To evaluate the repeatability, reproducibility, and agreement of thickness profile measurements of eight intra-retinal layers determined by an automated algorithm applied to optical coherence tomography (OCT) images from two different instruments.Twenty normal subjects  were enrolled. Imaging was performed with a custom built ultra-high resolution OCT instrument  and a commercial RTVue100 OCT (∼5 µm resolution) instrument. An automated algorithm was developed to segment the macular retina into eight layers and quantitate the thickness of each layer. The right eye of each subject was imaged two times by the first examiner using each instrument to assess intra-observer repeatability and once by the second examiner to assess inter-observer reproducibility. The intraclass correlation coefficient (ICC) and coefficients of repeatability and reproducibility (COR) were analyzed to evaluate the reliability.The ICCs for the intra-observer repeatability and inter-observer reproducibility of both SD-OCT instruments were greater than 0.945 for the total retina and all intra-retinal layers, except the photoreceptor inner segments, which ranged from 0.051 to 0.643, and the outer segments, which ranged from 0.709 to 0.959. The CORs were less than 6.73% for the total retina and all intra-retinal layers. The total retinal thickness measured by the UHR-OCT was significantly thinner than that measured by the RTVue100. However, the ICC for agreement of the thickness profiles between UHR-OCT and RTVue OCT were greater than 0.80 except for the inner segment and outer segment layers.Thickness measurements of the intra-retinal layers determined by the automated algorithm are reliable when applied to images acquired by the UHR-OCT and RTVue100 instruments. Evaluation of intra-retinal layer thickness plays an important role in the diagnosis and monitoring of various retinal diseases. For example, thinning of the retinal nerve fiber layer (RNFL) is often noted in glaucoma and myopia Optical coherence tomography (OCT) is a noninvasive, noncontact diagnostic tool that can provide in vivo cross-sectional images of the retina with high resolution The use of automated algorithms has undoubtedly enhanced the quantitative diagnosis of ophthalmic disease. To our best knowledge, however, there are few studies that test the reliability of the intra-retinal layer thickness measurements determined by automated segmentation of OCT images. Knowing the level of reliability of such measurements is very important for clinical applications. Thus, the goal of this study was to investigate the repeatability and reproducibility of thickness measurements determined by an automated segmentation algorithm applied to images of eight intra-retinal layers acquired by a custom-built UHR-OCT instrument and a commercially available RTVue100 OCT  instrument.This study followed the tenets of the Declaration of Helsinki and was approved by Ethics Committee of Wenzhou Medical University. Twenty normal subjects  were included, and each signed an informed consent. The inclusion criteria were as follows: no history of ocular or systemic disease, 20/20 or better visual acuity, range of refractive error between −2.00 diopter (D) and +0.50 D, no history of intraocular pressure higher than 21 mmHg, and a normal appearance of the macula.Retinal OCT imaging was performed with two SD-OCT instruments configured as shown in Before imaging, all eyes received an ocular examination including visual acuity testing, autorefraction, intraocular pressure, and ophthalmoscopic examination. After enrollment, all eyes were imaged without mydriasis. Two repeated measurements were performed in a short time on the same day by a single examiner (XL) using both SD-OCT instruments to test the intra-observer repeatability. The OCT imaging was also performed one time by another examiner (LL) using each OCT instrument on the same day to test inter-observer reproducibility. The order of OCT examinations was chosen randomly for each patient. During OCT imaging, the subjects were asked to move their head away from the headrest after each image acquisition, and after five minutes they were asked to reposition their head for the following measurement Custom software for automatic segmentation was developed to measure the thicknesses of eight intra-retinal layers on 2-D images produced by each OCT instrument. The image segmentation algorithm mainly employed graph theory and shortest-path search based on an optimization algorithm of dynamic programming technique as described in a previous study All statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS) software . Descriptive statistics were determined as means ± standard deviations. The intra-observer repeatability was measured with two OCT images obtained by the same operator, and the inter-observer reproducibility was measured with two OCT images obtained by two different operators. The overall mean thickness, the coefficients of repeatability and reproducibility (COR), and intraclass correlation coefficients (ICC) were calculated to evaluate the repeatability and reproducibility of the thickness measurements. The overall mean thicknesses of the eight intra-retinal layers along the central macular 6-mm scan length were determined as the average of the first and second measurement by the same examiner. The COR was defined as the standard deviation of differences between the two measurements divided by the mean value of two different measurements. The ICC was determined based on a mixed-model analysis of variance proposed by Bartko and Carpender The automated algorithm successfully segmented eight intra-retinal layers in the macular images obtained by the UHR-OCT and RTVue100 instruments. The algorithm also determined the thickness profiles of each layer along the 6-mm horizontal  and vertFor the twenty normal eyes, the overall mean total retinal thickness measured with UHR-OCT was 292.17±14.70 µm and 293.83±14.96 µm in horizontal and vertical meridians, respectively. The total retinal thickness measured with RTVue was 305.70±15.73 µm and 309.03±15.07 µm in horizontal and vertical meridians, respectively. The overall mean intra-retinal layers thicknesses ranged from 19.85 to 67.18 µm with UHR-OCT and 20.88 to 72.03 µm with the RTVue100.The overall mean thicknesses of intra-retinal layers measured by UHR-OCT were compared with RTVue100 . There wIt is very important in the clinical routine to know the repeatability and reproducibility of measurements. This information enables the clinician to evaluate if observed changes are due to fluctuations in the methods or if they are valid changes in the structures. This is especially true for the measurements of the intra-retinal layers, which are important morphometric parameters in the diagnosis of retinal and neurological diseases and the monitoring of the progression of these disorders. In this current study, we applied our automated algorithm to images from a custom-built UHR-OCT instrument and commercially available RTVue100 OCT instrument to yield the thickness profiles of eight intra-retinal layers. The intra-observer and inter-observer test results indicated that both instruments produced highly repeatable and reproducible measurements for most of the intra-retinal layers. The results of inter-instrument comparisons of the thickness measurements suggested that the thicknesses of intra-retinal layers obtained by the methods were not interchangeable between the two different SD-OCT instruments.There are many studies showing the repeatability and reproducibility of the retinal measurements for normal subjects. However due to limitations of the image processing software on the commercial OCT instruments, most of them only focus on the RNFL or the total retinal thickness In the current study, we found that the repeatability and reproducibility of the UHR-OCT instrument was better than that of the RTVue100. Additionally, the repeatability of the UHR-OCT with an axial resolution below 2 µm The thickness measurements of most of the intra-retinal layers and the total retina were different between the UHR-OCT and RTVue100. This result was consistent with previously reported studies in which the retinal thickness measurements differed significantly depending upon the OCT systems used. For example, Seibold et al. There are some limitations to the present study. One is the unequal ratio of males to females. Setaro Oeto et al. In conclusion, thickness measurements of the intra-retinal layers have good repeatability and reproducibility when determined by the automated algorithm applied to images acquired by the UHR-OCT and RTVue100 instruments. Use of this algorithm will be helpful in the diagnosis of retinal diseases and the evaluation of disease progression.


Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation of pulmonary artery smooth muscle cell (PASMC) and suppressed apoptosis. This phenotype has been associated with the upregulation of the oncoprotein survivin promoting mitochondrial membrane potential hyperpolarization (decreasing apoptosis) and the upregulation of growth factor and cytokines like PDGF, IL-6 and vasoactive agent like endothelin-1 (ET-1) promoting PASMC proliferation. Krüppel-like factor 5 (KLF5), is a zinc-finger-type transcription factor implicated in the regulation of cell differentiation, proliferation, migration and apoptosis. Recent studies have demonstrated the implication of KLF5 in tissue remodeling in cardiovascular diseases, such as atherosclerosis, restenosis, and cardiac hypertrophy. Nonetheless, the implication of KLF5 in pulmonary arterial hypertension (PAH) remains unknown. We hypothesized that KLF5 up-regulation in PAH triggers PASMC proliferation and resistance to apoptosis.We showed that KFL5 is upregulated in both human lung biopsies and cultured human PASMC isolated from distal pulmonary arteries from PAH patients compared to controls. Using stimulation experiments, we demonstrated that PDGF, ET-1 and IL-6 trigger KLF-5 activation in control PASMC to a level similar to the one seen in PAH-PASMC. Inhibition of the STAT3 pathway abrogates KLF5 activation in PAH-PASMC. Once activated, KLF5 promotes cyclin B1 upregulation and promotes PASMC proliferation and triggers survivin expression hyperpolarizing mitochondria membrane potential decreasing PASMC ability to undergo apoptosis.We demonstrated for the first time that KLF5 is activated in human PAH and implicated in the pro-proliferative and anti-apoptotic phenotype that characterize PAH-PASMC. We believe that our findings will open new avenues of investigation on the role of KLF5 in PAH and might lead to the identification of new therapeutic targets. Inteht exist . Indeed,ht exist . These fThe mechanisms accounting for KLF5 upregulation in PAH remain to be established. Recently, a study from Cheng et al  has suggAlthough our findings will need to be repeated in greater amount of patients and at different stages of PAH to have a better understanding of the role of KLF5 in PAH, nonetheless our study suggests for the first time the implication of KLF5 in the etiology of human PAH. Moreover we provide evidences that its activation might account for many feature seen in PAH, including PASMC proliferation, mitochondrial hyperpolarization, survivin expression and resistance to apoptosis. Because, pulmonary arterial hypertension is a rapidly lethal disease for which treatments are limited, we believe that our findings will open the door to new avenues of investigation and potentially future therapies for PAH.The authors declare that they have no competing interests.in vivo measurements and cell culture experiments. SP helps in manuscript criticism and collaborates for human tissue experiments. SB designed the study, supervised the overall study and wrote the manuscript. All authors have read and approved the manuscript.AC contributed in all the data experiments, analysis and elaborated the figures. VLT performed the Western blot, their analysis and contributed to immunofluorescence experiments. MB, JM and MHJ contributed in qRT-PCR analysis and amelioration of the manuscript. MC, MB, RP and CL contributed in analyzed,


In recent years, research on the association between physical environments and cardiovascular disease outcomes has gained momentum with growing attention being paid to Geographic Information Systems (GIS). This nationwide study is the first to examine the effect of neighbourhood physical environments on individual-level stroke, using GIS-based measures of neighbourhood availability of potentially health-damaging (fast food restaurants and pubs/bars) and health-promoting  resources.The study population comprised a nationwide sample of 2,115,974 men and 2,193,700 women aged 35–80 years who were followed between 1 December 2005 and 31 December 2007 in Sweden. Totally 42,270 first-ever strokes  were identified. Multilevel logistic regression models were used to estimate the association between neighbourhood availability of four different resources  and individual-level stroke.There were significant associations between neighbourhood availability of the four types of neighbourhood resources and individual-level stroke. The significant odds ratios varied between 1.06 and 1.12 for men and 1.07 and 1.24 for women. After adjustment for age, income, and neighbourhood-level deprivation, the increased odds remained statistically significant for neighbourhood availability of fast food restaurants in both men and women.Specific neighbourhood availability of resources were associated with individual-level stroke but most of these associations were explained by individual-level sociodemographic factors and neighbourhood-level deprivation. During the last decade, there have been great efforts to identify the effect of neighbourhood environments on cardiovascular disease outcomes neighbourhood rates of stroke individual-level stroke risk, using GIS-based measures of neighbourhood availability of potentially health-damaging (fast food restaurants and pubs/bars) and health-promoting  resources. The first aim of this nationwide multilevel study was to examine whether GIS-based measures of neighbourhood availability of health-damaging resources  or health-promoting resources  was associated with individual-level stroke. The second aim was to test if these possible associations remained significant after adjustment for neighbourhood-level deprivation and individual-level sociodemographic characteristics.Although previous research has analysed the possible associations between physical environments, assessed by GIS, and risk of coronary heart disease using multilevel analysis The study was approved by the Ethics Committee of Lund University. Data handling and analysis were performed by assigning serial numbers to data to preserve anonymity.The study population comprised a nationwide sample of 2,115,974 men and 2,193,700 women aged 35–80 years who were followed between 1 December 2005 and 31 December 2007 in Sweden. All individuals were included in a national Swedish research dataset, constructed at the Center for Primary Health Care Research at Lund University. This dataset contained nationwide individual-level medical diagnoses from the Swedish Hospital Discharge Register  and the Cause of Death Register. These data were linked to the Swedish Population Register (census) data obtained from Statistics Sweden, the Swedish government-owned statistics bureau. The Population Register includes individual-level data on sociodemographic factors such as age and income.The men and women were followed between 1 December 2005 (the start of follow-up) and 31 December 2007 for the outcome variable, i.e., first hospitalisation during the study period for stroke . The disease codes were based on the 10th version of the International Classification of Diseases (ICD-10) Four categories of neighbourhood resources that could be regarded as potentially health-damaging or health-promoting were selected The ready to-use nationwide GIS dataset of business contacts  for November 2005 was provided to us by the Swedish company Teleadress SAMS units are predefined areas and were used as proxies for neighbourhoods, as has been done previously For gender, analyses for men and women were conducted separately. Age was categorised as 35–44, 45–54, 55–64, 65–74 and 75–80 years. Family income was categorised as empirical quartiles based on the distribution. The family income variable took the number of people in the family into account as well as the ages of the family members (children were given lower consumption weights than adults).Neighbourhood-level deprivation was also included as a covariate because previous research has shown that neighbourhood deprivation is an important environmental disease determinant Age-standardised incidence proportions  were calculated separately for men and women by direct age standardisation using 10-year age groups, with the entire Swedish population of men or women aged 35–80 as the standard population.Multilevel logistic regression models were created with incidence proportions as the outcome variables. These models are a good approximation of multilevel Cox proportional hazards models under conditions such as ours: a large sample size, low incidence rates, risk ratios of moderate size and relatively short follow-up For women, a similar pattern was observed, with significantly higher ORs of stroke for those living in neighbourhoods with availability of fast food restaurants, bars/pubs, physical activity facilities and healthcare facilities (Model 1). The significant ORs varied between 1.07 and 1.24. These ORs remained significant after adjustment for neighbourhood deprivation (Model 2). After further adjustment for individual-level age and income (Model 3), only neighbourhood availability of fast food restaurants and health care facilities continued to be significantly associated with higher odds of stroke. For both men and women, the ORs that remained significant in the full model were only slightly increased and varied between 1.02 and 1.03.There were no interactions between neighbourhood-level availability of the four resources and neighbourhood-level deprivation (data not shown).-damaging as well as health-promoting neighbourhood resources imply higher odds of stroke. Although the ORs of the associations are not large, our study included more than four million people aged between 35 and 80 years.To the best of our knowledge, no study has examined the effect of neighbourhood availability of potentially health-damaging and health-promoting resources on individual-level stroke risk in a multilevel fashion. The main finding of this study is that specific potentially healthOur results are consistent with recent work from the United States, where an association between the number of fast food restaurants in the neighbourhood and stroke was found The reason why high neighbourhood availability to healthcare facilities was associated with stroke is, however, uncertain. It might be explained by lack of timeliness These results extend previous findings on the association between GIS-based measures of physical environments and stroke. A study that examined the number of fast food outlets and stroke from the United States used census tracts as a proxy for neighbourhoods -level stroke in a multi-level fashion. The large-scale design allowed us to include 42,270 cases of stroke. Moreover, our study included adjustment for individual-level and neighbourhood-level covariates in the multilevel framework. This study also has certain limitations. First, our data did not allow for the assessment of other important risk factors for stroke, such as smoking, poor dietary habits, and physical inactivity. Second, we had no data on whether those individuals with stroke actually utilized the examined resources in their neighbourhoods. Third, the follow-up period was only two years. However, this means that the potential changes during the study period in neighbourhood availability of resources were most likely minor and that most people probably remained in their neighbourhoods.The present study has several strengths. To our knowledge, this is the first large-scale study of an entire national population to examine the effect of potentially health-damaging and health-promoting neighbourhood resources on individualOur results suggest that specific neighbourhood-level resources are associated with individual-level stroke but that some associations seem to be explained by individual-level sociodemographic factors and neighbourhood-level deprivation. Caution is, however, warranted in the interpretation of these findings. Future studies should consider the actual use of neighbourhood resources in individuals with stroke and compare their use with healthy individuals.


