Mucinous cystadenocarcinoma of appendix is a rare entity. Differentiating mucinous cystadenocarcinoma from mucinous cystadenoma is very challenging and depends on establishing the presence of malignant cells in the appendix wall. The invasion may be very difficult to assess in some cases, especially in early stages of the disease, which could have devastating prognostic effects on patients. Therefore, it is necessary to develop an ancillary test that can differentiate the mucinous cystadenocarcinoma from mucinous cystadenoma. So far, there is no report available about the role of differentially expressed miRNAs in the diagnosis of appendiceal mucinous cystadenocarcinoma.Six confirmed mucinous appendiceal cystadenocarcinoma and twelve mucinous appendiceal cystadenoma cases were selected. The total RNAs were extracted from the formalin-fixed paraffin-embedded specimen of these cases. The comprehensive miRNA microarray expression profiling from pooled aliquots of RNA samples from these two entities were analyzed to detect the differentially expressed miRNAs in mucinous cystadenocarcinoma. The best seven differentially expressed miRNAs were validated in individual cases by quantitative reverse transcriptase PCR (qRT-PCR).miR-4328 was significantly down regulated in mucinous cystadenocarcinoma compared to the mucinous cystadenoma (p<0.05). On the other hand, the expression of miR-200b, miR-200c, miR-451, miR-223 and miR-21 were significantly upregulated in mucinous cystadenocarcinoma (p<0.05).The microarray miRNA expression profiling analysis revealed 646 miRNAs that were differentially expressed in the mucinous cystadenocarcinoma. Among these differentially expressed miRNAs, the expression of 80 miRNAs showed statistical difference (p<0.01). The quantitative RT-PCR validated that the expression of miR-1, The expression levels of miRNAs tested were significantly altered in the appendiceal mucinous cystadenocarcinoma samples compared to the mucinous cystadenoma. These data suggest that the miRNA expression in mucinous appendiceal neoplasm may help to supplement the morphological evaluation in distinguishing benign from malignant tumors. Mucinous cystadenocarcinoma of appendix is a very rare entity, which comprises only 4%-6% appendix neoplasm . The diaThe miRNAs are a class of small, non-coding RNAs. They are involved in biological and pathological process including cell differentiation, apoptosis, proliferation and metabolism by splicing and regulating mRNAs . Their pIn this study, we first compared the comprehensive microarray miRNA profiling between mucinous cystadenocarcinoma from mucinous cystadenoma. Next, we identified several deregulated miRNAs that distinguished the cystadenocarcinoma from cystadenoma of the appendix. Finally, the greatest differentially expressed miRNAs in mucinous cystadenocarcinoma were verified in individual cases by quantitative Real-Time PCR.The protocol was approved by the institutional review board at Wayne State University. The entire patient’s demographic information was obtained from patients’ electronic records. The cases were retrieved through copath from the database of the department of pathology at Wayne State University between 2000–2013. The slides were reviewed and the diagnoses were confirmed by a senior board certified pathologist. All the patients underwent surgical resection at Harper University hospital, Huron Valley-Sinai hospital, Sinai Grace Hospital and Karmanos Cancer Institute. A total of six mucinous adenocarcinoma and twelve of mucinous adenoma were selected for the study.The total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections using RNeasy Kit . The protocol was followed according to the instructions from the manufacturer, with minor modifications. Briefly, the foci of mucinous adenoma and mucinous adenocarcinoma were macro-dissected. The 4–10 pieces of tissue curls with 10 μm thickness were placed in micro tubes. Xylene was used to extract the tissue from the paraffin and then the tissue was washed with ethanol to remove xylene. The pellet was air dried and was suspended in 240 μl of PKD buffer with 10 μl proteinase K as described previously [The aliquots of purified RNA from each case were pooled into two groups: mucinous cystadenoma and mucinous cystadenocarcinoma. The pooled RNAs were submitted for analysis using service provider  for comprehensive miRNA microarray profiling. The miRNA microarray profiling was quantitatively analyzed using miRBase version 21 . Selected housekeeping genes were used to normalize the data. In addition, a web based bioinformatics tool, ingenuity pathway analysis software  was used to identify the target mRNAs from these differentially expressed miRNAs.miR-1, miR-4328, miR-200a, miR-200c, miR-21, miR-223, and miR-451 were validated using qRT-PCR. Briefly, 10 ng of total RNA were reverse transcribed using respective specific miRNA primers and Taqman miRNA reverse transcription kit . The resulting cDNA was used as input in real time PCR using miRNA specific probes mix and TaqMan Universal PCR Master Mixture kit  according to manufacturers instructions. All reactions were performed in triplicate. The relative expression of miRNAs was analyzed with Ct method and was normalized by RNU48 expression.The expression of the differentially expressed miRNAs such as The non-parametric Mann-Whitney test was used to assess the differences in the miRNA expression level between the mucinous cystadenoma and mucinous cystadenocarcinoma samples using GraphPad StatMate software (GraphPad Software Inc.). The p values that represent differences between the two groups are displayed in the graph.  