_{\mu \nu}}r = 8\pi g{t_{\mu \nu}},$$\end{document } where t is the matter energy - momentum tensor . general covariance of the matter action implies the equations of motion t = 0 ; varying i m with respect to m yields the matter field equations of the standard model . by virtue of the absence of prior - geometric elements , the equations of motion are also a consequence of the field equations via the bianchi identities g = 0 . the general procedure for deriving the post - newtonian limit of metric theories is spelled out in tegp 5.1 , and is described in detail for gr in tegp 5.2 . table 3metric theories and their ppn parameter values ( 3 = i = 0 for all cases ) . the parameters , , 1 , and 2 denote complicated functions of u and of the arbitrary constants . here is not the cosmological constant c , but is defined by equation ( 37).theoryarbitrary functions or constantscosmic matching parametersppn parameters 1 2 general relativitynonenone11000scalar - tensorbrans - dicke bd 0 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{1 + { \omega _ { { \rm{bd } } } } } \over { 2 + { \omega _ { { \rm{bd}}}}}}$\end{document } 1000generala( ) , v( ) 0 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{1 + \omega } \over { 2 + \omega}}$\end{document } 1+000vector - tensorunconstrained , c1 , c2 , c3 , c4 u 0 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 1\prime$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 2\prime$\end{document } einstein - therc1,c2,c3,c4none110 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 1\prime$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 2\prime$\end{document } rosen s bimetricnonec0 , c11100 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{{c_0 } } \over { { c_1 } } } - 1$\end{document } metric theories and their ppn parameter values ( 3 = i = 0 for all cases ) . the parameters , , 1 , and 2 denote complicated functions of u and of the arbitrary constants . here is not the cosmological constant c , but is defined by equation ( 37 ) . these theories contain the metric g , a scalar field , a potential function v( ) , and a coupling function a( ) ( generalizations to more than one scalar field have also been carried out ) . for some purposes , the action is conveniently written in a non - metric representation , sometimes denoted the einstein frame , in which the gravitational action looks exactly like that of gr : 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde{i } = { ( 16\pi g)^{- 1}}\int { \left [ { \tilde r - 2{{\tilde g}^{\mu \nu}}{\partial _ \mu}\varphi \;{\partial _ \nu}\varphi - v(\varphi ) } \right ] } { ( - \tilde g)^{1/2}}{d^4}x + { i_m}({\psi _ { \rm{m}}},{a^2}(\varphi){\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 r \equiv { \tilde g^{\mu \nu}}{\tilde r_{\mu \nu}}$\end{document } is the ricci scalar of the einstein 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}. ( apart from the scalar potential term v( ) , this corresponds to equation ( 28 ) with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde g(\varphi ) \equiv { ( 4\pi g)^{- 1}},u(\varphi ) \equiv 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}$\tilde m(\varphi ) \propto a(\varphi).)$\end{document}. this representation is a non - metric one because the matter fields m couple to a combination of and \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}. despite appearances , however , it is a metric theory , because it can be put into a metric representation by identifying the physical metric 34\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 } } \equiv { a^2}(\varphi){\tilde g_{\mu \nu}}.$$\end{document } the action can then be rewritten in the metric form 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = { ( 16\pi g)^{- 1}}\int { [ \phi r - { \phi ^{- 1}}\omega ( \phi){g^{\mu \nu}}{\partial _ \mu}\phi \;{\partial _ \nu}\phi - { \phi ^2}v ] } { ( - g)^{1/2}}{d^4}x + { i_m}({\psi _ { \rm{m}}},{g_{\mu \nu}}),$$\end{document } where 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \quad \quad \quad \;\phi \equiv a{{(\varphi)}^{- 2}},}\\ { 3 + 2\omega ( \phi ) \equiv \alpha { { ( \varphi)}^{- 2}},}\\ { \quad \quad \alpha ( \varphi ) \equiv { { d(\ln a(\varphi ) ) } \over { d\varphi}}.}\\ \end{array}$$\end{document } the einstein frame is useful for discussing general characteristics of such theories , and for some cosmological applications , while the metric representation is most useful for calculating observable effects . the field equations , post - newtonian limit and ppn parameters are discussed in tegp 5.3 , and the values of the ppn parameters are listed in table 3 . the parameters that enter the post - newtonian limit are 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\omega \equiv \omega ( { \phi _ 0}),\quad \lambda \equiv { \left [ { { { d\omega } \over { d\phi}}{{(3 + 2\omega)}^{- 2}}{{(4 + 2\omega)}^{- 1 } } } \right]_{\phi 0}},$$\end{document } where 0 is the value of today far from the system being studied , as determined by appropriate cosmological boundary conditions . in brans - dicke theory ( ( ) = bd = const . ) , the larger the value of bd , the smaller the effects of the scalar field , and in the limit bd ( 0 0 ) , the theory becomes indistinguishable from gr in all its predictions . in more general theories , the function ( ) could have the property that , at the present epoch , and in weak - field situations , the value of the scalar field 0 is such that is very large and is very small ( theory almost identical to gr today ) , but that for past or future values of , or in strong - field regions such as the interiors of neutron stars , and could take on values that would lead to significant differences from gr . it is useful to point out that all versions of scalar - tensor gravity predict that 1 ( see table 3 ) . damour and esposito - farse have adopted an alternative parametrization of scalar - tensor theories , in which one expands ln a( ) about a cosmological background field value 0 : 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ln a(\varphi ) = { \alpha _ 0}(\varphi - { \varphi _ 0 } ) + { 1 \over 2}{\beta _ 0}{(\varphi - { \varphi _ 0})^2 } + \ldots$$\end{document } a precisely linear coupling function produces brans - dicke theory , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 0 ^ 2 = 1/(2{\omega _ { { \rm{bd } } } } + 3)$\end{document } , or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1/(2 + { \omega _ { { \rm{bd } } } } ) = 2\alpha _ 0 ^ 2/(1 + \alpha _ 0 ^ 2)$\end{document}. the function ln a( ) acts as a potential for the scalar field within matter , and , if 0 > 0 , then during cosmological evolution , the scalar field naturally evolves toward the minimum of the potential , i.e. toward 0 0 , , or toward a theory close to , though not precisely gr [ 80 , 81 ] . estimates of the expected relic deviations from gr today in such theories depend on the cosmological model , but range from 10 to a few times 10 for | 1| . negative values of 0 correspond to a locally unstable scalar potential ( the overall theory is still stable in the sense of having no tachyons or ghosts ) . in this case , whereby the interior values of can take on values very different from the exterior values , through non - linear interactions between strong gravity and the scalar field , dramatically affecting the stars internal structure and leading to strong violations of sep . on the other hand , in the case 0 < 0 , one must confront that fact that , with an unstable potential , cosmological evolution would presumably drive the system away from the peak where 0 0 , toward parameter values that could be excluded by solar system experiments . scalar fields coupled to gravity or matter are also ubiquitous in particle - physics - inspired models of unification , such as string theory [ 254 , 176 , 85 , 82 , 83 ] . in some models , the coupling to matter may lead to violations of eep , which could be tested or bounded by the experiments described in section 2.1 . in many models the scalar field could be massive ; if the compton wavelength is of macroscopic scale , its effects are those of a fifth force . only if the theory can be cast as a metric theory with a scalar field of infinite range or of range long compared to the scale of the system in question ( solar system ) can the ppn framework be strictly applied . if the mass of the scalar field is sufficiently large that its range is microscopic , then , on solar - system scales , the scalar field is suppressed , and the theory is essentially equivalent to general relativity . these theories contain the metric g and a dynamical , typically timelike , four - vector field u. in some models , the four - vector is unconstrained , while in others , called einstein - ther theories it is constrained to be timelike with unit norm . the most general action for such theories that is quadratic in derivatives of the vector is given by 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = { ( 16\pi g)^{- 1}}\int { \left [ { ( 1 + \omega { u_\mu}{u^\mu})r - k_{\alpha \beta}^{\mu \nu}{\nabla _ \mu}{u^\alpha}{\nabla _ \nu}{u^\beta } + \lambda ( { u_\mu}{u^\mu } + 1 ) } \right ] } { ( - g)^{1/2}}{d^4}x + { i_m}({\psi _ { \rm{m}}},{g_{\mu \nu}}),$$\end{document } where 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k_{\alpha \beta}^{\mu \nu } = { c_1}{g^{\mu \nu}}{g_{\alpha \beta } } + { c_2}\delta _ \alpha ^\mu \delta _ \beta ^\nu + { c_3}\delta _ \beta ^\mu \delta _ \alpha ^\nu - { c_4}{u^\mu}{u^\nu}{g_{\alpha \beta}}.$$\end{document } the coefficients ci are arbitrary . in the unconstrained theories , 0 and is arbitrary . in the constrained theories , is a lagrange multiplier , and by virtue of the constraint uu = 1 , the factor uu in front of the ricci scalar can be absorbed into a rescaling of g ; equivalently , in the constrained theories , we can set = 0 . note that the possible term ur can be shown under integration by parts to be equivalent to a linear combination of the terms involving c2 and c3 . in addition to having up to four arbitrary parameters , they also left the magnitude of the vector field arbitrary , since it satisfies a linear homogenous vacuum field equation 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}${\mathcal l}{u^\mu } = 0({c_4 } = 0$\end{document } in all such cases studied ) . each theory studied corresponds to a special case of the action ( 39 ) , all with 0 : general vector - tensor theory ; , , , ( see tegp 5.4 ) the gravitational lagrangian for this class of theories had the form r+uu r+uu r ff + uu , where f = u u , corresponding to the values c1 = 2 , c2 = , c1 + c2 + c3 = , c4 = 0 . in these theories , , 1 , and 2 are complicated functions of the parameters and of u = uu , while the rest vanish.will-nordtvedt theory ( see ) this is the special case c1 = 1 , c2 = c3 = c4 = 0 . in this theory , the ppn parameters are given by = = 1 , 2 = u/(1 + u/2 ) , and zero for the rest.hellings-nordtvedt theory ; ( see ) this is the special case c1 = 2 , c2 = 2 , c1 + c2 + c3 = 0 = c4 . here , , 1 and 2 are complicated functions of the parameters and of u , while the rest vanish . general vector - tensor theory ; , , , ( see tegp 5.4 ) the gravitational lagrangian for this class of theories had the form r+uu r+uu r ff + uu , where f = u u , corresponding to the values c1 = 2 , c2 = , c1 + c2 + c3 = , c4 = 0 . in these theories , , 1 , and 2 are complicated functions of the parameters and of u = uu , while the rest vanish . will - nordtvedt theory ( see ) this is the special case c1 = 1 , c2 = c3 = c4 = 0 . in this theory , the ppn parameters are given by = = 1 , 2 = u/(1 + u/2 ) , and zero for the rest . hellings - nordtvedt theory ; ( see ) this is the special case c1 = 2 , c2 = 2 , c1 + c2 + c3 = 0 = c4 . here , , 1 and 2 are complicated functions of the parameters and of u , while the rest vanish . the einstein - ther theories were motivated in part by a desire to explore possibilities for violations of lorentz invariance in gravity , in parallel with similar studies in matter interactions , such as the sme . the general class of theories was analyzed by jacobson and collaborators [ 137 , 183 , 138 , 99 , 113 ] , motivated in part by . analyzing the post - newtonian limit , they were able to infer values of the ppn parameters and as follows : 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma = 1,$$\end{document } 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta = 1,$$\end{document } 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\xi = { \alpha _ 3 } = { \zeta _ 1 } = { \zeta _ 2 } = { \zeta _ 3 } = { \zeta _ 4},$$\end{document } 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\alpha _ 1 } = { { 8(c_3 ^ 2 + { c_1}{c_4 } ) } \over { 2{c_1 } - c_1 ^ 2 + c_3 ^ 2}},$$\end{document } 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\alpha _ 2 } = { { { { ( 2{c_{13 } } - { c_{14}})}^2 } } \over { { c_{123}}(2 - { c_{14 } } ) } } - { { 12{c_3}{c_{13 } } + 2{c_2}{c_{14}}(1 - 2{c_{14 } } ) + ( c_1 ^ 2 - c_3 ^ 2)(4 - 6{c_{13 } } + 7{c_{14 } } ) } \over { ( 2 - { c_{14}})(2{c_1 } - c_1 ^ 2 + c_3 ^ 2)}},$$\end{document } where c123 = c1 + c2 + c3 , c13 = c1 c3 , c14 = c1 c4 , subject to the constraints \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${c_{123 } } \neq 0,{c_{14 } } \neq 2,2{c_1 } - c_1 ^ 2 + c_3 ^ 2 \neq 0$\end{document}. by requiring that gravitational wave modes have real ( as opposed to imaginary ) frequencies , one can impose the bounds c1/(c1 + c4 ) 0 and ( c1 + c2 + c3)/(c1 + c4 ) 0 . considerations of positivity of energy impose the constraints c1 > 0 , c1 + c4 > 0 and c1 + c2 + c3 > 0 . with the ppn formalism in hand , we are now ready to confront gravitation theories with the results of solar - system experiments . in this section we focus on tests of the parameter , consisting of the deflection of light and the time delay of light . a light ray ( or photon ) which passes the sun at a distance d is deflected by an angle 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \theta = { 1 \over 2}(1 + \gamma){{4{m _ \odot } } \over d}{{1 + \cos \phi } \over 2}$$\end{document } ( tegp 7.1 ) , where m is the mass of the sun and is the angle between the earth - sun line and the incoming direction of the photon ( see figure 4 ) . for a grazing ray , d d , 0 , and 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \theta \approx { 1 \over 2}(1 + \gamma)1.{\prime\prime}7505,$$\end{document } independent of the frequency of light . another , more useful expression gives the change in the relative angular separation between an observed source of light and a nearby reference source as both rays pass near the sun : 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \theta = { 1 \over 2}(1 + \gamma)\left [ { - { { 4{m _ \odot } } \over d}\cos \chi + { { 4{m _ \odot } } \over { { d_{\rm{r}}}}}\left({{{1 + \cos { \phi _ { \rm{r } } } } \over 2 } } \right ) } \right],$$\end{document } where d and dr are the distances of closest approach of the source and reference rays respectively , r is the angular separation between the sun and the reference source , and is the angle between the sun - source and the sun - reference directions , projected on the plane of the sky ( see figure 4 ) . thus , for example , the relative angular separation between the two sources may vary if the line of sight of one of them passes near the sun ( d r , dr d , varying with time ) . it is interesting to note that the classic derivations of the deflection of light that use only the corpuscular theory of light ( cavendish 1784 , von soldner 1803 ) , or the principle of equivalence ( einstein 1911 ) , yield only the 1/2 part of the coefficient in front of the expression in equation ( 46 ) . but the result of these calculations is the deflection of light relative to local straight lines , as established for example by rigid rods ; however , because of space curvature around the sun , determined by the ppn parameter , local straight lines are bent relative to asymptotic straight lines far from the sun by just enough to yield the remaining factor /2 . the first factor 1/2 holds in any metric theory , thus , calculations that purport to derive the full deflection using the equivalence principle alone are incorrect . the prediction of the full bending of light by the sun was one of the great successes of einstein s gr . eddington s confirmation of the bending of optical starlight observed during a solar eclipse in the first days following world war i helped make einstein famous . however , the experiments of eddington and his co - workers had only 30 percent accuracy , and succeeding experiments were not much better : the results were scattered between one half and twice the einstein value ( see figure 5 ) , and the accuracies were low . figure 5measurements of the coefficient ( 1 + )/2 from light deflection and time delay measurements . the shapiro time - delay measurements using the cassini spacecraft yielded an agreement with gr to 10 percent , and vlbi light deflection measurements have reached 0.02 percent . measurements of the coefficient ( 1 + )/2 from light deflection and time delay measurements . the shapiro time - delay measurements using the cassini spacecraft yielded an agreement with gr to 10 percent , and vlbi light deflection measurements have reached 0.02 percent . however , the development of radio - interferometery , and later of very - long - baseline radio interferometry ( vlbi ) , produced greatly improved determinations of the deflection of light . these techniques now have the capability of measuring angular separations and changes in angles to accuracies better than 100 microarcseconds . early measurements took advantage of a series of heavenly coincidences : each year , groups of strong quasistellar radio sources pass very close to the sun ( as seen from the earth ) , including the group 3c273 , 3c279 , and 3c48 , and the group 0111 + 02 , 0119 + 11 , and 0116 + 08 . as the earth moves in its orbit , changing the lines of sight of the quasars relative to the sun , the angular separation between pairs of quasars varies ( see equation ( 48 ) ) . the time variation in the quantities d , dr , , and r in equation ( 48 ) is determined using an accurate ephemeris for the earth and initial directions for the quasars , and the resulting prediction for as a function of time is used as a basis for a least - squares fit of the measured , with one of the fitted parameters being the coefficient \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${1 \over 2}(1 + \gamma)$\end{document}. a number of measurements of this kind over the period 19691975 yielded an accurate determination of the coefficient \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${1 \over 2}(1 + \gamma)$\end{document}. a 1995 vlbi measurement using 3c273 and 3c279 yielded ( 1 + )/2 = 0.9996 0.0017 . in recent years , transcontinental and intercontinental vlbi observations of quasars and radio galaxies have been made primarily to monitor the earth s rotation ( vlbi in figure 5 ) . these measurements are sensitive to the deflection of light over almost the entire celestial sphere ( at 90 from the sun , the deflection is still 4 milliarcseconds ) . a 2004 analysis of almost 2 million vlbi observations of 541 radio sources , made by 87 vlbi sites yielded ( 1 + )/2 = 0.99992 0.00023 , or equivalently , 1 = ( 1.7 4.5 ) 10 . analysis of observations made by the hipparcos optical astrometry satellite yielded a test at the level of 0.3 percent . a vlbi measurement of the deflection of light by jupiter was reported ; the predicted deflection of about 300 microarcseconds was seen with about 50 percent accuracy . a radar signal sent across the solar system past the sun to a planet or satellite and returned to the earth suffers an additional non - newtonian delay in its round - trip travel time , given by ( see figure 4 ) 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta t = 2(1 + \gamma){m _ \left({{{({r _ \otimes } + { { \bf{x } } _ \otimes } \cdot { \bf{n}})({r_{\rm{e } } } + { { \bf{x}}_{\rm{e}}}\cdot{\bf{n } } ) } \over { { d^2 } } } } \right),$$\end{document } where xe ( x ) are the vectors , and re ( r ) are the distances from the sun to the source ( earth ) , respectively ( tegp 7.2 ) . for a ray which passes close to the sun , 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta t \approx { 1 \over 2}(1 + \gamma)\left({240 - 20\ln { { { d^2 } } \over r } } \right)\mu { \rm{s}},$$\end{document } where d is the distance of closest approach of the ray in solar radii , and r is the distance of the planet or satellite from the sun , in astronomical units . in the two decades following irwin shapiro s 1964 discovery of this effect as a theoretical consequence of gr , several high - precision measurements were made using radar ranging to targets passing through superior conjunction . since one does not have access to a newtonian signal against which to compare the round - trip travel time of the observed signal , it is necessary to do a differential measurement of the variations in round - trip travel times as the target passes through superior conjunction , and to look for the logarithmic behavior of equation ( 50 ) . in order to do this accurately however , one must take into account the variations in round - trip travel time due to the orbital motion of the target relative to the earth . this is done by using radar - ranging ( and possibly other ) data on the target taken when it is far from superior conjunction ( i.e. when the time - delay term is negligible ) to determine an accurate ephemeris for the target , using the ephemeris to predict the ppn coordinate trajectory xe(t ) near superior conjunction , then combining that trajectory with the trajectory of the earth x(t ) to determine the newtonian round - trip time and the logarithmic term in equation ( 50 ) . the resulting predicted round - trip travel times in terms of the unknown coefficient \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${1 \over 2}(1 + \gamma)$\end{document } are then fit to the measured travel times using the method of least - squares , and an estimate obtained for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${1 \over 2}(1 + \gamma)$\end{document}. the targets employed included planets , such as mercury or venus , used as passive reflectors of the radar signals ( passive radar ) , and artificial satellites , such as mariners 6 and 7 , voyager 2 , the viking mars landers and orbiters , and the cassini spacecraft to saturn , used as active retransmitters of the radar signals ( active radar ) . the results for the coefficient \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${1 \over 2}(1 + \gamma)$\end{document } of all radar time - delay measurements performed to date ( including a measurement of the one - way time delay of signals from the millisecond pulsar psr 1937 + 21 ) are shown in figure 5 ( see tegp 7.2 for discussion and references ) . a significant improvement was reported in 2003 from doppler tracking of the cassini spacecraft while it was on its way to saturn , with a result 1 = ( 2.1 2.3 ) 10 . this was made possible by the ability to do doppler measurements using both x - band ( 7175 mhz ) and ka - band ( 34316 mhz ) radar , thereby significantly reducing the dispersive effects of the solar corona . in addition , the 2002 superior conjunction of cassini was particularly favorable : with the spacecraft at 8.43 astronomical units from the sun , the distance of closest approach of the radar signals to the sun was only 1.6 r. from the results of the cassini experiment , we can conclude that the coefficient \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${1 \over 2}(1 + \gamma)$\end{document } must be within at most 0.0012 percent of unity . scalar - tensor theories must have > 40000 to be compatible with this constraint . in 2001 , kopeikin suggested that a measurement of the time delay of light from a quasar as the light passed by the planet jupiter could be used to measure the speed of the gravitational interaction . he argued that , since jupiter is moving relative to the solar system , and since gravity propagates with a finite speed , the gravitational field experienced by the light ray should be affected by gravity s speed , since the field experienced at one time depends on the location of the source a short time earlier , depending on how fast gravity propagates . according to his calculations , there should be a post - newtonian correction to the normal shapiro time - delay formula ( 49 ) which depends on the velocity of jupiter and on the velocity of gravity . on september 8 , 2002 , jupiter passed almost in front of a quasar , and kopeikin and fomalont made precise measurements of the shapiro delay with picosecond timing accuracy , and claimed to have measured the correction term to about 20 percent [ 112 , 153 , 148 , 149 ] . however , several authors pointed out that this 1.5pn effect does not depend on the speed of propagation of gravity , but rather only depends on the speed of light [ 14 , 288 , 232 , 49 , 233 ] . intuitively , if one is working to only first order in /c , then all that counts is the uniform motion of the planet , jupiter ( its acceleration about the sun contributes a higher - order , unmeasurably small effect ) . but if that is the case , then the principle of relativity says that one can view things from the rest frame of jupiter . in this frame , jupiter s gravitational field is static , and the speed of propagation of gravity is irrelevant . a detailed post - newtonian calculation of the effect was done using a variant of the ppn framework , in a class of theories in which the speed of gravity could be different from that of light , and found explicitly that , at first order in /c , the effect depends on the speed of light , not the speed of gravity , in line with intuition . effects dependent upon the speed of gravity show up only at higher order in /c . kopeikin gave a number of arguments in opposition to this interpretation [ 149 , 151 , 150 , 152 ] . on the other hand , the /c correction term does show a dependence on the ppn parameter 1 , which could be non - zero in theories of gravity with a differing speed cg of gravity ( see equation ( 7 ) of ) . but existing tight bounds on 1 from other experiments ( see table 4 ) already far exceed the capability of the jupiter vlbi experiment . n is a combination of other parameters given by n = 4 3 10/3 1 + 22/3 21/3 2/3.parametereffectlimitremarks1time delay2.3 10cassini trackinglight deflection4 10vlbi1perihelion shift3 10j2 = 10 from helioseismologynordtvedt effect2.3 10n = 4 3 assumed earth tides10gravimeter data 1 orbital polarization10lunar laser ranging2 10psr j2317 + 1439 2 spin precession4 10solar alignment with ecliptic 3 pulsar acceleration4 10pulsar statistics n nordtvedt effect9 10lunar laser ranging l 2 10combined ppn bounds 2 binary acceleration4 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\ddot p_{\rm{p}}}$\end{document } for psr 1913 + 16 3 newton s 3rd law10lunar acceleration 4 not independent ( see equation ( 58 ) ) current limits on the ppn parameters . here n is a combination of other parameters given by n = 4 3 10/3 1 + 22/3 21/3 2/3 . the explanation of the anomalous perihelion shift of mercury s orbit was another of the triumphs of gr . this had been an unsolved problem in celestial mechanics for over half a century , since the announcement by le verrier in 1859 that , after the perturbing effects of the planets on mercury s orbit had been accounted for , and after the effect of the precession of the equinoxes on the astronomical coordinate system had been subtracted , there remained in the data an unexplained advance in the perihelion of mercury . a number of ad hoc proposals were made in an attempt to account for this excess , including , among others , the existence of a new planet vulcan near the sun , a ring of planetoids , a solar quadrupole moment and a deviation from the inverse - square law of gravitation , but none was successful . general relativity accounted for the anomalous shift in a natural way without disturbing the agreement with other planetary observations . the predicted advance per orbit \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \tilde \omega$\end{document } , including both relativistic ppn contributions and the newtonian contribution resulting from a possible solar quadrupole moment , is given by 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \tilde \omega = { { 6\pi m } \over p}\left({{1 \over 3}(2 + 2\gamma - \beta ) + { 1 \over 6}(2{\alpha _ 1 } - { \alpha _ 2 } + { \alpha _ 3 } + 2{\zeta _ 2}){\mu \over m } + { { { j_2}{r^2 } } \over { 2mp } } } \right),$$\end{document } where m m1 + m2 and m1m2/m are the total mass and reduced mass of the two - body system respectively ; p a(1 e ) is the semi - latus rectum of the orbit , with the semi - major axis a and the eccentricity e ; r is the mean radius of the oblate body ; and j2 is a dimensionless measure of its quadrupole moment , given by j2 = ( c a)/m1r , where c and a are the moments of inertia about the body s rotation and equatorial axes , respectively ( for details of the derivation see tegp 7.3 ) . we have ignored preferred - frame and galaxy - induced contributions to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta \tilde \omega$\end{document } ; these are discussed in tegp 8.3 . the first term in equation ( 51 ) is the classical relativistic perihelion shift , which depends upon the ppn parameters and . the second term depends upon the ratio of the masses of the two bodies ; it is zero in any fully conservative theory of gravity ( 1 2 3 2 0 ) ; it is also negligible for mercury , since /m mmerc / m 2 10 . the third term depends upon the solar quadrupole moment j2 . for a sun that rotates uniformly with its observed surface angular velocity , so that the quadrupole moment is produced by centrifugal flattening this actually agrees reasonably well with values inferred from rotating solar models that are in accord with observations of the normal modes of solar oscillations ( helioseismology ) ; the latest inversions of helioseismology data give j2 = ( 2.2 0.1 ) 10 [ 207 , 211 , 230 , 184 ] . substituting standard orbital elements and physical constants for mercury and the sun we obtain the rate of perihelion shift \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\dot \tilde \omega}$\end{document } , in seconds of arc per century , 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot \tilde \omega = 42.{\prime\prime}98\left({{1 \over 3}(2 + 2\gamma - \beta ) + 3 \times { { 10}^{- 4}}{{{j_2 } } \over { { { 10}^{- 7 } } } } } \right).$$\end{document } now , the measured perihelion shift of mercury is known accurately : after the perturbing effects of the other planets have been accounted for , the excess shift is known to about 0.1 percent from radar observations of mercury between 1966 and 1990 . the solar oblateness effect is smaller than the observational error , so we obtain the ppn bound |2 1| < 3 10 . the next class of solar - system experiments that test relativistic gravitational effects may be called tests of the strong equivalence principle ( sep ) . in section 3.1.2 we pointed out that many metric theories of gravity ( perhaps all except gr ) can be expected to violate one or more aspects of sep . among the testable violations of sep are a violation of the weak equivalence principle for gravitating bodies that leads to perturbations in the earth - moon orbit , preferred - location and preferred - frame effects in the locally measured gravitational constant that could produce observable geophysical effects , and possible variations in the gravitational constant over cosmological timescales . in a pioneering calculation using his early form of the ppn formalism , nordtvedt showed that many metric theories of gravity predict that massive bodies violate the weak equivalence principle that is , fall with different accelerations depending on their gravitational self - energy . dicke argued that such an effect would occur in theories with a spatially varying gravitational constant , such as scalar - tensor gravity . for a spherically symmetric body , the acceleration from rest in an external gravitational potential u has the form 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \quad { \rm{a } } = { { { m_{\rm{p } } } } \over m}\nabla u,}\\ { { { { m_{\rm{p } } } } \over m } = 1 - { \eta _ { \rm{n}}}{{{m_{\rm{g } } } } \over m},}\\ { { \eta _ { \rm{n } } } = 4\beta - \gamma - 3 - { { 10 } \over 3}\xi - { \alpha _ 1 } + { 2 \over 3}{\alpha _ 2 } - { 2 \over 3}{\zeta _ 1 } - { 1 \over 3}{\zeta _ 2},}\\ \end{array}$$\end{document } where eg is the negative of the gravitational self - energy of the body ( eg > 0 ) . this violation of the massive - body equivalence principle is known as the nordtvedt effect . the effect is absent in gr ( n = 0 ) but present in scalar - tensor theory ( n= 1/(2 + ) + 4 ) . the existence of the nordtvedt effect does not violate the results of laboratory etvs experiments , since for laboratory - sized objects eg / m 10 , far below the sensitivity of current or future experiments . however , for astronomical bodies , eg / m may be significant ( 3.6 10 for the sun , 10 for jupiter , 4.6 10 for the earth , 0.2 10 for the moon ) . if the nordtvedt effect is present ( n 0 ) then the earth should fall toward the sun with a slightly different acceleration than the moon . this perturbation in the earth - moon orbit leads to a polarization of the orbit that is directed toward the sun as it moves around the earth - moon system , as seen from earth . this polarization represents a perturbation in the earth - moon distance of the form 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta r = 13.1{\eta _ { \rm{n}}}\cos ( { \omega _ 0 } - { \omega _ { \rm{s}}})t\;[{\rm{m}}],$$\end{document } where 0 and s are the angular frequencies of the orbits of the moon and sun around the earth ( see tegp 8.1 for detailed derivations and references ; for improved calculations of the numerical coefficient , see [ 201 , 89 ] ) . since august 1969 , when the first successful acquisition was made of a laser signal reflected from the apollo 11 retroreflector on the moon , the llr experiment has made regular measurements of the round - trip travel times of laser pulses between a network of observatories and the lunar retroreflectors , with accuracies that are at the 50 ps ( 1 cm ) level , and that may soon approach 5 ps ( 1 mm ) . these measurements are fit using the method of least - squares to a theoretical model for the lunar motion that takes into account perturbations due to the sun and the other planets , tidal interactions , and post - newtonian gravitational effects . the predicted round - trip travel times between retroreflector and telescope also take into account the librations of the moon , the orientation of the earth , the location of the observatories , and atmospheric effects on the signal propagation . the nordtvedt parameter n along with several other important parameters of the model are then estimated in the least - squares method . numerous ongoing analyses of the data find no evidence , within experimental uncertainty , for the nordtvedt effect [ 295 , 296 ] ( for earlier results see [ 95 , 294 , 192 ] ) . these results represent a limit on a possible violation of wep for massive bodies of about 1.4 parts in 10 ( compare figure 1 ) . however , at this level of precision , one can not regard the results of llr as a test of sep until one eliminates the possibility of a compensating violation of wep for the two bodies , because the chemical compositions of the earth and moon differ in the relative fractions of iron and silicates . to this end , the et - wash group carried out an improved test of wep for laboratory bodies whose chemical compositions mimic that of the earth and moon . the resulting bound of 1.4 parts in 10 [ 19 , 2 ] from composition effects reduces the ambiguity in the llr bound , and establishes the firm sep test at the level of about 2 parts in 10 . these results can be summarized by the nordtvedt parameter bound |n| = ( 4.4 4.5 ) 10 . in the future , the apache point observatory for lunar laser ranging operation ( apollo ) project , a joint effort by researchers from the universities of washington , seattle , and california , san diego , plans to use enhanced laser and telescope technology , together with a good , high - altitude site in new mexico , to improve the llr bound by as much as an order of magnitude . in gr , the nordtvedt effect vanishes ; at the level of several centimeters and below , a number of non - null general relativistic effects should be present . tests of the nordtvedt effect for neutron stars have also been carried out using a class of systems known as wide - orbit binary millisecond pulsars ( wbmsp ) , which are pulsar - white - dwarf binary systems with small orbital eccentricities . in the gravitational field of the galaxy , a non - zero nordtvedt effect can induce an apparent anomalous eccentricity pointed toward the galactic center , which can be bounded using statistical methods , given enough wbmsps ( see for a review and references ) . using data from 21 wbmsps , including obtained the bound < 5.6 10 , where = n(eg / m)ns . because ( eg / m)ns 0.1 for typical neutron stars , this bound does not compete with the bound on n from llr ; on the other hand , it does test sep in the strong - field regime because of the presence of the neutron stars . some theories of gravity violate sep by predicting that the outcomes of local gravitational experiments may depend on the velocity of the laboratory relative to the mean rest frame of the universe ( preferred - frame effects ) or on the location of the laboratory relative to a nearby gravitating body ( preferred - location effects ) . in the post - newtonian limit , preferred - frame effects are governed by the values of the ppn parameters 1 , 2 , and 3 , and some preferred - location effects are governed by ( see table 2 ) . the most important such effects are variations and anisotropies in the locally - measured value of the gravitational constant which lead to anomalous earth tides and variations in the earth s rotation rate , anomalous contributions to the orbital dynamics of planets and the moon , self - accelerations of pulsars , and anomalous torques on the sun that would cause its spin axis to be randomly oriented relative to the ecliptic ( see tegp 8.2 , 8.3 , 9.3 , and 14.3 ( c ) ) . an bound on 3 of 4 10 from the period derivatives of 21 millisecond pulsars was reported in [ 26 , 244 ] ; improved bounds on 1 were achieved using llr data , and using observations of the circular binary orbit of the pulsar j2317 + 1439 . negative searches for these effects have produced strong constraints on the ppn parameters ( see table 4 ) . most theories of gravity that violate sep predict that the locally measured newtonian gravitational constant may vary with time as the universe evolves . for the scalar - tensor theories listed in table 3 , the predictions for / g can be written in terms of time derivatives of the asymptotic scalar field . where g does change with cosmic evolution , its rate of variation should be of the order of the expansion rate of the universe , i.e. / g h0 , where h0 is the hubble expansion parameter and is given by h0 = 100 h km s mpc = 1.02 10 h yr , where current observations of the expansion of the universe give h 0.73 0.03 . several observational constraints can be placed on / g , one kind coming from bounding the present rate of variation , another from bounding a difference between the present value and a past value . the first type of bound typically comes from llr measurements , planetary radar - ranging measurements , and pulsar timing data . the second type comes from studies of the evolution of the sun , stars and the earth , big - bang nucleosynthesis , and analyses of ancient eclipse data . the bounds are dependent upon the theory of gravity in the strong - field regime and on neutron star equation of state . big - bang nucleosynthesis bounds assume specific form for time dependence of g.method/g ( 10 yr)referencelunar laser ranging4 9binary pulsar 1913 + 1640 50helioseismology0 16big bang nucleosynthesis0 4[65 , 21 ] constancy of the gravitational constant . for binary pulsar data , the bounds are dependent upon the theory of gravity in the strong - field regime and on neutron star equation of state . big - bang nucleosynthesis bounds assume specific form for time dependence of g. the best limits on a current / g come from llr measurements ( for earlier results see [ 95 , 294 , 192 ] ) . these have largely supplanted earlier bounds from ranging to the 1976 viking landers ( see tegp , 14.3 ( c ) ) , which were limited by uncertain knowledge of the masses and orbits of asteroids . however , improvements in knowledge of the asteroid belt , combined with continuing radar observations of planets and spacecraft , notably the mars global surveyor ( 19982003 ) and mars odyssey ( 2002present ) , may enable a bound on / g at the level of a part in 10 per year . for an initial analysis along these lines , it has been suggested that radar observations of the planned 2012 bepi - colombo mercury orbiter mission over a two - year integration with 6 cm rms accuracy in range could yield ( / g ) < 10 yr ; an eight - year mission could improve this by a factor 15 [ 187 , 17 ] . although bounds on / g from solar - system measurements can be correctly obtained in a phenomenological manner through the simple expedient of replacing g by g0 + 0(t t0 ) in newton s equations of motion , the same does not hold true for pulsar and binary pulsar timing measurements . the reason is that , in theories of gravity that violate sep , such as scalar - tensor theories , the mass and moment of inertia of a gravitationally bound body may vary with variation in g. because neutron stars are highly relativistic , the fractional variation in these quantities can be comparable to g / g , the precise variation depending both on the equation of state of neutron star matter and on the theory of gravity in the strong - field regime . the variation in the moment of inertia affects the spin rate of the pulsar , while the variation in the mass can affect the orbital period in a manner that can subtract from the direct effect of a variation in g , given by b / pb = 2 / g . thus , the bounds quoted in table 5 for the binary pulsar psr 1913 + 16 and others ( see also ) are theory - dependent and must be treated as merely suggestive . in a similar manner , bounds from helioseismology and big - bang nucleosynthesis ( bbn ) assume a model for the evolution of g over the multi - billion year time spans involved . for example , the concordance of predictions for light elements produced around 3 minutes after the big bang with the abundances observed indicate that g then was within 20 percent of g today . assuming a power - law variation of g t then yields a bound on / g today shown in table 5 . according to gr , moving or rotating matter should produce a contribution to the gravitational field that is the analogue of the magnetic field of a moving charge or a magnetic dipole . in particular , one can view the g0i part of the ppn metric ( see box 2 ) as an analogue of the vector potential of electrodynamics . in a suitable gauge , and dropping the preferred - frame terms , it can be written 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{0i } } = - { 1 \over 2}(4\gamma + 4 + { \alpha _ 1}){v_i}.$$\end{document } at pn order , this contributes a lorentz - type acceleration v bg to the equation of motion , where the gravitomagnetic field bg is given by bg = ( g0ie ) . gravitomagnetism plays a role in a variety of measured relativistic effects involving moving material sources , such as the earth - moon system and binary pulsar systems . nordtvedt [ 199 , 198 ] has argued that , if the gravitomagnetic potential ( 55 ) were turned off , then there would be anomalous orbital effects in llr and binary pulsar data . rotation also produces a gravitomagnetic effect , since for a rotating body , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bf{v } } = - { 1 \over 2}{\bf{x } } \times { \bf{j}}/{r^3}$\end{document } , where j is the angular momentum of the body . the result is a dragging of inertial frames around the body , also called the lense - thirring effect . a consequence is a precession of a gyroscope s spin s according to 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{\bf{s } } } \over { d\tau } } = { \omega _ { { \rm{lt } } } } \times { \bf{s}},\quad { \omega _ { { \rm{lt } } } } = - { 1 \over 2}\left({1 + \gamma + { 1 \over 4}{\alpha _ 1 } } \right){{{\bf{j } } - 3{\bf{n}}({\bf{n } } \cdot { \bf{j } } ) } \over { { r^3}}},$$\end{document } where n is a unit radial vector , and r is the distance from the center of the body ( tegp 9.1 ) . the relativity gyroscope experiment ( gravity probe b or gpb ) at stanford university , in collaboration with nasa and lockheed - martin corporation , recently completed a space mission to detect this frame - dragging or lense - thirring precession , along with the geodetic a set of four superconducting - niobium - coated , spherical quartz gyroscopes were flown in a polar earth orbit ( 642 km mean altitude , 0.0014 eccentricity ) , and the precessions of the gyroscopes relative to a distant guide star ( hr 8703 , i m pegasi ) were measured . for the given orbit , the predicted secular angular precession of the gyroscopes is in a direction perpendicular to the orbital plane at a rate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${1 \over 2}(1 + \gamma + { 1 \over 4}{\alpha _ 1 } ) \times 41 \times { 10^{- 3}}$\end{document } arcsec yr . the spacecraft was launched on april 20 , 2004 , and the mission ended in september 2005 , as scheduled , when the remaining liquid helium boiled off . it is too early to know whether the relativistic precessions were measured in the amount predicted by gr , because an important calibration of the instrument exploits the effect of the aberration of starlight on the pointing of the on - board telescope toward the guide star , and completing this calibration required the full mission data set . in addition , part of the measured effect includes the motion of the guide star relative to distant inertial frames . this was measured before , during and after the mission separately by radio astronomers at harvard / sao and elsewhere using vlbi , and the results of those measurements were to be strictly embargoed until the gpb team has completed its analysis of the gyro data . another way to look for frame - dragging is to measure the precession of orbital planes of bodies circling a rotating body . one implementation of this idea is to measure the relative precession , at about 31 milliarcseconds per year , of the line of nodes of a pair of laser - ranged geodynamics satellites ( lageos ) , ideally with supplementary inclination angles ; the inclinations must be supplementary in order to cancel the dominant ( 126 degrees per year ) nodal precession caused by the earth s newtonian gravitational multipole moments . unfortunately , the two existing lageos satellites are not in appropriately inclined orbits , and no concrete plans exist at present to launch a third satellite in a supplementary orbit . nevertheless , ciufolini and pavlis combined nodal precession data from lageos i and ii with improved models for the earth s multipole moments provided by two recent orbiting geodesy satellites , europe s champ ( challenging minisatellite payload ) and nasa s grace ( gravity recovery and climate experiment ) , and reported a 510 percent confirmation of gr . in earlier reports , had reported tests at the the 2030 percent level , without the benefit of the grace / champ data [ 55 , 57 , 54 ] . some authors stressed the importance of adequately assessing systematic errors in the lageos data [ 226 , 133 ] . a gyroscope moving through curved spacetime suffers a precession of its spin axis given by 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{\bf{s } } } \over { d\tau } } = { \omega _ { \rm{g } } } \times { \bf{s}},\quad { \omega _ { \rm{g } } } = \left({\gamma + { 1 \over 2 } } \right){\bf{v } } \times \nabla u,$$\end{document } where v is the velocity of the gyroscope , and u is the newtonian gravitational potential of the source ( tegp 9.1 ) . the predicted precession is about 2 arcseconds per century , an effect first calculated by de sitter . this effect has been measured to about 0.6 percent using llr data [ 95 , 294 , 295 ] . for the gpb gyroscopes orbiting the earth , a goal of gpb is to measure this effect to 8 10 ; if achieved , this could bound the parameter to a part in 10 , not competitive with the cassini bound . ppn parameters 1 , 2 , 3 , 4 , and 3 , only three , 2 , 3 , and 3 , have been constrained directly with any precision ; 1 is constrained indirectly through its appearance in the nordtvedt effect parameter n , equation ( 53 ) . there is strong theoretical evidence that 4 , which is related to the gravity generated by fluid pressure , is not really an independent parameter in any reasonable theory of gravity there should be a connection between the gravity produced by kinetic energy ( ) , internal energy ( ) , and pressure ( p ) . from such considerations , there follows the additional theoretical constraint 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$6{\zeta _ 4 } = 3{\alpha _ 3 } + 2{\zeta _ 1 } - 3{\zeta _ 3}.$$\end{document } a non - zero value for any of these parameters would result in a violation of conservation of momentum , or of newton an alternative statement of newton s third law for gravitating systems is that the active gravitational mass , that is the mass that determines the gravitational potential exhibited by a body , should equal the passive gravitational mass , the mass that determines the force on a body in a gravitational field . such an equality guarantees the equality of action and reaction and of conservation of momentum , at least in the newtonian limit . a classic test of newton s third law for gravitating systems was carried out in 1968 by kreuzer , in which the gravitational attraction of fluorine and bromine were compared to a precision of 5 parts in 10 . they noted that current understanding of the structure of the moon involves an iron - rich , aluminum - poor mantle whose center of mass is offset about 10 km from the center of mass of an aluminum - rich , iron - poor crust . the direction of offset is toward the earth , about 14 to the east of the earth - moon line . such a model accounts for the basaltic maria which face the earth , and the aluminum - rich highlands on the moon s far side , and for a 2 km offset between the observed center of mass and center of figure for the moon . because of this asymmetry , a violation of newton s third law for aluminum and iron would result in a momentum non - conserving self - force on the moon , whose component along the orbital direction would contribute to the secular acceleration of the lunar orbit . improved knowledge of the lunar orbit through llr , and a better understanding of tidal effects in the earth - moon system ( which also contribute to the secular acceleration ) through satellite data , severely limit any anomalous secular acceleration , with the resulting limit 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\vert { { { { { ( { m_{\rm{a}}}/{m_{\rm{p}}})}_{{\rm{al } } } } - { { ( { m_{\rm{a}}}/{m_{\rm{p}}})}_{{\rm{fe } } } } } \over { { { ( { m_{\rm{a}}}/{m_{\rm{p}}})}_{{\rm{fe } } } } } } } \right\vert < 4 \times { 10^{- 12}}.$$\end{document } according to the ppn formalism , in a theory of gravity that violates conservation of momentum , but that obeys the constraint of equation ( 58 ) , the electrostatic binding energy ee of an atomic nucleus could make a contribution to the ratio of active to passive mass of the form 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{\rm{a } } } = { m_{\rm{p } } } + { 1 \over 2}{\zeta _ 3}{e_{\rm{e}}}.$$\end{document } the resulting limit on 3 from the lunar experiment is 3 < 1 10 ( tegp 9.2 , 14.3 ( d ) ) . nordtvedt has examined whether this bound could be improved by considering the asymmetric distribution of ocean water on earth . another consequence of a violation of conservation of momentum is a self - acceleration of the center of mass of a binary stellar system , given by 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\bf{a}}_{{\rm{cm } } } } = - { 1 \over 2}({\zeta _ 2 } + { \alpha _ 3}){m \over { { a^2}}}{\mu \over a}{{\delta m } \over m}{e \over { { { ( 1 - { e^2})}^{3/2}}}}{{\bf{n}}_{\rm{p}}},$$\end{document } where m = m1 m2 , a is the semi - major axis , and np is a unit vector directed from the center of mass to the point of periastron of m1 ( tegp 9.3 ) . a consequence of this acceleration would be non - vanishing values for dp / dt , where p denotes the period of any intrinsic process in the system ( orbit , spectra , pulsar periods ) . the observed upper limit on dpp / dt of the binary pulsar psr 1913 + 16 places a strong constraint on such an effect , resulting in the bound |3 + 2| < 4 10 . since 3 has already been constrained to be much less than this ( see table 4 ) , we obtain a strong solitary bound on 2 < 4 10 . a number of advanced experiments or space missions are under development or have been proposed which could lead to significant improvements in values of the ppn parameters , of j2 of the sun , and of / g . llr at the apache point observatory ( apollo project ) could improve bounds on the nordvedt parameter to the level 3 10 and on / g to better than 10 yr . the proposed 2012 esa bepi - columbo mercury orbiter , in a two - year experiment , with 6 cm range capability , could yield improvements in to 3 10 , in to 3 10 , in 1 to 10 , in / g to 10 yr , and in j2 to 3 10 . an eight - year mission could yield further improvements by factors of 25 in , 1 , and j2 , and a further factor 15 in / g [ 187 , 17 ] . gaia is a high - precision astrometric orbiting telescope ( a successor to hipparcos ) , which could measure light - deflection and to the 10 level . lator ( laser astrometric test of relativity ) is a concept for a nasa mission in which two microsatellites orbit the sun on earth - like orbits near superior conjunction , so that their lines of sight are close to the sun . using optical tracking and an optical interferometer on the international space station , it may be possible to measure the deflection of light with sufficient accuracy to bound to a part in 10 and j2 to a part in 10 , and to measure the solar frame - dragging effect to one percent [ 259 , 260 ] . nordtvedt has argued that grand fits of large solar system ranging data sets , including radar ranging to mercury , mars , and satellites , and laser ranging to the moon , could yield substantially improved measurements of ppn parameters . in the solar system , gravity is weak , in the sense that the newtonian gravitational potential and related variables ( u(x , t ) systems are those for which the simple 1pn approximation of the ppn framework is no longer appropriate . this can occur in a number of situations : the system may contain strongly relativistic objects , such as neutron stars or black holes , near and inside which 1 , and the post - newtonian approximation breaks down . nevertheless , under some circumstances , the orbital motion may be such that the interbody potential and orbital velocities still satisfy 1 so that a kind of post - newtonian approximation for the orbital motion might work ; however , the strong - field internal gravity of the bodies could ( especially in alternative theories of gravity ) leave imprints on the orbital motion.the evolution of the system may be affected by the emission of gravitational radiation . the 1pn approximation does not contain the effects of gravitational radiation back - reaction . in the expression for the metric given in box 2 , radiation back - reaction effects do not occur until \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^{7/2}})$\end{document } in g00 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^3})$\end{document } in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${g_{{0_i}}}$\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 o}({\varepsilon ^{5/2}})$\end{document } in gij . consequently , in order to describe such systems , one must carry out a solution of the equations substantially beyond 1pn order , sufficient to incorporate the leading radiation damping terms at 2.5pn order . in addition , the ppn metric described in section 3.2 is valid in the near zone of the system , i.e. within one gravitational wavelength of the system s center of mass . as such it can not describe the gravitational waves seen by a detector.the system may be highly relativistic in its orbital motion , systems like this include the late stage of the inspiral of binary systems of neutron stars or black holes , driven by gravitational radiation damping , prior to a merger and collapse to a final stationary state . binary inspiral is one of the leading candidate sources for detection by a world - wide network of laser interferometric gravitational wave observatories nearing completion . a proper description of such systems requires not only equations for the motion of the binary carried to extraordinarily high pn orders ( at least 3.5pn ) , but also requires equations for the far - zone gravitational waveform measured at the detector , that are equally accurate to high pn orders beyond the leading quadrupole approximation . the system may contain strongly relativistic objects , such as neutron stars or black holes , near and inside which 1 , and the post - newtonian approximation breaks down . nevertheless , under some circumstances , the orbital motion may be such that the interbody potential and orbital velocities still satisfy 1 so that a kind of post - newtonian approximation for the orbital motion might work ; however , the strong - field internal gravity of the bodies could ( especially in alternative theories of gravity ) leave imprints on the orbital motion . the 1pn approximation does not contain the effects of gravitational radiation back - reaction . in the expression for the metric given in box 2 , radiation back - reaction effects do not occur until \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^{7/2}})$\end{document } in g00 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^3})$\end{document } in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${g_{{0_i}}}$\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 o}({\varepsilon ^{5/2}})$\end{document } in gij . consequently , in order to describe such systems , one must carry out a solution of the equations substantially beyond 1pn order , sufficient to incorporate the leading radiation damping terms at 2.5pn order . in addition , the ppn metric described in section 3.2 is valid in the near zone of the system , i.e. within one gravitational wavelength of the system s center of mass . as such it can not describe the gravitational waves seen by a detector . systems like this include the late stage of the inspiral of binary systems of neutron stars or black holes , driven by gravitational radiation damping , prior to a merger and collapse to a final stationary state . binary inspiral is one of the leading candidate sources for detection by a world - wide network of laser interferometric gravitational wave observatories nearing completion . a proper description of such systems requires not only equations for the motion of the binary carried to extraordinarily high pn orders ( at least 3.5pn ) , but also requires equations for the far - zone gravitational waveform measured at the detector , that are equally accurate to high pn orders beyond the leading quadrupole approximation . of course , some systems can not be properly described by any post - newtonian approximation because their behavior is fundamentally controlled by strong gravity . these include the imploding cores of supernovae , the final merger of two compact objects , the quasinormal - mode vibrations of neutron stars and black holes , the structure of rapidly rotating neutron stars , and so on . this field of numerical relativity is a rapidly growing and maturing branch of gravitational physics , whose description is beyond the scope of this review ( see [ 165 , 24 ] for reviews ) . another is black hole perturbation theory ( see [ 188 , 146 , 235 ] for reviews ) . when dealing with the motion and gravitational wave generation by orbiting bodies , one finds a remarkable simplification within gr . as long as the bodies are sufficiently well - separated that one can ignore tidal interactions and other effects that depend upon the finite extent of the bodies ( such as their quadrupole and higher multipole moments ) , then all aspects of their orbital behavior and gravitational wave generation can be characterized by just two parameters : mass and angular momentum . whether their internal structure is highly relativistic , as in black holes or neutron stars , or non - relativistic as in the earth and sun , furthermore , both quantities are measurable in principle by examining the external gravitational field of the bodies , and make no reference whatsoever to their interiors . general relativity satisfies sep because it contains one and only one gravitational field , the spacetime metric g. consider the motion of a body in a binary system , whose size is small compared to the binary separation . surround the body by a region that is large compared to the size of the body , yet small compared to the separation . because of the general covariance of the theory , one can choose a freely - falling coordinate system which comoves with the body , whose spacetime metric takes the minkowski form at its outer boundary ( ignoring tidal effects generated by the companion ) . there is thus no evidence of the presence of the companion body , and the structure of the chosen body can be obtained using the field equations of gr in this coordinate system . far from the chosen body , the metric is characterized by the mass and angular momentum ( assuming that one ignores quadrupole and higher multipole moments of the body ) as measured far from the body using orbiting test particles and gyroscopes . . a black hole of mass m and a planet of mass m would produce identical spacetimes in this outer region . the geometry of this region surrounding the one body must be matched to the geometry provided by the companion body . einstein s equations provide consistency conditions for this matching that yield constraints on the motion of the bodies . the motion of two planets of mass and angular momentum m1 , m2 , j1 , and j2 is identical to that of two black holes of the same mass and angular momentum ( again , ignoring tidal effects ) . this effacement does not occur in an alternative gravitional theory like scalar - tensor gravity . there , in addition to the spacetime metric , a scalar field is generated by the masses of the bodies , and controls the local value of the gravitational coupling constant ( i.e. g is a function of ) . now , in the local frame surrounding one of the bodies in our binary system , while the metric can still be made minkowskian far away , the scalar field will take on a value 0 determined by the companion body . this can affect the value of g inside the chosen body , alter its internal structure ( specifically its gravitational binding energy ) and hence alter its mass . effectively , each body can be characterized by several mass functions ma( ) , which depend on the value of the scalar field at its location , and several distinct masses come into play , such as inertial mass , gravitational mass , radiation mass , etc . the precise nature of the functions will depend on the body , specifically on its gravitational binding energy , and as a result , the motion and gravitational radiation may depend on the internal structure of each body . for compact bodies such as neutron stars and black holes these internal structure effects could be large ; for example , the gravitational binding energy of a neutron star can be 1020 percent of its total mass . at 1pn order , this is how the study of orbiting systems containing compact objects provides strong - field tests of gr . even though the strong - field nature of the bodies is effaced in gr , it is not in other theories , thus any result in agreement with the predictions of gr constitutes a kind of null test of strong - field gravity . the motion of bodies and the generation of gravitational radiation are long - standing problems that date back to the first years following the publication of gr , when einstein calculated the gravitational radiation emitted by a laboratory - scale object using the linearized version of gr , and de sitter calculated n - body equations of motion for bodies in the 1pn approximation to gr . it has at times been controversial , with disputes over such issues as whether einstein s equations alone imply equations of motion for bodies ( einstein , infeld , and hoffman demonstrated explicitly that they do , using a matching procedure similar to the one described above ) , whether gravitational waves are real or are artifacts of general covariance ( einstein waffled ; bondi and colleagues proved their reality rigorously in the 1950s ) , and even over algebraic errors ( einstein erred by a factor of 2 in his first radiation calculation ; eddington found the mistake ) . shortly after the discovery of the binary pulsar psr 1913 + 16 in 1974 , questions were raised about the foundations of the quadrupole formula for gravitational radiation damping ( and in some quarters , even about its quantitative validity ) . these questions were answered in part by theoretical work designed to shore up the foundations of the quadrupole approximation , and in part ( perhaps mostly ) by the agreement between the predictions of the quadrupole formula and the observed rate of damping of the pulsar s orbit ( see section 5.1 ) . damour gives a thorough historical and technical review of this subject up to 1986 . the problem of motion and radiation in gr has received renewed interest since 1990 , with proposals for construction of large - scale laser interferometric gravitational wave observatories , such as the ligo project in the us , virgo and geo600 in europe , and tama300 in japan , and the realization that a leading candidate source of detectable waves would be the inspiral , driven by gravitational radiation damping , of a binary system of compact objects ( neutron stars or black holes ) [ 1 , 256 ] . the analysis of signals from such systems will require theoretical predictions from gr that are extremely accurate , well beyond the leading - order prediction of newtonian or even post - newtonian gravity for the orbits , and well beyond the leading - order formulae for gravitational waves . this presented a major theoretical challenge : to calculate the motion and radiation of systems of compact objects to very high pn order , a formidable algebraic task , while addressing a number of issues of principle that have historically plagued this subject , sufficiently well to ensure that the results were physically meaningful . this challenge has been largely met , so that we may soon see a remarkable convergence between observational data and accurate predictions of gravitational theory that could provide new , strong - field tests of gr . here we give a brief overview of the problem of motion and gravitational radiation in gr . the einstein equations g = 8t are elegant and deceptively simple , showing geometry ( in the form of the einstein tensor g , which is a function of spacetime curvature ) being generated by matter ( in the form of the material stress - energy tensor t ) . however , this is not the most useful form for actual calculations . for post - newtonian calculations , a far more useful form is the so - called relaxed einstein equations : 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\square h^{\alpha \beta } } = - 16\pi { \tau ^{\alpha \beta}},$$\end{document } where /t + is the flat - spacetime wave operator , h is a gravitational tensor potential related to the deviation of the spacetime metric from its minkowski form by the formula h ( g ) g , g is the determinant of g , and a particular coordinate system has been specified by the de donder or harmonic gauge condition h/x = 0 ( summation on repeated indices is assumed ) . this form of einstein s equations bears a striking similarity to maxwell s equations for the vector potential a in lorentz gauge : a = 4j , a/x = 0 . there is a key difference , however : the source on the right hand side of equation ( 62 ) is given by the effective stress - energy pseudotensor 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tau ^{\alpha \beta } } = - ( - g){t^{\alpha \beta } } + { ( 16\pi)^{- 1}}{\lambda ^{\alpha \beta}},$$\end{document } where is the non - linear field contribution given by terms quadratic ( and higher ) in h and its derivatives ( see , eqs . the gravitational field itself generates gravity , a reflection of the nonlinearity of einstein s equations , and in contrast to the linearity of maxwell s equations . equation ( 62 ) is exact , and depends only on the assumption that spacetime can be covered by harmonic coordinates . it is called relaxed because it can be solved formally as a functional of source variables without specifying the motion of the source , in the form 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{\alpha \beta}}(t,{\bf{x } } ) = - 4\int\nolimits_{\mathcal c } { { { { \tau ^{\alpha \beta}}(t - \vert{\bf{x } } - { \bf{x}}{\prime}\vert , { \bf{x}}{\prime } ) } \over { \vert{\bf{x } } - { \bf{x}}{\prime}\vert } } } { d^3}x{\prime},$$\end{document } where the integration is over the past flat - spacetime null cone \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } of the field point ( t , x ) . the motion of the source is then determined either by the equation /x = 0 ( which follows from the harmonic gauge condition ) , or from the usual covariant equation of motion \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t^{\alpha \beta}}_{;\beta } = 0$\end{document } , where the subscript ; denotes a covariant divergence . this formal solution can then be iterated in a slow motion ( < 1 ) weak - field ( h < 1 ) approximation . one begins by substituting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_0^{\alpha \beta } = 0$\end{document } into the source in equation ( 64 ) , and solving for the first iterate \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_1^{\alpha \beta}$\end{document } , and then repeating the procedure sufficiently many times to achieve a solution of the desired accuracy . for example , to obtain the 1pn equations of motion , two iterations are needed ( i.e. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_2^{\alpha \beta}$\end{document } must be calculated ) ; likewise , to obtain the leading gravitational waveform for a binary system , two iterations are needed . at the same time , just as in electromagnetism , the formal integral ( 64 ) must be handled differently , depending on whether the field point is in the far zone or the near zone . for field points in the far zone or radiation zone , |x| > > |x| ( is the gravitational wavelength divided by 2 ) , the field can be expanded in inverse powers of r = |x| in a multipole expansion , evaluated at the retarded time t r. the leading term in 1/r is the gravitational waveform . for field points in the near zone or induction zone , |x| |x| < , the field is expanded in powers of |x x| about the local time t , yielding instantaneous potentials that go into the equations of motion . however , because the source contains h itself , it is not confined to a compact region , but extends over all spacetime . as a result , there is a danger that the integrals involved in the various expansions will diverge or be ill - defined . this consequence of the non - linearity of einstein s equations has bedeviled the subject of gravitational radiation for decades . the post - minkowskian method of blanchet , damour , and iyer [ 35 , 36 , 37 , 76 , 38 , 33 ] solves einstein s equations by two different techniques , one in the near zone and one in the far zone , and uses the method of singular asymptotic matching to join the solutions in an overlap region . the method provides a natural regularization technique to control potentially divergent integrals ( see for a thorough review ) . the direct integration of the relaxed einstein equations ( dire ) approach of will , wiseman , and pati [ 291 , 208 ] retains equation ( 64 ) as the global solution , but splits the integration into one over the near zone and another over the far zone , and uses different integration variables to carry out the explicit integrals over the two zones . in the dire method , itoh and futamase have used an extension of the einstein - infeld - hoffman matching approach combined with a specific method for taking a point - particle limit , while damour , jaranowski , and schfer have pioneered an adm hamiltonian approach that focuses on the equations of motion [ 139 , 140 , 77 , 79 , 78 ] . these methods assume from the outset that gravity is sufficiently weak that h < 1 and harmonic coordinates exists everywhere , including inside the bodies . thus , in order to apply the results to cases where the bodies may be neutron stars or black holes , one relies upon the sep to argue that , if tidal forces are ignored , and equations are expressed in terms of masses and spins , one can simply extrapolate the results unchanged to the situation where the bodies are ultrarelativistic . while no general proof of this exists , it has been shown to be valid in specific circumstances , such as at 2pn order in the equations of motion , and for black holes moving in a newtonian background field . methods such as these have resolved most of the issues that led to criticism of the foundations of gravitational radiation theory during the 1970s . among the results of these approaches are formulae for the equations of motion and gravitational waveform of binary systems of compact objects , carried out to high orders in a pn expansion . here for example , the relative two - body equation of motion has the form 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\bf{a } } = { { d{\bf{v } } } \over { dt } } = { m \over { { r^2}}}\{- { \bf{\hat n } } + { { \bf{a}}_{1{\rm{pn } } } } + { { \bf{a}}_{2{\rm{pn } } } } + { { \bf{a}}_{2.5{\rm{pn } } } } + { { \bf{a}}_{3{\rm{pn } } } } + { { \bf{a}}_{3.5{\rm{pn } } } } + \ldots \},$$\end{document } where m = m1 + m2 is the total mass , r = |x1 x2| , v = v1 v2 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bf{a}}_{n{\rm{pn}}}}$\end{document}. the notation anpn indicates that the term is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^n})$\end{document } relative to the newtonian term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- { \rm{\hat n}}$\end{document}. explicit and unambiguous formulae for non - spinning bodies through 3.5pn order have been calculated by various authors ( see for a review ) . here we quote only the first pn corrections and the leading radiation - reaction terms at 2.5pn order : 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\bf{a}}_{1{\rm{pn } } } } = \left\{{(4 + 2\eta){m \over r } - ( 1 + 3\eta){v^2 } + { 3 \over 2}\eta { { \dot r}^2 } } \right\}{\bf{\hat n } } + ( 4 - 2\eta)\dot r{\bf{v}},$$\end{document } 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\bf{a}}_{2.5{\rm{pn } } } } = - { 8 \over { 15}}\eta { m \over r}\left\{{\left({9{v^2 } + 17{m \over r } } \right)\dot r{\bf{\hat n } } - \left({3{v^2 } + 9{m \over r } } \right){\bf{v } } } \right\},$$\end{document } where = m1m2/(m1 + m2 ) . these terms are sufficient to analyze the orbit and evolution of the binary pulsar ( see section 5.1 ) . for example , the 1pn terms are responsible for the periastron advance of an eccentric orbit , given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot \omega = 6\pi { f_{\rm{b}}}m / a(1 - { e^2})$\end{document } , where a and e are the semi - major axis and eccentricity of the orbit , respectively , and fb is the orbital frequency , given to the needed order by kepler s third law 2fb = ( m / a ) . another product is a formula for the gravitational field far from the system , written schematically in the form 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{ij } } = { { 2 m } \over r}\left\{{{q^{ij } } + q_{0.5{\rm{pn}}}^{ij } + q_{1{\rm{pn}}}^{ij } + q_{1.5{\rm{pn}}}^{ij } + q_{2{\rm{pn}}}^{ij } + q_{2.5{\rm{pn}}}^{ij } + \ldots } \right\},$$\end{document } where r is the distance from the source , and the variables are to be evaluated at retarded time t r. the leading term is the so - called quadrupole formula 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h^{ij}}(t,{\bf{x } } ) = { { 2 m } \over r}{\ddot i^{ij}}(t - r),$$\end{document } where i is the quadrupole moment of the source , and overdots denote time derivatives . for a binary system this leads to 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${q^{ij } } = 2\eta \left({{\upsilon ^i}{\upsilon ^j } - { { m{{\hat n}^i}{{\hat n}^j } } \over r } } \right).$$\end{document } for binary systems , explicit formulae for the waveform through 2pn order have been derived ( see for a ready - to - use presentation of the waveform for circular orbits ; see for a full review ) . given the gravitational waveform , one can compute the rate at which energy is carried off by the radiation ( schematically d , the gravitational analog of the poynting flux ) . the lowest - order quadrupole formula leads to the gravitational wave energy flux 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot e = { 8 \over { 15}}{\eta ^2}{{{m^4 } } \over { { r^4}}}(12{v^2 } - 11{\dot r^2}).$$\end{document } this has been extended to 3.5pn order beyond the quadrupole formula ( see for a review ) . the 2.5pn radiation - reaction terms in the equation of motion ( 65 ) result in a damping of the orbital energy that precisely balances the energy flux ( 71 ) determined from the waveform . averaged over one orbit , this results in a rate of increase of the binary s orbital frequency given by 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \quad { { \dot f}_{\rm{b } } } = { { 192\pi } \over 5}f_{\rm{b}}^2{{(2\pi{\mathcal m}{f_{\rm{b}}})}^{5/3}}f(e)}\\ { f(e ) = { { ( 1 - { e^2})}^{- 7/2}}\left({1 + { { 73 } \over { 24}}{e^2 } + { { 37 } \over { 96}}{e^4 } } \right),}\\ \end{array}$$\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 so - called chirp mass , given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m } = { \eta ^{3/5}}m$\end{document}. notice that by making precise measurements of the phase (t ) = 2 f ( t ) dt of either the orbit or the gravitational waves ( for which f = 2fb for the dominant component ) as a function of the frequency , one in effect measures the chirp mass of the system . these formalisms have also been generalized to include the leading effects of spin - orbit and spin - spin coupling between the bodies [ 145 , 144 , 289 ] . another approach to gravitational radiation is applicable to the special limit in which one mass is much smaller than the other . one begins with an exact background spacetime of a black hole , either the non - rotating schwarzschild or the rotating kerr solution , and perturbs it according to \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}^{(0 ) } + { h_{\mu \nu}}$\end{document}. the particle moves on a geodesic of the background spacetime , and a suitably defined source stress - energy tensor for the particle acts as a source for the gravitational perturbation and wave field h. this method provides numerical results that are exact in , as well as analytical results expressed as series in powers of , both for non - rotating and for rotating black holes . for non - rotating holes , the analytical expansions have been carried to 5.5pn order , or beyond the quadrupole approximation . all results of black hole perturbation agree precisely with the m1 0 limit of the pn results , up to the highest pn order where they can be compared ( for reviews see [ 188 , 235 ] ) . a gravitational wave detector can be modelled as a body of mass m at a distance l from a fiducial laboratory point , connected to the point by a spring of resonant frequency 0 and quality factor q. from the equation of geodesic deviation , the infinitesimal displacement of the mass along the line of separation from its equilibrium position satisfies the equation of motion 73\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ddot \xi + { { 2{\omega _ 0 } } \over q}\dot \xi + \omega _ 0 ^ 2\xi = { l \over 2}\left({{f _ + } ( \theta , \phi , \psi){{\ddot h } _ + } ( t ) + { f _ \times}(\theta , \phi , \psi){{\ddot h } _ \times}(t ) } \right),$$\end{document } wheref+(,, ) and f ( ,, ) are beam - pattern factors that depend on the direction of the source ( , ) and on a polarization angle , and h+(t ) and h(t ) are gravitational waveforms corresponding to the two polarizations of the gravitational wave ( for a review , see ) . in a source coordinate system in which the x y plane is the plane of the sky and the z - direction points toward the detector , these two modes are given by 74\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h _ + } ( t ) = { 1 \over 2}(h_{{\rm{tt}}}^{xx}(t ) - h_{{\rm{tt}}}^{yy}(t)),\quad { h _ \times}(t ) = h_{{\rm{tt}}}^{xy}(t),$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h_{{\rm{tt}}}^{ij}$\end{document } represent transverse - traceless ( tt ) projections of the calculated waveform of equation ( 68 ) , given by 75\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h_{{\rm{tt}}}^{ij } = { h^{kl}}\left [ { \left({{\delta ^{ik } } - { { \hat n}^i}{{\hat n}^k } } \right)\left({{\delta ^{jl } } - { { \hat n}^j}{{\hat n}^l } } \right ) - { 1 \over 2}\left({{\delta ^{ij } } - { { \hat n}^i}{{\hat n}^j } } \right)\left({{\delta ^{kl } } - { { \hat n}^k}{{\hat n}^l } } \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}${{\hat n}^j}$\end{document } is a unit vector pointing toward the detector . the beam pattern factors depend on the orientation and nature of the detector . for a wave approaching along the laboratory z - direction , and for a mass whose location on the x y plane makes an angle with the x axis , the beam pattern factors are given by f+ = cos 2 and f = sin 2. for a resonant cylinder oriented along the laboratory z axis , and for source direction ( , ) , they are given by f+ = sin cos 2 , f = sin sin 2 ( the angle measures the relative orientation of the laboratory and source x - axes ) . for a laser interferometer with one arm along the laboratory x - axis , the other along the y - axis , and with defined as the differential displacement along the two arms , the beam pattern functions are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f _ + } = { 1 \over 2}(1 + { \cos ^2}\theta)\cos 2\phi \cos 2\psi - \cos \theta \sin 2\phi \sin 2\psi$\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}${f _ \times } = { 1 \over 2}(1 + { \cos ^2}\theta)\cos 2\phi \sin 2\psi + \cos \theta \sin 2\phi \cos 2\psi$\end{document}. the waveforms h+(t ) and h ( t ) depend on the nature and evolution of the source . for example , for a binary system in a circular orbit , with an inclination i relative to the plane of the sky , and the x - axis oriented along the major axis of the projected orbit , the quadrupole approximation of equation ( 70 ) gives 76\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h _ + } ( t ) = - { { 2{\mathcal m } } \over r}{(2\pi { \mathcal m}{f_{\rm{b}}})^{2/3}}(1 + { \cos ^2}i)\cos 2{\phi _ { \rm{b}}}(t),$$\end{document } 77\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h _ \times}(t ) = - { { 2{\mathcal m } } \over r}{(2\pi { \mathcal m}{f_{\rm{b}}})^{2/3}}2\cos i\cos 2{\phi _ { \rm{b}}}(t),$$\end{document } where b(t ) = 2 fb(t ) dt is the orbital phase . the 1974 discovery of the binary pulsar b1913 + 16 by joseph taylor and russell hulse during a routine search for new pulsars provided the first possibility of probing new aspects of gravitational theory : the effects of strong relativistic internal gravitational fields on orbital dynamics , and the effects of gravitational radiation reaction . for reviews of the discovery , see the published nobel prize lectures by hulse and taylor [ 132 , 252 ] . for reviews of the current status of pulsars , including binary and millisecond pulsars , the system consists of a pulsar of nominal period 59 ms in a close binary orbit with an as yet unseen companion . the orbital period is about 7.75 hours , and the eccentricity is 0.617 . from detailed analyses of the arrival times of pulses ( which amounts to an integrated version of the doppler - shift methods used in spectroscopic binary systems ) , extremely accurate orbital and physical parameters for the system have been obtained ( see table 6 ) . because the orbit is so close ( 1r ) and because there is no evidence of an eclipse of the pulsar signal or of mass transfer from the companion , it is generally agreed that the companion is compact . evolutionary arguments suggest that it is most likely a dead pulsar , while b1913 + 16 is a recycled pulsar . thus the orbital motion is very clean , free from tidal or other complicating effects . clean in the sense that by exploiting the intrinsic stability of the pulsar clock combined with the ability to maintain and transfer atomic time accurately using gps , the observers can keep track of pulse time - of - arrival with an accuracy of 13 s , despite extended gaps between observing sessions ( including a several - year gap in the middle 1990s for an upgrade of the arecibo radio telescope ) . the pulsar has shown no evidence of glitches in its pulse period . data taken from [ 18 , 272 ] . note that is not the same as the ppn parameter ( see equations ( 78)).parametersymbol ( units)value(i)physical parameters : right ascension 191527.99999(2)declination 160627.4034(4)pulsar periodpp ( ms)59.0299983444181(5)derivative of period p 8.62713(8 ) 10(ii)keplerian parameters : projected semimajor axisap sin i ( s)2.341774(1)eccentricity e 0.6171338(4)orbital periodpb ( day)0.322997462727(5)longitude of periastron0 ( )226.57518(4)julian date of periastront0 ( mjd)46443.99588317(3)(iii)post - keplerian parameters : mean rate of periastron advance\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { \dot \omega } \right\rangle$\end{document } ( yr)4.226595(5)redshift / time dilation ( ms)4.2919(8)orbital period derivativeb ( 1012)2.4184(9 ) parameters of the binary pulsar b1913 + 16 . data taken from [ 18 , 272 ] . note that is not the same as the ppn parameter ( see equations ( 78 ) ) . three factors make this system an arena where relativistic celestial mechanics must be used : the relatively large size of relativistic effects [ orbit ( m / r ) 10 ] , a factor of 10 larger than the corresponding values for solar - system orbits ; the short orbital period , allowing secular effects to build up rapidly ; and the cleanliness of the system , allowing accurate determinations of small effects . because the orbital separation is large compared to the neutron stars compact size , tidal effects can be ignored . just as newtonian gravity is used as a tool for measuring astrophysical parameters of ordinary binary systems , so gr is used as a tool for measuring astrophysical parameters in the binary pulsar . the observational parameters that are obtained from a least - squares solution of the arrival - time data fall into three groups : non - orbital parameters , such as the pulsar period and its rate of change ( defined at a given epoch ) , and the position of the pulsar on the sky;five keplerian parameters , most closely related to those appropriate for standard newtonian binary systems , such as the eccentricity e , the orbital period pb , and the semi - major axis of the pulsar projected along the line of sight , ap sin i ; andfive post - keplerian parameters . the five post - keplerian parameters are : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\langle \dot \omega \rangle$\end{document } , the average rate of periastron advance ; , the amplitude of delays in arrival of pulses caused by the varying effects of the gravitational redshift and time dilation as the pulsar moves in its elliptical orbit at varying distances from the companion and with varying speeds ; b , the rate of change of orbital period , caused predominantly by gravitational radiation damping ; and r and s = sin i , respectively the range and shape of the shapiro time delay of the pulsar signal as it propagates through the curved spacetime region near the companion , where i is the angle of inclination of the orbit relative to the plane of the sky . non - orbital parameters , such as the pulsar period and its rate of change ( defined at a given epoch ) , and the position of the pulsar on the sky ; five keplerian parameters , most closely related to those appropriate for standard newtonian binary systems , such as the eccentricity e , the orbital period pb , and the semi - major axis of the pulsar projected along the line of sight , ap sin i ; and five post - keplerian parameters . in gr , the five post - keplerian parameters can be related to the masses of the two bodies and to measured keplerian parameters by the equations ( tegp 12.1 , 14.6 ( a ) ) 78\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 } { \langle \dot \omega \rangle = 6\pi { f_{\rm{b}}}{{(2\pi m{f_{\rm{b}}})}^{2/3}}{{(1 - { e^2})}^{- 1}},}\\ { \;\;\gamma{\prime } = e{{(2\pi { f_{\rm{b}}})}^{- 1}}{{(2\pi m{f_{\rm{b}}})}^{2/3}}{{{m_2 } } \over m}\left({1 + { { { m_2 } } \over m } } \right),}\\ { \;{{\dot p}_{\rm{b } } } = - { { 192\pi } \over 5}{{(2\pi m{f_{\rm{b}}})}^{5/3}}f(e),}\\ { \;\;s = \sin i,}\\ { \;\;r = { m_2},}\\ \end{array}$$\end{document } where m1 and m2 denote the pulsar and companion masses , respectively . the formula for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\langle \dot \omega \rangle$\end{document } ignores possible non - relativistic contributions to the periastron shift , such as tidally or rotationally induced effects caused by the companion ( for discussion of these effects , see tegp 12.1 ( c ) ) . the formula for b includes only quadrupole gravitational radiation ; it ignores other sources of energy loss , such as tidal dissipation ( tegp 12.1 ( f ) ) . notice that , by virtue of kepler s third law , ( 2fb ) = m / a , ( 2mfb ) = m / a , thus the first two post - keplerian parameters can be seen as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}(\epsilon)$\end{document } , or 1pn corrections to the underlying variable , while the third is an \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal o}({\epsilon ^{5/2}})$\end{document } , or 2.5pn correction . the current observed values for the keplerian and post - keplerian parameters are shown in table 6 . the parameters r and s are not separately measurable with interesting accuracy for b1913 + 16 because the orbit s 47 inclination does not lead to a substantial shapiro delay . because fb and e are separately measured parameters , the measurement of the three post - keplerian parameters provide three constraints on the two unknown masses . the periastron shift measures the total mass of the system , b measures the chirp mass , and measures a complicated function of the masses . gr passes the test if it provides a consistent solution to these constraints , within the measurement errors . from the intersection of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\langle \dot \omega \rangle$\end{document } and constraints we obtain the values m1 = 1.44140.0002m and m2 = 1.38670.0002m. the third of equations ( 78 ) then predicts the value b = 2.40242 0.00002 10 . in order to compare the predicted value for b with the observed value of table 6 , it is necessary to take into account the small effect of a relative acceleration between the binary pulsar system and the solar system caused by the differential rotation of the galaxy . this effect was previously considered unimportant when b was known only to 10 percent accuracy . damour and taylor carried out a careful estimate of this effect using data on the location and proper motion of the pulsar , combined with the best information available on galactic rotation ; the current value of this effect is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot p_{\rm{b}}^{{\rm{gal } } } \simeq - ( 0.0128 \pm 0.0050 ) \times { 10^{- 12}}$\end{document}. subtracting this from the observed b ( see table 6 ) gives the corrected \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot p_{\rm{b}}^{{\rm{corr } } } = - ( 2.4056 \pm 0.0051 ) \times { 10^{- 12}}$\end{document } , which agrees with the prediction within the errors . in other words , 79\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\dot p_{\rm{b}}^{{\rm{corr } } } } \over { \dot p_{\rm{b}}^{{\rm{gr } } } } } = 1.0013 \pm 0.0021.$$\end{document } the consistency among the measurements is displayed in figure 6 , in which the regions allowed by the three most precise constraints have a single common overlap . uncertainties in the parameters that go into the galactic correction are now the limiting factor in the accuracy of the test of gravitational damping . figure 6constraints on masses of the pulsar and its companion from data on b1913 + 16 , assuming gr to be valid . the width of each strip in the plane reflects observational accuracy , shown as a percentage . an inset shows the three constraints on the full mass plane ; the intersection region ( a ) has been magnified 400 times for the full figure . constraints on masses of the pulsar and its companion from data on b1913 + 16 , assuming gr to be valid . the width of each strip in the plane reflects observational accuracy , shown as a percentage . an inset shows the three constraints on the full mass plane ; the intersection region ( a ) has been magnified 400 times for the full figure . a third way to display the agreement with gr is by comparing the observed phase of the orbit with a theoretical template phase as a function of time . if fb varies slowly in time , then to first order in a taylor expansion , the orbital phase is given by b ( t ) = 2fb0 t + b0 t . the time of periastron passage tp is given by (tp ) = 2n , where n is an integer , and consequently , the periastron time will not grow linearly with n. thus the cumulative difference between periastron time tp and n / fb0 , the quantities actually measured in practice , should vary according to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_{\rm{p } } } - n/{f_{{\rm{b}}0 } } = - { { \dot f}_{{\rm{b0}}}}{n^2}/2f_{{\rm{b0}}}^3 \approx - ( { { \dot f}_{{\rm{b0}}}}/2{f_{{\rm{b0}}}}){t^2}$\end{document}. figure 7 shows the results : the dots are the data points , while the curve is the predicted difference using the measured masses and the quadrupole formula for b0 . the gap during the middle 1990s was caused by a closure of arecibo for upgrading . the gap during the middle 1990s was caused by a closure of arecibo for upgrading . the consistency among the constraints provides a test of the assumption that the two bodies behave as point masses , without complicated tidal effects , obeying the general relativistic equations of motion including gravitational radiation . it is also a test of strong gravity , in that the highly relativistic internal structure of the neutron stars does not influence their orbital motion , as predicted by the sep of gr . recent observations [ 157 , 271 ] indicate variations in the pulse profile , which suggests that the pulsar is undergoing geodetic precession on a 300-year timescale as it moves through the curved spacetime generated by its companion ( see section 3.7.2 ) . the amount is consistent with gr , assuming that the pulsar s spin is suitably misaligned with the orbital angular momentum . unfortunately , the evidence suggests that the pulsar beam may precess out of our line of sight by 2025 . nine relativistic binary neutron star systems with orbital periods less than a day are now known . while some are less interesting for testing relativity , some have yielded interesting tests , and others , notably the recently discovered we describe some of the more interesting or best studied cases ; the parameters of the first four are listed in table 7 . b1534 + 12 this is a binary pulsar system in our galaxy [ 245 , 243 , 18 ] . its pulses are significantly stronger and narrower than those of b1913 + 16 , so timing measurements are more precise , reaching 3 s accuracy . the orbital plane appears to be almost edge - on relative to the line of sight ( i 80 ) ; as a result the shapiro delay is substantial , and separate values of the parameters r and s have been obtained with interesting accuracy . assuming gr , one infers that the two masses are m1 = 1.335 0.002 m and m2 = 1.344 0.002 m. the rate of orbit decay b agrees with gr to about 15 percent , but the precision is limited by the poorly known distance to the pulsar , which introduces a significant uncertainty into the subtraction of galactic acceleration . independently of b , measurement of the four other post - keplerian parameters gives two tests of strong - field gravity in the non - radiative regime . b2127 + 11c this system appears to be a clone of the hulse - taylor binary pulsar , with very similar values for orbital period and eccentricity ( see table 7 ) . the inferred total mass of the system is 2.706 0.011 m. but because the system is in the globular cluster m15 ( ngc 7078 ) , it suffers doppler shifts resulting from local accelerations , either by the mean cluster gravitational field or by nearby stars , that are more difficult to estimate than was the case with the galactic system b1913 + 16 . this makes a separate , precision measurement of the relativistic contribution to b essentially impossible . j0737 - 3039a , b this binary pulsar system , discovered in 2003 , was already remarkable for its extraordinarily short orbital period ( 0.1 days ) and large periastron advance ( 16.88 yr ) , but then the companion was also discovered to be a pulsar . because two projected semi - major axes can now be measured , one can obtain the mass ratio directly from the ratio of the two values of ap sin i , and thereby obtain the two masses by combining that ratio with the periastron advance , assuming gr . the results are ma = 1.3370.005 m and mb = 1.2500.005 m , where a denotes the primary ( first ) pulsar . from these values , one finds that the orbit is nearly edge - on , with sin i = 0.9991 , a value which is completely consistent with that inferred from the shapiro delay parameter ( see table 7 ) . in fact , the five measured post - keplerian parameters plus the ratio of the projected semi - major axes give six constraints on the masses ( assuming gr ) : all six overlap within their measurement errors . this system provides a unique opportunity for tight tests of strong - field and radiative effects in gr . furthermore , it is likely that galactic proper motion effects will play a significantly smaller role in the interpretation of b measurements than they did in b1913 + 16 . j1141 - 6545 this is a case where the companion is probably a white dwarf [ 20 , 18 ] . but because of the asymmetry in sensitivities ( sns 0.2 , swd 10 ) , there is the possibility , absent in the double neutronstar systems , to place a strong bound on scalar - tensor gravity ( see section 5.4 ) . j1756 - 2251 discovered in 2004 , this pulsar is in a binary system with a probable neutron star companion , with pb = 7.67 hr , e = 0.18 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot \omega = 2.585 \pm 0.002\deg { \rm{y}}{{\rm{r}}^{{\rm{- 1}}}}$\end{document } . it is a young , 144-ms pulsar in a relativistic orbit with pb = 3.98 hr , e = 0.085 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot \omega = 7.57 \pm 0.03\deg { \rm{y}}{{\rm{r}}^{{\rm{- 1}}}}$\end{document}. table 7parameters of other binary pulsars . references may be found in the text ; for an online catalogue of pulsars with reasonably up - to - date parameters , see .parameterb1534 + 12b2127 + 11cj11416545j07373039(a , b)(i)keplerian parameters : ap sin i ( s)3.7294626(8)2.520(3)1.85894(1)1.41504(2)/1.513(3 ) e 0.2736767(1)0.68141(2)0.171876(2)0.087779(5)pb ( day)0.420737299153(4)0.335282052(6)0.1976509587(3)0.102251563(1)(ii)post - keplerian parameters:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\left\langle { \dot \omega } \right\rangle$\end{document } ( yr)1.755805(3)4.457(12)5.3084(9)16.90(1) ( ms)2.070(2)4.670.72(3)0.382(5)b ( 10)0.137(3)3.940.43(10)1.21(6)r ( s)6.7(1.0)6.2(5)s = sin i0.975(7)0.9995(4 ) this is a binary pulsar system in our galaxy [ 245 , 243 , 18 ] . its pulses are significantly stronger and narrower than those of b1913 + 16 , so timing measurements are more precise , reaching 3 s accuracy . the orbital plane appears to be almost edge - on relative to the line of sight ( i 80 ) ; as a result the shapiro delay is substantial , and separate values of the parameters r and s have been obtained with interesting accuracy . assuming gr , one infers that the two masses are m1 = 1.335 0.002 m and m2 = 1.344 0.002 m. the rate of orbit decay b agrees with gr to about 15 percent , but the precision is limited by the poorly known distance to the pulsar , which introduces a significant uncertainty into the subtraction of galactic acceleration . independently of b , measurement of the four other post - keplerian parameters gives two tests of strong - field gravity in the non - radiative regime . this system appears to be a clone of the hulse - taylor binary pulsar , with very similar values for orbital period and eccentricity ( see table 7 ) . the inferred total mass of the system is 2.706 0.011 m. but because the system is in the globular cluster m15 ( ngc 7078 ) , it suffers doppler shifts resulting from local accelerations , either by the mean cluster gravitational field or by nearby stars , that are more difficult to estimate than was the case with the galactic system b1913 + 16 . this makes a separate , precision measurement of the relativistic contribution to b essentially impossible . this binary pulsar system , discovered in 2003 , was already remarkable for its extraordinarily short orbital period ( 0.1 days ) and large periastron advance ( 16.88 yr ) , but then the companion was also discovered to be a pulsar . because two projected semi - major axes can now be measured , one can obtain the mass ratio directly from the ratio of the two values of ap sin i , and thereby obtain the two masses by combining that ratio with the periastron advance , assuming gr . the results are ma = 1.3370.005 m and mb = 1.2500.005 m , where a denotes the primary ( first ) pulsar . from these values , one finds that the orbit is nearly edge - on , with sin i = 0.9991 , a value which is completely consistent with that inferred from the shapiro delay parameter ( see table 7 ) . in fact , the five measured post - keplerian parameters plus the ratio of the projected semi - major axes give six constraints on the masses ( assuming gr ) : all six overlap within their measurement errors . this system provides a unique opportunity for tight tests of strong - field and radiative effects in gr . furthermore , it is likely that galactic proper motion effects will play a significantly smaller role in the interpretation of b measurements than they did in b1913 + 16 . this is a case where the companion is probably a white dwarf [ 20 , 18 ] . but because of the asymmetry in sensitivities ( sns 0.2 , swd 10 ) , there is the possibility , absent in the double neutronstar systems , to place a strong bound on scalar - tensor gravity ( see section 5.4 ) . discovered in 2004 , this pulsar is in a binary system with a probable neutron star companion , with pb = 7.67 hr , e = 0.18 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot \omega = 2.585 \pm 0.002\deg { \rm{y}}{{\rm{r}}^{{\rm{- 1}}}}$\end{document } . it is a young , 144-ms pulsar in a relativistic orbit with pb = 3.98 hr , e = 0.085 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot \omega = 7.57 \pm 0.03\deg { \rm{y}}{{\rm{r}}^{{\rm{- 1}}}}$\end{document}. parameters of other binary pulsars . references may be found in the text ; for an online catalogue of pulsars with reasonably up - to - date parameters , see . soon after the discovery of the binary pulsar it was widely hailed as a new testing ground for relativistic gravitational effects . as we have seen in the case of gr , in most respects , the system has lived up to , indeed exceeded , the early expectations . in another respect , however , the system has only partially lived up to its promise , namely as a direct testing ground for alternative theories of gravity . the origin of this promise was the discovery that alternative theories of gravity generically predict the emission of dipole gravitational radiation from binary star systems . in gr , there is no dipole radiation because the dipole moment ( center of mass ) of isolated systems is uniform in time ( conservation of momentum ) , and because the inertial mass that determines the dipole moment is the same as the mass that generates gravitational waves ( sep ) . in other theories , while the inertial dipole moment may remain uniform , the gravity wave dipole moment need not , because the mass that generates gravitational waves depends differently on the internal gravitational binding energy of each body than does the inertial mass ( violation of sep ) . schematically , in a coordinate system in which the center of inertial mass is at the origin , so that mi,1x1 + mi,2x2 = 0 , the dipole part of the retarded gravitational field would be 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}$$h \sim { 1 \over r}{d \over { dt}}({m_{{\rm{gw,1}}}}{{\bf{x}}_1 } + { m_{{\rm{gw,2}}}}{{\bf{x}}_2 } ) \sim { { \eta m } \over r}{\bf{v}}\left({{{{m_{{\rm{gw,1 } } } } } \over { { m_{{\rm{i,1 } } } } } } - { { { m_{{\rm{gw,2 } } } } } \over { { m_{{\rm{i,2 } } } } } } } \right),$$\end{document } where v = v1 v2 and and m are defined using inertial masses . in theories that violate sep , the difference between gravitational wave mass and inertial mass is a function of the internal gravitational binding energy of the bodies . this additional form of gravitational radiation damping could , at least in principle , be significantly stronger than the usual quadrupole damping , because it depends on fewer powers of the orbital velocity , and it depends on the gravitational binding energy per unit mass of the bodies , which , for neutron stars , could be as large as 20 percent ( see tegp 10 for further details ) . as one fulfillment of this promise , will and eardley worked out in detail the effects of dipole gravitational radiation in the bimetric theory of rosen , and , when the first observation of the decrease of the orbital period was announced in 1979 , the rosen theory suffered a terminal blow . a wide class of alternative theories also fails the binary pulsar test because of dipole gravitational radiation ( tegp 12.3 ) . on the other hand , the early observations of psr 1913 + 16 already indicated that , in gr , the masses of the two bodies were nearly equal , so that , in theories of gravity that are in some sense close to gr , dipole gravitational radiation would not be a strong effect , because of the apparent symmetry of the system . the rosen theory , and others like it , are not close to gr , except in their predictions for the weak - field , slow - motion regime of the solar system . when relativistic neutron stars are present , theories like these can predict strong effects on the motion of the bodies resulting from their internal highly relativistic gravitational structure ( violations of sep ) . as a consequence , the masses inferred from observations of the periastron shift and may be significantly different from those inferred using gr , and may be different from each other , leading to strong dipole gravitational radiation damping . by contrast , the brans - dicke theory is close to gr , roughly speaking within 1/bd of the predictions of the latter , for large values of the coupling constant bd . thus , despite the presence of dipole gravitational radiation , the binary pulsar provides at present only a weak test of brans - dicke theory , not competitive with solar - system tests . making the usual assumption that both members of the system are neutron stars , and using the methods summarized in tegp 1012 , one can obtain formulas for the periastron shift , the gravitational redshift / second - order doppler shift parameter , and the rate of change of orbital period , analogous to equations ( 78 ) . these formulas depend on the masses of the two neutron stars , on their self - gravitational binding energy , represented by sensitivities s and * , and on the brans - dicke coupling constant bd . first , there is a modification of kepler s third law , given by 81\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2\pi { f_{\rm{b } } } = { \left({{{{\mathcal g}m } \over { { a^3 } } } } \right)^{1/2}},$$\end{document } then , the predictions for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\langle \dot \omega \rangle$\end{document } , and b are 82\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\langle \dot \omega \rangle = 6\pi { f_{\rm{b}}}{(2\pi m{f_{\rm{b}}})^{2/3}}{(1 - { e^2})^{- 1}}{\mathcal p}{{\mathcal g}^{- 4/3}},$$\end{document } 83\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma{\prime } = e{(2\pi { f_{\rm{b}}})^{- 1}}{(2\pi m{f_{\rm{b}}})^{2/3}}{{{m_2 } } \over m}{{\mathcal g}^{- 1/3}}(\alpha _ 2^\ast + { \mathcal g}{{{m_2 } } \over m } + \kappa _ 1^\ast\eta _ 2^\ast),$$\end{document } 84\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\dot p_{\rm{b } } } = - { { 192\pi } \over 5}{(2\pi { \mathcal m}{f_{\rm{b}}})^{5/3}}f{\prime}(e ) - 4\pi ( 2\pi \mu { f_{\rm{b}}})\xi { { \mathcal s}^2}g(e),$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m } \equiv { \chi ^{3/5}}{{\mathcal g}^{- 4/5}}{\eta ^{3/5}}m$\end{document } , and , to first order in ( 2 + bd ) , we have 85\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f{\prime}(e ) = f(e ) + { 5 \over { 144}}\xi { ( \gamma + 3\gamma{\prime})^2}\left({{1 \over 2}{e^2 } + { 1 \over 8}{e^4 } } \right){(1 - { e^2})^{- 7/2}},$$\end{document } 86\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g(e ) = { ( 1 - { e^2})^{- 5/2}}\left({1 + { 1 \over 2}{e^2 } } \right),$$\end{document } 87\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal s } = { s_1 } - { s_2},$$\end{document } 88\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal g } = 1 - \xi ( { s_1 } + { s_2 } - 2{s_1}{s_2}),$$\end{document } 89\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal p } = { \mathcal g}\left [ { 1 - { 2 \over 3}\xi + { 1 \over 3}\xi ( { s_1 } + { s_2 } - 2{s_1}{s_2 } ) } \right],$$\end{document } 90\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\alpha _ 2^\ast = 1 - \xi { s_2},$$\end{document } 91\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\eta _ 2^\ast = ( 1 - 2{s_2})\xi,$$\end{document } 92\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi = { { \mathcal g}^2}\left [ { 1 - { 1 \over 2}\xi + { 1 \over { 12}}\xi { \gamma ^2 } } \right],$$\end{document } 93\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma = 1 - 2{{{m_1}{s_2 } + { m_2}{s_1 } } \over m},$$\end{document } 94\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\gamma{\prime } = 1 - { s_1 } - { s_2},$$\end{document } where f(e ) is defined in equation ( 72 ) . the quantities sa and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k_{\rm{a}}^{\ast}$\end{document } are defined by 95\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{\rm{a } } } = - { \left({{{\partial ( \ln { m_{\rm{a } } } ) } \over { \partial ( \ln g ) } } } \right)_n},\quad \kappa _ { \rm{a}}^{\ast } = - { \left({{{\partial ( \ln { i_{\rm{a } } } ) } \over { \partial ( \ln g ) } } } \right)_n},$$\end{document } and measure the sensitivity of the mass ma and moment of inertia ia of each body to changes in the scalar field ( reflected in changes in g ) for a fixed baryon number n ( see tegp 11 , 12 and 14.6 ( c ) for further details ) . notice how the violation of sep in brans - dicke theory introduces complex structure - dependent effects in everything from the newtonian limit ( modification of the effective coupling constant in kepler s third law ) to gravitational radiation . in the limit 0 , we recover gr , and all structure dependence disappears . the first term in b ( see equation ( 84 ) ) is the combined effect of quadrupole and monopole gravitational radiation , while the second term is the effect of dipole radiation . unfortunately , because of the near equality of the neutron star masses in the binary pulsar , dipole radiation is suppressed , and the bounds obtained are not competitive with the cassini bound on , except for those generalized scalar - tensor theories , with 0 < 0 . bounds on the parameters 0 and 0 from solar system , binary pulsar , and gravitational wave observations ( see sections 5.1 and 6.3 ) are found in . alternatively , a binary pulsar system with dissimilar objects , such as a white dwarf or black hole companion , would provide potentially more promising tests of dipole radiation . in this regard , the recently discovered binary pulsar j1141 + 6545 , with an apparent white dwarf companion , may play an important role . here then , from equation ( 84 ) , it is straightforward to show that , if the timing reaches sufficient accuracy to determine b to an accuracy in agreement with the prediction of gr , then the resulting lower bound on bd would be 96\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ { { \rm{bd } } } } > 4 \times { 10 ^ 4}{{s_{{\rm{ns}}}^2 } \over \sigma}.$$\end{document } thus , for sns 0.2 , a 4 percent measurement would already compete with the cassini bound ( for further details , see [ 118 , 102 ] ) . some time in the next decade , a new opportunity for testing relativistic gravity will be realized , when a worldwide network of kilometer - scale , laser interferometric gravitational wave observatories in the u.s . ( ligo project ) , europe ( virgo and geo600 projects ) , and japan ( tama300 project ) begins regular detection and analysis of gravitational wave signals from astrophysical sources . these broad - band antennas will have the capability of detecting and measuring the gravitational waveforms from astronomical sources in a frequency band between about 10 hz ( the seismic noise cutoff ) and 500 hz ( the photon counting noise cutoff ) , with a maximum sensitivity to strain at around 100 hz of h l / l 10 ( rms ) , for the kilometer - scale ligo / virgo projects . the most promising source for detection and study of the gravitational wave signal is the inspiralling compact binary a binary system of neutron stars or black holes ( or one of each ) in the final minutes of a death spiral leading to a violent merger . such is the fate , for example , of the hulse - taylor binary pulsar b1913 + 16 in about 300 myr , or the ( around 2010 ) , which could see such sources out to many hundreds of megaparsecs , it has been estimated that from 40 to several hundred annual inspiral events could be detectable . other sources , such as supernova core collapse events , instabilities in rapidly rotating newborn neutron stars , signals from non - axisymmetric pulsars , and a stochastic background of waves , may be detectable ( for reviews , see [ 1 , 256 ] ; for updates on the status of various projects , see [ 114 , 45 ] ) . a similar network of cryogenic resonant - mass gravitational antennas have been in operation for many years , albeit at lower levels of sensitivity ( h 10 ) . while modest improvements in sensitivity may be expected in the future , these resonant detectors are not expected to be competitive with the large interferometers , unless new designs involving masses of spherical , or nearly spherical shape come to fruition . these systems are primarily sensitive to waves in relatively narrow bands about frequencies in the hundreds to thousands of hz range [ 206 , 123 , 32 , 217 ] , although future improvements in sensitivity and increases in bandwidth may be possible . in addition , plans are being developed for an orbiting laser interferometer space antenna ( lisa for short ) . such a system , consisting of three spacecraft orbiting the sun in a triangular formation separated from each other by five million kilometers , would be sensitive primarily in the very low frequency band between 10 and 10 hz , with peak strain sensitivity of order h 10 . in addition to opening a new astronomical window , the detailed observation of gravitational waves by such observatories may provide the means to test general relativistic predictions for the polarization and speed of the waves , for gravitational radiation damping and for strong - field gravity . a laser interferometric or resonant bar gravitational wave detector whose scale is small compared to the gravitational wavelength measures the local components of a symmetric 3 3 tensor which is composed of the electric components of the riemann curvature tensor , r0i0j , via the equation of geodesic deviation , given , for a pair of freely falling particles by = r0i0jx , where x denotes the spatial separation . in general there are six independent components , which can be expressed in terms of polarizations ( modes with specific transformation properties under rotations and boosts ) . three are transverse to the direction of propagation , with two representing quadrupolar deformations and one representing a monopolar breathing deformation . three modes are longitudinal , with one an axially symmetric stretching mode in the propagation direction , and one quadrupolar mode in each of the two orthogonal planes containing the propagation direction . figure 8 shows the displacements induced on a ring of freely falling test particles by each of these modes . general relativity predicts only the first two transverse quadrupolar modes ( a ) and ( b ) independently of the source ; these correspond to the waveforms h+ and h discussed earlier ( note the cos 2 and sin 2 dependences of the displacements ) . massless scalar - tensor gravitational waves can in addition contain the transverse breathing mode ( c ) . in massive scalar - tensor theories , the longitudinal mode ( d ) can also be present , but is suppressed relative to ( c ) by a factor ( /c ) , where is the wavelength of the radiation , and c is the compton wavelength of the massive scalar . more general metric theories predict additional longitudinal modes , up to the full complement of six ( tegp 10.2 ) . figure 8the six polarization modes for gravitational waves permitted in any metric theory of gravity . there is no displacement out of the plane of the picture . in ( a ) , ( b ) , and ( c ) , the wave propagates out of the plane ; in ( d ) , ( e ) , and ( f ) , the wave propagates in the plane . in gr , only ( a ) and ( b ) are present ; in massless scalar - tensor gravity , ( c ) may also be present . there is no displacement out of the plane of the picture . in ( a ) , ( b ) , and ( c ) , the wave propagates out of the plane ; in ( d ) , ( e ) , and ( f ) , the wave propagates in the plane . in gr , only ( a ) and ( b ) are present ; in massless scalar - tensor gravity , ( c ) may also be present . a suitable array of gravitational antennas could delineate or limit the number of modes present in a given wave . there are eight unknowns ( six polarizations and two direction cosines ) , but only six measurables ( r0i0j ) . if the direction can be established by either association of the waves with optical or other observations , or by time - of - flight measurements between separated detectors , then six suitably oriented detectors suffice to determine all six components . if the direction can not be established , then the system is underdetermined , and no unique solution can be found . however , if one assumes that only transverse waves are present , then there are only three unknowns if the source direction is known , or five unknowns otherwise . then the corresponding number ( three or five ) of detectors can determine the polarization . if distinct evidence were found of any mode other than the two transverse quadrupolar modes of gr , the result would be disastrous for gr . on the other hand , the absence of a breathing mode would not necessarily rule out scalar - tensor gravity , because the strength of that mode depends on the nature of the source . some of the details of implementing such polarization observations have been worked out for arrays of resonant cylindrical , disk - shaped , spherical , and truncated icosahedral detectors ( tegp 10.2 , for recent reviews see [ 169 , 266 ] ) ; initial work has been done to assess whether the ground - based or space - based laser interferometers ( or combinations of the two types ) could perform interesting polarization measurements [ 267 , 47 , 177 , 117 , 273 ] . unfortunately for this purpose , the two ligo observatories ( in washington and louisiana states , respectively ) have been constructed to have their respective arms as parallel as possible , apart from the curvature of the earth ; while this maximizes the joint sensitivity of the two detectors to gravitational waves , it minimizes their ability to detect two modes of polarization . in the binary pulsar , a test of gr was made possible by measuring at least three relativistic effects that depended upon only two unknown masses . the evolution of the orbital phase under the damping effect of gravitational radiation played a crucial role . another situation in which measurement of orbital phase can lead to tests of gr is that of the inspiralling compact binary system . the key differences are that here gravitational radiation itself is the detected signal , rather than radio pulses , and the phase evolution alone carries all the information . in the binary pulsar , the first derivative of the binary frequency b was measured ; here the full nonlinear variation of fb as a function of time is measured . broad - band laser interferometers are especially sensitive to the phase evolution of the gravitational waves , which carry the information about the orbital phase evolution . the analysis of gravitational wave data from such sources will involve some form of matched filtering of the noisy detector output against an ensemble of theoretical template waveforms which depend on the intrinsic parameters of the inspiralling binary , such as the component masses , spins , and so on , and on its inspiral evolution . how accurate must a template be in order to match the waveform from a given source ( where by a match we mean maximizing the cross - correlation or the signal - to - noise ratio ) ? in the total accumulated phase of the wave detected in the sensitive bandwidth , the template must match the signal to a fraction of a cycle . for two inspiralling neutron stars , around 16,000 cycles should be detected during the final few minutes of inspiral ; this implies a phasing accuracy of 10 or better . since 1/10 during the late inspiral , this means that correction terms in the phasing at the level of or higher are needed . more formal analyses confirm this intuition [ 67 , 105 , 68 , 214 ] . because it is a slow - motion system ( 10 ) , the binary pulsar is sensitive only to the lowest - order effects of gravitational radiation as predicted by the quadrupole formula . nevertheless , the first correction terms of order and to the quadrupole formula were calculated as early as 1976 ( see tegp 10.3 ) . but for laser interferometric observations of gravitational waves , the bottom line is that , in order to measure the astrophysical parameters of the source and to test the properties of the gravitational waves , it is necessary to derive the gravitational waveform and the resulting radiation back - reaction on the orbit phasing at least to 2pn order beyond the quadrupole approximation , and preferably to 3pn order . for the special case of non - spinning bodies moving on quasi - circular orbits ( i.e. circular apart from a slow inspiral ) , the evolution of the gravitational wave frequency f = 2fb through 2pn order has the form 97\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 } { \dot f = { { 96\pi } \over 5}{f^2}{{(\pi { \mathcal m}f)}^{5/3}}\left [ { 1 - \left({{{743 } \over { 336 } } + { { 11 } \over 4}\eta } \right){{(\pi mf)}^{2/3 } } + 4\pi ( \pi mf ) } \right.}\\ { \left . { \quad \quad \quad \quad \quad \quad \quad \quad \quad + \left({{{34103 } \over { 18144 } } + { { 13661 } \over { 2016}}\eta + { { 59 } \over { 18}}{\eta ^2 } } \right){{(\pi mf)}^{4/3 } } + { \mathcal o}[{{(\pi mf)}^{5/3 } } ] } \right],}\\ \end{array}$$\end{document } where = m1m2/m . the first term is the quadrupole contribution ( compare equation ( 72 ) ) , the second term is the 1pn contribution , the third term , with the coefficient 4 , is the tail contribution , and the fourth term is the 2pn contribution , first reported jointly by blanchet et al . the 2.5pn , 3pn and 3.5pn contributions have also been calculated ( see for a review ) . expressions for non - circular orbits have also been derived [ 121 , 75 ] . these losses react back on the orbit to circularize it and cause it to inspiral . the result is that the orbital phase ( and consequently the gravitational wave phase ) evolves non - linearly with time . it is the sensitivity of the broadband laser interferometric detectors to phase that makes the higher - order contributions to df / dt so observationally relevant . if the coefficients of each of the powers of f in equation ( 97 ) can be measured , then one again obtains more than two constraints on the two unknowns m1 and m2 , leading to the possibility to test gr . for example , blanchet and sathyaprakash [ 42 , 41 ] have shown that , by observing a source with a sufficiently strong signal , an interesting test of the 4 coefficient of the tail term could be performed . another possibility involves gravitational waves from a small mass orbiting and inspiralling into a ( possibly supermassive ) spinning black hole . a general non - circular , non - equatorial orbit will precess around the hole , both in periastron and in orbital plane , leading to a complex gravitational waveform that carries information about the non - spherical , strong - field spacetime around the hole . according to gr , this spacetime must be the kerr spacetime of a rotating black hole , uniquely specified by its mass and angular momentum , and consequently , observation of the waves could test this fundamental hypothesis of gr [ 231 , 213 ] . thirdly , the dipole gravitational radiation predicted by scalar - tensor theories will result in a modification of the gravitational radiation back - reaction , and thereby of the phase evolution . including only the leading quadrupole and dipole contributions , one obtains , in brans - dicke theory , 98\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot f = { { 96\pi } \over 5}{f^2}{(\pi { \mathcal m}f)^{5/3}}\left [ { 1 + b{{(\pi mf)}^{- 2/3 } } } \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}${\mathcal m } = ( { \chi ^{3/5}}{{\mathcal g}^{- 4/5}}){\eta ^{3/5}}m$\end{document } , and b is the coefficient of the dipole term , given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b = ( 5/48)({\chi ^{- 1}}{{\mathcal g}^{4/3}})\xi { { \mathcal s}^2}$\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 g},{\mathcal s}$\end{document } are given by equations ( 94 ) , and = 1/(2 + bd ) . double neutron star systems are not promising because the small range of masses available near 1.4 m results in suppression of dipole radiation by symmetry . for black holes , s = 0.5 identically , consequently double black hole systems turn out to be observationally identical in the two theories . however , a number of analyses of the capabilities of both ground - based and space - based ( lisa ) observatories have shown that observing waves from neutron - star - black - hole inspirals is not likely to bound scalar - tensor gravity at a level competitive with the cassini bound or with future solar - system improvements [ 283 , 161 , 236 , 292 , 27 , 28 ] . according to gr , in the limit in which the wavelength of gravitational waves is small compared to the radius of curvature of the background spacetime , the waves propagate along null geodesics of the background spacetime , i.e. they have the same speed c as light ( in this section , we do not set c = 1 ) . in other theories , the speed could differ from c because of coupling of gravitation to background gravitational fields . for example , in the rosen bimetric theory with a flat background metric , gravitational waves follow null geodesics of , while light follows null geodesics of g ( tegp 10.1 ) . another way in which the speed of gravitational waves could differ from c is if gravitation were propagated by a massive field ( a massive graviton ) , in which case g would be given by , in a local inertial frame , 99\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{v_{\rm{g}}^2 } \over { { c^2 } } } = 1 - { { m_{\rm{g}}^2{c^4 } } \over { { e^2}}},$$\end{document } where mg and e are the graviton rest mass and energy , respectively . the simplest attempt to incorporate a massive graviton into general relativity in a ghost - free manner suffers from the so - called van dam - veltman - zakharov ( vdvz ) discontinuity [ 263 , 299 ] . because of the 3 additional helicity states available to the massive spin-2 graviton , the limit of small graviton mass does not coincide with pure gr , and the predicted perihelion advance , for example , violates experiment . a model theory by visser attempts to circumvent the vdvz problem by introducing a non - dynamical flat - background metric . this theory is truly continuous with gr in the limit of vanishing graviton mass ; on the other hand , its observational implications have been only partially explored . braneworld scenarios predict a tower or a continuum of massive gravitons , and may avoid the vdvz discontinuity , although the full details are still a work in progress [ 91 , 66 ] . the most obvious way to test this is to compare the arrival times of a gravitational wave and an electromagnetic wave from the same event , e.g. , a supernova . for a source at a distance d , the resulting value of the difference 1 g / c is 100\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$1 - { { { v_{\rm{g } } } } \over c } = 5 \times { 10^{- 17}}\left({{{200\;{{\rm{m}}_{{\rm{pc } } } } } \over d } } \right)\left({{{\delta t } \over { 1\;{\rm{s } } } } } \right),$$\end{document } where t = ta ( 1 + z)te is the time difference , where ta and te are the differences in arrival time and emission time of the two signals , respectively , and z is the redshift of the source . in many cases , te is unknown , so that the best one can do is employ an upper bound on te based on observation or modelling . the result will then be a bound on 1 g / c . for a massive graviton , if the frequency of the gravitational waves is such that hf mgc , where h is planck s constant , then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\upsilon _ g}/c \approx 1 - { 1 \over 2}{(c/{\lambda _ g}f)^2}$\end{document } , where g = h / mgc is the graviton compton wavelength , and the bound on 1 g / c can be converted to a bound on g , given by 101\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda _ { \rm{g } } } > 3 \times { 10^{12}}{\rm{km}}{\left({{d \over { 200\;{{\rm{m}}_{{\rm{pc}}}}}}{{100\;{\rm{hz } } } \over f } } \right)^{1/2}}{\left({{1 \over { f\delta t } } } \right)^{1/2}}.$$\end{document } the foregoing discussion assumes that the source emits both gravitational and electromagnetic radiation in detectable amounts , and that the relative time of emission can be established to sufficient accuracy , or can be shown to be sufficiently small . however , there is a situation in which a bound on the graviton mass can be set using gravitational radiation alone . that is the case of the inspiralling compact binary . because the frequency of the gravitational radiation sweeps from low frequency at the initial moment of observation to higher frequency at the final moment , the speed of the gravitons emitted will vary , from lower speeds initially to higher speeds ( closer to c ) at the end this will cause a distortion of the observed phasing of the waves and result in a shorter than expected overall time ta of passage of a given number of cycles . furthermore , through the technique of matched filtering , the parameters of the compact binary can be measured accurately ( assuming that gr is a good approximation to the orbital evolution , even in the presence of a massive graviton ) , and thereby the emission time te can be determined accurately . roughly speaking , the phase interval ft in equation ( 101 ) can be measured to an accuracy 1/ , where is the signal - to - noise ratio . thus one can estimate the bounds on g achievable for various compact inspiral systems , and for various detectors . for stellar - mass inspiral ( neutron stars or black holes ) observed by the ligo / virgo class of ground - based interferometers , d 200 mpc , f 100 hz , and f t 1/10 . the result is g > 10 km . for supermassive binary black holes ( 10 to 10 m ) observed by the proposed laser interferometer space antenna ( lisa ) , d 3 gpc , f 10 hz , and f t 1/1000 . a full noise analysis using proposed noise curves for the advanced ligo and for lisa weakens these crude bounds by factors between two and 10 [ 285 , 292 , 27 , 28 ] . for example , for the inspiral of two 10 m black holes with aligned spins at a distance of 3 gpc observed by lisa , a bound of 2 10 km could be placed . other possibilities include using binary pulsar data to bound modifications of gravitational radiation damping by a massive graviton , and using lisa observations of the phasing of waves from compact white - dwarf binaries , eccentric galactic binaries , and eccentric inspiral binaries [ 69 , 142 ] . bounds obtainable from gravitational radiation effects should be compared with the solid bound g > 2.8 10 km derived from solar system dynamics , which limit the presence of a yukawa modification of newtonian gravity of the form 102\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v(r ) = { { gm } \over r}\exp ( - r/{\lambda _ { \rm{g}}}),$$\end{document } and with the model - dependent bound g > 6 10 km from consideration of galactic and cluster dynamics . one of the central difficulties of testing gr in the strong - field regime is the possibility of contamination by uncertain or complex physics . in the solar system , weak - field gravitational effects could in most cases be measured cleanly and separately from non - gravitational effects . the remarkable cleanliness of the binary pulsar permitted precise measurements of gravitational phenomena in a strong - field context . unfortunately , nature is rarely so kind . still , under suitable conditions , qualitative and even quantitative strong - field tests of gr could be carried out . one example is in cosmology . from a few seconds after the big bang until the present , the underlying physics of the universe is well understood , in terms of a standard model of a nearly spatially flat universe , 13.6 gyr old , dominated by dark matter and dark energy . some alternative theories of gravity that are qualitatively different from gr fail to produce cosmologies that meet even the minimum requirements of agreeing qualitatively with big - bang nucleosynthesis ( bbn ) or the properties of the cosmic microwave background ( tegp 13.2 ) . others , such as brans - dicke theory , are sufficiently close to gr ( for large enough bd ) that they conform to all cosmological observations , given the underlying uncertainties . the generalized scalar - tensor theories , however , could have small at early times , while evolving through the attractor mechanism to large today . one way to test such theories is through big - bang nucleosynthesis , since the abundances of the light elements produced when the temperature of the universe was about 1 mev are sensitive to the rate of expansion at that epoch , which in turn depends on the strength of interaction between geometry and the scalar field . because the universe is radiation - dominated at that epoch , uncertainties in the amount of cold dark matter or of the cosmological constant are unimportant . the nuclear reaction rates are reasonably well understood from laboratory experiments and theory , and the number of light neutrino families ( 3 ) conforms to evidence from particle accelerators . thus , within modest uncertainties , one can assess the quantitative difference between the bbn predictions of gr and scalar - tensor gravity under strong - field conditions and compare with observations . another example is the exploration of the spacetime near black holes and neutron stars via accreting matter . studies of certain kinds of accretion known as advection - dominated accretion flow ( adaf ) in low - luminosity binary x - ray sources may yield the signature of the black hole event horizon . the spectrum of frequencies of quasi - periodic oscillations ( qpo ) from galactic black hole binaries may permit measurement of the spins of the black holes . aspects of strong - field gravity and frame - dragging may be revealed in spectral shapes of iron fluorescence lines from the inner regions of accretion disks [ 225 , 224 ] . because of uncertainties in the detailed models , the results to date of studies like these are suggestive at best , but the combination of higher - resolution observations and better modelling could lead to striking tests of strong - field predictions of gr . one reason is that gravity is a fundamental interaction of nature , and as such requires the most solid empirical underpinning we can provide . another is that all attempts to quantize gravity and to unify it with the other forces suggest that the standard general relativity of einstein is not likely to be the last word . furthermore , the predictions of general relativity are fixed ; the theory contains no adjustable constants so nothing can be changed . thus every test of the theory is either a potentially deadly test or a possible probe for new physics . although it is remarkable that this theory , born 90 years ago out of almost pure thought , has managed to survive every test , the possibility of finding a discrepancy will continue to drive experiments for years to come .

the status of experimental tests of general relativity and of theoretical frameworks for analyzing them is reviewed . einstein s equivalence principle ( eep ) is well supported by experiments such as the etvs experiment , tests of special relativity , and the gravitational redshift experiment . ongoing tests of eep and of the inverse square law are searching for new interactions arising from unification or quantum gravity . tests of general relativity at the post - newtonian level have reached high precision , including the light deflection , the shapiro time delay , the perihelion advance of mercury , and the nordtvedt effect in lunar motion . gravitational wave damping has been detected in an amount that agrees with general relativity to better than half a percent using the hulse - taylor binary pulsar , and other binary pulsar systems have yielded other tests , especially of strong - field effects . when direct observation of gravitational radiation from astrophysical sources begins , new tests of general relativity will be possible .

Introduction Tests of the Foundations of Gravitation Theory Tests of Post-Newtonian Gravity Strong Gravity and Gravitational Waves: A New Testing Ground Stellar System Tests of Gravitational Theory Gravitational Wave Tests of Gravitational Theory Conclusions

einstein did calculate observable effects of general relativity , such as the perihelion advance of mercury , which he knew to be an unsolved problem , and the deflection of light , which was subsequently verified . the genesis ( 18871919 ) comprises the period of the two great experiments which were the foundation of relativistic physics the michelson - morley experiment and the etvs experiment and the two immediate confirmations of gr the deflection of light and the perihelion advance of mercury . examples include the use of laser - cooled atom and ion traps to perform ultra - precise tests of special relativity ; the proposal of a fifth force , which led to a host of new tests of the weak equivalence principle ; and recent ideas of large extra dimensions , which have motived new tests of the inverse square law of gravity at sub - millimeter scales . although special relativity itself never benefited from the kind of crucial experiments , such as the perihelion advance of mercury and the deflection of light , that contributed so much to the initial acceptance of gr and to the fame of einstein , the steady accumulation of experimental support , together with the successful merger of special relativity with quantum mechanics , led to its being accepted by mainstream physicists by the late 1920s , ultimately to become part of the standard toolkit of every working physicist . at the same time , tests of the inverse - square law of gravity were carried out by comparing variations in gravity measurements up tall towers or down mines or boreholes with gravity variations predicted using the inverse square law together with earth models and surface gravity data mathematically continued up the tower or down the hole . an example is rosen s bimetric theory.a large number of alternative theories of gravity predict gravitational wave emission substantially different from that of general relativity , in strong disagreement with observations of the binary pulsar ( see section 7).scalar - tensor modifications of gr have become very popular in unification schemes such as string theory , and in cosmological model building . in more general theories , the function ( ) could have the property that , at the present epoch , and in weak - field situations , the value of the scalar field 0 is such that is very large and is very small ( theory almost identical to gr today ) , but that for past or future values of , or in strong - field regions such as the interiors of neutron stars , and could take on values that would lead to significant differences from gr . nevertheless , under some circumstances , the orbital motion may be such that the interbody potential and orbital velocities still satisfy 1 so that a kind of post - newtonian approximation for the orbital motion might work ; however , the strong - field internal gravity of the bodies could ( especially in alternative theories of gravity ) leave imprints on the orbital motion.the evolution of the system may be affected by the emission of gravitational radiation . the 1974 discovery of the binary pulsar b1913 + 16 by joseph taylor and russell hulse during a routine search for new pulsars provided the first possibility of probing new aspects of gravitational theory : the effects of strong relativistic internal gravitational fields on orbital dynamics , and the effects of gravitational radiation reaction . the five post - keplerian parameters are : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\langle \dot \omega \rangle$\end{document } , the average rate of periastron advance ; , the amplitude of delays in arrival of pulses caused by the varying effects of the gravitational redshift and time dilation as the pulsar moves in its elliptical orbit at varying distances from the companion and with varying speeds ; b , the rate of change of orbital period , caused predominantly by gravitational radiation damping ; and r and s = sin i , respectively the range and shape of the shapiro time delay of the pulsar signal as it propagates through the curved spacetime region near the companion , where i is the angle of inclination of the orbit relative to the plane of the sky . in addition to opening a new astronomical window , the detailed observation of gravitational waves by such observatories may provide the means to test general relativistic predictions for the polarization and speed of the waves , for gravitational radiation damping and for strong - field gravity .

early cell membranes are thought to have been composed of fatty acids and related single - chain amphiphiles , in contrast to the phospholipid - based membranes of all modern cells . initial support for this hypothesis arose from the facile prebiotic synthesis of these molecules and the ability of fatty acids to spontaneously assemble into bilayer vesicles . fatty acids and other oxygenated alkanes can be synthesized via fischer tropsch - type chemistry , and membrane - forming samples of these molecules have been discovered in abiotic environments such as meteorites . more recently , the functional properties of fatty acid membranes have been studied and are consistent with the necessity for early cell membranes , prior to the evolution of transport machinery , to be permeable to polar nutrients . in addition , fatty acid vesicles have a striking ability to undergo intervesicle competition through exchange of monomers . these dynamic processes depend upon the rapid exchange of single - chain amphiphiles between membranes and the surrounding solution . the importance of these exchange processes motivated us to investigate the structural composition of fatty acid vesicle solutions . fatty acid membranes are only stable within a narrow ph range , from neutral to moderately alkaline ( ph 79 , depending on chain length ) , near the apparent pka of the fatty acid within the bilayer . this condition allows approximately equal proportions of protonated and ionized carboxylates to coexist , forming a bilayer - stabilizing hydrogen bonding network . under more alkaline conditions , fatty acids are fully ionized and aggregate into small soap micelles , as a result of the charge repulsion of the anionic head groups . under acidic conditions , fatty acids become fully protonated , lose their amphiphilicity , and condense into oil droplets . this ph dependence of fatty acid phase behavior has been extensively characterized by nmr , x - ray diffraction , and electron spin resonance ( esr ) . subsequent work has utilized the ph dependence of fatty acid aggregation to drive the de novo assembly of vesicles from micelles or the growth of pre - existing vesicles by introducing alkaline micelles into buffered suspensions of vesicles . supra - molecular self - assembly is intrinsically concentration dependent because of the entropic cost of aggregation . detergent solutions , for example , feature a critical micelle concentration ( cmc ) , below which only monomers are found and above which aggregation occurs . such self - assembly processes can be described as pseudophase equilibria , with critical concentrations being analogous to solubilities . critical aggregation concentrations ( cac ) have been observed for fatty acid vesicles , suggesting that monomers coexist with vesicles above the cac . because of their large size ( n > 10 ) , membrane vesicles have a higher entropic cost of formation than smaller ( n 50 ) micellar aggregates . we therefore asked if monomers , micelles , and vesicles could all coexist under certain conditions and whether the composition of the aggregate phase could be concentration dependent , with lower concentration solutions favoring micelles and higher concentrations favoring vesicles . these questions are of particular interest with regard to prebiotic scenarios , where membrane assembly may have frequently occurred in relatively dilute solutions of fatty acids , near the cac . to explore multiphase coexistence , we sought methods to quantitatively characterize the equilibrium between fatty acid monomers , micelles , and vesicles at low concentrations . we focused on a set of monounsaturated fatty acids , which serve as convenient laboratory models for the short - chain , saturated lipids expected to result from prebiotic synthesis . because of the techniques used , previous studies could only examine fatty acid aggregation behavior at concentrations an order of magnitude or more above the apparent cac . we distinguished between different aggregate phases using the fluorescent fatty acid analogue laurdan ( 6-dodecanoyl-2-dimethylaminonaphthalene ) , which undergoes an emission red shift with increasing solvent polarity . laurdan has been used extensively to study structural features of membranes , e.g. , lipid packing , membrane bending , and phase segregation . since micelles feature greater headgroup solvation than more tightly packed bilayers , we predicted that laurdan would be a sensitive means of distinguishing these two aggregate states . we used this assay alongside surface tension measurements , which can quantify monomer concentrations , to characterize the equilibrium between these states . our data support a micelle vesicle equilibrium above the cac in which dilute solutions are relatively enriched in micelles . we then used this multiphase coexistence to drive the growth of fatty acid vesicles by evaporative concentration , a process with potential prebiotic relevance to the growth of early cell membranes . we first characterized the fatty acid monomer to micelle phase transition by measuring solution surface tension at ph 10.5 , in which fatty acids aggregate into soap micelles . increasing concentrations of surfactant ( e.g. , fatty acid ) monomer reduce the effective surface tension of an interface ( e.g. , air water ) , but aggregates ( e.g. , micelles ) do not . we measured surface tensions of serial dilutions for a series of unsaturated fatty acids ranging from 14 to 18 carbons . from these surface tension plots ( figure s1 , supporting information ) , we calculated solution monomer concentrations by fitting to the szyszkowski equation , which relates surface tension to bulk concentration ( materials and methods for details ) . pseudophase equilibria feature abrupt transition points at the critical concentration , above which the monomer concentration stays constant and all additional lipids are incorporated into aggregates . as expected , this was the case for a series of unsaturated fatty acids at high ph , where micelles are the only aggregates that can form ( figure 1a ) . the critical micelle concentrations ( cmc s ) of these fatty acids scaled exponentially with chain length , a result of the linear dependence of the free energy of solvation on chain length via the hydrophobic effect . fatty acid monomer concentrations as a function of total concentration for a series of monounsaturated fatty acids at ph 10.5 ( a ) and 8.5 ( b ) . monomer concentrations were derived from surface tension plots since aggregates are not surface active . the plateau points in ( a ) correspond to critical micelle concentrations ( ma , 15 mm ; pa , 4 mm ; oa , 1 mm , in agreement with previous measurements ) . plateau points in ( b ) indicate critical aggregation concentrations ( ma , 2 mm ; pa , 0.2 mm ; oa , < 0.1 mm ) . ma , myristoleate ( c14:1 ) ; pa , palmitoleate ( c16:1 ) ; oa , oleate ( c18:1 ) . we then repeated the above experiments at ph 8.5 , where vesicles are expected to form . monomer concentrations in fatty acid solutions at this lower ph ( figure 1b ) also plateaued at critical concentrations , but not as abruptly as at ph 10.5 . this was somewhat surprising because vesicles contain very large numbers of monomers , and vesicle formation should therefore more closely resemble a pure phase transition . we reasoned that this effect could be due to fatty acids aggregating into multiples states , e.g. , vesicles and micelles , above the critical concentration . we also considered the alternative possibility that micelle aggregation occurred at a lower concentration than vesicle assembly at ph 8.5 ; i.e. , the system had two critical concentrations , as has been observed in cationic / anionic surfactant mixtures . however , when we used light scattering , which detects vesicle assembly but not the formation of much smaller micelles , the cac s we observed ( figure s2 , supporting information ) were at the same concentration as , or even slightly lower than , the critical concentrations obtained from surface tension plots . we therefore conclude that monomers do not aggregate into micelles at concentrations below that at which vesicle assembly occurs . these initial experiments motivated us to find experimental techniques that would allow us to detect and measure both micellar and lamellar aggregates in the same experiment . this is a challenge because of the large size difference between micelles and vesicles ( precluding microscopy or light scattering ) , the rapid exchange between these states ( precluding any sort of physical separation ) , and their similar internal chemical environments ( precluding dyes sensitive to nonpolar environments ) . laurdan is a c12 fatty acid analogue with a fluorescent naphthalene derivative that features an emission spectrum that is sensitive to the polarity of its environment . previous work in our laboratory had used laurdan to monitor structural changes during fatty acid membrane bending , and so we reasoned that it would also be sensitive to larger changes in aggregate structure ( figure s3 , supporting information ) . we observed a characteristic change in laurdan emission intensities when incubated as a minor component ( 1:400 ) in fatty acid micelles as compared to vesicles ( figure 2a ) . this is explained by the high curvature of the micelle surface , which results in increased water penetration compared to bilayers . we quantified this spectral shift using a unitless generalized polarization ( gp ) parameterwhere i430 and i500 are the emission intensities ( excitation 364 nm ) at 430 and 500 nm , respectively . we note that our expression for gp is inverted in sign from that generally used ( for phospholipid membranes ) due to the altered spectra of laurdan in fatty acid aggregates . in this form , larger gp values indicate a more solvated state of the dye , e.g. , as expected from micellar vs lamellar packing . ( a ) emission spectrum for 25 m laurdan ( excitation 364 nm ) in 10 mm oleate at ph 8.5 ( vesicles ) or ph 10.5 ( micelles ) . ( b ) dependence of laurdan gp on ph in 10 mm oleate with ( open squares ) or without ( closed circles ) 1% v / v triton x100 , which disrupts fatty acid aggregates . gp increased monotonically with ph until it plateaued above ph 10 ( figure 2b ) . this was consistent with a ph - dependent change from a lamellar to micellar phase , with intermediate values ( e.g. , at ph 9 ) reflecting coexisting vesicles and micelles . these ph - dependent changes in gp were not observed in the presence of detergent ( triton x100 ) , which disrupts all fatty acid aggregates . changes in laurdan gp thus were not caused by the ph change per se but rather by the structure of the resulting fatty acid aggregate . gp was also notably insensitive to vesicle radius and thus mean curvature , in extruded samples ( figure s4 , supporting information ) . this was consistent with our previous results on bending relaxation in fatty acid vesicles . we then asked if the laurdan gp is dependent on the concentration of the fatty acid solution . at concentrations below the aggregation concentrations , laurdan emission intensity and gp were low , likely reflecting the insolubility of the dye in the absence of hydrophobic aggregates ( figure s5 , supporting information ) . for solutions at ph 10.5 , gp remained constant with regard to concentration above the cmc for all three fatty acids tested ( figure 3a ) . therefore , the micelle aggregate is structurally consistent over this concentration range , though it is likely heterogeneous in nature . in contrast , solutions at ph 8.5 showed a dramatic dependence of gp on concentration ( figure 3b ) . concentrations just above the cac had a gp close to that for micelles , which decreased as the concentration increased , eventually plateauing at high concentrations . we interpreted this data to indicate a concentration dependence of the fatty acid aggregation state , with micelles favored in low concentration solutions . we also observed this effect in oleate solutions at ph 9.2 , with gp plateauing to an intermediate value reflecting a roughly equal mixture of micelles and vesicles ( figure 3c ) . ( a ) gp as a function of concentration for monounsaturated fatty acids at ph 10.5 . ( b ) gp as a function of concentration for monounsaturated fatty acids at ph 8.5 . gp drops monotonically once above the cac , reflecting a change in the aggregate composition . ( c ) gp as a function of concentration in oleate at ph 9.2 . dotted lines representing equivalent curves for ph 10.5 ( from a ) and 8.5 ( from b ) are provided for reference . assuming that laurdan partitions representatively between micelles and vesicles , its emission in a solution can be modeled as a weighted average between its characteristic micelle and vesicle emissions ( materials and methods ) . using this approach , we approximated the micelle to vesicle partition coefficient as a function of concentration in the systems tested ( figure 4 ) . these are relative partition coefficients with respect to the reference vesicle solutions at 30 or 50 mm and are thus expressed as apparent xm / xv , where xm and xv are the micelle and vesicle fractions , respectively . partition coefficients are calculated by equating measured emission intensities to weighted averages between reference vesicle and micelle solutions . partition coefficients are given as a function of concentration in vesicle solutions at ph 8.5 or 9.2 and show that low concentration solutions are enriched in micellar aggregates . we tested the concentration dependence of the micelle to vesicle ratio independently by measuring the turbidity of vesicle solutions that had been extruded to 50 nm to eliminate spurious effects due to variation in vesicle size . phospholipid ( dimyristoleoyl phosphocholine ) solutions , which only form vesicles , exhibited a linear increase in turbidity with concentration , corresponding to the expected linear increase in vesicle concentration ( figure 5a , black ) . in contrast , the absorbance of myristoleate solutions increased nonlinearly above the cac , with more dilute solutions depleted in vesicles ( figure 5a , green ) . from these absorbance values , we calculated apparent micelle to vesicle partition coefficients , assuming that all fatty acids not in vesicles were in the form of micelles ( materials and methods ) . these values corresponded well with the partition coefficients derived from laurdan measurements ( figure 5b ) . ( a ) turbidity of myristoleate solutions at ph 8.5 extruded to 50 nm ( green , left axis ) . dashed line is the expected absorbance if the vesicle concentration scaled linearly with myristoleate concentration , relative to the absorbance at 50 mm . in contrast , the turbidity of 50 nm phospholipid ( dimyristoleoyl phosphocholine , pc ) vesicles scales linearly with concentration ( black , right axis ) . ( b ) apparent micelle to vesicle partition coefficients for myristoleate at ph 8.5 as derived from absorbance readings ( green circles ) and from laurdan gp ( blue squares ) . a fitted single exponential decay ( k = 0.12 mm ) is shown and used to predict growth in figure 8 . our characterization of fatty acid phase behavior demonstrates that fatty acid incorporation into vesicles vs micelles increases with both increasing concentration and decreasing ph . the addition of alkaline micelles to buffered vesicles we therefore hypothesized that the concentration dependence of the micelle vesicle equilibrium could provide an alternative mechanism for the growth of pre - existing fatty acid vesicles . in this scenario , a rise in amphiphile concentration would cause the incorporation of excess micellar fatty acids into vesicles , and dilution would drive vesicle shrinkage ( as material leaves the lamellar phase and reforms micelles ) . we tested this possibility by monitoring changes in the membrane area of 100 nm vesicles using a frster resonance energy transfer ( fret ) growth assay . this assay quantitatively relates changes in fret to changes in dye concentrations and therefore membrane surface area ( materials and methods ) . upon dilution from 10 to 5 mm , we observed rapid shrinkage of 100 nm myristoleate vesicles ( figure 6 ) . dilution was performed with buffer containing 2 mm myristoleate , at the cac , so shrinkage was not due to general aggregate dissolution . this shrinkage was subsequently reversed by the addition of preformed 20 mm myristoleate vesicles , to raise the total myristoleate concentration back to 10 mm . we were thus able to demonstrate a full cycle of growth and shrinkage by modulating the fatty acid concentration in the solution . myristoleate vesicles , initially at 10 mm , shrink in surface area upon dilution to 5 mm . surface area grows back to near the initial value upon concentration via the addition of preformed vesicles and subsequently shrinks upon further dilution . changes in membrane area are tracked by fret between donor and acceptor phospholipids , which remain in the vesicles due to their insolubility . the rapid growth of large , multilamellar vesicles following the addition of excess micelles results in the transformation of initially spherical vesicles into extended filamentous vesicles . this pathway provides a straightforward route for protocell division due to the intrinsic fragility of filamentous vesicles , which break up into daughter vesicles in response to mild shear forces or photochemically induced pearling . we therefore asked whether concentration - driven vesicle growth is robust enough to drive the same filamentous shape transition . to test this possibility , we prepared large ( 4 um ) myristoleate vesicles by large pore extrusion and dialysis at a concentration of 5 mm . the initially spherical vesicles were brought to a concentration of 15 mm via the addition of preformed myristoleate vesicles and within 30 min had grown into long , thin filamentous vesicles ( figure 7 ) . the shape transition occurs because volume growth is osmotically limited by solute ( buffer ) permeation , geometrically necessitating high surface area morphologies . vesicles were labeled with a soluble fluorescent dye , which stayed encapsulated during the entire experiment . multilamellar ( 4 m ) myristoleate vesicles , initially mostly spherical ( top ) , grow into long , filamentous vesicles upon addition of concentrated preformed vesicles ( bottom ) . filamentous growth occurs because of the osmotically limited increase in vesicle volume and is similar to growth seen upon addition of alkaline micelles . the simplest prebiotic mechanism for increasing lipid concentration would be through solution evaporation . we therefore asked whether gentle evaporation would drive the growth of fatty acid vesicles as a result of the transfer of material from coexisting micelles into the preformed vesicles as the fatty acid concentration increased ( figure 8) . solutions of 100 nm myristoleate vesicles , initially at a concentration of 5 mm , were allowed to evaporate at 35 c with gentle agitation . membrane area was monitored by fret at discrete time points and approximately doubled over 24 h as the lipid concentration rose to 10 mm . this growth was similar in magnitude to that predicted ( dashed line ) from the previously measured apparent micelle vesicle partition coefficients ( figure 5b ; materials and methods ) and thus was consistent with our model for concentration - driven growth . growth was not observed for phospholipid vesicles , which do not feature a measurable coexisting solution phase of micelles or monomers and thus were not predicted to change in membrane area upon concentration . myristoleate ( ma ) vesicles , initially at 5 mm lipid concentration , were concentrated by gentle evaporation ( materials and methods ) and changes in surface area monitored by fret at time points of 3 , 10 , and 24 h. data are shown for two independent experiments ( solid circles , squares ) and are in agreement with that predicted from measured apparent micelle vesicle partition coefficients ( dashed line ) . an identical experiment with dimyristoleoyl phosphocholine ( pc ) we have used a combination of physical and spectroscopic assays to characterize the phase behavior of fatty acid solutions to better understand models for prebiotic membrane assembly and function . in the course of these experiments , we found laurdan to be a particularly useful fluorescent probe due to its ability to differentiate between fatty acid micelles and vesicles . this assay was complementary to standard approaches for measuring fatty acid monomer concentration ( via surface tension ) and vesicle concentration ( via light scattering ) . at a given temperature and pressure , there are two determinants of fatty acid phase behavior in our system : ph , which controls headgroup ionization , and concentration , which entropically regulates aggregate size . laurdan gp increases monotonically from ph 8.5 to ph 10 , which reflects the previously identified transition from vesicle to micelle aggregates as the fatty acids become fully ionized and thus favor a high curvature geometry . more surprisingly , we also found that gp at ph 8.5 decreases with concentration above the cac . vesicle equilibrium , with lower concentrations favoring the smaller micellar aggregates and higher concentrations favoring the much larger vesicle aggregates . this behavior is independent of the concentration - dependent transition from monomers to aggregates ( vesicles or micelles ) , which largely follows a pseudophase equilibrium that is characteristic of surfactant aggregation . our model for fatty acid aggregation solutions feature a pseudophase separation from monomers to aggregates , characterized by a cac ( dashed line ) that is dependent on ph . in addition , vesicle solutions feature a concentration - dependent vesicle micelle equilibrium , with higher concentrations favoring the larger vesicle aggregates . in contrast , alkaline solutions exhibit a single sharp pseudophase transition at the cmc . both ph and concentration - driven phase transitions can drive fatty acid vesicle growth . the predominance of micelles at lower concentrations can be rationalized entropically since micelles are much smaller than vesicles . the greater magnitude of this effect with shorter chain length lipids ( e.g. , myristoleate vs oleate ) supports this hypothesis : micelle aggregation number has a strong dependence on chain length , so shorter chain length lipids assemble into smaller micelles . we therefore expect this phenomenon to be broadly applicable to shorter , saturated single - chain lipids , which are the primary product of abiotic lipid synthesis . while the high working concentrations of such species precluded the quantitative fluorescence - based analysis introduced here , previous esr experiments have shown similar micelle vesicle coexistence in a decanoic acid ( c10 ) system . on the basis of the results described above , we present a potential scenario in which environmental fluctuations could drive repeated cycles of protocell growth and division . we begin by considering a small warm pond , containing dilute fatty acids and perhaps other single - chain amphiphiles , along with other organic compounds . we assume that fatty acids were present at a concentration sufficient to lead to the assembly of micelles and vesicles . evaporation , driven by solar or geothermal heat and wind , would lead to progressive concentration of the dissolved solutes and thus to vesicle growth as material in micelles redistributed into the pre - existing vesicles . if the increase in surface area caused by membrane growth occurred faster than the increase in vesicle volume , as could happen in the presence of slowly permeating solutes such as nucleotides , amino acids , and peptides , growth would result in the formation of fragile filamentous vesicles . our laboratory has previously demonstrated that such vesicles fragment easily in response to gentle shear forces , resulting in division into daughter vesicles . alternatively , photochemically induced membrane tension can drive vesicle division through a pearling instability , similarly resulting in daughter vesicles . after a cycle of growth and division , an influx of fresh water , for example , as a result of rainfall , would dilute the pond water , restoring initial concentrations . rapid mixing of fresh water with concentrated pond water would probably result in dissolution of many vesicles , while slower mixing would cause vesicle shrinkage ; both processes would increase the fraction of fatty acids present as micelles . vesicle division following growth into filamentous morphologies could also retard shrinkage , as membrane loss would be favored from undivided vesicles , which are characterized by excess membrane area . surviving vesicles would then be poised for another cycle of growth , driven by evaporation , and division , induced either by wave - induced shear forces or photochemically . although this model is quite speculative , it has the advantage that cycles of growth and division would be driven entirely by environmental fluctuations and could continue indefinitely in the absence of any additional input of fatty acids . if such a cycle could be coupled to the replication of encapsulated nucleic acids , the stage would be set for the emergence of darwinian evolution through the competitive advantage conferred by functional nucleic acids ( e.g. , ribozymes ) . the subsequent evolution of catalytic mechanisms to drive membrane growth , such as the assembly of double - chain lipids , would have eventually freed early cells from depending on environmental fluctuations to drive their cell cycle . fatty acids were obtained from nu - chek and phospholipids from avanti polar lipids . laurdan , nbd - pe ( n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine ) , and rhodamine - dhpe ( rhodamine b 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine ) were obtained from invitrogen . fatty acid vesicles were prepared by mixing the fatty acids ( as neat oil ) in 0.2 m bicine buffer titrated with naoh to ph 8.5 , unless otherwise noted . micelle solutions were prepared by dissolving the fatty acid in water and titrating with sodium hydroxide to ph 10.5 , unless otherwise noted . laurdan was incorporated into the solutions as a concentrated stock in ethanol either before or after addition of buffer . fret dyes were incorporated into fatty acid solutions by addition in chloroform to the neat oil , followed by rotary evaporation . large , multilamellar vesicles used for imaging were prepared with 2 mm 8-hydroxypyrene-1,3,6-trisulfonic acid ( hpts ) , a water - soluble dye , in the buffer and were isolated via extrusion through a 5 m filter followed by dialysis against a 3 m filter , as previously described . all other vesicle solutions were extruded 11 times through 100 nm filters with an avanti mini extruder . 50 nm vesicles were prepared with an additional 11 passes through a 50 nm filter . surface tensions were measured by the noy ring method on a fisher scientific surface tensiometer 21 . samples ( 5 ml ) were prepared by serially diluting a concentrated ( 100 mm ) vesicle / micelle stock and then allowed to equilibrate for at least 24 h before measuring . szyszkowski equationwhere is the measured surface tension ; 0 is the surface tension with no surfactant ( 72.8 dyn / cm ) ; k is the equilibrium constant for surface adsorption ; max is the maximum surface excess ; and c is the monomer concentration . therefore we obtained max from the maximum slope of the surface tension vs log([fatty acid ] ) plot according to the gibbs isotherm lastly , k was obtained by solving for c in the linear region below the cmc / cac . light scattering intensities of oleate and palmitoleate solutions were measured on a pddls / batch system ( precision detectors , bellingham , ma ) . absorbance readings of myristoleic acid solutions were taken on an amersham ultraspec 3100 uv / vis spectrophotometer . general polarization values were calculated from emission intensities at 500 and 430 nm upon excitation at 364 nm . micelle to vesicle partition coefficients were derived from measured laurdan intensities by equating observed gp to a weighted average of micelle and vesicle gps . characteristic vesicle ( i500v , i430v ) and micelle ( i500 m , i430 m ) laurdan intensities for each fatty acid were measured at ph 8.5 and 10.5 , respectively , and a concentration of either 30 mm ( oleate , palmitoleate ) or 50 mm ( myristoleate ) . the micelle / vesicle partition was calculated by solving for xm ( micelle aggregation fraction ) and xv ( vesicle aggregation fraction ) in the followingwhere gp is the measured laurdan polarization for the given sample . we note that this approach carries several assumptions : ( 1 ) the only existing aggregates are micelles or vesicles , with laurdan equally distributed between them on a molar basis and with minimal contribution from the monomer phase ; ( 2 ) laurdan emission ratios at high concentrations ( 30 or 50 mm ) at ph 8.5 or ph 10.5 approximate that in a pure vesicle or pure micelle solution , respectively . the latter assumption is limited by the excess light scattering of more concentrated vesicle solutions and the differing pka 's of the fatty acids , which likely result in a micelle : vesicle ratio of > 0 at ph 8.5 and the reference concentrations used . micelle / vesicle partition coefficients were also derived from absorbance at 400 nm ( abs400c ) . the vesicle fraction was calculated as proportional to the normalized absorbance for the concentration above the cac ( 2 mm for myristoleate ) . the micelle fraction was assumed to be the difference between this and the normalized vesicle absorbance at 50 mm ( abs400v ) , assuming xv 1 at 50 mm . thereforewhere k is the inverse of the absorption per unit concentration of 50 nm myristoleate vesicles and c is the solution concentration . this derivation involves the same assumptions used as for the laurdan partition coefficients and is therefore comparable . growth and shrinkage of 100 nm myristoleate vesicles was monitored as previously described . briefly , 10 mm myristoleate vesicles were prepared with equal fractions of rhodamine - dhpe and nbd - dhpe at a concentration of 0.2 mol % relative to total lipids . during experiments , fret was recorded on a cary eclipse fluorimeter ( excitation 430 nm ) by quantifying fret efficiency , fwhere ed is the emission of the donor ( 530 nm ) and ed, the emission of the donor at infinite dilution , which was measured via addition of 1% triton x100 at the end of the experiment . fret efficiency was equated to surface area using a standard curve of 10 mm myrisoleate with varying concentrations of fret dyes . growth of large vesicles was observed on a nikon te2000-s inverted microscope using a 60x extra long working distance objective . vesicle solutions ( 5 mm in 400 l of 0.1 m na bicine ) were agitated via a stir bar in opaque 5 ml vials at 35 c . agitation was used to keep solutions homogeneous and prevent films from forming on the side of the vials . identical experiments were also performed without evaporation ( using capped vials ) to confirm that there was no measurable bleaching and/or dye degradation in time scales up to 40 h. predicted relative surface area ( sa ) as a function of final concentration ( c ) was derived from the quadratic curve fit of the apparent xm / xv as a function of concentration in figure 5b using the followingwhere ( ( xm)/(xv)5 mm ) is the micelle vesicle partition at 5 mm ( initial concentration ) and ( ( xm)/(xv)c ) is the micelle vesicle partition at the final concentration .

the first protocell membranes may have assembled from fatty acids and related single - chain lipids available in the prebiotic environment . prior to the evolution of complex cellular machinery , spontaneous protocell membrane growth and division had to result from the intrinsic physicochemical properties of these molecules , in the context of specific environmental conditions . depending on the nature of the chemical and physical environment , fatty acids can partition between several different phases , including soluble monomers , micelles , and lamellar vesicles . here we address the concentration dependence of fatty acid aggregation , which is dominated by entropic considerations . we quantitatively distinguish between fatty acid phases using a combination of physical and spectroscopic techniques , including the use of the fluorescent fatty acid analogue laurdan , whose emission spectrum is sensitive to structural differences between micellar and lamellar aggregates . we find that the monomer aggregate transition largely follows a characteristic pseudophase model of molecular aggregation but that the composition of the aggregate phase is also concentration dependent . at low amphiphile concentrations above the critical aggregate concentration , vesicles coexist with a significant proportion of micelles , while more concentrated solutions favor the lamellar vesicle phase . we subsequently show that the micelle vesicle equilibrium can be used to drive the growth of pre - existing vesicles upon an increase in amphiphile concentration either through solvent evaporation or following the addition of excess lipids . we propose a simple model for a primitive environmentally driven cell cycle , in which protocell membrane growth results from evaporative concentration , followed by shear force or photochemically induced division .

Introduction Results and Discussion Conclusions Materials and Methods

early cell membranes are thought to have been composed of fatty acids and related single - chain amphiphiles , in contrast to the phospholipid - based membranes of all modern cells . initial support for this hypothesis arose from the facile prebiotic synthesis of these molecules and the ability of fatty acids to spontaneously assemble into bilayer vesicles . fatty acids and other oxygenated alkanes can be synthesized via fischer tropsch - type chemistry , and membrane - forming samples of these molecules have been discovered in abiotic environments such as meteorites . more recently , the functional properties of fatty acid membranes have been studied and are consistent with the necessity for early cell membranes , prior to the evolution of transport machinery , to be permeable to polar nutrients . subsequent work has utilized the ph dependence of fatty acid aggregation to drive the de novo assembly of vesicles from micelles or the growth of pre - existing vesicles by introducing alkaline micelles into buffered suspensions of vesicles . we therefore asked if monomers , micelles , and vesicles could all coexist under certain conditions and whether the composition of the aggregate phase could be concentration dependent , with lower concentration solutions favoring micelles and higher concentrations favoring vesicles . to explore multiphase coexistence , we sought methods to quantitatively characterize the equilibrium between fatty acid monomers , micelles , and vesicles at low concentrations . we focused on a set of monounsaturated fatty acids , which serve as convenient laboratory models for the short - chain , saturated lipids expected to result from prebiotic synthesis . we distinguished between different aggregate phases using the fluorescent fatty acid analogue laurdan ( 6-dodecanoyl-2-dimethylaminonaphthalene ) , which undergoes an emission red shift with increasing solvent polarity . we then used this multiphase coexistence to drive the growth of fatty acid vesicles by evaporative concentration , a process with potential prebiotic relevance to the growth of early cell membranes . the critical micelle concentrations ( cmc s ) of these fatty acids scaled exponentially with chain length , a result of the linear dependence of the free energy of solvation on chain length via the hydrophobic effect . laurdan is a c12 fatty acid analogue with a fluorescent naphthalene derivative that features an emission spectrum that is sensitive to the polarity of its environment . we interpreted this data to indicate a concentration dependence of the fatty acid aggregation state , with micelles favored in low concentration solutions . we tested the concentration dependence of the micelle to vesicle ratio independently by measuring the turbidity of vesicle solutions that had been extruded to 50 nm to eliminate spurious effects due to variation in vesicle size . the addition of alkaline micelles to buffered vesicles we therefore hypothesized that the concentration dependence of the micelle vesicle equilibrium could provide an alternative mechanism for the growth of pre - existing fatty acid vesicles . in this scenario , a rise in amphiphile concentration would cause the incorporation of excess micellar fatty acids into vesicles , and dilution would drive vesicle shrinkage ( as material leaves the lamellar phase and reforms micelles ) . the rapid growth of large , multilamellar vesicles following the addition of excess micelles results in the transformation of initially spherical vesicles into extended filamentous vesicles . we therefore asked whether gentle evaporation would drive the growth of fatty acid vesicles as a result of the transfer of material from coexisting micelles into the preformed vesicles as the fatty acid concentration increased ( figure 8) . an identical experiment with dimyristoleoyl phosphocholine ( pc ) we have used a combination of physical and spectroscopic assays to characterize the phase behavior of fatty acid solutions to better understand models for prebiotic membrane assembly and function . on the basis of the results described above , we present a potential scenario in which environmental fluctuations could drive repeated cycles of protocell growth and division . although this model is quite speculative , it has the advantage that cycles of growth and division would be driven entirely by environmental fluctuations and could continue indefinitely in the absence of any additional input of fatty acids . the subsequent evolution of catalytic mechanisms to drive membrane growth , such as the assembly of double - chain lipids , would have eventually freed early cells from depending on environmental fluctuations to drive their cell cycle .

in 1989 , the transient receptor potential ( trp ) protein was first identified as being encoded by the trp gene of drosophila . the trp protein superfamily consists of a diverse group of calcium ion ( ca)-permeable non - selective cation channels , and is found in most living organisms [ 2 - 4 ] . mammalian trp channels are currently divided into trpc ( canonical ) , trpv ( vanilloid ) , trpm ( melastatin ) , trpp ( polycystic kidney disease ) , trpml ( mucolipin ) and trpa ( ankyrin ) subfamilies , which consist of seven , six , eight , three , three and one members , respectively . trp channels have a tetrameric subunit stoichiometry , and each subunit contains cytoplasmic n- and c - terminal regions , six transmembrane ( tm ) domains and a pore - forming region between tm5 and tm6 . trp channels are sensitive to a variety of stimuli , including receptor stimulation , temperature , plant - derived compounds , environmental irritants , osmotic pressure , mechanical stress , ph and voltage from the extracellular and intracellular milieu , and are involved in diverse physiological and pathological processes [ 4 - 16 ] . several trp channels appear to respond well to mediators of oxidative stress , such as reactive oxygen species ( ros ) , reactive nitrogen species ( rns ) and other electrophiles [ 17 - 20 ] . while oxidative damage to dna , lipids and proteins is canonically known to cause cellular dysfunction , ros and rns are also increasingly recognized as cell signaling molecules [ 21 , 22 ] . the first identified ros - sensitive trp channel , trpm2 , is activated by hydrogen peroxide ( h2o2 ) and mediates several cellular responses , including cell death and chemokine production [ 23 - 26 ] . trpm7 , which can be modulated by both ros and rns , is an essential mediator of anoxic cell death [ 27 , 28 ] . some members of the trpc and trpv subfamily , including trpc5 and trpv1 , are activated by h2o2 , nitric oxide ( no ) and reactive disulfides . in addition , trpa1 is remarkably activated by various oxidants , including ros , rns , reactive disulfides and other electrophiles [ 30 - 33 ] . trpa1 proteins form a plasma membrane channel that contains many ankyrin repeats in its cytoplasmic n - terminal region [ 34 , 35 ] and can form a tetrameric assembly ( fig . trpa1 is expressed in a subset of nociceptive c - fiber neurons , including the dorsal root , trigeminal and nodose ganglion neurons [ 37 - 39 ] . it is targeted by environmental irritants , such as allyl isothiocyanate ( aitc ) from mustard oil and wasabi , cinnamaldehyde from cinnamon oil , allicin from garlic , and acrolein present in tear gas or vehicle exhaust [ 40 - 44 ] . these environmental irritants are electrophiles [ 30 , 31 ] , and further studies using trpa1 knockout mice have shown that trpa1 acts as a nociceptor for electrophilic environmental irritants to produce pain [ 42 , 45 - 48 ] . ros , rns and lipid peroxidation products also activate trpa1 , and can induce a trpa1-mediated pain sensation [ 49 - 53 ] . in terms of disorders , it is known that the activation of trpa1 by oxidative stress byproducts is reported to mediate both diabetic and anti - cancer medicine - induced neuropathic pain [ 54 - 57 ] . trpa1 is also involved in neurogenic inflammation , respiratory irritation and coughing elicited by electrophiles [ 49 , 51 , 58 - 62 ] . therefore , oxidative stress - sensitive trpa1 has been proposed as a potential drug target for the treatment of neurological diseases . in addition to the importance of trpa1 in neurological diseases , trpa1 activation also mediates vascular dilation [ 63 , 64 ] . furthermore , trpa1 activation induces both serotonin release from enterochromaffin cells and cholecystokinin release from a mouse intestinal neuroendocrine cell line [ 65 , 66 ] . trpa1 also regulates respiration by sensing oxygen ( o2 ) availability [ 67 , 68 ] . thus , a better understanding of the modulatory mechanisms of trpa1 by both inhibitors and activators is of high significance . a number of trpa1 modulators ( activators and inhibitors ) have been identified to date , including not only environmental electrophiles and oxidative stress mediators , but also non - electrophilic compounds [ 69 , 70 ] . some rodent models of neurological diseases respond positively to trpa1 inhibitors [ 71 - 73 ] , and some trpa1 inhibitors have reached the clinical trial stage as novel analgesic drugs . it is also reported that a novel trpa1 agonist exerts both anti - constipation and anti - abdominal pain actions . the mechanism of trpa1 modulation by oxidative mediators is thought to involve oxidative modification of cysteine residues , unlike trpa1 modulation by non - electrophilic compounds . trpa1 modulation by certain non - electrophilic compounds appears to be dependent on different chemical moieties within these compounds . furthermore , several critical sites have been identified in trpa1 that seem to be important for some non - electrophilic compounds to bind and modulate trpa1 . strikingly , the chemical structure seems to be important even for the activation of trpa1 by specific electrophiles . a novel no donor derived from the 7-azabenzobicyclo [ 2.2.1 ] heptane n - nitrosamines confer selectivity to modulatory action of no on trpa1 over the other no - sensitive trp channels . this subtype selectivity may be conferred through synergistic effects of two chemical processes : cysteine transnitrosylation and molecular recognition of a non - electrophilic moiety of the novel n - nitrosamine . this review will attempt to explore current molecular pharmacological knowledge of trpa1 modulation by small molecules . trpa1 senses various oxidative stress mediators and environmental compounds ( fig . 2 and table 1 ) . cysteine residues within a protein are emerging as direct targets for the oxidant signal reaction [ 77 , 78 ] . its activation by oxidants is proposed to be mediated via oxidative modification of the free sulfhydryl group of cysteine residues , as described for the activation of trpc5 and trpv1 [ 29 , 79 ] . for trpa1 , simultaneous mutation of three cysteine residues within the cytoplasmic n - terminus of human trpa1 ( cys621 , cys641 and cys665 ) decreases trpa1 channel activation by several exogenous cysteine - modifying electrophiles , such as isothiocyanates ( e.g. aitc ) , ,-unsaturated aldehyde compounds ( e.g. acrolein , n - methylmaleimide and cinnamaldehyde ) , and diallyl disulfide . three cysteine residues in mouse trpa1 ( cys415 , cys422 , and cys622 , conserved in the human homolog as cys414 , cys421 , and cys621 ) were independently identified as the target sites for aitc and cinnamaldehyde . systematic evaluation of trp channels was performed using a series of reactive disulfides , such as bis(5-nitro-2-pyridyl ) disulfide and diallyl disulfide . these compounds possess different electron acceptor ( oxidation ) capacity ( manifested as redox potential ) and these studies revealed that only trpa1 responds to the inert electrophile , diallyl disulfide . thus , trpa1 can sense inert oxidant o2 , and o2 activation of trpa1 is by oxidation of cys633 and/or cys856 , located intracellularly within , respectively , the n - terminal region and the putative linker region between tm4 and tm5 . in addition , trpa1 cysteine residues seem also to be critical for trpa1 activation by other exogenous compounds , including irritants ( tear gases , such as 2-chloroacetophenone [ 81 , 82 ] , and ,-unsaturated carbonyl - containing compounds , such as methylvinylketone [ 83 , 84 ] ) , some plant constituents ( umbellulone , ligustilide , hydroxy--sanshool ( -soh ) and 6-shogaol ) , and others ( the cyclooxygenase-2 inhibitor , etodolac ; the anti - diabetic drug , glibenclamide ; the gold - containing disease - modifying anti - rheumatic drug , auranofin ; cmp1 ( 4-methyl - n-2,2,2-trichloro-1-(4-nitro - phenylsulfanyl)-ethyl - benzamide ) ; and lidocaine ) . therefore , trpa1 is unarguably a receptor for oxidative exogenous electrophilic compounds . trpa1 is activated by h2o2 [ 32 , 50 , 51 , 93 ] , hypochlorite , ozone and the ros generated by ultraviolet light . in addition to ros , trpa1 is also activated by rns such as no [ 32 , 53 , 93 ] and peroxynitrite . functional characterization of site - directed mutants of trpa1 collectively demonstrates that specific cytoplasmic n - terminal cysteine residues and a lysine residue ( cys421 , cys621 , cys641 , cys665 and lys710 in human trpa1 ) are the primary targets of ros and rns [ 32 , 51 , 53 ] . in addition to ros and rns , other endogenous electrophilic mediators of oxidative stress modulate trpa1 activity . lipid peroxidation products such as 4-hydroxy-2-nonenal , 4-hydroxyhexenal , 4-oxo-2-nonenal , nitrooleic acid and 15-deoxy--prostaglandin j2 ( 15d - pgj2 ) activate trpa1 channels through oxidative modification of the cysteine residues [ 32 , 49 , 50 , 96 - 99 ] . labeling experiments using biotinylated 15d - pgj2 demonstrated that cys621 mediates the binding of 15d - pgj2 to human trpa1 . another electrophilic dicarbonyl compound , methylglyoxal ( mg ) , which is believed to be associated with the development of diabetic neuropathy , also activates trpa1 by hemithioacetal formation [ 54 , 55 ] . taken together , we can surmise that endogenous electrophilic products activate trpa1 channels by cysteine oxidation . to understand the modulatory mechanism of trpa1 channel activity recently , the structure of the capsaicin - sensitive polymodal receptor , trpv1 , was revealed at 3.4 resolution [ 100 , 101 ] . with regard to trpa1 it is suggested that cys414 and cys421 in the n - terminal ankyrin repeats from one subunit and cys621 located on an adjacent subunit form a ligand - binding pocket between the subunits . it is possible that covalent modification of cysteine residues around this pocket could alter the interaction between the subunits and the domains within each subunit , promoting conformational changes and leading to channel activation . as discussed above , oxidative stress mediators and environmental electrophiles activate trpa1 , but it has also been demonstrated that various other activators and inhibitors modulate trpa1 ( fig . 3 and table 1 ) . icilin , 2-aminoethyl diphenylborinate and carvacrol are compounds with no obvious reactivity towards cysteine residues and activate trpa1 in a way that is not disrupted by cysteine mutations [ 30 , 31 , 51 ] . trpa1 is also activated by non - reactive compounds including non - steroidal anti - inflammatory drugs , such as flufenamic acid ; general anesthetics , such as isoflurane ; farnesyl thiosalicylic acid ( fts ) ; and others [ 104 - 107 ] . the chloride channel blocker , nppb ( 5-nitro-2-(3-phenylpropylamino)benzoic acid ) , activates trpa1 , and a structure - activity relationship study using a group of nppb analogs indicates that its phenylalkane , carboxylic and nitro groups are critical for its activation of trpa1 . from mutagenesis studies , nppb and fts are suggested to have similar molecular mechanisms of action at trpa1 . thymol , 2,6-diisopropylphenol ( propofol ) and related simple alkyl phenols also activate trpa1 , and investigation with a series of alkyl phenol analogs indicates that bulky carbon substituents and high calculated logp values are correlated with an increased ability to activate trpa1 . trpa1 is also activated by polyunsaturated fatty acids , which should contain at least three double bonds and 18 carbon atoms , such as docosahexaenoic acid ( dha ) . arachidonic acid and its derivatives also activate trpa1 independently of cysteine oxidation . 6-paradol and 6-gingerol activate trpa1 , whereas the non - trpa1 agonist capsaicin does not , suggesting that a phenol core of these compounds is not sufficient to confer trpa1 activation [ 40 , 87 ] . moreover , capsiate , a non - pungent capsaicin analog , also activates trpa1 through a mechanism distinct from cysteine and histidine modification . therefore , trpa1 activation by non - reactive compounds is dependent on their chemical structures rather than cysteine oxidation . menthol and its derivatives , camphor [ 114 , 115 ] , nicotine , apomorphine , and propofol all activate trpa1 at low concentrations but show inhibitory effects at high concentrations . bimodal modulation of trpa1 is also a feature of certain cysteine - reactive compounds , such as cinnamaldehyde , aitc , umbellulone and ligustilide . inhibitors of trpa1 have been developed , exemplified by the synthetic inhibitors hc-030031 , chembridge-5861528 ( a derivative of hc-030031 ) , ap-18 , a-967079 ( a derivative of ap-18 ) , amg5445 and az868 [ 46 , 48 , 71 , 72 , 120 - 124 ] . another , adm_09 , is an antagonist of trpa1 with a putative dual - binding mode of action , which involves the synergic combination of ca - mediated binding of the carnosine group and disulfide - formation by its lipoic acid group . camphor and 1,8-cineol are naturally occurring inhibitors of human trpa1 , but 1,4-cineol is an activator . borneol is a more effective natural inhibitor than camphor and 1,8-cineol , and the hydroxyl group of borneol is suggested to contribute to its inhibitory action . the pharmacological profile of the human and rhesus monkey trpa1 is relatively distinct from mouse and rat trpa1 . importantly , findings of species - specific effects have helped to identify the critical region that determines trpa1 modulation ( fig . menthol is known to be a bimodal modulator of mouse trpa1 , whereas it does not inhibit human trpa1 , and drosophila trpa1 is insensitive to menthol . chimera and mutagenesis studies indicate that specific residues within tm5 ( notably ser876 and thr877 of mouse trpa1 , corresponding to ser873 and thr874 of human trpa1 ) are critical for menthol responsiveness . furthermore , the region from tm5 to tm6 in mouse and human trpa1 is the critical domain determining the inhibitory effects of menthol . the same two residues ( ser and thr within tm5 ) are also critical for the sensitivity of trpa1 to amg5445 , ap-18 and a-967079 [ 130 , 131 ] . ser873 , thr874 and tyr812 residues of human trpa1 are critical to the inhibitory effects of borneol , but not to camphor or 1,8-cineol . like menthol , propofol and lidocaine show bimodal effects on mouse trpa1 , but only work as activators of human trpa1 [ 92 , 118 ] . although the species - specific differences above were linked to the tm5 and tm6 region , the residues within tm5 are not involved in the action of propofol and lidocaine . dha sensitivity is limited to human and mouse trpa1 : drosophila trpa1 does not respond to dha . neither the cytoplasmic n - terminal region nor tm5 of trpa1 are directly involved in dha sensing . these compounds ( propofol , lidocaine and dha ) probably employ different or additional mechanisms to modulate trpa1 as compared with menthol . caffeine , which is not a reactive chemical reagent , activates mouse trpa1 , but suppresses human trpa1 . a met268pro mutation in the n - terminal cytoplasmic region of mouse trpa1 converts this residue to the human form and consequently changes caffeine action from activation to suppression . an electrophilic compound cmp1 , a structural analog of amg5445 , inhibits human trpa1 and activates rat trpa1 via modification of human cys621 and rat cys622 , respectively [ 91 , 134 ] . the specific mutations ala946ser and met949ile in the upper portion of the tm6 region of rat trpa1 change the effect of cmp1 from activatory to inhibitory . therefore , these studies demonstrate that specific regions and residues within trpa1 determine the trpa1 modulatory activity of non - electrophilic compounds , and that the key domains / residues vary between compounds . furthermore , while direct physical interaction of non - electrophilic compounds with trpa1 is likely to be critical for modulation , it is unclear whether or not these critical sites are involved in binding . isovelleral , a fungal natural product which contains an ,-unsaturated aldehyde moiety , activates trpa1 independently of cysteine oxidation . a major compound in extra - virgin olive oil , oleocanthal ( oc ) a structure - activity relationship study using synthetic oc analogs indicated that oc requires both aldehyde groups to activate trpa1 . trpa1 cysteine mutants show diminished responses to -soh , but further study with synthetic analogs of -soh indicated that the configuration of the cis c6 unsaturation in the alkylamides is also a determinant of the compound effect on trpa1 . the mouse cys622ser trpa1 mutant is still sensitive to umbellulone , albeit less so than wild type trpa1 . dihydroumbellulone , which lacks electrophilic properties because of reduction of the enone moiety , retains residual trpa1-activating capacity . zhong et al suggest that umbellulone is a mechanistically hybrid activator , apparently combining covalent interaction at a reactive cysteine with noncovalent interaction with a second site on trpa1 . thus , chemical structure recognition by trpa1 , a clearly distinct mechanism from cysteine oxidation , is supposed to be important even for trpa1 activation by some specific electrophiles . protein s - nitrosylation , the covalent attachment of an no moiety to the sulfur atom of cysteine residues to form s - nitrosothiol , regulates various protein functions to mediate no bioactivity . receptor - activated ( trpc5 , trpc1 and trpc4 ) and thermosensor ( trpv1 , trpv3 , trpv4 and trpa1 ) trp channels are activated by exogenous no - releasing donors through s - nitrosylation [ 29 , 32 ] , but with very limited trp subtype selectivity . recently , this problem was partly solved with our finding that the 7-azabenzobicyclo[2.2.1]heptane ( abbh ) n - nitrosamine selectively s - nitrosylates trpa1 through transnitrosylation without releasing no . although protein s - nitrosylation is widely accepted , questions regarding target selectivity of s - nitrosylation signaling are incompletely understood . no is produced in vivo by only three no synthase ( nos ) isoforms , and no is reactive and diffusible within cells . binding of nos to targets or their adaptors have been demonstrated to localize nitrosylation reactions , but there are many s - nitrosylated proteins ( > 1,000 ) [ 136 , 139 , 140 ] . recent studies have identified protein - protein transnitrosylation , the transfer of the no group from one protein to another in the absence of apparent no release , is a potentially important targeting pathway [ 140 - 142 ] . transnitrosylation has been reported between specific proteins , exemplified by transnitrosylation of x - linked inhibitor of apoptosis by sno - caspase-3 in apoptotic cell death [ 143 - 146 ] . a binding interaction between the two proteins is also required for transnitrosylation , because a binding - deficient mutant of one protein abrogates this protein - protein transnitrosylation [ 143 , 145 ] . to develop transnitrosylation - based subtype - selective activators of trp channels , it is necessary to first identify a synthetic no donor that has only the transnitrosylative reactivity . however , snap(s - nitroso - n - acetyl - dl - penicillamine ) and nor3 ( ( )-(e)-4-ethyl-2-(e)-hydroxyimino-5-nitro-3-hexenamide ) are no - releasing donors . s - nitrosoglutathione is known to be a biological transnitrosylating agent , but also releases no [ 148 , 149 ] . in contrast , the abbh n - nitrosamines constitute a new class of no donors that , at physiological ph and temperature , transnitrosylate thiols to generate s - nitrosothiols without releasing no [ 150 - 152 ] . surprisingly , our intracellular ca imaging measurements have demonstrated that n - nitroso-2-exo,3-exo - ditrifluoromethyl-7-azabenzobicyclo [ 2.2.1 ] heptane ( nno - abbh1 ) induces robust ca influx via recombinant human trpa1 channels , but not via other snap - activated trp channels , suggesting that nno - abbh1 selectively s - nitrosylates trpa1 ( fig . 4 ) . a modified labeling assay to biochemically identify protein s - nitrosothiol showed that snap s - nitrosylates both trpa1 and trpv1 , but nno - abbh1 s - nitrosylates only trpa1 . trpa1 activation by nno - abbh1 is suppressed by specific cysteine mutations but not by no scavenging , indicating that transnitrosylation underlies the activation of trpa1 by nno - abbh1 . this is supported by a positive correlation of n no bond reactivity and trpa1-activating potency in a congeneric series of abbh n - nitrosamines . cys540 , cys641 , and cys665 of human trpa1 are involved in its modification by nno - abbh1 . cys641 and cys665 are also required for responsiveness to snap [ 32 , 53 ] , indicating that cys540 may be a unique target for nno - abbh1 . to further explore this structure - activity relationship , we developed several non - electrophilic analogs of nno - abbh1 : n - h ( nh - abbh ) , n - formyl ( ncho - abbh ) , and n - methyl ( nme - abbh ) ( fig . they also did not cause s - nitrosylation of trpa1 and their activity was not affected by cysteine mutation of trpa1 , confirming that oxidative modification of cysteine residues is not critical for their mechanism of action . importantly , the dose - response relationship for nme - abbh - induced recombinant trpa1 activation was shifted to the left in the presence of snap ( 10 m ) , which by itself can not significantly activate trpa1 . this result supports the idea that trpa1 activation by these non - electrophilic analogs may be subject to positive synergistic interactions between nitrosylation and molecular recognition , indicating that nno - abbh1 may be a hybrid activator . it is reported that a non - electrophilic trpa1 activator flufenamic acid synergistically potentiates the activation of trpa1 by aitc . also , umbellulone has been proposed to activate trpa1 by combining covalent interaction at a reactive cystein with noncovalent interaction with a second site on trpa1 . it is important to note that trpc5 and trpv1 failed to respond to 300 m nme - abbh , suggesting that trpc5 and trpv1 , unlike trpa1 , may lack the molecular recognition sites for the non - electrophilic moiety of nno - abbh1 . thus , molecular recognition of chemical groups other than no may explain the subtype - selective activation of trpa1 by these compounds . despite evidence of synergistic effects between cysteine transnitrosylation and molecular recognition of the non - electrophilic moiety , it remains unclear how the transnitrosylation site and the non - electrophilic molecular recognition site converge in trpa1 . also , it is unknown whether nno - abbh1 and other non - electrophilic analogs have bimodal and/or species - specific effects on trpa1 . further detailed studies into trpa1 modulation by abbh n - nitrosamines will provide a basis for developing new drugs selectively targeting s - nitrosylation of trpa1 . in addition , these studies will present the opportunity for developing selective transnitrosylating modulators of other proteins . the findings of the selective activation of trpa1 by nno - abbh1 or its non - electrophilic derivatives suggest that the abbh skeleton imbues target protein selectivity via molecular recognition . thus , designing and evaluation of various derivatives of abbh could possibly be a strategy to find a derivative , which has an inhibitory effect specific on trpa1 activity . because trpa1 mediates neuropathic pain , vascular dilation and other functions , it has the potential to be an excellent drug target . therefore , it is important to understand the mechanisms of both activation and inhibition of trpa1 by small molecules . recent studies have revealed that trpa1 modulation by electrophiles is through cysteine oxidation , and that molecular recognition of chemical structures is a key determinant of trpa1 modulation not only by non - electrophilic compounds , but also by some specific electrophiles . a novel abbh n - nitrosamine induces selective s - nitrosylation of trpa1 probably through synergistic processes of cysteine oxidation and molecular recognition of non - electrophilic moiety . however , molecular bases of trpa1 modulation by non - electrophilic compounds are very poorly understood . similarly , further research is needed to define in detail the molecular mechanisms by which chemical ligands induce the activation of other trp channels , such as trpv1 and trpm8 [ 10 , 154 ] . trpa1 channel activity is also modulated by ca , receptor stimulation , ph , osmotic pressure and temperature [ 40 , 60 , 69 , 155 - 160 ] , so a better understanding of the complexities of its modulation is critical to the development of novel trpa1-specific drugs .

the transient receptor potential ( trp ) proteins are a family of ion channels that act as cellular sensors . several members of the trp family are sensitive to oxidative stress mediators . among them , trpa1 is remarkably susceptible to various oxidants , and is known to mediate neuropathic pain and respiratory , vascular and gastrointestinal functions , making trpa1 an attractive therapeutic target . recent studies have revealed a number of modulators ( both activators and inhibitors ) that act on trpa1 . endogenous mediators of oxidative stress and exogenous electrophiles activate trpa1 through oxidative modification of cysteine residues . non - electrophilic compounds also activate trpa1 . certain non - electrophilic modulators may act on critical non - cysteine sites in trpa1 . however , a method to achieve selective modulation of trpa1 by small molecules has not yet been established . more recently , we found that a novel n - nitrosamine compound activates trpa1 by s - nitrosylation ( the addition of a nitric oxide ( no ) group to cysteine thiol ) , and does so with significant selectivity over other no - sensitive trp channels . it is proposed that this subtype selectivity is conferred through synergistic effects of electrophilic cysteine transnitrosylation and molecular recognition of the non - electrophilic moiety on the n - nitrosamine . in this review , we describe the molecular pharmacology of these trpa1 modulators and discuss their modulatory mechanisms .