Recent increases in patient cost-sharing for health care have lent increasing importance to monitoring cost-related changes in health care use. Despite the widespread use of survey questions to measure changes in health care use and related behaviors, scant data exists on the reliability of such questions.We administered a cross-sectional survey to a stratified random sample of families in a New England health plan's high deductible health plan (HDHP) with ≥ $500 in annualized out-of-pocket expenditures. Enrollees were asked about their knowledge of their plan, information seeking, behavior change associated with having a deductible, experience of delay in care due in part to cost, and hypothetical delay in care due in part to cost. Initial respondents were mailed a follow-up survey within two weeks of each family returning the original survey. We computed several agreement statistics to measure the test-retest reliability for select questions. We also conducted continuity adjusted chi-square, and McNemar tests in both the original and follow-up samples to measure the degree to which our results could be reproduced. Analyses were stratified by self-reported income.The test-retest reliability was moderate for the majority of questions (0.41 - 0.60) and the level of test-retest reliability did not differ substantially across each of the broader domains of questions. The observed proportions of respondents with delayed or foregone pediatric, adult, or any family care were similar when comparing the original and follow-up surveys. In the original survey, respondents in the lower-income group were more likely to delay or forego pediatric care, adult care, or any family care. All of the tests comparing income groups in the follow-up survey produced the same result as in the original survey.In this population of HDHP beneficiaries, we found that survey questions concerning plan knowledge, information seeking, and delayed or foregone care were moderately reliable. Our results offer reassurance for researchers using survey information to study the effects cost sharing on health care utilization. Increases in health care costs have led to increases in patient cost-sharing arrangements such as high deductibles. The percentage of Americans insured by high deductible health plans (HDHPs) - also known as consumer-directed health plans (CDHPs) when combined with a health savings account or health reimbursement account - increased from 8% in 2006 to 17% in 2009[Some of the most important data on the effects of increases in cost-sharing for health care in the United States come from surveys -5. Reed Despite the widespread use of survey questions to measure changes in health care use and related behaviors, scant data exists on the reliability of such questions. If these questions do not produce reliable responses, this would blunt the ability of studies to identify true changes in health care use associated with different health insurance cost-sharing arrangements. A better understanding of the reliability of patients' responses to questions regarding their experiences delaying care and/or hypothetical health care seeking behavior would improve our ability to predict cost-related changes in health care use.We sought to measure the reliability of survey questions assessing changes in health care utilization and delayed or foregone care among a group of enrollees in HDHPs. While there is a vast body of literature reporting on the reliability and validity of various psychometric instruments -10, and In addition to evaluating the test-retest reliability of select survey questions, we also report aggregate differences in information use, health care utilization and delayed or foregone care in order to evaluate how potential changes, due in part to unreliability , might affect the findings of an analysis using a single cross-section of survey data.The study population consisted of enrollees in HDHPs offered by Harvard Pilgrim Health Care, a New England-based non-profit health insurer. A description of the population and plan characteristics has been previously published . The tarThe inclusion criteria also required: (1) continuous enrollment in an employer-sponsored HDHP for at least the previous 6 months; (2) at least one child < 18 years of age also enrolled in the plan; and (3) annualized family out-of-pocket costs (defined as outpatient visit and prescription drug co-pays) of at least $500 in an HDHP. This threshold of annualized OOP expenses included 54% of all families who met other inclusion criteria. We required at least $500 in OOP expenses to ensure that the questions concerning delayed or forgone care would be relevant to potential respondents. We reasoned that individuals who consumed some care would have some experience with their own plan and recent decisions to delay or forego care, and be more able to readily judge hypothetical decision making at different cost levels.versus other). Low income individuals were oversampled because nominal increases in cost sharing affect this population of enrollees disproportionately to their income; thus, these enrollees may be more likely to delay or forego care due to cost. Random sampling was performed in each of the two strata until surveys from approximately 200 families in each group were completed.We oversampled households living in low-income areas by stratifying families into two groups based on geocoded address information: (1) residence in a census block group with a median household income in the lowest quartile in the sample frame and (2) residence in a census block group with a median household income in the second, third and fourth quartiles of the sample frame . Families with incomes above this threshold were classified as higher-income.The survey included several domains: health plan characteristics, attitudes towards health care utilization, unexpected costs, information-seeking behaviors, delayed care, and demographic data. Survey domains and questions were developed based on a previous focus group study in this population and were in some cases drawn from existing national surveys . The draThe original (i.e. the "test") surveys were mailed between January and March 2009. The cover letter asked the adult in the family who is responsible for the family's health care decisions to complete the survey. We sent two mail waves followed by attempts at telephone administration. Respondents received a $30 gift card to their choice of 1 of 3 major retailers. Of the 750 surveys mailed, 229 (30.5%) were completed in the first wave, 130 (17.3%) were completed in the second wave, and 75 (10%) were completed by phone for a total of 434 completed surveys and an overall response rate of 58.1%.We attempted to administer the follow-up  survey to all 434 families who completed the original survey. The follow-up survey cover letter explained that, "the reason we are asking you to do these questions a second time is to test how reliable the questions are - whether they result in the same answers or different answers if asked at different times."Follow-up surveys were distributed (mail) or administered (telephone) within two weeks of each family responding to the original survey; however, individual response time varied. The follow-up surveys were all completed between February and July of 2009. We attempted the survey in the same format in which the respondents completed the original survey , using the same incentives and number of mailing waves. The questions were identical between the original and follow-up surveys. Five questions regarding demographic information  and two administrative questions  were omitted from the follow-up survey.The survey covered five broad themes: beneficiary knowledge of their health plan, information seeking, changes in behavior associated with having a deductible, experiences in delayed or foregone care due in part to cost, and hypothetical delays in care due in part to cost. Figure Our statistical analysis involved 3 components. We compared the agreement of (1) individual level responses between the original and follow-up surveys via percent agreement, absolute retraction, absolute initiation, tetrachoric correlation (binary responses) or polychoric correlation , and kappa statistics; (2) differences in responses between income groups - comparing the original versus follow-up responses; and (3) differences in responses between the original and follow-up responses, stratified by income group.We calculated the percentage agreement  for eachWe also calculated simple (for binary response) and weighted  kappa statistics for each question ,18. We u2 tests to investigate whether lower-income families with out-of-pocket expenditures in HDHPs were more likely than higher-income families to delay or forego health care services, have difficulties understanding and using their plans, or avoid discussing costly services with physicians. We repeated these analyses for the present study but restricted the original sample to individuals that responded to the follow-up survey in order to evaluate the extent to which our initial findings would be reproduced.In a previous study , we usedMcNemar's test [We compared the responses in the lower-income group (FPL < 300%) between the original and follow-up surveys using r's test ,34. The All statistical analyses were performed using SAS version 9.2 .Of the 434 original completed surveys, 387 completed the follow-up survey  and 47 did not respond giving a response rate of 89%. Twenty-four (6%) of the 387 follow-up surveys were completed in a different format than the original survey .Although follow-up surveys were fielded within two weeks of receiving the initial survey, time between receipt of the initial survey and receipt of the follow-up survey varied. The mean time between initial and follow-up survey receipt was 29.7 days 95% CI: 28.3, 31.0) and the median time was 26 days. Of the 387 follow-up surveys, 75% were returned within 31 days and 95% were returned within 56 days. A little less than 5% (n = 19) of follow-up surveys were returned between 57 and 142 days after the initial survey. Several studies have reported reliable