and 5The study cohort included twelve cases of mucinous cystadenoma and six cases of mucinous cystadenocarcinoma. The diagnoses of all cases were confirmed by a board certified pathologist. In twelve cases of mucinous cystadenoma, the ratio of male to female was 4:8 and the median age of the patients was 55 years old with range from 38 years old to 94 years old. In six cases of mucinous cystadenocarcinoma, the male to female ratio was 1:5 and the median age was 65 years old with range from 35 years old to 85 years old as depicted in The sizes of the mucinous cystadenoma varied with range from 0.5 cm to 11 cm. The tumors had cystic architecture filled with mucin and lined by mucinous epithelium with areas of papillary configuration or flattened mucinous epithelium without prominent cytological atypia . No invamiR-1 and miR-4328. The most significantly upregulated and interesting miRNAs were miR-200a, miR-200c, miR-451, miR-223 and miR-21. The log ratios of these miRNAs difference were around 2. The expressions of these selected miRNAs were further validated in individual cases using qRT-PCR as presented below.The comprehensive miRNAs microarray data of mucinous adenoma and mucinous adenocarcinoma have been compared and analyzed to identify the differentially expressed miRNAs in the mucinous adenocarcinoma. Expression profiling analysis revealed 646 miRNAs that are differentially expressed in the mucinous cystadenocarcinoma. Among these differentially expressed miRNAs, the expression of 80 miRNAs showed statistical difference (p<0.01). Of those 80 miRNAs, 38 miRNAs had high signal intensity (>500.00). The clustered heat map of miRNAs expression in In order to understand the pathways involved and their target mRNAs, ingenuity pathway analysis was carried out in mucinous adenocarcinoma. The miRNAs involved in the specific networks were generated based on their connectivity. We discovered the influence of many commonly studied pathways such as ERK 1/2, P38 MAPK, VEGF, Ras, Akt and IL 1/2 as depicted in miR-1 and miR-4328 were significantly down regulated in most of the samples of mucinous cystadenocarcinoma compared to the mucinous cystadenoma (p<0.05) confirmed by real time RT-PCR which is demonstrated in miR-200b, miR-200c, miR-223, miR-451 and miR-21 were significantly increased in mucinous adenocarcinoma compared to the mucinous adenoma (p<0.05) as presented in miR-451 was up regulated to a lesser degree compared to the rest of the 4 up regulated miRNAs.After identifying the differentially expressed miRNAs in mucinous cystadenocarcinoma using the microarray data, the expressions of seven important and interesting miRNAs were validated in individual cases of mucinous cystadenoma and mucinous cystadenocarcinoma as potential biomarker candidates. All the analyses were performed in parallel in order to avoid batch effects. The expression of The prognosis and treatment of the appendiceal mucinous neoplasm are greatly dependent on the diagnosis and classification of the tumor . The 4thmiR-1 expression in mucinous cystadenocarcinoma of appendix. This result is consistent with the findings of miRNAs expression profiling in colorectal cancer, in which the miR-1 expression was significantly decreased in CRC of all stages when compared to normal colonic mucosa [miR-1 expression is unknown in mucinous cystadenocarcinoma, there are many explanations about the role of miR-1 in various other tumor cells. The miR-1 was recently shown to be involved in the downstream effects of translocated EGFR signal transduction pathway as tumor suppressor and that loss of miR-1 expression promoted bone metastasis of prostate cancer [miR-1 targeted and regulated cell cycle proteins such as CDK4, CDK6, Caprin1 and Slug [miR-1 expression promoted cancer cell proliferation and metastasis. Restoring miR-1 expression reversed its inhibition of cell cycle progression [miR-1 with other down-regulated miRNAs could prove useful for mucinous cystadenocarcinoma patients, as this would reduce metastasis. Further mechanistic role of this miRNA in mucinous cystadenocarcinoma needs to be explored.In our study, we observed significant decrease (p<0.01) in c mucosa . Althouge cancer . In renaand Slug . In addigression . Hence, miR-4328 was significantly decreased in the keloid fibroblast [miR-4328 in cancer cells. Further studies are required to elucidate the relationship between the down regulation of miR-4328 and its role in the mucinous cystadenocarcinoma of appendix.Although the expression of broblast , no repomiRNA-200 family members in tumor are varied according to different types of diseases. Zuberi et al have demonstrated that the expression of miR-200a, 200b and 200c was significantly deregulated in epithelial ovarian cancer compared to matched normal control, which is correlated with advanced cancer stage and lymph node metastasis [miR-200a and miR-200c was significantly higher in ovarian cancer FFPE samples compared to its associated endometriosis [miR-200a level was