INTRODUCTION OXIDATION SENSITIVITY OF TRPA1 CHANNEL MODULATION OF TRPA1 BY OTHER ACTIVATORS AND INHIBITORS SUBTYPE SELECTIVE CONCLUSION CONFLICT OF INTEREST

in 1989 , the transient receptor potential ( trp ) protein was first identified as being encoded by the trp gene of drosophila . the trp protein superfamily consists of a diverse group of calcium ion ( ca)-permeable non - selective cation channels , and is found in most living organisms [ 2 - 4 ] . some members of the trpc and trpv subfamily , including trpc5 and trpv1 , are activated by h2o2 , nitric oxide ( no ) and reactive disulfides . in terms of disorders , it is known that the activation of trpa1 by oxidative stress byproducts is reported to mediate both diabetic and anti - cancer medicine - induced neuropathic pain [ 54 - 57 ] . a number of trpa1 modulators ( activators and inhibitors ) have been identified to date , including not only environmental electrophiles and oxidative stress mediators , but also non - electrophilic compounds [ 69 , 70 ] . the mechanism of trpa1 modulation by oxidative mediators is thought to involve oxidative modification of cysteine residues , unlike trpa1 modulation by non - electrophilic compounds . a novel no donor derived from the 7-azabenzobicyclo [ 2.2.1 ] heptane n - nitrosamines confer selectivity to modulatory action of no on trpa1 over the other no - sensitive trp channels . this subtype selectivity may be conferred through synergistic effects of two chemical processes : cysteine transnitrosylation and molecular recognition of a non - electrophilic moiety of the novel n - nitrosamine . its activation by oxidants is proposed to be mediated via oxidative modification of the free sulfhydryl group of cysteine residues , as described for the activation of trpc5 and trpv1 [ 29 , 79 ] . thus , trpa1 can sense inert oxidant o2 , and o2 activation of trpa1 is by oxidation of cys633 and/or cys856 , located intracellularly within , respectively , the n - terminal region and the putative linker region between tm4 and tm5 . in addition , trpa1 cysteine residues seem also to be critical for trpa1 activation by other exogenous compounds , including irritants ( tear gases , such as 2-chloroacetophenone [ 81 , 82 ] , and ,-unsaturated carbonyl - containing compounds , such as methylvinylketone [ 83 , 84 ] ) , some plant constituents ( umbellulone , ligustilide , hydroxy--sanshool ( -soh ) and 6-shogaol ) , and others ( the cyclooxygenase-2 inhibitor , etodolac ; the anti - diabetic drug , glibenclamide ; the gold - containing disease - modifying anti - rheumatic drug , auranofin ; cmp1 ( 4-methyl - n-2,2,2-trichloro-1-(4-nitro - phenylsulfanyl)-ethyl - benzamide ) ; and lidocaine ) . lipid peroxidation products such as 4-hydroxy-2-nonenal , 4-hydroxyhexenal , 4-oxo-2-nonenal , nitrooleic acid and 15-deoxy--prostaglandin j2 ( 15d - pgj2 ) activate trpa1 channels through oxidative modification of the cysteine residues [ 32 , 49 , 50 , 96 - 99 ] . as discussed above , oxidative stress mediators and environmental electrophiles activate trpa1 , but it has also been demonstrated that various other activators and inhibitors modulate trpa1 ( fig . to further explore this structure - activity relationship , we developed several non - electrophilic analogs of nno - abbh1 : n - h ( nh - abbh ) , n - formyl ( ncho - abbh ) , and n - methyl ( nme - abbh ) ( fig . they also did not cause s - nitrosylation of trpa1 and their activity was not affected by cysteine mutation of trpa1 , confirming that oxidative modification of cysteine residues is not critical for their mechanism of action . it is reported that a non - electrophilic trpa1 activator flufenamic acid synergistically potentiates the activation of trpa1 by aitc . it is important to note that trpc5 and trpv1 failed to respond to 300 m nme - abbh , suggesting that trpc5 and trpv1 , unlike trpa1 , may lack the molecular recognition sites for the non - electrophilic moiety of nno - abbh1 . despite evidence of synergistic effects between cysteine transnitrosylation and molecular recognition of the non - electrophilic moiety , it remains unclear how the transnitrosylation site and the non - electrophilic molecular recognition site converge in trpa1 . the findings of the selective activation of trpa1 by nno - abbh1 or its non - electrophilic derivatives suggest that the abbh skeleton imbues target protein selectivity via molecular recognition . recent studies have revealed that trpa1 modulation by electrophiles is through cysteine oxidation , and that molecular recognition of chemical structures is a key determinant of trpa1 modulation not only by non - electrophilic compounds , but also by some specific electrophiles . a novel abbh n - nitrosamine induces selective s - nitrosylation of trpa1 probably through synergistic processes of cysteine oxidation and molecular recognition of non - electrophilic moiety .

following pregnancy , low - income , minority women with a history of gdm or high bmis are at high risk for chronic illnesses , particularly obesity , type 2 diabetes mellitus ( dm ) , and postpartum depression [ 1 , 2 ] . racial / ethnic disparities exist across a variety of postpartum health outcomes , and these disparities may be widened by less postpartum clinical follow - up by hispanic / latina and african american patients . these disparities include a higher prevalence of gestational diabetes ( gdm ) during pregnancy , an increased risk for dm postpartum [ 4 , 5 ] , and a lack of postpartum diabetes screening for those with gdm . only one in five latina women with prior gdm returns for postpartum diabetes checkups , the lowest follow - up frequency of any group . these women are lost to follow - up despite recommendations for six - week postpartum screening as well as annual diabetes checks . in california , the prevalence of gdm is 5.7% among latina women , compared to 4% in non - hispanic whites [ 8 , 9 ] . women born outside the us make up a majority of pregnancies in california and may be at higher risk for gdm and subsequent dm for reasons including disruption to their normal eating habits , unhealthy dietary acculturation , barriers to physical activity , and rapid weight gain after migration to high - income countries [ 10 , 11 ] . results from the diabetes prevention program ( dpp ) suggest that the long - term risk for diabetes postpartum can be reduced through behavior change [ 1214 ] . current strategies to expand postpartum care management and to reduce chronic disease risk for ethnic minority women include tailored interventions that address health literacy and provide accessible resources or health coaching on lifestyle changes , but there are few programs that focus on delivering the dpp content for early postpartum women in the period immediately following birth . health information technology ( hit ) can be an important tool to tailor health communication efforts , and in particular if developed with end - users , it can be effective with low - literacy and low - income populations [ 1519 ] . a review of literacy - focused interventions shows that interventions using health information technologies have high potential to reach low - literacy , high risk populations since they have flexibility of access in the home and during convenient times [ 20 , 21 ] . interventions providing longitudinal care and support through health coaching or counseling have also been effective in reducing chronic illness . approximately 85% of adults in the us are cell phone users , and cell phone use does not vary by race or ethnicity . majority of low - income users have basic cell phones used for voice messaging and texting , and while this type of phone usage does not have the functionality of a smart phone ( web browsing and mobile applications ) , it provides access to the broadest range of users across ses status . to improve women 's health postpartum , several hit models are effective in enabling communication and reaching women when the clinic - based model is less convenient . postpartum low - income women may lose medi - cal eligibility , encounter barriers in accessing health care , and face new demands at home . postpartum calls can reinforce messages received in health care encounters as well as facilitate uptake of preventive services for both mother and child / children in the 69 months after delivery ( e.g. , 6-week glucose testing after delivery and vaccinations in infants ) . in the case of diabetes , there is growing literature indicating that diabetes self - management support can be improved with improvements in patient satisfaction as well as diabetes - specific health outcomes through health technology ( interactive voice messaging ; internet - based systems ) . our team has used a tailored combination of short ( 4 - 5 minutes ) automated calls with queries and narratives in diabetes self - management and have found such an approach , automated telephone self - management support ( atsm ) , effective for reaching and engaging patients with low health literacy and limited english proficiency and it can be cost effective and can improve health outcomes [ 2325 ] . another proven strategy for diabetes prevention health coaching involves counseling patients with chronic conditions to improve their own health by increasing their knowledge , skills , and confidence in managing their own health behaviors . the effectiveness of health coaching in motivating , empowering , and enabling patients to improve health behaviors is now well established [ 2731 ] . in particular , the peer health coach model has been successful in helping patients self - manage diabetes [ 32 , 33 ] . describe successfully using peer coaches , who were diabetic patients themselves , to provide support to other patients through three key roles : advisor , supporter , and role model . another study showed that the peer coach model is particularly effective among patients with worse medication adherence and higher hba1c levels . some evidence suggests no difference in patient outcomes when comparing peer coaches and health professionals in the counsel of diabetes patients . randomized patients with diabetes to a diabetes care group managed by a nurse practitioner either with a peer coach or with a nurse practitioner alone and found that participants with the peer coach had greater improvements in hba1c levels after intervention . despite the success of peer health coaching programs in primary care and clinical settings , there are few examples of similar programs implemented in community - based settings , such as federally funded programs or nonprofit organizations . there is also limited data on how such a model might address language and literacy challenges faced by low - income women with recent histories of migration to the us . one well - known and relevant community program that offers health coaching for this population is the women , infants , and children ( wic ) program , which provides supplemental foods , health care referrals , and nutrition education for low - income pregnant and postpartum women . in some wic locations , , we present a unique health coaching model , the star mama program ( support via telephone advice and resources / sistema telfonico de apoyo y recursos - mama ) , which combines hit - based queries and narratives , with follow - up by trained health coaches , to deliver adapted dpp content . the goals of this paper are to ( 1 ) illustrate a unique model of health coaching for high risk , low - income postpartum women which has relevance in community and clinic - based systems and ( 2 ) present case studies of exemplary star mama health coached participants stories . star mama is an ongoing randomized clinical trial comparing a hit - based health coaching program with a usual care arm providing an educational resource guide covering approaches to improving postpartum diabetes risk behaviors among gdm women based on dpp content . the study partnered with four key community sites : zuckerberg san francisco general ( zsfg ) , santa rosa community health centers ( vista and lombardi clinics ) in sonoma county , and san francisco and sonoma wic offices . we chose to partner with wic , as it is one of the few programs that provide services to low - income women spanning from pregnancy into the postpartum period and because migrant women can access most services on behalf of themselves or their infant . additionally , wic 's program content , which provides information and resources on healthy eating and referrals to health care , aligns with the main objectives of the star mama program . the star mama program is evaluated through a randomized control trial design in which women are assigned to one of two arms : ( 1 ) hit arm : participants receive weekly phone calls from the automated telemedicine self - support system on various diabetes preventive topics and are matched to a health coach for longitudinal follow - up ( 2 ) ; usual care / education arm : participants receive an education resource guide ( information and links to nearby resources ) about postpartum care for themselves and their baby along with community resources for diet , physical activity , and so forth . recruitment at clinic sites was monitored through a thorough list of gdm women who were approached based on eligibility criteria to enroll in the study . for the wic sites , women were targeted based on delivery dates and either having gdm or a high bmi status ( > 25 ) . women were randomized prior to conducting the baseline survey at their enrollment visit to one of the two study arms ( using an envelope sealed randomization assignment ) , following administration of the baseline survey . primary outcomes include self - reported weight , body mass index ( bmi ) based on chart review , receipt of recommended postpartum glucose testing , changes in dietary patterns , such as consumption of fruits and vegetables and foods high in fats or sugars , and physical activity ( minutes per week ) . star mama participants randomized to the intervention arm of the study receive weekly phone calls through our automated telemedicine self - support system and are paired with a health coach for longitudinal follow - up . a hit enabled model was selected at the outset because it allows participants to receive weekly content and health coaching support in their primary language while remaining in their homes , as traveling to appointments and group sessions is known to be a key barrier to receiving preventive services in community settings . the calls focus on dpp topics including diet , physical activity , encouraging partner support , balancing self - care postpartum , healthy eating tips , importance of receiving a blood sugar checkup , and baby care . for example , if a participant pressed 1 ( yes ) to a query asking if she was feeling stressed about her baby crying , she might hear a story about a new mom like her , facing similar challenges , reassuring her that it is ok to ask for help . the information from this question would then be delivered to her star mama health coach , who would also call her back and provide her with support . figure 1 describes the content and method of delivery of the range of star mama topics covered from weeks 1 through 20 . triggers are classified by predetermined values which determine whether a health coach callback is required . daily and weekly reports from the hit calls provide context for the health coaching call and motivational interviewing and help the coach understand what issues to focus on when developing an action plan or goals with the participant . a participant receiving the weekly phone calls is also matched to a health coach who monitors her response to the calls and regularly follows up with the participant on relevant issues . follow - up topics vary from specific concerns , longitudinal support , empowerment , or resources for the mother . for example , in the sixth week of calls , the participant is queried : if you have questions about feeding your baby or how to deal with the pressure you are feeling from your family and friends , press 1 and a health coach will call you back . if not , press 2 . health coaches at zsfgh were from bicultural backgrounds and had previous experience in the clinical setting as either health coaches or para - health professionals . coaches at the wic sites were trained nutritionists or registered dieticians already working in the wic system . at minimum , each health coach received a two - day training at the university of california san francisco ( ucsf ) center for excellence in primary care and was given a one - day star mama specific training that focused on delivery of the dpp , for a total of three days of health coaching training . ongoing health coaching training also included review of the health coaching manual adapted from the dpp and biweekly review of cases at staff meetings . additionally , coaches had ongoing meetings to discuss and reflect on common topics and support each other in the coaching process . health coaches kept detailed notes based on call summary and trigger reports , which all coaches could access for internal resources . the health coaching curriculum was codeveloped with relevant stakeholders , including participants and care providers familiar with the ethnic diversity of the local populations . the following list titled health coaching training topics , example query , narrative , and health coach script describes a summary of the health coaching training , an example of a star mama automated telephone call , and a guideline for the subsequent health coaching topics . health coaching training topics , example query , narrative , and health coach script health coach training topics communication ask - tell - ask , how to receive information from the participantclosing the loopsetting the agendaunderstanding your current health status , numbers motivational interviewing exploring patients motivations and barriers postpartum risk specific knowledge knowledge regarding risk of dm postpartum community and clinic specific resources community related risks : safety , access to affordable produce , and primary carecommunity - based resources : food banks , wic agencies , free health clinics , and physical activity groups ask - tell - ask , how to receive information from the participantclosing the loopsetting the agendaunderstanding your current health status , numbers ask - tell - ask , how to receive information from the participant understanding your current health status , numbers motivational interviewing exploring patients motivations and barriers exploring patients motivations and barriers postpartum risk specific knowledge knowledge regarding risk of dm postpartum knowledge regarding risk of dm postpartum community and clinic specific resources community related risks : safety , access to affordable produce , and primary carecommunity - based resources : food banks , wic agencies , free health clinics , and physical activity groups community related risks : safety , access to affordable produce , and primary care community - based resources : food banks , wic agencies , free health clinics , and physical activity groups health coaching example query to participant . in the last 7 days , how many days did you drink sweetened drinks like sodas , aguas frescas , fruit juices , coffee with sugar or condensed milk , sports drinks or energy drinks ? callback trigger 3 days ( this trigger is determined by the participant pressing 39 on her phone , as a response to the question about the number of days she drank sugar sweetened drinks ) a small can of soda can have as many as 10 sugar packets in it ! even aguas frescas can have extra calories and loads of sugar without giving you much nutrition . you do n't have to stop drinking them , but you can try having them less often and making your own with less sugar . for example , if you like preparing agua fresca with strawberries and usually add two large spoonfuls of sugar , try just adding one . at first it might taste less sweet but it is something that you and your family can get used to , and is part of showing them your commitment to health- theirs and yours ! health coach script and topic guide verify report on your call you answered that in the past 7 days you drank sweetened drinks _ _ _ _ days . on your call you answered that in the past 7 days you drank sweetened drinks _ open - ended question how many sugary drinks do you typically have in one day?what do you like about drinking sweetened drinks?is there something you do n't like about drinking them?how do you think drinking sweetened drinks affects your health?how do you think it could affect your risk of developing t2 dm ? how many sugary drinks do you typically have in one day ? what do you like about drinking sweetened drinks ? how do you think drinking sweetened drinks affects your health ? how do you think it could affect your risk of developing t2 dm ? provide education sugary drinks contribute to high calorie intake and can lead to obesity.drinks high in sugar make your blood sugar spike within a few minutes of drinking it.people who consume sugary drinks regularly1 to 2 cans a day or more have a greater risk of developing type 2 diabetes than people who rarely have such drinks . drinks high in sugar make your blood sugar spike within a few minutes of drinking it . people who consume sugary drinks regularly1 to 2 cans a day or more have a greater risk of developing type 2 diabetes than people who rarely have such drinks . help participant make action plan what step would you like to take to start reducing your intake of sugary drinks?when are you going to do it?how much are you going to decrease them?how often are you going to do it?on a scale of 110 , 1 being not sure at all and 10 being completely sure , how sure are you that you can _ _ _ _ _ _ _ by _ _ _ _ _ _ ? if less than 7 , encourage participant to modify action plan to make it achievable . what step would you like to take to start reducing your intake of sugary drinks ? when are you going to do it ? how much are you going to decrease them ? how often are you going to do it ? on a scale of 110 , 1 being not sure at all and 10 being completely sure , how sure are you that you can _ _ _ _ _ _ _ by _ _ _ _ _ _ ? while the automated phone calls provide participants with passive information and support through narratives , the health coaches directly reach participants , explore their needs , build on their strengths , and set goals to help them reach their health targets . as such , the health coach serves as a bridge between the participant and the primary care clinic and as a source of support , resources , and accountability . figure 2 illustrates the relationship between the health coach , the atsm service , primary care providers , the community , and the participant within the star mama study . in this patient - centered model , the health coach is an integral source of tangible preventive information and longitudinal communication with the participant and health care setting , community , or clinic . this paper includes a sample of women who have either completed star mama or are currently enrolled representing half of the targeted recruitment sample . eligibility criteria for star mama include 1839 years of age , at least 32 weeks pregnant , and either a gdm diagnosis or bmi > 25 . participants were recruited from our four community sites through either physician referral , wic referral , or direct communication during scheduled prenatal appointments . participant engagement was assessed using our online database tracking system , which monitors participant weekly call responses . different levels of engagement were determined based on measures in previous studies : ( 1 ) participant had completed at least one of the weekly phone calls ; ( 2 ) average number of calls completed out of the 20 weeks of calls , and ( 3 ) among those completing one or more call , the percentage of calls completed over the intervention period . all women receive a baseline visit , 3-month short phone survey and a 9-month postpartum follow - up survey and will have their medical charts reviewed over the study period . the follow - up surveys are used to assess feasibility , acceptability , and health related outcomes ( e.g. , weight loss , physical activity , consumption of healthy foods , breastfeeding , replacement of water for sugar sweetened drinks , and glucose screening ) . for the women enrolled in the health - it arm , engagement is tracked through the hit system , in which we monitor the calls women are responding to and the queries they trigger for . a health coach also monitors the participant 's progress through extended phone calls for resources and support . to achieve empirical results regarding the success of the program , key stakeholders in our partner sites were consulted to iteratively assess the implementation of the program . a group of regional and national advisors was assembled to help assess the challenges in integrating the star mama model and to critically evaluate the feasibility and acceptability of this hybrid hit and health coaching model in the community setting . the primary advisors were from wic , and they included research staff , management , and nutritionists . based on discussions , reflections , and informant interviews with our advisors and partners , we were able to articulate key barriers to our model and assess the scope of scalability . case studies were selected from wic sites to represent health coaching calls conducted in community settings . two participants from each the san francisco ( sf ) and sonoma county wic sites were chosen to reflect the diversity of women 's experiences and the diversity of coaching content . table 1 describes key demographic characteristics of the study population ( n = 86 ) . women enrolled were on average 30 years old ; 78% were identified as latina or hispanic and were not born in the us . migrant women lived an average of 10 years in the us and 63% listed spanish as their preferred language . the women had , on average , two children below the age of 18 currently living in their household . eighty - seven percent of women were diagnosed with gestational diabetes previously , of which 97% were diagnosed during their most recent pregnancy . thirty - six percent were obese , overweight , or experience unhealthy weight gain during their pregnancy . since the star mama program enrollment is ongoing , engagement data is representative of the first wave of enrollment , accounting for almost half of the recruitment target . among the study subsample ( n = 86 ) , twenty - eight women have completed all 20 weeks of the hit and health coaching program . of those women , 89% answered at least 1 phone call , with an average of 12 total phone calls answered out of 20 weekly calls . on average , the women answered 61% of calls during the intervention period . among all women randomized to the star mama hit and health coaching arm ( n = 43 ) , excluding those who withdrew from the study or are lost to follow - up , 86% have answered at least one phone call till date ( figure 4 ) . the women receiving the star mama hit program through weekly calls were at high risk and triggered for poor physical activity , high carbohydrate , fat or sugar consumption , signs of depression of feeling overwhelmed , and more . the length of the health coaching callbacks ranged from short follow - ups of 3 minutes to up to 45 minutes in some cases , with an average of 9 minutes per call . topics covered ranged from the health - it phone call narrative topics ( diet control , physical activity , depression , cutting back on high fat and sugary foods , breastfeeding , and bottle - feeding ) to miscellaneous health needs of the participants . in table 2 , we describe synopses of coaching calls with relevant actionable items , such as follow - up topics , and community or clinic implications . these stories illustrate accounts of coaching to women who are representative of the enrolled participants in the star mama program . it is evident that the health coach serves as a connection between the woman postpartum and critical resources , including information , basic knowledge and tips about postpartum care , and links to their preferred primary care system . moreover , the health coach is a key point of support not only for the health - it call - based topics , but also for miscellaneous questions the new mother has doubts about . figure 3 demonstrates how the hit component integrates with health coaching and how the coaches use the participant - driven triggers to direct coaching calls and discuss specific and relevant topics during a session . these vignettes illustrate the depth and breadth of issues covered by health coaches during their interactions with participants . they highlight major themes and barriers to self - management postpartum including ( but not limited to ) need for improved resources for child care after delivery , reinforcement for reduction of sugar and fat consumption , goal setting and action planning to improve physical activity , reminders about the importance of follow - up blood sugar testing postpartum and postpartum depression screening . because high risk and low - income postpartum women often do not receive the longitudinal care and support they need to reduce their risk of dm and other chronic illnesses , this is a critical window of opportunity to intervene and provide maximal resources , support , and tools for prevention of chronic illnesses such as diabetes and also help self - manage existing chronic conditions . in this paper , the example coaching calls suggest that health coaches in star mama act as a bridge between a participant and the primary care system to emulate a continuum of care even after delivery . scalable implementation of health coaching , a hit enabled model , or a hybrid hit and coaching model can have community and clinic specific benefits but there are several identified barriers to such an attempt at integration . we investigated these potential barriers with community programs through discussions with our regional advisors at san francisco and sonoma wic . in the following , we articulate three core limitations to health coaching first , community wic programs have boundaries regarding the services they can offer and may face restrictive funding for programs like health coaching and low capacity to train and hire coaches . in the context of wic , while there is a currently funded and high functioning peer coaching program ( loving support peer counseling ) it focuses primarily on breastfeeding and lactation support . as such , peer coaches within the wic infrastructure are not trained to coach women on critical postpartum topics , such as diet , physical activity , postpartum depression , healthy eating tips , and family support . moreover , not all counties within states have the funding allocated for the loving support peer counseling program , and absorbing a hit or health coaching component can strain their budget . a second major barrier for health coaching in community settings for example , within wic , those who provide nutrition counseling are often expert health professionals : dieticians , nurses , or trained diabetes educators . in most sites , though there are few of these expert health professionals available to receive additional training as health coaches for a broader set of concerns outside of nutrition . on the other hand there are often peer coaches , who are more numerous but less well trained , and they are not able to coach beyond a more limited scope , as with an emphasis on breastfeeding and lactation . lastly , a major limitation in prevention in such high risk , low - income populations is that they are very hard to reach and follow - up with [ 16 , 41 ] . women who are at most risk for chronic illnesses like type 2 diabetes , obesity , or depression postpartum are often from the most vulnerable populations , who historically have transient housing situations and may have difficulty engaging in such a program . even with a hit blended approach such as star mama , where a health coach counsels patients over the phone , reaching patients is the biggest barrier in coaching . in particular , women who start working after delivery are the most difficult to contact , with their irregular and often hectic schedules . however , the period after delivery is a very critical time , when women require the most support to adjust to their physical and mental changes after having a baby . it is critical to consider the positive impacts of health coaching and health - it interventions in the clinical settings and develop techniques to execute these strategies in the community . our preliminary conversations with key stakeholders ( county wic staff , star mama health coaches , and national wic advisors ) have outlined the scope of integration of these interventions in the community settings and have addressed the need to expand care through methods like telemedicine . a model like star mama may be daunting to implement within a community structure ; particularly when funding is limited , scopes of practice are restricted , or participants are hard to reach . however , frameworks of self - care management and behavior change using hit , health coaching , or both will be extremely beneficial in the future to improve preventive health practices in communities and potentially mitigate the disease burden that a safety net hospital or community clinic may face . we need more innovative programs to bridge counseling and resources between the patient and provider , facilitated by health information technology . while challenges persist , the flexibility of these interventions and evidence - based success in the clinical setting urges expanding care to community programs .

background . low - income minority women with prior gestational diabetes mellitus ( pgdm ) or high bmis have increased risk for chronic illnesses postpartum . although the diabetes prevention program ( dpp ) provides an evidence - based model for reducing diabetes risk , few community - based interventions have adapted this program for pgdm women . methods . star mama is an ongoing randomized control trial ( rct ) evaluating a hybrid hit / health coaching dpp - based 20-week postpartum program for diabetes prevention compared with education from written materials at baseline . eligibility includes women 1839 years old , 32 weeks pregnant , and gdm or bmi > 25 . clinic- and community - based recruitment in san francisco and sonoma counties targets 180 women . sociodemographic and health coaching data from a preliminary sample are presented . results . most of the 86 women included to date ( 88% ) have gdm , 80% were identified as hispanic / latina , 78% have migrant status , and most are spanish - speaking . women receiving the intervention indicate high engagement , with 86% answering 1 + calls . health coaching callbacks last an average of 9 minutes with range of topics discussed . case studies presented convey a range of emotional , instrumental , and health literacy - related supports offered by health coaches . discussion . the dpp - adapted hit / health coaching model highlights the possibility and challenge of delivering dpp content to postpartum women in community settings . this trial is registered with clinicaltrials.gov nct02240420 .

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

following pregnancy , low - income , minority women with a history of gdm or high bmis are at high risk for chronic illnesses , particularly obesity , type 2 diabetes mellitus ( dm ) , and postpartum depression [ 1 , 2 ] . results from the diabetes prevention program ( dpp ) suggest that the long - term risk for diabetes postpartum can be reduced through behavior change [ 1214 ] . current strategies to expand postpartum care management and to reduce chronic disease risk for ethnic minority women include tailored interventions that address health literacy and provide accessible resources or health coaching on lifestyle changes , but there are few programs that focus on delivering the dpp content for early postpartum women in the period immediately following birth . one well - known and relevant community program that offers health coaching for this population is the women , infants , and children ( wic ) program , which provides supplemental foods , health care referrals , and nutrition education for low - income pregnant and postpartum women . in some wic locations , , we present a unique health coaching model , the star mama program ( support via telephone advice and resources / sistema telfonico de apoyo y recursos - mama ) , which combines hit - based queries and narratives , with follow - up by trained health coaches , to deliver adapted dpp content . the goals of this paper are to ( 1 ) illustrate a unique model of health coaching for high risk , low - income postpartum women which has relevance in community and clinic - based systems and ( 2 ) present case studies of exemplary star mama health coached participants stories . star mama is an ongoing randomized clinical trial comparing a hit - based health coaching program with a usual care arm providing an educational resource guide covering approaches to improving postpartum diabetes risk behaviors among gdm women based on dpp content . at minimum , each health coach received a two - day training at the university of california san francisco ( ucsf ) center for excellence in primary care and was given a one - day star mama specific training that focused on delivery of the dpp , for a total of three days of health coaching training . the following list titled health coaching training topics , example query , narrative , and health coach script describes a summary of the health coaching training , an example of a star mama automated telephone call , and a guideline for the subsequent health coaching topics . health coaching training topics , example query , narrative , and health coach script health coach training topics communication ask - tell - ask , how to receive information from the participantclosing the loopsetting the agendaunderstanding your current health status , numbers motivational interviewing exploring patients motivations and barriers postpartum risk specific knowledge knowledge regarding risk of dm postpartum community and clinic specific resources community related risks : safety , access to affordable produce , and primary carecommunity - based resources : food banks , wic agencies , free health clinics , and physical activity groups ask - tell - ask , how to receive information from the participantclosing the loopsetting the agendaunderstanding your current health status , numbers ask - tell - ask , how to receive information from the participant understanding your current health status , numbers motivational interviewing exploring patients motivations and barriers exploring patients motivations and barriers postpartum risk specific knowledge knowledge regarding risk of dm postpartum knowledge regarding risk of dm postpartum community and clinic specific resources community related risks : safety , access to affordable produce , and primary carecommunity - based resources : food banks , wic agencies , free health clinics , and physical activity groups community related risks : safety , access to affordable produce , and primary care community - based resources : food banks , wic agencies , free health clinics , and physical activity groups health coaching example query to participant . eligibility criteria for star mama include 1839 years of age , at least 32 weeks pregnant , and either a gdm diagnosis or bmi > 25 . a group of regional and national advisors was assembled to help assess the challenges in integrating the star mama model and to critically evaluate the feasibility and acceptability of this hybrid hit and health coaching model in the community setting . the length of the health coaching callbacks ranged from short follow - ups of 3 minutes to up to 45 minutes in some cases , with an average of 9 minutes per call . because high risk and low - income postpartum women often do not receive the longitudinal care and support they need to reduce their risk of dm and other chronic illnesses , this is a critical window of opportunity to intervene and provide maximal resources , support , and tools for prevention of chronic illnesses such as diabetes and also help self - manage existing chronic conditions . our preliminary conversations with key stakeholders ( county wic staff , star mama health coaches , and national wic advisors ) have outlined the scope of integration of these interventions in the community settings and have addressed the need to expand care through methods like telemedicine .

in september of 1977 , gruentzig performed the first coronary angioplasty as a nonsurgical method for coronary artery revascularization on a 40-year - old patient in zurich , switzerland . the angioplasty in fact induces a controlled injury to the coronary vessel and has two major limitations - acute vessel closure ( 6%-8% ) and restenosis ( 30%-50% ) . the pathophysiology of acute vessel closure after angioplasty involves denudation of the endothelium of the coronary artery followed by rapid accumulation of fibrin and platelets , disruption of the atheromatous plaque with intimal dissection and medial tearing , and elastic recoil . restenosis involves smooth muscle proliferation and neointimal hyperplasia [ 2 , 3 ] . in an attempt to overcome these problems , bare metal stents ( bms ) were introduced into clinical practice in 1986 . bms are metallic scaffolds deployed within a diseased coronary artery segment to optimize the lumen integrity by tacking dissection flaps against the vessel wall and providing mechanical lumen patency . two large clinical trials , the belgium netherlands stent arterial revascularization therapies study ( benestent ) and the north american stent restenosis study ( stress ) [ 3 , 4 ] , showed bms significantly decrease the incidence of target - lesion revascularization from 25%-35% with percutaneous coronary angioplasty ( ptca ) to 10%-15% with stenting . the success in treatment of acute vessel closure came with a price - increased rates of acute ( 24h ) and subacute ( 24h to 30 days ) stent thrombosis , which was addressed with aggressive anticoagulation attempts . the benestent and the stress study reported subacute stent thrombosis of 3.5% and 3.4% respectively despite the complex anticoagulation regimens used ( dextran , aspirin , dipyridamole , heparin , and warfarin ) [ 3 , 4 ] . the introduction of intravascular ultrasound , high pressure balloons during stent deployment , and the establishment of dual antiplatelet therapy ( dapt ) after stent placement contributed to the decrease of bms thrombosis ( currently 1.2% ) [ 5 , 6 ] . the usefulness of a dual antiplatelet therapy was demonstrated by the pci - cure study in which 2658 patients with acute coronary syndrome ( acs ) underwent percutaneous coronary intervention ( pci ) . patients were randomly assigned to one - year treatment with clopidogrel and aspirin ( asa ) or placebo and asa . in this study , an overall 31% reduction ( p = 0.002 ) of cardiovascular mortality or myocardial infarction ( mi ) rate the difference between both groups appears during the first 3 months , and stays constant or slightly increases up to 12 months . the results of the pci - cure study provided the basis for recommending the institution of dapt of clopidogrel and asa for patients presenting with acs , including the patients treated with stents . the purpose of this review is to provide an overview of the changing culture of coronary artery stenting , in addition to discussing perioperative management strategies and controversies surrounding coronary stents and antiplatelet therapy . a comprehensive literature search of medline was conducted using as keywords : antiplatelet therapy , non - coronary surgery , drug - eluting stents , and stent thrombosis . over 250 relevant articles were found , 88 of which we have cited and discussed in this article based on their specific relevance to perioperative management , perioperative bleeding , and perioperative thrombosis in patients with drug eluting stents ( des ) on dapt presenting for invasive procedures , mechanisms of stent thrombosis . currently , there are three categories of antiplatelet agents in use : acetylsalicylic acid ( asa ) , platelet p2y12 receptor inhibitors ( clopidogrel , prasugrel , ticagrelor ) , and platelet gpiib - iiia inhibitors ( eptifibatide , tirofiban , abciximab ) . asa is recommended for primary prevention only for diabetic patients with risk of cardiovascular disease . when used for secondary prevention , asa is a lifelong therapy . in their meta - analyses of 50,279 patients for secondary prevention for coronary artery disease , biondi - zoccai et al . showed that the cardiac complication rate was three times higher after asa withdrawal and increased even more in patients with coronary stents [ 8 , 12 ] . there was , on average , a 10.6-day period between withdrawal from asa and thrombotic events ( 8.5 days for coronary symptoms ) . p2y12 inhibitors include the thienopyridines , clopidogrel and prasugrel , and the cyclopentyl triazolopyrimidine ticagrelor . clopidogrel , a pro - drug , is metabolized to active metabolite in the liver in a two - step process by cyp3a4/3a5 and cyp2b6/1a2/2c9/2c19 esterases . clopidogrel decreases the risk of mi in unstable angina by 18% and the risk of coronary stent thrombosis by 30% . addition of clopidogrel to asa decreases the relative risk in the combined end point of cardiovascular death , mi , or stroke by 20% . prasugrel , also a pro - drug , converts to active metabolite more rapidly , in only one step ( cyp3a4 dependent ) . the metabolite achieves 2.2 times higher level than that of clopidogrel . in phase iii clinical trials ( trilogy - acs ) , prasugrel , when compared to clopidogrel , produced a statistically significant reduction of 19% in the primary endpoint of cardiovascular death , nonfatal mi , or nonfatal stroke in the ua / nstemi population ( p=0.0004 ) . a 34% decline was observed in urgent target vessel revascularization ( p<0.001 ) and a 42% reduction in heart attack with subsequent death from cardiovascular causes ( p=0.02 ) . approval was based on the results of the plato ( platelet inhibition and patient outcomes ) trial , a large ( 18,624 patients in 43 countries ) head - to - head patient outcomes study of ticagrelor versus clopidogrel , both given in combination with asa and other standard therapy . plato showed treatment with ticagrelor for 12 months was associated with a 21% rrr ( relative risk reduction ) in death ( 4% vs. 5.1% ; 1.1% arr ; p=0.001 ) and a 16% rrr in mi compared to clopidogrel ( 5.8% vs. 6.9% ; 1.1% arr ( absolute risk reduction ) ; p<0.005 ) . was delayed due to lack of efficacy in the pre - specified subgroup of patients from north america . though analyses were not able to rule out the possibility of chance as an explanation for the north american subgroup , it was shown that high dose asa 300 mg / day was used far more often in the u.s . than in the rest of the world furthermore , the lowest risk of cardiovascular death , mi , or stroke with ticagrelor compared with clopidogrel is associated with a low maintenance dose of concomitant asa . as a result , the approval came with a black box warning stating that daily asa doses above 100 mg decrease effectiveness of the medication . it is also contraindicated in patients with a history of hemorrhagic stroke because of increased risk of bleeding . ticagrelor does not require metabolic activation for its clinical effects , has only one active metabolite . the food and drug administration ( fda ) recommends stopping ticagrelor 5 days prior to surgical procedures . even though it reversibly binds to platelet p2y12 receptors , there is currently no known reversible agent for ticagrelor and it is not expected to be dialyzable . dyspnea , requiring discontinuation of the treatment , was observed in 14% of the ticagrelor - treated patients compared with 8% in the clopidogrel group . in their new guidelines , the american college of chest physicians suggests ticagrelor 90 mg twice daily plus low dose asa over clopidogrel 75 mg plus low dose asa for patients in the first year after an acs who have undergone pci with stent placement . this is the first time that clinical treatment guidelines have specifically suggested the use of ticagrelor over clopidogrel . platelet gp iib / iiia inhibitors , including tirofiban , hydrochloride , and eptifibatide , block the cross - linking of platelets to fibrinogen , thus inhibiting formation of bridges between the activated platelets and thrombus formation . they are used for the prevention of immediate thrombosis of coronary stents in the first 24 - 48 hours after pci . transition from bms to des restenosis continues to be the weak point of the bms , occurring at a rate of 20%-25% within 6 months of implantation and peaking at 3 months after the stent implantation . it results in acs in about 35% of the patients and repeat revascularization of the restenotic lesions in 60%-80% . bms restenose because the stent struts traumatize the vascular wall and provoke an inflammatory response . this response is followed by an exaggerated proliferation within the media and adventitia , which produces significant neointimal proliferation and occlusion of the stent . in patients with co - morbidities and complex coronary lesions , techniques to treat stent restenosis include ptca , atherectomy , repeat stenting , and brachytherapy ( intra - coronary delivery of a radioactive isotope ) . failure using these techniques was almost 30% , with recurrent restenosis after in - stent ptca up to 85% and thrombotic occlusion after brachytherapy up to 15.6% . coating the bms with a polymer ( containing slowly released antiproliferative material that suppresses the neointimal hyperplasia ) decreased the in - stent restenosis from 20% to 4%-6% and significantly decreased of the rate of re - intervention [ 26 , 27 ] . the first generation des uses sirolimus and paclitaxel as antiproliferative agents to suppress the vascular smooth muscle cell migration and proliferation . sirolimus is completely released from the polymer within 4 to 6 weeks , while only 10% of the paclitaxel is released within 90 days ( the other 90% remains sequestered indefinitely ) . des were approved by the fda in april of 2003 ( sirolimus - eluting des ) and march of 2004 ( paclitaxel des ) . the fda approval was based on the results of randomized trials that involved selected patient populations . the indications included patients with symptomatic ischemic disease due to de novo lesions of length < 30 mm ( sirolimus eluting stents ) and < 28 mm ( paclitaxel eluting stents ) in native coronary arteries with reference vessel diameter of > 2.5 mm to < 3.5 mm ( < 3.7 mm for paclitaxel stents ) . clinical trials investigating the restenosis of stents found a 74% reduction of restenosis at 4 years of implantation . as a result , the use of des significantly increased ( up to 85% of all stents placed ) in the u.s . and the des were used off label in high - risk populations ( diabetics , patients with acs , low ejection fraction , or renal failure ) , high - risk lesions ( bifurcating lesions , long ones , small vessel lesions , in stent lesions , multiple lesions , left main disease , saphenous vein graft lesions ) , or other conditions that were excluded from the initial trials . stent thrombosis and des in 2003 , 290 cases of subacute stent thrombosis occurring after sirolimus des implantation were reported to the fda along with a 20% mortality rate . the goal of the basel stent kosten effektivitts trial - late thrombotic events ( basket - late ) study was to determine the true incidence of late stent thrombosis , mi , and death in 746 patients randomized to receive des or bms . patients on dapt for 6 months without any adverse cardiac events had clopidogrel stopped and were followed for an additional 12 months . results showed the following : late stent thrombosis - related events ( death and mi ) occurred two to three times more frequently in patients with des than those with bms;late stent thrombosis carried a four times higher risk of cardiac death / mi ( p < 0.00010);late stent thrombosis and its complications occurred up to 1 year after clopidogrel discontinuation . late stent thrombosis - related events ( death and mi ) occurred two to three times more frequently in patients with des than those with bms ; late stent thrombosis carried a four times higher risk of cardiac death / mi ( p < 0.00010 ) ; late stent thrombosis and its complications occurred up to 1 year after clopidogrel discontinuation . authors concluded that while des use in 100 patients avoids five target lesion revascularization events at 6 months , it unfortunately leads to 3.3 late ( within 18 months ) deaths or mi . at the 55world congress of cardiology , two meta - analyses were presented , showing a significant increase in the rate of total mortality and q wave mi in des compared to bms at after 12 months and up to 3 years [ 40 , 41 ] . experimental models of des demonstrate incomplete healing , fibrin deposition , and inflammatory cells , indicating a hypersensitivity reaction [ 2 , 42 ] while bms demonstrate complete endothelialization at 28 days . sirolimus and paclitaxel have shown to impair endothelial function both within the stent and in the distal coronary artery , leading to delayed arterial healing of the stent itself , as well as enhancing the risk for distal arterial ischemia and coronary occlusion . in addition , they enhance expression of the endothelial tissue factor , which creates a prothrombotic state . despite equal stenosis severity and follow - up duration , patients with des , compared to bms , more frequently have collaterals insufficient to prevent ischemia during occlusion . the most powerful histological predictor of stent thrombosis has been incomplete endothelial coverage of the stent . several studies and registries have identified clinical predictors for delayed endothelial coverage and thrombosis of des . acs , left ventricular ejection fraction < 30% , treatment of bifurcating lesions , renal insufficiency , and diabetes have shown to be strong predictors of stent thrombosis [ 41 , 45,46,47,48,49,50 ] . the concerns about late stent thrombosis resulted in an emergency fda advisory panel meeting in december 2006 , which reassured that des in the studied on - label settings appear safe and efficacious , but warned that data regarding safety and efficacy in off - label situations is not available and will likely not match results seen in the lower - risk on - label settings . multiple registry studies have since shown that off - label des use is indeed associated with a roughly two- to threefold increase in clinical adverse events , including stent thrombosis , compared with on - label use . despite these findings , it is clear that des remain superior to bms even in high - risk off - label situations . recently , a comprehensive meta - analysis of almost 10,000 randomized controlled trial patients and over 180,000 observational study patients confirmed the overall benefit of des over bms in both on - label and off - label populations . evidence showed trends to reduced ( randomized trials ) or significantly reduced ( observational studies ) death and mi , and dramatic significant reductions in target vessel revascularization ( regardless of study type ) . antiplatelet therapy and des controversy surrounding the des late thrombosis issue creates a controversy about the length of antiplatelet therapy in patients with des . the initial recommendations made by the fda / american college of cardiology ( acc)/american heart association ( aha)/society for cardiovascular angiography interventions ( scai ) and the stent manufacturers were completely arbitrary . they advised patients to remain on dapt for a minimum of 3 months after the implantation of sirolimus des and 6 months after paclitaxel des followed by life - long asa therapy . in 2005 , a focused update of the acc / aha / scai pci guidelines recommended that all patients who receive a des should be given clopidogrel for at least 12 months in the absence of an increased risk of bleeding . with the growing number of publications concerning the safety of des , the fda published a scientific advisory in january of 2007 , endorsed by 5 major professional societies : aha / acc / scai , the american college of surgeons ( acs ) , and the american dental association ( ada ) . once again , the importance of 12 months dapt after placement of des and life - long asa therapy was emphasized . once further , in the 2011 accf / aha / scai guidelines for pci , the role of dapt for prevention of thrombosis in patients with stents was strongly re - enforced with class ib recommendation for treatment with p2y12 inhibitor for at least 12 months after pci with des in addition to indefinite therapy with asa and class ii b recommendation for consideration of dapt beyond 12 months . dapt trial , which is currently ongoing , compares 12 versus 30 months of dapt among 15 000 patients treated with des . this trial is powered to assess the primary efficacy endpoints of differences in stent - thrombosis rates and major adverse cardiovascular / cerebrovascular events ( macce ) , with a primary safety endpoint of major bleeding . currently , around 60% of patients undergoing pci receive des and are placed on dapt for at least one year . it is suggested that 5% of these patients will require non - cardiac surgery during this time , posing a unique challenge during the perioperative period . des management - non - cardiac surgery the decision to stop antiplatelet medications before invasive procedures in order to decrease the risk of bleeding may expose patients with des to increased risk for stent thrombosis , mi , and cardiac death . conversely , continuing antiplatelet therapy in order to prevent stent thrombosis may expose patients to increased risk of bleeding and need for transfusions during invasive procedures . current literature on the use of antiplatelet agents in surgery reports the average surgical blood loss increase by asa is approximately 2.5% - 20% . in non - cardiac surgery , a meta analysis of 474 studies on the impact of low dose asa on surgical blood loss showed that asa alone increases the average intraoperative hemorrhagic risk by a factor of 1.5 , but does not increase mortality and morbidity . possible exceptions may be intracranial neurosurgery and transurethral prostatectomy where asa has been a contributing factor to fatal outcomes [ 12 , 59 ] . the major side effect of clopidogrel is increased risk of spontaneous hemorrhage by 38% ( incidence 1%-2% ) . in non - cardiac surgery increased bleeding has been described in transbronchial biopsy and pacemaker and defibrillator implantation [ 60 , 61 ] . prasugrel is a 10 times more potent platelet inhibitor than clopidogrel ; however , it is associated with a statistically significant increase in non - cabg ( coronary artery bypass grafting ) major bleeding ( 2.4% vs. 1.8% , p=0.03 ) and fatal bleeding ( 0.4% vs. 0.1% , p=0.002 ) compared to clopidogrel . although ticagrelor is a reversible p2y12 inhibitor , the plato trial showed higher incidence of non - cabg related bleeding ( rr 1.18 ) and significantly increased incidence of fatal intracranial bleeding ( rr 10.95 ) . with dapt , the bleeding time increases three- to fourfold over asa alone , and surgical blood loss increases by an average of 30% to 50% . significant bleeding in patients on dapt undergoing different non - cardiac surgical procedures has been described . however , other case reports and series have not found such association [ 57 , 67,68,69 ] . some surgical procedures are associated with significant mortality and morbidity if bleeding is encountered , such as intracranial or spine surgery , where even a small amount of bleeding can cause brain or cord compression and irreversible brain or cord damage . the surgical bleeding and transfusion rates in the rest of the surgical procedures , although increased by 50% , were not associated with an increase in mortality and morbidity . risk of perioperative thrombosis the incidence of major coronary adverse events after pci is estimated around 4% to 5% , and 20% to 45% of these events can result in death [ 56 , 70 ] . patients are most vulnerable immediately after a pci because the stenotic lesion is transformed into an unstable area due to the rupture of its endothelial covering . when undergoing surgery during this early period , the rate of mortality ( 30% ) and morbidity ( 20 - 40% ) is 5 to 10 times higher than matched patients undergoing the same operation under maximal medical therapy or after appropriate delay [ 68 , 71 ] . premature discontinuation of antiplatelet therapy has been found to be the strongest predictor of stent thrombosis , carrying significant risk of mortality and morbidity even when the discontinuation is not associated with surgery [ 38 , 48 , 72 ] . as long as des have not endothelialized , the patient is absolutely dependent on the antiplatelet medications for stent thrombosis prevention . surgery itself produces a prothrombotic and proinflammatory state that increases the risk of stent thrombosis . the stress response to surgery includes sympathetic activation and cytokine release that promotes shear stress on arterial plaques , enhanced vascular reactivity conductive of vasospasm , increased platelet activation , and increased hypercoagulability [ 73 , 74 ] . perioperative management currently , there is no definite standard of care for the perioperative management of patients with coronary stents , though deep understanding of the mechanisms of des and the indications for antiplatelet therapy could ensure patient safety and optimize outcome . a survey conducted on 374 interventional cardiologists found that although there is agreement among interventional cardiologists on the optimum delay for surgery after stenting , on the need for bms or balloon angioplasty alone if early noncardiac surgery is needed , and on treatment of perioperative thrombosis , there is significant inconsistency on the optimum antiplatelet therapy for patients who need surgery early after stent implantation . if the physicians most intimately involved with the management of patients with des do not agree on how to manage antiplatelet therapy perioperatively , we may speculate the chance of having consensus among anesthesiologists , surgeons , internists , and other interventional physicians on this topic is minimal . it is likely that the full implications of stopping antiplatelet therapy are not fully appreciated , and a reasonable fear of bleeding predominates . different algorithms for perioperative antiplatelet therapy management in patients with stents have been suggested [ 75 , 77,78,79,80,81 ] , but there are no prospective studies evaluating those algorithms . table 2 summarizes recent recommendations on the decision to proceed with antiplatelet therapy and/or surgical intervention , taking into account bleeding risk . until such studies are conducted and evidence - based guidelines are established , every institution should have well - publicized policies and guidelines to manage these patients . patients with stents , especially des , should be identified early in the preoperative work - up . each case should be managed on an individual basis and the risk and consequences of stent thrombosis should be weighed against those of perioperative bleeding . des = drug eluting stents ; asa = aspirin ; dapt = dual antiplatelet therapy . according to the 2011 accf / aha / scai guidelines , even before considering stent implantation , patients should be evaluated for possibility of surgery in the following 12 months and should not be treated with des if such possibility exists . percutaneous angioplasty or bms , requiring a minimum of 4 to 6 weeks of antiplatelet therapy , or medical management only if the surgery can not be delayed , should be considered instead . routine prophylactic coronary revascularization should not be performed in patients with stable coronary artery disease before non - cardiac surgery because of possible significant harm to the patient [ 55 , 56 ] . once patients present for surgery with a stent in place , consideration should be given to the electiveness of the surgical procedure . elective surgery should not be performedwithin 4 to 6 weeks of stent placement or within 12 months of des placement in patients who s antiplatelet therapy will need to be discontinued perioperatively [ 55 , 56 ] . assessed the perioperative outcome of patients undergoing non - cardiac surgery after coronary stent implantation in a single center registry . after multivariable analysis , the predictor of primary endpoint , defined as perioperative occurrence of major adverse events , was the time interval between stenting and surgery with a statistically significant increase in the number of events when surgery was performed within 6 weeks of bms placement . there was no difference in the major bleeding between the groups with different antiplatelet regimens . once again , these results supported the aha / acc recommendations on timing of non - cardiac surgery after stent implantation . if a patient with des presents for an elective procedure more than one year after the stent placement and still on dapt , the management of the antiplatelet medications will depend on the bleeding risk of the specific surgical procedure and on the complexities of the des ( thrombotic risk ) . grouped the surgical procedures into three groups according to their bleeding risk : low bleeding risk surgical procedures that usually do not require blood transfusion , such as peripheral , plastic , and minor general surgery ; biopsies ; minor orthopedic procedures ; minor ent ( ear - nose - throat ) procedures ; endoscopies ; anterior chamber of the eye surgeries ; or dental extractions and dental surgery.intermediate bleeding risk surgical procedures that frequently require blood transfusions . examples can be visceral surgery ; cardiovascular surgery ; major orthopedic , ent , and reconstructive surgery ; or endoscopic urology.high bleeding risk possible bleeding in a closed space such as intracranial neurosurgical procedures ; spinal canal surgery ; or posterior chamber of the eye surgery . low bleeding risk surgical procedures that usually do not require blood transfusion , such as peripheral , plastic , and minor general surgery ; biopsies ; minor orthopedic procedures ; minor ent ( ear - nose - throat ) procedures ; endoscopies ; anterior chamber of the eye surgeries ; or dental extractions and dental surgery . examples can be visceral surgery ; cardiovascular surgery ; major orthopedic , ent , and reconstructive surgery ; or endoscopic urology . high bleeding risk possible bleeding in a closed space such as intracranial neurosurgical procedures ; spinal canal surgery ; or posterior chamber of the eye surgery . there is a growing agreement that dapt needs to be continued indefinitely in patients with complex stent placing procedures , such as stenting of bifurcating lesions , left main stents , overlapping stents , stents within stents , small vessel stents , multiple stents , saphenous vein graft stents , chronically occluded stents , or in patients with co - morbidities , such as diabetes , low ejection fraction , end stage renal disease , malignancies , advanced age , or with resistance to antiplatelet medications and a history of stent occlusion / thrombosis . for surgical procedures with low risk for bleeding intermediate - bleeding - risk surgical procedures should be approached on a case - by - case basis . in patients with complex stent procedures and co - morbidities , the dapt should be continued despite the risk for increased bleeding . in all other patients , clopidogrel and ticagrelor can be stopped 5 days before surgery and prasugrel 7 days before surgery , while asa should be continued . dapt should be restarted as soon as possible postoperatively ( ideally within 24 hours ) with a loading dose of 300 mg - 600 mg clopidogrel , or prasugrel 60 mg , or ticagrelor 180 mg [ 57 , 83 ] . patients with 12 months completed of dapt and low risk for thrombosis of the coronary artery stents can stop clopidogrel and ticagrelor 5 days prior to surgery and prasugrel 7 days before surgery while continuing asa . asa discontinuation even more than one year after des placement may lead to stent thrombosis [ 83 , 84 ] . for patients with coronary stents with high risk for thrombosis presenting for high - risk bleeding surgical procedures , bridge therapy has been suggested . use of short - acting gp iib / iiia inhibitors , such as tirofiban or eptifibatide , has been proposed as a bridge between the time of the thienopyridine discontinuation and surgery . either medication is given as infusion and requires patient admission to the hospital 3 days after the discontinuation of thienopyridines . infusion is stopped 4 to 6 hours prior to surgical procedure and restarted as soon as possible after the surgery upon agreement between cardiology and surgery [ 85 , 86 ] . usually , heparin infusion commences , though there is no evidence supporting that heparin offers efficient protection in high - risk coronary situations . nonsteroidal anti - inflammatory drugs ( nsaids ) , such as ibuprofen , inhibit cox-1 , as asa does . since action is reversible and platelet function is completely recovered within 24 hours of their discontinuation , nsaids are suggested as an alternative to asa . however , all of these bridging techniques are controversial , with little data published to support use , and are associated with an increased cost . cangrelor , an investigational parenteral , reversible , direct p2y12 platelet inhibitor with its extremely short ( 5 to 9 minutes ) half - life , may present an alternative for bridge therapy in the near future . in the recently published results from the bridge ( maintenance of platelet inhibition with cangrelor ) trial , cangrelor was effective at maintaining platelet inhibition in patients on thienopyridines who required bypass surgery . this prospective , randomized , double - blind , placebo - controlled , multicenter trial evaluated 210 patients on thienopyridine therapy awaiting cabg . the thienopyridine was stopped and patients were randomized to treatment with cangrelor or placebo for at least 48 hours which then was stopped 1 to 6 hours prior to surgery . the cangrelor group had low levels of platelet reactivity throughout the treatment period compared to patients on placebo . excessive cabg related bleeding occurred in 11.8% of patients on cangrelor vs 10.4% in patients on placebo . there was no difference in major bleeding prior to cabg , although minor bleeding was slightly higher in the cangrelor group . in surgical procedures where even a small postoperative hemorrhage can have disastrous consequences , such as intracranial surgery , spinal surgery in the medullary canal , and surgery of the posterior chamber of the eye , both p2y12 inhibitors and asa may need to be discontinued 7 days prior to surgery . management of patients with stents in need of urgent surgical procedures depends on the time interval between des placement and the surgical procedure . if the time is less than one year ( or less than one month for bms ) , then the dapt has to be continued throughout , except for surgeries in enclosed spaces . less invasive surgical techniques and alternative treatments should be considered in such patients . if patients present for an urgent surgical procedure more than 12 months after the des placement , the same paths as elective surgical procedures should be followed . future developments of des management current efforts are directed towards creating reversible short - acting platelet inhibitors that can be used in the perioperative period , creating des with more predictable endothelialization , and developing point - of - care tests of platelet function that can help the perioperative management of patients on antiplatelet therapy . even though both the champion platform study and the champion pci trial , which compared clopidogrel and cangrelor , were terminated early for the lack of efficacy end points , cangrelor may prove valuable in the perioperative period as a bridge therapy as shown by the results of the bridge trial . although this path still involves hospital admission ahead of planned surgery and requires iv infusion which will be associated with increased cost , cangrelor may provide better platelet inhibition than that of the currently proposed combination of gpiib / iiia inhibitors and heparin . a new generation des was introduced in 2008 : zotarolimus des system , which uses phosphorylchlorine - base , biocompatible polymer and the everolimus des , approved in july 2008 . recently , the results of the isar - test trial were published showing that a polymer - free dual - drug sirolimus- and probucol - eluting stents are non - inferior to the second generation zotarolimus - eluting stent . new platforms ( cobalt - chromium and platinum - chromium ) , new delivery systems , new polymers allowing better biocompatibility and/or flexibility and will attempt to decrease the rate of late stent thrombosis and expand stent indications . in february 2012 , the fda approved the first des for use in patients with diabetes based on the results of the resolute trial - the resolute integrity zotarolimus stent . several days later , based on the results of the horizons - ami trial , the fda approved the first des for treatment of acute myocardial infarction - the ion paclitaxel eluting stent . alternatively to the traditional bms and des , a simple chemical coating may be effective in preventing thrombotic events , shortening the duration of required antiplatelet therapy , and allowing for safe perioperative discontinuation of antiplatelet agents . coatings such as titanium - nitride - oxide , dimethyl sulfoxide , cd34 antibodies and stents containing an integrilin - binding cyclic arg - gly - asp peptide are being explored . in recent years , there has also been a significant effort in developing point - of - care tests for assessment of the effects of the antiplatelet drugs for guidance of the perioperative management of patients on antiplatelet therapy . the gold standard is still light transmittance aggregometry , but the test requires significant sample preparation , special equipment , and trained personnel and is difficult to use in everyday practice . plateletworks , verifynow , and thromboelastography platelet mapping are some current available tests that are not as reliable as the light transmittance aggregometry and still require special equipment and trained personnel . understanding the risks of stopping antiplatelet therapy by the healthcare professionals is of paramount importance . use of a multidisciplinary team approach , including the interventional cardiologist , anesthesiologist , and surgeon , to guide perioperative management is imperative . invasive procedures on patients with des , at high risk for stent thrombosis , and dapt discontinued should be performed in centers with around - the - clock invasive cardiology services as the primary option . however , smaller institutions may not have such coverage , and should not be fully excluded from performing invasive procedures due to the overall high volume of stent implantation . outpatient surgery on such patients should not be performed , and ideally , patients should be kept in the hospital for at least 48 hours for cardiac monitoring or until antiplatelet medications are restarted .

significant advancements in percutaneous treatment of coronary artery disease have been achieved with the introduction of bare metal stents . they have two major drawbacks : acute / subacute stent thrombosis , successfully managed with antiplatelet therapy immediately after stent implantation ; and in - stent restenosis , prevention of which has been achieved with the development of drug - eluting stents . drug - eluting stents have become preferred therapy for patients undergoing coronary artery intervention , though reports of late stent thrombosis have led to uncertainty about the duration of antiplatelet therapy after drug - eluting stents placement . much controversy remains regarding perioperative management of patients with these devices , presenting for surgery or other invasive procedures . the purpose of this review is to provide an overview of the changing culture of coronary artery stenting , in addition to discussing perioperative management strategies and controversies surrounding coronary stents and antiplatelet therapy . a comprehensive literature search of medline was conducted using as keywords : antiplatelet therapy , non - coronary surgery , drug - eluting stents , and stent thrombosis . there is no definite standard of care for the perioperative management of drug - eluting stents in patients with drug - eluting stents . however , there is a growing understanding of the importance of continuation of drug - eluting stents in the perioperative period in order to prevent stent thrombosis along with a concern about the possibility of increased bleeding . appropriate timing of surgery after coronary artery stenting , team approach to the perioperative management of such patients with involvement of cardiologist , anesthesiologist , and surgeon , and development of an individual plan for each patient , weighing that patient s risk of thrombosis vs the risk of bleeding , could improve patient safety and optimize outcome .

Introduction

the introduction of intravascular ultrasound , high pressure balloons during stent deployment , and the establishment of dual antiplatelet therapy ( dapt ) after stent placement contributed to the decrease of bms thrombosis ( currently 1.2% ) [ 5 , 6 ] . the purpose of this review is to provide an overview of the changing culture of coronary artery stenting , in addition to discussing perioperative management strategies and controversies surrounding coronary stents and antiplatelet therapy . a comprehensive literature search of medline was conducted using as keywords : antiplatelet therapy , non - coronary surgery , drug - eluting stents , and stent thrombosis . over 250 relevant articles were found , 88 of which we have cited and discussed in this article based on their specific relevance to perioperative management , perioperative bleeding , and perioperative thrombosis in patients with drug eluting stents ( des ) on dapt presenting for invasive procedures , mechanisms of stent thrombosis . it is also contraindicated in patients with a history of hemorrhagic stroke because of increased risk of bleeding . they are used for the prevention of immediate thrombosis of coronary stents in the first 24 - 48 hours after pci . once further , in the 2011 accf / aha / scai guidelines for pci , the role of dapt for prevention of thrombosis in patients with stents was strongly re - enforced with class ib recommendation for treatment with p2y12 inhibitor for at least 12 months after pci with des in addition to indefinite therapy with asa and class ii b recommendation for consideration of dapt beyond 12 months . des management - non - cardiac surgery the decision to stop antiplatelet medications before invasive procedures in order to decrease the risk of bleeding may expose patients with des to increased risk for stent thrombosis , mi , and cardiac death . conversely , continuing antiplatelet therapy in order to prevent stent thrombosis may expose patients to increased risk of bleeding and need for transfusions during invasive procedures . premature discontinuation of antiplatelet therapy has been found to be the strongest predictor of stent thrombosis , carrying significant risk of mortality and morbidity even when the discontinuation is not associated with surgery [ 38 , 48 , 72 ] . perioperative management currently , there is no definite standard of care for the perioperative management of patients with coronary stents , though deep understanding of the mechanisms of des and the indications for antiplatelet therapy could ensure patient safety and optimize outcome . a survey conducted on 374 interventional cardiologists found that although there is agreement among interventional cardiologists on the optimum delay for surgery after stenting , on the need for bms or balloon angioplasty alone if early noncardiac surgery is needed , and on treatment of perioperative thrombosis , there is significant inconsistency on the optimum antiplatelet therapy for patients who need surgery early after stent implantation . if the physicians most intimately involved with the management of patients with des do not agree on how to manage antiplatelet therapy perioperatively , we may speculate the chance of having consensus among anesthesiologists , surgeons , internists , and other interventional physicians on this topic is minimal . routine prophylactic coronary revascularization should not be performed in patients with stable coronary artery disease before non - cardiac surgery because of possible significant harm to the patient [ 55 , 56 ] . assessed the perioperative outcome of patients undergoing non - cardiac surgery after coronary stent implantation in a single center registry . there is a growing agreement that dapt needs to be continued indefinitely in patients with complex stent placing procedures , such as stenting of bifurcating lesions , left main stents , overlapping stents , stents within stents , small vessel stents , multiple stents , saphenous vein graft stents , chronically occluded stents , or in patients with co - morbidities , such as diabetes , low ejection fraction , end stage renal disease , malignancies , advanced age , or with resistance to antiplatelet medications and a history of stent occlusion / thrombosis . future developments of des management current efforts are directed towards creating reversible short - acting platelet inhibitors that can be used in the perioperative period , creating des with more predictable endothelialization , and developing point - of - care tests of platelet function that can help the perioperative management of patients on antiplatelet therapy . alternatively to the traditional bms and des , a simple chemical coating may be effective in preventing thrombotic events , shortening the duration of required antiplatelet therapy , and allowing for safe perioperative discontinuation of antiplatelet agents . in recent years , there has also been a significant effort in developing point - of - care tests for assessment of the effects of the antiplatelet drugs for guidance of the perioperative management of patients on antiplatelet therapy . use of a multidisciplinary team approach , including the interventional cardiologist , anesthesiologist , and surgeon , to guide perioperative management is imperative .

functional networks can be defined by a set of elements with time - variant properties altogether that interact with each other identification of the constituents of a functional network is necessary in order to delineate it ( mcintosh , 1999 ; bressler and menon , 2010 ) . related to brain systems , for any brain region to be considered as a part of a functional neural network , it must be shown that , interacting with a specific group of areas , is engaged in a particular function or process ( bressler and menon , 2010 ) . in recent years , neuroscience has witnessed the appearance of numerous approaches for modeling functional brain networks ( guye et al . , 2008 ; bullmore and sporns , 2009 ; mcintosh , 2011 ) . connections among elements of a network can be characterized as undirected links , this is functional connectivity , or as directed links , this is effective connectivity ( sporns and tononi , 2007 ; friston , 2009 ; bressler and menon , 2010 ) . functional connectivity models establish symmetrical correlations between brain regions . in functional magnetic resonance imaging ( fmri ) usually involves the pairwise or partial correlation of bold signal among different voxels across the brain . in electrophysiology the phase - locking value ( plv ) and spectral coherence refer to the synchronization of the phases of oscillatory signals or the correlation between oscillatory signals ( combining power and phase ) , respectively ( castellanos et al . , 2011 ; fell and axmacher , 2011 ; cohen et al . , 2011 ) . let 's say , whether synchronization arise via master - slave or by mutual entrainment processes ( penny et al . , 2009 ) . these methods imply the specification of a causal model including structural parameters , which is not necessary in functional connectivity ( sporns and tononi , 2007 ; stephan and friston , 2007 ; friston , 2009 ; mcintosh , 2011 ; valdes - sosa et al . , 2011 ; although see vicente et al . , 2011 ) . most of the current conceptualizations of wm considers that it is a distributed system in which pfc and mtl interact with posterior cortices ( mainly inferior temporal cortex , itc ) , modulating its activity by enhancing relevant and suppressing irrelevant information - related activity ( ranganath and d'esposito , 2005 ) . from this perspective it is assumed that wm maintenance processes constitute the engagement of the same neural networks that support online processing ( fuster , 1995 ; ruchkin et al . , 2003 ; pasternak and greenlee , 2005 ; d'esposito , 2007 ) , and three core interactions can be highlighted : pfc - itc ; mtl - itc ; and pfc - mtl . thus , a pfc - itc interaction is modeled computationally as coupled attractor networks ( i.e. , network of neurons with excitatory interconnections that can settle into a stable pattern of firing rolls , 2010b ) , in which a wm module ( i.e. , pfc ) exerts a top - down modulatory influence in a perceptual module ( i.e. , itc ) ( renart et al . , 1999 , 2001 ) . interaction of hippocampus with content - specific regions in the itc via rhinal cortex ( fransn , 2005 ; chapman et al . , 2008 ; vitay and hamker , 2008 ; olsen et al . , 2009 ; zeithamova and preston , 2010 ; schwindel and mcnaughton , 2011 ) has been also suggested as a mechanism for the reinstatement of specific information processed or stored in those areas ( rolls , 2000 ; woloszyn and sheinberg , 2009 ; szatmary and izhikevich , 2010 ) . this interaction is proposed to be controlled by mtl based on short - term couplings from neural assemblies in mtl to corresponding assemblies in the neocortex ( cartling , 2001 ) . finally , recent evidence from animal studies suggest that the interplay between pfc and mtl , especially hippocampus , is key for normal wm function ( benchenane et al . , 2011 ; for review see colgin , 2011 ; gordon , 2011 ) , although there remains uncertainty about the functional role of this interaction ( gordon , 2011 ) . while the precise mechanism mediating the interaction between two brain regions is not known , synchronization of neural oscillatory activity may represent a mechanism for inter - regional communication ( for a review see fries , 2005 ; wang , 2010 ) . in networks of synchronized neurons , phase synchronization supports neural communication and induce synaptic plasticity between the interacting regions ( duzel et al . , 2010 ; jutras and buffalo , 2010 ; fell and axmacher , 2011 ) . recent models propose that theta oscillations play an important role in the interplay among the neural populations involved in memory processes by integrating widespread local circuits , that are organized in high frequencies , through phase synchronization ( canolty and knight , 2010 ; young , 2011 ; for reviews see jutras and buffalo , 2010 ; battaglia et al . , theta oscillations appear preferentially in the hippocampus , in the mtl and in other brain regions interconnected with the hippocampus ( dzel et al . we will review neuroimaging studies focusing on characterizing the functional interactions among pfc - mtl - posterior cortex , and how their findings adjudicate between the above mentioned proposals . in order to investigate the neural interactions underlying wm operations , most of these studies typically examine how connections among the elements of the network are modified when mnemonic demands are systematically modified , mainly parametric manipulation of memory load and/or the use of delay intervals of variable length . the rationale is that if certain interaction is significantly involved in wm , then the parameters of such interaction ( i.e. , strength or directionality of connection ) should be modulated by the systematic modification of task requirements ( van vugt et al . , 2010 ; colgin , 2011 ; fell and axmacher , 2011 ) . changes in network interactions as a function of wm load have been explored in several studies providing some hints about the dynamics of wm operations ( see table 1 ) . that pfc exerts a top - down modulation of the activity in content - representing posterior areas as a mechanism underlying maintenance of verbal or visual information is partially supported by findings showing load - dependent functional connectivity between pfc and itc ( fiebach et al . , 2006 ) , or enhanced phase synchronization between frontal and temporal regions in the gamma ( axmacher et al . , 2008a ) or theta range ( cashdollar et al . only a recent study made an exception to this observation ( rissman et al . , 2008 ) . hence , this load - induced enhancement could reflect a top - down selective strengthening of representation and maintenance of relevant information ( payne and kounios , 2009 ) . consistent with the role of pfc in modulating neural activity in posterior regions selectively related to relevant stimuli are findings from several studies using functional connectivity measures ( sauseng et al . , 2005 ; yoon et al . , 2006 ; gazzaley et al . , 2007 ; protzner and mcintosh , 2009 ; see also edin et al . , 2009 ) importantly , these findings were causally supported by recent studies using transcranial magnetic stimulation ( feredoes et al . , 2011 ; zanto et al . , 2011 ) . also related to this issue , complexity of manipulation performed on information actively maintained modulate prefrontal - posterior coupling in theta or alpha frequency ranges ( sauseng et al . , 2005 , 2007 ) , further supporting the idea of a top - down control process , and suggesting that phase coupling is a probable operational mechanism ( zanto et al . , 2011 ) . as we introduced above , functional coupling between pfc and mtl , 2009b ) or letters ( finn et al . , 2010 ) using a modified stenberg paradigm led to a greater connectivity strength between pfc and mtl structures , either hippocampus or parahippocampus . theta oscillation has been proposed as a potential neurophysiological mechanism mediating this interaction ( mitchell et al . , 2008 this proposal has been supported by studies demonstrating phase - locking of pfc spiking activity with the hippocampal theta cycle in animals during wm tasks that showed a positive correlation with performance or behavioral demands ( jones and wilson , 2005 ; siapas et al . , 2005 ; hyman et al . , 2010 ; brockmann et al . , 2011 ; fujisawa and buzsaki , 2011 ; for a review see colgin , 2011 ; gordon , 2011 ) . however , pfc - hippocampal communication through theta oscillations in human wm has not been directly observed thus far ( raghavachari et al . , 2006 ; but see anderson et al . , 2010 ) . , 2008a ) observed a decreased correlation of hippocampus with two contralateral frontal regions ( middle and inferior frontal gyrus ) with increasing memory demands . nonetheless , an elegant study combining functional and structural connectivity measures has reported direct empirical evidence that subjects with stronger structural connections between the hippocampus and the pfc have slower pfc oscillations , which correlated with better performance in a wm task ( cohen , 2011 ) . consistent with this finding , connectivity strength from pfc to mtl was positively correlated with performance in a verbal wm task ( campo et al . these data support the relevancy of pfc - mtl interaction in human wm by showing that individual differences in task performance could be related to differences in the strength of such interaction ( gordon , 2011 ) . increased wm load has also been shown to enhance connectivity strength and synchronization within mtl and between mtl , either hippocampus or parahippocampus , and itc ( axmacher et al . , 2008a ; rissman et al . , 2008 ) . a phase - modeling approach characterized this interaction as a top - down modulatory effect from mtl to itc in high frequency range ( axmacher et al . , 2008a ) . furthermore , connectivity strength from mtl to itc distinguished between healthy controls and patients with temporal lobe epilepsy of hippocampal origin during a verbal wm task ( see below campo et al . mtl could participate in reinstating wm contents in itc by means of this top - down interaction ( woloszyn and sheinberg , 2009 ) . these connectivity findings point to mtl - itc inter - regional interactions , probably mediated by cross - frequency coupling of beta / gamma amplitude to theta phase ( babiloni et al . , 2009 ; axmacher et al . , 2010 ; jutras and buffalo , 2010 ) , as a crucial neural process for wm encoding and maintenance ( fell and axmacher , 2011 ) . theta phase - locked gamma hippocampo - cortical loops may provide a coding scheme for structuring the maintenance of multiple items in wm ( lisman , 2005 ; moran et al . according to this scheme , individual memories represented by a cell assembly fires at a gamma subcycle in a certain theta phase . in this way , information contained in distributed populations across the neocortex can be transferred to the hippocampal networks . crucially , two recent studies ( fuentemilla et al . , 2010 ; poch et al . , 2011 ) reported evidence that the periodicity of distributed wm reactivations in the beta / gamma frequency range were phase - locked to the ongoing hipocampal and frontal theta activity , which was functionally relevant for the maintenance of configural - relational information . the interaction of theta and gamma oscillations may operates as the limited human brain resource that engenders limited wm capacity ( jensen and lisman , 1998 ; dzel et al . , 2010 ) . results from studies showing modulation among pfc - mtl - posterior cortex during the performance of a wm task as a function of parametric manipulation of mnemonic demands . dlpfc , dorsolateral prefrontal cortex ; dti , diffusion tensor imaging ; ieeg , intracranial eeg ; itc , inferior temporal cortex ; mtl , medial temporal lobe ; pfc , prefrontal cortex ; phc , parahippocampal cortex ; plv , phase - locking value ; ppi , psychophysical interaction ; sma , supplementary motor area ; tms , transcranial magnetic stimulation ; vac , visual associative cortex ; vlpfc , ventrolateral prefrontal cortex . finally , one interesting finding is that maintenance of an increasing number of items in wm led to smaller or more selectivity set of connections ( axmacher et al . proposed that this effect could be reflecting some properties of hippocampus sparse coding ( rolls , 2010a ; schwindel and mcnaughton , 2011 ) . data from animal and human studies have shown that neural activity in brain regions recruited during wm tasks can be influenced by the length of the mnemonic delay ( haxby et al . elliott and dolan , 1999 ; lee and kesner , 2003 ; picchioni et al . , 2007 ; yoon et al . , 2008 . however , very few studies have explored how functional interactions among the elements of the network varied as the length of the delay interval is manipulated . mcintosh and colleagues ( mcintosh et al . , 1996 ; mcintosh , 1999 ; see also grady et al . , 2001 ) the authors characterized a functional circuit encompassing inferior frontal regions ( ifcs ) , primary and multimodal sensory areas , anterior cingulate , and hippocampal gyrus . the interactions among the elements of this functional network were modulated ( expressed by the sign of connections , as well as some changes in strength ) by the manipulation of the temporal dimension of the task . specifically , the cortico - limbic interactions that characterized the short - delay networks showed a shift from right to left hemisphere with the increase of delay interval , and more effects from prefrontal regions ( ba47 ) as delay was extended . modulation of the interactions between hippocampus and pfc as a function of temporal wm constraints could be related to differential roles played by both structures during wm operations ( yoon et al . , 2008 ) . greater involvement of pfc in monitoring processes and prospective memory has been proposed ( floresco et al . , 1997 ; fuster , 2001 ; gilbert , 2011 ) , and thus extending the temporal requirements for information maintenance may require greater strength of connections from this region ( buchsbaum et al . , 2010 ; churchwell and kesner , 2011 ) . we have seen a number of studies showing that wm depends on a neural network including pfc , mtl , and posterior neocortical regions , whose interactions are modified to accommodate the increase of mnemonic demands . similar functional networks have been described for studies exploring patterns of connectivity during ltm processes ( rajah et al . , 1999 ; fell et al . , 2001 ; simons and spiers , 2003 ; addis and mcandrews , 2006 ; summerfield et al . , 2006 ; kahn et al . , 2008 ; babiloni et al . , 2009 ; anderson et al . , 2010 ) . these evidences constitute further support for the emerging view that the hippocampus and related structures form part of a neural network involved in both ltm and wm processes . however , despite the similarity between neural networks , the question emerges as to whether the connectivity among the elements of these apparently analogous functional networks differ qualitatively or quantitatively , thus allowing for a distinction between memory processes ( mcintosh , 1999 ; rajah and mcintosh , 2005 ) . accordingly , direct examinations of the pattern of interactions of mtl across tasks simultaneously tapping both wm and ltm process are needed in order to determine whether these interactions are reflecting a common functional architecture or not ( axmacher et al . , 2009a ; fell and axmacher , 2011 ) . using tasks designed under the influence of the framework of unitary memory systems ( cowan , 2001 ; oberauer , 2002 ; mcelree , 2006 ) could shed light on this question ( see figure 1 ) . these models consider that information could be represented in different states , either in the focus of attention or in an activated state with different accessibility ( lustig et al . , 2009 ; nee and jonides , 2011 ) . whether neural networks underlying these states were similar or could be differentiated was investigated in a recent fmri study ( nee and jonides , 2008 ) . in this study mechanisms of retrieval of items within the focus of attention or outside the focus information considered to be within the focus of attention was associated with stronger connectivity between itc and frontal and posterior parietal regions ( see also roth and courtney , 2007 ) . crucially , retrieval dynamics of information outside the focus ( lewis - peacock et al . , 2012 ) were characterized by increased coupling between ventrolateral prefrontal cortex ( vlpfc ) and mtl , as well as with other neocortical sites , as a function of retrieval demands ( see figure 1 ) . ( 2008 ) , can be interpreted under this framework . while increased pfc - itc coupling was observed during maintenance of low wm load ( i.e. , one face ) , increased involvement of mtl and pfc mediated coupling was observed at higher mnemonic loads ( i.e. , four faces ) . hence , these data support that processes of stm are similar to those implicated in ltm , suggesting common underlying mechanisms between stm and ltm . nonetheless , differentiation between retrieval and updating of information should be explored in order to refine the characterization of the neural bases reported by this study ( bledowski et al . , 2009 ; additionally , whether the information outside the focus of attention is better explained by two - state models or by three - state models are in need of further functional connectivity studies ( ztekin et al . , 2010 ; lewis - peacock et al . , 2012 ; nee and jonides , 2011 ) . a ) unitary memory models posit that perception , wm and ltm do not differ in the underlying neural substrates , but in the state of information representations . accordingly , two or three states are proposed : a focused of attention ( fa ) ; a region of direct access ( da ) ; and other information that is hypothesized to be an activated portion of ltm ( altm ) . the last two states are considered to be undistinguishable by some authors leading to two states models ( oberauer , 2002 ; mcelree , 2006 ; lustig et al . , 2009 ; ( b ) serial position task ( sp ) of 12 items used to explore differences among the three hypothesized states , and to investigate underlying neural networks . sp12 corresponds to fa ; sp911 corresponds to da ; sp18 corresponds to altm [ adapted from ( ztekin et al . , 2010 ) ] . ( c ) regions demonstrating functional connectivity increases with right itg related to the focus of attention [ adapted from nee and jonides ( 2008 ) ] . ( d ) some of the regions demonstrating enhanced connectivity with left mid - vlpfc related to altm [ adapted from nee and jonides ( 2008 ) ] . itg , inferior temporal gyrus ; mtl , medial temporal lobe ; oc , occipital cortex ; ppc , posterior parietal cortex ; stg , superior temporal gyrus ; vlpfc , ventrolateral prefrontal cortex . another line of research exploring whether the same neural mechanism underlies wm and ltm has focused on the interaction between both types of processes . several studies have shown that mtl wm maintenance - related activity contributes to subsequent ltm recognition ( schon et al . , 2004 ; ranganath et al . , 2005a ; axmacher et al . , 2007 , 2009a ; khader et al . , 2007 ) . , 2005b ) found that increase functional connectivity of hippocampus with a network of cortical regions including itc and frontal areas during the delay period of a complex visual wm task led to improved ltm recognition of the maintained items . likewise , a series of studies has refined these findings and identified ( axmacher et al . , 2008b , 2009b ) a parahippocampal ( phc ) functional network as the hallmark of wm - ltm interaction . using the right phc as the seed region ( rissman et al . , 2004 ) , it was shown that it was significantly correlated with the hippocampus , after correction for number of trials , in all possible conditions relating both types of memory . interestingly , the number of voxels which showed significant correlation with the seed region in the right phc was reduced when successful wm performance was followed by subsequent forgetting as compared to subsequent recognition . finally , subsequent ltm formation was facilitated by an increase in the number of regions engaged in a wm maintenance - related phc functional network , which was independent of wm performance . however , subsequent memory performance was modulated by wm load and was inversely related to the strength of the interaction between phc and medial frontal cortex . this is , ltm encoding and maintenance of multiple items in wm compete for the engagement of a pfc - mtl network ( axmacher et al . propose hippocampal theta coordination of posterior regions during maintenance as a potential mechanism facilitating ltm encoding . moreover , a recent study has found that slower oscillatory activity ( i.e. , theta range ) during wm maintenance and stronger structural connectivity between hippocampus and pfc was predictive of ltm recognition ( cohen , 2011 ) . considered together , these findings provide evidence for an interaction between wm and ltm in mtl networks . changes in network interactions as a function of wm load have been explored in several studies providing some hints about the dynamics of wm operations ( see table 1 ) . that pfc exerts a top - down modulation of the activity in content - representing posterior areas as a mechanism underlying maintenance of verbal or visual information is partially supported by findings showing load - dependent functional connectivity between pfc and itc ( fiebach et al . , 2006 ) , or enhanced phase synchronization between frontal and temporal regions in the gamma ( axmacher et al . , 2008a ) or theta range ( cashdollar et al . , only a recent study made an exception to this observation ( rissman et al . , 2008 ) . hence , this load - induced enhancement could reflect a top - down selective strengthening of representation and maintenance of relevant information ( payne and kounios , 2009 ) . consistent with the role of pfc in modulating neural activity in posterior regions selectively related to relevant stimuli are findings from several studies using functional connectivity measures ( sauseng et al . , 2005 ; yoon et al . , 2006 ; gazzaley et al . , 2007 ; protzner and mcintosh , 2009 ; see also edin et al . , importantly , these findings were causally supported by recent studies using transcranial magnetic stimulation ( feredoes et al . , 2011 ; zanto et al . , 2011 ) . also related to this issue , complexity of manipulation performed on information actively maintained modulate prefrontal - posterior coupling in theta or alpha frequency ranges ( sauseng et al . , 2005 , 2007 ) , further supporting the idea of a top - down control process , and suggesting that phase coupling is a probable operational mechanism ( zanto et al . , 2011 ) . as we introduced above , functional coupling between pfc and mtl 2009b ) or letters ( finn et al . , 2010 ) using a modified stenberg paradigm led to a greater connectivity strength between pfc and mtl structures , either hippocampus or parahippocampus . theta oscillation has been proposed as a potential neurophysiological mechanism mediating this interaction ( mitchell et al . , 2008 ; anderson et al . , 2010 this proposal has been supported by studies demonstrating phase - locking of pfc spiking activity with the hippocampal theta cycle in animals during wm tasks that showed a positive correlation with performance or behavioral demands ( jones and wilson , 2005 ; siapas et al . , 2005 ; hyman et al . , 2010 ; brockmann et al . , 2011 ; fujisawa and buzsaki , 2011 ; for a review see colgin , 2011 ; gordon , 2011 ) . however , pfc - hippocampal communication through theta oscillations in human wm has not been directly observed thus far ( raghavachari et al . , 2006 ; but see anderson et al . , , 2008a ) observed a decreased correlation of hippocampus with two contralateral frontal regions ( middle and inferior frontal gyrus ) with increasing memory demands . nonetheless , an elegant study combining functional and structural connectivity measures has reported direct empirical evidence that subjects with stronger structural connections between the hippocampus and the pfc have slower pfc oscillations , which correlated with better performance in a wm task ( cohen , 2011 ) . consistent with this finding , connectivity strength from pfc to mtl was positively correlated with performance in a verbal wm task ( campo et al . these data support the relevancy of pfc - mtl interaction in human wm by showing that individual differences in task performance could be related to differences in the strength of such interaction ( gordon , 2011 ) . increased wm load has also been shown to enhance connectivity strength and synchronization within mtl and between mtl , either hippocampus or parahippocampus , and itc ( axmacher et al . , 2008a ; rissman et al . , 2008 ) . a phase - modeling approach characterized this interaction as a top - down modulatory effect from mtl to itc in high frequency range ( axmacher et al . , 2008a ) . furthermore , connectivity strength from mtl to itc distinguished between healthy controls and patients with temporal lobe epilepsy of hippocampal origin during a verbal wm task ( see below campo et al . mtl could participate in reinstating wm contents in itc by means of this top - down interaction ( woloszyn and sheinberg , 2009 ) . these connectivity findings point to mtl - itc inter - regional interactions , probably mediated by cross - frequency coupling of beta / gamma amplitude to theta phase ( babiloni et al . , 2009 ; axmacher et al . , 2010 ; jutras and buffalo , 2010 ) , as a crucial neural process for wm encoding and maintenance ( fell and axmacher , 2011 ) . theta phase - locked gamma hippocampo - cortical loops may provide a coding scheme for structuring the maintenance of multiple items in wm ( lisman , 2005 ; moran et al . , 2010 ; fell and axmacher , 2011 ; freunberger et al . , 2011 ; lundqvist et al . , according to this scheme , individual memories represented by a cell assembly fires at a gamma subcycle in a certain theta phase . in this way , information contained in distributed populations across the neocortex can be transferred to the hippocampal networks . crucially , two recent studies ( fuentemilla et al . , 2010 ; poch et al . , 2011 ) reported evidence that the periodicity of distributed wm reactivations in the beta / gamma frequency range were phase - locked to the ongoing hipocampal and frontal theta activity , which was functionally relevant for the maintenance of configural - relational information . the interaction of theta and gamma oscillations may operates as the limited human brain resource that engenders limited wm capacity ( jensen and lisman , 1998 ; dzel et al . , 2010 ) . results from studies showing modulation among pfc - mtl - posterior cortex during the performance of a wm task as a function of parametric manipulation of mnemonic demands . dlpfc , dorsolateral prefrontal cortex ; dti , diffusion tensor imaging ; ieeg , intracranial eeg ; itc , inferior temporal cortex ; mtl , medial temporal lobe ; pfc , prefrontal cortex ; phc , parahippocampal cortex ; plv , phase - locking value ; ppi , psychophysical interaction ; sma , supplementary motor area ; tms , transcranial magnetic stimulation ; vac , visual associative cortex ; vlpfc , ventrolateral prefrontal cortex . finally , one interesting finding is that maintenance of an increasing number of items in wm led to smaller or more selectivity set of connections ( axmacher et al . , 2008a ) proposed that this effect could be reflecting some properties of hippocampus sparse coding ( rolls , 2010a ; schwindel and mcnaughton , 2011 ) . data from animal and human studies have shown that neural activity in brain regions recruited during wm tasks can be influenced by the length of the mnemonic delay ( haxby et al . , 1995 ; owen et al . , 1996 ; elliott and dolan , 1999 ; lee and kesner , 2003 ; picchioni et al . , 2007 ; yoon et al . , 2008 ; buchsbaum et al . , 2010 ) . however , very few studies have explored how functional interactions among the elements of the network varied as the length of the delay interval is manipulated . mcintosh and colleagues ( mcintosh et al . , 1996 ; mcintosh , 1999 ; see also grady et al . , 2001 ) the authors characterized a functional circuit encompassing inferior frontal regions ( ifcs ) , primary and multimodal sensory areas , anterior cingulate , and hippocampal gyrus . the interactions among the elements of this functional network were modulated ( expressed by the sign of connections , as well as some changes in strength ) by the manipulation of the temporal dimension of the task . specifically , the cortico - limbic interactions that characterized the short - delay networks showed a shift from right to left hemisphere with the increase of delay interval , and more effects from prefrontal regions ( ba47 ) as delay was extended . modulation of the interactions between hippocampus and pfc as a function of temporal wm constraints could be related to differential roles played by both structures during wm operations ( yoon et al . , 2008 ) . greater involvement of pfc in monitoring processes and prospective memory has been proposed ( floresco et al . , 1997 ; fuster , 2001 ; gilbert , 2011 ) , and thus extending the temporal requirements for information maintenance may require greater strength of connections from this region ( buchsbaum et al . , 2010 ; churchwell and kesner , 2011 ) . we have seen a number of studies showing that wm depends on a neural network including pfc , mtl , and posterior neocortical regions , whose interactions are modified to accommodate the increase of mnemonic demands . similar functional networks have been described for studies exploring patterns of connectivity during ltm processes ( rajah et al . , 1999 ; fell et al . , 2001 ; simons and spiers , 2003 ; addis and mcandrews , 2006 ; summerfield et al . , 2006 ; kahn et al . , 2008 ; babiloni et al . , 2009 ; anderson et al . , 2010 ) . these evidences constitute further support for the emerging view that the hippocampus and related structures form part of a neural network involved in both ltm and wm processes . however , despite the similarity between neural networks , the question emerges as to whether the connectivity among the elements of these apparently analogous functional networks differ qualitatively or quantitatively , thus allowing for a distinction between memory processes ( mcintosh , 1999 ; rajah and mcintosh , 2005 ) . accordingly , direct examinations of the pattern of interactions of mtl across tasks simultaneously tapping both wm and ltm process are needed in order to determine whether these interactions are reflecting a common functional architecture or not ( axmacher et al . using tasks designed under the influence of the framework of unitary memory systems ( cowan , 2001 ; oberauer , 2002 ; mcelree , 2006 ) could shed light on this question ( see figure 1 ) . these models consider that information could be represented in different states , either in the focus of attention or in an activated state with different accessibility ( lustig et al . , 2009 ; nee and jonides , 2011 ) . whether neural networks underlying these states were similar or could be differentiated was investigated in a recent fmri study ( nee and jonides , 2008 ) . in this study mechanisms of retrieval of items within the focus of attention or outside the focus information considered to be within the focus of attention was associated with stronger connectivity between itc and frontal and posterior parietal regions ( see also roth and courtney , 2007 ) . crucially , retrieval dynamics of information outside the focus ( lewis - peacock et al . , 2012 ) were characterized by increased coupling between ventrolateral prefrontal cortex ( vlpfc ) and mtl , as well as with other neocortical sites , as a function of retrieval demands ( see figure 1 ) . ( 2008 ) , can be interpreted under this framework . while increased pfc - itc coupling was observed during maintenance of low wm load ( i.e. , one face ) , increased involvement of mtl and pfc mediated coupling was observed at higher mnemonic loads ( i.e. , four faces ) . hence , these data support that processes of stm are similar to those implicated in ltm , suggesting common underlying mechanisms between stm and ltm . nonetheless , differentiation between retrieval and updating of information should be explored in order to refine the characterization of the neural bases reported by this study ( bledowski et al . , 2009 ; additionally , whether the information outside the focus of attention is better explained by two - state models or by three - state models are in need of further functional connectivity studies ( ztekin et al . , 2010 ; lewis - peacock et al . , 2012 ; nee and jonides , 2011 ) . unitary memory models and ( a ) unitary memory models posit that perception , wm and ltm do not differ in the underlying neural substrates , but in the state of information representations . accordingly , two or three states are proposed : a focused of attention ( fa ) ; a region of direct access ( da ) ; and other information that is hypothesized to be an activated portion of ltm ( altm ) . the last two states are considered to be undistinguishable by some authors leading to two states models ( oberauer , 2002 ; mcelree , 2006 ; lustig et al . , 2009 ; ( b ) serial position task ( sp ) of 12 items used to explore differences among the three hypothesized states , and to investigate underlying neural networks . sp12 corresponds to fa ; sp911 corresponds to da ; sp18 corresponds to altm [ adapted from ( ztekin et al . ( c ) regions demonstrating functional connectivity increases with right itg related to the focus of attention [ adapted from nee and jonides ( 2008 ) ] . ( d ) some of the regions demonstrating enhanced connectivity with left mid - vlpfc related to altm [ adapted from nee and jonides ( 2008 ) ] . itg , inferior temporal gyrus ; mtl , medial temporal lobe ; oc , occipital cortex ; ppc , posterior parietal cortex ; stg , superior temporal gyrus ; vlpfc , ventrolateral prefrontal cortex . another line of research exploring whether the same neural mechanism underlies wm and ltm has focused on the interaction between both types of processes . several studies have shown that mtl wm maintenance - related activity contributes to subsequent ltm recognition ( schon et al . , 2004 ; ranganath et al . , 2007 , 2009a ; khader et al . , 2007 ) . using this paradigm , ranganath and colleagues ( ranganath et al . , 2005b ) found that increase functional connectivity of hippocampus with a network of cortical regions including itc and frontal areas during the delay period of a complex visual wm task led to improved ltm recognition of the maintained items . likewise , a series of studies has refined these findings and identified ( axmacher et al . , 2008b , 2009b ) a parahippocampal ( phc ) functional network as the hallmark of wm - ltm interaction . using the right phc as the seed region ( rissman et al . , 2004 ) , it was shown that it was significantly correlated with the hippocampus , after correction for number of trials , in all possible conditions relating both types of memory . interestingly , the number of voxels which showed significant correlation with the seed region in the right phc was reduced when successful wm performance was followed by subsequent forgetting as compared to subsequent recognition . finally , subsequent ltm formation was facilitated by an increase in the number of regions engaged in a wm maintenance - related phc functional network , which was independent of wm performance . however , subsequent memory performance was modulated by wm load and was inversely related to the strength of the interaction between phc and medial frontal cortex . this is , ltm encoding and maintenance of multiple items in wm compete for the engagement of a pfc - mtl network ( axmacher et al . interestingly , recent findings ( cashdollar et al . , 2009 ) propose hippocampal theta coordination of posterior regions during maintenance as a potential mechanism facilitating ltm encoding . moreover , a recent study has found that slower oscillatory activity ( i.e. , theta range ) during wm maintenance and stronger structural connectivity between hippocampus and pfc was predictive of ltm recognition ( cohen , 2011 ) . considered together , these findings provide evidence for an interaction between wm and ltm in mtl networks . there is increasing agreement that cognitive impairments observed in neurological and neuropsychiatric disorders , whether they are characterized by localized focal lesion or not , are caused by abnormally organized or disconnected networks ( bassett et al . , 2009 ) . therefore , further understanding of the hippocampo - cortical wm network could be gained by exploring the impact that such disorders produce on the integrity of the system ( menon , 2011 ) . this is , how damage to one or more of its constituent elements lead to dynamic adjustments or reorganization of the remaining network in an attempt to compensate , and the efficacy of this change ( mcintosh , 1999 ; gazzaley et al . those pathological conditions in which the functioning of specific nodes of the network is disrupted , and consequently their connections , would provide the most valuable information . pathological disruption of the hippocampal formation has been primarily observed in alzheimer 's disease ( ad ) and temporal lobe epilepsy ( tle ) , although with different patterns of cell loss ( small et al . , 2011 ) . ad is characterized by gray matter atrophy and the formation of plaques , which are particularly prominent in the mtl and pcc ( posterior cingulate cortex ) ( braak and braak , 1997 ; thal et al . , 2002 ; sabuncu et al . , 2011 ; nickl - jockschat et al . , interestingly , these areas show significantly reduced network connectivity ( zhou et al . , 2008 ; yao et al . , 2010 ; pievani et al . , 2011 moreover , abnormalities in functional connectivity have been shown to overlap with structural atrophy and beta - amyloid ( -amyloid ) deposition ( buckner et al . hence , cognitive impairments associated with ad , and particularly wm deficits , can be examined considering the disruption of these connections ( uhlhaas and singer , 2006 ) . when comparing functional connectivity patterns between patients with ad and healthy controls during a wm task for faces , it was found that activity in pfc correlated with hippocampus and extrastriate cortex in controls , while it was restricted to other frontal regions in patients ( grady et al . , 2001 ) . the pathological nature of this pattern of connectivity is reinforced by recent findings showing an increased functional connectivity between hippocampus and diffuse areas of pfc which was negatively correlated with performance on a memory task in amnestic mild cognitive impairment patients showing hippocampal atrophy ( bai et al . , 2009 ) . these results are consistent with studies showing an increased connectivity within anterior sites and sparing of hubs in frontal regions ( babiloni et al . , 2006 ; 2011 ) , and reduced fronto - posterior interaction and impairment of hubs in temporal regions ( stam et al . , 2006 ; tle associated with hippocampal sclerosis ( hs ) ( wieser , 2004 ) , also referred to as mesial tle ( mtle ) , is typically considered a network disorder ( liao et al . , 2010 ) . several studies in mtle have shown abnormal patterns of functional connectivity during a resting state condition , the so - called default network ( raichle et al . , 2001 ) , revealing not only local network abnormalities within lesional mtl , but more widespread effects in network interactions ( liao et al . , 2010 , 2011 ; pereira et al . , 2010 ) . a recent study ( bettus et al . , 2009 ) has shown a decreased basal functional connectivity within the epileptogenic mtl which was correlated with the degree of connectivity increase in the contralateral mtl . interestingly , the strength of basal functional connectivity in the contralesional mtl was correlated with wm performance , in what was suggested to reflect a compensatory effect . further evidence of connectivity changes in mtl networks associated to hs comes from a study comparing dynamic causal models ( friston et al . , 2003 ) extracted from magnetoencephalographic recordings during verbal wm encoding ( campo et al . , in press ) . having established that the best model across subjects ( i.e. , bayesian model comparison , penny et al . , 2004 ) evidenced bilateral , forward and backward connections , coupling itc , ifcs , and mtl ( figure 2 ) , it was found that left hs patients were characterized by a decreased backward connectivity from left mtl to left itc , and an increased forward and backward pfc - hippocampal connectivity in the contralesional hemisphere . interestingly , performance was positively correlated with left pfc - mtl connectivity strength in controls ; while it was negatively correlated with right pfc - mtl across subjects . a critical role of hippocampal - dependent theta synchronization of activity in posterior regions for maintenance of relational visual information was demonstrated after showing that patients with bilateral hs exhibited impaired performance and selective loss of such synchronization when compared to a group of patients with tle but intact hippocampi ( cashdollar et al . , 2009 ) . dynamic causal models , extracted from magnetoencephalographic recordings during verbal wm encoding , were compared in tle patients ( with left hippocampal sclerosis ) and controls . ( a ) network architecture was specified on the basis of the inverse solutions for single subjects using multiple sparse priors . the models were constructed starting with simple architectures and adding hierarchical levels ( i.e. , sources and extrinsic connections ) . the simplest models only included the itc and ifc sources , while more complex models included mtl sources . models also differed in terms of their connections ; forward only or both forward and backward . ( b ) group level bayesian selection of the 12 tested models : random fixed effects ( rfx ) showing model expected probability . results indicate the best model is one with bilateral forward and backward connections comprising ifg , itc , and mtl ( model 12 ) . ( c ) group differences in effective connectivity assessed using subject - specific ( maximum a posteriori ) parameter estimates . adapted from campo et al . ( in press ) . a clearly distinct neuropathological pattern can be observed in schizophrenia , which is a complex disorder mainly associated with a profound deterioration of pfc function and characterized by several cognitive impairments ( lewis and gonzalez - burgos , 2006 ; bonilha et al . , 2008 ; pomarol - clotet et al . , 2010 ) . among these , wm dysfunction is regarded as a fundamental core of the disease ( goldman - rakic , 1994 ; lee and park , 2005 ; van snellenberg , 2009 ; haenschel and linden , 2011 ) and has been typically attributed to specific alterations in the neural microcircuits of the pfc ( perlstein et al . , 2001 ; manoach , 2003 ; arnsten et al . , 2010 ; banyai et al . , 2011 ; menon , 2011 ; anticevic et al . however , the disease is not considered to be caused by focal brain abnormalities ( friston , 1999 ; uhlhaas and singer , 2010 ) . the extent to which wm impairments in schizophrenia reflect dysregulations of the underlying neural networks has begun to be exploited recently ( uhlhaas and singer , 2006 ) . a number of studies have reported disturbed interactions between pfc and temporal regions , including hippocampus , in patients with schizophrenia and in healthy individuals at risk of developing the disease , although the nature of the altered connectivity is complex ( winterer et al . , 2003 ) . thus , mimicking animal models of genetic risk factors in which mutant mice showed reduced hippocampal - prefrontal connectivity during a wm task ( sigurdsson et al . , 2010 ) , functional connectivity studies have shown , for the most part , a decreased connectivity within pfc and between pfc and hippocampus and several posterior regions ( kim et al . , 2003 ; cho et al . 2010 ; banyai et al . , 2011 ; kang et al . , 2011 ) . however , some studies have observed an abnormal persistent coupling between pfc and hippocampus and temporal regions while engaged in wm tasks ( meyer - lindenberg et al . , 2001 , 2005 ; crossley et al . , 2009 ; esslinger et al . , 2009 ; several factors could be the cause of this inconsistency ( i.e. , chronicity , antipsychotic treatment , level of arousal , type of task ; manoach , 2003 ; winterer et al . , 2003 ) , but their influence has so far not been systematically investigated . thus , further research on the role of these factors in wm impairments and modulation of network interactions in schizophrenia is warranted . moreover , interaction of wm - related specific connectivity deficits with generalized task - independent connectivity dysfunction should be explored ( wolf et al . , 2009 ; lynall et al . additionally , it is also conceivable that altered early perceptual and encoding processes modulate wm dysfunction . more investigation on this relationship will help to clarify the complexity of the dynamics underlying the observed deficits ( haenschel and linden , 2011 ) . nonetheless , these data , along with data from structural connectivity studies ( karlsgodt et al . , 2008 ; spoletini et al . , 2009 ; for a meta - analysis see ellison - wright and bullmore , 2009 ) , support the proposal that abnormal functional connectivity between pfc and temporal structures might underlie wm dysfunction in schizophrenia ( friston , 1998 ; winterer et al . , 2003 ; stephan et al . , 2006 ) findings from connectivity studies we have reviewed thus far shed light on how the pattern of neural interactions of a functional network of brain regions , including mtl , varies as a result of wm dynamics . the picture that emerges is that connectivity of content - specific regions in posterior cortex with either pfc or mtl is enhanced during wm to adequate the system to mnemonic requirements . evidence that mtl connectivity is modulated by parametric manipulations of wm task demands , along with earlier findings from lesional and activation studies , bolster the view that mtl plays a critical role in wm processes . under the perspective of unitary models of memory it can be stated that which brain regions within the wm network are functionally linked , this is the neural context ( mcintosh , 1999 ) , depend on which specific processes are taking place . thus , different interactions support different levels of processing , from more attentional ( i.e. , pfc - itc ) to more mnemonic ( i.e. , mtl - itc , pfc - mtl ) ( nee and jonides , 2008 ; ztekin et al . , 2010 ; nee and jonides , 2011 ) . this modulatory feedback from pfc and mtl of the neural activity in posterior regions have been observed during the encoding , maintenance and recognition phases of wm , and with different types of stimuli . we have also seen how functional interactions of mtl during wm processes are predictive of how well the information is transferred to ltm ( axmacher et al . , 2008b ; cohen , 2011 ) . moreover , connectivity strength between parahippocampus and pfc modulated the encoding of items in ltm during simultaneous wm maintenance , thus pointing to mtl based networks as a site of wm - ltm interactions ( axmacher et al . , 2009b ) . the majority of these findings are derived from correlational methods , and can not be used to make strong statements about causality . thus , future studies should explore the directionality of the information flow using effective connectivity methods ( anderson et al . considered together , these data suggest that mtl participates in the wm network not only when stimuli are novel or relations between stimuli must be formed ( ranganath and blumenfeld , 2005 ) , but when the amount or complexity of to - be remembered stimuli can not be accomplished using attention - based maintenance process ( rissman et al . , 2008 ) . given that distinct sensory inputs arrive to specific regions of the mtl in virtue of their differential connectivity pathways ( rolls , 2000 ) , stimulus class effects in the pattern functional connectivity of different mtl regions ( i.e. , perirhinal and parahippocampal cortex ) should be explored in future experiments ( witter et al . , 2000 ; davachi , 2006 ; olsen et al . , 2009 ) . the compelling evidence provided by the studies we have reviewed about the involvement of mtl in wm should be reconciled with several demonstrations of wm - ltm dissociation and insensitivity of wm performance to hippocampal damage ( vallar and papagano , 1986 ; carlesimo et al . , 2001 ; tudesco ide et al . , 2010 ; jeneson et al . , 2011 ) . further research about of wm - ltm interplay and the role of mtl will be needed . even though the neural mechanism underlying information transfer between brain areas remains unknown , a prime candidate to play a key role in this process appears to be phase synchronization of oscillatory activity ( fries , 2005 ; fell and axmacher , 2011 ) . with regard to wm it has been shown that , consistent with neurophysiological and network models ( lisman and idiart , 1995 ; lundqvist et al . , 2011 ) , phase synchronization between slower and higher oscillations reflect the retention of information ( sauseng et al . , 2009 ; axmacher et al . , 2010 ; cohen , 2011 ) . interestingly , ltm processes are also governed by similar synchronization mechanisms ( nyhus and curran , 2010 ; fell and axmacher , 2011 ) . however , no studies have directly addressed how phase synchronization during wm modulate subsequent ltm recognition ( fell and axmacher , 2011 ) or the patterns of phase synchronization during different states of information processing under proceduralist models ( nee and jonides , 2011 ) . furthermore , the precise role of synchrony in support of inter - neuronal communication is far from being well understood ( aru et al . , 2011 ; fell and axmacher , 2011 ; gordon , 2011 ) . oscillatory coherence has been associated with several cognitive processes , from perceptual binding ( singer and gray , 1995 ) to rule learning ( benchenane et al . , 2010 ) , and , therefore , new experiments should explore the functional role of neural synchronization in wm and in wm - ltm interplay ( jutras and buffalo , 2010 ; benchenane et al . , 2011 ) . future studies should also explore how functional networks supporting wm interact with the anatomical network infrastructure ( guye et al . . additionally , greater understanding of the functional significance of neural synchronization would be obtained by investigating the relationship between neuromodulators and oscillatory coupling ( cartling , 2001 ; takahashi et al . , 2008 ; haenschel and linden , 2011 ; young , 2011 ) . finally , we have also seen how abnormalities in functional connectivity arising from more or less localized lesions can contribute to wm dysfunction observed in mtle , ad , and schizophrenia . particularly , abnormal communication between mtl and pfc has been observed after damage to one of these structures , probably due to tight structural and functional connection between them ( brockmann et al . , 2011 ) . the reviewed findings add to a growing body of evidence further supporting the involvement of mtl in the neural network underlying wm . characterizing dysfunctional networks not only provide a better understanding of a variety of disorders , but also critical inferences about the functional dynamics between particular elements of the wm network . relating patterns of altered oscillatory synchronization with histologically mapped patterns of cell loss ( i.e. , different degrees of cell death in ca1 in mtle ) could help in increasing our knowledge about the mechanisms mediating neural communication during wm ( colgin et al . the development of distinct patterns of connectivity following damage to the network underlying wm could help to develop better diagnostic and prognostic tools . evaluation of the integrity of memory structures in the presurgical evaluation of patients with mtle , and prognosis of surgical outcome , could benefit from network analysis . in pathologies such as ad , with a clear progress of dysfunction with time , the use of a longitudinal approach could increase our knowledge about disruptions in the network affecting certain elements in specific stages and its behavioral consequences . finally , network analysis could provide the neurophysiological basis for evaluating the efficacy of cognitive and pharmacological treatments ( colgin , 2011 ; haenschel and linden , 2011 ; kleen et al . , 2011 ) . 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 . 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 .

working memory ( wm ) is the ability to transiently maintain and manipulate internal representations beyond its external availability to the senses . this process is thought to support high level cognitive abilities and been shown to be strongly predictive of individual intelligence and reasoning abilities . while early models of wm have relied on a modular perspective of brain functioning , more recent evidence suggests that cognitive functions emerge from the interactions of multiple brain regions to generate large - scale networks . here we will review the current research on functional connectivity of wm processes to highlight the critical role played by neural interactions in healthy and pathological brain states . recent findings demonstrate that wm abilities are not determined solely by local brain activity , but also rely on the functional coupling of neocortical - hippocampal regions to support wm processes . although the hippocampus has long been held to be important for long - term declarative memory , recent evidence suggests that the hippocampus may also be necessary to coordinate disparate cortical regions supporting the periodic reactivation of internal representations in wm . furthermore , recent brain imaging studies using connectivity measures , have shown that changes in cortico - limbic interactions can be useful to characterize wm impairments observed in different neuropathological conditions . recent advances in electrophysiological and neuroimaging techniques to model network activity has led to important insights into how neocortical and hippocampal regions support wm processes and how disruptions along this network can lead to the memory impairments commonly reported in many neuropathological populations .

Functional brain networks Network dynamics between MTL and neocortex in the healthy brain Load-dependent functional interactions Time-dependent functional interactions A common neural network for working memory and long-term memory? Disturbed network dynamics between MTL and neocortex Concluding remarks and future directions Conflict of interest statement

connections among elements of a network can be characterized as undirected links , this is functional connectivity , or as directed links , this is effective connectivity ( sporns and tononi , 2007 ; friston , 2009 ; bressler and menon , 2010 ) . finally , recent evidence from animal studies suggest that the interplay between pfc and mtl , especially hippocampus , is key for normal wm function ( benchenane et al . we will review neuroimaging studies focusing on characterizing the functional interactions among pfc - mtl - posterior cortex , and how their findings adjudicate between the above mentioned proposals . consistent with the role of pfc in modulating neural activity in posterior regions selectively related to relevant stimuli are findings from several studies using functional connectivity measures ( sauseng et al . , 2011 ) reported evidence that the periodicity of distributed wm reactivations in the beta / gamma frequency range were phase - locked to the ongoing hipocampal and frontal theta activity , which was functionally relevant for the maintenance of configural - relational information . data from animal and human studies have shown that neural activity in brain regions recruited during wm tasks can be influenced by the length of the mnemonic delay ( haxby et al . specifically , the cortico - limbic interactions that characterized the short - delay networks showed a shift from right to left hemisphere with the increase of delay interval , and more effects from prefrontal regions ( ba47 ) as delay was extended . these evidences constitute further support for the emerging view that the hippocampus and related structures form part of a neural network involved in both ltm and wm processes . , 2005b ) found that increase functional connectivity of hippocampus with a network of cortical regions including itc and frontal areas during the delay period of a complex visual wm task led to improved ltm recognition of the maintained items . consistent with the role of pfc in modulating neural activity in posterior regions selectively related to relevant stimuli are findings from several studies using functional connectivity measures ( sauseng et al . theta phase - locked gamma hippocampo - cortical loops may provide a coding scheme for structuring the maintenance of multiple items in wm ( lisman , 2005 ; moran et al . , 2011 ) reported evidence that the periodicity of distributed wm reactivations in the beta / gamma frequency range were phase - locked to the ongoing hipocampal and frontal theta activity , which was functionally relevant for the maintenance of configural - relational information . data from animal and human studies have shown that neural activity in brain regions recruited during wm tasks can be influenced by the length of the mnemonic delay ( haxby et al . specifically , the cortico - limbic interactions that characterized the short - delay networks showed a shift from right to left hemisphere with the increase of delay interval , and more effects from prefrontal regions ( ba47 ) as delay was extended . these evidences constitute further support for the emerging view that the hippocampus and related structures form part of a neural network involved in both ltm and wm processes . , 2005b ) found that increase functional connectivity of hippocampus with a network of cortical regions including itc and frontal areas during the delay period of a complex visual wm task led to improved ltm recognition of the maintained items . there is increasing agreement that cognitive impairments observed in neurological and neuropsychiatric disorders , whether they are characterized by localized focal lesion or not , are caused by abnormally organized or disconnected networks ( bassett et al . , 2011 moreover , abnormalities in functional connectivity have been shown to overlap with structural atrophy and beta - amyloid ( -amyloid ) deposition ( buckner et al . the pathological nature of this pattern of connectivity is reinforced by recent findings showing an increased functional connectivity between hippocampus and diffuse areas of pfc which was negatively correlated with performance on a memory task in amnestic mild cognitive impairment patients showing hippocampal atrophy ( bai et al . thus , further research on the role of these factors in wm impairments and modulation of network interactions in schizophrenia is warranted . , 2006 ) findings from connectivity studies we have reviewed thus far shed light on how the pattern of neural interactions of a functional network of brain regions , including mtl , varies as a result of wm dynamics . evidence that mtl connectivity is modulated by parametric manipulations of wm task demands , along with earlier findings from lesional and activation studies , bolster the view that mtl plays a critical role in wm processes . under the perspective of unitary models of memory it can be stated that which brain regions within the wm network are functionally linked , this is the neural context ( mcintosh , 1999 ) , depend on which specific processes are taking place . we have also seen how functional interactions of mtl during wm processes are predictive of how well the information is transferred to ltm ( axmacher et al . characterizing dysfunctional networks not only provide a better understanding of a variety of disorders , but also critical inferences about the functional dynamics between particular elements of the wm network .

dentine hypersensitivity is a common condition variously reported to affect anywhere between 3 and 57% of the population depending on the method of diagnosis , geography , and population chosen [ 14 ] . it is characterised by a short sharp pain arising from exposed dentine in response to thermal , evaporative , tactile , osmotic , or chemical stimuli that can not be ascribed to any other form of dental defect [ 4 , 5 ] . although symptoms of the condition are acute and episodic they can persist for years . without proper clinical management dentine hypersensitivity can have a significant impact on a sufferers quality of life [ 6 , 7 ] . human dentine contains large numbers of fluid - filled tubules , typically 15 m in diameter , that run radially from the dentine - pulp junction to the surface of the dentine - enamel or dentine - cementum junction [ 8 , 9 ] . tubule lumens , that are unsclerosed , free of debris , and open at the dentine surface , facilitate transmission of the stimulus to the pain receptors present at the pulp / odontoblast interface . as a consequence , a key micromorphological factor influencing the severity of the sensitivity response is the extent to which the dentine surface and thus the tubules are occluded by a smear layer . the prevailing mechanistic description of stimulus transduction is the hydrodynamic theory elucidated by brannstrm over 40 years ago [ 1113 ] . in the event that externally applied stimuli significantly alter fluid flow within the dentinal tubule , this may be sufficient to trigger mechanoreceptors near the base of the tubule and firing of afferent nerves . aetiological factors relevant to the development of dentine hypersensitivity include erosive wear close to the gingival margin where the enamel is thinnest , its removal resulting in exposure of the underlying dentine . gingival recession resulting from periodontal disease or tooth brushing trauma has also been considered to be an aetiological factor in dentine hypersensitivity as this may result in the exposure of the tooth root and associated cementum . relative to enamel , the cementum is more susceptible to removal through erosive wear , a process that rapidly leads to exposure of the underlying dentine [ 14 , 15 ] . an implicit consequence of the hydrodynamic theory is the polarisation of treatment options into those that target desensitisation of the relevant intradental nerves , and those whose aim is to inhibit or prevent transmission of the stimulus itself . potassium salts including nitrate , chloride , and citrate are examples of the former , with dentifrice being the principal delivery format [ 16 , 17 ] . although clinical efficacy has been widely reported by hodosh and others [ 1619 ] , and the ability of potassium ions to suppress nerve firing demonstrated , the precise mode of action remains elusive [ 1922 ] . the second strategy for the treatment of dentine hypersensitivity is to employ occlusion agents to seal or at least partially occlude the patent tubules ; this may also be achieved through formation of a barrier layer on the surface of the dentine . although considered a nonphysiological approach , such treatments often rely on the reactivity of the desensitising agent to promote formation of mixed mineral deposits on and within the exposed dentine [ 22 , 23 ] . examples that have been widely reported to be effective in vivo include oxalate salts [ 2426 ] and strontium salts [ 2729 ] . their occlusion efficacy in vitro has also been widely studied using techniques that include measurement of dentine permeability [ 30 , 31 ] , scanning electron microscopy [ 28 , 32 ] , and radiopacity . the utility of polymer - based sealants as occlusion agents for treating dentine hypersensitivity has also been investigated [ 33 , 34 ] . a new occlusion - based technology has recently been commercialised in a dentifrice format for the treatment of dentine hypersensitivity . based on arginine , bicarbonate , and calcium carbonate , its utility both as an anticaries and desensitising active has previously been claimed [ 36 , 37 ] . in the latter case , the technology is reported to work through binding of positively charged agglomerates to exposed dentine surfaces and within the tubules themselves . the marketed dentifrice contains fluorine as sodium monofluorophosphate ( namfp ) , and its desensitising efficacy has been reported in a comparative clinical study against a dentifrice containing the safe and effective monographed desensitising agent potassium nitrate ( kno3 ) as the benchmark control [ 39 , 40 ] . as well as providing oral health benefits , a dentifrice should ideally meet basic aesthetic criteria such as acceptable taste and mouth feel , since this may affect compliance and consequently efficacy . it is relevant to note , therefore , that the recent development of gel - to - foam dentifrices wherein a volatile agent is incorporated into the formulation , that the foaming in vivo is significantly enhanced . a recently published clinical study employing a desensitising gel - to - foam dentifrice containing kno3 reported highly significant relief from tooth sensitivity at 4 , 8 , and 12 weeks . the improved in vivo dispersion associated with this novel format is believed to enhance delivery of the oral care actives in hard - to - reach areas . the aim of the present study was to compare the effectiveness of the two aforementioned dentifrices in an 8-week , two - arm , parallel group , longitudinal dentine hypersensitivity study . sensodyne multi action iso - active ( dentifrice a ) contained 50000 ppm kno3 and 1450 ppm fluoride as the sodium salt ( naf ) and was manufactured by glaxosmithkline consumer healthcare ( brentford , middlesex , uk ) . colgate sensitive pro - relief ( dentifrice b ) contained 80000 ppm arginine , bicarbonate , calcium carbonate , and 1450 ppm fluorine as namfp and was manufactured by colgate - palmolive ( guildford , surrey , uk ) . oral b indicator soft toothbrushes were sourced from procter & gamble ( weybridge , surrey , uk ) . test dentifrices were supplied to study volunteers in their original commercial packaging over - wrapped in opaque vinyl with a study - specific label detailing protocol number , product code , storage conditions , and precautionary information including an emergency contact telephone number . the study protocol was reviewed and approved by an independent ethics committee at wuhan university , china and conducted according to ich gcp guidelines . inclusion criteria required volunteers to be aged between 20 and 60 years of age , with at least 20 natural permanent teeth and pre existing self - reported and clinically diagnosed tooth sensitivity . subjects were required to present with test teeth exhibiting signs of facial / cervical erosion and/or abrasion and/or gingival recession . test teeth should , in the opinion of the examiner , otherwise exhibit good gingival health and should not exhibit any clinical mobility ( mobility score < 1 ) . subjects must have successfully completed a visual analogue scale ( vas ) instruction exercise at screening , whose purpose was to train subjects in the use of vas by requiring subjects to estimate how much of the total area of a series of 7 shapes had been shaded using a 100 mm line anchored at one end with no shading and the other with complete shading . subjects were excluded if they had received desensitising treatments or used a desensitising dentifrice within the previous 3 months . tactile and air sensitivity measurements were performed by two independent examiners . at the screening visit , the tactile sensitivity of each eligible tooth was determined using a calibrated yeaple probe ( xinix research inc . , portsmouth , nh , usa ) set at the equivalent microamperes to deliver a fixed force of 30 g. the probe tip was placed perpendicular to the buccal surface and moved in a slow distal - to - medial sweeping motion across the tooth surface in order to ensure application of the stimulus across the sensitive area of the exposed dentine . subsequent testing at weeks 1 , 2 , 4 , and 8 was performed by a single examiner on the two teeth selected at baseline . testing began at 10 g and increased by 10 g with each successive challenge until a yes response was obtained or the 50 g upper limit reached . the force setting eliciting the positive response was then repeated , but in the absence of a second positive response the force setting was increased by 10 g and continued until a force was found that elicited two consecutive yes the evaporative ( air ) assessment was taken after the determination of tactile sensitivity with a minimum of 10 min recovery time between each evaluation . it was felt that 10 mins would allow sufficient time for physiological and neurological recovery following the first set of stimuli . the test involved directing an air stream ( 60 5 psi ) at ambient temperature for 1 s from a triple air dental syringe toward the exposed dentine surface from a distance of approximately 1 cm ; adjacent teeth were shielded from the stimulus by the fingers of the examiner . each tooth was scored according to the four - point schiff air sensitivity scale , defined as follows . 0 subject does not respond to air stimulus . 1 subject responds to air stimulus , but does not request discontinuation of stimulus . 2 subject responds to air stimulus , considers stimulus to be painful , and requests discontinuation of the stimulus . at the screening visit , the teeth of subjects eliciting a score of 2 or 3 were recorded as qualifying as potential test teeth . at the baseline visit , subjects were also asked to rate the pain intensity associated with the evaporative ( air ) stimulus using a linear 100 mm vas end - anchored by no pain and worst pain imaginable . at least two incisors , canines , or premolars were required to provide an air sensitivity response of 25 mm based on the vas , a schiff score 2 , and a tactile pain threshold between 10 and 50 g. subject randomisation was performed using as stratification factors ; the number of air sensitive teeth at baseline based on the vas response ( < 6/6 ) and the mean vas score at baseline ( < 60 mm/60 mm ) . in total , 63 ( 57.3% ) of subjects had 6 or more sensitive teeth at baseline , 84 ( 76.4% ) had a mean baseline vas score 60 mm . a total of 110 eligible adult volunteers took part in this study after signing an informed written consent form . of these , 19 ( 17.3% ) were male and 91 ( 82.7% ) were female , with a mean ( sd ) age of 42.0 10.43 years ; all subjects were of chinese ethnicity . this was an 8-week , single - centre , examiner - blind , randomised , two - arm , parallel group study in otherwise healthy subjects experiencing dentine hypersensitivity . the clinical trial conformed to consensus design and conduct recommendations regarding population selection , sample size , test stimuli , assessment , and outcome variables . eligible subjects were randomised to the two treatment groups ( n = 55 per treatment group ) . two test teeth were selected on the basis of their eligibility criteria for both tactile and evaporative stimuli . subjects were given their assigned dentifrice and toothbrush to use in place of their regular oral hygiene products for the next 8 weeks . subjects were not permitted to use any oral care products after baseline other than the dentifrice and toothbrush provided . prior to the baseline visit , subjects were instructed to refrain from oral hygiene procedures and gum chewing for a minimum of 8 h before their scheduled appointment . subjects were reminded not to eat or drink for a minimum of 2 h nor take analgesics for at least 8 h prior to their scheduled appointments . dental scaling , prophylaxis , and bleaching were prohibited for the duration of the study . the test product was used for one - timed minute twice per day ( am / pm ) with subjects instructed to dispense a ribbon of paste to cover at least three quarters of the head of the toothbrush . during the treatment phase , subjects returned to the study site after 1 , 2 , 4 and 8 weeks following their baseline visit for evaluation of dentine hypersensitivity , oral health status , and continuing eligibility . the primary efficacy measure was observance of a statistically significant within treatment reduction from baseline in the subjects ' assessment of air sensitivity , measured using the vas , after 8 weeks . secondary efficacy measures included changes in schiff score and tactile pain threshold after 8 weeks . the definitions of reduction in sensitivity in each case are a decrease of at least 10 mm in mean vas score , a decrease in mean schiff score across the two teeth of at least 0.5 units , and a mean increase of at least 10 g in the tactile pain threshold determined with the yeaple probe . the safety profile of both dentifrices was assessed by reference to adverse events and oral soft tissue abnormalities . the subject level change from baseline was calculated as the mean change from baseline observed across the two selected teeth within a subject . a fixed effects analysis of covariance ( ancova ) model was used with treatment included as a fixed effect factors and baseline number of air sensitive teeth and mean baseline vas score across the two teeth included as covariates . all significance tests were two - sided and performed at the 5% level of significance with no adjustments for multiple comparisons . for analyses of subject level schiff data and subject level tactile pain threshold data , an ancova model was used with treatment and baseline vas stratification value and included as fixed effect factors . baseline number of air sensitive teeth and either mean baseline schiff score or mean baseline tactile pain threshold score across the two teeth were included as covariates , respectively . intent - to - treat ( itt ) and per protocol ( pp ) analyses were performed for within and between treatment assessments of change from baseline to weeks 1 , 2 , 4 , and 8 . two - sided significance tests were performed at the 5% level with no adjustments for multiple comparisons . tooth level analyses were also performed wherein each tooth individually contributed to the analyses , as opposed to the mean across the two teeth used for the subject level analyses . the statistical models were similar to those used for subject level data , but with the addition of subject included as a random effect . itt analyses were performed for within and between treatment assessments of change from baseline to weeks 1 , 2 , 4 , and 8 . two - sided significance tests were performed at the 5% level with no adjustments for multiple comparisons . one subject withdrew from the study and provided no postbaseline efficacy data leading to an itt population of 109 subjects . one subject who received antibiotics for gingival inflammation was excluded yielding a pp population of 108 subjects . other major protocol violations were reported for a further 13 subjects leading to the exclusion of specific data from pp analyses but not full exclusion of subjects . specifically , 8 subjects exhibited noncompliance with the study product , 3 were significantly noncompliant with the visit schedule and 2 subjects used a nonstudy toothbrush . a statistically significant reduction from baseline in tooth sensitivity was observed for both test products , for all three sensitivity parameters , at all four time points , including the primary analysis of sensitivity reduction at week 8 based on mean vas score as shown in table 1 . there were no statistically significant differences between the two study products in the reduction of sensitivity from baseline to any of the four time points , for any of the three sensitivity parameters , as shown in table 2 . the number and percentage of subjects experiencing a reduction in sensitivity from baseline after 8 weeks of treatment is summarised in table 3 for each sensitivity measure . of those subjects treated with dentifrice a , 50 ( 93% ) exhibited a reduction in air sensitivity measured using the vas , 53 ( 98% ) exhibited a reduction in air sensitivity based on schiff score , and 47 ( 87% ) exhibited a reduction in tactile sensitivity based on force - pain threshold . the equivalent numbers for subjects treated with dentifrice b were 44 ( 88% ) for the vas , 49 ( 98% ) for schiff score , and 42 ( 84% ) for the tactile pain threshold . the pp analyses performed on subject level data and additional itt analyses performed on tooth level data of within and between treatment outcomes yielded comparable results to those obtained using the primary subject level itt approach ( data not shown ) . the sole exception related to the pp analysis of vas score , where the reduction from baseline at 1 week for the treatment group using dentifrice b was not statistically significant ( p = 0.0645 ) . investigation of the relationship between efficacy variables yielded correlation coefficients of 0.74 , 0.55 , and 0.53 , respectively , for vas score versus schiff score , schiff score versus tactile pain threshold , and vas score versus tactile pain threshold . twelve subjects reported a total of 19 treatment emergent adverse events of which 11 were related to oral health status ; 3 were associated with the treatment group using dentifrice a and 8 with the group using dentifrice b. two adverse events were judged to be possibly related to the study treatment ( dentifrice b ) and were reported by the same subject , however both were mild in intensity and resolved at study completion . in this study , the efficacy of two new desensitising dentifrice formulations has been compared in a blinded , parallel group , controlled clinical study . the two dentifrices are very different both in terms of composition and their claimed mode of action . dentifrice a contains kno3 , a proven nerve desensitising active , and naf as the source of ionic fluoride . the formulation also contains 2-methyl butane , a nonsurfactant - based foaming agent with a boiling point of ca . 30c dentifrice b , in contrast , contains a novel dentine occlusion system that includes the dibasic amino acid arginine , bicarbonate , and calcium carbonate . the formulation does not contain fluoride due to the presence of calcium , but contains fluorine as namfp in order to confer anticaries activity . the results demonstrate comparable clinical effectiveness for the two test dentifrices with significant reductions in measures of tooth sensitivity observed across all measures , at all predefined time points . after 8 weeks of twice daily use , a 49% versus 45% reduction in the subjective outcome measure of evaporative vas score , and a 61% versus 61% reduction in examiner - based schiff score was observed for dentifrice a and b , respectively , versus baseline . large and clinically relevant improvements in the tactile pain threshold versus baseline were also observed for the two treatment groups . no significant between - treatment efficacy differences were apparent at any of the time points . given the comprehensive improvements in sensitivity relief in both treatment groups across all the efficacy measures , these results suggest a comparable level of performance between the test products with no apparent difference between the potassium - based novel gel - to - foam formulation ( dentifrice a ) and the arginine - based formulation ( dentifrice b ) . tooth - level analyses revealed one minor difference to the subject - level analysis wherein the evaporative vas score in the treatment group using the arginine - based dentifrice was not statistically different from baseline after 1 week of treatment ( p = 0.0645 ) . given the number of the statistical evaluations conducted , this specific outcome does not materially change the overall inferences drawn from these results of this clinical study . the correlation coefficients used to examine the relationship between the different efficacy variables showed a moderately strong correlation between the vas score and schiff score ( 0.78 ) . this is not unexpected given that the data contributing to both are elicited by the same evaporative air stimulus . comparisons of the schiff score versus tactile pain threshold and vas score versus tactile pain threshold elicited similar correlation coefficients ( 0.55 and 0.53 , resp . ) . that they are less strongly correlated than the vas and schiff scores is again to be expected . as previously indicated , two recently published 8-week dentine hypersensitivity studies reported superior efficacy for the occlusion - based dentifrice b containing the novel arginine / bicarbonate / calcium carbonate technology when compared with a conventional desensitising dentifrice containing kno3 [ 39 , 40 ] . in the aforementioned study , efficacy was determined using two outcome measures , mean schiff score , and mean tactile pain threshold and were found to be statistically superior for the arginine - based dentifrice at 2 , 4 , and 8 weeks ( p < 0.05 ) . the present study employed a similar clinical methodology that accords with published consensus recommendations , albeit the authors acknowledge that a negative control arm would have helped to contextualise the extent of the efficacy benefit offered by either of the treatments . the lack of negative control does make it is problematic to interpret the clinical relevance of the response measured in either treatment group in this specific study . future clinical studies on either product should look to incorporate a negative control arm to address this gap in the scientific design . 38% ) in comparison to the previously cited studies involving dentifrice b [ 39 , 40 ] , and additionally employed a subjective patient - assessed measure of product performance via the vas score . as such the current study was adequately powered to differentiate any clinically relevant differences in product performance . the results in the present study demonstrate no difference in efficacy for the two dentifrices in respect of the observed reductions of in self- and examiner - assessed dentine hypersensitivity at all observed time points ( 1 , 2 , 4 , and 8 weeks ) . one possible explanation for the different result observed in the present study compared to those reported previously for dentifrice b [ 39 , 40 ] may relate to differences in the sensory and aesthetic profiles of dentifrice a versus conventional potassium nitrate - based dentifrices . treatment with the gel - to - foam formulation has been previously shown to result in large and highly significant improvements in sensitivity relief . the formulation breaks down extremely quickly during brushing , thereby facilitating rapid dispersal of the constituent ingredients throughout the mouth . in addition , patient - based in - use sensory studies ( gsk - unpublished data ) indicate a strong preference for the gel - to - foam formulation compared to regular desensitising dentifrice formulations . this may be expected to promote greater compliance both in terms of frequency and duration of brushing . the prevalence of dentine hypersensitivity has been studied in a variety of different populations in a number of different countries across the world . direct comparison of the different epidemiological studies is often confounded by the use of different clinical methodologies and definitions . the lack of homogeneity in reporting dentine hypersensitivity does make characterising the size and progression of the condition a challenge . in this respect , consensus guidelines defining the most suitable epidemiological approach to take for assessing dentine hypersensitivity prevalence would be welcome . however , what is not in dispute is that millions of people are affected by dentine hypersensitivity and that for a number of patients the severity of the problem is such that it significantly impacts on their quality of life . it may therefore be speculated that the prevalence of dentine hypersensitivity will rise as lifespans increase and people retain their dentition for longer . prevalence levels may also be expected to increase in developing countries where increased affluence translates into behavioural adaptations that enhance susceptibility . despite the widespread incidence of dentine hypersensitivity , it is striking that the percentage of sufferers seeking clinical intervention , and/or using otherwise available desensitising treatments such as dentifrices , are comparatively small . in the latter case , there is anecdotal evidence to suggest that the negative organoleptics associated with the desensitising agent may be responsible for poor uptake and compliance . formulation strategies that improve product tolerability may promote their continued prescribed use , and potentially enhance therapeutic outcomes . in conclusion , this study demonstrates that the two study groups using either a potassium - based gel - to - foam dentifrice ( sensodyne multi action iso - active ) or an occlusion - based dentifrice ( colgate sensitive pro - relief ) over an 8 week duration experienced statistically significant reductions in self- or examiner - based dentine hypersensitivity versus baseline , however no differences in levels of hypersensitivity were observable between the two groups at any timepoint .

a comparison of the desensitising efficacy of two commercially available dentifrices with different modes of action was conducted in a randomised , examiner - blind , two - arm , parallel group , 8-week , longitudinal clinical study . dentifrice a , ( sensodyne multi action iso - active ) , contained 50000 ppm kno3 and 1450 ppm fluoride as naf . dentifrice b , colgate sensitive pro - relief , contained a combination of 80000 ppm arginine , bicarbonate , calcium carbonate , and 1450 ppm fluorine as namfp . subjects ( n = 110 ) , stratified into two groups ( n = 55 ) , brushed twice - daily for 60 s , over an 8-week period . sensitivity status , compliance , and safety were determined at 1 , 2 , 4 , and 8 weeks . a fixed - effects ancova statistical model was applied to the intent - to - treat population using a two - sided 5% significance level . after 8 weeks , the treatment groups using dentifrice a and dentifrice b exhibited mean reductions from baseline of 49% and 45% in air sensitivity visual analogue scale ( vas ) score , 61% ( both ) in examiner - based schiff sensitivity score , and clinically significant reductions in tactile pain threshold ; all reductions were statistically significant ( p < 0.0001 ) . both treatment groups also exhibited significant reductions across all sensitivity measures at 1 , 2 , and 4 weeks ( p 0.0059 , dentifrice a ; p 0.0137 , dentifrice b ) .

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

the aim of the present study was to compare the effectiveness of the two aforementioned dentifrices in an 8-week , two - arm , parallel group , longitudinal dentine hypersensitivity study . sensodyne multi action iso - active ( dentifrice a ) contained 50000 ppm kno3 and 1450 ppm fluoride as the sodium salt ( naf ) and was manufactured by glaxosmithkline consumer healthcare ( brentford , middlesex , uk ) . colgate sensitive pro - relief ( dentifrice b ) contained 80000 ppm arginine , bicarbonate , calcium carbonate , and 1450 ppm fluorine as namfp and was manufactured by colgate - palmolive ( guildford , surrey , uk ) . subjects must have successfully completed a visual analogue scale ( vas ) instruction exercise at screening , whose purpose was to train subjects in the use of vas by requiring subjects to estimate how much of the total area of a series of 7 shapes had been shaded using a 100 mm line anchored at one end with no shading and the other with complete shading . , portsmouth , nh , usa ) set at the equivalent microamperes to deliver a fixed force of 30 g. the probe tip was placed perpendicular to the buccal surface and moved in a slow distal - to - medial sweeping motion across the tooth surface in order to ensure application of the stimulus across the sensitive area of the exposed dentine . subsequent testing at weeks 1 , 2 , 4 , and 8 was performed by a single examiner on the two teeth selected at baseline . this was an 8-week , single - centre , examiner - blind , randomised , two - arm , parallel group study in otherwise healthy subjects experiencing dentine hypersensitivity . during the treatment phase , subjects returned to the study site after 1 , 2 , 4 and 8 weeks following their baseline visit for evaluation of dentine hypersensitivity , oral health status , and continuing eligibility . intent - to - treat ( itt ) and per protocol ( pp ) analyses were performed for within and between treatment assessments of change from baseline to weeks 1 , 2 , 4 , and 8 . itt analyses were performed for within and between treatment assessments of change from baseline to weeks 1 , 2 , 4 , and 8 . of those subjects treated with dentifrice a , 50 ( 93% ) exhibited a reduction in air sensitivity measured using the vas , 53 ( 98% ) exhibited a reduction in air sensitivity based on schiff score , and 47 ( 87% ) exhibited a reduction in tactile sensitivity based on force - pain threshold . the sole exception related to the pp analysis of vas score , where the reduction from baseline at 1 week for the treatment group using dentifrice b was not statistically significant ( p = 0.0645 ) . twelve subjects reported a total of 19 treatment emergent adverse events of which 11 were related to oral health status ; 3 were associated with the treatment group using dentifrice a and 8 with the group using dentifrice b. two adverse events were judged to be possibly related to the study treatment ( dentifrice b ) and were reported by the same subject , however both were mild in intensity and resolved at study completion . in this study , the efficacy of two new desensitising dentifrice formulations has been compared in a blinded , parallel group , controlled clinical study . 30c dentifrice b , in contrast , contains a novel dentine occlusion system that includes the dibasic amino acid arginine , bicarbonate , and calcium carbonate . after 8 weeks of twice daily use , a 49% versus 45% reduction in the subjective outcome measure of evaporative vas score , and a 61% versus 61% reduction in examiner - based schiff score was observed for dentifrice a and b , respectively , versus baseline . given the comprehensive improvements in sensitivity relief in both treatment groups across all the efficacy measures , these results suggest a comparable level of performance between the test products with no apparent difference between the potassium - based novel gel - to - foam formulation ( dentifrice a ) and the arginine - based formulation ( dentifrice b ) . in the aforementioned study , efficacy was determined using two outcome measures , mean schiff score , and mean tactile pain threshold and were found to be statistically superior for the arginine - based dentifrice at 2 , 4 , and 8 weeks ( p < 0.05 ) . the results in the present study demonstrate no difference in efficacy for the two dentifrices in respect of the observed reductions of in self- and examiner - assessed dentine hypersensitivity at all observed time points ( 1 , 2 , 4 , and 8 weeks ) . in conclusion , this study demonstrates that the two study groups using either a potassium - based gel - to - foam dentifrice ( sensodyne multi action iso - active ) or an occlusion - based dentifrice ( colgate sensitive pro - relief ) over an 8 week duration experienced statistically significant reductions in self- or examiner - based dentine hypersensitivity versus baseline , however no differences in levels of hypersensitivity were observable between the two groups at any timepoint .

incidence , prevalence , and risk have been found to be high in both europe and the us . the rotterdam study found that a man and a woman at age 55 have , respectively , a 33.0% and 28.5% chance of developing hf during their remaining lifetime , whereas the framingham heart study reported lifetime risks at age 50 of 20.9% for men and the rotterdam study further showed that the prevalence of hf increased with age ; in 1998 , 0.9% of subjects 55 - 64 years old had hf compared with 17.4% of those 85 years old or older . approximately 5.7 million americans have been diagnosed with the disease , and each year an additional 550,000 patients are diagnosed for the first time . this increasing clinical burden is expected to be matched with an increasing financial burden . by means of a prevalence - based approach , the economic burden of hf in the uk was estimated to be 905.3 million in 2000 , a 26% increase over 1995 estimates and equivalent to 1.91% of the total national health service expenditure . it is estimated that , in 2009 , over $ 37.2 billion was spent on costs of hf in the us . medicare expends more dollars for the diagnosis and treatment of hf than for any other diagnosis , as hf is the most frequent medicare diagnosis - related group . total mention deaths from hf in the us was 292,000 , which is higher than in 1995 ( 287,000 ) . although considerable advances have been made in the management of hf over the past few decades , hf remains a major public health issue with high prevalence and poor outcomes . an understanding of the pathophysiology and natural history of hf underpins the therapeutic approaches used to achieve the goals of treatment , which are to relieve symptoms , to avoid hospital admission , and to prolong life . on the basis of a large number of randomized controlled trials , drugs are the mainstay of treatment for all patients with hf and reduced left ventricular systolic function . device therapy and transplant surgery have carved out a respectable place in the field over the past decade . diuretics are essential for relief of dyspnea and signs of sodium and water retention ; they are needed in virtually all patients with symptomatic hf . these are best used flexibly and in the minimum dose needed to maintain euvolemia and avoid electrolyte disorders ( hypokalemia and hyponatremia ) , gout , and renal dysfunction . in advanced hf , high doses of loop diuretics and thiazide or thiazide - like diuretic ( metolazone ) might be needed to maintain dry weight. despite the widespread use of diuretics , no evidence exists to date to show that these agents prolong survival , and their use could activate key neurohormonal systems such as the renin angiotensin aldosterone system ( raas ) . raas is important for progression of the hf disease process ; conversely , attenuation of this system has yielded considerable benefit in the management of systolic hf . angiotensin - converting enzyme inhibitors ( aceis ) , by reducing the production of angiotensin ii and possibly by blocking the degradation of bradykinin , exert many biological effects that lead to improvement in symptoms , fewer admissions to the hospital , and prolonged survival in hf ; as a consequence , they are recommended for all patients with systolic dysfunction . reduced mortality was also noted with ace inhibition in people with recent myocardial infarction and left - ventricular systolic dysfunction , but without hf symptoms . the main causes of intolerance are cough , symptomatic hypotension , and renal dysfunction , which are exacerbated by overdiuresis and nonsteroidal anti - inflammatory drugs . angiotensin receptor blockers ( arbs ) seem to be a reasonable alternative for patients unable to tolerate ace inhibition secondary to cough . however , the use of ace inhibition in addition to arbs remains uncertain . several studies that looked at the combination of aceis and arbs in hf patients are worth mentioning . the valsartan heart failure trial ( val - heft ) included patients with a left ventricular ejection fraction ( lvef ) of less than 40% and new york heart association ( nyha ) class ii - iv ; 92% were on aceis and 35% were on beta blockers . rates of death from any cause during the entire trial were 19.7% in the valsartan group and 19.4% in the placebo group ( p = 0.8 ) . combined endpoint of death from any cause , hospitalization for hf , cardiac arrest with resuscitation , and intravenous therapy was statistically significant ( p = 0.009 ) ; however , it was driven mainly by a decline in hospitalization rate for hf ( 13.8% in the valsartan group and 18.2% in the placebo ) . patients were further subdivided into subgroups based on their background therapy ( aceis and beta blockers ) . among those who were receiving both drugs at baseline , valsartan had an adverse effect on mortality ( p = 0.009 ) and was associated with a trend toward an increase in combined endpoints of mortality and morbidity ( p = 0.1 ) . the candesartan in heart failure - assessment of reduction in mortality and morbidity trial ( charm - added ) was the only study that showed a reduction in cardiovascular mortality ( absolute risk reduction [ arr ] of 3.6% ) for combination therapy ; it also showed reduced hospitalization for hf ( arr of 4% ) . however , the all - cause mortality was not different between the groups . given the available data , it is safe to conclude that combination treatment in hf patients should be used with caution . both val - heft and charm - alternative confirm that arbs are appropriate substitutes for aceis when cough is the reason for intolerance . by contrast , use of the aldosterone receptor antagonist spironolactone in patients with advanced disease ( class iii or iv systolic hf ) yielded clear - cut survival benefits additional to background ace inhibition , although only a few people were receiving beta blockers . furthermore , the selective aldosterone receptor antagonist eplerenone was of benefit in individuals with systolic hf early after myocardial infarction . careful attention to the development of hyperkalemia during initiation is an essential safety measure when using aldosterone antagonists . furthermore , potassium supplements should be discontinued until the potassium levels reach equilibrium after several months of usage . chronic activation of the sympathetic nervous system - the cardiac effects of which are attenuated by beta blockers - has a key role in hf disease progression , including fibrosis , necrosis , apoptosis , and arrythmogenesis . the beneficial effects of beta blockers have largely been studied as additional to background ace inhibition , and therefore both are judged to be mandatory treatment . these effects have been shown with bisoprolol , carvedilol , and extended - release metoprolol in patients with stable systolic hf across a broad range of disease severities . beta blockers in hf patients should be initiated at low doses and gradually up - titrated to the target dosages proven effective in the major mortality trials ( carvedilol 25 mg twice daily , bisoprolol 10 mg daily , or metoprolol succinate 200 mg daily ) [ 15 - 17 ] . researchers in the cardiac insufficiency bisoprolol study iii ( cibis iii ) raised the hypothesis that the order of initiation of aceis and beta blockers might not be vital to outcomes provided that eventually the patient is receiving appropriate doses of both classes of drug in a timely manner . the combination of hydralazine and isosorbide dinitrate was the first treatment shown to improve survival in hf , but a subsequent study showed that it was less effective than an acei in direct comparisons . a strategy of adding a vasodilator combination to conventional treatment , including an acei , a beta blocker , and spironolactone , was shown to reduce mortality and admissions to hospital for hf in african - american patients , although this conclusion was based on a small number of events . although cardiac transplantation remains the ultimate surgical strategy for hf , the poor availability of suitable donor organs renders this option epidemiologically insignificant . other surgical approaches to hf include revascularization for ischemic hf , mitral valve repair to address functional mitral regurgitation associated with pathological ventricular remodeling , and surgical reconstruction of the size and shape of the failing left ventricle ( lv ) to render it more effective to pump . with the exception of the recently published surgical treatment for ischemic heart failure ( stich ) trial , most of these surgical techniques have not been adequately tested . the stich trial , funded by the national heart , lung , and blood institute , is a multicenter international randomized trial addressing two specific primary hypotheses : ( a ) coronary artery bypass grafting ( cabg ) with intensive medical therapy improves long - term survival compared with survival with medical therapy alone , and ( b ) in patients with anterior left ventricular dysfunction , surgical ventricular reconstruction to a more normal left ventricular size plus cabg improves survival free of subsequent hospitalization for cardiac cause when compared with cabg alone . subjects meeting the broad inclusion criteria of coronary artery disease ( cad ) amenable to cabg with an lvef of 0.35 or less without a specific exclusion were segregated into three strata , depending on investigator - determined suitability for continued medical therapy alone and eligibility for surgical ventricular restoration ( svr ) . eligibility for medical therapy alone was defined by the investigator but generally excluded patients with intraluminal left main coronary artery stenosis of 50% or more or severe disabling angina ( canadian cardiovascular society [ ccs ] class iii ) unresponsive to nonsurgical interventions . stratum a subjects are defined as suitable for medical therapy with or without cabg , and consenting patients are randomly assigned in a 1:1 ratio to medical therapy alone or medical therapy with cabg . stratum b subjects , defined as eligible for all three treatment options , are randomly assigned in a 1:1:1 ratio to medical therapy alone , medical therapy with cabg , or medical therapy with cabg and svr . subjects eligible for cabg and svr are randomly assigned in a 1:1 ratio to stratum c to either cabg or cabg with svr . this study included 1000 patients ( from 96 clinical centers ) who were randomly assigned to undergo either cabg alone ( 499 patients ) or cabg with svr ( 501 patients ) . the median age was 62 years , and 147 of the 1000 patients were women . the median left ventricular function was 28% , and the median left ventricular end systolic volume index ( lvesvi ) was 82 ml per square meter of body surface area . multivessel cad was present in 64% of patients in each group . although standard hf treatment was recorded at baseline and was encouraged throughout the trial , no information about the rates of its use over time is provided . the two strategies were equally successful in improvement of patients symptoms , with an average improvement in ccs class of 1.7 classes ( p = 0.84 ) and an average of 1 nyha class ( p = 0.7 ) . equivalent improvement in the 6-minute walk test , obtained at baseline and 4-month follow - up , was demonstrated between the two groups ( 48 m among patients who were assigned to cabg and 52 m among patients assigned to cabg and svr ; p = 0.8 ) . there was a greater reduction in lvesvi with combined procedure ( 16 ml per square meter of body surface area ) when compared with cabg alone ( 5 ml per square meter of body surface area ) . the primary outcome of death from any causes or hospitalization for cardiac causes was the same in cabg alone ( 59% ) when compared with cabg with svr ( 58% ) ( figure 1 ) . death occurring within 30 days after the procedure did not differ significantly between the two study groups . ( a ) the probability of the primary outcome ( death from any cause or hospitalization for cardiac causes ) , which did not differ significantly between the two groups . the primary outcome occurred in 292 patients ( 59% ) assigned to undergo coronary artery bypass grafting ( cabg ) alone and in 289 patients ( 58% ) assigned to undergo cabg with surgical ventricular reconstruction ( svr ) ( hazard ratio 0.99 , 95% confidence interval [ ci ] 0.84 - 1.17 ) . ( b ) the probability of death from any cause , which occurred in 141 patients ( 28% ) assigned to undergo cabg and in 138 patients ( 28% ) assigned to undergo cabg with svr ( hazard ratio 1.00 , 95% ci 0.79 - 1.26 ) . reproduced with permission from . the first stich trial hypothesis is addressing an important question of revascularization of patients with cad and lv systolic dysfunction . although specific clinical problems in this population , such as severe angina , are used to decide on revascularization strategies , the vast majority of patients with ischemic cardiomyopathy ( icm ) have limited or no angina and fall into a gray zone where clear evidence for adding cabg to optimal medical therapy is either absent or outdated . recent guidelines recommend cardiac resynchronization therapy ( crt ) in patients with an lvef of less than 35% , nyha class iii - iv symptoms , and a qrs duration of greater than 0.12 seconds . this represents a subgroup of hf patients with abnormal cardiac conduction and possible ventricular dyssynchronous contraction . dyssynchronous contraction results in suboptimal ventricular filling , reduced lv dp / dt ( rate of rise in lv pressure ) , prolonged duration and severity of mitral regurgitation , and paradoxical septal wall motion . crt is a biventricular pacemaker device that electrically paces the right and left ventricles in a synchronized mode and thus may improve ventricular contraction and diminish secondary mitral regurgitation . crt in conjunction with optimal medical therapy has been shown to significantly improve quality of life , functional class , exercise capacity and distance , and ejection fraction ( ef ) , as well as reduce hf hospitalizations by 32% and all - cause mortality by 25% ( in a meta - analysis ) . it was unknown whether these devices would be beneficial in asymptomatic or mildly symptomatic patients . madit - crt ( multicenter automatic defibrillator implantation trial with cardiac resynchronization therapy ) investigated whether crt - d ( crt + implantable cardioverter defibrillator [ icd ] device ) would reduce all - cause mortality and hf events ( need for intravenous diuretic therapy as an outpatient or augmented hf regimen during hospitalization ) in patients who qualify for icd but are nyha class i or ii . inclusion criteria were age of more than 21 years , an lvef of not more than 30% , stable optimal medical therapy , a qrs of at least 130 ms , normal sinus rhythm , and nyha class i or ii ( icm ) or class ii ( nonischemic cardiomyopathy ) . in a 3:2 ratio , 1820 patients were randomly assigned to either crt - d ( 1089 ) or icd ( 731 ) alone . successful device implantation occurred in 98.4% of patients , and 95.4% were in their originally assigned arm . the groups were well matched at baseline , and for the entire study group , icm occurred in about 55% of patients , approximately 10% had nyha class iii / iv symptoms more than 3 months prior to enrollment , mean lvef was 24% , with a mean 6-minute walk distance of about 361 ms , 70% had left bundle branch block , and 64% had a qrs width of at least 150 ms . medical therapy was optimized with 93% receiving beta blockers , 97% aceis or arbs , 31% aldosterone antagonist , 74% diuretics , 67% statin , and 7% amiodarone . the trial was terminated early after a mean follow - up of 2.4 years because of a significant difference in the combined primary endpoint . death or nonfatal hf events were 17.2% for crt - d versus 25.3% for icd ( hazard ratio 0.66 , 95% confidence interval [ ci ] 0.52 - 0.84 ; p = 0.001 ) ( figure 2 ) . reduced hf events ( 13.9% versus 22.8% , p < 0.001 ) drove the primary endpoint to significance , with no difference in all - cause mortality ( 6.8% versus 7.3% , p = 0.99 ) . in subgroup analysis , women and patients with a qrs duration of 150 ms or more showed a greater benefit . improvement of lvef occurred compared with baseline in the crt - d arm ( 0.11 versus 0.03 , p < 0.001 ) as measured by echocardiography in a subgroup of patients . lvesv and lvedv ( left ventricular end diastolic volume ) decreased significantly compared with baseline in the crt - d arm ( 57 versus 18 ml and 52 versus 15 ml , respectively ; p < 0.001 ) . more frequent device - related adverse outcomes occurred in the crt - d arm compared with the icd arm in the 30 days after implantation . there was a significant difference in the estimate of survival free of heart failure between the group that received cardiac resynchronization therapy plus an implantable cardioverter defibrillator ( crt - icd ) and the group that received an icd only ( unadjusted p < 0.001 by the log - rank test ) . reproduced with permission from . are that approximately 10% of patients qualified for crt with nyha class iii or iv more than 3 months prior to random assignment . also , patients were assessed by investigators who were not blinded to the patients device and thus there was a possible bias toward the crt - d arm . madit - crt results reveal that crt - d reduced the primary combined endpoint of death or hf events as compared with icd alone with optimal medical management in nyha class i and ii patients , driven largely by a reduction in hf events . while these effects may have been exaggerated due to the early termination of follow - up , it is also likely that a longer follow - up in these nyha i / ii patients would have shown a beneficial effect on mortality . as a result of this and other studies , device therapy has begun to play an even greater role in the management of hf patients . despite current treatments with optimal medical and device therapy including crt continuous intravenous inotropic assists in the short term only , and transplants are offered to a very small percentage of hf patients . thus , more permanent solutions with mechanical circulatory support devices such as left ventricular assist devices ( lvads ) are being evaluated . in 2002 , the landmark rematch ( randomized evaluation of mechanical assistance for the treatment of congestive heart failure ) trial was the first to randomly assign nontransplantable end - stage hf patients to optimal medical management or lvad plus optimal medical management . a 48% risk reduction in death from any cause was found in the lvad group as compared with medical therapy alone . one - year survival rate was 52% with lvad and 23% at 2 years versus 25% in the medically treated patients at 1 year and only 8% at 2 years . the older pulsatile - flow lvad used in rematch was compared with the new continuous - flow heartmate ii device in the advanced heart failure treated with continuous - flow left ventricular assist device ( heartmate ii ) trial . the continuous - flow device has fewer moving parts , is much smaller , and has less mechanical breakdown than the pulsatile - flow lvad . patients with advanced hf ( ef of less than 25% , peak oxygen consumption of less than 14 ml / kg per minute or less than 50% of the predicted value , and nyha class iiib or iv symptoms for at least 45 of the 60 days before enrollment ) , refractory to optimal medical management ( dependence on an intra - aortic balloon pump for 7 days or inotropes for at least 14 days before enrollment ) , and ineligible for transplantation were evaluated . random assignment occurred in a 2:1 ratio to undergo implantation of a continuous - flow device , heartmate ii ( 134 patients ) , or the currently approved pulsatile - flow device heartmate xve ( 66 patients ) . the primary composite endpoint was survival free from disabling stroke and reoperation to repair or replace the device at 2 years . secondary endpoints were survival , frequency of adverse events , quality of life , and functional capacity . the two treatment groups had similar preoperative characteristics , including a median age of 64 years ( range of 26 - 81 ) and a mean lvef of 17% , and almost 80% had received intravenous inotropic agents , more than 20% were fitted with an intra - aortic balloon pump , and greater than 60% failed crt . the primary composite endpoint occurred more frequently in patients with continuous - flow devices ( 62 of 134 [ 46% ] ) than with pulsatile - flow devices ( 7 of 66 [ 11% ] ; p < 0.001 ) . patients with continuous - flow devices had greater actuarial survival rates at 2 years ( 58% versus 24% ; p = 0.008 ) . estimated 1- and 2-year survival rates were 68% ( 95% ci 60 - 76% ) and 58% ( 95% ci 49 - 67% ) , respectively , with the continuous - flow device and 55% ( 95% ci 42 - 69% ) and 24% ( 95% ci 1 - 46% ) with the pulsatile - flow device . the better durability of the heartmate ii continuous - flow device was the primary determinant of the significant positive endpoint . one - third of patients with pulsatile - flow lvad required pump replacements , one required urgent transplantation , and three required device explantation . about 10% of patients with continuous - flow lvad required pump replacements due to breakage of the percutaneous lead , pump thrombosis , or outflow elbow disconnection . major adverse events were significantly reduced in patients with a continuous - flow lvad , including device- and non - device - related infection , right hf , respiratory failure , renal failure , and cardiac arrhythmia . there was a significant improvement in quality of life and functional capacity in both groups . limitations of the study include the potential bias despite random assignment of the devices because both patient and physician were aware that the device was being implanted . overall , the study found that continuous - flow lvad in patients with advanced hf significantly improved the probability of survival - free device failure at 2 years as compared with a pulsatile device . most importantly , the study demonstrated that the survival of patients with a continuous - flow device is twice that of the rematch trial pulsatile - flow group at 2 years , thus illustrating that newer technology and improved medical treatment have enhanced the survival of refractory hf patients in the past decade . although cardiac transplantation remains the ultimate surgical strategy for hf , the poor availability of suitable donor organs renders this option epidemiologically insignificant . other surgical approaches to hf include revascularization for ischemic hf , mitral valve repair to address functional mitral regurgitation associated with pathological ventricular remodeling , and surgical reconstruction of the size and shape of the failing left ventricle ( lv ) to render it more effective to pump . with the exception of the recently published surgical treatment for ischemic heart failure ( stich ) trial , most of these surgical techniques have not been adequately tested . the stich trial , funded by the national heart , lung , and blood institute , is a multicenter international randomized trial addressing two specific primary hypotheses : ( a ) coronary artery bypass grafting ( cabg ) with intensive medical therapy improves long - term survival compared with survival with medical therapy alone , and ( b ) in patients with anterior left ventricular dysfunction , surgical ventricular reconstruction to a more normal left ventricular size plus cabg improves survival free of subsequent hospitalization for cardiac cause when compared with cabg alone . subjects meeting the broad inclusion criteria of coronary artery disease ( cad ) amenable to cabg with an lvef of 0.35 or less without a specific exclusion were segregated into three strata , depending on investigator - determined suitability for continued medical therapy alone and eligibility for surgical ventricular restoration ( svr ) . eligibility for medical therapy alone was defined by the investigator but generally excluded patients with intraluminal left main coronary artery stenosis of 50% or more or severe disabling angina ( canadian cardiovascular society [ ccs ] class iii ) unresponsive to nonsurgical interventions . stratum a subjects are defined as suitable for medical therapy with or without cabg , and consenting patients are randomly assigned in a 1:1 ratio to medical therapy alone or medical therapy with cabg . stratum b subjects , defined as eligible for all three treatment options , are randomly assigned in a 1:1:1 ratio to medical therapy alone , medical therapy with cabg , or medical therapy with cabg and svr . subjects eligible for cabg and svr are randomly assigned in a 1:1 ratio to stratum c to either cabg or cabg with svr . this study included 1000 patients ( from 96 clinical centers ) who were randomly assigned to undergo either cabg alone ( 499 patients ) or cabg with svr ( 501 patients ) . the median age was 62 years , and 147 of the 1000 patients were women . the median left ventricular function was 28% , and the median left ventricular end systolic volume index ( lvesvi ) was 82 ml per square meter of body surface area . multivessel cad was present in 64% of patients in each group . although standard hf treatment was recorded at baseline and was encouraged throughout the trial , no information about the rates of its use over time is provided . the two strategies were equally successful in improvement of patients symptoms , with an average improvement in ccs class of 1.7 classes ( p = 0.84 ) and an average of 1 nyha class ( p = 0.7 ) . equivalent improvement in the 6-minute walk test , obtained at baseline and 4-month follow - up , was demonstrated between the two groups ( 48 m among patients who were assigned to cabg and 52 m among patients assigned to cabg and svr ; p = 0.8 ) . there was a greater reduction in lvesvi with combined procedure ( 16 ml per square meter of body surface area ) when compared with cabg alone ( 5 ml per square meter of body surface area ) . the primary outcome of death from any causes or hospitalization for cardiac causes was the same in cabg alone ( 59% ) when compared with cabg with svr ( 58% ) ( figure 1 ) . death occurring within 30 days after the procedure did not differ significantly between the two study groups . ( a ) the probability of the primary outcome ( death from any cause or hospitalization for cardiac causes ) , which did not differ significantly between the two groups . the primary outcome occurred in 292 patients ( 59% ) assigned to undergo coronary artery bypass grafting ( cabg ) alone and in 289 patients ( 58% ) assigned to undergo cabg with surgical ventricular reconstruction ( svr ) ( hazard ratio 0.99 , 95% confidence interval [ ci ] 0.84 - 1.17 ) . ( b ) the probability of death from any cause , which occurred in 141 patients ( 28% ) assigned to undergo cabg and in 138 patients ( 28% ) assigned to undergo cabg with svr ( hazard ratio 1.00 , 95% ci 0.79 - 1.26 ) . reproduced with permission from . the first stich trial hypothesis is addressing an important question of revascularization of patients with cad and lv systolic dysfunction . although specific clinical problems in this population , such as severe angina , are used to decide on revascularization strategies , the vast majority of patients with ischemic cardiomyopathy ( icm ) have limited or no angina and fall into a gray zone where clear evidence for adding cabg to optimal medical therapy is either absent or outdated . recent guidelines recommend cardiac resynchronization therapy ( crt ) in patients with an lvef of less than 35% , nyha class iii - iv symptoms , and a qrs duration of greater than 0.12 seconds . this represents a subgroup of hf patients with abnormal cardiac conduction and possible ventricular dyssynchronous contraction . dyssynchronous contraction results in suboptimal ventricular filling , reduced lv dp / dt ( rate of rise in lv pressure ) , prolonged duration and severity of mitral regurgitation , and paradoxical septal wall motion . crt is a biventricular pacemaker device that electrically paces the right and left ventricles in a synchronized mode and thus may improve ventricular contraction and diminish secondary mitral regurgitation . crt in conjunction with optimal medical therapy has been shown to significantly improve quality of life , functional class , exercise capacity and distance , and ejection fraction ( ef ) , as well as reduce hf hospitalizations by 32% and all - cause mortality by 25% ( in a meta - analysis ) . it was unknown whether these devices would be beneficial in asymptomatic or mildly symptomatic patients . madit - crt ( multicenter automatic defibrillator implantation trial with cardiac resynchronization therapy ) investigated whether crt - d ( crt + implantable cardioverter defibrillator [ icd ] device ) would reduce all - cause mortality and hf events ( need for intravenous diuretic therapy as an outpatient or augmented hf regimen during hospitalization ) in patients who qualify for icd but are nyha class i or ii . inclusion criteria were age of more than 21 years , an lvef of not more than 30% , stable optimal medical therapy , a qrs of at least 130 ms , normal sinus rhythm , and nyha class i or ii ( icm ) or class ii ( nonischemic cardiomyopathy ) . in a 3:2 ratio , 1820 patients were randomly assigned to either crt - d ( 1089 ) or icd ( 731 ) alone . successful device implantation occurred in 98.4% of patients , and 95.4% were in their originally assigned arm . the groups were well matched at baseline , and for the entire study group , icm occurred in about 55% of patients , approximately 10% had nyha class iii / iv symptoms more than 3 months prior to enrollment , mean lvef was 24% , with a mean 6-minute walk distance of about 361 ms , 70% had left bundle branch block , and 64% had a qrs width of at least 150 ms . medical therapy was optimized with 93% receiving beta blockers , 97% aceis or arbs , 31% aldosterone antagonist , 74% diuretics , 67% statin , and 7% amiodarone . the trial was terminated early after a mean follow - up of 2.4 years because of a significant difference in the combined primary endpoint . death or nonfatal hf events were 17.2% for crt - d versus 25.3% for icd ( hazard ratio 0.66 , 95% confidence interval [ ci ] 0.52 - 0.84 ; p = 0.001 ) ( figure 2 ) . reduced hf events ( 13.9% versus 22.8% , p < 0.001 ) drove the primary endpoint to significance , with no difference in all - cause mortality ( 6.8% versus 7.3% , p = 0.99 ) . in subgroup analysis , women and patients with a qrs duration of 150 ms or more showed a greater benefit . improvement of lvef occurred compared with baseline in the crt - d arm ( 0.11 versus 0.03 , p < 0.001 ) as measured by echocardiography in a subgroup of patients . lvesv and lvedv ( left ventricular end diastolic volume ) decreased significantly compared with baseline in the crt - d arm ( 57 versus 18 ml and 52 versus 15 ml , respectively ; p < 0.001 ) . more frequent device - related adverse outcomes occurred in the crt - d arm compared with the icd arm in the 30 days after implantation . there was a significant difference in the estimate of survival free of heart failure between the group that received cardiac resynchronization therapy plus an implantable cardioverter defibrillator ( crt - icd ) and the group that received an icd only ( unadjusted p < 0.001 by the log - rank test ) . reproduced with permission from . are that approximately 10% of patients qualified for crt with nyha class iii or iv more than 3 months prior to random assignment . also , patients were assessed by investigators who were not blinded to the patients device and thus there was a possible bias toward the crt - d arm . however , there was a blinded independent clinical events adjudication committee . madit - crt results reveal that crt - d reduced the primary combined endpoint of death or hf events as compared with icd alone with optimal medical management in nyha class i and ii patients , driven largely by a reduction in hf events . while these effects may have been exaggerated due to the early termination of follow - up , it is also likely that a longer follow - up in these nyha i / ii patients would have shown a beneficial effect on mortality . as a result of this and other studies , device therapy has begun to play an even greater role in the management of hf patients . despite current treatments with optimal medical and device therapy including crt , a number of hf patients will proceed to refractory hf . continuous intravenous inotropic assists in the short term only , and transplants are offered to a very small percentage of hf patients . thus , more permanent solutions with mechanical circulatory support devices such as left ventricular assist devices ( lvads ) are being evaluated . in 2002 , the landmark rematch ( randomized evaluation of mechanical assistance for the treatment of congestive heart failure ) trial was the first to randomly assign nontransplantable end - stage hf patients to optimal medical management or lvad plus optimal medical management . a 48% risk reduction in death from any cause one - year survival rate was 52% with lvad and 23% at 2 years versus 25% in the medically treated patients at 1 year and only 8% at 2 years . the older pulsatile - flow lvad used in rematch was compared with the new continuous - flow heartmate ii device in the advanced heart failure treated with continuous - flow left ventricular assist device ( heartmate ii ) trial . the continuous - flow device has fewer moving parts , is much smaller , and has less mechanical breakdown than the pulsatile - flow lvad . patients with advanced hf ( ef of less than 25% , peak oxygen consumption of less than 14 ml / kg per minute or less than 50% of the predicted value , and nyha class iiib or iv symptoms for at least 45 of the 60 days before enrollment ) , refractory to optimal medical management ( dependence on an intra - aortic balloon pump for 7 days or inotropes for at least 14 days before enrollment ) , and ineligible for transplantation were evaluated . random assignment occurred in a 2:1 ratio to undergo implantation of a continuous - flow device , heartmate ii ( 134 patients ) , or the currently approved pulsatile - flow device heartmate xve ( 66 patients ) . the primary composite endpoint was survival free from disabling stroke and reoperation to repair or replace the device at 2 years . secondary endpoints were survival , frequency of adverse events , quality of life , and functional capacity . the two treatment groups had similar preoperative characteristics , including a median age of 64 years ( range of 26 - 81 ) and a mean lvef of 17% , and almost 80% had received intravenous inotropic agents , more than 20% were fitted with an intra - aortic balloon pump , and greater than 60% failed crt . the primary composite endpoint occurred more frequently in patients with continuous - flow devices ( 62 of 134 [ 46% ] ) than with pulsatile - flow devices ( 7 of 66 [ 11% ] ; p < 0.001 ) . patients with continuous - flow devices had greater actuarial survival rates at 2 years ( 58% versus 24% ; p = 0.008 ) . estimated 1- and 2-year survival rates were 68% ( 95% ci 60 - 76% ) and 58% ( 95% ci 49 - 67% ) , respectively , with the continuous - flow device and 55% ( 95% ci 42 - 69% ) and 24% ( 95% ci 1 - 46% ) with the pulsatile - flow device . the better durability of the heartmate ii continuous - flow device was the primary determinant of the significant positive endpoint . one - third of patients with pulsatile - flow lvad required pump replacements , one required urgent transplantation , and three required device explantation . about 10% of patients with continuous - flow lvad required pump replacements due to breakage of the percutaneous lead , pump thrombosis , or outflow elbow disconnection . major adverse events were significantly reduced in patients with a continuous - flow lvad , including device- and non - device - related infection , right hf , respiratory failure , renal failure , and cardiac arrhythmia . there was a significant improvement in quality of life and functional capacity in both groups . limitations of the study include the potential bias despite random assignment of the devices because both patient and physician were aware that the device was being implanted . overall , the study found that continuous - flow lvad in patients with advanced hf significantly improved the probability of survival - free device failure at 2 years as compared with a pulsatile device . most importantly , the study demonstrated that the survival of patients with a continuous - flow device is twice that of the rematch trial pulsatile - flow group at 2 years , thus illustrating that newer technology and improved medical treatment have enhanced the survival of refractory hf patients in the past decade . medical therapy with optimal doses of acei and beta blocker based on patient tolerability and clinical trial evidence is essential for hf therapy . the judicious use of diuretics , in conjunction with a low sodium diet , helps to manage the volume overload of this condition . patients must follow appropriate lifestyle recommendations : no smoking , moderate exercise , avoidance of nonsteroidal anti - inflammatory drugs , and avoidance of alcohol . the careful addition of aldosterone antagonists or arbs ( or both ) can improve outcomes . finally , the appropriate use of bypass surgery and devices ( icds , crt - d , and lvads ) can beneficially impact morbidity and mortality for hf patients .

heart failure due to systolic dysfunction has enormous global impact . medical management based on an understanding of the pathophysiology of the disease as well as its neurohormonal mechanisms has greatly advanced over the past 25 years . below is a review of recent and emerging data on epidemiology and diagnosis of heart failure due to systolic dysfunction and the current and future management techniques to ameliorate this disease . at the end , we will highlight three significant trials in the field in 2009 that will impact heart failure care : stich , madit - crt , and heartmate ii .

Introduction and context Recent advances Surgical Treatment for Ischemic Heart Failure (STICH) trial Multicenter Automatic Defibrillator Implantation Trial with Cardiac Resynchronization Therapy (MADIT-CRT) Continuous-flow left ventricular assist device Implications for clinical practice Competing interests

although considerable advances have been made in the management of hf over the past few decades , hf remains a major public health issue with high prevalence and poor outcomes . an understanding of the pathophysiology and natural history of hf underpins the therapeutic approaches used to achieve the goals of treatment , which are to relieve symptoms , to avoid hospital admission , and to prolong life . device therapy and transplant surgery have carved out a respectable place in the field over the past decade . combined endpoint of death from any cause , hospitalization for hf , cardiac arrest with resuscitation , and intravenous therapy was statistically significant ( p = 0.009 ) ; however , it was driven mainly by a decline in hospitalization rate for hf ( 13.8% in the valsartan group and 18.2% in the placebo ) . crt in conjunction with optimal medical therapy has been shown to significantly improve quality of life , functional class , exercise capacity and distance , and ejection fraction ( ef ) , as well as reduce hf hospitalizations by 32% and all - cause mortality by 25% ( in a meta - analysis ) . there was a significant difference in the estimate of survival free of heart failure between the group that received cardiac resynchronization therapy plus an implantable cardioverter defibrillator ( crt - icd ) and the group that received an icd only ( unadjusted p < 0.001 by the log - rank test ) . madit - crt results reveal that crt - d reduced the primary combined endpoint of death or hf events as compared with icd alone with optimal medical management in nyha class i and ii patients , driven largely by a reduction in hf events . the older pulsatile - flow lvad used in rematch was compared with the new continuous - flow heartmate ii device in the advanced heart failure treated with continuous - flow left ventricular assist device ( heartmate ii ) trial . patients with advanced hf ( ef of less than 25% , peak oxygen consumption of less than 14 ml / kg per minute or less than 50% of the predicted value , and nyha class iiib or iv symptoms for at least 45 of the 60 days before enrollment ) , refractory to optimal medical management ( dependence on an intra - aortic balloon pump for 7 days or inotropes for at least 14 days before enrollment ) , and ineligible for transplantation were evaluated . most importantly , the study demonstrated that the survival of patients with a continuous - flow device is twice that of the rematch trial pulsatile - flow group at 2 years , thus illustrating that newer technology and improved medical treatment have enhanced the survival of refractory hf patients in the past decade . with the exception of the recently published surgical treatment for ischemic heart failure ( stich ) trial , most of these surgical techniques have not been adequately tested . crt in conjunction with optimal medical therapy has been shown to significantly improve quality of life , functional class , exercise capacity and distance , and ejection fraction ( ef ) , as well as reduce hf hospitalizations by 32% and all - cause mortality by 25% ( in a meta - analysis ) . madit - crt ( multicenter automatic defibrillator implantation trial with cardiac resynchronization therapy ) investigated whether crt - d ( crt + implantable cardioverter defibrillator [ icd ] device ) would reduce all - cause mortality and hf events ( need for intravenous diuretic therapy as an outpatient or augmented hf regimen during hospitalization ) in patients who qualify for icd but are nyha class i or ii . there was a significant difference in the estimate of survival free of heart failure between the group that received cardiac resynchronization therapy plus an implantable cardioverter defibrillator ( crt - icd ) and the group that received an icd only ( unadjusted p < 0.001 by the log - rank test ) . madit - crt results reveal that crt - d reduced the primary combined endpoint of death or hf events as compared with icd alone with optimal medical management in nyha class i and ii patients , driven largely by a reduction in hf events . the older pulsatile - flow lvad used in rematch was compared with the new continuous - flow heartmate ii device in the advanced heart failure treated with continuous - flow left ventricular assist device ( heartmate ii ) trial . patients with advanced hf ( ef of less than 25% , peak oxygen consumption of less than 14 ml / kg per minute or less than 50% of the predicted value , and nyha class iiib or iv symptoms for at least 45 of the 60 days before enrollment ) , refractory to optimal medical management ( dependence on an intra - aortic balloon pump for 7 days or inotropes for at least 14 days before enrollment ) , and ineligible for transplantation were evaluated . most importantly , the study demonstrated that the survival of patients with a continuous - flow device is twice that of the rematch trial pulsatile - flow group at 2 years , thus illustrating that newer technology and improved medical treatment have enhanced the survival of refractory hf patients in the past decade .

heart failure ( hf ) remains a significant burden on the health care industry in the united states . each year about 870,000 new cases are diagnosed in the united states , and currently , an estimated 5.7 million americans suffer from this disease . the mortality rate remains high , with one in five individuals dying within five years of diagnosis . additionally , hf remains one of the top diagnoses for hospital readmissions [ 2 , 3 ] , with total costs for hf treatment exceeding 30 billion dollars . in fact , hf is the most common diagnosis for hospitalized patients 65 years and older . previous research suggests that patients who maintain self - care behaviors , such as adhering to treatment regimen , have improved outcomes , including fewer hospital readmissions and improved survival . therefore , improving self - care among patients with hf is vital in influencing adverse events such as frequent hospitalizations and mortality . self - care involves an active process that includes physical and decision - making processes [ 6 , 7 ] . these physical activities are disease - specific and include adherence to medication , dietary and fluid restrictions , and regular exercise . decision - making processes used by individuals with hf include recognizing health changes , evaluating health status and decisions to take action , implementing potential solutions to address health - related issues , and evaluating the effectiveness of solutions . certain individual ( e.g. , race , gender , age , marital status , income , educational level , and number of people who live in a household ) and clinical ( e.g. , nyha class hf , length of time since diagnosis ) characteristics may influence self - care indirectly by impacting other factors ( i.e. , cognitive , psychosocial , physical , and sociocultural ) which influence self - care [ 913 ] . living with hf characteristics of hf symptoms may impact the ability to perform self - care activities and require utilization of coping resources , such as social support and social problem - solving . studies suggest patients experiencing severe symptoms have greater difficulty recognizing and responding to increased symptoms [ 15 , 16 ] . likewise , patients experiencing increased symptom frequency and severity are particularly vulnerable to dependency upon others for assistance with self - care . other research contradicts , suggesting more frequent symptoms of hf are associated with better self - care . nonetheless , studies examining the characteristics of hf symptoms ( i.e. , frequency , severity , and degree of symptom - related interference with activity and enjoyment of life ) are limited . thus , how symptom characteristics influence self - care behaviors in hf is relatively unknown , requiring more research . the relationship between social support and self - care in hf patients has been widely studied [ 2123 ] . while research indicates increased levels of social support are associated with better self - care [ 23 , 24 ] , the majority of these studies have investigated only perceived support . however , little is known about which type of perceived support ( i.e. , belonging , tangible , or appraisal ) is most beneficial in influencing self - care in patients with hf . similarly , few studies have investigated the influence of social networks ( e.g. , family and friends ) , despite research suggesting this type of support positively influences self - care in those with hf . hence , more research is needed to examine how both types of support influence self - care behaviors in this population . social problem - solving may also influence self - care behaviors in patients with hf . social problem - solving is defined as problem - solving in a real world environment and involves problem orientation and problem - solving style . published studies examining social problem - solving in those with hf are limited ; however , studies examining patients with diabetes mellitus suggest that the manner in which individuals solve problems directly influences self - care behaviors , specifically self - management [ 10 , 28 , 29 ] . furthermore , findings suggest that impulsivity / carelessness and avoidant problem - solving styles are associated with worse self - care in diabetic patients . to date , no published studies have examined the influence of problem orientation and problem - solving styles on hf self - care . information related to these relationships could aid in clinical management and patient education for those with hf . this study was guided by concepts from the theory of stress and coping , which describes how individuals adapt psychologically to stressful situations . stress is a relationship between individuals and their environment that when appraised is determined to exceed personal resources and serve as a threat to well - being . coping is a process utilized by individuals to manage a situation that is stressful and emotions that accompany the situation . individual and clinical characteristics may potentially influence disease - related stressors and coping resources , which affect disease - related outcomes . in this study , hf symptom frequency , severity , and degree of symptom - related interference with physical activity and enjoyment of life are viewed as potential stressors that influence self - care behaviors in individuals living with hf . social support and social problem - solving are potential coping resources used to manage hf symptomatology and improve hf self - care behaviors ( figure 1 ) . hence , the purpose of this study was to identify predictors of self - care behaviors from among characteristics of hf symptomatology ( i.e. , frequency , severity , and degree of symptom - related interference with physical activity and enjoyment of life ) , social support ( i.e. , belonging , tangible , and appraisal support ; social network ) , and social problem - solving ( i.e. , positive and negative problem orientation ; rational , impulsivity / carelessness , and avoidance problem - solving styles ) , in addition to individual and clinical characteristics in patients with hf of age 55 years and older . this study used a cross - sectional , correlational , predictive design to investigate predictors of self - care behaviors in a convenience sample of 201 outpatients with hf . a power analysis for multiple linear regression was conducted , using a medium effect size , 80% power , significance level of 0.05 , and variables identified in tables 1 and 2 [ 31 , 32 ] . patients with a diagnosis of hf , age 55 years and older , who resided in an outpatient setting were included in the study . the age range for inclusion in this study was limited to specifically examine predictors of self - care behaviors in middle - older age adults with hf . therefore , the telephone interview for cognitive status ( tics ) was used to screen for the potential of cognitive impairment . individuals with a score of 30 on the tics were excluded from study participation due to the possibility of impaired cognition . after institutional review board approval , potential participants were recruited using letters and flyers from three hospital - affiliated outpatient offices in north florida . those patients interested in the study contacted the primary investigator and underwent clinical and cognitive screening for inclusion over the telephone . following telephone screening for inclusion and exclusion , potential participants who met study criteria were then scheduled for an individual interview at their physician 's office . after obtaining informed consent , participants were interviewed using a set of self - report surveys presented in random order . a researcher - developed sociodemographic and clinical survey was used for clinical screening and to obtain information on participant characteristics . clinical information was gathered via self - report and included questions regarding the length of time since hf diagnosis and the severity of hf based upon the new york heart association ( nyha ) classification for hf . cognitive screening was conducted using the 11-item tics , which assesses orientation , attention , language , learning , and memory . a total of five instruments were used to measure study variables . the 14-item heart failure symptom survey ( hfss ) was used to measure four characteristics of hf symptomatology : frequency , severity , degree of interference with physical activity , and degree of interference with enjoyment of life based upon the last week . higher scores on a particular domain suggest more frequent and severe symptoms and greater interference with physical activity and enjoyment of life , respectively . previous studies support the validity and reliability ( > 0.80 ) of each domain . in this study , cronbach 's alphas were adequate for all domains , including frequency ( = 0.795 ) , severity ( = 0.857 ) , interference with physical activity ( = 0.853 ) , and interference with enjoyment of life ( = 0.878 ) . thus , in order to fully examine the concept , we used two measures of social support . the interpersonal support evaluation list-12 ( isel-12 ) was used to measure three types of perceived support ( i.e. , belonging , tangible , and appraisal support ) . scores range from 0 to 36 , with higher scores indicating a higher perception of available support with regard to the particular subscale . prior studies support adequate validity and reliability ( = 0.94 ) . internal consistency , using cronbach 's alpha , was adequate for all subscales ( > 0.70 ) in this study . the graven and grant social network survey ( ggsns ) is a 12-item survey that was used to measure social network . participants identify the number of people in their life who provide assistance and support , as well as rating their satisfaction with provided support . scores range from 12 to 84 , with higher scores suggesting higher levels of actual support . content and construct validity , as well as reliability ( = 0.93 ) , were established in a previous study . was measured using the social problem - solving inventory revised - short ( spsir - s ) . this 25-item survey represents both positive and negative problem orientation , as well as three problem - solving styles ( i.e. , rational , impulsivity / carelessness , and avoidance ) . adequate validity and reliability have been reported previously using a total score for adaptive and maladaptive items ( = 0.86 and 0.77 , resp . ) . with the exception of the positive problem orientation subscale ( = 0.672 ) , all other subscales were internally consistent with cronbach 's alphas greater than 0.80 . the european heart failure self - care behavior scale-9 ( ehfscbs-9 ) was used to measure self - care behaviors related to hf . this survey identifies nine activities specific to hf self - care and participants rate their level of agreement on a 5-point scale . total score ranges from 9 to 45 , with higher scores indicating worse self - care behaviors . prior research has supported its validity and reliability ( = 0.87 ) and in this study cronbach 's alpha was 0.67 . data were analyzed using the statistical package for the social sciences ( spss ) version 20 . descriptive statistics ( e.g. , mean , standard deviation , percent , and frequencies ) were conducted to examine sample characteristics and scores on all study variables . multiple linear regression with true stepwise variable selection was used to examine the impact of the study variables , using subscales versus total scores when applicable , on self - care behaviors , taking into account the potential influence of the other variables in the model . the model was fit with the total score on the ehfscbs-9 as the dependent variable . the addition / removal criteria in spss were used for stepwise variable selection , including a probability of f of 0.05 for addition and 0.10 for removal from the model . individual and clinical characteristics included in the initial model were gender , marital status , age , race , highest level of education attained , number of people in the household , annual income , nyha class hf , and length of time since hf diagnosis . additionally , the following variables were included as predictor variables : frequency and severity of hf symptoms ; degree of symptom - related interference with physical activity and enjoyment of life ; appraisal , tangible , and belonging support ; positive and negative problem orientation ; and rational , impulsivity / carelessness , and avoidance problem - solving styles . underlying assumptions for multiple regression were examined . telephone screening was conducted on 205 participants , with one scoring less than 30 on the tics and three which failed to follow up for the scheduled interview . thus , a total of 201 participants were included in the study ( table 1 ) . the average score on the ehfscbs-9 was 25.65 , indicating that the majority of the participants reported poor self - care behaviors . the majority of participants were not experiencing frequent ( 1.98 [ sd , 1.64 ] ) nor severe ( 1.69 [ sd , 1.62 ] ) symptoms of hf . there was little symptom - related interference with physical activity ( 1.20 [ sd , 1.17 ] ) and enjoyment of life ( sd , 1.14 [ 1.58 ] ) reported . on average , participants reported a larger social network ( 56.46 [ sd , 18.73 ] ) and above average appraisal ( 9.74 [ sd , 3.05 ] ) , tangible ( 10.30 [ sd , 2.47 ] ) , and belonging ( 9.05 [ sd , 3.02 ] ) support . likewise , greater - than - average scores were noted on all subscales of the spsir - s ( table 2 ) . using a level of significance set at 0.05 , the final model identified six significant predictors of self - care behaviors , including race ( = 4.362 ; p = 0.002 ) , frequency of hf symptoms ( = 1.888 ; p = 0.002 ) , hf symptom - related degree of interference with enjoyment of life ( = 1.394 ; p = 0.023 ) , nyha class hf ( = 1.180 ; p = 0.029 ) , rational problem - solving ( = 0.247 ; p = 0.025 ) , and social network ( = 0.095 ; p 0.001 ) . the overall significance of the model was p < 0.001 ( = 34.265 ) , with an adjusted r for the final model of 0.19 , indicating that the set of predictors accounted for approximately 19% of the variance in self - care ( table 4 ) . underlying assumptions for multiple linear regression were evaluated using ( 1 ) a normal probability plot of residuals , ( 2 ) a scatterplot of predicted values versus residuals , ( 3 ) collinearity statistics , and ( 4 ) reliability of study variables . based upon these , assumptions of multiple regression were not violated and no issues with multicollinearity were noted ( all tolerance and variance inflation factor values in the final model were within acceptable ranges [ tolerance > 0.1 ; vif < 10 ] ) . in this study , we focused on predictors of self - care behaviors in middle to older age patients with hf . regression analyses revealed six predictors of hf self - care behaviors , with race contributing the most to self - care behaviors in this study . while few studies have investigated race as a predictor of self - care , davis and colleagues found that nonblacks had higher self - care maintenance scores as compared to blacks . this is inconsistent with our findings , which indicated minorities have better self - care , scoring , on average , 4.37 points lower than nonminorities when controlling for the other variables . this finding was surprising , given that minority race has been associated with several negative prognostics of self - care , including lower health literacy , decreased quality of health care , and decreased knowledge of hf . in this sample , minorities reported a larger social network as compared to nonminorities ( 58.04 , sd 19.61 , versus 56.20 , sd 18.64 ) , perhaps contributing to this finding . prior studies investigating support in minorities have suggested that social network is a significant stress buffer and may have influenced self - care in this study , warranting further research . frequency of hf symptoms was found to contribute more than symptom - related interference with enjoyment of life to self - care behaviors . at this time , research is limited investigating whether symptom frequency predicts self - care in those with hf . research does , however , suggest that progression of hf symptoms influences an individuals ' ability to sustain adequate self - care behaviors [ 42 , 43 ] . no published studies , to date , have examined symptom - related interference with enjoyment of life as a predictor of self - care behaviors in individuals with hf . our findings suggest that symptom - related interference with enjoyment of life also predicts self - care behaviors . in fact , better self - care was noted in those patients whose symptoms interfered with their enjoyment of life , suggesting that individuals with hf may not report symptoms or seek treatment until symptoms significantly interfere with their enjoyment of life . thus , findings illustrate importance of a thorough symptom assessment at each health care visit , examining not only frequency and severity of symptoms , but also how symptoms impact daily life and leisure activities . in this study , approximately 50% of patients reported poor self - care behaviors , which is consistent with prior studies [ 38 , 44 ] . patients with nyha class iv hf reported worse self - care behaviors than those with nyha class i iii hf . additionally , a higher severity of hf , based upon nyha class , predicted worse self - care behaviors , when controlling for other variables in the model . findings in the literature related to the relationship between hf severity and self - care are inconsistent . while some studies indicate patients experiencing more symptoms and functional impairment practice better self - care [ 38 , 45 ] , qualitative research suggests that functional limitations and severe symptoms are actually barriers to effective self - care . our findings support that of riegel and carlson by implying that disease severity and subsequent functional limitations may actually hinder self - care behaviors . though more research is needed to investigate the relationship between disease severity and self - care , our results do provide a target for clinical assessment and management , as well as patient education . an important finding in this study was that rational problem - solving , a coping resource , contributed to better self - care behaviors . in this study , patients utilizing rational problem - solving strategies , such as planful problem - solving , active coping , and seeking social support , had better self - care than patients who used other strategies such as avoidance , denial , and behavioral / mental disengagement . to our knowledge , previous published studies have not studied rational problem - solving and self - care in individuals with hf . however , rational problem - solving and the development of problem - solving skills have been examined in diabetic patients , with use of these skills found to be independently associated with disease - specific self - management behaviors , such as diet and exercise . use of rational problem - solving skills , including problem identification and goal setting , has also been identified as a central concept involved in self - efficacy in those with chronic illnesses . findings of this study provide potential for intervention development ; still , more research is needed in this area to provide support of these findings in patients with hf . the availability of and satisfaction with one 's social network contributed the least to self - care behaviors , which may be due to other variables not examined in this study ( e.g. , family relationships ) . in this study , patients who reported higher social network scores had better self - care than those patients who reported lower social network scores . while studies investigating social network are limited [ 12 , 24 ] , this finding supports previous work , which suggests that a larger social network is related to hf self - care management and confidence , as well as fewer hospital readmissions . while the influence of family support has been well documented in the literature [ 7 , 17 , 25 ] , this study did not limit the investigation of social network to family but instead included anyone who might be involved in the provision of support and who may aid in effective coping ( e.g. , friends , neighbors , and church / social club members ) . this study provides important information related to predictors of self - care behaviors and the influence of symptomatology and coping resources . yet the low adjusted r suggests that an unmeasured variable may play an important role in the model and this finding should be considered in future studies . for example , an important component not measured in this study is health literacy . previous research , however , is not consistent with some studies identifying it as a potential barrier to effective hf self - care and others finding no relationship between health literacy and self - care in patients with hf . additionally , there may be other unmeasured variables that could potentially play a role in the prediction of self - care behaviors , such as comorbidities and cognitive status , warranting further research in this area . to our knowledge this study is the one of the first to examine the subcomponents of hf severity and social problem - solving in patients with hf , providing information useful for intervention development . the inclusion of social network , in addition to subcomponents of perceived social support , allows researchers to examine which component of social support is the stronger predictor of self - care , as most studies have previously only regressed perceived social support on self - care behaviors using a total score [ 21 , 22 , 24 ] . the majority of participants were nonminority men with nyha class ii hf , limiting generalizability of findings to similar individuals . in addition , this study included only patients 55 years and older , limiting examination of these variables in a younger population of hf patients . the cross - sectional nature of this study restricts our understanding of the influence of these variables on hf self - care and does not provide information regarding how these relationships may change over time as hf progresses . also , little variation with regard to the characteristics of hf symptomatology existed within the sample and may have contributed to measurement error . finally , the relatively asymptomatic nature of most participants may have impacted the reliability of the ehfscbs-9 , as many of the questions on this instrument were more appropriate for patients who were symptomatic . middle to older age patients with hf are susceptible to adverse outcomes related to poor self - care . this study advances our understanding of how symptomatology and coping resources may influence self - care behaviors in patients with hf , taking into account the limitations of this study . assessing the influence of race on self - care behaviors in middle to older age individuals with hf is important . monitoring symptom frequency , degree of symptom - related inference with enjoyment of life , and class of heart failure at clinical visits is recommended , as patients may not complain of symptoms until symptoms impact their enjoyment of life . similarly , appraisal and teaching of rational problem - solving skills to address heart - related issues may be useful in enhancing self - care behaviors in this population . evaluating the size of and satisfaction with one 's social network is appropriate when assessing self - care , with referral to community resources , if needed .

background . symptoms of heart failure ( hf ) and coping resources , such as social support and social problem - solving , may influence self - care behaviors . research regarding the influence of hf symptomatology characteristics and components of social support and social problem - solving on self - care is limited . objective . to identify predictors of hf self - care behaviors using characteristics of hf symptomatology , components of social support and social problem - solving , and demographic and clinical factors . methods . using a cross - sectional , correlational predictive design , a convenience sample ( n = 201 ) of outpatients with hf answered self - report surveys . multiple linear regression with stepwise variable selection was conducted . results . six predictors of hf self - care were identified : race , symptom frequency , symptom - related interference with enjoyment of life , new york heart association class hf , rational problem - solving style , and social network ( = 34.265 , r2 = 0.19 , p = 0.001 ) . conclusions . assessing the influence of race on self - care behaviors in middle to older age patients with hf is important . clinical assessment that focuses on symptom frequency , symptom - related interference with enjoyment of life , and hf class might also impact self - care behaviors in this population . rational problem - solving skills used and evaluation of the size of and satisfaction with one 's social network may be appropriate when assessing self - care .