estimates of retrospective and contemporaneous behaviors in a 2 to 10 week test-retest period .3, 31.0 -38.2 p = 0.028).We tested for differences in characteristics between follow-up survey respondents and non-respondents such as age, sex, minority status, education, family size, income, deductible level, enrollment time, choice of plan, and chronic illness among family members. The initial and follow-up surveys differed only with respect to the proportion of respondents with any adult chronic condition in the family . Of the 47 non-respondents, 37 (78.7%) had an adult in the family with a chronic condition compared to 57.8% of initial respondents  may also be higher than that for delayed or foregone care at "any other time" .The test-retest reliability as measured by kappa was moderate for the majority of questions (0.41 - 0.60). Similarly, 14 of the 18 correlations (TCC/PCC) are between 0.62 and 0.76 which could also be interpreted as moderate agreement. Interestingly, the level of test-retest reliability for questions concerning the experience of delays in care did not differ substantially from questions regarding hypothetical delays in care. The kappa statistic for delayed or foregone emergency department care was 0.67 , significantly higher than most questions related to behavior change associated with having a deductible . These results remain statistically significant using either kappa or the TCC/PCC.Table The results of difference of proportion tests comparing income groups are also largely similar. However, in two cases there were significant differences. The test comparing delayed or foregone ED care was statistically significant in the follow-up survey but not in the original survey. Also, the test comparing delayed or foregone operations/procedures was statistically significant in the original survey but not in the follow-up survey.Table Table In the original survey, 55.8% of respondents in the lower-income group reported feeling unprotected from OOP expenses versus 44.0% of those in the higher-income group (p = 0.043). The difference between income groups widened in the follow-up survey where 60.8% of lower-income respondents reported feeling unprotected versus 40.2% in the higher-income group (p < 0.0001). None of the other income group comparisons were significant and none of the proportions changed significantly between the original and follow-up surveys.Although surveys are widely used to measure self-reported and hypothetical use of health care by enrollees with cost sharing arrangements, the test-retest reliability of these surveys has not been adequately studied. A better understanding of the reliability of patients' responses to questions on health care seeking behavior is important for improving our ability to identify true changes in health care use associated with different health insurance cost-sharing arrangements.The test-retest reliability of self-reported plan knowledge, information seeking, and delayed or foregone care reported in this study can generally be characterized as "moderate". None of the questions with kappa statistics in the "substantial" range had confidence intervals that were completely within the substantial range. The apparent consistency of kappa statistics across the various domains is interesting because respondents were equally reliable answering questions about their experiences in delaying care as they were answering questions about hypothetical delays in care. However, readers should exercise care in interpreting differences in these kappa as the confidence intervals are conservatively wide.We found that most of the proportions of respondents reporting delayed or foregone care did not change significantly between the original and follow-up surveys. Only the proportion of higher-income respondents reporting delayed or foregone prescriptions medications changed. In comparing the lower-income and higher-income groups, only the results concerning delayed or foregone ED care and delayed or foregone operations/procedures changed between the original and follow-up surveys. These results suggest that we can be reasonably confident in our initial analyses and the propensity to delay or forego care in this population of beneficiaries.Results across the original and follow-up surveys show that 40 to 50 percent of respondents experienced unexpected costs or felt unprotected from OOP costs. Similarly, only 40 to 50 percent of respondents reported trying to discover whether or not they would have to pay for a service and/or how much they would have to pay for a service since joining their health plan. Although the proportions describing plan use and information seeking in each income group changed slightly between the original and follow-up surveys, none of these changes were statistically significant. Our analysis of the follow-up sample confirmed the results reported for the original study: a higher proportion of lower-income individuals reported feeling unprotected from OOP expenses. We also confirmed our previous analysis in that none of the other comprehension/information proportions were different in the lower-income group compared to the higher income group. These results suggest that we can be confident in our initial results comparing plan knowledge and information seeking.This study has two main strengths. This is the first test-retest reliability study of questions concerning beneficiaries' self-reported understanding and use of their HDHP benefits. Second, we repeated the analyses comparing lower-income and higher-income individuals in addition to calculating a variety of agreement statistics for the individual questions. Repeating the analyses by income allowed us to determine whether there were significant changes in the proportion of people reporting delayed or foregone care between the original and follow-up surveys. We found that we can be confident in our initial results using the original survey data because only one proportion out of eighteen changed despite the moderate level of reliability as captured by the agreement statistics.One limitation of our study is that the results may be biased by individuals' recall of their initial responses when completing the follow-up survey. Given the moderate level of test-retest reliability, the learning effect does not appear to have been strong. However, the absolute initiation among respondents with respect to experienced delays . As the proportion of Americans with cost-sharing arrangements for health care continues to increase, reliable self-reported information about their health care decision processes and use will become increasingly important.The authors declare that they have no competing interests.RP contributed to data analysis and wrote the manuscript. JK contributed to data analysis and interpretation and the writing of the manuscript. IM contributed to the design and conduct of the data analysis. AG contributed to data analysis and interpretation and the writing of the manuscript. VH collected the data and critically read the manuscript. TL designed the study, arranged funding, contributed to data analysis and interpretation, and critically revised the manuscript for intellectual content. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/11/133/prepub


Oral capecitabine is an oral prodrug of 5-fluorouracil that has been integrated into the management of multiple cancer types because of the convenience of administration and its efficacy compared with 5-fluorouracil. Capecitabine mimics the pharmacokinetics of intravenous 5-fluorouracil. While cardiac events associated with the use of 5-fluorouracil are a well-known side effect, capecitabine-induced cardiotoxicity has only been rarely reported.We present a case of a 46-year-old woman of Greek ethnicity who presented to our institution with an operated gastric sarcoma who experienced capecitabine-induced vasospastic angina. Primarily a clinical diagnosis of a possible acute coronary syndrome was proposed and the patient was admitted to the hospital for further investigation which was proved between normal limits. After a witnessed episode of angina, her prior history of capecitabine intake and an undertaken further imaging investigation we associated anginal symptoms and signs with vasospastic angina induced by capecitabine 36 hours prior to hospital admission.Cardiologists should be aware of the potential cardiac hazards of capecitabine, especially in patients with cardiovascular risk factors. Due to the increasing usage of capecitabine during the last years, patients should be warned for the possibility of chest pain, particularly during the first few days of capecitabine treatment. Specifically, patients developing acute coronary syndrome should not be retreated with capecitabine. On the other hand, due to its promising antitumoral efficacy, its use should not be discouraged. Oral capecitabine is an oral prodrug of 5-fluorouracil (5-FU) that has been integrated into the management of multiple cancer types. Capecitabine, a thymidine phosphorylase– activated fluoropyrimidine carbamate, is absorbed by the gastrointestinal tract and metabolized to 5-FU by a cascade of three enzymes. It is currently considered the only universally approved orally administered 5-FU prodrug. It belongs to a new generation of orally administered fluoropyrimidines. It has been developed because of the clinical need for efficient, tolerable and convenient agents that do not require continuous infusion. Capecitabine is not a cytotoxic drug itself; by a three-step enzymatic cascade, it is converted to 5-FU mainly within human cancer cells. Capecitabine is converted by carboxylesterase in the liver to 5′-deoxy-5-fluorocytidine and by cytidine deaminase in the liver and tumor tissue to 5′-deoxy-5-fluorouridine. Furthermore, it is converted by thymidine phosphorylase to 5-FU in tumor tissue. Thymidine phosphorylase is found in higher concentrations in tumor tissue than in normal tissue and is upregulated by radiation in tumor tissue, but not in normal tissue. Thus, oral capecitabine can result in higher intratumoral and lower systemic 5-FU concentration than bolus delivery of 