significantly lower compared to normal control patient [miR-200a, miR-200b and miR-200c was down regulated significantly. This decrease in expression level was associated with high histologic grade and the presence of intravascular invasion [miR-200b and miR-200c was significantly increased in mucinous adenocarcinoma. The mechanism of variability of miR-200 expression in different types of cancers is interesting and deserves to be dissected in future studies.Numerous reports on the expression of tastasis . In our  patient . In gastinvasion . In our miR-21 expression in mucinous adenocarcinoma in our study is consistent with reports from many different types of cancers [miR-21 miRNA may be used as biomarker that differentiates mucinous cystadenocarcinoma from cystadenoma. The miR-21 is one of the most common miRNAs that are up regulated in both solid and hematological malignancies [PDCD4, TPM1, PTEN and Maspin, which are involved in cell cycle progression and apoptosis [miR-21 was found to be significantly associated with the progression of the tumor [miR-21 could be one of the miRNA biomarker candidates in the prognosis of mucinous cystadenocarcinoma patients in the future studies.The significant up regulation of  cancers –25. Thisgnancies . It may poptosis . Recentlhe tumor . The rolhe tumor ,30. ThermiR-223 was interestingly increased in the mucinous adenocarcinoma, which is significantly different. Considering the miR-223 is the major negative modulator of myeloid differentiation, especially the granulocytic differentiation, the role of the miR-223 in the formation of mucinous adenocarcinoma is unclear and difficult to interpret. In the miR-223 null mice, the mice had expanded granulocytic compartment due to its increase of the granulocyte progenitors [miR-223 is the most abundant and important miRNA in the human peripheral blood neutrophil granulocytes [miR-223 levels were found to be correlated with the activity of the rheumatoid arthritis [miR-223 could be a biomarker for inflammatory activity instead of tumor biomarker. Thus, one explanation of up regulation of miR-223 in mucinous adenocarcinoma could be that it may be caused by the inflammatory cells that infiltrated within the tumor, not from tumor cell itself.In this report we observed that the expression of genitors . The miRulocytes . In the rthritis . TherefomiR-1, miR-4328, miR-200b, miR-200C, miR-223, miR-21 and mir-451 were significantly dysregulated in the appendiceal mucinous cystadenocarcinoma compared to the mucinous cystadenoma. Our findings based on limited number of cases analyzed in these relatively rare entities could benefit from similar analyses using a larger cohort to help confirming our potentially promising results and their implications in the future.In conclusion, the expression of


People who rate their health as poor experience higher all-cause mortality. Study of disease-specific association with self-rated health might increase understanding of why this association exists.To estimate the strength of association between self-rated health and fatal and non-fatal cardiovascular disease.A comprehensive search of PubMed MEDLINE, EMBASE, CINAHL, BIOSIS, PsycINFO, DARE, Cochrane Library, and Web of Science was undertaken during June 2013. Two reviewers independently searched databases and selected studies. Inclusion criteria were prospective cohort studies or cohort analyses of randomised trials with baseline measurement of self-rated health with fatal or non-fatal cardiovascular outcomes. 20 studies were pooled quantitatively in different meta-analyses. Study quality was assessed using Newcastle-Ottawa scales.2 = 68.61%), Self-rated health was not significantly associated with non-fatal cardiovascular disease outcomes (HR 1.66 (95% CI 0.96 to 2.87); 5 studies; I2 = 83.60%).‘Poor’ relative to ‘excellent’ self-rated health  was associated over a follow-up of 2.3–23 years with cardiovascular mortality in studies: where varying degrees of adjustments had been made for cardiovascular disease risk (HR 1.79 (95% CI 1.50 to 2.14); 15 studies, I2 = 71.24%), and in studies reporting outcomes in people with pre-existing cardiovascular disease or ischaemic heart disease symptoms (HR 2.42 (95% CI 1.32 to 4.44); 3 studies; I2 = 71.83%). ‘Poor’ relative to ‘excellent’ self rated health was also associated with the combined outcome of fatal and non-fatal cardiovascular events (HR 1.90 (95% CI 1.26 to 2.87); 5 studies; IPoor self rated health is associated with cardiovascular mortality in populations with and without prior cardiovascular disease. Those with current poor self-rated health may warrant additional input from health services to identify and address reasons for their low subjective health. Self-rated health (SRH), a simple measure of subjective health status, strongly and consistently predicts all-cause mortality across varied populations even after adjustment for demographic, biophysical, and behavioural risk factors. Studies of SRH have primarily studied its’ relationship with all-cause mortality.The relationship between SRH and mortality has been shown to vary by disease. In a unique analysis of 700,000 American National Health Interview Survey participants followed for over 20 years, self-rated health strongly predicted death from diabetes, infectious, and respiratory