1. Introduction 2. Conceptual Framework 3. Methods 4. Data Analysis 5. Results 6. Discussion 7. Conclusions

living with hf characteristics of hf symptoms may impact the ability to perform self - care activities and require utilization of coping resources , such as social support and social problem - solving . social problem - solving may also influence self - care behaviors in patients with hf . in this study , hf symptom frequency , severity , and degree of symptom - related interference with physical activity and enjoyment of life are viewed as potential stressors that influence self - care behaviors in individuals living with hf . social support and social problem - solving are potential coping resources used to manage hf symptomatology and improve hf self - care behaviors ( figure 1 ) . hence , the purpose of this study was to identify predictors of self - care behaviors from among characteristics of hf symptomatology ( i.e. , frequency , severity , and degree of symptom - related interference with physical activity and enjoyment of life ) , social support ( i.e. this study used a cross - sectional , correlational , predictive design to investigate predictors of self - care behaviors in a convenience sample of 201 outpatients with hf . the age range for inclusion in this study was limited to specifically examine predictors of self - care behaviors in middle - older age adults with hf . the 14-item heart failure symptom survey ( hfss ) was used to measure four characteristics of hf symptomatology : frequency , severity , degree of interference with physical activity , and degree of interference with enjoyment of life based upon the last week . multiple linear regression with true stepwise variable selection was used to examine the impact of the study variables , using subscales versus total scores when applicable , on self - care behaviors , taking into account the potential influence of the other variables in the model . additionally , the following variables were included as predictor variables : frequency and severity of hf symptoms ; degree of symptom - related interference with physical activity and enjoyment of life ; appraisal , tangible , and belonging support ; positive and negative problem orientation ; and rational , impulsivity / carelessness , and avoidance problem - solving styles . using a level of significance set at 0.05 , the final model identified six significant predictors of self - care behaviors , including race ( = 4.362 ; p = 0.002 ) , frequency of hf symptoms ( = 1.888 ; p = 0.002 ) , hf symptom - related degree of interference with enjoyment of life ( = 1.394 ; p = 0.023 ) , nyha class hf ( = 1.180 ; p = 0.029 ) , rational problem - solving ( = 0.247 ; p = 0.025 ) , and social network ( = 0.095 ; p 0.001 ) . in this study , we focused on predictors of self - care behaviors in middle to older age patients with hf . regression analyses revealed six predictors of hf self - care behaviors , with race contributing the most to self - care behaviors in this study . frequency of hf symptoms was found to contribute more than symptom - related interference with enjoyment of life to self - care behaviors . no published studies , to date , have examined symptom - related interference with enjoyment of life as a predictor of self - care behaviors in individuals with hf . our findings suggest that symptom - related interference with enjoyment of life also predicts self - care behaviors . in this study , patients utilizing rational problem - solving strategies , such as planful problem - solving , active coping , and seeking social support , had better self - care than patients who used other strategies such as avoidance , denial , and behavioral / mental disengagement . the availability of and satisfaction with one 's social network contributed the least to self - care behaviors , which may be due to other variables not examined in this study ( e.g. to our knowledge this study is the one of the first to examine the subcomponents of hf severity and social problem - solving in patients with hf , providing information useful for intervention development . the inclusion of social network , in addition to subcomponents of perceived social support , allows researchers to examine which component of social support is the stronger predictor of self - care , as most studies have previously only regressed perceived social support on self - care behaviors using a total score [ 21 , 22 , 24 ] . this study advances our understanding of how symptomatology and coping resources may influence self - care behaviors in patients with hf , taking into account the limitations of this study . assessing the influence of race on self - care behaviors in middle to older age individuals with hf is important . monitoring symptom frequency , degree of symptom - related inference with enjoyment of life , and class of heart failure at clinical visits is recommended , as patients may not complain of symptoms until symptoms impact their enjoyment of life . similarly , appraisal and teaching of rational problem - solving skills to address heart - related issues may be useful in enhancing self - care behaviors in this population . evaluating the size of and satisfaction with one 's social network is appropriate when assessing self - care , with referral to community resources , if needed .

the chemical and structural flexibility of the pyrochlore materials ( a2b2o7 ) has attracted significant interest in recent years . this has led to a variety of applications , including those related to nuclear fission , with pyrochlores and pyrochlore - based materials being proposed as both matrices for immobilization and as advanced inert matrix fuels . one of the key criteria for determining the applicability of a material for such applications is how it responds to the damaging effects of radiation , that is , its propensity to amorphise . an advantage of pyrochlores in this context is their ability to transform to different structures as the composition or conditions vary . the pyrochlore structure ( a , space group fd3 m ) shown in figure 1a is an ordered derivative of fluorite ( ao2 ) , with the removal of 1/8 of the oxygen atoms . this produces two types of cation sites : an eight - coordinate a site ( typically occupied by 2 + or 3 + cations ) , and a six - coordinate b site ( usually occupied by smaller 5 + or 4 + cations ) . the stability of this structure is indicated by the relative ratio of the a and b site cations , ra / rb , with pyrochlore stable when this is between 1.46 and 1.78 . below 1.46 a transformation to a defect fluorite structure ( with disorder on both cation and anion lattices ) is expected while , above 1.78 , materials are predicted to adopt a layered perovskite - related ( la2ti2o7 ) structure , as shown in figure 1b . there are four crystallographically distinct types of la and ti sites in this structure . materials that exhibit this structural type have been investigated for uses in photocatalysis and electrical applications that exploit their piezoelectric and ferroelectric properties . there has been some debate over the exact structure adopted by la2ti2o7 itself , with two suggested forms ; monoclinic ( p21 ) ( b , figure 1b ) and orthorhombic ( pna21 ) ( c , see supporting information , figure s1.1 ) . the structures have two similar unit cell lengths ( of 7.8 and 5.5 ) , with the remaining length twice as large in c than in b ( see the supporting information for more detail ) . although subsequent work appears to confirm the presence of a monoclinic structure , it is not clear whether an orthorhombic modification exists , or whether the twinned nature of the crystals studied led to an incorrect choice of symmetry in the earlier work . structure of ( a ) la2sn2o7 pyrochlore , a , and ( b ) the monoclinic form of la2ti2o7 layered perovskite , b ( with the four different ti sites highlighted ) . dashed lines indicate the unit cell . information on the orthorhombic model of la2ti2o7 ( c ) is given in the supporting information . the physical and chemical properties of ceramics are intimately linked to their local structure and disorder . however , the study of disordered materials can be challenging , particularly if more than one phase is present , with many diffraction - based approaches providing information only on the average structure . nuclear magnetic resonance ( nmr ) spectroscopy , with its sensitivity to the local structural environment , without the need for any long - range order , has proven a useful element - specific and complementary tool when studying disorder in solids , with both the isotropic chemical shift and the chemical shift anisotropy ( csa ) shown to be highly dependent on the number , nature , and position of the surrounding atoms . more recently , the simultaneous use of theoretical calculations alongside experiment has seen a significant increase in popularity , aiding the interpretation and assignment of the complex spectra that are frequently observed for disordered solids . this combined approach ( often termed nmr crystallography ) has previously been applied to the investigation of cation disorder in y2(sn , ti)2o7 pyrochlore solid solutions , demonstrating no significant mixing of the a and b site cations were observed , but that sn and ti were randomly distributed on the b sites . more recently , nmr crystallography was applied to the y2(sn , zr)2o7 compositional series , where a transition to a defect fluorite phase was expected for zr - rich compositions . nmr was able to demonstrate the presence of a significant two - phase region in this series , from y2sn1.8zr0.2o7 to y2sn0.4zr1.6o7 , with the compositions of the phases present shown to differ greatly . in this work , we utilize nmr spectroscopy and density functional theory ( dft ) calculations to study la2(sn , ti)2o7 , where a phase transition from a pyrochlore ( la2sn2o7 , where ra / rb = 1.68 ) to a layered perovskite phase ( la2ti2o7 , where ra / rb = 1.92 ) is expected . we exploit a combination of experiment and computation to consider the number and composition of the phases present for each of the nominal starting compositions , and how the phase transition progresses . we also use dft calculations to investigate the preferred position and distribution of the substituted cations in each of the two phases , and consider the consistency of our data with the suggested ( i.e. , b and c ) structural models for la2ti2o7 . this nmr crystallographic study provides more detailed structural information than is available from laboratory x - ray diffraction and demonstrates the future potential of this approach for the investigation of disordered solids . samples in the system la2sn2xtixo7 were prepared in two batches , the first batch consisting of 11 samples from x = 0 to x = 2 , in steps of 0.2 . subsequently , a second batch of samples from x = 1.8 to x = 1.95 ( with x varying in steps of 0.05 ) was prepared . both sets of samples were prepared under identical conditions , using stoichiometric amounts of la2o3 ( sigma - aldrich 99.9% ) , tio2 ( sigma - aldrich 99% ) , and sno2 ( sigma - aldrich 99.9% ) , which were predried overnight to remove co2 and h2o before weighing . these powders were then ball milled for 16 h in isopropanol with zirconia media , dried , sieved and ( uniaxially ) pressed into pellets . the pellets were then heated at 1673 k for 48 h , with a ramp rate of 5 k min . after cooling , the samples were ground for both x - ray diffraction and mas nmr analysis . structural analysis was undertaken by x - ray powder diffraction using a bruker d2 phaser , with weighted cu k ( = 1.54184 ) radiation . the angular range was 5 to 90 with a step size of 0.02 and a step duration of 0.4 s. powder patterns are shown in the supporting information , across two compositional ranges , with figure s2.1 showing the complete compositional range and figure s2.2 from la2ti2o7 la2ti1.6sn0.4o7 . nmr spectra were acquired using a bruker avance iii spectrometer , equipped with a 9.4 t widebore magnet operating at a larmor frequency of 149.2 mhz for sn . powdered samples were packed into a 4.0 mm zro2 rotor and rotated at a rate of 14 khz , using a conventional 4 mm hx probe . spectra were acquired using a radiofrequency field strength of 111 khz ( /2 2.25 s ) and a recycle interval of 30 s and are the result of averaging between 16 and 10688 transients . spectra were acquired using either a spin echo ( to ensure accurate acquisition of any broader components ) or a carr meiboom gill ( cpmg ) echo train to increase sensitivity . in the latter case , 50 echoes were typically acquired , with a frequency - domain spikelet spacing of between 70 and 100 hz . chemical shifts are shown ( in ppm ) relative to ( ch3)4sn , measured using a secondary reference of sno2 ( = 604.3 ppm ) . csa parameters were measured using slow mas ( la2sn2o7 , 2 khz mas ) or csa - amplified pass experiments ( la2sn2xtixo7 , with x = 0.2 , 0.4 , and 0.6 ) , using the pulse sequence of orr et al . pass - based experiments were carried out at an mas rate of 10 khz , and a total scaling factor , nt , of 6.67 , resulting in an apparent mas rate of 1.5 khz in the indirect dimension . spectra are the result of averaging between 78 and 182 transients for each of 16 rows , with a recycle interval of 30 s. fitting of the sideband patterns in the indirect dimension was carried out using simpson , by comparison to a one - dimensional mas spectrum ( assuming ideal pulses ) . the root - mean - square ( rms ) error quoted is that output by simpson , as described in the simpson manual . periodic dft calculations were carried out using the castep code ( version 8.0 ) and adopted the pbe exchange - correlation functional . valence interactions were described by ultrasoft pseudopotentials , taking zora scalar relativistic effects into account . optimization of atomic coordinates and unit / supercell parameters was carried out starting from literature crystal structures or following sn / ti substitutions where appropriate . nmr parameters were computed on the optimized structures employing the gauge - including projector augmented wave ( gipaw ) approach to reconstruct the all - electron wave function in the presence of a magnetic field . calculations were performed either on a cluster at the university of st andrews , consisting of 300 12-core intel westmere nodes , connected with qdr infiniband or , for larger systems , on archer , the uk high performance computing service , a cray xc30 mpp supercomputer with 4920 24-core intel ivy bridge nodes . diagonalization of the absolute shielding tensor ( ) yields the three principal components , 11 , 22 , and 33 , from which the principal components of the shift tensor , ii , can be generated using ii = (ii the reference shielding , ref ( assumed to be 1 ) , was determined ( by comparing the calculated absolute shielding in la2sn2o7 to experiment ) to be 3243.25 ppm for sn . the isotropic shift , iso , is then given by iso = ( 11 + 22 + 33)/3 . the anisotropy is defined by the span , = 11 33 , and the skew , = 3 ( 22 iso)/ , is a measure of the asymmetry of the tensor . computed structures and nmr parameters were processed using python scripts extending the ccp - nc magrespython module . cation substitution in la2sn2xtixo7 is expected to lead to a change from pyrochlore ( for sn - rich compositions ) to a layered perovskite structure as the ti content increases . this change is predicted , by simple radius ratio considerations , to occur at x 0.95 . figure 2 shows sn mas nmr spectra of la2(sn , ti)2o7 , acquired using a spin - echo pulse sequence . the spectrum of the end member , la2sn2o7 contains a single sharp resonance , at 642 ppm , in good agreement with the previous literature . this corresponds to six - coordinate sn , confirming that sn exclusively occupies the b site in the ordered pyrochlore structure . a spectrum of la2sn2o7 acquired using slow mas ( see the supporting information ) reveals that the sn is 43 ( 5 ) ppm and is 0.93 ( 5 ) . this is in reasonable agreement with values calculated using dft ( = 58 ppm and = 1.0 ) . sn ( 9.4 t , 14 khz mas ) nmr spectra of la2sn2xtixo7 , acquired using a spin echo pulse sequence . additional spectra acquired with cpmg experiments are shown ( as spikelets ) for x = 0.20 and 0.40 . when x = 0.2 , two additional sharp resonances are observed , at 647 and 653 ppm , most likely arising from substitution of ti into the next - nearest neighbor ( nnn ) b sites in the pyrochlore structure , a. the assignment of the resonances can be confirmed using dft calculations [ following the approach outlined in ref ( 18 ) ( described in more detail in the supporting information ) , where the local environment of one of the sn atoms in the unit cell of la2sn2o7 is systematically modified to include increasing numbers of ti cations on the six surrounding b sites ] . figure 3a shows the calculated sn iso as the number of sn and ti nnn are varied . the calculations confirm a decrease in the average sn iso ( of 5 ppm ) as the first ti is substituted into the nnn sites , although a range of shifts ( of 10 ppm ) is observed for each type of nnn environment , as a result of variation in the longer - range structure . a smaller decrease in the average sn iso is observed upon the substitution of a second ti , while further substitution appears to result in small shifts back to higher ( although it should be noted that relatively few points are available for environments with very high ti content , owing to the nature of the model used , i.e. , substitution into a unit cell of la2sn2o7 ) . while perhaps a little unexpected , this behavior is reminiscent of that observed for sn nmr of y2(sn , ti)2o7 and y2(sn , zr)2o7 pyrochlores in earlier work . a similar plot against calculated is shown in figure 3b and reveals a similar trend , with an increase in ( of 1015 ppm ) when one ti is substituted onto the nnn b sites , but a decrease in when a much greater number of ti are substituted . plot of the number of sn nnn against ( a ) calculated sn iso and ( b ) calculated sn for ti substitution into the pyrochlore b sites . owing to the difficulties associated with measuring parameters using slow mas experiments , particularly if the csa is small and if there is more than one site present , csa - amplified pass experiments were employed ( see the supporting information for more detail ) . for la2sn1.8ti0.2o7 , la2sn1.6ti0.4o7 , and la2sn1.4ti0.6o7 , sn species having sn6 an increase in was observed for resonances assigned as having sn5ti nnn , as predicted by the dft calculations . for all three materials , the peak corresponding to this environment appeared to consist of two overlapping contributions in the amplified pass experiments ( as shown in the supporting information ) probably arising from small differences in the longer - range structure . sideband manifolds extracted for both had similar values of 5460 ppm . owing to its low intensity , it was not possible to extract for the resonance at 653 ppm . although , in general , the calculated appears slightly overestimated when compared to experiment , the computed and experimental trends are in good agreement and so support the spectral assignment . previous y csa measurements on related pyrochlore materials applied a scaling factor to calculated values of for comparison to experiment . this approach has not been employed here owing to the relatively small amount ( and the small range ) of data available , and only the trends and variations in the measurements are considered . the calculations represented by figure 3 confirm that the resonances at 647 and 653 ppm can be assigned to sn species with sn5ti and sn4ti2 nnn environments , respectively . however , it is also suggested that it may be difficult to separate and assign resonances corresponding to sn with differing nnn environments in the pyrochlore phase as the ti content increases , hence , complicating any analysis . however , it should be noted that the relative intensity of the sharp pyrochlore resonances observed varies very little as x increases , perhaps suggesting that there is limited substitution of ti into this phase . the three sharp signals persist throughout the compositional range shown in figure 2 , through to x = 1.8 ( which , from radius ratio considerations , should exhibit a layered perovskite structure ) . this raises the question of whether sn signals from the layered perovskite - like phase simply appear at similar shifts to those from the pyrochlore or whether a pyrochlore phase is indeed present from x = 0.2 to x = 1.8 . as it is not possible to predict nmr parameters for the sn - free end member la2ti2o7 , to provide further insight dft calculations were performed for a monoclinic unit cell of la2sn0.125ti1.875o7 by substitution of one ti atom with one sn atom in b. as four crystallographically distinct ti sites are present ( ti1 , ti2 , ti3 and ti4 ) , four separate structural models , with sn placed on each of these sites , were constructed before performing geometry optimization and nmr calculations ( see experimental and computational methods ) . the predicted sn iso values are shown in figure 4 and are given in table 1 . the values range between 584 and 619 ppm , suggesting that the signal at 650 ppm does not result from the layered perovskite phase . the signal in this region can , therefore , be assumed to result from a pyrochlore phase , suggesting a significant two - phase region is present within the series . figure 4 and table 1 also show that sn / ti substitutions in c ( i.e. , la2sn0.125ti1.875o7 , with sites numbered for consistency with the monoclinic form , see the supporting information , figure s1.1 ) give a similar conclusion , with predicted shifts between 583 and 616 ppm . in light of these computational results , a second set of la2(sn , ti)2o7 samples was prepared , with x varying from 1.80 to 1.95 in steps of 0.05 . owing to the low level of sn in these materials , sn spectra were acquired using cpmg experiments , and are shown ( as spikelets ) in figure 5 . cpmg spectra provide greater peak - height signal , but can be less quantitative if there are any significant t2 differences between the signals . above x = 1.85 , no signal from the pyrochlore phase is observed between 640 and 655 ppm , indicating that this represents the upper limit of the two - phase region observed . plot of relative enthalpy ( h ) against calculated sn iso for sn substitutions in b ( * ) , b ( ) , and c ( + ) with overlay of the experimental sn mas nmr spectrum for the sample with nominal composition la2sn0.2ti1.8o7 . h values are relative to sn2/ti2 substitution for b and sn2/ti2 substitution in b for both b and c series . sn ( 9.4 t , 14 khz mas ) cpmg nmr spectra ( shown as spikelets ) of la2sn2xtixo7 , with x from 1.80 to 1.95 . h relative to sn / ti2 substitution in b. h relative to sn / ti2 substitution in b. interestingly , figure 4 shows that the predicted sn shifts for the layered perovskite phase agree with the resonance positions measured experimentally only when sn is placed on the perovskite - like ti1 and ti2 sites , suggesting there may be a preference for sn substitution into these sites ( alternative sites , ti3 and ti4 , are denoted as the calculated sn shifts are in reasonable agreement with experiment for single sn substitutions in both b and c forms and so can not prove conclusively which structural model is most accurate . in both cases , two of the unit cell dimensions are relatively short , corresponding to a repeat unit of just two tio6 octahedra . for b , substitution of one sn corresponds to a doping level of 12.5% ( i.e. , 1/8 of the ti sites in the unit cell ) , higher than that in many of the spectra shown in figure 2 . the periodic nature of the calculations also results in an artificially ordered structure with o - bridged , alternating sn and ti cations along the crystallographic a direction . in order to consider the effects of such ordering upon the calculated sn iso values ( by better isolating substituted sn cations ) , an additional set of dft calculations were also performed on a 2 1 1 supercell of b , termed b. sn / ti substitutions were made into each of the four distinct ti sites , giving la2sn0.125ti1.875o7 ( see the supporting information for more detail ) . as shown in figure 4 and table 1 , the calculated sn shifts are very similar between b and b , and match experiment only for sn1/ti1 and sn2/ti2 substitutions , again suggesting preferential substitution into these sites . this conclusion is also supported by the relative enthalpies , h , of the substituted models , as shown in figure 4 , for each of the three series of calculations . the range of relative enthalpies is relatively small , however , the most stable structures correspond to the same substitutions that produce sn shifts in agreement with experiment . enthalpy differences between the four structures calculated from b are given relative to the most thermodynamically stable structure , that is , that with sn2/ti2 . this is followed by substitution into ti1 ( h = 0.01 ev ) , ti3 ( h = 0.05 ev ) , and finally ti4 ( h = 0.11 ev ) . for the b and c models , since these have equivalent composition , h can be renormalized to sn2/ti2 substitution in b , placing both series on the same relative enthalpy scale . while the differences in h are again small , the same trend is seen as in the b series . for b and c , sn2/ti2 substitution is most favorable ( h = 0.00 and 0.04 ev , respectively ) , then sn1/ti1 substitution ( h = 0.02 and 0.05 ev respectively ) , sn3/ti3 ( h = 0.07 and 0.07 ev , respectively ) , and sn4/ti4 ( h = 0.13 and 0.14 ev for b and c , respectively ) . of further note is that equivalent substitutions in the c series are typically of higher enthalpy than in the b series , although sn substitution appears to amplify this difference , as undoped b is only 0.01 ev more stable than undoped c. the use of supercell calculations based on the monoclinic b structure ( and unit cell models based on c ) , with a single sn substitution , enabled a doping level of 6.25% to be considered computationally and resulted in sn atoms that are relatively isolated in the structure ( i.e. , snsn distances > 7 ) . however , given the suggestion of preferential site substitution raised by the predicted sn shifts , the possibility of a nonrandom spatial distribution of sn ( i.e. , sn clustering ) should also be considered . furthermore , a variation in the sn local environment ( e.g. , in nnn atoms ) might also result in a significant change in iso for sn on ti3 and ti4 sites , perhaps bringing these into better agreement with the experimentally measured shifts . to investigate this , a further series of supercell models , each based on b with two sn atom substitutions , was constructed . in separate supercells , a first sn / ti substitution was made for each of ti1 , ti2 , ti3 , and ti4 sites and a second sn was substituted into selected ti sites within a radius of about 6 of the first . to limit computational expense , the second sn atoms within a supercell were placed to account for one from each group of ti sites at similar distances ( and , therefore , assumed to give a similar effect ) . for example , in the case of sn1/ti1 and sn2/ti2 substitutions , within the adopted supercell , pairs of these sites lie at distances of 3.860 and 3.940 and at 5.694 and 5.700 ; here , only combinations at 3.940 and 5.700 were considered . a total of 16 disubstituted models were thus generated and were subjected to geometry optimization and nmr calculations ( see the supporting information , table s4.1 , for detailed results ) . calculated sn isotropic chemical shifts are shown in figure 6 and given in table s4.1 . plot of relative enthalpy ( h ) against calculated sn iso for mono- and disubstituted b models showing the spread associated with disubstituted structures . sn1 and sn2 models that fall outside their typical shift ranges are indicated ( see the main text for details ) . figure 6 shows that the variation in local environment introduced in the disubstituted structures results in a wider range of predicted shifts for each sn site compared to those predicted for monosubstituted b structures . when the sn nnn environment is varied , the majority of shifts for disubstituted models including sn / ti1 and sn2/ti2 substitutions remain in good agreement with the signal seen experimentally and with those for the monosubstituted models . for models with sn3/ti3 and sn4/ti4 all calculated shifts lie outside the signal seen experimentally . two cases of sn1/ti1 and sn2/ti2 substitutions appear to result in sn shifts outside the experimental range ( denoted by arrows in figure 6 ) . although this may be due to a systematic overestimation of the shifts in the calculations ( i.e. , all are shifted upfield with respect to experiment ) , this error is minimized by referencing . alternatively , the arrangement of sn atoms leading to these shifts may not arise in the experimental system ; for the sn1 shift at 610 ppm , the second sn is on the ti3 site , while for the sn2 shift at 611 ppm , the second sn is on the ti4 site . therefore , since sn appears to substitute preferentially into the ti1 and ti2 sites , these combinations may not occur at any significant level . finally , it is noted that the level of noise in the experimental spectrum is such that the presence of a low intensity signal around 610 ppm can not be completely ruled out . no correlations were found between predicted sn shifts or relative enthalpies and snsn internuclear distance and , therefore , no evidence for sn clustering was found . however , with the trends between iso and h and sn / ti substitution site noted above , it appears the particular site(s ) of sn / ti substitution , rather than the distance between a given pair of sn atoms , is the more significant factor affecting these quantities . figure 6 suggests that the signal seen at the highest sn chemical shifts experimentally results from sn2/ti2 substitution , and that at slightly lower shift to sn1/ti1 . however , figure 5 reveals that as x increases the relative intensity of the signals in these two regions varies , suggesting that , when present at very low concentrations , sn preferentially substitutes onto the ti2 site , with the amount of sn1/ti1 substitution increasing as the sn content rises . although the differences in the calculated enthalpies are small , it should be noted that sn2/ti2 substitution ( considering the substitution of one atom per cell ) does also have the lowest relative enthalpy for b and b models . although it is clear from figure 5 that a two - phase region extends in this series to x = 1.8 , it is not clear from the mas spectra in figure 2 exactly where the lower boundary of this region lies . to clarify this , sn cpmg spectra of samples with x = 0.2 and 0.4 were acquired , and are shown as insets in figure 2 . although a low intensity signal is observed for x = 0.4 at 580 ppm , no signal is seen for x = 0.2 ( after 24 h of acquisition ) , suggesting that the two - phase region is present from x = 0.4 to 1.8 . figure 7a plots the % sn in the pyrochlore and layered perovskite phases against the composition of the initial synthesis mixture . a small increase in the amount of sn in the layered perovskite phase is observed between x = 0.4 and 1.2 , before a more significant increase is seen from x = 1.4 . above note that the intensity of the peaks attributed to this phase is very low when x < 1 , resulting in a significant uncertainty in the measurement . the relative intensity of the two sets of peaks is not able to provide information directly on the proportion of the two phases present , since sn may not be distributed equally between them . however , the composition of the two phases and , therefore , their proportions can be obtained from the relative intensities of the peaks attributed to the pyrochlore phase . if it is assumed that the peaks at 641 , 647 , and 653 ppm arise from sn species with sn6 , sn5ti , and sn4ti2 nnn environments , their relative intensities can then provide information on the proportions of sn and ti present in this phase for each sample . from this , the starting composition and the relative intensities of the total peaks attributed to the pyrochlore and layered perovskite phases , it is then possible to determine both the composition of the layered perovskite phase and the relative proportions of the two phases present for any starting composition . the proportion ( expressed as % ) of the two phases present , and their relative compositions , is plotted in figure 7b and c , respectively . although no significant signal attributed to the layered perovskite phase can be seen when x = 0.2 , this analysis suggests a very small amount of this phase is present ( 3.5% ) but that it contains almost no sn . the proportion of the layered perovskite phase increases almost linearly as x increases , until it is the major phase ( after x = 1.0 ) and the only phase after x = 1.8 . there appears to be a relatively low solid solution limit for ti into the stannate pyrochlore , with a maximum of 78% ( i.e. , la2sn0.15ti1.85o7 ) , with the variation observed probably resulting from the errors in obtaining accurate relative intensities for compositions where the sn signal for any one phase is low . there is more variation in the composition of the layered perovskite phase , varying from 100% ti to 75% ti as x decreases , although the margin of error becomes significant when x < 1.0 owing to the low intensity of the resonance . plots showing ( a ) the proportion of sn found in the pyrochlore ( pyr ) and layered perovskite ( lp ) phases , ( b ) the total proportion of pyr and lp phases present in the sample , and ( c ) the composition of the two phases , as a function of the nominal composition used in the initial synthesis . in ( c ) , the percentages of the sn and of the ti in the pyrochlore phase ( or the layered perovskite phase ) should equal 100% . in order to undertake the analysis described and produce the results shown in figure 7 , it must be assumed that the pyrochlore peaks observed can be attributed solely to nnn environments of sn6 , sn5ti , and sn4ti2 . figure 3 predicted some overlap of signals from differing nnn environments at higher x although , as shown in figure 7 , the relatively low level of ti that appears to be substituted into this phase supports the validity of the assumption above . the approach used , however , also requires a very accurate analysis of the relative intensities of all three pyrochlore peaks . an alternative approach is possible , requiring only an accurate determination of the intensity of the peak ascribed to an environment with sn6 nnn ( relative to all other signals ) , but does then assume that there is a random distribution of the b site cations in the pyrochlore phase . if this is true , simple statistics shows that the probability of finding sn with 6 sn nnn is directly related to the proportion of sn in the material ( and , therefore , the composition ) , with p(6 sn nnn ) = ( x/2 ) ( see the supporting information for a more detailed explanation ) . the relative proportions of each phase present ( and their compositions ) determined using this alternative approach are almost identical to those determined previously , as shown in the supporting information , both ( i ) confirming the validity of the first method ( despite the more stringent requirement for accurate relative intensities for all peaks ) and ( ii ) suggesting that the low number of ti cations in the pyrochlore phase are , indeed , distributed randomly . sn nmr spectroscopy has been used to investigate la2(sn , ti)2o7 , providing detailed insight into the number and proportion of phases present , and the local atomic - level environments observed . a broad two - phase region is present in the series , with limited solid solution at each end of the compositional range . the conclusions are in broad agreement with powder xrd measurements ( shown in the supporting information ) . although the quality of these laboratory data is not sufficient to carry out multiple - phase refinements and determine the proportions and compositions of the phases present , it is clear that the reflection of the pyrochlore phase is first seen when x = 1.8 . similarly , figure s2.1 shows evidence of the reflection of a la2ti2o7 based phase when x = 0.4 , but not at x = 0.2 . this is in agreement with the sn nmr spectra in figure 2 , although detailed analysis of the spectral intensities of the pyrochlore phase suggest the possibility of a very small amount ( < 3% ) of layered perovskite phase present when x = 0.2 , but that this contains no sn . at the sn - rich end of the series , there appears to be a solid - solution limit of 78% of ti into the pyrochlore phase , and the relative intensities of the spectral resonances seen confirm that the cation substitution is random . this is not the case for ti - rich layered - perovskite phase , where dft calculations suggest preferential substitution of sn into just two of the four distinct ti sites . these correspond to the sites in the bulk of the perovskite layer , rather than those on the edge . as the sn content decreases further , substitution into ti2 appears to be preferred , with this arrangement giving the lowest energy ( from dft ) and producing calculated sn iso that are in best agreement with those seen experimentally . preferential substitution of y into la2ti2o7 was also observed in earlier work using y nmr spectroscopy . four distinct resonances could be identified within the nmr spectrum , attributed to y substitution onto the four distinct a sites present . the relative intensity of these varied with y content , suggesting preferential substitution of y onto two of the four sites at la - rich compositions . there appears to be a maximum of 2530% sn substitution into the layered perovskite phase in the current work , although the errors inherent in the accurate integration of the spectral resonances as the sn content increases are larger . the dft calculations predict very similar sn shifts for sn substitution into the two proposed models of la2ti2o7 , indicating it is not possible to distinguish between them by nmr spectroscopy . however , it should be noted that the relative energy of the sn - substituted orthorhombic phase was higher than that of substituted monoclinic cells , suggesting that the latter is a more accurate structural model , in agreement with the conclusions of a number of diffraction - based investigations . although we have focused on sn nmr in this work , as it has spin quantum number i = 1/2 , and high - resolution spectra can be easily obtained , it may well be possible to obtain further information from the other ( quadrupolar ) nuclei present in the materials . while wide - line nmr spectra for la and ti could be obtained , the challenges in measuring small changes in very broad lineshapes ( and the additional line broadening resulting from the distribution of local environments ) , however , o nmr spectroscopy offers an easier , and potentially more informative , approach for studying cation disorder , with o directly coordinated to the cations studied . we are currently investigating the conditions required for quantitative o enrichment of a range of pyrochlores and related ceramics ( including la2ti2o7 and la2sn2o7 ) , and the parameters required to ensure quantitative spectra can be obtained . once successful , we will consider whether o nmr spectroscopy can provide more detailed information on the cation disorder and preferential substitution in this system . in summary , this work highlights the detailed information that is available from accurate analysis of quantitative nmr spectra and confirms the advantages of a nmr crystallography approach for structural characterization , with the combination of nmr , xrd and dft calculations able to provide a detailed picture of phase distribution , composition , and cation ordering in ceramic oxides .

an nmr crystallographic approach , involving the combination of 119sn nmr spectroscopy , xrd , and dft calculations , is demonstrated for the characterization of la2sn2xtixo7 ceramics . a phase change from pyrochlore ( la2sn2o7 ) to a layered perovskite phase ( la2ti2o7 ) is predicted ( by radius ratio rules ) to occur when x 0.95 . however , the sensitivity of nmr spectroscopy to the local environment is able to reveal a significant two - phase region is present , extending from x = 1.8 to 0.2 , with limited solid solution at the two extremes , in broad agreement with powder xrd measurements . dft calculations reveal that there is preferential site substitution of sn in la2ti2o7 , with calculated shifts for sn substitution onto ti1 and ti2 sites ( in the bulk perovskite layers ) in better agreement with experiment than those for ti3 and ti4 ( edge sites ) . substitution onto these two sites also produces structural models with lower relative enthalpy . as the sn content decreases , there is a further preference for substitution onto sn2 . in contrast , the relative intensities of the spectral resonances suggest that ti substitution into the pyrochlore phase is random , although only a limited solid solution is observed ( up to 7% ti ) . dft calculations predict very similar 119sn shifts for sn substitution into the two proposed models of la2ti2o7 ( monoclinic ( p21 ) and orthorhombic ( pna21 ) ) , indicating it is not possible to distinguish between them . however , the relative energy of the sn - substituted orthorhombic phase was higher than that of substituted monoclinic cells , suggesting that the latter is the more likely structure .

Introduction Experimental and Computational Methods Results and Discussion Conclusions

in this work , we utilize nmr spectroscopy and density functional theory ( dft ) calculations to study la2(sn , ti)2o7 , where a phase transition from a pyrochlore ( la2sn2o7 , where ra / rb = 1.68 ) to a layered perovskite phase ( la2ti2o7 , where ra / rb = 1.92 ) is expected . when x = 0.2 , two additional sharp resonances are observed , at 647 and 653 ppm , most likely arising from substitution of ti into the next - nearest neighbor ( nnn ) b sites in the pyrochlore structure , a. the assignment of the resonances can be confirmed using dft calculations [ following the approach outlined in ref ( 18 ) ( described in more detail in the supporting information ) , where the local environment of one of the sn atoms in the unit cell of la2sn2o7 is systematically modified to include increasing numbers of ti cations on the six surrounding b sites ] . as it is not possible to predict nmr parameters for the sn - free end member la2ti2o7 , to provide further insight dft calculations were performed for a monoclinic unit cell of la2sn0.125ti1.875o7 by substitution of one ti atom with one sn atom in b. as four crystallographically distinct ti sites are present ( ti1 , ti2 , ti3 and ti4 ) , four separate structural models , with sn placed on each of these sites , were constructed before performing geometry optimization and nmr calculations ( see experimental and computational methods ) . above x = 1.85 , no signal from the pyrochlore phase is observed between 640 and 655 ppm , indicating that this represents the upper limit of the two - phase region observed . h relative to sn / ti2 substitution in b. h relative to sn / ti2 substitution in b. interestingly , figure 4 shows that the predicted sn shifts for the layered perovskite phase agree with the resonance positions measured experimentally only when sn is placed on the perovskite - like ti1 and ti2 sites , suggesting there may be a preference for sn substitution into these sites ( alternative sites , ti3 and ti4 , are denoted as the calculated sn shifts are in reasonable agreement with experiment for single sn substitutions in both b and c forms and so can not prove conclusively which structural model is most accurate . although a low intensity signal is observed for x = 0.4 at 580 ppm , no signal is seen for x = 0.2 ( after 24 h of acquisition ) , suggesting that the two - phase region is present from x = 0.4 to 1.8 . from this , the starting composition and the relative intensities of the total peaks attributed to the pyrochlore and layered perovskite phases , it is then possible to determine both the composition of the layered perovskite phase and the relative proportions of the two phases present for any starting composition . a broad two - phase region is present in the series , with limited solid solution at each end of the compositional range . although the quality of these laboratory data is not sufficient to carry out multiple - phase refinements and determine the proportions and compositions of the phases present , it is clear that the reflection of the pyrochlore phase is first seen when x = 1.8 . this is in agreement with the sn nmr spectra in figure 2 , although detailed analysis of the spectral intensities of the pyrochlore phase suggest the possibility of a very small amount ( < 3% ) of layered perovskite phase present when x = 0.2 , but that this contains no sn . at the sn - rich end of the series , there appears to be a solid - solution limit of 78% of ti into the pyrochlore phase , and the relative intensities of the spectral resonances seen confirm that the cation substitution is random . there appears to be a maximum of 2530% sn substitution into the layered perovskite phase in the current work , although the errors inherent in the accurate integration of the spectral resonances as the sn content increases are larger . the dft calculations predict very similar sn shifts for sn substitution into the two proposed models of la2ti2o7 , indicating it is not possible to distinguish between them by nmr spectroscopy . however , it should be noted that the relative energy of the sn - substituted orthorhombic phase was higher than that of substituted monoclinic cells , suggesting that the latter is a more accurate structural model , in agreement with the conclusions of a number of diffraction - based investigations .

the cytochromes p450 ( cyps ) constitute a superfamily of hemoprotein enzymes ( zhu and silverman 2008 ) . they are well recognized for their major roles in the synthesis , activation , and metabolism of many endogenous and xenobiotic compounds , such as hormones , fatty acids , drugs , and carcinogens ( spriet et al . it has been repeatedly reported that multiple p450s metabolize arachidonic acid ( ara ) ( devos et al . 2010 ; mitra et al . 2011 ; theken et al . 2011 ; xu et al . 2011 ) . ara is a fatty acid found in the human cell membrane , which is the precursor of eicosanoids that play important biological roles in the regulation of blood homeostasis and inflammation process ( imig 2012 ) . ara metabolites have also been reported to be involved in bone marrow haematopoiesis , and its abnormal regulation has been associated with the case of cardiovascular diseases ( lutton et al . 1989 ; abraham et al . 1991 ; pfister et al . ara is epoxidized to epoxyeicosatrienoic acids ( eets ) by cyp2c and cyp2j subfamilies , while cyp4a , 4f , and 1a subfamilies and cyp2u1 enzyme hydroxylate ara to 20-hete ( lasker et al . 2000 ; pearson et al . for example , cyp2j2 is expressed in cardiac tissues more predominantly than in other tissues ( wu et al . 1996 ) . furthermore , cyp2c subfamily is found in significantly higher quantities in the liver and gastrointestinal tract than in bone marrow ( bieche et al . these differences in ara - metabolizing p450 expression levels may lead to different levels of ara metabolites , thus affecting tissues biological regulation . megakaryocytes are the bone marrow cells responsible for the production of blood platelets . in humans , each megakaryocyte produces 1,0003,000 enucleate platelets through a thrombopoietin - mediated process ( deutsch and tomer 2006 ) . although thromboxane synthase ( cyp5a1 ) has been found to be highly expressed in megakaryocytes and platelets ( nakahata 2008 ) , other p450 expressions in megakaryocytes and their daughter platelets are , as yet , poorly understood . interestingly , ara and some of its metabolites have been identified in platelets ( sheppard et al . 1992 ) , suggesting the possible presence of ara - metabolizing p450s in megakaryocytes . since platelets , being cell fragments derived from the precursor megakaryocytes , can not proliferate in the cell culture system due to a lack of nucleus , there are no cell lines available for the molecular study of platelets . the megakaryocytic cell line ( dami cells ) has frequently been used for the study of platelets ( khetawat et al . 2011).the goal of the present study was to determine which p450s are expressed in dami cells with the functional activity for the metabolism of ara . iscove 's modified dulbecco 's medium , moloney murine leukemia virus reverse transcriptase , oligo(dt)18 primer , dithiothreitol ( dtt ) , and first strand buffer were obtained from invitrogen ( carlsbad , ca ) . d - taq polymerase , 10 d - taq buffer , and 2.5 mm dntp were purchased from sun gen ( daejeon , korea ) . primary antibodies for cyp1a1 , 2u1 , and 2j2 and the chemiluminescence kit were purchased from santa cruz biotechnology ( santa cruz , ca ) . the primary antibody for actin was obtained from cell signaling technology ( danvers , ma ) . resorufin , 7-ethoxyresorufin ( 7-er ) , skf-525 , protease inhibitor cocktail , and 3-methylenecholantheren ( 3-mc ) were obtained from sigma aldrich ( st . louis , mo ) . both 20-hete and 20-hete - d6 were obtained from cayman ( ann arbor , mi ) . all other chemicals and organic solvents for activity assays were of the highest grade available from commercial sources . the human megakaryocytic dami cell line was obtained from the american type culture collections ( rockville , md ) . dami cells were cultured in iscove 's modified dulbecco 's medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin . cultures were maintained in a humidified atmosphere of 5% co2 at 37c as previously described ( khetawat et al . dami cells were treated with 10 m of 3-mc for 72 h in order to test the induction of cyp1a1 and determine the effect of cyp1a1 induction on ara metabolism . for these experiments , 3-mc was dissolved in dimethyl sulfoxide ( dmso ) , and the final dmso concentration in the culture medium was 0.1% ( v / v ) . after 3-mc treatment , the cells were incubated with 100 m ara for 12 h ( rehfeldt et al . 1993 ) . rnas from dami cells , and human liver were extracted using trizol reagent ( invitrogen ) according to the manufacturer s instructions . liver tissue samples were obtained from the inje pharmacogenomics research center biobank ( inje university college of medicine , busan , korea ) . the research protocol for the usage of human liver tissues was approved by the institutional review board of busan paik hospital ( inje university , busan , korea ) . three micrograms of total isolated rna was added to a reaction mixture containing 100 pmol oligo(dt ) , 2.5 mm dntp , 0.1 m dtt , 5 first strand buffer , 200 u of m - mlv reverse transcriptase and rnaase - free water for synthesis of cdna . next , conventional pcr was performed by adding 330 ng of cdna to a mixture containing 10 mm dntps , 25 mm mgcl2 , 2 d - taq polymerase buffer , 10 pmoles from each of the forward and reverse primers ( table 1 ) , and 1.5 u of d - taq dna polymerase . the pcr products were separated on a 2% agarose gel and visualized using ethidium bromide staining.table 1.pcr primer sequences and amplified product sizes for rt - pcr analysisgeneforeword primer ( 5 to 3)reverse primer ( 5 to 3)band size ( bp)referencecyp1a1gtaatcagggcctcaagacgacattggtcactgatacc518finnstrom et al . 2001gene - specific primers were designed in the present study pcr primer sequences and amplified product sizes for rt - pcr analysis gene - specific primers were designed in the present study dami cell cultures were centrifuged at 1,000 rpm for 3 min . then , lysis buffer [ 50 mm tris hcl ( ph 7.4 ) , 150 mm nacl , 1% np40 , 0.25% na - deoxycholate , and protease inhibitor cocktail ] was added to the dami cell pellet . the mixture was vortexed vigorously , sonicated twice for 10 s , and the resulting lysate was incubated on ice for 50 min . , 35 g of protein was boiled at 95c for 5 min with 4 loading buffer containing 0.1 m tris hcl ( ph 6.8 ) , 4% sodium dodecyl sulfate ( sds ) , 1.5% bromophenol blue , 20% glycerol , and 5% -mercaptoethanol . polyacrylamide gel and then transferred to a nitrocellulose membrane in a buffer containing 25 mm tris hcl , 192 mm glycine , and 20% ( v / v ) methanol . the membrane was blocked by 5% skimmed milk in tris - buffered saline supplemented with 0.1% tween 20 solution . the membrane was probed with polyclonal goat anti - cyp1a1 igg and anti - cyp2u1 igg , and monoclonal mouse anti - cyp2j2 igg , separately , and reproved with monoclonal rabbit anti - beta - actin igg at 4c overnight . immunoreactive proteins were detected using the enhanced chemiluminescence method according to the manufacturer 's instruction ( ge healthcare bio - sciences , buckinghamshire , uk ) . intact cells were incubated with 2 g of 7-er in a tn assay buffer [ 0.1 m nacl , 50 mm tris ( ph 7.8 ) ] for 20 min . the amount of resorufin formed was measured at excitation / emission wavelengths of 545/575 nm ( perkin elmer victor 3v , mtx lab systems , vienna , va ) . the standard curve of known concentrations of resorufin was between 50 and 500 pmoles ( kennedy et al . , the cells were lysed , and the protein concentration was determined using the bradford dye method ( martin et al . ara metabolism in dami cells was investigated by incubation of 1 10 dami cells with 100 m ara for 12 h ( rehfeldt et al . ara metabolites were extracted from dami cells and cell media by ethyl acetate as previously reported ( zordoky et al . ara and its metabolites were identified and quantified using a liquid chromatography mass spectrometry ( lc - ms / ms ) system , with some modifications on the previous method ( shinde et al . briefly , metabolites were separated on a reverse - phase column atlantis dc18 ( 2.1 mm i.d . 150 mm , 3 m particle size ; waters , ireland ) with a solvent consisting of water ( a ) and acetonitrile containing 0.1% formic acid ( b ) . the flow rate of 0.25 ml / min with gradient system was as follow : 05 min , 5% b ; 5 min , 35% b ; 15 min , 65% b ; 20 min , 75% b ; 2428 min , 100% b ; and 28.01 min , 5% b. ara metabolites were identified using an api 5500 mass spectrophotometer ( applied biosystems , foster city , ca ) . peak areas for all compounds were integrated using analyst software ( version 1.2 ; applied biosystems ) , and 20-hete-6 ( 100 g / ml ) was used as internal standard . the detection limits of 14,15-eet , 14,15-dihydroxyeicosatrienoic acid ( 14,15-dhet ) and 20-hete were 20 , 10 , and 20 pg / ml , respectively . all statistical analyses were performed using the sas program ( version 9.1.3 ; sas institute , cary , nc ) . statistically significant differences as compared with the control groups are represented as * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 . using reverse transcriptase - polymerase chain reaction ( rt - pcr ) analysis , we screened the mrna expression of 15 major p450s which are reported to metabolize ara ( devos et al . the screened ara - p450s were as follows : cyp1a1 , 1a2 , 1b1 , 2c8 , 2c9 , 2c19 , 2d6 , 2j2 , 2u1 , 4a11 , 4f2 , 4f3 , 3a4 , and 3a5 in addition to cyp5a1 which is known to be expressed in platelets . we detected mrnas of cyp1a1 , 2u1 , and 2j2 in dami cells , as well as the mrna of cyp5a1 ( fig . 1 ) . there were no detectable bands in the pcr reactions performed with rna samples that were not treated with reverse transcriptase ; these reactions served as a control to monitor genomic dna contamination . immunobloting analysis showed the detection of cyp1a1 , 2u1 , and 2j2 proteins , with bands size of 5060 kda , in dami cells ( fig . the detected bands were in agreement with the theoretical size of 5060 kda for p450s and with the liver microsome sample used as a reference standard for the detection of p450s.figure 1.expression profiles of p450s in dami cells . ( a ) rt - pcr was used to amplify each p450 with the specific primers listed in table 1 . the targeted genes were amplified as a single product of the expected size ; no bands were detected in control samples in which the pcr was performed in rna samples without reverse transcriptase treatment to confirm that there was no genomic dna contamination . ( b ) detection of cyp1a1 , 2u1 , and 2j2 proteins in dami cells by immunublot analysis . total protein lysates ( 35 g ) from dami cells were loaded in each lane . proteins were separated on a 13% sds - polyacrylamide gel , transferred to nitrocellulose membrane , and detected by primary antibodies against cyp1a1 , 2u1 , and 2j2 . ( a ) rt - pcr was used to amplify each p450 with the specific primers listed in table 1 . the targeted genes were amplified as a single product of the expected size ; no bands were detected in control samples in which the pcr was performed in rna samples without reverse transcriptase treatment to confirm that there was no genomic dna contamination . ( b ) detection of cyp1a1 , 2u1 , and 2j2 proteins in dami cells by immunublot analysis . total protein lysates ( 35 g ) from dami cells were loaded in each lane . proteins were separated on a 13% sds - polyacrylamide gel , transferred to nitrocellulose membrane , and detected by primary antibodies against cyp1a1 , 2u1 , and 2j2 . the activity of resorufin formation was 112 10 nmol / min / mg protein . to confirm the activity of p450 , 40 m of the p450 inhibitor skf-525a was added to the reactions and compared with the non - skf-525a - treated samples . figure 2 shows that erod activity decreased significantly with the addition of skf-525a to dami cells ( 60% , p < 0.001 ) when compared to the control groups . because cyp1a1 ( this study ) and the nuclear aryl hydrocarbon receptor ( ahr ) ( lindsey and papoutsakis 2011 ) are both expressed in megakaryocytes , we investigated the inducibility of cyp1a1 in dami cells using 3-mc which is an ahr agonist , after confirming the expression of ahr in dami cells ( fig . immunoblot analysis showed that the cyp1a1 protein level increased in dami cells after 3-mc treatment ( fig . 3b ) , and that this increased protein level correlated with the increase in erod activity ( fourfold , p < 0.001 ) as compared with the dmso - treated groups ( fig . the intact cells were incubated with 2 g of 7-er in a tn assay buffer for 20 min . the amount of resorufin formed was measured at excitation / emission wavelengths of 545/575 nm by comparison with a standard curve of known concentrations of resorufin . statistically significant differences as compared with the control group are indicated as * * * p < 0.001 , based on a two - tailed student s t test . data are presented as the mean s.d . of reactions performed in triplicate.figure 3.the effect of 3-mc on cyp1a1 expression and erod activity . ( a ) detection of ah receptor by rt - pcr analysis in dami cells . rt - pcr contains a set of ahr - specific primers as shown in table 1 . a specific band of ahr is marked by the arrow ( 322 bp ) . dami cells were cultured with 10 m of 3-mc ( + 3mc ) for 72 h. the control group ( -3mc ) received the same volume of the solvent ( dmso ) without 3-mc . after 3-mc treatment , 35 g of total protein lysates from each group was loaded in a sds - polyacrylamide gel for western blot analysis of cyp1a1 . detailed procedures for the western blot analysis are explained in the materials and methods section . the intact dami cells were incubated with 2 g of 7-er in a tn assay buffer for 20 min . details regarding the erod assay are explained in the materials and methods section . statistically significant changes as compared with the dmso treatment group are indicated as * * * p < 0.001 . the intact cells were incubated with 2 g of 7-er in a tn assay buffer for 20 min . the amount of resorufin formed was measured at excitation / emission wavelengths of 545/575 nm by comparison with a standard curve of known concentrations of resorufin . statistically significant differences as compared with the control group are indicated as * * * p < 0.001 , based on a two - tailed student s t test . ( a ) detection of ah receptor by rt - pcr analysis in dami cells . rt - pcr contains a set of ahr - specific primers as shown in table 1 . a specific band of ahr is marked by the arrow ( 322 bp ) . dami cells were cultured with 10 m of 3-mc ( + 3mc ) for 72 h. the control group ( -3mc ) received the same volume of the solvent ( dmso ) without 3-mc . after 3-mc treatment , 35 g of total protein lysates from each group was loaded in a sds - polyacrylamide gel for western blot analysis of cyp1a1 . detailed procedures for the western blot analysis are explained in the materials and methods section . the intact dami cells were incubated with 2 g of 7-er in a tn assay buffer for 20 min . details regarding the erod assay are explained in the materials and methods section . statistically significant changes as compared with the dmso treatment group are indicated as * * * p < 0.001 . we found that 15 ara metabolites were detected by lc - ms / ms in dami cells ( fig . 4 ) , including 5-hete , 8-hete , 9-hete , 11-hete , 12-hete , 15-hete , 20-hete , 11,12-eet , 14,15-eet , 5,6-dhet , 11,12-dhet , 14,15-dhet , leukotriene b4 ( ltb4 ) , 5,6-lipoxin a4 ( lxa4 ) , and txb2 . among the detected ara metabolites in dami cells , 20-hete , 11,12-eet , and 14,15-eet have been reported to be mediated through the ara - p450-metabolizing pathway in the kidney , liver , and vascular tissues ( lasker et al . the expression of soluble epoxide hydrolase was confirmed by a specific rt - pcr ( fig 1a ) , indicating that the generation of dhets from eets would be from soluble epoxide hydrolase in dami cells . induction of cyp1a1 protein by 3-mc treatment significantly increased the levels of 20-hete , 14,15-eet , and 14,15-dhet than in the control group ( fig . these ara metabolites were significantly reduced with the treatment of the skf-525a ( p < 0.050.001).figure 4.identification of ara metabolites in dami cells . the cells were incubated with 100 m ara for 12 h. ara metabolites were then extracted by ethyl acetate ( ph 34 ) . ethyl acetate was evaporated , and the residue was dissolved in 100 l ethanol , followed by lc - ms / ms screening for ara metabolites . 20-hete-6 ( 100 g / ml ) was used as internal standard.figure 5.the effect of 3-mc on ara metabolism in dami cells . dami cells were treated with 10 m 3-mc for 72 h. the control group of dami cells received the same final volume of the solvent ( dmso ) without 3-mc . one group of dami cells ( 1 10 cells ) was incubated with 100 m ara for 12 h. the other group received 40 m of skf-525a in addition to ara . ara metabolites , 14,15-eet ( a ) , 14,15-dhet ( b ) , and 20-hete ( c ) were identified by lc - ms / ms after liquid / liquid extraction by ethyl acetate . statistically significant differences as compared with the dmso treatment group are indicated as * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , based on two - tailed student s t tests . the cells were incubated with 100 m ara for 12 h. ara metabolites were then extracted by ethyl acetate ( ph 34 ) . ethyl acetate was evaporated , and the residue was dissolved in 100 l ethanol , followed by lc - ms / ms screening for ara metabolites . 20-hete-6 ( 100 g / ml ) was used as internal standard . the effect of 3-mc on ara metabolism in dami cells . dami cells were treated with 10 m 3-mc for 72 h. the control group of dami cells received the same final volume of the solvent ( dmso ) without 3-mc . one group of dami cells ( 1 10 cells ) was incubated with 100 m ara for 12 h. the other group received 40 m of skf-525a in addition to ara . ara metabolites , 14,15-eet ( a ) , 14,15-dhet ( b ) , and 20-hete ( c ) were identified by lc - ms / ms after liquid / liquid extraction by ethyl acetate . . statistically significant differences as compared with the dmso treatment group are indicated as * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 , based on two - tailed student s t tests . data are presented as the mean s.d . of reactions performed in triplicate . although ara and its metabolites are important signal molecules in blood hemostasis , ara metabolism by p450s in megakaryocytes and megakaryocytic dami cells remains unclear . megakaryocytic dami cells have been used to study the biological function of megakaryocytes and platelets , because circulating platelets have no nucleus ( khetawat et al . 2012 ) . in the current study , we investigated ara - metabolizing p450s in dami cells . in addition to cyp5a1 , we found that cyp1a1 , 2u1 , and 2j2 were also expressed in dami cells . cyp1a1 , 2u1 , and 2j2 have been reported to metabolize ara and its derivatives ( devos et al . therefore , it can be suggested that these p450s may play a role in the metabolism of ara and its related eicosanoid compounds in the megakaryocytes and the platelets . the literature reports several instances of similarities in the p450 expression profiles of dami cells and bone marrow . for example , in human bone marrows , cyp2u1 and 1a1 are expressed at high levels , but cyp3a and 2c are not present ( bieche et al . similarly , in this study cyp1a1 and 2u1 were strongly expressed in dami cells , while cyp3a4 , 3a5 , 2c8 , 2c9 , and 2c19 were not detectable . the similar expression profiles of p450s in bone marrow tissues and dami cells may indicate that cyp2u1 and 1a1 in bone marrow could be derived , at least in part , from megakaryocytes ; however , it can not rule out the possibility that other bone marrow cell types can also express cyp1a1 and 2u1 . cyp1a1 expression was detected , and its expression was induced by 3-mc in dami cells . these results suggest that there could be variations in cyp1a1 expression levels in megakaryocytes induced by environmental stimuli , such as cigarette smoking and other aromatic hydrocarbons with the potential to alter the metabolism of ara as well as other cyp1a1 substrates . cyp1a1 metabolizes some drugs , such as theophylline and caffeine ( yang and lee 2008 ; amin et al . it is expressed more in cancer tissues and activates pro - carcinogenic compounds , such as polycyclic aromatic hydrocarbons ( levova et al . we found that erod activity was inhibited by 40 m skf-525a , confirming that the reaction was mediated by p450s in megakaryocytic dami cells . because erod activity is mediated by the cyp1a subfamily and cyp1b1 ( smith et al . 2011 ) , erod activity in dami cells appears to be a result of cyp1a1 . our results showed that 14,15-eet and 14,15-dhet were increased in dami cells after treatment with 3-mc . it is known that 14,15-eet is metabolized to 14 , 15-dhet by epoxide hydrolases ( seidegard et al . this finding is consistent with a previous report that 14,15-eet synthesis increases after induction of the cyp1a subfamily by different ahr nuclear receptor agonists in human hepg2 cells ( diani - moore et al . the expression of soluble epoxide hydrolase was evidenced in dami cells , and the experiment using the inhibitor of epoxide hydrolase enzyme would add additional information for further translation of the metabolic fate for eets and dhets . cyp4a and 4f subfamilies have been identified as the main p450s for 20-hete production ( lasker et al . 2000 ; pearson et al . 2009 ) , although multiple studies showed that cyp1a1 hydroxylases ara to 20-hete ( aboutabl et al . 2009 ; arnold et al . 2010 ) . furthermore , the formation of 20-hete is inhibited by 7-er in bone marrow ( abraham et al . these results potentially indicate an essential role for cyp1a1 in the formation of 20-hete in bone marrow . our results showed that induction of cyp1a1 by 3-mc in dami cells increased the metabolism of ara to 20-hete . these results indicate that altered levels of the metabolite may cause variation in megakaryocyte and platelet functions . the relatively high expression of cyp1a1 in megakaryocytes could increase our understanding of hematologic diseases and hematotoxicity . benzenes cause bone marrow toxicity and hematotoxicity ( hirabayashi and inoue 2010 ) , and the aryl hydrocarbon receptor ( ahr ) was found to mediate benzene - induced toxicity ( hayashibara et al . , we confirmed the expression of ahr in dami cells by rt - pcr and observed that 3-mc induced cyp1a1 expression , which is predicted due to the activation of ahr by 3-mc . polyaromatic hydrocarbons identified in cigarette smoke revealed to activate ahr and induce cyp1a1 ( roth et al . therefore , it is suggested to investigate the effect of cyp1a1 induction on platelet aggregation variations . the results of the present study indicate that dami cells may be a good cell line for studying cyp1a1 induction and metabolism for human megakaryocytes and human platelets , at least in part . pregnane x receptor ( pxr ) and constitutive androstane receptor ( car ) have been reported to be involved in the regulation of cyp2j2 ( siest et al . . however , car and pxr were not detected by rt - pcr in our hand ( data not shown ) , prompting us to study the induction of cyp1a1 in the present study . it is expressed more in the thyroid , brain , heart , and bone marrow than in the liver ( karlgren et al . information on the molecular regulation of cyp2u1 is very limited , and there is still no specific inhibitor and inducer of cyp2u1 . 2010 ) , suggesting that 20-hete , which was detected in dami cells , could be produced , at least in part by cyp2u1 . it affects blood hemostasis via the metabolism of ara to eets , which are known to decrease blood pressure and to inhibit platelet aggregation ( spiecker and liao 2006 ) . western blot analysis of cyp2j2 revealed one major band with a molecular weight of 5060 kda in dami cells . however , we noticed that cyp2j2 in liver tissue was revealed by two distinct bands . ( 1996 ) also showed two bands in the liver and major one band of cyp2j2 in other tissues in immunoblot analysis . these results may indicate that cyp2j2 in human heart , kidney , and the platelet precursor cells megakaryocytes share similar immunochemical reactions . cyp2j2 is suggested to contribute to the formation of epoxidation of ara ( wu et al . expression of cyp2j2 in megakaryocytes might play a role in the formation of ara metabolites , and they may be involved in the biological function of megakaryocytes and platelets . in summary , we identified ara - metabolizing p450s and confirmed their expressions with functional activities in dami cells . these p450s may play important roles in the metabolism of ara as well as other xenobiotic compounds in megakaryocytes . particularly , because cyp1a1 expression levels are largely induced by environmental polycyclic aromatic hydrocarbons , altered levels of cyp1a1 expression may cause variations in cyp1a1-mediated metabolism in megakaryocytes . the present information on p450 expressions in megakaryocytic dami cells would further extend our knowledge on the roles of p450s in megakaryocytes as well as platelets .

cytochrome p450s ( p450s ) are involved in the metabolism of arachidonic acid ( ara ) , and ara metabolites are associated with various cellular signaling pathways , such as blood hemostasis and inflammation . the present study demonstrates the expression of ara - metabolizing p450s in the human megakaryocytic dami cells using reverse transcriptase - polymerase chain reaction ( rt - pcr ) and immunublotting analysis followed by activity assays using ara as a substrate . in addition to the previously identified cyp5a1 , both protein and mrnas of cyp1a1 , 2u1 , and 2j2 bands were detected . ethoxyresorufin - o - deethylase ( erod ) activity was observed in dami cells , and its activity was significantly decreased after treatment with the p450 inhibitor skf-525a when compared to the control groups ( 60% reduction , p < 0.001 ) . cyp1a1 protein expression in dami cells was induced by 3-methylenecholantheren . this increase in cyp1a1 protein level was correlated with enhanced erod activity ( fourfold increase vs. the control ) , as well as with increased metabolites , such as 20-hydroxyeicosatrienoic acid ( 20-hete ) , 14 , 15-eet ( 14-,15-epoxyeicosatrienoic acid ) , and 14 , 15-dihydroxyeicosatrienoic acid ( 14 , 15-dhet ) . the expression of soluble epoxide hydrolase , an enzyme responsible for the synthesis of dhets from eets , was confirmed by rt - pcr . furthermore , 15 ara metabolites , including 8,9-eet , 14,15-eet , and 20-hete , were detected by lc - ms / ms in ara - treated dami cells , and their levels were decreased with the treatment of the skf-525a . the present data suggest the possibility that the p450s play a role in the metabolism of ara and other cyp - related substrates in human megakaryocytes and that p450 expression in megakaryocytic cell lines may predict their existences in platelets with functional activities .

Introduction Materials and Methods Results Discussion

2011).the goal of the present study was to determine which p450s are expressed in dami cells with the functional activity for the metabolism of ara . using reverse transcriptase - polymerase chain reaction ( rt - pcr ) analysis , we screened the mrna expression of 15 major p450s which are reported to metabolize ara ( devos et al . the screened ara - p450s were as follows : cyp1a1 , 1a2 , 1b1 , 2c8 , 2c9 , 2c19 , 2d6 , 2j2 , 2u1 , 4a11 , 4f2 , 4f3 , 3a4 , and 3a5 in addition to cyp5a1 which is known to be expressed in platelets . we detected mrnas of cyp1a1 , 2u1 , and 2j2 in dami cells , as well as the mrna of cyp5a1 ( fig . ( b ) detection of cyp1a1 , 2u1 , and 2j2 proteins in dami cells by immunublot analysis . figure 2 shows that erod activity decreased significantly with the addition of skf-525a to dami cells ( 60% , p < 0.001 ) when compared to the control groups . because cyp1a1 ( this study ) and the nuclear aryl hydrocarbon receptor ( ahr ) ( lindsey and papoutsakis 2011 ) are both expressed in megakaryocytes , we investigated the inducibility of cyp1a1 in dami cells using 3-mc which is an ahr agonist , after confirming the expression of ahr in dami cells ( fig . 3b ) , and that this increased protein level correlated with the increase in erod activity ( fourfold , p < 0.001 ) as compared with the dmso - treated groups ( fig . we found that 15 ara metabolites were detected by lc - ms / ms in dami cells ( fig . among the detected ara metabolites in dami cells , 20-hete , 11,12-eet , and 14,15-eet have been reported to be mediated through the ara - p450-metabolizing pathway in the kidney , liver , and vascular tissues ( lasker et al . the expression of soluble epoxide hydrolase was confirmed by a specific rt - pcr ( fig 1a ) , indicating that the generation of dhets from eets would be from soluble epoxide hydrolase in dami cells . these ara metabolites were significantly reduced with the treatment of the skf-525a ( p < 0.050.001).figure 4.identification of ara metabolites in dami cells . ara metabolites , 14,15-eet ( a ) , 14,15-dhet ( b ) , and 20-hete ( c ) were identified by lc - ms / ms after liquid / liquid extraction by ethyl acetate . ara metabolites , 14,15-eet ( a ) , 14,15-dhet ( b ) , and 20-hete ( c ) were identified by lc - ms / ms after liquid / liquid extraction by ethyl acetate . although ara and its metabolites are important signal molecules in blood hemostasis , ara metabolism by p450s in megakaryocytes and megakaryocytic dami cells remains unclear . in addition to cyp5a1 , we found that cyp1a1 , 2u1 , and 2j2 were also expressed in dami cells . therefore , it can be suggested that these p450s may play a role in the metabolism of ara and its related eicosanoid compounds in the megakaryocytes and the platelets . these results suggest that there could be variations in cyp1a1 expression levels in megakaryocytes induced by environmental stimuli , such as cigarette smoking and other aromatic hydrocarbons with the potential to alter the metabolism of ara as well as other cyp1a1 substrates . the expression of soluble epoxide hydrolase was evidenced in dami cells , and the experiment using the inhibitor of epoxide hydrolase enzyme would add additional information for further translation of the metabolic fate for eets and dhets . , we confirmed the expression of ahr in dami cells by rt - pcr and observed that 3-mc induced cyp1a1 expression , which is predicted due to the activation of ahr by 3-mc . however , car and pxr were not detected by rt - pcr in our hand ( data not shown ) , prompting us to study the induction of cyp1a1 in the present study . expression of cyp2j2 in megakaryocytes might play a role in the formation of ara metabolites , and they may be involved in the biological function of megakaryocytes and platelets .

fans of doctor who , the quintessential british science fiction television series , have been able to enjoy the adventures of its main character ( the doctor ) for over three decades . as a time lord from the planet gallifrey , the doctor can regenerate his body completely as he nears death . the doctor s regenerative abilities , however , are not without flaw : after he regenerates , he takes on different features and characteristics . this imperfect process enables the series to continue with a new actor portraying the doctor upon the departure of his predecessor . the doctor s slightly defective regenerative power is not only a boon for the show s fans ( and producers ) , but it is also a rare example of science fiction being outdone by the real world . on earth , a wide range of actual organisms can regenerate missing parts after injury . unlike the fictional doctor , these animals can rebuild new structures that are indistinguishable from those they are replacing . with the goal of understanding the factors that enable some organisms , but not others , to restore missing structures , biologists have been studying regeneration for well over two centuries ( morgan , 1901 ; lenhoff and lenhoff , 1986 ) . although the doctor s regenerative abilities remain a mystery , recent research on various earthlings has started to reveal the mechanisms leading to regeneration of complex structures . during the past decade the application of molecular genetic techniques , including double - stranded rna - mediated genetic interference ( rnai ) and transgenesis , has revitalized studies of classical animal models of regeneration ( poss , 2010 ) . here we highlight a sample of recent work addressing some of the critical questions facing regeneration researchers : what signals initiate regeneration ? the answers to these and related questions should help developmental biologists , tissue engineers , and clinicians to understand and one day overcome the limits on human regeneration . after amputation , local responses at the site of the wound play important roles in the initiation of regenerative processes . ion flux ( levin , 2009 ) , and interactions between the wound epidermis and the underlying tissue appear critical for regenerative outgrowth . recent work suggests that programmed cell death may play a role in triggering regenerative responses in many different organisms , including hydra ( bergmann and steller , 2010 ) . hydra , a freshwater polyp belonging to the phylum cnidaria ( which includes jellyfish , sea anemones , and corals ) , was the subject of the first scientific investigations of regeneration in animals ( lenhoff and lenhoff , 1986 ) . the hydra body consists of ectodermal and endodermal cell layers separated by an extracellular matrix ; neurons and interstitial cells ( stem cells that produce neurons , gland cells , and germ cells , among other cell types ) reside between these two layers . the animal is radially symmetric , and is polarized along the oral ( head and tentacles)/aboral ( foot ) axis ( fig . small fragments of hydra tissue can regenerate a complete organism ; even dissociated single cells can reaggregate , reestablish polarity , and form a new animal ( noda , 1971 ; gierer et al . , 1972 ) . ( a ) after mid - gastric bisection in hydra , mapk signaling leads to rapid activation of the transcription factor creb in fragments regenerating a head ( blue cells ) . mapk / creb activity is required for stimulating a wave of apoptosis in interstitial cells near the site of injury ( red cells ) . these apoptotic cells secrete wnt3 , inducing a zone of proliferation ( green cells ) below the region of apoptosis . ( b ) planarians and ( c ) xenopus larval tails also show a rapid , localized increase in apoptosis after amputation . in xenopus , like hydra , apoptosis may provide important signals during early phases of regeneration ; inhibiting apoptosis during the first 24 h post amputation ( hpa ) blocks regeneration . the fragment that will regenerate a new head displays a robust apoptotic response near the wound site , whereas the fragment regenerating a new foot does not ( fig . 2009 ) . inhibition of apoptosis blocks head regeneration , and activation of apoptosis at a foot - regenerating wound leads to ectopic head formation ( chera et al . , the apoptotic response stimulates a synchronous burst of proliferative activity in neighboring cells and leads to the establishment of a head organizer via secretion of wnt3 ligand by apoptotic interstitial cells ( chera et al . , 2009 ) . rnai knockdown of wnt3 prevents the proliferative burst induced by apoptosis , whereas treatment with exogenous wnt3 rescues proliferation when apoptosis is inhibited ( chera et al . , 2009 ) . the induction of apoptosis after injury appears to require the mitogen - activated protein kinase ( mapk ) pathway , including ribosomal s6 kinase ( rsk ) , and camp response element binding protein ( creb ) . rnai knockdown of rsk and creb blocks apoptosis at the head - regenerating tip , as does treatment with u0126 , a pharmacological inhibitor of mek , the kinase that phosphorylates mapk ( chera et al . , , the transcription factor creb is phosphorylated by rsk within minutes of amputation ( fig . intriguingly , mapk / creb - induced apoptosis is not required in fragments regenerating a foot , and cell proliferation is not up - regulated in foot regenerates . the exact nature of the signal that leads to asymmetric mapk activation ( and , thus , apoptosis ) at the site of injury in head - regenerating fragments remains to be determined . the apoptosis - induced compensatory proliferation ( fan and bergmann , 2008 ) observed in hydra may be a conserved mechanism for stimulating proper wound healing and regeneration . apoptotic cells have been observed during early phases of regeneration in several animals that can regenerate missing tissues : planarians ( fig . 1 b ) , xenopus ( fig . 1 c ) , and newts ( hwang et al . , 2004 ; vlaskalin et al . , 2004 ; tseng et al . , 2007 ; chera et al . , 2009 ; pellettieri et al . , although the requirement for apoptosis during regeneration has not been addressed in planarians and newts , treatment of xenopus larvae with caspase inhibitors during the first 24 h after amputation blocks tail regeneration ( tseng et al . as observed in hydra , inhibition of apoptosis impedes proliferation ; in addition , nerves fail to extend appropriately toward the site of amputation . because nerves release important regenerative signals ( see penultimate section ) , it is unclear whether reduced proliferation results directly from lack of signals derived from apoptotic cells , or indirectly from defective innervation . furthermore , because these assays rely on caspase inhibition it is possible that nonapoptotic roles for caspases ( kuranaga and miura , 2007 ) may be involved rather than apoptosis by itself . apoptotic cells have been shown to provide a number of signals that can regulate wound healing and regeneration . in drosophila , the larval imaginal discs are capable of regenerating : wing discs can produce appropriately sized adult wings after radiation - induced killing of over 50% of the cells ( haynie and bryant , 1977 ) . mapk signaling through jun kinase is important both for initiating apoptosis and production of wnt ( wg ) and bmp ( dpp ) mitogens ( prez - garijo et al . , 2009 ; bergantios et al . after radiation - induced apoptosis , neither dpp nor wg were required ( prez - garijo et al . , 2009 ) however , after ectopic expression of a pro - apoptotic gene , wg was required for the proliferative response ( smith - bolton et al . , 2009 ) . furthermore , wg is also required for disc regeneration after surgical transection ( schubiger et al . , 2010 ) . additional roles for apoptosis have been reported in epidermal wound healing and liver regeneration in mouse . caspase 3 and caspase 7 mutant mice have defects in both processes , and these mutants show reduced cell proliferation in these contexts ( li et al . , 2010 ) . caspases 3 and 7 can activate ca - independent phospholipase a2 , leading to production of arachidonic acid and prostaglandin in apoptotic cells , the latter of which can stimulate proliferation ( li et al . , 2010 ) . when cells of the adult drosophila midgut are injured by toxins or induced to undergo apoptosis , intestinal enterocytes secrete the cytokine unpaired , which stimulates proliferation of intestinal stem cells through activation of the jak / stat pathway ( jiang et al . similarly , in the mouse intestine massive induction of apoptosis ( via intestine - specific knockout of the p53 inhibitor mdm2 ) is eventually compensated in adults by increased proliferation and expansion of the stem cell pool ( valentin - vega et al . , 2008 ) . apoptotic cells also contribute to homeostasis in epithelia by lipid - based signaling ( sphingosine-1-phosphate ) that triggers actomyosin contraction in the surrounding cells , leading to the extrusion of the dying cells ( gu et al . , 2011 ) . these observations suggest many potential roles for dead and dying cells to alter cell behavior at sites of injury . in most regenerating organisms , replacing an amputated structure requires the production of new cells . therefore , one of the main functions of early signaling events after injury is to stimulate the production of additional cells that are capable of rebuilding lost structures . new cells coalesce near the site of injury , giving rise to a mass of undifferentiated cells called the regeneration blastema . subsequent signals then regulate outgrowth and patterning of the newly formed tissue . to understand how early signaling events initiate regeneration and stimulate blastema formation , it is crucial to identify the cells upon which these signals act . new cells can be generated in a variety of ways , including proliferation of a resident stem cell population , division of terminally differentiated cells , or dedifferentiation / transdifferentiation of mature cells to a stem cell like precursor or another cell type ( fig . 2 a ) . the extent to which each mode is used varies between species and even across tissues within the same species . cellular sources of regeneration . ( a ) the ability to regenerate amputated structures often requires the production of new cells . these new cells can be derived from amplification and differentiation of resident stem cells , proliferation of differentiated cells , dedifferentiation of cells to a more primitive state , or transdifferentiation of one cell type to another cell type . neoblasts are the only mitotic somatic cells and are defined by their high nuclear ( blue ) to cytoplasm ( green ) ratio and the presence of cytoplasmic ribonucleoprotein complexes called chromatoid bodies ( arrows ) . image courtesy of ana vieira ( university of illinois at urbana - champaign , urbana , il ) . ( c ) rescue of a lethally irradiated planarian by introduction of a single neoblast . s. mediterranea exists as two genetically distinct strains : an asexual strain ( brown ) that reproduces by transverse fission ; and a sexual , hermaphroditic strain ( gray ) that reproduces by cross - fertilization . wagner et al . ( 2011 ) have shown that lethally irradiated sexual animals can be rescued by injection of a single asexual neoblast . because the asexual donor neoblast is the only source of new cells , the sexual host is eventually converted into an asexual animal after repeated rounds of amputation , regeneration , and tissue turnover . these animals possess three distinct stem cell populations : ectodermal , interstitial , and endodermal stem cells ( galliot et al . , 2006 ) . development of transgenesis in hydra ( wittlieb et al . , 2006 ) has enabled in vivo tracking of the stem cell lineages : for example , investigating the differentiation of interstitial stem cells and migration of their progeny in intact animals ( khalturin et al . , 2007 ) . transgenesis has also aided the analysis of transdifferentiation . by expressing egfp in the zymogen gland cells ( a derivative of the interstitial stem cells ) , siebert et al . ( 2008 ) showed that these cells move up the body column and down - regulate the expression of a zymogen gland cell specific marker . by using histology , electron microscopy , and in situ hybridization with cell type specific markers , they identified cells with characteristics of both zymogen gland cells and another cell type ( granular mucous cells ) , suggesting that zymogen gland cells could convert to granular mucous cells as they were displaced up the body column and entered the head region ( siebert et al . , 2008 ) . the ability of hydra cells to transdifferentiate permits these animals to regenerate even in the absence of cell proliferation ( cummings and bode , 1984 ) . freshwater planarians ( another classical model for studying regeneration ) owe their amazing regenerative abilities to stem cells . in these animals , a population of mesenchymal stem cells , called neoblasts , is the source of cells for regeneration ( bagu et al . , 1989 ) . neoblasts are the only dividing somatic cells and have been defined by their high nuclear / cytoplasmic ratio and sensitivity to -radiation , as well as the expression of several post - transcriptional regulators and markers of proliferation ( fig . 2 b ; bagu et al . , 1989 ; newmark and snchez alvarado , 2000 ; orii et al . , 2005 ; reddien et al . , 2005 ; salvetti et al . , 2005 ; guo et al . , 2006 ; yoshida - kashikawa et al . , 2007 ; classical experiments showed that injection of neoblasts could restore regenerative abilities and long - term viability to lethally irradiated planarians ; furthermore , injection of neoblasts derived from a sexual planarian strain could transform lethally irradiated asexual individuals into sexuals ( bagu et al . , 1989 ) . because these injections used thousands of cells , neoblasts as a population however , whether individual neoblasts are pluripotent or if there are subsets of lineage - committed neoblasts has remained an open question . they injected single asexual donor neoblasts into lethally irradiated sexual hosts ( the irradiated sexual animals survive longer after irradiation than the asexuals do , permitting sufficient time for clonal expansion of the injected cells ) . single neoblast injections were able to restore viability to the sexual animals and convert them to an asexual mode of reproduction . restriction fragment length polymorphism and haplotype sequencing confirmed the genotypic conversion of the host to that of the donor strain ( fig . 2 c ; wagner et al . , 2011 ) . characterizing this pluripotent subset of the neoblast population will be an important avenue for future research . the source of regenerative cells in vertebrates varies between tissues and organisms , and in some cases remains a matter of continued debate . many vertebrate tissues contain adult stem cells that play important roles in tissue turnover and homeostasis . nonetheless , division , dedifferentiation , and transdifferentiation of differentiated cells contribute to regeneration in several different contexts . for example , whereas liver progenitor cells appear to be major sources of new hepatocytes under conditions of extreme damage or chronic disease , restoration of liver mass after partial hepatectomy or mild liver injury is largely accomplished through proliferation of remaining hepatocytes ( riehle et al . , 2011 ) . similarly , recent lineage - tracing experiments indicate that after damage to the zebrafish heart , existing cardiomyocytes undergo dedifferentiation and proliferate to generate new cardiomyocytes for replacing lost heart mass ( jopling et al . , 2010 ; kikuchi et al . , 2010 ) . pigmented epithelial cells in the newt dorsal iris can regenerate a new lens via transdifferentiation : these cells from the dorsal iris can dedifferentiate , reenter the cell cycle , and differentiate to produce new lens cells ( henry and tsonis , 2010 ) . dedifferentiation also contributes new cells during appendage regeneration in urodele amphibians ( newts and axolotls ) . near the site of amputation , syncytial skeletal myotubes fragment and produce mononucleate cells that reenter the cell cycle ( lo et al . skeletal muscle from adult newts also contains pax7-positive muscle stem cells ( satellite cells ) that become activated and contribute to new tissues during regeneration ( morrison et al . the respective contributions of muscle dedifferentiation and satellite cell activation during limb regeneration in the axolotl and newt remain open questions . in contrast , larval tail regeneration in xenopus laevis appears to be driven largely by progenitor cells ; in lineage - tracing experiments the extent of labeling of new muscle correlates with the amount of satellite cell labeling before amputation ( slack et al . lineage tracing experiments in both axolotl and xenopus indicate that multiple cell types contribute to formation of the blastema ( slack et al . , 2004 ; kragl et al . , however , it is still unclear whether these other tissues contribute cells through proliferation of progenitor cells or dedifferentiation of mature cells . such an understanding will be crucial for deciphering the mechanisms by which early regenerative signals trigger blastema formation . in addition to identifying the cellular sources of regeneration in different systems , researchers have been examining whether blastemal cells are pluripotent , multipotent , or have more limited potential . as discussed already , pluripotent neoblasts are the source of new cellular material that drives regeneration in planarians . however , cell proliferation is restricted largely to regions outside the blastema ( reddien and snchez alvarado , 2004 ; wenemoser and reddien , 2010 ) ; thus , post - mitotic neoblast progeny are the predominant cells within the blastema that rebuild lost tissues . ( 2008 ) have exploited the sensitivity of neoblasts to -radiation to identify numerous neoblast as well as early and late neoblast progeny markers . interestingly , not all early neoblast progeny express the same panel of markers ( fig . although this difference could reflect transient temporal alterations in gene expression in early neoblast progeny , it more likely indicates that these cells are in various early stages of lineage commitment and/or en route to different terminal cell types . this heterogeneity has also been suggested by gene expression profiling on individual neoblasts ( and their progeny ) isolated using fluorescence - activated cell sorting ( hayashi et al . , 2010 ) . a subset of radiation - sensitive cells with g2 dna content and expressing various neoblast markers also expressed a marker of muscle differentiation , suggesting that not all lineage - committed neoblasts are post - mitotic ( hayashi et al . , 2010 ) . consistent with this result , s phase or g2 neoblasts have been observed to express markers of differentiated cell types , including excretory and neuronal markers ( nishimura et al . , 2011 ; scimone et al . , ( a ) the planarian blastema is composed of a heterogeneous mixture of differentiating cells . the blastema is largely devoid of proliferating neoblasts , and instead is composed of cells expressing early and late neoblast progeny markers with distinct spatial distributions . although some cells coexpress early ( or late ) neoblast progeny markers , other cells show distinct expression profiles for various early neoblast progeny markers , suggesting early neoblast progeny may be en route to committing to terminal cell types ( adapted from eisenhoffer et al . , 2008 ) . ( b ) 3-d anterior blastemas ( top ) are capable of forming anterior structures , including photoreceptors and cephalic ganglia , after being surgically isolated from intact tissue and cultured in vitro . similarly , 3-d posterior blastemas ( bottom ) repigment and form muscle , but do not generate anterior structures , suggesting that by 3 d of regeneration head or tail specification has occurred . when 3-d anterior and posterior blastemas are juxtaposed ( middle ) they can generate a planarian containing mid - body structures ( including a pharynx ) , in addition to head and tail tissue . these results indicate that at least some of the post - mitotic neoblast progeny that compose the blastema are capable of altering their fates ( adapted from sengel , 1960 ) . in planarians , specification of blastema positional identity ( i.e. , as anterior or posterior ) likely occurs early during regeneration . sengel ( 1960 ) removed anterior blastemas after 3 d of regeneration and cultured them as explants in vitro ; these blastema explants produced only anterior tissues , including photoreceptors and cephalic ganglia ( fig . therefore , these early experiments suggested that specification of blastemas to produce heads or tails occurs during early stages of regeneration . this idea has been confirmed by recent molecular studies showing rapid up - regulation of polarity signals ( petersen and reddien , 2009 , 2011 ; gurley et al . , 2010 ) . surprisingly , when anterior blastemas were co - cultured together with posterior blastemas , they were now able to produce small planarians containing head , central body , and tail structures ( fig . these results suggest that , although neoblast progeny are specified early during regeneration , they may retain some developmental plasticity ; in response to altered position cues ( e.g. , juxtaposition of anterior and posterior tissues ) they may be able to change their fates and generate different cell types and tissues than they would have formed otherwise . alternatively , the blastema explants may have contained some neoblasts that responded to the juxtaposition of anterior and posterior tissues to generate distinct body structures . recent experiments using transgene - based lineage tracing suggest that vertebrate blastema cells can remember the tissue from which they are derived , and that their fates are largely restricted to forming similar tissues in the regenerate . for example , transgenesis in axolotl and xenopus has been combined with embryonic grafting to specifically label various tissues , including muscle , schwann cells , spinal cord , and dermis ; this tissue - specific labeling enables the contribution of various tissues to be analyzed during regeneration ( fig . 4 a ; gargioli and slack , 2004 ; kragl et al . , 2009 ) . these experiments revealed that regenerated muscle tissue is derived solely from muscles present in the limb before amputation . both of these tissues are derivatives of lateral plate mesoderm , suggesting that these tissues may dedifferentiate to produce progenitors restricted to lateral plate fates or that the dermis may contain an uncommitted stem cell population ( kragl et al . , 2009 ) . cellular memory during vertebrate limb regeneration . ( a ) although the regeneration blastema appears to be a homogeneous mass of undifferentiated cells , lineage - tracing experiments in the axolotl limb , the xenopus tail , and the zebrafish caudal fin indicate that blastema cells only contribute to tissues of similar developmental origin as that from which they are derived . although dermis ( gray ) can give rise to new skeletal elements , these tissues are both lateral plate mesoderm derivatives , suggesting only limited dedifferentiation . therefore , the blastema is composed of a heterogeneous mixture of lineage - restricted cells and is not a homogeneous population of multipotent cells . ( b ) blastema cells retain their proximo - distal identity : distal amputations produce blastemas that only regenerate distal structures , whereas more proximal amputations produce blastemas that regenerate medial and distal structures . when cells from a distal blastema ( green dots ) are transplanted into a proximal blastema , they contribute only to distal structures ; by contrast , cells from a proximal blastema ( green dots ) contribute to structures along the length of the proximo - distal axis . overexpression of the cell surface protein prod1 transforms distal blastema cells to more proximal fates . tu and johnson ( 2011 ) have used transposon - based clonal analysis to examine the potency of various cell lineages during development , growth , and regeneration ( tu and johnson , 2010 , 2011 ) . this method stably labels one to a few cells in the developing fin bud , enabling the progeny of single fin bud cells to be monitored . examination of hundreds of animals indicated that fin bud cells are greatly restricted in developmental potential , only generating one to a few cell types in the adult fin ( tu and johnson , 2011 ) . as in axolotl and xenopus , zebrafish caudal fin cells remain lineage committed during regeneration , and do not contribute to lineages other than that from which they are derived ( tu and johnson , 2011 ) . osteoblasts near the amputation site down - regulate osteoblast differentiation markers , lose their differentiated morphology , proliferate , and give rise to new bone in the regenerate ( knopf et al . , 2011 ) . these results reveal that regeneration in these contexts does not require dedifferentiation to a pluripotent state , and that the cells that make up the blastema are lineage restricted . these lineage - restricted progenitors even occupy distinct spatial domains in the blastema , indicating that the initial sorting and positioning of these cells may be crucial for producing a properly patterned appendage ( kragl et al . , although the lineage restrictions observed during limb regeneration ( kragl , et al . , 2009 ) are similar to those observed during limb development ( pearse et al . , 2007 ) , there are dramatic differences between development and regeneration . in addition to nerve dependence ( see next section ) and differences in scale of the structures being formed , limb regeneration differs significantly from limb development in that some components of the proximo - distal axis of the limb are retained after amputation . to prevent duplication or deletion of limb structures , cells at the site of amputation must be able to determine their position along the proximo - distal axis and use this information to regenerate only the structures that have been lost . interestingly , when distal ( wrist ) blastemas are transplanted onto proximal ( shoulder ) blastemas , the distal blastema cells contribute only to distal structures , suggesting that they retain memory of their proximo - distal origin ( fig . additionally , when proximal and distal blastemas are co - cultured , the proximal blastema encapsulates the distal blastema , suggesting that the adhesive properties of cells differ along the proximo - distal axis ( nardi and stocum , 1984 ; da silva et al . , lineage tracing and examination of proximal ( e.g. , nuclear localized meis ) and distal limb markers ( e.g. , hoxa13 expression ) indicate that although some cells ( such as cartilage ) retain positional identity , other cells ( such as schwann cells ) do not ( kragl et al . , because of the important role positional identity plays in limb regeneration , it is critical to understand the sources that provide positional values along the limb axes . based on its differential expression along the proximo - distal axis , da silva et al . ( 2002 ) identified a cell surface protein , referred to as prod1 , that is expressed at high levels proximally and low levels distally . treatment with anti - prod1 antibodies blocked the encapsulation of distal blastema by proximal blastema tissues , suggesting that prod1 plays a role in cell cell interactions that mediate identity along the proximo - distal axis . furthermore , when distal blastema cells overexpressing prod1 were transplanted into proximal blastemas , they contributed to proximal rather than distal structures ( echeverri and tanaka , 2005 ) . the mechanisms by which differences in prod1 expression along the proximo - distal axis translate into faithful regeneration of the limb are not completely clear ; however , prod1 has been found to interact with a secreted protein called newt anterior gradient ( nag ; see next section ) , providing a link between proximo - distal patterning and the role of innervation in regeneration ( kumar et al . , 2007a ) . the above , out - of - context quote ( rowling , 2003 ) introduces the important role that innervation plays during regeneration in many organisms . for example , earthworms in which a portion of the ventral nerve cord was removed near the amputation plane failed to regenerate new heads , whereas manipulations resulting in two anteriorly exposed nerve cords produced two heads ( morgan , 1901 ) . in another annelid , the polychaete spirographis spallanzani , deviation of the nerve cord toward the lateral body wall induced the formation of supernumerary structures : the anterior - directed cut nerve cord led to the formation of an ectopic head , whereas the posterior - directed cut nerve cord led to the production of an ectopic tail ( kiortsis and moraitou , 1965 ) . the nervous system also appears to be required for arm regeneration in echinoderms because removal of the radial nerve in the starfish prevents arm regeneration ( huet , 1975 ) . in planarians , the nervous system is required for proper regeneration of the gonads and accessory reproductive organs . ( 2010 ) identified a single neuropeptide that mediates the effects of the nervous system on the postembryonic development and maintenance of the reproductive organs . furthermore , the formation of ectopic cephalic outgrowths in planarians with improperly connected cephalic ganglia and ventral nerve cords suggests that discontinuities between the brain and nerve cords result in the production of signals that trigger proliferative outgrowth ( cebri and newmark , 2007 ) . classical experiments by singer showed that denervated limbs failed to regenerate , and that the amount of innervation was critical for proper regeneration ( carlson , 2007 ) . ( 2007b ) identified the nag protein as a ligand for prod1 , the cell surface protein involved in proximo - distal patterning . during regeneration nag is expressed in the schwann cells of the nerve sheath and , later , in gland cells of the wound epidermis . remarkably , introduction of a vector encoding nag into denervated limbs was able to rescue regeneration to the digit stage , thus restoring proximo - distal patterning . experiments in which nag was added to blastemal cells in culture revealed that nag exerts its effects upon regeneration by stimulating blastemal cell proliferation . the mechanism by which nag stimulates blastema cell proliferation remains an open question . however , when prod1 is expressed in cultured newt blastema cells , it leads to the activation of mmp9 expression and erk signaling ; it also interacts with the epidermal growth factor receptor ( blassberg et al . , 2011 ) . it will be important to determine whether nag plays a role in mediating these activities of prod1 . given the wide distribution of regenerative abilities throughout the animal kingdom , it has been postulated that some regenerative mechanisms have been conserved throughout evolution ( snchez alvarado and tsonis , 2006 ; brockes and kumar , 2008 ; bely , 2010 ; poss , 2010 ) . according to this view , however , prod1 provides an intriguing example of a molecule that has apparently evolved within a specific evolutionary lineage , the urodele amphibians ( garza - garcia et al . , 2010 ) . thus , it will also be important for investigators to consider the roles of such taxon - specific genes in regeneration in diverse organisms . ultimately , we need to know a great deal more about the cellular and molecular mechanisms operating during various regenerative processes to better understand the distribution of regenerative abilities throughout the metazoa . as discussed in this review , investigations of model organisms have begun to characterize the cellular sources of regeneration , define their potency , and identify molecules required for restorative events . thus , there is now a great opportunity for cell biological studies to link gene function to cellular behavior . for example , the cell biology of dedifferentiation is poorly understood : how is the complex cytoskeleton of cardiomyocytes or skeletal muscles reconfigured during the course of dedifferentiation to enable assembly of a mitotic spindle ? in addition , we need to understand how individual cell behaviors are integrated across tissues to permit a coordinated response to tissue loss . for example , the observation that tissues from proximal limb blastema encapsulate those from distal limb blastema suggests that cell cell interactions are critical for proper sorting and organization within the blastema ( nardi and stocum , 1984 ; da silva et al . , 2002 ) . similarly , it will be important to characterize how the extracellular environment is altered during regeneration , and how different extracellular matrices shape cell behaviors in the blastema ( calve et al . , 2010 ) . one of the long - term goals of regeneration research is to understand why humans have such limited regenerative potential and what , if anything , can be done to improve it . the knowledge gained from studying cell biological questions in model organisms should help drive such future efforts of regenerative medicine .

regeneration of complex structures after injury requires dramatic changes in cellular behavior . regenerating tissues initiate a program that includes diverse processes such as wound healing , cell death , dedifferentiation , and stem ( or progenitor ) cell proliferation ; furthermore , newly regenerated tissues must integrate polarity and positional identity cues with preexisting body structures . gene knockdown approaches and transgenesis - based lineage and functional analyses have been instrumental in deciphering various aspects of regenerative processes in diverse animal models for studying regeneration .

Regeneration: Sometimes truth is stranger than (science) fiction Back from the dead: Apoptosis and regeneration Conjuring up spare parts: Cellular sources of regeneration To thine own self be true: Cellular memory during regeneration Anythings possible if youve got enough nerve: Nerve dependence in regeneration Concluding thoughts

with the goal of understanding the factors that enable some organisms , but not others , to restore missing structures , biologists have been studying regeneration for well over two centuries ( morgan , 1901 ; lenhoff and lenhoff , 1986 ) . although the doctor s regenerative abilities remain a mystery , recent research on various earthlings has started to reveal the mechanisms leading to regeneration of complex structures . during the past decade the application of molecular genetic techniques , including double - stranded rna - mediated genetic interference ( rnai ) and transgenesis , has revitalized studies of classical animal models of regeneration ( poss , 2010 ) . after amputation , local responses at the site of the wound play important roles in the initiation of regenerative processes . ion flux ( levin , 2009 ) , and interactions between the wound epidermis and the underlying tissue appear critical for regenerative outgrowth . hydra , a freshwater polyp belonging to the phylum cnidaria ( which includes jellyfish , sea anemones , and corals ) , was the subject of the first scientific investigations of regeneration in animals ( lenhoff and lenhoff , 1986 ) . the animal is radially symmetric , and is polarized along the oral ( head and tentacles)/aboral ( foot ) axis ( fig . the induction of apoptosis after injury appears to require the mitogen - activated protein kinase ( mapk ) pathway , including ribosomal s6 kinase ( rsk ) , and camp response element binding protein ( creb ) . intriguingly , mapk / creb - induced apoptosis is not required in fragments regenerating a foot , and cell proliferation is not up - regulated in foot regenerates . furthermore , because these assays rely on caspase inhibition it is possible that nonapoptotic roles for caspases ( kuranaga and miura , 2007 ) may be involved rather than apoptosis by itself . apoptotic cells have been shown to provide a number of signals that can regulate wound healing and regeneration . additional roles for apoptosis have been reported in epidermal wound healing and liver regeneration in mouse . caspase 3 and caspase 7 mutant mice have defects in both processes , and these mutants show reduced cell proliferation in these contexts ( li et al . because the asexual donor neoblast is the only source of new cells , the sexual host is eventually converted into an asexual animal after repeated rounds of amputation , regeneration , and tissue turnover . the ability of hydra cells to transdifferentiate permits these animals to regenerate even in the absence of cell proliferation ( cummings and bode , 1984 ) . freshwater planarians ( another classical model for studying regeneration ) owe their amazing regenerative abilities to stem cells . , 2007 ; classical experiments showed that injection of neoblasts could restore regenerative abilities and long - term viability to lethally irradiated planarians ; furthermore , injection of neoblasts derived from a sexual planarian strain could transform lethally irradiated asexual individuals into sexuals ( bagu et al . the source of regenerative cells in vertebrates varies between tissues and organisms , and in some cases remains a matter of continued debate . nonetheless , division , dedifferentiation , and transdifferentiation of differentiated cells contribute to regeneration in several different contexts . however , cell proliferation is restricted largely to regions outside the blastema ( reddien and snchez alvarado , 2004 ; wenemoser and reddien , 2010 ) ; thus , post - mitotic neoblast progeny are the predominant cells within the blastema that rebuild lost tissues . recent experiments using transgene - based lineage tracing suggest that vertebrate blastema cells can remember the tissue from which they are derived , and that their fates are largely restricted to forming similar tissues in the regenerate . tu and johnson ( 2011 ) have used transposon - based clonal analysis to examine the potency of various cell lineages during development , growth , and regeneration ( tu and johnson , 2010 , 2011 ) . , hoxa13 expression ) indicate that although some cells ( such as cartilage ) retain positional identity , other cells ( such as schwann cells ) do not ( kragl et al . classical experiments by singer showed that denervated limbs failed to regenerate , and that the amount of innervation was critical for proper regeneration ( carlson , 2007 ) . experiments in which nag was added to blastemal cells in culture revealed that nag exerts its effects upon regeneration by stimulating blastemal cell proliferation . given the wide distribution of regenerative abilities throughout the animal kingdom , it has been postulated that some regenerative mechanisms have been conserved throughout evolution ( snchez alvarado and tsonis , 2006 ; brockes and kumar , 2008 ; bely , 2010 ; poss , 2010 ) . thus , it will also be important for investigators to consider the roles of such taxon - specific genes in regeneration in diverse organisms . ultimately , we need to know a great deal more about the cellular and molecular mechanisms operating during various regenerative processes to better understand the distribution of regenerative abilities throughout the metazoa .

the amazon basin has a drainage area of more than six million square kilometers , producing an average flow of around two hundred thousand cubic meters per second , ranging from about one hundred thousand cubic meters per second in the dry season and about three hundred thousand cubic meters per second in times of flood . the average flow rate corresponds to about one - fifth of all the fresh water that reaches the oceans coming from the continents . the river , stretching over more than six thousand kilometers from the foot of the andes to its mouth in the atlantic ocean , can be several hundred meters wide and can even reach a hundred feet deep . the city of manaus ( http://goo.gl/maps/ynyji ) is located around 1500 km from the mouth and is only sixty feet above sea level , leading to a slope that varies from thence to the mouth , between one and two inches per mile , between droughts and floods . the negro and solimes rivers meet in front of the city of manaus ( approximately 3s , 60w ( http://goo.gl/maps/kblyf ) ) , which is the capital of the brazilian state of amazonas ( http://goo.gl/maps/64k1o ) , to form the amazon river . the phenomenon of the separation of the waters of the two rivers is well known and occurs for several kilometers along the amazon river bed and such separation is attributed to several factors . for a long time , it was thought that the two rivers ran side by side , but the french brazilian research program hydrology and geochemistry of the amazon basin showed that the waters of the river solimes , which is most copious , move beside and below the waters of the river negro , until fully mixed . mention that a partial mixing of the waters of the two rivers is already evident from about twelve kilometers further from the initial meeting , but complete mixing occurs only about a hundred kilometers beyond the meeting point of two rivers . a quick visit to the nasa website or google maps allows a preview of the meeting of the waters ( http://www.nasa.gov/multimedia/imagegallery/image_feature_577.html ) and navigation on google maps allows the observation that the interface between the waters of the two rivers persists until itacoatiara ( http://goo.gl/maps/el1cp ) , about 150 kilometers down along the amazon river . figure 1 shows the region of the meeting of the waters , as it is known . the satellite image shows the city of manaus and the junction of the dark waters of the negro river , coming from the northwest , and the muddy waters of the solimes river , coming from the southwest , to form the amazon river , flowing eastward . laraque presents a very interesting comment , which reports that local fishermen utilize the meeting of the waters and the clear interface between the black waters of the negro river and the muddy waters of the solimes river to create a trap by placing the fish between the interface of the waters of the two rivers and their fishing nets . the project mentioned above revealed that the waters of the negro and solimes rivers have different speeds ( respectively , 0.3 m / s and 1.0 m / s ) , different conductivities ( respectively , 8 s / cm and 80 s / cm at 25c ) , different turbidity values ( respectively , 5 ntu and 80 ntu ) , different ph values ( respectively , 5.5 and 7.0 ) , and different temperatures , indicating a difference of 1c . provide an extensive study on the mixing of the waters of the two rivers in the region of their meeting , presenting measurements of concentration of various elements in various sections throughout the meeting of the waters , without conducting , however , temperature measurements . the authors report that complete mixing is achieved only about 25 km downstream from the meeting point and massive amounts of ca , mg , si , fe , and al present in particulate materials are lost without lateral contributions of these elements by groundwater or floodplain ponds . in the dissolved fraction the work also indicates a large loss of dissolved organic carbon in the mixture of waters of the two rivers . present vertical profiles in cross sections along the test region of the meeting of the waters , which together with the data provided by silva and pecly and data obtained from the database hydroweb allow knowing quite well the shape of the cross sections of the rivers negro and solimes and amazonas and the vertical distribution of speeds in the region of the meeting of the waters . the waters of the negro river , as indicated by its name , are darker and warmer , usually treated in the literature as black water , while the waters of the solimes river are more muddy and cooler , treated as clear water or white water . this difference appears even as a difference in the movement of air masses [ 6 , 7 ] . several national websites ( http://www.descobrindooamazonas.webs.com/encontrodasguas.htm ) ( http://www.portalamazonia.com.br/secao/amazoniadeaz/interna.php?id=242 ) related to tourism and natural heritage , approaching the meeting of the waters , mention temperatures of 22c to 24c for negro and 28c to 30c for solimes , a temperature difference of 4c to 8c . also , they mention speed of 2 km / h for the waters of the negro river and 4 to 6 km / h for the waters of the solimes river . the temperature difference between the waters of the two rivers can be harnessed to produce electricity through a system similar to that used in geothermal plants or plants that harness the thermal gradient of the ocean waters . a technological breakthrough to generate energy from small temperature gradients a project of the 70s prepared by the national institute for amazonian research ( inpa ) and the state university of campinas ( unicamp ) to obtain funds from the financier of studies and projects ( finep ) to quantify the difference in temperature between the rivers negro and solimes foresaw the development of temperature meters in depth and pointed out a temperature difference of 7c . the project was not funded by finep apparently because it was not associated with the development of a strategy for the transport of energy generated outside the region , for the major consuming centers of the country . this project presented an initial estimate of maximum available power of about 15 gw and a specific cost estimate of us$ 0.28/mw installed . the project compares this estimate with the cost of ocean thermal plants , from us$ 0.63/mw installed , and assigns the difference of these two values to greater difficulties associated with this type of use . the otec plants are subject to storms and corrosion and are installed on floating vessels and demand a reasonable amount of energy for pumping water from great depths . the author further argues that the availability of this power plant would allow the use of large - scale deposits of aluminum in the amazon region as well as the production of ammonia , nitrogen fertilizers , and hydrogen . brazil has faced problems over the past three decades in the energy sector , first with the oil crisis and then with the lack of installed power and energy stored in reservoirs to meet economic growth evidenced in the 90s . the difficulties have always encouraged projects of renewable resources and currently have increased the participation of wind farms and even of photovoltaic systems in the energy matrix . however , this energy resource in the amazon was not considered even in texts of nongovernmental organizations . this paper aims to present this energy resource for discussion and present a preliminary evaluation of the potential available for power generation from this untapped resource . it is clear that the temperature gradient between the rivers negro and solimes is significantly lower than the gradients available in geothermal and ocean thermal plants , but it is possible to provide reasonable power since the flows available for use are fairly high . this paper is a very preliminary study , based on theoretical considerations and without any concerns related to technological issues , intending only to present this previously unknown energetic potential . the newness of the subject justified the inclusion of the term river thermal energy in the keywords of this paper . the thermal gradient between the waters of the negro and solimes rivers can be harnessed to generate energy with a thermodynamic cycle similar to that used in thermal power plants . the difference appears in the intensity of this thermal gradient , which is very small , of the order of 5c to 8c . plants that use naturally available thermal gradients , as in geothermal and ocean thermal power plants , generate energy from thermal gradients with a few tens of degrees celsius . several authors [ 10 , 11 ] say that it is not economically feasible to generate power thermal gradients less than 15c . in fact , power generation from thermal gradients is so limited and the use of high flow rates will require research work for the selection of a suitable working fluid and the development of heat exchangers and the design of a plant that is appropriate to prevailing environmental concerns . figure 2 shows schematically the river thermal power plant placed near the meeting of the waters . the warm source is maintained by the waters of the negro and the cold source is maintained with the waters of the solimes river . the figure shows the water being returned to its rivers , after passing through the heat exchanger , but this issue is discussed later . the mechanical power pmec ( w ) that can be extracted in the system is given by ( 1 ) , where ( kg / m ) is the specific mass of water , c ( j / kgk ) is the specific heat capacity at constant pressure , q ( m / s ) is the flow rate , tw ( k ) is the temperature of the warm source , tc ( k ) is the temperature of the cold source , and is the efficiency . consider ( 1)pmec=cqtwtc. this power is ideal and actual machines operate closer to a rankine cycle . the performance in a carnot cycle is given by ( 2 ) , but this limit would be obtained only in a conservative system . a more realistic measure of efficiency can be obtained with ( 3 ) , which was obtained considering that the heat transfer processes are irreversible . the exploitation of a gradient of 6c between 32c and 26c , for example , would result in an efficiency of 0.998% and 8c gradient between 33c and 25c would result in an efficiency of 1.333% . very low efficiency values could be realized only under the conditions found in the amazon , with large flows . assuming smaller temperature gradients throughout most of the year , 3c between 30c and 27c would result in an efficiency of 0.499% and 3c gradient between 23c and 20c would result in an efficiency of 0.511% . a temperature gradient of 2c between 30c and 28c would result in 0.332% and between 22c and 20c would result in 0.341% . the hypothetical situation of the use of 1,000 m / s , with a temperature gradient of 6c , would lead to a theoretical maximum of about 240 mw of mechanical power available for conversion to electricity . as shown in the next chapter , the negro river flow varies between 30,000 m / s and 10,000 m / s , much higher than the value considered . the pumping of 1.000 m / s of water over 1,000 m with two pipes with diameters of 1 m would result in a power consumption of about 10 kw . the total energy consumption for pumping , considering warm water , cold water , and working fluid , would not exceed 2% of the power of 500 mw , available with this flow . the design problem of the thermodynamic cycle [ 1214 ] , operating with a thermal gradient so small [ 1518 ] , the working fluid selection [ 1921 ] , design of heat exchangers , and the location of the system components are very similar to those found in geothermal [ 22 , 23 ] and ocean thermal energy conversion systems [ 24 , 25 ] . the evaluation of the available potential requires knowledge of river flows and water temperatures of rivers and their variations over a year . the hydrological behavior in the region of the meeting of the waters can be characterized with data from three fluviometric stations , available online . one of the stations , jatuarana ( station jatuarana , code 15030000 , amazonas river , manaus , state of amazonas , responsibility of national agency of waters , with drainage area of 2.854.286 km ) , is located in the amazon river , downstream from the city of manaus and the junction of the rivers . the other two stations are located one on the negro river , serrinha ( station serrinha , code 14420000 , negro river , santa isabel do rio negro , state of amazonas , responsibility of national agency of waters , with drainage area of 279.945 km ) , and the other on the solimes , manacapuru ( station manacapuru , code 14100000 , solimes river , manaus , state of amazonas , responsibility of national agency of waters , with drainage area of 2.147.736 km ) . figure 3 shows the location of these stations . according to cappelaere et al . and guyot et al . , the water level in the city of manaus reaches its maximum annual value between may and july and lasts for several weeks around this maximum value manaus is situated immediately on the backwater of the meeting of the waters and waterline depends much more on the flow of the amazon river ( the sum of the flows of the two rivers ) than on the flow of the negro river . the waterline can also be influenced by the flow rate of the madeira river , whose mouth in the amazon river is less than 200 km downstream . looking at the data of these three stations , floods occur between may and august and the dry season extends from approximately the months of october and february . figure 4 shows the minimum , average , and maximum historical levels in manacapuru , serrinha , and jatuarana , corresponding , respectively , to series from january 1973 to december 2011 , january 1978 to december 2005 , and january 1978 to december 2005 . in solimes , there was variation between the maximum and minimum values of the average quota of 0,865 m ; at the station in negro river , there was a variation of 0.365 m , while at the amazon river there was a variation of 0.829 m. figure 5 shows the discharge data , for series from january 1974 to december 1985 , january 1978 to december 2012 , and january 2006 to december 2011 . in solimes , the maximum discharge was 137.462 m / s , in june , and the minimum was 68.218 m / s in november . in negro , the maximum discharge was 26.108 m / s in june , and the minimum was 10.883 m / s in february . in the amazon river , the highest discharge was 165.159 m / s in june , and the minimum was 83.549 m / s in november . it is also observed that the maximum values of monthly mean discharges were 160.444 m / s , 32.028 m / s , and 193.124 m / s , respectively , for stations in the solimes river , the negro river , and the amazon river , and the minimum values were 54.124 m / s , 3.599 m / s , and 56.303 m / s , respectively , for the three stations . the cross section of the solimes river in manacapuru has an average width of 3,300 m and depths in the central portion that can reach 40 m. in the negro river in serrinha , the average width is 1900 m and depths can reach about 20 m. the cross section of the amazon river in jatuarana has an average width of 2800 m and depths in the central portion that can reach 60 m. figure 7 shows average speeds estimates for the three stations . the estimated average speeds along the flow section varied between 0.90 m / s and 1.90 m / s , between 0.32 m / s and 1.30 m / s , and between 0.50 m / s and 1.85 m / s at stations , respectively , of the solimes river , the negro river , and the amazon river . the hydrological characteristics of the region of the meeting of the waters must be evaluated in order to determine the real energetic potential available for this type of energy resource , in order to know the available energy resource and to determine the best location for the power plant . the behavior of the temperature in the region of the meeting of the waters can be determined from the temperature data at five sites in the region , obtained from the work of fonseca . figure 8 shows monthly average values of temperature for three stations at negro river while figure 9 shows values for two stations at solimes river . in figure 9 , there are monthly average temperatures for the surface , to 1 meter and 2 meters deep . in figure 10 , there are monthly average temperatures for the surface to 1 meter to 2 meters deep and there are still temperature values obtained at the bottom of the solimes river . the stations on the negro river are lago cristalino , serraria , and janauary . the stations on the solimes river are castanho and so sebastio . none of these measurement points are located near to places of interest to the water intake for the generating system and these points are located away from the main flow of the two rivers , in sites with low speed water displacement . these measurements were not made with the intention of evaluating the energetic potential and were inserted into a project that had other goals . however , these temperature values allow a better understanding of the behaviour of monthly average temperatures over a year . in fact , the temperatures in these graphs do not differ substantially from each other . however , it can be seen that the temperatures obtained from the solimes river are lower and that the temperatures obtained in the bottom present reasonable thermal gradients in relation to the observed temperatures on the surface of negro river . the maintenance of the cold source should be done with taking water deep into the solimes river , approximately as it is done with the cold source in ocean thermal power plants . there are reports of people of the region about chunks of ice floating in the waters of the solimes river in some periods of the year , coming obviously of the highest points of the basin , located in the andes . the behavior of the thermal gradient between the waters of the two rivers over a year needs to be accurately determined on a project focused on power generation . a comprehensive study of the flow and thermal gradient available is needed to accurately provide the available energetic potential and provide information for the leasing of a river power plant and establish appropriate locations for water intake to keep the heat exchangers . the negro river has smaller flow rate than the solimes river and can be considered as a reference in the following remarks . clearly , equivalent amounts of water of the solimes river are also necessary . considering a hypothetical situation in which 100% of the flow of the negro river was harnessed for power generation , an average of 19,919.2 m / s , a total of 47,306.03 gwh per year , with a maximum of almost 20 gw and an average of 9,79 gw could be made available . figure 10 shows the mechanical power that can be provided depending on the utilized portion of the negro . these results already take into account the power consumption for pumping . considering the use of 20% of the flow of the negro river for power generation , equivalent to an average of 3,984 m / s , a total of 9,461 gwh per year , with a maximum of 1.96 gw and an average of 1.08 gw , could be made available . similarly , considering the use of 10% of the flow of the negro river for power generation , equivalent to an average of 1,992 m / s , a total of 541 gwh per year , with a maximum of 0.98 gw and an average of 0.54 gw , could be made available . note that these values would be available for conversion into mechanical energy and then into electricity . the performances of converting equipment were not considered in these projections because they are considered outside the scope of this work . the use of larger portions of the flow rate of the negro river will certainly involve larger hydraulic structures and potentially devastating environmental impacts . but the use of portions of the flow rate of the negro of approximately 10% to 20% seems much more reasonable while still providing significant amounts of power . the power currently installed in brazil is 124.33 gw and the addition of 0.54 gw to 1.08 gw , as discussed above , in a region that is not connected to the brazilian energy system would be a key support for the development of the region and certainly the ultimate justification for their interconnection . the meeting of the waters was listed ( http://portal.iphan.gov.br/portal/montardetalheconteudo.do?id=16243&sigla=noticia&retorno=detalhenoticia ) by the brazilian institute of national historical and artistic heritage ( http://portal.iphan.gov.br/ ) ( iphan ) in 2010 and there is a request to be listed as heritage of humanity by unesco . this tipping has serious implications on the possibility of power generation from thermal gradient between the two rivers . the size of the available energetic potential and the need for energy supplies can transform this energy resource into a strategic asset . that would be the only way to reverse this tipping , at least partially , for its use for power generation . in the case of the use of this energy resource to become a reality , the most obvious is that the amount of energy generated will establish the proportion that the phenomenon of the meeting of the waters will be dissipated . the use of 10% to 20% of the flow rate of the negro , as discussed above , also will play a far - reaching impact on the natural phenomenon . as a result , it may be important to properly choose a location for return of waters used in heat exchangers . this water may even be returned few miles downstream to reduce the impact on tourism . this review paper presented an energy resource that has not been discussed or considered as an alternative , present in the temperature gradient of the waters of the negro and solimes rivers , where they join to form the amazon river . the thermal gradient is very small , of approximately 5c to 8c , allowing the generation of energy just because of the high flow rates available in the amazon region . it is estimated that the use of 20% of the waters of the negro river , and equivalent amount of water of the solimes river , provides about annual average of 1.08 gw of gross energy . this amount of energy available in the amazon has fundamental role in the development of the region and would be definitive for interconnection to the brazilian energy system . concerns that are beyond the scope of this paper only intended to present this energy resource . environmental concerns such as the relationship between the energy and the extracted portion of the phenomenon of the meeting of the waters should be preserved and any other related to undertaking of power generation . recently , the meeting of the waters was listed as national natural heritage and this prevents the generation of energy from this resource unless its use assumes a strategic character that transcends heritage preservation .

the negro and solimoes rivers join in front of the brazilian city of manaus to form the amazon river . this meeting of the waters is a natural phenomenon of great aesthetic beauty that has been the focus of attention of researchers all over the world in various scientific fields . the waters of the negro are darker and warmer , while the waters of the solimoes are lighter and cooler . these waters have very different characteristics and remain without mixing , flowing side by side for several miles . some reports indicate a temperature gradient between the waters of the order of 6c , which can be used in conjunction with very high flow rates delivered by the two rivers , with a heat engine operating on a thermodynamic cycle to provide electricity . this review paper identifies this energy resource and presents a preliminary assessment of the potential for power generation . a realistic assessment of the potential points to an available power of about 1 gw . it is clear that further studies are needed to accurately assess the available thermal gradient and its variation over time , to move forward in the design of the power converter , and to establish an appropriate location for a power plant .

1. Introduction 2. Energy in a Small Thermal Gradient 3. Characterization of the Meeting of the Rivers Negro and Solimes 4. Energetic Potential 5. Concerns about Environmental Issues 6. Final Remarks

the negro and solimes rivers meet in front of the city of manaus ( approximately 3s , 60w ( http://goo.gl/maps/kblyf ) ) , which is the capital of the brazilian state of amazonas ( http://goo.gl/maps/64k1o ) , to form the amazon river . the phenomenon of the separation of the waters of the two rivers is well known and occurs for several kilometers along the amazon river bed and such separation is attributed to several factors . for a long time , it was thought that the two rivers ran side by side , but the french brazilian research program hydrology and geochemistry of the amazon basin showed that the waters of the river solimes , which is most copious , move beside and below the waters of the river negro , until fully mixed . a quick visit to the nasa website or google maps allows a preview of the meeting of the waters ( http://www.nasa.gov/multimedia/imagegallery/image_feature_577.html ) and navigation on google maps allows the observation that the interface between the waters of the two rivers persists until itacoatiara ( http://goo.gl/maps/el1cp ) , about 150 kilometers down along the amazon river . the satellite image shows the city of manaus and the junction of the dark waters of the negro river , coming from the northwest , and the muddy waters of the solimes river , coming from the southwest , to form the amazon river , flowing eastward . laraque presents a very interesting comment , which reports that local fishermen utilize the meeting of the waters and the clear interface between the black waters of the negro river and the muddy waters of the solimes river to create a trap by placing the fish between the interface of the waters of the two rivers and their fishing nets . provide an extensive study on the mixing of the waters of the two rivers in the region of their meeting , presenting measurements of concentration of various elements in various sections throughout the meeting of the waters , without conducting , however , temperature measurements . the waters of the negro river , as indicated by its name , are darker and warmer , usually treated in the literature as black water , while the waters of the solimes river are more muddy and cooler , treated as clear water or white water . the temperature difference between the waters of the two rivers can be harnessed to produce electricity through a system similar to that used in geothermal plants or plants that harness the thermal gradient of the ocean waters . it is clear that the temperature gradient between the rivers negro and solimes is significantly lower than the gradients available in geothermal and ocean thermal plants , but it is possible to provide reasonable power since the flows available for use are fairly high . the thermal gradient between the waters of the negro and solimes rivers can be harnessed to generate energy with a thermodynamic cycle similar to that used in thermal power plants . the hypothetical situation of the use of 1,000 m / s , with a temperature gradient of 6c , would lead to a theoretical maximum of about 240 mw of mechanical power available for conversion to electricity . the design problem of the thermodynamic cycle [ 1214 ] , operating with a thermal gradient so small [ 1518 ] , the working fluid selection [ 1921 ] , design of heat exchangers , and the location of the system components are very similar to those found in geothermal [ 22 , 23 ] and ocean thermal energy conversion systems [ 24 , 25 ] . one of the stations , jatuarana ( station jatuarana , code 15030000 , amazonas river , manaus , state of amazonas , responsibility of national agency of waters , with drainage area of 2.854.286 km ) , is located in the amazon river , downstream from the city of manaus and the junction of the rivers . , the water level in the city of manaus reaches its maximum annual value between may and july and lasts for several weeks around this maximum value manaus is situated immediately on the backwater of the meeting of the waters and waterline depends much more on the flow of the amazon river ( the sum of the flows of the two rivers ) than on the flow of the negro river . the hydrological characteristics of the region of the meeting of the waters must be evaluated in order to determine the real energetic potential available for this type of energy resource , in order to know the available energy resource and to determine the best location for the power plant . the behavior of the thermal gradient between the waters of the two rivers over a year needs to be accurately determined on a project focused on power generation . similarly , considering the use of 10% of the flow of the negro river for power generation , equivalent to an average of 1,992 m / s , a total of 541 gwh per year , with a maximum of 0.98 gw and an average of 0.54 gw , could be made available . this review paper presented an energy resource that has not been discussed or considered as an alternative , present in the temperature gradient of the waters of the negro and solimes rivers , where they join to form the amazon river .

to appreciate means of communication unique to humans , such as music , speech , or dance , the perceptual system needs to keep track of the dynamic information unfolding over time . beyond simple interval timing , current understanding of more complex temporal processes , such as rhythm and beat perception , is mainly derived from findings of music and speech in the auditory domain . this , however , overlooks the fact that amongst the abundant visually available information , human movements ( e.g. , walking ) are also often rhythmic , for which there is little knowledge how their temporal structure is visually perceived . in this study , we investigated timing mechanisms employed in visual perception of dance movements , a class of movements most immediately linked to musical rhythms . we aimed to establish whether mechanisms adopted for processing auditory rhythms would be similarly found for ecological visual stimuli . timing especially in the range of hundreds of milliseconds forms the basis for rhythm perception . in this range , purely perceptual timing without requiring a motor task implicates cortical motor systems , supporting the idea that sensory and motor timing share common mechanisms within the time scale that is relevant for movement execution [ 8 , 9 ] . in the same range , two modes of auditory timing have been distinguished , each subserved by a different motor circuitry that may work as a unified system : the duration - based mechanism , which times the absolute interval duration in a sequence without a perceivable beat , and the beat - based mechanism , which relies on a perceived beat in a sequence as reference for timing an interval . rhythm perception entails tracking the underlying periodicity , such as a beat or pulse , in a temporal pattern of ( often auditory ) events . the audio - motor link has been shown externally as body movements assisting pulse extraction and event timing in auditory rhythms . internally , beat perception implicates motor areas of the brain and is modulated by their connection to the auditory area . while beat - based mechanism is not superior to duration - based in timing a single auditory interval , the presence of a beat facilitates perception of an auditory rhythm ( consisting of successive intervals ) as a whole [ 13 , 14 ] : for example , the patterns of auditory rhythms with a perceivable beat can be more accurately reproduced or recalled than those without a clear beat . one explanation of the beat advantage is that beat - based rhythms effectively couple humans ' internal motor system , which in turn enhances rhythm perception . most studies in timing and rhythm perception converge to show auditory superiority compared to its visual counterpart , which may be attributed to a stronger link to the motor system in the former . however , recent findings point to possible visual rhythm and beat perception in moving stimuli [ 15 , 17 ] , particularly for periodic movements of a biological motion profile [ 18 , 19 ] . the significance of biological motion in timing is also supported by the literature that there seems to be a specialized timing mode for movements of biological kinematics compared to nonbiological ones [ 20 , 21 ] . furthermore , human movement kinematics facilitates temporal prediction of an action , compared to motions of artificial , linear velocity [ 22 , 23 ] , which is consistent with the internal motor simulation account during movement observation , as well as embodied theories of temporal processing . as such , questions arise as to whether the sensorimotor coupling underlying rhythmic timing can be strengthened by visual observation of temporally structured biological motion and whether this leads to visual timing behaviors similar to those found for auditory rhythms . one type of human movements , dance , provides suitable visual stimuli for addressing this issue , as dance is often performed in time with musical rhythms and may thus communicate visual spatiotemporal rhythms by observation . moreover , as dance entails whole - body movements , dance observation may activate internal motor representations more effectively than simple , artificial moving stimuli [ 15 , 17 ] , which can be useful for visual timing purposes . we designed the present study as a novel investigation of visual timing mechanisms during observation of realistic dance movements , focusing on possible beat - based advantages in this process . as periodic biological motion ( whole - body bouncing ) has been proposed to serve a visual beat [ 18 , 19 ] , we extended the idea to various movements in three experiments , using a duration reproduction task . this task was chosen for the reason that it has not been established whether and how rhythms are visually perceived when observing realistic human movements . as opposed to various perceptual tasks typically employed to measure auditory rhythmic timing , no visual paradigm involving complex movements we thus probed visual temporal perception of a movement sequence in which the feature in question , a potential beat , was embedded or not . the perceived sequence duration and how well it was encoded would likely reflect the movement information during the sequence . we hypothesized that when observing movements involving one or more body parts , periodic limb trajectories , such as recurrent hand clapping or foot tapping , would serve a visual beat . we expected such a visual beat to afford a beat - based mechanism that would benefit visual timing of the whole movement sequence . participants watched short silent videos of a dancer moving with the arms or with the legs . the movements consisted of periodic trajectories ( clapping or stepping ) , continuous and nonperiodic trajectories ( circular movements ) , or a mixture of both interspersed . we expected that movements with periodic trajectories would be better timed than those without . in experiment 2 , we presented movements performed by both the arms and the legs , each of which could contain periodic trajectories or not . we examined whether the arms , the legs , or both yielded the salient beat in visual timing of whole - body movements . in experiment 3 , we verified whether the beat advantage in visual timing was attributed to auditory imagery of the impact sounds , by presenting auditory interferences during the same visual task . if the beat advantage persisted , it would argue for visual beat - based timing that is not transformed into auditory representations . we examined whether the arm or the leg movements with periodic trajectories were better timed visually than those without and whether the effect varied across different tempi . for the purpose of cross - modal comparison , a similar auditory timing task was also included , in which an auditory sequence could either contain a beat or not . we expected similar patterns of results for the visual and the auditory tasks : namely , better temporal perception for sequences with a beat than those without , within each modality . twenty - two healthy volunteers ( eleven male , mean age 27 years , sd = 4 ) took part in this experiment . participants in all the experiments in this study were nave of the purpose , gave written informed consent prior to the experiment , and received an honorarium of 8 per hour for their participation . participants were not prescreened for musical or dance training , and the training duration ranged from zero to fifteen years for music and zero to six years for dance . eight participants had received music training ( all amateurs ) , and the learned instruments included piano / keyboard ( 4 ) , guitar ( 3 ) , and flute ( 1 ) . the study had been approved by the ethic commission of technical university of munich and was conducted in accordance with the ethical standards of the 1964 declaration of helsinki . the visual stimuli consisted of videos of six kinds of movement sequences derived from the flamenco dance repertoire . each movement sequence was performed in five different tempi , yielding thirty different videos . the movements were chosen based on the moving body part , the body positioning in space , and the direction of the body motion , following criteria similar to those employed by calvo - merino et al . . the rationale of employing specific flamenco movements was that ( 1 ) the chosen movements were not too complex for nondancers to imagine performing , thus minimizing potential effects of different sensorimotor experiences across expertise , and ( 2 ) , at the same time , the specific postures and kinematics performed by a trained dancer , preserving the characteristic movement accent ( in flamenco term ) , distinguished these movements from everyday actions , thus maintaining the ecological plausibility of a dance scenario . we defined a movement sequence here as a set of movements that lasted a certain duration from the start to the end posture . to generate the stimuli , we first choreographed each movement sequence with the knowledge of the second author , who holds a degree in flamenco dance . the sequences were choreographed based on the planned experimental variables , while keeping the movements as characteristic of flamenco as possible . we then recorded a professional flamenco dancer ( 15 years of training ) performing each of the six movement sequences paced by metronomes of five different tempi , corresponding to an interbeat interval ( ibi ) of 375 ms , 425 ms , 500 ms , 575 ms , and 625 ms ( i.e. , 500 ms 0 , 15% , and 25% ) . each complete movement sequence corresponded temporally to eight ibis at the respective tempo , equaling eight beats in a 4/4 musical meter . the dancer practiced each movement sequence until she could perform it fluently to all the metronome tempi . for each movement at each tempo , we recorded the dancer performing at least four cycles of the sequence continuously , one of which was selected later as the visual stimulus . the recordings were made with a camcorder ( panasonic hc - v500 ) at 25 frames per second in a dance studio against a white background . a spatial reference of 2.5 m 1.75 m was marked , creating a 6.5 m space in which every sequence was performed . the videos were later edited on a frame basis using the software imovie ( apple , inc . ) . for each movement sequence , we defined a starting posture and an end posture in the video as encompassing a complete movement cycle . we then selected one cycle ( corresponding to the eight - beat count ) at each tempo that yielded the highest consistency of start and end postures with the same movement at the other tempi , as well as the best match to the duration of eight ibis . given the natural variability in real human movements and the fact that the kinematics of the same movement varied slightly when performed in different tempi ( or speed ) , for each sequence we allowed five additional frames ( i.e. , 200 ms ) to the intended eight - beat duration to ensure that each selected movement cycle could be fully and consistently presented across all the tempi . the total duration of a sequence at each tempo , shown as video , was thus 3200 ms , 3600 ms , 4200 ms , 4800 ms , and 5200 ms , respectively . each sequence was exported as an .m4v file for playback in matlab ( 2012b ) . the movements varied according to two variables of interest : the limbs used to perform the movement ( arms or legs ) and the type of movement ( with or without periodic trajectories , or a mixture of both ) . in all the movements the dancer faced the front . in the arms - only movements , the dancer 's legs stood still with the feet separated by around 30 cm ( basic flamenco posture ) . in the legs - only movements , the dancer placed her hands on each side of the hips so that the arms did not move . regarding the movement type , movements containing periodic limb trajectories for labeling purpose , we termed this movement type discrete to reflect the brief moments of discrete contact . we termed movements that did not contain such recurrent contact points as continuous , as the limbs moved continuously in a circular manner . it should be noted that the dancer performed all types of movement paced by the metronome ; while it is more self - evident that discrete movement trajectories could be temporally segmented by the metronome beats , the dancer applied the same principle of segmentation in continuous movements , such that the limbs reached a defined body position at each given beat , regardless of movement tempo . the critical difference between these two movement types thus lied in the recurrent patterns , the absence of which made a movement continuous in our scenario . in the following sections , we describe each movement sequence with reference to the metronome beat count that was used to pace the dancer 's movements . see also table 1 for an overview of the movement and the limb displacement ( total traveled distance ) in each sequence . limb displacements were calculated for sequences at the middle tempo ( ibi = 500 ms ) , which should be most representative of the kinematics of each movement type . ( 1a ) the arm movement sequence was based on toque de palmas , where the dancer held her forearms in front of the face and clapped her hands on the left - frontal side of the body . the dancer started with the two hands in a closed position and clapped six times ( on beats 1 , 2 , and 3 and 5 , 6 , and 7 of the eight - beat count ) . zapateado , in which the dancer started with the standing position and made alternating foot taps on the ground ( without horizontal translational motion ) on beats 1 , 2 , and 3 and 5 , 6 , and 7 . the taps were made in the following order of the foot : right - right - left , left - left - right ( figure 1(b ) , 1st row ) . note that , in these discrete movements , the sequence was defined to start at the beginning of the limb trajectory leading to the first contact point ( beat 1 ) , instead of at the first contact point per se . the dancer started with both hands held above the head and moved the right hand downward to the hip level in a circular manner ( the arm trajectory similar to that of the arm of a clock ) and then upwards in front of the trunk until the two hands were joined above the head in the end . the arm movements were accompanied by hand gestures through wrist rotation that was typical of flamenco . see figure 1(a ) , 2nd row . ( 2b ) in the leg movement , the dancer started with both feet on the ground , lifted the right leg up and down to the ground again to the right side of the body while shifting the hip balance rightward ( beats 1 to 4 ) , and then drew a circle on the ground with the left leg in front of the body ( beats 5 to 8) that ended by the left foot joining the right ( figure 1(b ) , 2nd row ) . ( 3 ) mixed movements . segments of discrete and continuous movements were combined within a sequence . ( 3a ) the arm sequence started with two handclaps in front of the face ( toque de palmas , beats 1 and 2 ) , followed by a continuous trajectory of both arms drawing a circle in parallel in the frontal - coronal plane , stretching above the head and back to the face level ( bimanual variation of braceo , beats 3 to 6 ) , and ended with another two claps ( beats 7 and 8) in front of the face ( figure 1(a ) , 3rd row ) . ( 3b ) the leg sequence started with two taps on the ground by the left foot ( beats 1 and 2 ) , followed by the left leg drawing a full circle above the ground in the transverse plan ( beats 3 to 6 ) and back with two more taps on the ground ( beats 7 and 8) . the auditory stimuli consisted of two types of sound sequences , discrete or continuous , each lasting the same five durations as those of the visual stimuli . the continuous sequence was a tone lasting one of the five durations , made up of continuously frequency - modulated linear sine sweeps that went from 600 hz to 200 hz in the first half of the stimulus duration and from 200 hz back to 600 hz in the second half ( resembling a siren sound ) . in the discrete sequence , six discrete tones ( i.e. , six beats ) were embedded in the same continuous sequence as described above . the discrete tone was of a synthesized sound of the instrument clave with 43 ms tone duration . the beats followed the same temporal structure as the claps or the steps in the visual discrete movement , that is , occupying beats 1 , 2 , 3 , 5 , 6 , and 7 of an eight - beat count , with an ibi of 375 ms , 425 ms , 500 ms , 575 ms , and 625 ms for the respective sequence duration . the first beat always appeared at 200 ms after the onset of the continuous pitch sweeps . it should be noted that the discrete auditory sequence consisted of both a continuous sound and the discrete beats in parallel , the reason for which was to present comparable visual and auditory stimuli : the visual discrete movements contained continuously varying spatiotemporal information ( i.e. , velocity ) in the trajectory between successive contact points . we reasoned that this should be more closely mirrored in a continuous sound whose rate of frequency sweeps also scaled according to the sound duration , with discrete beats on top of it , instead of successive beats bordering empty temporal intervals . the experimental program was controlled by a customized matlab script using psychophysics toolbox version 3 routines running on a mac osx environment . the visual stimuli were displayed on a 17-inch crt monitor ( fujitsu x178 p117a ) with a frame frequency of 100 hz at a spatial resolution of 1024 768 pixels . sounds were presented at a sampling rate of 44,100 hz through closed studio headphones ( akg k271 mkii ) . two timing tasks were presented in a blocked manner : a visual task and an auditory task , with the former always preceding the latter . participants self - initiated each trial when they were ready . in the visual task , participants observed on each trial a short silent video of a dancer performing a movement sequence as described in the visual stimuli . we used the term speed , instead of tempo , as participants more easily understood the former where human movements were concerned . participants were required to attend to the sequence carefully and to memorize its entire duration . immediately following the video a reminder text was briefly shown ( please reproduce the duration now ! ) , after which an image of the dancer , taken from the first frame of the video , was displayed on the screen . as soon as this image was shown , participants were required to start reproducing the duration by mentally replaying the memorized movement sequence once . they were instructed to do so as closely to the movement speed of the video as possible . in the auditory task , participants underwent a similar procedure of duration reproduction with the auditory stimuli as described above . during auditory stimulus presentation and the reproduction phase , only a fixation cross was shown in the middle of the screen , which participants should fixate . for both the visual and auditory tasks , participants were especially instructed not to use any explicit strategies such as counting or moving along but should rather do so by mere observation and listening , respectively . at the end of the entire experiment , each participant was briefly interviewed for any strategies they had adopted to perform each task . the visual task followed a 2 ( limb type ) 3 ( movement type ) 5 ( tempo ) design , each with 10 repetitions ( see ) , and the total trials were presented in five blocks of about 15 minutes each . the auditory task followed a 2 ( sound type ) 5 ( tempo ) design , each with 10 repetitions , presented in five blocks of around 5 minutes each . all the conditions were presented in a balanced manner across blocks , with the order of conditions randomized within a block . participants underwent five practice trials prior to the visual and the auditory task , respectively . every participant completed the visual blocks before starting the auditory ones , as we intended to avoid introducing the idea of auditory imagery for the visual task . the entire experiment was completed in about two hours , and a break was required after each block . no participant reported substantial difficulty in carrying out the tasks . in the rare occasions where a response was given by mistake before the duration reproduction was carried out ( if a reproduced duration was shorter than 1500 ms , which exceeded three standard deviations from each within - participant mean ) , the trial was considered as errors and discarded from analyses . three parameters were analyzed individually for each condition to index the performance of duration reproduction : ( 1 ) absolute error ( ae ) , calculated as the absolute deviation of the reproduced interval from the presented one , in percentage . ( 2 ) ratio , calculated as the reproduced duration divided by the presented duration . a ratio of one signifies perfect reproduction , and a ratio smaller / larger than one represents underestimation / overestimation of the duration . ( 3 ) coefficient of variation ( cv ) , calculated for a given condition as the within - participant standard deviation of the reproduced intervals divided by his / her mean reproduced interval , shown in percentage . cv indexes the consistency of duration perception and reproduction ; a greater cv signifies more variable reproduction and thus poorer performance . as the present task required timing the durations of movement sequences with varying embedded temporal structures , the perceptual mechanism was expected to resemble that for timing the pattern of an auditory rhythm ( as opposed to timing a duration without content ) . while ae and ratio indexed how accurately a sequence was estimated in absolute terms , there could be systematic over- or underestimation due , for example , to vierordt 's law across sequence tempi [ 35 , 36 ] , or due to individual differences in the tendency to over- or underreproduce [ 13 , 37 ] , which is not necessarily associated with the presence or absence of a beat . in comparison , timing variability as indexed by cv may be more immune to these factors and able to reflect the rhythmicity of the movement . as such , along with ae and ratio that describe timing behaviors , cv would be taken as the more indicative measurement of the present task . data from one participant were excluded from further analyses , as the intervals were overall substantially underreproduced ( mean ratio = 0.63 and mean ae = 37% , which was the only case from the whole sample exceeding two standard deviations of the sample mean in both parameters ) . this suggests that the participant either did not fully understand the task or was hurrying through each trial without proper recall of the stimulus . for all the repeated - measures anovas and ancovas reported in this study , greenhouse - geisser correction was applied to the p values of effects of variables with more than two levels . first we provide an overview of the strategies participants ( n = 21 ) reported of adopting for the visual task : fourteen participants reported associating sounds along with visual imagery to aid mental replay , eight of whom used the auditory strategy only for the discrete movements ( i.e. , as if they could hear the impact sounds in their head ) . for each of the three parameters , we conducted a 2 ( limb type ) 3 ( movement type ) 5 ( tempo ) repeated - measures ancova of the individual means , with individual music or dance training duration entered as covariate in each analysis . we pulled together training in music and dance as one general category of rhythm - related expertise that may influence performance in the present task . only a significant main effect of movement type was found , f(2 , 38 ) = 3.95 , p = 0.028 , and p = 0.20 , and the post hoc tests showed that ae was lower for discrete than for either mixed , p = 0.02 , or ( almost ) continuous , p = 0.058 , while the latter two did not differ from each other . the interaction between limb type and tempo was significant , f(4 , 76 ) = 3.73 , p = 0.025 , and p = 0.16 , which was also modulated by the covariate of training duration , f(4 , 76 ) = 3.29 , p = 0.038 , and p = 0.15 . following this interaction , post hoc comparisons ( bonferroni corrected ) for the arm movements did not identify any difference amongst different tempi , all ps > 0.5 , while for the leg movements ae in the middle tempo ( ibi = 500 ms ) was lower than that in the two slowest tempi ( ibi = 575 and 625 ms ) , p = 0.028 and p = 0.014 , respectively . to examine how expertise modulated this effect , pearson 's correlations ( n = 21 ) were computed between training duration and ae of leg movement for the three slower tempi , which revealed a significant negative correlation between ae and training duration at the slowest tempo ( ibi = 625 ms ) , r = 0.44 and p = 0.04 , and marginally so at the next slowest ( ibi = 575 ms ) , r = 0.41 and p = 0.06 . no other significant effects were found : limb type , f(1 , 19 ) = 1.60 , p = 0.22 , and p = 0.078 , and tempo , f(4 , 76 ) = 1.06 , p = 0.34 , and p = 0.053 ( figure 2(a ) ) . training duration did not interact with any other effects , all ps > 0.2 and all p < 0.08 first , a main effect of tempo was found , f(4 , 76 ) = 39.73 , p < 0.001 , and p = 0.68 , with the post hoc tests showing that the reproduced ratio for the two fastest tempi was greater than that for the two slowest ones , all ps < 0.001 . the reproduced ratio for the middle tempo ( ibi = 500 ms ) also differed from those for the two fastest ones , both ps < 0.002 , as well as from those for the two slowest ones , both ps < 0.05 . on average , participants ' reproduced ratio descended across decreasing tempo , with overestimation for the faster ones and underestimation for the slower ones . main effects of limb type and movement type were not significant , f(1 , 19 ) = 2.81 , p = 0.11 , and p = 0.13 and f(2 , 38 ) = 0.91 , p = 0.40 and p = 0.046 . following a significant three - way interaction , f(8 , 152 ) = 4.42 , p = 0.002 , and p = 0.19 , follow - up two - way anovas were conducted for each limb type separately . for the arm movements , the movement type tempo interaction was significant , f(8 , 160 ) = 5.95 , p < 0.001 , and p = 0.23 , and the post hoc one - way anovas for each tempo separately showed that only at the fastest tempo was the ratio different between discrete and continuous movements , p = 0.008 ( bonferroni corrected ) , while no effect of movement type was found in all the other tempi . for the leg movements , a main effect of movement type was found , f(2 , 40 ) = 3.61 , p = 0.04 , and p = 0.15 , the post hoc test showing a trend of greater reproduced ratio for mixed than for continuous movements , p = 0.08 . the movement type tempo interaction was only marginally significant , f(8 , 160 ) = 2.32 , p = 0.054 , and p = 0.10 ( figure 3(a ) ) . training duration as covariate did not interact with any of the effects , all ps > 0.15 and all p < 0.08 the main effect of movement type was significant , f(2 , 38 ) = 17.40 , p < 0.001 , and p = 0.48 , with post hoc tests showing a lower cv for discrete than for either continuous or mixed movements , both ps < 0.005 , while the latter two did not differ from each other . the main effect of tempo was marginally significant , f(4 , 76 ) = 2.66 , p = 0.055 , and p = 0.12 . there was no effect of limb type , f(1 , 19 ) = 0.014 , p = 0.908 , and p = 0.001 ( figure 4(a ) ) . training duration as covariate did not interact with any of the effects , all ps > 0.16 and all p < 0.04 . for the auditory task , five participants reported visualizing the sounds , and two of them did so especially for continuous auditory sequences . individual means of each of the three parameters were submitted to a 2 ( sound type ) 5 ( tempo ) repeated - measures ancova , with training duration as covariate . the analysis did not reveal any significant effect of the variables , sound type , f(1 , 19 ) = 0.01 , p = 0.92 , and p = 0.001 , and tempo , f(4 , 76 ) = 1.36 , p = 0.26 , and p = 0.067 , or interaction , f(4 , 76 ) = 1.34 , p = 0.27 , and p = 0.066 . training duration did not interact with any variable , all ps > 0.2 and all p < 0.1 . only a significant main effect of tempo was found , f(4 , 76 ) = 21.09 , p < 0.001 , and p = 0.53 . post hoc comparisons showed that the reproduced ratio for the two fastest sequences was greater than that for the two slowest ones , all ps < 0.02 ( figure 3(b ) ) . effect of sound type was not significant , f(1 , 19 ) = 0.8 , p = 0.38 , and p = 0.04 , nor was its interaction with tempo , f(4 , 76 ) = 2.07 , p = 0.12 , and p = 0.098 . training duration did not interact with any variable , all ps > 0.2 and all p < 0.1 . on average , as found in the visual task , shorter durations were overestimated while longer ones were underestimated , although the extent of underestimation appeared smaller than in the visual task . the effect of sound type was not significant in the ancova analysis , f(1 , 19 ) = 1.76 , p = 0.2 , and p = 0.09 , though it was in the anova without covariate , f(1 , 20 ) = 6.0 , p = 0.02 , and p = 0.23 ( the covariate did not interact with sound type , f(1 , 19 ) = 0.66 , p = 0.43 , and p = 0.03 ) . no other effect was significant : tempo , f(4 , 76 ) = 1.75 , p = 0.16 , and p = 0.084 , and interaction , f(4 , 76 ) = 0.71 , p = 0.53 , and p = 0.036 . training duration only interacted with tempo , f(4 , 76 ) = 2.84 , p = 0.04 , and p = 0.13 ( figure 4(b ) ) . in sum , results of the visual task showed that , regardless of the limbs performing the movements and the movement tempo , discrete movements led to more accurate ( lower ae ) and more consistent ( lower cv ) temporal reproduction than both the continuous and the mixed movements , while performance in the latter two did not differ from each other . timing for the leg movements was more accurate ( lower ae ) in the middle tempo compared to the two slowest tempi ; this effect was modulated by expertise , such that longer training duration was associated with lower ae in the two slowest tempi . besides , faster movements tended to be overestimated and slower ones underestimated . for the auditory task , the pattern of ratio was similar to that in the visual task , with overestimation and underestimation for the faster and slower tempi . the effect of a discrete beat on auditory timing was not robust enough to survive the analysis with the covariate included . following results of experiment 1 , we examined whether the arms , the legs , or both , in a multi - limb movement sequence accounted for the beat advantage in visual timing . applying the same visual timing paradigm , we presented now movements performed by both the arms and the legs , each of which could be either discrete or continuous . thirteen participants had received music training ranging from three to seventeen years ( all amateurs ) , and the instruments included piano / keyboard ( 6 ) , guitar ( 4 ) , trumpet ( 1 ) , oboe ( 1 ) , and cello ( 1 ) . seven of the participants had participated in experiment 1 two to four weeks earlier . only visual stimuli were presented in this experiment , and they consisted of videos of four kinds of movement sequences derived from the flamenco repertoire . the sequences were performed by the same flamenco dancer across the same five tempi as in experiment 1 . the same procedures of movement recording and video editing and formatting were applied , yielding the same five sequence durations . the sequences now varied according to two variables : the arm movement type ( discrete or continuous ) and the leg movement type ( discrete or continuous ) . see also table 2 for an overview . the dancer made one tap on the ground with the left foot ( beat 1 ) , followed by three claps on the right - frontal side of the body ( toque de palmas , on beats 2 , 3 , and 4 ) , and then another tap with the right foot ( beat 5 ) , followed by three more claps on the left - frontal side of the body ( beats 6 , 7 , and 8) . the start of the sequence followed the same rule as previously described for the discrete movements . discrete claps were combined with the continuous leg movement as described in experiment 1 . the dancer held her arms at the head level and clapped three times along the right - frontal plane of the body ( beats 1 , 2 , and 3 ) , during which the left leg was lifted and stretched above the ground and down on the left side ( beats 1 to 4 ) , followed by another three claps on the left side of the body ( beats 5 , 6 , and 7 ) , during which the right leg drew a circle on the ground in front of the body ( beats 5 to 8) that ended by joining where the left foot was . the arm movement was similar to the continuous one in experiment 1 ( braceo ) , where the left arm moved downward ( beats 1 to 4 ) and upward again ( beats 5 to 8) in a circular manner to eventually join the right arm that was held above the head throughout . in parallel , the legs carried out discrete taps ( without horizontal translational motion ) derived from the movement marcaje , in which the first right tap ( beat 1 ) was followed by the left foot doing a front kick by sliding the shoe forward ( beat 2 ) , one back kick by sliding the shoe backwards ( beat 3 ) , and then one down kick by tapping the ground with the toe cap ( beat 4 ) and concluded by three successive left - right - left taps ( beats 5 to 7 ) . see figure 5(c ) . this movement combined similar continuous movements of the arms and the legs as in experiment 1 . the right arm carried out the circular movement ( braceo ) while the right leg drew a circle on the ground ( beats 1 to 4 ) , followed by the same movement pattern performed with the left arm and left leg ( beats 5 to 8) . participants performed the visual timing task following the same instruction and procedures as for the previous experiment , with special emphasis on observing the multi - limb movement as a whole instead of focusing on any specific body part . the experiment followed a 2 ( arm movement type ) 2 ( leg movement type ) 5 ( tempo ) design , each with 10 repetitions . the total trials were presented in five blocks of about 10 minutes each , with all the conditions presented in a balanced manner across blocks and the order of conditions randomized within a block . the whole experiment was completed within one hour , with a short break after each block . erroneous trials with too short intervals ( same criterion as in experiment 1 ) were discarded , which constituted on average only 0.6% of the trials . eight participants reported imagining the sounds along with visual imagery for the task , four of whom did so only when there were discrete movements . the majority of the participants reported adopting only a visual imagery strategy . the same three parameters as described in experiment 1 were analyzed individually , and the individual means of each parameter were submitted to a 2 ( arm movement type ) 2 ( leg movement type ) 5 ( tempo ) repeated - measures ancova , with training duration as covariate . no significant main effects or interactions were found , except for the marginally significant effect of arm movement , f(1 , 18 ) = 21.09 , p = 0.077 , and p = 0.16 , and the marginally significant three - way interaction , f(4 , 72 ) = 2.76 , p = 0.051 , and p = 0.13 . training did not interact with any of the variables ( figure 6(a ) ) . ratio . the main effect of arm movement was significant , f(1 , 18 ) = 19.22 , p < 0.001 , and p = 0.52 , showing a greater ratio in discrete than in continuous arm movements , but that of leg movement was not , f(1 , 18 ) = 0.024 , p = 0.88 , and p = 0.001 . the main effect of tempo was also significant , f(4 , 72 ) = 35.56 , p < 0.001 , and p = 0.66 , and the post hoc tests showed that while the ratio did not differ between the two fastest tempi or between the two slowest tempi , the two groups differed from each other , as well as from the ratio in the middle tempo , all ps < 0.02 . as found in experiment 1 , sequences of the faster tempi were on average more overestimated than those of the slower tempi ( figure 6(b ) ) . the arm leg interaction was marginally significant , f(1 , 18 ) = 3.31 , p = 0.086 , and p = 0.15 . there was a main effect of leg movement type , f(1 , 18 ) = 5.83 , p = 0.027 , and p = 0.25 , showing lower cv for discrete than for continuous leg movements , but not of arm movement type , f(1 , 18 ) = 1.16 , p = 0.69 , and p = 0.009 . the main effect of tempo was also significant , f(4 , 72 ) = 8.59 , p < 0.001 , and p = 0.32 ; post hoc tests showed that cv for the two fastest tempi was higher than that for the other three slower ones , all ps < 0.01 ( except for p = 0.08 between ibis of 425 ms and 575 ms ) . no interaction was significant , all ps > 0.2 and all p < 0.1 ( figure 6(c ) ) . training duration as covariate did not interact with any of the variables , all ps > 0.2 and all p < 0.1 . to summarize , regardless of the movement tempo , discrete leg movements led to more consistent timing ( lower cv ) than continuous leg movements , while arm movements did not influence cv . besides , movement tempo affected cv , which was not found in experiment 1 , such that faster movements led to lower consistency in timing than slower ones the first two experiments showed better visual timing for ( especially leg ) periodic trajectories marked by discrete contact points , possibly due to a sense of visual beat arising from observing these movements . here we verified whether this effect was attributed to internalized impact sounds , namely , whether the hypothesized visual beat was obligatorily encoded as auditory representation . we presented the discrete and the continuous leg movements either in silence , or with task - irrelevant auditory sequences that were temporally congruent or incongruent with the foot taps . if the beat had been encoded auditorily , incongruent interferences would have eliminated the timing advantage of discrete movements . if the beat percept remained visual , then the result pattern should persist despite auditory interferences . we included both congruent and incongruent sounds so that , should an auditory interference effect be observed , it could be determined whether it was caused by the temporal structure or the mere presence of the sounds . twenty healthy volunteers ( nine female , mean age 28 years , sd = 4.6 ) took part in this experiment , whose musical training duration ranged from zero to twenty years ( mean duration 4.9 years , sd = 5 ) . thirteen participants were musically trained ( all amateurs ) , and the instruments included piano / keyboard ( 6 ) , guitar ( 5 ) , trumpet ( 1 ) , and saxophone ( 1 ) . six and four participants had participated in experiments 1 and 2 , respectively , amongst whom two had participated in both . the visual stimuli here consisted of videos of two leg movement types as employed in experiment 1 : discrete and continuous . two from the five previously displayed tempi , corresponding to an ibi of 425 ms and 575 ms ( i.e. , the second fastest and the second slowest ) , were used here . the auditory interference in this task consisted of discrete tones of the same clave sound as used in experiment 1 . two kinds of auditory sequences were presented that were temporally congruent or incongruent with the timing of the discrete leg movement . the congruent sequence consisted of four discrete tones , which , when presented concurrently to the discrete leg movement , would temporally coincide with four of the six foot taps ( see the description of the discrete leg movement in experiment 1 ) . the incongruent sequence was initially constructed in the same way as the congruent one , but each tone was then advanced or delayed for a magnitude of 20% to 40% of the respective ibi . whether a tone was delayed or advanced , as well as the magnitude of this shift , the setup was the same , and participants performed the visual timing task following the same procedures as described before . in one - third of the trials , videos were presented in silence . in the other two - thirds , sounds were displayed through headphones during the video ; half of them were the congruent sequences , and the other half were the incongruent ones . participants received the same instruction as in experiment 1 and were additionally informed that they would sometimes hear sounds during the video , which were task irrelevant and should be ignored . the experiment followed a 2 ( leg movement type ) 2 ( tempo ) 3 ( auditory interference ) design , each condition with 10 repetitions . the whole experiment was completed in about half an hour , with a short break after each block . erroneous trials were discarded in which a response was accidentally given too quickly ( same criterion as before ) , which occurred rarely ( 0.5% of the trials on average ) . most participants reported having difficulty ignoring the sounds completely , despite the intention to comply with the instruction . as before , ae , ratio , and cv were analyzed individually and submitted to a 2 ( movement type ) 2 ( tempo ) 3 ( auditory interference ) repeated - measures ancova , with training duration entered as covariate . no significant effect of any factor was found , movement type , f(1 , 18 ) = 1.92 , p = 0.18 , and p = 0.097 ; tempo , f(1 , 18 ) = 0.15 , p = 0.70 , and p = 0.008 ; auditory interference , f(2 , 36 ) = 0.63 , p = 0.52 , and p = 0.034 , or any significant interaction ( figure 7(a ) ) . a significant effect of tempo was shown , f(1 , 18 ) = 39.86 , p < 0.001 , and p = 0.69 . similar to what was previously found , sequences of the faster tempo were overestimated ( mean ratio > 1 ) while those of the slower tempo were underestimated ( mean ratio < 1 ) . although there was a main effect of auditory interference , f(2 , 36 ) = 3.61 , p = 0.041 , and p = 0.17 , post hoc comparisons did not identify any significant difference between conditions , all ps > 0.15 ( figure 7(b ) ) . no other effects nor interactions were found significant , and training duration did not interact with any variable . there was a significant main effect of movement type , f(1 , 18 ) = 9.95 , p = 0.005 , and p = 0.36 , showing a lower cv for discrete than for continuous movements . the effect of tempo was only marginally significant , f(1 , 18 ) = 3.48 , p = 0.078 , and p = 0.16 , with a trend of higher cv for the faster tempo . the effect of auditory interference was again not significant , f(2 , 36 ) = 1.32 , p = 0.28 , and p = 0.068 ( figure 7(c ) ) . no significant interaction was found , nor did training duration interact with any variable . compared to when the visual task was performed in silence , the presence of an auditory interference sequence , regardless of its temporal structure , had no influence on any of the measured parameters . the result of more consistent timing in discrete than in continuous movements , as found in experiment 1 , persisted despite the auditory interferences . we investigated whether perceptual mechanisms similar to those previously found for auditory rhythms , such as beat - based strategies , were employed when observing temporally structured dance movements . in all three experiments , we found that periodic limb trajectories benefitted visual timing of a movement sequence , which was most consistently reflected in timing variability ( cv ) . when both the arms and the legs moved , only periodicities in the leg movement accounted for the timing advantage . this advantage persisted despite auditory interferences , suggesting that it was not attributed to internal representation of the impact sounds . we interpret the main result as evidence that observers extracted a visual beat from periodic trajectories , which facilitated temporal perception of the whole movement sequence . notably , the periodic trajectories ( handclaps or foot taps ) did not necessarily occur on every beat . their temporal structure resembled non - isochronous auditory rhythms that communicated an underlying beat [ 13 , 40 ] . our visual results are thus reminiscent of previous auditory findings that a perceived beat leads listeners to adopt a beat - based timing strategy that enhances rhythm perception [ 13 , 14 ] , suggesting similarities between auditory and visual rhythmic timing . the lack of a robust beat effect on improving auditory timing in experiment 1 might be due to several factors : for one , the auditory stimuli were not as rich and ecological as the visual ones . for another , in terms of contrasting conditions with and without a beat , the auditory stimuli might not have been optimally comparable to their visual counterpart . perhaps a closer resemblance to the visual discrete condition would have been , for example , successive ( shorter ) filled intervals yielding the same beat structure . the auditory beat effect might also have been attenuated by the learning effect , as the auditory task was always performed after the visual one . finally , whereas a picture of the dancer was presented to trigger participants ' recall in the visual task , no such rich cues were given prior to the auditory recall , which might have compromised the auditory performance . thus , different factors deserve consideration when comparing timing behaviors between dance movements and auditory rhythms : naturalistic content or biological motion [ 18 , 21 , 23 ] of visual stimuli may enhance beat advantage in real dance movements , compared to artificial sounds simulating the temporal structure of these movements . in addition , the compatibility of the visual and auditory stimuli yielding the same temporal structure appears critical and needs further verifications . the beat effect on timing was not modulated by music or dance expertise , suggesting the generality of this mechanism . while it seems fitting to explain our visual results borrowing the framework of auditory timing , with beat - based mechanism for discrete movements and duration - based mechanism for continuous ones , given the differences in paradigms and stimuli , we do not imply that these auditory mechanisms can be directly mapped onto visual timing of realistic human movements . whether these timing modes are indeed supramodal still warrants further investigations [ 2 , 42 ] . similarly , on the basis of shared perceptual and motor timing processes , our perceptual results ( for discrete versus continuous movements ) seem reminiscent of the dualistic motor timing in synchronization tasks : discrete movements ( e.g. , finger - tapping ) employ event - based timing , whereas continuous movements ( e.g. , circle drawing ) employ emergent timing . the former carry motor timing advantages over the latter due to their perceivable discrete events ( tap contact ) . it may be tentatively argued that the present timing advantages for beat - based movements arise from perceptual processes corresponding to , or even shared with , their motor counterparts . furthermore , it has been proposed that these two motor timing modes can not be combined , which seems consistent with our result that adding beat - based components to a non - beat - based movement ( mixed ) did not improve visual timing . timing difficulty in this case likely arose from the continuous trajectory , which deterred the perceptual system from adopting a beat - based strategy . one question may arise as to whether the observed advantage of a visual beat in timing was associated with possible counting strategies for discrete movements . this explanation was , however , not supported by the result that mixed movements , despite the presence of regular trajectories and thus the possibility of counting , were not better timed than continuous ones . in addition , counting or segmenting would also have been possible in a continuous movement based on positional cues and could thus not exclusively account for improved timing for discrete ones . similarly , one might discuss whether visual timing could have been influenced by stimulus factors such as total traveled distance of the limbs . as shown in experiment 1 ( table 1 ) , while differences in limb displacement were admittedly hard to control for in real human movements , there was no systematic difference across different movement types or limbs that would correspond to the obtained results ( e.g. , more consistent timing for discrete movements was not associated with more or less limb displacement across limb types ) . thus , performance in the present tasks was unlikely to be modulated by such stimulus features . our findings also reveal how different parts of a whole - body movement are timed in parallel . while a beat in either the arm or the leg movement assisted visual timing , in a multi - limb movement the beat - based benefit relied only on the legs . it would seem as if observers first oriented to the leg movement for a beat which , if found , enabled them to adopt beat - based timing . if not , however , observers did not resort to the arm movement either , even if a beat was available . this pattern suggests that temporal perception of multi - limb movements is somewhat different than can be explained by timing the upper or lower limbs alone , and a higher weight in timing is given to the lower limbs . the fact that the beat - based mechanism is driven by the leg movements seems to fit the action - perception coupling often proposed in rhythm perception : for example , preferred musical tempo corresponds to preferred frequency of locomotion , which concerns mainly the leg movements . thus , visual timing of dance movements may engage a common sensory - motor platform as for processing auditory rhythms , arguing for the multimodal nature of rhythm representations . it should be noted that the leg dominance in visual timing can not be explained by a preference for the lower visual field alone , as such a preference has mainly been established in goal - directed actions involving tools , and only when viewers are actively engaged in object manipulation , not during passive viewing . in addition , an upper visual field preference has also been found in a visual search task . thus , a general spatial bias regardless of the visual information does not seem to underlie our finding . contrary to earlier proposals that the temporal structures of simpler visual stimuli were obligatorily represented in auditory terms [ 16 , 39 ] , where task - irrelevant sounds were shown to impair visual timing , the present lack of auditory interference effect argues for the visual nature of beat - based timing , at least for rich , ecological movement information , an idea that has received increasing support [ 19 , 48 ] . the fact that also the congruent sounds had no effect on visual task performance suggests that either the auditory and visual streams were not integrated temporally , or the integration provided no additional assistance to the present task , as the sounds did not offer more beat - related information than the visual stimuli . it would be interesting for future studies to examine whether ( task irrelevant ) congruent and incongruent visual interferences would influence visual timing in this case . as several participants reported auditory imagery during the visual tasks , we can not rule out possible auditory co - representations of visual movement rhythms . although these co - representations may exist in parallel to the visual ones in fact , when movements became more complex ( as in experiment 2 ) , fewer participants reported using auditory strategies , indicating greater reliance on the visual representation . to what extent movement observation elicits auditory co - representations , how the tendency varies with movement complexity , and whether the two sensory representations interact remain interesting questions for follow - up research . movement tempo modulated visual timing of whole - body movements in experiment 2 , where slower movements were more consistently timed . as dance observation activates an internal motor program in the observers , greater difficulty in simulating these movements at faster tempi may increase difficulty in representing their temporal structures . this interpretation is supported by the fact that movement tempo did not affect visual timing consistency of simpler movements in experiment 1 , which could likely be simulated with equal ease across tempi . there might be a range of optimal tempi for each movement both in execution and in perception , such that movements considerably slower or faster than these tempi are less well represented and thus more difficult to time visually . movement tempo did , however , influence absolute timing accuracy ( ae ) of the leg movements in experiment 1 , with more deviation in the two slowest tempi than in the middle one , whereby those with longer music or dance training were less subject to such errors . thus , while beat - specific effects in visual timing were independent of expertise , training appeared to be beneficial for more general timing functions irrespective of beat , such as absolute duration estimation , in slower movements . finally , the effect of tempo on ratio observed in both experiments 1 and 2 , namely , more over- and underestimation for faster and slower sequences , respectively , can be explained by vierordt 's law . the fact that shorter and longer intervals tend to be over- and underestimated when presented in the same experiment has been repeatedly reported in the timing literature , which also applies to tempo in a rhythmic context [ 35 , 36 , 49 ] . in conclusion , we presented evidence of visual timing mechanisms for dancelike movements , showing a beat - based advantage that relies especially on the leg trajectories . while they appear similar to mechanisms of auditory rhythm perception found in previous studies , we demonstrated the visual nature of movement timing . these results have implications in how we approach multisensory rhythms in an ecological scenario , which may lead to new research linking action perception and rhythm perception in music and dance .

temporal mechanisms for processing auditory musical rhythms are well established , in which a perceived beat is beneficial for timing purposes . it is yet unknown whether such beat - based timing would also underlie visual perception of temporally structured , ecological stimuli connected to music : dance . in this study , we investigated whether observers extracted a visual beat when watching dance movements to assist visual timing of these movements . participants watched silent videos of dance sequences and reproduced the movement duration by mental recall . we found better visual timing for limb movements with regular patterns in the trajectories than without , similar to the beat advantage for auditory rhythms . when movements involved both the arms and the legs , the benefit of a visual beat relied only on the latter . the beat - based advantage persisted despite auditory interferences that were temporally incongruent with the visual beat , arguing for the visual nature of these mechanisms . our results suggest that visual timing principles for dance parallel their auditory counterparts for music , which may be based on common sensorimotor coupling . these processes likely yield multimodal rhythm representations in the scenario of music and dance .

1. Introduction 2. Experiment 1 3. Experiment 2 4. Experiment 3 5. Discussion

in this study , we investigated timing mechanisms employed in visual perception of dance movements , a class of movements most immediately linked to musical rhythms . in the same range , two modes of auditory timing have been distinguished , each subserved by a different motor circuitry that may work as a unified system : the duration - based mechanism , which times the absolute interval duration in a sequence without a perceivable beat , and the beat - based mechanism , which relies on a perceived beat in a sequence as reference for timing an interval . while beat - based mechanism is not superior to duration - based in timing a single auditory interval , the presence of a beat facilitates perception of an auditory rhythm ( consisting of successive intervals ) as a whole [ 13 , 14 ] : for example , the patterns of auditory rhythms with a perceivable beat can be more accurately reproduced or recalled than those without a clear beat . as such , questions arise as to whether the sensorimotor coupling underlying rhythmic timing can be strengthened by visual observation of temporally structured biological motion and whether this leads to visual timing behaviors similar to those found for auditory rhythms . we expected such a visual beat to afford a beat - based mechanism that would benefit visual timing of the whole movement sequence . participants watched short silent videos of a dancer moving with the arms or with the legs . in experiment 2 , we presented movements performed by both the arms and the legs , each of which could contain periodic trajectories or not . we examined whether the arms , the legs , or both yielded the salient beat in visual timing of whole - body movements . if the beat advantage persisted , it would argue for visual beat - based timing that is not transformed into auditory representations . in sum , results of the visual task showed that , regardless of the limbs performing the movements and the movement tempo , discrete movements led to more accurate ( lower ae ) and more consistent ( lower cv ) temporal reproduction than both the continuous and the mixed movements , while performance in the latter two did not differ from each other . for the auditory task , the pattern of ratio was similar to that in the visual task , with overestimation and underestimation for the faster and slower tempi . following results of experiment 1 , we examined whether the arms , the legs , or both , in a multi - limb movement sequence accounted for the beat advantage in visual timing . applying the same visual timing paradigm , we presented now movements performed by both the arms and the legs , each of which could be either discrete or continuous . in parallel , the legs carried out discrete taps ( without horizontal translational motion ) derived from the movement marcaje , in which the first right tap ( beat 1 ) was followed by the left foot doing a front kick by sliding the shoe forward ( beat 2 ) , one back kick by sliding the shoe backwards ( beat 3 ) , and then one down kick by tapping the ground with the toe cap ( beat 4 ) and concluded by three successive left - right - left taps ( beats 5 to 7 ) . besides , movement tempo affected cv , which was not found in experiment 1 , such that faster movements led to lower consistency in timing than slower ones the first two experiments showed better visual timing for ( especially leg ) periodic trajectories marked by discrete contact points , possibly due to a sense of visual beat arising from observing these movements . we investigated whether perceptual mechanisms similar to those previously found for auditory rhythms , such as beat - based strategies , were employed when observing temporally structured dance movements . in all three experiments , we found that periodic limb trajectories benefitted visual timing of a movement sequence , which was most consistently reflected in timing variability ( cv ) . when both the arms and the legs moved , only periodicities in the leg movement accounted for the timing advantage . we interpret the main result as evidence that observers extracted a visual beat from periodic trajectories , which facilitated temporal perception of the whole movement sequence . our visual results are thus reminiscent of previous auditory findings that a perceived beat leads listeners to adopt a beat - based timing strategy that enhances rhythm perception [ 13 , 14 ] , suggesting similarities between auditory and visual rhythmic timing . while a beat in either the arm or the leg movement assisted visual timing , in a multi - limb movement the beat - based benefit relied only on the legs . thus , visual timing of dance movements may engage a common sensory - motor platform as for processing auditory rhythms , arguing for the multimodal nature of rhythm representations . contrary to earlier proposals that the temporal structures of simpler visual stimuli were obligatorily represented in auditory terms [ 16 , 39 ] , where task - irrelevant sounds were shown to impair visual timing , the present lack of auditory interference effect argues for the visual nature of beat - based timing , at least for rich , ecological movement information , an idea that has received increasing support [ 19 , 48 ] . in conclusion , we presented evidence of visual timing mechanisms for dancelike movements , showing a beat - based advantage that relies especially on the leg trajectories . while they appear similar to mechanisms of auditory rhythm perception found in previous studies , we demonstrated the visual nature of movement timing .

esophageal cancer is the eighth most commonly diagnosed cancer worldwide , with squamous cell carcinoma and adenocarcinoma comprising the two main esophageal cancer histologies.[13 ] esophageal adenocarcinoma ( eac ) is the major histological type of esophageal cancer diagnosed in the western world today . risk factors associated with eac and the only known precursor lesion , barrett 's esophagus ( be ) , are still being unraveled ; however , persistent , symptomatic reflux of gastric and duodenal contents , known as gastro - esophageal reflux disease ( gerd ) , have long been known to correlate with the development of be and eac . obesity has also been reported to impart increased risk for eac and a 1.5 - 2-fold increased risk for gerd . rates of eac have increased at an alarming pace in the us and western europe in recent years.[811 ] esophageal cancer is the fourth leading cause of cancer - related mortality in uk males and the sixth leading cause of cancer - related deaths in uk females . in the us , 16,980 new incident cases of esophageal cancer are estimated to occur in 2011 and 14,710 deaths , representing the 7 leading cause of cancer - related deaths among us males . thus , there is a large population at increased risk for the development of barrett 's and eac , illustrating the potential global health significance of this growing problem . moreover , mortality figures closely parallel the incidence data , reflecting the poor 5-year survival rate of only 17% for esophageal cancer in the us . the latter is due to late stage of diagnosis coupled with ineffective screening , preventive and treatment options . chemoprevention with efficacious bioactive constituents derived from various food stuffs is an active area of investigation supported by the fact that plant - based diets rich in fruits and vegetables have generally been associated with reduced risk for eac and be.[1214 ] cranberries , for example , have been reported to have a multitude of positive health effects ranging from improved immune function and decreased infections to cardiovascular benefits , and more recently , cancer inhibition.[1517 ] our laboratory has specifically been investigating the ability of a proanthocyanidin - rich cranberry extract ( c - pac ) to inhibit cancers of the aerodigestive tract.[1820 ] in brief , in this report , we sought first to summarize the current findings on microrna ( mirna or mir ) expression patterns in be through eac pathologies and second , to identify c - pac induced alterations of mirnas following treatment of a panel of three validated eac human cell lines . the results summarize our knowledge of esophageal mir targets across varying histopathological categories and provide new insight regarding mir modulation following c - pac exposure and identify cancer - related pathways linked to mir modulation by the chemopreventive agent under evaluation in the context of eac . recent studies have linked dysregulation of specific mirnas to histological grade , neoplastic progression , metastatic potential , treatment responiveness and patient prognosis . briefly , cranberries were homogenized in 70% aqueous acetone , filtered and the pulp discarded . collected c - pac was concentrated under reduced pressure and the extract isolated using bioassay - directed fractionation as previously reported.[2224 ] methods including c nuclear magnetic resonance ( nmr ) , electrospray mass spectrometry , matrix - assisted laser desorption / ionization time - of - flight mass spectrometry , and acid - catalyzed degradation with phloroglucinol have all been utilized to confirm the presence of a - type linkages and the concentration of pacs present in the c - pac extract.[2224 ] purified proanthocyanidin extract was freeze - dried and stored at 80c until dissolved in media for individual experiments . a panel of three authenticated human eac cell lines was utilized in this series of experiments . specifically , jh - esoad1 , referred to as jhad1 , isolated in 1997 from a distal eac , stage iii , n0 ( johns hopkins university , baltimore , md , usa ) , oe33 cells isolated in 1993 from a distal eac , stage ii , n0 , ( ecacc , wiltshire , uk ) , and oe19 cells isolated in 1993 from an adenocarcinoma at the gastro - esophageal junction , stage iii , n1 ( ecacc , wiltshire , uk ) were utilized . cells were grown in roswell park memorial institute ( rpmi ) 1640 medium containing l - glutamine ( 2.0 mm ) , penicillin ( 10 units / ml ) , streptomycin ( 10 g / ml ) , sodium pyruvate ( 1 mm ) , and 010% fetal bovine serum ( fbs ) depending on the experiment . cells were maintained as monolayers at 37c with 95% air and 5% co2 throughout all studies . c - pac induced inhibition of cell viability was determined utilizing the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2h-5-tetrazolio]-1,3-benzene disulfonate ( wst-1 ) colorimetric assay ( roche applied science , indianapolis , in , usa ) as we previously reported . briefly , the viability assay is based on the ability of metabolically active cells to cleave the tetrazolium salt , wst-1 , into a formazen dye . jhad1 and oe19 cells were plated in sterile 96-well plates at 12e3 and 24e3 cells / well , respectively , and allowed to adhere for 35 hours ( 70% confluency ) prior to treatment with c - pac at concentrations of 25100 g / ml for 24 , 48 , 72 and 96 hours . complete phenol red - free rpmi medium with 5% fbs was utilized for viability assays . plates were processed according to the manufacturer 's instructions with spectrophotometer readings at 450 nm ( biotek synergy ht ) . a minimum of six wells were analyzed for each test condition and time - point . jhad1 , oe33 and oe19 ac cells were seeded at 5e6 , 14e6 and 14e6 cells ( 60% confluency ) , respectively , in t75 flasks and permitted to adhere for 30 hours prior to c - pac ( 50 g / ml ) treatment . cells were harvested 6 hours after c - pac treatment using trizol reagent ( invitrogen , carlsbad , ca , usa ) as per the manufacturer 's recommendations and rna was isolated utilizing standard phenol chloroform extraction procedures . rna quality was determined by nanodrop using the 8000 spectrophotometer ( thermo scientific , wilmington , nc , usa ) , and rna integrity and presence of the small rna fraction was determined using the bioanalyzer 2100 capillary electrophoresis system ( agilent , santa clara , ca , usa ) . sixty nanograms of total rna was reverse transcribed using the human megaplex primer pools a and b and the taqman mirna reverse transcription kit ( applied biosystems , foster city , ca , usa ) according to the manufacturer 's instructions . each sample was pre - amplified for 12 cycles using human pool a and b taqman megaplex preamp primers and preamp master mix ( applied biosystems ) according to the manufacturer 's instructions . for each sample , the preamplification reactions a and b were diluted and each reaction was combined with taqman gene - expression master mix ( applied biosystems ) divided into eight aliquots and each aliquot was added to one of the eight sample ports of the taqman array a or b ( v2.0 ) , respectively . the taqman array human mirna card set v2.0 enables detection of 667 human mirnas , 3 mirna endogenous reference controls and 1 mirna assay not related to human as a negative control . the real - time polymerase chain reaction ( pcr ) reactions were run according to the manufacturer 's instructions . realtime statminer software ( integromics , philadelphia , pa , usa ) was used to analyze the data . the global geometric mean of all expressed mirna assays was used to normalize the data . mirna targets were determined using mirwalk ( http://www.ma.uni-heidelberg.de/apps/zmf/mirwalk/ ) , a comprehensive database of human , mouse and rat mirnas on predicted and validated targets associated with specific genes , pathways , diseases or inherited disorders , organs , cell lines and transcription factors . the validated targets module of mirwalk was utilized to derive gene target information for mirs previously reported to be altered in be and eac compared to normal esophageal tissues and to determine gene targets altered in eac cell lines following c - pac treatment [ tables 14 ] . in addition , mirna predicted targets in mrna selected regions were determined and analyzed for the five common mirs altered in all three eac cell lines following c - pac treatment [ supplemental table 2 ] . next , gene targets were analyzed utilizing the database for annotation , visualization and integrated discovery ( david , v6.7 ) and signaling pathways were further investigated through use of the panther(http://www.pantherdb.org ) software , a system for inferring gene function based on published scientific experimental evidence and evolutionary relationships to predict function . panther utilizes gene family and subfamily information , gene ontology classes ( molecular function , biological process , and cellular component ) , evolutionary relationships , panther protein classes and pathway diagrams to classify genes based on their function . gene target lists were further subjected to both enrichment analysis and network analysis using genego 's metacore software ( http://www.genego.com ) which is an integrated knowledge base and pathway analysis tool based on a proprietary manually curated database of human protein protein , protein dna and protein compound interactions , metabolic and signaling pathways , all supported by proprietary ontologies . the enrichment analysis provided lists of biological pathways and functional ontologies ( go and genego ) statistically over - represented in each target list ( false discovery rate or fdr < 0.05 ) . we also used the genego analyze network algorithm with canonical pathways to build statistically significant biological networks from each target list ( at fdr < 0.05 ) . the results of this analysis serve to further investigate potential pathways altered by c - pac and to compare pathway results generated from panther and kegg , as well as provide additional information which may only be available through genego 's proprietary database . differential expression of mirnas in be and eac tissues as identified in 13 studies and c - pac induced alterations in eac cell lines common mirnas altered by c - pac treatment of jhad1 , oe19 , and oe33 eac cell lines , validated gene targets and resultant pathways canonical pathways enriched with targets of mirnas altered by c - pac treatment of eac cell lines panther pathways resulting from micornas altered in be and eac tissues or eac cell lines following c - pac treatment pathways resulting from c - pac altered predicted mir targets based on the five common mirs modulated in jhad1 , oe19 and oe33 eac cell lines results are presented as the mean value se for cell viability experiments . viability data were evaluated for statistical significance comparing media - treated versus c - pac - treated eac cells using the student 's t test ( two - sided , p < 0.05 ) . the mirna data were analyzed as described above with only common mirnas which were altered 2-fold in all three eac cell lines ( with p < 0.05 ) included in the analysis to derive validated targets and evaluate pathways impacted by c - pac treatment . a comprehensive review of the literature through july 1 , 2011 identified 13 original research studies evaluating dysregulation of mirnas in be or eac tissues relative to normal squamous epithelium ( nse),[3345 ] whereas only one study characterizing changes in eac or be cell lines relative to normal utilizing authenticated esophageal cell lines was identified . studies characterizing mirna alterations in esophageal squamous cell carcinoma were excluded , as were reports primarily focused on mirnas linked to prognosis , therapeutic response or other clinical outcomes . results of the 13 published studies reporting dysregulated mirnas in be , eac , or cell lines of be or eac origin are summarized in table 1 and supplemental table 1 , respectively . generally , we have included what the previous authors described as significantly modulated mirs based upon the methodology and criteria of the individual studies . however , mirs were excluded based upon a lack of significant difference in addition to a fold change value less than two - fold compared to normal or a lower grade of histopathological change . in addition , for all mirs altered in esophageal tissues , the mirwalk database was utilized to determine validated and predicted gene targets for further pathway analysis across the three databases as described above . differential expression of mirs in cell lines of be and eac origin as previously reported and c - pac induced mir alterations in eac cell lines briefly , cranberries were homogenized in 70% aqueous acetone , filtered and the pulp discarded . collected c - pac was concentrated under reduced pressure and the extract isolated using bioassay - directed fractionation as previously reported.[2224 ] methods including c nuclear magnetic resonance ( nmr ) , electrospray mass spectrometry , matrix - assisted laser desorption / ionization time - of - flight mass spectrometry , and acid - catalyzed degradation with phloroglucinol have all been utilized to confirm the presence of a - type linkages and the concentration of pacs present in the c - pac extract.[2224 ] purified proanthocyanidin extract was freeze - dried and stored at 80c until dissolved in media for individual experiments . a panel of three authenticated human eac cell lines was utilized in this series of experiments . specifically , jh - esoad1 , referred to as jhad1 , isolated in 1997 from a distal eac , stage iii , n0 ( johns hopkins university , baltimore , md , usa ) , oe33 cells isolated in 1993 from a distal eac , stage ii , n0 , ( ecacc , wiltshire , uk ) , and oe19 cells isolated in 1993 from an adenocarcinoma at the gastro - esophageal junction , stage iii , n1 ( ecacc , wiltshire , uk ) were utilized . cells were grown in roswell park memorial institute ( rpmi ) 1640 medium containing l - glutamine ( 2.0 mm ) , penicillin ( 10 units / ml ) , streptomycin ( 10 g / ml ) , sodium pyruvate ( 1 mm ) , and 010% fetal bovine serum ( fbs ) depending on the experiment . cells were maintained as monolayers at 37c with 95% air and 5% co2 throughout all studies . c - pac induced inhibition of cell viability was determined utilizing the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2h-5-tetrazolio]-1,3-benzene disulfonate ( wst-1 ) colorimetric assay ( roche applied science , indianapolis , in , usa ) as we previously reported . briefly , the viability assay is based on the ability of metabolically active cells to cleave the tetrazolium salt , wst-1 , into a formazen dye . jhad1 and oe19 cells were plated in sterile 96-well plates at 12e3 and 24e3 cells / well , respectively , and allowed to adhere for 35 hours ( 70% confluency ) prior to treatment with c - pac at concentrations of 25100 g / ml for 24 , 48 , 72 and 96 hours . complete phenol red - free rpmi medium with 5% fbs was utilized for viability assays . plates were processed according to the manufacturer 's instructions with spectrophotometer readings at 450 nm ( biotek synergy ht ) . a minimum of six wells were analyzed for each test condition and time - point . jhad1 , oe33 and oe19 ac cells were seeded at 5e6 , 14e6 and 14e6 cells ( 60% confluency ) , respectively , in t75 flasks and permitted to adhere for 30 hours prior to c - pac ( 50 g / ml ) treatment . cells were harvested 6 hours after c - pac treatment using trizol reagent ( invitrogen , carlsbad , ca , usa ) as per the manufacturer 's recommendations and rna was isolated utilizing standard phenol chloroform extraction procedures . rna quality was determined by nanodrop using the 8000 spectrophotometer ( thermo scientific , wilmington , nc , usa ) , and rna integrity and presence of the small rna fraction was determined using the bioanalyzer 2100 capillary electrophoresis system ( agilent , santa clara , ca , usa ) . sixty nanograms of total rna was reverse transcribed using the human megaplex primer pools a and b and the taqman mirna reverse transcription kit ( applied biosystems , foster city , ca , usa ) according to the manufacturer 's instructions . each sample was pre - amplified for 12 cycles using human pool a and b taqman megaplex preamp primers and preamp master mix ( applied biosystems ) according to the manufacturer 's instructions . for each sample , the preamplification reactions a and b were diluted and each reaction was combined with taqman gene - expression master mix ( applied biosystems ) divided into eight aliquots and each aliquot was added to one of the eight sample ports of the taqman array a or b ( v2.0 ) , respectively . the taqman array human mirna card set v2.0 enables detection of 667 human mirnas , 3 mirna endogenous reference controls and 1 mirna assay not related to human as a negative control . the real - time polymerase chain reaction ( pcr ) realtime statminer software ( integromics , philadelphia , pa , usa ) was used to analyze the data . the global geometric mean of all expressed mirna assays was used to normalize the data . mirna targets were determined using mirwalk ( http://www.ma.uni-heidelberg.de/apps/zmf/mirwalk/ ) , a comprehensive database of human , mouse and rat mirnas on predicted and validated targets associated with specific genes , pathways , diseases or inherited disorders , organs , cell lines and transcription factors . the validated targets module of mirwalk was utilized to derive gene target information for mirs previously reported to be altered in be and eac compared to normal esophageal tissues and to determine gene targets altered in eac cell lines following c - pac treatment [ tables 14 ] . in addition , mirna predicted targets in mrna selected regions were determined and analyzed for the five common mirs altered in all three eac cell lines following c - pac treatment [ supplemental table 2 ] . next , gene targets were analyzed utilizing the database for annotation , visualization and integrated discovery ( david , v6.7 ) and signaling pathways were further investigated through use of the panther(http://www.pantherdb.org ) software , a system for inferring gene function based on published scientific experimental evidence and evolutionary relationships to predict function . panther utilizes gene family and subfamily information , gene ontology classes ( molecular function , biological process , and cellular component ) , evolutionary relationships , panther protein classes and pathway diagrams to classify genes based on their function . gene target lists were further subjected to both enrichment analysis and network analysis using genego 's metacore software ( http://www.genego.com ) which is an integrated knowledge base and pathway analysis tool based on a proprietary manually curated database of human protein protein , protein dna and protein compound interactions , metabolic and signaling pathways , all supported by proprietary ontologies . the enrichment analysis provided lists of biological pathways and functional ontologies ( go and genego ) statistically over - represented in each target list ( false discovery rate or fdr < 0.05 ) . we also used the genego analyze network algorithm with canonical pathways to build statistically significant biological networks from each target list ( at fdr < 0.05 ) . the results of this analysis serve to further investigate potential pathways altered by c - pac and to compare pathway results generated from panther and kegg , as well as provide additional information which may only be available through genego 's proprietary database . differential expression of mirnas in be and eac tissues as identified in 13 studies and c - pac induced alterations in eac cell lines common mirnas altered by c - pac treatment of jhad1 , oe19 , and oe33 eac cell lines , validated gene targets and resultant pathways canonical pathways enriched with targets of mirnas altered by c - pac treatment of eac cell lines panther pathways resulting from micornas altered in be and eac tissues or eac cell lines following c - pac treatment pathways resulting from c - pac altered predicted mir targets based on the five common mirs modulated in jhad1 , oe19 and oe33 eac cell lines viability data were evaluated for statistical significance comparing media - treated versus c - pac - treated eac cells using the student 's t test ( two - sided , p < 0.05 ) . the mirna data were analyzed as described above with only common mirnas which were altered 2-fold in all three eac cell lines ( with p < 0.05 ) included in the analysis to derive validated targets and evaluate pathways impacted by c - pac treatment . a comprehensive review of the literature through july 1 , 2011 identified 13 original research studies evaluating dysregulation of mirnas in be or eac tissues relative to normal squamous epithelium ( nse),[3345 ] whereas only one study characterizing changes in eac or be cell lines relative to normal utilizing authenticated esophageal cell lines was identified . studies characterizing mirna alterations in esophageal squamous cell carcinoma were excluded , as were reports primarily focused on mirnas linked to prognosis , therapeutic response or other clinical outcomes . we regret if any studies meeting the inclusion criteria were omitted . results of the 13 published studies reporting dysregulated mirnas in be , eac , or cell lines of be or eac origin are summarized in table 1 and supplemental table 1 , respectively . generally , we have included what the previous authors described as significantly modulated mirs based upon the methodology and criteria of the individual studies . however , mirs were excluded based upon a lack of significant difference in addition to a fold change value less than two - fold compared to normal or a lower grade of histopathological change . in addition , for all mirs altered in esophageal tissues , the mirwalk database was utilized to determine validated and predicted gene targets for further pathway analysis across the three databases as described above . differential expression of mirs in cell lines of be and eac origin as previously reported and c - pac induced mir alterations in eac cell lines pre - treatment of jhad-1 and oe19 eac cells with c - pac at 25 , 50 , and 100 g / ml resulted in a concentration- and time - dependent significant inhibition of cellular viability . the data presented in figures 1a and 1b support an ic50 of 50 g / ml for c - pac . similar concentrations of c - pac have previously been found to inhibit the viability and proliferation of lung ( nci - h460 ) and colon cancer ( sw460 ) cell lines , increase the percentage of cells accumulating at the g1 checkpoint , induce apoptosis , modulate global gene expression profiles and alter select proteins linked to cell cycle regulation and apoptosis.[1820 ] in addition , c - pacs inhibitory effects are greater in cancer cells compared to normal het1a esophageal cells ( data not shown ) and c - pac shows superior inhibition of eac cell viability ( 8590% inhibition ) compared to black raspberry extract ( < 40% ) . c - pac has unique a - type chemical linkages found only in a limited number of fruits to date including cranberry , chokeberry , plums and avocado , which may account for some of the improved inhibitory capacity and other unique mechanisms by which c - pac inhibits cancer - related processes in eac cells . this unique a - type linkage is important for c - pacs anti - adhesion effects in the bladder as previously reported by dr . moreover , similar in vitro concentrations reported to block bacterial adhesion to the bladder wall inhibit cancer cell growth in vitro and these levels appear to be behaviorally achievable and efficacious in human cohorts for inhibiting urinary tract infections . ( a ) c - pac induced inhibition of jhad1 cellular viability over time . ( b ) c - pac induced inhibition of oe19 cellular viability over time . reported inhibition is relative to vehicle or media - treated esophageal adenocarcinoma cells . evaluations were in replicates of six per cell line , per experimental time point ( p < 0.05 , significantly different from media - treated controls by t - test ) table 1 summarizes aberrant mirna expression in be and eac tissues as identified in 13 original research studies.[3345 ] a total of 87 mirnas were identified from the previously published reports based on a thorough literature search through july 1 , 2011 . a total of 44 mirnas were altered in both eac and premalignant tissues as indicated by a superscript on the common mirna in table 1 . ten mirnas were uniquely altered in be tissues compared to nse with the upregulated including mir-10a , mir-144 , mir-148a , mir-451 and mir-548b-3p , and the downregulated including mir-222 , mir-370 , mir-509 , mir-543 , and mir-636 . references 3335 include analysis of patient tissues with varying degrees of histopathological alteration providing some insight into the potential mirnas linked to eac progression . the latter may prove particularly useful for patient stratification and for assessing select mirnas as biomarkers of early efficacy in chemopreventive intervention trials . the last column of table 1 includes mirnas altered in eac cell lines following a 6-hour treatment with c - pac . a total of 10 mirnas ( let-7b , mir-106b , mir-143 , mir-199a , mir-215 , mir-223 , mir-23b , mir-32 , mir-543 , and mir-7 ) were altered by c - pac inversely of at least one previously reported mirna aberration associated with the be or eac , supporting that c - pac may in part normalize mirna expression in eac . in addition , six of c - pac modulated mirs identified have been reported to be differentially expressed between eac compared to nse and also in premalignant pathologies , supporting that c - pac may hold cancer inhibitory potential at late stages of neoplastic transformation , as well as early during the development of esophageal premalignancy . importantly , let-7 family members and mir-143 are associated with tumor suppressor activity and both are downregulated in be and eac,[33353739 ] yet upregulated following c - pac treatment . mir-223 was recently linked to gastric cancer invasion and is documented to be upregulated in eac compared to normal esophageal tissues . c - pac treatment results in downregulation of mir-223 in oe33 eac cells , again supporting the potential benefits of c - pac against eac and precursor lesions . c - pac also inversely impacted let-7b , mir-136 , and mir-34a based upon be and eac cell line findings summarized in supplemental table 1 . a total of 52 mirnas were dysregulated in be or eac cells when compared to normal esophageal cells or a more normal histopathological\ state as previously reported by dr . additionally , 25 of the 52 ( 48.1% ) mirna alterations detected in esophageal cell lines were in common with those reported in tissue - based studies . overall , there are a relatively small number of validated be and eac cell lines and few studies characterizing mirnas alterations in those lines compared to normal cells . a 6-hour c - pac ( 50 g / ml ) treatment of jhad1 , oe19 and oe33 eac cells resulted in significant modulation of five common mirnas in all three cell lines . common mirnas significantly upregulated 2.0-fold included mir-410 and mir-520d-5p , whereas common downregulated 2.0-fold included mir-202 , mir-516a-3p , and mir-586 , as detailed in figure 2a . in addition , the venn diagrams in figures 2b and 2c show additional mir overlap between cell lines , with specific mirs listed in figure 2a . overall , the greatest number of c - pac induced mir alterations occurred in the jhad1 cell line ( n = 98 ) , followed by the oe33 ( n = 85 ) and oe19 ( n = 81 ) cell lines . we have found the oe19 cells to be the most tumorigenic in a mouse xenograft model and generally this cell line has been more resistant to treatment with cancer inhibitory agents compared to the oe33 and jhad1 cell lines . the five common mirs altered by c - pac treatment across all three cell lines resulted in identification of 26 validated gene targets utilizing the mirwalk database as detailed in table 2 . a number of gene targets have been implicated in cancer , including tumor suppressor genes ( p53 and p16 ) , oncogenes ( rb and erbb ) and inflammatory linked transcription factors ( nfkb ) . pathway analysis utilizing the target genes resulting from the five mirs altered by c - pac treatment in all three eac cell lines resulted in 23 kegg pathways detected as modulated . identified kegg pathways included pathways in cancer , a number of specific cancers ( bladder , nonsmall cell lung cancer , chronic myeloid leukemia , prostate cancer , glioma , melanoma , small cell lung cancer , endometrial cancer , basal cell carcinoma , renal cell cancer , colorectal cancer ) , cell cycle , p53 signaling , b - cell receptor signaling , focal adhesion , erbb signaling , apoptosis , toll - like receptor , t - cell receptor and wnt signaling . we have previously reported that c - pac has inhibitory effects in esophageal , colon and lung cancer cell lines;[1820 ] however , this is the first report evaluating the ability of c - pac to modulate mirna expression profiles . effects of c - pac treatment on mirna expression patterns in jhad1 , oe19 and oe33 esophageal adenocarcinoma cell lines . ( a ) details of commonly up and downregulated mirnas following a 6-hour c - pac treatment by cell line ; ( b ) venn diagram illustrating the number of c - pac upregulated mirnas in each cell line ; ( c ) venn diagram illustrating the number of downregulated mirnas associated with c - pac treatment next , panther pathway analysis was conducted to gain additional insight into metabolic and signaling pathways impacted by c - pac treatment . panther analysis resulted in the identification of five c - pac altered pathways in eac cells , with angiogenesis being the only unique addition beyond the kegg identified pathways as documented in table 2 . metacore pathway analysis was also applied utilizing the same 26 validated gene targets and resulted not only in a number of pathways previously identified by kegg and panther , but also in some unique pathways such as g - protein signaling , anti - angiogenic pigment epithelium - derived factor ( pedf ) signaling , dna damage , vascular endothelial growth factor ( vegf ) , mucin and immune response , among others . a number of the c - pac modulated pathways are logical targets for chemoprevention of eac , given the documented molecular alterations occurring on the continuum from be to eac.[5153 ] similarly , the top 20 canonical pathways identified via genego / metacore analysis of the targets resulting from the five mirs altered by c - pac in all three eac cell lines include not only p53 signaling , apoptosis , cell cycle , immune response , but also dna damage - related pathways as displayed in table 3 . metacore pathway analysis was also conducted utilizing validated targets derived from all 41 mirs altered in two or more eac cell lines . the top 20 canonical pathways included more gene ontology categories linked to immune response , cell cycle and epithelial - to - mesenchymal transition , but less involvement of dna damage pathways when compared to the targets derived form the five common mirs altered in all three cell lines . other unique categories resulting from the five common mir targets included proteolysis and mucin expression , whereas the 41 mirs dysregulated in two or more cell lines resulted in alterations in akt and pten signaling . these data support that c - pac modulated some common cancer - linked pathways across all three eac cell lines ; however , unique pathways were also detected between the eac cell lines in terms of c - pac induced changes likely reflecting differences in the molecular profiles of the individual cell lines . predicted targets derived from the five commonly altered mirs were also assessed and resulted in identification of 395 gene targets , 12 kegg and three panther pathways which are summarized in supplemental table 2 . interestingly , the toll - like receptor signaling pathway was the only one in common with pathways identified utilizing only validated targets . we found that utilizing multiple databases to assess mir targets provided additional information regarding potential mechanisms by which c - pac inhibits cancer cell growth ; however , additional validation of findings is required to better understand the true overlap between pathways identified via the various databases and actual molecular alterations . a recent paper by shmelkov et al , evaluated multiple pathway databases for identifying transcriptional regulatory targets and reported little overlap between experimentally obtained target genes compared to targets identified in transcriptional regulatory pathway databases . the authors did report that the metacore pathway database results intersected with experimental results in 84% of the cases compared to 24% utilizing the kegg database , for example ; panther was not included in the analysis comparing the various databases . in addition to the results varying by database choice , we detected little overlap between pathways identified as altered utilizing validated versus predicted gene targets . thus , both the types of targets and database choice are important considerations which stand to significantly impact interpretation of study results . based on the published literature , a total of 87 mirs have been reported to be dysregulated in eac and precursor lesions , as detailed in table 1 . utilizing the mirwalk database , 4335 validated gene targets depositing the gene entrez ids for the targets in david resulted in 1786 recognized annotations for conducting pathway analysis utilizing the panther database . next , to assess whether c - pac impacted panther pathways altered in eac tissues , 1665 validated gene targets were derived from the 41 mirs ( altered in two or more eac cell lines post c - pac treatment ) ; 981 of the 1665 targets were recognized annotations utilized for panther pathway analysis . based on the 87 dysregulated mirs reported in the published literature [ table 1 ] , a total of 26 panther pathways were identified as significantly altered , with angiogenesis , transforming growth factor ( tgf)--signaling , apoptosis , toll receptor signaling and p53 pathway feedback loops 2 representing the top five pathways . c - pac altered mirs ( 2 eac cell lines ) also resulted in the identification of 26 modulated pathways with 21 of the 26 overlapping with those identified as altered in eac or be tissues , supporting that c - pac may normalize altered mirna profiles in be and eac . this is also consistent with the data in table 1 showing that c - pac inversely modulated 10 mirs dysregulated in be or eac . pathways altered in eac or be which were not modulated by c - pac treatment included the ras pathway , insulin / insulin - like growth factor ( igf ) pathway - mitogen activated protein kinase / map kinase cascade , fibroblast growth factor ( fgf ) signaling , p38 pathway and integrin signaling . similarly , c - pac altered a small number of panther pathways not reported in eac tissues , including oxidative stress response , hypoxia response via hypoxia - inducible factor activation , dna replication and notch signaling pathways . these results and those detailed in tables 2 and 3 support that c - pac may hold promise as an inhibitor against other cancers with abberations in genes and pathways identified . as an example , mutations in p53 , p16 , pten , pik3ca , ras , and more recently , notch have all been linked to cancers of the head and neck and based on the results presented are targets potentially modulated by c - pac treatment . pre - treatment of jhad-1 and oe19 eac cells with c - pac at 25 , 50 , and 100 g / ml resulted in a concentration- and time - dependent significant inhibition of cellular viability . the data presented in figures 1a and 1b support an ic50 of 50 g / ml for c - pac . similar concentrations of c - pac have previously been found to inhibit the viability and proliferation of lung ( nci - h460 ) and colon cancer ( sw460 ) cell lines , increase the percentage of cells accumulating at the g1 checkpoint , induce apoptosis , modulate global gene expression profiles and alter select proteins linked to cell cycle regulation and apoptosis.[1820 ] in addition , c - pacs inhibitory effects are greater in cancer cells compared to normal het1a esophageal cells ( data not shown ) and c - pac shows superior inhibition of eac cell viability ( 8590% inhibition ) compared to black raspberry extract ( < 40% ) . c - pac has unique a - type chemical linkages found only in a limited number of fruits to date including cranberry , chokeberry , plums and avocado , which may account for some of the improved inhibitory capacity and other unique mechanisms by which c - pac inhibits cancer - related processes in eac cells . this unique a - type linkage is important for c - pacs anti - adhesion effects in the bladder as previously reported by dr . moreover , similar in vitro concentrations reported to block bacterial adhesion to the bladder wall inhibit cancer cell growth in vitro and these levels appear to be behaviorally achievable and efficacious in human cohorts for inhibiting urinary tract infections . ( a ) c - pac induced inhibition of jhad1 cellular viability over time . ( b ) c - pac induced inhibition of oe19 cellular viability over time . reported inhibition is relative to vehicle or media - treated esophageal adenocarcinoma cells . evaluations were in replicates of six per cell line , per experimental time point ( p < 0.05 , significantly different from media - treated controls by t - test ) table 1 summarizes aberrant mirna expression in be and eac tissues as identified in 13 original research studies.[3345 ] a total of 87 mirnas were identified from the previously published reports based on a thorough literature search through july 1 , 2011 . a total of 44 mirnas were altered in both eac and premalignant tissues as indicated by a superscript on the common mirna in table 1 . ten mirnas were uniquely altered in be tissues compared to nse with the upregulated including mir-10a , mir-144 , mir-148a , mir-451 and mir-548b-3p , and the downregulated including mir-222 , mir-370 , mir-509 , mir-543 , and mir-636 . references 3335 include analysis of patient tissues with varying degrees of histopathological alteration providing some insight into the potential mirnas linked to eac progression . the latter may prove particularly useful for patient stratification and for assessing select mirnas as biomarkers of early efficacy in chemopreventive intervention trials . the last column of table 1 includes mirnas altered in eac cell lines following a 6-hour treatment with c - pac . a total of 10 mirnas ( let-7b , mir-106b , mir-143 , mir-199a , mir-215 , mir-223 , mir-23b , mir-32 , mir-543 , and mir-7 ) were altered by c - pac inversely of at least one previously reported mirna aberration associated with the be or eac , supporting that c - pac may in part normalize mirna expression in eac . in addition , six of c - pac modulated mirs identified have been reported to be differentially expressed between eac compared to nse and also in premalignant pathologies , supporting that c - pac may hold cancer inhibitory potential at late stages of neoplastic transformation , as well as early during the development of esophageal premalignancy . importantly , let-7 family members and mir-143 are associated with tumor suppressor activity and both are downregulated in be and eac,[33353739 ] yet upregulated following c - pac treatment . mir-223 was recently linked to gastric cancer invasion and is documented to be upregulated in eac compared to normal esophageal tissues . c - pac treatment results in downregulation of mir-223 in oe33 eac cells , again supporting the potential benefits of c - pac against eac and precursor lesions . c - pac also inversely impacted let-7b , mir-136 , and mir-34a based upon be and eac cell line findings summarized in supplemental table 1 . a total of 52 mirnas were dysregulated in be or eac cells when compared to normal esophageal cells or a more normal histopathological\ state as previously reported by dr . additionally , 25 of the 52 ( 48.1% ) mirna alterations detected in esophageal cell lines were in common with those reported in tissue - based studies . overall , there are a relatively small number of validated be and eac cell lines and few studies characterizing mirnas alterations in those lines compared to normal cells . a 6-hour c - pac ( 50 g / ml ) treatment of jhad1 , oe19 and oe33 eac cells resulted in significant modulation of five common mirnas in all three cell lines . common mirnas significantly upregulated 2.0-fold included mir-410 and mir-520d-5p , whereas common downregulated 2.0-fold included mir-202 , mir-516a-3p , and mir-586 , as detailed in figure 2a . in addition , the venn diagrams in figures 2b and 2c show additional mir overlap between cell lines , with specific mirs listed in figure 2a . overall , the greatest number of c - pac induced mir alterations occurred in the jhad1 cell line ( n = 98 ) , followed by the oe33 ( n = 85 ) and oe19 ( n = 81 ) cell lines . we have found the oe19 cells to be the most tumorigenic in a mouse xenograft model and generally this cell line has been more resistant to treatment with cancer inhibitory agents compared to the oe33 and jhad1 cell lines . the five common mirs altered by c - pac treatment across all three cell lines resulted in identification of 26 validated gene targets utilizing the mirwalk database as detailed in table 2 . a number of gene targets have been implicated in cancer , including tumor suppressor genes ( p53 and p16 ) , oncogenes ( rb and erbb ) and inflammatory linked transcription factors ( nfkb ) . pathway analysis utilizing the target genes resulting from the five mirs altered by c - pac treatment in all three eac cell lines resulted in 23 kegg pathways detected as modulated . identified kegg pathways included pathways in cancer , a number of specific cancers ( bladder , nonsmall cell lung cancer , chronic myeloid leukemia , prostate cancer , glioma , melanoma , small cell lung cancer , endometrial cancer , basal cell carcinoma , renal cell cancer , colorectal cancer ) , cell cycle , p53 signaling , b - cell receptor signaling , focal adhesion , erbb signaling , apoptosis , toll - like receptor , t - cell receptor and wnt signaling . we have previously reported that c - pac has inhibitory effects in esophageal , colon and lung cancer cell lines;[1820 ] however , this is the first report evaluating the ability of c - pac to modulate mirna expression profiles . effects of c - pac treatment on mirna expression patterns in jhad1 , oe19 and oe33 esophageal adenocarcinoma cell lines . ( a ) details of commonly up and downregulated mirnas following a 6-hour c - pac treatment by cell line ; ( b ) venn diagram illustrating the number of c - pac upregulated mirnas in each cell line ; ( c ) venn diagram illustrating the number of downregulated mirnas associated with c - pac treatment next , panther pathway analysis was conducted to gain additional insight into metabolic and signaling pathways impacted by c - pac treatment . panther analysis resulted in the identification of five c - pac altered pathways in eac cells , with angiogenesis being the only unique addition beyond the kegg identified pathways as documented in table 2 . metacore pathway analysis was also applied utilizing the same 26 validated gene targets and resulted not only in a number of pathways previously identified by kegg and panther , but also in some unique pathways such as g - protein signaling , anti - angiogenic pigment epithelium - derived factor ( pedf ) signaling , dna damage , vascular endothelial growth factor ( vegf ) , mucin and immune response , among others . a number of the c - pac modulated pathways are logical targets for chemoprevention of eac , given the documented molecular alterations occurring on the continuum from be to eac.[5153 ] similarly , the top 20 canonical pathways identified via genego / metacore analysis of the targets resulting from the five mirs altered by c - pac in all three eac cell lines include not only p53 signaling , apoptosis , cell cycle , immune response , but also dna damage - related pathways as displayed in table 3 . metacore pathway analysis was also conducted utilizing validated targets derived from all 41 mirs altered in two or more eac cell lines . the top 20 canonical pathways included more gene ontology categories linked to immune response , cell cycle and epithelial - to - mesenchymal transition , but less involvement of dna damage pathways when compared to the targets derived form the five common mirs altered in all three cell lines . other unique categories resulting from the five common mir targets included proteolysis and mucin expression , whereas the 41 mirs dysregulated in two or more cell lines resulted in alterations in akt and pten signaling . these data support that c - pac modulated some common cancer - linked pathways across all three eac cell lines ; however , unique pathways were also detected between the eac cell lines in terms of c - pac induced changes likely reflecting differences in the molecular profiles of the individual cell lines . predicted targets derived from the five commonly altered mirs were also assessed and resulted in identification of 395 gene targets , 12 kegg and three panther pathways which are summarized in supplemental table 2 . interestingly , the toll - like receptor signaling pathway was the only one in common with pathways identified utilizing only validated targets . we found that utilizing multiple databases to assess mir targets provided additional information regarding potential mechanisms by which c - pac inhibits cancer cell growth ; however , additional validation of findings is required to better understand the true overlap between pathways identified via the various databases and actual molecular alterations . a recent paper by shmelkov et al . , evaluated multiple pathway databases for identifying transcriptional regulatory targets and reported little overlap between experimentally obtained target genes compared to targets identified in transcriptional regulatory pathway databases . the authors did report that the metacore pathway database results intersected with experimental results in 84% of the cases compared to 24% utilizing the kegg database , for example ; panther was not included in the analysis comparing the various databases . in addition to the results varying by database choice , we detected little overlap between pathways identified as altered utilizing validated versus predicted gene targets . thus , both the types of targets and database choice are important considerations which stand to significantly impact interpretation of study results . based on the published literature , a total of 87 mirs have been reported to be dysregulated in eac and precursor lesions , as detailed in table 1 . utilizing the mirwalk database , depositing the gene entrez ids for the targets in david resulted in 1786 recognized annotations for conducting pathway analysis utilizing the panther database . next , to assess whether c - pac impacted panther pathways altered in eac tissues , 1665 validated gene targets were derived from the 41 mirs ( altered in two or more eac cell lines post c - pac treatment ) ; 981 of the 1665 targets were recognized annotations utilized for panther pathway analysis . based on the 87 dysregulated mirs reported in the published literature [ table 1 ] , a total of 26 panther pathways were identified as significantly altered , with angiogenesis , transforming growth factor ( tgf)--signaling , apoptosis , toll receptor signaling and p53 pathway feedback loops 2 representing the top five pathways . c - pac altered mirs ( 2 eac cell lines ) also resulted in the identification of 26 modulated pathways with 21 of the 26 overlapping with those identified as altered in eac or be tissues , supporting that c - pac may normalize altered mirna profiles in be and eac . this is also consistent with the data in table 1 showing that c - pac inversely modulated 10 mirs dysregulated in be or eac . pathways altered in eac or be which were not modulated by c - pac treatment included the ras pathway , insulin / insulin - like growth factor ( igf ) pathway - mitogen activated protein kinase / map kinase cascade , fibroblast growth factor ( fgf ) signaling , p38 pathway and integrin signaling . similarly , c - pac altered a small number of panther pathways not reported in eac tissues , including oxidative stress response , hypoxia response via hypoxia - inducible factor activation , dna replication and notch signaling pathways . these results and those detailed in tables 2 and 3 support that c - pac may hold promise as an inhibitor against other cancers with abberations in genes and pathways identified . as an example , mutations in p53 , p16 , pten , pik3ca , ras , and more recently , notch have all been linked to cancers of the head and neck and based on the results presented are targets potentially modulated by c - pac treatment . pathway results across three database platforms utilizing gene targets derived from mirs altered in be and eac tissues or by c - pac treatment of eac cell lines detected multiple common pathways , but also uncovered a limited number of unique pathways per database illustrating the effect that the database choice has on study results . moreover , divergent pathways were detected when comparing validated mir targets to predicted mir targets , raising another important consideration for the interprtation of research results . overall , the data support that c - pac , a proanthocyanidin - rich cranberry extract , has potent inhibitory effects on the viability of eac cells , which is in part attributable to modulation of select mirnas , some of which are known to be altered in eac and precursor lesions . still , mechanistic information regarding the cancer inhibitory potential of c - pac and other cranberry constituents is limited , particularly in terms of in vivo research , compared to other berry types like black raspberries.[5559 ] our results support that future targeted interventions utilizing c - pac in cohorts at increased risk for eac progression or other cancers as idenitified by pathway analysis may prove promising . it is encouraging that several mirs modulated by c - pac in eac cell lines have also been reported to be inversely dysregulated in premalignant esophageal pathologies as well as eac [ table 1 ] , supporting that c - pac may hold chemopreventive potential at early stages during the development of esophageal premalignancy as well as later stages characterized by neoplastic transformation and progression to eac . the fact that c - pac also modulates angiogenesis and pedf signaling based on pathway analysis further supports this finding . interestingly , black raspberries have recently been reported to inhibit late stage carcinogenesis in a preclinical model for esophageal squamous cell carcinoma through modulation of pathways linked to proliferation , apoptosis , inflammation , angiogenesis , cyclooxygenase and lipoxygenase . thus , although the findings reported herein are limited by their descriptive nature , they are still informative for the identification of pathways linked to eac progression or pathways modulated by c - pac treatment , in turn serving to guide more mechanistically driven investigations . further in vitro , in vivo and clinical investigations are warranted to assess the true chemopreventive efficacy of c - pac toward reducing be and eac risk . improving our knowledge of dysregulated mirnas in specific cancers ( eac ) and premalignant conditions ( be ) will permit identification of potential mirna - based therapeutic or chemopreventive targets and support improved mechanistically informed interventions with the overarching goal of reducing cancer risk in high - risk cohorts . efficacious cancer risk reducing interventions are especially needed for esophageal and other cancers plagued with ineffective screening methods , late stage diagnosis , cancers with limited treatment options and those with poor prognosis . improving our understanding of specific mirnas altered by natural or dietary sources of cancer inhibitory agents , such as c - pac , is a relatively new area of investigation , but one that may lead to promising mutitargeted chemopreventive strategies utilizing single agents that modulate mutiple cancer - associated pathways or combinations of agents targeting complementary pathways as a risk - reducing strategy . laura ann kresty , department of epidemiology and public health , university of miami miller school of medicine and sylvester cancer center , miami , florida 33136 ; usa dr . jennifer clarke , department of epidemiology and public health , university of miami miller school of medicine and sylvester cancer center , miami , florida 33136 ; usa ms . kristin ezell , department of epidemiology and public health , university of miami miller school of medicine and sylvester cancer center , miami , florida 33136 ; usa ms . amy exum , department of epidemiology and public health , university of miami miller school of medicine and sylvester cancer center , miami , florida 33136 ; usa dr . amy b howell , marucci center for blueberry cranberry research , rutgers university , chatsworth , new jersey 08019 ; usa dr . toumy guettouche , institute for human genomics , university of miami miller school of medicine , miami , florida 33136 ; usa

background : aberrant expression of small noncoding endogenous rna molecules known as micrornas ( mirnas ) is documented to occur in multiple cancer types including esophageal adencarcinoma ( eac ) and its only known precursor , barrett 's esophagus ( be ) . recent studies have linked dysregulation of specific mirnas to histological grade , neoplastic progression and metastatic potential.materials and methods : herein , we present a summary of previously reported dysregulated mirnas in be and eac tissues as well as eac cell lines and evaluate a cranberry proanthocyanidin rich extract 's ( c - pac ) ability to modulate mirna expression patterns of three human eac cell lines ( jheso - ad-1 , oe33 and oe19).results : a review of 13 published studies revealed dysregulation of 87 mirnas in be and eac tissues , whereas 52 mirnas have been reported to be altered in be or eac cell lines , with 48% overlap with mirna changes reported in tissues . we report for the first time c - pac induced modulation of five mirnas in three eac cell lines resulting in 26 validated gene targets and identification of key signaling pathways including p53 , angiogenesis , t - cell activation and apoptosis . additionally , mutiple cancer related networks were ideintified as modulated by c - pac utilizing kyoto encyclopedia of genes and genomes ( kegg ) , protein analysis through evolutionary relationships ( panther ) , and metacore analysis tools.conclusions:study results support the cancer inhibitory potential of c - pac is in part attributable to c - pac 's ability to modify mirna profiles within eac cells . a number of c - pac modulated mirnas have been been identified as dysregulated in be and eac . further insights into mirna dysregulation and modulation by select cancer preventive agents will support improved targeted interventions in high - risk cohorts .