5-FU. The drug compares favorably with 5-FU in patients with colorectal cancer and breast cancer, and it has a better toxicity profile with mainly gastrointestinal and dermatologic effects. Capecitabine shows antineoplastic activity and synergy with other cytotoxic agents, including cyclophosphamide and docetaxel, in animal models. Bioavailability after oral administration is close to 100%. Although patients may receive the drug orally in the convenience of their own homes, the key to successful management of capecitabine is the clinician’s awareness of its severe, but low in incidence, adverse effects and the patient’s education, emphasizing compliance with the treatment plan, adverse effect prevention and timely recognition of the drug’s toxicities. This improved therapeutic index, along with more favorable pharmacokinetics (similar to those of protracted infusion of 5-FU) and convenient oral administration without the need for central venous access and an ambulatory infusion pump, make capecitabine particularly appealing to use.et al. compiled a review of reports in the literature describing adverse effects observed in capecitabine-treated patients [2 per day in two divided doses for 14 days followed by 1 week of rest. The authors concluded that this complication is more frequent in patients who have a history of coronary artery disease and recommended close monitoring for cardiac abnormalities during therapy. Herein we present the case of a 46-year-old woman with an operated gastric sarcoma who presented to our institution with capecitabine-induced vasospastic angina.In particular, although cardiac events associated with the use of 5-FU are a well- known side effect, capecitabine-induced cardiotoxicity has only rarely been reported -4. WalkoA 46-year-old woman of Greek ethnicity was admitted to the emergency room at our institution because of a retrosternal pain episode 90 minutes before admission that lasted approximately 10 minutes. Additionally, the patient reported two transient episodes of chest pain during the previous 24 hours. The duration of each episode was estimated to be between two and ten minutes, and spontaneous resolution was reported. During these episodes, the symptoms included chest discomfort with radiation to the back, malaise, nausea and sweating. Our patient had no prior history of cardiac disease, coagulation disorders or drug abuse, and she mentioned only one cardiovascular risk factor (smoking). From her medical history, we found that she is a heterozygous carrier of β-thalassemia. She reported having gastric sarcoma with metastatic peritoneal infiltrates. We had performed a subtotal gastrectomy on her for this reason 2 months prior to her current presentation. Treatment with oral capecitabine (1500mg twice daily) was initiated 48 hours before she was admitted to our hospital. The echocardiogram taken in the emergency room was within the normal limits, including a sinus heart rate of about 62 beats per minute (bpm) and physiological hemodynamic parameters and negative biochemical markers for myocardial necrosis (including cardiac troponin I (CTnI), creatine phosphokinase, creatine kinase isoenzyme MB, lactate dehydrogenase, and serum glutamic oxaloacetic transaminase). She was transferred to the coronary intensive care unit (ICU) for further observation. The echocardiogram, which was performed when the patient was pain-free, revealed normal ventricular volume, no segmentary wall hypokinesia with a left ventricular ejection fraction of 72%, normal diastolic heart function, normal valve flow and absence of pericardial effusion Figure . Daily Ein vitro compared with 5-FU [Capecitabine is an oral prodrug that is converted through a three-stage enzymatic reaction to 5-FU primarily in liver and tumor cells. Through the years, it has gained popularity as well as support from the oncologic community because of its efficacy, ease of administration and milder toxicity profile. The cardiotoxic effects of intravenously administered 5-FU pyrimidine analogues have been well-described in the literature, with a reported incidence ranging from 1% to 68% ,8, whereith 5-FU . Our preith 5-FU . Acute cith 5-FU .Capecitabine should be considered a drug with cardiotoxic potential , even inWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.CGo made substantial contributions to the conception and design of this report, drafting the manuscript and revising it critically. He gave final approval of the version to be published. GD made substantial contributions to the conception of this report, drafting the manuscript and revising it critically and analyzing and interpreting echocardiographic data. DD and CGr made substantial contributions to drafting the manuscript and performing analysis and interpretation of coronary angiography data. AT examined the patient and analyzed and interpreted the data regarding hospital admission. TB and ID made substantial contributions to the collection and acquisition of data and drafting the manuscript. KC made substantial contribution to the conception and design of this report; acquisition, analysis and interpretation of data. He was involved in revising the manuscript critically and gave final approval of the version to be published. All authors read and approved the final manuscript.


Arabidopsis thaliana ten years ago, various large-scale analyses of gene function have been performed in this model species. In particular, the availability of collections of lines harbouring random T-DNA or transposon insertions, which include mutants for almost all of the ~27,000 A. thaliana genes, has been crucial for the success of forward and reverse genetic approaches. In the foreseeable future, genome-wide phenotypic data from mutant analyses will become available for Arabidopsis, and will stimulate a flood of novel in-depth gene-function analyses. In this review, we consider the present status of resources and concepts for systematic studies of gene function in A. thaliana. Current perspectives on the utility of loss-of-function and gain-of-function mutants will be discussed in light of the genetic and functional redundancy of many A. thaliana genes.Since the sequencing of the nuclear genome of  Since its nuclear genome was completely sequenced 10 years ago [Arabidopsis research. Forward (from-phenotype-to-gene) and reverse (from-gene-to-phenotype) genetics have been the main workhorses en route to this goal. Even before the Arabidopsis Genome Initiative got underway, numerous forward genetic screens had identified mutants with abnormal phenotypes that resulted in the identification and functional characterization of many genes for specific plant functions. Identification of the mutated genes is usually the most labour-intensive step in classical forward genetics. With increasing knowledge of the sequences of Arabidopsis genes and their transcripts, reverse genetic screens have become possible, which complement the classical approach by permitting direct analysis of mutants for specific genes, selected for their potential impact on a biological function of interest. According to the Annual Report 2010 of The Multinational Coordinated Arabidopsis thaliana Functional Genomics Project , the experimentally verified functions of just over 9000 of the ~27,000 Arabidopsis genes had been annotated as of March 2010.Because of its small size, short life cycle and compact genome, In silico approaches can assess the functions of Arabidopsis genes whose homologs have been characterized in other species, or which code for proteins with conserved domains, and gene functions can be predicted by correlating data derived from functional genomics analyses, such as large-scale mRNA, protein or metabolite profiling [ Arabidopsis will require much further effort, and many projects have been initiated to create the resources required for this formidable task [Arabidopsis mutants bearing molecularly mapped insertions. Besides total inactivation of the gene function, some of the newly developed mutagenesis techniques allow one to alter the dosage or function of gene products, making refined analyses of gene-function relationships possible. Several databases are now available that permit researchers to scan the Arabidopsis genome for mutations in genes of interest and obtain the corresponding mutant lines from public stock centres.rofiling -4. Howevble task . TechnolArabidopsis genes requires ways to systematically assess a wide range of phenotypes, which can be applied in a high-throughput manner to large numbers of mutants. Saturated collections of loss-of-function and gain-of-function lines will also be very valuable. A further challenge arises from the recognition that the vast majority of gene knock-outs in Arabidopsis do not give rise to obvious phenotypes, and might require functional characterisation by –omics-type analyses or the simultaneous inactivation/down-regulation of more than one gene. In this review, we summarize the state of the art in systematic gene-function analyses and highlight tools and trends in the application of systematic forward and reverse genetics to Arabidopsis.To elucidate the function of the entire complement of 2Arabidopsis genome sequence was published most efforts have focused on generating loss-of-function lines for reverse genetics. In addition, methods for overexpressing genes have gained in importance more recently [Several methods have been developed that enable one to change the amount or the nature of gene products either by altering the original gene through the introduction of mutations or by introducing transgenes. Since the recently , 7.2.1The most straightforward approach to investigating the function of a gene is to characterize the phenotypic changes associated with its total inactivation . Loss of2.1.1Point mutations can be introduced with high frequency by mutagenization with chemicals such as ethyl methanesulfonate (EMS), but only a small fraction of such mutations actually lead to total loss of gene function, e.g. when they generate a premature stop codon or alter functionally critical amino acids . SimilarArabidopsis reference genome sequences (such as