diseases, , and a to a lesser extent from coronary heart disease (HR of 2.3) and cancer (HR of 1.6) This meta-analysis was undertaken to collate evidence from individual studies and examine the association across studies between SRH and cardiovascular disease-specific mortality and morbidity. Cardiovascular disease including stroke was chosen since it remains the most important cause of morbidity and mortality in many countries and because its epidemiology is well established, facilitating better measurement and control of covariates in study design and analysis. It has been speculated that the association of poor self-rated health with all-cause mortality may be driven by its association with cardiovascular diseases.A comprehensive search of the following electronic resources was undertaken during June 2013: PubMed MEDLINE, EMBASE, CINAHL, BIOSIS, PsycINFO, DARE, Cochrane Library, and Web of Science. Search strategies were tailored to each database using a combination of indexed headings and text words; a sample search is included in the Appendix. Two reviewers independently searched the databases, selected the studies and reviewed the contents of the manuscripts to determine whether they met the criteria for inclusion. Data were then independently extracted and quality assessment performed. When discrepancies occurred between reviewers in determining inclusion into the analyses, other authors were asked to evaluate the studies. No language or time restrictions were imposed. Authors of primary studies and experts in the field were contacted to answer questions about methodology or study results. For the systematic review, studies had to meet the following inclusion criteria: (a) adult populations (b) current self-rated health measured by a single question at the beginning of follow-up, with clearly defined and similar response categories (c) prospective cohort study, cohort analysis of randomised trials or incident (nested) case-control study (d) reporting non-fatal events and/or fatal outcomes for coronary heart disease, stroke, or combined CVD. Assessment of study quality was primarily concerned with identifying risk of bias, rather than merely examining the quality of article reporting Studies were sub-divided into those reporting: (i) fatal CVD events or mortality outcomes in both population studies and in those reporting outcomes in people with pre-existing cardiovascular disease or ischaemic heart disease symptoms (ii) those with non-fatal CVD events only (iii) and those with combined fatal and non-fatal CVD events.2-statistic, based on the DerSimonian-Laird estimator. Publication bias was assessed using a funnel plot and a rank correlation test for funnel plot asymmetry. We combined log-hazard ratios for appropriate subsets of individual studies using random-effects meta-analyses, and presented corresponding forest plots of hazard ratios with a diamond indicating the summary effect estimate and 95% confidence interval. Box sizes were drawn inversely proportional to the magnitude of the standard error for each study and indicate the relative weight of each study in the meta-analysis. Where there was more than one CVD outcome per study or analyses on two or more patient samples, the effect sizes for each outcome/patient sample were averaged using a fixed effects meta-analysis to generate a single effect size for each study. The meta-analysis focussed on the comparison of patients reporting poor health relative to excellent health, via hazard ratios. For studies analysing ordered categories, we used the categories at each end of the Likert scale . A number of studies collapsed their self-rated health categories into a dichotomous variable, likely to ensure adequate numbers for analysis. For these, the highest SRH category analysed was used as the “excellent” reference category and the comparison category labelled “poor” self-rated health. Heterogeneity between studies was assessed using the Q-test for heterogeneity and IP-values in the statistical tests. Sensitivity analyses are shown in Details of the 20 studies included in the various meta-analyses are presented in 2–statistic (I2 = 71.24%) and Q-test statistic (Q-test statistic of 48.7 (p<0.0001)). However, the I2 statistic was 0 if the meta-analysis was restricted to studies containing elderly populations, or to studies of men which were well controlled for covariates. In both cases, the summary estimate (hazard ratio) for CVD mortality for excellent to poor health was reduced.  (see 013) see . The Q-tOur meta-analysis demonstrates a significant association between SRH and CVD mortality. This was present in populations with pre-existing disease and also in those where efforts were made to control for disease. Compared with excellent health, poor self-rated health was associated with more than one and a half times the risk of CVD mortality even after maximal adjustment for risk factors . This is comparable to but somewhat less than the effect size observed for SRH with all-cause mortality in other studies ).These findings need to be interpreted in the light of the observed heterogeneity. Both the size of the association with cardiovascular mortality and the degree of heterogeneity was reduced if the analysis was restricted to elderly populations, or male populations with good control of