BACKGROUND MATERIALS AND METHODS Cranberry proanthocyanidin preparation Cell-lines and cultures conditions Cell viability assays RNA isolation and miRNA assay MiRNA targets and pathway analysis Statistical analysis Literature review RESULTS AND DISCUSSION Inhibition of EAC cellular viability by C-PAC MiRNA alterations in BE, EAC and esophageal cell lines miRNA modulation in JHAD1, OE19 and OE33 cell lines following C-PAC treatment Pathways resulting from miRNAs altered in BE and EAC and pathways altered in EAC cell lines by C-PAC treatment CONCLUSIONS AUTHOR'S PROFILE

risk factors associated with eac and the only known precursor lesion , barrett 's esophagus ( be ) , are still being unraveled ; however , persistent , symptomatic reflux of gastric and duodenal contents , known as gastro - esophageal reflux disease ( gerd ) , have long been known to correlate with the development of be and eac . recent studies have linked dysregulation of specific mirnas to histological grade , neoplastic progression , metastatic potential , treatment responiveness and patient prognosis . the validated targets module of mirwalk was utilized to derive gene target information for mirs previously reported to be altered in be and eac compared to normal esophageal tissues and to determine gene targets altered in eac cell lines following c - pac treatment [ tables 14 ] . differential expression of mirnas in be and eac tissues as identified in 13 studies and c - pac induced alterations in eac cell lines common mirnas altered by c - pac treatment of jhad1 , oe19 , and oe33 eac cell lines , validated gene targets and resultant pathways canonical pathways enriched with targets of mirnas altered by c - pac treatment of eac cell lines panther pathways resulting from micornas altered in be and eac tissues or eac cell lines following c - pac treatment pathways resulting from c - pac altered predicted mir targets based on the five common mirs modulated in jhad1 , oe19 and oe33 eac cell lines results are presented as the mean value se for cell viability experiments . the validated targets module of mirwalk was utilized to derive gene target information for mirs previously reported to be altered in be and eac compared to normal esophageal tissues and to determine gene targets altered in eac cell lines following c - pac treatment [ tables 14 ] . differential expression of mirnas in be and eac tissues as identified in 13 studies and c - pac induced alterations in eac cell lines common mirnas altered by c - pac treatment of jhad1 , oe19 , and oe33 eac cell lines , validated gene targets and resultant pathways canonical pathways enriched with targets of mirnas altered by c - pac treatment of eac cell lines panther pathways resulting from micornas altered in be and eac tissues or eac cell lines following c - pac treatment pathways resulting from c - pac altered predicted mir targets based on the five common mirs modulated in jhad1 , oe19 and oe33 eac cell lines viability data were evaluated for statistical significance comparing media - treated versus c - pac - treated eac cells using the student 's t test ( two - sided , p < 0.05 ) . in addition , six of c - pac modulated mirs identified have been reported to be differentially expressed between eac compared to nse and also in premalignant pathologies , supporting that c - pac may hold cancer inhibitory potential at late stages of neoplastic transformation , as well as early during the development of esophageal premalignancy . c - pac altered mirs ( 2 eac cell lines ) also resulted in the identification of 26 modulated pathways with 21 of the 26 overlapping with those identified as altered in eac or be tissues , supporting that c - pac may normalize altered mirna profiles in be and eac . in addition , six of c - pac modulated mirs identified have been reported to be differentially expressed between eac compared to nse and also in premalignant pathologies , supporting that c - pac may hold cancer inhibitory potential at late stages of neoplastic transformation , as well as early during the development of esophageal premalignancy . c - pac altered mirs ( 2 eac cell lines ) also resulted in the identification of 26 modulated pathways with 21 of the 26 overlapping with those identified as altered in eac or be tissues , supporting that c - pac may normalize altered mirna profiles in be and eac . pathway results across three database platforms utilizing gene targets derived from mirs altered in be and eac tissues or by c - pac treatment of eac cell lines detected multiple common pathways , but also uncovered a limited number of unique pathways per database illustrating the effect that the database choice has on study results . it is encouraging that several mirs modulated by c - pac in eac cell lines have also been reported to be inversely dysregulated in premalignant esophageal pathologies as well as eac [ table 1 ] , supporting that c - pac may hold chemopreventive potential at early stages during the development of esophageal premalignancy as well as later stages characterized by neoplastic transformation and progression to eac . improving our knowledge of dysregulated mirnas in specific cancers ( eac ) and premalignant conditions ( be ) will permit identification of potential mirna - based therapeutic or chemopreventive targets and support improved mechanistically informed interventions with the overarching goal of reducing cancer risk in high - risk cohorts .

gingivitis and periodontal disease are frequent concomitant phenomena of orthodontic treatment with fixed appliances . it seems that the main factor for an increased accumulation of dental plaque and inflammatory response is the appearance of new retentive places around the components of fixed appliances attached to the teeth . several studies have addressed the impact of fixed , removable , and myofunctional orthodontic / orthopedic appliances or retainers in relation to supragingival plaque accumulation and gingivitis . the quantity , as well as the quality of plaque , is influenced by many factors including surface characteristics , surface roughness and surface free energy and bracket design , frequency of sucrose exposition . the presence of gingival inflammation will further increase plaque growth . because of its outstanding aesthetic preconditions and its growing practicability , lingual orthodontics accounts for an ever - increasing percentage of orthodontic treatments . oral hygiene is even more important for therapy with lingual brackets than for therapy with labial brackets because control is more difficult from the lingual face than from the buccal face , and plaque accumulations , gingivitis , and demineralization are not detected by the patient . most microbiological investigations have been performed during orthodontic treatment but there are no studies dealing with the difference between buccal and lingual brackets . accordingly , the purpose of the present investigation was to evaluate the influence of buccal and lingual orthodontic appliances on microbiological and periodontal parameters of bonded teeth . the null hypothesis of the study was that there is no significant difference in terms of microbiological environment and clinical periodontal parameters between buccal and lingual brackets . twenty dental students ( 14 females and 6 males , caucasians aged between 20 and 32 years ) were involved in the study ( table 1 ) . they were given a written explanation of the background of the study and its objectives . after screening for their suitability and after good comprehension of the protocol , they all gave their written informed consent . during the experiment before the study , all students received oral hygiene instructions in order to ensure a healthy periodontal situation . study population with data on gender distribution , previous orthodontic treatment and age sd= standard deviation the initial placement of the brackets was performed via a randomized protocol by means of concealed envelopes . the students were selected to fulfill the following inclusion criteria : no smoking , absence of extensive dental restorations or adhesive - fixed partial dentures , a sulcus bleeding index of < 0.3 and no antibiotics during or up to 4 months before the study . the students were also asked whether they had already received an orthodontic treatment with fixed appliances because this might have consequences for smoothness of the buccal enamel and as such on the microbial adhesion in the early formation of a dental plaque film . the study had a randomized , examiner - blind , split - mouth design . in every student , the mouth was divided into four quadrants , two of which served as controls . one type of bracket ( 2d , forestadent , pforzheim , germany ) was bonded in two different sites : buccal and lingual . for the split - mouth comparison , 8 sites were defined , namely the canine and the first premolar of each quadrant . the first quadrant used for bracket placement and the order in which the brackets were placed were randomly chosen by means of concealed envelopes , the second one was at the other side of the mouth in the antagonistic jaw . the buccal and lingual bonded teeth were alternated , giving rise to four different experimental settings ( figure 1 ) : the four different clinical conditions of the splitmouth design -buccal position in the first quadrant , lingual position in the third quadrant ; -buccal position in the second quadrant , lingual position in the fourth quadrant ; -buccal position in the third quadrant , lingual position in the first quadrant ; -buccal position in the fourth quadrant , lingual position in the second quadrant . the teeth bonded with the different brackets were compared with each other and with the non - bonded control sites . during the study period , the students visited the clinic three times : t0 ( baseline ) : to record the status of the periodontium ( periodontal pocket depth : ppd ; bleeding on probing : bop ) , to collect samples of dental plaque from the teeth and to place the brackets ; t1 ( day 7 ) : to record the periodontal status ( ppd and bop ) and to collect from the test teeth and from control teeth ; t2 ( day 30 ) : to record the periodontal status ( ppd and bop ) , to collect samples of dental plaque from the test teeth and the control teeth , and to remove the brackets . the teeth were rinsed , dried with an oil - free air syringe , and etched with 37% phosphoric acid for 30 s. after a thorough washing , they were completely dried with an oil - free air syringe . then , stainless steel brackets ( 2d , forestadent , pforzheim , germany ) were bonded to the selected teeth with an adhesive system ( transbond xt , 3 m , monrovia , ca , usa ) , according to the manufacturer 's directions . after applying the primer on the etched enamel , a small amount of composite resin was placed on the mesh pad of the bracket . the bracket was positioned on the buccal or lingual surface of the teeth with sufficient pressure to squeeze excess adhesive , which was removed from the margins of the bracket base with an explorer before polymerization . bracket was then light - cured with a visible light - curing unit ( ortholux xt , 3 m unitek ) for 10 s on the mesial side of the bracket and for 10 s on the distal side ( total cure time 20 s ) . verbal and written instructions regarding the appliance care and hygiene protocols were issued to each patient , along with a specific request to return if a bracket became loose or if any problem arose with the appliance . ppd and bop were scored at baseline , day 7 and day 30 . to record the ppd , a millimeter probe ( hu - friedly pc puns , chicago , il , usa ) was inserted in the gingival sulcus . the pocket depths were measured at the buccal , lingual , mesial and distal sides of the tooth and rounded off to the nearest 0.5 mm . bop was recorded ( 0 : absent ; 1 : present ) 24 s after determination of ppd . seven and thirty microbial samples were also taken using a sterile curette ( sg 5/6 hu - friedy ) from the test and the control teeth . this was carried out without traumatizing the gingiva and without disturbing the plaque film on the remaining sites . the supragingival plaque samples were transferred into flip - capped vials containing 250 l of reduced transport fluid ( rtf ) . all samples were transferred to the laboratory and processed within 3 h. the samples were pooled in 250 l of rtf and serial ten - fold dilutions were prepared in the same medium . dilutions of 10 - 10 were plated in duplicate using a spiral plater onto three different media : mitis - salivarius ( ms ) agar was used to determine the total count of streptococci , mitis - salivarius - bacitracin ( msb ) agar was used as the selective medium for differentiation and enumeration quantification of s. mutans and cdc anaerobe 5% sheep blood agar ( bd ) for enumeration of total recoverable anaerobic bacteria . after 3 days of aerobic incubation at 37c for ms and msb agar plates and 6 days of anaerobic incubation ( gas pack ez system , bd ) at 37c for blood agar plates , the number of colony - forming units ( cfu ) was counted . the total count of microorganism was determined on countable ( from 30 to 300 colonies ) plates . the p values report concern the interaction between time and position of the bracket , included in mixed linear models fitted to the microbiological and clinical outcomes . the models included other possibly relevant fixed effects ( e.g. side of the mouth ) , and the patient identity as a random effect . the r statistical package ( r development core team , wien , austria ) was used for computation . twenty dental students ( 14 females and 6 males , caucasians aged between 20 and 32 years ) were involved in the study ( table 1 ) . they were given a written explanation of the background of the study and its objectives . after screening for their suitability and after good comprehension of the protocol , they all gave their written informed consent . during the experiment before the study , all students received oral hygiene instructions in order to ensure a healthy periodontal situation . study population with data on gender distribution , previous orthodontic treatment and age sd= standard deviation the initial placement of the brackets was performed via a randomized protocol by means of concealed envelopes . the students were selected to fulfill the following inclusion criteria : no smoking , absence of extensive dental restorations or adhesive - fixed partial dentures , a sulcus bleeding index of < 0.3 and no antibiotics during or up to 4 months before the study . the students were also asked whether they had already received an orthodontic treatment with fixed appliances because this might have consequences for smoothness of the buccal enamel and as such on the microbial adhesion in the early formation of a dental plaque film . the study had a randomized , examiner - blind , split - mouth design . in every student , the mouth was divided into four quadrants , two of which served as controls . one type of bracket ( 2d , forestadent , pforzheim , germany ) was bonded in two different sites : buccal and lingual . for the split - mouth comparison , 8 sites were defined , namely the canine and the first premolar of each quadrant . the first quadrant used for bracket placement and the order in which the brackets were placed were randomly chosen by means of concealed envelopes , the second one was at the other side of the mouth in the antagonistic jaw . the buccal and lingual bonded teeth were alternated , giving rise to four different experimental settings ( figure 1 ) : the four different clinical conditions of the splitmouth design -buccal position in the first quadrant , lingual position in the third quadrant ; -buccal position in the second quadrant , lingual position in the fourth quadrant ; -buccal position in the third quadrant , lingual position in the first quadrant ; -buccal position in the fourth quadrant , lingual position in the second quadrant . the teeth bonded with the different brackets were compared with each other and with the non - bonded control sites . during the study period , the students visited the clinic three times : t0 ( baseline ) : to record the status of the periodontium ( periodontal pocket depth : ppd ; bleeding on probing : bop ) , to collect samples of dental plaque from the teeth and to place the brackets ; t1 ( day 7 ) : to record the periodontal status ( ppd and bop ) and to collect from the test teeth and from control teeth ; t2 ( day 30 ) : to record the periodontal status ( ppd and bop ) , to collect samples of dental plaque from the test teeth and the control teeth , and to remove the brackets . the study had a randomized , examiner - blind , split - mouth design . in every student , the mouth was divided into four quadrants , two of which served as controls . one type of bracket ( 2d , forestadent , pforzheim , germany ) was bonded in two different sites : buccal and lingual . for the split - mouth comparison , 8 sites were defined , namely the canine and the first premolar of each quadrant . the first quadrant used for bracket placement and the order in which the brackets were placed were randomly chosen by means of concealed envelopes , the second one was at the other side of the mouth in the antagonistic jaw . the buccal and lingual bonded teeth were alternated , giving rise to four different experimental settings ( figure 1 ) : the four different clinical conditions of the splitmouth design -buccal position in the first quadrant , lingual position in the third quadrant ; -buccal position in the second quadrant , lingual position in the fourth quadrant ; -buccal position in the third quadrant , lingual position in the first quadrant ; -buccal position in the fourth quadrant , lingual position in the second quadrant . the teeth bonded with the different brackets were compared with each other and with the non - bonded control sites . during the study period , the students visited the clinic three times : t0 ( baseline ) : to record the status of the periodontium ( periodontal pocket depth : ppd ; bleeding on probing : bop ) , to collect samples of dental plaque from the teeth and to place the brackets ; t1 ( day 7 ) : to record the periodontal status ( ppd and bop ) and to collect from the test teeth and from control teeth ; t2 ( day 30 ) : to record the periodontal status ( ppd and bop ) , to collect samples of dental plaque from the test teeth and the control teeth , and to remove the brackets . the teeth were rinsed , dried with an oil - free air syringe , and etched with 37% phosphoric acid for 30 s. after a thorough washing , they were completely dried with an oil - free air syringe . then , stainless steel brackets ( 2d , forestadent , pforzheim , germany ) were bonded to the selected teeth with an adhesive system ( transbond xt , 3 m , monrovia , ca , usa ) , according to the manufacturer 's directions . after applying the primer on the etched enamel , a small amount of composite resin was placed on the mesh pad of the bracket . the bracket was positioned on the buccal or lingual surface of the teeth with sufficient pressure to squeeze excess adhesive , which was removed from the margins of the bracket base with an explorer before polymerization . bracket was then light - cured with a visible light - curing unit ( ortholux xt , 3 m unitek ) for 10 s on the mesial side of the bracket and for 10 s on the distal side ( total cure time 20 s ) . verbal and written instructions regarding the appliance care and hygiene protocols were issued to each patient , along with a specific request to return if a bracket became loose or if any problem arose with the appliance . ppd and bop were scored at baseline , day 7 and day 30 . to record the ppd , a millimeter probe ( hu - friedly pc puns , chicago , il , usa ) the pocket depths were measured at the buccal , lingual , mesial and distal sides of the tooth and rounded off to the nearest 0.5 mm . bop was recorded ( 0 : absent ; 1 : present ) 24 s after determination of ppd . at baseline , on days seven and thirty microbial samples were also taken using a sterile curette ( sg 5/6 hu - friedy ) from the test and the control teeth . this was carried out without traumatizing the gingiva and without disturbing the plaque film on the remaining sites . the supragingival plaque samples were transferred into flip - capped vials containing 250 l of reduced transport fluid ( rtf ) . all samples were transferred to the laboratory and processed within 3 h. the samples were pooled in 250 l of rtf and serial ten - fold dilutions were prepared in the same medium . dilutions of 10 - 10 were plated in duplicate using a spiral plater onto three different media : mitis - salivarius ( ms ) agar was used to determine the total count of streptococci , mitis - salivarius - bacitracin ( msb ) agar was used as the selective medium for differentiation and enumeration quantification of s. mutans and cdc anaerobe 5% sheep blood agar ( bd ) for enumeration of total recoverable anaerobic bacteria . after 3 days of aerobic incubation at 37c for ms and msb agar plates and 6 days of anaerobic incubation ( gas pack ez system , bd ) at 37c for blood agar plates , the number of colony - forming units ( cfu ) was counted . the total count of microorganism was determined on countable ( from 30 to 300 colonies ) plates . the p values report concern the interaction between time and position of the bracket , included in mixed linear models fitted to the microbiological and clinical outcomes . the models included other possibly relevant fixed effects ( e.g. side of the mouth ) , and the patient identity as a random effect . the r statistical package ( r development core team , wien , austria ) was used for computation . the numbers of streptococci , anaerobic and total cfu in supragingival plaque samples during the experimental period showed no significant differences days combined ( table 2 ) . buccal sites in general allowed equal plaque formation than the lingual sites ( p>0.05 ) . the upper part displays the means per site ; the lower part the differences between the sites with the corresponding p - values . sd= standard deviation in table 3 , the results are separately depicted per day and per material . buccal sites showed no significant differences from lingual sites ( p>0.05 ) for either streptococci , anaerobe or total cfu counts . the mean pocket depth ( ppd ) measurements in millimeters , displayed per day and per site with the corresponding standard deviations ( sd ) no significant inter - material differences in ppd were present among the various groups . no significant increase in ppd was recorded ( p>0.05 ) on days 7 and 30 for either buccal or lingual bracket position and for control sites . no significant differences ( p>0.05 ) in bop sites were detected among the different groups at different times . the numbers of streptococci , anaerobic and total cfu in supragingival plaque samples during the experimental period showed no significant differences days combined ( table 2 ) . buccal sites in general allowed equal plaque formation than the lingual sites ( p>0.05 ) . the upper part displays the means per site ; the lower part the differences between the sites with the corresponding p - values . sd= standard deviation in table 3 , the results are separately depicted per day and per material . buccal sites showed no significant differences from lingual sites ( p>0.05 ) for either streptococci , anaerobe or total cfu counts . the mean pocket depth ( ppd ) measurements in millimeters , displayed per day and per site with the corresponding standard deviations ( sd ) no significant inter - material differences in ppd were present among the various groups . no significant increase in ppd was recorded ( p>0.05 ) on days 7 and 30 for either buccal or lingual bracket position and for control sites . no significant differences ( p>0.05 ) in bop sites were detected among the different groups at different times . the present experiment with split - mouth design did not detect any significant difference in periodontal and microbiological parameters between the bonded teeth and the non - bonded control teeth or between the groups with brackets bonded onto buccal and lingual sides . a number of studies have investigated the influence of orthodontic therapy and appliances on the oral microbial flora . these changes could potentially have a significant impact on patient oral health , including gingival inflammation and demineralization of teeth . moreover , in literature , orthodontic therapy was associated with 0.03 millimeters of gingival recession , 0.13 mm of alveolar bone loss and 0.23 mm of increased pocket depth when compared with no treatment . the effects of orthodontic therapy on gingivitis and attachment loss are inconsistent across studies . ( 2002 ) evaluated microbial profile on metallic and ceramic bracket materials and found that composition of dental plaque formed on each bracket type is very similar between the two bracket types and may be of limited clinical significance . furthermore , the differences detected do not favor one bracket type over another with respect to bacterial accumulation . other authors evaluated influence of bracket design on microbiological and periodontal parameters showing both anaerobe and aerobe colony - forming units significantly higher for self - ligating brackets than for conventional brackets . microbial and periodontal parameters have been evaluated also for orthodontic bands , conventional stainless steel brackets , ceramic attachments , and self - ligating brackets . in literature these brackets have been investigated about their laboratory and clinical processes and for particular recommendations on oral hygiene . those authors showed that special emphasis should be placed on the oral hygiene of patients with lingual brackets and that excellent oral hygiene is possible in patients with lingual devices after instruction and motivation . the results of the present investigation showed no significant differences in total cfu , streptococci cfu and anaerobe cfu counts among buccal , lingual and control sites . this is in agreement with a previous investigation that evaluated cfu of buccal brackets versus control non - bonded sites and showed no significant differences between the two groups . there are also authors that found significant cfu count decrease or increase in bracket sites compared with control sites . this variability is probably due to the different testing conditions and multiple variables correlated with clinical researches . moreover , in the present study , ppd and bop measurements were analyzed and showed no significant differences among buccal , lingual and control sites . this is in agreement with previous studies that evaluated these periodontal parameters using conventional brackets : all of them showed no significant increase of bop and ppd after brackets placement . there are also studies that detected significant increase of ppd and bop after bracket placement . this variability could be due to different orthodontic bracket materials that have dissimilar clinical manifestations . the limits of the present study would be that the population of the present investigation was represented by dental students , who are expected to maintain better oral hygiene than the general population . hygiene regimen of subjects in the trial can be reached also in the general population only if adequately motivated . in fact , even if in recent decades , decreasing prevalence in dental caries has been observed worldwide correct teaching of hygiene protocols is crucial , especially during orthodontic treatment . therefore , even if in the present investigation buccal or lingual bracket position did not have significant impact on ppd and bop periodontal parameters , dental health promotion should be fully integrated into broadly based health - promoting strategies . oral hygiene another issue is represented by the presence of wires used during the regular orthodontic treatment . the wires and brackets work together to limit hygiene and therefore a further development of the present study could include also this aspect . future investigations should be performed to visualize the potentially different periodontal parameters correlated with different orthodontic bracket systems so that brackets can be designed to reduce plaque adhesion . this study demonstrated the following : 1 . buccal or lingual bracket position does not have a significant impact on ppd and bop periodontal parameters ; buccal or lingual bracket position does not have significant impact on streptococci , anaerobe and total cfu counts . the authors wish to thank 3 m and forestadent for providing the materials tested in this study .

objectivelingual orthodontics is becoming more popular in dental practice . the purpose of the present investigation was to compare plaque formation on teeth bonded with the same bracket onto buccal or lingual surface , with non - bonded control teeth , via an in vivo growth experiment over a 30-day period . material and methodsa randomized controlled trial with split - mouth design was set up enrolling 20 dental students . within each subject sites with buccal and lingual brackets and control sites were followed . clinical periodontal parameters ( periodontal pocket depth : ppd ; bleeding on probing : bop ) were recorded at baseline and on days 1 , 7 and 30 . microbiological samples were taken from the brackets and the teeth on days 1 , 7 and 30 to detect colony - forming units ( cfu ) . total cfu , streptococci cfu and anaerobe cfu were measured . resultsno significant differences ( p>0.05 ) were found between buccal and lingual brackets in terms of clinical periodontal parameters and microbiological values . conclusionbracket position does not have significant impact on bacterial load and on periodontal parameters .

INTRODUCTION MATERIAL AND METHODS Subjects Experimental procedure Experimental design Bracket placement/removal Periodontal parameters Culture techniques Statistical analysis RESULTS Microbiological parameters Periodontal parameters PPD BOP DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS

accordingly , the purpose of the present investigation was to evaluate the influence of buccal and lingual orthodontic appliances on microbiological and periodontal parameters of bonded teeth . the null hypothesis of the study was that there is no significant difference in terms of microbiological environment and clinical periodontal parameters between buccal and lingual brackets . the teeth bonded with the different brackets were compared with each other and with the non - bonded control sites . during the study period , the students visited the clinic three times : t0 ( baseline ) : to record the status of the periodontium ( periodontal pocket depth : ppd ; bleeding on probing : bop ) , to collect samples of dental plaque from the teeth and to place the brackets ; t1 ( day 7 ) : to record the periodontal status ( ppd and bop ) and to collect from the test teeth and from control teeth ; t2 ( day 30 ) : to record the periodontal status ( ppd and bop ) , to collect samples of dental plaque from the test teeth and the control teeth , and to remove the brackets . the bracket was positioned on the buccal or lingual surface of the teeth with sufficient pressure to squeeze excess adhesive , which was removed from the margins of the bracket base with an explorer before polymerization . the teeth bonded with the different brackets were compared with each other and with the non - bonded control sites . during the study period , the students visited the clinic three times : t0 ( baseline ) : to record the status of the periodontium ( periodontal pocket depth : ppd ; bleeding on probing : bop ) , to collect samples of dental plaque from the teeth and to place the brackets ; t1 ( day 7 ) : to record the periodontal status ( ppd and bop ) and to collect from the test teeth and from control teeth ; t2 ( day 30 ) : to record the periodontal status ( ppd and bop ) , to collect samples of dental plaque from the test teeth and the control teeth , and to remove the brackets . the teeth bonded with the different brackets were compared with each other and with the non - bonded control sites . during the study period , the students visited the clinic three times : t0 ( baseline ) : to record the status of the periodontium ( periodontal pocket depth : ppd ; bleeding on probing : bop ) , to collect samples of dental plaque from the teeth and to place the brackets ; t1 ( day 7 ) : to record the periodontal status ( ppd and bop ) and to collect from the test teeth and from control teeth ; t2 ( day 30 ) : to record the periodontal status ( ppd and bop ) , to collect samples of dental plaque from the test teeth and the control teeth , and to remove the brackets . after 3 days of aerobic incubation at 37c for ms and msb agar plates and 6 days of anaerobic incubation ( gas pack ez system , bd ) at 37c for blood agar plates , the number of colony - forming units ( cfu ) was counted . no significant increase in ppd was recorded ( p>0.05 ) on days 7 and 30 for either buccal or lingual bracket position and for control sites . no significant differences ( p>0.05 ) in bop sites were detected among the different groups at different times . no significant increase in ppd was recorded ( p>0.05 ) on days 7 and 30 for either buccal or lingual bracket position and for control sites . the present experiment with split - mouth design did not detect any significant difference in periodontal and microbiological parameters between the bonded teeth and the non - bonded control teeth or between the groups with brackets bonded onto buccal and lingual sides . the results of the present investigation showed no significant differences in total cfu , streptococci cfu and anaerobe cfu counts among buccal , lingual and control sites . therefore , even if in the present investigation buccal or lingual bracket position did not have significant impact on ppd and bop periodontal parameters , dental health promotion should be fully integrated into broadly based health - promoting strategies . buccal or lingual bracket position does not have a significant impact on ppd and bop periodontal parameters ; buccal or lingual bracket position does not have significant impact on streptococci , anaerobe and total cfu counts .

one of the key questions in understanding plant development is how single cells behave during organ differentiation and growth . ideally , cellular events , like gene expression patterns and intracellular protein localization , can be seen in the light of a larger context of the tissue . this objective poses technical challenges and requires whole organ observation with a high spatial as well as temporal resolution over prolonged periods of time , which may cause photo - toxicity effects . since plants quickly adapt to environmental changes , the growing conditions must be tightly controlled . in order to do long - term imaging without interfering with the physiological state of the plant , three things have to be ensured , 1 ) growing conditions in the sample chamber , 2 ) stable sample mounting over long periods of time , and 3 ) the imaging with low light intensities to avoid photo - damage and non - physiological conditions . physiological growing conditions in the microscope specimen chamber are crucial for long - term experiments . there are a number of protocols available that describe imaging growth chambers for confocal microscopes . however , confocal microscopy introduces high light - intensity to the plant , which can cause stress responses and usually inhibits the growth . in addition , most conventional microscopes allow only horizontal positioning of the sample , which is not optimal for plants since they try to reorient themselves and grow towards the vector of gravity . over the past ten years , light - sheet microscopy has emerged as a powerful tool to capture the development of large specimens at cellular resolution for time periods of up to several days . light - sheet microscopy allows positioning the specimen vertically and is increasingly used in plant research studying root development , recently reviewed by berthet and maizel . many of the mentioned studies were optimized and conducted in the laboratory of ernst h.k . stelzer employing a special way of sample mounting characterized by growing the root on the surface of a gel . in these studies , a custom - made microscope was used , in which the plant is held from the bottom . in contrast , the majority of broadly available light - sheet microscopes hold the sample from the top . the method presented here provides a protocol for the well - established on - surface mounting method applicable for the openspim , an open access platform for applying and enhancing selective plane illumination microscopy ( spim ) . the overall goal of this protocol is to enable long - term imaging of arabidopsis roots in the openspim light - sheet microscope . this is accomplished by growing a plant upright on the surface of a gel with the roots in a liquid medium while the leaves remain in the air . in order to ensure photosynthetic activity of the plant , a custom - made lighting system illuminates the leaves but not the roots ( figure 1 ) . prepare ms medium ( half - strength murashige and skoog medium ) by adding 2.15 g ms - medium , 10 g sucrose , 0.97 g mes ( 2-(n - morpholino)ethanesulfonic acid ) and 1 l ddh2o ( double - distilled water ) into a 1 l bottle . adjust the ph to 5.8 using koh . add 15 g / l gellan gum to the ms medium and pour 30 ml of the hot medium into square petri dishes ( 245 x 245 x 25 mm ) to create a layer of gel with a thickness of about 2 mm . let the dishes cool down to room temperature to allow the medium to solidify . put sterilized arabidopsis seeds into a 1.5 ml reaction tube containing 1 ml sterile h2o . pick up the seeds using a glass pipet or a 1,000 l pipet tip and sow them onto the surface of the gel . cultivate the plate in a growth incubator , e.g. at 22 c in a 16/8 h day / night cycle with 120 - 140 mol / m/s amount of light for 6 days . note : the sample holder can be either 3d printed or hand made in a machine workshop using the dimensions depicted in figure 2c . see the link for ordering the 3d print in the material list . add 5 g low - melt agarose into a 50 ml bottle containing 50 ml ms medium and autoclave it for 20 min at 121 c . store at 4 c ( can be used for at least two months ) . melt an aliquot of 1%% low - melt agarose at 80 c and let it cool down to 33 c . clean the sample holder in an ultrasound unit . sterilize the sample holder with 70% ethanol and wash with sterile water . cut the gel around the plant using a scalpel . lift the block with a flat spatula and slide it carefully on the sample holder using a second spatula . glue the gel on the sample holder with 1% agarose ( at 33 c ) using a 100 l pipette . use a stereo microscope to verify that the leaves are not covered with gel . do not position the gel directly on the region of interest . to prevent the plant from drying out , insert the sample holder into a 1,000 l pipet tip whenever possible . use the pipet tip as a cap and slide it carefully over one end of the sample holder where the plant is located . put the sample holder in a pipette tip box and prepare more plants if necessary . the technical details required to build the led - ring can be found in figure 3 and the material list . screw or glue ( e.g. double sided tape ) the led ring on the lower side of the openspim x / y / z/-stage arm . connect the led ring with an adjustable power supply ( 0 - 30 v , max 2 a ) . adjust the voltage to the desired light intensity ( for arabidopsis , 120 - 140 mol / m / s , figure 3d ) . connect the perfusion tubes to the sample chamber in a one - way arrangement . put a 1 l bottle containing fresh ms medium and another empty bottle next to the peristaltic perfusion pump . connect the bottle with medium with the lower inlet of the sample chamber using one perfusion tube . connect the upper outlet of the sample chamber with the empty bottle to trash the used medium using another perfusion tube . set the speed of flow to 1 ml / min . note : to not overspill the sample chamber , it is important to have a higher outflow than the inflow . either increase the pumping rate or use a tube with a larger inner diameter for the outflow . cut a black plastic foil into 3 mm small squares , wash with 70% ethanol and let them dry before placing them in the sample chamber on the water surface . remove the pipet tip from the sample holder and insert the sample holder in the sample chamber . if the newly manufactured sample holder does not fit into the stage arm , use a fine sandpaper to make it thinner or use an o - ring ( 6 mm ) in case it is too thin . to create the lids , cut the black aluminum foil into two 50 x 25 mm pieces . close the sample chamber with the two lids by putting the lids on top of the sample chamber with the triangle indentations facing the sample holder . make sure that the plant leaves are not in shade of the lids and receive light from the illumination system . find the region of interest using the x / y / z and rotation stage to position an emerging lateral root in the field of view . before recording , setup the image acquisition . set a stack of 217 images with 3 m z - spacing ( 650 m ) and set the time lapse with 15 min imaging interval for a total period of 17 h. note : a detailed documentation on how to operate the openspim software can be found on ( http://openspim.org/acquisition#acquiring_a_stack ) . set a stack of 217 images with 3 m z - spacing ( 650 m ) and set the time lapse with 15 min imaging interval for a total period of 17 h. note : a detailed documentation on how to operate the openspim software can be found on ( http://openspim.org/acquisition#acquiring_a_stack ) . prepare ms medium ( half - strength murashige and skoog medium ) by adding 2.15 g ms - medium , 10 g sucrose , 0.97 g mes ( 2-(n - morpholino)ethanesulfonic acid ) and 1 l ddh2o ( double - distilled water ) into a 1 l bottle . adjust the ph to 5.8 using koh . add 15 g / l gellan gum to the ms medium and pour 30 ml of the hot medium into square petri dishes ( 245 x 245 x 25 mm ) to create a layer of gel with a thickness of about 2 mm . let the dishes cool down to room temperature to allow the medium to solidify . put sterilized arabidopsis seeds into a 1.5 ml reaction tube containing 1 ml sterile h2o . pick up the seeds using a glass pipet or a 1,000 l pipet tip and sow them onto the surface of the gel . cultivate the plate in a growth incubator , e.g. at 22 c in a 16/8 h day / night cycle with 120 - 140 mol / m/s amount of light for 6 days . note : the sample holder can be either 3d printed or hand made in a machine workshop using the dimensions depicted in figure 2c . see the link for ordering the 3d print in the material list . add 5 g low - melt agarose into a 50 ml bottle containing 50 ml ms medium and autoclave it for 20 min at 121 c . store at 4 c ( can be used for at least two months ) . melt an aliquot of 1%% low - melt agarose at 80 c and let it cool down to 33 c . clean the sample holder in an ultrasound unit . sterilize the sample holder with 70% ethanol and wash with sterile water . cut the gel around the plant using a scalpel . lift the block with a flat spatula and slide it carefully on the sample holder using a second spatula . glue the gel on the sample holder with 1% agarose ( at 33 c ) using a 100 l pipette . use a stereo microscope to verify that the leaves are not covered with gel . do not position the gel directly on the region of interest . to prevent the plant from drying out , use the pipet tip as a cap and slide it carefully over one end of the sample holder where the plant is located . put the sample holder in a pipette tip box and prepare more plants if necessary . the technical details required to build the led - ring can be found in figure 3 and the material list . screw or glue ( e.g. double sided tape ) the led ring on the lower side of the openspim x / y / z/-stage arm . connect the led ring with an adjustable power supply ( 0 - 30 v , max 2 a ) . adjust the voltage to the desired light intensity ( for arabidopsis , 120 - 140 mol / m / s , figure 3d ) . connect the perfusion tubes to the sample chamber in a one - way arrangement . put a 1 l bottle containing fresh ms medium and another empty bottle next to the peristaltic perfusion pump . connect the bottle with medium with the lower inlet of the sample chamber using one perfusion tube . connect the upper outlet of the sample chamber with the empty bottle to trash the used medium using another perfusion tube . set the speed of flow to 1 ml / min . note : to not overspill the sample chamber , it is important to have a higher outflow than the inflow . either increase the pumping rate or use a tube with a larger inner diameter for the outflow . cut a black plastic foil into 3 mm small squares , wash with 70% ethanol and let them dry before placing them in the sample chamber on the water surface . remove the pipet tip from the sample holder and insert the sample holder in the sample chamber . if the newly manufactured sample holder does not fit into the stage arm , use a fine sandpaper to make it thinner or use an o - ring ( 6 mm ) in case it is too thin . to create the lids , close the sample chamber with the two lids by putting the lids on top of the sample chamber with the triangle indentations facing the sample holder . make sure that the plant leaves are not in shade of the lids and receive light from the illumination system . find the region of interest using the x / y / z and rotation stage to position an emerging lateral root in the field of view . before recording , setup the image acquisition . set a stack of 217 images with 3 m z - spacing ( 650 m ) and set the time lapse with 15 min imaging interval for a total period of 17 h. note : a detailed documentation on how to operate the openspim software can be found on ( http://openspim.org/acquisition#acquiring_a_stack ) . set a stack of 217 images with 3 m z - spacing ( 650 m ) and set the time lapse with 15 min imaging interval for a total period of 17 h. note : a detailed documentation on how to operate the openspim software can be found on ( http://openspim.org/acquisition#acquiring_a_stack ) . this sample preparation method allows the cultivation of the plant inside the microscope sample chamber while observing the root system with a light - sheet microscope ( figure 1 ) . the plant grows on the surface of a layer of gel ( ms medium containing 1.5% gellan gum ) mounted on a custom designed sample holder ( figure 2 ) . the roots are immersed in liquid ( ms medium ) , which is continuously refreshed by a perfusion system . the leaves remain in the air and are continuously illuminated with a light intensity of 130 mol / m/s coming from blue and red leds that are arranged in a ring above the plant ( figure 1a , b and figure 3a - c ) . the led ring is manufactured in our machine workshop and we provide technical details on how to build the led - ring in the figure 3 and the material list . the light intensity can be continuously adjusted ranging from 30 - 250 mol / m / s ( figure 3d ) . the root system is shaded by small sheets of a black plastic foil covering the water surface ( figure 1 ) . any stray light from the illumination that is collected by the detection lens is filtered by the gfp filter ( figure 3e ) . with this setup , a time lapse of a growing arabidopsis lateral root was recorded for 17 h using a 20x/0.5 lens ( figure 4 ) . the lateral root has its origin in the pericycle cell layer , which is located deep inside the primary root . in order to demonstrate the imaging capabilities even deeper inside of a tissue for prolonged time periods , a higher magnification ( 40x/0.75 ) was used to capture the formation of a lateral root from the first stage primordium until the emergence out of the primary root within a time period of 38 h ( figure 5 ) . this recording allows us to follow the dynamics of lateral root formation in 3d ( figure 5a ) with a cellular resolution . the plant is growing upright on the surface of a gel , mounted on a custom built sample holder ( see also figure 2 ) . the roots are growing in a liquid medium , which is continuously exchanged by a perfusion system . the plant leaves grow in air and are illuminated by red and blue leds ( see also figure 3 ) . the root system is shaded with small sheets of a black plastic foil covering the water surface . a lid made of two pieces of black aluminum foil further reduces the amount of light below the water surface and maintains humidity in the sample chamber . the magnified panel on the right highlights the plant growing on the surface of a block of gel immersed in the liquid medium . numbers ( 1)-(10 ) in a and b represent : ( 1 ) : x / y / z/-stage with led ring , ( 2 ) : sample holder , ( 3 ) : lid , ( 4 ) : arabidopsis thaliana , ( 5 ) : sheets of black plastic foil , ( 6 ) : perfusion system , ( 7 ) : detection objective lens , ( 8) : liquid medium , ( 9 ) : sample chamber , ( 10 ) : illumination objective lens . b ) photograph of 3d prints using different materials ( 1)-(3 ) : transparent acrylic plastics , ( 4 ) and ( 5 ) : resin , ( 6 ) : transparent resin . pairs of leds can be switched on / off individually for directional lightning . led : light - emitting diode , r : resistance , t : transistor , jp : pinhead . b ) the final design of the illumination lamp was drawn using a pcb - software ( pcb : printed circuit board ) . d ) the range of voltage can be adjusted between 3.5 v and 14.0 v. resistances were used to reach the amount of light ranging from 30 - 250 mol / m / s ( r1 - 8 : 220 ohm , r9 - 12 : 1,220 ohm ) . the 5 days old seedling expresses a membrane marker ( pubq10::yfp - pip1;4 ) and a nuclear reporter ( pgata23::nls - gus - gfp ) specifically marking pericycle cells that develop into a lateral root . a stack of 217 images ( 3 m z - spacing ) was captured every 15 min for 17 h recording using a 20x/0.5 lens . a ) four time points out of 69 are shown in a maximum intensity projection . b ) six out of 217 single slices of a z - stack of one time point are shown . the 6 days old seedling expresses a membrane marker ( pubq10::yfp - pip1;4 ) and a nuclear reporter ( pgata23::nls - gus - gfp ) specifically marking pericycle cells that develop into a lateral root . a stack of 200 images ( 1.5 m z - spacing ) was captured every 15 min for 38 h recording using a 40x/0.75 lens . a ) 3d rendering of four time points , the numbers in the grid represent m , b ) single slice through the central plane of the main root . light sheet fluorescence microscopy has the great advantage to combine low phototoxicity and ultrafast acquisition speed , which can be used to capture a large volume with a high spatio - temporal resolution while keeping the sample in a physiological state . the resolution of a light sheet microscope can be compared to that of a confocal microscope . however , light scattering and absorption occurs along the excitation and emission path individually and the overall image quality can be significantly lower inside opaque tissues compared to the surface . to circumvent this complication one can use the possibility to rotate the sample along the vertical axis and observe the same volume from different directions . but this is not always advantageous , e.g. lateral roots emerge on one side of the root and imaging from behind results in a low image quality without gaining more information . however , the rotation can be principally used to position the sample in the best way . the classic horizontal arrangement of the objective lenses allows for new ways of sample mounting . , the " on the surface of the gel " mounting method has several advantages compared to other mounting methods such as embedding the root inside of a gel . it can also be used to rapidly exchange the entire volume of the sample chamber to apply different media or drugs . 2 ) prior to sample preparation plants grow as they are used to grow in laboratories . plants can be selected under a fluorescence microscope and only the desired plants need to be prepared . 3 ) the plant is transferred from the petri dish to the sample holder without being touched . thereby the plant can further develop on the same gel it was growing on in the growth incubator and mechanical stress is reduced to a minimum . 4 ) the view on the specimen is unobstructed and optical aberrations are minimized because the space between the sample and the detection objective is solely filled with medium and no other materials with differing refractive indices . in order to perform long - term imaging , the plant illumination system is necessary to ensure photosynthetic activity of the plant . in most laboratories plants this may cause different responses to their environment and induces changes in their biochemistry and development . in order to reduce the amount of light on the root system , black plastic foil was used to cover the water surface as well as a lid made of black aluminum foil covered the sample chamber . light can reach the plant leaves through the central hole in the lid . in this setup , no increase in background light was observed , suggesting that the amount of stray light from the red and blue leds was significantly reduced by the gfp filter and the shading approaches . this allowed keeping the light turned on during image acquisition without increasing the camera background noise . the sample holder is designed for 3d printing . however , the choice of material is crucial as several plastics that were tested were not 100% stable , resulting in a drift of the sample . therefore , it is recommended to use resins instead or build the sample holder by milling a polyethylene ( pep ) rod . when using a light sheet microscope setup with double - sided illumination system the sample holder might interfere with one of the light sheets depending on the rotation angle . to reduce mechanical stress during scooping the plant from the plate , use a flat angle of the spatula . the plant can quickly dry out and experience air flow for the very first time . try to avoid any air draft ( rapid movements , air - condition flow ) , work uninterruptedly and slide the sample holder into a 1,000 l pipet tip whenever possible . inside the microscope , it is crucial to not dip the whole plant in liquid and keep the leaves dry . the technique is ideal for imaging early stages of lateral root formation . when performing long term imaging of mature root tips one must keep in mind that arabidopsis roots grow with 100 - 300 m / h rapidly out of the field of view . a very useful future implementation could be an automated tracking algorithm , which would allow following root tip growth over prolonged periods of time . the ability to control environmental conditions such as light and nutrient composition of the medium during the acquisition process allows investigating how plants adapt to changes . the root is in direct contact with the liquid medium , which can be used to apply drugs to chemically activate gene expression , for example using the dexamethasone inducible or the -estradiol inducible system . however , it takes time to exchange the entire volume of the sample chamber to wash out a drug . the setup could be improved by minimizing the volume of the sample chamber to accelerate medium exchange . nevertheless , this technique has a great potential . the combination of mounting procedure , standardized growing conditions and the gentle image acquisition using light - sheet microscopy allows long - term studies of plant development with high resolution at a physiological level .

one of the key questions in understanding plant development is how single cells behave in a larger context of the tissue . therefore , it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time , which may cause photo - toxic effects . this protocol shows a plant sample preparation method for light - sheet microscopy , which is characterized by mounting the plant vertically on the surface of a gel . the plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air . in order to ensure photosynthetic activity of the plant , a custom - made lighting system illuminates the leaves . to keep the roots in darkness the water surface is covered with sheets of black plastic foil . this method allows long - term imaging of plant organ development in standardized conditions .

Introduction Protocol 1. Arabidopsis Culturing Prior to Imaging 2. Plant Sample Preparation Method 3. Set up the Microscope Representative Results Discussion Disclosures

one of the key questions in understanding plant development is how single cells behave during organ differentiation and growth . ideally , cellular events , like gene expression patterns and intracellular protein localization , can be seen in the light of a larger context of the tissue . this objective poses technical challenges and requires whole organ observation with a high spatial as well as temporal resolution over prolonged periods of time , which may cause photo - toxicity effects . in order to do long - term imaging without interfering with the physiological state of the plant , three things have to be ensured , 1 ) growing conditions in the sample chamber , 2 ) stable sample mounting over long periods of time , and 3 ) the imaging with low light intensities to avoid photo - damage and non - physiological conditions . over the past ten years , light - sheet microscopy has emerged as a powerful tool to capture the development of large specimens at cellular resolution for time periods of up to several days . stelzer employing a special way of sample mounting characterized by growing the root on the surface of a gel . in these studies , a custom - made microscope was used , in which the plant is held from the bottom . the overall goal of this protocol is to enable long - term imaging of arabidopsis roots in the openspim light - sheet microscope . this is accomplished by growing a plant upright on the surface of a gel with the roots in a liquid medium while the leaves remain in the air . in order to ensure photosynthetic activity of the plant , a custom - made lighting system illuminates the leaves but not the roots ( figure 1 ) . cut a black plastic foil into 3 mm small squares , wash with 70% ethanol and let them dry before placing them in the sample chamber on the water surface . cut a black plastic foil into 3 mm small squares , wash with 70% ethanol and let them dry before placing them in the sample chamber on the water surface . this sample preparation method allows the cultivation of the plant inside the microscope sample chamber while observing the root system with a light - sheet microscope ( figure 1 ) . the plant grows on the surface of a layer of gel ( ms medium containing 1.5% gellan gum ) mounted on a custom designed sample holder ( figure 2 ) . the leaves remain in the air and are continuously illuminated with a light intensity of 130 mol / m/s coming from blue and red leds that are arranged in a ring above the plant ( figure 1a , b and figure 3a - c ) . the root system is shaded by small sheets of a black plastic foil covering the water surface ( figure 1 ) . in order to demonstrate the imaging capabilities even deeper inside of a tissue for prolonged time periods , a higher magnification ( 40x/0.75 ) was used to capture the formation of a lateral root from the first stage primordium until the emergence out of the primary root within a time period of 38 h ( figure 5 ) . the plant is growing upright on the surface of a gel , mounted on a custom built sample holder ( see also figure 2 ) . the roots are growing in a liquid medium , which is continuously exchanged by a perfusion system . the root system is shaded with small sheets of a black plastic foil covering the water surface . a lid made of two pieces of black aluminum foil further reduces the amount of light below the water surface and maintains humidity in the sample chamber . the magnified panel on the right highlights the plant growing on the surface of a block of gel immersed in the liquid medium . numbers ( 1)-(10 ) in a and b represent : ( 1 ) : x / y / z/-stage with led ring , ( 2 ) : sample holder , ( 3 ) : lid , ( 4 ) : arabidopsis thaliana , ( 5 ) : sheets of black plastic foil , ( 6 ) : perfusion system , ( 7 ) : detection objective lens , ( 8) : liquid medium , ( 9 ) : sample chamber , ( 10 ) : illumination objective lens . light sheet fluorescence microscopy has the great advantage to combine low phototoxicity and ultrafast acquisition speed , which can be used to capture a large volume with a high spatio - temporal resolution while keeping the sample in a physiological state . , the " on the surface of the gel " mounting method has several advantages compared to other mounting methods such as embedding the root inside of a gel . in order to perform long - term imaging , the plant illumination system is necessary to ensure photosynthetic activity of the plant . in order to reduce the amount of light on the root system , black plastic foil was used to cover the water surface as well as a lid made of black aluminum foil covered the sample chamber . a very useful future implementation could be an automated tracking algorithm , which would allow following root tip growth over prolonged periods of time . the combination of mounting procedure , standardized growing conditions and the gentle image acquisition using light - sheet microscopy allows long - term studies of plant development with high resolution at a physiological level .

however , transplantation is limited not only by organ shortage but also by its unfavorable long - term success due to chronic allograft injury ( cai ) [ 1 , 2 ] . chronic allograft vasculopathy ( cav ) , glomerulosclerosis , and interstitial fibrosis and tubular atrophy ( ifta ) are the dominating histopathological findings in cai [ 1 , 2 ] . its pathogenesis is still poorly understood , but acute rejection episodes were defined as an important risk factor and experimental data suggest that they play an important pathogenetic role [ 15 ] . transplantation of kidneys in fischer-344 ( f344 ) to lewis rat strain combination is a well - established experimental model for human cai . we and others thoroughly characterized a variant of this model , where immunosuppression is omitted [ 710 ] . this model allows investigation of a severe but reversible acute rejection episode , which peaks 9 days after allogenic transplantation and precedes the development of cai . acute rejection of f344 to lewis renal allografts is characterized by a strong mononuclear infiltration of the allograft interstitium . at the same time , numerous leukocytes accumulate in the arterial , capillary , and venous blood vessels . after resolution of acute rejection , the number of intravascular leukocytes decreases but remains chronically elevated compared to healthy kidneys . a majority of blood leukocytes in day 9 allografts are monocytes displaying a unique state of partial activation regarding cell surface antigen and cytokine expression . these cells interact with graft endothelial cells and might trigger cav , which is characterized by intimal hyperplasia . to identify factors contributing to the pathogenesis of cai during reversible acute rejection , we compared the transcriptome of mononuclear leukocytes isolated from renal allografts to isografts 9 days after transplantation . in this gene array experiment , tissue transglutaminase ( transglutaminase 2 , tgm2 ) was found among the genes , which were upregulated by mononuclear leukocytes accumulating in the lumina of allograft blood vessels . transglutaminases catalyze posttranscriptional modifications of proteins , resulting in cross - linking of proteins such as cytoskeleton or extracellular matrix ( ecm ) , as well as in modifications via amine incorporation into proteins and via deamidation [ 11 , 12 ] . these modifications can have drastic consequences including increased mechanical stability , formation of autoantigens , and profound changes in protein function [ 11 , 13 , 14 ] . among the 9 transglutaminases present in humans , tgm2 is most intensively investigated and a bewildering functional diversity is described . it acts as classical ca - dependent transglutaminase , as g - protein , as disulfide isomerase , and as protein kinase [ 16 , 17 ] . in addition , it interacts with a plethora of other proteins and is involved in numerous cellular processes such as apoptosis and cell survival , phagocytosis , cell adhesion and migration , and cell signaling . tgm2 is ubiquitously expressed and localizes to virtually all compartments of the cell including the cytoplasm , the nucleus , and mitochondria . in addition , tgm2 can be attached to the surface and is secreted into the extracellular space , where it can cross - link itself to extracellular proteins [ 15 , 18 ] . we are interested in tgm2 in the context of reversible acute rejection and subsequent chronic deterioration of renal allografts . tgm2 might exert essential functions involved in rejection such as antigen presentation and t cell activation via dendritic cells , activation of the nfb pathway , and enabling the expression of proinflammatory factors , leukocyte adhesion to vascular endothelial cells , and leukocyte migration . during resolution of acute rejection , its role in apoptosis , phagocytosis , and release of tgf- may be of importance [ 21 , 22 ] . finally , during the development of cai , tgm2 might protect graft blood vessels as already evidenced in the context of atherosclerosis . alternatively , tgm2 could contribute to vascular remodeling by increasing vascular stiffness and by promoting epithelial / endothelial to mesenchymal transition [ 24 , 25 ] , which was suggested to contribute to ifta . in this study , we investigate tgm2 mrna and protein expression by mononuclear blood leukocytes isolated from lewis to lewis isografts and f344 to lewis allografts and by respective renal tissue tgm2 expressions compared to activated caspase-3 , a protease critically involved in apoptosis . leukocytes and grafts are studied during acute rejection on day 9 after transplantation and after resolution of acute rejection on day 42 . furthermore , allograft recipients were treated with cystamine during the first month after transplantation to assess the role of early extracellular transglutaminase activity in the pathogenesis of cai . male lewis ( rt1 ) ( janvier , st . berthevin , france ) and f344 ( rt1 ) ( harlan winkelmann , borchen , germany ) rats weighing 270300 g were used for transplantation . animals were kept under conventional conditions and received humane care according to the german law on the protection of animals and the principles of laboratory animal care formulated by the national society for medical research as well as the nih guide for the care and use of laboratory animals . experiments were approved by the local authorities ( permit number gi 20/10 number 23/2008 ; gi 20/27 number 51/2010 ) . kidneys were transplanted orthotopically to totally nephrectomized lewis recipients as described previously , except that the ureter was anastomosed end to end . for allogenic transplantation , f344 rats and , for isogenic transplantation , 30 g ampicillin after surgery ( ratiopharm , ulm , germany ) ; no immunosuppressant was given . total ischemic times remained below 30 min . on day 9 or 42 after transplantation application of 60 mg / kg sodium pentobarbital ( narcoren , merial , hallbergmoos , germany ) and grafts were removed , cut in small pieces , and snap frozen in liquid nitrogen until use . allograft recipients were treated with the transglutaminase inhibitor cystamine dihydrochloride ( sigma - aldrich , taufkirchen , germany ) for 28 days starting at the day of transplantation . cystamine was diluted in sterile saline and delivered continuously using subcutaneously implanted osmotic minipumps ( 2ml4 alzet , cupertino , ca ) at a concentration of 12 mg per day . animals were sacrificed 12 weeks after transplantation ; grafts were removed , fixed in 4% buffered paraformaldehyde , and embedded in paraffin . to study renal function , isolation of mononuclear blood cells was described previously in detail [ 28 , 29 ] . injection of 200 u of heparin ( liquemin n 5000 , roche , basel , switzerland ) , and kidneys were intensively perfused with cold ca- and mg - free phosphate buffered saline ( paa , pasching , austria ) , supplemented with 2.7 mm edta and 0.1% bovine serum albumin ( serva , heidelberg , germany ) . to deplete erythrocytes and granulocytes total rna was isolated from 30 mg kidney tissue or from 5 10 mononuclear leukocytes harvested from the blood vessels of control kidneys , isografts , or allografts on day 9 or 42 after transplantation . rna extraction was performed using the rneasy mini kit ( qiagen , hilden , germany ) according to the instructions of the supplier . one g of total rna was reversely transcribed using the m - mlv h reverse transcriptase and 1 g of random hexamer primers ( promega , mannheim , germany ) . the reaction was carried out at 40c for 1 h. real - time rt - pcr was used to assess the mrna gene expression of tgm2 . reactions were performed in an abi 7900 sequence detection system ( applied biosystems , foster city , ca ) using platinum sybr green qpcr super mix - udg ( invitrogen , karlsruhe , germany ) . the relative gene expression values were calculated by the equation 2 , where ct is the difference in ct values between the porphobilinogen deaminase ( pbgd ) housekeeping gene and tgm2 . data for each experimental group are expressed in relation to the expression in the healthy control , which was set to 1 arbitrary unit . all primers for real - time rt - pcr were synthesized by mwg biotech ( ebersberg , germany ) . primer sequences are as follows : for tgm2 , 5-cca gcg tgg aca gac tta ca-3 ( sense ) , 5-ctg ctc cac atc gtc aga ca-3(antisense ) and for pbgd , 5-ggc gca gct aca gag aaa gt-3(sense ) , 5-agc cag gat aat ggc act ga-3(antisense ) . pcr conditions included denaturation for 5 min at 95c , followed by 45 cycles of 20 s at 95c , 20 s at 60c , and 10 s at 72c . to confirm the production of a single amplicon with the expected molecular mass , in addition , each product was sequenced ( seqlab , gttingen , germany ) to confirm the specificity of the pcr . a negative control , where cdna was omitted , was included in every run . in negative controls , protein concentrations were determined using micro bca protein assay kit ( pierce biotechnology , rockford , il ) . equal amounts of protein ( 40 g of tissue extract or 8 g of cellular extract ) were resolved on 8% or 15% reducing sds - polyacrylamide gels and transferred onto polyvinylidene difluoride membranes ( millipore , billerica , ma ) . membranes were blocked in 1x roti - block solution ( roth , karlsruhe , germany ) diluted in 50 mm tris - hcl , ph 7.6 , and 0.9% nacl for 60 min at rt and then incubated overnight at 4c with mouse monoclonal antibodies ( mabs ) to tgm2 ( clone tg100 ) ( thermo fisher scientific , fremont , ca ) diluted 1 : 4000 or 1 : 6000 for cellular extract or kidney tissue , respectively . optionally , membranes were incubated overnight with rabbit anti - active caspase-3 abs ( abcam , cambridge , uk ) diluted 1 : 1000 in roti - block solution . to ensure equal protein loading , membranes were blocked with 5% milk and incubated with mabs to gapdh ( novus biologicals , littleton , co ) , 1 : 20000 . bound antibodies were visualized with horseradish peroxidase - conjugated secondary abs ( dako , glostrup , denmark ) , 1 : 5000 , using the chemiluminescent reagent lumi - light western blotting substrate ( roche , mannheim , germany ) . densitometric analyses were performed using a digital gel documentation system ( biozym , hessisch oldendorf , germany ) . all data of individual samples were divided by the values obtained for gapdh on the same blot . the mean of the tgm2/gapdh or active caspase-3/gapdh ratio of the controls was set to 1 arbitrary unit and each individual value including control values was calculated accordingly . histological sections were stained with acidic orcein 12 weeks after transplantation to visualize the internal and external elastic lamina of arteries and to evaluate a possible effect of cystamine on arterial remodelling . at least 10 arteries of the muscular type were investigated per section . to estimate the relative thickness of arterial media and intima , the ratio of the total vessel diameter , including media and intima , and the diameter of the lumen of the artery were determined . additionally , the percentage of arteries exhibiting intimal hyperplasia was analyzed . histological sections were stained with azocarmine / aniline blue ( azan ) 12 weeks after transplantation and tunica adventitia was measured . to estimate relative thickness of the tunica adventitia , the diameter of outer arterial diameter ( including tunica adventitia , media , intima , and lumen ) was determined and divided by diameter of inner arterial diameter ( including media , intima , and lumen ) . sections of 6 m were dewaxed and rehydrated . to unmask tgm2 immunoreactivity in the fixed tissue , antigen retrieval was performed in 0.01 m sodium citrate buffer ( ph 6.0 ) for 15 min at 120c in a steamer . sections were pretreated with protease xiv ( sigma - aldrich ) for 15 min at rt for detection of cd163 or active caspase-3 . after washing in pbs , ph 7.2 , the paraffin sections were incubated for 30 min at rt with pbs supplemented with 1% bsa and 0.1% nan3 . thereafter , mouse mabs to tgm2 diluted 1 : 1500 or ed2 ( anti - cd163 , serotec , oxford , uk ) abs diluted 1 : 200 or polyclonal rabbit abs to active caspase-3 ( abcam ) diluted 1 : 100 in pbs supplemented with 1% bsa ( serva ) were applied and incubated at 4c overnight . bound , primary tgm2 abs were detected using rabbit anti - mouse immunoglobulin and goat anti - rabbit hrp labeled abs ( dako ) and 3,3-diaminobenzidine ( dab ) ( sigma - aldrich ) . to amplify the signal for ed2 abs , additionally , anti - rabbit envision peroxidase system ( dako ) was applied according to the manufacturer 's instructions . to detect abs bound to active caspase-3 , anti - rabbit envision peroxidase system ( dako ) was used . sections were weakly stained with hemalum and coverslipped with pertex ( medite , burgdorf , germany ) . to detect tgm2-positive monocytes / macrophages , sections were first stained with mabs to tgm2 as described , followed by protease xiv treatment for 15 min at rt and overnight incubation at 4c with mab ed1 directed to a cd68-like antigen ( serotec ) diluted 1 : 500 in pbs supplemented with 1% bsa and 0.1% nan3 . to detect bound primary abs , rabbit anti - mouse immunoglobulin and alkaline phosphatase anti - alkaline phosphatase ( apaap ) ( both from dako ) were used in combination with fast blue ( sigma - aldrich ) as chromogen . sections were evaluated with an olympus bx51 microscope and analysis software ( olympus , hamburg , germany ) . data were analyzed , where applicable , by nonparametric kruskal - wallis test followed by the mann - whitney rank sum test using spss software ( spss software , munich , germany ) . renal transplantation in the f344 to lewis rat strain combination results in accumulation of mononuclear leukocytes in allograft blood vessels [ 9 , 31 ] . to isolate these cells , kidneys were intensively perfused on days 9 and 42 after transplantation . in agreement with the previously published data , we observed a strong increase in the number of mononuclear leukocytes accumulating in the vasculature of allografts on day 9 compared to isografts . furthermore , the number of intravascular leukocytes was reduced on day 42 ; however , it remained still increased compared to day 42 isografts . the difference in cell number was also observed between perfusates from control kidneys and isografts on days 9 and 42 ( figure 1 ) . the cellular composition of intravascular leukocytes obtained by perfusion on day 9 and day 42 was published before [ 9 , 31 ] . the mrna expression of tgm2 was analyzed by real - time rt - pcr in mononuclear leukocytes isolated from renal blood vessels of control animals and isogenic and allogenic renal transplants . real - time rt - pcr led to a single amplicon of the expected molecular mass in all samples analyzed . in negative controls , where cdna was omitted , interestingly , an elevated level of tgm2 mrna was detected in cells collected from allografts on day 9 , as well as on day 42 after transplantation , when compared to the respective isografts ( figure 2(a ) ) . tgm2 mrna expression was also increased in day 9 and day 42 allograft tissue compared to respective isografts . in contrast to graft blood leukocytes , where the expression was stronger on day 9 compared to day 42 , tgm2 tissue expression did not differ between day 9 and day 42 ( figure 2(b ) ) . protein expression of tgm2 was investigated by immunoblotting of lysates of mononuclear intravascular leukocytes isolated from control animals and isogenic or allogenic renal transplants ( figures 3(a ) and 4(a ) ) as well as of total tissue lysates ( figures 3(c ) and 4(c ) ) . expression of tgm2 was detected in all groups investigated as a major band with expected molecular mass of ~70 kda . on day 42 densitometrical analyses revealed elevated levels of tgm2 expression in blood mononuclear leukocytes isolated from isografts on day 9 compared to cells from control kidneys . furthermore , an elevated expression level of tgm2 was detected in perfusates from allografts on day 9 compared to respective isograft samples ( figure 3(b ) ) . in contrast to the results from real - time rt - pcr , no difference in the expression of tgm2 was detected in perfusates on day 42 after transplantation ( figure 4(b ) ) as well as in total kidney tissue on day 9 and day 42 after transplantation ( figures 3(d ) and 4(d ) ) . so far , the expression of tgm2 was analyzed in the total intravascular population of mononuclear leukocytes . to examine tgm2 expression by graft monocytes , we performed immunohistochemical staining on paraffin sections from control kidneys , isografts , and allografts explanted on day 9 after transplantation ( figures 5(a)5(d ) ) . in line with previously published data describing accumulation of blood leukocytes , tgm2-positive cells were most abundant in the vasculature of day 9 allografts ( figure 5(c ) ) . in addition to intravascular leukocytes , blood plasma exhibited tgm2 immunoreactivity , predominantly in allografts . to identify monocytes expressing tgm2 , double - staining experiments with mab ed1 detecting cd68-like antigen were performed ( figure 5(d ) ) . in isograft tissue , tgm2 immunoreactivity was detected in the vascular endothelium of some arteries , veins , and capillaries . in the media of arteries , smooth muscle cells were immunopositive in the adventitia extracellular material and cells with a fibroblast - like shape . in the renal parenchyma , renal tubules , most probably proximal tubules , collecting ducts , and the outer layer of bowman 's capsule were stained with antibodies to tgm2 . the staining was identical to the pattern seen in isografts but it was more intense all over . in the leukocytic infiltrate , no conspicuous additional staining was detected ( data not shown ) . tgm2 was described as a marker for m2 macrophages ; we investigated if graft monocytes also express the classical m2 cell surface marker cd163 . to detect cd163-positive cells , immunohistochemical staining with mab ed2 as expected , we detected a very low number of positive cells in isografts ( figure 5(e ) ) . in contrast , cd163-positive cells were more frequent in allografts ( figure 5(f ) ) . in both groups analyzed , no cd163-positive cells were detected in the lumina of blood vessels . therefore , we analyzed if caspase-3 , a key protease directly involved in apoptosis , is also expressed by intravascular graft leukocytes . to examine activation of caspase-3 during reversible acute rejection , detection of active caspase-3 in renal mononuclear graft leukocytes as well as in graft tissue on days 9 and 42 after transplantation was performed by immunoblotting ( figures 6(a ) , 6(c ) , 7(a ) , and 7(c ) ) . the appearance of the active p19 ( ~19 kda ) and p17 ( ~17 kda ) fragments of caspase-3 was detected . densitometric analyses revealed increased levels of active caspase-3 in mononuclear blood leukocytes from allografts on day 9 ( figure 6(b ) ) . active caspase-3 was also detected in perfusates from day 42 isografts and allografts , but no difference was observed among them ( figures 7(a ) and 7(b ) ) . also , in allograft tissue , elevated levels of active caspase-3 were measured on days 9 and 42 compared to respective isografts ( figures 6(c ) and 7(c ) ) . furthermore , these results were corroborated by immunohistochemical staining of graft tissue on day 9 after transplantation . active caspase-3-positive cells were detected in the graft blood vessels predominantly in allografts veins , whereas caspase-3 immunoreactivity was less prominent in leukocytes accumulating in arteries ( figure 6(d ) ) . to examine the role of tgm2 in the pathogenesis of cai , renal allograft recipients were treated with cystamine , a general inhibitor of tgm , or placebo . creatinine clearance was monitored 4 , 8 , and 12 weeks after transplantation to determine renal function . no differences were observed between placebo- ( 3.4 1.5 at 4 weeks ; 5.1 0.7 at 8 weeks ; 3.0 0.7 at 12 weeks ; n = 6 per group ; data are given as mean standard deviation , ml / min ) and cystamine - treated animals ( 3.9 1.2 at 4 weeks ; 4.4 0.6 at 8 weeks ; 3.5 0.5 at 12 weeks ; n = 6 per group ; data are given as mean standard deviation , ml / min ) independent of the time point investigated . at the end of the study , at 12 weeks after transplantation , animals were sacrificed and relative thickness of arteries was quantified on sections stained with acidic orcein . no significant difference was observed between both experimental groups ( figures 8(a ) , 8(c ) , and 8(d ) ) . furthermore , arteries with intimal hyperplasia were detected in the sham - treated group , whereas cystamine treatment had no effect on intimal remodeling ( figures 8(b ) , 8(c ) , and 8(d ) ) . additionally , histological sections were stained with azocarmine / aniline blue ( azan ) 12 weeks after transplantation to detect interstitial fibrosis . we demonstrate in this study that tgm2 mrna and protein are overexpressed by mononuclear leukocytes , predominantly by activated monocytes , which accumulate in the vascular bed of rat renal allografts ( f344 to lewis ) on day 9 after transplantation . after resolution of acute rejection on day 42 , tgm2 mrna levels are lower but still moderately increased compared to respective isografts , which is not reflected in increased protein levels . tgm2 mrna expression was also induced in graft tissue upon allogenic transplantation . on the protein level , however , no quantitative changes were detected . we focused on the well - characterized population of mononuclear leukocytes , which accumulate in large numbers in the blood vessels of experimental allografts during acute rejection . the number of leukocytes , which were isolated from control kidneys , isografts , and allografts 9 and 42 days after transplantation by intensively perfusing renal blood vessels , nicely corroborated data published previously [ 9 , 31 ] . the acute rejection episode , which peaks at around day 9 after transplantation , is of major interest for the understanding of cai , since clinical and experimental data suggest that acute rejection is an important triggering factor [ 15 ] . as cav , a hallmark of cai , is characterized by arterial intimal hyperplasia , leukocytes interacting with allogenic endothelial cells were suggested to play a pivotal pathogenic role . the number of intravascular leukocytes decreases after reversion of acute rejection in the f344 to lewis rat strain combination but remains elevated for several weeks . first hints regarding the overexpression of tgm2 by mononuclear leukocytes isolated from the blood vessels of allografts on day 9 after transplantation came from gene array data . these data were confirmed by real - time rt - pcr and immunoblotting using antibodies , which detected a major band of the expected molecular mass . immunohistochemistry with the same antibodies revealed that intravascular leukocytes in renal allografts were strongly tgm2-immunoreactive . on tissue sections double - stained with mab ed1 directed to a cd-68-like antigen , an established marker for rat monocytes / macrophages , we identified graft monocytes as strongly tgm2-immunopositive . it might be argued that the increase in tgm2 expression in mononuclear blood leukocytes from day 9 allografts is due to the increase in the proportion of monocytes , which is indeed seen . however , isograft blood leukocytes contain about 50% monocytes and allografts about 70% , whereas tgm2 mrna expression levels increase by about 10-fold , suggesting that the increased proportion of monocytes is not the only explanation for the observed increase in tgm2 mrna levels . interactions of monocytes with graft endothelial cells , which is a prerequisite for intravascular accumulations , may induce tgm2 expression as described previously for monocytes adhering to endothelial cells in vitro . in addition , proinflammatory cytokines produced during acute rejection are known inducers of tgm2 expression [ 14 , 15 , 33 , 34 ] . in graft tissue , which was investigated for comparison , mrna levels were elevated on both days 9 and 42 . tgm2 protein was detected by immunoblotting but , in contrast to tgm2 mrna , no significant increase in protein signals was seen . however , some weak additional bands of lower molecular mass that were only detected in allograft perfusates and tissue would be compatible with an accelerated degradation of tgm2 in allografts . another explanation for the discrepancy between mrna and protein is based on reports in which tgm2 acts as a substrate of itself and forms insoluble cross - linked complexes , which are not detected in conventional immunoblots . for the detection of tgm2 covalently bound to extracellular matrix , unfixed cryostat sections should be stained in future studies . in line with this idea and with our mrna data , tissue sections of renal day the renal expression pattern of tgm2 was in line with other publications on rodent and human tgm2 expression . recently , shrestha et al . reported upregulation of tgm2 mrna , protein , and transglutaminase activity in cai kidneys . their model also is based on the f344 to lewis rat strain combination but , in contrast to our model , includes a 10-day course of suboptimal immunosuppression , which interferes with early acute rejection , followed by delayed contralateral nephrectomy . our study and the study by these authors complement each other , as they predominantly investigate end - stage cai , whereas we focus on the early phase posttransplantation . evidence increased mrna levels of tgm2 as well as increased tgm2 immunopositivity in allograft tissue sections compared to isografts . of note , these authors also demonstrate that , in addition to blood leukocytes investigated only in our study , renal tubules and glomeruli also overexpress tgm2 in allografts . during the pathogenesis of cai , urinary secretion of the transglutaminase cross - link product (-glutamyl)-lysine is increased . in end - stage cai , this product is detected more abundantly in allografts compared to isografts . from both studies , however , it is difficult to conclude if tgm2 is a marker of cai reflecting changes in the cellular composition of the graft or actively contributes to the pathogenesis of cai as shown for diverse models of experimental renal fibrosis [ 3740 ] . tgm2 was recently described to be a reliable marker of m2 macrophages , an anti - inflammatory subpopulation of differentiated macrophages . to the best of our knowledge , tgm2 expression by blood monocytes in vivo has not been described before , although , similarly to macrophages , blood monocytes can be activated and express cytokine patterns and cell surface molecules resembling proinflammatory m1 macrophages and anti - inflammatory m2 macrophages . we previously demonstrated that blood monocytes of f344 to lewis renal allografts are activated , acquire an intermediate state of differentiation , and express both cytokines , typical for m1 and for m2 macrophages , during acute rejection . we do not know , however , if this population is a mixture of m1- and m2-like monocytes or if individual monocytes are characterized by an intermediate phenotype . to further characterize these monocytes , we investigated expression of cd163 , a typical m2 cell surface marker , which was not detected on graft monocytes during the peak of acute rejection . hence , we conclude that tgm2 can be expressed by activated monocytes but we can not decide if , similarly to macrophages , tgm2 expression is confined to m2-like monocytes . the functional relevance of tgm2 expression by m2 macrophages , however , is still elusive and similarly its role in monocytes is unclear . we described before that monocytes isolated from the blood vessels of renal allografts during fatal acute rejection undergo apoptosis when cultivated ex vivo . as tgm2 is more strongly expressed in apoptotic cells [ 11 , 13 , 44 ] , we investigated if levels of activated caspase-3 correlate with expression of tgm2 in perfusate cells . indeed , a stronger induction of activated caspase-3 was seen in leukocytes isolated from day 9 allografts compared to respective isografts . this suggests that tgm2 expression by blood mononuclear cells might be linked to apoptosis . in line with the idea that tgm2 enhances apoptosis depending on a prolonged contact with endothelial cells in the inflamed allograft , activation of caspase-3 was more visible in veins compared to arteries . tgm2 plays a dual role in apoptosis : expression of tgm2 can induce apoptosis but it also stabilizes the cytoskeleton of dying cells , which prevents cell rupture and release of proinflammatory cytoplasmic components [ 13 , 4446 ] . as a large number of monocytes accumulate in allograft vessels , their apoptosis might be a tremendous anti - inflammatory stimulus capable of reverting rejection . this issue certainly deserves further investigation . in the tissue of day 9 and day 42 allografts , immunohistochemistry , however , evidenced that tgm2 not only is present in the cytoplasm of graft blood monocytes but was also detected in the surrounding blood plasma . due to rejection - associated vascular damage and the high number of leukocytes accumulating in the blood vessels of allografts , we assume that blood flow slowed down considerably in allografts and that endothelial cells and underlying extracellular matrix are in intensive and prolonged contact with products secreted locally . in the context of hypertension , tgm2 and other active transglutaminases were shown to contribute to the pathogenesis of vascular remodeling [ 4750 ] . active transglutaminases were suggested to cross - link extracellular matrix proteins and to result in an increased rigidity of vascular walls , which precedes inward remodeling of arteries [ 4750 ] . in line with this idea , patients suffering from celiac disease , which typically form autoantibodies to tgm2 , are less likely to have a diagnosis of hypertension . it is not known if tgm2 plays a similar role in the pathogenesis of cav . we performed animal experiments to investigate this hypothesis and chronically applied biologically meaningful concentrations of cystamine , an inhibitor of transglutaminase activity , for 4 weeks after transplantation . however , our hypothesis was not supported . within 84 days after surgery , allografts developed first hallmarks of cai , such as ifta , cav , and renal dysfunction , irrespective of cystamine treatment . we can , however , not exclude that extracellular transglutaminase activity is involved in both remodeling and re - remodeling of graft arteries as a protective effect of tgm2 was reported for experimental atherosclerosis [ 23 , 53 , 54 ] . the experimental rat model is characterized by a minor mismatch in the mhc class-1 locus and does not include immunosuppression , which is not typical for clinical transplantation . in spite of these disadvantages , it is a favorable , experimental model to investigate an acute rejection episode preceding cai . we investigate neither transglutaminase activity in the graft nor excretion of its products , and animal experiments were performed using a single dose of transglutaminase inhibitor , which was applied for just one month . we ignore if treatment with other concentrations of cystamine or later time - points would lead to allograft protection . to evaluate the effect of cystamine , we focus on renal function and graft remodeling within 84 days after transplantation . we do not know if , after longer periods of time , differences would have developed among the experimental groups . in addition , cystamine is a weak and rather unspecific inhibitor of tgm2 as it also inhibits other members of the transglutaminase family as well as certain proteases . more specific inhibitors for tgm2 are commercially available . finally , apart from its role in apoptosis , there is still a plethora of potential functions of tgm2 [ 11 , 15 ] , some of which are enumerated in the introduction , that are awaiting investigation in the context of acute and chronic allograft rejection . only a part of these functions are inhibited by exogenous cystamine . in conclusion , this is the first study to demonstrate expression of tgm2 by monocytes activated in vivo during a reversible acute rejection episode . potentially , the function of monocytic tgm2 is induction of apoptosis , which in turn might contribute to the reversion of acute rejection in this experimental model . our data do not support but also do not falsify the hypothesis that tgm2 expressed by graft monocytes during reversible acute rejection contributes to an early induction of cai .

acute rejection is a major risk factor for chronic allograft injury ( cai ) . blood leukocytes interacting with allograft endothelial cells during acute rejection were suggested to contribute to the still enigmatic pathogenesis of cai . we hypothesize that tissue transglutaminase ( tgm2 ) , a multifunctional protein and established marker of m2 macrophages , is involved in acute and chronic graft rejection . we focus on leukocytes accumulating in blood vessels of rat renal allografts ( fischer-344 to lewis ) , an established model for reversible acute rejection and cai . monocytes in graft blood vessels overexpress tgm2 when acute rejection peaks on day 9 after transplantation . concomitantly , caspase-3 is activated , suggesting that tgm2 expression is linked to apoptosis . after resolution of acute rejection on day 42 , leukocytic tgm2 levels are lower and activated caspase-3 does not differ among isografts and allografts . cystamine was applied for 4 weeks after transplantation to inhibit extracellular transglutaminase activity , which did , however , not reduce cai in the long run . in conclusion , this is the first report on tgm2 expression by monocytes in vivo . tgm2 may be involved in leukocytic apoptosis and thus in reversion of acute rejection . however , our data do not support a role of extracellular transglutaminase activity as a factor triggering cai during self - limiting acute rejection .

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

however , transplantation is limited not only by organ shortage but also by its unfavorable long - term success due to chronic allograft injury ( cai ) [ 1 , 2 ] . to identify factors contributing to the pathogenesis of cai during reversible acute rejection , we compared the transcriptome of mononuclear leukocytes isolated from renal allografts to isografts 9 days after transplantation . in this gene array experiment , tissue transglutaminase ( transglutaminase 2 , tgm2 ) was found among the genes , which were upregulated by mononuclear leukocytes accumulating in the lumina of allograft blood vessels . in this study , we investigate tgm2 mrna and protein expression by mononuclear blood leukocytes isolated from lewis to lewis isografts and f344 to lewis allografts and by respective renal tissue tgm2 expressions compared to activated caspase-3 , a protease critically involved in apoptosis . leukocytes and grafts are studied during acute rejection on day 9 after transplantation and after resolution of acute rejection on day 42 . furthermore , allograft recipients were treated with cystamine during the first month after transplantation to assess the role of early extracellular transglutaminase activity in the pathogenesis of cai . in contrast to graft blood leukocytes , where the expression was stronger on day 9 compared to day 42 , tgm2 tissue expression did not differ between day 9 and day 42 ( figure 2(b ) ) . in contrast to the results from real - time rt - pcr , no difference in the expression of tgm2 was detected in perfusates on day 42 after transplantation ( figure 4(b ) ) as well as in total kidney tissue on day 9 and day 42 after transplantation ( figures 3(d ) and 4(d ) ) . to examine tgm2 expression by graft monocytes , we performed immunohistochemical staining on paraffin sections from control kidneys , isografts , and allografts explanted on day 9 after transplantation ( figures 5(a)5(d ) ) . we demonstrate in this study that tgm2 mrna and protein are overexpressed by mononuclear leukocytes , predominantly by activated monocytes , which accumulate in the vascular bed of rat renal allografts ( f344 to lewis ) on day 9 after transplantation . after resolution of acute rejection on day 42 , tgm2 mrna levels are lower but still moderately increased compared to respective isografts , which is not reflected in increased protein levels . the acute rejection episode , which peaks at around day 9 after transplantation , is of major interest for the understanding of cai , since clinical and experimental data suggest that acute rejection is an important triggering factor [ 15 ] . as cav , a hallmark of cai , is characterized by arterial intimal hyperplasia , leukocytes interacting with allogenic endothelial cells were suggested to play a pivotal pathogenic role . from both studies , however , it is difficult to conclude if tgm2 is a marker of cai reflecting changes in the cellular composition of the graft or actively contributes to the pathogenesis of cai as shown for diverse models of experimental renal fibrosis [ 3740 ] . to the best of our knowledge , tgm2 expression by blood monocytes in vivo has not been described before , although , similarly to macrophages , blood monocytes can be activated and express cytokine patterns and cell surface molecules resembling proinflammatory m1 macrophages and anti - inflammatory m2 macrophages . we previously demonstrated that blood monocytes of f344 to lewis renal allografts are activated , acquire an intermediate state of differentiation , and express both cytokines , typical for m1 and for m2 macrophages , during acute rejection . the functional relevance of tgm2 expression by m2 macrophages , however , is still elusive and similarly its role in monocytes is unclear . in the tissue of day 9 and day 42 allografts , immunohistochemistry , however , evidenced that tgm2 not only is present in the cytoplasm of graft blood monocytes but was also detected in the surrounding blood plasma . we performed animal experiments to investigate this hypothesis and chronically applied biologically meaningful concentrations of cystamine , an inhibitor of transglutaminase activity , for 4 weeks after transplantation . we can , however , not exclude that extracellular transglutaminase activity is involved in both remodeling and re - remodeling of graft arteries as a protective effect of tgm2 was reported for experimental atherosclerosis [ 23 , 53 , 54 ] . in conclusion , this is the first study to demonstrate expression of tgm2 by monocytes activated in vivo during a reversible acute rejection episode . our data do not support but also do not falsify the hypothesis that tgm2 expressed by graft monocytes during reversible acute rejection contributes to an early induction of cai .

aggregation or sex pheromones produced by males have been identified for 12 species of longhorned beetles of the subfamily cerambycinae ( coleoptera : cerambycidae ; lacey et al . 2004 , 2007b ; hanks et al . 2007 ; ray et al . 2009a , b , and references therein ) . pheromone components of most of these species are typically six to ten carbons long with hydroxyl or carbonyl groups at c2 and c3 ( lacey et al . minor components that are different from this general structural motif may be important synergists in the pheromone ( fettkther et al . 1995 ; reddy et al . 2005 ; lacey et al . , we describe investigations of the chemical ecology of two sympatric and synchronic cerambycine species of the tribe clytini , xylotrechus colonus ( f. ) and sarosesthes fulminans ( f. ) . larvae of both species develop in woody tissues of stressed , moribund , and damaged ( often wind - thrown ) trees of a variety of hardwood species ( especially of the genera caryae and quercus ; for general biology , see linsley 1964 ) . both species adults are active in spring and summer and are crepuscular , flying from 16:00 to 22:00 h ( esl , personal observation ) . adult male and female x. colonus commonly aggregate , in numbers that may exceed 30 individuals per group , on larval hosts . adult s. fulminans may be present on the same larval hosts , but in much smaller numbers ( esl , personal observation ) . adult male x. colonus and s. fulminans have pores on the surface of the prothorax that are characteristic of pheromone production in other species of cerambycines ( ray et al . male x. colonus sometimes also display a characteristic posture that has been associated with pheromone release in other cerambycine species ( the push - up stance ; lacey et al . we tested the hypotheses that male x. colonus and s. fulminans produce aggregation pheromones that conform to the structural motif of other cerambycines and that complete blends of synthetic pheromone components are more attractive to beetles than individual components . we also tested the responses of both species to traps baited with blends of all regio- and stereoisomers of the main pheromone components . these blends are more economical to synthesize than enantiomerically enriched pheromone components and have proven effective as attractants for other cerambycine species ( hanks et al . 2007 ) . source of insects adult x. colonus and s. fulminans were collected by hand on 6 june 2004 from a wind - thrown white oak , quercus alba l. , at allerton park ( piatt co. , il , usa ) , a 600-ha mixed hardwood forest owned by the university of illinois . adults were housed individually in the laboratory ( 12:12 h l : d , 20c , 50% rh ) in cylindrical cages , constructed of aluminum window screen , with plastic petri dishes at top and bottom , and provided with 10% sucrose in water . olfactometer bioassays we tested for volatile attractants in both species by using a horizontal glass y - tube olfactometer ( 6 cm diameter , main tube 26 cm long , arm length 22 cm , and 70 angle between arms ) . we conducted olfactometer studies outdoors in partial shade because all cerambycine species that we have bioassayed to date did not respond in y - tube olfactometer bioassays conducted under laboratory conditions ( see lacey et al . 2004 , 2007b , 2008 ) . a 2-l plastic chamber , containing a cylinder of aluminum screen for a perch , was attached to each arm of the y - tube . when bioassaying x. colonus , one chamber held six males and the other held six females . only three s. fulminans of each sex were used as odor sources in their respective bioassay , because this species was less abundant . ambient air was pulled through the olfactometer ( air speed 2.5 m s ) with a 0.75 kw vacuum cleaner connected to a variable voltage power supply . for each trial , a beetle was released at the base of the y - tube and allowed 10 min to respond ( crossing a line 18 cm down one arm ) to an odor source . chambers were alternated between y - tube arms every three trials to control for positional bias . the chambers and olfactometer were washed with unscented laboratory detergent ( alconox powder , alconox , inc . we bioassayed 20 x. colonus of each sex between 17:0020:00 h on 9 , 10 , and 13 june 2004 and 13 female and seven male s. fulminans during the same hours on 11 and 12 june 2004 ( skies clear , air temperatures 2430c ) . for each species , numbers of each sex responding to treatments were compared with the goodness - of - fit test corrected for continuity ( sokal and rohlf 1995 ) . identification of pheromone components volatile compounds produced by adult x. colonus and s. fulminans were collected by placing five adult females and males in separate glass vacuum traps ( 0.3 l , manufactured by the glass shop , school of chemical sciences , uiuc ) that were lined with aluminum screen to provide perches . a glass tube ( 6 cm long 9.5 o.d . 4 mm i.d . ) containing 100 mg of 80/100 mesh superq ( alltech associates , deerfield , il , usa ) was attached to one nipple of each chamber with an 8-cm long section of teflon tubing . charcoal - purified air was pulled through the apparatus with a water aspirator ( 1 l min ) . males and females of each species were aerated simultaneously on a laboratory windowsill from 16:00 to 22:00 h : x. colonus on 11 , 12 , and 13 june and s. fulminans on 14 , 15 , and 16 june 2004 . we selected these time periods for collecting volatiles from beetles because they corresponded to activity periods in the field ( esl , personal observation ) . # 27114 , supelco , bellefonte , pa , usa ) with three 0.5-ml aliquots of methylene chloride . the resulting extracts were analyzed with a hewlett - packard ( sunnyvale , ca , usa ) 6890 gas chromatograph ( gc ) coupled to a 5973 mass selective detector ( ms ) . the gc was fitted with a db5-ms column ( 30 m 0.25 mm , 25 m film thickness ; agilent technologies , santa clara , ca , usa ) and programmed from 40c ( held for 1 min ) to 250c at 10c min ( held for 15 min ) , with an injector temperature of 250c . injections were made in the splitless mode . when it became clear that one or more of the compounds in the extract might be thermally unstable ( see leal et al . 2007b ) , the injector temperature was lowered to 100c , and the temperature program was changed to 20c ( held for 2 min ) to 250c at 10c min ( held for 15 min ) . absolute configurations of the insect - produced compounds were determined by analysis of aliquots on a cyclodex - b gc column ( 30 m 0.25 mm , 0.25 micron film thickness , j&w scientific , folsom ca , usa ) with the gc programmed from 50c ( held for 1 min ) to 200c at 5c min , injector 100c , detector 200c . field bioassays of synthetic pheromone in experiment 1 , bioassays of reconstructed blends of male - specific volatiles for each of the two species , as well as of the individual components , were conducted on 9 days between 11 june and 27 july 2006 and 5 days between 29 may and 14 june 2007 . bioassays were carried out at allerton park and brownfield woods ( champaign co. , il ; skies clear to partly cloudy , maximum air temperatures 2230c , wind speed 820 kph ) , the latter a 26-ha mixed deciduous forest owned by the university of illinois . we used two different sites because we observed that trap catches of cerambycine species at individual sites decline over time , possibly because trapping depletes local populations ( esl , unpublished data ) . both of the sites had populations of x. colonus and s. fulminans ( esl , personal observation ) and replications of the experiment were assigned randomly to sites.traps were black cross - vane flight intercept panel traps ( 1.2 m tall 0.30 m wide , intercept , model pt , aptiv , inc . , portland , or , usa ) . pvc pipe ( d1785 , schedule 40 , charlotte pipe and foundry co. , charlotte , nc , usa ) , with a 1.5 m long upright connected with a t fitting to a 20-cm long arm with a loop of wire at the end to which the trap was attached . the upright was mounted on a 1.5 m section of 1.27 cm diameter steel reinforcing bar driven into the ground . traps were positioned 10 m apart in a straight line approximately perpendicular to the prevailing wind direction . traps were set up at 14:00 h and beetles were removed from traps at 11:00 h the following day.enantiomerically enriched hydroxyhexanones ( 94% ee ; lacey et al . 2007a , b ) and hexanediols ( > 98% stereoisomerically pure , lacey et al . we randomly assigned each of the following nine treatments to traps : ( 1 ) x. colonus blend ( see results ) : ( r)-3-hydroxyhexan-2-one ( 8 mg ) , ( s)-3-hydroxyhexan-2-one ( 1.25 mg ) , ( 2r,3r)-2,3-hexanediol ( 2.5 mg ) , and ( 2 s,3 s)-2,3-hexanediol ( 0.5 mg ) ; ( 2 ) s. fulminans blend ( see results ) : ( r)-3-hydroxyhexan-2-one ( 8 mg ) and ( 2 s,3r)-2,3-hexanediol ( 1.25 mg ) ; ( 3 ) ( r)-3-hydroxyhexan-2-one ( 8 mg ) ; ( 4 ) ( s)-3-hydroxyhexan-2-one ( 8 mg ) ; ( 5 ) ( 2 s,3 s)-2,3-hexanediol ( 8 mg ) ; ( 6 ) ( 2r,3r)-2,3-hexanediol ( 8 mg ) ; ( 7 ) ( 2 s,3r)-2,3-hexanediol ( 8 mg ) ; ( 8) ethanol control ( 1 ml ) ; and ( 9 ) blank control ( empty emitter).pheromones were loaded into release devices that consisted of modified 1.5-ml microcentrifuge tubes ( # 05 - 406 - 16 ; fisher scientific , pittsburg , pa , usa ) . a section of polyethylene tubing ( 25 mm long , 4 mm o.d . , 2 mm i.d . ) was inserted through a 4 mm hole drilled through the tube cap and cemented in place with quick setting epoxy glue such that 8 mm of tubing extended beyond the cap . a 5-cm long section of pipe cleaner ( cotton fiber and steel wire , 2.5 mm diameter ) was inserted through the tubing such that one end reached the bottom of the microcentrifuge tube and the other end extended 5 mm beyond the end of the polyethylene tubing ( modified from hanks et al . 2007 ) and the lures released 1 ml of ethanol solution over the course of 1 day under field conditions ( unpublished data ) . lures were hung with wire in the open central slot of panel traps.differences among treatments in numbers of beetles captured ( sexes combined ) were tested with the nonparametric friedman s test ( blocked by site and day ; proc freq with cmh option , sas institute , 2001 ) because assumptions of analysis of variance were violated by heteroscedasticity ( sokal and rohlf 1995 ) . differences between pairs of means were tested with the regwq means - separation test to control maximum experiment - wise error rates ( sas institute , 2001 ) . replications that captured fewer than two beetles overall ( n = 9 ) were excluded from the statistical analyses for a given species . differences between numbers of females and males captured within treatments were tested with the goodness - of - fit test ( sokal and rohlf 1995).we conducted a further trial ( experiment 2 ) to test the responses of x. colonus and s. fulminans to blends of stereoisomers of the chemicals to determine whether unnatural regio- and/or stereoisomers had an inhibitory effect . this bioassay was conducted on 25 days between 13 june31 august 2006 and 18 days between 5 may15 august 2007 at allerton park and brownfield woods using the methods described above . ( 2007 ) and diluted to 1 ml with absolute ethanol ) : ( 1 ) 1:1:1:1 blend of enantiomers of 3-hydroxyhexan-2-one and 2-hydroxyhexan-3-one ( 100 mg total load , 25 mg of each isomer ; henceforward referred to as generic hydroxyhexanones ) ; ( 2 ) 1:1:1:1 blend of all four stereoisomers of 2,3-hexanediol ( 100 mg total load , 25 mg of each isomer ; henceforward generic hexanediols ) ; ( 3 ) 1:1 combination of generic hydroxyhexanones and generic hexanediols ( 200 mg total load ) ; ( 4 ) ethanol control ( 1 ml ) ; and ( 5 ) blank control ( empty emitter).the mixtures were loaded into lures and differences among treatments in numbers of beetles captured per species were tested as in the previous bioassay . replications that captured fewer than two beetles of a given species were excluded from statistical analysis ( n = 28 ) . on days when enantiomerically enriched pheromones and generic pheromones were bioassayed simultaneously , trap lines were separated by > 1 km . olfactometer bioassays in olfactometer bioassays , 17 female x. colonus were attracted to odors emitted by live males , compared to only two that responded to odors of live females ( = 11.8 , p < 0.001 ) . male x. colonus showed a similar response , with 16 responding to odors of live males compared to only one responding to odors from live females ( = 13.2 ; p < 0.001 ) . both sexes of s. fulminans were attracted to odors produced by male conspecifics , with 12 females responding to males and only one responding to females ( = 9.31 ; p < 0.01 ) and all seven males responding to males ( = 7.00 ; p < 0.05 ) . these findings confirmed that males of both species produce volatile pheromones that attract both sexes . identification of pheromone components gc - ms analyses of volatiles collected from male x. colonus revealed two major peaks in the total ion chromatogram that were absent in the analogous headspace of females . the retention time and mass spectrum of the larger peak ( base peak at m / z 55 , other significant fragments at m / z 43 [ 78% ] , 73 [ 53% ] , and 45 [ 26% ] ) matched those of a synthetic standard of 3-hydroxyhexan-2-one . the retention time and mass spectrum of the smaller peak ( base peak at m / z 55 , other significant fragments at m / z 75 [ 10% ] , 73 [ 61% ] , 72 [ 34% ] , 45 [ 26% ] , and 43 [ 35% ] ) matched those of a synthetic standard of 2,3-hexanediol . the stereoisomeric compositions of the insect - produced compounds were determined to be 70% ( r)- and 10% ( s)-3-hydroxyhexan-2-one and 17% ( 2 s,3 s)- and 3% ( 2r,3r)-2,3-hexanediol by analysis on the cyclodex - b column , with the enantiomers being resolved to baseline ( for elution order of all hydroxyketones and 2,3-hexanediols , see ray et al . analogous collection and analysis of headspace volatiles from both sexes of s. fulminans revealed that males produced 83% ( r)-3-hydroxyhexan-2-one and 17% ( 2 s,3r)-2,3-hexanediol as the only detectable male - specific components ( identified as described above ) . field bioassays of synthetic pheromone in experiment 1 , testing chiral synthetic pheromone components , traps captured 79 adult x. colonus of both sexes , but only ten adult s. fulminans ( table 1 ) . treatments differed significantly in numbers of x. colonus captured ( fig . 1 ; friedman s q8,79 = 52.3 , p < 0.001 ) . only traps baited with ( r)-3-hydroxyhexan-2-one alone or in a blend ( i.e. , the x. colonus and s. fulminans blends ) were significantly attractive compared to ethanol and blank controls ( fig . 1 ) . the blend of pheromone components specific for x. colonus captured the greatest number of beetles ( fig . 1 ) . too few adult s. fulminans were captured for statistical analysis of treatments ; however , all ten beetles were captured with lures that contained the most abundant component of its volatile collection , ( r)-3-hydroxyhexan-2-one ( table 1 ) . table 1results of experiment 1 xylotrechus colonussarosesthes fulminansneoclytus a. acuminatustreatment(1 ) x. colonus blend17132143(2 ) s. fulminans blend9932(3 ) ( r)-3-hydroxyhexan-2-one1072(4 ) ( s)-3-hydroxyhexan-2-one53(5 ) ( r , r)-2,3-hexanediol1(6 ) ( s , r)-2,3-hexanediol11(7 ) ( s , s)-2,3,-hexanediol32214(8 ) ethanol control11(9 ) blank controltotal4633732718numbers of cerambycid beetles of three species caught in traps baited with enantiomerically enriched candidate pheromone components ( n = 9 replicates for xylotrechus colonus and sarosesthes fulminans ; n = 10 replicates for n. a. acuminatus ) . the blend of components specific to x. colonus contained ( r)- and ( s)-3-hydroxyhexan-2-one and ( 2r,3r)- and ( 2 s,3 s)-2,3-hexanediol . the s. fulminans blend contained ( r)-3-hydroxyhexan-2-one and ( 2 s,3r)-2,3-hexanediol . treatment numbers correspond to treatments in fig . 1 . sex ratio of total numbers within species were not significantly different from 1:1 ( , p > 0.05).fig . 1mean ( se ) numbers of adult xylotrechus colonus ( n = 9 trials ) captured per trap ( sexes combined ) with respect to composition of the lure ( n = 9 trials ) . the blend of components specific for x. colonus contained ( r)- and ( s)-3-hydroxyhexan-2-one with ( 2r,3r)- and ( 2 s,3 s)-2,3-hexanediol , the sarosesthes fulminans blend contained ( r)-3-hydroxyhexan-2-one and ( 2 s,3r)-2,3-hexanediol . treatment numbers correspond to treatments in table 1 : ( 1 ) x. colonus blend , ( 2 ) s. fulminans blend , ( 3 ) ( r)-3-hydroxyhexan-2-one , ( 4 ) ( s)-3-hydroxyhexan-2-one , ( 5 ) ( 2r,3r)-2,3-hexanediol , ( 6 ) ( 2 s,3r)-2,3-hexanediol , ( 7 ) ( 2 s,3 s)-2,3-hexanediol , ( 8) ethanol control , and ( 9 ) blank control . means with different letters are significantly different ( regwq test ; p < 0.05)during experiment 1 , we also trapped adults of another cerambycine species , neoclytus acuminatus acuminatus ( f. ) ( table 1 ) , the male - produced pheromone of which is composed solely of ( 2 s,3 s)-2,3-hexanediol ( lacey et al . treatments differed in numbers of n. a. acuminatus captured ( friedman s q8,45 = 64.2 , p < 0.001 , ten replicates ) , with only traps baited with ( 2 s,3 s)-2,3-hexanediol catching significantly more beetles ( 3.60 0.54 , sexes combined ) than ethanol and blank controls ( means = 0 for both).in experiment 2 , testing the generic blends of pheromone stereoisomers , we captured 111 adult x. colonus of both sexes ( table 2 ) but only eight s. fulminans . treatments again differed in numbers of adult x. colonus captured ( fig . 2 , top ; friedman s q4,111 = 70.98 , p < 0.001 ) , with significantly greater numbers in traps baited with generic hydroxyketones , alone or in combination with generic hexanediols , than in solvent or blank controls . again , too few adult s. fulminans were captured for statistical analysis , although all were captured in traps with lures that contained generic hydroxyketones ( table 2 ) . hydroxyhexanones2930322213(2 ) hexanediols27381(3 ) ketones + diols232421223295(4 ) ethanol control232(5 ) blank controltotal54575349723119*numbers of cerambycid beetles of four species caught in traps baited with generic pheromone blends ( n = 31 , 28 , 28 , and 18 replicates for xylotrechus colonus , sarosesthes fulminans , n. a. acuminatus , and n. m. mucronatus , respectively ) . the blend of hydroxyhexanones contained equal amounts of ( r)- and ( s)-3-hydroxyhexan-2-one and ( r)- and ( s)-2-hydroxyhexan-3-one , and the blend of hexanediols contained approximately equal amounts of ( 2r,3r)- ( 2 s,3 s)- ( 2r,3 s)- and ( 2 s,3r)-2,3-hexanediol . the treatment designated ketones + diols contained equal amounts of the blends of hydroxyhexanones and hexanediols . 2.*p < 0.05 ( ) next to total number of males indicates sex ratio of beetles captured within species that is significantly different from 1:1fig . 2mean ( se ) numbers of adults ( sexes combined ) of three species of cerambycid beetles captured with respect to composition of the lure : top , xylotrechus colonus ( n = 31 trials ) ; middle , neoclytus a. acuminatus ( n = 28 trials ) ; and bottom , neoclytus m. mucronatus ( n = 18 trials ) . treatment numbers correspond to treatments in table 2 : ( 1 ) generic hydroxyhexanones ; ( 2 ) generic hexanediols ; ( 3 ) 1:1 mixture of generic hydroxyhexanones and hexanediols ; ( 4 ) ethanol control ; and ( 5 ) blank control . means with different letters are significantly different ( regwq test ; p < 0.05)in experiment 2 , traps baited with generic pheromone lures also captured 121 adult n. a. acuminatus of both sexes , as well as 50 adult males and females of a congener , neoclytus m. mucronatus ( f. ) ( table 2 ) . the male - produced pheromone of n. m. mucronatus is composed of only ( r)-3-hydroxyhexan-2-one ( lacey et al . treatments differed in numbers of n. a. acuminatus and n. m. mucronatus captured ( fig . 2 ; friedman s q4,121 = 89.3 , p < 0.001 ; q4,50 = 36.3 p < 0.001 , respectively ) . significantly more n. a. acuminatus were captured in traps baited with generic hexanediols , alone or in combination with hydroxyhexanones ( fig . 2 , middle ) , than in control traps or traps baited with generic hydroxyketones . conversely , adult n. m. mucronatus were only significantly caught in traps baited with generic hydroxyhexanones ( fig . 2 , bottom).in both field bioassays , traps baited with the synthetic pheromone components also captured small numbers ( total catch < 8 individuals ) of an additional 11 species of cerambycids , including seven species in the subfamily cerambycinae ( anelaphus pumilus [ newman ] , cyrtophorus verrucosus [ olivier ] , knulliana cincta [ drury ] , neoclytus caprea [ say ] , neoclytus scutellaris [ olivier ] , phymatodes amoenus [ say ] , xylotrechus convergens leconte ) , three species of the subfamily lamiinae ( liopinus alpha [ say ] , psenocerus supernotatus [ say ] , and urographis fasciatus [ degeer ] ) , and one species of subfamily parandrinae ( neandra brunnea [ f. ] ) . traps captured few insects other than the species reported here . results of experiment 1 numbers of cerambycid beetles of three species caught in traps baited with enantiomerically enriched candidate pheromone components ( n = 9 replicates for xylotrechus colonus and sarosesthes fulminans ; n = 10 replicates for n. a. acuminatus ) . the blend of components specific to x. colonus contained ( r)- and ( s)-3-hydroxyhexan-2-one and ( 2r,3r)- and ( 2 s,3 s)-2,3-hexanediol . the s. fulminans blend contained ( r)-3-hydroxyhexan-2-one and ( 2 s,3r)-2,3-hexanediol . sex ratio of total numbers within species were not significantly different from 1:1 ( , p > 0.05 ) . mean ( se ) numbers of adult xylotrechus colonus ( n = 9 trials ) captured per trap ( sexes combined ) with respect to composition of the lure ( n = 9 trials ) . the blend of components specific for x. colonus contained ( r)- and ( s)-3-hydroxyhexan-2-one with ( 2r,3r)- and ( 2 s,3 treatment numbers correspond to treatments in table 1 : ( 1 ) x. colonus blend , ( 2 ) s. fulminans blend , ( 3 ) ( r)-3-hydroxyhexan-2-one , ( 4 ) ( s)-3-hydroxyhexan-2-one , ( 5 ) ( 2r,3r)-2,3-hexanediol , ( 6 ) ( 2 s,3r)-2,3-hexanediol , ( 7 ) ( 2 s,3 s)-2,3-hexanediol , ( 8) ethanol control , and ( 9 ) blank control . means with different letters are significantly different ( regwq test ; p < 0.05 ) results of experiment 2 numbers of cerambycid beetles of four species caught in traps baited with generic pheromone blends ( n = 31 , 28 , 28 , and 18 replicates for xylotrechus colonus , sarosesthes fulminans , n. a. acuminatus , and n. m. mucronatus , respectively ) . the blend of hydroxyhexanones contained equal amounts of ( r)- and ( s)-3-hydroxyhexan-2-one and ( r)- and ( s)-2-hydroxyhexan-3-one , and the blend of hexanediols contained approximately equal amounts of ( 2r,3r)- ( 2 s,3 s)- ( 2r,3 s)- and ( 2 s,3r)-2,3-hexanediol . the treatment designated ketones + diols contained equal amounts of the blends of hydroxyhexanones and hexanediols . p < 0.05 ( ) next to total number of males indicates sex ratio of beetles captured within species that is significantly different from 1:1 mean ( se ) numbers of adults ( sexes combined ) of three species of cerambycid beetles captured with respect to composition of the lure : top , xylotrechus colonus ( n = 31 trials ) ; middle , neoclytus a. acuminatus ( n = 28 trials ) ; and bottom , neoclytus m. mucronatus ( n = 18 trials ) . treatment numbers correspond to treatments in table 2 : ( 1 ) generic hydroxyhexanones ; ( 2 ) generic hexanediols ; ( 3 ) 1:1 mixture of generic hydroxyhexanones and hexanediols ; ( 4 ) ethanol control ; and ( 5 ) blank control . means with different letters are significantly different ( regwq test ; p < 0.05 ) attraction of both sexes of x. colonus and s. fulminans to odors from conspecific males in olfactometer bioassays provided the first evidence that males of both species produce aggregation pheromones . traps baited with synthetic pheromone captured similar numbers of male and female x. colonus , confirming that males produce an aggregation pheromone ( tables 1 and 2 ) . the small number of s. fulminans captured is consistent with its scarcity in the habitats where we conducted our bioassays ( esl , personal observation ) . the structure of the pheromone components of x. colonus and the putative pheromone compounds of s. fulminans are consistent with the ketone / diol structural motif of other cerambycine species ( see introduction ) and provide further support for the hypothesis that this structural motif has been conserved within the cerambycinae ( see lacey et al . nevertheless , recent studies have shown that some cerambycine species have pheromone components of entirely different chemical classes ( hanks et al . our study also provides further evidence that the presence of gland pores on the prothorax of males ( in x. colonus , s. fulminans , n. a. acuminatus , and n. m. mucronatus ; ray et al . 2007b ) and calling behavior of males ( in x. colonus , n. a. acuminatus , and n. m. mucronatus ; lacey et al . 2007a , b ) reliably indicate that volatile pheromones play a role in mate location in cerambycine species . in both field experiments , all treatments that contained ( r)-3-hydroxyhexan-2-one attracted significant numbers of adult x. colonus ( figs . 1 and 2 ( top ) ) . however , x. colonus were caught in greatest numbers in traps baited with its species - specific blend ( fig . 1 ) , suggesting that the full blend is necessary for optimal attraction . in the absence of ( r)-3-hydroxyhexan-2-one , adult x. colonus responded weakly , or did not respond ( fig . 1 ) to the minor components of the blend produced by males [ including ( s)-3-hydroxyhexan-2-one and ( 2r,3r)- and ( 2 s,3 s)-2,3-hexanediols ] . unnatural isomers apparently did not inhibit attraction of x. colonus to ( r)-3-hydroxyhexan-2-one , as evidenced by the significant number of beetles captured in traps baited with the s. fulminans lure or the generic hydroxyketones ( figs . 1 and 2 ( top ) ) . attraction of adult males and females of a congener , xylotrechus nauticus ( mannerheim ) , to synthetic pheromones also is not inhibited by unnatural isomers ( hanks et al . ( r)-3-hydroxyhexan-2-one now has been shown to be an important component of male - produced pheromones of 14 species of cerambycine beetles ( including x. colonus and possibly s. fulminans ; see hanks et al . 2007 ) . overlap in pheromone composition among sympatric cerambycine species can result in cross - attraction ( hanks et al . cross - attraction was evident in the present study , with both sexes of x. colonus caught in traps baited with treatments representative of reconstructed blends of the pheromone produced by s. fulminans ( fig . 1 ) , as well as the other species caught in traps baited with compounds identified in the two focal species of this study . x. colonus , s. fulminans , n. a. acuminatus , and n. m. mucronatus are sympatric , overlap in phenology , and share larval hosts ( linsley 1964 ) . in fact , adults of all four species may aggregate on the same downed host tree ( esl , personal observation ) and adult x. colonus and n. m. mucronatus , at least , are attracted to odors emanating from dying hosts ( ginzel and hanks 2005 ) . larvae of all four species require hosts that are severely weakened or moribund ( linsley 1964 ) and competition for these resources ( with other insect species and saprophytes ) , results in rapid degradation of hosts ( hanks 1999 ) . species that rely on such ephemeral hosts may be under selective pressure to locate hosts quickly and oviposit ( hanks 1999 ) . thus , these species may exploit the pheromones of other species in their feeding guild as kairomones . closely related species of bark beetles ( curculionidae : scolytinae ) , as well as closely related species of sap beetles ( nitidulidae ) , share pheromone components , respectively , resulting in cross - attraction among species ( e.g. , zilkowski and bartelt 1999 ; haberkern and raffa 2003 ) . perhaps not coincidentally , males of these bark and sap beetle species also produce aggregation pheromones ( zilkowski and bartelt 1999 ; haberkern and raffa 2003 ) such as those produced by male cerambycine beetles . the responses of beetles of three species , x. colonus , n. a. acuminatus , and n. m. mucronatus , to generic lures ( table 2 , fig . 2 ) again demonstrates that these blends could be used to collect live specimens of various cerambycine species , for analysis and identification of the pheromone blends that they actually produce . in this context , even weak attraction to generic blends may be sufficient for collecting adults for pheromone identification or even monitoring the distribution and phenology of economically important species of interest for which a pheromone has yet to be identified .

adults of both sexes of the cerambycid beetles xylotrechus colonus ( f. ) and sarosesthes fulminans ( f. ) were attracted to odors produced by male conspecifics in olfactometer bioassays . analyses of headspace volatiles from adults revealed that male x. colonus produced a blend of ( r)- and ( s)-3-hydroxyhexan-2-one and ( 2 s,3 s)- and ( 2r,3r)-2,3-hexanediol , whereas male s. fulminans produced ( r)-3-hydroxyhexan-2-one and ( 2 s,3r)-2,3-hexanediol . all of these compounds were absent in the headspace of females . two field bioassays were conducted to confirm the biological activity of the synthesized pheromones : ( 1 ) enantiomerically enriched pheromone components were tested singly and in species - specific blends and ( 2 ) four - component mixture of racemic 3-hydroxyhexan-2-one plus racemic 2-hydroxyhexan-3-one and the four - component blend of the stereoisomers of 2,3-hexanediols were tested separately and as a combined eight - component blend . in these experiments , adult male and female x. colonus were captured in greatest numbers in traps baited with the reconstructed blend of components produced by males , although significant numbers were also captured in traps baited with ( r)-3-hydroxyhexan-2-one alone or in blends with other compounds . too few adult s. fulminans were captured for a statistical comparison among treatments , but all were caught in traps baited with lures containing ( r)-3-hydroxyhexan-2-one . in addition to these two species , adults of two other species of cerambycid beetles , for which pheromones had previously been identified , were caught : neoclytus a. acuminatus ( f. ) and its congener neoclytus m. mucronatus ( f. ) . cross - attraction of beetles to pheromone blends of other species , and to individual pheromone components that are shared by two or more sympatric species , may facilitate location of larval hosts by species that compete for the same host species .

Introduction Methods and Materials Results Discussion

we randomly assigned each of the following nine treatments to traps : ( 1 ) x. colonus blend ( see results ) : ( r)-3-hydroxyhexan-2-one ( 8 mg ) , ( s)-3-hydroxyhexan-2-one ( 1.25 mg ) , ( 2r,3r)-2,3-hexanediol ( 2.5 mg ) , and ( 2 s,3 s)-2,3-hexanediol ( 0.5 mg ) ; ( 2 ) s. fulminans blend ( see results ) : ( r)-3-hydroxyhexan-2-one ( 8 mg ) and ( 2 s,3r)-2,3-hexanediol ( 1.25 mg ) ; ( 3 ) ( r)-3-hydroxyhexan-2-one ( 8 mg ) ; ( 4 ) ( s)-3-hydroxyhexan-2-one ( 8 mg ) ; ( 5 ) ( 2 s,3 s)-2,3-hexanediol ( 8 mg ) ; ( 6 ) ( 2r,3r)-2,3-hexanediol ( 8 mg ) ; ( 7 ) ( 2 s,3r)-2,3-hexanediol ( 8 mg ) ; ( 8) ethanol control ( 1 ml ) ; and ( 9 ) blank control ( empty emitter).pheromones were loaded into release devices that consisted of modified 1.5-ml microcentrifuge tubes ( # 05 - 406 - 16 ; fisher scientific , pittsburg , pa , usa ) . table 1results of experiment 1 xylotrechus colonussarosesthes fulminansneoclytus a. acuminatustreatment(1 ) x. colonus blend17132143(2 ) s. fulminans blend9932(3 ) ( r)-3-hydroxyhexan-2-one1072(4 ) ( s)-3-hydroxyhexan-2-one53(5 ) ( r , r)-2,3-hexanediol1(6 ) ( s , r)-2,3-hexanediol11(7 ) ( s , s)-2,3,-hexanediol32214(8 ) ethanol control11(9 ) blank controltotal4633732718numbers of cerambycid beetles of three species caught in traps baited with enantiomerically enriched candidate pheromone components ( n = 9 replicates for xylotrechus colonus and sarosesthes fulminans ; n = 10 replicates for n. a. acuminatus ) . the blend of components specific for x. colonus contained ( r)- and ( s)-3-hydroxyhexan-2-one with ( 2r,3r)- and ( 2 s,3 s)-2,3-hexanediol , the sarosesthes fulminans blend contained ( r)-3-hydroxyhexan-2-one and ( 2 s,3r)-2,3-hexanediol . treatment numbers correspond to treatments in table 1 : ( 1 ) x. colonus blend , ( 2 ) s. fulminans blend , ( 3 ) ( r)-3-hydroxyhexan-2-one , ( 4 ) ( s)-3-hydroxyhexan-2-one , ( 5 ) ( 2r,3r)-2,3-hexanediol , ( 6 ) ( 2 s,3r)-2,3-hexanediol , ( 7 ) ( 2 s,3 s)-2,3-hexanediol , ( 8) ethanol control , and ( 9 ) blank control . the blend of hydroxyhexanones contained equal amounts of ( r)- and ( s)-3-hydroxyhexan-2-one and ( r)- and ( s)-2-hydroxyhexan-3-one , and the blend of hexanediols contained approximately equal amounts of ( 2r,3r)- ( 2 s,3 s)- ( 2r,3 s)- and ( 2 s,3r)-2,3-hexanediol . the blend of components specific for x. colonus contained ( r)- and ( s)-3-hydroxyhexan-2-one with ( 2r,3r)- and ( 2 s,3 treatment numbers correspond to treatments in table 1 : ( 1 ) x. colonus blend , ( 2 ) s. fulminans blend , ( 3 ) ( r)-3-hydroxyhexan-2-one , ( 4 ) ( s)-3-hydroxyhexan-2-one , ( 5 ) ( 2r,3r)-2,3-hexanediol , ( 6 ) ( 2 s,3r)-2,3-hexanediol , ( 7 ) ( 2 s,3 s)-2,3-hexanediol , ( 8) ethanol control , and ( 9 ) blank control . the blend of hydroxyhexanones contained equal amounts of ( r)- and ( s)-3-hydroxyhexan-2-one and ( r)- and ( s)-2-hydroxyhexan-3-one , and the blend of hexanediols contained approximately equal amounts of ( 2r,3r)- ( 2 s,3 s)- ( 2r,3 s)- and ( 2 s,3r)-2,3-hexanediol . cross - attraction was evident in the present study , with both sexes of x. colonus caught in traps baited with treatments representative of reconstructed blends of the pheromone produced by s. fulminans ( fig .

dentine sensitivity is defined as short , sharp pain from exposed dentine in response to thermal , tactile , osmotic , or chemical stimuli that can not be ascribed to any other dental defect or disease . clinically the presence of dentine sensitivity creates challenges for both patients and dental practitioners ; in addition to causing patient discomfort , dentine sensitivity can complicate the provision of restorative treatment and the treatment of periodontal tissues [ 3 , 4 ] . dentine sensitivity is related to the exposure of dentine tubules resulting from either loss of tooth enamel or loss of periodontal tissue ( gingival recession ) [ 5 , 6 ] . for dentine sensitivity to be incited , dentine tubules must be open at the dentine surface as well as remaining patent to the dental pulp [ 6 , 7 ] . at the microscopic level dentine sensitivity is currently still described by the hydrodynamic theory proposed by brannstrom [ 8 , 9 ] , which states that following dentine exposure to stimuli such as physical touch , temperature alteration , sweet liquid , and acidic liquid , changes in intratubular fluid movement and intratubular pressure occur . these changes can cause activation of intratubular nerve fibres of pulpal origin ; this nerve excitation causes pain to be experienced [ 8 , 10 ] . to manage dentine sensitivity products have been developed for at home use functioning primarily through two modalities : suppressing the excitability of intratubular nerve fibres or reducing the patency of exposed dentine tubules [ 2 , 11 , 12 ] . when acting to suppress the excitability of nerve fibres within dentine tubules , the most common approach is to elevate the extracellular potassium concentration within the dental pulp . this can be achieved through a patient applying a dentifrice containing potassium ions in a relatively soluble form to exposed dentine . movement of potassium ions through patent dentinal tubules will act to raise the response threshold of pulpal nociceptors and reduce their ability to fire when provoked [ 13 , 14 ] . when considering the effectiveness of agents that reduce dentine sensitivity through reduction in the patency of exposed dentine tubules , the most successful outcomes have been achieved through application of fluoride based gels to dentine [ 2 , 11 , 12 ] . it is well documented that application of dentifrices containing stannous fluoride [ 1517 ] and sodium fluoride [ 1821 ] can promote the deposition of mineral precipitates within open dentinal tubules , thereby reducing fluid flow in dentine tubules following exposure to stimuli . a dentifrice containing functionalised tricalcium phosphate and 950 ppm sodium fluoride has been shown to enable dentine tubule occlusion in vitro ; application of this dentifrice to extracted bovine teeth following ph cycling was observed to reduce both dentine tubule opening and tubule diameter in comparison to pretreatment levels . this finding is notable , as not only there is limited data demonstrating that 1000 ppmf dentifrices can effectively reduce dentine sensitivity , but the availability of such a dentifrice could lower the cost and complexity of at home treatment undertaken by a patient suffering from dentine sensitivity , a dentifrice containing 1000 ppmf being appropriate for the control of dental caries in low caries risk patients [ 23 , 24 ] . importantly , while assessment of the anticaries benefits of this dentifrice has been reported , no assessment of the efficacy of this dentifrice to reduce dentine sensitivity in vivo has taken place . the aim of the present study was therefore to assess the effectiveness of a dentifrice containing 950 ppm fluoride and functionalised tricalcium phosphate ( ftcp ) in the reduction of dentine sensitivity in vivo . the null hypothesis was that the effectiveness of the dentifrice containing ftcp and 950 ppm fluoride would not be different to the effectiveness of a dentifrice containing 1000 ppm fluoride in reducing dentine sensitivity . the present study was a single centre , parallel group , blinded ( subjects and examiners ) randomised controlled clinical trial conducted at the westmead centre for oral health , westmead hospital , sydney , australia . ethical approval from the western sydney local health network human research ethics committee was obtained prior to commencement of the study ( sac2010/11/4.6 ( 3223 ) hrec/10/wmead/202 ) and the trial was registered with the australia new zealand clinical trials register . subjects included within the study were from the pool of patients eligible to receive treatment at the westmead centre for oral health . from this patient pool , during an initial off waiting list examination , eight hundred and fifty individuals indicated they suffered from dentine hypersensitivity and were willing to participate in the study . of these 850 individuals a total of 71 subjects were recruited for the study having met the strict inclusion criteria . all subjects which participated in the study consented to participation prior to the inclusion . as part of the consent process , patients were made aware of adverse effects of dentifrices , namely , abrasion and staining of hard and soft tissues and the detrimental sequelae of excessive fluoride ingestion . to be considered for this study , subjects were required to meet all of the following inclusion criteria : being aged between 18 and 70 years;demonstrating good general health with no history of chronic illness;possession of at least 2 teeth with an exposed root surface which are responsive on probing ( 50 g force ) or a 1 s duration cold air blast ( 70 psi ) ; these teeth were not to exhibit diagnosed caries , defective restorations , or signs of fracture on initial assessment ; these teeth were not to have had dental restorations , periodontal surgery , or orthodontics that has resulted in postoperative pain in the immediate past 3 months;a willingness to read , understand , and sign the consent form;a capability and willingness to brush teeth at least 2 times a day for 2 minutes on each occasion . being aged between 18 and 70 years ; demonstrating good general health with no history of chronic illness ; possession of at least 2 teeth with an exposed root surface which are responsive on probing ( 50 g force ) or a 1 s duration cold air blast ( 70 psi ) ; these teeth were not to exhibit diagnosed caries , defective restorations , or signs of fracture on initial assessment ; these teeth were not to have had dental restorations , periodontal surgery , or orthodontics that has resulted in postoperative pain in the immediate past 3 months ; a willingness to read , understand , and sign the consent form ; a capability and willingness to brush teeth at least 2 times a day for 2 minutes on each occasion . a subject was excluded from participation in the study if any of the following conditions applied : use of a desensitising agent in the 3 months prior to the study;undertaking regular medical treatment involving anti - inflammatory or analgesic use;being pregnant or nursing;exhibiting a known allergy to any ingredients in the examined dentifrices;suffering from conditions which could increase the level of acid within the oral cavity : bulimia , gastric reflux disease;excessive dietary exposure to acids ; lemons 2 times per day , raw tomatoes 2 times per day , acidic drinks 1 litre per day ( sports drinks , energy drinks , or fruit juice ) , wine 3 standard glasses per day;an inability to read the oral hygiene instructions provided to each participant . use of a desensitising agent in the 3 months prior to the study ; undertaking regular medical treatment involving anti - inflammatory or analgesic use ; being pregnant or nursing ; exhibiting a known allergy to any ingredients in the examined dentifrices ; suffering from conditions which could increase the level of acid within the oral cavity : bulimia , gastric reflux disease ; excessive dietary exposure to acids ; lemons 2 times per day , raw tomatoes 2 times per day , acidic drinks 1 litre per day ( sports drinks , energy drinks , or fruit juice ) , wine 3 standard glasses per day ; an inability to read the oral hygiene instructions provided to each participant . allocation of subjects to four study groups was randomised through use of a computer algorithm to limit the impact of age , gender , diet , and current level of oral hygiene on the study results . group a. this group brushed teeth twice daily with a dentifrice containing 1000 ppm fluoride ions ( mfp ) : colgate cavity protection ( colgate - palmolive , new york , ny , usa , ) . group b. this group brushed teeth twice daily with a dentifrice containing 1000 ppm fluoride ions ( naf ) + 19300 ppm potassium ions ( kno3 ) : sensodyne total care ( glaxosmithkline , sydney , nsw , australia ) . group c. this group brushed teeth twice daily with a dentifrice containing ftcp and 950 ppm fluoride ions ( naf ) : clinpro tooth crme ( 3 m espe , st . group d. this group brushed teeth twice daily with a dentifrice containing ftcp and 950 ppm fluoride ions ( naf ) : clinpro tooth crme ( 3 m espe , st . paul , mn , usa ) and a directed pea sized topical application of clinpro tooth crme onto sensitive teeth before sleeping without rinsing . at no time during the trial were subjects made aware of what test group they belonged to or which product they were using ; all packaging was discarded prior to distribution and toothpaste tubes were wrapped in generic sticky white labelling . at the study commencement , all subjects were provided with a new toothbrush and dental floss and were given the same oral hygiene instructions verbally and as a take home pamphlet . subjects were also provided with a two - minute timer in a bid to maintain strict and consistent adherence to the prescribed two - minute brushing time twice daily . patients were assessed at three time points during the study : baseline , 6 weeks , and 10 weeks . the clinical assessment was completed by three senior dental officers at westmead centre for oral health to limit the effect of clinician variation on study results . standardisation was also maintained through each clinician recalibrating the process of stimulus provision to sensitive surfaces at the commencement of each assessment day . examiners remained blinded to test groups to which participants belonged at all examination points throughout the study . at each assessment , each sensitive tooth surface was exposed to three different stimuli that were applied directly onto the identified sensitive tooth . the three stimuli included an air blast , tactile stimulation , and application of a hypertonic solution . the identified tooth surface was exposed to air delivered from a standard dental unit triplex syringe from an operating distance of approximately 1 cm for a period of 1 s at an operating temperature of 21c ( 5c ) . a pressure gauge was mounted to the dental unit and calibrated prior to each assessment day to ensure air was delivered at a standard pressure of 70 psi . the identified tooth surface was stroked for 3 s using a standard dental explorer probe that was held perpendicular to the surface using a force of 50 g. hypertonic solution . a 70% hypertonic sucrose solution was applied to the identified tooth surface for 3 s. the solution was at room temperature at the time of application . following application of each stimulus patients rated the pain / sensitivity experienced using an 11-point numbered pain rating scale ( nrs-11 ; figure 1 ) . these scores were recorded on clinical test forms . following the 6-week examination patients returned the study dentifrice wash - out period to elapse before reexamination at 10 weeks to quantify any prolonged actions of each test dentifrice ; 6-week usage of a dentifrice has been shown to provide sufficient time to allow maximum benefit of a desensitising product . patient compliance in the use of the study dentifrice was established at the 6-week examination . patients were asked to return products which were issued and each tube was weighed to calculate the total amount of toothpaste used over the 6-week period . for each test group the mean pain score for each stimulus modality ( air blast , tactile , and hypertonic solution ) was calculated by patient and by tooth at each assessment point ( baseline , 6 weeks , and 10 weeks ) . additionally , the mean pain scores for each test group for each stimulus were combined and divided by 3 to give a total combined modalities sensitivity ( cms ) score at each assessment point . the percentage change relative to baseline in mean pain score for each stimulus and the percentage change in cms relative to baseline were calculated for each test group . ancova ( with baseline as the covariate ) with a tukey hsd post hoc test was used for between group comparisons . of the 850 individuals from the westmead centre for oral health patient pool who indicated suffering from dentine sensitivity , 80 individuals satisfied the inclusion criteria . these 80 individuals were randomly allocated to the four study groups ( a d ) . the study population exhibited a mean age of 40.9 and a range of 1767 years of age . seventy - one of the 80 subjects completed the 10-week clinical study and complied with the protocol given . of the participants who completed the study , there were 54 females and 17 males . no adverse soft tissue or hard tissue effects were observed by the assessing clinicians during the study ; however one participant withdrew from the study reporting an allergic reaction to colgate cavity protection . the 9 patients that did not complete the study did so as they no longer wanted to attend the recall appointments on the basis of convenience . at 6 weeks ( end of the treatment phase ) all groups showed a reduction in evaporative sensitivity score from baseline , with colgate cavity protection showing a 12% reduction , sensodyne total care a 19% reduction , clinpro tooth crme ( brushing only ) a 45% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 43% reduction . the reduction in evaporative sensitivity scores from base line of both clinpro tooth crme groups at 6 weeks was significantly greater ( p 0.05 , 95% ci ) than the reduction in evaporative sensitivity scores of both the positive control group ( sensodyne total care ) and the negative control group ( colgate cavity protection ) . there was no significant difference ( p 0.05 , 95% ci ) in the reduction of evaporative sensitivity scores from baseline when comparing the sensodyne total care and colgate cavity protection groups at the end of the 6-week treatment phase ( tables 1 and 2 and figure 2 ) . at 10 weeks ( four weeks after cessation of treatment ) , all four groups demonstrated a reduction in evaporative sensitivity score from baseline , with colgate cavity protection showing a 18% reduction , sensodyne total care a 40% reduction , clinpro tooth crme ( brushing only ) a 24% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 54% reduction . the reduction in evaporative sensitivity scores at 10 weeks for clinpro tooth crme ( brushing + topical application ) and sensodyne total care was significantly greater ( p 0.05 , 95% ci ) than the reduction in evaporative sensitivity demonstrated by the negative control colgate cavity protection . there was no significant difference ( p 0.05 , 95% ci ) in the reduction in evaporative sensitivity scores at 10 weeks from baseline when comparing groups using sensodyne total care , clinpro tooth crme ( brushing only ) , and clinpro tooth crme ( brushing + topical application ) . at 6 weeks ( end of the treatment phase ) all groups showed a reduction in tactile sensitivity from baseline , with colgate cavity protection showing a 20% reduction , sensodyne total care a 39% reduction , clinpro tooth crme ( brushing only ) a 44% reduction , and clinpro tooth crme ( brushing + topical application ) a 62% reduction . at 6 weeks the only group to show a significant reduction ( p 0.05 , 95% ci ) in tactile sensitivity scores in comparison to the negative control group ( colgate total protection ) was clinpro tooth crme ( brushing + topical application ) . there were no other comparisons that displayed significantly different sensitivity scores ; both clinpro tooth crme groups were not significantly different ( p 0.05 , 95% ci ) from the positive control group , sensodyne total care ( tables 1 and 2 and figure 3 ) . at 10 weeks ( four weeks after cessation of treatment ) , not all groups showed a reduction in tactile sensitivity from baseline , with colgate cavity protection showing a 6% increase in sensitivity . however , sensodyne total care showed a 37% reduction in tactile sensitivity , clinpro tooth crme ( brushing only ) a 14% reduction , and clinpro tooth crme ( brushing + topical application ) a 64% reduction from baseline . a significant reduction ( p 0.05 , 95% ci ) in tactile sensitivity at 10 weeks from baseline for clinpro tooth crme ( brushing + topical application ) and sensodyne total care in comparison to colgate cavity protection was observed . no significant difference ( p 0.05 , 95% ci ) in the reduction in tactile sensitivity scores from baseline to 10 weeks was observed between clinpro tooth crme ( brushing only ) and sensodyne total care . additionally , no significant difference ( p 0.05 , 95% ci ) in tactile sensitivity score reduction at 10 weeks was observed between clinpro tooth crme ( brushing only ) and colgate cavity protection ( tables 1 and 2 and figure 3 ) . at 6 weeks all groups exhibited a reduction in hypertonic sensitivity from baseline , with colgate cavity protection showing a 32% reduction , sensodyne total care a 41% reduction , clinpro tooth crme ( brushing only ) a 40% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 61% reduction . at 6 weeks the difference in % reduction from baseline was not significant ( p 0.05 , 95% ci ) for any of the four groups ( tables 1 and 2 and figure 4 ) . at 10 weeks , all groups exhibited a reduction in hypertonic sensitivity from baseline , with colgate cavity protection showing a 42% reduction , sensodyne total care a 56% reduction , clinpro tooth crme ( brushing only ) a 22% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 66% reduction . the only groups to exhibit a significant difference ( p 0.05 , 95% ci ) in hypertonic sensitivity reduction at 10 weeks compared to baseline were sensodyne total care and clinpro tooth crme ( brushing + topical application ) . there was no significant difference ( p 0.05 , 95% ci ) in hypertonic sensitivity reduction at 10 weeks between the groups using colgate cavity protection , sensodyne total care , and clinpro tooth crme ( brushing + topical application ) ( tables 1 and 2 and figure 4 ) . at 6 weeks ( end of the treatment phase ) all groups exhibited a reduction in the combined sensitivity score ( cms ) from baseline , with colgate cavity protection showing a 20% reduction , sensodyne total care a 30% reduction , clinpro tooth crme ( brushing only ) a 42% , and clinpro tooth crme ( brushing + topical application ) showing a 52% reduction . at the end of the 6-week treatment phase , both clinpro tooth crme study groups showed a significant reduction ( p 0.05 , 95% ci ) in cms when compared to the negative control group using colgate cavity protection . clinpro tooth crme when used with an additional topical application also demonstrated a significant reduction ( p 0.05 , 95% ci ) in dentine sensitivity compared to the positive control , sensodyne total care , after 6 weeks ( tables 1 and 2 and figure 5 ) . at 10 weeks ( 4 weeks after cessation of treatment ) , all groups showed a reduction in combined sensitivity score ( cms ) from baseline , with colgate cavity protection showing an 18% reduction , sensodyne total care a 40% reduction , clinpro tooth crme ( brushing only ) a 24% reduction , and clinpro tooth crme ( brushing + topical application ) a 54% reduction . the reduction in cms for clinpro tooth crme ( brushing + topical application ) and sensodyne total care was significantly greater ( p 0.05 , 95% ci ) than the reduction in cms from baseline of the negative control colgate cavity protection at four weeks after cessation of treatment . there was no significant difference ( p 0.05 , 95% ci ) in cms reduction from baseline to 10 weeks between groups using sensodyne total care , clinpro tooth crme ( brushing only ) , and clinpro tooth crme ( brushing + topical application ) ( tables 1 and 2 and figure 5 ) . the ability for fluoride ions sourced from a dentifrice to form fluoride - calcium precipitates that occlude dentine tubules with the consequence of reducing dentine sensitivity is well documented [ 1821 , 27 , 28 ] . this ability occurs due to the negative electric charge of fluoride ions , which results in their binding with calcium cations . once bound to calcium cations present within a dentifrice the fluoride ions are rendered incapable of combining with cations present at the tooth surface . to overcome the effect of reduced fluoride ion availability at the tooth surface caused by intradentifrice fluoride - calcium bonding , dentifrice manufacturers have traditionally acted to increase the concentration of fluoride ions within a dentifrice . in recent times , however , altering the chemical structure of calcium complexes within a dentifrice to reduce the bonding affinity between calcium cations and fluoride anions has been undertaken as an alternative solution to simple fluoride ion concentration increase . one such example of this has been the development and incorporation of altered calcium phosphate complexes , such as functionalised tricalcium phosphate , which is currently incorporated within clinpro tooth crme . functionalised tricalcium phosphate ( ftcp ) is produced through beta tricalcium phosphate ( tcp ) complexes being milled with sodium lauryl sulfate ( sls ) . the tcp crystal structure exhibits several reactive sites including calcium - oxygen clusters ( cao3 , cao7 , and cao8 ) and lattice defects . these reactive sites are available to undergo chemical interaction with anions such as fluoride ions . to reduce the reactivity of these sites within b - tcp complexes the incorporation of sls within the tcp structure therefore impedes fluoride ions from combining with calcium ions in the dentifrice , so in turn potentially increasing the concentration of both calcium and fluoride to tooth surfaces . [ 25 , 3234 ] have reported a synergistic relationship between ftcp and fluoride with regard to enamel remineralisation ; enamel that is remineralised through a fluoride / ftcp combination demonstrates significantly greater surface and subsurface rehardening following ph cycling compared to that achieved by fluoride application alone . notably , the results of the present study suggest that this remineralisation synergy between fluoride and ftcp may also produce a benefit in terms of enhancing dentine tubule occlusion ; the two test groups that utilised clinpro tooth crme ( 950 ppm f ) demonstrated a greater level of sensitivity relief for all stimuli except the hypertonic test at 10 weeks ( group c ) in comparison to the group using colgate total , despite the fact that colgate total contains a greater concentration of fluoride ions ( 1000 ppm f ) . previous microscopic analysis showing the ability of clinpro tooth crme to reduce the diameter of tubule openings following ph cycling to a greater degree than sensodyne nupro 5000 and topex renew supports this possibility . within the present study a greater reduction in dentine sensitivity was observed when an additional topical application of clinpro tooth crme was placed to supplement application provided through brushing alone . this finding is in contrast to reports in the literature stating that there is no evidence to suggest that additional topical application increases the effectiveness of a desensitizing dentifrice . first an intentional topical application of a dentifrice potentially allows a greater concentration of fluoride ions to be applied to a tooth surface in comparison to that applied through unintentional brushing alone . secondly , through an additional topical application , fluoride ions can remain at a sensitive surface for a longer duration than following brushing and rinsing and thirdly through increasing the duration of fluoride ion presence upon a sensitive surface , the propensity for fluoride migration and tubule occlusion is raised . the greater success of clinpro tooth crme when applied as an additional topical application rather than brushing alone may also be a result of greater salivary fluoride concentration as a by - product of topical application and no rinsing following application . this possibility is consistent with studies that have examined the effect of patient activity following dentifrice application on the level of salivary fluoride . nordstrm and birkhed determined that a topical application of dentifrice to a tooth surface combined with regular brushing resulted in a greater concentration of salivary fluoride than when compared to brushing alone . sjgren and melin identified that a single post - brushing rinse with fluoridated water decreased fluoride concentration by a factor of two when compared to no rinsing , while rinsing with fluoridated water two times following brushing decreased salivary concentration by a factor of 5 when compared to no rinsing . the reduction in cms of the negative control group over the study duration can be in part attributed to the placebo effect which is well documented in dentine sensitivity studies [ 7 , 38 , 39 ] . additionally the hawthorne effect , which describes a positive response to noninterventional treatment , should also be assumed to have had some impact on the study results . improved oral hygiene in patients enrolled in the study could have also reduced perceived dentine sensitivity across all groups as a reduction in tooth surface plaque facilitated increased dentifrice access to dentine tubules . these reasons may also account for the antihypersensitivity effects continuing for the month following the cessation of the use of the treatment dentifrices . an identified limitation of the study was the numbers of patients enrolled in each study group . due to the breadth of the potential patient pool , patients eligible for treatment at westmead centre for oral health , it was anticipated that including 40 subjects per group would be an achievable goal over a 3-year study period . as a result of the very strict exclusion criteria , especially the requirement that the use of a desensitising agent in the 3 months prior to the study excluded an individual from being a subject , patient recruitment was extremely difficult . however despite the smaller numbers than originally forecast per group , the results of the present study provide useful information . not only are the numbers sufficient to demonstrate statistical significance , but when tooth number rather than patient number is used for analysis the subject number exceeds 50 for each group and the statistical outcomes remain the same . the addition of ftcp to a dentifrice suitable for daily oral hygiene can enhance the ability of dentifrice fluoride to reduce dentine sensitivity . in the present study twice daily brushing with clinpro tooth crme resulted in a similar reduction in dentine sensitivity to that achieved through brushing with a dentifrice containing kno3 + f ( sensodyne total care ) . if clinpro tooth crme is used twice daily for brushing in combination with a nightly topical application , it can be more effective in reducing dentine sensitivity than twice daily brushing with sensodyne total care .

background . to assess the clinical efficacy of a dentifrice containing fluoride and functionalised tricalcium phosphate ( ftcp ) in reducing dentine sensitivity . methods . a 10-week parallel blind randomised control trial was conducted . subjects were assigned to one of four groups and instructed to brush twice daily : a : colgate cavity protection ( 1000 ppmf - mfp ) ; b : sensodyne total care ( 1000 ppmf - naf + 19300 ppmk+-kno3 ) ; c : clinpro tooth crme ( 950 ppmf - naf + ftcp ) ; and d : clinpro tooth crme ( brushing + additional topical application ) . seventy - one patients were assessed at baseline , 6 weeks , and 10 weeks for cold , tactile , and hypertonic sensitivity using the nrs-11 pain rating scale . a combined modalities sensitivity score ( cms ) was calculated . results . at 6 weeks , patients reported the following reduction in cms : a ( 20% ) ; b ( 30% ) ; c ( 42% ) ; d ( 52% ) . at 10 weeks , patients reported the following reduction in cms : a ( 18% ) , b ( 40% ) , c ( 24% ) , and d ( 54% ) . the only cms comparisons to show a significant difference ( p < 0.05 ) were between groups a and d ( 6 and 10 weeks ) . conclusions . addition of ftcp to a dentifrice enhances the ability of dentifrice fluoride in reducing dentine sensitivity . using clinpro tooth crme twice daily for brushing can be as effective to reduce dentine sensitivity as twice daily brushing using sensodyne total care . however , additional nightly topical application of ftcp , in addition to twice daily brushing , showed an enhanced reduction in dentine sensitivity .

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

at 6 weeks ( end of the treatment phase ) all groups showed a reduction in evaporative sensitivity score from baseline , with colgate cavity protection showing a 12% reduction , sensodyne total care a 19% reduction , clinpro tooth crme ( brushing only ) a 45% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 43% reduction . at 10 weeks ( four weeks after cessation of treatment ) , all four groups demonstrated a reduction in evaporative sensitivity score from baseline , with colgate cavity protection showing a 18% reduction , sensodyne total care a 40% reduction , clinpro tooth crme ( brushing only ) a 24% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 54% reduction . the reduction in evaporative sensitivity scores at 10 weeks for clinpro tooth crme ( brushing + topical application ) and sensodyne total care was significantly greater ( p 0.05 , 95% ci ) than the reduction in evaporative sensitivity demonstrated by the negative control colgate cavity protection . there was no significant difference ( p 0.05 , 95% ci ) in the reduction in evaporative sensitivity scores at 10 weeks from baseline when comparing groups using sensodyne total care , clinpro tooth crme ( brushing only ) , and clinpro tooth crme ( brushing + topical application ) . at 6 weeks ( end of the treatment phase ) all groups showed a reduction in tactile sensitivity from baseline , with colgate cavity protection showing a 20% reduction , sensodyne total care a 39% reduction , clinpro tooth crme ( brushing only ) a 44% reduction , and clinpro tooth crme ( brushing + topical application ) a 62% reduction . at 6 weeks the only group to show a significant reduction ( p 0.05 , 95% ci ) in tactile sensitivity scores in comparison to the negative control group ( colgate total protection ) was clinpro tooth crme ( brushing + topical application ) . a significant reduction ( p 0.05 , 95% ci ) in tactile sensitivity at 10 weeks from baseline for clinpro tooth crme ( brushing + topical application ) and sensodyne total care in comparison to colgate cavity protection was observed . additionally , no significant difference ( p 0.05 , 95% ci ) in tactile sensitivity score reduction at 10 weeks was observed between clinpro tooth crme ( brushing only ) and colgate cavity protection ( tables 1 and 2 and figure 3 ) . at 6 weeks all groups exhibited a reduction in hypertonic sensitivity from baseline , with colgate cavity protection showing a 32% reduction , sensodyne total care a 41% reduction , clinpro tooth crme ( brushing only ) a 40% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 61% reduction . at 10 weeks , all groups exhibited a reduction in hypertonic sensitivity from baseline , with colgate cavity protection showing a 42% reduction , sensodyne total care a 56% reduction , clinpro tooth crme ( brushing only ) a 22% reduction , and clinpro tooth crme ( brushing + topical application ) showing a 66% reduction . the only groups to exhibit a significant difference ( p 0.05 , 95% ci ) in hypertonic sensitivity reduction at 10 weeks compared to baseline were sensodyne total care and clinpro tooth crme ( brushing + topical application ) . there was no significant difference ( p 0.05 , 95% ci ) in hypertonic sensitivity reduction at 10 weeks between the groups using colgate cavity protection , sensodyne total care , and clinpro tooth crme ( brushing + topical application ) ( tables 1 and 2 and figure 4 ) . at 6 weeks ( end of the treatment phase ) all groups exhibited a reduction in the combined sensitivity score ( cms ) from baseline , with colgate cavity protection showing a 20% reduction , sensodyne total care a 30% reduction , clinpro tooth crme ( brushing only ) a 42% , and clinpro tooth crme ( brushing + topical application ) showing a 52% reduction . at 10 weeks ( 4 weeks after cessation of treatment ) , all groups showed a reduction in combined sensitivity score ( cms ) from baseline , with colgate cavity protection showing an 18% reduction , sensodyne total care a 40% reduction , clinpro tooth crme ( brushing only ) a 24% reduction , and clinpro tooth crme ( brushing + topical application ) a 54% reduction . the reduction in cms for clinpro tooth crme ( brushing + topical application ) and sensodyne total care was significantly greater ( p 0.05 , 95% ci ) than the reduction in cms from baseline of the negative control colgate cavity protection at four weeks after cessation of treatment . there was no significant difference ( p 0.05 , 95% ci ) in cms reduction from baseline to 10 weeks between groups using sensodyne total care , clinpro tooth crme ( brushing only ) , and clinpro tooth crme ( brushing + topical application ) ( tables 1 and 2 and figure 5 ) .

atrial fibrillation ( af ) is the most common sustained arrhythmia and an important source of mortality and morbidity on a population level . more than 6 million people in europe are affected and this is expected to double during the next 3050 years . the estimated lifetime risk of developing af is one in four for the population having reached the age of 55 . it is associated with a doubled risk on death , a five - fold increased risk of stroke , increased risk of heart failure and hospitalization , a reduced exercise capacity and left ventricular function , and an impaired quality of life which may be worse in women than in men . despite the clear association between af and death , stroke , and other cardiovascular events all published studies have shown that rate control is not inferior to rhythm control for the prevention of mortality and morbidity ( tables 1 and 2 ) . this disappointing outcome may relate to the low long - term maintenance rate of sinus rhythm in the rhythm control groups of these studies , being 63% after 5 years in the atrial fibrillation follow - up investigation of rhythm management ( affirm ) trial and 39% after 2.3 years of follow - up in the rate control versus electrical cardioversion ( race ) trial . also the atrial fibrillation and congestive heart failure trial ( af - chf ) observed no difference in cardiovascular mortality ( primary outcome ) between patients with a left ventricular ejection fraction ( lvef ) 35% , symptoms of congestive heart failure and a history of af randomized to rate or rhythm control , nor in the secondary outcomes including death from any cause and worsening of heart failure . in addition , a post - hoc time - dependent analysis did not show that sinus rhythm was associated with improved outcome . the negative outcome of rhythm control therapy may also be a consequence of positive patient selection. the enrolled patients were selected by not having severe af - related symptoms and having survived a phase of af - related complications . furthermore , according to the guidelines used when the rate vs. rhythm control studies were performed , anticoagulant therapy was often withdrawn from patients in the rhythm control arms based on the assumption that sinus rhythm was present , resulting in a potentially avoidable excess risk of ischaemic stroke . on the other hand , it should be noted that during rate control therapy morbidity and mortality are still significant . even with oral anticoagulation the residual stroke or systemic embolism rate in patients with af remains relatively high , ranging between 1.1 and 2.4% , depending on the presence of risk factors . although recent studies showed a trend towards reduction of events including stroke ( figure 1 ) , further improvement of therapy to reduce af - associated events clearly is warranted . table 1characteristics of rhythm control and rate control trials in patients with atrial fibrillation ( adapted from camm et al . with permission)patients reaching primary endpoint ( n)trialpatients ( n)mean age ( years)mean length of follow - up ( years)inclusion criteriaprimary endpointrate controlrhythm controlppiaf25261.01.0persistent af ( 7360 days)symptomatic improvement76/125 ( 60.8%)70/127 ( 55.1%)0.32affirm406069.73.5paroxysmal af or persistent af , age 65 years or older , or risk of stroke or deathall - cause mortality310/2027 ( 25.9%)356/2033 ( 26.7%)0.08race52268.02.3persistent af or flutter for < 1 year and 1 to 2 cardioversions > 2 years and oral anticoagulationcomposite : cardiovascular death , chf , severe bleeding , pm implantation , thromboembolic events , severe adverse effects of antiarrhythmic drugs44/256 ( 17.2%)60/266 ( 22.6%)0.11staf20066.01.6persistent af ( > 4 weeks and < 2years ) , left atrial size > 45 mm , chf nyha ii iv , lvef < 45%composite : overall mortality , cerebrovascular complications , cpr , embolic events10/100 ( 10.0%)9/100 ( 9.0%)0.99hot caf20560.81.7first clinically overt persistent af ( 7 and < 2 years ) , 5075-year oldcomposite : death , thromboembolic events ; intracranial/ major haemorrhage1/101 ( 1.0%)4/104 ( 3.9%)>0.71af - chf1376663.1lvef35% , symptoms of chf , history of af ( 6 h or ecv < last 6 months)cardiovascular death175/1376 ( 25%)182/1376 ( 27%)0.59af , atrial fibrillation ; affirm , atrial fibrillation follow - up investigation of rhythm management ; chf , congestive heart failure ; cpr , cardiopulmonary resuscitation ; ecv , electrical cardioversion ; hot cafe , how to treat chronic atrial fibrillation ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; piaf , pharmacological intervention in atrial fibrillation ; pm , pacemaker ; race , rate control versus electrical cardioversion for persistent atrial fibrillation ; staf , strategies of treatment of atrial fibrillation . table 2comparison of adverse outcomes in rhythm control and rate control trials in patients with atrial fibrillation ( adapted from camm et al . with permission)trialdeaths of all causes ( in rate / rhythm)deaths from cardiovascular causesdeaths from non - cardiovascular causesstrokethromboembolic eventsbleedingpiaf41/11ndndndaffirm666 ( 310/356)167/164113/16577/80nd107/96race3618/18ndnd14/2112/9staf12 ( 8/4)8/30/11/5nd8/11hot caf4 ( 1/3)0/21/10/3nd5/8af - chf228/217175/18253/3511/9ndndaf , atrial fibrillation ; affirm , atrial fibrillation follow - up investigation of rhythm management ; hot cafe , how to treat chronic atrial fibrillation ; nd , not determined ; piaf , pharmacological intervention in atrial fibrillation ; race , rate control versus electrical cardioversion for persistent atrial fibrillation ; and staf , strategies of treatment of atrial fibrillation.total number of patients not reported . figure 1yearly cardiovascular morbidity and mortality rate in the rate control versus electrical cardioversion ( race ) i study ( published in 2002 ) and the rate control efficacy in permanent atrial fibrillation ( race ) ii study ( published in 2010 ) . characteristics of rhythm control and rate control trials in patients with atrial fibrillation ( adapted from camm et al . with permission ) af , atrial fibrillation ; affirm , atrial fibrillation follow - up investigation of rhythm management ; chf , congestive heart failure ; cpr , cardiopulmonary resuscitation ; ecv , electrical cardioversion ; hot cafe , how to treat chronic atrial fibrillation ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; piaf , pharmacological intervention in atrial fibrillation ; pm , pacemaker ; race , rate control versus electrical cardioversion for persistent atrial fibrillation ; staf , strategies of treatment of atrial fibrillation . comparison of adverse outcomes in rhythm control and rate control trials in patients with atrial fibrillation ( adapted from camm et al . with permission ) af , atrial fibrillation ; affirm , atrial fibrillation follow - up investigation of rhythm management ; hot cafe , how to treat chronic atrial fibrillation ; nd , not determined ; piaf , pharmacological intervention in atrial fibrillation ; race , rate control versus electrical cardioversion for persistent atrial fibrillation ; and staf , strategies of treatment of atrial fibrillation . yearly cardiovascular morbidity and mortality rate in the rate control versus electrical cardioversion ( race ) i study ( published in 2002 ) and the rate control efficacy in permanent atrial fibrillation ( race ) ii study ( published in 2010 ) . interestingly , a subgroup analysis of the affirm trial demonstrated an association between sinus rhythm maintenance and improved survival . this , together with the results of the recently published athena trial , supports the idea that the presence of af is one of the modifiable factors associated with death and cardiovascular morbidity in af patients . apart from the beneficial effect of dronedarone on a composite endpoint , predominantly driven by cardiovascular hospitalizations in the athena trial , there are no other controlled data that show a benefit of rhythm control therapy beyond improved quality of life . hence , current guidelines for the treatment of af base the decision to add rhythm control therapy to the management of af on individual factors interpreted by the physician and the patient . these factors include the severity of complaints and how these will affect the individual patients , and the severity of af , i.e. how successful rhythm control is expected to be . further elucidation of the mechanisms and signals involved in the process of sustaining af might ultimately improve therapeutic strategies and outcome of rhythm control therapy both for maintenance of sinus rhythm and for prevention of morbidity and mortality . age , hypertension , congestive heart failure , valve disease , and diabetes are all well - known risk factors for the development of af . less well - known risk factors include , among others , endurance training , obesity , sleep apnoea syndrome , and chronic obstructive pulmonary disease . these risk factors together with an altered metabolism , autonomic changes , and genetic and environmental factors cause marked changes in the molecular function and structure of the atria , which is called structural remodelling . the induced molecular and structural changes in the atria include cellular calcium overload , activation of the reninangiotensinaldosterone system , inflammation , oxidative stress , enlarged atria , hypertrophy , fibrosis , dedifferentiation , apoptosis , myolysis , and amyloidosis ( figure 2 ) . structural remodelling ultimately creates a substrate for af due to electrical dissociation between muscle bundles and local conduction heterogeneities facilitating the initiation and perpetuation of af . once af develops , it causes marked changes in atrial electrophysiology ( electrical remodelling ) and further deteriorates the structural remodelling process . thus , atrial remodelling in patients with af is caused by both the associated diseases and af itself and may contribute to af - related complications . ultimately , due to ongoing remodelling , patients progress to permanent af . figure 2flow chart showing the series of events caused by stretch . hypothetical scheme of stretch induced by hypertension , heart failure and possibly extreme endurance exercise leading to calcium overload , activation of the reninangiotensinaldosterone system and release of different factors , resulting in structural remodelling and finally in atrial fibrillation ( adapted with permission from de jong et al . ) . hypothetical scheme of stretch induced by hypertension , heart failure and possibly extreme endurance exercise leading to calcium overload , activation of the reninangiotensinaldosterone system and release of different factors , resulting in structural remodelling and finally in atrial fibrillation ( adapted with permission from de jong et al . ) . the remodelling changes may still be reversible during early phases of the arrhythmia , probably even more if the duration of the underlying disease also is not too long , but may provoke relevant and permanent atrial damage during later stages of af and associated diseases . this may explain the disappointing outcome of rhythm control therapy in prior studies , both for the prevention of recurrent af and for cardiovascular morbidity and mortality . most trials included patients in whom the extent of remodelling was severe and even irreversible due to a long history of both af and the underlying heart disease . since the underlying disease is also a major contributor to the remodelling process , in some patients a first episode of af may already be untreatable , even with aggressive therapy , due to the presence of substantial structural changes . in patients with a shorter history of both af and the underlying disease , the remodelling processes are assumingly less advanced , which may provide more opportunities for rhythm control strategies to be effective . by successfully eliminating af the remodelling process may become less progressive , reducing the extent of fibrosis , inflammation , atrial hypertrophy , and other adaptation processes . hypothetically , this may also lower the risk of complications associated with af , like stroke and heart failure . the poor outcome of rhythm control relates to the severity of the atrial substrate for af not only due to the underlying atrial remodelling process but also due to the poor efficacy and adverse events of the currently available ion - channel antiarrhythmic drugs and ablation techniques . while catheter ablation was not incorporated into the rate vs. rhythm control trials , today it is increasingly performed in patients with symptomatic af . this is performed with one long , encircling lesion around the right and another lesion around the left pulmonary veins . several prospective randomized trials comparing ablation vs. antiarrhythmic drugs to maintain sinus rhythm consistently show that ablation therapy is significantly more effective in maintaining sinus rhythm compared with antiarrhythmic drugs with an overall risk reduction of af recurrence by 65 to 70% at 1-year follow - up , keeping in mind that most of these trials have shortcomings in detecting recurrent af . a recent meta - analysis showed a single - procedure success rate of ablation off antiarrhythmic drugs of 57% , a multiple procedure success rate off antiarrhythmic drugs of 71% , and a multiple procedure success rate on antiarrhythmic drugs of 77% . in comparison , whether these short - term success rates may be extrapolated to long - term success is yet unclear . recently reported on 123 consecutive patients with paroxysmal or persistent af who underwent pulmonary vein isolation and were free from af and without antiarrhythmic drugs 1 year after ablation . long - term ablation success , defined as freedom from af off antiarrhythmic drugs after a single - ablation procedure , was 85% at 3 years and 71% at 5 years , with an 7% per year late recurrence rate after the first year . predictors for late recurrences were the known risk factors for af ( progression ) , including higher age , hypertension , larger left atrial size , as well as more af triggers being present during the electrophysiological procedure , and patients who present with persistent af . arrhythmia - free survival following the last catheter ablation procedure was , in 100 patients undergoing ablation in 20012002 in their experienced hands , 87 , 81 , and 63% at 1 , 2 , and 5 years , respectively . thus , although most recurrences transpire over the first 612 months , a slow but steady decline in arrhythmia - free survival is noted thereafter , even after three or more years of apparent arrhythmia control . nevertheless , catheter ablation for af appears to be more effective in maintaining sinus rhythm compared with antiarrhythmic drug therapy . it might be even more effective when considering the fact that most ablation studies included patients with a relatively long history of af and the associated disease who had failed serial antiarrhythmic drug testing . thus , success might be further improved by a better selection of patients and more efficacious and safe ablation techniques . the highest efficacy of catheter ablation is observed in younger patients with less severe atrial remodelling . feared complications include tamponade , stroke , pulmonary vein stenosis , permanent diaphragmatic paralysis , and oesophageal fistula . only one small study investigated catheter ablation and antiarrhythmic drugs as first - line therapy in patients with a duration of af of > 8 months . duration of the associated disease was not reported . at 1 year of follow - up it is , however , unknown yet as to whether an early ablation therapy and a further optimization of ablation procedures could further improve success rate and reduce complications associated with the ablation procedure , and , in turn , may improve cardiovascular outcomes like mortality , cardiovascular hospitalizations , worsening of heart failure , and stroke . safer and more effective antiarrhythmic drugs may also improve the success rate of a rhythm control strategy . ion channel - blocking antiarrhythmic drugs may counteract the electrical remodelling , but leave other mechanisms like the structural remodelling process untouched . in addition , these drugs all carry a relatively high risk of adverse events including life - threatening arrhythmias like torsades de pointes . for that reason however , most of these drugs were abandoned before approval due to the risk of proarrhythmia . dronedarone , a novel benzofuran derivative structurally related to amiodarone , has recently been approved and may improve the outcome of rhythm control therapy . it has a beneficial safety profile both in patients without structural heart disease and in those with stable mild - to - moderate heart disease and seems to carry a very low risk for proarrhythmia . however , it is contraindicated in patients with impaired left ventricular function [ new york heart association ( nyha ) class iii / iv ] and haemodynamic instability because of the data of the andromeda study . this study investigated the effect of dronedarone on the risk of hospitalization for progressive heart failure in a placebo - controlled study in patients with nyha class iii or iv congestive heart failure , and a lvef < 35% . after 2 months this trial was terminated because of a higher mortality rate in the dronedarone treatment group due to progressive heart failure . similar to sotalol , propafenone , and flecainide , dronedarone is less effective to maintain sinus rhythm compared with amiodarone , but its efficacy has not been tested in patients with early af . data from the recently published athena trial on outcome in patients with af showed a reduction of the primary composite outcome driven by cardiovascular hospitalizations ( hazard ratio 0.74 , 95% confidence interval 0.690.84 , p < 0.0001 ) . comparable beneficial outcome effects have been demonstrated for amiodarone , but this beneficial effect is counteracted by a high rate of non - cardiac adverse events . adverse effects associated with dronedarone have also been reported but seem to be less harmful . thyroid , ocular , or pulmonary side effects in these studies were not significantly different from placebo - treated patients . similar to amiodarone , however , dronedarone is associated with an increase in serum creatinine , which are assumed to be the result of inhibition of tubular secretion , independent of renal function . substrate - oriented antiarrhythmic drug therapy that modifies the structural atrial remodelling process may also improve the outcome of rhythm control . upstream therapy refers to the use of non - ion channel antiarrhythmic drugs that modify the atrial substrate to prevent the occurrence of new onset af or recurrence of the arrhythmia . it includes treatment with reninangiotensinaldosterone system ( raas ) blockers [ angiotensin - converting enzyme inhibitors ( ace - inhibitor ) , angiotensin receptor blockers , aldosterone receptor antagonists ] , statins , and omega-3 polyunsaturated fatty acids . in addition , these drugs improve haemodynamics by lowering of blood pressure and reduction of left ventricular and atrial wall stress , which also may have beneficial effects on the remodelling process . statins , known for their lipid - lowering capacities , have a variety of pleiotropic properties including attenuation of inflammation through anti - atherogenic and antioxidant actions . results of upstream therapy for the prevention of af in animal experiments , hypothesis - generating small clinical studies , and retrospective analyses in selected patient categories have been encouraging . larger prospective randomized trials , however , did fail to show any protective benefit against af in patients with and without structural heart disease , while patients with known left ventricular dysfunction or with diabetes mellitus and left ventricular hypertrophy experience less new onset af on ace - inhibitor or sartans compared with placebo or beta - blockers . this suggests that inhibition of the reninangiotensin system may be helpful to prevent af in patients whose atria are exposed to marked volume or pressure overload by systolic or diastolic dysfunction . the randomized trials so far included patients in whom the extent of remodelling was severe and even irreversible due to a longer history of af and underlying heart disease . in patients with a shorter history of af and the underlying disease , remodelling processes are assumingly less advanced , providing greater opportunity for upstream therapies to be effective . atrial fibrillation is responsible for a five - fold increase in the risk of ischaemic stroke . therefore , oral anticoagulation therapy is the cornerstone for the treatment of af patients with an increased risk of thromboembolic complications . such treatment is needed independently from the therapeutical strategy decided , rate , or rhythm control . but even with oral anticoagulation the residual stroke or systemic embolism rate in patients with af remains relatively high . the presence of af seems one of the modifiable factors associated with death and cardiovascular morbidity in af patients . we can therefore hypothesize that if effective and safe methods for maintaining sinus rhythm with fewer adverse effects become available rhythm control therapy may become the first choice therapy in more patients . a promising strategy might be catheter ablation combined with safe antiarrhythmic drugs and substrate - oriented antiarrhythmic drugs with beneficial effects on outcome parameters . catheter ablation is nowadays an effective therapy but only retrospective evidence supports the notion that catheter ablation may result in reduced mortality . therefore , prospective randomized trials that include catheter ablation and new antiarrhythmic drugs for rhythm control are needed to reaffirm the concept that sinus rhythm maintenance may improve outcome . these trials preferably should be performed in patients with a short history of af and the underlying disease , i.e. in patients with less severe remodelled atria . patients with a short history of af and the underlying heart disease have not been studied before . it is fair to assume that the abolishment of af in these patients is more successful and possibly also safer , which could translate into a prognostic benefit of early rhythm control therapy . several trials are now investigating whether aggressive early rhythm control therapy can reduce cardiovascular morbidity and mortality and increase the maintenance of sinus rhythm . the radiofrequency ablation versus antiarrhythmic drugs for atrial fibrillation treatment ( raaft ) study ( clinical trials.gov number nct00393054 ) randomized 130 patients nave to antiarrhythmic drugs to either atrial ablation or antiarrhythmic drugs as first - line treatment of symptomatic af . their primary endpoint is time to first recurrence of electrocardiographically documented symptomatic af lasting > 30 s. a second trial , the catheter ablation versus anti - arrhythmic drug therapy for atrial fibrillation trial ( cabana , clinical trials.gov number nct00911508 ) currently randomizes patients to left atrial endocardial catheter ablation or current state - of - the - art therapy with either rate or rhythm control drugs . the routine versus aggressive upstream rhythm control for the prevention of early atrial fibrillation in heart failure study ( race 3 , nct00877643 ) includes patients with early af ( total af history < 2 years , total persistent af duration < 6 months , and 1 previous electrical cardioversion ) , and mild - to - moderate early heart failure ( total heart failure history < 1 year ) . patients are randomized to aggressive upstream therapy with structured physical activity or routine rhythm control as described in the 2010 af guidelines . the primary endpoint of the study is sinus rhythm after 1 year of follow - up , defined as sinus rhythm during 6/7th of assessable time of continuous 7 days holter monitoring during the last week of the study . finally , the early treatment of atrial fibrillation for stroke prevention trial ( east , isrctn - nr : 04708680 ) will soon start to include high - risk patients with shortlasting af ( known history of af < 1 year ) . this trial will randomize 3150 patients to early rhythm control therapy either by atrial catheter ablation or antiarrhythmic drugs ( preferably dronedarone , and in case of a recurrence both modalities ) , or usual care as described in the 2010 af guidelines . the primary outcome is cardiovascular mortality , stroke , transient ischaemic attack , and hospitalization due to worsening of heart failure or acute coronary syndrome . the above - mentioned studies will give us new insight into whether slowing down the progression of af may prevent af - related complications . i.c.v.g . received research grants from netherlands heart foundation , interuniversity cardiology institute the netherlands , astra zeneca , biotronik , medtronic , sanofi - aventis , boehringer ingelheim , st jude medical , boston scientific . l.s . received research grants from polish ministry of science and higher education , european union ( eu ) , biotronik . l.m . received research grants from biosense webster , medtronic ; boston scientific , st jude medical . p.k . received research grants from 3 m medica / meda pharma , cardiovascular therapeutics , medtronic , omron , sanofi - aventis , st jude medical , german federal ministry for education and research ( bmbf ) , fondation leducq , german research foundation ( dfg ) , european union ( eu ) . funding to pay the open access publication charges conflict of interest : i.c.v.g . has received consulting fees / honoraria from medtronic , msd , sanofi - aventis , and boehringer ingelheim ; travel support to attend meetings from the european society of cardiology ( esc ) and the european heart rhythm association ( ehra ) , a registered branch of esc ; and research grants from the netherlands heart foundation , the interuniversity cardiology institute of the netherlands , astra zeneca , biotronik , medtronic , sanofi - aventis , boehringer ingelheim , st jude medical , and boston scientific . a.b . has received consulting fees / honoraria from biotronik , boehringer ingelheim , bristol - myers squibb , medtronic , msd , sanofi - aventis , and st jude medical . has received consulting fees / honoraria from sanofi aventis , meda pharma , glaxosmithkline , bayer , boehringer ingelheim , bms - pfizer , medtronic , biotronik , and st jude medical . has received consulting fees / honoraria from abbott , biosense webster , biotronik , boehringer ingelheim , boston scientific , cardiofocus , ge healthcare , hansen medical , medtronic , pfizer , sanofi - aventis , siemens healthcare , and st jude medical , and travel support to attend meetings from the european society of cardiology ( esc ) , the european heart rhythm association ( ehra ) , and from the german atrial fibrillation competence network ( afnet ) . has received consulting fees / honoraria from medtronic , biotronik , biosense webster , st jude medical , and sanofi - aventis ; travel support from the european heart rhythm association and the european society of cardiology ; and research grants from the polish ministry of science and higher education , the european union , and biotronik . has received consulting fees / honoraria from bard , biosense webster , medtronic , boston scientific , st jude medical , sanofi - aventis , biotronik , and sorin group , and research grants from biosense webster , medtronic , boston scientific , and st jude medical . has received consulting fees / honoraria from biosence webster and sanofi - aventis , and research grants from st jude medical . has received consulting fees / honoraria from 3 m medica , meda pharma , astrazeneca , bayer healthcare , boehringer ingelheim , medtronic , merck , msd , otsuka pharma , pfizer / bms , sanofi - aventis , servier , siemens , and takeda ; travel support to attend meetings from the european society of cardiology ( esc ) , the european heart rhythm association ( ehra ) , a registered branch of esc , and from the german , atrial fibrillation competence network ( afnet ) ; and research grants from 3 m medica / meda pharma , cardiovascular therapeutics , medtronic , omron , sanofi - aventis , st jude medical , german federal ministry for education and research ( bmbf ) , fondation leducq , german research foundation ( dfg ) , and the european union .

atrial fibrillation ( af ) is the most common sustained arrhythmia and an important source for mortality and morbidity on a population level . despite the clear association between af and death , stroke , and other cardiovascular events , there is no evidence that rhythm control treatment improves outcome in af patients . the poor outcome of rhythm control relates to the severity of the atrial substrate for af not only due to the underlying atrial remodelling process but also due to the poor efficacy and adverse events of the currently available ion - channel antiarrhythmic drugs and ablation techniques . data suggest , however , an association between sinus rhythm maintenance and improved survival . hypothetically , sinus rhythm may also lead to a lower risk of stroke and heart failure . the presence of af , thus , seems one of the modifiable factors associated with death and cardiovascular morbidity in af patients . patients with a short history of af and the underlying heart disease have not been studied before . it is fair to assume that abolishment of af in these patients is more successful and possibly also safer , which could translate into a prognostic benefit of early rhythm control therapy . several trials are now investigating whether aggressive early rhythm control therapy can reduce cardiovascular morbidity and mortality and increase maintenance of sinus rhythm . in the present paper we describe the background of these studies and provide some information on their design .