the Col-0 ecotype) are already available, new generation sequencing of mutant genomes will be time and cost effective. Another approach to detect small changes within the genome is an adaptation of the microarray technology, which detects single nucleotide polymorphisms (SNPs) through hybridizing genomic DNA to oligonucleotides representing the entire genome spotted on chips [The identification of individual, randomly induced point mutations or short insertions/deletions within the genome of a mutant is a laborious process involving map-based cloning , althougon chips . With co1).For reverse genetic approaches employing EMS-induced mutations, the “Targeting Induced Local Lesions IN Genomes” (TILLING) method has been developed . Here, t2). The recent release of the genome sequences of 80 different Arabidopsis ecotypes (with more to come when the 1001 Genomes project is completed in 2011) makes this an important tool for understanding natural variation of sequences and correlating them with evolutionary processes [2).Most point mutations do not cause complete gene knock-out, but rather a decrease in gene expression  or a change in the function of the gene product due to alterations in its sequence. Amino acid exchanges can lead to hypomorphic alleles displaying a range of phenotypes, allowing the analysis of important domains of the protein. Such variants can be pinpointed by searching the TILLING database /Dissociation (Ds) system - are usually employed as insertional mutagens [1) which now provide nearly genome-wide coverage - about 96% of all Arabidopsis genes according to the Annual Report 2010 of The Multinational Coordinated Arabidopsis thaliana Functional Genomics Project . The regions flanking the insertions  in over 325,000 lines have been sequenced by several laboratories and mapped to the Arabidopsis reference genome to identify the precise insertion sites. These lines have been indexed, deposited in public stock centres and are accessible for Internet-based searches (Table 2).Insertional mutagenesis has two major advantages: the mutations are labelled by the inserted fragment of known sequence (‘tag’) and insertions within the coding region have a high probability of eliminating the gene function. In mutagens . In an iBecause stock centres distribute T-DNA or transposon lines usually as populations that segregate for the mutation, a genotyping step is necessary to identify homozygous plants for the analysis of phenotypes. Recently, populations of homozygous SALK lines were generated, which can be used directly for phenotypic screens . To date2.2The utility of loss-of-function lines is limited when mutations result in lethality, or in cases of genetic redundancy, when more than one copy of the gene exists as a result of tandem or segmental duplications or the evolution of multigene families. To overcome these drawbacks, transgene-mediated gene silencing can be used to reduce but not completely abolish (“knock-down”) the expression of gene(s) of interest. Such “knock-down” lines allow one simultaneously to down-regulate several, sequence-related genes and thus avoid the complications associated with genetic redundancy. Because knock-down lines are not null mutants and show a diverse pattern of phenotypes due to differences in the levels of activity remaining, the more subtle phenotypes induced by the partial or conditional inactivation of essential genes can also be studied.via small RNAs that act in a sequence-specific manner by base pairing to complementary mRNA molecules. Various strategies for small RNA-based gene silencing have been developed [Arabidopsis with RNAi constructs is being systematically conducted by the AGRIKOLA  Consortium to create a collection of silenced Arabidopsis lines which are available through the Nottingham Arabidopsis Stock Centre (NASC) [1 and 2). A newer approach involves the use of artificial microRNAs (amiRNAs) [Arabidopsis (as of June 2010), providing an important tool for future functional genomics studies (Table 1). The Chimeric Repressor Silencing Technology (CRES-T) represents a novel method for creating loss-of-function mutations for the analysis of redundant plant transcription factors [Arabidopsis is stored in the Web-based interface FioreDB [1 and 2).Silencing is normally achieved by post-transcriptional down-regulation of transcript accumulation eveloped . The fireveloped . Later teveloped . Transfoe (NASC)  (Tables amiRNAs) , 24, 25. factors . With CR FioreDB   collection now makes large numbers of such cDNA clones available. The RAFL collection was generated by inserting full-length cDNAs in the correct orientation between the CaMV 35S promoter and the NOS terminator and verifying their sequences [Arabidopsis to generate so-called ‘FOX’ (Full-length cDNA Over-eXpressing gene) lines [2).Overexpression of a gene driven by a strong promoter can result in gain-of-function phenotypes, especially when expression is also ectopic. This strategy requires the use of full-length cDNA clones. The RIKEN equences . About 1e) lines . The FOXloxP sites, and pCre lines that contain the CRE gene [In the activation tagging approach, DNA sequences carrying enhancers (e.g. four tandemly arranged copies of the CaMV 35S enhancer) are inserted randomly into the genome . Large nCRE gene . By crosDs-tagged lines of Arabidopsis, each of which contains within the Ds-tagging cassette a CaMV 35S::luciferase gene  and a lac operator repeat . The selected lines will be used to evaluate transgene position effects and how these are affected by developmental or externally applied cues; since position effects on gene expression arise from alterations in chromatin architecture, this approach provides insights into global epigenetic control [1).The Chromatin Charting Project has set itself the task of producing over 15,000  control , 43 , have been screened for visually discernible phenotypes. In all, 1262 lines exhibited visible phenotypes  and sequence information is available for 1172 mutant loci (Table 2). Of four dominant mutants that showed hyponastic leaves, downward-pointing flowers and decreased apical dominance, two were caused by activation of the gene ASYMMETRIC LEAVES2 (AS2) and two by activation of ASL1/LBD36 [The RIKEN Activation Tagged Lines, comprising about 50,000 independently transformed is lines , 40 (seeL1/LBD36 .1) were also screened for abnormal morphologies, fertility and leaf coloration. A total of 1487 candidate morphological mutants were found among 15,547 transformants, and of 115 pale green T1 mutants, 59 lines displayed the mutant phenotypes in more than 50% of the T2 progeny, suggesting that the phenotypes were caused by gain of function mutations (see Section 2.3). Two leaf coloration mutants were further characterized and one mutant was found to result from up-regulation of AtPDH1 (Arabidospsis prokaryotic DEVH box helicase1); knock-out of this same gene resulted in an albino phenotype [The FOX lines  genes required for embryo or seed development, and the December 2007 release of its database SeedGenes (Table 2) includes information on 358 genes required for seed development and 605 mutant alleles with known disruptions in these genes. EMB genes encode proteins involved in basic cellular processes, such as DNA replication, RNA processing and protein synthesis, as well as chloroplast proteins, indicating that a functional chloroplast is needed for normal embryo development in Arabidopsis.The Seed GenesProject -47 was dFurthermore, over 500 seedling-lethal mutants were identified in a set of 38,000 insertion mutants, of which 54 were molecularly characterized and 22 mutants were described in detail . Many ofTo isolate mutants defective in early steps of meiotic recombination, a specialized two-step genetic screen employing 55,000 T-DNA insertion lines has been carried out . In the 3.3The life cycle of plants alternates between a haploid gametophyte and a diploid sporophyte phase. Mutations that eliminate or disrupt the function of the haploid pollen  or of the embryo sac  can only be maintained in the heterozygous state, because such mutations cannot be transmitted through the defective gamete (reviewed in -52). In Arabidopsis, due to the widely used “floral dipping” transformation protocol the primary target of T-DNA integration is the embryo sac. Therefore, it can be difficult to recover T-DNA insertional knock-out lines, in which female gametophyte function is severely compromised [Ds transposable elements are mobilized during the sporopyhtic stage of a plant’s life cycle and most Ds transposon insertion lines possess further advantages like single-copy insertions without generating DNA rearrangements or truncations [ Ds insertion lines containing either a Ds gene trap or an enhancer trap element were screened and 130 Arabidopsis mutants with defects in female gametophyte development and function were identified [Ds element could be followed by the linked antibiotic resistance marker. Given the dominant mode of inheritance of the resistance phenotype, a segregation of 3:1 is expected in the progeny of a heterozygous Ds insertion line. Aberrant segregation ratios lower than 2:1 are often the result of a gametophytic defect. For 1.38% of the screened lines a segregation ratio of 1.5:1 or less was found and a wide variety of mutant phenotypes were observed, such as defects in different stages of embryo sac development and in processes such as pollen tube guidance, fertilization or early embryo development. Besides female gametophytic mutants, 109 lines showing predominant defects in male gametophyte transmission were also identified in this collection [Visual inspection of siliques for 50% or more desiccated ovules, as well as screens for aberrant transmission of an antibiotic resistance marker gene, led to the identification of gametophytic mutants -61. In Apromised . To overncations . 