covariates. This suggests that our summary estimates of the association of SRH with cardiovascular mortality were probably inflated through inadequate adjustment for different participant characteristics in some of the studies. Heterogeneity in studies included in the meta-analysis of SRH and non-fatal CVD outcomes was considerably reduced by excluding van der Linde et al (2013), which found the strongest association between SRH and non-fatal CVD outcomes ), but a weaker association with cardiovascular mortality than the other studies. The van der Linde study however was of high quality. It was carried out in a large population of over 16,000 men and women with over 10 years of follow-up, and with particular efforts made to exclude from the analyses those with pre-existing disease . Adjustments were also made for cardiovascular risk at baseline including behavioural factors and dietary measures, and outcomes were robustly defined and measured. Other studies had not excluded prevalent disease as thoroughly, or had included older cohorts where results were more likely to reflect current ill health. van der Linde also had more complete ascertainment of fatal CVD and of confirmed diagnosis of non-fatal CVD outcomes through hospital admission identified from participants’ National Health Service number through data linkage with Health Authority databases. This study therefore perhaps provides the most reliable estimates of the association of SRH with confirmed cardiovascular morbidity. Indeed, a more recent study not included in the meta-analysis due to statistical non-conformity with other included studies, also found a significant association between SRH and onset of non-fatal CVD (HR 1.25 for CHD and 1.54 for stroke p< = .001). In this study the strength of the association was closer to the other studies in this meta-analysis than to van der Linde, perhaps because it relied on a weaker method of ascertaining outcome (self report of physician diagnosis). There are still surprisingly few high quality studies in this area. We found only 5 cohorts reporting on the incidence or recurrence of non-fatal CVD, only 5 studies reporting combined fatal and non-fatal CVD events, and only 3 studies of fatal CVD events in those with pre-existing cardiovascular disease or ischaemic heart disease symptoms. There were methodological problems with many of the included studies, including poor measurement of baseline risk and CVD status (e.g. self-report versus objectively diagnosed), lack of detail of study methods and poor ascertainment of disease status and severity. We used a random-effects method of analysis because of the heterogeneity, but this results in smaller studies being given a greater relative weight than if a fixed effects method was performed.The association of SRH with CVD mortality found in our meta-analysis is likely to explain some but not all of the relationship between SRH and all-cause mortality. It nevertheless remains an important finding, in particular in view of the fact that in many of the studies included in the meta-analysis, adequate adjustment for covariates including traditional Framingham CV risk factors was undertaken. The most important hypothesis to explain the relationship between SRH and all-cause or disease-specific mortality is that subjective assessment of SRH is also a sensitive measure of objective health status. If this is so, precision of measurement of disease correlates, and better adjustment should decrease the strength of the association, which studies including better measurement of covariates may account for. Our meta-analysis did confirm that the strength of the association was reduced (but remained significant) if only well-controlled studies for cardiovascular risk were included. Secondly the association between SRH and mortality outcomes may reflect a personal predisposition to better or worse health, not related to objective health status at the time of questioning. The predisposition might be rooted in a range of health-protective factors, including biophysical factors; immune responses or neuro-endocrine homoeostasis, behavioural factors; diet, physical activity, smoking - usually adjusted for in studies, and psycho-social factors . This study suggests that standard CVD risk factors and disease severity measures may underestimate risks of clinical cardiac events and that self-ratings may convey additional knowledge that may not be completely captured by epidemiological or available clinical measurement. The use of SRH as an additional risk factor in traditional cardiovascular risk prediction models or as part of a simple non-invasive risk score acceptable to patients in primary care therefore requires further investigation. It is uncertain when would be the best time to measure SRH for purposes of accurate outcome prediction. There is also uncertainty around whether SRH is in fact modifiable and if any improvements in SRH may result in improved cardiovascular outcomes. Nevertheless, those with poor self-rated health at cardiovascular risk in both non-clinical and clinical populations may warrant additional input from health services to identify and address reasons for their low subjective health.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.File S1Sample search strategy.(DOCX)Click here for additional data file.