IntroductionScope of the problem Potential benefit of early rhythm control therapy New modalities for safe and relatively effective rhythm control therapies: ablation, new antiarrhythmic drugs, and upstream therapy The need for staged therapy Perspective: slowing down the progression of atrial fibrillation to prevent atrial fibrillation-related complications Funding

atrial fibrillation ( af ) is the most common sustained arrhythmia and an important source of mortality and morbidity on a population level . despite the clear association between af and death , stroke , and other cardiovascular events all published studies have shown that rate control is not inferior to rhythm control for the prevention of mortality and morbidity ( tables 1 and 2 ) . this disappointing outcome may relate to the low long - term maintenance rate of sinus rhythm in the rhythm control groups of these studies , being 63% after 5 years in the atrial fibrillation follow - up investigation of rhythm management ( affirm ) trial and 39% after 2.3 years of follow - up in the rate control versus electrical cardioversion ( race ) trial . also the atrial fibrillation and congestive heart failure trial ( af - chf ) observed no difference in cardiovascular mortality ( primary outcome ) between patients with a left ventricular ejection fraction ( lvef ) 35% , symptoms of congestive heart failure and a history of af randomized to rate or rhythm control , nor in the secondary outcomes including death from any cause and worsening of heart failure . with permission)patients reaching primary endpoint ( n)trialpatients ( n)mean age ( years)mean length of follow - up ( years)inclusion criteriaprimary endpointrate controlrhythm controlppiaf25261.01.0persistent af ( 7360 days)symptomatic improvement76/125 ( 60.8%)70/127 ( 55.1%)0.32affirm406069.73.5paroxysmal af or persistent af , age 65 years or older , or risk of stroke or deathall - cause mortality310/2027 ( 25.9%)356/2033 ( 26.7%)0.08race52268.02.3persistent af or flutter for < 1 year and 1 to 2 cardioversions > 2 years and oral anticoagulationcomposite : cardiovascular death , chf , severe bleeding , pm implantation , thromboembolic events , severe adverse effects of antiarrhythmic drugs44/256 ( 17.2%)60/266 ( 22.6%)0.11staf20066.01.6persistent af ( > 4 weeks and < 2years ) , left atrial size > 45 mm , chf nyha ii iv , lvef < 45%composite : overall mortality , cerebrovascular complications , cpr , embolic events10/100 ( 10.0%)9/100 ( 9.0%)0.99hot caf20560.81.7first clinically overt persistent af ( 7 and < 2 years ) , 5075-year oldcomposite : death , thromboembolic events ; intracranial/ major haemorrhage1/101 ( 1.0%)4/104 ( 3.9%)>0.71af - chf1376663.1lvef35% , symptoms of chf , history of af ( 6 h or ecv < last 6 months)cardiovascular death175/1376 ( 25%)182/1376 ( 27%)0.59af , atrial fibrillation ; affirm , atrial fibrillation follow - up investigation of rhythm management ; chf , congestive heart failure ; cpr , cardiopulmonary resuscitation ; ecv , electrical cardioversion ; hot cafe , how to treat chronic atrial fibrillation ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; piaf , pharmacological intervention in atrial fibrillation ; pm , pacemaker ; race , rate control versus electrical cardioversion for persistent atrial fibrillation ; staf , strategies of treatment of atrial fibrillation . this , together with the results of the recently published athena trial , supports the idea that the presence of af is one of the modifiable factors associated with death and cardiovascular morbidity in af patients . further elucidation of the mechanisms and signals involved in the process of sustaining af might ultimately improve therapeutic strategies and outcome of rhythm control therapy both for maintenance of sinus rhythm and for prevention of morbidity and mortality . the poor outcome of rhythm control relates to the severity of the atrial substrate for af not only due to the underlying atrial remodelling process but also due to the poor efficacy and adverse events of the currently available ion - channel antiarrhythmic drugs and ablation techniques . the presence of af seems one of the modifiable factors associated with death and cardiovascular morbidity in af patients . patients with a short history of af and the underlying heart disease have not been studied before . it is fair to assume that the abolishment of af in these patients is more successful and possibly also safer , which could translate into a prognostic benefit of early rhythm control therapy . several trials are now investigating whether aggressive early rhythm control therapy can reduce cardiovascular morbidity and mortality and increase the maintenance of sinus rhythm .

nerve lesions developing due to trauma are the most common ones in practice1 . magnetic fields and swimming exercises have been shown to affect nerve regeneration , soft tissue , and particularly the growth of all nerve tissue in many studies done about electromagnetic fields and swimming2,3,4,5 . the aim of the this study was to reveal the effects of swimming exercise and a pulsed electromagnetic field applied after a crush - type injury , which is a neuropathy model , with electrophysiologic methods . this study was conducted after ethics committee approval had been obtained from the local ethics committee for animal experimentation at adnan menderes university ( ad - hadyek 050.04/2010/090 ) . this study was conducted in the department of physiology of veterinary medicine , adnan menderes university , between 2010 and 2012 . a total of 28 male wistar albino rats aged 3 months were used in the study . the influences of swimming exercise and a pulsed electromagnetic field ( pemf ) applied for 4 weeks on healing were detected by an electroneuromyographic method in the regeneration processes of the rat sciatic nerve after a crush - type injury , which was selected as a neuropathy model . group 1 was the control group , group 2 was the injury control group ( sciatic nerve injury ) , group 3 was the injury + pemf group ( sciatic nerve injury and pemf ) , and group 4 was the injury + swimming group ( sciatic nerve injury and swimming exercise ) . each group was kept in macrolon cages . rats were kept in a semi - climatized room at 222 c with a 12/12 h dark and light cycle and fed standard rat feed ; they were given feed and water ad libitum . rats were applied anesthesia with ketamine ( 90 mg / kg ) + xylazine ( 10 mg / kg ) , and the left sciatic nerve was removed at the middle femoral level by blunt dissection under aseptic conditions . the sciatic nerve was squeezed and crushed with fine forceps for 30 sec as 3 notch ( the severity of the clamping forceps ) . afterwards , the skin in the incision region was closed routinely , and the operation was terminated . a superficial wound was created only on the skin surface of the animals in control group . a regular magnetic field was formed in the axial center of two equal regular coils with diameter of 6.5 cm set 6.5 cm apart . according to the known basic electronic principles , the formula for calculating the magnetic field b= ( 8.99 10 ) ni / r is used6 . here , n is the number of wire winding of a coil , i is the effective value of serial , equal flow passing from the coils , and r is the mean diameter of the coils . at 2 hz ( 120 ppm ) , when power stage is at 8 , the effective value of the coil flow is 119 ma and feeding tension of the coils is 0,4 v. the measured and calculated homogenous magnetic field density are both 0.3 mt . pemf application was performed with a two - channel electromagnetic therapy system ( emts digital bm 3006s ) . the pulsed electromagnetic field system was composed of a pair of coils and a pulsed power source . pemf was performed with 0,3 mt ( 3 g ) power with a frequency of 2 hz and a sinusoidal wave form . pemf application was performed by placing the rats between the coils in a specifically prepared apparatus with the rats in a restrainer . application was performed for hour a day , five days a week , for four weeks and was performed concurrent to the swimming exercise in the swimming exercise group . the rats in the swimming exercise group were put into individual pools with a depth of 30 cm and diameter of 41 cm containing water at 351 c and were induced to swim . swimming exercise was applied one hour a day , five days a week , for four weeks . this was performed concurrent to the pemf application in the electromagnetic field treatment group . animals were administered anesthesia comprising a combination of 50 mg / kg ketamine ( alfamine ) + 10 mg / kg xylazine ( alfazine ) . the field to be measured was shaved , cleaned with alcohol , and dried . animals were placed on thermal pads in order to avoid potential effects of low body temperature on nerve conduction , and body temperatures were kept within normal physiologic ranges as best as possible7 . the gastrocnemius muscle was used for emg recording . an active electrode was placed in the middle part of the muscle , reference electrode was place in the tendon region , and ground electrode was placed on the tail . a stimulus electrode was placed at the trochanter major level of the sciatic nerve . a viasys nicolet viking quest ( usa ) h - reflex and m - wave responses were determined by elevating the stimulus intensity 12 v until reaching the maximum amplitude level . the average stimulus intensity for the motor nerve conduction velocity ( mncv ) was 12.03 v for the control group , 12.17 v for the injury group , 12.48 v for the injury + electromagnetic field group , and 12.50 v for the injury + swimming group . the mean stimulus intensity for the h - reflex was determined to be 7.55 v for the control group , 7.83 v for the injury group , 7.73 v for the injury + electromagnetic field group , and 8.68 v for the injury + swimming group . the latency period was taken as the duration between beginning of the stimulus and the beginning of the action potential . the shapiro - wilk test was used to determine the normality of the distribution of data . logarithmic or square root transformation was applied to data not showing a normal distribution . significance of the difference between mean values was determined with two - way analysis of variance for repeated measurements ( two - way anova ) . duncan s post hoc test was used to determine from which group a difference arose . tukey s hsd post hoc test was performed to determine from when or from which group a difference arose in data with interaction8 ) . one - way analysis of variance ( one - way anova ) was performed using duncan s post hoc test in order to determine from which time interval and from which group an effect arose in data where there was no time difference and group - time interaction but there was a group effect . the paired t - test was performed to compare data obtained from the right and left legs at the same measurement time points . although there were time - dependent variations in all groups in terms of body weights and left side mncv and latency values compared with the output values for four weeks ( p<0.05 ) , there was no effect of any groups ( p>0.05 ) or group - time interaction ( p>0.05 ) . the left mncv values at the first week were lower in the injury group compared with the control group ; however , the difference was not statistically significant ( p>0.05 ) . the increase in mncv in the swimming and electromagnetic field groups were not statistically significant when compared with the control and injury groups . when the right and left mncv values were analyzed for all groups , a significant reduction was detected in initial values at the time of injury in the pemf and swimming groups and in initial values in the injury group and in the left leg compared with the right leg in week one measurements ( p<0.05 ) . no difference was detected in the control group in terms of right and left leg pemf values during the experiment ( table 2table 1.left leg latency ( msec ) and cmap ( mv ) values of ratsgroupnweeksoutput values ( 0)1st week2nd week 3rd week4th weekleft leg latency ( msec ) control*71.190.011.140.011.210.021.170.021.150.02injury control*71.200.011.140.011.190.021.180.021.160.02pemf*71.200.011.180.011.180.021.190.021.180.02swimming*71.190.011.180.011.200.021.160.021.180.02left leg cmap ( mv)control77.696.018.645.1610.984.628.243.3611.026.28injury control71.911.382.471.644.612.544.042.394.632.26pemf74.223.504.332.042.402.823.332.465.034.45swimming72.511.744.703.504.012.183.272.202.062.17different symbols within a column indicate statistical significance ( p<0.05 ) , * statistically significant ( there were time - dependent variations in all groups ) ( p<0.05 ) . control , control group ( group 1 ) ; injury control , injury control group ( group 2 ) ; pemf , injury + pemf ( group 3 ) ; swimming , injury + swimming ( group 4).table 2.left and right leg mncv ( m / sec ) and latency ( msec ) ratios of ratsgroup / weeknmncv ( m / sec ) output values ( 0)1st week2nd week3rd week4th weekleftrightleftrightleftrightleftrightleftrightcontrol732.701.6033.590.5038.071,0536.061.4633.581.5034.730.7032.1407334.201.3534.721.2335.011.07injury control729.231.21 * 33.891.0234.371.48 * 37.141.3034.581.9935.631.1234.680.9435.660.7334.461.3934.440.64pemf729.741.54 * 33.441.2134.070.6633.691.1731.951.9332.670.7333.260.9934.920.7933.051.7533.771.76swimming730.281.57 * 34.901.0633.620.7634.551.3633.121.1934.191.2936.560.3535.881.5533.221.86 * 36.240.97latency ( msec)control71.410.021.330.041.300.031.330.411.450.021.350.021.360.011.410.021.310.031.160.02injury control71.450.02 * 1.310.031.300.031.340.021.420.03 * 1.310.021.400.051.270.021.350.041.150.03pemf71.430.041.420.041.380.021.350.021.400.061.440.011.420.051.320.021.400.031.170.03swimming71.410.03 * 1.300.031.400.021.340.031.440.03 * 1.320.0421.350.01 * 1.240.011.400.0651.160.03*statistical significance compared with the right leg ( p<0.05 ) , different symbols within a column indicate statistical significance ( p<0.05 ) . control , control group ( group 1 ) ; injury control , injury control group ( group 2 ) ; pemf , injury + pemf ( group 3 ) ; swimming , injury + swimming ( group 4 ) . ) . different symbols within a column indicate statistical significance ( p<0.05 ) , * statistically significant ( there were time - dependent variations in all groups ) ( p<0.05 ) . control , control group ( group 1 ) ; injury control , injury control group ( group 2 ) ; pemf , injury + pemf ( group 3 ) ; swimming , injury + swimming ( group 4 ) . * statistical significance compared with the right leg ( p<0.05 ) , different symbols within a column indicate statistical significance ( p<0.05 ) . control , control group ( group 1 ) ; injury control , injury control group ( group 2 ) ; pemf , injury + pemf ( group 3 ) ; swimming , injury + swimming ( group 4 ) . right side latency values were prolonged in the electromagnetic field group compared with the injury and swimming groups during the entire experiment ( p<0.05 ) . when the right and left leg latency values were analyzed for all measurement time points , significant elongation was detected in left side latency values compared with right side latency values at the beginning and in the second week in the injury group and at the beginning and in the second and third weeks in the swimming group ( p<0.05 ) ( table 2 ) . in the control and pemf groups , no difference was detected in terms of the rigt and left leg latency values ( p>0.05 ) ( table 2 ) . a group effect was detected in terms of the left leg compound muscle action potential ( cmap ) ( p<0.001 ) . a statistically significant decrease was detected in the injury group in terms of the cmap value compared with the control group at output and in the first week ( p<0.05 ) . in the second week , the cmap values of the control group were higher than those in all other groups ( p<0.01 ) . the cmap values were significantly lower in the injury and swimming groups compared with the control group in week 3 and were significantly lower in the swimming group only in week 4 ( p<0.01 ) ( table 1 ) . the right and left leg cmap values were found to be significantly different from each other at all measurement times points in all groups ( p<0.05 ) ( table 3table 3.left and right amplitudes of cmap ( mv ) and hmax / mmax ratios of ratsgroup / weekncmap ( mv)output values ( 0)1st week2nd week3rd week4th weekleftrightleftrightleftrightleftrightleftrightcontrol73.210.35 * 6.821.227.861.65 * 7.890.6116.093.04 * 5.860.338.121.18 * 9.632.1412.501.77 * 12.591.77injury control71.890.84 * 5.652.932.220.44 * 3.580.524.941.01 * 11.172.314.120.08 * 14.224.135.320.85 * 6.150.52pemf76.250.68 * 34.302.343.990.31 * 5.011.611.400.46 * 13.070.883.560.75 * 14.474.193.301.03 * 5.301.24swimming72.650.47 * 14.314.492.000.80 * 21.204.404.780.64 * 26.613.134.030.60 * 2.350.561.320.52 * 1.470.50hmax / mmax ratioscontrol70.180.040.220.080.220.110.250.110.170.080.220.060.140.090.440.650.180.110.200.12injury control7 * 0.490.200.230.01 * 0.430.140.230.05 * 0.700.280.240.03 * 0.570.350.260.05 * 0.540.220.210.03pemf7 * 0.560.180.230.04 * 0.450.170.280.13 * 0.780.300.230.07 * 0.390.200.220.09 * 0.370.130.200.04swimming7 * 0.630.260.180.04 * 0.440.230.170.03 * 0.790.330.210.07 * 0.390.300.180.06 * 0.310.090.190.08*statistical significance compared with the right leg ( p<0.05 ) , different symbols within a column indicate statistical significance ( p<0.05 ) . control , control group ( group 1 ) ; injury control , injury control group ( group 2 ) ; pemf , injury + pemf ( group 3 ) ; swimming , injury + swimming ( group 4 ) . ) . * statistical significance compared with the right leg ( p<0.05 ) , different symbols within a column indicate statistical significance ( p<0.05 ) . control , control group ( group 1 ) ; injury control , injury control group ( group 2 ) ; pemf , injury + pemf ( group 3 ) ; swimming , injury + swimming ( group 4 ) . when data in the injury field were analyzed in terms of h - reflex latency values , time ( p<0.001 ) , group ( p<0.001 ) , and group - time interaction found ( p<0.001 ) . in within - group comparison for the pemf and swimming groups , while there was a significant improvement in h - reflex latency values beginning from the first week , this improvement was detected beginning from the second week in the injury group . when groups were compared with each other , the treatment application groups reached the control group values in the second week , and the injury group reach the control group values in the third week . in within - group comparisons for the injury , pemf , and swimming groups in terms of the right and left leg h - reflex latency values , the lef - side h - reflex latency values were seen to decrease in the injury , pemf , and swimming groups ( p<0.05 ) ; however , this decrease was found in the right - side h - reflex latency values ( healthy side ) ( table 4table 4.left and right leg h - reflex latency ( msec ) valuesgroup / weeknh - reflex latency values ( msec)output values ( 0)1st week2nd week3rd week4th weekleftrightleftrightleftrightleftrightleftrightcontrol75.290.155.050.105.250.224.950.135.200.064.870.105.380.094.970.08 * 5.140.104.940.08injury control76.920.194.710.07 * 6.200.234.870.08 * 5.780.164.840.19 * 5.780.225.070.145.480.154.710.05*pemf77.260.214.750.08 * 6.010.124.910.11 * 5.500.134.910.09 * 5.460.164.810.08 * 5.450.184.800.09*swimming76.850.114.700.14 * 6.010.154.500.11 * 5.720.094.670.13 * 5.260.144.940.055.470.184.670.08**statistical significance compared with the right leg ( p<0.05 ) . control , control group ( group 1 ) ; injury control , injury control group ( group 2 ) ; pemf , injury + pemf ( group 3 ) ; swimming , injury + swimming ( group 4 ) . ) . control , control group ( group 1 ) ; injury control , injury control group ( group 2 ) ; pemf , injury + pemf ( group 3 ) ; swimming , injury + swimming ( group 4 ) . while there were no significant differences in left hmax / mmax values for time and group factors ( p<0.001 ) , group - time interaction was not found ( p>0.05 ) . the left hmax / mmax values of the control group were lower than those of the other groups at output and in the second week ( p<0.01 ) . the left hmax / mmax values of the control group were lower than those of the other groups in the first week ( p<0.05 ) . the left hmax / mmax values in the third and fourth weeks were lower than those of the groups ; however , this difference was only significant in the injury group ( p<0.05 ) . when the rigth and left leg hmax / mmax ratios were compared , while the left leg hmax / mmax ratios of the injury , pemf , and swimming groups were higher than those of the right leg hmax / mmax ratios during the experiment ( p<0.05 ) , no difference was found in the control group ( p>0.05 ) ( table 3 ) . weight gain in the injury group was found to be particularly more prominent after the second week . this result suggests that injured animals could have stayed immobile and therefore desired to move less , and their weight gain may have arise from this . van meeteren et al.9 revealed that chronic - pulsed stress application reduced live weight gain in sciatic nerve injury - induced rats . similarly , the live weight gains in the magnetic field and swimming groups being less than that of the control group indicates that both swimming exercise and pemf application act as stress factors in animals . in addition , the reduction in live weight in the pemf - applied animals during the first two weeks suggests that magnetic field application is a more effective stress factor compared with swimming in animals . in our study , the mean mncv values of the control group were 3238 m / sec at all measurement time points . in the study of hseyinolu et al.10 investigating nerve regeneration in wistar albino rats , the mean sciatic nerve mncv value was found to be 59.08 m / sec . hseyinolu et al.10 obtained records by stimulating the nerve from two different points using a bipolar needle electrode . in our study , a bipolar superficial electrode was used for nerve stimulation . the distance between the anode and cathode of this electrode was about 2 cm . reference points that could most easily reach the nerve were used in accordance with the literature11 . the anatomic injury and measurement technique made determination of mncv using a reference point mandatory . in other words , mncv was calculated using the distance between the stimulus point and record point through only distal latency . this difference in methods also caused a lower nerve conduction velocity to be obtained than the mean nerve conduction velocity reported in the literature . data were also found to be similar in the experiment groups , as measurements were performed similarly in all groups . in this study , squeezing the sciatic nerve for 30 sec led to a reduction in nerve conduction velocity . however , this reduction was not significant in the intergroup comparison . on the other hand , when data obtained from healthy and injured legs of rats were compared , the difference was significant five days after injury . this difference remained in the next measurement interval in the injury group ( on day 12 after injury ) . however , the mncv is seen to normalize within the day seven after the fifth day that injury is determined in the swimming and pemf groups . this finding indicates that the swimming and pemf applications caused normalization of the mncv one week earlier compared with the control group . this five - day period , wound healing in rats , and is necessary for the formation of wallerian degeneration . in addition , previous studies revealed that wallerian degeneration started within the hours following injury , and this process could change depending on the type and duration of injury formation12,13,14 . myelin destruction after axotomy in rats begins from the second day , and the number of myelin - phagocytosing macrophages reaches the maximum on day 7 . both destruction and renewal processes take approximately two weeks15 , 16 . considering that the process results in axonotmesis , a similar process may also be possible in a squeeze injury model in which cell bodies stay durable . given that the injury was limited to only the myelin sheath , the healing process takes an average of two weeks without any interventions . in the swimming and pemf groups , this significance disappeared after the first measurement time point , and functional recovery after sciatic nerve injury is similar in mice and rats , and sensory and motor functions normalize within approximately three weeks in both types of injury17 . the myelin sheath thickness is significantly increased in injured axons at the end of 6 weeks of swimming exercise started just after sciatic nerve injury and beginning 14 days after injury18 . however , functional evaluations revealed that conduction velocities returned to normal in the injury group within two weeks . regarding the latency values , no significance was detected when the injury and control groups were compared to each other at the first measurement time point . when the right and left leg latency values were analyzed for all measurement times points , significance was found between the initial measurements and those at 2 weeks in the injury group and between the measurements for the second and third weeks . no change was detected in the control and pemf groups in terms of latency values . english et al.19 found that m - response and h - reflex latencies were longer compared with control groups after incision . navarro and valero cabre20 also found similar results . in their study , the latency values were less prolonged in the crush group compared with the incision and implant groups . when recovery rates were evaluated , the latency values were found to be long in the crush group compared with the control even after 90 days . the latency values reached the same values as the controls in all groups at the end of 4 weeks . udina et al.21 found that active and passive exercise caused prolongation of the latency value at the beginning of reinnervation and that this difference decreased with time , and they did not detect a difference between control and exercise groups in terms of latency . based on this finding , they advocated that exercise did not increase the reinnervation ratio and that it only shortened this process . when the data of these researchers were evaluated , the results were found to be similar to ours in terms of the latency values , although they applied a different exercise . in our study , the latency values were found to be prolonged but the difference was not significant compared with the control . however , when healthy and injured legs of the same animals were compared , latency prolongation was found to be important at the first measurement time point after injury for the injury and swimming groups . latency may change due to differences in anatomic factors like different sizes and extremity lengths of the animals and variations in measurement site ( stimulus point and record point ) . however , performance of measurement for both the right and left legs of the same animal causes these variations to affect the groups by the same degree and consequently gives us the opportunity to see the effect of the intervention . significant prolongation was seen at day 5 after injury in the swimming and injury groups when compared with healthy legs . these changes are more prominent and longer compared with the latency values when evaluated in terms of conduction velocity data and h - reflex latency . this prolongation was not found to be significant , although it was also observed in the pemf group . detection of changes in latency values at week 3 in animals in the swimming group suggests that one hour of exercise may be a mild stress factor in animals . the severity of the stress factor was also found to be important in this regression . regarding the cmap values , amplitude values of the healthy leg were seen to be higher than in the injured leg when the right and left leg cmap values were analyzed . the second week in the injury group , first week in the control group , initial value and second week in the pemf group , and initial value and first and second weeks in the swimming group were found to be significant . however , the variations among the data were large ( not normally distributed ) , making statistically significant differences difficult to find . the h - reflex is a monosynaptic or oligosynaptic spinal reflex involving motor and sensory fibers . when data for the injured region were evaluated in terms of h - reflex latency values , time factors being statistically significant and the presence of group - time interaction indicated that the treatment applications were effective . in the within - group comparisons for the pemf and swimming groups , it was found that the h - reflex latency values showed significant improvement beginning from the first week , and this improvement was seen beginning from the second week in the injury group . in the intergroup comparison , the control group values were reached in the second week in the treatment application groups and in only third week in the injury group . so it can be seen that the h - reflex latency values were normalized one week earlier compared with the control group and other groups , which is similar to the findings for the mncv values . the hmax / mmax ratios were found to be higher in all other groups compared with the control group beginning from the first measurement time point , and this continued to be the case for all measurement time points . however , the difference was less in the injury group , and it decreased compared with the injury group in the pemf and swimming groups and approached that of the control group . even this change was found to be more prominent in the fourth week in the swimming group . the h - reflex is highly facilitated in the early stages of the reinnervation process . as a result this is the indicator of increased synaptic responses of motor neurons to electrical stimulation of healthy afferents20 . this increase in the reinnervation of the muscle reverses this facilitator effect , and the m wave amplitude increases , enabling the hmax /mmax ratio to return to normal values . , it was found that electromagnetic field application and swimming caused mild stress in animals within the first two weeks and that this was reflected in the hmax / mmax values as a negative effect on recovery . however , it was also found that the values of the pemf and swimming groups approached those of the control group rather than those of the injury group within the next two weeks , but the presence of no significant difference between the swimming and pemf groups and both the control and injury groups indicates that complete recovery could not be achieved . in conclusion , the hmax / mmax values indicate that the regeneration period after sciatic nerve injury is longer than four weeks . the present study revealed that pemf and swimming exercise after sciatic nerve injury had positive effects on the recovery process and increased regeneration . when evaluated in terms of application time , a four - week treatment period was no found to be enough for complete regeneration in both adjunctive treatment options . all these data indicate that swimming exercise may be a better option due to its positive effects and due to findings about the biologic effects and side effects of electromagnetic fields being still a source of debate . in addition , it revealed that the duration of application and application protocol of a rehabilitation program may be as important as the treatment option .

[ purpose ] the current study aimed to reveal the therapeutic effects of a pulsed electromagnetic field and swimming exercises on rats with experimental sciatic nerve injury , which was induced with crush - type neuropathy model damage , using electrophysiological methods . [ subjects ] in the current study , the sample consisted of 28 adult male wistar albino rats . [ methods ] the rats were randomized into four groups ( n=7 ) . swimming exercise and pemf ( 2 hz and 0.3 mt ) were applied one hour a day , five days a week , for four weeks . electroneuromyographic ( enmg ) measurements were taken on day 7 . [ results ] when the data were evaluated , it was found that the 4 weeks of pemf and swimming exercises led to an increase in motor conduction rates and a decrease in latency values , but the changes were not significant in comparison with the control and injury groups . the compound muscle action potential ( cmap ) values of the left leg were lower in weeks 2 , 3 , and 4 in the swimming exercise group in comparison with the control group , although for the pemf group , the cmap values of the left leg reached the level observed in the control group beginning in week 3 . [ conclusion ] pemf and swimming exercise made positive contributions to nerve regeneration after week 1 , and regeneration was enhanced .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

the aim of the this study was to reveal the effects of swimming exercise and a pulsed electromagnetic field applied after a crush - type injury , which is a neuropathy model , with electrophysiologic methods . the influences of swimming exercise and a pulsed electromagnetic field ( pemf ) applied for 4 weeks on healing were detected by an electroneuromyographic method in the regeneration processes of the rat sciatic nerve after a crush - type injury , which was selected as a neuropathy model . group 1 was the control group , group 2 was the injury control group ( sciatic nerve injury ) , group 3 was the injury + pemf group ( sciatic nerve injury and pemf ) , and group 4 was the injury + swimming group ( sciatic nerve injury and swimming exercise ) . application was performed for hour a day , five days a week , for four weeks and was performed concurrent to the swimming exercise in the swimming exercise group . swimming exercise was applied one hour a day , five days a week , for four weeks . the increase in mncv in the swimming and electromagnetic field groups were not statistically significant when compared with the control and injury groups . in the second week , the cmap values of the control group were higher than those in all other groups ( p<0.01 ) . the cmap values were significantly lower in the injury and swimming groups compared with the control group in week 3 and were significantly lower in the swimming group only in week 4 ( p<0.01 ) ( table 1 ) . in within - group comparisons for the injury , pemf , and swimming groups in terms of the right and left leg h - reflex latency values , the lef - side h - reflex latency values were seen to decrease in the injury , pemf , and swimming groups ( p<0.05 ) ; however , this decrease was found in the right - side h - reflex latency values ( healthy side ) ( table 4table 4.left and right leg h - reflex latency ( msec ) valuesgroup / weeknh - reflex latency values ( msec)output values ( 0)1st week2nd week3rd week4th weekleftrightleftrightleftrightleftrightleftrightcontrol75.290.155.050.105.250.224.950.135.200.064.870.105.380.094.970.08 * 5.140.104.940.08injury control76.920.194.710.07 * 6.200.234.870.08 * 5.780.164.840.19 * 5.780.225.070.145.480.154.710.05*pemf77.260.214.750.08 * 6.010.124.910.11 * 5.500.134.910.09 * 5.460.164.810.08 * 5.450.184.800.09*swimming76.850.114.700.14 * 6.010.154.500.11 * 5.720.094.670.13 * 5.260.144.940.055.470.184.670.08**statistical significance compared with the right leg ( p<0.05 ) . when the rigth and left leg hmax / mmax ratios were compared , while the left leg hmax / mmax ratios of the injury , pemf , and swimming groups were higher than those of the right leg hmax / mmax ratios during the experiment ( p<0.05 ) , no difference was found in the control group ( p>0.05 ) ( table 3 ) . similarly , the live weight gains in the magnetic field and swimming groups being less than that of the control group indicates that both swimming exercise and pemf application act as stress factors in animals . regarding the cmap values , amplitude values of the healthy leg were seen to be higher than in the injured leg when the right and left leg cmap values were analyzed . in the within - group comparisons for the pemf and swimming groups , it was found that the h - reflex latency values showed significant improvement beginning from the first week , and this improvement was seen beginning from the second week in the injury group . however , the difference was less in the injury group , and it decreased compared with the injury group in the pemf and swimming groups and approached that of the control group . however , it was also found that the values of the pemf and swimming groups approached those of the control group rather than those of the injury group within the next two weeks , but the presence of no significant difference between the swimming and pemf groups and both the control and injury groups indicates that complete recovery could not be achieved .

their assessment was detailed in a previous report.11 after approximately 6 years , 15 of the subjects with cfs , aged 34.06 8.77 ( mean sd ) at first evaluation , and 10 nc subjects , aged 30.5 7.93 at first evaluation , could be contacted and agreed to participate in a repeat longitudinal evaluation . the time intervals between the two evaluations for cfs and ncs were 6.43 0.57 and 6.21 0.31 years , respectively . no cfs or nc subject had developed a significant medical , including hypertension , or psychiatric illness in the intervening years . one cfs and one nc subject had remained on thyroxine to treat hypothyroidism , which predated their involvement in the initial study . three cfs subjects were taking antidepressants and were requested to stop these for 1 week before their mri scans . there were four cfs males ( male to female ratio = 0.36 ) and two nc males ( male to female ratio = 0.25 ) . there was no significant difference in age ( p = 0.20 ) or time interval ( p = 0.27 ) between the cfs and nc groups . cfs severity was measured by three scores : the bell cfs disability score,15 a somatic symptom score ( somatic ss ) , and a neurological symptom score ( neuro ss).14 the somatic and neuro ss were derived from selfscores of the 10 most significant symptoms.16 the somatic ss was the sum of the six selfscores for severity of fatigue , change in sleeping patterns , dizziness on standing , pain in muscles , stomach symptoms , and overall level of function . the neuro ss was the sum of four selfscores for change in concentration , change in shortterm memory , headaches , and experience of emotional swings . all three symptom scores are inversely proportional to the severity of the disease , i.e. , lower scores indicate more severe disease . to determine their levels of depression and anxiety , all subjects completed the hospital anxiety and depression scale ( hads ) questionnaire.17 the hads questionnaire was used in this study because it has been proved to be a reliable and simple test and patients have no difficulty in understanding the reason to answer the questionnaire . the study protocol was approved by the human research ethics committee of the queen elizabeth hospital in compliance with the australia national statement on ethical conduct in human research , and all subjects gave informed written consent for both examinations . mr images at both time points were acquired on a philips 1.5 tesla ( t ) intera mr scanner ( philips , eindhoven , the netherlands ) with a body transmit coil and birdcage receive coil . transverse anatomic images were acquired using a threedimensional spoiled gradientecho sequence with tr = 5.76 ms , te = 1.9 ms , flip angle = 9 , resolution = 0.938 0.938 1 mm . transverse t1 weighted spin echo mr images with contiguous slices of 0.82 0.82 3 mm were acquired with tr = 600 ms , te = 15 ms , flip angle = 90. transverse t2 weighted spin echo mr images with contiguous slices of 0.82 0.82 3 mm were acquired with tr = 4000 ms , te = 80 ms , flip angle = 90. the threedimensional anatomic images were segmented into gm , wm , csf , and extracerebral tissue using spm12 ( wellcome trust centre for neuroimaging , london , uk ) , in which a unified probabilistic framework that combined image segmentation , tissue classification , and bias correction within the same generative model was implemented.18 in spm12 , the unified segmentation incorporates a few iterations of a simple markov random field ( mrf ) cleanup procedure to remove isolated voxel assignments . optimization of the userspecified parameter that controls the strength of the mrf is described in the supplementary material , which is available online . as a result anatomical images from cfs and nc wm volumes were computed in spm12 by summing all voxel values in the gm and wm images accounting for the voxel volumes . longitudinal changes in each group and intergroup differences in the clinical measures ( bell cfs disability score , somatic ss , neuro ss , anxiety and depression from hads ) and global gm and wm volumes were tested using spss22 ( ibm , new york ) . paired two tailed ttests were used to test for equality of means and to compare longitudinal changes in the clinical measures , global gm volumes , and global wm volumes in both the cfs and nc groups . correlation coefficients between time point 1 and time point 2 values for the clinical measures , global gm volumes , and global wm volumes , were tested for a significant difference from zero . bonferroni correction for multiple comparisons of the seven characteristics ( five symptom measures and two brain volumes ) was used to determine uncorrected ( p < 0.05 ) and corrected ( p < 0.0071 ) statistical significance . the clinical measures and global gm and wm volumes of the cfs and nc groups pooled over the two times were compared using an independent two group ttest . twotailed tests for equality of means with unequal group variances were followed by bonferroni correction for multiple comparisons of eight characteristics ( five symptom measures , one age , and two brain volumes ) to determine uncorrected ( p < 0.05 ) and corrected ( p < 0.0062 ) . pearson correlations between each clinical measure and global gm and wm volumes from all subjects were calculated . bivariate tests of significant difference in correlation coefficient from zero with two tails were followed with bonferroni correction for multiple comparisons of 21 pairs to test for uncorrected ( p < 0.05 ) and corrected ( p < 0.0023 ) statistical significance . vbm derives images of relative gm and wm volumes from structural mr images , which can then be subjected to voxelwise statistical analysis in crosssectional or longitudinal studies . vbm is a wellestablished tool to examine patterns of regional brain changes in neurological diseases and neuroanatomical correlates of cns diseases . although much vbm preprocessing and analysis is automated in spm , many methodological options remain for user selection,19 including selection or creation of templates , registration algorithms , and comparison strategies . to optimize and test the vbm methodology options , we used an atrophy simulation tool with a topology preserving transformation model.20 mr images from 25 normal controls were used for the simulation study . tool inputs were the original image , its segmentation , location of the atrophy center , and the radius of the atrophy sphere . the tool simulates atrophy by finding a dense warping deformation that produces the specified levels of volumetric loss on the labelled tissue using an energy minimization strategy . 1 ) . we selected the atrophy center on the axial slice at the level of the inferior colliculus and , within that slice , in wm at the anterior pole of the left cerebral peduncle . this region was chosen to mimic the atrophy reported in one of our previous studies.11 atrophy spheres ( radius = 5 mm ) with three levels of atrophy ( 5% , 3% , and 1% ) were applied . a representative axial slice at the center of the atrophy sphere is shown with and without simulated atrophy . the circle radius has no relationship to the radius of the atrophy sphere or atrophy rate . a , e , c , g : respectively , the original raw and wm images without change from two subjects . b , f , d , h : respectively , the raw and wm images with 5% simulated atrophy in the left midbrain . atrophy can be seen at the edge of segmented wm images while topologies of deformed structures were preserved ( f , h ) . the original images and images with simulated atrophy represent images at time points 1 and 2 in a longitudinal study . smoothing was performed with an 8 mm 8 mm 8 mm fullwidth halfmaximum gaussian filter for all options . dartel dn directly normalizes individual images into mni ( montreal neurological institute ) space ( supp . ( a ) all 3d anatomic images were segmented into gm and wm partitions using a unified segmentation framework18 with the optimized mrf parameter . ( b ) for each individual , the deformation of their gm image to mni space was computed using dartel ( diffeomorphic anatomical registration through exponentiated lie algebra ) nonlinear registration and recorded as flow field image.21 this procedure was implemented in spm12 by means of run dartel ( existing template ) . ( c ) the flow field images were then used to normalize gm and wm images to mni space and generate smoothed , spatially normalized , and modulated images . the latter encode the local volume changes associated with the nonlinear deformation , by means of jacobian scaling , into image intensities . ( d ) finally , the preprocessed wm images at time point 1 were compared with those from time point 2 using an spm12 ( parametric ) paired ttest.dartel st ( study specific template ) is the same as option 1 except that a studyspecific gm template was created in ( b ) ( supp . this involves iteratively matching all images to a template generated from their own means , it was implemented by means of run dartel ( create template ) in spm12 . two deformations , the flow field images representing individual image to studyspecific template and the transformations of the template to mni space , were combined and encoded in modulated images.shoot st is the same as option 2 except that the two implementations of dartel were replaced by a diffeomorphic registration using geodesic shooting and gaussnewton optimization22 ( supp . s1).dartel snpm is the same as option 2 except that a nonparametric paired ttest23 was used in step ( d ) ( supp . ( a ) raw images from the 1 and 2 time points were aligned to mni space using a rigid body registration . ( b ) for each subject , these aligned images were averaged to create the subject template . ( c ) raw images from 1 and 2 time points were then aligned to this subject template . ( d ) aligned images from step ( c ) were preprocessed using the same procedures as option 2 . ( e ) difference images were calculated by subtracting the preprocessed image of time point 2 from that of time point 1 . ( f ) difference images from step ( e ) were compared using a one sample parametric ttest.ljd ( longitudinal jacobian difference ) creates a withinsubject template differently to option 5 ( supp . ( a ) a symmetric diffeomorphic modelling of longitudinal data24 was used to create a midpoint image by estimating the optimal mapping between the template and each of the images by means of pairwise longitudinal registration in spm12 . a jacobian difference image records the difference of the jacobian map for deformation from the midpoint image to the first scan and this to the second scan . ( b ) the midpoint ( mean ) image was segmented into gm , wm and csf images . ( c ) gm images from step ( b ) were used to create a studyspecific template using dartel . ( d ) the jacobian difference images from step ( a ) were multiplied by the gm image from step ( b ) . ( e ) the difference images from step ( d ) were normalized to mni space and smoothed . ( f ) difference images from step ( e ) were compared using a one sample spm12 ( parametric ) ttest . dartel dn directly normalizes individual images into mni ( montreal neurological institute ) space ( supp . ( a ) all 3d anatomic images were segmented into gm and wm partitions using a unified segmentation framework18 with the optimized mrf parameter . ( b ) for each individual , the deformation of their gm image to mni space was computed using dartel ( diffeomorphic anatomical registration through exponentiated lie algebra ) nonlinear registration and recorded as flow field image.21 this procedure was implemented in spm12 by means of run dartel ( existing template ) . ( c ) the flow field images were then used to normalize gm and wm images to mni space and generate smoothed , spatially normalized , and modulated images . the latter encode the local volume changes associated with the nonlinear deformation , by means of jacobian scaling , into image intensities . ( d ) finally , the preprocessed wm images at time point 1 were compared with those from time point 2 using an spm12 ( parametric ) paired ttest . dartel st ( study specific template ) is the same as option 1 except that a studyspecific gm template was created in ( b ) ( supp . this involves iteratively matching all images to a template generated from their own means , it was implemented by means of run dartel ( create template ) in spm12 . two deformations , the flow field images representing individual image to studyspecific template and the transformations of the template to mni space , were combined and encoded in modulated images . shoot st is the same as option 2 except that the two implementations of dartel were replaced by a diffeomorphic registration using geodesic shooting and gaussnewton optimization22 ( supp . dartel snpm is the same as option 2 except that a nonparametric paired ttest23 was used in step ( d ) ( supp . ( a ) raw images from the 1 and 2 time points were aligned to mni space using a rigid body registration . ( b ) for each subject , these aligned images were averaged to create the subject template . ( c ) raw images from 1 and 2 time points were then aligned to this subject template . ( d ) aligned images from step ( c ) were preprocessed using the same procedures as option 2 . ( e ) difference images were calculated by subtracting the preprocessed image of time point 2 from that of time point 1 . ( f ) difference images from step ( e ) were compared using a one sample parametric ttest . ljd ( longitudinal jacobian difference ) creates a withinsubject template differently to option 5 ( supp . ( a ) a symmetric diffeomorphic modelling of longitudinal data24 was used to create a midpoint image by estimating the optimal mapping between the template and each of the images by means of pairwise longitudinal registration in spm12 . a jacobian difference image records the difference of the jacobian map for deformation from the midpoint image to the first scan and this to the second scan . ( b ) the midpoint ( mean ) image was segmented into gm , wm and csf images . ( c ) gm images from step ( b ) were used to create a studyspecific template using dartel . ( d ) the jacobian difference images from step ( a ) were multiplied by the gm image from step ( b ) . ( e ) the difference images from step ( d ) were normalized to mni space and smoothed . ( f ) difference images from step ( e ) were compared using a one sample spm12 ( parametric ) ttest . the simulated atrophy images were analyzed using spm12 with the above six vbm methodology options . to evaluate the sensitivity of each option , different randomly selected sample sizes ( n = 25 , 20 , 15 , 10 ) for each level of atrophy were compared with the original images from the same subjects with the six vbm options . statistical significance was determined using family wise error ( fwe ) corrected voxel level p < 0.05 . significant clusters with more than 25 voxels and less than 25 voxels that overlapped the simulated atrophy were defined as true positive ( tp ) and small true positive ( stp ) , respectively . significant clusters that were found at different locations from the simulated atrophy were defined as false positive ( fp ) . failure to detect simulated atrophy was defined as false negative ( fn ) ( supp . anatomic mr images from cfs and nc were preprocessed using the optimum vbm ld methodological option ( 5a5e ) . images of t1 and t2 spin echo intensities were first coregistered to their corresponding anatomic images . second , coregistered t1w and t2w images were normalized to the mni atlas using the deformation from 5f above and smoothed . the global signal levels were computed for each subject as the mean voxel value in a mask ( brain region ) generated using the voxelbased iterative sensitivity ( vbis ) method.25 each image was first normalized either by the total gm volume , wm volume , global t1w level , or global t2w level . to perform vbm comparisons of longitudinal difference between cfs patients and ncs , temporal difference images were divided by the corresponding time between the two scan dates to generate rateofchange images . rateofchange images from cfs patients were compared with those from ncs with a two sample ttest . the significance of rateofchange difference was tested using fwe corrected cluster level p < 0.05 . to perform crosssectional comparisons between groups of cfs images and nc images pooled over the two time points , regional brain differences were determined with a two sample ttest . the significance of inter group difference was tested using fwe corrected cluster level p < 0.05 . in addition to group comparisons , spm regressions were performed against clinical measures of bell cfs disability score , somatic ss , and neuro ss ) . all analyses incorporated the covariates of age , the appropriate global value ( total gm volume , wm volume , global t1w level , or global t2w level ) , and hads depression and anxiety scores . the significance of correlation was tested using family wise error ( fwe ) corrected cluster level p < 0.05 . the subjects were recruited based on availability , from 25 subjects with cfs and 25 ncs from a previous crosssectional study.14 the initial recruitment of the cfs group was from communitybased specialist and general practice . their assessment was detailed in a previous report.11 after approximately 6 years , 15 of the subjects with cfs , aged 34.06 8.77 ( mean sd ) at first evaluation , and 10 nc subjects , aged 30.5 7.93 at first evaluation , could be contacted and agreed to participate in a repeat longitudinal evaluation . the time intervals between the two evaluations for cfs and ncs were 6.43 0.57 and 6.21 0.31 years , respectively . no cfs or nc subject had developed a significant medical , including hypertension , or psychiatric illness in the intervening years . one cfs and one nc subject had remained on thyroxine to treat hypothyroidism , which predated their involvement in the initial study . three cfs subjects were taking antidepressants and were requested to stop these for 1 week before their mri scans . there were four cfs males ( male to female ratio = 0.36 ) and two nc males ( male to female ratio = 0.25 ) . there was no significant difference in age ( p = 0.20 ) or time interval ( p = 0.27 ) between the cfs and nc groups . cfs severity was measured by three scores : the bell cfs disability score,15 a somatic symptom score ( somatic ss ) , and a neurological symptom score ( neuro ss).14 the somatic and neuro ss were derived from selfscores of the 10 most significant symptoms.16 the somatic ss was the sum of the six selfscores for severity of fatigue , change in sleeping patterns , dizziness on standing , pain in muscles , stomach symptoms , and overall level of function . the neuro ss was the sum of four selfscores for change in concentration , change in shortterm memory , headaches , and experience of emotional swings . all three symptom scores are inversely proportional to the severity of the disease , i.e. , lower scores indicate more severe disease . to determine their levels of depression and anxiety , all subjects completed the hospital anxiety and depression scale ( hads ) questionnaire.17 the hads questionnaire was used in this study because it has been proved to be a reliable and simple test and patients have no difficulty in understanding the reason to answer the questionnaire . the study protocol was approved by the human research ethics committee of the queen elizabeth hospital in compliance with the australia national statement on ethical conduct in human research , and all subjects gave informed written consent for both examinations . mr images at both time points were acquired on a philips 1.5 tesla ( t ) intera mr scanner ( philips , eindhoven , the netherlands ) with a body transmit coil and birdcage receive coil . transverse anatomic images were acquired using a threedimensional spoiled gradientecho sequence with tr = 5.76 ms , te = 1.9 ms , flip angle = 9 , resolution = 0.938 0.938 1 mm . transverse t1 weighted spin echo mr images with contiguous slices of 0.82 0.82 3 mm were acquired with tr = 600 ms , te = 15 ms , flip angle = 90. transverse t2 weighted spin echo mr images with contiguous slices of 0.82 0.82 3 mm were acquired with tr = 4000 ms , te = 80 ms , flip angle = 90. the threedimensional anatomic images were segmented into gm , wm , csf , and extracerebral tissue using spm12 ( wellcome trust centre for neuroimaging , london , uk ) , in which a unified probabilistic framework that combined image segmentation , tissue classification , and bias correction within the same generative model was implemented.18 in spm12 , the unified segmentation incorporates a few iterations of a simple markov random field ( mrf ) cleanup procedure to remove isolated voxel assignments . optimization of the userspecified parameter that controls the strength of the mrf is described in the supplementary material , which is available online . as a result anatomical images from cfs and nc wm volumes were computed in spm12 by summing all voxel values in the gm and wm images accounting for the voxel volumes . longitudinal changes in each group and intergroup differences in the clinical measures ( bell cfs disability score , somatic ss , neuro ss , anxiety and depression from hads ) and global gm and wm volumes were tested using spss22 ( ibm , new york ) . paired two tailed ttests were used to test for equality of means and to compare longitudinal changes in the clinical measures , global gm volumes , and global wm volumes in both the cfs and nc groups . correlation coefficients between time point 1 and time point 2 values for the clinical measures , global gm volumes , and global wm volumes , were tested for a significant difference from zero . bonferroni correction for multiple comparisons of the seven characteristics ( five symptom measures and two brain volumes ) was used to determine uncorrected ( p < 0.05 ) and corrected ( p < 0.0071 ) statistical significance . the clinical measures and global gm and wm volumes of the cfs and nc groups pooled over the two times were compared using an independent two group ttest . twotailed tests for equality of means with unequal group variances were followed by bonferroni correction for multiple comparisons of eight characteristics ( five symptom measures , one age , and two brain volumes ) to determine uncorrected ( p < 0.05 ) and corrected ( p < 0.0062 ) . pearson correlations between each clinical measure and global gm and wm volumes from all subjects were calculated . bivariate tests of significant difference in correlation coefficient from zero with two tails were followed with bonferroni correction for multiple comparisons of 21 pairs to test for uncorrected ( p < 0.05 ) and corrected ( p < 0.0023 ) statistical significance . vbm derives images of relative gm and wm volumes from structural mr images , which can then be subjected to voxelwise statistical analysis in crosssectional or longitudinal studies . vbm is a wellestablished tool to examine patterns of regional brain changes in neurological diseases and neuroanatomical correlates of cns diseases . although much vbm preprocessing and analysis is automated in spm , many methodological options remain for user selection,19 including selection or creation of templates , registration algorithms , and comparison strategies . to optimize and test the vbm methodology options , we used an atrophy simulation tool with a topology preserving transformation model.20 mr images from 25 normal controls were used for the simulation study . tool inputs were the original image , its segmentation , location of the atrophy center , and the radius of the atrophy sphere . the tool simulates atrophy by finding a dense warping deformation that produces the specified levels of volumetric loss on the labelled tissue using an energy minimization strategy . 1 ) . we selected the atrophy center on the axial slice at the level of the inferior colliculus and , within that slice , in wm at the anterior pole of the left cerebral peduncle . this region was chosen to mimic the atrophy reported in one of our previous studies.11 atrophy spheres ( radius = 5 mm ) with three levels of atrophy ( 5% , 3% , and 1% ) were applied . a representative axial slice at the center of the atrophy sphere is shown with and without simulated atrophy . the circle radius has no relationship to the radius of the atrophy sphere or atrophy rate . a , e , c , g : respectively , the original raw and wm images without change from two subjects . b , f , d , h : respectively , the raw and wm images with 5% simulated atrophy in the left midbrain . atrophy can be seen at the edge of segmented wm images while topologies of deformed structures were preserved ( f , h ) . the original images and images with simulated atrophy represent images at time points 1 and 2 in a longitudinal study . smoothing was performed with an 8 mm 8 mm 8 mm fullwidth halfmaximum gaussian filter for all options . dartel dn directly normalizes individual images into mni ( montreal neurological institute ) space ( supp . ( a ) all 3d anatomic images were segmented into gm and wm partitions using a unified segmentation framework18 with the optimized mrf parameter . ( b ) for each individual , the deformation of their gm image to mni space was computed using dartel ( diffeomorphic anatomical registration through exponentiated lie algebra ) nonlinear registration and recorded as flow field image.21 this procedure was implemented in spm12 by means of run dartel ( existing template ) . ( c ) the flow field images were then used to normalize gm and wm images to mni space and generate smoothed , spatially normalized , and modulated images . the latter encode the local volume changes associated with the nonlinear deformation , by means of jacobian scaling , into image intensities . ( d ) finally , the preprocessed wm images at time point 1 were compared with those from time point 2 using an spm12 ( parametric ) paired ttest.dartel st ( study specific template ) is the same as option 1 except that a studyspecific gm template was created in ( b ) ( supp . this involves iteratively matching all images to a template generated from their own means , it was implemented by means of run dartel ( create template ) in spm12 . two deformations , the flow field images representing individual image to studyspecific template and the transformations of the template to mni space , were combined and encoded in modulated images.shoot st is the same as option 2 except that the two implementations of dartel were replaced by a diffeomorphic registration using geodesic shooting and gaussnewton optimization22 ( supp . s1).dartel snpm is the same as option 2 except that a nonparametric paired ttest23 was used in step ( d ) ( supp . ( a ) raw images from the 1 and 2 time points were aligned to mni space using a rigid body registration . ( b ) for each subject , these aligned images were averaged to create the subject template . ( c ) raw images from 1 and 2 time points were then aligned to this subject template . ( d ) aligned images from step ( c ) were preprocessed using the same procedures as option 2 . ( e ) difference images were calculated by subtracting the preprocessed image of time point 2 from that of time point 1 . ( f ) difference images from step ( e ) were compared using a one sample parametric ttest.ljd ( longitudinal jacobian difference ) creates a withinsubject template differently to option 5 ( supp . fig . ( a ) a symmetric diffeomorphic modelling of longitudinal data24 was used to create a midpoint image by estimating the optimal mapping between the template and each of the images by means of pairwise longitudinal registration in spm12 . a jacobian difference image records the difference of the jacobian map for deformation from the midpoint image to the first scan and this to the second scan . ( b ) the midpoint ( mean ) image was segmented into gm , wm and csf images . ( c ) gm images from step ( b ) were used to create a studyspecific template using dartel . ( d ) the jacobian difference images from step ( a ) were multiplied by the gm image from step ( b ) . ( e ) the difference images from step ( d ) were normalized to mni space and smoothed . ( f ) difference images from step ( e ) were compared using a one sample spm12 ( parametric ) ttest . dartel dn directly normalizes individual images into mni ( montreal neurological institute ) space ( supp . ( a ) all 3d anatomic images were segmented into gm and wm partitions using a unified segmentation framework18 with the optimized mrf parameter . ( b ) for each individual , the deformation of their gm image to mni space was computed using dartel ( diffeomorphic anatomical registration through exponentiated lie algebra ) nonlinear registration and recorded as flow field image.21 this procedure was implemented in spm12 by means of run dartel ( existing template ) . ( c ) the flow field images were then used to normalize gm and wm images to mni space and generate smoothed , spatially normalized , and modulated images . the latter encode the local volume changes associated with the nonlinear deformation , by means of jacobian scaling , into image intensities . ( d ) finally , the preprocessed wm images at time point 1 were compared with those from time point 2 using an spm12 ( parametric ) paired ttest . dartel st ( study specific template ) is the same as option 1 except that a studyspecific gm template was created in ( b ) ( supp . this involves iteratively matching all images to a template generated from their own means , it was implemented by means of run dartel ( create template ) in spm12 . two deformations , the flow field images representing individual image to studyspecific template and the transformations of the template to mni space , were combined and encoded in modulated images . shoot st is the same as option 2 except that the two implementations of dartel were replaced by a diffeomorphic registration using geodesic shooting and gaussnewton optimization22 ( supp . dartel snpm is the same as option 2 except that a nonparametric paired ttest23 was used in step ( d ) ( supp . ( a ) raw images from the 1 and 2 time points were aligned to mni space using a rigid body registration . ( b ) for each subject , these aligned images were averaged to create the subject template . ( c ) raw images from 1 and 2 time points were then aligned to this subject template . ( d ) aligned images from step ( c ) were preprocessed using the same procedures as option 2 . ( e ) difference images were calculated by subtracting the preprocessed image of time point 2 from that of time point 1 . ( f ) difference images from step ( e ) were compared using a one sample parametric ttest . ljd ( longitudinal jacobian difference ) creates a withinsubject template differently to option 5 ( supp . ( a ) a symmetric diffeomorphic modelling of longitudinal data24 was used to create a midpoint image by estimating the optimal mapping between the template and each of the images by means of pairwise longitudinal registration in spm12 . a jacobian difference image records the difference of the jacobian map for deformation from the midpoint image to the first scan and this to the second scan . ( b ) the midpoint ( mean ) image was segmented into gm , wm and csf images . ( c ) gm images from step ( b ) were used to create a studyspecific template using dartel . ( d ) the jacobian difference images from step ( a ) were multiplied by the gm image from step ( b ) . ( e ) the difference images from step ( d ) were normalized to mni space and smoothed . ( f ) difference images from step ( e ) were compared using a one sample spm12 ( parametric ) ttest . the simulated atrophy images were analyzed using spm12 with the above six vbm methodology options . to evaluate the sensitivity of each option , different randomly selected sample sizes ( n = 25 , 20 , 15 , 10 ) for each level of atrophy were compared with the original images from the same subjects with the six vbm options . statistical significance was determined using family wise error ( fwe ) corrected voxel level p < 0.05 . significant clusters with more than 25 voxels and less than 25 voxels that overlapped the simulated atrophy were defined as true positive ( tp ) and small true positive ( stp ) , respectively . significant clusters that were found at different locations from the simulated atrophy were defined as false positive ( fp ) . failure to detect simulated atrophy was defined as false negative ( fn ) ( supp . anatomic mr images from cfs and nc were preprocessed using the optimum vbm ld methodological option ( 5a5e ) . images of t1 and t2 spin echo intensities were first coregistered to their corresponding anatomic images . second , coregistered t1w and t2w images were normalized to the mni atlas using the deformation from 5f above and smoothed . the global signal levels were computed for each subject as the mean voxel value in a mask ( brain region ) generated using the voxelbased iterative sensitivity ( vbis ) method.25 each image was first normalized either by the total gm volume , wm volume , global t1w level , or global t2w level . to perform vbm comparisons of longitudinal difference between cfs patients and ncs , temporal difference images were divided by the corresponding time between the two scan dates to generate rateofchange images . rateofchange images from cfs patients were compared with those from ncs with a two sample ttest . the significance of rateofchange difference was tested using fwe corrected cluster level p < 0.05 . to perform crosssectional comparisons between groups of cfs images and nc images pooled over the two time points , regional brain differences were determined with a two sample ttest . the significance of inter group difference was tested using fwe corrected cluster level p < 0.05 . in addition to group comparisons , spm regressions were performed against clinical measures of bell cfs disability score , somatic ss , and neuro ss ) . all analyses incorporated the covariates of age , the appropriate global value ( total gm volume , wm volume , global t1w level , or global t2w level ) , and hads depression and anxiety scores . the significance of correlation was tested using family wise error ( fwe ) corrected cluster level p < 0.05 . the spm12 segmentation with mrf of 4 rendered gm images that were closest to the manual segmentation results , i.e. , highest median and smallest interquartile range for the dice coefficient ( supp . fig . table 1 summarizes the performance of the six vbm options . for sample sizes less than or equal to 15 , the longitudinal approaches ( ld and ljd ) have higher sensitivity in preventing fn than dartel dn , dartel st , and dartel snpm . the ld option was , therefore , optimal and was used here in vbm analysis of cfs and nc images . performance of vbm methodological options in detection of simulated atrophya if not specified otherwise , default parameters / procedures were used . default parameters / procedures include unified segmentation , creation of study specific templates , normalization to montreal neurological institute ( mni ) space , encoding of deformations as volume changes , smoothing with gaussian kernel of 8 mm 8 mm 8 mm , and parametric comparison . for an example , methodological option of dartel st uses dartel registration to create a study specific template , normalizes final template mni space , warps segmented images into mni space , modulates volumetrics into intensities , smooths , and compares parametrically . n = sample size ; dartel = diffeomorphic anatomical registration through exponentiated lie algebra ; dn = direct normalization ; st = studyspecific template ; shoot = geodesic shooting registration snpm = statistical nonparametric mapping ; ld = longitudinal difference ; ljd = longitudinal jacobian difference ; tp = true positive ; stp = small true positive ; fn = false negative ; fp = false positive . for patients with cfs , mean longitudinal changes and correlations between time 1 and time 2 values for clinical measures and global gm and wm volumes are summarized in table 2 ( see supp . there are significant decreases in total gm volume and increases in total wm volumes in both the cfs and nc groups ( table 2 and supp . table s1 ) , although these longitudinal changes in total gm and wm volumes did not differ significantly between cfs and nc groups . mean changes and correlations between values at time 1 and time 2 for clinical measures , global gm volumes , and global wm volumes in cfsa changes were calculated as values at time 1 minus values at time 2 for each patient of chronic fatigue syndrome ( cfs ) . gm and wm represent global gray matter ( gm ) and white matter ( wm ) volumes . neuro ss and somatic ss are neurological symptom score and somatic symptom score , respectively . ci = confidence level ; p = significance level ; r = pearson correlation coefficient . table 3 summarizes means of clinical measures and total gm and wm volumes for pooled cfs and nc groups . there is no significant difference in total gm and wm volumes and ages between cfs and nc groups . bivariate correlations between any pair of clinical measures or total gm and wm volumes are summarized in table 4 . pairs that are significantly correlated to each other are : total gm & wm volumes , depression and anxiety , and neuro ss and somatic ss . in addition , total wm volumes were correlated with somatic ss with uncorrected p < 0.05 . means of clinical measures and global gm and wm volumes of pooled cfs and nc groups gm = gray matter ; wm = white matter ; cfs = chronic fatigue syndrome ; nc = normal control ; m = mean value ; p = significance level ; neuro ss = neurological symptom score ; somatic ss = somatic symptom score . bivariate correlations among clinical measures and the total gm and wm volumes gm = gray matter ; wm = white matter ; neuro ss = neurological symptom score ; somatic ss = somatic symptom score . the rateofchange of regional wm volumes in cfs patients was significantly different from that in ncs in the left posterior part of the inferior frontooccipital fasciculus ( ifof ) and/or arcuate fasciculus ( fig . wm volume relative to global wm volume decreased with time in the cfs group while in ncs it was unchanged . voxelbased morphometry ( vbm ) of white matter ( wm ) longitudinal change rate difference between chronic fatigue syndrome ( cfs ) and normal control ( nc ) subjects . the significant cluster is superimposed on sections through its peak voxel of the average 3d t1 weighted image from this study . the plot shows the mean modulated wm volume at time points 1 and 2 in the cfs and nc groups . pfwe_corr_cluster is the family wise error ( fwe ) corrected cluster p value ; k is the size of cluster in voxels ; t statistic value is the rate difference relative to the temporal change rate variance . the regional differences of gm and wm volumes and t1w and t2w signal intensities between pooled cfs and nc groups are summarized in figure 3 . in cfs , gm volumes were significantly decreased in the right inferior temporal gyrus and increased in the right supplementary motor area . the cfs group showed a significant decrease in wm volume in the left posterior part of ifof / arcuate fasciculus . in the cfs group t1w signal intensities were significantly higher on the right in the parahippocampal gyrus , inferior temporal gyrus , and ifof / arcuate fasciculus . t2w signal intensities were significantly higher in the right ifof / arcuate fasciculus and inferior frontal gyrus regions in the cfs group . regional differences of gray matter ( gm ) and white matter ( wm ) volumes and t1w and t2w signal intensities between cfs and nc groups . maximum intensity projections of t statistic clusters that are significantly different between cfs group and ncs were superimposed on a 3d rendering of t1 weighted images in montreal neurological institute ( mni ) space . the 3d rendered image was set to transparent to reveal underlaying clusters : top view ( a ) , right view ( b ) , left view ( c ) , and posterior view ( d ) . characteristics of significant clusters are summarized in the side table , in which pfwe is the family wise error ( fwe ) corrected cluster p value ; k is the size of the cluster in voxels ; t is the difference relative to the variation ; and mni refers to the mni coordinates . regional gm volume was significantly correlated with neuro ss in the right fusiform gyrus positively and left superior parietal lobule negatively . regional gm volume in the culmen regional wm volume was significantly and positively correlated with somatic ss in wm regions inferior to the left precentral sulcus . clusters from regressions of regional gray matter ( gm ) and white matter ( wm ) volumes and t1w and t2w signal intensities versus clinical measures in the pooled cfs group . maximum intensity projections of t statistic clusters that are significantly correlated with clinical measures were superimposed on a 3d rendering of t1 weighted images in montreal neurological institute ( mni ) space . the 3d rendered image was set to transparent to reveal underlaying clusters : top view ( a ) , right view ( b ) , left view ( c ) , and posterior view ( d ) . characteristics of significant clusters are summarized in the side table , in which pfwe is the family wise error ( fwe ) corrected cluster p value ; k is the size of cluster in voxels ; t is the correlation coefficient relative to the standard error ; and mni refers to the mni coordinates ; + and represent positive and negative correlations , respectively . the spm12 segmentation with mrf of 4 rendered gm images that were closest to the manual segmentation results , i.e. , highest median and smallest interquartile range for the dice coefficient ( supp . fig . table 1 summarizes the performance of the six vbm options . for sample sizes less than or equal to 15 , the longitudinal approaches ( ld and ljd ) have higher sensitivity in preventing fn than dartel dn , dartel st , and dartel snpm . the ld option was , therefore , optimal and was used here in vbm analysis of cfs and nc images . performance of vbm methodological options in detection of simulated atrophya if not specified otherwise , default parameters / procedures were used . default parameters / procedures include unified segmentation , creation of study specific templates , normalization to montreal neurological institute ( mni ) space , encoding of deformations as volume changes , smoothing with gaussian kernel of 8 mm 8 mm 8 mm , and parametric comparison . for an example , methodological option of dartel st uses dartel registration to create a study specific template , normalizes final template mni space , warps segmented images into mni space , modulates volumetrics into intensities , smooths , and compares parametrically . n = sample size ; dartel = diffeomorphic anatomical registration through exponentiated lie algebra ; dn = direct normalization ; st = studyspecific template ; shoot = geodesic shooting registration snpm = statistical nonparametric mapping ; ld = longitudinal difference ; ljd = longitudinal jacobian difference ; tp = true positive ; stp = small true positive ; fn = false negative ; fp = false positive . for patients with cfs , mean longitudinal changes and correlations between time 1 and time 2 values for clinical measures and global gm and wm volumes are summarized in table 2 ( see supp . there are significant decreases in total gm volume and increases in total wm volumes in both the cfs and nc groups ( table 2 and supp . table s1 ) , although these longitudinal changes in total gm and wm volumes did not differ significantly between cfs and nc groups . mean changes and correlations between values at time 1 and time 2 for clinical measures , global gm volumes , and global wm volumes in cfsa changes were calculated as values at time 1 minus values at time 2 for each patient of chronic fatigue syndrome ( cfs ) . gm and wm represent global gray matter ( gm ) and white matter ( wm ) volumes . neuro ss and somatic ss are neurological symptom score and somatic symptom score , respectively . ci = confidence level ; p = significance level ; r = pearson correlation coefficient . table 3 summarizes means of clinical measures and total gm and wm volumes for pooled cfs and nc groups . there is no significant difference in total gm and wm volumes and ages between cfs and nc groups . bivariate correlations between any pair of clinical measures or total gm and wm volumes are summarized in table 4 . pairs that are significantly correlated to each other are : total gm & wm volumes , depression and anxiety , and neuro ss and somatic ss . in addition , total wm volumes were correlated with somatic ss with uncorrected p < 0.05 . means of clinical measures and global gm and wm volumes of pooled cfs and nc groups gm = gray matter ; wm = white matter ; cfs = chronic fatigue syndrome ; nc = normal control ; m = mean value ; p = significance level ; neuro ss = neurological symptom score ; somatic ss = somatic symptom score . bivariate correlations among clinical measures and the total gm and wm volumes gm = gray matter ; wm = white matter ; neuro ss = neurological symptom score ; somatic ss = somatic symptom score . the rateofchange of regional wm volumes in cfs patients was significantly different from that in ncs in the left posterior part of the inferior frontooccipital fasciculus ( ifof ) and/or arcuate fasciculus ( fig . wm volume relative to global wm volume decreased with time in the cfs group while in ncs it was unchanged . voxelbased morphometry ( vbm ) of white matter ( wm ) longitudinal change rate difference between chronic fatigue syndrome ( cfs ) and normal control ( nc ) subjects . the significant cluster is superimposed on sections through its peak voxel of the average 3d t1 weighted image from this study . the plot shows the mean modulated wm volume at time points 1 and 2 in the cfs and nc groups . pfwe_corr_cluster is the family wise error ( fwe ) corrected cluster p value ; k is the size of cluster in voxels ; t statistic value is the rate difference relative to the temporal change rate variance . the regional differences of gm and wm volumes and t1w and t2w signal intensities between pooled cfs and nc groups are summarized in figure 3 . in cfs , gm volumes were significantly decreased in the right inferior temporal gyrus and increased in the right supplementary motor area . the cfs group showed a significant decrease in wm volume in the left posterior part of ifof / arcuate fasciculus . in the cfs group t1w signal intensities were significantly higher on the right in the parahippocampal gyrus , inferior temporal gyrus , and ifof / arcuate fasciculus . t2w signal intensities were significantly higher in the right ifof / arcuate fasciculus and inferior frontal gyrus regions in the cfs group . regional differences of gray matter ( gm ) and white matter ( wm ) volumes and t1w and t2w signal intensities between cfs and nc groups . maximum intensity projections of t statistic clusters that are significantly different between cfs group and ncs were superimposed on a 3d rendering of t1 weighted images in montreal neurological institute ( mni ) space . the 3d rendered image was set to transparent to reveal underlaying clusters : top view ( a ) , right view ( b ) , left view ( c ) , and posterior view ( d ) . characteristics of significant clusters are summarized in the side table , in which pfwe is the family wise error ( fwe ) corrected cluster p value ; k is the size of the cluster in voxels ; t is the difference relative to the variation ; and mni refers to the mni coordinates . regional gm volume was significantly correlated with neuro ss in the right fusiform gyrus positively and left superior parietal lobule negatively . regional gm volume in the culmen regional wm volume was significantly and positively correlated with somatic ss in wm regions inferior to the left precentral sulcus . t2 signal intensities in left and right anterior ifof / arcuate fasciculus were significantly and negatively correlated with neuro ss . clusters from regressions of regional gray matter ( gm ) and white matter ( wm ) volumes and t1w and t2w signal intensities versus clinical measures in the pooled cfs group . maximum intensity projections of t statistic clusters that are significantly correlated with clinical measures were superimposed on a 3d rendering of t1 weighted images in montreal neurological institute ( mni ) space . the 3d rendered image was set to transparent to reveal underlaying clusters : top view ( a ) , right view ( b ) , left view ( c ) , and posterior view ( d ) . characteristics of significant clusters are summarized in the side table , in which pfwe is the family wise error ( fwe ) corrected cluster p value ; k is the size of cluster in voxels ; t is the correlation coefficient relative to the standard error ; and mni refers to the mni coordinates ; + and represent positive and negative correlations , respectively . the strength of the study is that we first validated spm segmentation against manual segmentation and optimized vbm methodology for longitudinal analysis . we found for the first time that , over a period of 6 years , regional wm volume decreases in the left ifof / arcuate fasciculus in cfs patients while remaining stable in ncs . this longitudinal wm finding was consolidated by crosssectional analysis of the pooled cfs and nc groups . intergroup comparison found decreased gm and wm volumes and increased t1w and increased t2w intensities ( which may reflect decreased blood volumes and ischemia ) near this ilof / arcuate area both ipsi and contralaterally . furthermore , regional gm and t2w ( blood volumes ) were positively correlated with the neuro and somatic symptom scores in the same areas and elsewhere . vbm is a commonly used automated tool for studying patterns of brain change in neurological diseases and neuroanatomical correlates of subject characteristics . although most vbm processing and analysis is automated in spm , several methodological options remain for user specification,19 especially in analysis of longitudinal image data . inconsistent vbm findings in cfs patients in previous studies6 , 7 , 12 , 14 motivated us here to validate segmentation and optimize vbm methodology . the vbm atrophy simulation experiment demonstrated ( 1 ) that normalization to a study specific template performs better than direct normalization to the default template provided in spm12 ; ( 2 ) when the sample size is small , a random effects onesample ttest of longitudinal difference images , either by means of the jacobian difference method or the direct difference between different time points , has higher sensitivity than a paired ttest of images from two time points ; ( 3 ) that creation of a single image representative of the two time points ( withinsubject template ) for the purpose of the shared final spatial normalization should involve similar normalizations of both raw images and the same processing steps . this study detected continuing shrinkage of wm in the left ifof in patients with cfs , but not in ncs . this result was consolidated by the pooled inter group comparisons revealing decreased regional wm volumes in adjacent regions and decreased gm and blood volumes in contralateral regions and by regression analysis showing significant correlations of wm and gm volumes and t2w intensities with cfs symptom scores in those regions . the superficial and dorsal subcomponent of the ifof connects the frontal lobe with the superior parietal lobe and the ventral subcomponent of the ifof connects the frontal lobe with the inferior occipital lobe and temporobasal area.26 therefore , our findings are consistent with the decreased intrinsic connectivity in cfs within left frontoparietal networks ( fpn ) found in a restingstate functional connectivity study.27 similarly , a diffusion tensor imaging ( dti ) study reported bilateral white matter atrophy and increased fractional anisotropy in the arcuate fasciculus , possibly reflecting degeneration of cross fibers.8 the ifof connects networks of cognitive control , attention , language processing , and working memory28 and links the bilateral insular regions and anterior cingulate cortex.29 considerable evidence suggested that the ifof plays a pivotal role as the connectivity substrate in goaldirected cognition , mediating the dynamic balance between default mode network and dorsal attention networks.30 therefore , symptoms experienced by cfs patients , such as impaired concentration , working memory loss , inability to focus vision , and poor motor coordination could be explained , in part at least , by our finding of continuing shrinkage of wm in the ifof . our finding of ifof wm shrinkage in cfs , together with consistent findings by us and others above , warrants more investigations to understand the pathomechanism of this deficit . compared with nc , cfs patients also showed decreased gm volume and increased t1w and t2w signal intensities near the contralateral ilof . the absence of corresponding longitudinal findings in the right hemisphere may be a result of lower sensitivity in longitudinal studies and/or because the changes in cfs occurred soon after onset ( before the first scan ) and did not progress appreciably thereafter . the increased t2w signal intensities suggest possible decreased blood volume in these regions.31 indeed , previous studies have found decreased regional cerebral blood flow in frontal and temporal lobes32 and decreased frontal oxygenation33 in patients with cfs . thus , a gradual and chronic hypoperfusion of the brain may contribute to this continuing wm shrinkage . additionally , chronic functional hypoxia due to dysfunction of the neurovascular unit could also cause neurodegeneration.34 of interest , a recent study found seventeen single nucleotide polymorphisms ( snps ) were significantly associated with cfs.35 nine of these snps were associated with muscarinic acetylcholine receptors and eight with nicotinic ach receptors ( nachrs ) . ach , a neuromodulator in the brain , changes the state of neuronal networks throughout the brain and modifies their response to internal and external inputs . control of synaptic ca concentration following nachr stimulation is a major pathway for ach to influence neuronal networks.36 furthermore , nachrs are also present in the cerebral vascular endothelium and smooth muscles.37 thus , aberrant achr function may impair cerebrovascular autoregulation and cause chronic functional hypoxia . distribution of moderate nachr density in temporal parietal cortices and low levels in white matter tracts could make ifof more vulnerable to aberrant achr function . furthermore , the ifof is in a brain region that undergoes continued organization in early adulthood38 and is particularly vulnerable to vascular risk factors.39 regression analysis also showed several image measures correlated with symptom scores in regions other than the ifof . for example , regional gm volumes are positively correlated with neuro ss in the right fusiform gyrus , which is involved with brain recognition functions . similarly , low regional gm volumes in the culmen of cerebellum and low regional wm volumes inferior to precentral sulcus are correlated with impaired somatic status . the intergroup comparison and regression analysis showed that cfs patients had increased regional gm volume in the supplementary motor area and this regional gm volume is negatively correlated with neuro ss , i.e. , higher regional gm volume with more severe symptoms . these findings are in line with the clinical observation that cfs patients need extra effort for motor coordination , with fmri results that cfs patients recruit more cerebral regions for tasks,40 and with increased cortical thickness in cfs in the precentral gyrus.8 we postulate that these increased regional gm volumes may be a result of brain plasticity compensating for communication deficits because of wm shrinkage . although several tracts could be affected in this area , we postulated that the ifof was mainly involved based on previous fmri studies.27 therefore , it is still necessary to identify and confirm deficits in wm fiber tracts using dti . indeed , we are currently acquiring dti and fmri data to identify affected wm tracts and to investigate functional consequences of these deficits . two symptom severity scores , somatic and neuro ss , used in this study are based on the most frequent cfs symptoms16 and , although not validated , have much in common with validated scores , such as the center for disease control cfs symptom inventory . the study was designed to investigate progressive brain changes with an interval between the two scans of 6 years . the first scan included 25 cfs patients and 25 ncs , but because of the relatively long interval , only 15 cfs patients and 10 ncs returned for the second scan . although we detected progressive wm atrophy in the ifof region in cfs patients , because of our small sample size and low detection power , we can not rule out progressive changes elsewhere that are specific to cfs . in conclusion , we optimized vbm for longitudinal analysis and for the first time found continuing wm shrinkage in the left posterior ifof in cfs patients but not ncs , using mr data acquired with a 6year gap . the longitudinal finding was consolidated by the decreased regional gm , wm , and blood volumes in adjacent regions . the results suggest that cfs is a chronic illness with abnormal connections among brain regions and wm deficits which continue to deteriorate post onset . this study warrants further investigations to understand the pathomechanism of wm deficits in the ifof in cfs . additional supporting information may be found in the online version of this article . supporting information

purposeto examine progressive brain changes associated with chronic fatigue syndrome ( cfs).materials and methodswe investigated progressive brain changes with longitudinal mri in 15 cfs and 10 normal controls ( ncs ) scanned twice 6 years apart on the same 1.5 tesla ( t ) scanner . mr images yielded gray matter ( gm ) volumes , white matter ( wm ) volumes , and t1 and t2weighted signal intensities ( t1w and t2w ) . each participant was characterized with bell disability scores , and somatic and neurological symptom scores . we tested for differences in longitudinal changes between cfs and nc groups , inter group differences between pooled cfs and pooled nc populations , and correlations between mri and symptom scores using voxel based morphometry . the analysis methodologies were first optimized using simulated atrophy.resultswe found a significant decrease in wm volumes in the left inferior frontooccipital fasciculus ( ifof ) in cfs while in ncs it was unchanged ( family wise error adjusted cluster level p value , p fwe < 0.05 ) . this longitudinal finding was consolidated by the group comparisons which detected significantly decreased regional wm volumes in adjacent regions ( p fwe < 0.05 ) and decreased gm and blood volumes in contralateral regions ( p fwe < 0.05 ) . moreover , the regional gm and wm volumes and t2w in those areas showed significant correlations with cfs symptom scores ( p fwe < 0.05).conclusionthe results suggested that cfs is associated with ifof wm deficits which continue to deteriorate at an abnormal rate . j. magn . reson . imaging 2016;44:13011311 .

Materials and Methods Subjects MRI Acquisition Segmentation and Validation Statistical Analysis of Longitudinal Changes in Clinical Measures and Global Volumes Assessment of VBM Options Using Simulated Atrophy VBM Preprocessing of Longitudinal MR Data From CFS Patients and NCs Image Comparison Results Optimization of Segmentation and VBM Options Comparison of Clinical Measures and Global Volumes Different Longitudinal MRI Changes Between CFS and NC Crosssectional Differences in Pooled MR Images Correlations of Pooled MR Images With Symptom Scores in CFS Discussion Supporting information

paired two tailed ttests were used to test for equality of means and to compare longitudinal changes in the clinical measures , global gm volumes , and global wm volumes in both the cfs and nc groups . paired two tailed ttests were used to test for equality of means and to compare longitudinal changes in the clinical measures , global gm volumes , and global wm volumes in both the cfs and nc groups . table s1 ) , although these longitudinal changes in total gm and wm volumes did not differ significantly between cfs and nc groups . mean changes and correlations between values at time 1 and time 2 for clinical measures , global gm volumes , and global wm volumes in cfsa changes were calculated as values at time 1 minus values at time 2 for each patient of chronic fatigue syndrome ( cfs ) . gm and wm represent global gray matter ( gm ) and white matter ( wm ) volumes . there is no significant difference in total gm and wm volumes and ages between cfs and nc groups . means of clinical measures and global gm and wm volumes of pooled cfs and nc groups gm = gray matter ; wm = white matter ; cfs = chronic fatigue syndrome ; nc = normal control ; m = mean value ; p = significance level ; neuro ss = neurological symptom score ; somatic ss = somatic symptom score . the rateofchange of regional wm volumes in cfs patients was significantly different from that in ncs in the left posterior part of the inferior frontooccipital fasciculus ( ifof ) and/or arcuate fasciculus ( fig . voxelbased morphometry ( vbm ) of white matter ( wm ) longitudinal change rate difference between chronic fatigue syndrome ( cfs ) and normal control ( nc ) subjects . the regional differences of gm and wm volumes and t1w and t2w signal intensities between pooled cfs and nc groups are summarized in figure 3 . regional differences of gray matter ( gm ) and white matter ( wm ) volumes and t1w and t2w signal intensities between cfs and nc groups . clusters from regressions of regional gray matter ( gm ) and white matter ( wm ) volumes and t1w and t2w signal intensities versus clinical measures in the pooled cfs group . table s1 ) , although these longitudinal changes in total gm and wm volumes did not differ significantly between cfs and nc groups . mean changes and correlations between values at time 1 and time 2 for clinical measures , global gm volumes , and global wm volumes in cfsa changes were calculated as values at time 1 minus values at time 2 for each patient of chronic fatigue syndrome ( cfs ) . gm and wm represent global gray matter ( gm ) and white matter ( wm ) volumes . there is no significant difference in total gm and wm volumes and ages between cfs and nc groups . means of clinical measures and global gm and wm volumes of pooled cfs and nc groups gm = gray matter ; wm = white matter ; cfs = chronic fatigue syndrome ; nc = normal control ; m = mean value ; p = significance level ; neuro ss = neurological symptom score ; somatic ss = somatic symptom score . the rateofchange of regional wm volumes in cfs patients was significantly different from that in ncs in the left posterior part of the inferior frontooccipital fasciculus ( ifof ) and/or arcuate fasciculus ( fig . the regional differences of gm and wm volumes and t1w and t2w signal intensities between pooled cfs and nc groups are summarized in figure 3 . regional differences of gray matter ( gm ) and white matter ( wm ) volumes and t1w and t2w signal intensities between cfs and nc groups . clusters from regressions of regional gray matter ( gm ) and white matter ( wm ) volumes and t1w and t2w signal intensities versus clinical measures in the pooled cfs group . intergroup comparison found decreased gm and wm volumes and increased t1w and increased t2w intensities ( which may reflect decreased blood volumes and ischemia ) near this ilof / arcuate area both ipsi and contralaterally . this result was consolidated by the pooled inter group comparisons revealing decreased regional wm volumes in adjacent regions and decreased gm and blood volumes in contralateral regions and by regression analysis showing significant correlations of wm and gm volumes and t2w intensities with cfs symptom scores in those regions . the longitudinal finding was consolidated by the decreased regional gm , wm , and blood volumes in adjacent regions . the results suggest that cfs is a chronic illness with abnormal connections among brain regions and wm deficits which continue to deteriorate post onset .

lung cancer is the third - most commonly diagnosed cancer and the leading cause of cancer - related deaths in the united states . over 220,000 new cases of lung cancer are expected to be diagnosed in 2015 while more than 158,000 people are expected to die from lung cancer that same year . worldwide , lung cancer is the leading cause of both new cancer diagnoses and cancer - related deaths with nearly 1.8 million new cases and 1.6 million deaths estimated in 2012 ( the most recent year for which data is available ) . most ( approximately 85% ) of lung cancers are of the non - small - cell type ( nsclc ) , with 2530% of nsclc being squamous histology type . unlike nonsquamous nsclc , squamous nsclc rarely harbors epidermal growth factor receptor ( egfr ) and anaplastic lymphoma kinase ( alk ) mutations for which there are directed therapies , and until the recent approval of immunotherapies for squamous nsclc , a limited number of traditional cytotoxic chemotherapy drugs have been fda - approved for use in the treatment of advanced and metastatic squamous nsclc . the programmed cell death-1 receptor ( pd-1 ) is found on cytotoxic t cells and t - regulatory cells and is expressed when t cells become activated in response to inflammation or infection in peripheral tissues . binding of the pd-1 ligand to its receptor inactivates the t cell in order to limit the immune response to the stimuli , thus causing an immune suppression . cancer cells induce pd-1 expression , enhancing the immunosuppressive action of this pathway , ultimately allowing the cancer to be hidden from natural immune attack . anti - pd-1 therapies disrupt this pathway by preventing the pd-1 ligand from binding to its receptor , leaving activated cytotoxic t cells available to attack the cancer cells . immunotherapies directed at the pd-1 or its ligand ( pd - l1 ) have demonstrated efficacy in both nonsquamous and squamous cell nsclc . notably , while egfr , kirsten rat sarcoma viral oncogene homolog ( kras ) , and alk mutation status guide drug therapy selection and provide more treatment options for patients who harbor these mutations ( typically nonsquamous nsclc ) , patients with squamous cell disease with larger tumors or positive lymph nodes who express pd - l1 may have improved survival compared with their pd - l1-deficient counterparts . it is not known , however , what minimum level of pd - l1 expression is necessary to predict treatment outcomes with anti - pd-1 therapies because while patients who are pd - l1 positive have higher response rates to treatments across various tumor types , it has been shown that even patients who test negative for pd-1 can respond to these therapies . a platinum - based combination chemotherapy regimen has been the standard first - line treatment for all nsclc since cisplatin was first fda - approved in 1978 . carboplatin is frequently substituted for cisplatin for patients who have poor renal function or who experience toxicities from cisplatin ( most notably , nausea and vomiting ) . taxanes , especially paclitaxel in the first - line setting or docetaxel in refractory patients , commonly complete the standard two - drug backbone of platinum - based chemotherapy for the first - line treatment of nsclc . in combination , platinum with a taxane frequently causes myelosuppression , nausea , vomiting , alopecia , paronychia , stomatitis , fatigue , taste changes , and , particularly when docetaxel is used as part of the regimen , febrile neutropenia and subsequent risk of systemic infections . gemcitabine , etoposide , vincristine , vinorelbine , and pemetrexed have also been evaluated in combination with a platinum agent in the first line - setting for nsclc . of note , patients with nonsquamous cell disease have improved survival with cisplatin and pemetrexed combination therapy in the first - line setting compared with combination cisplatin and gemcitabine , whereas patients with squamous cell disease have improved survival with cisplatin and gemcitabine combination treatment . while cisplatin and pemetrexed have demonstrated higher rates of severe nausea compared with cisplatin and gemcitabine , patients treated with cisplatin and gemcitabine experienced higher rates of severe neutropenia , anemia , thrombocytopenia , febrile neutropenia , and alopecia . for patients with squamous cell nsclc , several traditional cytotoxic chemotherapy options are available for subsequent treatment , including single - agent docetaxel or gemcitabine ( if not used in the first - line setting ) or combination gemcitabine with the vascular endothelial growth factor receptor monoclonal antibody ramucirumab . the rare squamous cell nsclc patient with wild - type egfr status may be offered erlotinib or gefitinib if not used first - line . both docetaxel ramucirumab and single - agent nivolumab have shown improved survival for squamous cell nsclc patients in the second - line setting compared with single - agent docetaxel ; however , nivolumab has been given a category 1 recommendation by the national comprehensive network s clinical practice guideline for non - small - cell lung cancer for the second - line treatment of squamous cell disease after failure of first - line platinum - based treatment . because of their novel mechanism of action and because the traditional cytotoxic agents have been available for so long with known efficacy and toxicities , the remainder of this review will focus on the clinical trial experience of the anti - pd-1 therapies nivolumab and pembrolizumab , currently the only immunotherapies that are fda - approved for the treatment of nsclc . these therapies represent a novel approach to treating nsclc and spare the patient from the myelosuppresive and emetic effects of the traditional cytotoxic chemotherapy described above . nivolumab is currently fda - approved only for squamous cell nsclc ; most recently , pembrolizumab received fda - approval for any histological type of nsclc as long as the tumor tests positive for pd-1 expression . in contrast , nivolumab was not approved with the condition that the tumor test positive for pd-1 expression . when the pd-1 ligand binds to its receptor on activated t cells , the t cell is unable to exert its immunologic effects on antigens , including cancer cells . many cancer cells , including those of nsclc , express the pd-1 ligand , and the ligand receptor binding essentially allows the cancer to hide from the t cell . current smokers are significantly more likely to express the pd-1 receptor than nonsmokers . in fact , current / former smokers with nsclc were significantly more likely to respond to anti - pd-1 therapy with pembrolizumab than never - smokers ; however , once corrected for pd-1 expression of at least 50% , smoking status was not as robust of a predictor of response to treatment . current / former smokers with squamous nsclc were also significantly more likely to respond to nivolumab than docetaxel compared with never - smokers ; in this trial , pd-1 expression did not predict response to treatment ( though the maximum expression level measured was 10% ) . drugs that block the binding of the pd-1 ligand to its pd-1 receptor , such as nivolumab and pembrolizumab , allow activated t cells to identify and attack cancer cells . in clinical trials that have evaluated nivolumab and pembrolizumab activity against various cancers , including nsclc , an apparent pseudoprogression of the cancer has been noted because of the infiltration of the t cells into the site of the primary tumor , which on imaging makes the mass appear to have increased in size . it may take up to 6 months to have a clear picture of a patient s true response to treatment because of this t - cell infiltration , and some have called for an alternative to the response evaluation criteria in solid tumors ( recist ) criteria and other parameters for monitoring and measuring response to treatment because of this phenomenon . checkmate 063 was a phase 2 , single - arm trial conducted in europe and the united states of nivolumab 3 mg / kg iv over 1 hour every 2 weeks until disease progression or unacceptable toxicity in 117 patients with stage iiib or iv squamous cell nsclc that had progressed on at least two prior treatment regimens a platinum - doublet therapy and one additional drug therapy regimen ( one - third of patients were previously treated with an anti - egfr tyrosine kinase inhibitor ) . patients who had apparent disease progression on radiographic images were allowed to continue treatment at the discretion of their physician if it was determined that the patient was experiencing a clinical benefit because of the known pseudo - progression that can occur with anti - pd-1 therapy . patients with stable brain metastases were eligible for enrollment in the trial , which is notable because such patients are frequently excluded from clinical trials , particularly when they have squamous histology , because of the tendency for this histology to bleed . in this trial , two patients ( 2% ) had brain metastases . other interesting eligibility criteria for this trial include that while patients with interstitial pneumonitis were excluded , patients with a history of pneumonitis were not ; of note , pneumonitis was one of the three most common national cancer institute common terminology criteria for adverse events ( nci ctc ) grade 34 adverse events reported in the trial . another notable detail regarding the study procedures was that dose modifications were not allowed , although criteria were established for dose delays . this is an important aspect of the protocol for clinicians to be aware because dose modifications in clinical oncology practice are frequent and sometimes empiric . pd - l1 status was assessed on pretreatment , formalin - fixed , and paraffin - embedded tumor specimens and was considered positive if 5% of the cells stained positive by a validated immunohistochemical ( ihc ) assay . as noted earlier , the relevance and exact lower threshold for meaningful pd - l1 positivity is an area of clinical controversy , and the keynote trial of pembrolizumab used a much higher breakpoint ( 50% ) for pd - l1 expression [ 911 ] . a median of six doses of nivolumab were administered to patients in checkmate 063 with a median treatment duration of 2.3 months ( 95% ci , 1.42.8 months ) . the most common reason for treatment discontinuation was disease progression ( 67% of patients ) . patients were assessed by radiographic imaging at baseline , 8 weeks after the start of treatment , and then every 6 weeks until disease progression . on this imaging schedule , median time to response was reported as 3.3 months and median duration of response had not been reached ( minimum follow - up for response was 11 months and median follow - up for overall survival was 8 months ) . the median duration of stable disease was 6 months . although survival was a secondary end point in this phase 2 trial , the reported median progression - free survival was 1.9 months ( 95% ci , 1.83.2 months ) and median overall survival was 8.2 months ( 95% ci , 6.110.9 months ) . patients with greater than or less than the 5% threshold for pd - l1 positivity responded to treatment , although those with at least 5% positivity had numerically higher rates of partial response ( 24 vs 14% ) and lower rates of progressive disease ( 44 vs 49% ) compared with those with less than 5% pd - l1 positivity . the most common adverse events of any nci ctc grade reported in checkmate 063 were fatigue ( 33% ) , decreased appetite ( 19% ) , nausea ( 15% ) , asthenia ( 12% ) , rash ( 11% ) , and diarrhea ( 10% ) ; the most common severe ( nci ctc grade 3 or 4 ) adverse events reported were fatigue ( 4% ) , pneumonitis ( 3% ) , and diarrhea ( 3% ) . immune - related toxicities were anticipated in the trial because of the mechanism of action of the drug ; however , most were of low grade with most being skin or gastrointestinal in nature . high - dose oral corticosteroids are the general management strategy for the immune - related adverse events . given the activity of nivolumab in this phase 2 trial with tolerable / manageable toxicities , nivolumab was a candidate for more rigorous evaluation in a phase 3 trial . the same dose and schedule of nivolumab was further evaluated in comparison with standard docetaxel 75 mg / m iv every 3 weeks in checkpoint 017 , a phase 3 , randomized , open - label , international study of 272 patients with stage iiib or iv squamous cell nsclc who failed only one prior platinum - containing treatment . similar to the phase 2 checkpoint 063 , patients with treated , stable brain metastases were eligible for enrollment , and dose reductions in nivolumab were not allowed . a total of 6% of patients in the trial had cns metastasis ( 7% of nivolumab patients and 6% of docetaxel patients ) . unlike checkpoint 063 , however , no nivolumab patients and only 1% of docetaxel patients had been treated with anti - egfr therapy ( only cetuximab use was reported for checkpoint 017 , whereas the oral tyrosine kinase inhibitors against egfr were reported for checkpoint 063 ) . the most common first - line chemotherapies used with a platinum in checkpoint 017 were gemcitabine ( 48% ) , paclitaxel ( 34% ) , and vinorelbine ( 16% ) . checkpoint 017 was initially designed around the primary end point of response rate ; however , the protocol was amended after interim results were reported from the mdx-1106 - 03 trial to make overall survival the primary end point . the study was stopped early because a prespecified interim analysis showed that overall survival was significantly improved on the nivolumab arm compared with the docetaxel arm . at the time that the study closed , median overall survival was 9.2 months ( 95% ci , 7.313.3 months ) on nivolumab compared with 6.0 months ( 95% ci , 5.17.3 months ) on docetaxel , with a 41% reduction in the risk of death on the nivolumab arm ( hr , 0.59 ; 95% ci , 0.440.49 ; p<0.001 ) . objective response was also significantly higher on the nivolumab arm compared with docetaxel ( 20% [ 95% ci , 1428% ] vs 9% [ 95% ci , 515% ] ; p=0.008 ) , and it took approximately 2 months on both arms to observe a response ( patients were monitored with radiographic imaging at baseline , week 9 , and every 6 weeks thereafter ) . statistically , the median progression - free survival was not different between the arms with 3.5 months ( 95% ci , 2.14.9 months ) on nivolumab compared with 2.8 months ( 95% ci , 2.13.5 months ) on docetaxel , although the cumulative risk of progressive disease over the course of the trial was significantly improved with nivolumab with a 38% reduction in the risk of progression ( hr , 0.62 ; 95% ci , 0.470.81 ; p<0.001 ) . in this trial , no level of pd - l1 positivity by ihc staining ( 1 , 5 , and 10% levels were evaluated ) predicted response or was prognostic for survival . 85% of nivolumab patients received at least 90% of their planned dose intensity , compared with only 69% of docetaxel patients . patients were also more likely to discontinue treatment due to adverse events of docetaxel ( 10% of patients ) than of nivolumab ( 3% of patients ) . furthermore , 58% of patients on nivolumab experienced any nci ctc grade of adverse event compared with 86% of docetaxel patients , 7% of which were grade 3 or 4 on nivolumab compared with 55% on docetaxel . the most common adverse events on the nivolumab arm were fatigue ( 16% ) , decreased appetite ( 11% ) , and asthenia ( 10% ) , similar to what was seen in checkpoint 063 . fatigue , decreased appetite , and leukopenia were the only grade 3 or 4 adverse events reported on nivolumab , each occurring in only 1% of nivolumab - treated patients . pembrolizumab is another anti - pd - l1 , monoclonal antibody that , like nivolumab , is approved for refractory metastatic melanoma patients and recently received accelerated fda - approval for the treatment of any histological type of nsclc after failure of first - line therapy that includes a platinum or an anti - egfr or anti - alk therapy in patients with appropriate mutations . it has been evaluated in the phase 1 keynote-001 trial at doses of 2 or 10 mg / kg every 3 weeks or 10 mg / kg every 2 weeks ( in all cases administered as a 30-minute infusion ) in 495 patients who were either treatment - naive or treatment - experienced with any histologic type of stage iiib or iv nsclc to determine the preliminary safety and efficacy of pembrolizumab in this population . patients were monitored for response by traditional radiographic imaging and recist criteria every 9 weeks as well as investigator - assessed , immune - related response criteria . biopsy samples had to be available for enrollment , and in the biomarker validation component of the trial , those samples with at least 1% of cells expressing pd - l1 by ihc staining were considered pd - l1 positive . previously untreated patient were more likely to respond to therapy ( 24.8 ; 95% ci , 16.734.3% ) and to have longer median duration of response ( 23.3 months ; 95% ci , 1.023.3 months ) than those patients who were treatment - experienced ( 18.0% response rate ; 95% ci , 14.422.2% ; median duration of response 10.4 months ; 95% ci , 1.010.4 months ) . there were no differences in the response rate based on histologic subtype ( squamous vs nonsquamous ) or based on the dose or schedule of pembrolizumab . current / former smokers had higher response rates ( 22.5% ) compared with never - smokers ( 10.3% ) . median overall survival reported in this phase 1 trial was also longer for patients receiving pembrolizumab as first - line therapy ( 16.2 months ; 95% ci , 16.2not reached ) compared with those receiving pembrolizumab after failing at least one prior regimen ( 9.3 months ; 95% ci , 9.314.7 months ) . the keynote-001 investigators selected 50% pd - l1 expression as the cutoff for pd - l1 positivity , which was found in almost one - quarter of the population ; at this level , the overall response rate was 45.2% ( 95% ci , 33.557.3% ) , and differences in response between previously treated and previously untreated patients were no longer apparent ( 43.9% response rate [ 95% ci , 30.757.6% ] in previously treated patients compared with 50.0% response rate [ 95% ci , 24.775.3% ] in previously untreated patients ) . side effects that were seen with pembrolizumab were of similar nature as those seen with nivolumab , although some seem numerically higher than that reported in the nivolumab trial . more grade 35 dyspnea ( 3.8% ) and pneumonitis ( 1.8% ) and more overall pruritis ( 10.7% ) , rash ( 9.7% ) , arthralgia ( 9.1% ) , diarrhea ( 8.1% ) , and nausea ( 7.5% ) were reported with pembrolizumab in this phase 1 trial . additionally , 6.9% of patients experienced any grade of hypothyroidism ( only 1 patient ( 0.2% ) experienced grade 35 hypothyroidsim ) . the most common adverse events of any severity in keynote-001 were fatigue , pruritus , decreased appetite , rash , arthralgia , diarrhea , nausea , and hypothyroidism ( all occurring in more than 5% of patients ) and the most common grade 35 adverse events were dyspnea ( 3.8% ) , pneumonitis ( 1.8% ) , decreased appetite ( 1% ) , and asthenia ( 1% ) . both nivolumab and pembrolizumab offer an important drug therapy option for selected patients in the second - line treatment of nsclc . pembrolizumab received accelerated fda - approval based on the outcomes of a phase 1 trial and is awaiting confirmatory results from phase 3 trials to establish its effects on overall and progression - free survival . in contrast , nivolumab received standard fda - approval based on the outcomes of a phase 3 trial demonstrating improved survival in comparison with docetaxel , an accepted second - line therapy for patients who fail platinum - doublet therapy . because of their similar mechanism of action against the pd - l1/pd-1 pathway , both drugs have similar toxicity profiles related to immune - mediated adverse reactions that can generally be monitored and managed with oral corticosteroids . pembrolizumab requires less frequent administration at a dose of 2 mg / kg iv over 30 minutes every 3 weeks , compared with nivolumab 3 mg / kg iv over 60 minutes every 2 weeks . while nivolumab received fda - approval strictly for squamous cell nsclc , clinical trials demonstrated that both squamous and nonsquamous histologies respond to both nivolumab and pembrolizumab . finally , pembrolizumab was approved for patients who test positive for pd - l1 ( defined in the keynote-001 trial as at least 50% ) based on differences in response rates for patients who meet this threshold compared with those who fall below it ; nivolumab was not approved with a pd - l1 expression contingency , though the checkmate 063 trial only used 5% and the checkmate 017 trial only used 10% pd - l1 threshold for determining response ( and subsequently determined that pd - l1 expression using these definitions did not predict response ) . while smokers seem to be more likely to express pd - l1 and to respond to anti - pd - l1 therapies , using the 50% threshold for pd - l1 positivity more accurately predicts response to these therapies and therefore smoking status should not be used to select patients for treatment . neither drug requires dose adjustment for renal or hepatic impairment unless immune - mediated nephritis or hepatitis occurs after the start of treatment . the decision of which anti - pd - l1 therapy to use in the second - line setting will generally depend currently on the histology of the patient , possibly pd - l1 expression , and the preference of the treating clinician to select therapy based on the efficacy reports from phase 3 compared with phase 1 trials . in most cases , second - line therapy options that do not include anti - pd - l1 therapy are cytotoxic chemotherapy ; patients who may be unlikely to tolerate the typical adverse effects of these other traditional agents may be best suited for second - line anti - pd - l1 therapy . anti - pd - l1/pd-1 therapies are the first immunotherapies to demonstrate clinical benefit in nsclc . these are particularly important in the nsclc population with squamous histology as the treatment options for these patients have been generally limited to traditional cytotoxic chemotherapies because of the infrequent presence of biomarker expression for which there are targeted therapies . for all nsclc , validation of pd - l1 or pd-1 assays and a defined level of pd - l1/pd-1 expression to predict those most likely to benefit from treatment will be an important area of ongoing research . published trials of nivolumab have evaluated its use in refractory patients , and it is currently unknown if there is clinical benefit to using this therapy in the first - line setting for all or a subset of nsclc patients . checkmate 026 ( nci clinical trial number nct02041533 ) and keynote-042 ( nci clinical trial number nct02220894 ) are phase 3 trials that are currently recruiting patients for the first - line treatment of nsclc with nivolumab compared with investigator s choice chemotherapy ( checkmate 026 ) or pembrolizumab compared with platinum - based chemotherapy ( keynote-042 ) in pd - l1-positive nsclc with results expected in august 2016 ( checkmate 026 ) and june 2018 ( keynote-042 ) . keynote-024 ( nci clinical trial number nct02142738 ) is also recruiting treatment - naive patients with strongly positive pd - l1-positive nsclc in a phase 3 trial of pembrolizumab compared with platinum - based chemotherapy . an additional area of clinical interest is the use of the anti - pd-1 therapies in combination with currently available treatments . the currently published trials of nivolumab and pembrolizumab have evaluated their use as single agents ; trials are currently ongoing to evaluate these therapies in combination with other therapies , including chemotherapy , the anti - ctla4 immunotherapy ipilimumab , and anti - egfr targeted agents . checkmate 227 ( nci clinical trial number nct02477826 ) opened in august 2015 and is currently recruiting patients to evaluate nivolumumab with or without ipilimumab compared with standard platinum - doublet therapies in the first - line treatment of advanced or metastatic nsclc for any histology , with preliminary results expected in may 2017 . keynote-021 ( nci clinical trial number nct02039674 ) is currently recruiting patients to a randomized , open - label , multiarm safety / efficacy study of pembrolizumab in combination with standard therapies with a control arm of standard platinum - doublet chemotherapy . additional trials are also ongoing with other anti - pdl1 therapies , including avelumab ( msb0010718c ) , mpdl3280a , and medi4736 . standard platinum - doublet therapy remains the primary drug therapy approach to treating patients with advanced/ metastatic squamous cell nsclc . recent advances in anti - pdl1 immunotherapy have expanded the treatment armamentarium for this population of patients in the second - line setting . phase 13 trials have demonstrated improved response rates and survival outcomes with acceptable levels of toxicity when nivolumab or pembrolizumab is used as monotherapy in this setting . there is a need for further research to better define the role of pd - l1 expression as a biomarker predictive of response to these treatments as well as how these treatments may be used in the first - line setting and/or in combination with existing standard therapies . ongoing clinical trials evaluating these questions will help aid the clinician in determining the optimal approach to the medical management of advanced / metastatic squamous nsclc .

lung cancer is the third most commonly diagnosed cancer and the leading cause of cancer - related death in the united states . unlike non - squamous nsclc , squamous nsclc rarely harbor epidermal growth factor receptor ( egfr ) and anaplastic lymphoma kinase ( alk ) mutations for which there are directed therapies , and until the recent approval of immunotherapies for squamous nsclc , a limited number of traditional cytotoxic chemotherapy drugs have been fda - approved for use in the treatment of advanced and metastatic squamous nsclc . immunotherapies directed at the programmed cell death-1 receptor ( pd-1 ) or its ligand ( pd - l1 ) ( nivolumab and pembrolizumab ) have demonstrated efficacy in both nonsquamous and squamous cell nsclc . because of their similar mechanism of action against the pd - l1/pd-1 pathway , both drugs have similar toxicity profiles related to immune - mediated adverse reactions that can generally be monitored and managed with oral corticosteroids . this paper provides an overview of drug therapy options for squamous cell nsclc with a focus on the evidence and clinical application of the anti - pd1 therapies . a comparison of the dosing , administration , indications , and differences in the measurement of pd - l1 expression in the clinical trials of nivolumab and pembrolizumab is also provided .

Introduction Drug therapy overview Anti-PD-L1 immunotherapy for squamous NSCLC Treatment selection: nivolumab compared with pembrolizumab Future directions Summary

lung cancer is the third - most commonly diagnosed cancer and the leading cause of cancer - related deaths in the united states . worldwide , lung cancer is the leading cause of both new cancer diagnoses and cancer - related deaths with nearly 1.8 million new cases and 1.6 million deaths estimated in 2012 ( the most recent year for which data is available ) . unlike nonsquamous nsclc , squamous nsclc rarely harbors epidermal growth factor receptor ( egfr ) and anaplastic lymphoma kinase ( alk ) mutations for which there are directed therapies , and until the recent approval of immunotherapies for squamous nsclc , a limited number of traditional cytotoxic chemotherapy drugs have been fda - approved for use in the treatment of advanced and metastatic squamous nsclc . immunotherapies directed at the pd-1 or its ligand ( pd - l1 ) have demonstrated efficacy in both nonsquamous and squamous cell nsclc . for patients with squamous cell nsclc , several traditional cytotoxic chemotherapy options are available for subsequent treatment , including single - agent docetaxel or gemcitabine ( if not used in the first - line setting ) or combination gemcitabine with the vascular endothelial growth factor receptor monoclonal antibody ramucirumab . because of their novel mechanism of action and because the traditional cytotoxic agents have been available for so long with known efficacy and toxicities , the remainder of this review will focus on the clinical trial experience of the anti - pd-1 therapies nivolumab and pembrolizumab , currently the only immunotherapies that are fda - approved for the treatment of nsclc . checkmate 063 was a phase 2 , single - arm trial conducted in europe and the united states of nivolumab 3 mg / kg iv over 1 hour every 2 weeks until disease progression or unacceptable toxicity in 117 patients with stage iiib or iv squamous cell nsclc that had progressed on at least two prior treatment regimens a platinum - doublet therapy and one additional drug therapy regimen ( one - third of patients were previously treated with an anti - egfr tyrosine kinase inhibitor ) . pembrolizumab is another anti - pd - l1 , monoclonal antibody that , like nivolumab , is approved for refractory metastatic melanoma patients and recently received accelerated fda - approval for the treatment of any histological type of nsclc after failure of first - line therapy that includes a platinum or an anti - egfr or anti - alk therapy in patients with appropriate mutations . the keynote-001 investigators selected 50% pd - l1 expression as the cutoff for pd - l1 positivity , which was found in almost one - quarter of the population ; at this level , the overall response rate was 45.2% ( 95% ci , 33.557.3% ) , and differences in response between previously treated and previously untreated patients were no longer apparent ( 43.9% response rate [ 95% ci , 30.757.6% ] in previously treated patients compared with 50.0% response rate [ 95% ci , 24.775.3% ] in previously untreated patients ) . because of their similar mechanism of action against the pd - l1/pd-1 pathway , both drugs have similar toxicity profiles related to immune - mediated adverse reactions that can generally be monitored and managed with oral corticosteroids . while nivolumab received fda - approval strictly for squamous cell nsclc , clinical trials demonstrated that both squamous and nonsquamous histologies respond to both nivolumab and pembrolizumab . finally , pembrolizumab was approved for patients who test positive for pd - l1 ( defined in the keynote-001 trial as at least 50% ) based on differences in response rates for patients who meet this threshold compared with those who fall below it ; nivolumab was not approved with a pd - l1 expression contingency , though the checkmate 063 trial only used 5% and the checkmate 017 trial only used 10% pd - l1 threshold for determining response ( and subsequently determined that pd - l1 expression using these definitions did not predict response ) . the decision of which anti - pd - l1 therapy to use in the second - line setting will generally depend currently on the histology of the patient , possibly pd - l1 expression , and the preference of the treating clinician to select therapy based on the efficacy reports from phase 3 compared with phase 1 trials . these are particularly important in the nsclc population with squamous histology as the treatment options for these patients have been generally limited to traditional cytotoxic chemotherapies because of the infrequent presence of biomarker expression for which there are targeted therapies . the currently published trials of nivolumab and pembrolizumab have evaluated their use as single agents ; trials are currently ongoing to evaluate these therapies in combination with other therapies , including chemotherapy , the anti - ctla4 immunotherapy ipilimumab , and anti - egfr targeted agents .

the national mandate for health systems to migrate to icd-10-cm in october 2015 has an impact on all research activities that rely on these codes . further , many current and ongoing investigations will need to manage and analyze data sets that define conditions of interest ( i.e. , clinical phenotypes ) using both icd-9-cm and icd-10-cm codes . the growing availability of electronic health record ( ehr ) data ( encoded in icd-10-cm ) will increasingly be leveraged to support pragmatic clinical trials and quality improvement studies that learning health care comprises . longitudinal studies in progress and retrospective studies will use icd-9-cm - based population definitions and will need to understand how those relate to definitions based upon icd-10-cm . a common challenge for researchers and health administrators moving forward , then , is the need to translate icd-9-cm - based phenotype definitions , which can include hundreds of codes , into icd-10-cm and to ensure that the populations retrieved with those codes are clinically equivalent . although the centers for medicare & medicaid services ( cms ) has produced general equivalent maps ( gems ) , their use is not straightforward , and different methods for using the gems can result in different outcomes . in the context of pragmatic clinical trials , we explore the use of publicly available mapping files to convert clinical phenotype definitions from icd-9-cm to icd-10-cm , compare the outcome of different approaches , and suggest preferred strategies for using the gems in automated translation . in addition to the phenotype definitions , we also make use of the value sets defined for electronic quality measurement as an additional way to evaluate the mapping methods . quality measurement value sets are lists of codes from standard terminologies used to identify sub - populations of patients sharing certain demographic and clinical characteristics , as defined by a clinical quality measure . these value sets are very similar in their function to phenotype definitions . as part of the cms meaningful use of ehr program , certified systems have to demonstrate the electronic submission of data for some selected clinical quality measures . value sets are published to allow automatic computation of the numerator and denominator of a quality measure . the tremendous costs associated with traditional clinical trials limit their use in addressing the majority of clinical questions and treatment decisions that are based upon insufficient evidence.14 further , the limited generalizability of results inherent in clinical trials has stimulated interest in alternative research models , including observational research and pragmatic trials , to support patient - centered outcomes research.5,6 these alternative research models depend upon access to ehr data collected by health systems as part of the patient care process . the hmo research network ( hmorn ) and other networks have used electronic health care and claims data to advance our understanding of disease.7,8 while electronic claims data have been used in observational research for decades , the growing adoption of ehrs brings the potential to support more sophisticated research activities , such as cohort selection and randomization , to facilitate prospective and interventional research studies.9,10 the routine use of ehr data is a vital component of the envisaged learning health care system , and has become feasible with the widespread adoption and meaningful use of ehrs in health care systems.11 pragmatic trials are those conducted in actual patient care settings and in cooperation with health care systems.6 the national institutes of health ( nih ) health care systems research collaboratory is funded by the nih common fund to strengthen the national capacity for implementing cost - effective , large - scale research studies that engage health care delivery organizations as research partners , with the assumption that this will make research results more relevant to providers and , ultimately , patients.12 the collaboratory includes a number of pragmatic trial demonstration projects that are multisite , often cluster - randomized , intervention studies.13 these demonstration projects have developed explicit and reproducible definitions ( i.e. , clinical phenotypes ) using icd-9-cm and other standardized code systems to identify patients with precise clinical attributes from various organizations and heterogeneous ehrs . these clinical phenotype definitions support a number of research activities , including cohort identification and describing the baseline characteristics ( e.g. , the proportion of patients with diabetes or hypertension ) of different patient populations . the phenotype definitions of the nih collaboratory projects currently include codes from icd-9-cm , but investigators need to adapt them to icd-10-cm since health care systems transitioned to it by october 1 , 2015 . rather , it is a radical transformation , involving major changes in not only the size of the terminology , but also in the organization , granularity , and semantics ( or meaning ) of terms.14 the more than 68,000 possible terms in icd-10-cm more than quadruple the 14,000 terms in icd-9-cm . because the collaboratory demonstration projects are all multiyear studies that span this national icd-10-cm transition period , investigators need to address both icd-9-cm and icd-10-cm in their research data sets . to ease the burden of researchers who need to translate their cohort or clinical phenotype definitions from icd-9-cm to icd-10-cm , we explored the use of published maps between icd-9-cm and icd-10-cm for automatic conversion . the general equivalent maps ( gems ) are created and maintained by the centers for medicare & medicaid services ( cms ) and the centers for disease control and prevention ( cdc ) , and serve as a tool for the conversion of data between icd-9-cm and icd-10-cm.15 the gems are often also referred to as crosswalks since they provide important information linking codes from one system with codes in the other system.16 users are cautioned against using the gems for actual coding as they have not been completely validated for clinical use . however , the conversion of data for quality measures and research is specifically listed among the applicable use cases.16 the gems are directional and therefore have two types : the forward maps convert icd-9-cm codes into icd-10-cm , and the backward maps convert icd-10-cm codes into icd-9-cm . because the relationships between icd-9-cm and icd-10-cm codes are often complex and not one - to - one , the use of gems is complicated and requires informed consideration.1720 while the impact of icd-10-cm transition has been explored in various health care and research settings,2124 there are few studies on the evaluation of automated translation of codes between icd-9-cm and icd-10-cm in the context of phenotype definitions for pragmatic trials . the forward and backward gems are not simple mirror images of each other , as the names may suggest . they are independent maps that differ significantly in scope and coverage ( table 1 ) . the majority of icd-10-cm codes are not represented in the forward map , and a significant portion of icd-9-cm codes ( 25 percent ) are not represented in the backward map . the backward map provides 78,034 unique pairs of icd-9-cm and icd-10-cm codes ( over three times more than the forward map ) , of which only 18,484 pairs ( 23.7 percent ) are also found in the forward map . users of the gems often find that they need to apply the forward and backward maps iteratively in order to obtain code maps ( or links ) that would otherwise be missed . according to boyd et al.,25 36 percent of the icd-9-cm codes are involved in so - called convoluted mappings , meaning that they are not simple one - to - one , one - to - many , or many - to - one maps to icd-10-cm codes . in these complex cases , iterative application of the forward and backward maps will discover more and more links from an icd-9-cm source code to icd-10-cm targets ( see methods section ) . as an example , consider the icd-9-cm code 648.82 abnormal glucose tolerance of mother , delivered , with mention of postpartum complication . using either the forward or the backward gem alone , one will find the target icd-10-cm code o99.815 abnormal glucose complicating the puerperium . with the iterative use of the two gems , three additional relevant icd-10-cm target codes can be found : o24.430 gestational diabetes mellitus in the puerperium , diet controlledo24.434 gestational diabetes mellitus in the puerperium , insulin controlledo24.439 gestational diabetes mellitus in the puerperium , unspecified control . o24.430 gestational diabetes mellitus in the puerperium , diet controlled o24.434 gestational diabetes mellitus in the puerperium , insulin controlled o24.439 gestational diabetes mellitus in the puerperium , unspecified control . first , it may take many iterations to exhaust all mapping relationships because some of the convoluted mappings are open - ended . second , some of the additional codes discovered in this way are not relevant . the aim of this study is to determine the optimal way to use the gems in the context of icd-9-cm code translation in phenotypic definition . the tremendous costs associated with traditional clinical trials limit their use in addressing the majority of clinical questions and treatment decisions that are based upon insufficient evidence.14 further , the limited generalizability of results inherent in clinical trials has stimulated interest in alternative research models , including observational research and pragmatic trials , to support patient - centered outcomes research.5,6 these alternative research models depend upon access to ehr data collected by health systems as part of the patient care process . the hmo research network ( hmorn ) and other networks have used electronic health care and claims data to advance our understanding of disease.7,8 while electronic claims data have been used in observational research for decades , the growing adoption of ehrs brings the potential to support more sophisticated research activities , such as cohort selection and randomization , to facilitate prospective and interventional research studies.9,10 the routine use of ehr data is a vital component of the envisaged learning health care system , and has become feasible with the widespread adoption and meaningful use of ehrs in health care systems.11 pragmatic trials are those conducted in actual patient care settings and in cooperation with health care systems.6 the national institutes of health ( nih ) health care systems research collaboratory is funded by the nih common fund to strengthen the national capacity for implementing cost - effective , large - scale research studies that engage health care delivery organizations as research partners , with the assumption that this will make research results more relevant to providers and , ultimately , patients.12 the collaboratory includes a number of pragmatic trial demonstration projects that are multisite , often cluster - randomized , intervention studies.13 these demonstration projects have developed explicit and reproducible definitions ( i.e. , clinical phenotypes ) using icd-9-cm and other standardized code systems to identify patients with precise clinical attributes from various organizations and heterogeneous ehrs . these clinical phenotype definitions support a number of research activities , including cohort identification and describing the baseline characteristics ( e.g. , the proportion of patients with diabetes or hypertension ) of different patient populations . the phenotype definitions of the nih collaboratory projects currently include codes from icd-9-cm , but investigators need to adapt them to icd-10-cm since health care systems transitioned to it by october 1 , 2015 . rather , it is a radical transformation , involving major changes in not only the size of the terminology , but also in the organization , granularity , and semantics ( or meaning ) of terms.14 the more than 68,000 possible terms in icd-10-cm more than quadruple the 14,000 terms in icd-9-cm . because the collaboratory demonstration projects are all multiyear studies that span this national icd-10-cm transition period , investigators need to address both icd-9-cm and icd-10-cm in their research data sets . to ease the burden of researchers who need to translate their cohort or clinical phenotype definitions from icd-9-cm to icd-10-cm , we explored the use of published maps between icd-9-cm and icd-10-cm for automatic conversion . the general equivalent maps ( gems ) are created and maintained by the centers for medicare & medicaid services ( cms ) and the centers for disease control and prevention ( cdc ) , and serve as a tool for the conversion of data between icd-9-cm and icd-10-cm.15 the gems are often also referred to as crosswalks since they provide important information linking codes from one system with codes in the other system.16 users are cautioned against using the gems for actual coding as they have not been completely validated for clinical use . however , the conversion of data for quality measures and research is specifically listed among the applicable use cases.16 the gems are directional and therefore have two types : the forward maps convert icd-9-cm codes into icd-10-cm , and the backward maps convert icd-10-cm codes into icd-9-cm . because the relationships between icd-9-cm and icd-10-cm codes are often complex and not one - to - one , the use of gems is complicated and requires informed consideration.1720 while the impact of icd-10-cm transition has been explored in various health care and research settings,2124 there are few studies on the evaluation of automated translation of codes between icd-9-cm and icd-10-cm in the context of phenotype definitions for pragmatic trials . the forward and backward gems are not simple mirror images of each other , as the names may suggest . they are independent maps that differ significantly in scope and coverage ( table 1 ) . the majority of icd-10-cm codes are not represented in the forward map , and a significant portion of icd-9-cm codes ( 25 percent ) are not represented in the backward map . the backward map provides 78,034 unique pairs of icd-9-cm and icd-10-cm codes ( over three times more than the forward map ) , of which only 18,484 pairs ( 23.7 percent ) are also found in the forward map . users of the gems often find that they need to apply the forward and backward maps iteratively in order to obtain code maps ( or links ) that would otherwise be missed . according to boyd et al.,25 36 percent of the icd-9-cm codes are involved in so - called convoluted mappings , meaning that they are not simple one - to - one , one - to - many , or many - to - one maps to icd-10-cm codes . in these complex cases , iterative application of the forward and backward maps will discover more and more links from an icd-9-cm source code to icd-10-cm targets ( see methods section ) . as an example , consider the icd-9-cm code 648.82 abnormal glucose tolerance of mother , delivered , with mention of postpartum complication . using either the forward or the backward gem alone , one will find the target icd-10-cm code o99.815 abnormal glucose complicating the puerperium . with the iterative use of the two gems , three additional relevant icd-10-cm target codes can be found : o24.430 gestational diabetes mellitus in the puerperium , diet controlledo24.434 gestational diabetes mellitus in the puerperium , insulin controlledo24.439 gestational diabetes mellitus in the puerperium , unspecified control . o24.430 gestational diabetes mellitus in the puerperium , diet controlled o24.434 gestational diabetes mellitus in the puerperium , insulin controlled o24.439 gestational diabetes mellitus in the puerperium , unspecified control . first , it may take many iterations to exhaust all mapping relationships because some of the convoluted mappings are open - ended . the aim of this study is to determine the optimal way to use the gems in the context of icd-9-cm code translation in phenotypic definition . in this study , we compared four progressively more aggressive methods for using the gems ( figure 1 ) . the goal of each method was to identify , for each icd-9-cm code ( the source code ) , one or more corresponding icd-10-cm codes ( the target codes ) . for all methods , we used a combination of the forward and backward gems to discover linkages between icd-9-cm and icd-10-cm codes . we treated the linkages in the forward and backward gems as the same , ignoring the stated directionality of the maps . in increasing order of aggressiveness , the methods are the following : simple forward map ( sfm ) : all icd-10-cm codes linked to an icd-9-cm code in the forward gem are used as targets.forward backward map ( fbm ) : uses direct links from both the forward and backward gems . this includes all maps in sfm , plus additional map targets identified by the links between icd-9-cm and icd-10-cm codes in the backward gem.secondary map ( sm ) : uses all maps in fbm , plus additional target codes identified by secondary icd-9-cm codes.the following are the steps to generate sm : based on fbm , identify secondary icd-9-cm codes , which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code . in figure 1 , consider the ( primary ) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm . since a and b share the same target x , b is identified as a secondary code of a.add the targets of the secondary codes in fbm to the list of targets for the primary source code . in figure 1 , x and y are added as targets for source code a.tertiary map ( tm ) : uses all maps in sm , plus additional target codes identified by tertiary icd-9-cm codes.the following are the steps to generate tm : based on fbm , identify tertiary icd-9-cm codes , which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code ( identified in the generation of sm ) . in figure 1 , b has been identified as a secondary code to primary source code a. in fbm , b has targets x and y , while c has targets y and z. since b and c share the same target y , c is identified as a tertiary code of a.add the targets of the tertiary codes in fbm to the list of targets for the primary source code . in figure 1 , y and z are added as targets for source code a. simple forward map ( sfm ) : uses only direct links from the forward gem . all icd-10-cm codes linked to an icd-9-cm code in the forward gem are used as targets . forward backward map ( fbm ) : uses direct links from both the forward and backward gems . this includes all maps in sfm , plus additional map targets identified by the links between icd-9-cm and icd-10-cm codes in the backward gem . secondary map ( sm ) : uses all maps in fbm , plus additional target codes identified by secondary icd-9-cm codes . the following are the steps to generate sm : based on fbm , identify secondary icd-9-cm codes , which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code . in figure 1 , consider the ( primary ) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm . since a and b share the same target x , b is identified as a secondary code of a.add the targets of the secondary codes in fbm to the list of targets for the primary source code . in figure 1 , x and y are added as targets for source code a. based on fbm , identify secondary icd-9-cm codes , which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code . in figure 1 , consider the ( primary ) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm . since a and b share the same target x , b is identified as a secondary code of a. add the targets of the secondary codes in fbm to the list of targets for the primary source code . in figure 1 , x and y are added as targets for source code a. tertiary map ( tm ) : uses all maps in sm , plus additional target codes identified by tertiary icd-9-cm codes . the following are the steps to generate tm : based on fbm , identify tertiary icd-9-cm codes , which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code ( identified in the generation of sm ) . in figure 1 , b has been identified as a secondary code to primary source code a. in fbm , b has targets x and y , while c has targets y and z. since b and c share the same target y , c is identified as a tertiary code of a.add the targets of the tertiary codes in fbm to the list of targets for the primary source code . in figure 1 , y and z are added as targets for source code a. based on fbm , identify tertiary icd-9-cm codes , which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code ( identified in the generation of sm ) . in figure 1 , b has been identified as a secondary code to primary source code a. in fbm , b has targets x and y , while c has targets y and z. since b and c share the same target y , c is identified as a tertiary code of a. add the targets of the tertiary codes in fbm to the list of targets for the primary source code . in figure 1 , y and z are added as targets for source code a. we chose these four methods for a number of reasons . the sfm and fbm are the most common ways to use the gems , and will provide a baseline measure of mapping performance . sm corresponds to the method used in the online transition tool provided by boyd s group.26 given their experience and commentary , we hypothesized that additional iterations will increase the number of icd-10-cm target codes and may enhance mapping performance . therefore tm was included to assess whether additional iterations are indeed beneficial . to evaluate the performance of the four mapping methods , we used a convenience sample of 32 phenotypes ( developed to identify research cohorts , characterize risk factors , or define outcomes ) from three different pragmatic trials collaborative care for chronic pain in primary care ( ppact ) , strategies and opportunities to stop colorectal cancer in priority populations ( stop crc ) , and a pragmatic trial of population - based programs to prevent suicide attempt that were defined by icd-9-cm codes . the icd-9-cm codes were translated to icd-10-cm codes using the four mapping methods based on the 2014 version of the gems . one generalist nurse practitioner ( kp ) and an md domain expert for each trial ( bg , ap , and mc ) reviewed the phenotype name and the icd-10-cm code sets generated by the maps to determine if each icd-10-cm code semantically fit into the named phenotype condition , based on their understanding of that phenotype and its intent . for example , for the phenotype active alcohol abuse the reviewer was asked to look at the icd-10-cm codes and determine ( yes or no ) if those codes were appropriate for inclusion in that heading . reviewers were provided the original phenotype definition ( i.e. , the set of icd-9-cm codes that constitute the specified condition ) as a reference on the same review sheet . to limit the scope and time for the evaluation , the reviewers were asked to review only the icd-10-cm codes generated by the different mapping methods . they were not asked to search for additional icd-10-cm codes that should have been included . to shorten the list of icd-10-cm codes for review , we derived an algorithm to roll up codes to their parents , as long as the total number of codes in the list was reduced . for example , if the list contained m47.10 , m47.11 , m47.12 , m47.13 , m47.15 , m47.16 , which were all children of m47.1 , we converted it into m47.1 exclusion : m47.14 because m47.14 was the only child of m47.1 not included in the list . we did this iteratively until no further reduction in the number of codes was possible . to evaluate the roll up algorithm , one cohort definition from each demonstration project with at least 10 icd-10-cm codes was manually reviewed to make sure that the final list of codes represented the meaning of the original codes . to obtain the quality measurement value sets , we used the value set authority center ( vsac ) launched by the united states national library of medicine ( nlm ) in 2012 to provide access to all official versions of value sets.27,28 in the vsac , we identified all value sets for 2014 clinical quality measures that were dually defined with both icd-9-cm and icd-10-cm code lists . we applied the four mapping methods to the icd-9-cm code lists , and evaluated the resulting icd-10-cm target codes against the icd-10-cm codes listed for that measure , using the latter as the gold standard . since the value sets differed considerably in their sizes , we also analyzed the effect of value set size on the mapping performance . to evaluate the performance of each mapping method , we calculated the recall , precision and f - score of each method for every phenotype definition and quality measure value set . note that for the phenotype definitions , we did not measure the true recall because the reviewers were not asked to look for missing icd-10-cm codes . to give an estimate of recall for the phenotype definitions , we assumed that the most aggressive method ( tm ) contained all the correct icd-10-cm codes . we used the f - score ( the harmonic mean between recall and precision ) as an overall indicator of performance of each mapping method . based on the distribution of the f - scores , we used the anova test to check the statistical significance of the difference between the four methods . in this study , we compared four progressively more aggressive methods for using the gems ( figure 1 ) . the goal of each method was to identify , for each icd-9-cm code ( the source code ) , one or more corresponding icd-10-cm codes ( the target codes ) . for all methods , we used a combination of the forward and backward gems to discover linkages between icd-9-cm and icd-10-cm codes . we treated the linkages in the forward and backward gems as the same , ignoring the stated directionality of the maps . in increasing order of aggressiveness , the methods are the following : simple forward map ( sfm ) : all icd-10-cm codes linked to an icd-9-cm code in the forward gem are used as targets.forward backward map ( fbm ) : uses direct links from both the forward and backward gems . this includes all maps in sfm , plus additional map targets identified by the links between icd-9-cm and icd-10-cm codes in the backward gem.secondary map ( sm ) : uses all maps in fbm , plus additional target codes identified by secondary icd-9-cm codes.the following are the steps to generate sm : based on fbm , identify secondary icd-9-cm codes , which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code . in figure 1 , consider the ( primary ) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm . since a and b share the same target x , b is identified as a secondary code of a.add the targets of the secondary codes in fbm to the list of targets for the primary source code . in figure 1 , x and y are added as targets for source code a.tertiary map ( tm ) : uses all maps in sm , plus additional target codes identified by tertiary icd-9-cm codes.the following are the steps to generate tm : based on fbm , identify tertiary icd-9-cm codes , which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code ( identified in the generation of sm ) . in figure 1 , b has been identified as a secondary code to primary source code a. in fbm , b has targets x and y , while c has targets y and z. since b and c share the same target y , c is identified as a tertiary code of a.add the targets of the tertiary codes in fbm to the list of targets for the primary source code . in figure 1 , y and z are added as targets for source code a. simple forward map ( sfm ) : uses only direct links from the forward gem . all icd-10-cm codes linked to an icd-9-cm code in the forward gem are used as targets . forward backward map ( fbm ) : uses direct links from both the forward and backward gems . this includes all maps in sfm , plus additional map targets identified by the links between icd-9-cm and icd-10-cm codes in the backward gem . secondary map ( sm ) : uses all maps in fbm , plus additional target codes identified by secondary icd-9-cm codes . the following are the steps to generate sm : based on fbm , identify secondary icd-9-cm codes , which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code . in figure 1 , consider the ( primary ) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm . since a and b share the same target x , b is identified as a secondary code of a.add the targets of the secondary codes in fbm to the list of targets for the primary source code . in figure 1 , x and y are added as targets for source code a. based on fbm , identify secondary icd-9-cm codes , which are defined as icd-9-cm codes that share the same target icd-10-cm code as the primary icd-9-cm source code . in figure 1 , consider the ( primary ) icd-9-cm source code a. it has targets w and x in fbm ; while another icd-9-cm code b has targets x and y in fbm . since a and b share the same target x , b is identified as a secondary code of a. add the targets of the secondary codes in fbm to the list of targets for the primary source code . in figure 1 , x and y are added as targets for source code a. tertiary map ( tm ) : uses all maps in sm , plus additional target codes identified by tertiary icd-9-cm codes . the following are the steps to generate tm : based on fbm , identify tertiary icd-9-cm codes , which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code ( identified in the generation of sm ) . in figure 1 , b has been identified as a secondary code to primary source code a. in fbm , b has targets x and y , while c has targets y and z. since b and c share the same target y , c is identified as a tertiary code of a.add the targets of the tertiary codes in fbm to the list of targets for the primary source code . in figure 1 , y and z are added as targets for source code a. based on fbm , identify tertiary icd-9-cm codes , which are defined as icd-9-cm codes that share the same icd-10-cm code as the secondary icd-9-cm code ( identified in the generation of sm ) . in figure 1 , b has been identified as a secondary code to primary source code a. in fbm , b has targets x and y , while c has targets y and z. since b and c share the same target y , c is identified as a tertiary code of a. add the targets of the tertiary codes in fbm to the list of targets for the primary source code . in figure 1 , y and z are added as targets for source code a. we chose these four methods for a number of reasons . the sfm and fbm are the most common ways to use the gems , and will provide a baseline measure of mapping performance . sm corresponds to the method used in the online transition tool provided by boyd s group.26 given their experience and commentary , we hypothesized that additional iterations will increase the number of icd-10-cm target codes and may enhance mapping performance . to evaluate the performance of the four mapping methods , we used a convenience sample of 32 phenotypes ( developed to identify research cohorts , characterize risk factors , or define outcomes ) from three different pragmatic trials collaborative care for chronic pain in primary care ( ppact ) , strategies and opportunities to stop colorectal cancer in priority populations ( stop crc ) , and a pragmatic trial of population - based programs to prevent suicide attempt that were defined by icd-9-cm codes . the icd-9-cm codes were translated to icd-10-cm codes using the four mapping methods based on the 2014 version of the gems . one generalist nurse practitioner ( kp ) and an md domain expert for each trial ( bg , ap , and mc ) reviewed the phenotype name and the icd-10-cm code sets generated by the maps to determine if each icd-10-cm code semantically fit into the named phenotype condition , based on their understanding of that phenotype and its intent . for example , for the phenotype active alcohol abuse the reviewer was asked to look at the icd-10-cm codes and determine ( yes or no ) if those codes were appropriate for inclusion in that heading . reviewers were provided the original phenotype definition ( i.e. , the set of icd-9-cm codes that constitute the specified condition ) as a reference on the same review sheet . to limit the scope and time for the evaluation , the reviewers were asked to review only the icd-10-cm codes generated by the different mapping methods . they were not asked to search for additional icd-10-cm codes that should have been included . to shorten the list of icd-10-cm codes for review codes to their parents , as long as the total number of codes in the list was reduced . m47.10 , m47.11 , m47.12 , m47.13 , m47.15 , m47.16 , which were all children of m47.1 , we converted it into m47.1 exclusion : m47.14 because m47.14 was the only child of m47.1 not included in the list . we did this iteratively until no further reduction in the number of codes was possible . to evaluate the roll up algorithm , one cohort definition from each demonstration project with at least 10 icd-10-cm codes was manually reviewed to make sure that the final list of codes represented the meaning of the original codes . to obtain the quality measurement value sets , we used the value set authority center ( vsac ) launched by the united states national library of medicine ( nlm ) in 2012 to provide access to all official versions of value sets.27,28 in the vsac , we identified all value sets for 2014 clinical quality measures that were dually defined with both icd-9-cm and icd-10-cm code lists . we applied the four mapping methods to the icd-9-cm code lists , and evaluated the resulting icd-10-cm target codes against the icd-10-cm codes listed for that measure , using the latter as the gold standard . since the value sets differed considerably in their sizes , we also analyzed the effect of value set size on the mapping performance . to evaluate the performance of each mapping method , we calculated the recall , precision and f - score of each method for every phenotype definition and quality measure value set . note that for the phenotype definitions , we did not measure the true recall because the reviewers were not asked to look for missing icd-10-cm codes . to give an estimate of recall for the phenotype definitions , we assumed that the most aggressive method ( tm ) contained all the correct icd-10-cm codes . we used the f - score ( the harmonic mean between recall and precision ) as an overall indicator of performance of each mapping method . based on the distribution of the f - scores , we used the anova test to check the statistical significance of the difference between the four methods . the selected pragmatic trials used 32 cohort definitions with 3161 ( median 4 ) icd-9-cm codes per definition ( table 2 ) . there were altogether 536 unique icd-9-cm codes , all of which could be mapped by the four different methods . the size of the resulting icd-10-cm code sets progressively increased as more aggressive mapping methods were used . overall for sfm , the median size of the icd-10-cm code sets was comparable to their icd-9-cm counterparts . there was a sharp increase from sfm to fbm , and also from fbm to sm . our roll up algorithm reduced the review workload to around 2,000 codes . to ensure that the shortened list of codes represented the same meaning as the original codes , we reviewed three cohort definitions ( pelvic and abdominal pain , alcohol abuse , and colon cancer ) with 14 , 40 , and 80 leaf level icd-10-cm codes respectively . our roll up algorithm collapsed the lists to 5 codes ( 1 higher level code , 4 leaf codes ) , 11 codes ( 4 high level codes , 2 leaf codes , 5 exclusion codes ) and 20 codes ( 11 high level codes , 9 leaf codes ) respectively . by comparing the meaning of the original codes to the shortened lists , we confirmed that the shortened lists were semantically the same as the original lists . the recall , precision , and f score values are the means for the code sets in a demonstration project . fbm was better than sfm in all three metrics ( precision , recall , and f score ) . as expected , the more aggressive methods sm and tm resulted in higher recall at the expense of precision . using the overall mean f score as a single indicator of performance , fbm was the best ( f=0.67 ) , but was close to sm ( f=0.62 ) and tm ( f=0.60 ) . based on the distribution of individual f scores in each method , the overall difference between the four methods was statistically significant ( one - way anova , f=5.749 , p=0.001 ) . pairwise comparison between adjacent pairs of methods by paired samples t - test showed that the difference between sfm and fbm was statistically significant ( t=6.184 , p<0.0001 ) , while the differences for fbm versus sm and sm versus tm were not . a total of 202 quality measure value sets defined by both icd-9-cm and icd-10-cm code sets were retrieved from the vsac . there were altogether 5,545 unique icd-9-cm codes , of which 2 codes could not be mapped by our selected methods because they were not included in either the forward or backward gem . the performance of the mapping methods in relation to the size of the icd-9-cm code sets is summarized in table 4 . the recall , precision , and f - score values shown are the means for the value sets within a particular size range . based on the overall f - score , the overall best performing mapping method was fbm , followed by sm , tm , and sfm . based on the distribution of f - scores for each value set , the difference in the performance of the four methods was statistically significant ( one - way anova , f=40.889 , p<0.0005 ) . pairwise comparisons between adjacent methods ( sfm versus fbm , fbm versus sm and sm versus tm ) by paired samples t - test were all statistically significant ( all with p<0.0001 ) . the number of icd-9-cm codes in the value sets varied considerably from 1 to 1,212 ( mean 58.6 , median 6 ) . smaller value sets generally had better recall , precision , and f - scores , regardless of mapping method . for fbm , value sets with 20 or fewer codes had almost perfect recall ( 0.97 ) and precision ( 0.93 ) . the selected pragmatic trials used 32 cohort definitions with 3161 ( median 4 ) icd-9-cm codes per definition ( table 2 ) . there were altogether 536 unique icd-9-cm codes , all of which could be mapped by the four different methods . the size of the resulting icd-10-cm code sets progressively increased as more aggressive mapping methods were used . overall for sfm , the median size of the icd-10-cm code sets was comparable to their icd-9-cm counterparts . there was a sharp increase from sfm to fbm , and also from fbm to sm . our roll up algorithm reduced the review workload to around 2,000 codes . to ensure that the shortened list of codes represented the same meaning as the original codes , we reviewed three cohort definitions ( pelvic and abdominal pain , alcohol abuse , and colon cancer ) with 14 , 40 , and 80 leaf level icd-10-cm codes respectively . our roll up algorithm collapsed the lists to 5 codes ( 1 higher level code , 4 leaf codes ) , 11 codes ( 4 high level codes , 2 leaf codes , 5 exclusion codes ) and 20 codes ( 11 high level codes , 9 leaf codes ) respectively . by comparing the meaning of the original codes to the shortened lists , we confirmed that the shortened lists were semantically the same as the original lists . the recall , precision , and f score values are the means for the code sets in a demonstration project . fbm was better than sfm in all three metrics ( precision , recall , and f score ) . as expected , the more aggressive methods sm and tm resulted in higher recall at the expense of precision . using the overall mean f score as a single indicator of performance , fbm was the best ( f=0.67 ) , but was close to sm ( f=0.62 ) and tm ( f=0.60 ) . based on the distribution of individual f scores in each method , the overall difference between the four methods was statistically significant ( one - way anova , f=5.749 , p=0.001 ) . pairwise comparison between adjacent pairs of methods by paired samples t - test showed that the difference between sfm and fbm was statistically significant ( t=6.184 , p<0.0001 ) , while the differences for fbm versus sm and sm versus tm were not . a total of 202 quality measure value sets defined by both icd-9-cm and icd-10-cm code sets were retrieved from the vsac . there were altogether 5,545 unique icd-9-cm codes , of which 2 codes could not be mapped by our selected methods because they were not included in either the forward or backward gem . the performance of the mapping methods in relation to the size of the icd-9-cm code sets is summarized in table 4 . the recall , precision , and f - score values shown are the means for the value sets within a particular size range . based on the overall f - score , the overall best performing mapping method was fbm , followed by sm , tm , and sfm . based on the distribution of f - scores for each value set , the difference in the performance of the four methods was statistically significant ( one - way anova , f=40.889 , p<0.0005 ) . pairwise comparisons between adjacent methods ( sfm versus fbm , fbm versus sm and sm versus tm ) by paired samples t - test were all statistically significant ( all with p<0.0001 ) . the number of icd-9-cm codes in the value sets varied considerably from 1 to 1,212 ( mean 58.6 , median 6 ) . smaller value sets generally had better recall , precision , and f - scores , regardless of mapping method . for fbm , value sets with 20 or fewer codes had almost perfect recall ( 0.97 ) and precision ( 0.93 ) . after several false starts and delays , the transition from icd-9-cm to icd-10-cm finally happened in 2015 . health care providers have adopted the new coding system to ensure continued revenue ; researchers and other secondary users of health care data must be prepared to adapt to this change . after october 1 , 2015 , phenotype definitions that use icd-9-cm codes to identify cohorts of patients have to shift to icd-10-cm codes if they are applied to newly - collected data . these icd-9-cm - based phenotype definitions can include hundreds of codes . translating them into icd-10-cm entails significant effort , and automated methods to support these translations reduce this burden . the use of the gems is not straightforward because it includes two independent maps in both directions . different methods for using the gems will result in different outcomes , and our findings can inform optimal approaches to using the maps for automated translation . in this study , we compare four progressively aggressive methods of using the gems to translate icd-9-cm codes used in phenotype definitions to icd-10-cm codes : ( 1 ) simple forward map ( sfm ) ; ( 2 ) forward backward map ( fbm ) ; ( 3 ) secondary map ( sm ) ; and ( 4 ) tertiary map ( tm ) . the papers and online tool from boyd et al . seem to favor an approach similar to sm , but they did not explain why , nor did they compare the various mapping methods quantitatively.21,25,29 in our study , the different methods are compared quantitatively , and their strengths and weakness are highlighted . the poor results from the simple forward map should caution novice users of the gems , who may believe that using the forward map alone will be sufficient to translate icd-9-cm codes to icd-10-cm . since the majority of icd-10-cm codes ( 75 percent ) are not reachable by the forward map , it is not surprising that the performance of sfm is the worst . the two gems together include 13,478 ( 93 percent ) of icd-9-cm codes and 69,154 ( 99 percent ) of icd-10-cm codes . this is an absolute limitation for any mapping method relying on the gems alone , which means that there is a small percentage of icd-9-cm ( 7 percent ) and icd-10-cm ( 1 percent ) codes that will not be covered . boyd et al . demonstrated that the majority of the icd-9-cm to icd-10-cm translations are complex , convoluted , and nonreciprocal.29 this is why one needs to apply the forward and backward maps iteratively to obtain more complete results . in our study , sm ( the first iteration ) identified several times more icd-10-cm codes than did fbm . a common source of error related to composite concepts involving more than one medical condition . for example , starting from the icd-9-cm code 716.80 other specified arthropathy , site unspecified the fbm found e08.618 diabetes mellitus due to underlying condition with other diabetic arthropathy , which was a correct target . however , in the sm , e08.618 led to the identification of the secondary icd-9-cm code 249.80 secondary diabetes mellitus with other specified manifestations , not stated as uncontrolled , or unspecified . this secondary icd-9-cm code led to additional icd-10-cm targets , such as e10.621 type 1 diabetes mellitus with foot ulcer , which were completely unrelated to the primary icd-9-cm source code . such examples highlight the need for thoughtfulness and manual review of mappings generated by aggressive iterative mapping methods . based on the f - scores , the fbm was the best performing among all methods ( the complete fbm list is available as appendix a ) . however , the sm was a close second . for the clinical phenotype use case , sm had a better recall ( 0.89 ) over fbm ( 0.68 ) , but precision dropped considerably ( from 0.76 to 0.56 ) . the median number of icd-10-cm target codes increased six times from fbm to sm , and only one - third of the additional icd-10-cm codes identified were correct . in practice , the optimal method will depend upon the specific use case , particularly whether higher recall is considered more important than precision or vice versa . in our limited sample of medical conditions , it appears that for clinically distinct and homogenous conditions , such as colorectal cancer , the recall of fbm is already very good , and there is no need to go to more aggressive methods . conditions with heterogeneous pathology involving multiple organ systems , e.g. , chronic pain , might require more aggressive mapping methods . in general , the performance of all mapping methods dropped considerably with diverse and heterogeneous conditions . in such cases , manual review of the map - generated codes and search for missing codes will be essential . users of the gems should be aware that only billable codes ( leaf codes ) are included in the gems . if their code lists include nonbillable ( high level codes ) , such as some code lists used in quality metrics , they should expand the high level codes to the leaf codes before applying the maps to ensure a more complete translation . the performance of gem - based mapping also depends on the extent of the changes between icd-9-cm and icd-10-cm . some chapters such as mental and behavioral disorders and diseases of the skin and subcutaneous tissues have undergone major reorganization . the definitions in the suicide prevention project mostly fall within mental health disorders . as a result , all mapping methods performed more poorly for this group . , this condition has four codes depending on whether it is continuous , episodic , in remission , or unspecified . icd-10-cm does not distinguish between the time course ( which explains why the sfm maps have less icd-10-cm codes than icd-9-cm codes ) , but has added a new axis of classification about the effects of the abuse ( e.g. , sleep disorder , sexual dysfunction ) . in the chapters that have changed significantly , more intense scrutiny of gem - based translations is warranted , and users may need to look outside the gems for codes that are not reachable through the gems . regardless of the mapping method , our results suggest that automatic translation is not perfect and validation by human review is recommended . however , it is likely that automated translation will save time by reducing the scope of review . the burden of manual review is a real concern , especially in codes sets with hundreds of codes . very often , all descendants of a subbranch are included in a phenotype definition , so it saves significant time for reviewers if codes are rolled up to their parents . with our roll up algorithm , we managed to reduce the number of codes requiring expert review by 72 percent . while clinical quality measurement and pragmatic clinical trials are distinct activities , they both rely on code sets to identify their relevant subpopulations of patients , and there is clear overlap in the function between the phenotype definition code sets and quality measurement value sets . for example , there is a phenotype code set for colon cancer in the nih collaboratory , and a quality measure value set for malignant neoplasm of colon , and both have exactly the same icd-9-cm codes . because of this , we have included the quality measure value sets as an additional evaluation of the mapping methods . for the quality measure value sets , the performance of the four mapping methods followed essentially the same trend as in phenotype code sets . based on the overall f - scores , fbm performed best , followed by sm , tm , and sfm . the more aggressive methods ( sm and tm ) resulted in only marginal increase in recall with considerable drop in precision . therefore , if there is a need to use the gems to translate icd-9-cm code sets for clinical quality measurement , it would seem appropriate to use the fbm mapping method . note that mapping performance is generally better with smaller value sets . one possible explanation is that smaller value sets may involve more distinct and homogenous conditions ( e.g. , malignant neoplasm of colon , 13 codes ) , and that larger value sets tend to be more heterogeneous ( e.g. , immunocompromised conditions , 149 codes ) . as we found in cohort definitions , generally , for small value set ( fewer than 20 codes ) involving homogeneous conditions that are not in the chapters known to have undergone major reorganization in icd-10-cm ( e.g. , mental disorders ) , the fbm mappings are expected to perform very well , and only minimal manual review will be required . the existence of code sets used for phenotype definition and quality measurement raises the interesting possibility of cross - fertilization . it is conceivable that , in some cases , the same set of codes can serve both functions , as in the colon cancer example above . indeed , the icd-10-cm codes in the colon cancer value set are all considered appropriate for phenotype definition by the reviewers . so instead of defining new icd-10-cm code sets from scratch , the researchers may be able to find quality value sets defined with icd-10-cm codes that they can reuse . however , to do that one has to search through the thousands of value sets in vsac . to narrow the search , one can use some similarity measure ( e.g. , jaccard coefficient ) between the icd-9-cm phenotype code sets and icd-9-cm value sets in vsac.30 in the future , this kind of cross - fertilization between various secondary uses of clinical codes will become more important and perhaps encourage health care organizations to participate in pragmatic trials and nationally coordinated biomedical and health services research , such as hmorn and the patient centered outcomes research network ( pcornet ) . the phenotype knowledge base ( phekb)31 and other repositories of phenotypes should consider partnerships with vsac and investigate formal linkages between research phenotypes and quality measurement value sets . some of the phenotype definitions used in this study are posted on phekb . the use of common value sets for clinical research and quality measurement can enable the generation of evidence from health care organizations and facilitate the vision of learning health care.32,33 for future work , we can explore ways to improve the performance of the mapping methods . there is additional information in the gems , such as flags for approximate or exact maps , and indicators of combination codes , which can be exploited to refine the mapping algorithms . boyd et al . showed that the mapping relationships for codes from different icd-9-cm chapters varied considerably.28 this is because the difference between icd-9-cm and icd-10-cm is not uniform across all medical specialties . chapters that do not change radically may require a less aggressive mapping approach . outside the use of the gems , two additional mapping resources first , the international health terminology standards development organisation ( ihtsdo ) publishes a map from systematized nomenclature of medicine clinical terms ( snomed ct ) to icd-9-cm , and the nlm publishes a map from snomed ct to icd-10-cm . therefore , it is possible to map from icd-9-cm to icd-10-cm using snomed ct as an intermediary . second , the unified medical language system ( umls ) has been found to be useful in interterminology mapping.34,35 mapping relations between icd-9-cm and icd-10-cm can be discovered by exploring the synonymy and other relationships within the umls . these relationships can then be used to corroborate or supplement the maps derived from the gems . in the future snomed ct is a better clinical terminology than icd because of its coverage , granularity , clinical orientation , and logical underpinning.36 many quality value sets are already defined in snomed ct codes . although it is true that icd codes are more commonly found in ehrs at present , snomed ct codes will become more ubiquitous with the meaningful use initiative . the collaboratory demonstration projects we used were a convenience sample and are not representative of all pragmatic trials . the phenotype definitions in this study were developed to support a number of purposes for very specific research studies and might not be generalizable or appropriate for other research or quality measurement use cases related to those conditions . although we did use two reviewers for each mapping relationship , the reviews by clinical experts have not been independently corroborated . for example , medication or laboratory values can be used to identify more records for potentially complete recall sets . a future related activity would be to examine a known subset of patients with chronic diseases before and after the icd-10-cm transition , and to contrast the assigned icd-10-cm codes with historical icd-9-cm codes . obviously , any practical migration of phenotyping algorithms from icd-9-cm to icd-10-cm will ultimately require the use and synthesis of other data types for validation and human review , including a more rigorously defined characterization of a gold standard diagnosis . even with rigorous gems mapping approaches , real - world application would require some level of human review to consider phenotype definitions fully validated . it is important to mention that although we focused our investigation on the translation of icd - based phenotype definitions , most phenotyping methods include other data , such as laboratory test results , medications , demographics , and natural language processing , in addition to diagnostic code value sets . while most researchers recognize that icd code sets by themselves are not sufficient for research phenotyping , these codes are widely used and remain an important component of virtually all of the phenotype definitions posted on phekb . given the national impact of implementation to icd-10-cm in october 2015 , specific scrutiny of public gems tools is warranted to clarify the research implications of the icd-9-cm to icd-10-cm transition . after several false starts and delays , the transition from icd-9-cm to icd-10-cm finally happened in 2015 . health care providers have adopted the new coding system to ensure continued revenue ; researchers and other secondary users of health care data must be prepared to adapt to this change . after october 1 , 2015 , phenotype definitions that use icd-9-cm codes to identify cohorts of patients have to shift to icd-10-cm codes if they are applied to newly - collected data . these icd-9-cm - based phenotype definitions can include hundreds of codes . translating them into icd-10-cm entails significant effort , and automated methods to support these translations reduce this burden . the use of the gems is not straightforward because it includes two independent maps in both directions . different methods for using the gems will result in different outcomes , and our findings can inform optimal approaches to using the maps for automated translation . in this study , we compare four progressively aggressive methods of using the gems to translate icd-9-cm codes used in phenotype definitions to icd-10-cm codes : ( 1 ) simple forward map ( sfm ) ; ( 2 ) forward backward map ( fbm ) ; ( 3 ) secondary map ( sm ) ; and ( 4 ) tertiary map ( tm ) . the papers and online tool from boyd et al . seem to favor an approach similar to sm , but they did not explain why , nor did they compare the various mapping methods quantitatively.21,25,29 in our study , the different methods are compared quantitatively , and their strengths and weakness are highlighted . the poor results from the simple forward map should caution novice users of the gems , who may believe that using the forward map alone will be sufficient to translate icd-9-cm codes to icd-10-cm . since the majority of icd-10-cm codes ( 75 percent ) are not reachable by the forward map , it is not surprising that the performance of sfm is the worst . the two gems together include 13,478 ( 93 percent ) of icd-9-cm codes and 69,154 ( 99 percent ) of icd-10-cm codes . this is an absolute limitation for any mapping method relying on the gems alone , which means that there is a small percentage of icd-9-cm ( 7 percent ) and icd-10-cm ( 1 percent ) codes that will not be covered . boyd et al . demonstrated that the majority of the icd-9-cm to icd-10-cm translations are complex , convoluted , and nonreciprocal.29 this is why one needs to apply the forward and backward maps iteratively to obtain more complete results . in our study , sm ( the first iteration ) identified several times more icd-10-cm codes than did fbm . a common source of error related to composite concepts involving more than one medical condition . for example , starting from the icd-9-cm code 716.80 other specified arthropathy , site unspecified the fbm found e08.618 diabetes mellitus due to underlying condition with other diabetic arthropathy , which was a correct target . however , in the sm , e08.618 led to the identification of the secondary icd-9-cm code 249.80 secondary diabetes mellitus with other specified manifestations , not stated as uncontrolled , or unspecified . this secondary icd-9-cm code led to additional icd-10-cm targets , such as e10.621 type 1 diabetes mellitus with foot ulcer , which were completely unrelated to the primary icd-9-cm source code . such examples highlight the need for thoughtfulness and manual review of mappings generated by aggressive iterative mapping methods . based on the f - scores , the fbm was the best performing among all methods ( the complete fbm list is available as appendix a ) . however , the sm was a close second . for the clinical phenotype use case , sm had a better recall ( 0.89 ) over fbm ( 0.68 ) , but precision dropped considerably ( from 0.76 to 0.56 ) . the median number of icd-10-cm target codes increased six times from fbm to sm , and only one - third of the additional icd-10-cm codes identified were correct . in practice , the optimal method will depend upon the specific use case , particularly whether higher recall is considered more important than precision or vice versa . in our limited sample of medical conditions , it appears that for clinically distinct and homogenous conditions , such as colorectal cancer , the recall of fbm is already very good , and there is no need to go to more aggressive methods . conditions with heterogeneous pathology involving multiple organ systems , e.g. , chronic pain , might require more aggressive mapping methods . in general , the performance of all mapping methods dropped considerably with diverse and heterogeneous conditions . in such cases , manual review of the map - generated codes and search for missing codes will be essential . users of the gems should be aware that only billable codes ( leaf codes ) are included in the gems . if their code lists include nonbillable ( high level codes ) , such as some code lists used in quality metrics , they should expand the high level codes to the leaf codes before applying the maps to ensure a more complete translation . the performance of gem - based mapping also depends on the extent of the changes between icd-9-cm and icd-10-cm . some chapters such as mental and behavioral disorders and diseases of the skin and subcutaneous tissues have undergone major reorganization . the definitions in the suicide prevention project mostly fall within mental health disorders . as a result , all mapping methods performed more poorly for this group . , this condition has four codes depending on whether it is continuous , episodic , in remission , or unspecified . icd-10-cm does not distinguish between the time course ( which explains why the sfm maps have less icd-10-cm codes than icd-9-cm codes ) , but has added a new axis of classification about the effects of the abuse ( e.g. , sleep disorder , sexual dysfunction ) . in the chapters that have changed significantly , more intense scrutiny of gem - based translations is warranted , and users may need to look outside the gems for codes that are not reachable through the gems . regardless of the mapping method , our results suggest that automatic translation is not perfect and validation by human review is recommended . however , it is likely that automated translation will save time by reducing the scope of review . the burden of manual review is a real concern , especially in codes sets with hundreds of codes . very often , all descendants of a subbranch are included in a phenotype definition , so it saves significant time for reviewers if codes are rolled up to their parents . with our roll up algorithm , we managed to reduce the number of codes requiring expert review by 72 percent . while clinical quality measurement and pragmatic clinical trials are distinct activities , they both rely on code sets to identify their relevant subpopulations of patients , and there is clear overlap in the function between the phenotype definition code sets and quality measurement value sets . for example , there is a phenotype code set for colon cancer in the nih collaboratory , and a quality measure value set for malignant neoplasm of colon , and both have exactly the same icd-9-cm codes . because of this , we have included the quality measure value sets as an additional evaluation of the mapping methods . for the quality measure value sets , the performance of the four mapping methods followed essentially the same trend as in phenotype code sets . based on the overall f - scores , fbm performed best , followed by sm , tm , and sfm . the more aggressive methods ( sm and tm ) resulted in only marginal increase in recall with considerable drop in precision . therefore , if there is a need to use the gems to translate icd-9-cm code sets for clinical quality measurement , it would seem appropriate to use the fbm mapping method . note that mapping performance is generally better with smaller value sets . one possible explanation is that smaller value sets may involve more distinct and homogenous conditions ( e.g. , malignant neoplasm of colon , 13 codes ) , and that larger value sets tend to be more heterogeneous ( e.g. , immunocompromised conditions , 149 codes ) . as we found in cohort definitions , generally , for small value set ( fewer than 20 codes ) involving homogeneous conditions that are not in the chapters known to have undergone major reorganization in icd-10-cm ( e.g. , mental disorders ) , the fbm mappings are expected to perform very well , and only minimal manual review will be required . the existence of code sets used for phenotype definition and quality measurement raises the interesting possibility of cross - fertilization . it is conceivable that , in some cases , the same set of codes can serve both functions , as in the colon cancer example above . indeed , the icd-10-cm codes in the colon cancer value set are all considered appropriate for phenotype definition by the reviewers . so instead of defining new icd-10-cm code sets from scratch , the researchers may be able to find quality value sets defined with icd-10-cm codes that they can reuse . however , to do that one has to search through the thousands of value sets in vsac . to narrow the search , one can use some similarity measure ( e.g. , jaccard coefficient ) between the icd-9-cm phenotype code sets and icd-9-cm value sets in vsac.30 in the future , this kind of cross - fertilization between various secondary uses of clinical codes will become more important and perhaps encourage health care organizations to participate in pragmatic trials and nationally coordinated biomedical and health services research , such as hmorn and the patient centered outcomes research network ( pcornet ) . the phenotype knowledge base ( phekb)31 and other repositories of phenotypes should consider partnerships with vsac and investigate formal linkages between research phenotypes and quality measurement value sets . the use of common value sets for clinical research and quality measurement can enable the generation of evidence from health care organizations and facilitate the vision of learning health care.32,33 for future work , we can explore ways to improve the performance of the mapping methods . there is additional information in the gems , such as flags for approximate or exact maps , and indicators of combination codes , which can be exploited to refine the mapping algorithms . boyd et al . showed that the mapping relationships for codes from different icd-9-cm chapters varied considerably.28 this is because the difference between icd-9-cm and icd-10-cm is not uniform across all medical specialties . outside the use of the gems , two additional mapping resources may be worthy of consideration . first , the international health terminology standards development organisation ( ihtsdo ) publishes a map from systematized nomenclature of medicine clinical terms ( snomed ct ) to icd-9-cm , and the nlm publishes a map from snomed ct to icd-10-cm . therefore , it is possible to map from icd-9-cm to icd-10-cm using snomed ct as an intermediary . second , the unified medical language system ( umls ) has been found to be useful in interterminology mapping.34,35 mapping relations between icd-9-cm and icd-10-cm can be discovered by exploring the synonymy and other relationships within the umls . these relationships can then be used to corroborate or supplement the maps derived from the gems . in the future snomed ct is a better clinical terminology than icd because of its coverage , granularity , clinical orientation , and logical underpinning.36 many quality value sets are already defined in snomed ct codes . although it is true that icd codes are more commonly found in ehrs at present , snomed ct codes will become more ubiquitous with the meaningful use initiative . the collaboratory demonstration projects we used were a convenience sample and are not representative of all pragmatic trials . the phenotype definitions in this study were developed to support a number of purposes for very specific research studies and might not be generalizable or appropriate for other research or quality measurement use cases related to those conditions . although we did use two reviewers for each mapping relationship , the reviews by clinical experts have not been independently corroborated . in the future for example , medication or laboratory values can be used to identify more records for potentially complete recall sets . a future related activity would be to examine a known subset of patients with chronic diseases before and after the icd-10-cm transition , and to contrast the assigned icd-10-cm codes with historical icd-9-cm codes . obviously , any practical migration of phenotyping algorithms from icd-9-cm to icd-10-cm will ultimately require the use and synthesis of other data types for validation and human review , including a more rigorously defined characterization of a gold standard diagnosis . even with rigorous gems mapping approaches , real - world application would require some level of human review to consider phenotype definitions fully validated . it is important to mention that although we focused our investigation on the translation of icd - based phenotype definitions , most phenotyping methods include other data , such as laboratory test results , medications , demographics , and natural language processing , in addition to diagnostic code value sets . while most researchers recognize that icd code sets by themselves are not sufficient for research phenotyping , these codes are widely used and remain an important component of virtually all of the phenotype definitions posted on phekb . given the national impact of implementation to icd-10-cm in october 2015 , specific scrutiny of public gems tools is warranted to clarify the research implications of the icd-9-cm to icd-10-cm transition . the transition from icd-9-cm to icd-10-cm creates a heavy burden of code translation for clinical researchers using icd codes in identifying patient cohorts based on clinical criteria . although national reference mappings and tools exist to support icd-9-cm to icd-10-cm conversion , their use is not straightforward . different approaches yield different sets of icd-10-cm codes , and users should be aware of the pros and cons of each approach . in most cases , automatic code translation is not accurate enough on its own , and should be used as an auxiliary tool to assist human reviewers . variation in the migration of phenotype definitions can have an impact on the consistency of definition of cohorts and data collection over time , and can potentially have an impact on study findings if not addressed .

background : the national mandate for health systems to transition from icd-9-cm to icd-10-cm in october 2015 has an impact on research activities . clinical phenotypes defined by icd-9-cm codes need to be converted to icd-10-cm , which has nearly four times more codes and a very different structure than icd-9-cm.methods:we used the centers for medicare & medicaid services ( cms ) general equivalent maps ( gems ) to translate , using four different methods , condition - specific icd-9-cm code sets used for pragmatic trials ( n=32 ) into icd-10-cm . we calculated the recall , precision , and f score of each method . we also used the icd-9-cm and icd-10-cm value sets defined for electronic quality measure as an additional evaluation of the mapping methods.results:the forward - backward mapping ( fbm ) method had higher precision , recall and f - score metrics than simple forward mapping ( sfm ) . the more aggressive secondary ( sm ) and tertiary mapping ( tm ) methods resulted in higher recall but lower precision . for clinical phenotype definition , fbm was the best ( f=0.67 ) , but was close to sm ( f=0.62 ) and tm ( f=0.60 ) , judging on the f - scores alone . the overall difference between the four methods was statistically significant ( one - way anova , f=5.749 , p=0.001 ) . however , pairwise comparisons between fbm , sm , and tm did not reach statistical significance . a similar trend was found for the quality measure value sets.discussion:the optimal method for using the gems depends on the relative importance of recall versus precision for a given use case . it appears that for clinically distinct and homogenous conditions , the recall of fbm is sufficient . the performance of all mapping methods was lower for heterogeneous conditions . since code sets used for phenotype definition and quality measurement can be very similar , there is a possibility of cross - fertilization between the two activities.conclusion:different mapping approaches yield different collections of icd-10-cm codes . all methods require some level of human validation .

Introduction Pragmatic Clinical Trials and International Classification of Diseases (ICD) Codes Automatic Code Translation by the General Equivalent Maps (GEMs) Methods Generation of the Target ICD-10-CM Codes Evaluation of the Target ICD-10-CM Codes Results Phenotype Definitions Quality Measure Value Sets Discussion Use of Automatic Code Translation in Phenotype Definitions Synergism Between Quality Measurement and Cohort Definition Future Research Conclusion

the national mandate for health systems to migrate to icd-10-cm in october 2015 has an impact on all research activities that rely on these codes . although the centers for medicare & medicaid services ( cms ) has produced general equivalent maps ( gems ) , their use is not straightforward , and different methods for using the gems can result in different outcomes . the general equivalent maps ( gems ) are created and maintained by the centers for medicare & medicaid services ( cms ) and the centers for disease control and prevention ( cdc ) , and serve as a tool for the conversion of data between icd-9-cm and icd-10-cm.15 the gems are often also referred to as crosswalks since they provide important information linking codes from one system with codes in the other system.16 users are cautioned against using the gems for actual coding as they have not been completely validated for clinical use . the general equivalent maps ( gems ) are created and maintained by the centers for medicare & medicaid services ( cms ) and the centers for disease control and prevention ( cdc ) , and serve as a tool for the conversion of data between icd-9-cm and icd-10-cm.15 the gems are often also referred to as crosswalks since they provide important information linking codes from one system with codes in the other system.16 users are cautioned against using the gems for actual coding as they have not been completely validated for clinical use . to evaluate the performance of the four mapping methods , we used a convenience sample of 32 phenotypes ( developed to identify research cohorts , characterize risk factors , or define outcomes ) from three different pragmatic trials collaborative care for chronic pain in primary care ( ppact ) , strategies and opportunities to stop colorectal cancer in priority populations ( stop crc ) , and a pragmatic trial of population - based programs to prevent suicide attempt that were defined by icd-9-cm codes . to evaluate the performance of each mapping method , we calculated the recall , precision and f - score of each method for every phenotype definition and quality measure value set . to evaluate the performance of the four mapping methods , we used a convenience sample of 32 phenotypes ( developed to identify research cohorts , characterize risk factors , or define outcomes ) from three different pragmatic trials collaborative care for chronic pain in primary care ( ppact ) , strategies and opportunities to stop colorectal cancer in priority populations ( stop crc ) , and a pragmatic trial of population - based programs to prevent suicide attempt that were defined by icd-9-cm codes . to evaluate the performance of each mapping method , we calculated the recall , precision and f - score of each method for every phenotype definition and quality measure value set . using the overall mean f score as a single indicator of performance , fbm was the best ( f=0.67 ) , but was close to sm ( f=0.62 ) and tm ( f=0.60 ) . based on the distribution of individual f scores in each method , the overall difference between the four methods was statistically significant ( one - way anova , f=5.749 , p=0.001 ) . based on the distribution of f - scores for each value set , the difference in the performance of the four methods was statistically significant ( one - way anova , f=40.889 , p<0.0005 ) . using the overall mean f score as a single indicator of performance , fbm was the best ( f=0.67 ) , but was close to sm ( f=0.62 ) and tm ( f=0.60 ) . based on the distribution of individual f scores in each method , the overall difference between the four methods was statistically significant ( one - way anova , f=5.749 , p=0.001 ) . based on the distribution of f - scores for each value set , the difference in the performance of the four methods was statistically significant ( one - way anova , f=40.889 , p<0.0005 ) . in this study , we compare four progressively aggressive methods of using the gems to translate icd-9-cm codes used in phenotype definitions to icd-10-cm codes : ( 1 ) simple forward map ( sfm ) ; ( 2 ) forward backward map ( fbm ) ; ( 3 ) secondary map ( sm ) ; and ( 4 ) tertiary map ( tm ) . in our limited sample of medical conditions , it appears that for clinically distinct and homogenous conditions , such as colorectal cancer , the recall of fbm is already very good , and there is no need to go to more aggressive methods . in this study , we compare four progressively aggressive methods of using the gems to translate icd-9-cm codes used in phenotype definitions to icd-10-cm codes : ( 1 ) simple forward map ( sfm ) ; ( 2 ) forward backward map ( fbm ) ; ( 3 ) secondary map ( sm ) ; and ( 4 ) tertiary map ( tm ) . in our limited sample of medical conditions , it appears that for clinically distinct and homogenous conditions , such as colorectal cancer , the recall of fbm is already very good , and there is no need to go to more aggressive methods .

asthma is a major health problem that affects 26 million individuals in the usa.1 respiratory - related emergency department ( ed ) visits along with hospitalizations due to exacerbations impose significant health care resource utilization ( hru ) burden among patients with asthma.2,3 the economic burden of asthma is large and is attributable to patients with poorly controlled disease , highlighting the importance of maintaining disease control and minimizing the frequency of exacerbations.1,4 recent estimates suggest that the average cost per asthma - related hospital stay increased from $ 5,200 to $ 6,600 ( 2010 us dollars)5 and for an outpatient ( op ) ed visit averaged $ 1,502 ( 2008 us dollars).6 in addition , costs due to ed visits and hospitalization disproportionately account for a major portion of the total health care costs of asthma.47 the treatment goals for asthma are primarily driven by patient - centered outcomes such as relieving symptoms , preventing disease progression and exacerbations , and optimizing health status and quality of life.8,9 clinical practice guidelines for asthma emphasize the importance of using preventer / controllers with anti - inflammatory properties ( e.g. , inhaled corticosteroids [ icss ] ) as first - line treatment in persistent asthma.8,9 despite guidelines , many patients may use their preventer / controller inhalers intermittently and wait for asthma flare ups to seek medication prescriptions . proair hydrofluoroalkane ( hfa ) ( albuterol sulfate inhalation aerosol ; teva pharmaceuticals , inc . , frazer , pa ) is a short - acting beta agonist ( saba ) rescue / relief agent indicated for the treatment of bronchospasm with reversible obstructive airway disease and for the prevention of exercise - induced bronchospasm.10 as a rescue / relief agent , albuterol sulfate inhalation aerosol is used to improve the symptoms of asthma while the preventer / controller agents are being titrated.11,12 accurate tracking of the administered dose is , therefore , critically important for optimal asthma control and for potentially reducing the rates of unscheduled health care utilization , and thus the cost of care , in patients with asthma.1316 the advent of integrated dose counters ( idcs ) led to a logistical shift in the effective management of patients with asthma.1721 idcs may add value as they monitor rescue inhaler use ; yet , they are not a standard feature across all rescue inhalers . idcs indicate the number of actuations remaining in the canister , allowing patients to determine the number of doses available . the use of idcs may contribute to improvements in the control of respiratory disease and respiratory - related health care utilization and costs . results from a real - world study demonstrated reduced incidence of respiratory - related ed visits in patients using rescue inhalers with idc compared to those with no dose counter on their inhalers.22 however , there is paucity of data on the real - world impact of proair hfa equipped with dose counters on hru among patients with asthma . therefore , this study was conducted to evaluate health care resource use including hospitalizations , ed visits , and op visits for lower respiratory tract infection ( lrti ) among users of proair hfa with idc compared to without idc in patients with asthma . a retrospective , observational study was conducted using patient - level administrative claims data from the truven health marketscan commercial claims and encounters , and medicare supplemental and coordinated benefits databases . the commercial and medicare supplemental databases contain administrative claims data for over 35 million covered lives ( in 2013 alone ) from ~150 large employers and health plans across the usa . data included medical claims for health care services performed in both the inpatient and op settings along with enrollment data including member demographic information , eligibility , and benefits data . the medical claims files included service dates , provider reimbursement amounts , patient copayment , and deductible amounts . data are fully compliant with the health insurance portability and accountability act of 1996 , and because this study did not involve the collection , use , or transmittal of individually identifiable data , institutional review board review or approval was not required . patients between 4 and 64 years of age who received at least one new prescription for proair hfa with or without idc and had at least one nonrule - out diagnosis indicative of asthma ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] diagnosis code 493.xx except 493.20 , 493.21 , 493.22 , 493.81 ) between january 1 , 2011 through july 31 , 2014 , were included in the study . patients having continuous enrollment with medical and prescription drug benefits for 6 months pre- and post - index ( including the index date ) period were included in the analysis , and patients with a proair hfa prescription in the 6 months pre - index period were not included in the results time frame . in addition , patients having a prescription for other brands of albuterol or sabas during the pre- and post - index period were excluded . the index date was defined as the date of the first prescription fill for either proair hfa without idc between january 1 , 2011 , and december 31 , 2012 ( a 2-year period without idc ) , or for proair hfa with idc between july 1 , 2013 and july 31 , 2014 ( a 1-year period with idc ) . the period from january 1 , 2013 to june 30 , 2013 ( as idc use was implemented for proair hfa ) was not examined , because it was a time period when proair hfa was transitioning from not having an idc to having an idc . therefore , patients included in the study utilized proair hfa ( one or more prescriptions ) during the 6-month post - index period . patient demographic characteristics including age , gender , geographic location ( us census division ) , population density ( urban / rural ) , and type of health plan insurance were recorded on the index date . clinical characteristics , including the comorbid conditions ( based on the presence of icd-9-cm diagnosis and procedure codes ) and levels of utilization of oral / injectable corticosteroids and other asthma medications ( based on healthcare common procedure coding system codes and national drug codes ) , were measured during the 6-month pre - admission period . in addition , the number and duration of inpatient hospitalizations and ed visits were examined . data on hru , mainly respiratory - related hospitalizations and ed visits , were collected during the follow - up period . hospitalizations were defined as respiratory - related if there was a claim with at least one of the following icd-9-cm codes in the primary or secondary positions : 464 , 466 , 476 , 480488 , 490496 , 500508 , or 510519 . the mean number and proportion of patients with hospital stays or ed visits , and the average number and percent of patients with op visits for lrti treated with antibiotics were determined . in addition , the mean number and proportion of patients with more than one albuterol inhalation aerosol prescription during the follow - up period were recorded . descriptive statistics were used to test differences in demographic and clinical characteristics , as well as the health care utilization outcomes between users of albuterol inhalation aerosol with and without idc , stratified by age . the means and standard deviations were reported for the continuous variables , and the counts and percentages were reported for the dichotomous or categorical variables . chi - squared tests were used to evaluate the statistical significance of differences for dichotomous or categorical variables ; student s t - tests or wilcoxon rank sum tests were used for comparison of continuous variables . an a priori p<0.05 multivariate generalized linear models ( glms ) and logistic regressions were used to control for potential confounding bias due to differences in pre - index patient demographics , clinical characteristics , and concomitant medications . glms with logit link and binomial error distribution were used to obtain the adjusted odds of post - index respiratory - related hospitalizations and ed visits among patients using albuterol inhalation aerosol with and without idc . mean number of hospitalizations and ed visits was modeled using a glm with log link and a negative binomial error distribution . model covariates included age , gender , pre - index comorbid conditions , pre - index medication use ( oral / injectable corticosteroids , ics , long - acting beta agonists [ labas ] , long - acting muscarinic antagonists [ lamas ] , short - acting muscarinic antagonists [ samas ] , leukotriene receptor antagonists [ ltras ] , and other medications ) , and pre - index all - cause health care costs . all analyses were conducted using sas version 9.4 ( sas institute , inc . , cary , nc , usa ) . a retrospective , observational study was conducted using patient - level administrative claims data from the truven health marketscan commercial claims and encounters , and medicare supplemental and coordinated benefits databases . the commercial and medicare supplemental databases contain administrative claims data for over 35 million covered lives ( in 2013 alone ) from ~150 large employers and health plans across the usa . data included medical claims for health care services performed in both the inpatient and op settings along with enrollment data including member demographic information , eligibility , and benefits data . the medical claims files included service dates , provider reimbursement amounts , patient copayment , and deductible amounts . data are fully compliant with the health insurance portability and accountability act of 1996 , and because this study did not involve the collection , use , or transmittal of individually identifiable data , institutional review board review or approval was not required . patients between 4 and 64 years of age who received at least one new prescription for proair hfa with or without idc and had at least one nonrule - out diagnosis indicative of asthma ( international classification of diseases , ninth revision , clinical modification [ icd-9-cm ] diagnosis code 493.xx except 493.20 , 493.21 , 493.22 , 493.81 ) between january 1 , 2011 through july 31 , 2014 , were included in the study . patients having continuous enrollment with medical and prescription drug benefits for 6 months pre- and post - index ( including the index date ) period were included in the analysis , and patients with a proair hfa prescription in the 6 months pre - index period were not included in the results time frame . in addition , patients having a prescription for other brands of albuterol or sabas during the pre- and post - index period were excluded . the index date was defined as the date of the first prescription fill for either proair hfa without idc between january 1 , 2011 , and december 31 , 2012 ( a 2-year period without idc ) , or for proair hfa with idc between july 1 , 2013 and july 31 , 2014 ( a 1-year period with idc ) . the period from january 1 , 2013 to june 30 , 2013 ( as idc use was implemented for proair hfa ) was not examined , because it was a time period when proair hfa was transitioning from not having an idc to having an idc . therefore , patients included in the study utilized proair hfa ( one or more prescriptions ) during the 6-month post - index period . patient demographic characteristics including age , gender , geographic location ( us census division ) , population density ( urban / rural ) , and type of health plan insurance were recorded on the index date . clinical characteristics , including the comorbid conditions ( based on the presence of icd-9-cm diagnosis and procedure codes ) and levels of utilization of oral / injectable corticosteroids and other asthma medications ( based on healthcare common procedure coding system codes and national drug codes ) , were measured during the 6-month pre - admission period . in addition , the number and duration of inpatient hospitalizations and ed visits were examined . data on hru , mainly respiratory - related hospitalizations and ed visits , were collected during the follow - up period . hospitalizations were defined as respiratory - related if there was a claim with at least one of the following icd-9-cm codes in the primary or secondary positions : 464 , 466 , 476 , 480488 , 490496 , 500508 , or 510519 . the mean number and proportion of patients with hospital stays or ed visits , and the average number and percent of patients with op visits for lrti treated with antibiotics were determined . in addition , the mean number and proportion of patients with more than one albuterol inhalation aerosol prescription during the follow - up period were recorded . descriptive statistics were used to test differences in demographic and clinical characteristics , as well as the health care utilization outcomes between users of albuterol inhalation aerosol with and without idc , stratified by age . the means and standard deviations were reported for the continuous variables , and the counts and percentages were reported for the dichotomous or categorical variables . chi - squared tests were used to evaluate the statistical significance of differences for dichotomous or categorical variables ; student s t - tests or wilcoxon rank sum tests were used for comparison of continuous variables . an a priori p<0.05 multivariate generalized linear models ( glms ) and logistic regressions were used to control for potential confounding bias due to differences in pre - index patient demographics , clinical characteristics , and concomitant medications . glms with logit link and binomial error distribution were used to obtain the adjusted odds of post - index respiratory - related hospitalizations and ed visits among patients using albuterol inhalation aerosol with and without idc . mean number of hospitalizations and ed visits was modeled using a glm with log link and a negative binomial error distribution . model covariates included age , gender , pre - index comorbid conditions , pre - index medication use ( oral / injectable corticosteroids , ics , long - acting beta agonists [ labas ] , long - acting muscarinic antagonists [ lamas ] , short - acting muscarinic antagonists [ samas ] , leukotriene receptor antagonists [ ltras ] , and other medications ) , and pre - index all - cause health care costs . all analyses were conducted using sas version 9.4 ( sas institute , inc . , cary , nc , usa ) . a total of 422,548 patients with asthma were included in the analysis . of these , 135,305 ( 32% ) patients used albuterol inhalation aerosol with idc , and 287,243 ( 68% ) patients received albuterol inhalation aerosol without idc ( figure 1 ) . baseline demographic and clinical characteristics stratified by cohorts and age are presented in table 1 . the mean age ( 32.9 vs 32.5 years ) and the proportion of female patients ( 57.3% vs 57.9% ) were similar across both cohorts . females made up the majority in both cohorts with an exception of those in the 17 years subcohorts . the presence of rhinitis , gastroesophageal reflux disease ( gerd ) , and eczema was modestly higher among the cohort with idc compared to the cohort without idc ( all p < 0.05 ) . in general , baseline markers of asthma treatment were higher and all - cause hospitalizations and ed visits were lower in patients in the albuterol inhalation aerosol with idc cohort compared to those in the cohort without idc . overall , both cohorts contained an asthma population with a higher level of severity : 17.5%20.5% of patients needed oral or injectable corticosteroids , 14.7%15.4% had ed visits , and 3.1%3.3% had hospitalizations in the pre - index 6 months even though they had not filled albuterol pre - index prescriptions . only 28.2%33.5% of patients were using maintenance ics in any form ( 12.5%14.7% were on labas with ics ) at baseline . the proportion of patients in the albuterol inhalation aerosol cohort without idc who were not receiving any asthma medications at baseline was higher than for the cohort with idc ( 66.5% vs 59.8% ; p<0.05 ) . patients in the idc cohort had higher baseline use of oral or injectable corticosteroids ( 20.5% vs 17.5% ) , ics ( 33.5% vs 28.2% ) , labas ( 14.7% vs 12.5% ) , lamas ( 0.4% vs 0.3% ) , and other respiratory medications ( 16.1% vs 12.3% ) ( all p<0.05 ) . table 2 summarizes the unadjusted health care utilization of study patients by treatment and age . the proportion of patients experiencing respiratory - related hospitalizations ( 2.1% vs 2.3% ) , ed visits ( 7.1% vs 8% ) , and lrti - related op visits treated with antibiotics ( 13.3% vs 13.7% ) was significantly lower among patients using albuterol inhalation aerosol with idc relative to the cohort without idc . in addition , mean total numbers of respiratory - related hospitalizations and ed visits were significantly lower for the albuterol inhalation aerosol with idc cohort compared to the cohort without idc ( all p<0.05 ) ( table 2 ) . those with idc also had a higher proportion of patients refill their albuterol prescription versus the cohort without idc ( 35% vs 31.1% ; p<0.05 ) . these differences in health care utilization were generally seen across all age groups : 17 , 1839 , and 40 + ( table 2 ) . after adjusting for baseline confounding factors ( pre - index patient demographics , clinical characteristics , and concomitant medications ) , the odds ratio ( or ) for experiencing a respiratory - related hospitalization ( or=0.92 ; 95% confidence interval [ ci ] 0.880.96 ) or ed visit ( or=0.92 ; 95% ci 0.900.94 ) was significantly lower among patients using albuterol inhalation aerosol with idc compared to those without idc ( table 3 ) . similarly , the adjusted mean total numbers of respiratory - related hospitalizations and ed visits were significantly lower ( p<0.05 ) among the cohort with idc than the cohort without idc ( figure 2 ) . other factors potentially leading to higher risk of hospitalizations ( significant relative risk over 1.2 ) included older age groups ( 1839 or 40 + vs < 18 years ) , female gender , presence of ischemic heart disease , or other respiratory disease . for ed visits , potential protective factors ( significant relative risks below 0.9 ) included presence of rhinitis ( for hospitalizations and ed visits ) , eczema ( for hospitalizations ) , ics ( for hospitalizations ) or labas with ics ( for ed visits ) , lamas ( for ed visits ) , and other respiratory medication use ( for hospitalizations ) . a total of 422,548 patients with asthma were included in the analysis . of these , 135,305 ( 32% ) patients used albuterol inhalation aerosol with idc , and 287,243 ( 68% ) patients received albuterol inhalation aerosol without idc ( figure 1 ) . baseline demographic and clinical characteristics stratified by cohorts and age are presented in table 1 . the mean age ( 32.9 vs 32.5 years ) and the proportion of female patients ( 57.3% vs 57.9% ) were similar across both cohorts . females made up the majority in both cohorts with an exception of those in the 17 years subcohorts . the presence of rhinitis , gastroesophageal reflux disease ( gerd ) , and eczema was modestly higher among the cohort with idc compared to the cohort without idc ( all p < 0.05 ) . in general , baseline markers of asthma treatment were higher and all - cause hospitalizations and ed visits were lower in patients in the albuterol inhalation aerosol with idc cohort compared to those in the cohort without idc . overall , both cohorts contained an asthma population with a higher level of severity : 17.5%20.5% of patients needed oral or injectable corticosteroids , 14.7%15.4% had ed visits , and 3.1%3.3% had hospitalizations in the pre - index 6 months even though they had not filled albuterol pre - index prescriptions . only 28.2%33.5% of patients were using maintenance ics in any form ( 12.5%14.7% were on labas with ics ) at baseline . the proportion of patients in the albuterol inhalation aerosol cohort without idc who were not receiving any asthma medications at baseline was higher than for the cohort with idc ( 66.5% vs 59.8% ; p<0.05 ) . patients in the idc cohort had higher baseline use of oral or injectable corticosteroids ( 20.5% vs 17.5% ) , ics ( 33.5% vs 28.2% ) , labas ( 14.7% vs 12.5% ) , lamas ( 0.4% vs 0.3% ) , and other respiratory medications ( 16.1% vs 12.3% ) ( all p<0.05 ) . table 2 summarizes the unadjusted health care utilization of study patients by treatment and age . the proportion of patients experiencing respiratory - related hospitalizations ( 2.1% vs 2.3% ) , ed visits ( 7.1% vs 8% ) , and lrti - related op visits treated with antibiotics ( 13.3% vs 13.7% ) was significantly lower among patients using albuterol inhalation aerosol with idc relative to the cohort without idc . in addition , mean total numbers of respiratory - related hospitalizations and ed visits were significantly lower for the albuterol inhalation aerosol with idc cohort compared to the cohort without idc ( all p<0.05 ) ( table 2 ) . those with idc also had a higher proportion of patients refill their albuterol prescription versus the cohort without idc ( 35% vs 31.1% ; p<0.05 ) . these differences in health care utilization were generally seen across all age groups : 17 , 1839 , and 40 + ( table 2 ) . after adjusting for baseline confounding factors ( pre - index patient demographics , clinical characteristics , and concomitant medications ) , the odds ratio ( or ) for experiencing a respiratory - related hospitalization ( or=0.92 ; 95% confidence interval [ ci ] 0.880.96 ) or ed visit ( or=0.92 ; 95% ci 0.900.94 ) was significantly lower among patients using albuterol inhalation aerosol with idc compared to those without idc ( table 3 ) . similarly , the adjusted mean total numbers of respiratory - related hospitalizations and ed visits were significantly lower ( p<0.05 ) among the cohort with idc than the cohort without idc ( figure 2 ) . other factors potentially leading to higher risk of hospitalizations ( significant relative risk over 1.2 ) included older age groups ( 1839 or 40 + vs < 18 years ) , female gender , presence of ischemic heart disease , or other respiratory disease . for ed visits , potential protective factors ( significant relative risks below 0.9 ) included presence of rhinitis ( for hospitalizations and ed visits ) , eczema ( for hospitalizations ) , ics ( for hospitalizations ) or labas with ics ( for ed visits ) , lamas ( for ed visits ) , and other respiratory medication use ( for hospitalizations ) . this study is the largest retrospective analysis to assess real - world respiratory - related health care utilization in asthma patients ( n=422,548 ) indexed to albuterol sulfate inhalation aerosol with idc compared to similar patients who received albuterol sulfate inhalation aerosol without idc during a previous time period in the usa . the results of this analysis demonstrate that respiratory - related ed visits and hospitalizations were both ~8% lower in association with idc after controlling for baseline characteristics . the mean numbers of total ed visits and total hospitalizations were also reduced significantly ( p<0.05 ) in idc users after controlling for potential confounders . baseline markers of asthma treatment were higher and all - cause hospitalizations and ed visits were lower in the idc group . these findings likely represent differences in the two unique patient populations with respect to asthma severity and may also suggest that asthma care strategies may have changed from 20112012 to the later 20132014 period when patients received an albuterol inhalation aerosol prescription at the index date . in addition , it is important to note that the usa implemented the affordable care act , which increased access for ~8 million patients beginning january 2014 , which may have impacted these rates . assessment of the patients therapeutic profiles at baseline suggests that many selected patients may have had seasonal asthma or asthma triggered by infection as their predominant disease state . similar results were seen when patients in each treatment cohort were stratified by age , with patients using albuterol inhalation aerosol with idc having significantly lower rates and mean numbers of hospitalizations and ed visits compared to their non - idc counterparts . consistent with our findings , a historical cohort study of 75,787 patients with asthma aged 464 years ( 53,964 using albuterol with a dose counter and 21,823 using albuterol without a dose counter ) by price et al demonstrated that patients using an inhaler with a dose counter had a 51% lower incidence of respiratory - related ed visits ( adjusted rate ratio : 0.49 ; 95% ci 0.410.59).22 the authors speculated that the albuterol dose counters may have enabled patients to determine when their rescue medication was empty , and/or when additional controller adherence was needed , thus preventing them from using an empty inhaler during an asthma exacerbation.22 the addition of a dose counter can help to reduce ed visits , thereby reducing health care costs associated with asthma . when using saba metered - dose inhalers ( mdis ) without idcs , it may be difficult for the patient to gauge increasing use of rescue / relief medication as a sign of worsening asthma control , or to determine the remaining number of effective doses of rescue / relief medication.13,20,23 this study was not designed to determine whether the utilization benefit of rescue / relief inhaled delivery devices tagged to an effective idc is the result of increased patient insight into disease status ( i.e. , an early warning regarding impending loss of control ) , increased awareness of drug content ( i.e. , impending empty saba inhaler ) , or both . several study findings suggest , however , that the ability of an idc to optimize controller therapy by targeting patients with increasing saba use is at least a partial factor in the utilization benefit . these results include : 1 ) the finding at baseline of relatively low usage rates of ics controller therapy ( 28.2%33.5% ) coupled with relatively high levels of acuity ( 14.7%15.4% having ed visits ) ; 2 ) the finding of no asthma therapy as a primary risk factor for ed visits ; and 3 ) the finding that the use of an ics controller is a potential protective factor for hospitalizations . the small but significant utilization reduction associated with an idc in this study should be understood in the context of the potentially low penetrance of idc engagement by patients and providers in the real - world setting . for example , in a phone survey , only 36% of bronchodilator users reported ever having been told to keep track of mdi doses.14 in the future , newer technologies may improve patient engagement with their asthma therapy , and gains in disease management may be possible if data for rescue dosing are better integrated into practice . in a recent study , telemonitoring of saba use via a patient - facing smartphone application , with dose reporting to providers , was associated with decreased use of rescue medication and improved asthma control among those adults initially lacking asthma control.24 there are some aspects of the retrospective analysis design that may impact the study results . this study analyzed asthma patients newly treated with albuterol inhalation aerosol who had not received any other sabas in the pre - index period ; these criteria may have resulted in the selection of an intermittent , poorly adherent , or seasonally exacerbating asthma population . baseline data showed somewhat increased use of asthma controllers ( ics , labas , and lamas ) in the later 20132014 period compared to the earlier 20112012 cohort of patients , which may have reflected a national incentive for enhanced quality of care , as the affordable care act , with widespread use of electronic medical records , and health quality tracking became implemented during late 2013 and 2014 . the inconsistent use of asthma medications , with only 28%34% of patients utilizing ics , may have influenced ed and hospitalization rates . baseline medications were included in the multivariate models ; therefore , the confounding effects of these variables were controlled in the analysis . future prospective studies could evaluate patients enrolled at a single point in time and randomly assign patients to albuterol with or without idc to prospectively monitor outcomes occurring over concurrent time frames . the marketscan databases rely on administrative claims data for clinical detail ; therefore , the data may be subject to data coding limitations and data entry error . this analysis was conducted over a relatively short follow - up time frame ( e.g. , 6 months post - index ) , and hospitalizations and ed visits in general are a relatively rare outcome . finally , this study was limited to asthma patients who utilized health care services and continuously enrolled with commercial or private medicare supplemental coverage , thereby limiting the generalizability of the findings to all asthma patients , especially those with other insurance or without health insurance coverage . the methodology of this study is not able to identify the degree to which the improvements seen reflect general improvements in health care delivery for asthma during 20112014 or the extent to which the use of an idc device contributed to a reduction in health care utilization . notable strengths of this analysis include the fact that a very large patient population drawn from administrative claims data across the usa was evaluated . in addition , this study provides real - world data on respiratory - related health care utilization ( hospitalizations , ed visits , and lrti - related op visits ) among asthma patients indexed to albuterol inhalation aerosol with or without idc in a geographically diverse population . in a real - world setting during 20132014 , patients with asthma using albuterol sulfate inhalation aerosol with idc experienced significantly fewer hospitalizations and ed visits compared to a cohort of patients using albuterol inhalation aerosol without idc in 20112012 . dosage information provided by idcs may improve treatment outcomes by decreasing the likelihood that the canister will be empty when needed , thereby enhancing disease management and reducing health care utilization , specifically respiratory - related hospitalizations and ed visits.3,13,1517 therefore , idcs may be of value for long - term health care cost savings , which is in line with key national health policy objectives . long - term studies in patients with all levels of asthma severity are warranted to validate the findings of this study .

backgroundaccurate tracking of the administered dose of asthma rescue inhalers is critical for optimal disease management and is related to reductions in rates of unscheduled health care utilization in asthma patients . there are few published data on the real - world impact of rescue inhalers with integrated dose counters ( idcs ) on health care resource utilization ( hru ) for asthma patients . this study evaluates hru among users of proair hydrofluoroalkane ( hfa ) ( albuterol sulfate inhalation aerosol ) , with idc versus without idc , in asthma patients.methodsthis was a retrospective administrative claims study of asthma patients receiving a new prescription for albuterol inhalation aerosol without idc during 2 years ( january 2011december 2012 ) or with idc during the first full year after idc implementation in the usa ( july 2013july 2014 ) . six months of continuous enrollment with medical and prescription drug benefits were required before and after the first prescription during the study period . data on respiratory - related hospitalizations and emergency department ( ed ) visits were collected during the follow - up period.resultsa total of 135,305 ( 32% ) patients used albuterol inhalation aerosol with idc , and 287,243 ( 68% ) patients received albuterol inhalation aerosol without idc . after adjusting for baseline confounding factors , the odds ratio ( or ) for experiencing a respiratory - related hospitalization ( or=0.92 ; 95% confidence interval [ ci ] 0.880.96 ) or ed visit ( or=0.92 ; 95% ci 0.900.94 ) was significantly lower among patients using albuterol inhalation aerosol with idc versus without idc.conclusionin a real - world setting , asthma patients using proair hfa with idc experienced significantly fewer hospitalizations and ed visits compared with patients using proair hfa without idc . dosage information provided by idcs may allow providers to better understand patients disease severity and aid in titrating controller medications and also decrease the likelihood that the canister will be empty when needed , thereby enhancing disease management and reducing hru .