24,000 entified . The inhllection .3.42) [In a screen of the TAMARA gain-of-function collection  for muta2) .A small subset of the FOX lines has also been assessed for mutants affecting stress-inducible transcription factors. Among 43 cDNAs tested in a transgenic plant library, the authors identified salt-tolerant lines, all of which overexpressed the same bZIP-type transcription factor, AtbZIP60 .The COS (controlled cDNA overexpression) system is based on a cDNA expression library that is driven by an inducible promoter. Sets of 20,000 to 40,000 transgenic seeds/seedlings were screened in three different ways, namely for ABA insensitivity, salt tolerance and for activation of a stress-responsive alcohol dehydrogenase-luciferase reporter system , 68. Twe3.5Arabidopsis EMS mutants were screened with pyrabactin and a new component of the ABA pathway, the PYRABACTIN RESISTANCE 1 (PYR1) gene was identified. It codes for a START protein, which was characterized as the long-elusive ABA receptor [Treatment of mutant collection with a chemical compound to elucidate gene functions and signal transduction pathways are widely applied . Limitatreceptor . Arabidopsis natural accessions are a rich source for applying chemical genetic screens. When Arabidopsis natural accessions were subjected to a library of more than 10,000 compounds, twelve accession-specific molecules were identified [Besides EMS or insertion mutants multiple entified . The nathttp://pubchem.ncbi.nlm.nih.gov/).Chemical molecules that inhibit a specific protein function result in a similar phenotype like corresponding loss-of-function mutants. Vice versa, plants treated with a compound that activates a protein function resemble gain-of-function mutants. Variation in concentration of the chemical compound can act similar to allelic series of mutations. If a chemical is able to interfere with the function of all members of a protein family, it can substitute for mutants of a higher order. The ability to perturb protein functions only in the presence of a chemical and therefore for a limited time allows the study of essential genes that would display a lethal phenotype if permanently mutated. Nevertheless, currently only a limited number of chemical compounds with a defined function are available to be applied for biological questions. Helpful are databases such as ChemMine  or Pubch3.6Ideally, high-throughput screens should not damage the plants investigated so that they can be used directly for the next steps in forward genetics. Besides visual screens for altered growth or coloration phenotypes, other non-invasive screening procedures have been employed. For instance, systematic measurements of chlorophyll fluorescence have been used to determine the efficiency of photosynthetic electron flow. To this end, image analysis , 75 and qrt1-2 mutant background - a mutant, which maintains the four male meiotic products in a tetrad [hapless (hap) mutations was isolated, which impairs the development or function of haploid gametophytes [Some of the lines generated within the SAIL project were transformed with a vector that carries the LAT52:GUS reporter gene, a visible cell-autonomous marker for pollen grains and tubes . This poa tetrad  and whictophytes . http://www.plantsci.cam.ac.uk/Haseloff/geneControl/catalogFrame.html) and are very useful to identify enhancers for a specific cell type. The screen has been successfully applied and resulted in the isolation of Dof elements, which are involved in the regulation of guard cell gene expression [The collection of GAL4 enhancer trap lines harbors a GAL4-responsive GFP gene as a marker to tag specific cell types and to reveal developmental transitions . The linpression . In a fupression .4Arabidopsis have benefitted from previous experience with the model organisms Caenorhabditis elegans [Saccharomyces cerevisiae [Arabidopsis genome are members of gene families [Efficient systematic reverse genetics requires the availability of gene-indexed mutant populations, and the use of robust, high-throughput assays for phenotypic characterizations. Studies of this type in  elegans  and Saccrevisiae . In partrevisiae . The majfamilies , so that1) are usually combined, in order to achieve maximum coverage of the target genes. In addition, for genes that are not covered by existing mutant collections, partial loss-of-function lines are often generated by the knock-down approach (see Section 2.2).In the course of systematic reverse genetic screens, lines from different collections (see Table 4.13). One of the first systematic screens with insertion-tagged populations was performed more than 20 years ago. A collection of 8,000 Arabidopsis plants carrying 48,000 insertions of the maize transposable element En-1 was screened for knock-out alleles of genes involved in flavonoid biosynthesis, utilizing a PCR-based screening protocol [Numerous small- and medium-scale reverse genetic screens have been performed which have addressed specific biological functions or multigene families. Only a small selection can be discussed here . A larger screen was conducted for subtilisin-like serine proteases (subtilases), which comprises 56 members in Arabidopsis. Here 144 T-DNA insertion lines for 55 subtilase genes were identified. With the exception of SDD1, none of the lines displayed an obvious visible phenotype in the homozygous state [For reverse genetic analysis of the nine members of the 1-aminocyclopropane-1-carboxylate synthase (ACS) gene family, seven T-DNA mutants and two amiRNA lines were employed  . The opquartet (qrt) background containing a LAT52 promoter driven GUS reporter gene [For instance, it was shown that nuclear genes for chloroplast proteins involved in photosynthesis or plastid gene expression indeed exhibit a high level of co-expression at the transcript level . This fiter gene . Indeed,ter gene . Other eter gene . HoweverArabidopsis lines over-expressing 91 P450 cytochrome oxidases. In the course of this screen two P450 cytochrome oxidases that hydroxylate the therapeutic compound 8-methoxypsorelen were identified [An example of chemical genetic screening in combination with reverse genetics involves entified . This de4.22). Among the confirmed mutants obtained by the Chloroplast 2010 Project are mutants for At1g10310  that were found to have an altered fatty acid desaturation phenotype. Single insertions in the gene Acyl Carrier Protein4 (ACP4) were responsible for defects in growth, fatty acid profiles and chlorophyll fluorescence, previously associated with the knock-down of the Acyl Carrier Protein gene family. However, the case studies of the Chloroplast 2010 Project also stressed the difficulty of securely establishing genotype-phenotype relationships. It became clear, that - as in traditional reverse genetic screens - it is essential to confirm the genotype in advance and to exclude the possibility that a secondary mutation causes the phenotype. A second independent collection of 3246 Ds/Spm- or T-DNA-tagged homozygous lines for 1369 nuclear-encoded chloroplast proteins has been initiated [http://rarge.psc.riken.jp/chloroplast/). In the course of the Chloroplast 2010 Project mutations in nuclear genes that were computationally predicted to encode chloroplast-targeted proteins were subjected to a diverse set of phenotypic screens , 106. Dunitiated . Phenoty55.1Arabidopsis genome would make it possible to systematically test the phenotypic consequences of gene loss under a wide variety of conditions by forward genetics [Arabidopsis genes can be tested relatively quickly. But loss-of-function lines have their limitations. For instance, they provide only the first step in the analysis of essential genes (see Section 2.2). Secondly, the function of many genes will be very difficult to assess because of their apparent phenotypic silence in many assays. Thirdly, genetic redundancy will continue to hamper attempts to assign functions to members of gene families (see Section 5.2).The isolation of two homozygous insert lines for every gene in the genetics . The SALgenetics -111. The5.2Arabidopsis genes? In the simplest case, when two unlinked genes code for the same protein, the systematic generation of double mutants will make such genes manageable. In fact, in the course of the German Plant Genome Initiative (GABI), one ongoing project is dedicated to the systematic generation of segmentally duplicated genes. Segmental duplications account for the duplication of around 2900 genes in the Arabidopsis genome and can encompass chromosomal segments with up to about 300 genes which have been duplicated as blocks [https://www.gabi-kat.de/duplo.html/).How can the problem of genetic redundancy be solved for thousands of s blocks , 113. InClosely linked gene duplications, including tandem duplications as well as gene families containing more than two closely related genes, can only be handled by gene silencing approaches, and here the AGRIKOLA collection or amiRNA lines (see Section 2.2) promises to meet this need.5.3Arabidopsis, but no routine approach is available yet. Recently, zinc-finger nucleases (ZFNs) were used for targeted gene inactivation in Arabidopsis [Site-directed mutagenesis remains a significant technical challenge in higher plants. Many attempts have been made to establish targeted mutagenesis in bidopsis . In thisvia the type III secretion pathway found in many plant pathogenic Xanthomonas spp. Once inside, they may contribute to disease or trigger resistance by binding to DNA and turning on TALE-specific host genes. Recent advances in understanding of the mechanistic basis of specific DNA binding by TALEs [The use of transcription activator-like effectors (TALEs) represents another approach with great potential. TALEs are injected into plant cells by TALEs  should pby TALEs ).5.42; [Arabidopsis literature since 2005. As of July 2009 the TAIR database contained 7352 distinct free-text phenotypes associated with 11,381 distinct genotypes derived from almost 1500 publications.Because very many research groups generate data from forward and reverse genetics experiments, a central depository for documentation of mutant phenotypes is highly desirable. To this end, The Arabidopsis Information Resource (TAIR) recently began to assign a phenotype to each mutant allele and made pictorial records of the mutant phenotypes available online . CuratoArabidopsis Phenome Information Database (RAPID) (Table 2), which