This study aims to assess the measurement invariance (MI) of the KIDSCREEN-27 questionnaire across girl-parent and boy-parent dyad to clarify how child gender affects the agreement between children’s and parents’ perception of the meaning of the items in the questionnaire. The child self-reports and parent proxy-reports of the KIDSCREEN-27 were completed by 1061 child-parent dyad. Multiple group categorical confirmatory factor analysis (MGCCFA) was applied to assess MI. The non-invariant items across girl-parent dyad were mostly detected in the psychological well-being and the social support and peers domains. Moreover, the boys and their parents differed mainly in the autonomy and parent relation domain. Detecting different non-invariant items across the girl-parent dyad compared to the boy-parent dyad underlines the importance of taking the child’s gender into account when assessing measurement invariance between children and their parents and consequently deciding about children’s physical, psychological or social well-being from the parents’ viewpoint. The importance of pediatric health-related quality of life (HRQOL) assessment to identify children being at risk of health problems has recently received much attention . Over thHowever, the potential influence of child gender has been rarely assessed in the literature and no consistent results have been reported . For exaHowever, in the literature cited above the agreement between child self-reports and parent proxy-reports was simply evaluated by comparing the mean of HRQOL scores. For a meaningful interpretation of mean differences, an essential assumption known as measurement invariance (MI) should be established . MI showTo explore MI, various statistical techniques are available, which have their own advantages and disadvantages . MultiplThe KIDSCREEN-27 is a well-known international generic HRQOL instrument with parallel child self-reports and parent proxy-reports. Although the MI of the KIDSCREEN have been established across different European countries , this isThe target population was composed of 287551 school children aged 8-18 years and their parents in Shiraz, in the academic year 2011-2012. The participants were selected based on a two-stage cluster random sampling technique from the four educational districts of Shiraz, southern Iran. In the first stage, 6 middle and 7 high schools in each educational district were chosen randomly by cluster sampling. Within the selected 24 middle schools and 28 high schools, 40 and 50 classes were chosen respectively by stratified sampling. Finally, in each class a simple random sample of students was selected. Both child self-reports and parent proxy-reports of the Persian version of the KIDSCREEN-27 questionnaire and informed parent consent forms were distributed in each classroom by a trained researcher asking the students to take them home to their parents. About 75% of parents signed the informed consent and agreed to participate in the study with their children. Only one parent (mother or father) completed the proxy-reports, but there was no information on the gender of proxy rater in this study. The proxy-reports of the questionnaire were signed by parents to ensure that parents actually completed the questionnaire. All the children and their parents in our sampling population spoke Persian. They independently filled in the self- and proxy-reports at home. Finally 1061 completed child self-reports and parent proxy-reports of the questionnaire were returned to school by students. Of the 1061 children 593 (55.89%) were boys. The mean (± SD) age of boys was 13.65 ± 2.11 years and that of girls was 12.70 ± 2.65 years.The KIDSCREEN is one of the international instruments for measuring HRQOL in children and adolescents. Both child self-reports and parent proxy-reports of this questionnaire are available in 3 versions including 52, 27 and 10 items . AcceptaThis instrument encompasses five subscales of physical well-being (5 items), psychological well-being (7 items), autonomy and parent relation (7 items), social supports and peers (4 items), and school environment (4 items). All items were scored on a 5-point Likert scale from 1=never to 5=always or from 1=not at all to 5=extremely. For ease of interpretation, rating scale categories of negatively worded items were reversed so that higher scores showed better HRQOL.In this study, the MI of each domain of the KIDSCREEN-27 across girl-parent and boy-parent dyad was assessed by MGCCFA technique. An advantage of MGCCFA is that it can appropriately model the ordered-categorical responses by including the threshold structure . InteresIn the present study, four levels of invariance including configural, metric, strong, and strict invariance were tested by setting four types of constraint on a set of parameters in an increasingly hierarchical order. Configural invariance investigates whether an underlying construct of interest is measured by the same set of items across two groups i.e., children and their parents use the same conceptual framework in their appraisal of underlying construct of interest . In thisIn this study the concept of partial measurement invariance is applied. When a certain level of invariance are not satisfied for all the items in a specific domain, the equality constraints on the parameters of the items demonstrating non-invariance between groups are removed one at a time and they will be allowed to be estimated freely . Assessi2) statistic, and for testing relative fit of two nested model, the change in it (∆χ2) can be used. However, this well-known statistics is not a practical test of model fit due to the detection of even trivial differences under large sample size (2 (with df) and (∆χ2) were reported, other fit indices like comparative fit index (CFI), ΔCFI , and root mean square error of approximation (RMSEA) were considered for a final decision about accepting or rejecting the hypothesis of interest owing to their more reliable results. Because 2, value of CFI≥0.95, ΔCFI≤0.01, and RMSEA≤0.06 can support acceptable model fit , in which the value of modification indices (not shown here) indicated that items 2 (Been in a good mood) and 7 (Been happy with the way you are) had different factor loadings among the groups. This result suggests that the associations of all items with their corresponding underlying constructs are equivalent across the groups except for the two cited items (items 2 and 7). After relaxing the equality constraints of the factor loadings of these two items across groups, partial metric invariance was accepted .Moreover, the full strong invariance was held for the physical well-being and the school environment domains across girl-parent dyad  and 3  in the physical well-being domain, partial strict invariance was verified  across child-parents dyad did not consider child gender . HoweverIn this case, it is important to develop a new or revised version of the instrument with invariant items for meaningful cross-group comparisons. However, non-invariance of an item should not per se lead to its elimination, especially when the instrument has high convergent and discriminant validity . RegardiOur study has some limitations that merit attention when interpreting our findings. First, the effect of clustered data  has not been taken into account which may result in increased likelihood of finding measurement non-invariant items . Hence, In conclusion, detecting different non-invariant items across the girl-parent dyad compared to the boy-parent dyad underlines the importance of taking the child’s gender into account when assessing measurement invariance between children and their parents and consequently deciding about children’s physical, psychological or social well-being from the parents’ viewpoint. Therefore, future studies should replicate or extend our findings from the analysis of KIDSCREEN-27 to other pediatric HRQOL measures and also in other cultures to provide a questionnaire with measurement invariant items which is also stable regarding child gender.