Introduction Methods Data source Patient selection and study period Study covariates Outcome measures Statistical analysis Results Study population Patient characteristics Health care utilization Discussion Conclusion

asthma is a major health problem that affects 26 million individuals in the usa.1 respiratory - related emergency department ( ed ) visits along with hospitalizations due to exacerbations impose significant health care resource utilization ( hru ) burden among patients with asthma.2,3 the economic burden of asthma is large and is attributable to patients with poorly controlled disease , highlighting the importance of maintaining disease control and minimizing the frequency of exacerbations.1,4 recent estimates suggest that the average cost per asthma - related hospital stay increased from $ 5,200 to $ 6,600 ( 2010 us dollars)5 and for an outpatient ( op ) ed visit averaged $ 1,502 ( 2008 us dollars).6 in addition , costs due to ed visits and hospitalization disproportionately account for a major portion of the total health care costs of asthma.47 the treatment goals for asthma are primarily driven by patient - centered outcomes such as relieving symptoms , preventing disease progression and exacerbations , and optimizing health status and quality of life.8,9 clinical practice guidelines for asthma emphasize the importance of using preventer / controllers with anti - inflammatory properties ( e.g. , frazer , pa ) is a short - acting beta agonist ( saba ) rescue / relief agent indicated for the treatment of bronchospasm with reversible obstructive airway disease and for the prevention of exercise - induced bronchospasm.10 as a rescue / relief agent , albuterol sulfate inhalation aerosol is used to improve the symptoms of asthma while the preventer / controller agents are being titrated.11,12 accurate tracking of the administered dose is , therefore , critically important for optimal asthma control and for potentially reducing the rates of unscheduled health care utilization , and thus the cost of care , in patients with asthma.1316 the advent of integrated dose counters ( idcs ) led to a logistical shift in the effective management of patients with asthma.1721 idcs may add value as they monitor rescue inhaler use ; yet , they are not a standard feature across all rescue inhalers . results from a real - world study demonstrated reduced incidence of respiratory - related ed visits in patients using rescue inhalers with idc compared to those with no dose counter on their inhalers.22 however , there is paucity of data on the real - world impact of proair hfa equipped with dose counters on hru among patients with asthma . of these , 135,305 ( 32% ) patients used albuterol inhalation aerosol with idc , and 287,243 ( 68% ) patients received albuterol inhalation aerosol without idc ( figure 1 ) . the proportion of patients experiencing respiratory - related hospitalizations ( 2.1% vs 2.3% ) , ed visits ( 7.1% vs 8% ) , and lrti - related op visits treated with antibiotics ( 13.3% vs 13.7% ) was significantly lower among patients using albuterol inhalation aerosol with idc relative to the cohort without idc . after adjusting for baseline confounding factors ( pre - index patient demographics , clinical characteristics , and concomitant medications ) , the odds ratio ( or ) for experiencing a respiratory - related hospitalization ( or=0.92 ; 95% confidence interval [ ci ] 0.880.96 ) or ed visit ( or=0.92 ; 95% ci 0.900.94 ) was significantly lower among patients using albuterol inhalation aerosol with idc compared to those without idc ( table 3 ) . of these , 135,305 ( 32% ) patients used albuterol inhalation aerosol with idc , and 287,243 ( 68% ) patients received albuterol inhalation aerosol without idc ( figure 1 ) . after adjusting for baseline confounding factors ( pre - index patient demographics , clinical characteristics , and concomitant medications ) , the odds ratio ( or ) for experiencing a respiratory - related hospitalization ( or=0.92 ; 95% confidence interval [ ci ] 0.880.96 ) or ed visit ( or=0.92 ; 95% ci 0.900.94 ) was significantly lower among patients using albuterol inhalation aerosol with idc compared to those without idc ( table 3 ) . this study is the largest retrospective analysis to assess real - world respiratory - related health care utilization in asthma patients ( n=422,548 ) indexed to albuterol sulfate inhalation aerosol with idc compared to similar patients who received albuterol sulfate inhalation aerosol without idc during a previous time period in the usa . in a real - world setting during 20132014 , patients with asthma using albuterol sulfate inhalation aerosol with idc experienced significantly fewer hospitalizations and ed visits compared to a cohort of patients using albuterol inhalation aerosol without idc in 20112012 . dosage information provided by idcs may improve treatment outcomes by decreasing the likelihood that the canister will be empty when needed , thereby enhancing disease management and reducing health care utilization , specifically respiratory - related hospitalizations and ed visits.3,13,1517 therefore , idcs may be of value for long - term health care cost savings , which is in line with key national health policy objectives .

receptor tyrosine kinases ( rtks ) constitute a large family of single - spanning membrane proteins found only in metazoans ( robinson et al , 2000 ) . their primary role is to mediate intercellular communication by recognizing extracellular ligands and translating that information into an appropriate cellular response ( schlessinger , 2000 ) . the intracellular region of an rtk contains a tyrosine kinase domain as well as several tyrosine residues that are phosphorylated upon receptor activation . these phosphotyrosines act as relays for information transmission , and the sequences surrounding these sites define signal specificity . intracellular signaling proteins bind to these sites of tyrosine phosphorylation through src homology 2 ( sh2 ) or phosphotyrosine - binding ( ptb ) domains , initiating a variety of signaling cascades within the cell . rtks can elicit diverse and even opposing phenotypic responses , ranging from adhesion to migration , differentiation to proliferation , and survival to apoptosis ( schlessinger , 2000 ; yarden and sliwkowski , 2001 ) . although no two receptors feature identical sequences surrounding their ptyr sites , there is considerable qualitative overlap in the pathways they activate ( fambrough et al , 1999 ; simon , 2000 ) . the ability of rtks to signal through common pathways , yet to induce diverse phenotypic responses , has largely been attributed to differences in cellular context , as signaling proteins are differentially expressed in different cell types ( jordan et al , 2000 ; simon , 2000 ) . for example , fibroblast growth factor receptor 1 ( fgfr1 ) induces differentiation in neuronal cells , but induces proliferation in fibroblasts ( marshall , 1995 ; lin et al , 1996 ) . when expressed in the same cellular background , however , different rtks have also been shown to elicit different phenotypic responses . for example , activation of epidermal growth factor receptor ( egfr ) induces proliferation in pc12 neuronal cells , whereas activation of fgfr1 induces differentiation ( pollock et al , 1990 ; lin et al , 1996 ) . how , then , are intrinsic differences between rtks manifested within the same cell type ? how is that information processed ? to address these questions , we expressed six diverse rtks in the same cellular background and monitored their signaling properties by quantitative immunoblotting . we found that although they activated many of the same signaling proteins , they did so to different degrees . we then used protein microarrays to define a quantitative interaction map for each receptor by measuring the affinity of almost every human sh2 and ptb domain for phosphopeptides representing ptyr sites on the receptors . using partial least - squares regression ( plsr ) , we found that the relative phosphorylation levels of upstream signaling proteins could be accurately predicted using linear combinations of receptor - docking affinities , and that much of the predictive information resides in the docking sites for two central signaling proteins : phosphoinositide 3-kinase ( pi3k ) and shc1 . we also found that the relative phosphorylation levels of downstream proteins could not be predicted using linear models , suggesting that rtk signaling can be segmented into discrete upstream and downstream layers . to determine at a quantitative level how different rtks behave when placed in the same cellular background , we selected six well - studied and phylogenetically diverse rtks : egfr , fgfr1 , insulin - like growth factor 1 receptor ( igf1r ) , hepatocyte growth factor receptor ( met ) , neurotrophic tyrosine kinase receptor type 2 ( ntrk2 ) , and platelet - derived growth factor receptor ( pdgfr ) . six stable cell lines were generated by transfecting the full - length coding region for each receptor into human embryonic kidney flp - in-293 cells , which do not normally express these receptors at appreciable levels ( figure 1a ) . the resulting cell lines grew normally and , in each case , the receptor was produced at 10 copies per cell and activated by its cognate ligand in a dose - dependent manner ( supplementary figure s1 ) . to obtain a broad and quantitative view of how each receptor activates intracellular signaling proteins , the six cell lines were serum - starved for 24 h and stimulated for 5 min with saturating levels of the appropriate ligand . this early time point was chosen because many signaling proteins peak in their phosphorylation levels within the first 10 min of stimulation and because we wanted to capture immediate , receptor - dependent signaling events without additional complications arising from feedback loops and other forms of network regulation . quantitative immunoblotting was then used to measure the relative phosphorylation levels of a wide range of proteins that have previously been implicated in rtk signaling ( figure 1b and c ) . in total , we queried 65 sites of phosphorylation on 57 proteins and observed growth factor - induced phosphorylation of 24 sites on 23 proteins ( supplementary table si ) . to compare the phosphorylation levels of a given protein across the six cell lines , lysate concentrations were normalized , basal phosphorylation was subtracted , and each level was calculated relative to the maximum observed level for that site ( figure 1b ; supplementary figure s2 ; supplementary information ) . some proteins , such as shp-2 and cbl , were phosphorylated in as few as two of the cell lines , while others , such as crkl and p90rsk , were phosphorylated in all six ( figure 1b ) . for every site of phosphorylation , quantitative differences were observed across the six cell lines and the rank order varied depending on the site . thus , although these six receptors have previously been shown to activate many of the same pathways , they do so to different degrees when placed in the same cellular context . what , then , accounts for these differences ? as rtks initiate signaling by recruiting proteins to sites of tyrosine phosphorylation ( schlessinger , 2000 ) , we asked whether there was information in the recruitment properties of the ptyr sites on these receptors that could explain the observed differences . sites of tyrosine phosphorylation are recognized by either sh2 ( sadowski et al , 1986 ) or ptb domains ( kavanaugh and williams , 1994 ) . to obtain a genome - wide , unbiased , and quantitative measure of the recruitment potential of each receptor , we prepared protein microarrays comprising nearly every sh2 and ptb domain encoded in the human genome ( figure 1d ; supplementary table sii ) ( jones et al , 2006 ) . we then probed these arrays with fluorescently labeled , 18-residue phosphopeptides with sequences derived from every known site of tyrosine phosphorylation on each of the six receptors ( supplementary table siii ) . equilibrium dissociation constants ( kd values ) were obtained by probing the arrays with eight concentrations of each peptide and fitting the resulting fluorescence data ( supplementary information ) to an equation that describes saturation binding ( figure 1d ) ( jones et al , 2006 ) . in total , we queried 96 sh2 domains and 37 ptb domains with 47 phosphopeptides and observed 652 interactions with kd2 m ( supplementary table siv ) . when we repeated this process , duplicate kd measurements were in close agreement ( r=0.85 ; supplementary figure s3b ) and the mean kd was used for subsequent analyses . of the 131 domains , 112 domains representing 74 different proteins bound at least one phosphopeptide . as anticipated , there was considerable qualitative overlap between the six receptors : 50 of these proteins recognized peptides from at least three receptors and 21 of them recognized peptides from at least five receptors ( supplementary figure s4 ) . in general , the domains that recognized the most receptors are those found in well - studied signaling proteins , including the lipid - modifying enzyme pi3k ; the transcription factors stat1 and stat2 ; the non - receptor tyrosine kinases src and abl1 ; the guanine nucleotide exchange factor vav2 ; the adaptor proteins crk , crkl , and nck ; and the scaffold proteins shc1 and grb7 . thus , if viewed in strictly binary terms , these phylogenetically diverse receptors differ very little in their recruitment properties with respect to these core signaling proteins . at the quantitative level , however , they differ substantially . for example , although five of the six receptors feature docking sites for the regulatory subunit of pi3k , there is only one low - affinity site ( kd=590 nm ) on igf1r , but there are five sites , including one high - affinity site ( kd=10 nm ) , on pdgfr. quantitative differences in both the number of docking sites and the binding affinities at these sites may therefore explain the observed differences in signaling elicited by each receptor . to test this hypothesis , we represented each phosphopeptide as a row vector of association constants , ka , with each element in the vector corresponding to a different sh2 or ptb domain - containing protein ( figure 1e ) . for proteins that contained two domains that bound the same peptide , the larger ka was used . in addition , the three isoforms of the regulatory subunit of pi3k were treated as a single protein as their sh2 domains behaved similarly . the binding vector for a given receptor was then defined as the sum of its phosphopeptide vectors to take into account the number of docking sites , as well as the affinities at each site . the implicit assumption in adding the phosphopeptide vectors is that multiple docking sites for the same protein within a given receptor act independently of each other . while this is probably not always true , it is the simplest way to combine the data and is a reasonable approximation . in addition , the phosphopeptide vectors were all weighted equally as the relative stoichiometry of phosphorylation at each ptyr site was not known . thus , the intrinsic signaling capabilities of the six receptors was captured in the matrix x , which comprises six rows , one for each receptor , and 74 columns , one for each sh2 or ptb domain - containing protein ( figure 1f ) . in a similar manner , the cellular activity of the rtks was captured in the matrix y , which comprises six rows , one for each receptor , and 24 columns ( y1 y24 ) , one for each phosphorylation site that was monitored by immunoblotting ( figure 1c ) . the simplest connection between the in vitro binding data and the cellular phosphorylation data is a one - to - one relationship in which the degree to which an sh2/ptb - containing protein is phosphorylated correlates linearly with its docking affinities . of the eight proteins for which both microarray and immunoblotting data were obtained , significant correlations were observed for two : shc1 ( r=0.82 , p=0.045 ) and pi3k ( r=0.94 , p=0.0059 ) ( figure 2 ) . these correlations depend heavily on the number of shc1- and pi3k - docking sites on each receptor . if the number of docking sites is taken into account but the affinities are ignored , the correlation actually improves for shc1 ( r=0.99 , p=0.0001 ) , but gets slightly worse for pi3k ( r=0.91 , p=0.013 ) ( figure 2 ) . if the quantitative information is ignored and the interactions are treated as binary , correlations become meaningless as each protein recognized five of the six receptors . these results are consistent with a model in which shc1 and pi3k interact directly with the activated receptors and are not influenced substantially by other docking proteins . the same is not true , however , for the other sh2/ptb - containing proteins that were monitored by immunoblotting ; significant correlations were not observed ( figure 2 ) . for these proteins , the reductionist assumption that they bind directly to the receptor and act independently is too simplistic . some proteins that contain sh2 or ptb domains have been shown to compete with each other for the same ptyr sites ( zhang et al , 2003 ) , and many have been shown to interact with each other and with components of the cell membrane ( schlessinger and lemmon , 2003 ) . are their relationships complex and nonlinear , or can they be approximated using relatively simple models that depend on combinations of docking affinities , rather than on single affinities alone ? the simplest multivariate model is one in which the phosphorylation levels of a given protein , yi , can be predicted using a linear combination of docking affinities . as the number of variables ( docking affinities ) exceeds the number of observations ( rtks ) , we used plsr to regress each yi against x. plsr reduces the dimensionality of x by decomposing it into a small number of orthogonal components that capture most of the covariance between x and yi . each component is a linear combination of docking affinities , weighted by how much they contribute to predicting each immunoblot ( yi ) . we found that four components were sufficient to capture 90% of the covariance with each yi . to guard against overfitting and to assess the predictive value of the docking affinities , we built our models using leave - one - out cross - validation : each model was trained using data from five receptors and then used to predict the immunoblotting data for the sixth receptor based on its docking affinities . this procedure was performed in all six combinations and the cross - validated residual between these predictions and the observed data , q , was calculated . to assess the significance of these predictions , we repeated our calculations 2000 times for each phosphorylation site using randomized x matrices and then calculated p - values for each model . models were built using all of the microarray data , as well as subsets of the data that included only the sh2/ptb - containing proteins that bound at least two receptors , at least three receptors , at least four receptors , or at least five receptors . similar results were obtained in every case , but the significance of the results increased as the number of variables was reduced ( supplementary figure s5 ) . most of the information content in x resides in the 21 sh2/ptb - containing proteins that recognize at least five receptors and hence the results presented below are based on these data alone . of the 24 phosphorylation sites that we monitored by immunoblotting , nine were accurately predicted using linear combinations of docking affinities ( q0.9 ; figure 3a and b ; supplementary figure s6 ) . of these , six passed significance testing ( p0.05 and false discovery rate 0.1 ) . interestingly , all nine of these sites are found on proteins that contain sh2 or ptb domains and therefore represent upstream signaling events ( figure 3b ; supplementary figure s6 ) . moreover , only two phosphorylation sites that occur on sh2/ptb - containing proteins had a q value less than 0.9 : ptyr239/240 of shc1 and pser727 of stat3 . as noted earlier , the relative phosphorylation levels of shc1 can be explained using only the number of shc1-docking sites on each receptor ( figure 2 ) ; combinations of docking affinities are not required . this serine residue is phosphorylated in a protein kinase c - dependent fashion and so represents a downstream signaling event ( aziz et al , 2007 ) . in contrast , tyr705 of stat3 can be phosphorylated by the rtk itself and so represents an upstream event ( hwang et al , 2003 ) ; its phosphorylation is accurately predicted ( q=0.99 ; p=0.03 ; figure 3a ) . similar to pser727 of stat3 , the other 13 downstream signaling events could not be predicted using linear models ( figure 3b ; supplementary figure s6 ) . thus , the phosphorylation sites that we monitored by immunoblotting naturally segregate into two groups : upstream phosphorylation events that are accurately predicted and downstream phosphorylation events that are not . from this , we submit that information processing by rtk signaling networks can be segmented into an upstream layer comprising proteins that are activated in an approximately linear manner through combinations of receptor - docking affinities and a downstream layer comprising proteins that are activated in a nonlinear manner . we note , however , that this result does not prove that the upstream step is linear mechanistically , but rather that this step can be approximated using relatively simple linear models . both the number of docking sites and the docking affinities are important for predicting upstream signaling events . if only the number of docking sites is taken into account , the models perform less well and the results are less significant ( figure 3c ) . if only binary information is used ( proteins are described either to interact or not interact with a receptor ) , all predictions fail as , at this level , the receptors are very similar ( x is close to singular ) . to determine where most of the predictive information resides , we assessed the contribution of each sh2/ptb - containing protein to each plsr model by calculating their variable importance in the projection ( vip ; see materials and methods ) . for all of the upstream signaling events , reduced models that included only the four most important variables performed almost as well as the full plsr models ( figure 3c ) . on average , the two most important variables were pi3k and shc1 ( figure 4a ) . in other words , much of the information needed to predict the relative phosphorylation levels of upstream signaling proteins resides in the number and affinity of pi3k- and shc1-docking sites on the rtk . as these models are statistical in nature , this observation does not necessarily mean that pi3k and shc1 have a causative function in determining the strength of signaling through other upstream proteins . pi3k- and shc1-binding sites may have co - evolved with some other feature of rtks that determines their ability to activate upstream proteins , such as kinase specificity or localization of the receptors to different membrane microdomains . nevertheless , it is possible that these proteins do have a causative function in determining the extent to which other signaling proteins are activated . this hypothesis can not be tested by altering the abundance of pi3k or shc1 , as altering the composition of the cell would change the parameter values in the models . it is possible , however , to alter the catalytic activity of pi3k without altering its abundance using the small molecule inhibitor ly294002 . if pi3k activity has a causative function in determining the degree to which upstream proteins are phosphorylated , we would expect ly294002 treatment to have the largest effect on proteins that have high plsr coefficients for pi3k ( figure 4b ) , and on receptors with the strongest recruitment potential for pi3k ( figure 4c ) . we therefore stimulated all six cell lines in the presence or absence of ly294002 and assessed the relative phosphorylation levels of the upstream signaling proteins by immunoblotting ( figure 4d and e ; supplementary figure s7 ; supplementary information ) . interestingly , the relative phosphorylation levels of src , which has a low coefficient for pi3k ( figure 4b ) , were minimally affected by ly294002 treatment ( figure 4d ) , whereas the relative phosphorylation levels of stat3 , which has a high positive coefficient for pi3k ( figure 4b ) , were affected in a manner consistent with the number and affinity of pi3k - docking sites on the six rtks ( figure 4e ) . this result suggests that , at least for stat3 , the contribution of pi3k in the plsr model is , in part , dependent on its kinase activity . the same result was not observed , however , for all of the upstream signaling proteins ( supplementary figure s7 ) . the stat3 result is not easily explained based on our current rtk wiring diagrams and a mechanistic understanding of this observation will require further investigation . the overall importance of pi3k and shc1 in rtk signaling was recently highlighted in a comprehensive map of the erbb network , which revealed that a large fraction of information converges on a small number of signaling molecules , all of which can be modulated by pi3k and shc1 ( oda et al , 2005 ) . interestingly , when we examined the sequences surrounding all known sites of tyrosine phosphorylation on human rtks as reported in the phospho . elm database ( diella et al , 2008 ) , we observed a distinct and significant ( p<0.05 ) bias for sites that feature the consensus binding sequences for the ptb domain of shc1 ( npxpy ) ( songyang et al , 1995 ) and the sh2 domains of pi3k ( pyxxm ) ( songyang et al , 1993 ; yaffe et al , 2001 ) ( figure 5a and b ) . this bias is not observed in known sites of tyrosine phosphorylation derived from all other human proteins ( figure 5c and d ) . thus , we find that , despite activating many of the same proteins , intrinsic differences between rtks are manifested in the degree to which they activate upstream signaling proteins and that much of this information resides in the number and affinity of docking sites for pi3k and shc1 . as these proteins lie upstream of the akt and map kinase signaling pathways , and as these two pathways have been found repeatedly to have a central function in rtk biology , it is likely that our observations are not specific to hek flp - in-293 cells , but extend to more physiological settings as well . recently , miller - jensen et al ( 2007 ) showed that the phenotypic response of cells to external stimuli can be predicted using models that rely on linear combinations of a common set of downstream signaling proteins . coupled with our results , this suggests that different rtks may be able to elicit different phenotypic responses in the same cell type by activating a common set of signaling proteins , but to different quantitative degrees . in addition , our study , coupled with that of miller - jensen et al , supports a model in which information processing by rtk signaling networks can be segmented into three discrete layers : an upstream layer comprising proteins that are activated in a linear manner through combinations of receptor - docking affinities ; an intermediate layer in which these signals are processed in a nonlinear manner ; and a downstream layer in which integrators of signaling combine in a linear manner to determine cellular outcome . we submit that the difficult task of constructing mathematical models of rtk signaling can be parsed into discrete problems and that our greatest challenge lies in dissecting the middle layer . stable cell lines were generated by co - transfecting flp - in-293 cells ( invitrogen , carlsbad , ca ) with the plasmid pef5/frt / v5-dest bearing the open reading frame for each rtk and the accessory plasmid pog44 according to the manufacturer 's directions ( invitrogen ) . cells were maintained in dulbecco 's modified eagle 's medium supplemented with 10% ( v / v ) fetal bovine serum , 2 mm glutamine , 100 iu / ml penicillin , 100 g / ml streptomycin , and 150 g / ml hygromycin b. all cell culture and immunoblotting experiments were performed using standard procedures . rabbit - derived primary antibodies were from cell signaling technologies ( beverly , ma ; supplementary table si ) . for quantitative immunoblots , bands were detected with irdye 680-labeled goat anti - rabbit igg ( li - cor biosciences , lincoln , ne ) and imaged using an odyssey infrared imaging system ( li - cor biosciences ) . expression levels of the rtks were determined using elisa kits from invitrogen for egfr and met , and from r&d systems ( minneapolis , mn ) for ntrk2 and pdgfr. all protein microarray experiments were performed as described earlier ( jones et al , 2006 ; kaushansky et al , 2008 ) . to define the receptor - docking affinity matrix , x , the matrix of kd values ( supplementary table siv ) was converted to a matrix of ka values ( ka=1/kd ) . if a protein contained two domains that bound the same peptide , the higher ka value was used . each phosphopeptide was then expressed as a row vector of ka values : and each receptor was defined as the sum of its constituent phosphopeptide vectors : the raw receptor - docking affinity matrix , xraw , was then assembled from the six receptor vectors : the raw matrix was adjusted such that every sh2/ptb - containing protein ( i.e. every column ) was mean - centred and weighted according to its average affinity . this yielded the final receptor - docking affinity matrix , x. for the models presented in figure 3 , proteins that bound fewer than five receptors were removed from the matrix . models obtained using all of the data or increasingly smaller subsets are shown in supplementary figure s5 . relative phosphorylation levels of signaling proteins , as measured by immunoblotting , were calculated by first subtracting the level observed in the mock - treated , parental flp - in-293 cell line and then dividing each value by the maximum observed value for that site across the six cell lines . each phosphorylation site was treated as a separate vector , y , and each y was mean - centred and variance - normalized . a plsr ( geladi and kowalski , 1986 ) was then performed separately on each y. for cross - validation , each receptor ( row i ' ) was removed once from both x and y , the regression was performed , and the resulting model was used to predict the value of yi . the residual , q , of this prediction was then compared with residuals generated from randomly shuffling x 2000 times . the distribution of these residuals was used to calculate the p - value of the observed q. the weighted sum of squares ( also known as the vip ) for each variable , k , was calculated according to equation ( 5 ) : where kt is the total number of variables , a is the principal component , and ssa is the sum of squares for that component . experimentally determined sites of tyrosine phosphorylation in human proteins were acquired from the phospho.elm database ( diella et al , 2008 ) . of the 1397 identified sites , the amino - acid frequencies at positions upstream and downstream of ptyr sites were calculated and then normalized to the expected frequency of each amino acid in all human proteins ( echols et al , 2002 ) . the resulting histograms of observed / expected frequencies were fit to a log - normal distribution from which p - values were calculated . stable cell lines were generated by co - transfecting flp - in-293 cells ( invitrogen , carlsbad , ca ) with the plasmid pef5/frt / v5-dest bearing the open reading frame for each rtk and the accessory plasmid pog44 according to the manufacturer 's directions ( invitrogen ) . cells were maintained in dulbecco 's modified eagle 's medium supplemented with 10% ( v / v ) fetal bovine serum , 2 mm glutamine , 100 iu / ml penicillin , 100 g / ml streptomycin , and 150 g / ml hygromycin b. all cell culture and immunoblotting experiments were performed using standard procedures . rabbit - derived primary antibodies were from cell signaling technologies ( beverly , ma ; supplementary table si ) . for quantitative immunoblots , bands were detected with irdye 680-labeled goat anti - rabbit igg ( li - cor biosciences , lincoln , ne ) and imaged using an odyssey infrared imaging system ( li - cor biosciences ) . expression levels of the rtks were determined using elisa kits from invitrogen for egfr and met , and from r&d systems ( minneapolis , mn ) for ntrk2 and pdgfr. all protein microarray experiments were performed as described earlier ( jones et al , 2006 ; kaushansky et al , 2008 ) . to define the receptor - docking affinity matrix , x , the matrix of kd values ( supplementary table siv ) was converted to a matrix of ka values ( ka=1/kd ) . if a protein contained two domains that bound the same peptide , the higher ka value was used . each phosphopeptide was then expressed as a row vector of ka values : and each receptor was defined as the sum of its constituent phosphopeptide vectors : the raw receptor - docking affinity matrix , xraw , was then assembled from the six receptor vectors : the raw matrix was adjusted such that every sh2/ptb - containing protein ( i.e. every column ) was mean - centred and weighted according to its average affinity . this yielded the final receptor - docking affinity matrix , x. for the models presented in figure 3 , proteins that bound fewer than five receptors were removed from the matrix . models obtained using all of the data or increasingly smaller subsets are shown in supplementary figure s5 . relative phosphorylation levels of signaling proteins , as measured by immunoblotting , were calculated by first subtracting the level observed in the mock - treated , parental flp - in-293 cell line and then dividing each value by the maximum observed value for that site across the six cell lines . each phosphorylation site was treated as a separate vector , y , and each y was mean - centred and variance - normalized . a plsr ( geladi and kowalski , 1986 ) was then performed separately on each y. for cross - validation , each receptor ( row i ' ) was removed once from both x and y , the regression was performed , and the resulting model was used to predict the value of yi . the residual , q , of this prediction was then compared with residuals generated from randomly shuffling x 2000 times . the distribution of these residuals was used to calculate the p - value of the observed q. the weighted sum of squares ( also known as the vip ) for each variable , k , was calculated according to equation ( 5 ) : where kt is the total number of variables , a is the principal component , and ssa is the sum of squares for that component . experimentally determined sites of tyrosine phosphorylation in human proteins were acquired from the phospho.elm database ( diella et al , 2008 ) . of the 1397 identified sites , the amino - acid frequencies at positions upstream and downstream of ptyr sites were calculated and then normalized to the expected frequency of each amino acid in all human proteins ( echols et al , 2002 ) . the resulting histograms of observed / expected frequencies were fit to a log - normal distribution from which p - values were calculated . supplementary information : this supplementary file includes : supplementary methods ; supplementary references ; supplementary figures 6 , 7 , 8 , 9 , 10 , 11 , 12 ; supplementary tables 1 , 2 , 3 supplementary table 4 : equilibrium dissociation constants for the binding of phosphopeptides to human sh2 or ptb domains .

receptor tyrosine kinases ( rtks ) process extracellular cues by activating a broad array of signaling proteins . paradoxically , they often use the same proteins to elicit diverse and even opposing phenotypic responses . binary , on off ' wiring diagrams are therefore inadequate to explain their differences . here , we show that when six diverse rtks are placed in the same cellular background , they activate many of the same proteins , but to different quantitative degrees . additionally , we find that the relative phosphorylation levels of upstream signaling proteins can be accurately predicted using linear models that rely on combinations of receptor - docking affinities and that the docking sites for phosphoinositide 3-kinase ( pi3k ) and shc1 provide much of the predictive information . in contrast , we find that the phosphorylation levels of downstream proteins can not be predicted using linear models . taken together , these results show that information processing by rtks can be segmented into discrete upstream and downstream steps , suggesting that the challenging task of constructing mathematical models of rtk signaling can be parsed into separate and more manageable layers .

Introduction Results and discussion Materials and methods Cell culture, immunoblotting, ELISA, and protein microarray experiments PLSR Amino-acid frequencies near sites of tyrosine phosphorylation Supplementary Material

rtks can elicit diverse and even opposing phenotypic responses , ranging from adhesion to migration , differentiation to proliferation , and survival to apoptosis ( schlessinger , 2000 ; yarden and sliwkowski , 2001 ) . when expressed in the same cellular background , however , different rtks have also been shown to elicit different phenotypic responses . to address these questions , we expressed six diverse rtks in the same cellular background and monitored their signaling properties by quantitative immunoblotting . we found that although they activated many of the same signaling proteins , they did so to different degrees . using partial least - squares regression ( plsr ) , we found that the relative phosphorylation levels of upstream signaling proteins could be accurately predicted using linear combinations of receptor - docking affinities , and that much of the predictive information resides in the docking sites for two central signaling proteins : phosphoinositide 3-kinase ( pi3k ) and shc1 . we also found that the relative phosphorylation levels of downstream proteins could not be predicted using linear models , suggesting that rtk signaling can be segmented into discrete upstream and downstream layers . to determine at a quantitative level how different rtks behave when placed in the same cellular background , we selected six well - studied and phylogenetically diverse rtks : egfr , fgfr1 , insulin - like growth factor 1 receptor ( igf1r ) , hepatocyte growth factor receptor ( met ) , neurotrophic tyrosine kinase receptor type 2 ( ntrk2 ) , and platelet - derived growth factor receptor ( pdgfr ) . thus , although these six receptors have previously been shown to activate many of the same pathways , they do so to different degrees when placed in the same cellular context . the simplest multivariate model is one in which the phosphorylation levels of a given protein , yi , can be predicted using a linear combination of docking affinities . of the 24 phosphorylation sites that we monitored by immunoblotting , nine were accurately predicted using linear combinations of docking affinities ( q0.9 ; figure 3a and b ; supplementary figure s6 ) . as noted earlier , the relative phosphorylation levels of shc1 can be explained using only the number of shc1-docking sites on each receptor ( figure 2 ) ; combinations of docking affinities are not required . from this , we submit that information processing by rtk signaling networks can be segmented into an upstream layer comprising proteins that are activated in an approximately linear manner through combinations of receptor - docking affinities and a downstream layer comprising proteins that are activated in a nonlinear manner . in other words , much of the information needed to predict the relative phosphorylation levels of upstream signaling proteins resides in the number and affinity of pi3k- and shc1-docking sites on the rtk . we therefore stimulated all six cell lines in the presence or absence of ly294002 and assessed the relative phosphorylation levels of the upstream signaling proteins by immunoblotting ( figure 4d and e ; supplementary figure s7 ; supplementary information ) . thus , we find that , despite activating many of the same proteins , intrinsic differences between rtks are manifested in the degree to which they activate upstream signaling proteins and that much of this information resides in the number and affinity of docking sites for pi3k and shc1 . recently , miller - jensen et al ( 2007 ) showed that the phenotypic response of cells to external stimuli can be predicted using models that rely on linear combinations of a common set of downstream signaling proteins . coupled with our results , this suggests that different rtks may be able to elicit different phenotypic responses in the same cell type by activating a common set of signaling proteins , but to different quantitative degrees . in addition , our study , coupled with that of miller - jensen et al , supports a model in which information processing by rtk signaling networks can be segmented into three discrete layers : an upstream layer comprising proteins that are activated in a linear manner through combinations of receptor - docking affinities ; an intermediate layer in which these signals are processed in a nonlinear manner ; and a downstream layer in which integrators of signaling combine in a linear manner to determine cellular outcome . we submit that the difficult task of constructing mathematical models of rtk signaling can be parsed into discrete problems and that our greatest challenge lies in dissecting the middle layer . relative phosphorylation levels of signaling proteins , as measured by immunoblotting , were calculated by first subtracting the level observed in the mock - treated , parental flp - in-293 cell line and then dividing each value by the maximum observed value for that site across the six cell lines . relative phosphorylation levels of signaling proteins , as measured by immunoblotting , were calculated by first subtracting the level observed in the mock - treated , parental flp - in-293 cell line and then dividing each value by the maximum observed value for that site across the six cell lines .

chemicals and fish.trichlorfon ( > 90% pure ) was purchased from shanghai biochemical reagent , shanghai , china . age of the trichlorfon used was 6 months . prior to the experiments , the crucian carp ( weight , 74.56 0.02 g [ mean se ] length , 17.03 0.13 cm ) were acclimatized to laboratory conditions in glass tanks ( 0.85 0.55 0.45 m ) for 2 weeks . experimental design.after acclimation , 75 fish were divided into five groups and for each group the experiment was conducted in triplicate ( three tanks per group ) . one of these groups was maintained in natural tap water and used as a control . the experimental aquaria were aerated , and chlorine concentrations in the water were measured daily and were below 0.05 mg / l . fish in the other aquaria were exposed to 0.5 , 1.0 , 2.0 , and 4.0 mg / l of trichlorfon . a concentration of 4.0 mg / l was 10% of the concentration lethal to 50% of a test population ( lc50 ) in 96 h for crucian carp . the water was changed every 12 h during the first 24-h exposure period to avoid adsorption on exposed surfaces ; the water was then changed daily until the end of the experiments ( tessier et al . the renewed water contained nominal exposure concentrations to ensure the desired concentrations of trichlorfon in the water . the half - life time of trichlorfon in water was 2.5 d , and the dissipation time of 95% of trichlorfon in water was 10.2 d ( lopes et al . composition ( ingredients ) and nutrition levels of basic diet of crucian carp ( based on dry matter ) . provided per kilogram of premix : copper sulfate 2.0 g , iron vitriol 25 g , zinc sulfate 22 g , manganous sulfate 7 g , sodium selenite 0.04 g , kalium iodide 0.026 g , cobaltous chloride 0.1 g , vitamin a 900,000 iu , vitamin d 200,000 iu , vitamin e 4,500 mg , vitamin k3 220 mg , vitamin b1 320 mg , vitamin b2 1,090 mg , vitamin b5 2,000 mg , vitamin b6 500 mg , vitamin b12 1.6 mg , vitamin c 10,000 mg , pantothenate ( vitamin b3 ) 1,000 mg , folic acid ( vitamin b11 ) 165 mg . fish were fed the designated diet at a ration of 3% wet body mass , twice daily for 30 d ( mattson et al . 1988 ; shakoori et al . 1996 ; ibrahim and el - gamal 2003 ) , and no fish mortality occurred during these exposures . fish were reared under the following water quality conditions : ph of 7.07.4 , photoperiod of 14 h dark : 10 h light , total ammonia nitrogen of 0.020.04 mg / l , and temperature of 2224c . sample preparation.at the end of the experimental period , two fish from each tank were caught randomly . blood was taken from the caudal vein with 2-ml heparinized syringes ( sodium heparin ) and centrifuged at 1,160 g for 15 min at 4c to separate the plasma . the hepatic sample for biochemical analysis was fully excised , washed with cold saline ( 0.85% nacl ) , and homogenated in 0.1 m tris - hcl buffer ( 1:9 w / v ) using a glass homogenizer at 4c . the homogenate was centrifuged at 2,370 g for 10 min at 4c , and the resultant supernatant was used as the enzyme source for the estimation of all enzyme activities . the hepatic samples for triglyceride contents and enzyme activities were carefully weighed and homogenized ( 1:10 dilution ) in ice - cold buffer with a teflon pestle attached to a motor - driven tissue - cell disrupter . the homogenization buffer solution was 0.02 m tris-0.01 m phosphate , ph 7.0 , in 50% ( v / v ) glycerol ( moro et al . the extract was later centrifuged at 2,500 rpm at 4c for 10 min , and the supernatant was used as the enzyme source . the hepatic samples for enzyme - linked immunosorbent assay ( elisa ) analysis were homogenated with 0.2 ml of 20 mm hepes buffer containing protease inhibitor ( phenylmethanesulfonyl fluoride , 1 mmol / l ) ( 1:4 w / v ) and disrupted using a glass tissue grinder . homogenates were centrifuged at 9,500 g for 10 min at 4c , and the resulting supernatants were transferred to 0.5-ml conical tubes . the hepatic sample for electron microscope observation was fixed with 0.25% glutaraldehyde in phosphate - buffered saline ( pbs ) ( ph 7.2 ) . biochemical analysis.plasma insulin was measured by radioimmunoassay ( ria ) using bonito ( bluefin tuna thunnus thynnus ) insulin as the standard and rabbit anti - bonito insulin as antiserum ( gutierrez et al . 1984 ) and expressed as iu / ml . briefly , 100 l aliquots of each sample and standard were added to tubes coated on the inner surface with an insulin antibody following the procedure similar to a triiodothyronine assay except that the tracer solution was i - labeled insulin and samples were incubated for 2 h. sample insulin levels were later determined according to the standard curve generated after a parallelism test ( li et al . 2012 ) . hepatic hormone - sensitive lipase ( hsl ) activity was assayed continuously by ph - stat titration of free fatty acid ( ffa ) release ( nilsson and belfrage 1979 ) and expressed as ng / mg protein . plasma and hepatic triglyceride contents were assayed by enzymatic procedures using an automatic biochemical analyzer ( hitachi 7170 , tokyo , japan ) and expressed as iu / ml ( souza et al . total proteins in liver and plasma were determined with coomassie brilliant blue g-250 staining according to the classical bradford method ( bradford 1976 ) . all detection kits were purchased from nanjing jian cheng biology company , nanjing , china . hepatic cyclic adenosine 3 , 5-monophosphate ( camp ) , very - low - density lipoprotein ( vldl ) , apolipoprotein b100 ( apo b100 ) and hsl contents were determined by elisa ( xu et al . 2009 ) using a double antibody sandwich method . detection kits were purchased from r&d systems china , shanghai , china . according to the manufacturer 's protocol , the optical density ( od ) of each well was determined by using an elisa reader at 450 nm and a cytochrome c calibration curve , and was expressed as ng / mg protein . hepatocyte morphological assay.for transmission electron microscope ( tem ) observations after treatment of fish with trichlorfon , the hepatic samples were fixed with 0.25% glutaraldehyde in pbs ( ph 7.2 ) for 4 h at 25c , then washed in cacodylate buffer and postfixed with 1% osmium tetroxide solution , dehydrated in a graded series of ethanol , infiltrated with propylene oxide , and embedded in epon . ultrathin sections were prepared , counterstained with 4% uranyl acetate and lead citrate ( shirpoor et al . 2009 ) , and observed using an h-7650 tem ( hitachi high - technologies , tokyo , japan ) . statistical analysis.data are expressed as means se . after testing for homogeneity of variance , statistical differences between the treatment and control groups were determined by a single - factor one - way anova followed by least - significant - difference multiple comparison tests when variances were homogeneous . when there was variance heterogeneity , multiple pairwise comparisons were made using tamhane 's t2 test . the concentration - response relationship for plasma and hepatic triglycerides are shown in figure 1a , b. plasma triglyceride contents were significantly ( p < 0.05 ) decreased in fish exposed to 1.0 , 2.0 , and 4.0 mg / l trichlorfon compared with the control and 0.5 mg / l trichlorfon . significant ( p < 0.05 ) increases in insulin content occurred in fish after treatments of 0.5 and 1.0 mg / l trichlorfon . in 2.0-mg / l treatments , plasma insulin contents were increased slightly ( p > 0.05 ) . as shown in table 2 , the concentration - response relationships of hepatic vldl , apo b100 , and camp were reduced in fish exposed to trichlorfon . hepatic vldl content was markedly ( p < 0.05 ) decreased at 2.0 mg / l trichlorfon compared with the control . no significant ( p > 0.05 ) differences in liver content of vldl in fish treated with 0.5 and 1.0 mg / l trichlorfon were observed . compared with the control , hepatic apo b100 contents tended ( p > 0.05 ) to decrease when trichlorfon concentrations were 0.5 , 1.0 , and 4.0 mg / l . no significant differences in the apo b100 contents were found in fish treated with 0.5 , 1.0 , and 4.0 mg / l trichlorfon . hepatic apo b100 content was significantly ( p < 0.05 ) decreased in fish exposed to 2.0 mg / l trichlorfon compared with the control . hepatic camp content was significantly ( p < 0.05 ) decreased at 2.0 and 4.0 mg / l trichlorfon compared with the control . effect of trichlorfon on hepatic very - low - density lipoprotein ( vldl ) , apolipoprotein b100 ( apo b100 ) , and cyclic adenosine 3,5-monophosphate ( camp ) contents in crucian carp . values ( mean se ) in the same column sharing different letters denote results significantly different from control ( p < 0.05 ) . effects of trichlorfon on crucian carp : ( a ) plasma triglyceride ( tg ) content ; ( b ) hepatic tg content ; ( c ) plasma insulin ( ins ) content . the changes in hepatic hsl content and activity are shown in figure 2a , b. there were no significant differences in hepatic hsl content in fish between the trichlorfon treatments and the control ( figure 2a ) . in the low trichlorfon concentration ( 0.5 mg / l ) , no significant difference in hsl activity was observed compared with the control . however , hsl activities in fish from the other treatment groups ( 1.0 , 2.0 and 4.0 mg / l ) were not detected ( figure 2b ) . effects of trichlorfon on crucian carp : ( a ) hepatic hormone - sensitive lipase ( hsl ) content ; ( b ) hepatic hsl activity . the cytomembrane was intact , and mitochondria and rough endoplasmic reticulum ( rer ) were abundant in the cytoplasm . rough endoplasmic reticulum was continuous with the external nuclear membrane and was concentrated in the perinuclear region . after treatment with 0.5 mg / l trichlorfon , dilatation of the mitochondrial matrix was observed ( figure 3b ) . in the 1.0-mg / l trichlorfon treatment , mitochondria were vacuolated and mitochondrial cristae were lost ( figure 3c ) . in the 2.0-mg / l trichlorfon treatment , swollen mitochondria and rer dilatation were present ( figure 3d ) . at 4.0 mg / l trichlorfon , broken mitochondrial membranes , mitochondrial vacuolization , loss of cytoplasm , and pyknotic nuclei were observed ( figure 3e ) . transmission electron microscope ( tem ) images of crucian carp hepatocyte structural organization after trichlorfon treatment . ( a ) control cells , nucleus ( n ) , mitochondria ( mi ) , and rough endoplasmic reticulum ( rer ) in cytoplasm ( tem , bar = 1 m ) ; ( b ) 0.5 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) ( tem , bar = 500 nm ) ; ( c ) 1.0 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) ( tem , bar = 1 m ) ; ( d ) 2.0 mg / l trichlorfon treatment , swelling of the mitochondria ( arrow up ) , dilatation of rer ( arrow right ) ( tem , bar = 1 m ) ; ( e ) 4.0 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) , loss of cytoplasm ( arrow down ) , and pyknotic nuclei ( arrow right ) ( tem , bar = 1 m ) . the concentration - response relationship for plasma and hepatic triglycerides are shown in figure 1a , b. plasma triglyceride contents were significantly ( p < 0.05 ) decreased in fish exposed to 1.0 , 2.0 , and 4.0 mg / l trichlorfon compared with the control and 0.5 mg / l trichlorfon . significant ( p < 0.05 ) increases in insulin content occurred in fish after treatments of 0.5 and 1.0 mg / l trichlorfon . in 2.0-mg / l treatments , plasma insulin contents were increased slightly ( p > 0.05 ) . as shown in table 2 , the concentration - response relationships of hepatic vldl , apo b100 , and camp were reduced in fish exposed to trichlorfon . hepatic vldl content was markedly ( p < 0.05 ) decreased at 2.0 mg / l trichlorfon compared with the control . no significant ( p > 0.05 ) differences in liver content of vldl in fish treated with 0.5 and 1.0 mg / l trichlorfon were observed . compared with the control , hepatic apo b100 contents tended ( p > 0.05 ) to decrease when trichlorfon concentrations were 0.5 , 1.0 , and 4.0 mg / l . no significant differences in the apo b100 contents were found in fish treated with 0.5 , 1.0 , and 4.0 mg / l trichlorfon . hepatic apo b100 content was significantly ( p < 0.05 ) decreased in fish exposed to 2.0 mg / l trichlorfon compared with the control . hepatic camp content was significantly ( p < 0.05 ) decreased at 2.0 and 4.0 mg / l trichlorfon compared with the control . effect of trichlorfon on hepatic very - low - density lipoprotein ( vldl ) , apolipoprotein b100 ( apo b100 ) , and cyclic adenosine 3,5-monophosphate ( camp ) contents in crucian carp . values ( mean se ) in the same column sharing different letters denote results significantly different from control ( p < 0.05 ) . effects of trichlorfon on crucian carp : ( a ) plasma triglyceride ( tg ) content ; ( b ) hepatic tg content ; ( c ) plasma insulin ( ins ) content . the changes in hepatic hsl content and activity are shown in figure 2a , b. there were no significant differences in hepatic hsl content in fish between the trichlorfon treatments and the control ( figure 2a ) . in the low trichlorfon concentration ( 0.5 mg / l ) , no significant difference in hsl activity was observed compared with the control . however , hsl activities in fish from the other treatment groups ( 1.0 , 2.0 and 4.0 mg / l ) were not detected ( figure 2b ) . effects of trichlorfon on crucian carp : ( a ) hepatic hormone - sensitive lipase ( hsl ) content ; ( b ) hepatic hsl activity . hepatocytes in control fish had a normal ultrastructure as seen in figure 3a . in normal hepatocytes , the cytomembrane was intact , and mitochondria and rough endoplasmic reticulum ( rer ) were abundant in the cytoplasm . rough endoplasmic reticulum was continuous with the external nuclear membrane and was concentrated in the perinuclear region . after treatment with 0.5 mg / l trichlorfon , dilatation of the mitochondrial matrix was observed ( figure 3b ) . in the 1.0-mg / l trichlorfon treatment , mitochondria were vacuolated and mitochondrial cristae were lost ( figure 3c ) . in the 2.0-mg / l trichlorfon treatment , swollen mitochondria and rer dilatation were present ( figure 3d ) . at 4.0 mg / l trichlorfon , broken mitochondrial membranes , mitochondrial vacuolization , loss of cytoplasm , and pyknotic nuclei were observed ( figure 3e ) . transmission electron microscope ( tem ) images of crucian carp hepatocyte structural organization after trichlorfon treatment . ( a ) control cells , nucleus ( n ) , mitochondria ( mi ) , and rough endoplasmic reticulum ( rer ) in cytoplasm ( tem , bar = 1 m ) ; ( b ) 0.5 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) ( tem , bar = 500 nm ) ; ( c ) 1.0 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) ( tem , bar = 1 m ) ; ( d ) 2.0 mg / l trichlorfon treatment , swelling of the mitochondria ( arrow up ) , dilatation of rer ( arrow right ) ( tem , bar = 1 m ) ; ( e ) 4.0 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) , loss of cytoplasm ( arrow down ) , and pyknotic nuclei ( arrow right ) ( tem , bar = 1 m ) . nearly all the reactions related to lipid metabolism and lipid transport occur in the liver ( fabbrini et al . the aim of the present study was to determine the effect of trichlorfon on lipid metabolism and lipid transport in crucian carp . the results showed that crucian carp hepatic concentration of triglycerides increased , and hepatic hsl activity and hepatic camp , vldl and apo b100 contents decreased in trichlorfon - exposure treatments . it is well known that the accumulation of lipid in hepatocytes represents a complex interaction , which includes a balance between triglyceride synthesis ( lipogenesis ) , hydrolysis ( lipolysis ) , and transport ( fabbrini et al . 2010 ) . during these processes , both metabolic enzyme activities and the formation of lipoprotein influence lipid deposition . ( 2007 ) found that gene expression of hepatic lipase and lipoprotein lipase ( lpl ) resulted in the release of ffas from lipolysis of circulating triglyceride , which contributed to hepatocellular ffa accumulation . triglycerides are hydrolyzed by cyclic amp - regulated lipases in the mitochondrial matrix ( yeaman 1990 ) in liver . hormone - sensitive lipase is a lipolysis rate limiting enzyme in lipid metabolism and mobilizes triglyceride and cholesterol ester stores in several tissues ( yeaman 2004 ) . in this experiment , hsl activities were inhibited significantly , and in the 1.0- , 2.0- , and 4.0-mg / l trichlorfon treatments , the activities were not detected in liver . however , there was no change in the hsl content in liver . the results illustrated that phosphorylation of hsl was inhibited , resulting in a decrease in hsl activity . hormone - sensitive lipase activity is triggered by the hormones epinephrine , norepinephrine , glucagon , and adrenocorticotropic hormone , and is transduced through the camp signaling pathway ( aboulaich et al . an increased level of camp stimulates protein kinase a , which activates the hsl by phosphorylating it ( berg et al . in contrast , insulin can inhibit camp synthesis , which results in phosphorylation of hsl degradation ( aboulaich et al . in addition to influencing hsl activity , insulin also activates lipoprotein lipases , promotes the entry of triglyceride into hepatocytes , and increases lipid deposition ( hillgartner et al . insulin may also induce zymoprotein synthesis , increase the related lipase activity , and increase lipid synthesis ( sessler et al . this resulted in a decrease in hepatic camp concentration followed by inhibition of hsl activity . however , the precise biological action in trichlorfon - induced disorders of insulin is unclear . ( 2007 ) studied that trichlorfon was involved in hormonal disruption , inhibited camp , protein kinase ( pka ) , follicle - stimulating hormone , and subsequent reproductive dysfunctions . additionally , hepatic lipid accumulation is also caused by an inability to form the lipoproteins responsible for transporting lipids out of the liver . water - insoluble triglycerides depend on apolipoprotein to be packaged into lipoprotein transport particles , and then re - exported to other tissues or stored in adipose tissue ( fabbrini et al . very - low - density lipoproteins are complex lipoprotein particles that are produced by the liver and secreted into the systemic circulation . these particles are mainly composed of triglyceride and apo b100 ( stryer 2002 ) . therefore , the quantities of apolipoprotein and vldl present are associated with lipid transport ( liang et al . related research has also indicated that fatty liver is associated with the change in apolipoprotein quantity ( fabbrini et al . our results showed that hepatic apo b100 content decreased with increasing trichlorfon concentration , which resulted in a decrease in the hepatic vldl content . hepatic triglyceride can not then be transported from the liver and is accumulated in the liver . ibrahim and el - gamal ( 2003 ) determined that diazinon may interfere with lipid metabolism in mammals . they found that the high - density lipoprotein cholesterol ( hdl - c ) and phospholipids ( pl ) were decreased , but the low - density lipoprotein cholesterol ( ldl - c ) and triglyceride were increased . a recent study showed that increased reactive oxygen species ( ros ) led to dna damage and generalized oxidative damage in all mitochondrial components , e.g. , oxidative mitochondrial dna ( mtdna ) damage ( franco et al . trichlorfon induced hepatocyte apoptosis and caused mitochondrial vacuolization and lipid droplet accumulation in the hepatocytes of crucian carp in vitro ( xu et al . ( 2009 ) , dilatation of the mitochondrial matrix , mitochondrial vacuolation , rer dilatation , and pyknotic nuclei were present , which might be caused by lpo . when hepatic ros production exceeds the antioxidant defense capacity of the cell , increased ros leads to lipid peroxidations and protein oxidations . these peroxidations can not be hydrolyzed by lipases and are accumulated in hepatocytes , subsequently leading to changes in hepatocyte ultrastructure , such as cytomembrane breakage , mitochondrial vacuolization , dilatation of the rer , and nuclei pyknotion ( xu et al . 2009 ) . ( 2009 ) studied that trichlorfon could cause fish ( pacu piaractus mesopotamicus ) hepatocyte fusion and loss of normal cellular shape . with trichlorfon concentration increase , there were also necrotic hepatic cells with pycnotic nuclei and decreased cytoplasmatic affinity for eosin , and the liver changes were more severe with the passage of time . the same lesions were described in the liver of curimbat prochilodus lineatus ( rodrigues et al . 2001 ) exposed to trichlorfon , 0.2 l / l ) and in callychthidae ( peppered corydoras corydoras paleatus ) ( fanta et al . the histological alterations in crucian carp livers suggest that the fish may face a metabolic crisis caused by tissue damage . hepatocellular ultrastructure damage could disturb cellular function and also be associated with apo b100 expression level , which could result in a reduction in apo b100 content . clinical studies indirectly support the fact that oxidant stress plays a substantial role in regulation of the output of apo b100 from hepatocytes ( pan et al . 2004 ) . karami - mohajeri and abdollahi ( 2011 ) reviewed the effect of pesticides , including organochlorine and carbamate compounds , on metabolic disorders and the underlying mechanism . results indicated that organophosphorus impairs the enzymatic pathways involved in metabolism of carbohydrates , fats , and protein within cytoplasm , mitochondria , and proxisomes . organochlorines mostly affect lipid metabolism in the adipose tissues and change glucose pathway in other cells . as a shared mechanism , all organophosphorus , organochlorine , and carbamate compounds induce cellular oxidative stress via affecting mitochondrial function and therefore disrupt the neuronal and hormonal status of the body . however , the precise biological action and molecular mechanism of organophosphorus in fish lipid metabolism are still unclear and need to be elucidated in further studies . trichlorfon influences hepatic pathways of metabolism and transportation and the ultrastructure of hepatocytes in crucian carp , which results in lipid accumulation in the liver . the dosage of trichlorfon used to eradicate ectoparasites varies from 0.1 to 1.0 mg / l in ponds , and the findings of this study show that lipid metabolism disorders can occur in crucian carp as a result of long - term exposure to low concentrations of trichlorfon in residual water .

this study evaluated the toxic effects of the organophosphate pesticide trichlorfon on hepatic lipid accumulation in crucian carp carassius auratus gibelio . seventy - five fish were divided into five groups ( each group in triplicate ) , and then exposed to 0 , 0.5 , 1.0 , 2.0 , and 4.0 mg / l of trichlorfon and fed with commercial feed for 30 d. at the end of the experiment , plasma and hepatic lipid metabolic biochemical status were analyzed . triglyceride contents were significantly ( p < 0.05 ) increased in liver but decreased in plasma after 1.0 , 2.0 , and 4.0 mg / l trichlorfon treatments . plasma insulin contents were markedly ( p < 0.05 ) increased when trichlorfon concentrations were 0.5 , 1.0 , and 4.0 mg / l . there were no significant differences in hepatic hormone - sensitive lipase contents between the trichlorfon - treated fish and the controls . hepatic cyclic adenosine 3 , 5-monophosphate , very - low - density lipoprotein , and apolipoprotein b100 contents were decreased in the fish when trichlorfon concentration was 2.0 mg / l . furthermore , electron microscope observations showed rough endoplasmic reticulum dilatation and mitochondrial vacuolization in hepatocytes with trichlorfon exposure . on the basis of morphological and physiological evidence , trichlorfon influenced crucian carp hepatic pathways of lipid metabolism and hepatocellular ultrastructure , which resulted in lipid accumulation in the liver .

Methods Results Plasma and Hepatic Triglyceride and Plasma Insulin Contents Hepatic VLDL, Apo B100, and cAMP Contents Hepatic HSL Content and Activity Hepatocellular Ultrastructure Discussion Conclusion

fish in the other aquaria were exposed to 0.5 , 1.0 , 2.0 , and 4.0 mg / l of trichlorfon . hepatic cyclic adenosine 3 , 5-monophosphate ( camp ) , very - low - density lipoprotein ( vldl ) , apolipoprotein b100 ( apo b100 ) and hsl contents were determined by elisa ( xu et al . the concentration - response relationship for plasma and hepatic triglycerides are shown in figure 1a , b. plasma triglyceride contents were significantly ( p < 0.05 ) decreased in fish exposed to 1.0 , 2.0 , and 4.0 mg / l trichlorfon compared with the control and 0.5 mg / l trichlorfon . compared with the control , hepatic apo b100 contents tended ( p > 0.05 ) to decrease when trichlorfon concentrations were 0.5 , 1.0 , and 4.0 mg / l . no significant differences in the apo b100 contents were found in fish treated with 0.5 , 1.0 , and 4.0 mg / l trichlorfon . hepatic camp content was significantly ( p < 0.05 ) decreased at 2.0 and 4.0 mg / l trichlorfon compared with the control . effect of trichlorfon on hepatic very - low - density lipoprotein ( vldl ) , apolipoprotein b100 ( apo b100 ) , and cyclic adenosine 3,5-monophosphate ( camp ) contents in crucian carp . ( a ) control cells , nucleus ( n ) , mitochondria ( mi ) , and rough endoplasmic reticulum ( rer ) in cytoplasm ( tem , bar = 1 m ) ; ( b ) 0.5 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) ( tem , bar = 500 nm ) ; ( c ) 1.0 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) ( tem , bar = 1 m ) ; ( d ) 2.0 mg / l trichlorfon treatment , swelling of the mitochondria ( arrow up ) , dilatation of rer ( arrow right ) ( tem , bar = 1 m ) ; ( e ) 4.0 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) , loss of cytoplasm ( arrow down ) , and pyknotic nuclei ( arrow right ) ( tem , bar = 1 m ) . the concentration - response relationship for plasma and hepatic triglycerides are shown in figure 1a , b. plasma triglyceride contents were significantly ( p < 0.05 ) decreased in fish exposed to 1.0 , 2.0 , and 4.0 mg / l trichlorfon compared with the control and 0.5 mg / l trichlorfon . compared with the control , hepatic apo b100 contents tended ( p > 0.05 ) to decrease when trichlorfon concentrations were 0.5 , 1.0 , and 4.0 mg / l . no significant differences in the apo b100 contents were found in fish treated with 0.5 , 1.0 , and 4.0 mg / l trichlorfon . hepatic camp content was significantly ( p < 0.05 ) decreased at 2.0 and 4.0 mg / l trichlorfon compared with the control . effect of trichlorfon on hepatic very - low - density lipoprotein ( vldl ) , apolipoprotein b100 ( apo b100 ) , and cyclic adenosine 3,5-monophosphate ( camp ) contents in crucian carp . ( a ) control cells , nucleus ( n ) , mitochondria ( mi ) , and rough endoplasmic reticulum ( rer ) in cytoplasm ( tem , bar = 1 m ) ; ( b ) 0.5 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) ( tem , bar = 500 nm ) ; ( c ) 1.0 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) ( tem , bar = 1 m ) ; ( d ) 2.0 mg / l trichlorfon treatment , swelling of the mitochondria ( arrow up ) , dilatation of rer ( arrow right ) ( tem , bar = 1 m ) ; ( e ) 4.0 mg / l trichlorfon treatment , vacuolization of mitochondria ( arrow up ) , loss of cytoplasm ( arrow down ) , and pyknotic nuclei ( arrow right ) ( tem , bar = 1 m ) .

there are few events in life that could be so dramatic and that could change a life in a second . spinal cord injury ( sci ) is a major trauma that affects people s lives . in the last few decades , extreme sports , violence , increased number of patients with tumors and chronic illnesses , general aging of population and the fact that elderly persons are injured easily , along with the significant technological development , make important risk factor for occurrence of sci . incidence rate ranges between 10.4 and 83 cases per million in one year , worldwide . in europe , the incidence is from 10.4 per million per year to 29.7 per million per year , while 27.1 was reported in asia ( 1 ) . recently published data indicate the incidence of 10.5 per million per year in tehran , iran ( 2 ) . incidence between 27.1 per million per year and 83 per million per year was observed in northern america ( 3 , 4 ) . the segment that is injured and the impairment severity will determine which body functions will be compromised or lost . consequently , temporary or permanent change in normal motor , sensory , or autonomic functions will occur . besides that , secondary complications such as diabetes , decubitus , thrombosis , hypertension and hypotension more frequently occur ( 5 ) . considering high rate of incidence , the increased survival rates following acquired injury , extended life expectance and expanded age limit related to the time of injury , the focus of outcomes in rehabilitation treatment have been redirected to the lifetime monitoring of the quality of life ( qol ) in persons with sci in the last few decades . despite this change in focus , the researches indicated that qol of persons with sci was not equivalent to those in their peers from typical population ( 6 ) . it has also been established that qol in persons with sci differ from country to country . in the research from 2010 , the authors published data about qol in persons with sci in australia , brazil , canada , israel , south africa and usa , and obtained results showed variations in qol from country to country ( 7 ) . it can be defined as the individual s personal perception of overall well - being and contentment in life including both psychosocial and physical or health - related domains ( 8 , 9 ) . as most people emphasize the health as the most important quality above others , there is a clear need for its evaluation in relation to the perception of qol . the health - related quality of life ( hrqol ) is perceived as a multidisciplinary concept that in addition to physical symptoms associated with the disease , should also embrace physical , physiological and social functioning ( 10 ) . having in mind the general recommendation of world health organization ( who ) to use new ways of assessment , hrqol was measured using the short form-36 health survey ( sf-36 ) ( 11 , 12 ) . moreover , the spinal cord injury quality of life questionnaire ( sci ql-23 ) ( 13 ) was used to assess qol in persons with sci in our study . up to date , there are no studies focused at qol of persons with sci in republic of serbia , except of partial studies of the certain diseases that lead to physical impairments ( 14 ) . by reviewing available literature , it was noted that population of persons with sci consisted mostly of young , especially active men in productive age ( 2 , 1520 ) . therefore , studies of this kind could contribute significantly to both developing and implementing of preventive measures , the improvement of rehabilitation interventions , as well as social planning . the aim of our study was to determine the difference in perceiving of qol between the participants with various levels of injury and the participants of typical population . this cross - sectional study included 100 participants of both gender , aged 18 - 65 years , residing in the territory of the republic of serbia . the general inclusion criteria for all participants were as follows : age from 18 to 65 years , negative history of any chronic medical problem , brain injury , significant congenital diseases , psychiatric disorders or somatic diseases . the general criterion for exclusion for all participants was subject s unwillingness to participate in the study . the total sample consisted of two subgroups of participants with sci and one control group . the first group ( e-1 ) consisted of 23 participants with paraplegia . specific inclusion criterion for e-1 group was diagnosed injury at thoracic , lumbar or sacral level of spinal cord ( diagnosis of paraplegia ) . specific inclusion criterion for e-2 group was diagnosed injury at cervical level of spinal cord ( diagnosis of tetraplegia ) . common criterion for both e-1 and e-2 group was related to the period of time from injury occurrence to conduction of present study which had to be longer than one year , whereas all participants should had been treated in an inpatient ward for rehabilitation after sci for at least six months . in addition to the above mentioned general inclusion and exclusion criteria , a method of purposive or convenience sampling of control group was used . therefore , control group was not representative of the general population . control group included 56 participants of typical population ( healthy participants without sci or any other physical impairment ) . the study was conducted during november and december 2013 at the clinic for rehabilitation dr miroslav zotovic and the health centre dr simo milosevic in belgrade , serbia . the general inclusion and exclusion criteria for all research groups were applied , as well as the specific inclusion criterion for two subgroups of participants with sci . all patients that were present at the clinic for rehabilitation and the health centre during the time period of this research and met the criteria listed above were included . medical records were reviewed in order to exclude potential participants with medical conditions , impairments or illness that could affect the research results . participation was voluntary , and written informed consent was obtained from all participants . after the sample was formed , data on health status , time and cause of injury , age , gender , and diagnosis of paraplegia or tetraplegia ( for both e-1 and e-2 group ) were extracted from medical records . in the second phase , each participant was provided a peaceful and quiet setting in a separate room within the clinic . participants responded to questions individually , in one session that took no more than 90 minutes . if needed , the time was extended or divided into two time intervals . assistance of the first was available all the time . for assessing of hrqol , the serbian version of sf-36 was used ( 21 ) . as a generic measure of hrqol , this questionnaire provides an acceptable , psychometrically correct and efficient way of measuring qol from the patient s point of view . the sf-36 consists of thirty - six items grouped in eight scaled scores and measures eight dimensions of different health domains . the scale sections include physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional and mental health . in accordance to recommendation scores can range from zero ( worst possible health state ) to 100 ( best possible health state ) , and higher values indicate better functioning and well - being . the sci ql-23 is self - report questionnaire designed for assessing of qol in persons with sci . this questionnaire is derived from a comprehensive battery of general and specific questionnaires applied in numerous studies of persons with sci ( 13 , 2224 ) . the sci ql-23 consists of 23 statements / questions , 22 of which contain three variables of physical , mental and social functioning . the sci ql-23 includes the functioning scale ( ten items assessing physical and social functional limitations in mobility , body care , movement and social interaction ) , the mood scale ( six items concerning anxiety and depressive feelings ) , the problems related to the injury ( six items describing the perception with loss of physical independence , sci - related complications and associated social stigma ) , and global quality of life ( indicates overall rating of life situations ) . the entire sci ql-23 questionnaire was applied only on participants in e-1 and e-2 groups . it was used in order to measure the level of qol and determine differences in perceived qol among participants with sci . the study protocol was approved by the ethical committee of clinic for rehabilitation dr miroslav zotovic , the ethical issues in this study included introducing the researcher to each participant and giving necessary explanation on study purpose and content of questionnaires . participants were also informed that they could withdraw from the study at any time , and they were ensured about confidentiality and privacy of information . statistical analysis of data collected from the documentation , survey and assessment of persons with sci , consisted of a simple descriptive analysis ( calculation of frequencies , means , and standard deviation ) and inferential statistics . mann - whitney u - test for planned comparison between groups , as a nonparametric technique , was applied for the reason that basic assumptions underlying t - test and anova had not been fulfilled ( sample size , distribution normality , linearity ) . in addition , test was used in order to determine differences between research groups according to gender and age of participants . a p value of < 0.05 was considered statistically significant . in the part of preliminary analysis , the cronbach s alpha coefficients were calculated . all analyses were completed using statistical package for the social sciences for windows ( spss ) version 19.0 . this cross - sectional study included 100 participants of both gender , aged 18 - 65 years , residing in the territory of the republic of serbia . the general inclusion criteria for all participants were as follows : age from 18 to 65 years , negative history of any chronic medical problem , brain injury , significant congenital diseases , psychiatric disorders or somatic diseases . the general criterion for exclusion for all participants was subject s unwillingness to participate in the study . the total sample consisted of two subgroups of participants with sci and one control group . the first group ( e-1 ) consisted of 23 participants with paraplegia . specific inclusion criterion for e-1 group was diagnosed injury at thoracic , lumbar or sacral level of spinal cord ( diagnosis of paraplegia ) . specific inclusion criterion for e-2 group was diagnosed injury at cervical level of spinal cord ( diagnosis of tetraplegia ) . common criterion for both e-1 and e-2 group was related to the period of time from injury occurrence to conduction of present study which had to be longer than one year , whereas all participants should had been treated in an inpatient ward for rehabilitation after sci for at least six months . in addition to the above mentioned general inclusion and exclusion criteria , a method of purposive or convenience sampling of control group was used . therefore , control group was not representative of the general population . control group included 56 participants of typical population ( healthy participants without sci or any other physical impairment ) . dr miroslav zotovic and the health centre dr simo milosevic in belgrade , serbia . the general inclusion and exclusion criteria for all research groups were applied , as well as the specific inclusion criterion for two subgroups of participants with sci . all patients that were present at the clinic for rehabilitation and the health centre during the time period of this research and met the criteria listed above were included . medical records were reviewed in order to exclude potential participants with medical conditions , impairments or illness that could affect the research results . participation was voluntary , and written informed consent was obtained from all participants . after the sample was formed , data on health status , time and cause of injury , age , gender , and diagnosis of paraplegia or tetraplegia ( for both e-1 and e-2 group ) were extracted from medical records . in the second phase , each participant was provided a peaceful and quiet setting in a separate room within the clinic . participants responded to questions individually , in one session that took no more than 90 minutes . if needed , the time was extended or divided into two time intervals . for assessing of hrqol , the serbian version of sf-36 was used ( 21 ) . as a generic measure of hrqol , this questionnaire provides an acceptable , psychometrically correct and efficient way of measuring qol from the patient s point of view . the sf-36 consists of thirty - six items grouped in eight scaled scores and measures eight dimensions of different health domains . the scale sections include physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional and mental health . in accordance to recommendation scores can range from zero ( worst possible health state ) to 100 ( best possible health state ) , and higher values indicate better functioning and well - being . the sci ql-23 is self - report questionnaire designed for assessing of qol in persons with sci . this questionnaire is derived from a comprehensive battery of general and specific questionnaires applied in numerous studies of persons with sci ( 13 , 2224 ) . the sci ql-23 consists of 23 statements / questions , 22 of which contain three variables of physical , mental and social functioning . the sci ql-23 includes the functioning scale ( ten items assessing physical and social functional limitations in mobility , body care , movement and social interaction ) , the mood scale ( six items concerning anxiety and depressive feelings ) , the problems related to the injury ( six items describing the perception with loss of physical independence , sci - related complications and associated social stigma ) , and global quality of life ( indicates overall rating of life situations ) . the entire sci ql-23 questionnaire was applied only on participants in e-1 and e-2 groups . it was used in order to measure the level of qol and determine differences in perceived qol among participants with sci . the study protocol was approved by the ethical committee of clinic for rehabilitation dr miroslav zotovic , belgrade , serbia . the ethical issues in this study included introducing the researcher to each participant and giving necessary explanation on study purpose and content of questionnaires . participants were also informed that they could withdraw from the study at any time , and they were ensured about confidentiality and privacy of information . statistical analysis of data collected from the documentation , survey and assessment of persons with sci , consisted of a simple descriptive analysis ( calculation of frequencies , means , and standard deviation ) and inferential statistics . mann - whitney u - test for planned comparison between groups , as a nonparametric technique , was applied for the reason that basic assumptions underlying t - test and anova had not been fulfilled ( sample size , distribution normality , linearity ) . in addition , test was used in order to determine differences between research groups according to gender and age of participants . a p value of < 0.05 was considered statistically significant . in the part of preliminary analysis , the cronbach s alpha coefficients were calculated . all analyses were completed using statistical package for the social sciences for windows ( spss ) version 19.0 . of these , 56 participants were from the general population ( without sci ) , 23 participants had an injury at thoracic , lumbar or sacral level of spinal cord ( paraplegia ) , while 21 participants had cervical sci and functional diagnosis of tetraplegia . the presence of male participants was dominant in all three groups : 90.5% ( e-2 ) , 73.9% ( e-1 ) , and 75.1% ( controls ) , yet without statistically significant difference ( =2.422 ; df=2 ; p=0.298 ) . at the time of study , the average age of participants in control group was 39.20 years ( sd=14.88 ) . the average age of participants in e-1 and e-2 group was 45.43 years ( sd=10.56 ) and 41.38 years ( sd=12.89 ) , respectively . there was no statistically significant difference between this three research groups in relation to age groups [ =5.124 ; df=6 ; p=0.528 ] . distribution of participants according to gender and age groups the most common cause of sci in e-1 group was tumors ( 34.8% ) . unidentified causes such as injuries on work , electric shocks , etc . accounted for 26.1% of cases . the most common cause of sci in e-2 group was water diving ( 42.9% ) , followed by traffic accidents ( 28.6% ) and tumors ( 19% ) , while violence and falls due to weakness or aging process were etiological factors in 4.8% of cases . the value of 0.848 was recorded on sci ql-23 , while the value of 0.869 was found on sf-36 , both indicating a high reliability . table 2 shows the sf-36 score values of participants with and without sci . in the group of participants with sci , the lowest mean score was noted in physical functioning domain ( 10.1111.49 ) , as opposed to the group of healthy controls in which the highest score was found in that same qol domain ( 89.7315.51 ) . participants with sci reported the highest score of qol in domain of emotional problems ( 72.7336.85 ) . on the other hand , in the group of healthy controls , the lowest mean statistically significant differences were confirmed on the following subscales : physical functioning ( u=5.000 , z=-8.643 , p<0.001 ) , role - physical ( u=571.000 , z=-5.023 , p<0.001 ) , bodily pain ( u=663.000 , z=-4.011 , p<0.001 ) , vitality ( u=799.500 , z=-3.017 , p<0.005 ) , social functioning ( u=799.000 , z=-3.059 , p<0.005 ) , and mental health ( u=816.500 , z=-2.894 , p<0.005 ) . however , the statistical significance of the difference was not confirmed in domain of general health ( u=1168.500 , z=-0.447 , p=0.655 ) , and role - emotional ( u=1063.000 , z=-1.372 , p=0.170 ) . the greatest extent of range of responses was noted in both groups on role - physical and role - emotional subscale . in addition , in the group of participants with spinal cord injury , the greatest extent of range of responses was also found on bodily pain subscale and social functioning subscale . comparisons of the short form-36 health survey ( sf-36 ) subscale scores between participants with and without spinal cord injury when it comes to a global measure of physical and mental functioning , participants with sci reported lower score in physical domain ( 29.576.57 ) when compared to score in domain of mental functioning ( 55.118.83 ) ( table 3 ) . on the other hand , participants without sci reported similar scores in overall physical and mental domains , 50.406.57 and 49.927.27 , respectively . results obtained by mann - whitney u - test indicated that the groups of participants with sci and participants without sci differed with statistical significance in both overall physical domain ( u=48.000 , z=-8.222 , p<0.001 ) and mental domain ( u=767.000 , z=-3.229 , p<0.005 ) . however , the extent of responses in overall physical domain in the groups of participants with sci was lower when compared with the range of responses of healthy controls . on the other hand , the extent of responses in overall mental ability in the groups of healthy controls was lower when compared with the range of responses of participants with sci . comparisons of the short form-36 health survey ( sf-36 ) summary scores between participants with and without spinal cord injury as shown in table 4 , the low mean scores found on the mood scale of sci ql-23 in groups of both participants with tetraplegia and paraplegia ( 31.2216.05 and 36.2325.12 , respectively ) reflect greater perceived qol in this domain . in contrast , qol was perceived as lower in domain of functioning and in the area of problems related to the injury . however , the only one statistically significant difference between participants with tetraplegia and participants with paraplegia was noted on the functioning scale ( u=103.000 , z=-3.256 , p<0.005 ) . the mean score in the subgroup of participants with tetraplegia was 73.37 ( sd=20.80 ) , while the mean score in the subgroup of participants with paraplegia was 44.07 ( sd=29.11 ) . no statistically significant difference was found on other tree subscales ( mood scale , problems related to the injury scale , global quality of life ) . on all subscales of sci ql-23 , participants with paraplegia had scores in the range that was wider than the range of values of answers of participants with tetraplegia . comparisons of the spinal cord injury quality of life questionnaire ( sci ql-23 ) subscales scores participants with tetraplegia and participants with paraplegia table 5 presents the scores of global quality of life scale of sci ql-23 . the mean score noted in the groups of participants with sci was lower than the mean score in healthy controls , 52.276.57 and 81.856.57 , respectively . it is also important to note that scores in the group of participants with sci ranged from 0.00 to 83.33 , while the extent of this range in the group of healthy controls was lesser ( from 43.33 to 100.00 ) . comparisons of global quality of life scale scores between participants with and without spinal cord injury in this study , the differences between perception of qol among participants with sci and participants without sci have been observed , as well as among participants with different levels of injury . by analyzing the differences between sf-36 scores obtained in the group of participants with sci and participants without sci ( table 2 ) , a negative influence of sci was detected on six of the eight sf-36 subscales : physical functioning , role - physical , bodily pain , vitality , social functioning and mental health . on the other hand , subscales related to general health and emotional role did not show statistically significant differences between groups . statistically significant results were not found only on two subscales ( general health and role - emotional subscales ) . most studies involved in hrqol research , usually with the sf-36 , show that persons with sci have expectedly lower scores in physical functioning compared to general population . similar data have been detected when it comes to bodily pain , social functioning and mental health ( 20 ) . however , unlike other studies , participants with sci identified general health and emotional issues approximately equal as those from the typical population in present research . similar results were obtained in a study conducted in sweden and australia in which the same methodology was used . specifically , kreuter , sisteen , erkholm et al . ( 22 ) identified the highest difference in both physical functioning and role - physical subscales between group of participants with sci and group of participants without sci , which were results equivalent to results presented in this study . in addition , statistically significant differences were observed on both social functioning and mental health subscales . however , on the role - emotional subscale , the answers of participants from serbia did not match the answers of participants from sweden and australia . next , the physical component summary and the mental component summary , which represent total physical and mental abilities , were compared between group of participants with sci and control group . arithmetic means have reached different values ( table 3 ) , therefore resulting with differences in total physical domain and overall mental abilities . moreover , the results obtained on the scale of mental component indicated that the minimum score in sci group was higher than the same of control group ( 30.34 versus 27.50 ) . similarly , the maximal score achieved in sci group was higher in comparison to control group ( 68.56 versus 59.83 ) . such a distribution of scores had further caused occurrence of greater differences in favor of sci group . these results could be interpreted by the fact that questionnaire sf-36 represents a self - assessment of qol of participants , more precisely of both physical and mental components of their qol . the participants with sci were satisfied to a greater extent with their own participation in life situations , socializing , going out , etc . in contrast , participants of typical population considered that they were not satisfied with their qol in these domains , which further suggested that their wishes and demands were rather higher or different compared to population of person with sci . the additional assessment of qol the degree of differences in qol within participants with sci in relation to the level of injury was determined . 4 related to the self - assessment of the general qol , i.e. global qol . as presented in table 4 , there were no statistically significant differences between participants with tetraplegia and participants with paraplegia on the mood scale , the problems related to the injury scale and the global qol scale . in contrast , on the functioning scale , groups of participants with tetraplegia had achieved the mean value of 73.37 ( sd=20.80 ) , whereas the group of participants with paraplegia achieved the mean of 44.07 ( sd=29.11 ) which indicated a statistically significant difference ( u=103.000 , z=-3.256 , p<0.005 ) . the obtained results demonstrated that participants with tetraplegia showed lower results on the subscale of physical and social dysfunctions than participants with ( paraplegia . persons with preserved function of upper limbs , as it was expected , showed higher level of both physical and social independence , compared to persons who did not have the upper or lower limbs functionality . regarding mood , independence and general qol , all participants with sci showed approximate values , which indicates that the level of injury is related to the minimal connection with the qol assessment , which was detected in similar previous researches ( 20 , 22 , 25 , 26 ) . when it comes to general qol assessed by global qol subscale , participants with sci had lower observed mean scores in comparison to controls ( 52.26.57 and 81.86.57 , respectively ) . this difference reached a level of statistical significance ( u=415.000 , z=-5.804 , p<0.000 ) . considering that higher global qol scores reflect greater perceived qol , and based on the obtained results , it can be concluded that participants without sci perceived their qol at a higher level compared to those with sci . the same questionnaire was used in assessing of qol of persons with sci and control group from typical population the study conducted in australia and sweden ( 22 , 27 ) . the obtained results were equivalent to research results presented here because both groups of sci participants ( sweden and australia ) estimated their global qol on a significantly higher level in relation to the control group . in addition , it should be noted that statistically significant difference ( p<0.0001 ) between the group of participants with sci and control group from australia and sweden was detected on the mood scale , whereas such a difference was not confirmed in this study . the concept of qol contains different domains . however , it is obvious that not all of them are equally affected when it comes to population of persons with sci . research results presented here indicate that when the impairment occurs as a consequence of acquired sci , perceived qol is changed . the participants of typical population perceived their general qol on a higher level in comparison to participants with sci . in addition , when it comes to hrqol , the group of participants of typical population perceived their qol at a higher level in most cases . thus , differences are not as big as it might be expected . by observing the participants in relation to the level of injury , it can be concluded that level of injury could determine the self - perception of qol , yet in a small degree . differences were evident on the physical and social dysfunction scales , as expected , and clearly defined by the level of injury . even so , in all other areas of functioning , no relationship was found between level of injury and the perceived qol . in other words , self - reported qol is not necessarily determined by the level of sci . considering that this study was focused on initial exploration of self - reported health status and qol in persons with sci , and given that in the republic of serbia , up to date , no studies have dealt with this topic , it should be emphasized that forming of an overall picture represents only a starting point for future studies . first , sample was relatively small and therefore did not allow detailed examination of the differences . a more detailed picture of connections between different domains of qol in addition , all data were self - reported , thus individuals may not have always expressed all of their perceptions . moreover , regardless of given diagnosis of paraplegia and tetraplegia , there is a general heterogeneity in population of persons with sci . this heterogeneity limits generalizability of findings . as recently reported , newly injured persons with sci encounter numerous barriers during the first year post injury ( 28 ) . perception of barriers to community reintegration that persons with sci experience should be included in future studies , as well as the availability of facilities for persons with sci . finally , future research should include other factors that could influence qol of persons with sci , such as place of residence , age , marital status , family cohesion , occupation and employment , income and economic conditions , different factors of social protection and welfare , social support , etc . regardless of the injury level , person with sci perceive their qol and hrqol at a lower level in comparison to healthy population . the results of this study suggest that a comprehensive rehabilitation should include focuses on increasing of physical functioning and vitality level , enhancing of independence within the activities of daily living , reducing of bodily pain , and improving of both social functioning and mental health . this study has important practical implications in the design of further interventions in rehabilitation and reintegration of persons with sci . considering the growing population of people with sci , more studies are needed in order to provide a good basis for developing long - term strategies of improving the qol . the contribution is evident in the development of methods of post - traumatic rehabilitation , and in the prevention of occurrence of injuries , in general , as well as consequential social isolation . 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 .

abstractbackgroundduring the last few decades , focus of rehabilitation outcome has been redirected to the lifetime monitoring of quality of life . the purpose of this study was to investigate the differences in quality of life perceptions between participants with spinal cord injury and participants of typical population.methodsthis cross - sectional controlled study of 100 adults aged 18 - 65 years was based on two questionnaires , short form-36 health survey ( sf-36 ) and spinal cord injury quality of life questionnaire ( ql-23 ) , completed by 23 participants with paraplegia , 21 participants with tetraplegia , and 56 participants of typical population . mann - whitney u - test for planned comparison between groups and 2 test were used to analyze the differences between research groups.resultsparticipants from control group perceived their general quality of life at higher level in comparison to participants with spinal cord injury ( u=415.000 , z=-5.804 , p<0.000 ) . negative influence of spinal cord injury was detected in six domains ( physical functioning , physical role , bodily pain , vitality , social functioning , mental health ) . statistical differences between participants with paraplegia and participants with tetraplegia only in domain of functional limitations ( u=103.000 , z=-3.256 , p<0.005).conclusionthe participants with spinal cord injury perceived both health - related and general quality of life at a lower level in comparison to controls . however , the injury level only partially determined the estimated quality of life .

Introduction Methods Participants Procedure Measures Ethical notes Statistics Results Discussion Conclusion Ethical considerations

considering high rate of incidence , the increased survival rates following acquired injury , extended life expectance and expanded age limit related to the time of injury , the focus of outcomes in rehabilitation treatment have been redirected to the lifetime monitoring of the quality of life ( qol ) in persons with sci in the last few decades . the scale sections include physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional and mental health . the sci ql-23 includes the functioning scale ( ten items assessing physical and social functional limitations in mobility , body care , movement and social interaction ) , the mood scale ( six items concerning anxiety and depressive feelings ) , the problems related to the injury ( six items describing the perception with loss of physical independence , sci - related complications and associated social stigma ) , and global quality of life ( indicates overall rating of life situations ) . mann - whitney u - test for planned comparison between groups , as a nonparametric technique , was applied for the reason that basic assumptions underlying t - test and anova had not been fulfilled ( sample size , distribution normality , linearity ) . the scale sections include physical functioning , role - physical , bodily pain , general health , vitality , social functioning , role - emotional and mental health . mann - whitney u - test for planned comparison between groups , as a nonparametric technique , was applied for the reason that basic assumptions underlying t - test and anova had not been fulfilled ( sample size , distribution normality , linearity ) . on the other hand , in the group of healthy controls , the lowest mean statistically significant differences were confirmed on the following subscales : physical functioning ( u=5.000 , z=-8.643 , p<0.001 ) , role - physical ( u=571.000 , z=-5.023 , p<0.001 ) , bodily pain ( u=663.000 , z=-4.011 , p<0.001 ) , vitality ( u=799.500 , z=-3.017 , p<0.005 ) , social functioning ( u=799.000 , z=-3.059 , p<0.005 ) , and mental health ( u=816.500 , z=-2.894 , p<0.005 ) . comparisons of the short form-36 health survey ( sf-36 ) subscale scores between participants with and without spinal cord injury when it comes to a global measure of physical and mental functioning , participants with sci reported lower score in physical domain ( 29.576.57 ) when compared to score in domain of mental functioning ( 55.118.83 ) ( table 3 ) . results obtained by mann - whitney u - test indicated that the groups of participants with sci and participants without sci differed with statistical significance in both overall physical domain ( u=48.000 , z=-8.222 , p<0.001 ) and mental domain ( u=767.000 , z=-3.229 , p<0.005 ) . comparisons of the short form-36 health survey ( sf-36 ) summary scores between participants with and without spinal cord injury as shown in table 4 , the low mean scores found on the mood scale of sci ql-23 in groups of both participants with tetraplegia and paraplegia ( 31.2216.05 and 36.2325.12 , respectively ) reflect greater perceived qol in this domain . however , the only one statistically significant difference between participants with tetraplegia and participants with paraplegia was noted on the functioning scale ( u=103.000 , z=-3.256 , p<0.005 ) . comparisons of the spinal cord injury quality of life questionnaire ( sci ql-23 ) subscales scores participants with tetraplegia and participants with paraplegia table 5 presents the scores of global quality of life scale of sci ql-23 . comparisons of global quality of life scale scores between participants with and without spinal cord injury in this study , the differences between perception of qol among participants with sci and participants without sci have been observed , as well as among participants with different levels of injury . by analyzing the differences between sf-36 scores obtained in the group of participants with sci and participants without sci ( table 2 ) , a negative influence of sci was detected on six of the eight sf-36 subscales : physical functioning , role - physical , bodily pain , vitality , social functioning and mental health . in contrast , on the functioning scale , groups of participants with tetraplegia had achieved the mean value of 73.37 ( sd=20.80 ) , whereas the group of participants with paraplegia achieved the mean of 44.07 ( sd=29.11 ) which indicated a statistically significant difference ( u=103.000 , z=-3.256 , p<0.005 ) . the participants of typical population perceived their general qol on a higher level in comparison to participants with sci .

solution x - ray scattering techniques are important for studying the conformations of molecules in solution . time - resolved x - ray scattering has been developed to study the structural dynamics of molecular reactions . in the experiment , a reaction is triggered and scattering is recorded as a function of reaction time . time - resolved data are displayed and interpreted as difference scattering patterns , where the scattered intensity of a reference state is subtracted from that of a certain time point during the reaction . initially , time - resolved scattering studies focused on photoreactions of small molecules . first , time - resolved scattering during carboxy - hemoglobin photolysis and the photoreaction of bacteriorhodopsin and proteorhodopsin were reported . several other investigations were conducted on the structural dynamics of , for example , myoglobin , photoactive yellow protein , and a phytochrome . these studies were carried out at synchrotron sources and covered time scales from 100 ps to seconds . the availability of ultrashort x - ray bursts at free electron lasers has enabled time - resolved scattering studies with femtosecond time resolution . today , time - resolved x - ray scattering experiments on proteins can be carried out reliably , but they are usually limited by data interpretation . difference scattering reflects a difference in pair distribution functions and thus encodes the structural change of the molecules . however , the information content in the one - dimensional spherically averaged ( isotropic ) scattering curves is inherently low . as in conventional small - angle x - ray scattering ( saxs ) , a curve typically contains only tens of independent data points . unlike saxs , difference x - ray scattering has the advantage that the protein hydration shell typically only gives a minor contribution to the scattering signal . a drawback is that a resting state structure , the starting point of structural change , is required for structural interpretation . structural rearrangements can be uniquely identified from difference scattering data , when only a few atoms participate in the reaction . for proteins with many hundreds , often thousands , of atoms , fitting atomistic models without additional structural information earlier attempts to surmount this challenge used rigid body refinement , dynamic annealing of pseudo atoms , and selection of suitable frames from molecular simulations . in all cases , many candidate structures are generated , the scattering of each is computed , and the best fits to the experimental scattering data are identified . since the sampling in conformational space is usually limited , uniqueness of the solutions is not guaranteed . the ideal method to identify structural change from difference scattering data would be to harvest its limited information content while simultaneously taking chemical information , such as bonded and nonbonded interactions , into account . biomolecular force fields encode such chemical information , and are used in molecular dynamics ( md ) or monte carlo ( mc ) simulations that sample thermal ensembles of proteins in solution . in structural biology , many experimental techniques are increasingly aided by molecular simulation for data interpretation . refinement by simulation of protein structure based on x - ray diffraction ( xrd ) data is now commonplace . in nmr spectroscopy , the use of md with experimental restraints has been even more instrumental , since such experiments have historically not supplied a sufficient amount of data to determine a protein structure from scratch . the use of data from small - molecule crystal structures , restraints found from sequence and chemical shift homology , and molecular mechanics force fields supplied the missing information . more recently , orientational restraints from residual dipolar couplings have been used to refine protein structures from nmr data and to evaluate the performance of unrestrained simulations . conventional saxs data are usually interpreted by ab initio reconstruction of low - resolution envelopes or by assembly of rigid high - resolution elements . they also do not use chemical knowledge beyond rudimentary considerations to identify likely conformations . a notable exception is the attempt by grishaev et al . to improve nmr refinement by also taking saxs intensities into account . as with nmr data , absolute saxs intensities have also been used for after - the - fact validation of md trajectories . to our knowledge , no systematic attempts to interpret difference x - ray scattering data with atomistic force fields have been published to date . here , we describe and implement x - ray scattering - guided molecular dynamics simulations ( xs - guided md ) , which is a technique that refines structures against difference scattering data . the method can be used to guide proteins in md simulations from one structure to another , and therefore is well - suited for elucidating the structural dynamics of biomolecules . in what follows , we shall only be concerned with difference x - ray scattering data , and as explained and justified below , neglect the scattering contribution from the protein s solvation layer . the method uses an additional pseudo - energy term , based on the debye scattering equation , which guides the simulation toward states that reproduce target data . the energy landscape of the simulation is biased toward the experimentally observed conformation and distinctions can be made between alternative conformations with similar free energies . as a result , conformers , which are not normally seen in simulations either because they are transient species observed in dynamic experiments , because they are kinetically inaccessible or because they are incorrectly disfavored by the force field can potentially be observed and characterized in md simulations . the algorithm reduces the computational time to determine the structures that agree with experiment , and retains the chemical knowledge contained in md force fields . the method is implemented in the popular and freely available gromacs simulations package and is open to further development . identical but randomly oriented collections of spherical scatterers give rise to a scattered intensity described by the debye formula.1here , fi is the scattering factor for scatterer i , q is the magnitude of the scattering vector ( q = 4 sin / , with 2 the scattering angle ) , and rij = |rij| = |ri rj| , with ri the coordinate vector for particle i. for molecules , the sums typically run over all atoms . for an actual protein solution , one typically measures the excess scattering of the solution compared to the solvent . in such cases , eq equation 1 ignores the fact that the solvent and electrolyte densities around a macromolecule are generally different from their bulk values , which , in principle , leads to additional scattering terms . to our knowledge , taking this into account involves either ( i ) making extensive and explicit simulations for each scattering evaluation or ( ii ) making ad hoc assumptions about excess surface densities . we ignore this effect , which is an approximation that appears to be reasonable when the data are recorded as differences between two states , s(q ) = s(q ) then , solvation contributions largely cancel . for biasing md simulations toward configurations that agree with experimental scattering curves , following ahn et al . , an additional term ( uxs ) is added to the usual md potential energy ( umd):4here , k and q are weighting factors for the scattering bias and for every q - point , respectively . scalc(q ) is the scattering computed from the current state of the simulation . for refinement against difference data , sexp(q ) is the experimental difference curve , sref(q ) is the scattering from the starting structure and is the fraction of the observed sample that undergoes conformational change . for refinement against absolute data , sexp(q ) the total force on a particle k is5the first term on the right is given by the force field , so the present problem reduces to finding k . the initial structure and experimental data are the same throughout a simulation , so sref(q ) and sexp(q ) are constant and ksref(q ) = ksexp(q ) = 0 . thus,6the last step is to determine the vector kscalc(q ) , which , by taking the appropriate derivatives of eq 1 , comes out as7finally , the force contribution from the scattering energy term acting on particle k is obtained by combining eqs 5 , 6 , and 7:8calculating these forces amounts to evaluating two double sums over all scatterers , first for { scalc(q ) sref(q ) } , as in eq 1 , then for the forces , as in eq 8 . for convenience , we define9which gives10 when evaluating the debye equation , the resulting intensity carries units given by the atomic scattering factors . , that is , as relative scattering amplitudes referenced to a classical unit point charge . if concentrations , path lengths , incoming x - ray intensities , and detector efficiencies are precisely known , experimentally measured intensities can be converted to such units . however , this is often not the case in practice , and experimental data are instead scaled to predicted curves . for absolute data , this is straightforward . either the experimental curve is scaled to each calculated profile , or it is rescaled such that the experimental extrapolation of s(q = 0 ) matches the calculated value . the latter is independent of conformation ( see eq 1 ) . for time - resolved difference measurements scaling is more problematic and involves a parameter ( eq 4 ) . if the experimental system has pure initial and final states with i(q ) = si and sf , and is initially distributed so that the relative populations over these states are 0 and 1 0 , then with a relative yield ,provided that the input sexp(q ) has meaningful units ( this can be approximately achieved by scaling the detector reading to scalc(q ) and then scaling sexp(q ) by the same number ) , the parameter in eq 4 can be identified with the relative yield of the difference experiment . xs - guided md is implemented in gromacs 5 and , therefore , profits from the efficient core of gromacs . moreover , it can , in principle , be used in combination with any other featured algorithm , for example , with the simulated annealing method or replica exchange method , to improve sampling . the debye scattering terms are represented as bonded interactions of a new type ( pairs type 3 ) . any atom or virtual interaction site can act as a scatterer , leaving it up to the user to design an appropriate simulation . the debye summation is an n problem that becomes computationally demanding for large biomolecules . in some cases , the code allows for combinations of these choices , and we provide a tool to expedite topology construction . to speed up simulations further , the slowly varying scattering forces can be calculated less often than the other forces . we have implemented a dual time - step algorithm , as described in detail in the gromacs 5.0 manual . we provide implementations of atomic scattering factors with and without a displaced - solvent correction , commonly used in saxs intensity prediction tools . for flexibility , we also implement the library - averaged and coarse - grained scattering factors calculated by niebling et al . for amino acid and martini - bead scatterers . in addition to structural refinement , the user can calculate debye sums from single frames or of entire trajectories , using a tool shipped with the code . all simulations were performed using gromacs 5.0 , with the xs - guided md implementation added . a constant temperature of 300 k was achieved in all simulations using a modified berendsen thermostat with = 0.5 . dibutyl ether was simulated in vacuum , using the opls / aa force field . a periodic box was used , with electrostatic cutoffs and a time step of 2 fs . the 10 ns equilibrium simulation was run directly from the initial model with velocities drawn from a maxwell distribution corresponding to 300 k. the first 500 ps were considered equilibration and removed from the analysis . xs - guided md simulations were run with atomistic vacuum form factors against scattering data predicted using the debye equation ( eq 1 ) . simulations started from crystal structures of the apo and holo forms of lao , with pdb codes 2lao and 1lst , respectively . missing atoms were filled in using the what if web server ( http://swift.cmbi.ru.nl/whatif/ ) . a periodic box was used , and electrostatics were treated with pme , using fourth - order interpolation and a grid spacing of 0.12 nm . the initial models were converted and protonated with the gromacs tool pdb2gmx . using cubic simulation boxes initially 2 nm larger than the protein in each direction , the starting structures were first energy - minimized to a maximum force of 2000 kj / mol / nm . the systems were energy - minimized again after the addition of tip3p water and ions corresponding to 50 mm nacl ( in addition to two or three cations to neutralize the protein ) . the simulation boxes were equilibrated under nvt conditions for 50 ps , then subsequently under npt conditions for 500 ps using the parrinello all non - hydrogen atoms were position - restrained to their initial positions during equilibration ( force constants = 1000 kj / mol / nm ) . equilibrium simulations covering 100 ns were carried out for the apo and holo forms , the latter both with and without bound lysine . the xs - guided md simulations were carried out in a a larger cubic box , with dimensions of 13.5 nm , separately minimized and equilibrated as above . amino acid based scattering factors were used , centered on virtual interaction sites representing the center of mass of each residue . the target scattering was calculated from the crystal structures using the debye equation ( eq 1 ) . xs - guided md simulations of phytochrome photoconversion started from the crystal structure with pdb code 4o01 . the charmm27 force field was used , with the chromophore co - factor added by manually adapting the parameters published by kaminski et al . to the gromacs format . the initial structure was energy - minimized , solvated , and equilibrated as described for lao above , in a 25 nm cubic box , with ions corresponding to 100 mm nacl in addition to the 48 cations necessary to neutralize the protein . xs - guided md simulations used amino - acid scattering factors centered on the c - beta atoms , and were run against previously published experimental difference scattering data . the implementation of xs - guided md described above was validated against three test systems . the first is an illustrative theoretical experiment which explores how the conformational distribution of a small organic molecule can be biased toward reproducing theoretical scattering curves . the second is more complex , as a real protein movement is reproduced by providing theoretical scattering data corresponding to two observed crystal structures . the third case involves actual difference scattering data , and shows that xs - guided md reproduces earlier results that were based on a knowledge - based ad hoc approach . in the following , we review the results and discuss the limitations and possibilities of the method . the linear dibutyl ether molecule was first simulated without experimental constraints under vacuum for 10 ns , and the degree of openness of the carbon figure 1a shows examples of the conformations encountered , and the distribution of end - to - end distances ( rc1c8 ) is shown in figures 1b and 1c ( black curve ) . dibutyl ether guided toward an open or a closed conformation based on calculated x - ray scattering . ( a ) example conformations from an unrestrained simulation , with various end - to - end distances in , chosen at random for each distance . ( b , c ) distance distributions of an unrestrained simulation ( black ) and of xs - guided md simulations aiming at an open conformation ( panel ( b ) , 9.8 ) and a closed conformation ( panel ( c ) , 5.0 ) with k values as indicated ( blue ) . insets show average calculated scattering profiles for each k , as a difference relative to the open conformation . there , red curves correspond to different k values , while target curves are shown in black . after calculating the theoretical scattering curves of an open dibutyl ether conformation ( rc1c8 = 9.8 ) and of a closed ( rc1c8 = 5.0 ) dibutyl ether conformation , these curves were used as input for xs - guided md . figures 1b and 1c show that the artificial scattering energy ( uxs ) has a dramatic effect on the end - to - end distance distribution . for both simulations , increasing the coupling coefficient k skews the distance distribution toward the target structure . at high coupling , all conformations that do not display the degree of openness of the target conformation are avoided . meanwhile , the calculated scattering curves also approach their targets , as shown in the insets of figures 1b and 1c . this establishes that the method and implementation work for this simple test system , where one degree of freedom essentially determines the scattering profile . the lysine / arginine / ornithine - binding protein ( lao ) from salmonella typhimurium undergoes large - scale domain movements when binding ligands . specifically , lysine binding causes one of its domains to rotate 52 about an axis formed by two hinge points located on adjacent beta strand termini . constitutes a more complex , albeit hypothetical , test case for the xs - guided md method . unrestrained md simulations starting from equilibrated crystal structures show that both the lysine - bound holo and the unbound apo forms are stable on the 100 ns time scale ( figure 2a ) . in contrast , with the lysine ligand removed , the holo form becomes ill - defined and deviates from its initial conformation without reaching the apo state ( figure 2a , bottom panel ) . we ran simulations guided by theoretical scattering profiles toward the apo state , starting from the lysine - free holo structure . the target scattering data was computed as the scattering of the apo minus the holo state . figure 2b shows how the structural similarity to both states , as well as the energy term uxs , develop over time at several coupling strengths k. the figure shows that guiding the simulation toward the theoretical target scattering data causes the holo - to - apo transition to readily occur . as expected , increasing the coupling parameter k causes the transition to happen earlier in the simulation . the final root - mean - square deviation ( rmsd ) , with respect to the apo state , is consistent with the equilibrium apo simulation ( figure 2a ) , at 2 . figure 2c confirms that typical difference x - ray scattering patterns from the simulations agree well with the target data . figure 2d gives a structural view of the xs - guided md simulation and , again , confirms the holo - to - apo transition suggested by the rmsd traces . method validation against the lysine / arginine / ornithine - binding protein ( lao ) . ( a ) unrestrained simulations of the apo , holo , and lysine - free holo states . ( b ) xs - guided md trajectories aiming at the apo state , starting from the lysine - free holo state , with various coupling strengths k. the plot shows rmsd : s compared to initial and target structures , as well as the evolution of the scattering energy . ( c ) 25 scattering curves , extracted from the second half of the 30 kj / mol run , together with the target curve ( thick line ) . ( d ) graphical representation of the apo ( red ) and holo ( orange ) conformations . the right - hand model also shows a trajectory view over the second half of the 30 kj / mol run . the hinge axis is indicated . interestingly , if even stronger coupling to scattering data is imposed , the simulation fails to find the apo state ( data not shown ) . this indicates that the scattering energy term contains barriers that become insurmountable on the time scale simulated here , if k is set too high . the ability of xs - guided md to quickly reproduce the apo state of lao , based solely on a difference scattering curve , shows that the curve contains enough information to bias the macromolecular force field toward the correct conformation . it also shows that xs - guided md provides sufficient sampling to find the correct structure in a relatively short simulation , at least for this particular system . as a final test system , we applied the xs - guided md algorithm to the bacterial phytochrome from deinococcus radiodurans . the dimeric photosensory module of this light - sensing protein was recently shown to undergo dramatic structural change during photoconversion . upon illumination with red light , the dark - adapted form , labeled the pr state , undergoes a series of ngstrm - scale structural transformations that amplify and ultimately cause the homodimer to partially open in a nanometer - scale movement , forming the so - called pfr state . as a result , the distance between the globular phy ( phytochrome - specific ) domains on opposing monomers increases . in ref ( 10 ) , we reported dark and illuminated crystal and solution structures , which showed that the opening motion is larger in solution than in the crystals . simulations guided by x - ray scattering were run starting from the illuminated crystal structure of the photosensory core from the d. radiodurans phytochrome . the simulations were biased toward an experimental difference scattering curve corresponding to the pr - to - pfr transition , by comparing the calculated scattering profiles of the simulation to that of the proposed pr solution structure . this data was previously used to propose the solution structure for the illuminated pfr state . figure 3a shows that guiding the simulation toward experimental scattering data causes the distance between the phytochrome dimer s opposing phy domains to grow . as the phy phy distance approaches the value previously found , the global conformation of the dimer reproduces the pfr solution structure . indeed , the structural overlap shown in figure 3c illustrates that these structures are identical at the low - resolution level . thus , xs - guided md simulations are capable of refining solution structures based on an initial model and experimental x - ray difference scattering data . ( a ) the center - of - mass distances between the globular phy domains ( residues 330445 and 480503 ) on opposing monomers , as well as the evolution of the scattering energy term ; the initial scattering energy was 300 kj / mol . ( b ) theoretical difference scattering during the second half of the 4 ns run . ( c ) structural views of the pr and pfr solution structures , and a trajectory view over the second half of the run , superimposed on the pfr target structure . we note that , while the application to phytochrome photoconversion validates the xs - guided md method as such , and it lends confidence to the nature of the previously proposed structural change , the magnitude of the opening between the phy domains can not be rigorously defined at very large openings . this was already recognized in ref ( 10 ) and does not affect the conlusions drawn in that paper ; however , it does illustrate a potential problem in refinement against difference x - ray solution scattering data . the parameter , which describes the experimental conversion efficiency and is needed for the correct scaling of calculated to experimental data , was estimated based on the original analysis , where was arbitrarily scaled for best fit to data . in fact , the absolute size of the difference signal , and therefore the precise value of , affects the degree to which the phytochrome dimer opens up in xs - guided md refinement . this is illustrated in figure 4 , which shows predicted peak positions for difference scattering curves based on the previously published trajectories . as the dimer opening increases , the peak first shifts along the q - axis , and then , after an initial opening to 5.5 nm , instead grows in magnitude . thus , experimental determination of the yield parameter is important for successful structural refinement . magnitude and position of the s peak at q 1/nm for various phyphy distances . using three test cases , ranging from small and theoretical to large and experimental , we have shown that xs - guided md simulations may serve as a tool to structurally interpret difference solution x - ray scattering data . we have focused on the use of difference scattering data , as encountered in time - resolved x - ray scattering experiments of proteins in solution . however , the method could , in principle , be equally well - applied to absolute data , provided that attention is paid to the limitations of the debye equation ( eq 1 ) in predicting absolute x - ray scattering . in contrast to macromolecular crystallography and saxs , which have seen the development of rigorous methodologies in the last decades , there is no established way of structurally interpreting difference scattering data . the applicability of the method relies on its ability ( i ) to calculate x - ray scattering with adequate accuracy , ( ii ) to distinguish between the true target conformation and other conformations using the combined knowledge of the chemical force field and the experimental data , and ( iii ) to sample enough conformations to find the target structure within a reasonable simulation time . we found that requirement ( iii ) is satisfied for all test systems tried here , usually after simulating 10 ns . regarding requirement ( i ) , it is noted that a shortcoming of the debye equation ( eq 1 ) , where the sums run only over the protein atoms , is that it does not account for the solvation layer scattering . however , when computing difference x - ray scattering data , this effect can often be neglected . the second requirement , uniqueness of the target structure , is more critical . first , we note that a certain feature in a difference scattering pattern is more likely to reflect a unique structural rearrangement when it occurs at q values that describe the molecular envelope ( for proteins , this region is typically described by q 2 nm ) . difference scattering in this q - range reflects large - scale protein motions . all the test cases used here can be described by collective motions along a small number of degrees of freedom . for example , the phytochrome rearrangement can be thought of as an increase in distance between the phy domains , while the structural change of lao can be seen as rotation around an axis . these structural processes affect the overall shape of the protein and the difference scattering pattern of lao is dominated by peaks at q 2 nm , while the phytochrome difference scattering has a distinct feature at q = 1 nm . consequently , unique structural fits can be obtained . at higher q values , multiple candidate structures are more likely to produce overlapping features in difference scattering patterns and they must be discriminated using geometrical or chemical constraints . in the intermediate q range ( 2 nm < q < 8 nm ) , typical structural dynamics would involve the rearrangement of secondary or tertiary structural elements . geometric constraints , such as used in crystallography for dihedrals , angles , and bond lengths , are not likely to be effective in discriminating candidate structures . instead , the full range of covalent , dispersion , and electrostatic interactions described by molecular force fields must be considered , and the resulting energy landscape sampled in the appropriate ensemble . in previous reports , for example , helix movements , hand - picked based on previous knowledge , were refined against difference scattering features at 2 nm < q < 6 nm . in the approach presented here , we consider this to be an important step toward unbiased structural interpretation of time - resolved x - ray scattering data . finally , we note that some protein structural changes may be difficult to observe with x - ray scattering . for example , the conformational change upon photolysis of the hemoglobin carbon monoxide bond corresponds to an rmsd value of 4 , but has a relatively small effect on the molecular envelope . therefore , it affects the x - ray scattering signal at low q only weakly . we have tested a refinement of the hemoglobin structural change against the difference scattering patterns published in ref ( 6 ) ; however , even though the scattering energy decreased and stabilized rapidly , the rmsd observables did not reach the target values . this indicates that structures exist , which describe the difference scattering data of hemoglobin well , but which do not agree with structural changes derived from crystallographic structures . the situation arises because the experimental data contain too little information , and the demands on the force field and sampling become too high . in principle , molecular force fields should be able to distinguish between artificial and true target structures ; however , in practice , this would require long and repeated simulations , beyond what is practically possible .

time - resolved x - ray solution scattering is an increasingly popular method to measure conformational changes in proteins . extracting structural information from the resulting difference x - ray scattering data is a daunting task . we present a method in which the limited but precious information encoded in such scattering curves is combined with the chemical knowledge of molecular force fields . the molecule of interest is then refined toward experimental data using molecular dynamics simulation . therefore , the energy landscape is biased toward conformations that agree with experimental data . we describe and verify the method , and we provide an implementation in gromacs .

Introduction Theory and Methods Results Discussion

time - resolved x - ray scattering has been developed to study the structural dynamics of molecular reactions . the availability of ultrashort x - ray bursts at free electron lasers has enabled time - resolved scattering studies with femtosecond time resolution . today , time - resolved x - ray scattering experiments on proteins can be carried out reliably , but they are usually limited by data interpretation . however , the information content in the one - dimensional spherically averaged ( isotropic ) scattering curves is inherently low . unlike saxs , difference x - ray scattering has the advantage that the protein hydration shell typically only gives a minor contribution to the scattering signal . biomolecular force fields encode such chemical information , and are used in molecular dynamics ( md ) or monte carlo ( mc ) simulations that sample thermal ensembles of proteins in solution . to our knowledge , no systematic attempts to interpret difference x - ray scattering data with atomistic force fields have been published to date . here , we describe and implement x - ray scattering - guided molecular dynamics simulations ( xs - guided md ) , which is a technique that refines structures against difference scattering data . in what follows , we shall only be concerned with difference x - ray scattering data , and as explained and justified below , neglect the scattering contribution from the protein s solvation layer . the energy landscape of the simulation is biased toward the experimentally observed conformation and distinctions can be made between alternative conformations with similar free energies . the algorithm reduces the computational time to determine the structures that agree with experiment , and retains the chemical knowledge contained in md force fields . for biasing md simulations toward configurations that agree with experimental scattering curves , following ahn et al . if concentrations , path lengths , incoming x - ray intensities , and detector efficiencies are precisely known , experimentally measured intensities can be converted to such units . xs - guided md is implemented in gromacs 5 and , therefore , profits from the efficient core of gromacs . in some cases , the code allows for combinations of these choices , and we provide a tool to expedite topology construction . the first is an illustrative theoretical experiment which explores how the conformational distribution of a small organic molecule can be biased toward reproducing theoretical scattering curves . figure 2c confirms that typical difference x - ray scattering patterns from the simulations agree well with the target data . ( c ) 25 scattering curves , extracted from the second half of the 30 kj / mol run , together with the target curve ( thick line ) . simulations guided by x - ray scattering were run starting from the illuminated crystal structure of the photosensory core from the d. radiodurans phytochrome . thus , xs - guided md simulations are capable of refining solution structures based on an initial model and experimental x - ray difference scattering data . this was already recognized in ref ( 10 ) and does not affect the conlusions drawn in that paper ; however , it does illustrate a potential problem in refinement against difference x - ray solution scattering data . using three test cases , ranging from small and theoretical to large and experimental , we have shown that xs - guided md simulations may serve as a tool to structurally interpret difference solution x - ray scattering data . we have focused on the use of difference scattering data , as encountered in time - resolved x - ray scattering experiments of proteins in solution . however , the method could , in principle , be equally well - applied to absolute data , provided that attention is paid to the limitations of the debye equation ( eq 1 ) in predicting absolute x - ray scattering . the applicability of the method relies on its ability ( i ) to calculate x - ray scattering with adequate accuracy , ( ii ) to distinguish between the true target conformation and other conformations using the combined knowledge of the chemical force field and the experimental data , and ( iii ) to sample enough conformations to find the target structure within a reasonable simulation time . however , when computing difference x - ray scattering data , this effect can often be neglected . instead , the full range of covalent , dispersion , and electrostatic interactions described by molecular force fields must be considered , and the resulting energy landscape sampled in the appropriate ensemble . in the approach presented here , we consider this to be an important step toward unbiased structural interpretation of time - resolved x - ray scattering data . finally , we note that some protein structural changes may be difficult to observe with x - ray scattering . therefore , it affects the x - ray scattering signal at low q only weakly .

topoisomerases are ubiquitous proteins found in all three domains of life ( bacteria , archaea and eukarya ) . they are capable of changing the topology of dna via transient breaks on one or two of the dna strands to allow passage of either a single or double dna strand through the break . topoisomerases have been involved in several cellular processes , such as transcription , replication and recombination , and the importance of their cellular role is underscored by the fact that they are the target of several cancer chemotherapeutic agents and antibiotics [ for a review , see ( 1 ) ] . topoisomerases are classified into two types : type i enzymes cleave one dna strand and pass either one or two strands through the break before resealing it , while type ii molecules cleave both dna strands in concert and pass another double strand through the break followed by religation of the double strand break . type i enzymes do not require any high - energy cofactor for activity ; the reaction is driven by the energy stored in the supercoiled dna as torsional strain . type ii enzymes also do not require an external source of energy for the cleavage / religation part of the reaction , but they do utilize atp hydrolysis to drive conformational changes in the protein during the reaction cycle . type i enzymes have been further classified into three different families : types ia , ib and ic ( 2 ) . however , members of one family show no sequence or structural similarities to members of the other families . the atomic mechanisms employed for cleavage and religation of the dna by different families have only superficial similarities . type i enzymes operate by forming a transient phosphotyrosine covalent bond with one end of the broken dna strand , either the 5 or the 3 end , followed by passage of the unbroken strand through the break , and ultimately resealing of the break . in this context , only two possible mechanisms of relaxation are possible : enzyme - bridged strand passage , where the protein mediates the opening of one strand to allow passage of the other , and swiveling , which involves the rotation of the dna strands . one important difference between these mechanisms is the need to hold one or both ends of the broken dna strand . during enzyme - bridged strand passage , it is necessary to non - covalently hold one end of the broken strand while the other end is covalently attached to the active site tyrosine . on the other hand , the swiveling mechanism requires the non - covalently bound end of the dna to move around freely and is not attached to the protein . it is clear that each family of type i enzymes has to employ one of the two relaxation mechanisms and form either a 5 or 3 covalent intermediate . it is now established that type ia enzymes form transient 5 covalent intermediates and relax dna through an enzyme - bridged strand - passage mechanism , while type ib and ic enzymes both form 3 covalent intermediates and relax dna by swiveling , although the details of how this is accomplished are probably very different . type i topoisomerases change the topology of dna without the need of an external source of energy , such as atp hydrolysis . so far , the only exception is reverse gyrase , which introduces positive supercoils with the aid of atp hydrolysis , although the way the strand passage and atp hydrolysis steps are coupled is still not clear . to relax dna , the way the stored energy in the dna is transformed into changes in the protein as the reaction proceeds is still not understood and remains one of the major future challenges in understanding not only type i topoisomerases , but all topoisomerases . type ia enzymes have been found in bacteria , archaea and eukarya and there are clear sequence similarities among all members , suggesting a strong structural and mechanistic conservation ( 3 ) . they form a phosphotyrosine linkage to the 5 end of the broken dna strand while non - covalently holding the free 3-end hydroxyl group . relaxation activity occurs in the absence of a high - energy cofactor , but in the presence of divalent cations . the exact role of these cations has not been fully determined , however they are needed for dna relaxation ( 4 ) . although they are an attractive target for chemotherapeutic agents , there are no known inhibitors or drugs that target type ia enzymes . type ia topoisomerases relax only negatively supercoiled dna and require single - stranded dna regions for activity ( 5 ) . the requirement of single - stranded dna for activity provides a plausible mechanism for sensing the overall topological state of dna as single - stranded regions are only found in negatively supercoiled or underwound dna but not in positively supercoiled or overwound dna . thus , the presence of single - stranded regions , a property of dna that can be monitored locally , could serve as an indicator of the global topological state of dna . the core structure of type ia topoisomerases , typically of around 67 kda in molecular weight , has a characteristic toroidal fold formed by four domains . the toroidal fold was first observed in escherichia coli topoisomerase i ( 6 ) and later in e. coli topoisomerase iii ( 7 ) , thermotoga maritima topoisomerase i ( 8) , and reverse gyrase from archaeoglobus fulgidus , a hyperthermophilic archaeal organism ( 9 ) ( figure 1 ) . reverse gyrase is unique as it consists of a topoisomerase domain fused to a helicase domain at the n - terminus of the protein . sequence comparison of the c - terminal region extending from the core domain of the type ia enzymes shows that this region does not present the same level of conservation as the core . the c - terminal domains can vary greatly in size and play a role in dna / protein interactions ( 1012 ) . in the case of e. coli topoisomerase iii , deletion of this c - terminal domain is deleterious , although its removal does not abolish activity completely ( 13 ) . in the case of e. coli topoisomerase i , the c - terminal domain is essential for activity ( 14,15 ) . in both cases , the c - terminal domain is responsible for single - stranded dna binding and confers higher dna binding affinity ( 10,12,13 ) . the structures of the c - terminal domain of e. coli topoisomerase i ( 16 ) and the intact t. maritima topoisomerase i ( 8) reveal the presence of zinc - ribbon single - stranded dna binding motif . the structure of the t. maritima topoisomerase i shows the way the core domain and the c - terminal domain are arranged relative to each other , but the structural bases for the role of this domain in the overall reaction are still unknown . e. coli topoisomerase iii is shown to illustrate the overall structure of a type ia topoisomerase and the typical toroidal fold observed in all members of this type . ( a ) diagram showing the structure of the apo - enzyme [ pdb 1d6 m ( 7 ) ] . in the absence of dna , the active site , found at the intersection of domains ( b ) diagram showing the structure of a complex with single - stranded dna [ pdb 1i7d ( 23 ) ] . note the movement of domains that occurs in order to accommodate dna . in both diagrams , the four major domains of the protein are colored red , blue , purple and green for domains i , ii , iii and iv , respectively . the single - stranded dna binding groove , shown circled in black , extends from domain iv to the active site . the active site residues as well as the single - stranded dna in the complex are shown in a ball and stick representation . e. coli topoisomerase iii is shown to illustrate the overall structure of a type ia topoisomerase and the typical toroidal fold observed in all members of this type . ( a ) diagram showing the structure of the apo - enzyme [ pdb 1d6 m ( 7 ) ] . in the absence of dna , the active site , found at the intersection of domains ( b ) diagram showing the structure of a complex with single - stranded dna [ pdb 1i7d ( 23 ) ] . note the movement of domains that occurs in order to accommodate dna . in both diagrams , the four major domains of the protein are colored red , blue , purple and green for domains i , ii , iii and iv , respectively . the single - stranded dna binding groove , shown circled in black , extends from domain iv to the active site . the active site residues as well as the single - stranded dna in the complex are shown in a ball and stick representation . a combination of biochemical , biophysical and structural studies has helped reveal the atomic mechanism of the type ia enzymes . a detailed model on how type ia enzymes perform complex topological rearrangements was proposed several years ago based on the structure of e. coli topoisomerase i ( 6 ) . subsequent biochemical studies using a mutant enzyme capable of forming a disulfide bridge trapped some of the proposed intermediates and confirmed the general features of the mechanism of action ( 17 ) , in particular that the enzyme opens up through a large conformational change and sequesters the passing dna strand inside the protein . recent single molecule experiments ( 18,19 ) have further confirmed the general features of the proposed mechanism . these experiments , in conjunction with the wealth of existing biochemical data , confirmed that the enzymes change the linking number in steps of one , as is mandated in an enzyme - bridged strand - passage mechanism . the proposed mechanism of action involves several steps ( figure 2 ) , and crystal structures for some of these intermediate steps have been obtained ( 2022 ) . the structures revealed that the enzyme and the dna have to change conformation during the catalytic cycle in a coordinated manner . structures of the apo - state of e. coli topoisomerase i and iii ( 6,7 ) , t. maritima topoisomerase i ( 8) and a. fulgidus reverse gyrase ( 9 ) showed the enzymes in a closed conformation with the active site not fully formed . the structure of e. coli topoisomerase iii in complex with a short oligonucleotide shows that type ia enzymes have to undergo conformational changes to form the active site and that these changes are not only confined to the relative movement of the domains , but also involve small but important rearrangements in some of the domains of the molecule ( 20,23 ) , in particular domain iv , which is directly involved in forming a single - stranded dna binding groove . additionally , the structure of a fragment of e. coli topoisomerase i ( 21 ) indicated that the enzyme can undergo dramatic rearrangements of one of its domains ( domain ii ) , while , the structure of e. coli topoisomerase i in complex with a short oligonucleotide ( 22 ) shows the structure of an intermediate where the active site is not yet fully formed , but some movements of secondary structure elements in domain iv have started to occur . there is still no structure of a covalent intermediate that shows the crucial interactions with the 5 and 3 ends of the dna , but recent studies of wild - type e. coli topoisomerase iii in complex with dna showed for the first time a wild - type enzyme in complex with dna perfectly poised for dna cleavage ( 20 ) ( figure 3 ) . this structure confirms that the formation of a competent active site depends on several rearrangements of the enzyme . the sequence of the steps and the intermediates are hypothetical and more states are likely to be involved in the cycle . processivity by the enzyme requires that after one relaxation event the protein continues to another relaxation cycle without releasing the dna . in the diagram , ( a ) apo - structure , where the protein is in a closed conformation and the active site is buried between domains i and iii [ pdb 1d6 m ( 7 ) ] . ( b ) intermediate structure , where the dna starts entering the active site but it is not yet positioned properly for cleavage / religation [ pdb 2o59 ( 20 ) ] . ( c ) fully formed active site in the wild - type enzyme , with the single - stranded dna positioned and the active site tyrosine poised for cleavage [ pdb 2o19 ( 20 ) ] . the diagrams illustrate the way the active site is assembled and the interactions with single - stranded dna . some of the side chains forming the active site are shown , as well as some possible hydrogen bonds . the sequence of the steps and the intermediates are hypothetical and more states are likely to be involved in the cycle . processivity by the enzyme requires that after one relaxation event the protein continues to another relaxation cycle without releasing the dna . in the diagram , ( a ) apo - structure , where the protein is in a closed conformation and the active site is buried between domains i and iii [ pdb 1d6 m ( 7 ) ] . ( b ) intermediate structure , where the dna starts entering the active site but it is not yet positioned properly for cleavage / religation [ pdb 2o59 ( 20 ) ] . ( c ) fully formed active site in the wild - type enzyme , with the single - stranded dna positioned and the active site tyrosine poised for cleavage [ pdb 2o19 ( 20 ) ] . the diagrams illustrate the way the active site is assembled and the interactions with single - stranded dna . some of the side chains forming the active site are shown , as well as some possible hydrogen bonds . an initial identification of the residues comprising the active site was based on the structure of the apo - enzyme ( 6 ) and sequence conservation ( 24 ) . the structure of an e. coli topoisomerase iii - single - stranded dna complex ( 23 ) showed that a conformational change occurs upon single - stranded dna binding and this leads to the creation of a competent active site by bringing together residues that are distant in the apo - enzyme . in the complex structure , the active site tyrosine , an arginine , a lysine and a glutamate form direct contacts to the dna near the scissile bond . in addition , a second layer of highly conserved amino acids surrounds the active site , including three highly conserved acidic residues that have been shown to be involved in magnesium binding ( 25 ) . due to the conformational changes that occur upon binding dna ( figure 3 ) , some of the residues that in the apo - structure were observed to be distant from the putative active site an example is glu7 in topoisomerase iii , which is completely conserved and interacts directly with dna , but is located several angstroms away from the active site in the apo - structure and whose exact role could not be surmised from biochemical studies . several biochemical studies , mostly using site - directed mutagenesis [ for example ( 2632 ) ] have confirmed the role of many of the residues identified by these structures . due to their dynamic nature , type ia topoisomerases are a very good example of proteins where understanding the catalytic mechanism in the absence of structures is almost impossible . the chemistry of the mechanism of cleavage and religation , the manner in which the free 3-end interacts with the protein during the reaction , the role of magnesium , and the atomic mechanism of strand passage are some of the questions that remain to be answered . our understanding of type ia enzymes will be incomplete until we elucidate the atomic details of the movement of one strand past the other and how the torsional strain in dna directs conformational changes in the protein . additional structural information on the intermediate steps in the reaction cycle , especially those with large conformational changes both in the protein and the dna , is essential to address these difficult questions . type ib enzymes have been identified in eukaryotes , poxviruses and bacteria , but not in archaea . eukaryotic type ib molecules are large , typically over 90 kda in molecular weight , while viral and bacterial type ib molecules are relatively small , around 36 kda . despite their differences in size , all type ib molecules share a common fold around the active site region and a common catalytic mechanism ( 3,33,34 ) ( figure 4 ) . interestingly , these similarities extend to the tyrosine recombinase family suggesting a common ancestor for both type ib topoisomerases and tyrosine recombinases ( 33 ) . type ib enzymes form a transient 3 phosphotyrosine covalent linkage with the broken end of the dna . they can relax both negatively and positively supercoiled dna without requiring divalent ions or atp . by breaking transiently one dna strand , as long as there is torsional strain to drive the swiveling , the reaction will proceed until the dna is fully relaxed . because of this , type ib enzymes do not have to sense directly the global topological state of dna , they simply provide the means to relieve torsional strain if some is present , even if it is only in a local supercoiled domain . ( a ) structure of human topoisomerase i in non - covalent complex with dna [ pdb 1a35 ( 34 ) ] . the protein is composed of two domains : a core domain , shown in red and blue , and a c - terminal domain , shown in green . ( b ) structure of variola virus topoisomerase i in covalent complex with dna [ pdb 2h7f ( 51 ) ] . the viral and bacterial type ib enzymes are composed of two domains : an n - terminal domain , shown in red , and a larger , c - terminal domain , shown in blue . note the marked similarities between the human and poxviral proteins despite their great disparity in size . in the case of human topoisomerase i , the core and c - terminal domains are joined by a linker domain , which is not present in this structure but that has been observed in other structures ( 44 ) . ( a ) structure of human topoisomerase i in non - covalent complex with dna [ pdb 1a35 ( 34 ) ] . the protein is composed of two domains : a core domain , shown in red and blue , and a c - terminal domain , shown in green . ( b ) structure of variola virus topoisomerase i in covalent complex with dna [ pdb 2h7f ( 51 ) ] . the viral and bacterial type ib enzymes are composed of two domains : an n - terminal domain , shown in red , and a larger , c - terminal domain , shown in blue . note the marked similarities between the human and poxviral proteins despite their great disparity in size . in the case of human topoisomerase i , the core and c - terminal domains are joined by a linker domain , which is not present in this structure but that has been observed in other structures ( 44 ) . they all contain a highly conserved pentad of residues forming the active site ( tyr , arg , arg , lys and his / asn ) with an identical architecture around the active site region ( figure 5 ) . eukaryotic and viral enzymes have a histidine in the pentad , while in bacterial type ib enzymes the histidine is replaced by an asparagine . experiments show that the histidine can be replaced by an asparagine in both viral ( 35 ) and eukaryotic ( 36 ) topoisomerases , suggesting that a histidine or an asparagine can play the same role and that the overall mechanism of cleavage and religation is identical in all type ib enzymes . the mechanism of cleavage by type ib enzymes has been extensively studied and the role of different residues in catalysis has been characterized in great detail ( 37 ) . the sequence specificity of the poxviral topoisomerases offers an unique advantage over other topoisomerases for biochemical studies , which has allowed exquisitely detailed biochemical studies that elucidated the role of dna ( 38,39 ) and different amino acids ( 37,4042 ) in the cleavage reaction . ( a ) active site in the structure of a non - covalent complex of human topoisomerase i with dna [ pdb 1a35 ( 34 ) ] . note the presence of the amino acids forming the catalytic pentad , arg488 , lys532 , arg590 , his632 and tyr723 ( in this structure the tyrosine was mutated to a phenylalanine ) . ( b ) active site in the structure of a covalent complex of variola virus topoisomerase i with dna [ pdb 2h7f ( 51 ) ] . note the equivalent pentad of amino acids in the active site and the formation of the covalent intermediate between the tyrosine and the 3 end of the broken strand . the diagrams serve to illustrate the high similarity between the active sites of the smaller and larger type ib enzymes and also the structures of non - covalent and covalent complexes ( a ) active site in the structure of a non - covalent complex of human topoisomerase i with dna [ pdb 1a35 ( 34 ) ] . note the presence of the amino acids forming the catalytic pentad , arg488 , lys532 , arg590 , his632 and tyr723 ( in this structure the tyrosine was mutated to a phenylalanine ) . ( b ) active site in the structure of a covalent complex of variola virus topoisomerase i with dna [ pdb 2h7f ( 51 ) ] . note the equivalent pentad of amino acids in the active site and the formation of the covalent intermediate between the tyrosine and the 3 end of the broken strand . the diagrams serve to illustrate the high similarity between the active sites of the smaller and larger type ib enzymes and also the structures of non - covalent and covalent complexes . eukaryotic type ib topoisomerases are large proteins that contain multiple structural components that have no counterpart in the viral and bacterial topoisomerases . the central or core domain together with a c - terminal domain can reconstitute a fully functional enzyme ( 43 ) . these two domains are joined by a short linker domain formed by two long helices ( 43 ) . the c - terminal domain contains the catalytic tyrosine while all other residues in the catalytic pentad reside in the core domain . structures of the fully functional enzyme , formed by the core and c - terminal domains and in some instances the linker domain , in complex with dna shows a c - shaped protein clamp around duplex dna ( 34,44 ) ( figure 4 ) . the two regions that close the c - clamp consist of loops that meet in a non - covalent interaction to envelop the target dna . in contrast to the eukaryotic enzymes , the poxviral and bacterial type ib topoisomerases contain two distinct domains ( 45,46 ) . the smaller amino terminal domain is involved in interactions with dna ( 45,47,48 ) , while the larger domain contains the active site and retains full enzymatic activity ( 49 ) . the structures of both domains of vaccinia virus topoisomerase i , the best characterized and studied viral type ib enzyme , are known ( 33,45 ) as well as the structure of the intact deinococcus radiodurans topoisomerase ib ( 50 ) . the structure of variola virus topoisomerases i in complex with dna revealed an important point : all type ib enzymes encircle dna in an identical fashion , despite their differences in size . in addition , poxviral type ib enzymes are unique in that they have a preference for binding to specific dna sequences . hence , the structure of variola virus topoisomerase i in complex with dna ( 51 ) helps to understand the atomic basis for the sequence specificity . overall , the structure of all these type ib enzymes confirms that eukaryotic , viral and bacterial type ib topoisomerases are similar despite their dissimilarities in size . even though the n - terminal domain in the bacterial and viral enzymes is less conserved , there are marked commonalities with the equivalent region in the larger enzymes . clearly , the smaller type ib enzymes represent an abbreviated form of the larger eukaryotic topoisomerases where all the essential regions are present . the mechanisms of cleavage / religation and dna relaxation are , without any doubt , identical . type ib enzymes relax dna by a mechanism termed controlled rotation , where one dna strand swivels around the other . a swiveling mechanism predicts that the enzymes relax dna in steps of n , and this has been confirmed recently by single - molecule experiments on vaccinia virus , yeast and human topoisomerase ib ( 52,53 ) . the single - molecule experiments also indicate that the swiveling is slowed down by friction created by the interaction between the enzyme and the dna , consistent with the embracing of the dna by the protein as observed in the structures of human and variola virus topoisomerase i in complex with dna ( 34,51 ) . they are the target of important anticancer compounds such as camptothecins ( 5457 ) , indolocarbazoles ( 58 ) and indenoisoquinolines ( 5961 ) . although type ib enzymes are not widespread in bacteria , they are present in some important human pathogens , such as pseudomonas aeuroginosa and bordetella parapertussis . hence , bacterial type ib enzymes could serve as targets of chemotherapeutic agents , especially now that structures are starting to reveal the atomic differences between human , viral and bacterial enzymes . the structures of ternary complexes of the enzyme , dna and drugs ( 6265 ) reveal the drug interactions at the atomic level and this may be helpful in the development of better type ib poisons . given the importance of type ib enzymes in cancer therapeutics , it is likely that this will continue to be a fruitful study area and that the information garnered will also shed light on the mechanism of action of the enzymes . as is the case with the type ia enzymes , there are still many details of the mechanism of type ib enzymes that need to be elucidated . unlike type ia enzymes , the structures of both covalent and non - covalent intermediates of type ib enzymes are available . together with the wealth of biochemical information , these structures have helped us understand the cleavage and religation mechanism in great detail . unfortunately , there is scant atomic information on the way that type ib enzymes drive dna relaxation . the changes that occur in the protein as the dna swivels inside the c - clamp are yet to be elucidated . even though the single - molecule experiments suggest the presence of atomic friction due to the interaction of the protein and dna , the details of this process are largely unknown . capturing intermediates in the relaxation process is not an easy task , but they are required in order to truly understand the atomic mechanism of action . the third family , type ic topoisomerases , was recently defined with the realization that topoisomerase v is distinct from all other topoisomerases . dna topoisomerase v was originally found in the hyperthermophile methanopyrus kandleri isolated from a deep - water later on its presence was confirmed in other methanopyrus isolates from around the world ( a. slesarev , personal communication ) . in common with type ib enzymes , topoisomerase v cleaves one dna strand , forms a transient phosphotyrosine bond with the 3 end of the broken strand , can relax positive and negative supercoils , and does not require the presence of magnesium or atp ( 67 ) . despite these biochemical similarities topoisomerase v is a large enzyme , over 100 kda in molecular weight , with unusual characteristics . not only can it relax dna identically to other topoisomerases ( 67 ) , but it is also involved in dna repair ( 68 ) . the two different catalytic activities are found in the same polypeptide : topoisomerase activity resides in the n - terminus of the protein , while the c - terminus of the protein possesses apurinic / apyrimidinic ( ap ) site - processing activity , normally associated with base excision dna repair ( 69 ) . sequence analysis indicates that the protein contains 24 helix - hairpin - helix ( hhh ) dna binding motifs arranged in 12 tandem ( hhh)2 domains ( 70 ) that follow the topoisomerase domain ( 68 ) . n - terminal constructs as small as 30 kda comprising the topoisomerase domain have full topoisomerase activity . the ap site - processing activity resides in the c - terminal 34 kda fragment of the protein ( 69 ) , which also contains several putative hhh motifs ( 68 ) . the minimal construct with both topoisomerase and dna repair activities is a 78 kda n - terminal fragment ( 69 ) . it has maximal topoisomerase activity at around 108c and is still active at 122c ( 71 ) . it is also active in a wide range of conditions , up to 0.65 m nacl or kcl and up to 3.1 m potassium glutamate . these properties have been attributed to the hhh motifs , as fragments containing fewer motifs show optimal activity in a narrower set of conditions and also a marked loss of processivity ( 69 ) . the structure of a 61 kda n - terminal fragment of topoisomerase v comprising the topoisomerase domain and eight hhh motifs reveals a fold that is not related to the folds observed in any other topoisomerase , tyrosine recombinase , or other known protein ( 72 ) ( figure 6a ) . the composition of the active site of topoisomerase v is similar to the one in type ib molecules and includes the active site tyrosine , two arginines , a lysine and a histidine ( figure 6b ) . the key difference between the active site of type ib and type ic enzymes is that the spatial arrangement of these residues is different . in addition , an acidic residue in topoisomerase v has no equivalent in type ib enzymes , suggesting an alternative mechanism of catalysis . interestingly , type ia enzymes do contain an acidic residue in the active site , but it is not known whether the acidic amino acids play a similar role in catalysis . furthermore , the structure suggests that a large conformational change in the protein is needed for activity , which could alter the conformation and position of the residues in the active site , similarly to what was observed in type ia enzymes ( see above ) . limited mutagenesis experiments suggest that topoisomerase v employs a different mechanism from type ib enzymes ( 72 ) as mutation of equivalent residues in both enzymes does not produce similar reductions in relaxation activity . further studies are needed to elucidate the exact mechanism of catalysis , the residues involved , and their role in the cleavage / religation reaction . topoisomerase v also has dna repair activity , but the structure of the domain containing the ap site processing activity is not known yet and the residues involved in dna repair have not been identified . figure 6.structure of a type ic topoisomerase . ( a ) diagram showing the structure of a 61 kda fragment of m. kandleri topoisomerase v [ pdb 2csb ( 72 ) ] . the fragment comprises the topoisomerase domain and four ( hhh)2 domains , which are likely to be involved in dna binding . the topoisomerase domain , shown in red , and the ( hhh)2 domains , shown in tones of blue , are joined by a linker helix , shown in orange . the active site , shown as ball and stick representation , is buried at the interface of the topoisomerase domain and one of the ( hhh)2 domains . to access the active site , the protein has to change conformation , probably by separating the sub - domains . ( b ) the putative amino acids forming the topoisomerase v active site are shown . aside from the tyrosine , two arginines , a lysine , a histidine and a glutamate form the putative active site . mutagenesis studies show that removal of the arginines has a marked detrimental effect on activity . removal of the histidine and lysine has a modest effect on activity ( 72 ) . ( a ) diagram showing the structure of a 61 kda fragment of m. kandleri topoisomerase v [ pdb 2csb ( 72 ) ] . the fragment comprises the topoisomerase domain and four ( hhh)2 domains , which are likely to be involved in dna binding . the topoisomerase domain , shown in red , and the ( hhh)2 domains , shown in tones of blue , are joined by a linker helix , shown in orange . the active site , shown as ball and stick representation , is buried at the interface of the topoisomerase domain and one of the ( hhh)2 domains . to access the active site , the protein has to change conformation , probably by separating the sub - domains . ( b ) the putative amino acids forming the topoisomerase v active site are shown . aside from the tyrosine , two arginines , a lysine , a histidine and a glutamate form the putative active site . mutagenesis studies show that removal of the arginines has a marked detrimental effect on activity . removal of the histidine and lysine has a modest effect on activity ( 72 ) . the relaxation mechanism of this new topoisomerase is not nearly as well understood as that of the other topoisomerases . recent single - molecule experiments established that the mechanism of action of topoisomerase v is similar to the controlled rotation mechanism proposed for type ib enzymes , where relaxation occurs in steps of n ( 73 ) . furthermore , the topoisomerase domain is active in the absence of any of the hhh repeats , suggesting that the topoisomerase domain may be able to relax dna without having to completely encircle it . these observations strongly suggest that although type ib topoisomerases and topoisomerase v have overall mechanistic similarities , the way they perform the reaction may be very different at the atomic level . the similarities between topoisomerases v and type ib enzymes in the overall relaxation mechanism are striking : both work by swiveling and form 3 covalent intermediates . even more remarkable are the differences : no sequence or structural similarity and a different catalytic mechanism for cleavage and religation . this clearly shows that the two types of enzymes represent two different solutions to the same overall problem . this observation is even more interesting when viewed in the context of all other topoisomerases . there appears to be two general mechanisms employed by topoisomerases for dna relaxation : ( a ) type ia and type ii enzymes employ an enzyme - bridged strand - passage mechanism , with biochemical similarities in the way they cleave / religate dna , and structural similarities in various domains , such as the toprim domain ( 3 ) , ( b ) type ib enzymes all share a common fold and employ the same swiveling mechanism for dna relaxation . topoisomerase v illustrates a completely different solution to the same topological problem without any structural , sequence or biochemical similarity to either type ib or type ia / type ii enzymes . over the last few years , the elucidation of the structures of various members of all the topoisomerase types has helped to bring our understanding of the mechanism of action of these important enzymes to the atomic level . in the case of type i enzymes , structures of the apoenzymes , along with enzyme dna complexes have provided exquisitely detailed snapshots of the enzymes during their catalytic cycle . the structures of type ia enzymes in different stages of the catalytic cycle illustrate the way these proteins recognize and bind dna , and also the changes that occur in both substrate and enzyme as they interact with each other . in particular , the mechanism of cleavage and religation is now very well dissected by a combination of structural and functional studies . finally , type ic topoisomerases , the newest family of type i enzymes , are not as well understood as only a few structures are available and the biochemical work is not as advanced . nevertheless , the available information indicates that type ic enzymes evolved a completely different way of performing essentially the same reaction . overall , the structural work on type i enzymes is now maturing and reaching the stage where many important questions have been answered , but many more remain unanswered and need to be addressed if we truly want to have a complete atomic picture of their mechanism . national institutes of health ( gm51350 to a.m. ) . funding for open access charge : national institutes of health ( gm51350 to a.m. ) .

topoisomerases are ubiquitous proteins found in all three domains of life . they change the topology of dna via transient breaks on either one or two of the dna strands to allow passage of another single or double dna strand through the break . topoisomerases are classified into two types : type i enzymes cleave one dna strand and pass either one or two dna strands through the break before resealing it , while type ii molecules cleave both dna strands in concert and pass another double strand through the break followed by religation of the double strand break . here we review recent work on the structure of type i enzymes . these structural studies are providing atomic details that , together with the existing wealth of biochemical and biophysical data , are bringing our understanding of the mechanism of action of these enzymes to the atomic level .

INTRODUCTION TYPE IA TOPOISOMERASES TYPE IB TOPOISOMERASES TYPE IC TOPOISOMERASES CONCLUSIONS FUNDING

topoisomerases are ubiquitous proteins found in all three domains of life ( bacteria , archaea and eukarya ) . they are capable of changing the topology of dna via transient breaks on one or two of the dna strands to allow passage of either a single or double dna strand through the break . topoisomerases are classified into two types : type i enzymes cleave one dna strand and pass either one or two strands through the break before resealing it , while type ii molecules cleave both dna strands in concert and pass another double strand through the break followed by religation of the double strand break . the atomic mechanisms employed for cleavage and religation of the dna by different families have only superficial similarities . type i enzymes operate by forming a transient phosphotyrosine covalent bond with one end of the broken dna strand , either the 5 or the 3 end , followed by passage of the unbroken strand through the break , and ultimately resealing of the break . in this context , only two possible mechanisms of relaxation are possible : enzyme - bridged strand passage , where the protein mediates the opening of one strand to allow passage of the other , and swiveling , which involves the rotation of the dna strands . on the other hand , the swiveling mechanism requires the non - covalently bound end of the dna to move around freely and is not attached to the protein . it is clear that each family of type i enzymes has to employ one of the two relaxation mechanisms and form either a 5 or 3 covalent intermediate . type i topoisomerases change the topology of dna without the need of an external source of energy , such as atp hydrolysis . a combination of biochemical , biophysical and structural studies has helped reveal the atomic mechanism of the type ia enzymes . subsequent biochemical studies using a mutant enzyme capable of forming a disulfide bridge trapped some of the proposed intermediates and confirmed the general features of the mechanism of action ( 17 ) , in particular that the enzyme opens up through a large conformational change and sequesters the passing dna strand inside the protein . these experiments , in conjunction with the wealth of existing biochemical data , confirmed that the enzymes change the linking number in steps of one , as is mandated in an enzyme - bridged strand - passage mechanism . the structure of e. coli topoisomerase iii in complex with a short oligonucleotide shows that type ia enzymes have to undergo conformational changes to form the active site and that these changes are not only confined to the relative movement of the domains , but also involve small but important rearrangements in some of the domains of the molecule ( 20,23 ) , in particular domain iv , which is directly involved in forming a single - stranded dna binding groove . there is still no structure of a covalent intermediate that shows the crucial interactions with the 5 and 3 ends of the dna , but recent studies of wild - type e. coli topoisomerase iii in complex with dna showed for the first time a wild - type enzyme in complex with dna perfectly poised for dna cleavage ( 20 ) ( figure 3 ) . the chemistry of the mechanism of cleavage and religation , the manner in which the free 3-end interacts with the protein during the reaction , the role of magnesium , and the atomic mechanism of strand passage are some of the questions that remain to be answered . our understanding of type ia enzymes will be incomplete until we elucidate the atomic details of the movement of one strand past the other and how the torsional strain in dna directs conformational changes in the protein . the structures of both domains of vaccinia virus topoisomerase i , the best characterized and studied viral type ib enzyme , are known ( 33,45 ) as well as the structure of the intact deinococcus radiodurans topoisomerase ib ( 50 ) . given the importance of type ib enzymes in cancer therapeutics , it is likely that this will continue to be a fruitful study area and that the information garnered will also shed light on the mechanism of action of the enzymes . as is the case with the type ia enzymes , there are still many details of the mechanism of type ib enzymes that need to be elucidated . together with the wealth of biochemical information , these structures have helped us understand the cleavage and religation mechanism in great detail . in common with type ib enzymes , topoisomerase v cleaves one dna strand , forms a transient phosphotyrosine bond with the 3 end of the broken strand , can relax positive and negative supercoils , and does not require the presence of magnesium or atp ( 67 ) . recent single - molecule experiments established that the mechanism of action of topoisomerase v is similar to the controlled rotation mechanism proposed for type ib enzymes , where relaxation occurs in steps of n ( 73 ) . over the last few years , the elucidation of the structures of various members of all the topoisomerase types has helped to bring our understanding of the mechanism of action of these important enzymes to the atomic level . finally , type ic topoisomerases , the newest family of type i enzymes , are not as well understood as only a few structures are available and the biochemical work is not as advanced .

the need to decarbonize the global economy is widely recognized and , in the uk , it is enshrined in legislation in the form of the 2008 climate change act , which mandates an 80% reduction in carbon emissions by 2050 relative to 1990 . to achieve this , a rapid transformation of infrastructure is required , a process which , as described by the national infrastructure plan published by the uk treasury in 2012 , is aligned with the needs to overhaul and retrofit the national infrastructure to support economic growth . the infrastructure transition envisioned by the uk government includes the adoption of new , low - carbon technologies ( e.g. , electric vehicles for transport and wind turbines for electricity generation ) at unprecedented levels . these new technologies contain a material mix that is very different to that of the current infrastructure stock , potentially introducing a reliance on critical materials at risk of supply disruption ( e.g. , rare earth elements , cobalt , and lithium ) . owing to the huge scale of infrastructure , changes thereto are likely to cause a step change in the demand for such materials . currently , policies planning the deployment of low carbon technologies are based primarily on carbon abatement potential and economic cost . the impacts in terms of new material demands , along with changing infrastructure stocks and future waste treatment , are not considered although these will significantly impact both the sustainability and resilience of future infrastructure systems . introducing technologies into infrastructure that rely on critical materials should prompt a greater effort into understanding and quantifying the changed material . the materials included in current technologies will remain embedded in infrastructure for many years , since infrastructure remains in use for lengths of time ranging from a few years to several decades or even centuries . the resulting delay between materials being introduced into infrastructure and becoming waste complicates the potential for a circular ( or closed - loop ) economy , a concept that has been gaining traction in policy internationally . when the materials embedded in infrastructure are critical materials that may suffer from supply shortfalls , understanding where , when , and how these materials and technologies within which they are embedded may be recovered ( or better , reused ) becomes important , first for enabling a closed - loop system of material use , and second to ensure the continuing functioning of basic infrastructure . to this purpose , infrastructure planning should include estimates of material demands : these estimates should be dynamic , including deployment and end - of - life , and take into account a variety of different technologies and components and their recovery and recycling potentials . by integrating such an analysis into infrastructure transition planning , scenarios can be developed that make optimal use of material resources , minimizing the risk of supply disruptions and making the best use of future end - of - life material . this article presents such a planning tool : a dynamic stocks and flows model for technologies embedded in infrastructure . previous work on dynamic material flow forecasting has established stocks and flows modeling ( sfm ) as a robust and useful tool for predicting future demand , in - use stocks , and waste of material resources . the approach of deriving future material demand from a service demand scenario has found wide use , with the calculation of future waste based on the lifetime characteristics of stocks in - use . past implementations of this approach , however , have limited the lifetime dynamics calculations to a single layer in the model . this is typified by the model of modaresi and mller which includes a nesting of three classes of stocks : the total vehicle stock , three different drive technologies , and three different materials . within this model , the technology stocks and flows alone this approach may be appropriate where the materials have a one - to - one correspondence with the relevant infrastructure , e.g. , concrete used in building stock but falls short when we want to study materials in technologies embedded within infrastructure , where one technology relies on subcomponents , each with their own in - use dynamics and lifetime characteristics . this complex technological structure results in material flow dynamics that are difficult to predict , yet must be understood in order for supply bottle - necks to be averted , and to take advantage of recovery possibilities . in this paper , we present a novel sfm that projects the stocks and flows of technologies and materials based on low - carbon technology deployment scenarios . technology components and materials are explicitly incorporated in the model , each with their own stock and flow dynamics . this allows the projection of material dynamics to include technology components with diverse lifetimes , the recovery and reuse of technology components , and the recycling of materials , an analysis that is often discussed qualitatively in the circular economy literature but has not previously been studied quantitatively . the model provides a methodology to assist planning of technology roll - out , define the materials demand profile and potential bottlenecks , and avoid or diminish supply risks through the planning of recovery and recycling : in short , to manage critical materials in infrastructure . quantifying the potential for recycling and reuse can also act as a driver for the adoption of better material stewardship practices in a circular economy . we demonstrate the approach on the transition in personal transportation vehicles in the uk from internal combustion engine to electric vehicles . this transition involves the potential introduction of large quantities of lithium and cobalt ( in electric vehicle batteries ) and the rare - earth metal neodymium ( in electric vehicle motors ) , while releasing the platinum in catalytic converters , unnecessary in electric vehicles . all four of these materials have been included in previous assessment of material criticality at national and eu scale and found to be of interest . with our model , we show how the demand for these materials and their anthropogenic stocks and waste flows change over time and how different recovery scenarios affect these stocks and flows . the model we present has been designed to study the relationships between the attributes of technologies that make up infrastructure and the stock and flow dynamics of the material contained in these technologies . to enable this , the model has three key features : first , a dynamic representation of stocks and flows ; second , a focus on infrastructure transitions with the adoption of new technologies ; third , the potential for recovery and substitution to occur at the level of technology as well as materials . the first feature is already included by the methodology for dynamic material flow modeling developed by mller , and it is this approach that we build on . to incorporate the second and third feature technologies and their components are explicitly included with their own dynamic stocks and flows . as the purpose of this model is to study the relation between infrastructure and technology dynamics and material flows , the focus is on the in - use phase of the material life - cycle , including the recovery of end - of - life stock for reuse or recycling . furthermore , the purpose of this model is not to produce a perfect representation of every processing step involved in the waste management phases of the infrastructure lifecycle . rather , we design the model to allow us to identify the availability of end - of - life stocks for reuse or recycling . elements of the hierarchical stocks and flows model , from infrastructure stocks to technological structures and components and their material requirements , each resulting in in- and outflows . the model separates an infrastructure system into three distinct classes of stocks:1.infrastructure stocks represent the service level an infrastructure provides , e.g. , vehicles providing transportation . this stock does not refer directly to physical objects , and hence , no physical flows are necessary.2.technology stocks represent the technologies that provide the infrastructure service and are further disaggregated into technology structures , which directly provide the service ( e.g. , vehicles that provide transportation services ) , and their components ( e.g. , batteries , motors , magnets ) , which can be nested to any depth.3.material stocks that are contained in the technology stocks described above , e.g. , lithium contained in an electric vehicle li - ion battery . infrastructure stocks represent the service level an infrastructure provides , e.g. , vehicles providing transportation . this stock does not refer directly to physical objects , and hence , no physical flows are necessary . technology stocks represent the technologies that provide the infrastructure service and are further disaggregated into technology structures , which directly provide the service ( e.g. , vehicles that provide transportation services ) , and their components ( e.g. , batteries , motors , magnets ) , which can be nested to any depth . material stocks that are contained in the technology stocks described above , e.g. , lithium contained in an electric vehicle li - ion battery . in any implementation of the model an infrastructure service could be provided by any number of different technology structures , each of which could be made up of multiple components . a single component stock can also be shared by more than one structure , as would be the case in two types of electric vehicle that share a common motor design . the same is true for materials , where one material stock can represent material contained in several technology components and structures . each of the stocks in the model has its own properties and associated inflows and outflows ( see supporting information 2 for a full stocks and flows diagram ) . this means each structure and component can have different lifetimes , and the outflows from each stock will depend on its own lifetime as well as the dynamics of the stocks in which it is contained . this interaction can result in significant differences to stocks and flows dynamics compared to a simpler model that only considers the lifetime of either vehicle or battery ( see the supporting information for an illustration of the difference ) . the separate representation of each stock requires a separate treatment of these stocks at end - of - life . any of the structures , components , or materials can potentially be recovered at end - of - life and reused or recycled to displace virgin inflow . the calculation of the stocks and flows is done for each class of stocks from the top of the hierarchy down . infrastructure stocks are determined from historical data and a deployment scenario for future dates . the technology structure stocks follow from a combination of the infrastructure stocks and a technology mix that describes what split of technologies is used to provide the infrastructure service . technology component stocks must then match the stock levels of the structures ( or other components ) of which they are constituent parts . finally , the material stocks are determined by the material intensities of the technology stocks which they make up . central to the calculation of every stock and flow in the model is the balance equation1where km(t ) is the stock amount ( kapital ) of structure , component , or material m at time t and im(t ) and om(t ) are the corresponding inflows and outflows of m , respectively . from this balance equation , the determination of stock and flow time - series can proceed through either a flow driven or a stock driven approach . a flow driven approach is appropriate where the inflow and outflow are known or straightforward to model . this would be the case particularly with consumable objects that do not have a complex or long - lived in - use phase , such as aluminum cans or plastic cups . a stock driven approach determines the inflow and outflow from known stock levels and the dynamics of in - use stocks . this is more appropriate where the in - use dynamics are more complicated , for example , in the case of infrastructure where technologies and materials remain in use for long periods and there is a long delay between the inflow of material and it becoming available for recycling . following the stock driven approach , the outflow of any stock is determined by a lifetime function that determines the fraction of the stock added at any previous time that reaches end - of - life at the current time , i.e.,2where lm(,t ) is the lifetime function that gives the fraction of stock added in year that reaches end - of - life in year t and the integral goes over all historical inputs to the current time . the lifetime function is assumed to take the form of a gaussian ; for further details , see the supporting information . given this and the required stock level , km(t ) , the calculations are complicated by the additional outflow due to a parent stock reaching end - of - life . the potential for future reuse or recycling is handled in the model by a recovery process that is described in technical detail in the supporting information . technology structures and components that reach end - of - life have the potential to be reused at the same system level or lost as a waste stream . reuse of technology will usually involve a remanufacturing process that , like primary production , is not included in our model . lithium - ion batteries for example would require remanufacturing before reuse in vehicles is practical , whereas ndfeb magnets could be reused directly without a remanufacturing step . for consistency , we use the term reuse to refer to both possibilities . what was termed reuse for technology structures and component , we call recycling for materials ; recycling is thus defined as a process that occurs strictly at the same system level and is distinct from down - cycling ( without a measure of the function or quality of materials and components , it is difficult to define down - cycling anyway ) . both components and materials can also be contained in parent structures or components that are reused at end - of - life . the model tracks these as embedded stocks . for both technology structures and components , and materials , the term waste is used in the sense of no longer being of use for its previous purpose . this does not preclude the stock being down - cycled and its constituent components or materials being reused or recycled . these definitions of reuse , recycling , and waste are intended to be compatible with the definitions commonly used in circular economy literature and eu waste framework and end - of - life vehicle directives . in the model , the split of end - of - life stock into waste and reuse flows is supplied to the model as a recovery scenario . the purpose of this approach to reuse and recycling is not to give an accurate representation of the end - of - life treatment of technology but to account for the availability of end - of - life stock for recovery and highlight the potential impact of adopting different recycling policies . the transition to low carbon personal transportation is vital to the uk meeting its carbon emission reduction targets , as the transport sector accounts for almost a quarter of ghg emissions in the uk . the department of energy and climate change ( decc ) publishes a series of scenarios for decarbonization that would achieve emissions reduction targets and has an online pathways the scenarios are published by decc on five year increments ; to get a realistic timeline , we apply a cubic interpolation algorithm to calculate consistent yearly increments . we use one of the core scenarios from this work as the driver for infrastructure service and technology roll - out in our model . the scenarios detail the fleets of internal combustion engine vehicles ( icevs ) , plug - in hybrid electric vehicles ( phevs ) , and fully electric vehicles ( evs ) . we use the renewables scenario , which is biased toward the adoption of renewable energy production . this scenario forecasts a decline of icevs starting in 2020 with phevs becoming the most common technology around 2030 and evs taking over in about 2040 , as illustrated in figure 2 . total in - use stocks of vehicles for the uk deployment of electric vehicles under the decc pathway analysis renewables scenario used in the model . the materials we are interested in tracking in this study , lithium , neodymium , cobalt , and platinum , are contained in the batteries , motors , and catalytic converters of vehicles . we take most of the material intensities of these components from the us department of energy as shown in table 1 , and they are consistent with a number of similar studies . the range of material intensities for li - ion batteries is due to uncertainty in the battery chemistry that will be used . a number of candidates exist , each of which has their own advantages and drawbacks , well described by gaines et al . in the model , these ranges the lifetime of all three vehicle types is assumed to remain at 13 years with a standard deviation of 3 years ( c.f . supporting information ) . catalytic converters and electric motors in modern cars are designed to last for the full lifetime of the vehicle and will not usually be replaced , so there is no lifetime for these separate from the vehicle lifetime . the li - ion batteries found in both phevs and evs today are sold with a warranty of 8 years . due to the relatively recent introduction of these batteries , there is no reliable statistical data for their lifetimes . we hence use a lifetime of 8 years with a variance of 2 years for these batteries in the model . we note that the model has the capability for both material intensities of technology and technology lifetimes to change over time ; however , due to a lack of reliable forecasts for what technological changes may bring , we make the conservative estimate that they will remain constant . the supporting information includes a sensitivity analysis for both the vehicle and battery lifetimes . recycling of materials from end - of - life vehicle stock are limited by both the efficiency of recycling processes and collection efficiency . a combination of these two factors leads to realistic recycling rates of 70% for lithium , 90% for cobalt , 70% for platinum , and 80% for neodymium ( for detailed sources , see the supporting information ) . the limits to lithium recycling lie mostly in the difficulty of chemical separation of battery material whereas cobalt and neodymium recycling are limited mostly by collection efficiencies ( which we assume to be very high due to the eu end - of - life vehicle directive ) . the reuse of remanufactured li - ion batteries we analyze in this paper is highly speculative . there is currently no commercial activity in this direction , although research projects are beginning to investigate the possibility . the reuse of ndfeb permanent magnets in motors is also failing to see commercial application , although the process is much simpler as magnets degrade only negligibly over the lifetime of a vehicle . in the absence of more detailed information , we assume an optimistic rate of 95% for both of these , to demonstrate the model s potential . more detailed justifications of recycling and reuse rates are given in the supporting information . figure 3 shows the stocks and flows of cobalt , lithium , neodymium , and platinum under the renewables scenario assuming no recycling or reuse of any material or technology . for the three materials found in low carbon transport technologies ( cobalt , lithium , and neodymium ) , we see the expected steep increase in in - use stock from 2020 . for cobalt and lithium , there is a very steep increase between about 2020 and 2025 , which then continues to increase at a slower rate . neodymium use , in contrast , shows a very rapid increase between 2020 and 2025 and then stabilizes after 2025 . this difference can be explained by the lower material intensity of cobalt and lithium in phev batteries compared to ev batteries . neodymium , however , has equal material intensity in both types of vehicles ; hence , a switch from phevs to evs results in no further increase in material inflow . solid lines indicate the high estimate for material intensity with the low estimate shown as fainter lines ( the low estimate for cobalt being zero ) . use stocks are initially high and drop from 2020 onward , mirroring the decline in icev stock . materials ( cobalt , lithium , and neodymium ) is that the uk demand for these materials for the transport sector will increase rapidly from 2020 . by 2030 , the uk would require over 30 kilotons of cobalt , between 10 and 45 kilotons of lithium , and between 0.7 and 1.5 kilotons of neodymium per year ( depending on the technologies used ) . to provide a sense of scale , in 2010 world production was 88 megatons of cobalt , 28.1 kilotons of lithium , and 22 kilotons of neodymium . this puts the high estimate scenario results in 2030 at 0.03% , 160% , and 7% of 2010 world production for cobalt , lithium , and neodymium , respectively . while it is important to note that we make no forecast for future world mine production , which would be required for fair comparison , or any assessment of any other factors that would lead to potential material criticality , these results are still significant . the relatively short time horizon of the step - change in demand for lithium from 2020 and the scale of this step - change being on the order of 2010 world production is enough to suggest that concern for the supply of lithium is warranted . the scale of the step - change for neodymium is also of concern , especially in context of the wider uses for neodymium which include other low - carbon technologies such as permanent magnet wind turbines . cobalt , however , from a purely supply vs demand perspective does not appear to be particularly critical . there is a step change in demand due to uk electric vehicle estimates , but it is not significant compared to global production . the results from this model are clearly a good starting point for highlighting the potential risks to infrastructure from critical materials . the outflows for all materials ( shown in figure 3 ) and technology components are split in the model into waste flow , recycling / reuse flow , and embedded flow ( material or components that are embedded in reused technology ) . to illustrate the potential for recycling or reuse to displace virgin material or component inflow , we model a set of possible recovery scenarios . three scenarios are modeled with an initial recycling / reuse rate of zero that increases , beginning in 2015 , 2025 , and 2035 , to a set maximum linearly over a period of ten years . the scenario beginning in 2015 represents a highly optimistic roll - out of recycling infrastructure and almost immediate adoption of design standards for reuse . the 2025 scenario represents a more realistic estimate assuming still ambitious targets for policy action and subsequent changes in practice . the 2035 scenario represents late action , with a time scale that will miss most of the alarming demand projections identified above . for lithium , cobalt , and neodymium , the maximum recycling rates are 70% , 90% , and 80% , respectively . for li - ion batteries and ndfeb , the results of applying these scenarios for lithium and neodymium are shown in figure 4 . the result for cobalt is not shown because the shape is identical to the lithium result ; only the scale is different . recovery scenarios for material recycling and component recovery applied to lithium and neodymium in the renewables scenario with a high estimate for material intensity . graphs show the virgin inflow , reuse inflow ( recycled material ) , and embedded inflow ( in reused components ) along with the recovery fraction which is also indicated on the graph by the year recovery begins . the material recycling results for lithium show that a large reduction in virgin inflow is possible with the use of recovered material . it is not possible to completely displace virgin inflow as there will not be enough secondary stock , but the volume of recycled material can be greater than virgin material by 2030 if recycling facilities are in place before then . the peak requirement of about 15 kilotons by 2024 is unavoidable , as there is no vehicle end - of - life stock available for recycling at that time but the no - recovery level of 25 kilotons in 2030 can be reduced to around 15 kilotons . the reductions grow progressively more significant toward 2050 where the requirement for virgin material disappears completely . the effect of reuse of li - ion batteries on the demand for virgin lithium is very similar to the material recycling option , despite the much higher recovery fraction ( 70% to 95% ) . the reason for this is that batteries from phevs are not the same as those needed for evs . this highlights the higher flexibility in recycling materials than trying to reuse more specific technology components . the results for neodymium recycling are similar to those of lithium , i.e. , an unavoidable peak in demand in 2024 . the timing of the impacts of recovery are different because of the longer lifetime of the ndfeb motor compared to li - ion batteries , so there is little difference between the 2025 and the 2035 recovery scenarios . the second peak in demand of 1400 tons seen around 2039 ( figure 3 ) can be reduced to a peak of just 400 tons by material recycling . the effect of ndfeb motor reuse has the potential to reduce this peak even further to 220 tons , a reduction of over 80% . the application of material recycling on platinum is obvious already from the results in figure 3 . this shows that outflows of platinum are significantly greater than inflows in almost every year . recovery and recycling of platinum with a modest recycling rate would thus clearly reduce virgin platinum requirements to almost zero as soon as it is applied ( for details , see the supporting information ) . the remaining recycled platinum surplus would likely also find use in other technologies , given the high value and demand for platinum . the comparison of material recycling and technology reuse allowed by this model enables two important results . first , the explicit inclusion of components with distinct lifetimes in the model is needed for an accurate prediction of the availability of end - of - life resources for recovery . this accounts for the earlier impact of lithium recycling compared to neodymium recycling because the battery lifespan is only 8 years as compared to a 13 year vehicle lifespan . second , the incompatibility of li - ion batteries between phevs and evs is representative of a general feature of remanufacturing and reuse of components : there is a loss of flexibility which must be balanced against the potentially lower efficiency and higher cost of disassembly and material recycling . this type of trade - off is evident only through using technology - specific dynamic modeling , such as the model presented here . the transition from internal combustion engine to electric vehicles , as shown in the decc scenarios , has the potential to make a significant contribution to the planned uk transition to low carbon personal transport . we have now seen that this technology change will be accompanied by the introduction of lithium and neodymium into infrastructure in amounts that will significantly increase uk demand for these materials . the possibility of shortages in supply of these materials constraining a successful transition to low carbon transport should prompt policy actions to mitigate against this . by including the possibilities for material and technology recovery , we have shown how the potential for reuse can be used to mitigate potential supply bottlenecks and support a circular economy , as well as when this option is not viable , due to a lack of available secondary resources . in the case of lithium ( and less critically cobalt ) for li - ion batteries , our results indicate that there exists a trade - off between a battery remanufacturing approach and a lithium recycling approach . the difficulty in component reuse is likely to be compounded by the variety of different li - ion battery chemistries that exist . the implication is that a policy of encouraging the development of material recycling from li - ion batteries is likely to be more fruitful in the medium term , with benefits most significantly felt if recycling infrastructure is in place by 2025 . although material recycling is effective in this case , the higher rate component reuse leads to greater demand reductions . furthermore , the properties of permanent magnets are such that reuse could require only minimal remanufacturing , given appropriate design for reuse . material recycling provides no significant flexibility advantage as magnet technology is relatively mature and uniform ( reflected by the smaller range in material intensity ) . the appropriate policy intervention in this case would be to enable efficient reuse of magnets in ndfeb motors through high collection efficiency and design standards that allow reuse without remanufacturing . in the example of a transition to electric vehicles for personal transportation , these results highlight the need for an evidence based material stewardship policy . understanding where materials go into infrastructure , when they will reach end - of - life , the potential for either material recycling or technology remanufacturing and reuse , and when to prioritize one over the other is crucial to achieving a circular economy . beyond personal transportation , the model could also be applied to wider infrastructure transitions involving many more technologies and materials . a nation - wide study involving interdependent infrastructure systems which share common material bases would have the potential to highlight the full scale of nationally relevant supply bottlenecks and identify significant reuse opportunities for technologies and materials . the trade - off between higher efficiency component remanufacturing and reuse and the lower efficiency , more flexible material recycling , that allows materials to be recycled between different technologies and infrastructures , could be extended in such a study to the reuse of components between different infrastructures , such as ev batteries reused for grid - attached storage . quantifying this trade - off for specific infrastructure systems and technologies where the recovery efficiencies are known could thus inform policies that foster industrial strategies toward optimizing material efficiency . in a broader context , the results from this model bring into focus challenges in the transitions to a low carbon economy . these transitions are often discussed with reference to two separate ideas : the use of low carbon technologies and the move to a circular economy . there is a fundamental short - to - medium term conflict between these two ideals : low carbon technologies have a radically different material mix compared to existing infrastructure stock . for these critical materials , a truly circular economy is therefore not possible until the infrastructure has reached a low - carbon equilibrium state where end - of - life stock is available to substitute for virgin material demand .

the transition to low carbon infrastructure systems required to meet climate change mitigation targets will involve an unprecedented roll - out of technologies reliant upon materials not previously widespread in infrastructure . many of these materials ( including lithium and rare earth metals ) are at risk of supply disruption . to ensure the future sustainability and resilience of infrastructure , circular economy policies must be crafted to manage these critical materials effectively . these policies can only be effective if supported by an understanding of the material demands of infrastructure transition and what reuse and recycling options are possible given the future availability of end - of - life stocks . this article presents a novel , enhanced stocks and flows model for the dynamic assessment of material demands resulting from infrastructure transitions . by including a hierarchical , nested description of infrastructure technologies , their components , and the materials they contain , this model can be used to quantify the effectiveness of recovery at both a technology remanufacturing and reuse level and a material recycling level . the model s potential is demonstrated on a case study on the roll - out of electric vehicles in the uk forecast by uk department of energy and climate change scenarios . the results suggest policy action should be taken to ensure li - ion battery recycling infrastructure is in place by 2025 and ndfeb motor magnets should be designed for reuse . this could result in a reduction in primary demand for lithium of 40% and neodymium of 70% .