is a searchable database describing morphological phenotypes of ~4,000 Ds transposon mutant lines [2) is a database for exploring gene functions based on available phenotypes and for screening data from mutant, transgenic and wild-type organisms. It currently contains some project-specific phenotypic data for mutants under various abiotic stresses. BAP DB also allows users to upload their own assay and phenotypic data.Phenotypic databases already exist for individual mutant collections. These include, for instance, the Riken nt lines . Phenoty5.5Arabidopsis, a total of 108,000 individual plants would have to be planted to analyse the completed unimutant collection (see Section 5.1). When grown in standard trays, a total greenhouse area of 420 m2 would be needed - a requirement which becomes feasible if plants are screened consecutively and not in parallel. A space-saving alternative are screens which can be performed on seedlings using either media plates or liquid assays in media-containing multi-well plates, to which different compounds to generate abiotic or biotic stresses can be easily added. Moreover, because the unimutant collection would need to be complemented with double mutants for segmentally duplicated genes (like the GABI-DUPLO lines) and RNAi lines for members of very similar members of multigene families (like AGRIKOLA or amiRNA lines), the numbers listed above might have to be increased by 25% to screen a uni-multimutant collection. Exhaustive characterisation of such populations can only be achieved by the collaborations involving many laboratories, each covering a specific set of phenotypic screens according to their specific expertise. Such screens would in many cases be repetitions of already established phenotypic assays but, because of the large number of plants to be considered, automated, innovative and non-invasive screens will have to be developed. Such screens will combine the capture of ‘classical’ visible phenotypes , specialized non-invasive assays (such as the ones to measure photosynthesis) and ‘chemical’ phenotyping . In addition, the response of plants to different environmental conditions will have to be assessed. Finally, data collection and processing has to be streamlined to allow efficient and reliable assignment of phenotypes to individual plants. This will have to include the capability of following and documenting the fate (and phenotype) of each individual plant from sowing to disposal, preferably in an automated way. New resources for the computational analysis of images and data sets are important. In addition, standardized storage of data that allows systematic interpretation of the phenotyping results is necessary. The use of ontology terms to describe the phenotypes - like the use of Gene Ontology to describe biological processes, molecular functions and cellular components - will have to be employed. Assuming that one needs to screen a minimum of two homozygous individuals for two different mutant alleles for each of the approximately 27,000 genes in Arabidopsis does not appear to be all that far-fetched. Its realisation will no doubt stimulate a wealth of in-depth gene-function analyses.In spite of these many challenges, the prediction that genome-wide phenotypic data will soon become available for


To determine seropositivity of herpes simplex virus type 2 (HSV-2) infection and associated risk factors among female sex workers (FSWs) in Guangxi, China.A convenience sample of FSWs was recruited from different types of sex work venues in two cities (Wuzhou and Hezhou) in Guangxi. Blood specimens were collected for ELISA-based detection of HSV-2 antibodies to examine the seropositivity of HSV-2 infection. Socio-demographic and behavioral data were collected through a structured questionnaire interview. Association of HSV-2 seropositivity with socio-demographic and behavioral characteristics and HIV status was analyzed.The overall prevalence of HSV-2 seropositivity among 2453 FSWs was 54.9% . The HSV-2 seropositivity was independently associated with older age, low education level, non-Han minority, migration status, working in lower-tier venues and positive HIV status.The study indicates a high prevalence of HSV-2 infection among FSWs, particularly in those working in low-tier venues in study areas, suggesting the needs to further emphasize the inclusion of HSV-2 in surveillance and intervention programs in this population. Herpes simplex virus type 2 (HSV-2) infections is one of the most prevalent sexually transmitted infections (STIs) in the world and is a major cause of genital ulcers China's HIV and STI epidemic varies widely across the country. Six provinces with the most reported cases, including Guangxi are classified as high prevalence areas, and account for 70–80% of total reports nationwide The study protocol was reviewed and approved by the Medical Ethics Committee at Institute of Dermatology, the Chinese Academy of Medical Sciences & Peking Union Medical College and National Center for Sexually Transmitted Disease Control, Nanjing, China. Written informed consent was obtained from all the participants or the caretakers of the minors/children participants involved in our study.The current study was a cross-sectional study to recruit a convenience sample from different types of sex work venues in two cities (Wuzhou and Hezhou) in Guangxi Zhuang Autonomous Region from June to September, 2009. Sex work venues were mapped to determine the nature and location and classified into three types  of venues. FSWs were categorized into three subgroups based on the sex work venues where FSWs solicited clients. High-tier FSW were those who solicited clients from karaoke bars, or hotels; middle-tier FSWs were those from hair salons or barber shops, massage parlors, foot bathing shops, roadside shops, guesthouses, or roadside restaurants; and low-tier FSWs were those on the street or in public outdoor places.FSWs were sampled from each of the three categories, with oversampling from middle- and low-tier venues. Participants eligible for this study were females who were older than 15 years and who reported having commercial sex with clients in the past three months, and gave an informed consent. This survey was integrated into the local routine outreach services in the study sites, and the study details have been reported elsewhere Sera were separated from the blood samples and stored at -70°C prior to the laboratory testing. Enzyme-linked immunosorbent assay (ELISA)  was used to detect IgG antibodies to HSV-2. Sera were screened for HIV antibodies by ELISA  and a western blot  was used to confirm initially reactive ELISA results. HIV infection was defined as positivity to both ELISA and western blot, and HSV-2 seropositivity was defined as having detectable IgG antibodies to HSV-2. For those specimens with ambiguous test results, a repeat test was performed to determine the positivity. If the result was still ambiguous, the sample was considered to be negative. The HIV testing was conducted locally with quality control from the National STD Reference Laboratory and the HSV-2 testing was conducted in the Reference Laboratory.2) test to determine the association of HSV-2 infection with individual socio-demographic and behavioral factors. The variables that were significant at the level of P<0.05 in the univariate analysis were included in the multivariate logistic regression model to evaluate the factors associated with the infection and to estimate adjusted odds ratio (AOR) and 95% confidence intervals (95%CI) for each factor. Variables reaching p<0.05 was considered statistically significant. SPSS version 13.0  was used for the statistical analysis.All participants who answered the questionnaire survey and provided blood specimens were included into final analysis. All data from the questionnaires and laboratory tests were double entered into a computer using EpiData software  by two research assistants. The univariate analysis was performed by using Chi-square . As shown in In the multivariate logistic model , HSV-2 iSeveral different studies conducted in different parts of China have reported varying HSV-2 seropositivity among FSWs in China  [10], [1We found that HSV-2 prevalence varied among FSWs from different tiers of sex work venues, probably indicating different levels of risk in their sexual behaviors. FSWs working in low-tier venues tend to offer a lower cost service, but have a greater number of clients The findings in our study show a significant association between HSV-2 infection and older age and this is consistent with that reported in previous study Consistent use of condoms has been reported to be associated with lower rates of infection with HSV-2 in previous studies The current study has some strengths, including recruitment of FSWs from different categories of sex work venues. However, some limitations in the study should be addressed. First, a convenience sampling rather than a random sampling was used to recruit participants from each venues may result in the sample bias and possible poor representativeness. Second, reporting and recall bias may have occurred during the questionnaire interview as it was based on self reported information. Third, although the current study investigated a few of common risk behaviors to be possibly related to the infection, more information about the risk behaviors, including the number of sexual partners, frequency of sexual intercourse, duration of commercial sex work, and sexual debut time were not included, and this has limited the exploration of relationship between the infections with potential risk factors. These limitations should be addressed in the further studies in the future.It can be concluded from the study that prevalence of HSV-2 seropositivity is high among FSWs, particularly those women working in low-tier sex work venues. In addition, HSV-2 seropositivity was also found to be associated with HIV infection and some socio-demographic characteristics. These findings will be helpful for designing the STI surveillance programs and developing the innovative interventions among FSW population in China.