Drosophila melanogaster genome were analyzed in vivo, confirming 15 active enhancer regions. A comparison with Drosophila pseudoobscura sequences revealed that conservation of binding-site clusters accurately discriminates functional regions from non-functional ones. 27 predicted gene-regulatory regions in the  Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters.The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns.We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: Measuring conservation of sequence features closely linked to function - such as binding-site clustering - makes better use of comparative sequence data than commonly used methods that examine only sequence identity. The transcription of protein-coding genes in distinct temporal and spatial patterns plays a central role in the differentiation and development of animal embryos. Decoding how the unique expression pattern of every transcript is encoded in DNA is essential to understanding how genome sequences specify organismal form and function.cis-acting sequences that control transcription, identifying which trans-acting factors act on each regulatory sequence, and determining how these interactions affect the timing and organization of transcription. The first step in this process is by no means straightforward. Regulatory regions are often large and complex. Functional cis-acting sequences are found 5' and 3' of transcripts and in introns, and can act over short or long distances. Most of the described animal regulatory sequences were identified by experimental dissection of a locus, and astonishingly few of these are well characterized.Understanding gene regulation requires discovering the cis-regulatory modules (CRMs) - tend to contain functional binding sites for several different transcription factors, often with multiple sites for each factor.Despite the paucity of good examples, as multiple regulatory sequences from different organisms were identified and characterized, some common features became apparent ,2. Most in silico methods to identify regulatory sequences on the basis of binding-site clustering have been developed and applied to animal genomes [in vivo regulatory activity [As the first animal genome sequences were completed -6, resea genomes -10. Someactivity -17, yet Drosophila development is perhaps the best system in which to apply and evaluate these methods. Development of the Drosophila embryo is arguably better understood than that of any other animal. Sophisticated genetic screens [in situ expression studies [The transcriptional regulatory network governing early  screens ,19 have  screens  and in s studies . TranscrDrosophila melanogaster genome with unusually high densities of predicted binding sites for the early-acting transcription factors Bicoid (BCD), Hunchback (HB), Krüppel (KR), Knirps (KNI) and Caudal (CAD). As nine of these regions overlapped previously known CRMs, we proposed the remaining 28 as predicted CRMs (pCRMs). We tested one of the previously untested pCRMs for enhancer activity in a standard reporter gene assay [giant (gt) in a posterior stripe. Here, we report the systematic testing of the remaining 27 untested pCRMs for enhancer activity, resulting in collections of both bona fide positive and false-positive predictions, allowing us to develop and evaluate methods to improve the accuracy of methods for identifying functional cis-regulatory sequences.In a previous study , we idenne assay ,23 and sD. pseudoobscura  was rec species ,26, and Drosophila suggest that similar methods will be valuable in insects as well [D. melanogaster and D. pseudoobscura showed that known regulatory regions are only slightly more conserved than the rest of the non-coding genome [Most methods used to identify regulatory sequences from interspecies sequence comparison are fairly simple. They identify 'conserved' non-coding sequences (CNSs), operationally defined as islands of non-coding sequence with relatively high conservation flanked by regions of low conservation, and assume that this conservation reflects regulatory function. Although crude, these methods have been remarkably effective in identifying mammalian regulatory sequences ,28, and  as well . However as well ,31. Simig genome , highligrunt (run), even-skipped (eve), hairy (h), knirps (kni) and hunchback (hb). To determine whether any of the remaining 28 pCRMs also function as enhancers, we generated P-element constructs containing the pCRM sequence with minimal flanking sequence on both sides fused to the eve basal promoter and a lacZ reporter gene . As the margins of the tested sequences do not precisely correspond to the margins of the clusters, we assigned a unique identifier (of the form CEXXXX) to each tested fragment .The 37 pCRMs are shown in Table We successfully generated multiple independent transgenic fly lines for 27 of the 28 pCRMs. We repeatedly failed to generate transgenes containing CE8007. This sequence contains five copies of an approximately 358 base-pair (bp) degenerate repeat. One additional pCRM (CE8002) also contains