Introduction Methodology Results and Discussion

these new technologies contain a material mix that is very different to that of the current infrastructure stock , potentially introducing a reliance on critical materials at risk of supply disruption ( e.g. the impacts in terms of new material demands , along with changing infrastructure stocks and future waste treatment , are not considered although these will significantly impact both the sustainability and resilience of future infrastructure systems . when the materials embedded in infrastructure are critical materials that may suffer from supply shortfalls , understanding where , when , and how these materials and technologies within which they are embedded may be recovered ( or better , reused ) becomes important , first for enabling a closed - loop system of material use , and second to ensure the continuing functioning of basic infrastructure . to this purpose , infrastructure planning should include estimates of material demands : these estimates should be dynamic , including deployment and end - of - life , and take into account a variety of different technologies and components and their recovery and recycling potentials . by integrating such an analysis into infrastructure transition planning , scenarios can be developed that make optimal use of material resources , minimizing the risk of supply disruptions and making the best use of future end - of - life material . this article presents such a planning tool : a dynamic stocks and flows model for technologies embedded in infrastructure . this allows the projection of material dynamics to include technology components with diverse lifetimes , the recovery and reuse of technology components , and the recycling of materials , an analysis that is often discussed qualitatively in the circular economy literature but has not previously been studied quantitatively . the model provides a methodology to assist planning of technology roll - out , define the materials demand profile and potential bottlenecks , and avoid or diminish supply risks through the planning of recovery and recycling : in short , to manage critical materials in infrastructure . as the purpose of this model is to study the relation between infrastructure and technology dynamics and material flows , the focus is on the in - use phase of the material life - cycle , including the recovery of end - of - life stock for reuse or recycling . rather , we design the model to allow us to identify the availability of end - of - life stocks for reuse or recycling . in the model , the split of end - of - life stock into waste and reuse flows is supplied to the model as a recovery scenario . the purpose of this approach to reuse and recycling is not to give an accurate representation of the end - of - life treatment of technology but to account for the availability of end - of - life stock for recovery and highlight the potential impact of adopting different recycling policies . total in - use stocks of vehicles for the uk deployment of electric vehicles under the decc pathway analysis renewables scenario used in the model . the limits to lithium recycling lie mostly in the difficulty of chemical separation of battery material whereas cobalt and neodymium recycling are limited mostly by collection efficiencies ( which we assume to be very high due to the eu end - of - life vehicle directive ) . materials ( cobalt , lithium , and neodymium ) is that the uk demand for these materials for the transport sector will increase rapidly from 2020 . for li - ion batteries and ndfeb , the results of applying these scenarios for lithium and neodymium are shown in figure 4 . the timing of the impacts of recovery are different because of the longer lifetime of the ndfeb motor compared to li - ion batteries , so there is little difference between the 2025 and the 2035 recovery scenarios . first , the explicit inclusion of components with distinct lifetimes in the model is needed for an accurate prediction of the availability of end - of - life resources for recovery . by including the possibilities for material and technology recovery , we have shown how the potential for reuse can be used to mitigate potential supply bottlenecks and support a circular economy , as well as when this option is not viable , due to a lack of available secondary resources . the implication is that a policy of encouraging the development of material recycling from li - ion batteries is likely to be more fruitful in the medium term , with benefits most significantly felt if recycling infrastructure is in place by 2025 . understanding where materials go into infrastructure , when they will reach end - of - life , the potential for either material recycling or technology remanufacturing and reuse , and when to prioritize one over the other is crucial to achieving a circular economy . for these critical materials , a truly circular economy is therefore not possible until the infrastructure has reached a low - carbon equilibrium state where end - of - life stock is available to substitute for virgin material demand .

using content from a variety of questionnaires used in previous studies ( 1720 , 2233 ) and recommendations from the aamc ( 11 , 34 , 35 ) and acgme ( 3 ) , physician staff from the national board of osteopathic medical examiners ( nbome ) compiled a list of specific procedural skills and advanced communication skills . recommendations from nbome 's clinical skills testing advisory committee ( 8 members ) and strategic planning committee ( 16 members ) were incorporated into the survey . members from both committees are considered experts in medical education and assessment ; members include representatives from undergraduate medical education ( deans , associate deans , faculty ) , graduate medical education ( acgme and aoa - accredited residency program directors , directors of medical education ) , clinicians , medical educators , and psychometricians . after receiving input from these expert panels , items were reviewed , and further enhancements were made by nbome 's research advisory committee ( 12 members ) composed of experts in assessment , education , and research . the final instrument ( found in supplemental content ) addressed 28 procedural skills and 18 advanced communication skills , was pretested by physician staff , and distributed using survey monkey . the web - delivered survey was distributed to 3,443 acgme and aoa - accredited residency program directors with valid email addresses contained in nbome 's residency program director database . program directors were randomly divided into two groups ; each group received one of two versions of the survey ( one with procedural skills presented first and another with advanced communication skills presented first ) . institutional review board approval was granted by the center for the advancement of healthcare education and delivery ( c - ahead ) to collect , analyze and report these data for this study . intraclass correlations ( icc ) , which describe the degree of group agreement , were calculated to examine the disparity in responses of program directors of different specialties . using content from a variety of questionnaires used in previous studies ( 1720 , 2233 ) and recommendations from the aamc ( 11 , 34 , 35 ) and acgme ( 3 ) , physician staff from the national board of osteopathic medical examiners ( nbome ) compiled a list of specific procedural skills and advanced communication skills . recommendations from nbome 's clinical skills testing advisory committee ( 8 members ) and strategic planning committee ( 16 members ) were incorporated into the survey . members from both committees are considered experts in medical education and assessment ; members include representatives from undergraduate medical education ( deans , associate deans , faculty ) , graduate medical education ( acgme and aoa - accredited residency program directors , directors of medical education ) , clinicians , medical educators , and psychometricians . after receiving input from these expert panels , items were reviewed , and further enhancements were made by nbome 's research advisory committee ( 12 members ) composed of experts in assessment , education , and research . the final instrument ( found in supplemental content ) addressed 28 procedural skills and 18 advanced communication skills , was pretested by physician staff , and distributed using survey monkey . the web - delivered survey was distributed to 3,443 acgme and aoa - accredited residency program directors with valid email addresses contained in nbome 's residency program director database . program directors were randomly divided into two groups ; each group received one of two versions of the survey ( one with procedural skills presented first and another with advanced communication skills presented first ) . institutional review board approval was granted by the center for the advancement of healthcare education and delivery ( c - ahead ) to collect , analyze and report these data for this study . intraclass correlations ( icc ) , which describe the degree of group agreement , were calculated to examine the disparity in responses of program directors of different specialties . a total of 347 program directors completed the survey , representing a response rate of 10.1% . program directors from a wide range of disciplines responded to the survey , and specialty distributions were reflective of national data ( table 1 ) ( 36 , 37 ) . for instance , 45 surgery and surgical subspecialty program directors were included in the sample ( 13% of the sample ) , compared to 946 in the national sample ( 19.9% of all residencies ) . for instance , 100 family medicine residency program directors were included in the sample ( 28.8% of the sample ) , compared to 636 in the national sample ( 13.4% of all residencies ) . among the 347 respondents , 44 were identified as surrogates, program director - selected surrogates ( e.g. , assistant / associate residency program directors ) who completed the survey on behalf of the residency program director . among those who completed the survey , 293 respondents completed the section on procedural skills and residency program director respondents in comparison with national sample data , by specialty ( n=347 ) abbreviations : acgme , accreditation council on graduate medical education ; aoa , american osteopathic association ; obgyn , obstetrics and gynecology ; pm & r , physical medicine and rehabilitation . eleven program directors reported to have more than one specialty so that each of the total percentages corresponding to sample subgroups may exceed 1 . sixty - four of the acgme - accredited residency programs are also certified by the aoa . program directors considered a number of procedures to be important to assess ( sum of important and extremely important responses ) : sterile technique ( 93.8% ) , advanced cardiovascular life support ( acls ) ( 91.1% ) , basic life support ( bls ) ( 90.0% ) , interpretation of ekg ( 89.4% ) , and interpretation of blood gas ( 88.7% ) . skills such as osteopathic manipulative treatment ( 35.6% ) , obtaining a blood culture ( 37.0% ) , and ppd placement ( 38.4% ) were considered less important . the values reflect responses to the survey question in your opinion , how important is it for each of the following skills to be assessed? values reflect the sum of responses to important and extremely important. the skills are sorted in the descending order of importance rated by all program directors ( n=293 ) . with regard to the importance of assessing procedures , agreement varied among program directors of different specialties . icc is an index representing proportion of the total variance explained by group effects with higher icc values indicating larger group variation ( lower agreement between specialty groups ) . signifying disagreement between program directors from different specialties , high levels of disparity between specialty groups ( icc values > 0.30 ) were found for central line access , lumbar puncture , incision and drainage , splinting / casting , child birth ( vaginal ) and pelvic exam . signifying agreement between program directors from different specialties , low levels of disparity ( icc values < 0.10 ) were found for nasogastric tube placement , obtaining blood culture , cardiac resuscitation ( bls ) , phlebotomy , sterile technique , and injection ( im / sc ) . some procedural skills , such as sterile technique and cardiac resuscitation ( bls ) , displayed both low group disparity and high importance ratings . presented in fig . program directors overwhelmingly reported that each of the procedures should be assessed in a formative fashion , followed by a combination of both formative and summative assessment . compared to other procedures , acls ( 24.2% ) , bls ( 23.6% ) , neonatal advanced life support ( nals ) ( 21.3% ) , pediatric advanced life support ( pals ) ( 20.7% ) , phlebotomy ( 15.2% ) , sterile technique ( 14.9% ) , injection ( 14.7% ) and intravenous placement ( 14.7% ) were considered to be procedures amenable to summative assessment . the values reflect responses to the survey question in your opinion , please mark whether the assessments should be summative ( e.g. , used for advancement purposes ) , formative ( e.g. , used for feedback and teaching purposes ) , both or neither. procedural skills are presented in the descending order of responses to the majority of program directors reported residency program faculty to be the most appropriate for assessing procedural skills ( table 2 ) . only for the phlebotomy skill a small number of program directors reported that resuscitation ( acls , bls , pals and nals ) could be evaluated in a high - stakes testing environment ( 28.7 , 27.6 , 27.6 and 27.0% , respectively ) . program directors perception of who should be evaluating procedural skillsa the values reflect responses to the survey question in your opinion , who would be the most appropriate to make such judgments? the skills are sorted in the descending order of importance rated by all program directors ( n=293 ) ( fig . 1 ) . as for the most appropriate time to assess the procedural skills ( table 3 ) , program directors reported that assessment of most procedures should be completed at the end of the first year of residency or later . of the responses for end of the first year of residency, the largest rates were reported for suturing ( 62.8% ) , lumbar puncture ( 61.1% ) , and incision and drainage ( 60.8% ) . a small number of skills were considered important to assess prior to the start of residency : bls ( 68.9% ) , sterile technique ( 67.2% ) , acls ( 65.9% ) , and phlebotomy ( 63.5% ) . program directors perception of when procedural skills should be assesseda the values reflect responses to the survey question in your opinion , when would be the most appropriate to make such judgments? the skills are sorted in the descending order of importance rated by all program directors ( n=293 ) ( fig . 1 ) . figure 3 displays program directors opinions about the importance of assessing 18 advanced communication skills . program directors considered most communication skills important to assess ( sum of important and extremely important responses ) . responses were the highest for demonstrating professionalism ( 99.6% ) , respectfulness ( 98.9% ) , good listening skills ( 98.6% ) , communication with nursing / ancillary staff ( 98.6% ) , and empathy ( 97.9% ) . the remaining skills each received ratings of importance higher than 78% . the icc coefficients examining group agreement between program directors of different specialties ranged from near 0 to 0.13 . the values reflect responses to the survey question in your opinion , how important is it for each of the following skills to be assessed? values reflect the sum of responses to important and extremely important. the skills are sorted in the descending order of importance rated by all program directors ( n=284 ) . regarding the 18 communication skills , program directors overwhelmingly reported that integrative evaluations using both summative and formative assessment should be utilized ( fig . the majority of the program directors reported residency program faculty to be the most appropriate to assess advanced communication skills ( table 4 ) . the values reflect responses to the survey question in your opinion , please mark whether the assessments should be summative ( e.g. , used for advancement purposes ) , formative ( e.g. , used for feedback and teaching purposes ) , both or neither. advanced communication skills are presented in the descending order of responses to program directors perception of who should be evaluating advanced communication skillsa the values reflect responses to the survey question in your opinion , who would be the most appropriate to make such judgments? the skills are sorted in the descending order of importance rated by all program directors ( n=284 ) ( fig . 3 ) . for all communication skills , except demonstrating respectfulness , program directors reported the end of first year of residency to be the most appropriate time for evaluation ( table 5 ) . of the responses for end of the first year of residency, the largest rates were for handoffs ( 83.1% ) , referral to consultants - oral ( 76.4% ) and dictation of medical record ( 77.5% ) . program directors perception of when advanced communication skills should be assesseda the values reflect responses to the survey question in your opinion , when would be the most appropriate to make such judgments? the skills are sorted in the descending order of importance rated by all program directors ( n=284 ) ( fig . figure 1 presents program directors opinions about the importance of assessing 28 procedural skills . program directors considered a number of procedures to be important to assess ( sum of important and extremely important responses ) : sterile technique ( 93.8% ) , advanced cardiovascular life support ( acls ) ( 91.1% ) , basic life support ( bls ) ( 90.0% ) , interpretation of ekg ( 89.4% ) , and interpretation of blood gas ( 88.7% ) . skills such as osteopathic manipulative treatment ( 35.6% ) , obtaining a blood culture ( 37.0% ) , and ppd placement ( 38.4% ) were considered less important . the values reflect responses to the survey question in your opinion , how important is it for each of the following skills to be assessed? values reflect the sum of responses to important and extremely important. the skills are sorted in the descending order of importance rated by all program directors ( n=293 ) . with regard to the importance of assessing procedures , agreement varied among program directors of different specialties . icc is an index representing proportion of the total variance explained by group effects with higher icc values indicating larger group variation ( lower agreement between specialty groups ) . icc values ranged from 0.04 to 0.51 . signifying disagreement between program directors from different specialties , high levels of disparity between specialty groups ( icc values > 0.30 ) were found for central line access , lumbar puncture , incision and drainage , splinting / casting , child birth ( vaginal ) and pelvic exam . signifying agreement between program directors from different specialties , low levels of disparity ( icc values < 0.10 ) were found for nasogastric tube placement , obtaining blood culture , cardiac resuscitation ( bls ) , phlebotomy , sterile technique , and injection ( im / sc ) . some procedural skills , such as sterile technique and cardiac resuscitation ( bls ) , displayed both low group disparity and high importance ratings . presented in fig . program directors overwhelmingly reported that each of the procedures should be assessed in a formative fashion , followed by a combination of both formative and summative assessment . compared to other procedures , acls ( 24.2% ) , bls ( 23.6% ) , neonatal advanced life support ( nals ) ( 21.3% ) , pediatric advanced life support ( pals ) ( 20.7% ) , phlebotomy ( 15.2% ) , sterile technique ( 14.9% ) , injection ( 14.7% ) and intravenous placement ( 14.7% ) were considered to be procedures amenable to summative assessment . the values reflect responses to the survey question in your opinion , please mark whether the assessments should be summative ( e.g. , used for advancement purposes ) , formative ( e.g. , used for feedback and teaching purposes ) , both or neither. procedural skills are presented in the descending order of responses to the majority of program directors reported residency program faculty to be the most appropriate for assessing procedural skills ( table 2 ) . only for the phlebotomy skill a small number of program directors reported that resuscitation ( acls , bls , pals and nals ) could be evaluated in a high - stakes testing environment ( 28.7 , 27.6 , 27.6 and 27.0% , respectively ) . program directors perception of who should be evaluating procedural skillsa the values reflect responses to the survey question in your opinion , who would be the most appropriate to make such judgments? the skills are sorted in the descending order of importance rated by all program directors ( n=293 ) ( fig . 1 ) . as for the most appropriate time to assess the procedural skills ( table 3 ) , program directors reported that assessment of most procedures should be completed at the end of the first year of residency or later . of the responses for end of the first year of residency, the largest rates were reported for suturing ( 62.8% ) , lumbar puncture ( 61.1% ) , and incision and drainage ( 60.8% ) . a small number of skills were considered important to assess prior to the start of residency : bls ( 68.9% ) , sterile technique ( 67.2% ) , acls ( 65.9% ) , and phlebotomy ( 63.5% ) . program directors perception of when procedural skills should be assesseda the values reflect responses to the survey question in your opinion , when would be the most appropriate to make such judgments? the skills are sorted in the descending order of importance rated by all program directors ( n=293 ) ( fig . figure 3 displays program directors opinions about the importance of assessing 18 advanced communication skills . program directors considered most communication skills important to assess ( sum of important and extremely important responses ) . responses were the highest for demonstrating professionalism ( 99.6% ) , respectfulness ( 98.9% ) , good listening skills ( 98.6% ) , communication with nursing / ancillary staff ( 98.6% ) , and empathy ( 97.9% ) . the icc coefficients examining group agreement between program directors of different specialties ranged from near 0 to 0.13 . the values reflect responses to the survey question in your opinion , how important is it for each of the following skills to be assessed? values reflect the sum of responses to important and extremely important. the skills are sorted in the descending order of importance rated by all program directors ( n=284 ) . regarding the 18 communication skills , program directors overwhelmingly reported that integrative evaluations using both summative and formative assessment should be utilized ( fig . the majority of the program directors reported residency program faculty to be the most appropriate to assess advanced communication skills ( table 4 ) . the values reflect responses to the survey question in your opinion , please mark whether the assessments should be summative ( e.g. , used for advancement purposes ) , formative ( e.g. , used for feedback and teaching purposes ) , both or neither. advanced communication skills are presented in the descending order of responses to program directors perception of who should be evaluating advanced communication skillsa the values reflect responses to the survey question in your opinion , who would be the most appropriate to make such judgments? the skills are sorted in the descending order of importance rated by all program directors ( n=284 ) ( fig . 3 ) . for all communication skills , except demonstrating respectfulness , program directors reported the end of first year of residency to be the most appropriate time for evaluation ( table 5 ) . of the responses for end of the first year of residency, the largest rates were for handoffs ( 83.1% ) , referral to consultants - oral ( 76.4% ) and dictation of medical record ( 77.5% ) . program directors perception of when advanced communication skills should be assesseda the values reflect responses to the survey question in your opinion , when would be the most appropriate to make such judgments? the skills are sorted in the descending order of importance rated by all program directors ( n=284 ) ( fig . program directors reported that all advanced communication tasks and some procedural tasks are important to assess during medical training . although their responses were consistent across disciplines when considering communication tasks , there was variability among groups when asked about procedures . high levels of agreement between program directors of different specialties were seen for nasogastric tube placement , obtaining a blood culture , cardiac resuscitation , phlebotomy , sterile technique and injections . strong agreement is likely explained by the fact that these procedures are common to all physicians , not just those of a particular discipline . identifying consistency among program directors of different disciplines is important , given the recent growth of specialization in graduate medical education ( 3840 ) . however , of these procedures with high levels of agreement , only cardiac resuscitation , sterile technique and injection were considered important to assess . in a similar survey of program directors , 89.7% expected competency three months into residency with regard to bls , and 74.4% with regard to acls ( 18 ) . for both advanced communication and procedural skills , program directors reported that assessments should include a combination of formative and summative evaluation . this was particularly true for advanced communication tasks , demonstrated by a small number of program directors advocating for exclusive summative assessment . compared to the other procedural skills , cardiac resuscitation , phlebotomy , sterile technique , injection and intravenous placement were considered amenable to summative assessment . program directors reported that most clinical skills should be assessed at the end of the first year of residency ( or later ) and not before graduation from medical school . exceptions to this include demonstration of respectfulness , sterile technique , cardiac resuscitation , and phlebotomy ; these were considered important to assess before the start of residency . this is a departure from the recommended procedures specified in aamc 's msop report , which advocates that students demonstrate the ability to complete the following eight procedures : venipuncture , inserting an intravenous catheter , arterial puncture , thoracentesis , lumbar puncture , inserting a nasogastric tube , inserting a foley catheter , and suturing lacerations ( 11 ) . among this list from the msop , only venipuncture ( or phlebotomy ) was considered important to assess at the end of medical school in our study , and the remainder were considered important to assess during the first year of residency or later . consistent with raymond 's findings that few residents report performing specific procedures early in residency ( 13 ) , our study of program directors supports that most clinical procedures should be assessed at the end of first year of residency ( or later ) . similarly , many of the clinical skills tasks assessed by the medical council of canada qualifying examination part ii ( mccqe part ii ) necessitate clinical experience during residency , and therefore examinees are required to complete a minimum of 12 months of postgraduate training before taking the clinical skills exam ( 7 ) . first , although the sample of program directors includes the largest sample of physicians from different institutions and disciplines than any other study we could locate addressing communication and procedural skills ( 347 program directors ) , the survey response rate was 10.1% , and a higher response rate may provide additional information . second , the program directors rationale for their responses was not elicited , and future study could be improved by complementing the survey with focus group discussion . third , we did not solicit responses from medical school faculty ( e.g. , clerkship directors ) . perspectives of medical school faculty is important to incorporate in future study , particularly since significant differences of opinion have been reported regarding which skills should be taught in medical school ( 1724 ) and residency training ( 18 , 25 , 26 ) . fourth , the study did not include a formal resident task analysis with verification of completion of procedures ; obtaining primary verification ( such as a review of credentialing logs ) would provide valuable information . ideally , assessments used for licensure should measure clinical skills considered important to assess among residency program directors across all disciplines and amenable to summative high - stakes assessment . as usmle and comlex - usa examination programs begin to augment and adapt current examinations to comply with a two decision point model for licensure , clarifying which skills should be assessed at specific levels of training ( entry into supervised practice and entry into unsupervised practice ) becomes particularly important . results from this study support that assessing procedural skills such as cardiac resuscitation , sterile technique , and phlebotomy would be important to assess at the end of medical school ( entry into supervised practice ) , but that the assessment of most procedural and advanced communications skills would be more suited at the end of the first year of residency training or later ( entry into unsupervised practice ) . gathering data from residency program directors provides support for examination development as new assessment tools are considered for high - stakes licensing examinations . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

backgroundhigh stakes medical licensing programs are planning to augment and adapt current examinations to be relevant for a two - decision point model for licensure : entry into supervised practice and entry into unsupervised practice . therefore , identifying which skills should be assessed at each decision point is critical for informing examination development , and gathering input from residency program directors is important.methodsusing data from previously developed surveys and expert panels , a web - delivered survey was distributed to 3,443 residency program directors . for each of the 28 procedural and 18 advanced communication skills , program directors were asked which clinical skills should be assessed , by whom , when , and how . descriptive statistics were collected , and intraclass correlations ( icc ) were conducted to determine consistency across different specialties.resultsamong 347 respondents , program directors reported that all advanced communication and some procedural tasks are important to assess . the following procedures were considered important or extremely important to assess : sterile technique ( 93.8% ) , advanced cardiovascular life support ( acls ) ( 91.1% ) , basic life support ( bls ) ( 90.0% ) , interpretation of electrocardiogram ( 89.4% ) and blood gas ( 88.7% ) . program directors reported that most clinical skills should be assessed at the end of the first year of residency ( or later ) and not before graduation from medical school . a minority were considered important to assess prior to the start of residency training : demonstration of respectfulness ( 64% ) , sterile technique ( 67.2% ) , bls ( 68.9% ) , acls ( 65.9% ) and phlebotomy ( 63.5%).discussionresults from this study support that assessing procedural skills such as cardiac resuscitation , sterile technique , and phlebotomy would be amenable to assessment at the end of medical school , but most procedural and advanced communications skills would be amenable to assessment at the end of the first year of residency training or later.conclusionsgathering data from residency program directors provides support for developing new assessment tools in high - stakes licensing examinations .

Methods Instrument (survey to residency program directors) Sample Analysis Results Procedural skills Advanced communication and interpersonal skills Discussion Conclusions Conflict of interest and funding

program directors considered a number of procedures to be important to assess ( sum of important and extremely important responses ) : sterile technique ( 93.8% ) , advanced cardiovascular life support ( acls ) ( 91.1% ) , basic life support ( bls ) ( 90.0% ) , interpretation of ekg ( 89.4% ) , and interpretation of blood gas ( 88.7% ) . signifying agreement between program directors from different specialties , low levels of disparity ( icc values < 0.10 ) were found for nasogastric tube placement , obtaining blood culture , cardiac resuscitation ( bls ) , phlebotomy , sterile technique , and injection ( im / sc ) . compared to other procedures , acls ( 24.2% ) , bls ( 23.6% ) , neonatal advanced life support ( nals ) ( 21.3% ) , pediatric advanced life support ( pals ) ( 20.7% ) , phlebotomy ( 15.2% ) , sterile technique ( 14.9% ) , injection ( 14.7% ) and intravenous placement ( 14.7% ) were considered to be procedures amenable to summative assessment . as for the most appropriate time to assess the procedural skills ( table 3 ) , program directors reported that assessment of most procedures should be completed at the end of the first year of residency or later . a small number of skills were considered important to assess prior to the start of residency : bls ( 68.9% ) , sterile technique ( 67.2% ) , acls ( 65.9% ) , and phlebotomy ( 63.5% ) . program directors considered a number of procedures to be important to assess ( sum of important and extremely important responses ) : sterile technique ( 93.8% ) , advanced cardiovascular life support ( acls ) ( 91.1% ) , basic life support ( bls ) ( 90.0% ) , interpretation of ekg ( 89.4% ) , and interpretation of blood gas ( 88.7% ) . signifying agreement between program directors from different specialties , low levels of disparity ( icc values < 0.10 ) were found for nasogastric tube placement , obtaining blood culture , cardiac resuscitation ( bls ) , phlebotomy , sterile technique , and injection ( im / sc ) . compared to other procedures , acls ( 24.2% ) , bls ( 23.6% ) , neonatal advanced life support ( nals ) ( 21.3% ) , pediatric advanced life support ( pals ) ( 20.7% ) , phlebotomy ( 15.2% ) , sterile technique ( 14.9% ) , injection ( 14.7% ) and intravenous placement ( 14.7% ) were considered to be procedures amenable to summative assessment . as for the most appropriate time to assess the procedural skills ( table 3 ) , program directors reported that assessment of most procedures should be completed at the end of the first year of residency or later . a small number of skills were considered important to assess prior to the start of residency : bls ( 68.9% ) , sterile technique ( 67.2% ) , acls ( 65.9% ) , and phlebotomy ( 63.5% ) . program directors reported that all advanced communication tasks and some procedural tasks are important to assess during medical training . program directors reported that most clinical skills should be assessed at the end of the first year of residency ( or later ) and not before graduation from medical school . exceptions to this include demonstration of respectfulness , sterile technique , cardiac resuscitation , and phlebotomy ; these were considered important to assess before the start of residency . among this list from the msop , only venipuncture ( or phlebotomy ) was considered important to assess at the end of medical school in our study , and the remainder were considered important to assess during the first year of residency or later . consistent with raymond 's findings that few residents report performing specific procedures early in residency ( 13 ) , our study of program directors supports that most clinical procedures should be assessed at the end of first year of residency ( or later ) . as usmle and comlex - usa examination programs begin to augment and adapt current examinations to comply with a two decision point model for licensure , clarifying which skills should be assessed at specific levels of training ( entry into supervised practice and entry into unsupervised practice ) becomes particularly important . results from this study support that assessing procedural skills such as cardiac resuscitation , sterile technique , and phlebotomy would be important to assess at the end of medical school ( entry into supervised practice ) , but that the assessment of most procedural and advanced communications skills would be more suited at the end of the first year of residency training or later ( entry into unsupervised practice ) . gathering data from residency program directors provides support for examination development as new assessment tools are considered for high - stakes licensing examinations .

niemann - pick disease type c ( np - c ) is a rare , autosomal recessive disease caused by mutations in the npc1 ( 95 % of cases ) or npc2 genes . the disease is characterized by heterogeneous and oligosymptomatic presentation of visceral , neurological , and psychiatric manifestations , making for difficult and often delayed diagnosis ( patterson et al . 2012 ; vanier 2010 ; wraith et al . 2009 ; wraith and imrie 2009 ) . current estimated incidence is 0.85 per 100,000 live births ( orphanet report series 2011 ) . diagnosis of np - c is made via physical assessment of the patient , biochemical tests involving filipin staining of skin fibroblasts , and genetic sequencing of the npc1 and npc2 mutations ( wraith et al . filipin staining and genetic analysis are recommended as the first - line diagnostic tests , to be carried out in parallel if possible in order to obtain complementary information ( patterson et al . 2012 ) . however , the choice of first - line test depends on the local availability of techniques and expertise . miglustat , the first and currently only specific treatment for pediatric and adult patients with np - c , was approved for use in the european union in 2009 ( zavesca , actelion pharmaceuticals ltd , allschwil , switzerland ) ( european medicines agency 2012 ; patterson et al . . early diagnosis of np - c is essential so that uninterrupted miglustat therapy can start at the onset of neurological manifestations ( wraith et al . 2009 ; pineda et al . 2010 ; vanier 2010 ; wraith and imrie 2009 ; chien et al . the np - c suspicion index ( si ) screening tool , developed by an international panel of np - c clinical experts , aids physicians unfamiliar with np - c in early identification of patients with suspicion of np - c ( www.npc-si.com ; wijburg et al . is calculated by the presence of key clinical manifestations of np - c within and across three domains plus family history information . due to the disease variability at age of onset and presentation , this manuscript reports subanalyses of the original study data to investigate the discriminatory power of the si tool by age and to investigate associations by the presence and absence of four leading np - c manifestations . subanalyses were performed on data arising from a published study reporting the development of the np - c si tool ( wijburg et al . the published study involved a retrospective chart review of 216 patients in seven specialist np - c centers in europe and australia . data were collected from two patient groups who were tested for np - c : 71 np - c - positive cases , confirmed by classical or variant np - c filipin staining , with subsequent identification of at least one mutation in npc1 or npc2 genes ; and 64 suspected np - c cases but in whom filipin staining was negative . data for np - c - positive cases and suspected cases were collected up to the decision to refer for a filipin test . data were also collected from 81 control patients who were without suspicion for np - c but who presented with at least one symptom associated with np - c and , where possible , from the same outpatient clinic as the np - c - positive and suspected cases . collected data included demographics such as age and gender , presence of individual manifestations within the three domains ( visceral , neurological , and psychiatric ) , and patient s first- or second - degree family history . the scoring system of the si tool assigned points to individual np - c manifestations within each domain . in addition , points were assigned for manifestations presenting across two or three domains and for familial history . rps 70 indicated high suspicion of np - c and recommendation for immediate testing at an np - c specialist center . rps score between 40 and 69 indicated moderate suspicion for np - c and recommendation for follow - up observation as well as further discussion with an np - c referral center . subanalyses involved categorization of all patients by age : infantile patients < 4 years ( n = 23 for np - c - positive patients , n = 23 for np - c - suspected cases , and n = 7 for control patients ) , juvenile patients 416 years ( n = 18 for np - c - positive patients , n = 16 for np - c - suspected patients , and n = 39 for control patients ) , and adolescent patients > 16 years ( n = 30 for np - c - positive patients , n = 25 for np - c - suspected patients , and n = 35 for control patients ) . frequency distribution of manifestations within and across domains was tabulated for each patient age group . the performance of the total and individual rps for each domain was investigated via logistic regression within each age group using the outcome np - c - positive cases versus combined np - c - suspected cases and controls as binary dependent variables and the rps as an independent variable . receiver operating characteristic ( roc ) curve ( sensitivity versus 1-specificity ) analysis was performed , and the area under the curve ( auc ) was estimated . frequency distribution of manifestations by rps ( disease severity ) categories < 70 points , 70150 points , and > 150 points was tabulated and also presented graphically to show the scatter of rps by age , where age was transformed to a logarithmic scale to improve visual assessment in the infantile group . association ( co - occurrence ) of manifestations by the presence and absence of four leading np - c manifestations ataxia , cognitive decline , psychosis , and splenomegaly were examined descriptively by frequency distribution of manifestations and graphical presentation in patients > 4 years . these leading manifestations are moderate or strong indicators for np - c in each domain , and due to their high frequency of occurrence , the presence of one or more of these manifestations is most often the reason for referral for np - c testing . other specific np - c symptoms , such as vertical supranuclear gaze palsy ( vsgp ) , cataplexy , and epilepsy , were excluded as leading symptoms because their presence is rarely the initial cause for referral for np - c testing due to their lower frequency , generally later onset , and in some cases such as vsgp the most frequent ( > 50 % ) manifestations displayed in infantile patients ( < 4 years of age ) were prolonged neonatal jaundice , splenomegaly , and delayed developmental milestones . the majority of manifestations in this patient group appeared across the visceral and neurological domains ( fig . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of three domains ( visceral , neurological , and psychiatric ) at each age group ( infantile < 4 years , juvenile 416 years , and adolescent > 16 years ) individual manifestation association in each domain by age group . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of three domains ( visceral , neurological , and psychiatric ) at each age group ( infantile < 4 years , juvenile 416 years , and adolescent > 16 years ) visceral manifestations were less frequent in adolescent patients ( > 16 years of age ) , as shown in fig . 1 ; however , findings within the neurological domain , in particular , vsgp , dystonia , and dysarthria / dysphagia , became more frequent . the frequency of cognitive decline , a strong psychiatric indicator of np - c , noticeably increased in juvenile and adolescent patients . the frequency of other manifestations within the psychiatric domain , including psychotic symptoms , treatment - resistant psychiatric symptoms , disruptive or aggressive behavior , and other psychiatric disorders also increased in patients > 16 years of age . as in the infantile patient group , juvenile and adolescent patients displayed manifestations clustered across the visceral and neurological domains . additionally , patients > 4 years of age displayed an increased frequency of combined manifestations across the visceral / psychiatric , visceral / neurological , and neurological / psychiatric domains . patients with a total rps < 70 points demonstrated a noticeable lack of psychiatric findings and lower levels of neurological involvement , indicating a more visceral phenotype ( fig . 2individual manifestation association in each domain by total risk prediction score ( rps ) . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of the three domains ( visceral , neurological , and psychiatric ) at each rps level ( < 70 points , 70150 points , and > 150 points ) individual manifestation association in each domain by total risk prediction score ( rps ) . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of the three domains ( visceral , neurological , and psychiatric ) at each rps level ( < 70 points , 70150 points , and > 150 points ) increased presence of vsgp , splenomegaly , ataxia , and all psychiatric manifestations were associated with high suspicion of np - c ( rps > 150 points ) . increased frequency of manifestations both within the same category , but more so across categories , greatly increased the rps . the mean rps for infantile patients < 4 years of age was much lower than rps for juvenile and adolescent patients ( mean rps of 79.2 points compared with 190 and 179 , respectively , fig . 3 ) . as shown in fig . 4 , patients with an rps score of < 70 tended to be infantile patients < 4 years of age.fig . 3mean total risk prediction score ( rps ) by age and scoring for individual and combined domains . mean total rps for each age category and the score for manifestations within individual and combined domainsfig . 4total risk prediction score ( rps ) versus age ( years ) for niemann - pick disease type c ( np - c)-positive cases . total rps and age for each np - c - positive case ( < 4 years n = 23 , 416 years n = 18 , > 16 years n = 30 ) mean total risk prediction score ( rps ) by age and scoring for individual and combined domains . mean total rps for each age category and the score for manifestations within individual and combined domains total risk prediction score ( rps ) versus age ( years ) for niemann - pick disease type c ( np - c)-positive cases . total rps and age for each np - c - positive case ( < 4 years n = 23 , 416 years n = 18 , > 16 years n = 30 ) the leading neurological manifestation of ataxia ( n = 41 , 85.4 % of patients ) , a moderate indicator of np - c according to the si tool , most commonly occurred with other manifestations within the same category , such as dystonia and dysarthria / dysphagia , but also with cognitive decline , which is a psychiatric domain manifestation ( fig . 5 ) . the two leading psychiatric manifestations were cognitive decline ( a strong indicator of np - c ) and psychosis ( a moderate indicator of np - c ) . np - c - positive patients showing manifestations of cognitive decline ( n = 42 , 87.5 % ) displayed increased levels of psychosis and slightly elevated frequency of treatment - resistant psychiatric symptoms , which are within the same domain category . these symptoms include ataxia , gelastic cataplexia , dystonia , and seizures ( fig . 5 ) . psychosis ( n = 15 , 31.3 % ) was associated with other illness features , including treatment - resistant psychiatric symptoms and across - category neurological findings , including dysarthria / dysphagia and dystonia ( fig . 6).fig . 5manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of ataxia and cognitive decline . frequency ( % ) of positive np - c manifestations in each of three domains ( visceral , neurological , and psychiatric ) associated with and without ataxia and with and without cognitive decline ( n = 48)fig . association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of psychosis and splenomegaly . frequency ( % ) of positive np - c manifestations in each of the three domains ( visceral , neurological , and psychiatric ) associated with and without psychosis and with and without splenomegaly ( n = 48 ) manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of ataxia and cognitive decline . frequency ( % ) of positive np - c manifestations in each of three domains ( visceral , neurological , and psychiatric ) associated with and without ataxia and with and without cognitive decline ( n = 48 ) manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of psychosis and splenomegaly . frequency ( % ) of positive np - c manifestations in each of the three domains ( visceral , neurological , and psychiatric ) associated with and without psychosis and with and without splenomegaly ( n = 48 ) the leading manifestation in the visceral domain was splenomegaly ( n = 26 , 54.2 % ) , a strong indicator of np - c . there was little difference between manifestation association within and across domains for patients with or without splenomegaly ( fig . 6 ) . patients without splenomegaly ( n = 22 ) showed a slight increase in frequency of prolonged neonatal jaundice , ataxia , and dysarthria / dysphagia compared with patients with splenomegaly . discriminatory power of the np - c si for np - c - positive cases versus combined np - c - suspected cases and controls was stronger for juvenile ( aged 416 years ) and adolescent ( > 16 years of age ) patients than for infantile ( < 4 years of age ) patients . the estimated roc auc values were 0.981 and 0.964 , respectively ( fig . 7 ) . the domain with highest discriminatory power for np - c in these age groups was the neurological domain , with roc auc values of 0.994 ( 416 years ) and 0.873 ( > 16 years ) , respectively . the discriminatory performance of the np - c si in infantile patients ( < 4 years of age ) was poor , with roc auc value of 0.562 for the total rps , with related poor performance in all three individual domains ( visceral auc = 0.597 , neurological auc = 0.615 , and psychiatric auc = 0.501).fig . niemann - pick disease type c ( np - c)-positive versus combined np - c - suspected cases and controls by age . roc curves that show higher sensitivity and specificity for np - c - positive cases versus combined np - c - suspected cases and controls in age groups 416 years ( juvenile ) and > 16 years ( adolescent ) compared with infantile patients ( < 4 years ) . niemann - pick disease type c ( np - c)-positive versus combined np - c - suspected cases and controls by age . roc curves that show higher sensitivity and specificity for np - c - positive cases versus combined np - c - suspected cases and controls in age groups 416 years ( juvenile ) and > 16 years ( adolescent ) compared with infantile patients ( < 4 years ) . the most frequent ( > 50 % ) manifestations displayed in infantile patients ( < 4 years of age ) were prolonged neonatal jaundice , splenomegaly , and delayed developmental milestones . the majority of manifestations in this patient group appeared across the visceral and neurological domains ( fig . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of three domains ( visceral , neurological , and psychiatric ) at each age group ( infantile < 4 years , juvenile 416 years , and adolescent > 16 years ) individual manifestation association in each domain by age group . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of three domains ( visceral , neurological , and psychiatric ) at each age group ( infantile < 4 years , juvenile 416 years , and adolescent > 16 years ) visceral manifestations were less frequent in adolescent patients ( > 16 years of age ) , as shown in fig . 1 ; however , findings within the neurological domain , in particular , vsgp , dystonia , and dysarthria / dysphagia , became more frequent . the frequency of cognitive decline , a strong psychiatric indicator of np - c , noticeably increased in juvenile and adolescent patients . the frequency of other manifestations within the psychiatric domain , including psychotic symptoms , treatment - resistant psychiatric symptoms , disruptive or aggressive behavior , and other psychiatric disorders also increased in patients > 16 years of age . as in the infantile patient group , juvenile and adolescent patients displayed manifestations clustered across the visceral and neurological domains . additionally , patients > 4 years of age displayed an increased frequency of combined manifestations across the visceral / psychiatric , visceral / neurological , and neurological / psychiatric domains . patients with a total rps < 70 points demonstrated a noticeable lack of psychiatric findings and lower levels of neurological involvement , indicating a more visceral phenotype ( fig . 2individual manifestation association in each domain by total risk prediction score ( rps ) . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of the three domains ( visceral , neurological , and psychiatric ) at each rps level ( < 70 points , 70150 points , and > 150 points ) individual manifestation association in each domain by total risk prediction score ( rps ) . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of the three domains ( visceral , neurological , and psychiatric ) at each rps level ( < 70 points , 70150 points , and > 150 points ) increased presence of vsgp , splenomegaly , ataxia , and all psychiatric manifestations were associated with high suspicion of np - c ( rps > 150 points ) . increased frequency of manifestations both within the same category , but more so across categories , greatly increased the rps . the mean rps for infantile patients < 4 years of age was much lower than rps for juvenile and adolescent patients ( mean rps of 79.2 points compared with 190 and 179 , respectively , fig . , patients with an rps score of < 70 tended to be infantile patients < 4 years of age.fig . 3mean total risk prediction score ( rps ) by age and scoring for individual and combined domains . mean total rps for each age category and the score for manifestations within individual and combined domainsfig . 4total risk prediction score ( rps ) versus age ( years ) for niemann - pick disease type c ( np - c)-positive cases . total rps and age for each np - c - positive case ( < 4 years n = 23 , 416 years n = 18 , > 16 years n = 30 ) mean total risk prediction score ( rps ) by age and scoring for individual and combined domains . mean total rps for each age category and the score for manifestations within individual and combined domains total risk prediction score ( rps ) versus age ( years ) for niemann - pick disease type c ( np - c)-positive cases . total rps and age for each np - c - positive case ( < 4 years n = 23 , 416 years n = 18 , > 16 years n = 30 ) the leading neurological manifestation of ataxia ( n = 41 , 85.4 % of patients ) , a moderate indicator of np - c according to the si tool , most commonly occurred with other manifestations within the same category , such as dystonia and dysarthria / dysphagia , but also with cognitive decline , which is a psychiatric domain manifestation ( fig . 5 ) . the two leading psychiatric manifestations were cognitive decline ( a strong indicator of np - c ) and psychosis ( a moderate indicator of np - c ) . np - c - positive patients showing manifestations of cognitive decline ( n = 42 , 87.5 % ) displayed increased levels of psychosis and slightly elevated frequency of treatment - resistant psychiatric symptoms , which are within the same domain category . these symptoms include ataxia , gelastic cataplexia , dystonia , and seizures ( fig . 5 ) . patients without cognitive decline showed increased findings of other types of psychiatric disorders . psychosis ( n = 15 , 31.3 % ) was associated with other illness features , including treatment - resistant psychiatric symptoms and across - category neurological findings , including dysarthria / dysphagia and dystonia ( fig . 5manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of ataxia and cognitive decline . frequency ( % ) of positive np - c manifestations in each of three domains ( visceral , neurological , and psychiatric ) associated with and without ataxia and with and without cognitive decline ( n = 48)fig . association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of psychosis and splenomegaly . frequency ( % ) of positive np - c manifestations in each of the three domains ( visceral , neurological , and psychiatric ) associated with and without psychosis and with and without splenomegaly ( n = 48 ) manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of ataxia and cognitive decline . frequency ( % ) of positive np - c manifestations in each of three domains ( visceral , neurological , and psychiatric ) associated with and without ataxia and with and without cognitive decline ( n = 48 ) manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of psychosis and splenomegaly . frequency ( % ) of positive np - c manifestations in each of the three domains ( visceral , neurological , and psychiatric ) associated with and without psychosis and with and without splenomegaly ( n = 48 ) the leading manifestation in the visceral domain was splenomegaly ( n = 26 , 54.2 % ) , a strong indicator of np - c . there was little difference between manifestation association within and across domains for patients with or without splenomegaly ( fig . patients without splenomegaly ( n = 22 ) showed a slight increase in frequency of prolonged neonatal jaundice , ataxia , and dysarthria / dysphagia compared with patients with splenomegaly . discriminatory power of the np - c si for np - c - positive cases versus combined np - c - suspected cases and controls was stronger for juvenile ( aged 416 years ) and adolescent ( > 16 years of age ) patients than for infantile ( < 4 years of age ) patients . the domain with highest discriminatory power for np - c in these age groups was the neurological domain , with roc auc values of 0.994 ( 416 years ) and 0.873 ( > 16 years ) , respectively . the discriminatory performance of the np - c si in infantile patients ( < 4 years of age ) was poor , with roc auc value of 0.562 for the total rps , with related poor performance in all three individual domains ( visceral auc = 0.597 , neurological auc = 0.615 , and psychiatric auc = 0.501).fig . niemann - pick disease type c ( np - c)-positive versus combined np - c - suspected cases and controls by age . roc curves that show higher sensitivity and specificity for np - c - positive cases versus combined np - c - suspected cases and controls in age groups 416 years ( juvenile ) and > 16 years ( adolescent ) compared with infantile patients ( < 4 years ) . niemann - pick disease type c ( np - c)-positive versus combined np - c - suspected cases and controls by age . roc curves that show higher sensitivity and specificity for np - c - positive cases versus combined np - c - suspected cases and controls in age groups 416 years ( juvenile ) and > 16 years ( adolescent ) compared with infantile patients ( < 4 years ) . this study was carried out to further determine the association of manifestations and the discriminatory power of the si in different age groups of patients with np - c . infantile patients displayed frequent manifestations of prolonged neonatal jaundice , splenomegaly , and delayed developmental milestones . this was as expected due to these signs being highly characteristic of np - c in this age group ( vanier 2010 ) . infants < 4 years of age also demonstrated fewer cross - domain manifestations compared with juvenile and adolescent patients , particularly the near absence of psychiatric manifestations . vsgp and gelastic cataplexia are the two strongest neurological indicators for np - c according to the si tool . these manifestations are usually present in low frequencies in infantile patients , if found at all . if vsgp is present , it may not be recognized , and the presence of gelastic cataplexia usually appears during the late infantile period ( > 3 years of age ) ( patterson et al . common neurological manifestations in infantile patients < 4 years of age can include delays in developmental motor milestones , hypotonia , and language / speech delay ( vanier 2010 ; patterson et al . whereas visceral manifestations appeared less frequently in adolescent patients ( > 16 years of age ) with np - c , neurological manifestations , including vsgp , dystonia , and dysarthria / dysphagia , showed an increased frequency compared with infant and juvenile patients . this could be an indicator of illness severity ; mild , slowly progressive cases with less frequent or severe epilepsy may survive into adolescence and adulthood , whereas severe cases die during childhood . co - occurrence of manifestations both within and across domains greatly increases the suspicion level of np - c , as evidenced by a high total rps . cross - domain manifestations in all three domains were experienced in higher proportions in patients > 4 years of age than in infantile patients < 4 years of age . manifestations found across the visceral / psychiatric and visceral / neurological domains were the strongest indicators of np - c , resulting in a higher point score within the np - c si tool than manifestations found across the neurological / psychiatric domains . the frequency of manifestations within the neurological / psychiatric domains increased in juvenile and adolescent patients , most noticeably , cognitive decline and ataxia . the np - c si tool had strong discriminatory power ( high roc auc ) within and across the three separate domains for patients aged 416 years ( juvenile ) and > 16 years of age ( adolescent ) for np - c - positive cases versus combined np - c - suspected cases and controls , confirming the rps 70 points suspicion threshold identified in the previously published study ( wijburg et al . the si tool is less able to discriminate np - c in infants < 4 years of age . this could be due to an inability to detect specific neurological manifestations and the lack of development of psychiatric manifestations in this age group . the individual domain with the weakest roc auc value in this age group was the visceral domain . there is a need to develop an si that is well suited to infantile patients . protocol development is currently underway to collect more data for infantile patients with np - c in order to develop a pediatric - specific si tool . discrimination between specific ( e.g. , vsgp , cataplexy ) and sensitive ( e.g. , ataxia , icterus prolonganus , cognitive decline ) symptoms as strong indicators of np - c , as well as the likelihood of any given symptom co - occurring with symptoms from other domains , was built into the np - c si tool during its initial construction when the individual rps of each individual symptom was calculated and validated ( wijburg et al . multivariable analysis of the si tool to examine relationships between individual manifestations within categories was not possible due to insufficient data ( occurrence of zero frequencies ) for several binary predictors . other types of disease can present with similar visceral , neurological , and psychiatric manifestations to that of np - c . acid - sphingomyelinase - deficient niemann - pick disease , sandhoff disease , and gaucher disease type 3 can all present with visceral manifestations of hepatosplenomegaly / splenomegaly and neurological signs including ataxia and seizures . patients with huntington s disease or progressive supranuclear gaze palsy can display neurological manifestations , including dystonia , vsgp , psychiatric symptoms , and cognitive decline ( patterson et al . however , suspicion for np - c should be greatly increased when patients present clinical manifestations not only within , but more importantly across , multiple domains . therefore , analysis of manifestations association based on the si tool concentrated on the complexity of manifestations across , rather than within , each domain . the np - c si tool is designed to allow physicians to objectively assess the likelihood of np - c in undiagnosed patients . as such , it is not , and should not be , used as a differential diagnosis tool . it is possible that patients with other neurological disorders may score high on the np - c si ( e.g. alzheimer s disease patients ) , but the precise diagnosis of these patients should be confirmed by further routine testing ; it is also expected that incorrect diagnoses will become less common as physicians gain more experience using the si tool . a strength of the original published study , and therefore of these subanalyses , is that control cases were selected from the same outpatient clinics , where possible , as the np - c - positive cases and suspected cases . one notable limitation is the retrospective data being collected from a small group of np - c - positive infantile patients ( < 4 years of age , n = 23 ) . as np - c is a rare disease , it is difficult for investigators to incorporate sufficient numbers of patients in studies , thereby making meaningful analysis a challenge . the current si tool categorizes delayed developmental milestones in the ancillary category ( allocated one point ) ; however , this manifestation could be a stronger indicator of np - c for this age group . the si tool can only be as good as the quality of patient data captured in the original study . this does not include patients who were never detected due to the atypical or nonspecific presentation of manifestations . as the disease is progressive , changing from visceral to neuropsychiatric in nature , the cross - sectional , retrospective nature of the data set makes it difficult to assess the usefulness of the si in aiding early detection and predicting progression of np - c in these patients . further retrospective and future prospective , longitudinal studies may help to determine how useful the np - c si tool is in clinical practice and also to refine its use . a strength of the original published study , and therefore of these subanalyses , is that control cases were selected from the same outpatient clinics , where possible , as the np - c - positive cases and suspected cases . one notable limitation is the retrospective data being collected from a small group of np - c - positive infantile patients ( < 4 years of age , n = 23 ) . as np - c is a rare disease , it is difficult for investigators to incorporate sufficient numbers of patients in studies , thereby making meaningful analysis a challenge . the current si tool categorizes delayed developmental milestones in the ancillary category ( allocated one point ) ; however , this manifestation could be a stronger indicator of np - c for this age group . the si tool can only be as good as the quality of patient data captured in the original study . this does not include patients who were never detected due to the atypical or nonspecific presentation of manifestations . as the disease is progressive , changing from visceral to neuropsychiatric in nature , the cross - sectional , retrospective nature of the data set makes it difficult to assess the usefulness of the si in aiding early detection and predicting progression of np - c in these patients . further retrospective and future prospective , longitudinal studies may help to determine how useful the np - c si tool is in clinical practice and also to refine its use . the np - c si tool is useful for providing information regarding association of manifestations within and across visceral , neurological , and psychiatric domains in infantile , juvenile , and adolescent patients with np - c . the screening tool provides strong predictive power for suspicion of np - c in patients > 4 years of age but it is not as useful for infantile patients < 4 years of age . the study was funded by a support grant from actelion pharmaceuticals ltd , allschwil , switzerland . the authors confirm independence from the sponsor ; the content of the article has not been influenced by the sponsor .

objectivethe suspicion index ( si ) screening tool was developed to identify patients suspected of having niemann - pick disease type c ( np - c ) . the si provides a risk prediction score ( rps ) based on np - c manifestations within and across domains ( visceral , neurological , and psychiatric ) . the aim of these subanalyses was to further examine the discriminatory power of the si by age and manifestation associations by np - c suspicion - level and leading manifestations.methodsthe original retrospectively collected data were split into three patient age groups , where np - c - positive cases were > 16 years ( n = 30 ) , 416 years ( n = 18 ) , and < 4 years ( n = 23 ) , and patients rps were analyzed by logistic regression . co - occurrence of manifestations within groups of suspicion level ( low , medium , high ) and leading manifestations ( presence / absence of ataxia , cognitive decline , psychosis , and splenomegaly ) were analyzed descriptively.resultsnp-c-positive cases versus controls showed strong discriminatory power of rps . area under the receiver operating characteristic curve was 0.964 ( > 16 years ) and 0.981 ( 416 years ) but weaker 0.562 for infants ( < 4 years ) . patients with rps < 70 were characterized by a lack of psychiatric manifestations and low levels of neurological involvement , suggestive of a preneurological phase of the disease . in patients > 4 years , prominent leading manifestation associations were ataxia with dystonia , dysarthria / dysphagia , and cognitive decline . psychosis was associated with dysarthria / dysphagia but also with cognitive decline and treatment - resistant psychiatric symptoms.conclusionsthe si tool maintains strong discriminatory power in patients > 4 years but is not as useful for infants < 4 years . the si is also informative regarding the association and co - occurrence of manifestations in patients with np - c .

Introduction Methods Results Frequency and association of manifestations by age group Frequency and association of manifestations by RPS Association by presence/absence of leading manifestations in patients >4years Discriminatory power by age Discussion Study strengths and limitations Conclusions Study sponsorship and funding Competing interest

frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of three domains ( visceral , neurological , and psychiatric ) at each age group ( infantile < 4 years , juvenile 416 years , and adolescent > 16 years ) individual manifestation association in each domain by age group . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of three domains ( visceral , neurological , and psychiatric ) at each age group ( infantile < 4 years , juvenile 416 years , and adolescent > 16 years ) visceral manifestations were less frequent in adolescent patients ( > 16 years of age ) , as shown in fig . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of the three domains ( visceral , neurological , and psychiatric ) at each rps level ( < 70 points , 70150 points , and > 150 points ) individual manifestation association in each domain by total risk prediction score ( rps ) . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of the three domains ( visceral , neurological , and psychiatric ) at each rps level ( < 70 points , 70150 points , and > 150 points ) increased presence of vsgp , splenomegaly , ataxia , and all psychiatric manifestations were associated with high suspicion of np - c ( rps > 150 points ) . total rps and age for each np - c - positive case ( < 4 years n = 23 , 416 years n = 18 , > 16 years n = 30 ) the leading neurological manifestation of ataxia ( n = 41 , 85.4 % of patients ) , a moderate indicator of np - c according to the si tool , most commonly occurred with other manifestations within the same category , such as dystonia and dysarthria / dysphagia , but also with cognitive decline , which is a psychiatric domain manifestation ( fig . frequency ( % ) of positive np - c manifestations in each of the three domains ( visceral , neurological , and psychiatric ) associated with and without psychosis and with and without splenomegaly ( n = 48 ) manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of ataxia and cognitive decline . frequency ( % ) of positive np - c manifestations in each of three domains ( visceral , neurological , and psychiatric ) associated with and without ataxia and with and without cognitive decline ( n = 48 ) manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of psychosis and splenomegaly . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of three domains ( visceral , neurological , and psychiatric ) at each age group ( infantile < 4 years , juvenile 416 years , and adolescent > 16 years ) individual manifestation association in each domain by age group . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of three domains ( visceral , neurological , and psychiatric ) at each age group ( infantile < 4 years , juvenile 416 years , and adolescent > 16 years ) visceral manifestations were less frequent in adolescent patients ( > 16 years of age ) , as shown in fig . frequency ( % ) of positive niemann - pick disease type c ( np - c ) manifestations in each of the three domains ( visceral , neurological , and psychiatric ) at each rps level ( < 70 points , 70150 points , and > 150 points ) individual manifestation association in each domain by total risk prediction score ( rps ) . total rps and age for each np - c - positive case ( < 4 years n = 23 , 416 years n = 18 , > 16 years n = 30 ) the leading neurological manifestation of ataxia ( n = 41 , 85.4 % of patients ) , a moderate indicator of np - c according to the si tool , most commonly occurred with other manifestations within the same category , such as dystonia and dysarthria / dysphagia , but also with cognitive decline , which is a psychiatric domain manifestation ( fig . frequency ( % ) of positive np - c manifestations in each of the three domains ( visceral , neurological , and psychiatric ) associated with and without psychosis and with and without splenomegaly ( n = 48 ) manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of ataxia and cognitive decline . frequency ( % ) of positive np - c manifestations in each of three domains ( visceral , neurological , and psychiatric ) associated with and without ataxia and with and without cognitive decline ( n = 48 ) manifestation association in patients 4 years with niemann - pick disease type c ( np - c ) by presence / absence of psychosis and splenomegaly .

in light of climate change , recent years have seen a rapid adoption of renewable energy production . because of their inherently variable nature , renewables have placed considerable strain on the power grid which must match energy production to demand . a possible solution to this semisolid flow batteries ( ssfbs ) , a recently developed configuration , are considered to be especially promising for such applications . ssfbs use two fluid electrodes , an anolyte and a catholyte , in place of traditional solid electrodes . the use of fluid electrodes decouples the energy of a ssfb , which depends on the size of storage tanks , from its power , which depends on the size of the reactor . additionally , ssfbs may allow easy lifecycle management through the modification or replacement of their fluid electrodes . ssfb electrodes are mixtures of conductive nanoparticles ( cnps ) and electrochemically active particles ( eaps ) dispersed in an electrolyte solution . in most ssfbs , the eaps intercalate and deintercalate lithium whereas the cnps wire the eaps to the current collectors . the continuous phase , a mixture of linear and cyclic carbonate solvents with a high concentration of dissolved lithium salt , provides an ion - conducting medium and a source of lithium ions . the cnps used in ssfbs are typically superconductive carbon blacks such as ketjen black ( kb ) or timcal superp . these carbon blacks are submicrometer - sized permanently fused aggregates of hollow spherical subunits . the self - assembly of the cnps , which depends on their morphology , colloidal interactions , and shear conditions , is of crucial importance to both the electrical and mechanical performance of ssfbs . for colloidal carbon black ( cb ) units in ssfb media , the van der waals attractions should be dominant because their electrostatic interactions are strongly screened . as a result , cb particles tend to form large cohesive structures . at rest ( assuming a high enough concentration ) , they form a space - filling network that can conduct electrons and suspend eaps against gravity through yield stress . in flow ( e.g. , pumping and stirring ) , this network will be broken down into agglomerates . henceforth in this article we differentiate a permanently fused primary aggregate from a reversibly flocculated cluster of these particles , which we will call an agglomerate . given the novelty of ssfbs , significant developments are still required to optimize their performance . mechanical protocols should be a part of this optimization : fluids containing adhesive particles generally produce nonequilibrium structures that can depend on mechanical history . in this article , we focus on how shear and its history influence the electrical and rheological properties of cb suspensions in an ssfb solvent . although the case of cb in an ssfb solvent is rather new , we anticipate that some existing insights into the behavior of cb suspensions will also be applicable to our system . suspensions of reversibly agglomerating colloids , including those of cb , are known to be strongly shear- and history - sensitive . the storage modulus and yield stress of cb gels in oils have even been found to depend predictably on the preshear used to prepare them . in flow , the viscosity of agglomerating colloidal suspensions typically decreases in time after an increase in shear stress or rate , a well - studied property known as thixotropy . the opposite effect , a temporal increase in viscosity after an increase in shear stress or rate , known as rheopexy or antithixotropy , is much less common but has been observed in cb suspensions . on a mechanistic level , rheopexy is thought to be caused by flow - induced flocculation or by the ability of fractal structures to rearrange ( when shear is lowered ) into more densified agglomerates . the electrical impedance of cb suspensions in shear flow has been studied much less often , but also here a dependence on the shear rate was found . the study of the suspension s rheology and impedance in conjunction capitalizes on the tight connection of both behaviors to the ( dynamic ) microstructure of the agglomerates , which can be difficult to measure with optical or scattering techniques , especially for concentrated suspensions . the present study makes use of a home - built rheo - impedance setup to characterize the influence of the mechanical history on both rheological and electrical properties . this approach addresses both the practical aspect of optimizing ssfb performance via mechanical protocols and the more fundamental aspect of understanding the underlying processes . both stepwise changes in shear rate and prolonged shear are explored to identify transient responses . the cessation of shear is included as a special case , where the system is left with only brownian forces to possibly reorganize its structure . our analysis of the structural changes at the microscale is supported by a theoretical model in which the viscosity of effective hard - sphere agglomerates is combined with an effective medium theory for the conductivity . ethylene carbonate ( ec ) and dimethyl carbonate ( dmc ) were obtained from sigma - aldrich ( anhydrous , 99%+ purity ) . ketjen black ec 600jd powder ( kb ) was obtained from akzonobel ( the netherlands ) . sem samples were dispersed in acetone , dried on silica wafers , and then imaged . all other sample preparations and experiments were carried out in an mbraun argon - filled glovebox ( o2 , h2o below 5 ppm ) . the two types of cb used in this study differ in morphology ( see also figure 8) : for n990 , the unit particles are more or less compact spheres , whereas for kb , they are more fractal - like as a result of fusion between spherical subunits . in this article , we designate the kb units as aggregates , whereas clusters formed via reversible flocculation are called agglomerates . all suspensions of kb ( of key interest for this work ) were prepared at a concentration of 1% by weight ( wf = 0.01 ) . because the kb aggregates are highly porous and their subunits are hollow shells , the occupied volume fraction in suspension is much higher than wf . a crude estimate can be obtained by multiplying wf by the ratio of the density of graphite ( 2267 kg / m ) to the tap density of kb ( 100 kg / m ) , giving a volume fraction of 23% . particles were first wetted by the pure ( binary ) solvent in polypropylene containers . after 8 h , lipf6 salt was added via a concentrated solution to bring its concentration to 1 m ( viscosity 4 mpas ) . after hand shaking , at least 8 h was allowed to let the particles equilibrate . samples were then homogenized by rotor stator mixing ( ultraturrax ) at 15 000 rpm for 2 min and subsequently loaded into the rheometer . in exploratory experiments , we found similar trends as reported in this article . however , the quantitative behavior of kb suspensions in ssfb media appeared to be sensitive to preparation protocols . this could be related to the poor wetting by the solvents , the sensitivity of the media , and the strong dependence of macroscopic properties on the volume fraction ( s.i . thus , for kb we performed all reported measurements ( except those in figure 4 ) on the same sample . parallel rheological and electrical measurements on cb suspensions were performed on a stress - controlled haake rheostress rs600 rheometer with a home - built adaptation for electrical impedance spectroscopy ( eis ) measurements ( figure 1 ) . a 60-mm - diameter parallel plate geometry was designed with both stainless steel plates also acting as electrodes . a frictionless low - noise electrical connection to the rotating plate , a mercury - based solvent trap was designed . a glass disk was used to electrically isolate the lower plate from the rheometer body . because of its high thermal conductivity , glass also allows accurate temperature control ( at 25 c for all experiments ) . the top plate was excited by a sinusoidal voltage , and the bottom was connected to the virtual ground of a transimpedance preamplifier ( hf2ta , zurich instruments ) . a buffer preamplifier ( hf2ca , zurich instruments ) was used to measure the potential difference between the plates . an impedance spectroscope ( hf2is , zurich instruments ) was used to extract the complex impedance from the current and voltage signals . ac frequency sweeps were performed from 10 to 100 mhz ( total time 550 s ) with a maximum applied voltage amplitude of 100 mv , which was within the linear response range of the samples . the frequency - dependent impedance of the empty measurement setup was calibrated out using the open short technique . suspensions of cb have been reported to exhibit nonideal rheometric behaviors such as wall slip , shear banding , and vorticity alignment of flocs , especially during start up and in low - shear - rate regimes . we avoided these effects in shear flow experiments by employing relatively high shear rates , where the samples behave as low - viscosity liquids . to obtain a reproducible starting state ( figure 2 ) , the suspensions were presheared at 10 000 s for 200 s , after which the shear rate was slowly ramped down to 316 s and then held for 200 s. step changes in shear rate were followed by a dwell time ( ) that was different per type of experiment . schematic of the general experimental protocol for preshearing and subsequent measurements of the electrical impedance and yield stress . measurements of the impedance during shear were made at a frequency of 0.5 hz to allow real - time monitoring of the electronic resistance ( section 2.3 ) . measurements after the cessation of shear were made after a 200 s rest period . impedance spectra were measured before the yield stress . in the measurement of the latter , the shear stress was ramped up at 62 s per stress decade while measuring the strain . the log(strain ) versus log(stress ) curve was fitted with two straight lines , and the stress at the intersection was taken to be the yield stress . because flow battery electrodes contain conductive carbon in an electrolyte solution , they are capable of both ionic and electronic conduction . to monitor the kb network electrically , the kb suspensions have two ionic charge carriers ( li , pf6 ) and an electron - conducting carbon network . during measurements , the sample is in contact with electrodes that are ionically blocking but electronically reversible . ignoring geometric capacitance , the response of this system can be simplified to that of an ionic resistor and an ionic double layer capacitor , both in parallel to the electronic resistance of the carbon black network . thus , the high - frequency real impedance gives us a parallel combination of both resistances whereas the low - frequency real impedance gives the electronic resistance . the data in figure 3 illustrate that the behavior of our kb suspension at rest can be analyzed with this approach . the very high frequencies ( figure 3 insets ) are affected by uncompensated parasitics . nyquist with equivalent circuit ( left ) and bode ( right ) plots of the impedance response of the kb suspension . the insets show a zoom around high - frequency data ( > 10 khz ) . open symbols : kb under 1000 s shear ( only two frequencies measured ) . ( for low shear rates , dynamic percolation may be possible . ) however , a measurement at two frequencies on the same kb suspension , but now under high shear ( open symbols of figure 3 ) , shows that the low - frequency real impedance is finite whereas the imaginary impedance is close to zero . our measurements on other carbon black samples show that the overall impedance response under shear is similar in shape to that at rest ( figure s2 in s.i . ) , justifying our use of a single low frequency to probe the electronic resistance . additionally , this is in qualitative agreement with earlier findings on carbon black filled polymers , which are known to conduct electrons via two mechanisms . ( at high volume fractions , when the network is continuous , conduction is graphitic . when the network is broken , the electrons can tunnel across small gaps ( < 10 nm ) modulated by thermal voltage fluctuations . for larger gaps we first consider the mechanical properties of our material in flow . to characterize kb , we measured a series of so - called intrinsic flow curves where each of the curves corresponds to a unique preshear rate ( psr ) that was maintained for 1000 s in order to reach a steady state . data points within each curve were then obtained by stepping from the psr to the shear rate of interest and recording the viscosity quickly . ( as shown later , the viscosity almost immediately reached an extremum ; it was this extremum that was recorded . ) are strongly shear - thinning , as expected for agglomerating suspensions . intrinsic flow curve of agglomerates formed at different indicated shear rates ( lines to guide the eye ) . the data point indicated by ( x ) represents the viscosity at 10 000 s after prolonged shearing . their dependence on the psr reveals interesting information that is specific to our kb system . first , the three curves with the highest psr coincide , indicating that a psr of 1000 s or more brings the material to a similar microstructural state . this suggests that the agglomerates are largely broken down under these conditions : generally for agglomerating systems a stronger shear flow causes more breakup . after prolonged ( 2000 s ) shearing at the highest accessible shear rate of 10 000 s ( from an arbitrary starting state ) , the sample showed a similar viscosity to the corresponding points of the intrinsic flow curves for psr 1000 s : this again suggests that the agglomerates are largely broken down ( i.e. , into primary aggregates ) by prolonged shear above 1000 s. second , it becomes clear that in order to reach a steady state even the highest preshears need to be maintained for a significant duration , i.e. , much longer than the data acquisition time ( of o(1 s ) ) of the intrinsic flow curve . this is shown by the curves with psr 316 s , for which the viscosities are different from the curves at higher psrs , even for measurement shear rates 1000 s. third , the fact that the intrinsic flow curves with smaller psrs have lower viscosities is remarkable . in earlier suggested mechanistic pictures , where shear leads to both breakdown and the densification of agglomerates into smaller units , a lower psrs should have resulted in higher viscosities , which is the opposite of what we observe . this points to a mechanism that can enhance the viscosity via agglomerate breakup in kb suspensions . the remarkable fact that the lowest possible viscosity ( 21 mpas ) for our system was measured after the smallest preshear rate ( 100 s ) also points in this direction . to examine this intriguing behavior , we performed experiments in which the material s response to shear rate steps was followed over time . both the viscosity and the electronic resistance ( section 2.3 ) were measured in parallel . all steps in the shear rate were taken from the same reference condition , for which we chose 316 s , based on figure 4 . some typical results of these measurements are found in figure 5 , where both steps up ( to 562 and 1000 s ) and down ( to 177 and 100 s ) are illustrated . evolution of viscosity and low - frequency real impedance of the kb suspension after a shear rate step from 316 to 100 ( black ) , 177 ( red ) , 562 ( green ) , and 1000 s ( blue ) . the inset is a close - up view of the response of the electronic resistance around the step . the initial response , a large increase in viscosity and a small decrease in electronic resistance for a step down in rate ( transition from region i to region ii ) is fast , with a time scale of o(1 s ) . it is not resolvable , considering the short time that the rheometer needs to step the shear rate and the low frequency of the impedance measurement . the secondary response ( region ii ) is significantly slower o ( 1001000 s ) . the slow changes in the viscosity and resistance appear to have similar time scales , suggesting that they both probe changes in the microstructure of the kb . remarkably , for both signals , the secondary response opposes the direction of the initial response . for the viscosity , this amounts to a relatively small correction ( rheopexy , shear thinning ) , but for the electronic resistance , the secondary effect is strong and causes a reversal of the overall effect . the shape of the decays of the viscosity and electronic resistance is well described by stretched exponentials ; such behavior has been found for the viscosity of thixotropic systems . the opposing change in the second stage is found irrespective of whether the shear rate is increased or decreased . yet the direction in which the shear rate is changed still determines the sign of all changes . this is consistent with a picture in which all structural transitions can be reversed via the shear rate . we interpret our findings in figures 4 and 5 with a picture in which kb reversibly agglomerates via two distinct mechanisms ( each with its own time scale and influence on the microstructure ) . both the viscosity and electronic resistance depend on the concentration and morphology of the agglomerates , but in different ways . flow curves of weakly agglomerating suspensions have been successfully described by modeling the agglomerates along with their immobilized solvent as effective hard spheres . shear thinning is then explained via a lowering of the effective hard sphere volume fraction . as the shear rate is increased , higher shear stress causes the agglomerates to break down into smaller structures . because of the fractal build up , the latter then occupy less total volume ( figure 6 ) . although ( strictly speaking ) perfectly fractal scaling is rarely observed , it is has often been found that the structure of agglomerates can be fairly well described with this concept . cartoon of fractal agglomeration and imperfect percolation ( left ) in shear flow . to understand the ( changes in ) electronic conductivity of the suspension under flow , we need to take into account the formation of ( transient ) pathways with missing links ( figure 6 , left ) . as such gaps dominate the resistance ( section 2.3 ) , the macroscopic electronic resistance of the sheared suspension should be determined by the concentration of agglomerates and their typical distance of closest approach . we now proceed to a qualitative interpretation of figure 5 , focusing first on the instant process . the jumps in viscosity and electronic resistance may be explained by agglomeration due to lower shear forces when the rate is stepped down . the immediate sticking between colliding fractal entities leads to more open agglomerates that enclose ( and hence immobilize ) more solvent , causing the hydrodynamic volume fraction and thus the suspension viscosity to increase . ( note : if the shear step were small , then a binary collision would lead to an unstable agglomerate , which would be quickly eroded . ) the formation of fewer but larger agglomerates after a step down in shear rate diminishes both the average gap between agglomerates and the number of gaps an electron needs to traverse along a conductive path . however , they are counteracted by a decrease in the number of electronic pathways . is continued after the step down in rate , the population of agglomerates is rejuvenated continually : bonds between kb units are incessantly broken and formed by shear . this provides a means for the agglomerates to overcome kinetic barriers and discover more stable states . as the unit particles ( and their agglomerates ) are ( roughly ) fractal in nature , the randomness of collisions will occasionally cause two particles to interpenetrate ( figure 7 ) . because of the enhanced contact area and generally lower aspect ratio , these agglomerates will be more stable ( compared to those formed by peripheral contacts ) . however , if the shear stress gets high enough , they can be torn apart again , making the process reversible . clearly , a consolidated interpenetration should lead to an overall densification of agglomerates , leading to a lower hydrodynamic volume fraction and hence a reduced viscosity . it also follows from mass conservation that the average gap between agglomerates will increase in this scenario . both effects should lead to the observed increase in the electronic resistance ( after a stepdown in shear rate ) . the dependence of the interpenetrating agglomeration on shear rate can be understood as follows : the stability of interpenetrating kb units will depend on how deeply they are lodged within the agglomerate because only the peripheral zone will be exposed to local shear flow . this explains why a complete reversal of the process can only be achieved at high shear rates : here , the shear stresses are high and the agglomerates are small , thus exposing all kb units to shear . ( this is also why we defined our samples by preshearing them at 10000 s. ) in our proposed mechanism of interpenetrating agglomeration , the shape of the kb units plays an important role . to test this hypothesis , we performed an experiment comparable to that in figure 5 but now with spherical unit particles ( n990 ) . a weight fraction of 30% was chosen to achieve a high shear viscosity similar to that of kb . assuming that the hydrodynamic volume fractions of both suspensions are comparable when they are broken down to the unit particles , the only relevant difference should be the shape of the unit particle ( figure 8) . the electrical resistance of the n990 suspensions turned out to be too high to be accessible at experimental frequencies ( < 10 mhz ) . as shown in figure 9 , similar to kb the n990 suspension also exhibits an immediate jump in viscosity when the shear rate is stepped down , consistent with instant open agglomeration . however , when shear is continued , the viscosity slightly increases ( thixotropy , shear thinning ) , in contrast to kb where it decreases . this supports our explanation that the fractal morphology of kb is responsible for the occurrence of interpenetrating agglomeration . evolution of the viscosity of an n990 suspension after a shear rate step from 316 s. the colors correspond to shear rate steps described in figure 5 . one tick corresponds to 1 mpas . to further examine the mechanistic explanations proposed so far ( as sketched in figures 6 and 7 ) , we combined previously developed concepts for describing the viscosity of agglomerating systems and the conductivity of filled polymers into a simplistic theoretical scheme . the scheme uses the measured viscosity and electronic resistance as inputs , and the collision radius and fractal dimension of the agglomerates ( along with other derived geometrical properties ) are output . the krieger dougherty equation is used to calculate the hydrodynamic volume fraction from the relative viscosity , while part of the potanin model is used to set a relation between the agglomerate radius and the fractal dimension . an effective medium approach for the electronic resistance is used to construct an additional relation between the agglomerate radius and the fractal dimension . in this approach , agglomerates are assumed to occupy centers in a lattice . the resistivity of the suspension then depends on the resistance between adjacent occupied lattice sites and depends asymptotically on the fraction of occupied sites . because the agglomerates have a fractal architecture , the resistance of a conductive contact depends not only on proximity but also on the local ( sub)unit particle density in the outer shells of the agglomerate . the above relations are solved numerically for each measured viscosity and electronic resistance combination to yield a solution ( fractal dimension , collision radius ) . application of the scheme to the measurements of figure 5 leads to the data shown in figure 10 . we observe that the trends used in our qualitative interpretation are reproduced by the model . for steps down in shear rate , the fast restructuring response leads to larger and less dense agglomerates while reducing both their concentration and spacing . during the slow interpenetrating agglomeration process , densification leads to a strong increase in the interagglomerate distance ( and hence electronic resistance ) . for steps up in shear rate , shear rate steps are made from 316 to 100 ( black ) , 177 ( red ) , 562 ( green ) , and 1000 s ( blue ) . we revisit the experiment shown in figure 5 , now focusing on the last stage ( region iii ) in which the flow is terminated after 1000 s of maintained shear at a constant rate . the ( still monitored ) electronic resistance shows an immediate sharp drop , indicating the formation of a space - filling network . after 200 s , the resistance reaches a steady state , justifying measurements of the full impedance spectra ( figure s3 in the s.i . ) and the yield stress . remarkably , both the low - frequency real impedance and the yield stress show a systematic dependence on the previously applied shear rate , with higher preshear leading to a higher yield stress and a lower electronic resistance ( figure 11 ) . this effect is of strong significance to ssfbs where a high yield stress and low electronic resistance are both required for efficient operation . rest impedance and yield stress of kb suspensions after preshearing for 1000 s at indicated rates . , we propose that the agglomerate structures as defined by the shear history are the building blocks of the solid network that forms after shear cessation . in other words , the agglomerates remain intact up to the point where they connect with other agglomerates to form a space - filling network . this freezing - in effect is plausible , as agglomerates can still undergo brownian motion while thermal forces are too weak to break bonds between the kb units . the qualitative dependences of the electronic resistance and yield stress on the preshear rate can be rationalized using the same shear ( history ) induced changes in the agglomerate structure as discussed earlier . high preshears result in small agglomerates , which assemble into a highly branched network when shear is stopped . conversely , preshearing at low rates leads to networks with fewer and thicker branches because the agglomerates were larger in size and fewer in number . the contact areas between the agglomerates that form the low preshear gel should be similar ( peripheral contacts ) and thus no more effective at transferring charge and mechanical force than those of the high preshear gel . however , in the latter material , the number density of branches will be higher , leading to a higher yield stress and a lower electronic resistance . to further examine the role of the secondary response ( region ii in figure 5 ) in light of the results of figure 11 , we explored the effect of shear cessation at different times ( figure 12 left ) . as before , a 200 s rest time was given between the cessation of shear and the measurement of the impedance spectrum ( complete spectrum in s.i . as shown in figure 12 right , both the final electronic resistance and yield stress were found to evolve in an s shape from their starting value . ( in the case of the step to 100 s , steady state is not completely reached even after 1000 s. ) remarkably , their values for just 1 s of shear after the step are fairly similar to those without the step . this indicates that the structures of the gelled networks must also be very similar , despite the instant agglomeration ( viscosity and resistance jumps ) that occurred during the 1 s step . this suggests that the instant agglomeration process and gel formation on shear cessation have similar mechanisms . additionally , it shows that the process responsible for the secondary response ( interpenetration ) is almost exclusively responsible for the change in rest network properties . ( left ) evolution of viscosity and low - frequency real impedance after a shear rate step from 316 to 1000 ( up ) or 100 s ( down ) with shear cessation at different ( indicated ) times . ( right ) rest network resistance and yield stress measured after shear cessation at the indicated time . the influence of shear and its history on the rheology and electrical impedance of ketjen black suspensions in ssfb solvent was studied with two objectives : to understand the microscopic origin of the shear - history - dependent behaviors and to identify the implications for the performance of ssfbs . both aspects revolve around the microstructure of the agglomerated suspension and how it adapts to mechanical conditions . given the difficulty of measuring relevant structural properties directly via microscopy or scattering , our simultaneous study of the rheology and electrical impedance provided a good alternative method . the fast and slow transient responses to a change in shear rate clearly indicate that kb agglomerates exhibit two buildup ( breakup ) mechanisms after decreasing ( increasing ) the shear rate . the fast response was attributed to open agglomeration , and the slower response was ascribed to an interpenetration of the fractal - like kb units . the combination of these processes leads to a shear- and time - dependent agglomerate structure that gets frozen in on cessation of flow , with a higher preshear leading to a stronger gel with a lower electronic resistance , both critical to ssfb performance . an implication of our findings is that for ssfbs operated in the absence of flow , a high preshear ( through fast pumping or stirring ) would be the best for minimizing the electronic resistance and maximizing the yield stress . for ssfbs operated in continuous flow , a high flow rate would minimize the electronic resistance . we expect that our findings will be of relevance not just to ssfbs but also to other systems that use fractal carbon blacks such as flow supercapacitors . additionally , we foresee electrochemical cycling to play an important role in the rheo - impedance of fluid electrodes ( and vice versa ) and are currently investigating the same .

we studied the effects of shear and its history on suspensions of carbon black ( cb ) in lithium ion battery electrolyte via simultaneous rheometry and electrical impedance spectroscopy . ketjen black ( kb ) suspensions showed shear thinning and rheopexy and exhibited a yield stress . shear step experiments revealed a two time scale response . the immediate effect of decreasing the shear rate is an increase in both viscosity and electronic conductivity . in a much slower secondary response , both quantities change in the opposite direction , leading to a reversal of the initial change in the conductivity . stepwise increases in the shear rate lead to similar responses in the opposite direction . this remarkable behavior is consistent with a picture in which agglomerating kb particles can stick directly on contact , forming open structures , and then slowly interpenetrate and densify . the fact that spherical cb particles show the opposite slow response suggests that the fractal structure of the kb primary units plays an important role . a theoretical scheme was used to analyze the shear and time - dependent viscosity and conductivity . describing the agglomerates as effective hard spheres with a fractal architecture and using an effective medium approximation for the conductivity , we found the changes in the derived suspension structure to be in agreement with our qualitative mechanistic picture . this behavior of kb in flow has consequences for the properties of the gel network that is formed immediately after the cessation of shear : both the yield stress and the electronic conductivity increase with the previously applied shear rate . our findings thus have clear implications for the operation and filling strategies of semisolid flow batteries .

Introduction Materials and Methods Results and Discussion Conclusions

our analysis of the structural changes at the microscale is supported by a theoretical model in which the viscosity of effective hard - sphere agglomerates is combined with an effective medium theory for the conductivity . the log(strain ) versus log(stress ) curve was fitted with two straight lines , and the stress at the intersection was taken to be the yield stress . evolution of viscosity and low - frequency real impedance of the kb suspension after a shear rate step from 316 to 100 ( black ) , 177 ( red ) , 562 ( green ) , and 1000 s ( blue ) . the initial response , a large increase in viscosity and a small decrease in electronic resistance for a step down in rate ( transition from region i to region ii ) is fast , with a time scale of o(1 s ) . it is not resolvable , considering the short time that the rheometer needs to step the shear rate and the low frequency of the impedance measurement . the slow changes in the viscosity and resistance appear to have similar time scales , suggesting that they both probe changes in the microstructure of the kb . for the viscosity , this amounts to a relatively small correction ( rheopexy , shear thinning ) , but for the electronic resistance , the secondary effect is strong and causes a reversal of the overall effect . the opposing change in the second stage is found irrespective of whether the shear rate is increased or decreased . this is consistent with a picture in which all structural transitions can be reversed via the shear rate . we interpret our findings in figures 4 and 5 with a picture in which kb reversibly agglomerates via two distinct mechanisms ( each with its own time scale and influence on the microstructure ) . both the viscosity and electronic resistance depend on the concentration and morphology of the agglomerates , but in different ways . this explains why a complete reversal of the process can only be achieved at high shear rates : here , the shear stresses are high and the agglomerates are small , thus exposing all kb units to shear . ( this is also why we defined our samples by preshearing them at 10000 s. ) in our proposed mechanism of interpenetrating agglomeration , the shape of the kb units plays an important role . to further examine the mechanistic explanations proposed so far ( as sketched in figures 6 and 7 ) , we combined previously developed concepts for describing the viscosity of agglomerating systems and the conductivity of filled polymers into a simplistic theoretical scheme . the scheme uses the measured viscosity and electronic resistance as inputs , and the collision radius and fractal dimension of the agglomerates ( along with other derived geometrical properties ) are output . an effective medium approach for the electronic resistance is used to construct an additional relation between the agglomerate radius and the fractal dimension . because the agglomerates have a fractal architecture , the resistance of a conductive contact depends not only on proximity but also on the local ( sub)unit particle density in the outer shells of the agglomerate . remarkably , both the low - frequency real impedance and the yield stress show a systematic dependence on the previously applied shear rate , with higher preshear leading to a higher yield stress and a lower electronic resistance ( figure 11 ) . , we propose that the agglomerate structures as defined by the shear history are the building blocks of the solid network that forms after shear cessation . however , in the latter material , the number density of branches will be higher , leading to a higher yield stress and a lower electronic resistance . to further examine the role of the secondary response ( region ii in figure 5 ) in light of the results of figure 11 , we explored the effect of shear cessation at different times ( figure 12 left ) . as before , a 200 s rest time was given between the cessation of shear and the measurement of the impedance spectrum ( complete spectrum in s.i . the influence of shear and its history on the rheology and electrical impedance of ketjen black suspensions in ssfb solvent was studied with two objectives : to understand the microscopic origin of the shear - history - dependent behaviors and to identify the implications for the performance of ssfbs . the combination of these processes leads to a shear- and time - dependent agglomerate structure that gets frozen in on cessation of flow , with a higher preshear leading to a stronger gel with a lower electronic resistance , both critical to ssfb performance .

the standard therapy for type 1 diabetes consists of exogenous insulin administration , either by multiple daily injections or with continuous subcutaneous insulin infusion ( csii ) through the insulin pump , adjusted according to self - monitored blood glucose ( smbg ) levels 34 times per day . however , in the past 1015 years , new possibilities in diabetes therapy have emerged thanks to continuous glucose monitoring ( cgm ) and csii , which substitute smbg and multiple daily injection therapy , respectively . minimally invasive cgm devices can measure , in real time , interstitial glucose ( ig ) concentrations in continuous time for up to several days . csii uses the subcutaneous route and administers insulin with a basal / bolus strategy , i.e. , continuously deliver insulin over 24 h and inject boluses . in order to determine the appropriate meal insulin bolus , it would be important to know the subject - specific insulin sensitivity , i.e. , the ability of insulin to stimulate glucose utilization and inhibit glucose production . indices of insulin sensitivity include those based on hyperinsulinemic - euglycemic clamp ( 1 ) , intravenous glucose tolerance test ( 2 ) , and , more recently , meal and oral glucose tolerance test . these indices include the oral glucose minimal model ( 3,4 ) , a minimal model based integral formula ( 5 ) , and surrogate measures ( 68 ) . however , all the above require the measurement of both plasma glucose and insulin concentrations and therefore can not be used in everyday life . in the outpatient setting , the only usable approach is the risk method proposed by breton and kovatchev ( 9 ) , which uses smbg data collected over a period of 26 weeks and some patient parameters . this index measures the average insulin sensitivity in the preceding 2 weeks and thus can not be used to assess the intraday variability of insulin sensitivity . here we propose a method to estimate insulin sensitivity from cgm sensor and insulin pump data and validate it against the oral minimal model , which uses plasma glucose and insulin concentrations . twelve type 1 diabetic subjects ( 5 females , aged 39.5 14.2 years , bmi 25.7 3.8 kg / m , hba1c 8.5% or 69 mmol / mol ) were studied for 3 days in the clinical research unit of the mayo clinic center for translational science activities as part of a separate study to determine diurnal patterns of insulin sensitivity ( 10 ) . briefly , once per day , a triple - tracer mixed - meal study protocol was performed during breakfast , lunch , or dinner in latin square design . blood samples were collected at 180 , 30 , 0 , 5 , 10 , 20 , 30 , 60 , 90 , 120 , 150 , 180 , 240 , 300 , and 360 , with t = 0 corresponding to meal time , for measurement of plasma glucose and insulin concentrations in order to estimate si with the oral minimal model ( 3 ) , here considered as reference . subjects also wore both subcutaneous insulin pump ( medtronic or insulet omnipod ) and cgm ( dexcom seven plus ) . within - subject mean absolute relative difference between cgm readings and reference was equal to 12.1 3.3% , and no systematic variation over time was found ( mean [ sd ] within subjects was 5.3 3.7% ) . ( 10 ) since they had the complete cgm and csii data required for the calculation described below . of note glucose data are measured by cgm ( left - hand panel , black line ) , with some smbg data available for sensor recalibration ( left - hand panel , gray dots ) . insulin data are the basal infusion and boluses administered by insulin pump ( right - hand panel , black line and gray triangles , respectively ) . the starting point is the derivation of insulin sensitivity by integrating the oral minimal model equations ( 3 ) , as described by caumo et al . , the calculation is adapted to allow the use of cgm and csii data , instead of plasma glucose and insulin concentrations . the oral minimal model ( 3 ) iswhere g is plasma glucose concentration ( milligrams per deciliter ) , with gb denoting its basal value ; x is insulin action ( min ) ; i is plasma insulin concentration ( microunits per milliliter ) , with ib denoting its basal value ; rag is posthepatic appearance of meal glucose ( milligrams per kilograms per minute ) ; vg is glucose distribution volume ( deciliters per kilogram ) ; p2 is speed of rise and decay of insulin action ( min ) ; and p3 is its size ( min per microunit per milliliter ) . insulin sensitivity is defined asby substituting and into eq . 1 , we obtainby integrating the differential equation ( eq . 3 ) from the time of the meal ingestion ( tmeal ) to the end of the experiment ( tend ) and rearranging , one hasthe first integral in the numerator can be rewritten aswhere d ( milligrams ) is the amount of glucose ingested during the meal and f(tend ) is the fraction of the ingested dose , which at t = tend has reached plasma . 4 is usually not available because model parameters are unknown , thus the denominator of eq . 4 is substituted with the average of glucose excursion times the overall insulin stimulus : where |g| is above basal plasma glucose concentration . thus si is given bywhere auc is the area under the curve calculated from the start of the meal ( tmeal ) to the end of the experiment ( tend ) . subject - specific parameters used by the formula are the body weight ( bw ) ( kilograms ) , height ( meters ) , and age ( years ) . together with height and age , used for the calculation of plasma insulin clearance ( cl ) , as discussed below . parameter fixed to population values are glucose effectiveness at zero insulin gezi [ deciliters per kilogram per minute ; fixed to 0.01 dl / kg / min for diabetic subjects ( 10,12 ) ] and volume of glucose distribution vg [ deciliters per kilogram ; fixed to 1.45 dl / kg , according to dalla man et al . g , g , and i are not directly available but can be derived from cgm and subcutaneous insulin delivery as detailed below . cgm measures the ig concentration , which is related to plasma glucose by a first - order differential equation , i.e. , ig is a delayed version of plasma glucose : thus , with a well - calibrated device , assuming ig(tend ) = ig(tmeal ) , one hasi.e.,similarly , for the above basal glucose signal , one hasthus one can safely assume that , in the presence of a well - calibrated device , auc(cgm ) and auc(|cgm| ) are good approximations of auc(g ) and auc(|g| ) , respectively . in presence of a noncalibrated device , if at least two smbg samples are available , it is possible to offline recalibrate the cgm profile ( 13,14 ) . for the calculation of auc(i ) , we assume that insulin is not degraded locally in the site of infusion . in this case , the integral of plasma insulin can be obtained from subcutaneous insulin infusion divided by the plasma insulin clearance cl . in fact , plasma insulin kinetics can be described with a single compartment model ( 15):where i(t ) is the plasma insulin concentration , rai(t ) is the insulin rate of appearance in plasma , vi is the insulin volume of distribution , and n the fractional insulin clearance rate ( n = cl / vi ) . 12 from the time of the premeal bolus ( tmeal ) to the end of the observation period ( tend ) and assuming that insulin is back to its initial value at the end of the experiment , one hasfinally , under the assumption that all infused insulin eventually reaches the circulation , one hasthus we can compute auc(i ) from the amount of insulin infused subcutaneously and cl : where basal(t ) is the basal insulin infusion rate during the integration period , bolus(tk ) is the premeal or correction bolus administered at t = tk , and cl ( liters per minute ) is the plasma insulin clearance , which can be calculated from subject s height , bw , and age by using the population model proposed by campioni et al . in addition , if correction boluses are administered before the start of the meal , one has to consider that part of that injected insulin could be still active . the residual active insulin can be determined by adopting the same algorithm presented by patek et al . ( 17 ) , which uses the insulin on board ( iob ) curves adapted from ellingsen et al . this quantity ( iob[tmeal ] ) must be added to the previously estimated auc(i ) . moreover , if correction boluses are administered before the end of the considered interval , iob is used to evaluate the active insulin at the end of the study ( iob[tend ] ) , which is subtracted from the previously estimated auc(i ) . in summary , as evident from eq . 7 , an accurate calculation of insulin sensitivity requires the knowledge of the amount of carbohydrates entering the circulation in the integration interval [ df(tend ) ] . in this study , meals were provided at 7:00 a.m. , 1:00 p.m. ; and 7:00 p.m. each day , thus the time interval between the meals was at least 6 h. however , this may not always be the case . furthermore , not all the carbohydrates ingested with a meal may be fully absorbed before the ingestion of a second meal . thus , to account for unabsorbed carbohydrates , e.g. , frequent meals close to each other , the concept of carbohydrates on board ( cob ) is introduced . similarly to iob , cob is a function that , at each time t , quantifies the fraction of the ingested carbohydrates that has not yet appeared in the circulation . cob is based on the model of gastrointestinal tract ( 19 ) : given the amount of carbohydrates ingested at time tm , cob provides , at each time t > tm , the percentage of carbohydrates not yet absorbed , while for t tm>360 min , it is assumed that the carbohydrate absorption is almost completed . the fraction of the ingested dose that has reached plasma at time t , f(t ) , can be calculated as the ratio between the auc of the meal rate of appearance and the ingested dose , d ( fig . 2 , left panel ) , assuming that , at the end of the meal , the fraction of the meal appearing in plasma is f = 0.9 ( fig . time course of meal rate of appearance , rag ( left - hand panel ) , and fraction of meal that appears in plasma , f ( right - hand panel ) . to better grasp the use of f(t ) and cob(t ) for the calculation of insulin sensitivity , let us consider the following example : suppose that a subject eats 50 g of carbohydrates at t = 0 min and another 40 g at t = 180 min then fasts for more than 360 min . one can calculate two values of si , one for each meal ingestion ( si and si ) . for the calculation of si , the amount of ingested glucose to be considered is 50f(180 ) g of carbohydrates , while for the calculation of the second , si , one should use 50cob(180)+40f. generalizing , the amount of carbohydrates to use in the formula for the ith meal iswhere aoc is the amount of carbohydrates , d(tmeal ) is the amount of carbohydrates ingested at the time of the ith meal ( tmeal ) , tend is the time at which the ith meal ends ( corresponding to the time of ingestion of the [ i+1]th meal ) , and cob(tend ) the cob at the end of the ( i1)th meal , which contained d(tmeal ) amount of carbohydrates . incorporating the above derivations into eq . 7 , one can estimate insulin sensitivity from sensor and pump data ( si ) for the ith meal ( 20):it is important to define the domain of validity of eq . when cgm is still high 6 h after meal ingestion , si can become negative . we thus recommend to use the formula only if the recalibrated cgm is lower than 150 mg / dl 6 h after meal ingestion . as a matter of fact , one of our subjects ( subject 4 ) during lunch has recalibrated cgm greater than 150 mg / dl 6 h after meal ingestion , and si was negative . this subject was thus excluded in the following analysis . the oral glucose minimal model ( 3,4 ) was used to estimate insulin sensitivity , from plasma glucose and insulin concentrations , in 12 subjects studied three times ( 10 ) . here we consider these measures as reference values ( si ) to which si is compared for validation . in our experimental protocol , meals were well spaced ( at least 6 h ) and thus completely absorbed before the next meal . thus it is important to assess the performance of the method in case of incompletely absorbed meals . we did so by comparing estimates of si obtained with a 360-min interval to those obtained from shorter intervals ( up to 180 min ) , both with and without using the cob function . when proposing a new metric like si , it is important to assess its reproducibility , i.e. , if it provides similar values when repeatedly applied to the same subject under the same experimental conditions . however , due to the large intrasubject variability of insulin sensitivity ( 10 ) , si will likely change if the same meal is administered in two occasions to a subject . we simulated a 7-day scenario for 100 in silico subjects with three meals per day ( breakfast from 6:00 to 8:00 a.m. , lunch from 11:30 a.m. to 1:30 p.m. , and dinner from 6:00 to 8.30 p.m. ) with different amounts ( breakfast 0.7 0.9 g / kg , lunch 0.8 1.0 g / kg , and dinner 0.8 1.3 g / kg ) , while subject - specific insulin sensitivity was maintained constant for the whole simulation . a total of 21 si were thus calculated for each subject ( three values per 7 days ) . to assess the repeatability of the index , we calculated the average ( mean ) , the sd , and the coefficient of variation ( cv ; sd / mean ) of the 21 estimates of si in each subject . twelve type 1 diabetic subjects ( 5 females , aged 39.5 14.2 years , bmi 25.7 3.8 kg / m , hba1c 8.5% or 69 mmol / mol ) were studied for 3 days in the clinical research unit of the mayo clinic center for translational science activities as part of a separate study to determine diurnal patterns of insulin sensitivity ( 10 ) . briefly , once per day , a triple - tracer mixed - meal study protocol was performed during breakfast , lunch , or dinner in latin square design . blood samples were collected at 180 , 30 , 0 , 5 , 10 , 20 , 30 , 60 , 90 , 120 , 150 , 180 , 240 , 300 , and 360 , with t = 0 corresponding to meal time , for measurement of plasma glucose and insulin concentrations in order to estimate si with the oral minimal model ( 3 ) , here considered as reference . subjects also wore both subcutaneous insulin pump ( medtronic or insulet omnipod ) and cgm ( dexcom seven plus ) . within - subject mean absolute relative difference between cgm readings and reference was equal to 12.1 3.3% , and no systematic variation over time was found ( mean [ sd ] within subjects was 5.3 3.7% ) . ( 10 ) since they had the complete cgm and csii data required for the calculation described below . of note glucose data are measured by cgm ( left - hand panel , black line ) , with some smbg data available for sensor recalibration ( left - hand panel , gray dots ) . insulin data are the basal infusion and boluses administered by insulin pump ( right - hand panel , black line and gray triangles , respectively ) . the starting point is the derivation of insulin sensitivity by integrating the oral minimal model equations ( 3 ) , as described by caumo et al . , the calculation is adapted to allow the use of cgm and csii data , instead of plasma glucose and insulin concentrations . the oral minimal model ( 3 ) iswhere g is plasma glucose concentration ( milligrams per deciliter ) , with gb denoting its basal value ; x is insulin action ( min ) ; i is plasma insulin concentration ( microunits per milliliter ) , with ib denoting its basal value ; rag is posthepatic appearance of meal glucose ( milligrams per kilograms per minute ) ; vg is glucose distribution volume ( deciliters per kilogram ) ; p2 is speed of rise and decay of insulin action ( min ) ; and p3 is its size ( min per microunit per milliliter ) . insulin sensitivity is defined asby substituting and into eq . 1 , we obtainby integrating the differential equation ( eq . 3 ) from the time of the meal ingestion ( tmeal ) to the end of the experiment ( tend ) and rearranging , one hasthe first integral in the numerator can be rewritten aswhere d ( milligrams ) is the amount of glucose ingested during the meal and f(tend ) is the fraction of the ingested dose , which at t = tend has reached plasma . x in eq . 4 is usually not available because model parameters are unknown , thus the denominator of eq . 4 is substituted with the average of glucose excursion times the overall insulin stimulus : where |g| is above basal plasma glucose concentration . thus si is given bywhere auc is the area under the curve calculated from the start of the meal ( tmeal ) to the end of the experiment ( tend ) . subject - specific parameters used by the formula are the body weight ( bw ) ( kilograms ) , height ( meters ) , and age ( years ) . together with height and age , used for the calculation of plasma insulin clearance ( cl ) , as discussed below . parameter fixed to population values are glucose effectiveness at zero insulin gezi [ deciliters per kilogram per minute ; fixed to 0.01 dl / kg / min for diabetic subjects ( 10,12 ) ] and volume of glucose distribution vg [ deciliters per kilogram ; fixed to 1.45 dl / kg , according to dalla man et al . g , g , and i are not directly available but can be derived from cgm and subcutaneous insulin delivery as detailed below . cgm measures the ig concentration , which is related to plasma glucose by a first - order differential equation , i.e. , ig is a delayed version of plasma glucose : thus , with a well - calibrated device , assuming ig(tend ) = ig(tmeal ) , one hasi.e.,similarly , for the above basal glucose signal , one hasthus one can safely assume that , in the presence of a well - calibrated device , auc(cgm ) and auc(|cgm| ) are good approximations of auc(g ) and auc(|g| ) , respectively . in presence of a noncalibrated device , if at least two smbg samples are available , it is possible to offline recalibrate the cgm profile ( 13,14 ) . for the calculation of auc(i ) , we assume that insulin is not degraded locally in the site of infusion . in this case , the integral of plasma insulin can be obtained from subcutaneous insulin infusion divided by the plasma insulin clearance cl . in fact , plasma insulin kinetics can be described with a single compartment model ( 15):where i(t ) is the plasma insulin concentration , rai(t ) is the insulin rate of appearance in plasma , vi is the insulin volume of distribution , and n the fractional insulin clearance rate ( n = cl / vi ) . 12 from the time of the premeal bolus ( tmeal ) to the end of the observation period ( tend ) and assuming that insulin is back to its initial value at the end of the experiment , one hasfinally , under the assumption that all infused insulin eventually reaches the circulation , one hasthus we can compute auc(i ) from the amount of insulin infused subcutaneously and cl : where basal(t ) is the basal insulin infusion rate during the integration period , bolus(tk ) is the premeal or correction bolus administered at t = tk , and cl ( liters per minute ) is the plasma insulin clearance , which can be calculated from subject s height , bw , and age by using the population model proposed by campioni et al . ( in addition , if correction boluses are administered before the start of the meal , one has to consider that part of that injected insulin could be still active . the residual active insulin can be determined by adopting the same algorithm presented by patek et al . ( 17 ) , which uses the insulin on board ( iob ) curves adapted from ellingsen et al . this quantity ( iob[tmeal ] ) must be added to the previously estimated auc(i ) . moreover , if correction boluses are administered before the end of the considered interval , iob is used to evaluate the active insulin at the end of the study ( iob[tend ] ) , which is subtracted from the previously estimated auc(i ) . in summary , as evident from eq . 7 , an accurate calculation of insulin sensitivity requires the knowledge of the amount of carbohydrates entering the circulation in the integration interval [ df(tend ) ] . in this study , meals were provided at 7:00 a.m. , 1:00 p.m. ; and 7:00 p.m. each day , thus the time interval between the meals was at least 6 h. however , this may not always be the case . furthermore , not all the carbohydrates ingested with a meal may be fully absorbed before the ingestion of a second meal . thus , to account for unabsorbed carbohydrates , e.g. , frequent meals close to each other , the concept of carbohydrates on board ( cob ) is introduced . similarly to iob , cob is a function that , at each time t , quantifies the fraction of the ingested carbohydrates that has not yet appeared in the circulation . cob is based on the model of gastrointestinal tract ( 19 ) : given the amount of carbohydrates ingested at time tm , cob provides , at each time t > tm , the percentage of carbohydrates not yet absorbed , while for t tm>360 min , it is assumed that the carbohydrate absorption is almost completed . the fraction of the ingested dose that has reached plasma at time t , f(t ) , can be calculated as the ratio between the auc of the meal rate of appearance and the ingested dose , d ( fig . 2 , left panel ) , assuming that , at the end of the meal , the fraction of the meal appearing in plasma is f = 0.9 ( fig . time course of meal rate of appearance , rag ( left - hand panel ) , and fraction of meal that appears in plasma , f ( right - hand panel ) . to better grasp the use of f(t ) and cob(t ) for the calculation of insulin sensitivity , let us consider the following example : suppose that a subject eats 50 g of carbohydrates at t = 0 min and another 40 g at t = 180 min then fasts for more than 360 min . one can calculate two values of si , one for each meal ingestion ( si and si ) . for the calculation of si , the amount of ingested glucose to be considered is 50f(180 ) g of carbohydrates , while for the calculation of the second , si , one should use 50cob(180)+40f. generalizing , the amount of carbohydrates to use in the formula for the ith meal iswhere aoc is the amount of carbohydrates , d(tmeal ) is the amount of carbohydrates ingested at the time of the ith meal ( tmeal ) , tend is the time at which the ith meal ends ( corresponding to the time of ingestion of the [ i+1]th meal ) , and cob(tend ) the cob at the end of the ( i1)th meal , which contained d(tmeal ) amount of carbohydrates . incorporating the above derivations into eq . 7 , one can estimate insulin sensitivity from sensor and pump data ( si ) for the ith meal ( 20):it is important to define the domain of validity of eq . when cgm is still high 6 h after meal ingestion , si can become negative . we thus recommend to use the formula only if the recalibrated cgm is lower than 150 mg / dl 6 h after meal ingestion . as a matter of fact , one of our subjects ( subject 4 ) during lunch has recalibrated cgm greater than 150 mg / dl 6 h after meal ingestion , and si was negative . cgm measures the ig concentration , which is related to plasma glucose by a first - order differential equation , i.e. , ig is a delayed version of plasma glucose : thus , with a well - calibrated device , assuming ig(tend ) = ig(tmeal ) , one hasi.e.,similarly , for the above basal glucose signal , one hasthus one can safely assume that , in the presence of a well - calibrated device , auc(cgm ) and auc(|cgm| ) are good approximations of auc(g ) and auc(|g| ) , respectively . in presence of a noncalibrated device , if at least two smbg samples are available , it is possible to offline recalibrate the cgm profile ( 13,14 ) . for the calculation of auc(i ) , we assume that insulin is not degraded locally in the site of infusion . in this case , the integral of plasma insulin can be obtained from subcutaneous insulin infusion divided by the plasma insulin clearance cl . in fact , plasma insulin kinetics can be described with a single compartment model ( 15):where i(t ) is the plasma insulin concentration , rai(t ) is the insulin rate of appearance in plasma , vi is the insulin volume of distribution , and n the fractional insulin clearance rate ( n = cl / vi ) . 12 from the time of the premeal bolus ( tmeal ) to the end of the observation period ( tend ) and assuming that insulin is back to its initial value at the end of the experiment , one hasfinally , under the assumption that all infused insulin eventually reaches the circulation , one hasthus we can compute auc(i ) from the amount of insulin infused subcutaneously and cl : where basal(t ) is the basal insulin infusion rate during the integration period , bolus(tk ) is the premeal or correction bolus administered at t = tk , and cl ( liters per minute ) is the plasma insulin clearance , which can be calculated from subject s height , bw , and age by using the population model proposed by campioni et al . in addition , if correction boluses are administered before the start of the meal , one has to consider that part of that injected insulin could be still active . the residual active insulin can be determined by adopting the same algorithm presented by patek et al . ( 17 ) , which uses the insulin on board ( iob ) curves adapted from ellingsen et al . ( 18 ) . this quantity ( iob[tmeal ] ) must be added to the previously estimated auc(i ) . moreover , if correction boluses are administered before the end of the considered interval , iob is used to evaluate the active insulin at the end of the study ( iob[tend ] ) , which is subtracted from the previously estimated auc(i ) . in summary , an accurate calculation of insulin sensitivity requires the knowledge of the amount of carbohydrates entering the circulation in the integration interval [ df(tend ) ] . in this study , meals were provided at 7:00 a.m. , 1:00 p.m. ; and 7:00 p.m. each day , thus the time interval between the meals was at least 6 h. however , this may not always be the case . furthermore , not all the carbohydrates ingested with a meal may be fully absorbed before the ingestion of a second meal . thus , to account for unabsorbed carbohydrates , e.g. , frequent meals close to each other , the concept of carbohydrates on board ( cob ) is introduced . similarly to iob , cob is a function that , at each time t , quantifies the fraction of the ingested carbohydrates that has not yet appeared in the circulation . cob is based on the model of gastrointestinal tract ( 19 ) : given the amount of carbohydrates ingested at time tm , cob provides , at each time t > tm , the percentage of carbohydrates not yet absorbed , while for t tm>360 min , it is assumed that the carbohydrate absorption is almost completed . the fraction of the ingested dose that has reached plasma at time t , f(t ) , can be calculated as the ratio between the auc of the meal rate of appearance and the ingested dose , d ( fig . 2 , left panel ) , assuming that , at the end of the meal , the fraction of the meal appearing in plasma is f = 0.9 ( fig . time course of meal rate of appearance , rag ( left - hand panel ) , and fraction of meal that appears in plasma , f ( right - hand panel ) . to better grasp the use of f(t ) and cob(t ) for the calculation of insulin sensitivity , let us consider the following example : suppose that a subject eats 50 g of carbohydrates at t = 0 min and another 40 g at t = 180 min then fasts for more than 360 min . one can calculate two values of si , one for each meal ingestion ( si and si ) . for the calculation of si , the amount of ingested glucose to be considered is 50f(180 ) g of carbohydrates , while for the calculation of the second , si , one should use 50cob(180)+40f. generalizing , the amount of carbohydrates to use in the formula for the ith meal iswhere aoc is the amount of carbohydrates , d(tmeal ) is the amount of carbohydrates ingested at the time of the ith meal ( tmeal ) , tend is the time at which the ith meal ends ( corresponding to the time of ingestion of the [ i+1]th meal ) , and cob(tend ) the cob at the end of the ( i1)th meal , which contained d(tmeal ) amount of carbohydrates . incorporating the above derivations into eq . 7 , one can estimate insulin sensitivity from sensor and pump data ( si ) for the ith meal ( 20):it is important to define the domain of validity of eq . when cgm is still high 6 h after meal ingestion , si can become negative . we thus recommend to use the formula only if the recalibrated cgm is lower than 150 mg / dl 6 h after meal ingestion . as a matter of fact , one of our subjects ( subject 4 ) during lunch has recalibrated cgm greater than 150 mg / dl 6 h after meal ingestion , and si was negative . the oral glucose minimal model ( 3,4 ) was used to estimate insulin sensitivity , from plasma glucose and insulin concentrations , in 12 subjects studied three times ( 10 ) . here we consider these measures as reference values ( si ) to which si is compared for validation . in our experimental protocol , meals were well spaced ( at least 6 h ) and thus completely absorbed before the next meal . thus it is important to assess the performance of the method in case of incompletely absorbed meals . we did so by comparing estimates of si obtained with a 360-min interval to those obtained from shorter intervals ( up to 180 min ) , both with and without using the cob function . when proposing a new metric like si , it is important to assess its reproducibility , i.e. , if it provides similar values when repeatedly applied to the same subject under the same experimental conditions . however , due to the large intrasubject variability of insulin sensitivity ( 10 ) , si will likely change if the same meal is administered in two occasions to a subject . we simulated a 7-day scenario for 100 in silico subjects with three meals per day ( breakfast from 6:00 to 8:00 a.m. , lunch from 11:30 a.m. to 1:30 p.m. , and dinner from 6:00 to 8.30 p.m. ) with different amounts ( breakfast 0.7 0.9 g / kg , lunch 0.8 1.0 g / kg , and dinner 0.8 1.3 g / kg ) , while subject - specific insulin sensitivity was maintained constant for the whole simulation . a total of 21 si were thus calculated for each subject ( three values per 7 days ) . to assess the repeatability of the index , we calculated the average ( mean ) , the sd , and the coefficient of variation ( cv ; sd / mean ) of the 21 estimates of si in each subject . the correlation between the two indices was very good ( r = 0.825 ; p < 10 ; fig . 3 , right - hand panel ) , and diurnal pattern was similar , indicating that , apart from a scale factor , si closely mirrors si . si and si have been estimated in the 12 subjects at breakfast , lunch , and dinner . 5.63 dl / kg / min per u / ml ; p < 10 ; fig . 3 , left - hand panel ) . mean values ( left - hand panel ) and correlation plot ( right - hand panel ) between si and si insulin sensitivity indices for breakfast , lunch , and dinner ( white , striped , and black bars , respectively ) . * p < 10 with paired sample t test . when si was calculated for incompletely absorbed meals , i.e. , relying on reduced integration intervals , mean values of si were virtually the same ( fig . the correlation between si calculated at the end of the experiment and that obtained from reduced integration intervals decreases only slightly ( fig . 4 , bottom panels ) both with and without using cob . finally , in silico reproducibility of si was 23 6% . sensitivity analysis of si with ( left - hand panel ) and without ( right - hand panel ) accounting for cob for different time integration intervals : mean values of si ( top ) , correlation indices ( middle ) , and absolute relative error ( bottom ) , calculated with respect to si , i.e. , estimated at t = 360 min . insulin sensitivity is a key parameter of the metabolic status of an individual , which could be beneficial also for optimizing insulin therapy in type 1 diabetes . in fact , the knowledge of patient - specific si and its daily variation can truly help in determining the optimal insulin bolus to be administered to cover the ingested carbohydrates . however , all methods available for the estimation of insulin sensitivity rely on plasma glucose and insulin measurements and thus can not be used in everyday life of a patient with type 1 diabetes . in this article , we have proposed an index of insulin sensitivity , si , which can be estimated in patients with type 1 diabetes wearing a cgm sensor and an insulin pump . we have demonstrated that it is similar to the one obtained with the oral minimal model , which requires plasma glucose and insulin data . the method uses retrospective subcutaneous sensor and insulin delivery data with some anthropometric parameters for each subject and provides , for each meal , the patient s insulin sensitivity by an integral formula . thus si is not exactly the same index derived with the minimal model ( si ) , which represents the ability of insulin to suppress endogenous glucose production and stimulate glucose uptake . in other words however , the correlation between the two indices was excellent ( r = 0.825 ; p < 10 ) . a robust estimate of insulin sensitivity can be obtained for each meal whenever meals are well spaced ( 56 h ) . however , we also tested the method in case of incompletely absorbed meals . to deal with this situation similarly to iob ( 18 ) , cob represents , at each time t , the amount of ingested glucose that has not yet been absorbed . thus one can define different cob curves for different types of meal , i.e. , fast or slow carbohydrates . we used cob in the si calculation to evaluate the correct amount of carbohydrates in relation with the observed cgm profiles in a given time interval . we demonstrated the need of using cob ; in case of a short time interval between consecutive meals , a robust estimation of si can only be obtained if carbohydrates absorption is taken into account ( fig . possible applications of the new index include its use for assessing intraday and interday variability ( e.g. , existence of diurnal patterns ) of insulin sensitivity in large cohorts of subjects with type 1 diabetes in normal life conditions . for instance , the method is currently being applied to the sensor - augmented pump therapy for a1c reduction ( star ) 3 data of type 1 diabetic subjects wearing a sensor - augmented insulin pump ( 22 ) . this will provide , in each subject , the si time course during several months and will allow testing for the existence of subject - specific si daily patterns and correlation of such variation with lifestyle and other factors . following validation using star 3 data , a clinical application would be the use of si to calculate the optimal insulin - to - carbohydrate ratio ( cr ) ( 23,24 ) . thus optimizing cr based on recent cgm and csii data collected 12 weeks prior to a clinical visit may be useful for physicians to improve patient - specific meal bolus insulin therapy and thus a major component of glucose control . in addition , the knowledge of patient cr daily pattern may help in the design of optimal closed - loop control algorithms relying on patient - specific open - loop insulin therapy at the time of transition from open- to closed - loop therapy . a natural progression of this work would be the development of real - time adaptation of open- or closed - loop therapy based on the presence of well - developed metrics such as the one developed here . further development of diabetes technology hardware and algorithms should make this a reality over the next several years . the method relies on data provided by a cgm device that can occasionally suffer from inaccuracies . for instance , the assumption that auc(cgm ) is a good approximation of auc(g ) becomes critical if the device is not well calibrated . however , to improve the quality of cgm measurements , some algorithms can be used to recalibrate cgm traces ( 13,14 ) . another possible limitation is the need to fix some parameters ( gezi and vg ) to population values ( 3,10,12 ) and others calculated from population models ( cl ) using anthropometric data ( 16 ) . in order to test the effect of fixing these parameters , we also calculated si using individualized gezi , estimated with the oral glucose minimal model ( 3 ) and cl , directly estimated from the data in each patient ; we obtained values very similar to those obtained with fixed parameters and a slightly higher correlation with si . insulin sensitivity is an important element in the daily life of patients with type 1 diabetes and could be useful to optimize insulin therapy . however , methods to estimate this index by emerging technologies , such as subcutaneous cgm sensor and insulin pump , has never been proposed . we have presented a method that estimates insulin sensitivity from cgm and insulin pump data . future studies involving a larger databases that include larger cohorts of subjects studied for a longer time are needed to better define the applicability in free - living conditions .

objectivethe goal was to develop a new index of insulin sensitivity in patients with type 1 diabetes estimated from continuous glucose monitoring ( cgm ) and subcutaneous insulin delivery data under carefully controlled conditions.research design and methodsthe database consists of 12 subjects with type 1 diabetes , studied during breakfast , lunch , and dinner , in a clinical research unit , wearing both subcutaneous insulin pump and cgm device . frequent blood samples were drawn for measurements of plasma glucose and insulin concentrations in order to estimate insulin sensitivity with the oral minimal model ( simm ) . the new index of insulin sensitivity ( sisp ) was calculated with a simple algebraic formula for each meal , using only cgm and insulin pump data and compared with simm.resultssisp was well correlated with simm ( r = 0.825 ; p < 108 ) , and diurnal pattern was also similar to simm.conclusionsa novel method for estimating insulin sensitivity in subjects with type 1 diabetes on sensor - augmented insulin pump therapy has been presented . this new index correlates well with the reference oral minimal model estimate of insulin sensitivity . the knowledge of patient - specific insulin sensitivity and its diurnal variation can help in optimizing insulin therapy in type 1 diabetes and could also inform next - generation closed - loop control systems .

Introduction RESEARCH DESIGN AND METHODS Database and Protocol Basis Glucose Signal From CGM Insulin Signal From CSII Accounting for Carbohydrates on Board Insulin Sensitivity From Sensor and Pump Data Minimal Model Insulin Sensitivity and Validation of Assessment in Case of Incompletely Absorbed Meals Reproducibility Statistical Analysis Results Conclusions

however , in the past 1015 years , new possibilities in diabetes therapy have emerged thanks to continuous glucose monitoring ( cgm ) and csii , which substitute smbg and multiple daily injection therapy , respectively . here we propose a method to estimate insulin sensitivity from cgm sensor and insulin pump data and validate it against the oral minimal model , which uses plasma glucose and insulin concentrations . blood samples were collected at 180 , 30 , 0 , 5 , 10 , 20 , 30 , 60 , 90 , 120 , 150 , 180 , 240 , 300 , and 360 , with t = 0 corresponding to meal time , for measurement of plasma glucose and insulin concentrations in order to estimate si with the oral minimal model ( 3 ) , here considered as reference . the oral glucose minimal model ( 3,4 ) was used to estimate insulin sensitivity , from plasma glucose and insulin concentrations , in 12 subjects studied three times ( 10 ) . we simulated a 7-day scenario for 100 in silico subjects with three meals per day ( breakfast from 6:00 to 8:00 a.m. , lunch from 11:30 a.m. to 1:30 p.m. , and dinner from 6:00 to 8.30 p.m. ) with different amounts ( breakfast 0.7 0.9 g / kg , lunch 0.8 1.0 g / kg , and dinner 0.8 1.3 g / kg ) , while subject - specific insulin sensitivity was maintained constant for the whole simulation . blood samples were collected at 180 , 30 , 0 , 5 , 10 , 20 , 30 , 60 , 90 , 120 , 150 , 180 , 240 , 300 , and 360 , with t = 0 corresponding to meal time , for measurement of plasma glucose and insulin concentrations in order to estimate si with the oral minimal model ( 3 ) , here considered as reference . the oral minimal model ( 3 ) iswhere g is plasma glucose concentration ( milligrams per deciliter ) , with gb denoting its basal value ; x is insulin action ( min ) ; i is plasma insulin concentration ( microunits per milliliter ) , with ib denoting its basal value ; rag is posthepatic appearance of meal glucose ( milligrams per kilograms per minute ) ; vg is glucose distribution volume ( deciliters per kilogram ) ; p2 is speed of rise and decay of insulin action ( min ) ; and p3 is its size ( min per microunit per milliliter ) . the oral glucose minimal model ( 3,4 ) was used to estimate insulin sensitivity , from plasma glucose and insulin concentrations , in 12 subjects studied three times ( 10 ) . we simulated a 7-day scenario for 100 in silico subjects with three meals per day ( breakfast from 6:00 to 8:00 a.m. , lunch from 11:30 a.m. to 1:30 p.m. , and dinner from 6:00 to 8.30 p.m. ) with different amounts ( breakfast 0.7 0.9 g / kg , lunch 0.8 1.0 g / kg , and dinner 0.8 1.3 g / kg ) , while subject - specific insulin sensitivity was maintained constant for the whole simulation . the correlation between the two indices was very good ( r = 0.825 ; p < 10 ; fig . mean values ( left - hand panel ) and correlation plot ( right - hand panel ) between si and si insulin sensitivity indices for breakfast , lunch , and dinner ( white , striped , and black bars , respectively ) . insulin sensitivity is a key parameter of the metabolic status of an individual , which could be beneficial also for optimizing insulin therapy in type 1 diabetes . in fact , the knowledge of patient - specific si and its daily variation can truly help in determining the optimal insulin bolus to be administered to cover the ingested carbohydrates . however , all methods available for the estimation of insulin sensitivity rely on plasma glucose and insulin measurements and thus can not be used in everyday life of a patient with type 1 diabetes . in this article , we have proposed an index of insulin sensitivity , si , which can be estimated in patients with type 1 diabetes wearing a cgm sensor and an insulin pump . we have demonstrated that it is similar to the one obtained with the oral minimal model , which requires plasma glucose and insulin data . the method uses retrospective subcutaneous sensor and insulin delivery data with some anthropometric parameters for each subject and provides , for each meal , the patient s insulin sensitivity by an integral formula . in addition , the knowledge of patient cr daily pattern may help in the design of optimal closed - loop control algorithms relying on patient - specific open - loop insulin therapy at the time of transition from open- to closed - loop therapy . in order to test the effect of fixing these parameters , we also calculated si using individualized gezi , estimated with the oral glucose minimal model ( 3 ) and cl , directly estimated from the data in each patient ; we obtained values very similar to those obtained with fixed parameters and a slightly higher correlation with si . insulin sensitivity is an important element in the daily life of patients with type 1 diabetes and could be useful to optimize insulin therapy .

this cross - sectional study included 49 patients with graves ' ophthalmopathy who were followed at the department of ophthalmology , yonsei university college of medicine between july 2008 and december 2008 ( referred to as the graves ' group ) . we excluded patients who were not biochemically euthyroid to eliminate the possible effect of dysthyroidism on quality of life . the control group was composed of healthy volunteers who were age- and sex - matched to the study group . written informed consent was obtained from all participants after gaining approval from the institutional review board at yonsei university college of medicine . graves ' ophthalmopathy was defined as the presence of typical eye symptoms and signs in a patient with autoimmune graves ' disease . a complete ophthalmic investigation was performed , and an assessment of the severity and activity of graves ' ophthalmopathy was performed . proptosis and eye muscle involvement were assessed using a hertel exophthalmometer and the gorman diplopia scale . a gorman score of 0 - 1 was considered low ; a score of 2 - 3 was considered high . severity of graves ' ophthalmopathy was scored according to the european group on graves ' orbitopathy ( eugogo ) classification system . mild graves ' ophthalmopathy was defined as minor lid retraction ( < 2 mm ) , mild soft tissue involvement , exophthalmos < 21 mm , or transient ( gorman score 1 ) or no diplopia ( gorman score 0 ) . moderately severe graves ' ophthalmopathy was defined as lid retraction 2 mm , moderate or severe soft tissue involvement , exophthalmos 21 mm , or inconstant ( gorman score 2 ) or constant diplopia ( gorman score 3 ) . very severe , sight - threatening graves ' ophthalmopathy was defined as dysthyroid optic neuropathy or corneal breakdown . patients with a history of optic neuropathy but who were without optic neuropathy at the time of the survey were included in the moderately severe group . optic neuropathy was defined as the presence of disc swelling or pallor , a visual field defect , or relative afferent pupillary defect , or if visual acuity less than 0.3 in the absence of other reasons for sight loss . inflammation activity was determined using a clinical activity score based on seven signs of inflammation of the orbit , with each scored as absent ( 0 ) or present ( 1 ) , with a maximum possible score of 7 . at an outpatient clinic visit , patients completed a questionnaire on demographics and duration and previous treatments of graves ' ophthalmopathy and graves ' thyroid disease . quality of life was assessed by the sf-36 , and depressive status was evaluated by the bdi . patients who could not fill out the survey themselves were provided support from those conducting the research . the korean version of the sf-36 was used to evaluate the health - related quality of life of the study participants . the sf-36 has been previously shown to be a credible and reliable instrument to measure the quality of life in study groups . the eight categories are : 1 ) physical functioning ( walking , lifting ) ; 2 ) role function - physical ( limitations in ability to perform usual activities ) ; 3 ) bodily pain ( level of bodily pain or discomfort ) ; 4 ) general health perceptions ( global evaluation of health ) ; 5 ) vitality ( energy level or fatigue ) ; 6 ) social functioning ( impact of health or emotional problems on social activities ) ; 7 ) role function - emotional ( impact of emotional problems on work or daily activities ) ; and 8) mental health ( anxiety , depression , sense of psychological well - being ) . the first four categories are grouped as the physical component summary , while the last four categories are grouped as the mental component summary . these eight categories and two summaries were converted into scores and evaluated with respect to the sociodemographic and clinical characteristics of the participants . depression in patients with graves ' ophthalmopathy was evaluated with the bdi , a standardized questionnaire of the cognitive , affective , and somatic symptoms of depression . the bdi was developed to assess the type and severity of depression based on clinical symptoms . the korean version of the bdi has high reliability , and a score of 16 or higher has been suggested as the optimal cutoff score for the diagnosis of major depression in korea . all measures of the sf-36 were compared between the graves ' and control groups using independent two - sample t - tests . quality of life ( by sf-36 scores ) and depressive status ( by bdi scores ) were compared among patients with mild , moderately severe , and sight - threatening graves ' ophthalmopathy using one - way analysis of variance ( anova ) and the pearson chi - square test for continuous and categorical variables , respectively . sf-36 and bdi scores were also compared between the high and low gorman score groups using the same statistical methods . characteristics , including age , sex , duration of graves ' disease and graves ' ophthalmopathy , hertel exophthalmos values , current activity score , and severity stage by eugogo classification were compared between the patients with high ( 16 ) and low ( < 16 ) bdi scores , using the independent two - sample t - test and the pearson chi - square test for continuous and categorical variables , respectively . the relationship between the bdi and sf-36 scores and age , sex , duration of graves ' disease and graves ' ophthalmopathy , gorman score , severity , and activity were examined by the pearson correlation or spearman rank correlation test . this cross - sectional study included 49 patients with graves ' ophthalmopathy who were followed at the department of ophthalmology , yonsei university college of medicine between july 2008 and december 2008 ( referred to as the graves ' group ) . we excluded patients who were not biochemically euthyroid to eliminate the possible effect of dysthyroidism on quality of life . the control group was composed of healthy volunteers who were age- and sex - matched to the study group . written informed consent was obtained from all participants after gaining approval from the institutional review board at yonsei university college of medicine . graves ' ophthalmopathy was defined as the presence of typical eye symptoms and signs in a patient with autoimmune graves ' disease . a complete ophthalmic investigation was performed , and an assessment of the severity and activity of graves ' ophthalmopathy was performed . proptosis and eye muscle involvement were assessed using a hertel exophthalmometer and the gorman diplopia scale . a gorman score of 0 - 1 was considered low ; a score of 2 - 3 was considered high . severity of graves ' ophthalmopathy was scored according to the european group on graves ' orbitopathy ( eugogo ) classification system . mild graves ' ophthalmopathy was defined as minor lid retraction ( < 2 mm ) , mild soft tissue involvement , exophthalmos < 21 mm , or transient ( gorman score 1 ) or no diplopia ( gorman score 0 ) . moderately severe graves ' ophthalmopathy was defined as lid retraction 2 mm , moderate or severe soft tissue involvement , exophthalmos 21 mm , or inconstant ( gorman score 2 ) or constant diplopia ( gorman score 3 ) . very severe , sight - threatening graves ' ophthalmopathy was defined as dysthyroid optic neuropathy or corneal breakdown . patients with a history of optic neuropathy but who were without optic neuropathy at the time of the survey were included in the moderately severe group . optic neuropathy was defined as the presence of disc swelling or pallor , a visual field defect , or relative afferent pupillary defect , or if visual acuity less than 0.3 in the absence of other reasons for sight loss . inflammation activity was determined using a clinical activity score based on seven signs of inflammation of the orbit , with each scored as absent ( 0 ) or present ( 1 ) , with a maximum possible score of 7 . at an outpatient clinic visit , patients completed a questionnaire on demographics and duration and previous treatments of graves ' ophthalmopathy and graves ' thyroid disease . quality of life was assessed by the sf-36 , and depressive status was evaluated by the bdi . patients who could not fill out the survey themselves were provided support from those conducting the research . the korean version of the sf-36 was used to evaluate the health - related quality of life of the study participants . the sf-36 has been previously shown to be a credible and reliable instrument to measure the quality of life in study groups . the eight categories are : 1 ) physical functioning ( walking , lifting ) ; 2 ) role function - physical ( limitations in ability to perform usual activities ) ; 3 ) bodily pain ( level of bodily pain or discomfort ) ; 4 ) general health perceptions ( global evaluation of health ) ; 5 ) vitality ( energy level or fatigue ) ; 6 ) social functioning ( impact of health or emotional problems on social activities ) ; 7 ) role function - emotional ( impact of emotional problems on work or daily activities ) ; and 8) mental health ( anxiety , depression , sense of psychological well - being ) . the first four categories are grouped as the physical component summary , while the last four categories are grouped as the mental component summary . these eight categories and two summaries were converted into scores and evaluated with respect to the sociodemographic and clinical characteristics of the participants . depression in patients with graves ' ophthalmopathy was evaluated with the bdi , a standardized questionnaire of the cognitive , affective , and somatic symptoms of depression . the bdi was developed to assess the type and severity of depression based on clinical symptoms . the korean version of the bdi has high reliability , and a score of 16 or higher has been suggested as the optimal cutoff score for the diagnosis of major depression in korea . the korean version of the sf-36 was used to evaluate the health - related quality of life of the study participants . the sf-36 has been previously shown to be a credible and reliable instrument to measure the quality of life in study groups . the eight categories are : 1 ) physical functioning ( walking , lifting ) ; 2 ) role function - physical ( limitations in ability to perform usual activities ) ; 3 ) bodily pain ( level of bodily pain or discomfort ) ; 4 ) general health perceptions ( global evaluation of health ) ; 5 ) vitality ( energy level or fatigue ) ; 6 ) social functioning ( impact of health or emotional problems on social activities ) ; 7 ) role function - emotional ( impact of emotional problems on work or daily activities ) ; and 8) mental health ( anxiety , depression , sense of psychological well - being ) . the first four categories are grouped as the physical component summary , while the last four categories are grouped as the mental component summary . these eight categories and two summaries were converted into scores and evaluated with respect to the sociodemographic and clinical characteristics of the participants . depression in patients with graves ' ophthalmopathy was evaluated with the bdi , a standardized questionnaire of the cognitive , affective , and somatic symptoms of depression . the bdi was developed to assess the type and severity of depression based on clinical symptoms . the korean version of the bdi has high reliability , and a score of 16 or higher has been suggested as the optimal cutoff score for the diagnosis of major depression in korea . all measures of the sf-36 were compared between the graves ' and control groups using independent two - sample t - tests . quality of life ( by sf-36 scores ) and depressive status ( by bdi scores ) were compared among patients with mild , moderately severe , and sight - threatening graves ' ophthalmopathy using one - way analysis of variance ( anova ) and the pearson chi - square test for continuous and categorical variables , respectively . sf-36 and bdi scores were also compared between the high and low gorman score groups using the same statistical methods . characteristics , including age , sex , duration of graves ' disease and graves ' ophthalmopathy , hertel exophthalmos values , current activity score , and severity stage by eugogo classification were compared between the patients with high ( 16 ) and low ( < 16 ) bdi scores , using the independent two - sample t - test and the pearson chi - square test for continuous and categorical variables , respectively . the relationship between the bdi and sf-36 scores and age , sex , duration of graves ' disease and graves ' ophthalmopathy , gorman score , severity , and activity were examined by the pearson correlation or spearman rank correlation test . the 49 patients included 38 women ( 77.6% ) and 11 men ( 22.4% ) , with a mean age of 41.4 years and an average duration of graves ' ophthalmopathy of 12 months . nine patients ( 18.4% ) had a history of optic neuropathy , and four patients ( 8.1% ) had optic neuropathy at the time of the survey . the patients were asked " what is the main cause of emotional distress now ? " with three possible answers : disfiguring change , functional impairment , or both equally distressful . twenty patients ( 40.8% ) responded with disfiguring change , 14 patients ( 28.6% ) responded with functional impairment , and 15 patients ( 30.6% ) reported both . the control group included 48 individuals ages 23 to 76 years ( mean age , 40.2 years sex ratio , m : f=11:37 ) . 1 , the graves ' group scored lower on all categories of the sf-36 , as compared to the control group ( p<0.05 ) . in comparing the sf-36 and bdi scores among the mild , moderately severe , and sight - threatening graves ' groups ( table 2 ) , the four patients with sight - threatening graves ' ophthalmopathy had significantly lower scores for physical functioning , role function - physical , and the physical component summary than the 28 patients with mild and 17 patients with moderately severe graves ' ophthalmopathy ( p<0.05 ) . patients with mild and moderately severe graves ' ophthalmopathy had similar scores for most sf-36 items . a higher bdi score was observed in patients with sight - threatening graves ' ophthalmopathy , as compared to patients with mild or moderately severe graves ' ophthalmopathy , and a higher proportion of patients with sight - threatening graves ' had depression ( bdi score 16 ) than did patients with mild and moderately severe graves ' ( although this difference was not statistically significant ) . nine patients who were previously diagnosed with optic neuropathy but recovered after orbital decompression surgery or high - dose steroid treatment were included in the moderately severe group . no significant difference in sf-36 and bdi scores were observed between the 9 patients with previously diagnosed optic neuropathy and the remaining 8 patients in the moderately severe category ( data now shown ) . the 17 patients with a high gorman score had a significantly lower physical component summary score on the sf-36 than the 32 patients with a low gorman score ( p=0.035 ) ( table 3 ) . compared to the low gorman score group , the high gorman score group also had lower subscale scores on the sf-36 for all measures but vitality , the mental component summary score , and mean bdi score ( albeit without statistical significance ) . sixteen patients ( 32.7% ) had bdi scores 16 ( indicative of clinical depression ) . these patients had significantly lower scores on the eight subscales and two summary scores of the sf-36 , as well as higher clinical activity scores , as compared to the 33 patients with low bdi scores ( p<0.05 ) ( table 4 ) . no significant differences were observed between the high and low bdi score groups with respect to age , sex , duration of graves ' disease or graves ' ophthalmopathy , or hertel exophthalmos values . there were no significant differences between patients with high and low bdi scores in terms of current severity stage by eugogo classification , main cause of distress , or history of optic neuropathy ( p>0.05 ) . although not statistically significant , it is interesting that 50% of patients in the depressed group ( bdi16 ) reported that both functional impairment and facial disfigurement equally affected their emotional distress , whereas only 18.2% of patients in the non - depressed group ( bdi<16 ) reported that both parameters were equally distressing ( p=0.058 ) ( table 4 ) . in addition , no significant correlation was observed between bdi score and age ( r=-0.025 , p=0.863 ) , sex ( r=0.090 , p=0.538 ) , duration of graves ' disease ( r=0.113 , p=0.441 ) , duration of graves ' ophthalmopathy ( r=-0.863 , p=0.556 ) , gorman score ( r=0.203 , p=0.163 ) , severity ( r=0.209 , p=0.150 ) , or disease activity ( r=0.201 , p=0.166 ) . however , the bdi scores had significant negative correlations with all subscale scores on the sf-36 ( data not shown , p<0.05 ) . because the goal of the treatment of graves ' ophthalmopathy is to improve functioning and make patients look and feel better , the functional capacity and quality of life should be evaluated . the impact of graves ' ophthalmopathy on quality of life has been the subject of several studies . the first article on quality of life in patients with graves ' ophthalmopathy was published by gerding et al . using a general quality of life questionnaire . in that study , the patient group was found to have lower score compared to a large reference group ; however , the correlations between scores on general quality of life questionnaires and duration , severity , or activity of graves ' ophthalmopathy were low . an australian group conducted a cross - sectional study using a modified instrument to measure quality of life in patients with graves ' ophthalmopathy , finding a significant relationship between impaired quality of life and severity of ophthalmopathy , as measured by nospecs . yeatts found a reduction in quality of life for both physical and mental health in patients with graves ' ophthalmopathy using national eye institute visual functioning questionnaires ( nei - vfq ) . used the nei - vfq to evaluate patients with graves ' ophthalmopathy and found that these patients were especially impaired on the mental health subscale . farid et al . used the profile of mood state survey in 48 patients with graves ' ophthalmopathy and reported that patients with moderate to severe graves ' ophthalmopathy exhibited significantly greater emotional distress than did patients with mild graves ' ophthalmopathy . to our knowledge , this is the first study that assesses both quality of life and the frequency and severity of depressive symptoms in korean patients with graves ' ophthalmopathy , although we did not use a graves ' ophthalmopathy - specific questionnaire . the results show that patients with graves ' ophthalmopathy had significantly lower quality of life scores than individuals in the age - and sex - matched control group , and that these scores were comparable to those of patients with other chronic diseases [ 21 - 23 ] . the prevalence of depression in this study was 32.7% , much higher than the 10% prevalence in the general population . this is higher than the prevalence of depression found in patients with retinitis pigmentosa and similar to patients with diabetes , cancer , or myocardial infarction . studies by kahaly et al . showed that 45% of patients with graves ' ophthalmopathy have anxiety or depression , and patients with graves ' ophthalmopathy score significantly lower than controls on measures of emotional role limitation and mental health . we assumed that both cosmetic disfigurement and functional impairment play key roles in causing emotional distress ( including depression ) in patients with graves ' ophthalmopathy . recently , paik and yang showed that patients with moderate to severe ophthalmopathy reported significantly greater emotional distress than patients with mild ophthalmopathy , according to the korean profile of mood states survey . in particular , patients whose disfiguring signs were predominantly due to proptosis had significantly greater emotional stress , as compared to patients in whom diplopia was predominant . in our study , however , no differences in hertel exophthalmos values were observed between the high and low bdi score groups . as cosmetic disfigurement is acknowledged differently between patients and because there are other determinants of cosmetic disfigurement beyond proptosis ( such as lid retraction , lid swelling , and conjunctival injection ) , we surveyed results from somewhat subjective self - questionnaires regarding the main cause of distress . more patients in the depressed group ( bdi 16 , 50% ) reported that both functional impairment and facial disfigurement equally contribute to their emotional distress than did those in the non - depressed group ( bdi < 16 , 18.2% ) with borderline significance ( p=0.058 ) . results from the current study are not consistent with the recent report by paik and yang possibly because of different survey instruments and methods of analysis . we found that patients with graves ' ophthalmopathy with depression had significantly lower quality of life ( with lower sf-36 scores for all subscales ) , as compared to those without depression . additionally , the bdi scores in patients with graves ' ophthalmopathy were negatively correlated with the sf-36 score . interestingly , patients with graves ' ophthalmopathy and depression had significantly higher clinical activity scores , as compared with those without depression , indicating that the severity of depression is associated with current inflammatory symptoms , such as pain , swelling , and chemosis ( p<0.05 ) . patients in the high gorman score group had significantly lower physical component summary scores than the patients in the low gorman score group . although the patients in the high gorman score group had a higher mean bdi score than patients in the low gorman score group , this difference was not statistically significant . patients with bothersome diplopia are significantly impaired in their social and vocational functions because they can not establish single binocular vision in primary gaze and create the largest possible field of single binocular vision . hatt et al . found that median scores on the psychosocial subscale were significantly lower in patients with strabismus , as compared to visually normal adults.successful treatment of strabismus , although not easy , significantly improves quality of life . we found that patients with sight - threatening graves ' ophthalmopathy had lower sf-36 scores and higher bdi scores than those with mild and moderately severe graves ' ophthalmopathy . the sf-36 and bdi scores were not significantly different between the nine patients who were previously diagnosed with optic neuropathy but had recovered after orbital decompression surgery or high - dose steroid treatment and the others in the moderately severe group . although patients may have low quality of life and high depressive status due to sight - threatening severity , prompt and proper treatment will lead to better quality of life and eliminate depression . the sf-36 is very broadly based , containing items to measure general aspects of health - related quality of life . it may miss relevant items for a specific disease and may not be able to measure small but clinically important changes for graves ' ophthalmopathy . therefore , a korean version of a disease - specific quality of life questionnaire for patients with graves ' ophthalmopathy is warranted . we did not diagnose depression using conventional diagnostic criteria , instead measuring symptoms of depression in patients with graves ' ophthalmopathy using the bdi self - reported questionnaire , as the bdi is a good screening method for depression . this study demonstrates that graves ' ophthalmopathy can be a risk factor for depression and reduced quality of life . patients with graves ' ophthalmopathy that is sight - threatening or associated with significant diplopia showed the greatest impairment in quality of life . our findings suggest the importance of assessing and managing the psychosocial burden and impairments in quality of life in patients with graves ' ophthalmopathy and suggest that they should be considered part of standard follow - up . psychological support or treatment is critical in the patient with graves ' ophthalmopathy to eliminate depression and improve quality of life . hence , a multidisciplinary approach , including ophthalmology , endocrinology , and psychiatry , is critical for patients with graves ' ophthalmopathy . such an integrated approach will help to define the extent of the problem and how patients adapt to living with the disease . in the future , extensive and randomized clinical studies are necessary for the assessment of the clinical effect of this multidisciplinary approach .

purposeto assess quality of life and depressive status in korean patients with graves ' ophthalmopathy.methodsa cross - sectional study of 49 patients ( mean age , 41 years ; sex ratio , m : f=11:38 ) with graves ' ophthalmopathy ( referred to as the graves ' group ) and 48 age - matched and sex - matched controls ( mean age , 40.2 years ; sex ratio , m : f=11:37 ) was performed using the korean version of the 36-item short - form general health survey ( sf-36 ) questionnaire and the beck depression inventory ( bdi ) . survey data was compared among patients with mild , moderately severe , and sight - threatening graves ' ophthalmopathy and between patients with low ( 0 or 1 ) or high ( 2 or 3 ) gorman scores.resultsthose in the graves ' group scored significantly lower on all categories of the sf-36 , as compared to the control group ( p<0.05 ) . the 4 patients with sight - threatening graves ' had significantly lower scores for physical functioning , role limitations due to physical health , and the physical component summary of the sf-36 , when compared with the 28 patients with mild graves ' and the 17 patients with moderately severe graves ' ( p<0.05 ) . the 17 patients in the high gorman score group had lower physical component summary scores than the 32 patients in the low gorman score group ( p=0.03 ) . the 16 patients with bdi scores 16 had significantly lower scores on the sf-36 and higher clinical activity scores , as compared to the 33 patients with bdi scores < 16 ( p<0.05).conclusionspatients with graves ' ophthalmopathy had reduced health - related quality of life and were more likely to be depressed , especially those with a sight threatening condition or significant diplopia . it is important to identify these patients to provide the necessary psychological support .

Materials and Methods Study design and recruitment of participants Survey instruments 1) Medical outcome study SF-36 2) BDI Statistical analysis Results Discussion

this cross - sectional study included 49 patients with graves ' ophthalmopathy who were followed at the department of ophthalmology , yonsei university college of medicine between july 2008 and december 2008 ( referred to as the graves ' group ) . quality of life ( by sf-36 scores ) and depressive status ( by bdi scores ) were compared among patients with mild , moderately severe , and sight - threatening graves ' ophthalmopathy using one - way analysis of variance ( anova ) and the pearson chi - square test for continuous and categorical variables , respectively . characteristics , including age , sex , duration of graves ' disease and graves ' ophthalmopathy , hertel exophthalmos values , current activity score , and severity stage by eugogo classification were compared between the patients with high ( 16 ) and low ( < 16 ) bdi scores , using the independent two - sample t - test and the pearson chi - square test for continuous and categorical variables , respectively . this cross - sectional study included 49 patients with graves ' ophthalmopathy who were followed at the department of ophthalmology , yonsei university college of medicine between july 2008 and december 2008 ( referred to as the graves ' group ) . quality of life ( by sf-36 scores ) and depressive status ( by bdi scores ) were compared among patients with mild , moderately severe , and sight - threatening graves ' ophthalmopathy using one - way analysis of variance ( anova ) and the pearson chi - square test for continuous and categorical variables , respectively . characteristics , including age , sex , duration of graves ' disease and graves ' ophthalmopathy , hertel exophthalmos values , current activity score , and severity stage by eugogo classification were compared between the patients with high ( 16 ) and low ( < 16 ) bdi scores , using the independent two - sample t - test and the pearson chi - square test for continuous and categorical variables , respectively . the control group included 48 individuals ages 23 to 76 years ( mean age , 40.2 years sex ratio , m : f=11:37 ) . 1 , the graves ' group scored lower on all categories of the sf-36 , as compared to the control group ( p<0.05 ) . in comparing the sf-36 and bdi scores among the mild , moderately severe , and sight - threatening graves ' groups ( table 2 ) , the four patients with sight - threatening graves ' ophthalmopathy had significantly lower scores for physical functioning , role function - physical , and the physical component summary than the 28 patients with mild and 17 patients with moderately severe graves ' ophthalmopathy ( p<0.05 ) . a higher bdi score was observed in patients with sight - threatening graves ' ophthalmopathy , as compared to patients with mild or moderately severe graves ' ophthalmopathy , and a higher proportion of patients with sight - threatening graves ' had depression ( bdi score 16 ) than did patients with mild and moderately severe graves ' ( although this difference was not statistically significant ) . the 17 patients with a high gorman score had a significantly lower physical component summary score on the sf-36 than the 32 patients with a low gorman score ( p=0.035 ) ( table 3 ) . compared to the low gorman score group , the high gorman score group also had lower subscale scores on the sf-36 for all measures but vitality , the mental component summary score , and mean bdi score ( albeit without statistical significance ) . these patients had significantly lower scores on the eight subscales and two summary scores of the sf-36 , as well as higher clinical activity scores , as compared to the 33 patients with low bdi scores ( p<0.05 ) ( table 4 ) . an australian group conducted a cross - sectional study using a modified instrument to measure quality of life in patients with graves ' ophthalmopathy , finding a significant relationship between impaired quality of life and severity of ophthalmopathy , as measured by nospecs . the results show that patients with graves ' ophthalmopathy had significantly lower quality of life scores than individuals in the age - and sex - matched control group , and that these scores were comparable to those of patients with other chronic diseases [ 21 - 23 ] . we found that patients with graves ' ophthalmopathy with depression had significantly lower quality of life ( with lower sf-36 scores for all subscales ) , as compared to those without depression . interestingly , patients with graves ' ophthalmopathy and depression had significantly higher clinical activity scores , as compared with those without depression , indicating that the severity of depression is associated with current inflammatory symptoms , such as pain , swelling , and chemosis ( p<0.05 ) . patients in the high gorman score group had significantly lower physical component summary scores than the patients in the low gorman score group . we found that patients with sight - threatening graves ' ophthalmopathy had lower sf-36 scores and higher bdi scores than those with mild and moderately severe graves ' ophthalmopathy .

graft - versus - host disease ( gvhd ) is a leading cause of mortality and morbidity following allogeneic hematopoietic cell transplantation ( hct ) . it is caused by mature donor t lymphocytes , which recognize the tissues of the recipient as foreign , causing a severe inflammatory disease most often characterized by rash , diarrhea , and liver disease . gvhd is classified into acute or chronic forms by its symptoms , the amount of damage it has caused to the liver , skin and mucosa and gastrointestinal tract as well as its timing . while acute gvhd usually refers to disease presenting within the first 100 days post - transplant , chronic gvhd symptoms may occasionally present as early as 50 - 60 days post - transplant and symptoms may overlap , typified by broader tissue damages often involving connective tissues and exocrine glands . gvhd is particularly virulent when there is a mismatch of major histocompatibility complex ( mhc ) class i or mhc class ii antigen between the donor and the host . mhc molecules are highly polymorphic glycoproteins that present peptide antigens to t cells . while mhc class i molecules present peptides generated in the cytosol to cd8 t cells , mhc class ii molecules present peptides degraded in the intracellular vesicles to cd4 t cells . thus , it is commonly recommended that hct be undertaken , when there is a human leukocyte antigen ( hla ) ( mhc of human ) match between the donor and the host . however , gvhd can also occur in the context of disparities between minor histocompatibility antigens , which are peptides of polymorphic cellular proteins bound to mhc molecules that can lead to graft rejection when they are recognized by t cells . one set of proteins that induce minor histocompatibility responses is encoded on the male - specific y chromosome and the responses induced by these proteins are called h - y . female anti - male minor histocompatibility response can occur in female transplant recipients , since y chromosome - specific genes are not expressed in females . because gvhd is characterized by tissue damage caused by t - cell mediated immune attack , immunosuppression is commonly used in the setting of hct . the use of immunosuppressive agents is to reduce the severity of gvhd with the potential to induce donor - specific tolerance and avoid increased risk of infection and cancer in transplant recipient . donor t cell activation requires two signaling events including binding of the t - cell receptor ( tcr ) to an allogeneic peptide presented by the host mhc as well as co - stimulatory signals delivered by antigen presenting cells ( apcs ) . co - stimulatory signals involve the interaction of cd28 on the t cell with cd80 or cd86 on the apcs . cd28 activates a signal transduction pathway , acting through phosphoinositide 3-kinase ( pi3k ) to provide its co - stimulatory signal for t cell activation . dendritic cells or macrophages are major hematopoietic originated apcs and a capacity of these cells to activate antigen presentation is determined by the innate immune mechanisms that sense various types of microbial ligands or endogenous danger signals . with the discovery of toll - like receptors ( tlrs ) and nucleotide binding oligomerization domain ( nod)-like receptors ( nlrs ) , the role of innate immunity in the regulation of adaptive immunity and modulation of immune tolerance has become an intense area of current investigation in the field of immunology . tlrs have been extensively studied for their ability to activate signaling pathways in response to microbial or viral infection and to provide a link between innate and adaptive immunity , as tlr signals augment antigen presentation by innate immune cells . following ligand recognition or cellular disruption , these receptors activate downstream signaling pathways , such as nuclear factor kappa b ( nf-b ) , mitogen activated protein kinase ( mapk ) and type i interferon ( ifn ) pathways , which result in the up - regulation of pro - inflammatory cytokines and chemokines that are important in inflammatory and antimicrobial responses . in addition to tlrs , nlrs also function to recognize pathogens and form a multi - protein intracellular complex with caspase-1 , known as inflammasome , which lead to release of interleukin-1 ( il-1 ) , interleukin-18 ( il-18 ) and interleukin-33 ( il-33 ) . exploration of the potential role of tlrs / nlrs in the outcome of gvhd is in its early stages . in addition to their role in host defense against pathogenic microorganisms , tlrs / nlrs also sense endogenous ligands / danger signals and this danger sensing may play a major role in survival and maintenance of the grafted organs . furthermore , studies of gastrointestinal gvhd show that the loss of intestinal homeostasis or tissue integrity contributed significantly to increased systemic severity of gvhd and this could be mediated by tlr4 or nod2 . given that polymorphisms in tlrs / nlrs are associated with increased risk of severe gvhd , understanding the mechanisms by which tlrs / nlrs play a role in gvhd pathophysiology is essential . recent advances in our understanding of how innate immune receptors recognize pathogen - associated molecular patterns ( pamps ) as well as danger - associated molecular patterns ( damps ) and trigger adaptive immune response may provide a new insight into molecular mechanisms by which a specific modulation of these pathways can be used as novel therapeutic approaches for gvhd . we discuss our current knowledge of the biology of the tlrs / nlrs and the mechanisms of their pathogen recognition as well as non - pathogen recognition function . furthermore , we focus on their potential role in pathophysiology of gvhd and discuss future therapeutic targets . toll - like receptors ( tlrs ) are innate immune receptors capable of sensing various components of microbial organisms , such as lipid , carbohydrates , peptides , and nucleic acid . as evolutionarily conserved molecules , tlrs were first described in vertebrates as homologous proteins to insect molecule , toll that stimulates the secretion of antimicrobial peptides in drosophila melagaster . to date , 11 members of tlrs have been identified in human ( 13 tlrs in mouse ) and their expression patterns are quite diverse in that they are expressed on both innate and adaptive immune cells . while some tlrs are expressed extracellularly , expression of other tlrs ( such as 3,7/8,9 ) are limited to endocytic or intracellular compartments . most tlrs have transmembrane domains , composed of n - terminal extracellular leucine - rich repeats ( lrrs ) that are responsible for recognition of specific pathogen components , a membrane - spanning domain and a c - terminal cytoplasmic domain similar to the cytoplasmic region of the interleukin-1 ( il-1 ) receptor , known as the toll / il-1 receptor ( tir ) domain , which is required for downstream signaling . tlrs ( except for tlr3 ) , as well as il-1r and il-18r family members utilize myd88 as an adaptor molecule for downstream signaling . tlrs recognize diverse microbial patterns and have broad specificity , detecting many related molecular structures . tlr2 can either dimerize with tlr1 or tlr6 . while tlr2/6 heterodimers recognize diacyl lipoproteins from mycoplasma species , tlr1/2 heterodimers recognize triacyl lipoproteins from various bacteria , including b. burgdorferi . tlr3 , which is found in endosomal compartments , recognizes double - stranded rna ( dsrna ) . tlr4 mainly recognizes lipopolysaccharides ( lps ) of gram - negative bacteria and interacts with cd14 and md2 [ 12 , 13 ] . both tlr7 and 8 sense single - stranded rna ( ssrna ) , whereas tlr9 is responsible for the recognition of unmethylated cpg nucleotides [ 16 , 17 ] . tlr11 detects profilin and plays an important role in host defense against uropathogenic bacterial infection [ 18 , 19 ] . tlrs utilize myd88 as an adaptor molecule for signaling , leading to nf-b - dependent cytokine production . on the other hand , tlr3 ( and tlr4 ) uses a myd88-independent signaling pathway that involves the adaptor molecule called tir - containing adaptor inducing ifn- ( trif ) , which can either promote the activation of nf-b pathway or the induction of type i interferon ( ifn-/ ) . signaling pathways downstream of tlrs are shown in fig . 1 . in the case of myd88-dependent signaling pathways , tir domains of tlrs interact with myd88 that recruits members of the il-1r - associated kinase ( irak ) family . irak activation results in targeting downstream tnf receptor - associated factor 6 ( traf6 ) , through the recruitment of transforming growth factor activated kinase 1 ( tak1 ) and tak1-binding protein 2 ( tab2 ) . these molecules ultimately lead to activation of the upstream kinases for mitogen - activated protein kinases ( mapk ) and activation of nf-b occurs , when ib kinases ( ikk ) complex phosphorylates ib , which leads to nuclear translocation of nf-b . in addition to nf-b activation , the trif - dependent pathway can also lead to the induction of type i interferon through phosphorylation and activation of the transcription factors , interferon regulatory factor 3 ( irf3 ) and irf7 [ 20 , 21 ] . trif is used by tlr3 or tlr4 and can directly bind to traf6 via its traf6-binding motifs in the n - terminal region and traf6 then activates tak1 in a manner similar to that in the myd88-dependent pathway [ 22 , 23 ] . trif can also recruit a signaling complex involving the non - canonical ikks , tbk1 and ikki , which induce irf3 activation , mediated by traf3 . a crucial role for tlrs in regulating the immune response is implicated in many cellular processes and diseases . signaling via tlrs can initiate inflammatory cascades in response to pathogens or cellular stress , and trigger the activation of adaptive immune responses by regulating expression of co - stimulatory molecules on apcs , such as cd40 or cd80/86 , and thereby enhancing antigen presentation . recent findings indicate that tlrs are involved in the process of acute allograft rejection and that their activation can prevent transplantation tolerance . we will review the expression of tlrs and the impact of tlr signaling on grafted organs in studies using both animal experiments and human clinical genetic association results . we then discuss possible mechanisms by which tlrs are involved in the rejection phenomenon and lead to increased alloimmune responses during gvhd . efficient priming of adaptive immune response depends on antigen presentation and stimulation of co - stimulatory molecules . tlrs regulate the expression of these co - stimulatory molecules and subsequently control the adaptive immune response , thereby influencing t cell activities . thus , because of tlrs ' role in the control of adaptive immune response , it was hypothesized that tlr signals may influence the activation of donor t lymphocytes and thus exacerbate the outcome of gvhd . moreover , tlrs have been shown to be involved in regulatory mechanisms for ischemia - reperfusion injury ( iri ) , which is a major cause of delayed allograft function in solid organ transplantation ( sot ) and an important inducer of acute and chronic transplant rejection [ 26 - 30 ] . evidences suggesting a role for tlrs in immune tolerance against allograft in sot is based on studies from both animal experiments using knockout mice and human genetic single nucleotide polymorphism ( snp ) analysis . lps is a tlr4 ligand and lps - mediated tlr4 activation leads to pro - inflammatory cytokine release . the finding that lps signals could influence the outcome of gvhd facilitated the use of lps antagonist at the time of transplantation to reduce the risk of gvhd . the importance of tlr signaling in graft intolerance using animal transplantation model was confirmed when goldstein et al . reported the tolerance of myd88-deficient mice towards h - y minor histocompatibility antigen - mismatched transplant , suggesting that the gvhd in h - y minor histocompatibility antigen - mismatched transplant in mice is dependent on the presence of tlr signaling . the authors further investigated whether the absence of acute allograft rejection from the myd88-deficient recipients could be restored by the adoptive transfer of wt - activated spleen cells . wt cells could re - activate the alloimmune response , confirming that tlr signaling exacerbates the alloimmune tolerance . thus , these results suggest that the defect in the myd88-deficient recipients most likely arises in the initiation phase of the alloimmune response because there are reduced numbers of mature dendritic cells migrating into draining lymph nodes , resulting in myd88-deficient mice 's inability to generate alloreactive t cells and thus cause the intolerance . furthermore , consistent with this study , even for mhc - mismatched sot , tlr signaling was found to be important for dendritic cell function and th1 cell response , which influence the outcome of transplantation . in addition to animal studies described above , the implication that tlr signaling may influence the severity and incidence of acute gvhd is also shown by human genetic association studies [ 34 , 35 ] . an association between tlr4 mutation and increased risk for gvhd was observed for patients who received organ grafts from human leukocyte antigen ( hla)-matched donor siblings , and increased risk for gram - negative bacteremia as a result of tlr4 mutation was also reported in these patients . however , these associations are not statistically significant in recipients of hla - matched sibling marrow transplants , prophylactically treated for infections and gvhd , suggesting that tlr4 polymorphisms may not cause more severe gvhd . however , another study indicates that there is a significant influence of mutations in tlr4 on the incidence and severity of acute gvhd in patients who underwent allogenic transplantation . interestingly , specific single nucleotide polymorphisms ( snp ) in the coding region of the tlr4 gene in both the patient or donor side were associated with an increased risk for severe gvhd and intestinal gvhd . based on the possibility that microbiota may contribute to gvhd outcome , the use of antibiotics during gvhd was evaluated and metronidazole and ciprofloxacin to decontaminate the intestine were found to be helpful for reducing the incidence of severe acute gvhd . it is possible that differential composition of the intestinal microbiota from the recipients of hct possibly contributes to the outcome of graft survival and maintenance . the finding that suggests a role for tlrs in the control of intestinal microbiota environment could be also a contributing factor that influences the occurrence and severity of gvhd . besides tlr4 , several other tlrs have been also implicated to play a role in gvhd pathophysiology . as mentioned earlier , tlr7/8 are known to recognize single - stranded rna and induce anti - viral response . the expression of tlr7/8 is present on plasmacytoid dendritic cells ( pdcs ) , which function as major anti - viral apcs . show that pdcs are capable of antigen presentation in gvhd and express immunosuppressive enzyme , indoleamine 2,3-dioxygenase ( ido ) , that influences gvhd pathophysiology . interestingly , administration of tlr7/8 agonist induced ido expression and reduced gvhd pathogenecity . in addition , tlr9 , a receptor that recognizes cpg dna , was also shown to be involved in gvhd outcome . tlr9 ligation of recipient apcs by cpg dna markedly accelerated gvhd lethality in a mouse transplantation model . one possibility is that tlr - mediated activation of recipient apcs may lead to maturation and migration of apcs , thereby indirectly causing the activation of donor t lymphocytes . t cell activation is likely to be triggered by stimulation of tlr pathway , thereby promoting the maturation and migration of alloantigen - expressing dcs to draining lymphoid organs . transplantation studies with myd88-deficient or myd88/trif - deficient mice supported the hypothesis that tlr function on apcs is important for t cell activity and gvhd outcome . defective tlr signals from either myd88-deficient or myd88/trif - deficient mice led to prolonged graft survival and this was consistent with diminished migration of donor cells to draining lymph nodes and subsequently , with delayed infiltration of host t cells into the grafted tissue [ 32 , 41 ] . although previous studies mentioned above focused on tlr - mediated stimulatory effect on apcs that indirectly affect t cell response , there are studies to suggest direct effects of tlrs on t cell functions . given that the expression of tlrs is not limited to innate immune cells , but is extended in different t cell subsets , including conventional t cells , regulatory t cells , and t cells as well as natural killer t cells , we can speculate that tlrs may be significant for induction of effector and regulatory function of t cells . stimulation of tlr ligand with purified cd4 t cells can directly promote activated cd4 t cell survival , suggesting that tlrs on t cells can directly modulate adaptive immune response [ 43 , 44 ] . furthermore , tlrs can act as a co - stimulatory receptor via t cell receptor ( tcr ) engagement and can enhance the priming or survival of alloreactive t cells . another evidence that tlr stimulation provides a signal for t cell activation comes from the study using the administration of the tlr9 agonist cpg or of the tlr2 ligand pam3csk4 at the time of skin graft transplantation . this co - administration resulted in increased production of interferon ( ifn)- by alloantigen - stimulated splenocytes and impaired intra - graft regulatory t cells ( treg ) recruitment , suggesting that innate immune signals prevented alloimmune tolerance against the graft . in addition to tlrs ' indirect or direct role in activation of t lymphocytes , tlrs may also participate in the control of intestinal microbiota , thereby contributing to the gvhd pathophysiology . intestinal microbiota and endotoxin were proposed to have a significant impact on the apc activation and this could be a crucial and initiating step in the induction of alloreactions [ 47 - 49 ] . compared with mice who died from acute gvhd , grown under conventional conditions early after transplantation , germ - free mice were resistant to developing acute gvhd , suggesting that intestinal microbiota could determine the susceptibility for gvhd severity . recent findings suggest that there is a significant association of tlr - mediated control of microbiota and the outcome of gvhd . the beneficial role of the diversity of the intestinal microbiota on the outcome of gvhd has been suggested by gerbitz et al . in that alteration of the intestinal microbiota may play an important role in the initiation of acute gvhd and the use of probiotics can ameliorate the outcome of gvhd . understanding the mechanisms by which the interaction of tlr - mediated innate immune activation with intestinal microbiota may shape the outcome of gvhd remains to be investigated in the future . in summary , both animal transplantation model and human clinical genetic studies show that tlr - mediated signaling is likely to play an important role in the pathogenesis gvhd . tlrs induce antigen presentation , together with tlr - mediated expression of co - stimulatory molecules and inflammatory cytokines , and instruct development of antigen specific adaptive immunity , especially helper t cells . therefore , alloimmune t cell responses can be enhanced by microbial and/or endogenous tlr ligands induced by iri and tissue stress / injury at the time of transplantation . elucidation of the factors controlling tlr - mediated events after transplantation could ultimately lead to novel therapeutic strategies that target tlr signals to reduce the severity of gvhd . together with tlrs , nlrs represent central platforms of innate immunity that link sensing of microbial pathogens and metabolic stress to the activation of pro - inflammatory cytokines , such as il-1 , il-18 and il-33 . nlr signaling results in the formation of large molecular scaffold complexes ( called inflammasome ) , which are intricately linked with inflammation / autoimmunity [ 52 , 53 ] and crosstalk with tlr - mediated signaling events . mutations / polymorphisms in nlrs have been linked to auto - inflammatory or autoimmune diseases [ 52 , 55 ] and thus this suggest that nlrs function beyond the recognition of pathogens and act as central components of immune system . since the discovery of inflammasome , nlrs - related studies have been focused on identifying molecules that are components of inflammasome and understanding how these molecules recognizes danger signals . activation of caspase-1 through autoproteolytic maturation is thought to be a mechanism by which the processing and secretion of il-1 occurs , however , further studies are required to determine the relative contribution of each of inflammasome mechanisms to innate and adaptive immune responses in response to danger signals . more than 20 members of nlr family have been identified in mammals so far , however , the ligands and functions of many of these receptors remain undefined . in addition to tlrs , nlrs function as intracellular sensors that recognize pathogen - associated molecular patterns ( pamps ) as well as danger - associated molecular patterns ( damps ) including cellular stress , and tissue damage / injury . nlrs are characterized by c - terminal leucine rich domain , an intermediate nod domain and an n - terminal effector region , comprising a protein - protein interaction domain , such as the caspase activation recruitment domain ( card ) , pyrin ( pyd ) or baculovirus inhibitor of apoptosis repeat domain ( bir ) domain . nlrs have been grouped into several subfamilies on the basis of the effector domains : nod , nalps , and ipaf / naips . nods and ipaf contain card effector domains , whereas nalps and naips contain pyrin ( pyd ) effector domains and three bir domains , respectively . nod2 was first described as a crohn 's disease susceptibility gene [ 57 , 58 ] and later , nod1/2 have been shown to be important for anti - microbial host response against bacterial pathogens and they are known to recognize peptidoglycans ( pgn ) , cell wall components of bacteria . nod1 is a sensor for d--glutamyl - meso - dap dipeptide ( ie - dap ) , which is found in pgn of all gram - negative and certain gram - positive bacteria , whereas nod2 recognizes the muramyl dipeptide ( mdp ) . loss of nod1 caused increased susceptibility to helicobacter pylori , while nod2 deficient mice were more susceptible to listeria monocytogenes infection [ 62 , 63 ] . nod2 is also regarded as a pivotal sensor molecule of the intestinal barrier , contributing to maintenance of intestinal homeostasis . once activated , nod1 and nod2 oligomerize and recruit the nf-b activating kinase rip2 through homotypic card - card interactions involving their n - terminal card motifs . rip2 interacts with the regulatory nf-b subunit , nemo / ikk- , triggering ib phosphorylation and nf-b activation [ 62 , 65 ] ( fig . 2 ) . nalps ( nlrps ) are characterized by the presence of pyd effector domains and so far , include 14 members . the function of many nlrps still remains unknown , however , several nalps ( nlrp1 , nlrp3 , and nlrc4 ) have been shown to participate in the formation of inflammasome . nalp1 ( nlrp1 ) was shown to form cytoplasmic multicomplex with the adaptor called , apoptosis - associated speck - like protein containing a caspase recruitment pycard ( asc ) and activate caspase-5 . nlrp1 can be activated by two molecules , muramyl dipeptide ( mdp ) and the anthrax toxin [ 66 , 67 ] . nalp3 ( nlrp3 ) is the best studied nlr member , which has been shown to mediate caspase-1 activation in response to a variety source of stimulus ; bacterial components [ 68 - 74 ] , endogenous danger signals released by damaged cells or tissues [ 75 - 77 ] , and pore - forming toxins . nlrp3 also interacts with asc to activate caspase-1 and nlrp3 signaling requires two stimulus , a cell priming signal from transcriptionally active tlr , nlr , or cytokine receptor , prior to activation of nlrp3 with pore - forming toxins , atp or various endogenous danger signals . similar to tlr5 , ipaf ( nlrc4 ) has been shown to respond to bacterial flagellin , a main component of the bacterial flagellum , restricting the proliferation of intracellular bacteria such as salmonella typhimurium , shigella flexneri , and legionella pneumophila [ 79 - 82 ] . these bacteria use type iii secretion system ( ttss ) to secrete flagellin into cytoplasmic compartment where ipaf senses flagellin . nlrc4 lacks a pyd domain , and it could activate pro - caspase-1 directly , however , asc is still required for full activation to induce the cleavage and secretion of mature il-1 . recent studies focused on highlighting the role of nlrs in broader control of adaptive immune response and various disease states . many nlrs have been identified and proposed to function as sensors for pamps as well as damps , released through damaged mucosal barriers or skin . specifically , damps - stimulated nlr activation may participate in enhancing the severity of gvhd , similar to the role of tlrs , thus , it is important to understand potential contribution of nlrs to gvhd outcome . below , we summarize the current understanding of the function of nlrs in gvhd pathophysiology . similar to tlrs , nlrs were also shown to mediate the immune response to iri [ 85 - 87 ] . previous studies indicate an important role for nod2 in contributing to susceptibility to gvhd after hct [ 6 , 35 , 88 - 91 ] and mutations in nlrp2 and nlrp3 have been proposed to be prognostic markers for the clinical outcomes of allogenic transplantation . in addition to the strong genetic association between nlr polymorphisms / mutations and gvhd susceptibilities by human snp analysis , animal experiments using nod2-deficient mice suggested a role of nod2 in gvhd pathophysiology . in contrast to tlrs , which drove donor t lymphocyte activation and severe gvhd outcome , the absence of nod2 from the bone marrow transplant donor mice had no significant impact on the development of gvhd and did not regulate alloactivation of donor t cells . instead , nod2 deficiency in allo - bone marrow transplantation recipients caused increased gvhd occurrence in both mhc - mismatched and mhc - matched models [ 93 , 94 ] . furthermore , the proliferation and activation of donor t cells were enhanced by the absence of nod2 from all bone marrow transplant recipients . this suggests that nod2 plays a role in the regulation of host apcs , as evident by nod2-deficient dcs displayed a higher activation status and increased ability to induce t cell proliferation during gvhd . several studies demonstrated a significant association between nod2 snps and increased gvhd incidence / severity [ 6 , 35 , 88 - 91 ] . in contrast to these studies , several clinical findings did not find a significant impact of nod2 snps on the incidence of gvhd [ 95 - 98 ] . multiple factors could explain the conflicting results on the role of nod2 and these may include differences in study population with nod2 snp frequency , overall incidence of gvhd , t cell activities , donor and recipient immune differences , and variation in environmental factors . nonetheless , these data give new insight into the potential role of nod2 in gvhd pathophysiology . many cytokines have been identified to be risk factors for predicting the outcome and severity of gvhd . il-1 is one of key inflammatory cytokines involved in gvhd pathophysiology and increased production of il-1 is found to be associated with worse outcomes of gvhd . given that il-1 is a major pro - inflammatory cytokine released following the activation of inflammasome , a genetic association study was conducted to evaluate whether mutations in nlrp genes are associated with gvhd pathogenecity . the genotypes of 133 patients undergoing hla - identical sibling allogenic stem cell transplantation ( allo - sct ) were analyzed and a strong association between common genetic variants in nlrp2 and nlrp3 genes and clinical outcome of hla - identical allo - sct was reported . despite increasing evidence , suggesting the importance of common variants in nod2 or nlrp genes on the clinical outcome of gvhd , the mechanisms underlying these associations remain unidentified . cellular damage or stress can ultimately lead to release of danger signals , which cause excessive immune - mediated tissue destruction , often found in acute gvhd . adenosine-5-triphosphate ( atp ) has been recognized as an endogenous danger signal during gvhd [ 4 , 101 , 102 ] . extracellular atp and its receptor p2x7r are expressed on several immune cell types and have been shown to be an important signal for activating nlrp3 inflammasome . increased concentration of atp was reported during the development of gvhd and atp - mediated stimulation of apcs led to increased expression of co - stimulatory molecules , such as cd80 and cd86 and further activated a variety of pro - inflammatory signaling pathways leading to expansion of donor t cells but reduction of regulatory t cells . this data gives a new insight into the mechanisms by which danger signals enhance gvhd . overall , studies characterizing the role of endogeneous or exogenous danger signals as well as their innate immune receptors in gvhd may hopefully help to develop a potential therapeutic target to bring a better clinical outcome for patients undergoing sot or hct . innate immune receptors significantly contribute to the inflammatory processes that consequently lead to the recruitment of alloactivated t cells as well as tissue damage in gvhd target organs . the generation of adaptive immunity can be triggered by activation of tlr - mediated apcs engulfing microbial pathogens or sensing danger signals in the peripheral tissues . subsequently , apcs migrate to the draining lymph nodes to present the processed peptides to nave t cells in the context of mhc molecules . thus , tlrs provide signals necessary for this migration or could act directly on t cell function . to date , most therapeutic and prophylactic efforts to prevent gvhd have been relied on immunosuppression mechanisms via t cell depletion or inhibition of t cell activation , proliferation , or effector function . alternatively , immunosuppressive therapeutic strategies targeting key innate immune receptor signaling pathways may also decrease the severity and incidence of gvhd . anti - il-1 therapies have been successfully used in treating auto - inflammatory diseases and the use of il-1 antagonist to reduce the severity of gvhd has been investigated . further studies on the role of tlrs / nlrs control of subsequent adaptive immune response will enhance our knowledge of the overall host response to transplant grafts and hopefully lead to the development of strategies to modify this response and to improve clinical outcomes of sot or hct .

graft - versus - host disease ( gvhd ) is a serious complication of allogeneic hematopoietic cell transplantation ( hct ) and this occurs as donor t lymphocytes , activated by recipient antigen presenting cells ( apc ) , attack the host tissues or organs . this apc activation is a crucial initial step of influencing the outcome of gvhd and is mediated by innate immune signaling . toll - like receptors ( tlrs ) and nucleotide binding oligomerization domain ( nod)-like receptors ( nlrs ) are important components of innate immunity ; both families of receptors are known for sensing various microbial ligands or danger signals . signaling through tlrs / nlrs regulate activities of apcs , through phagocytosis , cytokine and chemokine release , delivery of apcs from peripheral tissues to draining lymph nodes , and antigen presentation . several tlrs / nlrs have been identified and their ligands and signaling pathways have been described . recent findings suggest a significant association of tlr / nlr polymorphisms with the increased risk for severe gvhd . therefore , these tlr / nlr pathways likely contributing to immune response for gvhd may serve as novel therapeutic targets to facilitate allograft tolerance . this review summarizes the role of tlrs / nlrs innate immune receptors and signaling in gvhd pathophysiology .

INTRODUCTION INTRODUCTION TO TOLL-LIKE RECEPTORS (TLRs) THE ROLE OF TLRs IN GVHD INTRODUCTION TO NLRs ROLE OF NLRs IN GVHD CONCLUSION

graft - versus - host disease ( gvhd ) is a leading cause of mortality and morbidity following allogeneic hematopoietic cell transplantation ( hct ) . donor t cell activation requires two signaling events including binding of the t - cell receptor ( tcr ) to an allogeneic peptide presented by the host mhc as well as co - stimulatory signals delivered by antigen presenting cells ( apcs ) . dendritic cells or macrophages are major hematopoietic originated apcs and a capacity of these cells to activate antigen presentation is determined by the innate immune mechanisms that sense various types of microbial ligands or endogenous danger signals . with the discovery of toll - like receptors ( tlrs ) and nucleotide binding oligomerization domain ( nod)-like receptors ( nlrs ) , the role of innate immunity in the regulation of adaptive immunity and modulation of immune tolerance has become an intense area of current investigation in the field of immunology . tlrs have been extensively studied for their ability to activate signaling pathways in response to microbial or viral infection and to provide a link between innate and adaptive immunity , as tlr signals augment antigen presentation by innate immune cells . following ligand recognition or cellular disruption , these receptors activate downstream signaling pathways , such as nuclear factor kappa b ( nf-b ) , mitogen activated protein kinase ( mapk ) and type i interferon ( ifn ) pathways , which result in the up - regulation of pro - inflammatory cytokines and chemokines that are important in inflammatory and antimicrobial responses . exploration of the potential role of tlrs / nlrs in the outcome of gvhd is in its early stages . given that polymorphisms in tlrs / nlrs are associated with increased risk of severe gvhd , understanding the mechanisms by which tlrs / nlrs play a role in gvhd pathophysiology is essential . recent advances in our understanding of how innate immune receptors recognize pathogen - associated molecular patterns ( pamps ) as well as danger - associated molecular patterns ( damps ) and trigger adaptive immune response may provide a new insight into molecular mechanisms by which a specific modulation of these pathways can be used as novel therapeutic approaches for gvhd . toll - like receptors ( tlrs ) are innate immune receptors capable of sensing various components of microbial organisms , such as lipid , carbohydrates , peptides , and nucleic acid . to date , 11 members of tlrs have been identified in human ( 13 tlrs in mouse ) and their expression patterns are quite diverse in that they are expressed on both innate and adaptive immune cells . thus , because of tlrs ' role in the control of adaptive immune response , it was hypothesized that tlr signals may influence the activation of donor t lymphocytes and thus exacerbate the outcome of gvhd . moreover , tlrs have been shown to be involved in regulatory mechanisms for ischemia - reperfusion injury ( iri ) , which is a major cause of delayed allograft function in solid organ transplantation ( sot ) and an important inducer of acute and chronic transplant rejection [ 26 - 30 ] . interestingly , specific single nucleotide polymorphisms ( snp ) in the coding region of the tlr4 gene in both the patient or donor side were associated with an increased risk for severe gvhd and intestinal gvhd . show that pdcs are capable of antigen presentation in gvhd and express immunosuppressive enzyme , indoleamine 2,3-dioxygenase ( ido ) , that influences gvhd pathophysiology . recent findings suggest that there is a significant association of tlr - mediated control of microbiota and the outcome of gvhd . understanding the mechanisms by which the interaction of tlr - mediated innate immune activation with intestinal microbiota may shape the outcome of gvhd remains to be investigated in the future . specifically , damps - stimulated nlr activation may participate in enhancing the severity of gvhd , similar to the role of tlrs , thus , it is important to understand potential contribution of nlrs to gvhd outcome . increased concentration of atp was reported during the development of gvhd and atp - mediated stimulation of apcs led to increased expression of co - stimulatory molecules , such as cd80 and cd86 and further activated a variety of pro - inflammatory signaling pathways leading to expansion of donor t cells but reduction of regulatory t cells . overall , studies characterizing the role of endogeneous or exogenous danger signals as well as their innate immune receptors in gvhd may hopefully help to develop a potential therapeutic target to bring a better clinical outcome for patients undergoing sot or hct . further studies on the role of tlrs / nlrs control of subsequent adaptive immune response will enhance our knowledge of the overall host response to transplant grafts and hopefully lead to the development of strategies to modify this response and to improve clinical outcomes of sot or hct .

atrial fibrillation ( af ) is the most common cardiac arrhythmia in clinical practice , with an estimated 70 % of cases classified as nonvalvular af . a large us prospective cohort study estimated the lifetime risk of af in people 50 years of age to be 25.9 % for men and 23.2 % for women . the prevalence of af in the united states in 2012 was estimated at 5.8 million based on data from a large database of us health insurance claims , and estimates based on medicare data from 2007 to 2008 put the 2010 prevalence of nonvalvular af at 5.3 % of the us medicare population . this figure is expected to increase over time , and estimates of the prevalence of af in the united states in 2050 range from 5.61 million to 15.9 million [ 4 , 5 ] . in the framingham heart study , the risk of stroke in individuals with af was five - fold greater than in those without af , and was substantially higher than risks for other cardiovascular conditions . ischemic strokes account for 10 % of all deaths occurring within the first 4 months after a diagnosis of af , and 7 % of deaths occurring after this period [ 7 , 8 ] . in addition , the prevalence of af and the risk of stroke increase with advancing age [ 6 , 9 ] . risk of stroke attributed to af rises from 1.5 % at 5059 years of age to 23.5 % at 8089 years . strokes in individuals with af are generally more severe and are associated with greater stroke - related mortality [ 10 , 11 ] . af - related stroke also raises the risks of recurrence , functional impairment , and being subsequently bedridden [ 1012 ] . thus , survivors of af - related stroke are more likely to have disability and require greater inpatient and long - term care than stroke survivors without af , all of which impact medical costs . both ischemic and hemorrhagic strokes increase ongoing medical costs in patients with af relative to patients with af who have not experienced stroke , and the cost of care may remain elevated for several years after the event . patients with af and additional risk factors for stroke have higher cardiovascular - related inpatient costs and higher rates of rehospitalization , with readmissions incurring greater costs than the initial admission for af . the total us healthcare cost of all strokes was estimated at $ 53.9 billion in 2010 . in turn , the cost of af - related strokes was estimated to be as high as $ 13 billion . limited information is available comparing patient outcomes following stroke in patients with and without af . the aim of this study was to evaluate and quantify the impact of af on the discharge status , length of hospital stay , and associated costs for patients hospitalized for either ischemic or hemorrhagic stroke using data from a large us healthcare database . patients were identified from the premier alliance database ( premier , inc . , charlotte , nc , usa ) , a large nationally representative database ( premier is an alliance of community - based hospitals with over 2,700 hospital members ) accounting for 5 million discharges annually . the analysis included all adult patients hospitalized with a primary diagnosis of ischemic stroke ( icd-9 : 433.xx , 434.xx ) or hemorrhagic stroke ( icd-9 : 430.xx , 431.xx , 432.xx ) between january 2006 and june 2011 ( patients with multiple hospitalizations for stroke were excluded ) . patients included in the analysis were identified as with or without af , based on the presence or absence of a secondary listed diagnosis of af ( icd-9 : 427.3x ) . patients were identified from the premier alliance database ( premier , inc . , charlotte , nc , usa ) , a large nationally representative database ( premier is an alliance of community - based hospitals with over 2,700 hospital members ) accounting for 5 million discharges annually . the analysis included all adult patients hospitalized with a primary diagnosis of ischemic stroke ( icd-9 : 433.xx , 434.xx ) or hemorrhagic stroke ( icd-9 : 430.xx , 431.xx , 432.xx ) between january 2006 and june 2011 ( patients with multiple hospitalizations for stroke were excluded ) . patients included in the analysis were identified as with or without af , based on the presence or absence of a secondary listed diagnosis of af ( icd-9 : 427.3x ) . this was a retrospective analysis investigating clinical characteristics , hospital discharge status ( to home or continuing care ) , mortality , length of hospital stay , and patient costs associated with stroke in patients with or without af . data on length of hospital stay were analyzed using a poisson regression model ; patient cost and discharge status data were analyzed using generalized linear models with gamma distribution and log link , and multinomial distribution , respectively . the absolute differences in length of hospital stay and patient cost between af and non - af patients were the differences of least square means . demographic variables included sex , race ( white , black , hispanic , other ) , and age ( 1839 , 4049 , 5059 , 6069 , 7079 , and 80 + years ) . clinical variables that may have been associated with the index stroke event rather than preexisting stroke risk factors , such as hemiplegia and cerebrovascular disease , were excluded . additional variables included hospital size ( number of beds , categorized as 099 , 100199 , 200299 , 300399 , 400499 , 500599 , 600699 , 700799 , 800899 , and 900 + ) , hospital type ( urban or rural ; teaching or nonteaching ) , and geographic region ( midwest , northeast , south , and west ) . using a simplified version of the discharge / mortality code included in the premier alliance database , status was characterized as continuing care was defined as discharge to skilled nursing facility , other hospital or hospital department , hospice , or other medical department or facility . patient costs ( calculated as the actual cost to treat the patient ) included all supplies , labor , depreciation of equipment , etc . , based on the sum of variable costs ( direct ) plus all fixed costs ( overhead ) as obtained from the premier alliance database . data on length of hospital stay were analyzed using a poisson regression model ; patient cost and discharge status data were analyzed using generalized linear models with gamma distribution and log link , and multinomial distribution , respectively . the absolute differences in length of hospital stay and patient cost between af and non - af patients were the differences of least square means . demographic variables included sex , race ( white , black , hispanic , other ) , and age ( 1839 , 4049 , 5059 , 6069 , 7079 , and 80 + years ) . clinical variables that may have been associated with the index stroke event rather than preexisting stroke risk factors , such as hemiplegia and cerebrovascular disease , were excluded . additional variables included hospital size ( number of beds , categorized as 099 , 100199 , 200299 , 300399 , 400499 , 500599 , 600699 , 700799 , 800899 , and 900 + ) , hospital type ( urban or rural ; teaching or nonteaching ) , and geographic region ( midwest , northeast , south , and west ) . using a simplified version of the discharge / mortality code included in the premier alliance database , continuing care was defined as discharge to skilled nursing facility , other hospital or hospital department , hospice , or other medical department or facility . patient costs ( calculated as the actual cost to treat the patient ) included all supplies , labor , depreciation of equipment , etc . , based on the sum of variable costs ( direct ) plus all fixed costs ( overhead ) as obtained from the premier alliance database . a total of 351,601 patients ( mean age 70.29 years ) who experienced a stroke were identified for inclusion in this study , of whom 71,483 ( 20 % ) had a secondary diagnosis of af and 280,118 ( 80 % ) had no record of af . a summary of patient demographics , key clinical characteristics , and information relating to the health providers is reported in table 1 . among patients hospitalized with ischemic stroke , af was listed as a secondary diagnosis in 21 % ( n = 59,172 ) and not listed as a secondary diagnosis in 79 % ( n = 220,518 ) . among patients hospitalized with hemorrhagic stroke , 17 % ( n = 12,311 ) and 83 % ( n = 59,600 ) had and did not have a secondary diagnosis of af , respectively . among patients included in the study , there was a high prevalence of hypertension ( 63.568.2 % ) and diabetes ( 21.229.6 % ) in both af and non - af patient groups . however , while congestive heart failure ( chf ) was present in ~31 % of patients with af , only ~10 % of patients who did not have af had this comorbidity.table 1demographic characteristics , comorbidities , and information relating to healthcare providersstroke type , n ( % ) ischemichemorrhagicnon - af ( n = 220,518)af ( n = 59,172 ) p value*non - af ( n = 59,600)af ( n = 12,311 ) p value*age , years 18395,179 ( 2.4)116 ( 0.2)<0.00013,525 ( 5.9)45 ( 0.4)<0.0001 404914,554 ( 6.6)559 ( 0.9)7,309 ( 12.3)170 ( 1.4 ) 505934,523 ( 15.7)2,393 ( 4.0)11,801 ( 19.8)653 ( 5.3 ) 606950,877 ( 23.1)6,917 ( 11.7)11,247 ( 18.9)1,717 ( 14.0 ) 707956,588 ( 25.7)15,987 ( 27.0)11,824 ( 19.8)3,738 ( 30.4 ) 80 + 58,797 ( 26.7)33,200 ( 56.1)13,894 ( 23.3)5,988 ( 48.6)sex female109,500 ( 49.7)33,936 ( 57.4)<0.000130,480 ( 51.1)5,954 ( 48.4)<0.0001 race white141,317 ( 64.1)43,000 ( 72.7)<0.000133,984 ( 57.0)8,765 ( 71.2)<0.0001 black33,594 ( 15.2)4,515 ( 7.6)10,047 ( 16.9)847 ( 6.9 ) hispanic9,421 ( 4.3)1,874 ( 3.2)3,771 ( 6.3)524 ( 4.3 ) other36,186 ( 16.4)9,783 ( 16.5)11,798 ( 19.8)2,175 ( 17.7)comorbidities myocardial infarction20,824 ( 9.4)7,274 ( 12.3)<0.00013,604 ( 6.1)1,300 ( 10.6)<0.0001 congestive heart failure23,146 ( 10.5)19,052 ( 32.2)<0.00014,729 ( 7.9)3,558 ( 28.9)<0.0001 diabetes65,191 ( 29.6)15,793 ( 26.7)<0.000112,655 ( 21.2)3,353 ( 27.2)<0.0001 hypertension15,0402 ( 68.2)38,079 ( 64.4)<0.000137,820 ( 63.5)8,064 ( 65.5)<0.0001provider characteristics rural21,781 ( 9.9)5,722 ( 9.7)0.13313,928 ( 6.6)939 ( 7.6)<0.0001 urban198,737 ( 90.1)53,450 ( 90.3)55,672 ( 93.4)11,372 ( 92.4 ) teaching hospital88,392 ( 40.1)23,752 ( 40.1)0.802331,297 ( 52.5)6,186 ( 50.3)<0.0001 beds 0995,892 ( 2.7)1,688 ( 2.9)<0.0001559 ( 0.9)172 ( 1.4)<0.0001 10019923,697 ( 10.8)6,515 ( 11.1)4,015 ( 6.7)930 ( 7.6 ) 20029933,521 ( 15.2)9,139 ( 15.4)6,013 ( 10.1)1,411 ( 11.5 ) 30039950,575 ( 22.9)13,423 ( 22.7)11,568 ( 19.4)2,484 ( 20.2 ) 40049938,481 ( 17.5)10,167 ( 17.2)11,008 ( 18.5)2,261 ( 18.4 ) 50059925,217 ( 11.4)6,718 ( 11.4)7,777 ( 13.1)1,683 ( 13.7 ) 60069920,398 ( 9.3)5,277 ( 8.9)9,684 ( 16.3)1,647 ( 13.4 ) 70079910,048 ( 4.6)2,446 ( 4.1)3,216 ( 5.4)572 ( 4.7 ) 8008994,252 ( 1.9)1,317 ( 2.2)2,186 ( 3.7)459 ( 3.7 ) 900 + 8,437 ( 3.8)2,482 ( 4.2)3,574 ( 6.0)692 ( 5.6)provider region midwest40,585 ( 18.4)11,220 ( 19.0)<0.00019,626 ( 16.2)2,160 ( 17.6)<0.0001 northeast40,941 ( 18.6)12,313 ( 20.8)13,039 ( 21.9)2,993 ( 24.3 ) south98,649 ( 44.7)23,100 ( 39.0)25,123 ( 42.2)4,602 ( 37.4 ) west40,343 ( 18.3)12,539 ( 21.2)11,812 ( 19.8)2,556 ( 20.8 ) * chi squared values for the variable , by presence of af af atrial fibrillation demographic characteristics , comorbidities , and information relating to healthcare providers * chi squared values for the variable , by presence of af af atrial fibrillation a summary of discharge status and mortality results is shown in table 2 . after adjusting for all covariates , patients with af were more likely than patients without af to be discharged to a continuing care facility following hospitalization for ischemic stroke ( odds ratio [ or ] , 1.92 ; 95 % confidence interval [ ci ] , 1.881.97 ; p < 0.0001 ) and hemorrhagic stroke ( or , 1.38 ; 95 % ci , 1.291.47 ; p < 0.0001 ) . patients with af were also significantly more likely to die during the index hospitalization rather than be discharged home , irrespective of type of stroke ( ischemic : or , 3.49 ; 95 % ci , 3.353.63 ; p < 0.0001 ; hemorrhagic : or , 1.58 ; 95 % ci , 1.471.69 ; p < 0.0001).table 2discharge status for patients with ischemic and hemorrhagic stroke in the premier alliance databaseeffectcomparison discharged to continuing caredied during hospitalizationodds ratio95 % wald confidence limits p valueodds ratio95 % wald confidence limits p valueischemic stroke afaf vs non - af1.921.881.97<0.00013.493.353.63<0.0001 age , years1839 vs 80 + 0.200.190.21<0.00010.260.230.30<0.00014049 vs 80 + 0.220.210.23<0.00010.220.200.25<0.00015059 vs 80 + 0.240.240.25<0.00010.240.220.26<0.00016069 vs 80 + 0.270.260.28<0.00010.240.220.25<0.00017079 vs 80 + 0.390.380.40<0.00010.340.330.36<0.0001 sexmale vs female0.700.690.71<0.00010.720.690.75<0.0001 miyes vs no0.980.951.010.12551.631.551.71<0.0001 chfyes vs no1.621.581.66<0.00012.362.262.47<0.0001 diabetesyes vs no1.281.251.30<0.00011.191.141.24<0.0001 hypertensionyes vs no1.071.051.09<0.00010.730.700.76<0.0001 racehispanic vs black0.700.670.74<0.00010.850.760.94 0.002other vs black0.590.570.61<0.00010.700.650.75<0.0001white vs black0.530.510.54<0.00010.610.580.65<0.0001hemorrhagic stroke afaf vs non - af1.381.291.47<0.00011.581.471.69<0.0001 age , years1839 vs 80 + 0.140.130.15<0.00010.150.140.17<0.00014049 vs 80 + 0.190.170.20<0.00010.210.190.23<0.00015059 vs 80 + 0.240.220.26<0.00010.270.250.29<0.00016069 vs 80 + 0.320.300.35<0.00010.340.310.36<0.00017079 vs 80 + 0.520.480.55<0.00010.540.500.58<0.0001 sexmale vs female0.790.750.82<0.00010.780.750.81<0.0001 miyes vs no1.241.131.36<0.00011.521.391.68<0.0001 chfyes vs no1.611.491.75<0.00011.481.361.61<0.0001 diabetesyes vs no1.241.181.30<0.00011.181.121.25<0.0001 hypertensionyes vs no1.371.311.43<0.00011.151.091.21<0.0001 racehispanic vs black0.750.680.82<0.00010.770.700.86<0.0001other vs black0.800.750.86<0.00010.920.851.000.0449white vs black0.750.700.79<0.00010.860.800.92<0.0001odds ratios from multinomial regression . data for hospital type and geographic region are not shown af atrial fibrillation , chf congestive heart failure , mi myocardial infarction patients classified as discharged to home were used as the reference group discharge status for patients with ischemic and hemorrhagic stroke in the premier alliance database odds ratios from multinomial regression . data for hospital type and geographic region are not shown af atrial fibrillation , chf congestive heart failure , mi myocardial infarction patients classified as discharged to home were used as the reference group among patients with an ischemic stroke event , the duration of hospital stay was significantly longer for patients with af ( adjusted mean length of stay , 1.63 days longer ; p < 0.0001 ) . this was also true for patients who experienced hemorrhagic stroke ( adjusted mean length of stay , 0.95 days longer ; p < 0.0001 ) . table 3 shows a summary of the observed effect of various baseline characteristics and selected common cardiovascular comorbidities on the length of hospital stay.table 3length of stay for patients with ischemic and hemorrhagic stroke in the premier alliance databaseeffectcomparisondifference ( days ) 95 % ci p valueischemic stroke afaf vs non - af1.6261.6141.637<0.0001 age , years1839 vs 80 + 1.1121.0991.125<0.00014049 vs 80 + 0.5360.5320.540<0.00015059 vs 80 + 0.3410.3390.343<0.00016069 vs 80+0.1820.181 to 0.183<0.00017079 vs 80+0.2590.258 to 0.260<0.0001 sexmale vs female0.1150.115 to 0.116<0.0001 miyes vs no0.2980.2970.300<0.0001 chfyes vs no1.2651.2601.271<0.0001 diabetesyes vs no0.3050.3040.306<0.0001 hypertensionyes vs no0.2850.284 to 0.287<0.0001 racehispanic vs black0.1750.173 to 0.176<0.0001other vs black0.7120.708 to 0.716<0.0001white vs black1.3131.307 to 1.320<0.0001hemorrhagic stroke afaf vs non - af0.9470.9430.951<0.0001 age , years1839 vs 80 + 4.1494.0994.201<0.00014049 vs 80 + 4.7274.6834.771<0.00015059 vs 80 + 4.1354.1014.169<0.00016069 vs 80 + 2.6702.6482.692<0.00017079 vs 80 + 1.5301.5181.542<0.0001 sexmale vs female0.2730.2720.275<0.0001 miyes vs no0.1300.1290.1320.0015 chfyes vs no2.2532.2362.270<0.0001 diabetesyes vs no0.6230.6190.627<0.0001 hypertensionyes vs no0.5330.5300.536<0.0001 racehispanic vs black0.6610.654 to 0.669<0.0001other vs black0.8630.855 to 0.870<0.0001white vs black1.7001.688 to 1.712<0.0001estimates from poisson regression model . data for hospital type and geographic region are not shown af atrial fibrillation , chf congestive heart failure , ci confidence interval , mi myocardial infarction absolute differences in length of hospital stay between af and non - af patients are given as the differences of least square means length of stay for patients with ischemic and hemorrhagic stroke in the premier alliance database estimates from poisson regression model . data for hospital type and geographic region are not shown af atrial fibrillation , chf congestive heart failure , ci confidence interval , mi myocardial infarction absolute differences in length of hospital stay between af and non - af patients are given as the differences of least square means patients with af had an adjusted mean patient cost associated with an ischemic stroke event that was $ 2,997.18 ( 95 % ci , $ 2,937.97$3,057.58 ) higher than for patients without af . for patients with af having a hemorrhagic stroke event , this figure was $ 3,229.73 ( 95 % ci , $ 3,207.69$3,251.91 ) higher than for those without af . a summary of the effect of baseline characteristics and selected common cardiovascular comorbidities on patient costs associated with an index hospitalization for stroke is shown in table 4.table 4patient costs for ischemic and hemorrhagic stroke in the premier alliance databaseeffectcomparisondifference ( $ ) 95 % ci p valueischemic stroke afaf vs non - af2,997.182,937.973,057.58<0.0001age , years1839 vs 80 + 4,542.614,452.694,634.34<0.00014049 vs 80 + 2,651.132,618.012,684.68<0.00015059 vs 80 + 2,009.551,991.552,027.71<0.00016069 vs 80 + 1,235.461,226.001,244.99<0.00017079 vs 80 + 745.56740.34750.82<0.0001 sexmale vs female308.80307.14310.47<0.0001 miyes vs no1,228.621,217.791,239.56<0.0001 chfyes vs no2,338.772,320.622,357.07<0.0001 diabetesyes vs no473.68470.87476.50<0.0001 hypertensionyes vs no536.92533.38 to 540.49<0.0001 racehispanic vs black1,188.851,170.981,206.99<0.0001other vs black591.89585.92 to 597.93<0.0001white vs black1,087.181,078.27 to 1,096.15<0.0001hemorrhagic stroke afaf vs non - af3,229.733,207.693,251.91<0.0001 age , years1839 vs 80 + 20,898.3520,156.9121,667.05<0.00014049 vs 80 + 20,204.5019,671.1220,752.35<0.00015059 vs 80 + 16,538.7316,169.1716,916.74<0.00016069 vs 80 + 11,945.5911,687.1412,209.76<0.00017079 vs 80 + 6,090.325,967.326,215.86<0.0001 sexmale vs female116.29114.64117.960.45 miyes vs no1,500.941,459.121,543.97<0.0001 chfyes vs no6,485.876,336.966,638.27<0.0001 diabetesyes vs no1,100.251,081.291,119.55<0.0001 hypertensionyes vs no198.47195.18201.820.269 racehispanic vs black1,707.931,649.181,768.78<0.0001other vs black1,136.941,107.81 to 1,166.830.0001white vs black2,494.882,442.19 to 2,548.70<0.0001estimates from the generalized linear model with gamma distribution and log link . data for hospital type and geographic region are not shown af atrial fibrillation , chf congestive heart failure , ci confidence interval , mi myocardial infarction the absolute differences in patient cost between af and non - af patients are given as the differences of least square means patient costs for ischemic and hemorrhagic stroke in the premier alliance database estimates from the generalized linear model with gamma distribution and log link . data for hospital type and geographic region are not shown af atrial fibrillation , chf congestive heart failure , ci confidence interval , mi myocardial infarction the absolute differences in patient cost between af and non - af patients are given as the differences of least square means although the focus of this study was on the effects of af , several other variables included in the analysis were associated with notable effects on patient outcomes . among patients in the ischemic stroke group , hypertension was associated with a decreased likelihood of dying while in care versus being discharged home ( or , 0.73 ; p < 0.0001 ) , as well as with a significantly decreased duration of stay ( p < 0.0001 ) and patient costs ( p < 0.0001 ) . conversely , in patients who experienced hemorrhagic strokes , hypertension was associated with an increased duration of stay ( p < 0.0001 ) and an increased probability of dying while in care ( or , 1.15 ; p < 0.0001 ) , although there was no significant effect on patient costs ( p = 0.269 ) . patient costs and duration of hospital stay decreased with increasing patient age , which also correlated with a general trend toward an increasing or of being discharged to care and of dying during the initial hospitalization . chf was associated with an increased or for dying while in care in both the ischemic stroke group ( or , 2.36 , 95 % ci , 2.262.47 ; p < 0.0001 ) and the hemorrhagic stroke group ( or , 1.48 ; 95 % ci , 1.361.61 ; p < 0.0001 ) . this study analyzed a data source covering 4.5 years of hospital discharges , with over 350,000 strokes in 71,483 patients with af and 280,118 patients without af . both ischemic and hemorrhagic stroke events in patients with af were associated with a greater likelihood of being discharged to a continuing care facility , an increased risk of death from the stroke , a longer duration of hospital stay , and increased patient costs compared with stroke events in patients without af . hypertension was associated with reduced duration of hospital stay and reduced costs in patients in the ischemic stroke group , but had the opposite effect on these outcomes in patients who experienced hemorrhagic stroke . for example , an elevated blood pressure could be beneficial in helping to maintain blood supply during an ischemic event , but deleterious during a hemorrhagic or bleeding event . these opposite effects of hypertension might also help to confirm the validity of the data . after controlling for other variables , increasing age was associated with a decrease in patient costs and duration of stay , irrespective of the type of stroke . however , it was also associated with an increased likelihood of dying while in care or of being discharged to further care rather than to home , both of which would result in the observed reduction in length of stay for the index hospitalization event . additionally , as costs associated with additional care after the index hospitalization event were not included in this study , the shorter duration of the initial hospital stay for both outcomes could at least partly explain the apparent effect on patient costs . considering the aging population and that age is an important risk factor for stroke , the incidence of stroke is projected to increase over the next 2030 years ; this effect will be further magnified because age is a risk factor for af [ 4 , 5 ] . the synergistic combination of these two effects will likely lead to a greater incidence of stroke and a disproportionately greater incidence of af - related strokes . given the results of this study , increasing rates of af - related stroke would increase the demand for nursing homes , skilled nursing facilities , and hospices , and raise the question of who will bear the financial burden of this trend . under current medicare guidelines , the cost of a skilled nursing facility is reimbursed as long as patients require follow - up care after a qualifying hospital stay , but only for up to 100 days . if the stay extends beyond that , patients may use supplemental insurance coverage or would need to draw from personal resources until they meet medicaid eligibility requirements . thus , it is likely that an increase in af - related strokes will impact a variety of payers , from the federal medicare and state medicaid programs to private insurers and patients themselves . stroke risk is affected by nonmodifiable risk factors , such as age and sex , and modifiable risk factors , such as hypertension and high cholesterol levels . widespread use of low cost antihypertensives and statins , following a stricter implementation of current guidelines for these medications , could provide an excellent social return on investment with reduced rates of stroke and other cardiovascular events [ 21 , 22 ] . af is associated with an increased risk of stroke and more severe stroke outcomes than strokes unrelated to af , and appropriate thromboprophylaxis with warfarin or one of the new oral anticoagulants ( dabigatran , rivaroxaban , and apixaban ) has been shown to significantly reduce stroke risk in patients with af [ 2326 ] . therefore , a more rigorous implementation of current guidelines for anticoagulant use in af could potentially offer substantial reductions in stroke events and associated costs in this patient population . this analysis focused on stroke - associated outcomes in the presence or absence of af , and no direct statistical comparisons were made between patients who experienced ischemic or hemorrhagic strokes . a key strength of this study was the large number of patients included in the database ; however , there were also several inherent limitations regarding the level of detailed information available . first , whereas all patients included in the af group had a history of af prior to or on the date of their documented stroke , some cases of undiagnosed af may have been included in the group that did not have af . second , characterization of comorbidities was limited to data captured during the hospitalization period ; hence , the presence of pre - existing chronic conditions , such as valvular heart disease , diabetes , or hypertension would have been underestimated in the study population unless these conditions were specifically coded during the hospitalization event . therefore , the impact of risk - modifying drug therapies used in an outpatient setting prior to hospitalization on the outcomes of interest could not be assessed , either . third , the database was unclear as to whether the hospitalizations included primary admissions for stroke or whether patient transfers from other hospitals were included . fourth , treatment differences were not taken into account and may have existed between groups , both before and after the stroke event . fifth , the dataset did not include electronic medical record data on important clinical variables such as blood pressure , cholesterol levels or echocardiographic parameters , which could be explored in future research . finally , information on duration of stay in continuing care facilities following another potential limitation that the study was retrospective and not prospective could conceivably have introduced bias into the results . for example , chf was seen in a higher proportion of patients with af than without af . this could be expected , as chf is thought to predispose patients to af , af may predispose patients to chf , and the prevalence of both is known to increase with age . however , although the lack of a randomization step did result in some differences between categories , this was adjusted for as part of the multivariate model used in the analysis . among patients hospitalized with either ischemic or hemorrhagic stroke , af was associated with a higher mortality rate . patient care was extended beyond the initial hospitalization in a greater proportion of patients with af than without af , and length of hospital stay and patient costs were significantly higher in patients with af in both stroke subgroups . further research is needed to determine how modifiable risk factors can be managed to improve outcomes in the us population .

this retrospective analysis investigated the impact of baseline clinical characteristics , including atrial fibrillation ( af ) , on hospital discharge status ( to home or continuing care ) , mortality , length of hospital stay , and treatment costs in patients hospitalized for stroke . the analysis included adult patients hospitalized with a primary diagnosis of ischemic or hemorrhagic stroke between january 2006 and june 2011 from the premier alliance database , a large nationally representative database of inpatient health records . patients included in the analysis were categorized as with or without af , based on the presence or absence of a secondary listed diagnosis of af . irrespective of stroke type ( ischemic or hemorrhagic ) , af was associated with an increased risk of mortality during the index hospitalization event , as well as a higher probability of discharge to a continuing care facility , longer duration of stay , and higher treatment costs . in patients hospitalized for a stroke event , af appears to be an independent risk factor of in - hospital mortality , discharge to continuing care , length of hospital stay , and increased treatment costs .

Introduction Methods Data source and study population Study design Statistical analysis Results Discussion Conclusions

the aim of this study was to evaluate and quantify the impact of af on the discharge status , length of hospital stay , and associated costs for patients hospitalized for either ischemic or hemorrhagic stroke using data from a large us healthcare database . the analysis included all adult patients hospitalized with a primary diagnosis of ischemic stroke ( icd-9 : 433.xx , 434.xx ) or hemorrhagic stroke ( icd-9 : 430.xx , 431.xx , 432.xx ) between january 2006 and june 2011 ( patients with multiple hospitalizations for stroke were excluded ) . patients included in the analysis were identified as with or without af , based on the presence or absence of a secondary listed diagnosis of af ( icd-9 : 427.3x ) . the analysis included all adult patients hospitalized with a primary diagnosis of ischemic stroke ( icd-9 : 433.xx , 434.xx ) or hemorrhagic stroke ( icd-9 : 430.xx , 431.xx , 432.xx ) between january 2006 and june 2011 ( patients with multiple hospitalizations for stroke were excluded ) . patients included in the analysis were identified as with or without af , based on the presence or absence of a secondary listed diagnosis of af ( icd-9 : 427.3x ) . this was a retrospective analysis investigating clinical characteristics , hospital discharge status ( to home or continuing care ) , mortality , length of hospital stay , and patient costs associated with stroke in patients with or without af . patients with af were also significantly more likely to die during the index hospitalization rather than be discharged home , irrespective of type of stroke ( ischemic : or , 3.49 ; 95 % ci , 3.353.63 ; p < 0.0001 ; hemorrhagic : or , 1.58 ; 95 % ci , 1.471.69 ; p < 0.0001).table 2discharge status for patients with ischemic and hemorrhagic stroke in the premier alliance databaseeffectcomparison discharged to continuing caredied during hospitalizationodds ratio95 % wald confidence limits p valueodds ratio95 % wald confidence limits p valueischemic stroke afaf vs non - af1.921.881.97<0.00013.493.353.63<0.0001 age , years1839 vs 80 + 0.200.190.21<0.00010.260.230.30<0.00014049 vs 80 + 0.220.210.23<0.00010.220.200.25<0.00015059 vs 80 + 0.240.240.25<0.00010.240.220.26<0.00016069 vs 80 + 0.270.260.28<0.00010.240.220.25<0.00017079 vs 80 + 0.390.380.40<0.00010.340.330.36<0.0001 sexmale vs female0.700.690.71<0.00010.720.690.75<0.0001 miyes vs no0.980.951.010.12551.631.551.71<0.0001 chfyes vs no1.621.581.66<0.00012.362.262.47<0.0001 diabetesyes vs no1.281.251.30<0.00011.191.141.24<0.0001 hypertensionyes vs no1.071.051.09<0.00010.730.700.76<0.0001 racehispanic vs black0.700.670.74<0.00010.850.760.94 0.002other vs black0.590.570.61<0.00010.700.650.75<0.0001white vs black0.530.510.54<0.00010.610.580.65<0.0001hemorrhagic stroke afaf vs non - af1.381.291.47<0.00011.581.471.69<0.0001 age , years1839 vs 80 + 0.140.130.15<0.00010.150.140.17<0.00014049 vs 80 + 0.190.170.20<0.00010.210.190.23<0.00015059 vs 80 + 0.240.220.26<0.00010.270.250.29<0.00016069 vs 80 + 0.320.300.35<0.00010.340.310.36<0.00017079 vs 80 + 0.520.480.55<0.00010.540.500.58<0.0001 sexmale vs female0.790.750.82<0.00010.780.750.81<0.0001 miyes vs no1.241.131.36<0.00011.521.391.68<0.0001 chfyes vs no1.611.491.75<0.00011.481.361.61<0.0001 diabetesyes vs no1.241.181.30<0.00011.181.121.25<0.0001 hypertensionyes vs no1.371.311.43<0.00011.151.091.21<0.0001 racehispanic vs black0.750.680.82<0.00010.770.700.86<0.0001other vs black0.800.750.86<0.00010.920.851.000.0449white vs black0.750.700.79<0.00010.860.800.92<0.0001odds ratios from multinomial regression . both ischemic and hemorrhagic stroke events in patients with af were associated with a greater likelihood of being discharged to a continuing care facility , an increased risk of death from the stroke , a longer duration of hospital stay , and increased patient costs compared with stroke events in patients without af .

nowadays , the thermochemical processes such as pyrolysis , gasification and combustion are the most attractive and practical as far as the energy recovery from the biomass is concerned , being the combustion responsible for about a 97 % of the production of bioenergy in the world . in spain , the thermal processes are the option most widely used for the treatment of the waste coming from the industries for the transformation of agricultural products , specially interesting are the wastes from the olive oil mills , owing to the big amount produced and its elevated heating value between 20 kj / kg and 23 kj / kg dry basis . to carry out a correct design of the systems for thermochemical treatment and control them properly , it is important to know deeply the different states and complex transitions that take place in the biomass wastes as the temperature varies . in the case of the biomass , and similarly to the carbon , the reaction rates are influenced by the composition and size of the particle size . therefore , and both in the operation conditions and in the design procedure of the plant , the knowledge of the reactivity and the combustion kinetics of the fuels gives a valuable information for the combustion system of the fuel . considering also that the combustion of the biomass waste differs notably from the combustion of the conventional solid fuels , it is evident that the system used optimally for these ones are not suitable for biomass fuels . another factor to consider is the heterogeneity existing within the different biomass wastes , which does not allow to generalize when it comes down to the convenience of thermochemical treatment systems and general characteristics and dimensions of the equipment involved . therefore , it is essential to carry out a specific thermal analysis of the biomass to be used , in order to predict more accurately how it is going to behave in real systems . the limitations of these methods regarding their generalization have to be taken into account , since they are carried out in controlled laboratory conditions and over small samples in comparison with those ones used in real life . the thermogravimetric analysis ( tga ) is one of the main techniques used for the study of the thermal behaviour of carbonous materials and the kinetics of the thermal decomposition reactions of different solid fuels [ 58 ] , etc . the analysis of the characteristics of the combustion allows obtaining the " burning profile " of the fuel , defined as the representation of weight loss versus temperature in an oxidizing atmosphere . from these results the reactivity can be determined as an indicator of the combustion potential of a carbonous material , . other authors , , indicate that the information obtained from the burning profiles in the tga in the case of fuels like carbon can be used to estimate the behaviour of the combustion in industrial scale . in this work , tga techniques and other characterization tests have been used to evaluate the thermal behaviour in oxidizing atmosphere of different olive wastes , such as olive pit , pulp , residual olive cake and concentrated olive mill waste water ( comww ) . the behavior of the wastes has been compared in terms of emissions of nitrogen oxides , soiling phenomenons , corrosion and ash melting problem . also , it will be shown the thermal behavior of the wastes and its reactivity in an air atmosphere . in addition , the combustion in an industrial plant of the different fractions obtained has been studied in order to assess the viability of this process . the origin of each of these fractions in the plant is the following one : the two - phases olive mill solid waste ( two phases omsw ) comes from the olive mill and , once in the installation , is firstly processed to extract its pits . once the olive pit has been separated , the two - phases omsw is processed in a three - phases continuous system , obtaining wet olive cake and olive mill waste water ( omww ) . then , the lightest fraction , called pulp , is separated from the dry olive cake by a pneumatic system , remaining another fraction consisted of a smaller waste of olive pit and the most solid part of the pulp , called residual olive cake . the omww is taken to a concentration tower in which the residual heat from the drying process can be used for the reduction of the moisture . the characterization of the four fractions analyzed has been carried out : heating value , ultimate analysis , proximate analysis , chemical analysis , chemical composition of ashes and ash melting analysis . c , h , n , s contents in the four fuels were analyzed using a eurovector ea 3000 elemental analyzer and chlorine was determined by the eschka method . the moisture content of the samples was obtained by drying till constant weight in a holelab oven . ash content and volatile matter were determined after slow combustion of the samples in a hobersal hd-230 furnace . cellulose , hemicellulose and lignin were analyzed acording to standards lap-002 , lap-003 , lap-010 and lap-005 . chemical composition of ashes in the four samples was measured using x - ray fluorescence spectrometer philips pw 2400 under standard conditions and ash melting analysis were carried out in an oxidizing atmosphere by a leco af-600 . all the analysis were measured according to the normalized methods astm . for the determination of the combustion profiles of the different wastes , dynamic experiments have been done in air atmosphere in a tg / dta thermal analyzer meter toledo tga / sdta 851 , with horizontal oven and an only arm . the precision for the tga is 0.1 g and for the sdta is 0.005 c . the temperature of the oven in the thermobalance is controlled so that the sample follows the desired profile . it is important to remark that in the case of particles smaller than 1 mm the chemical reaction controls the process , whilst in the case of bigger particles heat and mass transfer phenomenons [ 1316 ] also take part in the control process . taking this into account , samples of olive pit , pulp and residual olive cake have been prepared in a centrifuge mill retsch zm 100 , choosing a sieve that guarantees a sample granulometry lower than 1 mm . to ensure the uniformity of the temperature in the sample , samples between 1030 mg have been chosen in the case of dry waste and 50 mg in the case of the comww . to obtain a noiseless signal therefore , a constant gas flow of 100 ml / min is used to feed the system in all the tests . the most common range of ramp reported by the literature for tga characterization of biomass appears to be 1050 c / min [ 1922 ] , thus 30 c / min was chosen as an average value in order to make this analysis . samples are heating from ambient temperature to 800 c , in order to ensure no significant further changes above this top limit . for each experimental point , the reproducibility was checked by at least a duplicate run . in all the cases the next parameters are determined : initial temperature , corresponding to the point in which the rate of weight loss is > 1%/min after the initial moisture loss peak ; maximum temperature , corresponding to the point in which the weight loss rate of the sample due to the combustion is maximum ( maximum combustion rate ) , and ending temperature corresponding to the point in which the char 's combustion is finished ( ending temperature was extrapolated from the slope of the combustion profile in a section where calcination had not started yet ) . apart from all these parameters mentioned , a detailed analysis of the combustion profile is carried out . tables 1 , 2 and 3 present the results obtained for the four fractions regarding their heating values , ultimate analysis , proximate analysis , chemical analysis , chemical composition of the ashes and their melting temperature . as observed in table 1 , the pulp is the by - product with higher heating value ( hv ) , being the olive pit the one with lower hv . from the ultimate analysis , it is remarkable the presence of high percentages of chlorine in all the wastes , except for the case of the olive pit . this fact could produce soiling phenomenons and the formation of dioxins if the combustion temperatures are low . the admissible percentages of chlorine for the proper use of the biomass are : 0.1% , to avoid corrosion problems and emissions of hcl ; and 0.3 % , to avoid the formation of dioxins and furans . from the results obtained for the different wastes , and except for the case of the olive pit , all the by - products can present problems of corrosion and emissions during their combustion . however , the formation of dioxins and furans is not expected a priori , since the combustion temperature must be higher than 500 c in all the cases ( see table 4 ) . for the rest of wastes , the values obtained are around 2 % , being able the formation of oxides from the nitrogen of the fuel . according to van loo and koppejan , percentages of nitrogen higher than 0.6 % can cause problems related to the emissions of nitrogen oxides . finally , as far as the emissions of co2 are concerned , though the variations are not very significant , the higher h / c ratio in the comww ( see figure 1 ) indicates a lower level of co2 in the gases per unit energy produced . pulp and residual olive cake present a similar chemical composition , showing cellulose and hemicelluloses proportions lower than that found on the olive pits and higher than the value measured on the comww . additionally , the highest amount of lignin can be found on the comww , being followed by pulp , residual olive cake and olive pits , respectively . this result confirmed that the ash free basis heating value of the wastes increases along with the lignin proportion on the sample . from the ultimate analysis , the first value will show the need of using an additional fuel for this by - product or the need of a previous drying process . as for the ash content and its composition , the high presence of water - soluble metals such as sodium or potassium , favours its accumulation in the omww and , later , in the comww . this fact affects notably the ash melting , as can be observed in the analysis of table 2 and in figure 2 , which shows the deformation temperatures , the percentages of ashes and their k2o content . literature reports that the increase of k2o amount on the ashes decreases the melting point . other elements such as silicon can decrease either this temperature , appearing on the ashes as sio2 . despite the melting point of this oxide is around 1450 c , it can form a eutectics mixture with the k2o showing a lower melting point around 800c . taking into account that the comww is also the waste with higher levels of ash , it can be expected that the fusion and ash removal processes for this by - product cause several problems . figures 3 , 4 and 5 show the weight loss velocity with respect to the temperature in oxidizing atmosphere in the case of the olive pit , the pulp and the residual olive cake . the first peak observed corresponds to the moisture loss and happens at temperatures between 70 c and 90 c , depending on the waste . the olive pit presents the maximum values of weight loss rate corresponding to moisture lost , being the waste with higher percentage of moisture and with the lower ash content ( 0.56 % dry basis ) . after the drying process , a mass loss in the sample can come about due to the emission of gases absorbed and the loss of very light volatiles . for temperatures between 180 c and 200 c a sudden mass loss happens in the samples , due to the beginning of the emission of some volatiles and their combustion . in the region of fast combustion the loss velocity becomes slower immediately for temperatures between 300 c and 400 c for the pulp and the residual olive cake . after this point the burning ratio decreases , and after a short period it increases again , more significantly in some wastes than in others , producing continuous mass losses until over 500 c due to the oxidation of the char . for higher temperatures , reductions in the mass can occur , which can be attributed to volatile metal loss and carbonate decomposition . however , as can be observed in the dta curves of figures 3 , 4 and 5 , the absence of increments in the temperature indicates that the combustion of the char has already concluded before the beginning of this last period . table 4 shows the different parameters that characterize the combustion profile of the three solid fractions studied and the comww . the initial temperature is determined from the profile of the samples , obtaining values of 220 c , 183 c and 181 c for the olive pit , the pulp and the residual olive cake , respectively . this shows that the olive pit is the waste that takes longer to react with the temperature , needing an increase of 40 c with respect to other wastes . haykiri - ama presents values of the initial temperature for different biomass wastes , concretely 202c for the sunflower shell , 150 c for the colza seed , 202 c for the pine cone , 150 c for the cotton refuse and 200 c for the olive refuse . garca presents values of the starting temperature between 179 c and 193 c for different samples of dry omsw , very close to the ones obtained for the samples analyzed . the value of the initial temperature for coals with different ranges is between 235 c and 441 c , corresponding the higher values to the carbons with higher carbon percentage in their composition . in the case of the waste studied here this effect does not take place , since the olive pit , the waste with lower carbon content , begins to react at the highest temperature . in the case of the lignocelluloses material , the temperature of degradation and the thermal degradation rate of the hemicelluloses and lignin are lower than the values that can be found on the cellulose . therefore , the initial temperature of degradation is higher in the olive pits , which present a high amount of cellulose in comparison with the pulp , olive cake and comww as the literature reports . in addition , it should be pointed out that a fuel with a higher percentage of ashes and impurities in his composition , presents a lower initial temperature of degradation , this effect takes place in the case of the pits , due to a lower amount of ashes on the wastes . as commented previously , the point of the burning profile in which the maximum weight loss comes about due to the combustion is called peak temperature . the peak temperature for the different samples are 292 c , 267 c and 295 c for the olive pit , the pulp and the residual olive cake , respectively . the pulp is the waste with higher value of dtgmax ( 63.01% min ) followed by the olive pit ( 39.82 % min ) and finally by the residual olive cake ( 22.81 % min ) . from the results corresponding to the analysis of the samples , it is observed that the olive cake is the waste with lower initial temperature and the pulp is the waste with higher maximum weight loss velocity . haykiri - ama determines the values for the maximum combustion velocity of different biomass wastes , that is , 5.5 mg min for the sunflower shell , 2.8 mg min for the colza seed , 5.2 mg min for the pine cone , 3.7 mg min for the cotton refuse and 3.4 mg min for the olive refuse , being the initial mass of the sample 40 mg . in all the cases , haykiri - ama obtains much lower values than those obtained in our study . garca determines the maximum burning velocity of two samples of dry omsw for three different heating ramps . with a ramp of 20 c min , values of 2.6 mg min and 4.3 mg min are obtained for samples with an initial weight of 10 mg , approximately . these values are close to the ones obtained for the olive pit and the residual olive cake , being lower than the ones obtained when the pulp sample gets oxidized . as far as the conventional solid fuels are concerned , benfell et al . obtained maximum burning velocities between 25 % min and 31 % min for different types of carbon with a heating ramp of 15 c min and samples of 5 mg , approximately . this fact shows the higher reactivity of the olive pit , pulp and residual olive cake in comparison with the carbon . taking into account that the reactivity of a fuel can be considered directly proportional to its maximum weight loss velocity and inversely proportional to the value of temperature for which it happens , it is interesting to introduce a parameter relating both magnitudes and giving an average value of the reactivity of the fuel . this parameter can be defined according to equation ( 1 ) and represents the weight loss per unit temperature . this factor confirms that the waste with better combustion properties is the pulp , followed by the olive pit and finally by the residual olive cake . another important parameter to consider is the burning temperature , which allows to obtain a qualitative information regarding the necessary residence time of the fuel that minimizes the unburnts . carpenter shows that , in the case of the carbon and from the tga results , the residence time of the fuel in a large scale installation can be estimated . if the sample has a low value of the burning temperature , the presence of unburnts is reduced , whilst samples with higher values of this temperature are more difficult to burn and will need higher residence times and higher temperatures for their complete combustion . it is necessary to take into account that tga and dtg curves , compared with the real combustion , present lower heating velocity and , therefore , the emission of volatiles happens at a lower temperature . according to su et al . , this fact reduces both the amount of volatile matter that can react with the fuel and the burning potential . this way , the results obtained from the tga will show poorer burning conditions than the ones in a real plant . in the case of the waste analyzed , the burning temperatures obtained are 530 c , 509 c , and 519 c for the olive pit , the pulp and the residual olive cake , respectively . these values are close to each other but slightly lower than in the case of the pulp , which indicates that the residence time of this fuel will be predictably shorter than in the case of the other wastes . in all the cases burning temperatures lower than those for the fossil fuels are obtained . present values between 530 c and 810 c for the coal of low - high rank in tests carried out with a ramp of 25c min . to study the thermal behaviour of the comww , a first test has been carried out in which an original sample with moisture of 73 % is processed by a heating program of 30 c / min . the presence of a big amount of water impedes the analysis of the data obtained , since the moisture loss takes place until high temperatures , hiding the degradation reactions and producing an important thermal demand . for that reason a previous drying program in the thermobalance has been carried out , long enough to guarantee the total dryness of the sample , minimizing the possibility of liberation of other products . this way , it is determined that this objective is fulfilled with a previous heating of the sample during 35 min at 100 c and followed by a ramp of 30 c / min . as can be observed , the mass loss begins when the temperature of the sample increases over 150c , due to the beginning of the liberation of some volatiles and their combustion . in the fast combustion region , a maximum of mass loss occurs . the mass loss velocity becomes lower immediately at temperatures close to 350c and increases slightly , again , between 400c and 500c . alter that point , the burning ratio decreases and , after a short period it increases significantly , obtaining the maximum value of the burning velocity due to the oxidation of the char . the sample presents an initial temperature of 161 c , lower than the one obtained for el olive pit , the pulp and the residual olive cake , with values of 220 c , 183 c , and 181 c , respectively . the peak temperature , temperature with the maximum burning velocity , has a value of 653 c , much higher than for the rest of wastes , with values of 267295 c for the solid olive residues . this temperature takes place in combustion zone of the char , not in the volatiles liberation zone , as happens in the case of the solid wastes . the comww presents a dtgmax value of 10.27 % min , being the velocity for the residual olive cake two times this value , three times higher for the olive pit and more than six times higher in the case of the pulp . the values obtained for this parameter confirms the lower reactivity of this byproduct in comparison with the solid wastes . the average value of reactivity for this waste is 1.11% mink , being 7.05 % mink , 11.66 % mink and 4.01 % mink for the olive pit , the pulp and the residual olive cake , respectively . finally , as far as the burning temperature is concerned , it is important to remark the high value of this parameter in the case of the comww , 743 c , much higher than those obtained in the case of the solid wastes , 507 c , 503 c , and 475 c , for the olive pit , the pulp and the residual olive cake , respectively . this effect can be due to the highest amount of lignin presents on the comww , that increases the char yield and consequently the residence time , and to the percentage and composition of the ashes . . showed that the demineralised samples presented lower burnout temperatures than those of the parent fuels . therefore , the residence time of this by - product in the combustion chamber has to be longer than in the case of the solid wastes and the temperatures for the complete combustion has to be higher . tables 1 , 2 and 3 present the results obtained for the four fractions regarding their heating values , ultimate analysis , proximate analysis , chemical analysis , chemical composition of the ashes and their melting temperature . as observed in table 1 , the pulp is the by - product with higher heating value ( hv ) , being the olive pit the one with lower hv . from the ultimate analysis , it is remarkable the presence of high percentages of chlorine in all the wastes , except for the case of the olive pit . this fact could produce soiling phenomenons and the formation of dioxins if the combustion temperatures are low . the admissible percentages of chlorine for the proper use of the biomass are : 0.1% , to avoid corrosion problems and emissions of hcl ; and 0.3 % , to avoid the formation of dioxins and furans . from the results obtained for the different wastes , and except for the case of the olive pit , all the by - products can present problems of corrosion and emissions during their combustion . however , the formation of dioxins and furans is not expected a priori , since the combustion temperature must be higher than 500 c in all the cases ( see table 4 ) . for the rest of wastes , the values obtained are around 2 % , being able the formation of oxides from the nitrogen of the fuel . according to van loo and koppejan , percentages of nitrogen higher than 0.6 % can cause problems related to the emissions of nitrogen oxides . finally , as far as the emissions of co2 are concerned , though the variations are not very significant , the higher h / c ratio in the comww ( see figure 1 ) indicates a lower level of co2 in the gases per unit energy produced . pulp and residual olive cake present a similar chemical composition , showing cellulose and hemicelluloses proportions lower than that found on the olive pits and higher than the value measured on the comww . additionally , the highest amount of lignin can be found on the comww , being followed by pulp , residual olive cake and olive pits , respectively . this result confirmed that the ash free basis heating value of the wastes increases along with the lignin proportion on the sample . from the ultimate analysis , the first value will show the need of using an additional fuel for this by - product or the need of a previous drying process . as for the ash content and its composition , the high presence of water - soluble metals such as sodium or potassium , favours its accumulation in the omww and , later , in the comww . this fact affects notably the ash melting , as can be observed in the analysis of table 2 and in figure 2 , which shows the deformation temperatures , the percentages of ashes and their k2o content . literature reports that the increase of k2o amount on the ashes decreases the melting point . other elements such as silicon can decrease either this temperature , appearing on the ashes as sio2 . despite the melting point of this oxide is around 1450 c , it can form a eutectics mixture with the k2o showing a lower melting point around 800c . taking into account that the comww is also the waste with higher levels of ash , it can be expected that the fusion and ash removal processes for this by - product cause several problems . figures 3 , 4 and 5 show the weight loss velocity with respect to the temperature in oxidizing atmosphere in the case of the olive pit , the pulp and the residual olive cake . the first peak observed corresponds to the moisture loss and happens at temperatures between 70 c and 90 c , depending on the waste . the olive pit presents the maximum values of weight loss rate corresponding to moisture lost , being the waste with higher percentage of moisture and with the lower ash content ( 0.56 % dry basis ) . after the drying process , a mass loss in the sample can come about due to the emission of gases absorbed and the loss of very light volatiles . for temperatures between 180 c and 200 c a sudden mass loss happens in the samples , due to the beginning of the emission of some volatiles and their combustion . in the region of fast combustion the loss velocity becomes slower immediately for temperatures between 300 c and 400 c for the pulp and the residual olive cake . after this point the burning ratio decreases , and after a short period it increases again , more significantly in some wastes than in others , producing continuous mass losses until over 500 c due to the oxidation of the char . for higher temperatures , reductions in the mass can occur , which can be attributed to volatile metal loss and carbonate decomposition . however , as can be observed in the dta curves of figures 3 , 4 and 5 , the absence of increments in the temperature indicates that the combustion of the char has already concluded before the beginning of this last period . table 4 shows the different parameters that characterize the combustion profile of the three solid fractions studied and the comww . the initial temperature is determined from the profile of the samples , obtaining values of 220 c , 183 c and 181 c for the olive pit , the pulp and the residual olive cake , respectively . this shows that the olive pit is the waste that takes longer to react with the temperature , needing an increase of 40 c with respect to other wastes . haykiri - ama presents values of the initial temperature for different biomass wastes , concretely 202c for the sunflower shell , 150 c for the colza seed , 202 c for the pine cone , 150 c for the cotton refuse and 200 c for the olive refuse . garca presents values of the starting temperature between 179 c and 193 c for different samples of dry omsw , very close to the ones obtained for the samples analyzed . the value of the initial temperature for coals with different ranges is between 235 c and 441 c , corresponding the higher values to the carbons with higher carbon percentage in their composition . in the case of the waste studied here this effect does not take place , since the olive pit , the waste with lower carbon content , begins to react at the highest temperature . in the case of the lignocelluloses material , the temperature of degradation and the thermal degradation rate of the hemicelluloses and lignin are lower than the values that can be found on the cellulose . therefore , the initial temperature of degradation is higher in the olive pits , which present a high amount of cellulose in comparison with the pulp , olive cake and comww as the literature reports . in addition , it should be pointed out that a fuel with a higher percentage of ashes and impurities in his composition , presents a lower initial temperature of degradation , this effect takes place in the case of the pits , due to a lower amount of ashes on the wastes . as commented previously , the point of the burning profile in which the maximum weight loss comes about due to the combustion is called peak temperature . the peak temperature for the different samples are 292 c , 267 c and 295 c for the olive pit , the pulp and the residual olive cake , respectively . the pulp is the waste with higher value of dtgmax ( 63.01% min ) followed by the olive pit ( 39.82 % min ) and finally by the residual olive cake ( 22.81 % min ) . from the results corresponding to the analysis of the samples , it is observed that the olive cake is the waste with lower initial temperature and the pulp is the waste with higher maximum weight loss velocity . haykiri - ama determines the values for the maximum combustion velocity of different biomass wastes , that is , 5.5 mg min for the sunflower shell , 2.8 mg min for the colza seed , 5.2 mg min for the pine cone , 3.7 mg min for the cotton refuse and 3.4 mg min for the olive refuse , being the initial mass of the sample 40 mg . in all the cases , haykiri - ama obtains much lower values than those obtained in our study . garca determines the maximum burning velocity of two samples of dry omsw for three different heating ramps . with a ramp of 20 c min , values of 2.6 mg min and 4.3 mg min are obtained for samples with an initial weight of 10 mg , approximately . these values are close to the ones obtained for the olive pit and the residual olive cake , being lower than the ones obtained when the pulp sample gets oxidized . as far as the conventional solid fuels are concerned , benfell et al . obtained maximum burning velocities between 25 % min and 31 % min for different types of carbon with a heating ramp of 15 c min and samples of 5 mg , approximately . this fact shows the higher reactivity of the olive pit , pulp and residual olive cake in comparison with the carbon . taking into account that the reactivity of a fuel can be considered directly proportional to its maximum weight loss velocity and inversely proportional to the value of temperature for which it happens , it is interesting to introduce a parameter relating both magnitudes and giving an average value of the reactivity of the fuel . this parameter can be defined according to equation ( 1 ) and represents the weight loss per unit temperature . this factor confirms that the waste with better combustion properties is the pulp , followed by the olive pit and finally by the residual olive cake . another important parameter to consider is the burning temperature , which allows to obtain a qualitative information regarding the necessary residence time of the fuel that minimizes the unburnts . carpenter shows that , in the case of the carbon and from the tga results , the residence time of the fuel in a large scale installation can be estimated . if the sample has a low value of the burning temperature , the presence of unburnts is reduced , whilst samples with higher values of this temperature are more difficult to burn and will need higher residence times and higher temperatures for their complete combustion . it is necessary to take into account that tga and dtg curves , compared with the real combustion , present lower heating velocity and , therefore , the emission of volatiles happens at a lower temperature . according to su et al . , this fact reduces both the amount of volatile matter that can react with the fuel and the burning potential . this way , the results obtained from the tga will show poorer burning conditions than the ones in a real plant . in the case of the waste analyzed , the burning temperatures obtained are 530 c , 509 c , and 519 c for the olive pit , the pulp and the residual olive cake , respectively . these values are close to each other but slightly lower than in the case of the pulp , which indicates that the residence time of this fuel will be predictably shorter than in the case of the other wastes . in all the cases burning temperatures lower than those for the fossil fuels present values between 530 c and 810 c for the coal of low - high rank in tests carried out with a ramp of 25c min . to study the thermal behaviour of the comww , a first test has been carried out in which an original sample with moisture of 73 % is processed by a heating program of 30 c / min . the presence of a big amount of water impedes the analysis of the data obtained , since the moisture loss takes place until high temperatures , hiding the degradation reactions and producing an important thermal demand . for that reason a previous drying program in the thermobalance has been carried out , long enough to guarantee the total dryness of the sample , minimizing the possibility of liberation of other products . this way , it is determined that this objective is fulfilled with a previous heating of the sample during 35 min at 100 c and followed by a ramp of 30 c / min . as can be observed , the mass loss begins when the temperature of the sample increases over 150c , due to the beginning of the liberation of some volatiles and their combustion . in the fast combustion region , a maximum of mass loss occurs . the mass loss velocity becomes lower immediately at temperatures close to 350c and increases slightly , again , between 400c and 500c . alter that point , the burning ratio decreases and , after a short period it increases significantly , obtaining the maximum value of the burning velocity due to the oxidation of the char . the sample presents an initial temperature of 161 c , lower than the one obtained for el olive pit , the pulp and the residual olive cake , with values of 220 c , 183 c , and 181 c , respectively . the peak temperature , temperature with the maximum burning velocity , has a value of 653 c , much higher than for the rest of wastes , with values of 267295 c for the solid olive residues . this temperature takes place in combustion zone of the char , not in the volatiles liberation zone , as happens in the case of the solid wastes . the comww presents a dtgmax value of 10.27 % min , being the velocity for the residual olive cake two times this value , three times higher for the olive pit and more than six times higher in the case of the pulp . the values obtained for this parameter confirms the lower reactivity of this byproduct in comparison with the solid wastes . the average value of reactivity for this waste is 1.11% mink , being 7.05 % mink , 11.66 % mink and 4.01 % mink for the olive pit , the pulp and the residual olive cake , respectively . finally , as far as the burning temperature is concerned , it is important to remark the high value of this parameter in the case of the comww , 743 c , much higher than those obtained in the case of the solid wastes , 507 c , 503 c , and 475 c , for the olive pit , the pulp and the residual olive cake , respectively . this effect can be due to the highest amount of lignin presents on the comww , that increases the char yield and consequently the residence time , and to the percentage and composition of the ashes . . showed that the demineralised samples presented lower burnout temperatures than those of the parent fuels . therefore , the residence time of this by - product in the combustion chamber has to be longer than in the case of the solid wastes and the temperatures for the complete combustion has to be higher . according to the results obtained and the analysis of the characterization of the wastes , it can be concluded that : the high moisture of the comww ( around 73% ) makes it inviable to keep the combustion process , being necessary the use of an additional fuel . the percentage of carbon in all the fractions studied is within the normal range for biomass wastes . as for the nitrogen , chlorine , sodium and potassium contents , the values obtained show that , except for the case of the olive pit , problems related to the emissions nitrogen oxides , corrosion and soiling are likely to appear . as far as the behaviour of the ashes is concerned , the comww is the most problematic fraction , since it presents higher amounts of ashes and lower melting point . moreover , taking into account the different combustion profiles of the wastes , it can be concluded that : in the case of the comww , the highest mass loss velocity takes place in the combustion zone of the char , instead of in the volatiles liberation zone , which indicates that the combustion of the comww will be slower than in the case of the other wastes , being also necessary a proper amount of air for the combustion of the char formed . the burning temperatures obtained for the solid fractions are similar to each other . for the comww , the burning temperature increases considerably , liberating the most significant amount of heat in the last period of reaction . therefore , it will be necessary to guarantee appropriate values of temperature and residence times in order to take advantage of its heating value . the reactivity parameters determined show that the waste with better combustion properties is the pulp , followed by the olive pit , the residual olive cake and finally , with much lower index , by the comww . considering the rm parameter as an average value of the reactivity , it can be confirmed that the solid wastes present a rm parameter about 4 to 10 times higher than the value observed on the comww .

the thermogravimetric analysis ( tga ) techniques and concretely the study of the burning profile provide information that can be used to estimate the behaviour of the combustion of carbonous materials . commonly , these techniques have been used for the study of carbons , but are also interesting for the analysis of biomass wastes , due to the different species present on the wastes affect directly to its thermal properties . in this work , techniques of thermal analysis have been applied to compare the behaviour of different wastes coming from olive oil mills . from these results , it is remarkable that the concentrated olive mill waste water ( comww ) presents more unfavourable conditions for its combustion .

1. Introduction 2. Materials and Methods 3. Results and Discussion 3.1. Physical and Chemical Characteristics 3.2. Thermogravimetric analysis of the olive pit, pulp and residual olive cake 3.3. Thermogravimetric analysis of the COMWW 4. Conclusions

in spain , the thermal processes are the option most widely used for the treatment of the waste coming from the industries for the transformation of agricultural products , specially interesting are the wastes from the olive oil mills , owing to the big amount produced and its elevated heating value between 20 kj / kg and 23 kj / kg dry basis . considering also that the combustion of the biomass waste differs notably from the combustion of the conventional solid fuels , it is evident that the system used optimally for these ones are not suitable for biomass fuels . therefore , it is essential to carry out a specific thermal analysis of the biomass to be used , in order to predict more accurately how it is going to behave in real systems . the thermogravimetric analysis ( tga ) is one of the main techniques used for the study of the thermal behaviour of carbonous materials and the kinetics of the thermal decomposition reactions of different solid fuels [ 58 ] , etc . other authors , , indicate that the information obtained from the burning profiles in the tga in the case of fuels like carbon can be used to estimate the behaviour of the combustion in industrial scale . in this work , tga techniques and other characterization tests have been used to evaluate the thermal behaviour in oxidizing atmosphere of different olive wastes , such as olive pit , pulp , residual olive cake and concentrated olive mill waste water ( comww ) . for the determination of the combustion profiles of the different wastes , dynamic experiments have been done in air atmosphere in a tg / dta thermal analyzer meter toledo tga / sdta 851 , with horizontal oven and an only arm . from the ultimate analysis , it is remarkable the presence of high percentages of chlorine in all the wastes , except for the case of the olive pit . in addition , it should be pointed out that a fuel with a higher percentage of ashes and impurities in his composition , presents a lower initial temperature of degradation , this effect takes place in the case of the pits , due to a lower amount of ashes on the wastes . as commented previously , the point of the burning profile in which the maximum weight loss comes about due to the combustion is called peak temperature . from the results corresponding to the analysis of the samples , it is observed that the olive cake is the waste with lower initial temperature and the pulp is the waste with higher maximum weight loss velocity . taking into account that the reactivity of a fuel can be considered directly proportional to its maximum weight loss velocity and inversely proportional to the value of temperature for which it happens , it is interesting to introduce a parameter relating both magnitudes and giving an average value of the reactivity of the fuel . finally , as far as the burning temperature is concerned , it is important to remark the high value of this parameter in the case of the comww , 743 c , much higher than those obtained in the case of the solid wastes , 507 c , 503 c , and 475 c , for the olive pit , the pulp and the residual olive cake , respectively . from the ultimate analysis , it is remarkable the presence of high percentages of chlorine in all the wastes , except for the case of the olive pit . in addition , it should be pointed out that a fuel with a higher percentage of ashes and impurities in his composition , presents a lower initial temperature of degradation , this effect takes place in the case of the pits , due to a lower amount of ashes on the wastes . as commented previously , the point of the burning profile in which the maximum weight loss comes about due to the combustion is called peak temperature . from the results corresponding to the analysis of the samples , it is observed that the olive cake is the waste with lower initial temperature and the pulp is the waste with higher maximum weight loss velocity . taking into account that the reactivity of a fuel can be considered directly proportional to its maximum weight loss velocity and inversely proportional to the value of temperature for which it happens , it is interesting to introduce a parameter relating both magnitudes and giving an average value of the reactivity of the fuel . finally , as far as the burning temperature is concerned , it is important to remark the high value of this parameter in the case of the comww , 743 c , much higher than those obtained in the case of the solid wastes , 507 c , 503 c , and 475 c , for the olive pit , the pulp and the residual olive cake , respectively . according to the results obtained and the analysis of the characterization of the wastes , it can be concluded that : the high moisture of the comww ( around 73% ) makes it inviable to keep the combustion process , being necessary the use of an additional fuel . moreover , taking into account the different combustion profiles of the wastes , it can be concluded that : in the case of the comww , the highest mass loss velocity takes place in the combustion zone of the char , instead of in the volatiles liberation zone , which indicates that the combustion of the comww will be slower than in the case of the other wastes , being also necessary a proper amount of air for the combustion of the char formed .

dna polymerases make and repair dna , thereby controlling both the stability and variability of genetic information . whereas both replication and repair employ dna polymerases that maximize faithful dna synthesis , other tasks such as lesion bypass come at the price of lower fidelity and are performed by the so - called sloppy dna polymerases ( goodman and tippin , 2000 ) . in humans , 16 different dna - synthesizing enzymes are dedicated to replication , repair , damage tolerance , and variability of nuclear dna ( reviewed in hbscher et al . , 2002 ) . in stark contrast , only one dna polymerase has been described in mitochondria ( bolden et al . , 1977 ) , dna polymerase gamma ( pol ) , which has long been presumed to mediate every dna synthesis reaction related to the replication and repair of mitochondrial dna ( mtdna ) . none of the known 17 human dna polymerases can initiate dna synthesis , and , because rna polymerases can polymerize ribonucleotides from a single nucleoside 5-triphosphate ( ntp ) , rna is widely used to prime dna synthesis . in most cases , such rna primers are produced by specialized polymerases or primases ( frick and richardson , 2001 ) . primases can be divided in two evolutionarily unrelated families : dnag - like ( bacteria ) and aep - like ( archaea and eukaryotes ) ( aravind et al . , 1998 ; iyer et al . the human primase operating at nuclear dna is a heterodimer ( pri1 + pri2 ) wherein pri1 is the small catalytic subunit belonging to the aep family . during nuclear dna replication , the primase interacts with pol , forming a very stable complex ( pol-primase ) that triggers the initiation of dna synthesis . the few primers made at ori sequences are further elongated by the leading - strand dna polymerase ( pol ) ; conversely , other primers are synthesized repeatedly , much like those used to initiate the synthesis of okazaki fragments in the antiparallel lagging strand . exceptionally , rna primers can be generated by the transcription activity of rna polymerases , as in plasmid cole1 ( itoh and tomizawa , 1980 ) and during the replication of mtdna ( chang and clayton , 1985 ; fust et al . , 2010 ) . the replication of mtdna ( a small circle of about 17 kb in human cells ) is a complex process involving multiple mechanisms ( holt and reyes , 2012 ) . at the replication fork , the mitochondrial dna helicase twinkle opens the duplex , and the mitochondrial single - stranded binding protein ( mtssb ) stabilizes the unwound dna ( falkenberg et al . , 2007 ) , although the extent of coating of the lagging - strand template with mtssb may be minimal because of the incorporation of processed transcripts ( reyes et al . , 2013 ) . mitochondrial rna polymerase ( polrmt ) primes at one major initiation site on each strand , orih and oril ( fust et al . , 2010 and the references therein ) , and primer elongation is performed by pol ( falkenberg et al . , 2007 ) . however , minor replication products of coupled leading- and lagging - strand mtdna synthesis ( holt et al . , 2000 ) would need frequent priming on the lagging strand . as long ago as 1985 , a primase activity distinct from polrmt was detected in human mitochondria , although the protein responsible has not yet been identified ( wong and clayton , 1985 ) . here , ccdc111 , a putative primase based on in silico predictions ( iyer et al . , 2005 ) , is shown to have both dna primase and dna polymerase activities ( and , thus , is renamed primpol ) and a striking capacity to tolerate dna damage . primpol was shown to be present at both nuclear and mitochondrial dna compartments but is absent in mitochondria derived from a mouse primpol knockout ( ko ) model . rnai experiments in human cells and analysis of primpol ko mouse embryonic fibroblasts ( mefs ) indicated that primpol plays a key role in mtdna synthesis . a plausible hypothesis is that this primase / polymerase facilitates replication restart after programmed or damage - induced fork arrest . human gene ccdc111 ( also known as flj33167 ) , which is located on chromosome 4q35.1 and codes for coiled coil domain - containing ( ccdc111 ) protein , was predicted to be a member of the aep family ( iyer et al . , 2005 ) . ccdc111 contains the active - site motifs of aep primases and a putative zn finger similar to that of herpesvirus ul52 primase and other aep - like enzymes ( figures 1a and 1b ; figure s1 available online ) . iterative blast searches with human ccdc111 and putative homologs indicated the presence of ccdc111 in a broad range of unicellular and multicellular eukaryotes , including animals , plants , unicellular microalgae , protists , choanoflagellates , fungi , and picoeukaryotes such as ostreococcus ( the smallest known free - living eukaryote with a single chloroplast and a single mitochondrion ) . alignment of putative ccdc111 homologs ( figure s1 ) revealed 14 conserved regions , including the zn finger domain and the motifs a , b , and c shared with aep primases , nonhomologous end - joining primases , primpols , and herpes and poxvirus - like primases ( figures 1a , 1b , and s1 ) . all members of the ccdc111 family have the motif a variant dxe ( figures 1b and s1 ) instead of the dxd motif present in most members of the aep superfamily . the dxe variant is also found in the primpol of plasmid prn1 from sulfolobus islandicus , which has both primase and dna polymerase activities ( lipps et al . , 2003 ) . in agreement with in silico predictions ( iyer et al . , 2005 ) , purified human ccdc111 ( figure s2a ) displayed rna primase activity on a single - stranded circular dna template ( m13 ssdna ) ( figure 1c , left ) . interestingly , the protein was dependent on manganese for rna synthesis , yielding remarkably long primers ( > 100 nt ) that were not generated in the presence of magnesium ( figures s2b and s3a , left ) . strikingly , a similar primase activity was observed when ntps were replaced by deoxynucleoside 5-triphosphates ( dntps ) ( figures 1c and s3a , right ) . in general , primases make rna primers , but the aep - related primases of archea and some bacteria are exceptions to this rule ( lao - sirieix et al . this unusual rna and dna primase activity is not present in escherichia coli and was demonstrated to be inherent to human ccdc111 by ablating the activity with a double mutation ( axa ) in the two potential metal ligands ( asp and glu , motif dxe ) at the predicted active site ( figure 1c ) , which was in agreement with enzymatic and structural studies of the primpol from plasmid prn1 ( lipps et al . , 2004 ) . next , we evaluated the primase activity on a template oligonucleotide in which a potential recognition sequence ( gtcc ) was flanked by thymine residues ( cavanaugh and kuchta , 2009 ) . such a tract of pyrimidines is the preferred template context for several viral , prokaryotic , and eukaryotic rna primases ( frick and richardson , 2001 ) . again , mn - activated synthesis of dna primers and their extensive elongation were observed with purified human ccdc111 ( figure s2c ) . ccdc111 primase recognized the 3 gtcc-5 sequence , preferentially forming initiating dinucleotides 5 a - dg-3 or 5 da - dg-3 ( figures 1d and 1e ) . there was no difference in the efficiency of initiation when providing either a ribo- or a deoxynucleotide at the 5position ( figure 1d ) . again , mutant axa was inactive in this assay , supporting the intrinsic nature of the primase activity attributable to ccdc111 protein . in respect to the second nucleotide , ccdc111 displayed a marked preference for a dntp ( figure 1e , left ) . mn - activated formation of a ribo - deoxy - initiating dimer ( occurring at mn concentrations as low as 50 m ; figure s3b ) is a hallmark of ccdc111 , given that several other dna synthesizing enzymes were unable to catalyze this reaction , and a conventional primase such as bacillus subtilis dnag displayed only a modest activity ( figure 1e , right ) . unlike eukaryotic primases , archeal primases are able to carry out the initiation and extension of both rna and dna chains of up to 1 or 7 kb , respectively ( lao - sirieix and bell , 2004 ; lao - sirieix et al . , 2005 ; hence , these enzymes are both primases and polymerases , or primpols ( lipps et al . , 2003 ) . therefore , we tested whether , in addition to its dna and rna primase activity , ccdc111 possessed dna - dependent polymerase activity . ccdc111 was able to extend a dna primer hybridized to m13 ssdna by polymerizing dntps ( figure 2a ) . again , mn was the preferred metal ( figure 2a , compare panels 1 and 2 ) , allowing the nonprocessive elongation of dna chains up to 5 kb ( data not shown ) . the enzyme also utilized ntps , albeit with a much lower efficiency ( figure 2a , panel 3 ) . as with the primase activity , the dna polymerase activity was abolished in the axa mutant ( figure 2a , panel 4 ) . hence , the primase and polymerase activities of ccdc111 ( compared in figure 2b as a function of enzyme concentration ) appear to share the same active site . thus , in light of its identified activities , we propose that ccdc111 should be renamed primpol . primpol efficiently catalyzed dna polymerization within a wide range of mn concentrations ( figure s3c ) . even at the lowest mn concentration tested ( 10 m ) , primpol was more efficient than using a physiological concentration of mg ( 10 mm ) . thus , mn is a more than 1,000-fold better metal activator than mg , suggesting that it is likely to be the physiological cofactor of primpol . a similar behavior has been described for human pol ( blanca et al . , 2003 ) . independent of the metal cation , primpol behaved as a dna - instructed dna polymerase , preferentially inserting the complementary nucleotide dictated by the first available templating base ( figure 2c ) . primpol provided a 1,000-fold bias toward correct watson - crick base pairs when extending a correctly - paired primer terminus ( figure 2d , top ) . primpol could not excise a mismatched primer ( figure 2d , bottom ) , demonstrating the absence of a proofreading 3-5 exonuclease activity . this is in agreement with the lack of exoi , exoii , and exoiii consensus motifs , which form an evolutionarily conserved 3-5exonuclease active site in several dna polymerase families ( bernad et al . , 1989 ) . however , the extension of a mismatched primer terminus by primpol is about 1,000-fold less efficient than the extension of a correctly paired primer terminus ( figure 2d ) . given that primpol is a dna primase / polymerase ( figures 1c1e , 2 , s2 , and s3 ) , it should logically operate in the nucleus and/or mitochondrion . biochemical fractionation of hela cells revealed that primpol was distributed between the cytosol ( 47% ) , mitochondria ( 34% ) , and nuclear ( 19% ) compartments ( figure 3a ) . in order to determine whether the mitochondrial - associated primpol was inside the organelles , mitochondria from human embryonic kidney ( hek ) 293 t cells were treated with trypsin and exposed to hypotonic buffer or digitonin ( figure 3b ) . as expected , a mitochondrial protein located in the outer membrane ( tom20 ) proved sensitive to trypsin , as did the portion of an inner membrane protein facing the intermembrane space ( tim23 ) after rupturing of the outer membrane ( figure 3b ) , whereas matrix proteins ( tfam and hsp60 ) and primpol were resistant to trypsin in all cases ( figure 3b ) . hence , the fraction of primpol associated with mitochondria is inferred to be in the matrix . fractionation of detergent - solubilized mitochondria of hek 293 t cells on an iodixanol gradient ( ig ) separated the majority of primpol and rnase - h1 from mtdna and mtdna maintenance proteins such as twinkle and tfam ( figure 3c ) . formaldehyde crosslinking increased the amount of primpol and rnase - h1 cofractionating with mtdna but not other mitochondrial proteins , such as vdac1 or etfb ( figure 3d ) . these data suggest that primpol , like rnase - h1 , interacts with mtdna transiently and that this interaction may be disrupted by the conditions used to lyse mitochondria . importantly , in the absence of crosslinking , ig sedimentation completely separated primpol from the mitochondrial rna polymerase polrmt ( figure 4a ) , thereby enabling the activities of the two enzymes to be assessed independently . when the different fractions from the igs were subjected to a dna primase assay with the use of oligo dt25 as template , those fractions enriched in mtdna ( 1012 ) yielded a mixed pattern of products ( figure 4a ) that can be attributed to the activities of pol alone or in conjunction with polrmt , terminal transferase(s ) , and poly - a polymerase ; in contrast , fractions enriched in primpol ( 1416 ) displayed a precise pattern of oligonucleotide products of 212 nt ( figure 4a , filled arrowhead , see also the zoomed - in area in figure 4a ) , whose relative abundance correlated with the amount of protein detected by immunoblotting and that could be further extended by klenow dna polymerase ( figure 4a , right ) . to establish that primpol was responsible for the observed dna primase activity , we subjected mitochondrial fractions from mefs possessing ( + /+ ) or lacking ( / ) the primpol gene ( figure s4 ) to the dna primase assay . in primpol mef mitochondria , short dna products of 212 nt were detected in gradient fractions 15 and 16 ( figure 4b ) ; i.e. , the portion of the gradient where human primpol migrates ( figure 4a ) . no such products were detected in mitochondria from primpol cells ( figure 4c ; empty arrowhead , compare the zoomed - in areas in figures 4b and 4c ) . in addition , selected mitochondrial fractions from hek 293 t cells were tested for the primpol - specific primase activity with oligo d(t20gtcct36 ) as a template , atp , and dgtp ( see the scheme in figure 4d ) . fraction 15 of the ig ( where primpol is concentrated ) displayed the same initiation specificity and preference for mn as purified primpol , yielding the same products in the same ratios ( figure 4d ) , whereas ig fraction 11 ( containing pol and polrmt ) lacked such an activity . by virtue of its primase activity , primpol might facilitate dna replication by restarting dna synthesis downstream of pauses or fork arrest due to damaged dna , as has been shown to occur in bacteria , yeast , and human cells ( heller and marians , 2006 ; lopes et al . on the other hand , the distributive pattern of dna polymerization and the lack of a proofreading exonuclease suggest that primpol might also perform translesion synthesis ( tls ) in order to facilitate elongation across a damaged template . considering its presence in mitochondria , tls by primpol was tested on synthetic templates containing either an oxidized form of guanine ( 8-oxoguanine or 8oxog ) or an abasic ( ap ) site , given that these are the most frequent forms of damage occurring in mtdna as a consequence of oxidative stress ( berquist and wilson , 2012 ) . as a control , and as previously documented ( graziewicz et al . , 2007 ) , pol dealt poorly with an 8oxog lesion in the template ( figure s5a ) . in contrast , primpol gave rise to similar elongation products with both damaged and undamaged templates ( figure 5a ) , indicating that it was not hindered by the presence of the 8oxog lesion . by using an oligonucleotide whose primer terminus was juxtaposed to the lesion ( standing start conditions ) , the efficiency and fidelity of nucleotide insertion opposite the 8oxog site was studied ( figure 5b ) . in agreement with an efficient tls capacity , the error - free insertion of dc opposite 8oxog by primpol paralleled that opposite an undamaged dg template , and the misinsertion of da opposite 8oxog was slightly more efficient ( 1.5-fold ) . interestingly , primpol could also use cytidine 5-triphosphate ( ctp ) or atp to bypass 8oxog , although , in this case , the error - free insertion ( figure 5c ) was 10-fold more efficient than the error - prone one ( figure 5a ) . mitochondrial pol was unable to bypass ap sites ( figure s5b ) , given that its 3-5 exonuclease favors primer degradation near the site of the lesion . conversely , human primpol efficiently polymerized across the ap site ( indicated with an x in figure 5c ) , although the accumulation of the + 3 elongation product indicated some delay at this point . primpol continued polymerization beyond the ap site in 80% of the molecules , which contrasts with the null bypass observed with pol. interestingly , the size of the longest elongation product ( + 16 ) was 1 nt shorter than that obtained on an equivalent template with no damage ( + 17 ) , suggesting that primpol does not insert a nucleotide opposite the ap site but skips the lesion , copying the next template base available ( figures 5c and 5d ) . moreover , the size of the main ( + 11 ) and other minor elongated products ( + 14 ) suggested that primer extension was occurring by a different mechanism . strikingly , the ap site was flanked by a 4 nt ( ctac ) repeat ( gray boxes in figure 5d ) . given the difficulty to copy the ap site , the microhomology provides an opportunity for a partially extended ( + 3 ) primer terminus to be realigned ahead of the lesion . further elongation of the relocated primer terminus would explain the most abundant + 11 product obtained ( see figure 5d for additional details ) . this peculiar capacity of primpol to bypass a lesion in the template probably reflects its features as a primase ( avidity for ssdna regions and flexibility to accept very limited primers [ even a single nucleotide ] ) . thus , the marked ability of primpol to bypass oxidative stress dna lesions suggests that its dna polymerase activity may have an important role in dna damage tolerance during the replication of mtdna . in a first approach , we measured deoxynucleotide polymerization using circular m13 ssdna as a template , an assay which strictly requires a primase activity in order to allow the start of dna synthesis ( figure 5e ) ; by only providing primpol , some incorporation was observed ( figure 5e , lanes 2 and 5 ) , but incorporation was not observed when pol was independently provided ( figure 5e , lanes 3 and 6 ) . the simultaneous presence of both enzymes had a synergistic effect ( figure 5e , lanes 4 and 7 ) , particularly when a physiological concentration of gtp ( 100 m ) was provided in order to maximize the primase activity of primpol ( figure 5e , lane 7 ) . moreover , under these optimized conditions ( figure 5f ) , much lower amounts of primpol ( 50 nm ; figure 5e , lane 5 ) or a very low concentration of manganese ions ( 10 m ; figure 5e , lane 9 ) were sufficient to detect the synergistic effect of primpol and pol , strongly suggesting their functional coupling . very similar results were obtained when the synergy of primpol and pol was evaluated ( figure 5 g ) . these experiments demonstrate that , during restart of dna synthesis , the dna primers made by primpol ( and most likely its tls products ) can be efficiently extended by the mitochondrial and nuclear replicative polymerases pol and pol , respectively ( figure 5h ) . the presence of primpol in mitochondria suggested that it might play a role in mtdna replication and maintenance ; therefore , we investigated the effect of gene - silencing primpol on mtdna metabolism . primpol small interfering rna ( sirna ) in human cells caused mtdna levels to decrease to half those of controls ( figure 6a ) , similar to twinkle sirna ( tyynismaa et al . , 2004 ) , and prevented the recovery of mtdna copy number after transient drug - induced mtdna depletion ( figure 6b ) . both results suggest that mtdna replication is impaired when there is an abrupt loss of primpol . we also analyzed mtdna replication intermediates , given that the perturbation of dna replication in mitochondria is typically associated with changes in their abundance and properties ( holt et al . although rna - containing intermediates ( yasukawa et al . , 2006 ) increased after 48 hr of primpol sirna , they returned to normal levels at later time points ( figures s6a and s6b ) . because mtdna copy number was decreasing between 48 and 96 hr of primpol gene silencing , we inferred that replication was not progressing normally . to determine the extent of mtdna replication after 72 hr of primpol gene silencing , we analyzed brdu incorporation into mtdna in double - stranded rna ( dsrna)-transfected cells after preincubation with aphidicolin , an inhibitor of nuclear dna replication . almost all the control cells ( 96% ) incorporated brdu into mtdna after 24 hr of labeling , whereas the figure was 13% for primpol - depleted cells ( p < 0.0001 ) ( figure 6c ) . hence , primpol gene silencing in human cultured cells caused a profound arrest of mtdna synthesis . notwithstanding these results , the contribution of primpol to mtdna maintenance is redundant , given that it proved possible to derive a viable primpol mouse ( figure s4 ) . although the mouse has adapted to the absence of primpol , mtdna replication was adversely affected in cells derived from the primpol ko mouse , given that the rate of restoring mtdna copy number after drug - induced transient depletion was 2.3-fold lower than in mouse cells with primpol ( figure 6d ) . human ccdc111 ( or primpol ) possesses dna and rna primase and dna - dependent dna and rna polymerase activities ( figures 1 , 2 , s2 , and s3 ) , demonstrating its functional relationship to archeal primpol enzymes and its role as a unique human enzyme capable of de novo dna synthesis solely with dntps . another remarkable feature of primpol is its capacity to tolerate lesions such as 8oxog and ap sites in dna . some primpol is present in the nucleus , but a larger fraction is located inside mitochondria . primpol gene silencing in human cultured cells has multiple adverse affects on mtdna , and mouse cells lacking primpol display inefficient mtdna replication . however , given that a mouse lacking the gene is viable , primpol is not essential for mtdna maintenance . polrmt , and not primpol , is believed to provide the primers for leading - strand mtdna synthesis at the heavy strand origin of replication in the major noncoding region and at the major site of second strand mtdna synthesis known as oril ( falkenberg et al . , 2007 ) , and this is compatible with primpol being a nonessential gene . instead , primpol might participate in priming at disparate sites when mtdna replication pauses or stalls due to damaged dna . in respect of damaged dna , primpol , as a tls dna polymerase ( figure 7b ) , could assist pol in reading template lesions ( e.g. , 8oxog ) , and it has the potential to bypass unreadable lesions ( e.g. , ap sites ) by realigning the stalled primer terminus to an alternative template position ahead of the lesion ( figure 7c ) . alternatively or additionally , primpol could exploit both its dna primase and dna polymerase activities in order to facilitate replication fork progression by acting as a specific tls primase in order to reinitiate replication at pause sites or downstream of lesions that block continuous synthesis by replicative dna polymerases ( figure 7d ) . primpol would be very well suited for this task , given that using deoxynucleotides for priming would leave little or no rna to be further processed . hence , the properties and behavior of primpol fit well with the proposed roles in mtdna metabolism . finally , primpol might contribute to multipriming events ( figure 7a ) occurring on the lagging strand during coupled leading- and lagging - strand mtdna replication ( holt et al . it has only recently been recognized that leading - strand dna synthesis is not always continuous . genotoxic damage has been linked to discontinuities in leading - strand synthesis that entail skipping the obstacle , resuming fork progression , and leaving a gap behind ( langston and odonnell , 2006 ; lopes et al . , 2006 ; heller and marians , 2006 ; yeeles and marians , 2011 ) . daughter - strand gap formation after dna damage induction has been detected in yeast and mammals , suggesting that repriming is a universal mechanism for bypassing dna lesions ( meneghini , 1976 ; prakash , 1981 ) . in this regard , we believe that primpol 's primase may play a direct role in replication restart during nuclear dna replication , further supported by the functional coupling shown here between primpol and human pol. we have recently found that primpol is required for repriming synthesis at stalled replication forks in the nucleus and its downregulation in cells produced persistent replication stress and genome instability ( s.m . , s. rodrguez - acebes , m.i.m .- j . , s.g .- g . , e.s.c . , l.b . , and j.m . these findings give additional support to our hypothesis of a similar function for primpol in mitochondria , given that replicative stress is constitutive during mtdna replication , most likely due to a highly oxidative environment . in summary , we identify and characterize a dna primase / polymerase in human cells endowed with the unique ability to initiate synthesis using dntps and a remarkable capacity for translesion synthesis . our initial analysis of its function in mitochondria suggests that primpol plays a key role in dna metabolism , pointing to a greater level of complexity in mtdna replication than previously recognized and implying that pol is not the sole dna polymerase in mammalian mitochondria . the presence of primpol in human mitochondria suggests that it represents an ancestral cellular response to genotoxic insults , including ap sites and oxidized bases , contributing to tolerance of the environmental damage inside mitochondria . now , the challenge is to tease out its precise contribution to dna damage tolerance in the two dna - containing compartments of the cell . the generation of primpol mice is described in detail in the supplemental information . all experiments with mice were performed according to spanish and european regulations for the use and treatment of experimental animals and with the approval of the ethics committee of the centro de biologa molecular severo ochoa . the reaction ( 20 l ) was carried out in buffer r ( 50 mm tris - hcl [ ph 7.5 ] , 75 mm nacl , 1 mm mncl2 , 1 mm dithiothreitol , 2.5% glycerol , and 0.1mg / ml bsa ) and [ -p]ctp ( 16 nm ; 3,000 ci / mmol ) + atp , gtp , and utp ( 100 m ) or [ -p]dctp ( 16 nm ; 3,000 ci / mmol ) + datp , dgtp , and dttp ( 100 m ) in the presence of primpol ( 400 nm ) wild - type ( wt ) or mutant axa . various concentrations of mgcl2 or mncl2 were used to define the range of metal activation . after 60 min at 30c , reactions were stopped by the addition of formamide loading buffer ( 10 mm edta , 95% v / v formamide , and 0.3% w / v xylen - cyanol ) and loaded in 8 m urea - containing 20% polyacrylamide sequencing gels . after electrophoresis , de novo synthesized polynucleotides were detected by autoradiography . to test the productive coupling of primpol with pol and pol , a similar assay was conducted in the presence of both mgcl2 and mncl2 along with 16 nm [ -p]dgtp and 100 m da , dt , and dctp . when indicated , the nucleotide mix was supplemented with 1 m dgtp or 100 m gtp . human pol was kindly provided by zachary f. pursell ( tulane university school of medicine ) . after incubation for 30 min at 37c , de novo synthesized products ( priming + elongation ) , associated to the m13 ssdna template , were detected by autoradiography after electrophoresis on neutral agarose gels . as an alternative to m13 dna , 60-mer oligonucleotide 5-t36cctgt20 - 3 or a 29-mer 5-t15cctgt10 - 3 , both containing a putative herpes virus priming initiation site ( cavanaugh and kuchta , 2009 ) , were used as templates . p]-labeled oligonucleotide primer ( 5-gttttcccagtcacgac-3 ) was hybridized to m13 ssdna . the reaction mixture ( 20 l ) contained buffer r , 1 mm mncl2 or 5 mm mgcl2 , 1.5 nm primed m13 ssdna , 1 m dntp or ntps , and 400 nm purified primpol , wt , or axa mutant . after the indicated incubation time at 30c , reactions were stopped and analyzed as described above . when indicated , alternative templates were prepared by hybridization of a 5[p]-labeled 15-mer oligonucleotide primer ( 5-gatcacagtgagtac-3 ) to a 28-mer oligonucleotide template ( 5-agaagtgtatct(x)gtactcactgtgatc-3 where x stands for any of the four bases ) . the reaction mixture ( 20 l ) contained buffer r , 2.5 nm [ -p]-labeled template / primer dna , the specified dntp at the concentration indicated , and purified human primpol ( 200 nm ) . when indicated , primpol was used at different concentrations . also when indicated , various concentrations of either mgcl2 or mncl2 were used to define the range of metal activation . after 60 min of incubation at 30c , reactions were stopped and analyzed as described above . elongation of a 3 terminally mismatched primer ( 16-mer , 5-gatcacagtgagtacc-3 ) hybridized to a 22-mer template ( 5-tctatcgtactcactgtgatc-3 ) was evaluated under the same conditions . for running start experiments , a 5 [ p]-labeled 12-mer oligonucleotide ( 5-gatcacagtgag-3 ) was hybridized to either the control ( undamaged ) 28-mer oligonucleotide ( 5-agaagtgtatctggtactcactgtgatc-3 ) template or an oligonucleotide template of the same sequence with the g in 13 position ( bold ) substituted with 8oxog ( obtained from eurogentec ) . for standing start experiments designed to evaluate the nucleotide inserted opposite 8oxog , a 5[p]-labeled 15-mer oligonucleotide ( 5-ctgcagctgatgcgc-3 ) was hybridized to either the control ( undamaged ) 34-mer oligonucleotide ( 5-gtacccggggatccgtacggcgcatcagctgcag-3 ) template or an oligonucleotide template of the same sequence with the g in 19 position ( bold ) substituted with 8oxog ( eurogentec ) . alternatively , a 5 [ p]-labeled 13-mer oligonucleotide primer ( 5-cactgactgtatg-3 ) was hybridized to either control ( undamaged ) 30-mer oligonucleotide template ( 5-ctcgtcagcatcttcatcatacagtcagtg-3 ) or damaged templates ( 30- or 31-mer ) containing an ap site ( 5-ctcgtcagcatct(ap)catcatacagtcagtg-3 ) . the synthetic ap nucleotide was obtained from linktech and used to customize oligonucleotide synthesis . the reaction mixture ( 20 l ) contained buffer r , 2.5 nm template / primer dna , dntps or ntps at the indicated concentration , and either purified human primpol ( 200 nm ) or human pol ( 20 nm , kindly provided by maria falkenberg s lab ) . in the latter case after incubation for either 60 min at 30c ( primpol ) or 15 min at 37c ( pol ) , primer extension products were analyzed as described above . hela cells were lysed and fractionated with a mitosciences cell fractionation kit ( ab109719 ) , which allowed the isolation of mitochondrial , cytoplasmic , and nuclear fractions . two different markers were used to assess the purity of each fraction : cytoplasm ( mek2 and -tubulin ) , mitochondria ( atp synthase and hsp60 ) , and nucleus ( ctcf and histone h3 ) . subcellular fractionation of hek 293 t cells was performed as described previously ( reyes et al . , 2011 ) , and sucrose - gradient purified mitochondria were left untreated or subjected to hypotonic or digitonin treatment ( ohsato et al . , 2002 ) followed by incubation with 100 g / ml trypsin ( 30 min at room temperature [ rt ] ) and heat inactivation of trypsin at 95c for 5 min in 0.1% sds . for ig , sucrose - gradient - purified mitochondria ( 2 mg / ml ) were treated with 100 g / ml of trypsin at rt for 30 min . n - dodecyl - - maltoside and centrifuged ( 10 min at 1,000 gmax ) ; the supernatant was loaded on a 20%42.5% ig and centrifuged at 100,000 gmax for 14 hr . in some cases , mitochondria were crosslinked with 1% formaldehyde for 30 min at rt prior to lysis . when indicated , hek 293 t cells were treated with 100 ng / ml ethidium bromide for 72 hr or exposed to uv light ( 20 j / m ) and allowed to recover for 18 hr prior to mitochondrial extraction and further ig fractionation . nucleic acid was extracted from a portion of each fraction of the gradient and after southern blotting and hybridized to a radiolabeled probe . a complete list of the antibodies used in this study can be found in the supplemental information . for sirna experiments , hek 293 t and human osteosarcoma ( hos ) cells were transfected with lipofectamine rnaimax ( invitrogen ) and 10 nm stealth rna as described previously ( reyes et al . , mitochondrial dna copy number was estimated by comparing the abundance of the mitochondrial cox2 gene to that of the nuclear app gene , as described previously ( tyynismaa et al . , 2004 ) . parental or primpol - silenced hos cells were incubated with 20 m aphidicolin for 3 hr in order to block nuclear dna replication before the addition of brdu ( 1:1,000 ; roche ) . after 24 hr , brdu - labeled cells were stained with 60 nm mitotracker orange ( invitrogen ) , fixed , and sequentially incubated with anti - brdu and anti - mouse igg fluorescein according to the manufacturer s indications ( roche ) . slides were mounted in dabco ( air products and chemicals ) supplemented with dapi ( 200 ng / ml ) and visualized in a zeiss lsm 510 confocal microscope .

summarywe describe a second primase in human cells , primpol , which has the ability to start dna chains with deoxynucleotides unlike regular primases , which use exclusively ribonucleotides . moreover , primpol is also a dna polymerase tailored to bypass the most common oxidative lesions in dna , such as abasic sites and 8-oxoguanine . subcellular fractionation and immunodetection studies indicated that primpol is present in both nuclear and mitochondrial dna compartments . primpol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from primpol knockout mice . primpol gene silencing or ablation in human and mouse cells impaired mitochondrial dna replication . on the basis of the synergy observed with replicative dna polymerases pol and pol , primpol is proposed to facilitate replication fork progression by acting as a translesion dna polymerase or as a specific dna primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear dna replication .

Introduction Results Discussion Experimental Procedures Author Contributions

, 1977 ) , dna polymerase gamma ( pol ) , which has long been presumed to mediate every dna synthesis reaction related to the replication and repair of mitochondrial dna ( mtdna ) . during nuclear dna replication , the primase interacts with pol , forming a very stable complex ( pol-primase ) that triggers the initiation of dna synthesis . primpol was shown to be present at both nuclear and mitochondrial dna compartments but is absent in mitochondria derived from a mouse primpol knockout ( ko ) model . rnai experiments in human cells and analysis of primpol ko mouse embryonic fibroblasts ( mefs ) indicated that primpol plays a key role in mtdna synthesis . the dxe variant is also found in the primpol of plasmid prn1 from sulfolobus islandicus , which has both primase and dna polymerase activities ( lipps et al . this unusual rna and dna primase activity is not present in escherichia coli and was demonstrated to be inherent to human ccdc111 by ablating the activity with a double mutation ( axa ) in the two potential metal ligands ( asp and glu , motif dxe ) at the predicted active site ( figure 1c ) , which was in agreement with enzymatic and structural studies of the primpol from plasmid prn1 ( lipps et al . independent of the metal cation , primpol behaved as a dna - instructed dna polymerase , preferentially inserting the complementary nucleotide dictated by the first available templating base ( figure 2c ) . given that primpol is a dna primase / polymerase ( figures 1c1e , 2 , s2 , and s3 ) , it should logically operate in the nucleus and/or mitochondrion . to establish that primpol was responsible for the observed dna primase activity , we subjected mitochondrial fractions from mefs possessing ( + /+ ) or lacking ( / ) the primpol gene ( figure s4 ) to the dna primase assay . fraction 15 of the ig ( where primpol is concentrated ) displayed the same initiation specificity and preference for mn as purified primpol , yielding the same products in the same ratios ( figure 4d ) , whereas ig fraction 11 ( containing pol and polrmt ) lacked such an activity . by virtue of its primase activity , primpol might facilitate dna replication by restarting dna synthesis downstream of pauses or fork arrest due to damaged dna , as has been shown to occur in bacteria , yeast , and human cells ( heller and marians , 2006 ; lopes et al . primpol continued polymerization beyond the ap site in 80% of the molecules , which contrasts with the null bypass observed with pol. moreover , under these optimized conditions ( figure 5f ) , much lower amounts of primpol ( 50 nm ; figure 5e , lane 5 ) or a very low concentration of manganese ions ( 10 m ; figure 5e , lane 9 ) were sufficient to detect the synergistic effect of primpol and pol , strongly suggesting their functional coupling . these experiments demonstrate that , during restart of dna synthesis , the dna primers made by primpol ( and most likely its tls products ) can be efficiently extended by the mitochondrial and nuclear replicative polymerases pol and pol , respectively ( figure 5h ) . to determine the extent of mtdna replication after 72 hr of primpol gene silencing , we analyzed brdu incorporation into mtdna in double - stranded rna ( dsrna)-transfected cells after preincubation with aphidicolin , an inhibitor of nuclear dna replication . hence , primpol gene silencing in human cultured cells caused a profound arrest of mtdna synthesis . primpol gene silencing in human cultured cells has multiple adverse affects on mtdna , and mouse cells lacking primpol display inefficient mtdna replication . in respect of damaged dna , primpol , as a tls dna polymerase ( figure 7b ) , could assist pol in reading template lesions ( e.g. alternatively or additionally , primpol could exploit both its dna primase and dna polymerase activities in order to facilitate replication fork progression by acting as a specific tls primase in order to reinitiate replication at pause sites or downstream of lesions that block continuous synthesis by replicative dna polymerases ( figure 7d ) . in summary , we identify and characterize a dna primase / polymerase in human cells endowed with the unique ability to initiate synthesis using dntps and a remarkable capacity for translesion synthesis . our initial analysis of its function in mitochondria suggests that primpol plays a key role in dna metabolism , pointing to a greater level of complexity in mtdna replication than previously recognized and implying that pol is not the sole dna polymerase in mammalian mitochondria .

since the earliest studies on glial cells in the 19th century , ramn y cajal , camillo golgi , and their contemporary colleagues have described astrocytes as very particular cells in intimate contact with neurons and capillaries . based on these observations , they made different hypotheses on their physiological function , ranging from passive space filling in the neuropil to active energy supply for neurons . almost 150 years later , the neurophysiological role of astrocytes is still a subject of intense debate , although increasing data suggest that they are active players in mechanisms of synaptic transmission and plasticity . peripheral astrocytic processes , as it is often difficult to distinguish , with light microscopy , their exact position with regard to different neuropil elements . however , in this review we will mostly focus on the data , obtained with various techniques , concerning fine astrocytic processes that are in close association with synaptic contacts , and thus the term close structural relationship between synaptic structures and paps makes astrocytes an important partner of neurons in the organization and functioning of synaptic connections . astrocytes take up glutamate from the synaptic cleft , control the amount of glutamate spillover that activates extrasynaptic receptors or enables intersynaptic crosstalk , control the ion and water homeostasis through selective transmembrane movements of inorganic and organic molecules and the equilibration of osmotic gradients , and provide energy substrates to neurons in the form of lactate . in addition , recent studies have revealed astrocytic processes as structural entities able to modulate neuronal function at the synaptic level through the calcium - dependent release of gliotransmitters [ 9 , 10 ] . indeed , astrocytes are able to sense neuronal activity by elevating their intracellular calcium concentration through gq - coupled protein mechanisms . even if this calcium excitability phenomenon is well accepted , its exact role is still controversial . finally , paps have been described by several studies as plastic structures able to change their morphology within minutes , thus modifying their coverage of pre- and postsynaptic elements . we review here these observations and discuss the physiological role that structural changes in paps could have on both synaptic and astrocytic function . a main structural characteristic of astrocytes is the star - shaped arborization of their stem processes . however , at the ultrastructural level , protoplasmic astrocytes have much more complex shapes consisting of thousands of very fine elaborated protrusions that make them look more like sponges than stars [ 1416 ] . detailed morphological descriptions and quantitative analyses provided by electron microscopy ( em ) , especially those based on three - dimensional ( 3d ) reconstructions from serial ultrathin sections , have allowed the identification of astroglia and their interactions with neurons and synapses . big stem processes of astrocytes containing typical bundles of intermediate filaments ( 8 - 9 nm in diameter ) that have glial fibrillary acidic protein ( gfap ) as their main constituent represent around 15% of the total volume of an astrocyte . these processes ramify progressively to finally generate a dense matrix of thin elaborate terminal processes , which infiltrate brain tissue and closely associate with neuropil elements and particularly with synapses . these fine astrocytic processes account for 7080% of the astrocytic plasma membrane and often surround spine synapses , sometimes completely encapsulating them ( figure 1 ) . astrocytic processes are found in close proximity of synapses at all synapses investigated so far with em in different brain regions . these small astrocytic processes are devoid of gfap filaments , have a cytoplasm that is light in appearance , are highly irregular in outline , forming flat or lamellar sheets or conforming to the shapes of surrounding neuropil elements ( figure 1 ) , and have large surface / volume ratios , as shown with 3d em reconstructions [ 19 , 20 ] ( reviewed in ) . in these very fine processes , mitochondria , microtubules , and endoplasmic reticulum they also contain actin filaments , together with actin - binding proteins [ 4 , 23 , 24 ] . the contacts between astrocytic processes and dendritic spines can be quite tight , sometimes with puncta adhaerens between the nonsynaptic surface of dendritic spines and astrocytic processes . paps are found in all brain regions , but the proportion of synapses having them and the level of synaptic coverage vary significantly . for example , paps embrace most of the synapses in cerebellum but only 2956% of the synapses in the neocortex , according to different em estimations [ 18 , 26 ] . in layer iv of adult mouse somatosensory cortex , 3d em demonstrated high heterogeneity of synaptic coverage , with around 10% of spine synapses having no contact with paps , while the rest of the spines have varying portion of their surface enwrapped , with a majority of them ( around 68% ) surrounded by astrocytic process at the axon - spine interface ( asi ; figure 1 ) . in ca1 they are present near approximately 62% of synapses , with a preference towards large synapses , especially those with perforated ( complex ) psds that are considered as morphological correlates of strengthened synapses ( up to 90% ; ) . some ca1 synapses are completely devoid of paps , while others could be covered only around the dendritic spine ( 7% ) , or around the presynaptic bouton ( 8% ) , or , most often , around the asi . in ca1 area of rat organotypic hippocampal slice cultures , the majority ( 85% ) of spine synapses are also contacted by glial processes that do not cover synaptic surface completely . 97% of big spine synapses with a complex psd are partially enveloped by glia but only 78% of simple synapses . in rat hippocampus , less than half ( 43% ) of the synaptic perimeter is surrounded by paps , and this parameter is highly variable from synapse to synapse (; see figures 1(b ) and 1(c ) ) . in general , 99.5% of ca1 synapses have some portions of asi free from glial processes , and only a fraction of the asi perimeter ( on average , 38% ) is enwrapped by astroglia . in contrast , in ca3 hippocampal region , large mossy fiber synapses are surrounded by astrocytic processes that physically isolate these synaptic complexes from the neighboring synapses , although paps never reach individual active zones . in cerebellum , a mossy fiber ( mf ) excitatory terminal has hundreds of release sites that establish synaptic contacts with different granule cell dendrites positioned close together forming a multisynaptic arrangement or glomerulus . only a small fraction ( around 15% ) of these excitatory terminals in contrast , excitatory inputs to purkinje cell ( pc ) spines are much better isolated by the bergman glia ( bg ) processes . synapses formed by climbing fibers ( cf ) have the highest level of astrocytic coverage , with an average degree of enwrapping around 87% . in more than half of these synapses , at least 90% of their surface is covered by paps . parallel fiber ( pf ) synapses have a bit less of perisynaptic glial coverage ( around 65% of the synapse perimeter ) . serial 3d em of labeled lateral bg processes that represent up to 90% of the bg total membrane surface area demonstrated their very complex and irregular morphology , with high surface to volume ratio . another peculiar morphological organization revealed by serial 3d em of labeled lateral bg processes is the so - called bg microdomain , a repetitive unit on the stem process consisting of a thin stalk and a complex cabbage - like head packed with thin leaflets that wrap around individual synapses or groups of synapses and constitutes an autonomous functional unit . in the nucleus tractus solitarii ( nts ) , a sensory nucleus in the caudal medulla that receives primary afferent visceroceptive inputs , but also afferent fibers from various brain regions , volume fraction of astrocytic processes and the density of astrocyte membranes are rather high , equal to 15% and 2.8 m/m , respectively . this nucleus contains simple one - to - one , mostly axodendritic , synapses but is also enriched in divergent ( one axonal terminal innervating several postsynaptic dendrites or spines ) and convergent ( several axon terminals making clustered synaptic contacts on the same dendritic segment ) multisynaptic arrangements or glomeruli . like in other brain regions , astrocytic coverage of synapses is generally not complete and highly variable , ranging from no coverage to almost complete enwrapping , and constitutes , on average , 47% of synapse perimeter . at simple glutamatergic synapses , astrocytic processes cover 58% of their perimeter , while , in multisynaptic arrangements , glial coverage is restricted to the nonshared , outer part of synaptic perimeters and amounts around 50% of this outer part . for more examples of pap distribution and synaptic ensheathment although electron microscopy provides the most precise estimations of pap morphology , distribution , localization , and degree of synaptic coverage , this technique is limited to static observations as it requires fixation and embedding of tissue samples . current light microscopy approaches use pap labeling with different fluorescent probes , static or time - lapse observations with confocal microscopy followed by off - line image treatment ( e.g. , deconvolution helping to overcome the limits of confocal resolution , often lower than the size of the smallest paps ) , and 3d/4d reconstructions [ 34 , 35 ] . development of superresolution light microscopy imaging techniques will further improve the precision of pap observations . the distribution of astrocytic processes shows high variability between different brain regions and even subregions . one of the first indications was provided by a light microscopy study that assessed the orientation of hippocampal astrocytic processes . is almost perpendicular to the stratum pyramidale , while in stratum lacunosum - moleculare they transiently orient parallel to the fissure . confocal and electron microscopy studies in hippocampus [ 17 , 38 ] and neocortex have shown that astrocytes cover their own exclusive spatial domains where they interact with neuronal elements , with only limited overlap in narrow peripheral zones . however , in some cortical regions this organization is compromised . in posterior piriform cortex ( ppc ) of adult rats , surface - associated astrocytes ( saa ) are directly opposed to the cortical surface , send large - caliber processes into layer i , and give rise to an extensive network of superficial processes that form a continuous sheet at the surface of ppc and lack the domain organization typical of neocortical astrocytes . in ppc , connectivity and patterns of activation by individual odorants are widely distributed , overlap extensively , and are modulated by context and behavior , and thus saa also form extensively overlapping processes . in hippocampus , 99.5% of ca1 synapses have some portions of asi free from glial processes , thus favoring glutamate spillover and synaptic crosstalk . interestingly , the fraction of synaptic perimeter covered with paps is inversely proportional to the synapse size , so that large synapses , often with complex psds , have more of asi open and are more susceptible to the transmitter spillover ( [ 27 , 29 ] ; see figures 1(b ) and 1(c ) ) . importantly , only 33% of the neighboring synapse pairs in the neuropil have an astrocytic process intruding along the shortest path between them , thus further favoring spillover and crosstalk . in ca1 synapses , a significant asymmetry characterizes the distribution of perisynaptic glia with paps being threefold more present on the postsynaptic side than on the presynaptic side . 3d modeling using these em data predicted that extrasynaptic actions of glutamate near these synapses would favor presynaptic feedback and preserve specificity transfer of information to the postsynaptic site . in cerebellum , the morphology of mossy fiber divergent glomeruli , mostly without glial fingers between the granule cell dendrites contacting an mf excitatory terminal , favors glutamate spillover . this organization enhances the efficacy of fast synaptic transmission and contributes to the time course of mf synaptic currents and to the desensitization of postsynaptic ampa receptors ( ampar ) during short - term depression . bergman glia constitutes a large volume fraction in cerebellum ( 33% ) , more than three times that of hippocampus and cortex [ 29 , 40 , 41 ] . unlike neocortex and hippocampus , functional microdomains from different bg cells intermingle within a given space of the neuropil [ 19 , 42 ] . intermingling of microdomains from different bg cells covering pf and cf synapses may provide an independent microenvironment designed to compartmentalize calcium signals , contribute to independent activation of individual dendritic spines or ensembles of spines on pc , and ensure selectivity in synapse modulation and efficient clearance through glutamate transporters expressed in the fine bg processes [ 19 , 43 ] . in the nts synapses , incomplete glial ensheathment of all synaptic types would provide enough routes for glutamate spillover , although the crosstalk between distant synapses is probably reduced , due to the low synaptic density and high volume fraction of astrocytic processes . at the same time , individual synapses in multisynaptic glomeruli in nts can strongly influence each other thanks to the direct apposition of neighboring synaptic elements mostly devoid of intervening glial processes that generally cover nonshared , outer part of synaptic perimeters . consequently , these glomeruli may be viewed as individual computing units providing processing of visceral information already at this level , especially given that sensory afferent terminals in the nts are frequently involved in divergent arrangements . on the other hand , convergent multisynaptic arrangements may also add to information processing by occlusion phenomenon between sensory inputs converging onto the same nts neuron . these examples demonstrate that the astrocytic spatial organization varies in different brain regions and may be related to the corresponding patterns of neuronal connectivity helping to establish specific functional and structural architectures . this suggests that astrocytes show sensitivity to neuronal activity and could adapt their morphology to the activity in their environment . the heterogeneity of paps around different synapses described above suggests the ability of astrocytes to control the level of synaptic coverage and , consequently , implies that paps might be as plastic as their neuronal counterparts . indeed , the first examples of pap structural plasticity were described a long time ago , in em studies of oxytocin ( oxt ) secreting magnocellular neurons in supraoptic ( son ) and paraventricular ( pvn ) nuclei of hypothalamus . under basal conditions , somata and dendrites of magnocellular secretory neurons are mostly separated by neuropil elements and especially by fine processes of both stellate and radial - like astrocytes [ 4547 ] . under conditions of stimulation , including parturition , lactation , osmotic stimulation , and stress , astrocytes respond by rapid , within few hours , and reversible structural remodeling leading to a reduced coverage [ 46 , 48 ] . even more striking rhythmic ultrastructural rearrangements take place in suprachiasmatic nucleus ( scn ) of the rat hypothalamus where glial and neuronal structural plasticity follows 24 hours light / dark ( l / d ) cycles . a technique of choice to observe real time movements of astrocytes is time - lapse light microscopy . in cultured hippocampal or cortical astrocytes , highly motile astrocytic filopodia - like processes were detected , moving or growing in the time course of minutes or even seconds [ 4 , 24 , 50 ] . whether these filopodia - like structures are comparable to in situ paps is uncertain , although they contain the actin binding protein ezrin that is specifically localized to paps . thus , the mechanisms driving filopodia movements in cultured astrocytes could be common with those of paps in vivo . using a transgenic mice line expressing green fluorescent protein ( gfp ) under a gfap promoter , hirrlinger et al . were able to image astrocytic processes adjacent to synapses in acute brain slices and however , because of the use of a nonmembrane targeted gfp that limited the possibility to detect very fine astrocytic processes in this and other studies [ 52 , 53 ] , it was difficult to identify whether these processes were indeed paps . in situ studies using membrane targeted fluorescent protein transfected biolistically in organotypic cultures allowed to visualize very fine astrocytic structures in a time - lapse manner and confirmed that those structures were dynamic . unfortunately , in this study astrocytic membranes were not coimaged with synaptic elements , again limiting a conclusion as to whether these processes were indeed paps . haber and coworkers were the first to demonstrate convincing movements of paps in situ by performing dual labeling of dendritic spines and astrocytic processes through viral gene delivery of membrane targeted fluorescent proteins . they nicely showed that paps are plastic structures that can engage and disengage from a dendritic spine in hippocampal slices . pap motility have been confirmed by other researchers in hippocampus (; see also figure 2 ) as well as in bg contacting pc in acute slices of cerebellum [ 54 , 55 ] . time - lapse confocal studies have definitively demonstrated that paps are highly dynamic as they can modify their structure in a time course of minutes . pap movements were observed in primary and organotypic cultures as well as in the acute slice model . whether pap motility occurs only during development and synaptogenesis or is persistent in adulthood and how this remodeling occurs in vivo remain , however , to be determined . already early em observations of astrocytic structural plasticity in rat hypothalamus suggested a dependence on environmental cues and stimulation , such as parturition , lactation , osmotic stimulation , and stress [ 4448 ] . an em study in the visual cortex of rats reared in a complex environment revealed a specific and significant increase in the ensheathment of synapses by astrocytic processes that accompanied structural and functional synaptic changes . chronic whisker stimulation in adult mice induced pap structural plasticity in the corresponding barrel of the somatosensory cortex . serial em analyses have revealed that stimulation leads to a significant increase of the perimeter of excitatory synapse covered by paps , particularly at the axon - spine interface . in dentate gyrus of hippocampus , the spatial relationship between astrocytic processes and synapses was analyzed at different time - points after induction of long - term potentiation ( ltp ) in adult rats in vivo . this study reported a significant increase in the number and surface density of astrocytic processes 8 h after induction of ltp . at this time - point , synaptic complexes became increasingly more enveloped by paps . in organotypic hippocampal slice cultures , the morphological remodeling of ca1 excitatory spine synapses underlying induction of synaptic potentiation by theta burst stimulation of schaffer collaterals was accompanied by an increase in the glial coverage of both pre- and postsynaptic elements , particularly of the large spine synapses with complex psds . importantly , the increase in coverage was nmda receptor - dependent . in the same hippocampal pathway , a growth of astrocytic processes related to kindling and ischemic additionally , em studies in both hippocampus and cortex revealed that paps are preferentially present near synapses with big and often complex - shaped psds that are considered as morphological correlates of strengthened synapses [ 2729 ] . these data , together with an observation that the proportion of cortical astrocyte coverage is correlated with the size of the psd also strongly suggest that pap structural organization can be regulated by synaptic activity . the em data available so far show that structural and functional pre- and postsynaptic changes due to experience - related paradigms and ltp ( read ) are accompanied by structural changes in adjacent paps . pap movements observed with time - lapse confocal microscopy in hippocampal slices appeared to be coordinated with the extent of spine movements suggesting that the two phenomena are linked . because spine dynamics is dependent upon neuronal activity , the same assumption could then be made for paps . interestingly , by assessing pap movements around spines using an index of motility , haber and coworkers revealed a high heterogeneity of this parameter , reminiscent of the synaptic coverage heterogeneity described above . the fast motility of paps could then represent the movements accomplished by paps to increase or decrease synaptic coverage . increase of neuronal activity in hippocampal slices by application of the gabar inhibitor bicuculline failed to elevate pap motility , and pap movements still occurred in the presence of ttx . nevertheless , glutamate application induced pap remodeling , and inhibition of glutamate transporter under ttx prevented coordinated pap - spine movements , suggesting , as shown in the cultures , that glutamate could be involved in pap structural plasticity in situ . a well - established feature of astrocytes is their ability to respond with an intracellular calcium elevation ( cai ) to neurotransmitters such as glutamate , gaba , atp , endocanabinoids , and others . this increase can be evoked by neuronal activity both in situ and in vivo [ 64 , 65 ] . glutamate stimulates filopodia outgrowth on cultured astrocytes and these effects are mimicked by both kainate and quisqualate agonists , as well as mglur agonist . mglur antagonists abolish this structural remodeling , while an nmdar agonist does not influence it . astrocytes were shown to express ca - permeable ampar [ 66 , 67 ] and mglur , the latter specifically in paps . this suggests the involvement of non - nmda ionotropic glutamate receptors ( possibly ampar ) and mglur in the mechanisms driving astrocytic filopodia outgrowth . consistent with this , bg retracted from pc spines when ca - impermeant ampar were overexpressed in bg . however , it should be mentioned that the subunit composition of ampar is regulated developmentally , with ca - permeable ampar subunits ( glua2 ) decreasing during development . moreover , the question about the glial cell type that expresses ampar is still open , as ng2 cells and astrocytes share common gfap marker . mglur 3 and 5 subtypes are localized in both filopodia from cultured astrocytes and in paps of hippocampal astrocytes in situ . although astrocytes are lacking voltage - gated channels , are not electrically excitable , and can not generate action potentials , they can sense neuronal activity through calcium excitability , particularly thanks to the close apposition of paps to synapses [ 9 , 10 ] . the most widely accepted mechanism for cai signaling is the gq protein coupled receptor - induced calcium release from intracellular stores , specifically from the endoplasmic reticulum . interestingly , calcium could be central to the mechanisms that control actin movements in astrocytes in the vicinity of the synapse [ 24 , 72 ] . calcium uncaging experiments in astrocytic cultures confirmed that ca elevation triggers filopodia outgrowth . in acute slices from transgenic mice that have an attenuated ip3 pathway , em analysis of the hippocampus from these mice revealed reduced pap coverage of synapses as well as an elevated proportion of uncovered synapses . this suggests that ip3-mediated ca signals may be part of the mechanism driving morphological changes in paps . actin - based movements and remodeling are sensitive to ca changes which are affecting a plethora of actin binding proteins . fine astrocytic processes contain mostly actin cytoskeleton , and the actin - binding protein alpha - actinin aggregates at the tip of growing filopodia in cultured astrocytes . filopodia growth requires the redistribution of cytoskeletal proteins but no de novo synthesis or degradation of respective proteins , suggesting that astrocytes are capable of rapid movements in response to their environment . in both cultures and slices [ 52 , 53 ] , astrocytic processes can adapt their morphology through a mechanism involving the rac-1 member of the ras superfamily of gtpases . interestingly , a dominant negative form of the actin binding protein profilin-1 abolished ca - dependent filopodia outgrowth and movement . immunocytochemistry in rat brain sections demonstrated that the actin - binding protein ezrin and the mglurs 3 and 5 are compartmentalized to paps but not to the main processes containing gfap . the experiments using ezrin sirna or dominant - negative ezrin in primary astrocytes indicated that filopodia formation and motility require ezrin . even if swelling is often associated with pathological conditions , subtle volumetric changes can occur under physiological conditions , typically during osmotic pressure changes following synaptic transmission . thus , the swelling- and actin - based mechanisms for pap structural plasticity could occur simultaneously . recent data indicate also the existence of extracellular mechanisms that could influence synapse - related pap plasticity . several astrocyte - neuron adhesion molecules were described , although there is still a lack of information about the presence of these molecules at the pap - synapse interface . among astrocyte - neuron adhesion molecules that could be regulated by activity and potentially localized at synapses the best candidate is the epha4 receptor tyrosine kinase that is enriched in dendritic spines of hippocampal pyramidal neurons ( reviewed in ) . the epha4 ligand , ephrin - a3 , appears to be localized on pap membranes . binding of glial ephrin - a3 to neuronal epha4 maintains normal spine morphology while preventing the interaction results in unstable spines with disrupted shapes . interestingly , exogenous and endogenous ephrin - a induce astrocytic processes outgrowth and extension through their binding to astrocytic epha ligands . concomitantly , calcium signals in astrocytes are perturbed by epha , suggesting the implication of intracellular calcium in the mechanism regulating epha4/ephrin - a3 adhesion and the subsequent regulation of individual dendritic spines . neuroligins are adhesion molecules known to be associated with synapses . the expression of neuroligins 1 and 2 in the cns is restricted to excitatory and inhibitory synapses , respectively . neuroligin 3 is expressed by neurons but also by glial cells , in particular in the ensheathing glia in olfactory bulb as well as in retinal astrocytes . mutations of the genes coding for neuroligins and neurexins are associated with autism , a brain disorder characterized by anomalies of dendritic spine morphology . it was shown recently that astrocytes in autistic patients also exhibit significantly altered morphology , particularly of their processes . this is suggesting implication of neuroligins in the maintenance of synaptic structures , including the astrocytic component . very little is known about intracellular interaction partners of neuroligin 3 . however , at glutamatergic synapses , neuroligin 1 binds to postsynaptic scaffolding protein psd-95 that links to gkap which in turn binds shank , and this complex may further recruit other postsynaptic proteins to the excitatory synaptic junctions ( read for a review on neuroligins ) . typically , mglur receptors as well as ionotropic glutamate receptors are associated with shank at postsynaptic sites , thereby providing a functional link between synaptic activity , intracellular calcium , and neuroligins ( read for a review on shank ) . another potential candidate is the neuron - glia cell adhesion molecule ( ng - cam ) . ng - cam has been implicated in binding between neurons and between neurons and glia . syncam1 , an adhesion molecule involved in synaptic differentiation and organization , is also expressed in astroglial cells where it mediates astrocyte - to - astrocyte and glial - neuronal adhesive communication . ncam is probably the most extensively studied cell adhesion molecule and is thought to intervene in most cell interactions via modulation of cell adhesivity and intracellular signaling . n - cadherin adhesion molecule is implicated in the establishment and stability of neuronal connections through activity - dependent mechanisms [ 86 , 87 ] . the most important mechanism is the modification in extracellular calcium concentration , which affects the structure of the ectodomain and cadherin - mediated cell adhesion . also , the cytoplasmic domain binds to various cytosolic and membrane proteins , including heterotrimeric g proteins . moreover , the c - terminal domain mainly binds to catenin and anchors the protein to the actin cytoskeleton . interestingly , a member of the cadherin family , protocadherin - gammac5 that is implicated in activity - regulated synaptic stability , is expressed in both astrocytes and neurons . in the brain , significant numbers of pcdh - gammac5 clusters are located at contact points between neuronal synaptic components and paps , suggesting that pcdh - gammac5 is involved in neuron - astrocyte interactions at the synaptic level . together , these data suggest that perisynaptic astrocytic processes possess the machinery to both sense neuronal activity and remodel their actin filaments in an activity - dependent manner . these mechanisms are regulated by intracellular calcium , through the ip3 pathway , and could directly contribute to activity - dependent pap structural plasticity . the data on adhesion molecules expressed in paps suggest their participation in remodeling , repositioning , and stabilization of perisynaptic astrocytic arrangements , although further research is needed to elucidate their exact roles . astrocytic ensheathment of the synapses may constitute a physical barrier for transmitter spillover from the synaptic cleft leading to extra- and heterosynaptic signaling . the consequences of the heterogeneity of the synaptic coverage by paps observed so far in different brain regions have been discussed in several studies . this heterogeneity suggests that glutamate or other neurotransmitters escape nonuniformly from synapses in a given brain area and that at certain synapses and/or under certain conditions synaptic crosstalk will be favored through a better spillover . structural plasticity of paps might significantly contribute to these mechanisms . a good example is provided by the rapid and reversible structural remodeling of astrocytes in hypothalamus under conditions of stimulation , leading to a reduced coverage of the somata and dendrites of magnocellular secretory neurons [ 46 , 48 ] . for example , in the son of lactating rats astrocytic coverage undergoes extensive reduction that leads to an increase in directly juxtaposed surfaces of oxt magnocellular neurons [ 91 , 92 ] and the change of both tortuosity and volume fraction of ecs . consequently , the number of synaptic contacts is increased and diffusion of neuroactive substances is facilitated . at the same time , glutamate clearance is delayed resulting in an increased negative feedback of glutamate on its own release and facilitated heterosynaptic depression of gaba release through glutamate spillover [ 93 , 94 ] . also , astrocytes in son provide d - serine , an endogenous ligand for nmdars , so that astrocytic withdrawal in lactating animals leads to a decrease in synaptic responses mediated by nmdars and in availability of these receptors for activation , thus influencing the direction and magnitude of long - term synaptic plasticity . similar structural modifications of neuronal - glial interactions under conditions of enhanced neurosecretion occur in parallel in the neurohypophysis , the major projection site of magnocellular neurons , leading to facilitation of hormone release to blood circulation . in the scn of the hypothalamus , em analysis of glial and axonal coverage of the somata and dendrites of the neurons expressing vasoactive intestinal peptide ( vip ) or arginine vasopressin ( avp ) , the two main sources of scn efferents , revealed significant variations during the l / d cycle . the glial coverage of vip dendrites increases at night , in concomitance with a decrease in the coverage of the somata and dendrites of these neurons by axon terminals . conversely , glial coverage of the avp dendrites drops during nighttime with no change in axonal coverage but with a striking increase in the somatal and dendritic appositions thus helping nocturnal facilitation of the intercellular synchronization involving these neurons . synapse modeling studies focused on the glial perisynaptic environment suggested profound effects of glial coverage on activation profiles of perisynaptic receptors . rusakov has estimated that if one - half of the synaptic perimeter is covered with paps , it leads to almost a twofold increase of the glutamate concentration inside the glial sheath and two- to fourfold decrease outside . if around 95% of the synapse is covered , the concentration difference between inner and outer sides can reach 10- to 100-fold . synaptic coverage may have stronger effects on synaptic transmission and ca depletion in the cleft at smaller synapses , especially with slower kinetics of perisynaptic ion transients . obviously , modifications of synaptic coverage due to pap structural plasticity may have significant impact on spillover effects . quantitative topological analysis of the extracellular space ( ecs ) based on 3d em reconstructions of the ca1 neuropil of the adult rat allowed to decompose ecs into sheets and tunnels that are built by the surface patches of the axonal / dendritic / glial membranes . these sheets and tunnels are distributed nonuniformly , with axons surrounded by more tunnel - like esc , astrocytic processes accompanied by more sheet - like volumes , while around dendritic spines , the ecs is more tunnel - like than elsewhere . diffusion simulations indicated that release of bioactive molecules to ecs would produce higher concentration peaks and maximum rate of concentration change , as seen by cell surface receptors , in the narrower sheet regions than in the larger tunnel spaces . these spatial irregularities would imply that ecs sheets may be specialized into enhancing signaling through concentration changes , while volume transmission of large molecules would be ensured through tunnels . given that glia swelling under different physiological and pathological conditions may modulate extracellular volume fraction and that astrocytic processes are preferentially accompanied by ecs sheets , astroglia are well positioned to regulate volume communication in neuropil and provide a selective and specialized control of perisynaptic ecs volume around a synapse . as shown by light and em immunochemistry , perisynaptic astrocytic processes ( paps ) surrounding excitatory spine synapses express a plethora of different proteins , among which glutamine synthetase , astrocytic glutamate transporters , and aquaporins that may regulate adaptive swelling of paps , as well as actin associated molecules [ 4 , 23 ] , metabotropic glutamate receptors , and cell adhesion molecules . thus , by enwrapping a synapse , astroglia not only constitute a physical barrier for transmitter diffusion and help to retain a high level of neurotransmitter around a synapse but also , through the expression of specific proteins in perisynaptic processes , can participate in both sensing and responding to synaptic activity , actively take up glutamate from the synaptic cleft , control the amount of glutamate spillover that activates extrasynaptic receptors or enable inter - synaptic crosstalk , buffer potassium following neuronal depolarization , and provide energy substrates to neurons . remarkably , astrocytes are also able to release so - called gliotransmitters in a calcium - dependent manner , among which are d - serine , glutamate , and atp . there is now extensive evidence that astrocytes can modulate synaptic transmission and plasticity through gliotransmission [ 62 , 95 , 107115 ] . however , the exact mechanisms taking place during calcium - dependent neuron - glia interactions are still a matter of debate as studies using different approaches to selectively increase astrocytic cai yielded conflicting results [ 12 , 116 , 117 ] . intracellular calcium is also known to take part in the mechanism of neurometabolic coupling with neurons by either modulating the energetic response or participating to the propagation of metabolic signals across the astrocytic network . as discussed above , calcium signaling , often related to synaptic activity , could be central to the mechanisms that control pap remodeling in the vicinity of the synapse [ 24 , 72 ] , which could in turn optimize the positioning of glial glutamate transporters to provide more efficient clearance of glutamate , modify the positioning of the release sites of gliotransmitters close to extrasynaptic neuronal receptors or release of energetic substrates such as lactate in front of neuronal monocarboxylate transporters , and regulate ionic content of the extrasynaptic space . another aspect of pap structural plasticity is its possible impact on the synaptic structure . in concert with this concept , it was shown that in hippocampal slices the probability of a spine to disappear is higher when paps are not present around it . inhibition of astrocytic processes motility through interference with rac1 results in an increase in dendritic filopodia , the protrusions known to be immature and nonpersistent . interfering with epha4 or ephrin - a3 induces growth of astrocytic processes in cell culture . the same treatments in slice cultures perturb the contact - dependent ephrin / eph signaling by astrocytes , shorten the lifetime of dendritic protrusions contacted by astrocytic processes , and decrease the probability for the newly formed spines to be stabilized . consistent with this , bg coverage of pc spines seems to be required for synapse formation and regulation [ 54 , 55 ] . some hippocampal dendritic spines are able to extend spine head protrusions to neighboring functional presynaptic boutons . interestingly , the volume overlap between spines and paps decreased during the formation of spine head protrusion , suggesting an inverse correlation between spine coverage by paps and the creation of new synaptic connections . astrocytes are well positioned to influence synaptic transmission as they send fine processes that ensheath a synapse , forming a tripartite complex with pre- and postsynaptic neuronal components . about 60% of neuronal synaptic structures are partially enwrapped by paps , and the level of synaptic coverage increases with neuronal activity , ltp , and during experience - related paradigms . moreover , paps can display rapid remodeling by extending and retracting from dendritic spines in a time scale of minutes . this rapid astrocytic motility appears to be influenced by neuronal activity and is coordinated with morphological changes in spines . in hippocampus , cortex , and cerebellum , glial processes exhibit two distinct structural plasticity phenomena : a highly dynamic movement referred to as motility and changes in their coverage of a synapse . whether these two forms of plasticity are linked is still unclear , as well as the precise impact of such structural plasticity on synaptic function , structure , and plasticity . one hypothesis that we would like to propose is that the activity - dependent structural plasticity of paps may allow astrocytes to control both the structural and functional properties of the synapse . astrocytic contacts around synapses may provide structural support to a synapse while controlling the diffusion of molecules within the ecs and participating in extracellular homeostasis . astrocytic processes in the vicinity of the synapse may control the amount of glutamate spillover that activates extrasynaptic receptors , enabling intersynaptic crosstalk and allowing for modified or synchronized neurotransmission . similarly , it could help to locally provide energy substrates to neurons during energy demanding processes such as plasticity . there are however many questions that still remain concerning the mechanisms of pap structural plasticity , about how it is governed and controlled . recent data provide indications that it could be controlled by neuronal activity , probably through glial metabotropic receptors and intracellular calcium . as ca signaling in astrocytic processes is synaptically evoked and can occur locally in the perisynaptic astrocytic environment without propagating to the cell body or other processes , pap motility might be required to control the established tripartite connections in place and to coordinate their plastic adaptations . moreover , synaptic calcium signals in astrocytes associated with pap structural plasticity could indicate that a given astrocyte can differentiate between the around 100 000 synapses it is enwrapping . thus , paps could be seen as structural as well as functional entities able to integrate synaptic signals . in the cns , excitatory synapses exhibit multiple forms of plasticity that play a central role in information processing by neural networks . they involve changes in synaptic strength but also structural reorganization of synaptic connections that can occur during the whole lifetime . these mechanisms of synaptic structural plasticity are now believed to be crucial for memory processes in the cns . consistent with this , abnormal synaptic morphologies are linked to various developmental psychiatric diseases including mental retardation , schizophrenia , and autism . an important question therefore is to better understand the mechanisms regulating these structural aspects of plasticity . as pap remodelling seems to be important for synapse function , formation , and stability , astrocytes might play an important role in these processes . further studies , however , are needed to evaluate the functional and structural consequences of pap structural plasticity in cns function and disease .

the function and efficacy of synaptic transmission are determined not only by the composition and activity of pre- and postsynaptic components but also by the environment in which a synapse is embedded . glial cells constitute an important part of this environment and participate in several aspects of synaptic functions . among the glial cell family , the roles played by astrocytes at the synaptic level are particularly important , ranging from the trophic support to the fine - tuning of transmission . astrocytic structures are frequently observed in close association with glutamatergic synapses , providing a morphological entity for bidirectional interactions with synapses . experimental evidence indicates that astrocytes sense neuronal activity by elevating their intracellular calcium in response to neurotransmitters and may communicate with neurons . the precise role of astrocytes in regulating synaptic properties , function , and plasticity remains however a subject of intense debate and many aspects of their interactions with neurons remain to be investigated . a particularly intriguing aspect is their ability to rapidly restructure their processes and modify their coverage of the synaptic elements . the present review summarizes some of these findings with a particular focus on the mechanisms driving this form of structural plasticity and its possible impact on synaptic structure and function .

1. Introduction 2. Astrocytic Processes Embrace Synapses 3. PAP Distribution Is Related to the Patterns of Neuronal Connectivity in Different Brain Regions 4. PAPs Exhibit Significant Structural Plasticity 5. Driving Force and Mechanisms of PAP Structural Plasticity 6. What Are the Consequences for a Synapse? 7. Discussion

almost 150 years later , the neurophysiological role of astrocytes is still a subject of intense debate , although increasing data suggest that they are active players in mechanisms of synaptic transmission and plasticity . however , in this review we will mostly focus on the data , obtained with various techniques , concerning fine astrocytic processes that are in close association with synaptic contacts , and thus the term close structural relationship between synaptic structures and paps makes astrocytes an important partner of neurons in the organization and functioning of synaptic connections . astrocytes take up glutamate from the synaptic cleft , control the amount of glutamate spillover that activates extrasynaptic receptors or enables intersynaptic crosstalk , control the ion and water homeostasis through selective transmembrane movements of inorganic and organic molecules and the equilibration of osmotic gradients , and provide energy substrates to neurons in the form of lactate . indeed , astrocytes are able to sense neuronal activity by elevating their intracellular calcium concentration through gq - coupled protein mechanisms . finally , paps have been described by several studies as plastic structures able to change their morphology within minutes , thus modifying their coverage of pre- and postsynaptic elements . in layer iv of adult mouse somatosensory cortex , 3d em demonstrated high heterogeneity of synaptic coverage , with around 10% of spine synapses having no contact with paps , while the rest of the spines have varying portion of their surface enwrapped , with a majority of them ( around 68% ) surrounded by astrocytic process at the axon - spine interface ( asi ; figure 1 ) . at simple glutamatergic synapses , astrocytic processes cover 58% of their perimeter , while , in multisynaptic arrangements , glial coverage is restricted to the nonshared , outer part of synaptic perimeters and amounts around 50% of this outer part . interestingly , the fraction of synaptic perimeter covered with paps is inversely proportional to the synapse size , so that large synapses , often with complex psds , have more of asi open and are more susceptible to the transmitter spillover ( [ 27 , 29 ] ; see figures 1(b ) and 1(c ) ) . at the same time , individual synapses in multisynaptic glomeruli in nts can strongly influence each other thanks to the direct apposition of neighboring synaptic elements mostly devoid of intervening glial processes that generally cover nonshared , outer part of synaptic perimeters . in organotypic hippocampal slice cultures , the morphological remodeling of ca1 excitatory spine synapses underlying induction of synaptic potentiation by theta burst stimulation of schaffer collaterals was accompanied by an increase in the glial coverage of both pre- and postsynaptic elements , particularly of the large spine synapses with complex psds . a well - established feature of astrocytes is their ability to respond with an intracellular calcium elevation ( cai ) to neurotransmitters such as glutamate , gaba , atp , endocanabinoids , and others . interestingly , calcium could be central to the mechanisms that control actin movements in astrocytes in the vicinity of the synapse [ 24 , 72 ] . however , at glutamatergic synapses , neuroligin 1 binds to postsynaptic scaffolding protein psd-95 that links to gkap which in turn binds shank , and this complex may further recruit other postsynaptic proteins to the excitatory synaptic junctions ( read for a review on neuroligins ) . a good example is provided by the rapid and reversible structural remodeling of astrocytes in hypothalamus under conditions of stimulation , leading to a reduced coverage of the somata and dendrites of magnocellular secretory neurons [ 46 , 48 ] . the glial coverage of vip dendrites increases at night , in concomitance with a decrease in the coverage of the somata and dendrites of these neurons by axon terminals . thus , by enwrapping a synapse , astroglia not only constitute a physical barrier for transmitter diffusion and help to retain a high level of neurotransmitter around a synapse but also , through the expression of specific proteins in perisynaptic processes , can participate in both sensing and responding to synaptic activity , actively take up glutamate from the synaptic cleft , control the amount of glutamate spillover that activates extrasynaptic receptors or enable inter - synaptic crosstalk , buffer potassium following neuronal depolarization , and provide energy substrates to neurons . another aspect of pap structural plasticity is its possible impact on the synaptic structure . in hippocampus , cortex , and cerebellum , glial processes exhibit two distinct structural plasticity phenomena : a highly dynamic movement referred to as motility and changes in their coverage of a synapse . whether these two forms of plasticity are linked is still unclear , as well as the precise impact of such structural plasticity on synaptic function , structure , and plasticity . as pap remodelling seems to be important for synapse function , formation , and stability , astrocytes might play an important role in these processes .

obstructive sleep apnea ( osa ) , a common condition in older adults affecting more males than females,1 causes intermittent nocturnal hypoxemia and is associated with inflammation , metabolic abnormalities , endothelial dysfunction , and poor control of diabetes mellitus and hypertension , which may lead to micro and macrovascular disease.2 , 3 , 4 osa has been associated with higher risk of clinical cardiovascular disease ( cvd ) , including ischemic stroke and coronary heart disease , independent of traditional risk factors and severity of atherosclerosis.4 , 5 it is unknown whether this relationship is related to microvascular disease.6 , 7 some investigators have suggested microvascular disease may be a potential mediator for the association between osa and clinical cvd . however , current evidence for the association of osa with microvascular disease is conflicting . some brain imaging studies revealed inconsistent results for the association between osa and cerebral smallvessel disease.8 , 9 in addition , although some cardiac perfusion scans showed that osa is associated with impaired myocardial microcirculation in patients with acute myocardial infarction , this finding may be attributable to the production of lipid microemboli from atheroma plaque rupture.10 , 11 the retinal microvasculature is structurally and functionally similar to microvasculature elsewhere in the body.12 previous studies have demonstrated the relationship between retinal microvascular signs and cardiovascular risk factors and shown that these signs predict a range of cvd , including stroke , coronary heart disease , and congestive heart failure.13 , 14 retinal arteriolar narrowing is strongly related to hypertension and age,15 while retinal venular widening is related to cigarette smoking , inflammation , atherosclerosis , and metabolic abnormalities such as obesity , hyperglycemia , and diabetes mellitus.13 , 16 retinopathy signs , specifically microaneurysms , hemorrhages , and cotton wool spots found in patients with diabetes mellitus are also prevalent in the general population ( 5% to 10% ) , and may be related to age , obesity , hyperglycemia , inflammation , and elevated blood pressure ( bp).13 several retinal imaging studies have been used to evaluate the association between sleepdisordered breathing and retinal microvascular signs.17 , 18 , 19 , 20 , 21 these studies showed conflicting results . notably , results from a recent analysis of the multiethnic study of atherosclerosis ( mesa ) data visit 2 ( 20022004 ) , in which a sample of 5803 participants underwent both a selfadministered sleep history questionnaire and retinal photography , found that physiciandiagnosed sleep apnea ( pdsa ) was associated with retinal arteriolar narrowing in women only.21 there was no association between pdsa and retinal vascular calibers in men . the clinical course of osa differs between men and women with women on average having less severe sleep apnea than men at younger ages , with differences narrowing after menopause.22 prior research suggests a sex difference in the associations between osa and clinical cvd.23 therefore , the association between osa and retinal microvascular signs may differ by sex . since pdsa was not an objective measure of osa severity in the previous mesa study,21 we used rigorously collected data from polysomnography ( psg ) and retinal photography in a large multiethnic general population in the mesa visit 5 ( 20102012 ) to quantify the sexspecific association between osa and retinal microvascular signs . the study design for mesa has been published elsewhere.24 in brief , mesa is a longitudinal cohort study designed to investigate the prevalence , correlates , and progression of subclinical cvd in individuals without clinical cvd at baseline . the cohort includes 6814 women and men aged 4584 years old recruited from 6 us communities ( baltimore , md ; chicago , il ; forsyth county , nc ; los angeles county , ca ; northern manhattan , ny ; and st . the participants were 38% white , 28% african american , 22% hispanic , and 12% chinese . this study was approved by the institutional review board of each study site , and written informed consent was obtained from all participants . in conjunction with the fifth mesa examination ( april 2010february 2012 ) , all mesa participants were offered fundus photography , and except for those reporting regular use of oral devices , nocturnal oxygen , or nightly positive airway pressure devices , all participants were also invited to participate in the mesa sleep ancillary study , which included inhome psg . of 4077 participants approached for psg , 147 ( 3.6% ) were ineligible and 141 lived too far away to participate . of the remaining 3789 participants , 2261 participated in the sleep exam ( 59.7% ) and 2060 had successful psg data . we further excluded those with poor retinal image quality in both eyes , and those with missing data on relevant variables , leaving a sample of 1808 individuals ( 87.8% ) aged 54 to 93 years for the analysis of retinal vascular calibers and another sample of 1831 individuals ( 88.9% ) for the analysis of retinopathy signs ( figure ) . flow diagram to select the eligible mesa sleepeye cohort , 20102012 . mesa indicates multiethnic study of atherosclerosis . a 15channel psg recording ( compumedics somte system ; compumedics ltd . , abbotsville , au ) including electroencephalography , electrooculography , chin electromyography , oxyhemoglobin saturation ( finger pulse oximetry ) , chest and abdominal excursion ( inductance plethysmography ) , airflow ( oral and nasal thermocouple ) , leg movements , body position , and ambient light was taken in the participant 's home . the psg data were transmitted to the centralized reading center in the brigham and women 's hospital ( boston , ma ) for review and scoring by a certified polysomnologist masked to all other data . sleep stages and electroencephalography ( cortical ) arousals were scored based on published guidelines and adapted for unattended studies using methods from the sleep heart health study , as detailed.25 apnea was scored when the thermocouple signal flattened or nearly flattened for > 10 s. hypopnea was scored if the amplitude of the sum of the abdominal and thoracic inductance signals or the nasal pressure flow signal decreased by 30% or more for 10 s. apneas were classified as obstructive or central based on absence or presence of respiratory effort during the event . specialized software was used to link apnea and hypopnea with data from the oxygen saturation and electroencephalography signals , allowing each event to be characterized according to the degree of associated desaturation and arousal . in this analysis , hypopnea index ( ahi ) defined as the average number of obstructive apneas plus hypopneas associated with a 4% desaturation per hour of objectively measured sleep.26 intraclass correlation coefficients for within and betweenscorer reliability exceed 0.94 . fundus photography was performed in both eyes of each participant according to a standardized protocol using a 45 digital nonmydriatic camera.27 all images were sent to the ocular epidemiology reading center at the university of wisconsin ( madison , wi ) and were evaluated by trained graders masked to participants characteristics . retinopathy was considered present if the graders found any lesions as defined by the early treatment diabetic retinopathy study severity scale , including retinal hemorrhages , microaneurysms , cotton wool spots , intraretinal microvascular abnormalities , hard exudates , venous beading , retinal neovascularization , and other lesions of proliferative diabetic retinopathy.28 retinal vascular caliber was measured with a computerbased program following a detailed protocol.28 for each photograph , all retinal arterioles and venules coursing through a zone between 0.5 and 1disc diameter away from the optic disc margin were measured as the central retinal arteriolar equivalents and venular equivalents.29 , 30 the average of the right and left eye measurements was taken for both central retinal arteriolar equivalents and central retinal venular equivalents in each participant . if retinal vascular diameter could not be measured in both eyes , the eye with the available photograph was used . the reproducibility of retinal vascular measurements has been reported previously with intra and intergrader intraclass correlation coefficients ranging from 0.78 to 0.99.14 , 15 all participants underwent an interview and were assessed for cvd risk factors at the fifth examination . body mass index ( bmi ) was calculated as weight ( kg)/height squared ( m ) . packyears of cigarette smoking were estimated from age of starting to quitting ( or current age among current smokers)(cigarettes per day/20 ) . hypertension was defined as systolic bp 140 mm hg , or diastolic bp 90 mm hg , or use of antihypertensive medication . diabetes mellitus was defined as fasting glucose 126 mg / dl or the use of hypoglycemic medications . hba1c was measured on the tosoh a1c 2.2 plus glycohemoglobin analyzer ( tosoh medics , inc , hercules , ca ) using automated highperformance liquid chromatography . glomerular filtration rate was estimated according to the chronic kidney disease epidemiology collaboration equation.31 micro and macroalbuminuria were defined by urinary albumin creatinine ratios of 30 to 299 mg / g and 300 mg / g , respectively . total cholesterol and highdensity lipoprotein cholesterol were measured from blood samples obtained after a 12hour fast . the severity of osa was classified by ahi ( events / h ) as none ( < 5 ) , mild ( 514.9 ) , moderate ( 1530 ) , and severe ( 30 ) . demographic characteristics of participants in each of the osa groups were compared using either analysis or anova and reported as meansd or percent for continuous and categorical variables , respectively . the effect of osa severity on retinal vascular calibers was estimated by using ancova , and the results were presented as means and se of retinal arteriolar and venular calibers for men and women . the overall effect of osa severity was tested and in the presence of a significant overall test , pairwise comparisons of no osa with mild and with moderate / severe osa were performed , respectively . logistic regression was used to determine the odds of retinal arteriolar narrowing ( the narrowest quartile versus others ) , retinal venular widening ( the widest quartile versus others ) , and specific retinopathy signs in association with osa severity in men and women . the wald test from the type 3 analysis of effects was used to determine the overall significance of osa severity . linear regression was also used to determine the associations of osa with retinal arteriolar and venular calibers in men and women . in comparison with the categorical osa severity , we analyzed the linear association of ahi with retinal vascular caliber by ahi : 0 to 14.9 ( nonetomild osa ) and ahi 15 ( moderatetosevere osa ) in a piecewise fashion . we constructed 3 models : model 1 included adjustments for age and race / ethnicity ; model 2 included additional adjustments for bmi , systolic bp , diabetes mellitus status , serum total cholesterol , highdensity lipoprotein cholesterol , smoking status , alcohol intake , antihypertensive therapy , and lipidlowering therapy . model 3 included comprehensive adjustments for age , race / ethnicity , bmi , packyears of cigarette smoking , alcohol intake , duration of hypertension and diabetes mellitus ( duration was defined as none , longer , or shorter than an average 9.5year period between the first and the fifth visit of mesa ) , hba1c , total cholesterol , highdensity lipoprotein cholesterol , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , medication with blockers , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . these potential confounders in models were chosen based on prior published associations with osa or retinal microvascular signs.32 , 33 , 34 formal testing for interactions between men and women regarding the associations of osa severity with retinal microvascular signs was performed . a 2tailed value of p<0.025 was considered significant for the overall test of osa severity in men and women separately . all statistical analyses were performed using sas version 9.4 ( sas institute , cary , nc ) . the study design for mesa has been published elsewhere.24 in brief , mesa is a longitudinal cohort study designed to investigate the prevalence , correlates , and progression of subclinical cvd in individuals without clinical cvd at baseline . the cohort includes 6814 women and men aged 4584 years old recruited from 6 us communities ( baltimore , md ; chicago , il ; forsyth county , nc ; los angeles county , ca ; northern manhattan , ny ; and st . the participants were 38% white , 28% african american , 22% hispanic , and 12% chinese . this study was approved by the institutional review board of each study site , and written informed consent was obtained from all participants . in conjunction with the fifth mesa examination ( april 2010february 2012 ) , all mesa participants were offered fundus photography , and except for those reporting regular use of oral devices , nocturnal oxygen , or nightly positive airway pressure devices , all participants were also invited to participate in the mesa sleep ancillary study , which included inhome psg . of 4077 participants approached for psg , 147 ( 3.6% ) were ineligible and 141 lived too far away to participate . of the remaining 3789 participants , 2261 participated in the sleep exam ( 59.7% ) and 2060 had successful psg data . we further excluded those with poor retinal image quality in both eyes , and those with missing data on relevant variables , leaving a sample of 1808 individuals ( 87.8% ) aged 54 to 93 years for the analysis of retinal vascular calibers and another sample of 1831 individuals ( 88.9% ) for the analysis of retinopathy signs ( figure ) . flow diagram to select the eligible mesa sleepeye cohort , 20102012 . a 15channel psg recording ( compumedics somte system ; compumedics ltd . , abbotsville , au ) including electroencephalography , electrooculography , chin electromyography , oxyhemoglobin saturation ( finger pulse oximetry ) , chest and abdominal excursion ( inductance plethysmography ) , airflow ( oral and nasal thermocouple ) , leg movements , body position , and ambient light was taken in the participant 's home . the psg data were transmitted to the centralized reading center in the brigham and women 's hospital ( boston , ma ) for review and scoring by a certified polysomnologist masked to all other data . sleep stages and electroencephalography ( cortical ) arousals were scored based on published guidelines and adapted for unattended studies using methods from the sleep heart health study , as detailed.25 apnea was scored when the thermocouple signal flattened or nearly flattened for > 10 s. hypopnea was scored if the amplitude of the sum of the abdominal and thoracic inductance signals or the nasal pressure flow signal decreased by 30% or more for 10 s. apneas were classified as obstructive or central based on absence or presence of respiratory effort during the event . specialized software was used to link apnea and hypopnea with data from the oxygen saturation and electroencephalography signals , allowing each event to be characterized according to the degree of associated desaturation and arousal . in this analysis , hypopnea index ( ahi ) defined as the average number of obstructive apneas plus hypopneas associated with a 4% desaturation per hour of objectively measured sleep.26 intraclass correlation coefficients for within and betweenscorer reliability exceed 0.94 . fundus photography was performed in both eyes of each participant according to a standardized protocol using a 45 digital nonmydriatic camera.27 all images were sent to the ocular epidemiology reading center at the university of wisconsin ( madison , wi ) and were evaluated by trained graders masked to participants characteristics . retinopathy was considered present if the graders found any lesions as defined by the early treatment diabetic retinopathy study severity scale , including retinal hemorrhages , microaneurysms , cotton wool spots , intraretinal microvascular abnormalities , hard exudates , venous beading , retinal neovascularization , and other lesions of proliferative diabetic retinopathy.28 retinal vascular caliber was measured with a computerbased program following a detailed protocol.28 for each photograph , all retinal arterioles and venules coursing through a zone between 0.5 and 1disc diameter away from the optic disc margin were measured as the central retinal arteriolar equivalents and venular equivalents.29 , 30 the average of the right and left eye measurements was taken for both central retinal arteriolar equivalents and central retinal venular equivalents in each participant . if retinal vascular diameter could not be measured in both eyes , the eye with the available photograph was used . the reproducibility of retinal vascular measurements has been reported previously with intra and intergrader intraclass correlation coefficients ranging from 0.78 to 0.99.14 , 15 all participants underwent an interview and were assessed for cvd risk factors at the fifth examination . body mass index ( bmi ) was calculated as weight ( kg)/height squared ( m ) . packyears of cigarette smoking were estimated from age of starting to quitting ( or current age among current smokers)(cigarettes per day/20 ) . hypertension was defined as systolic bp 140 mm hg , or diastolic bp 90 mm hg , or use of antihypertensive medication . diabetes mellitus was defined as fasting glucose 126 mg / dl or the use of hypoglycemic medications . hba1c was measured on the tosoh a1c 2.2 plus glycohemoglobin analyzer ( tosoh medics , inc , hercules , ca ) using automated highperformance liquid chromatography . glomerular filtration rate was estimated according to the chronic kidney disease epidemiology collaboration equation.31 micro and macroalbuminuria were defined by urinary albumin creatinine ratios of 30 to 299 mg / g and 300 mg / g , respectively . total cholesterol and highdensity lipoprotein cholesterol were measured from blood samples obtained after a 12hour fast . the severity of osa was classified by ahi ( events / h ) as none ( < 5 ) , mild ( 514.9 ) , moderate ( 1530 ) , and severe ( 30 ) . demographic characteristics of participants in each of the osa groups were compared using either analysis or anova and reported as meansd or percent for continuous and categorical variables , respectively . the effect of osa severity on retinal vascular calibers was estimated by using ancova , and the results were presented as means and se of retinal arteriolar and venular calibers for men and women . the overall effect of osa severity was tested and in the presence of a significant overall test , pairwise comparisons of no osa with mild and with moderate / severe osa were performed , respectively . logistic regression was used to determine the odds of retinal arteriolar narrowing ( the narrowest quartile versus others ) , retinal venular widening ( the widest quartile versus others ) , and specific retinopathy signs in association with osa severity in men and women . the wald test from the type 3 analysis of effects was used to determine the overall significance of osa severity . linear regression was also used to determine the associations of osa with retinal arteriolar and venular calibers in men and women . in comparison with the categorical osa severity , we analyzed the linear association of ahi with retinal vascular caliber by ahi : 0 to 14.9 ( nonetomild osa ) and ahi 15 ( moderatetosevere osa ) in a piecewise fashion . we constructed 3 models : model 1 included adjustments for age and race / ethnicity ; model 2 included additional adjustments for bmi , systolic bp , diabetes mellitus status , serum total cholesterol , highdensity lipoprotein cholesterol , smoking status , alcohol intake , antihypertensive therapy , and lipidlowering therapy . model 3 included comprehensive adjustments for age , race / ethnicity , bmi , packyears of cigarette smoking , alcohol intake , duration of hypertension and diabetes mellitus ( duration was defined as none , longer , or shorter than an average 9.5year period between the first and the fifth visit of mesa ) , hba1c , total cholesterol , highdensity lipoprotein cholesterol , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , medication with blockers , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . these potential confounders in models were chosen based on prior published associations with osa or retinal microvascular signs.32 , 33 , 34 formal testing for interactions between men and women regarding the associations of osa severity with retinal microvascular signs was performed . a 2tailed value of p<0.025 was considered significant for the overall test of osa severity in men and women separately . all statistical analyses were performed using sas version 9.4 ( sas institute , cary , nc ) . the baseline characteristics of the study cohort shown by severity of osa in men and women are provided in table 1 . as compared to those with no osa , those with mild or moderate or severe osa were older and had greater proportions of those with obesity , hypertension , diabetes mellitus , dyslipidemia , and cvd . characteristics of the population by the severity of osa estimated by ahi in men and women ahi indicates apnea hypopnea index ( events / h ) ; bmi , body mass index ; bp , blood pressure ; hdlc , highdensity lipo protein cholesterol ; ldlc , lowdensity lipoprotein cholesterol ; osa , obstructive sleep apnea . mild osa was found in 31.7% and 32.3% of men and women , respectively , whereas moderate / severe osa occurred in 43.0% of men and 23.2% of women ( table 2 ) . as compared with those with no osa , the mean value of retinal arteriolar caliber was significantly smaller in men but not in women with osa . in contrast , the mean value of retinal venular caliber was greater in both men and women with more severe osa . relationship of osa severity with retinal vascular calibers mean ( se ) for retinal vascular caliber estimated using analysis of covariance with adjustments for age , race / ethnicity , and fellow retinal vascular caliber ( m ) . table 3 shows the results of the logistic regression model of the relationship of mild and moderate / severe osa with retinal arteriolar narrowing and retinal venular widening as compared with no osa in men and women . after adjusting for age and race / ethnicity , mild and moderate / severe osa were associated with increased odds of retinal arteriolar narrowing in men ( odds ratios [ or ] : 2.26 and 1.91 , and overall p value : 0.003 ) , but not in women . after additionally adjusting for bmi , systolic bp , diabetes mellitus status , total cholesterol , highdensity lipoprotein cholesterol , smoking status , alcohol intake , antihypertensive therapy , and lipidlowering therapy , the overall osa association remained significant ( p=0.014 ) : mild osa was significant ( or : 2.08 , p=0.006 ) and moderate / severe osa was marginally significant ( or : 1.66 , p=0.052 ) for retinal arteriolar narrowing in men . the overall osa association was not significant in women ( overall p=0.87 , or : 0.95 and 1.09 , respectively ; p value for interaction of sex : 0.06 ) . the associations between osa severity and retinal vascular calibers for men and women were consistent in the model 3 linear regression analyses despite the p values for interaction of sex being not significant ( table 4 ) . notably , ahi levels were linear inversely associated with retinal arteriolar calibers in men only if ahi < 15 , the maximal threshold of mild osa ( =0.25 , p : 0.034 , table 5 ) , in model 3 . on the contrary , ahi levels were not associated with retinal vascular calibers in women ( table 5 ) . table 6 , the exploratory analysis , shows that severe osa was associated with higher risk of retinal microaneurysms in women in model 3 ( or : 3.22 and p value : 0.025 ) . in addition , severe osa was marginally associated with retinal arteriovenous nicking and soft exudates ( or : 1.61 [ 95% ci : 0.992.60 ] and 2.80 [ 95% ci : 0.997.92 ] , and p values : 0.053 and 0.054 , respectively ; data not shown ) in women in model 1 ; however , the associations were attenuated and became nonsignificant after adjusting for traditional vascular risk factors , bmi and diabetes mellitus status . the associations of severe osa with retinal microaneurysms , arteriovenous nicking , and soft exudates in men were reversed but nonsignificant in model 3 ( or : 0.59 , 0.78 , and 0.87 , respectively ) . logistic regression of retinal vascular calibers by osa severity data shown are odds ratios ( 95% ci ) . model 2 : model 1 plus further adjustments for systolic blood pressure , antihypertensive therapy , body mass index , diabetes mellitus , total and highdensity lipoprotein cholesterol , lipidlowering therapy , cigarette smoking status , and alcohol intake status . model 3 : model 1 plus further adjustments for body mass index , duration of hypertension and diabetes mellitus ( longer or shorter than an average 9.5year period between mesa visit 1 and 5 ) , hba1c , packyears cigarette smoking , alcohol status , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , blocker medication , total and highdensity lipoprotein cholesterol , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . retinal arteriolar narrowing was defined as the narrowest quartile of retinal arteriolar calibers in the overall cohort . retinal venular widening was defined as the widest quartile of retinal venular calibers in the overall cohort . linear regression model of osa severity with retinal vascular calibers in men and women linear regression model adjusted for age , race , body mass index , duration of hypertension and diabetes mellitus ( longer or shorter than an average 9.5year period between mesa visit 1 and visit 5 ) , hba1c , packyears cigarette smoking , alcohol status , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , blockers medication , total and highdensity lipoprotein cholesterol , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . model for retinal arteriolar caliber was adjusted for retinal venular caliber , and vice versa . ahi indicates apnea hypopnea index ; mesa , multiethnic study of atherosclerosis ; osa , obstructive sleep apnea . linear regression model of ahi with retinal vascular calibers in men and women linear regression model adjusted for age , race / ethnicity , body mass index , duration of hypertension and diabetes mellitus ( none , longer or shorter than an average 9.5year period between mesa visit 1 and visit 5 ) , hba1c , packyears cigarette smoking , alcohol status , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , blockers medication , total and highdensity lipoprotein cholesterol , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . model for retinal arteriolar caliber was adjusted for retinal venular caliber , and vice versa . logistic regression models comparing osa severity against no osa by specific retinopathy sign , separately in men and women data shown are odds ratios ( 95% ci ) . model 2 : model 1 plus further adjustments for systolic blood pressure , antihypertensive therapy , body mass index , diabetes mellitus , total and highdensity lipoprotein cholesterol , lipidlowering therapy , cigarette smoking status , and alcohol intake status . model 3 : model 1 plus further adjustments for body mass index , duration of hypertension and diabetes mellitus ( longer or shorter than an average 9.5year period between mesa visit 1 and 5 ) , hba1c , packyears cigarette smoking , alcohol status , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , blocker medication , total and highdensity lipoprotein cholesterol , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . mesa indicates multiethnic study of atherosclerosis ; na , not available ; ne , not estimable ; osa , obstructive sleep apnea . pvalue for interaction of sex for retinal microaneurysms : 0.09 , blot hemorrhages : 0.56 , soft exudate : 0.45 , arteriovenous nicking : 0.20 , focal arteriolar narrowing : 0.56 , and any retinopathy sign : 0.13 . any retinopathy sign included retinal hemorrhages , microaneurysms , cotton wool spots , intraretinal microvascular abnormalities , hard exudates , venous beading , retinal neovascularization , or other lesions of proliferative diabetic retinopathy . our principal finding was that mild and moderate / severe osa was associated with narrower retinal arterioles , and moderate / severe osa was associated with wider retinal venules in men . however , there was no association between osa severity and retinal vascular calibers in women . in contrast , in exploratory analyses the association of severe osa with retinal microaneurysms was greater in women than men . the sexspecific osa associations with retinal vascular calibers and retinopathy were independent of bmi and traditional cvd risk factors : age , hypertension and diabetes mellitus status ( severity and duration ) , lipid profile , cigarette smoking status , alcohol intake , micro / macroalbuminuria , renal function , and medications . two crosssectional studies have examined the association between sleepdisordered breathing and retinal vascular signs in the general population . in the sleep heart health study,17 boland et al reported no association between ahi and most retinal microvascular abnormalities , with the exceptions of an association with microaneurysms and smaller retinal arterioletovenule ratio , where the inverse linear relationship was blunted when the ahi was > 10 . in the wisconsin sleep cohort study,18 shankar et al subsequently reported that moderate / severe sleepdisordered breathing ( ahi 15 ) was associated with retinal venular widening , but not with retinal arteriolar narrowing in men or women . rudrappa et al showed that osa may promote diabetic retinopathy , whereas banerjee et al reported no association.19 , 20 in line with sleep heart health study and wisconsin sleep cohort study,17 , 18 our analysis revealed a nonlinear inverse association between ahi and retinal arteriolar calibers , an association between moderate / severe osa and retinal venular widening in men , and an association between severe osa with retinal microaneurysms in women . those findings from the earlier study in mesa appear to contradict the present study results.21 although pdsa may be a useful surrogate for more severe osa in mesa,35 it is likely those with unrecognized osa might be misclassified to the unaffected group . in fact , the median ahi levels , obtained by the psg in the mesa visit 5 for men selfreporting in mesa visit 2 as unaffected , habitual snoring , and pdsa , were 10.8 , 15.3 , and 25.9 , respectively . more than half of the men selfreporting that they were unaffected at visit 2 actually had osa when objectively measured by psg at visit 5 . since the association of osa with retinal arteriolar calibers was not dose dependent in men , a negative result using selfreport data may be erroneous and a direct result of osa misclassification in the reference group . furthermore , unrecognized sleep apnea may be more common in women , in whom a pdsa may identify the most severely affected individuals . in contrast to the 70 women with pdsa by selfreport at mesa visit 2 ( 2.3% of the total female cohort in the prior mesa analysis ) , in the present study , there were 87 women with severe osa and 229 women with moderate or severe osa , accounting for 8.8% and 23.1% of the total female cohort , respectively . in the earlier analysis , women with pdsa were older and had more severe osa resulting in a positive association between pdsa and retinal arteriolar narrowing . several mechanisms may account for the nonlinear inverse association between osa severity and narrower retinal arterioles in men . retinal arterioles are dilated in response to hypoxia.36 once reoxygenation occurs , retinal arterioles are constricted due to elevated concentrations of endothelin1 acting on eta receptors.37 , 38 endothelin1 concentrations may be increased proportionally to osa severity,39 and have a dosedependent effect on retinal arteriolar constrictions.40 the effect of endothelin1 may be modified by antihypertensive therapy in osa . for example , use of blockers could reduce the secretion of endothelin1 from endothelium,41 and calcium channel blockers attenuate the effect of endothelin1 on arteriolar constrictions.42 therefore , the blunting ahi association with smaller retinal arterioles in men may be explained by multiantihypertensive medications with highdose use for resistant hypertension in severe osa . the sensitivity of eta receptors to endothelin1 is much stronger in men than in women,43 which may explain the observed sex difference in the association between osa and retinal arteriolar narrowing . additionally , an animal study suggested that the sensitivity to endothelin1 may decline with age , especially in females.44 on the other hand , men but not women with severe osa have been reported with higher risk of cardiac remodeling such as congestive heart failure , which elevates pulmonary wedge pressure and may affect retinal venular widening.23 systemic inflammation and hyperglycemia , which are related to severe osa , may cause diabetic retinopathy.3 , 13 , 45 however , men with severe osa seemed to have lower risk of diabetic retinopathy . this finding could be explained partly by the hypothesis that among individuals with severe osa , retinal arteriolar dilatation occurred less in men , which may prevent blood overflow to the retina , possibly lowering the risk of retinal microaneurysms presenting as capillary leak despite chronic inflammation status.46 in addition , some data indicate more endothelial dysfunction in women with osa compared to men,47 possibly increasing the risk of retinal microaneurysms in women with osa . the strengths of our study include an objective classification of osa , the ethnic diversity of the mesa sleep cohort , the availability of digitally acquired retinal data on a variety of specific retinal signs , and the availability of a wide range of data on other covariates . almost all of the women in the current study were postmenopausal , which may mitigate the effect of sex hormones on the associations of osa with retinal signs . data on some potential confounders such as inflammatory markers were not available and , as a result , we were not able to adjust fully for degree of inflammation . only 50.5% of mesa visit 5 participants contributed psg data for this analysis , possibly leading to a selection bias , although the baseline profile of characteristics among those who contributed psg data and those who did not were similar . since psg was only obtained at a single visit , our analysis was crosssectional and could not address the temporal association between osa and retinal microvascular signs . finally , the present study performed many tests , and we are aware this could inflate overall type i error rate . due to the exploratory nature of this research problem , we selected the minimal requirement of multiple comparison procedure to satisfy statistical rigor and used p<0.025 as the significance level for the overall test of osa severity . however , in some comparisons , there were borderline significant results that need to be clarified in further large studies that have sufficient power to detect the statistical difference between groups . in mesa , the prevalence of osa differed by sex as did osa severity . similarly , the associations between osa severity and retinal microvascular signs may differ by sex . moderate / severe osa was associated with retinal vascular calibers in men , but not in women . in contrast , severe osa was associated with retinal microaneurysms in women but not men . further studies are necessary to explain the mechanisms underlying these sexspecific associations . a longitudinal followup for both osa status and retinal microvascular changes will help to clarify any temporal associations . moreover , whether sexspecific effects of osa manifest on the microvasculature in other sites , including arterioles , venules , and vasa vasorum , also deserves investigation . this research was supported by contracts n01hc95159 through n01hc95169 from the national heart , lung , and blood institute at the national institutes of health , by grants ul1tr000040 , ul1rr025005 from the national center for research resources , r01hl098433 ( mesa sleep , r21 hl121348 ) and t32hl069764 . support for the visit 5 retinal component was provided by the intramural research program at the national eye institute , national institutes of health ( ziaey000403 ) .

backgroundobstructive sleep apnea ( osa ) is a common condition affecting more men than women . the relationship of osa with microvascular disease is unclear , complicated by possible sex difference . assessment of the relationship of osa with retinal microvascular signs in men and women may provide insights into such a relationship.methods and resultswe examined the sexspecific crosssectional association of osa severity with retinal vascular calibers in 1808 participants , and with specific retinopathy signs in 1831 participants from a sample of 2060 participants aged 54 to 93 years who underwent successful polysomnography in the multiethnic study of atherosclerosis , 20102012 . osa severity was defined by the apnea hypopnea index ( events / h ) as none ( < 5 ) , mild ( 514.9 ) , moderate ( 1529.9 ) , and severe ( 30 ) . as compared to no osa , moderate / severe osa in men was associated with retinal arteriolar narrowing ( odds ratio [ or ] and 95% ci for the narrowest quartile : 1.65 [ 1.002.71 ] ) and retinal venular widening ( 1.80 [ 1.073.04 ] for the widest quartile ) , but not in women ( odds ratio : 1.10 [ 0.671.81 ] and 0.91 [ 0.581.43 ] , respectively ) after adjusting for age , race / ethnicity , body mass index , packyears of cigarette smoking , alcohol intake , hypertension duration , diabetes mellitus duration , hba1c levels , lipid profile , micro/macroalbuminuria , estimated glomerular filtration rate , blockers use , antihypertensive therapy , and lipidlowering therapy . in contrast , severe osa was associated with retinal microaneurysms in women , but not in men ( odds ratio : 3.22 [ 1.168.97 ] and 0.59 [ 0.271.30 ] , respectively).conclusionsthe associations of osa severity with retinal microvascular signs may differ by sex . whether these findings were related to sex differences in osa exposure needs further investigation .

Introduction Methods Study Population and Data Collection PSG and OSA Definition Retinal Photography and Retinal Grading Assessment of Risk Factors for OSA and Retinal Microvascular Signs Statistical Analysis Results Discussion Conclusions Sources of Funding Disclosures

notably , results from a recent analysis of the multiethnic study of atherosclerosis ( mesa ) data visit 2 ( 20022004 ) , in which a sample of 5803 participants underwent both a selfadministered sleep history questionnaire and retinal photography , found that physiciandiagnosed sleep apnea ( pdsa ) was associated with retinal arteriolar narrowing in women only.21 there was no association between pdsa and retinal vascular calibers in men . the severity of osa was classified by ahi ( events / h ) as none ( < 5 ) , mild ( 514.9 ) , moderate ( 1530 ) , and severe ( 30 ) . model 3 included comprehensive adjustments for age , race / ethnicity , bmi , packyears of cigarette smoking , alcohol intake , duration of hypertension and diabetes mellitus ( duration was defined as none , longer , or shorter than an average 9.5year period between the first and the fifth visit of mesa ) , hba1c , total cholesterol , highdensity lipoprotein cholesterol , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , medication with blockers , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . the severity of osa was classified by ahi ( events / h ) as none ( < 5 ) , mild ( 514.9 ) , moderate ( 1530 ) , and severe ( 30 ) . model 3 included comprehensive adjustments for age , race / ethnicity , bmi , packyears of cigarette smoking , alcohol intake , duration of hypertension and diabetes mellitus ( duration was defined as none , longer , or shorter than an average 9.5year period between the first and the fifth visit of mesa ) , hba1c , total cholesterol , highdensity lipoprotein cholesterol , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , medication with blockers , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . after adjusting for age and race / ethnicity , mild and moderate / severe osa were associated with increased odds of retinal arteriolar narrowing in men ( odds ratios [ or ] : 2.26 and 1.91 , and overall p value : 0.003 ) , but not in women . after additionally adjusting for bmi , systolic bp , diabetes mellitus status , total cholesterol , highdensity lipoprotein cholesterol , smoking status , alcohol intake , antihypertensive therapy , and lipidlowering therapy , the overall osa association remained significant ( p=0.014 ) : mild osa was significant ( or : 2.08 , p=0.006 ) and moderate / severe osa was marginally significant ( or : 1.66 , p=0.052 ) for retinal arteriolar narrowing in men . model 3 : model 1 plus further adjustments for body mass index , duration of hypertension and diabetes mellitus ( longer or shorter than an average 9.5year period between mesa visit 1 and 5 ) , hba1c , packyears cigarette smoking , alcohol status , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , blocker medication , total and highdensity lipoprotein cholesterol , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . linear regression model of osa severity with retinal vascular calibers in men and women linear regression model adjusted for age , race , body mass index , duration of hypertension and diabetes mellitus ( longer or shorter than an average 9.5year period between mesa visit 1 and visit 5 ) , hba1c , packyears cigarette smoking , alcohol status , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , blockers medication , total and highdensity lipoprotein cholesterol , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . linear regression model of ahi with retinal vascular calibers in men and women linear regression model adjusted for age , race / ethnicity , body mass index , duration of hypertension and diabetes mellitus ( none , longer or shorter than an average 9.5year period between mesa visit 1 and visit 5 ) , hba1c , packyears cigarette smoking , alcohol status , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , blockers medication , total and highdensity lipoprotein cholesterol , lipidlowering therapy , and current hormone replacement therapy ( for women only ) . model 3 : model 1 plus further adjustments for body mass index , duration of hypertension and diabetes mellitus ( longer or shorter than an average 9.5year period between mesa visit 1 and 5 ) , hba1c , packyears cigarette smoking , alcohol status , estimated glomerular filtration rate , micro/macroalbuminuria , antihypertensive therapy , blocker medication , total and highdensity lipoprotein cholesterol , lipidlowering therapy , and current hormone replacement therapy ( for women only ) .

the positron emission tomography ( pet ) tracer 3-deoxy-3-[f]fluorothymidine ( [ f]flt ) , a thymidine analogue that is a marker of tumour proliferation , is a promising tracer for monitoring treatment response and predicting outcome . it has been shown that [ f]flt uptake strongly correlates with the proliferation index as measured by ki-67 immunohistochemistry in lung and breast tumours , as well as with thymidine kinase-1 expression in lung tumours . the gold standard for analysing tracer uptake in tissue is by non - linear regression ( nlr ) of operational equations based on compartmental models . in case of [ f]flt , a two - tissue compartment model is used and , in general , it is assumed that phosphorylated tracer is irreversibly trapped [ 410 ] . when scan durations longer than 60 min are used , dephosphorylation can no longer be neglected and a two - tissue reversible compartment model may be preferred . alternatively , a basis function approach , not requiring prior assumptions about the exact underlying compartment model , can be used . a good correlation between net uptake rate ki of [ f]flt and although full kinetic analysis is , in principle , the most accurate method for determining net uptake of [ f]flt , it is also relatively complex , at the expense of clinical applicability . two simplified methods often are used to ( semi-)quantitatively assess [ f]flt uptake : graphical ( patlak ) analysis and standardised uptake values ( suv ) . patlak analysis assumes irreversible trapping in tissue , and its accuracy thus depends on the assumption that no significant dephosphorylation occurs within the time course of the study . both nlr and patlak measure net uptake of [ f]flt , taking into account the concentration of tracer in plasma during the course of the study . only nlr , however , allows for measurements of individual rate constants between compartments and for an implicit correction for blood volume in the tissue of interest . suv is the ratio of tissue concentration and injected activity at a certain time after administration of the tracer . it does not take tracer kinetics into account but has the advantage that it is a single - scan procedure that does not require plasma data . previous studies have shown a good correlation between [ f]flt suv and net uptake calculated using patlak analysis in for example untreated lung cancer and breast cancer patients , as well as in patients with recurrent glioma [ 8 , 11 , 13 ] . kenny and co - workers showed that both suv and patlak - derived ki predicted response to chemotherapy for breast cancer after the first cycle of chemotherapy with good reproducibility . they did not specifically address the relationship of changes in suv with those in nlr - derived ki . for simplified uptake measures to be valid for monitoring response or predicting outcome , their relationship with the more accurate outcome measures of full kinetic analysis must be similar before and after therapy . chemotherapy , however , might alter the correlations between nlr , patlak and suv , as has previously been shown for 2-deoxy-2-[f]fluoro - d - glucose . this could be due to changes in tumour blood flow , blood volume or plasma clearance of the tracer . the changes are accounted for in full kinetic analysis ( nlr ) , but not in the use of suv . in those cases , therefore , the aim of the present study was to validate simplified [ f]flt uptake measures for monitoring response to chemotherapy in locally advanced breast cancer ( labc ) by evaluating their relationships with nlr before and after chemotherapy . data were used from 15 labc patients participating in an ongoing response monitoring study , the protocol of which was approved by the medical ethics review committee of the vu university medical centre and for which all patients had given written informed consent . patients underwent an [ f]flt scan shortly before the start of chemotherapy and again 3 weeks later , shortly before the second cycle of chemotherapy in case of traditional , three - weekly schemes ( n = 12 ) or before the fourth cycle in case of weekly schemes ( n = 3 ) . the median delay between baseline pet scan and pet scans were performed using an ecat exact hr+ scanner ( siemens / cti , knoxville , tn ) . first , a 10-min transmission scan over the tumour area was performed using three retractable ge / ga line sources . a 60-min dynamic emission scan ( 6 5 , 6 10 , 3 20 , 5 30 , 5 60 , 8 150 , 6 300 s ) was subsequently performed in 2d acquisition mode after bolus injection of ~370 mbq [ f]flt . during the [ f]flt scan , six venous samples were drawn at set times , both for immediate measurement of whole blood and plasma radioactivity concentrations and for measurement of metabolite fractions using solid - phase extraction chromatography to separate flt from flt - glucuronide . this solution was brought onto a seppak vac 6-cc ( 1 g ) c18 cartridge ( waters corporation , milford , ma ) . the eluate was collected , after which the cartridge was rinsed with 5 ml water to collect polar metabolites , primarily being [ f]flt - glucuronide . the cartridge was then rinsed with 5 ml ethanol 96% to collect the parent compound . all fractions and the cartridge were counted using a wallac 1480 wizard well counter ( perkin - elmer life science , zaventem , belgium ) , and the percentage parent within each plasma sample calculated . scan data were normalised and corrected for dead time , decay , scattered radiation , random coincidences and photon attenuation , and were reconstructed using filtered back projection ( fbp ) with a hanning filter ( cut - off 0.5 cycles / pixel ) . this resulted in a transaxial spatial resolution of approximately 7 mm full width at half maximum ( fwhm ) . for region of interest ( roi ) definition , the last three frames ( i.e. , 4560 min p.i . ) were summed and reconstructed using attenuation - weighted ordered subset expectation maximisation with two iterations and 16 subsets , followed by post smoothing using a 5-mm fwhm gaussian filter to obtain the same resolution as the dynamic images reconstructed with fbp . volumes of interest ( vois ) were defined semi - automatically over the tumour by applying a threshold of 70% of the maximum pixel value within the lesion . arterial tacs were measured using 1.5-cm - diameter circular rois manually defined over the aortic arch , ascending aorta , left atrium and left ventricle in the frame of the fbp - reconstructed dynamic image in which the injected bolus was best seen passing through these structures [ 16 , 17 ] . absolute radioactivity concentration in the arterial tacs was verified using the radioactivity concentrations measured in the blood samples . arterial tacs were then converted to image - derived input functions by first multiplying them with a single exponential fit to plasma to whole blood ratio data and subsequently with a sigmoid function fit to parent fraction data . data were analysed using in - house developed software written in matlab ( natick , ma ) . the following analytical methods were applied : net uptake rate ( ki ) was determined by nlr , using reversible or irreversible two - tissue compartment models with four ( nlr4k ) or three ( nlr3k ; k4 = 0 ) parameters , respectively , and a blood volume parameter . the presence of a fourth rate constant k4 and the need to include this in the nlr model were assessed by comparing residual sum of squares of fits with and without a k4 parameter using the akaike and schwarz criteria .patlak analysis , giving net uptake rate ki , using the 1060-min post - injection data , suv for the 5060-min interval normalised to body weight , andtumour - to - whole blood ratio ( tbr ) , i.e. , tumour suv normalised to whole blood suv at 5060 min p.i . , obtained from the arterial tac . net uptake rate ( ki ) was determined by nlr , using reversible or irreversible two - tissue compartment models with four ( nlr4k ) or three ( nlr3k ; k4 = 0 ) parameters , respectively , and a blood volume parameter . the presence of a fourth rate constant k4 and the need to include this in the nlr model were assessed by comparing residual sum of squares of fits with and without a k4 parameter using the akaike and schwarz criteria . patlak analysis , giving net uptake rate ki , using the 1060-min post - injection data , suv for the 5060-min interval normalised to body weight , and tumour - to - whole blood ratio ( tbr ) , i.e. , tumour suv normalised to whole blood suv at 5060 min p.i . , correlation and agreement between all measures of [ f]flt uptake and nlr were assessed using orthogonal regression , spearman s correlation coefficient and intraclass correlation coefficients ( icc ) . relative and absolute changes in patlak - derived ki , suv and tbr were compared with nlr3k - derived ki using orthogonal regression . confidence intervals of regression parameters were estimated by bootstrapping using 1,000 resamples obtained by random sampling with replacement from the measured data . data were used from 15 labc patients participating in an ongoing response monitoring study , the protocol of which was approved by the medical ethics review committee of the vu university medical centre and for which all patients had given written informed consent . patients underwent an [ f]flt scan shortly before the start of chemotherapy and again 3 weeks later , shortly before the second cycle of chemotherapy in case of traditional , three - weekly schemes ( n = 12 ) or before the fourth cycle in case of weekly schemes ( n = 3 ) . the median delay between baseline pet scan and pet scans were performed using an ecat exact hr+ scanner ( siemens / cti , knoxville , tn ) . first , a 10-min transmission scan over the tumour area was performed using three retractable ge / ga line sources . a 60-min dynamic emission scan ( 6 5 , 6 10 , 3 20 , 5 30 , 5 60 , 8 150 , 6 300 s ) was subsequently performed in 2d acquisition mode after bolus injection of ~370 mbq [ f]flt . during the [ f]flt scan , six venous samples were drawn at set times , both for immediate measurement of whole blood and plasma radioactivity concentrations and for measurement of metabolite fractions using solid - phase extraction chromatography to separate flt from flt - glucuronide . this solution was brought onto a seppak vac 6-cc ( 1 g ) c18 cartridge ( waters corporation , milford , ma ) . the eluate was collected , after which the cartridge was rinsed with 5 ml water to collect polar metabolites , primarily being [ f]flt - glucuronide . the cartridge was then rinsed with 5 ml ethanol 96% to collect the parent compound . all fractions and the cartridge were counted using a wallac 1480 wizard well counter ( perkin - elmer life science , zaventem , belgium ) , and the percentage parent within each plasma sample calculated . scan data were normalised and corrected for dead time , decay , scattered radiation , random coincidences and photon attenuation , and were reconstructed using filtered back projection ( fbp ) with a hanning filter ( cut - off 0.5 cycles / pixel ) . this resulted in a transaxial spatial resolution of approximately 7 mm full width at half maximum ( fwhm ) . for region of interest ( roi ) definition , the last three frames ( i.e. , 4560 min p.i . ) were summed and reconstructed using attenuation - weighted ordered subset expectation maximisation with two iterations and 16 subsets , followed by post smoothing using a 5-mm fwhm gaussian filter to obtain the same resolution as the dynamic images reconstructed with fbp . volumes of interest ( vois ) were defined semi - automatically over the tumour by applying a threshold of 70% of the maximum pixel value within the lesion . arterial tacs were measured using 1.5-cm - diameter circular rois manually defined over the aortic arch , ascending aorta , left atrium and left ventricle in the frame of the fbp - reconstructed dynamic image in which the injected bolus was best seen passing through these structures [ 16 , 17 ] . absolute radioactivity concentration in the arterial tacs was verified using the radioactivity concentrations measured in the blood samples . arterial tacs were then converted to image - derived input functions by first multiplying them with a single exponential fit to plasma to whole blood ratio data and subsequently with a sigmoid function fit to parent fraction data . data were analysed using in - house developed software written in matlab ( natick , ma ) . the following analytical methods were applied : net uptake rate ( ki ) was determined by nlr , using reversible or irreversible two - tissue compartment models with four ( nlr4k ) or three ( nlr3k ; k4 = 0 ) parameters , respectively , and a blood volume parameter . the presence of a fourth rate constant k4 and the need to include this in the nlr model were assessed by comparing residual sum of squares of fits with and without a k4 parameter using the akaike and schwarz criteria .patlak analysis , giving net uptake rate ki , using the 1060-min post - injection data , suv for the 5060-min interval normalised to body weight , andtumour - to - whole blood ratio ( tbr ) , i.e. , tumour suv normalised to whole blood suv at 5060 min p.i . , obtained from the arterial tac . net uptake rate ( ki ) was determined by nlr , using reversible or irreversible two - tissue compartment models with four ( nlr4k ) or three ( nlr3k ; k4 = 0 ) parameters , respectively , and a blood volume parameter . the presence of a fourth rate constant k4 and the need to include this in the nlr model were assessed by comparing residual sum of squares of fits with and without a k4 parameter using the akaike and schwarz criteria . patlak analysis , giving net uptake rate ki , using the 1060-min post - injection data , suv for the 5060-min interval normalised to body weight , and tumour - to - whole blood ratio ( tbr ) , i.e. , tumour suv normalised to whole blood suv at 5060 min p.i . , obtained from the arterial tac . correlation and agreement between all measures of [ f]flt uptake and nlr were assessed using orthogonal regression , spearman s correlation coefficient and intraclass correlation coefficients ( icc ) . relative and absolute changes in patlak - derived ki , suv and tbr were compared with nlr3k - derived ki using orthogonal regression . confidence intervals of regression parameters were estimated by bootstrapping using 1,000 resamples obtained by random sampling with replacement from the measured data . for one patient , [ f]flt scans were excluded because no good fit could be obtained for the second scan , probably due to patient movement . the mean fraction of parent [ f]flt at 60 min p.i . was 79% and 80% for baseline and post - chemotherapy measurements , respectively , with a range of 7185% at baseline and of 7484% after chemotherapy . there were no significant differences between pre- and post - therapy values ( p = 0.68 ) . were significantly lower for post - chemotherapy [ f]flt scans than for baseline scans ( whole blood : mean ( sd ) suv 0.57 ( 0.09 ) versus 0.61 ( 0.09 ) , p = 0.05 , and plasma : mean ( sd ) suv 0.66 ( 0.12 ) versus 0.72 ( 0.10 ) , p = 0.02 ) . the nlr3k model provided better fits than the nlr4k model in 17 out of 28 ( 61% ) [ f]flt scans , respectively , according to the akaike criterion , with similar results for the schwarz criterion . based on these criteria , a fourth rate constant could not be reliably identified and therefore nlr3k was used in the remainder of this study . figure 1 shows net uptake rates of [ f]flt as measured using patlak analysis versus those measured using nlr3k . no significant differences between baseline and post - chemotherapy slopes of the relationships between patlak and nlr were found.fig . nlr3k - derived ki at baseline ( closed circles , solid line ) and post - chemotherapy ( open circles , dashed line ) . the lines are orthogonal regressions.table 1[f]flt simplified measures versus nlrnlr3kki patlak kinlr3kki suvnlr3kki tbrspearman s rho0.980.960.96icc0.98n.a.n.a.slope baseline ( ci)1.01 ( 0.901.13)101 ( 90113)140 ( 119164)intercept baseline ( ci)0.003 ( 0.005 to 0.000)0.34 ( 0.00 to 0.75)0.16 ( 0.42 to 0.64)slope post - therapy ( ci)1.00 ( 0.861.17)108 ( 95142)147 ( 136179)intercept post - therapy ( ci)0.002 ( 0.006 to 0.001)0.09 ( 0.49 to 0.37)0.15 ( 0.41 to 0.41)n.a . not available correlation of patlak - derived kiversus nlr3k - derived ki at baseline ( closed circles , solid line ) and post - chemotherapy ( open circles , dashed line ) . [ f]flt simplified measures versus nlr figure 2 shows correlations between suv , tbr and nlr3k - derived ki . a non - significant post - chemotherapy increase in slope between nlr3k - derived ki and suv of about 7% was found.fig . 2correlation of a suv and b tbr versus nlr3k - derived ki , at baseline ( closed circles , solid line ) and post - chemotherapy ( open circles , dashed line ) . correlation of a suv and b tbr versus nlr3k - derived ki , at baseline ( closed circles , solid line ) and post - chemotherapy ( open circles , dashed line ) . . figures 3 and 4 and table 2 show relative changes in simplified uptake measures versus those obtained for nlr3k - derived ki after chemotherapy . the slope of suv versus ki was significantly smaller than one ( 0.69 , confidence interval ci 0.57 to 0.88 ) , and a significant negative bias of 0.12 ( ci 0.16 to 0.05 ) in suv was seen . absolute changes in suv showed a significant bias of 0.20 ( ci 0.37 to 0.03 ) as well . the slope of tbr versus ki was not significantly different from one ( 0.82 , ci 0.561.13 ) , and bias was smaller and non - significant ( 0.03 , ci 0.12 to 0.09 ) as well , although the correlation between tbr and ki ( rho = 0.93 ) was slightly lower than between suv and ki ( rho = 0.96).fig . the solid line is an orthogonal regression ; the dashed lines show the 95% confidence interval of the regression line.fig . the solid lines are orthogonal regressions ; the dashed lines show the 95% confidence intervals of the regression lines.table 2[f]flt : relative change in simplified measures versus nlrpatlak kisuvtbrspearman s rho0.810.960.93icc0.890.900.88slope ( ci)1.14 ( 0.851.39)0.69 ( 0.570.88)0.82 ( 0.561.13)intercept ( ci)0.06 ( 0.08 to 0.20)0.12 ( 0.16 0.05)0.03 ( 0.12 to 0.09 ) correlation between relative change in patlak ki and nlr3kki . the solid line is an orthogonal regression ; the dashed lines show the 95% confidence interval of the regression line . correlation between relative change in a suv and b tbr versus nlr3kki . the solid lines are orthogonal regressions ; the dashed lines show the 95% confidence intervals of the regression lines . for one patient , [ f]flt scans were excluded because no good fit could be obtained for the second scan , probably due to patient movement . the mean fraction of parent [ f]flt at 60 min p.i . was 79% and 80% for baseline and post - chemotherapy measurements , respectively , with a range of 7185% at baseline and of 7484% after chemotherapy . there were no significant differences between pre- and post - therapy values ( p = 0.68 ) . were significantly lower for post - chemotherapy [ f]flt scans than for baseline scans ( whole blood : mean ( sd ) suv 0.57 ( 0.09 ) versus 0.61 ( 0.09 ) , p = 0.05 , and plasma : mean ( sd ) suv 0.66 ( 0.12 ) versus 0.72 ( 0.10 ) , p = 0.02 ) . the nlr3k model provided better fits than the nlr4k model in 17 out of 28 ( 61% ) [ f]flt scans , respectively , according to the akaike criterion , with similar results for the schwarz criterion . based on these criteria , a fourth rate constant could not be reliably identified and therefore nlr3k was used in the remainder of this study . figure 1 shows net uptake rates of [ f]flt as measured using patlak analysis versus those measured using nlr3k . no significant differences between baseline and post - chemotherapy slopes of the relationships between patlak and nlr were found.fig . 1correlation of patlak - derived kiversus nlr3k - derived ki at baseline ( closed circles , solid line ) and post - chemotherapy ( open circles , dashed line ) . the lines are orthogonal regressions.table 1[f]flt simplified measures versus nlrnlr3kki patlak kinlr3kki suvnlr3kki tbrspearman s rho0.980.960.96icc0.98n.a.n.a.slope baseline ( ci)1.01 ( 0.901.13)101 ( 90113)140 ( 119164)intercept baseline ( ci)0.003 ( 0.005 to 0.000)0.34 ( 0.00 to 0.75)0.16 ( 0.42 to 0.64)slope post - therapy ( ci)1.00 ( 0.861.17)108 ( 95142)147 ( 136179)intercept post - therapy ( ci)0.002 ( 0.006 to 0.001)0.09 ( 0.49 to 0.37)0.15 ( 0.41 to 0.41)n.a . not available correlation of patlak - derived kiversus nlr3k - derived ki at baseline ( closed circles , solid line ) and post - chemotherapy ( open circles , dashed line ) . [ f]flt simplified measures versus nlr figure 2 shows correlations between suv , tbr and nlr3k - derived ki . a non - significant post - chemotherapy increase in slope between nlr3k - derived ki and suv of about 7% was found.fig . 2correlation of a suv and b tbr versus nlr3k - derived ki , at baseline ( closed circles , solid line ) and post - chemotherapy ( open circles , dashed line ) . correlation of a suv and b tbr versus nlr3k - derived ki , at baseline ( closed circles , solid line ) and post - chemotherapy ( open circles , dashed line ) . the lines are orthogonal regressions . figures 3 and 4 and table 2 show relative changes in simplified uptake measures versus those obtained for nlr3k - derived ki after chemotherapy . the slope of suv versus ki was significantly smaller than one ( 0.69 , confidence interval ci 0.57 to 0.88 ) , and a significant negative bias of 0.12 ( ci 0.16 to 0.05 ) in suv was seen . absolute changes in suv showed a significant bias of 0.20 ( ci 0.37 to 0.03 ) as well . the slope of tbr versus ki was not significantly different from one ( 0.82 , ci 0.561.13 ) , and bias was smaller and non - significant ( 0.03 , ci 0.12 to 0.09 ) as well , although the correlation between tbr and ki ( rho = 0.93 ) was slightly lower than between suv and ki ( rho = 0.96).fig . the solid line is an orthogonal regression ; the dashed lines show the 95% confidence interval of the regression line.fig . the solid lines are orthogonal regressions ; the dashed lines show the 95% confidence intervals of the regression lines.table 2[f]flt : relative change in simplified measures versus nlrpatlak kisuvtbrspearman s rho0.810.960.93icc0.890.900.88slope ( ci)1.14 ( 0.851.39)0.69 ( 0.570.88)0.82 ( 0.561.13)intercept ( ci)0.06 ( 0.08 to 0.20)0.12 ( 0.16 0.05)0.03 ( 0.12 to 0.09 ) correlation between relative change in patlak ki and nlr3kki . the solid line is an orthogonal regression ; the dashed lines show the 95% confidence interval of the regression line . correlation between relative change in a suv and b tbr versus nlr3kki . the solid lines are orthogonal regressions ; the dashed lines show the 95% confidence intervals of the regression lines . in the present study , the use of simplified uptake measures for measuring breast cancer treatment response using [ f]flt was compared to non - linear regression , i.e. , to full tracer kinetic analysis . although previous studies have shown a good correlation between [ f]flt suv and net uptake calculated using nlr and patlak analysis in untreated lung cancer and breast cancer patients [ 8 , 11 ] , the present study also addresses correlation between responses as measured using nlr and simplified methods . therapy - induced changes in suv were negatively biased compared to changes in nlr - derived ki , with no change in ki corresponding to an 11% decrease in suv . as [ f]flt uptake is primarily mediated by tk-1 activity , it can be argued that k3 may be a more accurate predictor of tumour proliferation than ki , which is also dependent on other factors ( i.e. , blood flow ) . unfortunately , however , the accuracy of nlr in determining micro parameters such as k3 is far lower than that of ki , with more than half of the individual k3 measurements showing standard errors of 20% or more . since previous studies have shown good correlation between nlr - derived ki and the proliferation marker ki-67 , as determined by immunohistochemistry , this ki was chosen as the standard in the present study . the use of a fourth parameter has been suggested for [ f]flt based on the potentially reversible behaviour of the tracer . however , apart from the three - parameter model being preferred by the akaike and schwartz criteria in the present study , ki values derived from the reversible model showed a much higher uncertainty than those determined using the irreversible model . in addition , patlak plots of the 1060-min interval were linear , which suggests that in the present study no dephosphorylation of [ f]flt occurs during the first 60-min p.i . as shown previously [ 5 , 10 , 21 ] , results of the patlak analysis correlated very well with nlr3k . in addition , no differences in pre- and post - chemotherapy relationships with nlr3k were found , and there was a high correlation between changes in the patlak and nlr3k - derived ki values . however , as patlak analysis still requires dynamic scanning and a plasma input function , it is generally not considered to be a measure that can be used in routine clinical practice . the fact that a significant negative bias in suv compared to ki was found for [ f]flt suggests that less tracer was available for uptake into malignant tissue , possibly because of higher post - chemotherapy uptake in other ( normal ) tissues as a result of the treatment . this is confirmed by a significantly lower plasma radioactivity concentration at 60 min p.i . a reduction in tumour perfusion would cause a decrease in both suv and ki and can therefore not explain the preferential reduction of suv . figure 4 suggests that suv is considerably less sensitive than nlr3k in detecting therapy - induced changes in tumour metabolism , with a slope of suv versus ki of about 0.7 . however , previous studies have shown that test retest variability of [ f]flt suv is considerably better than that of ki [ 14 , 20 ] . in general , an approximately 30% larger change in ki than in suv had to be found for it to be considered as a response . the lower sensitivity but better reproducibility of suv suggests that suv and ki have comparable sensitivity in response monitoring , with suv showing a negative bias . potentially , tbr , the change of which does not show a negative bias relative to ki , could be a better measure for treatment response than suv , provided its test retest variability is comparable to or better than that of suv . the clinical relevance of the differences between suv and tbr , as found in the present work , will be assessed in a clinical study . in the present work , tbr was calculated using the mean value of the arterial tac between 50 and 60 min . in 11 out of 14 patients , a venous whole blood sample taken between 55 and 60 min p.i . was available . for these 11 patients , correlation and agreement between tbr and ki were better when tbr was based on blood sample data than on image - derived blood data ( pearson s r 0.92 versus 0.81 ; spearman s rho 0.95 for both cases ) , whilst the relation between suv or image - based tbr and ki was similar for these 11 patients as for the complete group of 14 patients ( fig . 5 ) . this suggests that , probably because of the noisy nature of the arterial tac at 5060 min p.i . , use of a blood sample might produce more robust tbr values . although tbr , as opposed to suv , does take post - therapy changes in whole blood clearance into account , it can not account for therapy - induced changes in metabolism . if major changes in metabolism are observed for a certain type of chemotherapy , the validity of tbr has to be addressed by comparison to full kinetic modelling using nlr with a metabolite - corrected plasma input function , and full kinetic modelling may be preferable.fig . 5correlation between relative change in tbr versus nlr3kki , using a blood sample at 5560 min p.i . for calculation of tbr instead of an ascending aorta voi . the solid line is an orthogonal regression ; the dashed lines show the 95% confidence interval of the regression line . correlation between relative change in tbr versus nlr3kki , using a blood sample at 5560 min p.i . for calculation of tbr instead of an ascending aorta voi . the solid line is an orthogonal regression ; the dashed lines show the 95% confidence interval of the regression line . for [ f]flt , change in suv was negatively biased compared to change in nlr3k - derived ki , with no change in ki corresponding to a significant decrease in suv . use of tbr did not show this bias and has a similar correlation to nlr3k - derived ki . therefore , tumour - to - whole blood ratio may be preferred to suv as a simplified measure for monitoring response to chemotherapy in labc when using [ f]flt . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

purposepositron emission tomography using 3-deoxy-3-[18f]fluorothymidine ( [ 18f]flt ) has been suggested as a means for monitoring response to chemotherapy . the aim of this study was to evaluate the validity of simplified uptake measures for assessing response to chemotherapy using [ 18f]flt in locally advanced breast cancer ( labc).proceduresfifteen labc patients underwent dynamic [ 18f]flt scans both prior to and after the first cycle of chemotherapy with fluorouracil , epirubicin or doxorubicin , and cyclophosphamide . the net uptake rate constant of [ 18f]flt , ki , determined by non - linear regression ( nlr ) of an irreversible two - tissue compartment model was used as the gold standard . in addition to patlak graphical analysis , standardised uptake values ( suv ) and tumour - to - whole blood ratio ( tbr ) were used for analysing [ 18f]flt data . correlations and relationships between simplified uptake measures and nlr before and after chemotherapy were assessed using regression analysis.resultsno significant differences in both pre- and post - chemotherapy relationships between any of the simplified uptake measures and nlr were found . however , changes in suv between baseline and post - therapy scans showed a significant negative bias and slope less than one , while tbr did not.conclusionsin labc , tbr instead of suv may be preferred for monitoring response to chemotherapy with [ 18f]flt .

Introduction Materials and Methods Patients PET Acquisition Protocol Image Reconstruction and Processing Data Analysis Results Patients and Scans Data Analysis Discussion Conclusion Conflict of Interest Open Access

the gold standard for analysing tracer uptake in tissue is by non - linear regression ( nlr ) of operational equations based on compartmental models . kenny and co - workers showed that both suv and patlak - derived ki predicted response to chemotherapy for breast cancer after the first cycle of chemotherapy with good reproducibility . in those cases , therefore , the aim of the present study was to validate simplified [ f]flt uptake measures for monitoring response to chemotherapy in locally advanced breast cancer ( labc ) by evaluating their relationships with nlr before and after chemotherapy . the following analytical methods were applied : net uptake rate ( ki ) was determined by nlr , using reversible or irreversible two - tissue compartment models with four ( nlr4k ) or three ( nlr3k ; k4 = 0 ) parameters , respectively , and a blood volume parameter . the presence of a fourth rate constant k4 and the need to include this in the nlr model were assessed by comparing residual sum of squares of fits with and without a k4 parameter using the akaike and schwarz criteria .patlak analysis , giving net uptake rate ki , using the 1060-min post - injection data , suv for the 5060-min interval normalised to body weight , andtumour - to - whole blood ratio ( tbr ) , i.e. net uptake rate ( ki ) was determined by nlr , using reversible or irreversible two - tissue compartment models with four ( nlr4k ) or three ( nlr3k ; k4 = 0 ) parameters , respectively , and a blood volume parameter . patlak analysis , giving net uptake rate ki , using the 1060-min post - injection data , suv for the 5060-min interval normalised to body weight , and tumour - to - whole blood ratio ( tbr ) , i.e. the following analytical methods were applied : net uptake rate ( ki ) was determined by nlr , using reversible or irreversible two - tissue compartment models with four ( nlr4k ) or three ( nlr3k ; k4 = 0 ) parameters , respectively , and a blood volume parameter . the presence of a fourth rate constant k4 and the need to include this in the nlr model were assessed by comparing residual sum of squares of fits with and without a k4 parameter using the akaike and schwarz criteria .patlak analysis , giving net uptake rate ki , using the 1060-min post - injection data , suv for the 5060-min interval normalised to body weight , andtumour - to - whole blood ratio ( tbr ) , i.e. net uptake rate ( ki ) was determined by nlr , using reversible or irreversible two - tissue compartment models with four ( nlr4k ) or three ( nlr3k ; k4 = 0 ) parameters , respectively , and a blood volume parameter . patlak analysis , giving net uptake rate ki , using the 1060-min post - injection data , suv for the 5060-min interval normalised to body weight , and tumour - to - whole blood ratio ( tbr ) , i.e. there were no significant differences between pre- and post - therapy values ( p = 0.68 ) . no significant differences between baseline and post - chemotherapy slopes of the relationships between patlak and nlr were found.fig . the slope of suv versus ki was significantly smaller than one ( 0.69 , confidence interval ci 0.57 to 0.88 ) , and a significant negative bias of 0.12 ( ci 0.16 to 0.05 ) in suv was seen . no significant differences between baseline and post - chemotherapy slopes of the relationships between patlak and nlr were found.fig . the slope of suv versus ki was significantly smaller than one ( 0.69 , confidence interval ci 0.57 to 0.88 ) , and a significant negative bias of 0.12 ( ci 0.16 to 0.05 ) in suv was seen . in the present study , the use of simplified uptake measures for measuring breast cancer treatment response using [ f]flt was compared to non - linear regression , i.e. in addition , no differences in pre- and post - chemotherapy relationships with nlr3k were found , and there was a high correlation between changes in the patlak and nlr3k - derived ki values . the fact that a significant negative bias in suv compared to ki was found for [ f]flt suggests that less tracer was available for uptake into malignant tissue , possibly because of higher post - chemotherapy uptake in other ( normal ) tissues as a result of the treatment . therefore , tumour - to - whole blood ratio may be preferred to suv as a simplified measure for monitoring response to chemotherapy in labc when using [ f]flt .