tandem repeats. While we were able to generate transgenes for CE8002 and assay its expression, these two tandem repeat-containing pCRMs (CE8007 and CE8002) were excluded from subsequent analyses.in situ RNA hybridization to the lacZ transcript in embryos at different stages in at least three independent transformant lines. Nine of the 27 transgenes showed mRNA expression during embryogenesis  of gt expression in the blastoderm as previously described [CE8001 is 5' of the gene for the gap transcription factor escribed .nub). The CRM recapitulates the endogenous blastoderm expression pattern of nub, first detected as a broad band extending from 50 to 75% egg length. Although nub expression continues in later embryonic stages, CE8011 expression is limited to the blastoderm stage.CE8011 is 5' of the gene for the POU-homeobox transcription factor nubbin (odd-skipped (odd) and drives expression of two of its seven stripes: stripe 3 at 55% and stripe 6 at 75% egg length. This CRM also has the ability to drive later, more complex, patterns of expression. During stages 6 and 7, expression is detected in the procephalic ectoderm anlage and in the primordium of the posterior midgut. By stage 13, expression is also detected in the anterior cells of the midgut which will give rise to the proventriulus, the first midgut constriction, the posterior midgut and microtubule primordial as well as cells in the hindgut, all similar to portions of the pattern of wildtype odd protein expression previously described [CE8010 is 5' of the pair-rule gene escribed .fushi-tarazu (ftz) and drives expression of two of its stripes: stripe 1 at 35% and stripe 5 at 65% egg length. Using a similar CRM reporter assay, this pattern of expression was also detected by [CE8024 is 3' of the pair-rule gene POU domain protein 2 (pdm2) and appears to completely recapitulate its stage-12 expression pattern, which is limited to a subset of the developing neuroblasts and ganglion mother cells of the developing central nervous system. A similar pattern of expression was previously described for the protein product of pdm2 [nub.CE8012 is in the third intron of  of pdm2 . It is wsqz) and recapitulates the wild-type expression pattern of sqz RNA in a subset of cells in the neuroectoderm at stage 12. The wild-type sqz expression pattern was previously described [CE8027 is 3' of the gene for the Zn-finger transcription factor squeeze (escribed .CG9650) that is maternally expressed and deposited in the embryo. This strong maternal expression potentially obscures a zygotic expression pattern. Two additional adjacent genes, CG32725 and CG1958, showed no expression in whole-mount in situ hybridization of embryos.The remaining three active pCRMs cannot be easily associated with a specific gene. CE8005 drives expression in the ventral region of the embryo. It is 3' of a gene encoding a ubiquitously expressed Zn-finger containing protein , which is expressed maternally, possibly obscuring an early zygotic expression pattern (a few in situ images show a hint of striping). sbb is also expressed later in development in the ventral nervous system. An additional potential target, Otefin (Ote), is also expressed maternally and relatively ubiquitously through germ-band extension. All other nearby genes displayed in Figure in situ hybridization or by microarray.CE8016 drives a seven-stripe expression pattern in the blastoderm. It is in the first intron of ftz stripe and two stripes at 39% and 87%. It is in the first intron of ome (CG32145), which is not expressed maternally and has no blastoderm expression, but is expressed late in salivary gland, trachea, hindgut and a subset of the epidermis. All other nearby genes displayed in Figure in situ hybridization or by microarray.CE8020 drives an atypical four-stripe pattern in the blastoderm - two stripes at 7% and 26% that are anterior to the first  ome CG3245, whichWith these results, and the nine previously known enhancers, at least 15 of the 37 highest density clusters of the five transcription factors used in our initial screen have early-embryonic enhancer activity. The remainder of this paper examines 35 of the original 37 clusters, with the two tandem repeat-containing clusters excluded. We divide these 35 into three categories - 15 positives (the nine overlapping previously known enhancers plus the six new enhancers identified here), three ambiguous (the three positives without a clear regulated gene), and 17 negatives (see Table reaper), so out of 16 pCRMs located within 20 kb of a gene with appropriate expression, 15 (94%) are active enhancers.All 15 positives are within 20 kb of the transcription start site  of transcripts expressed in spatiotemporal patterns consistent with regulation by the maternal and gap transcription factors used in our screen . Only one of the 17 negatives was located within 20 kb of a plausible target (PCE8021 is 7 kb upstream of p = 0.017). The positives have a slightly higher density of binding sites, but this difference was not significant. The binding site composition of the positives and negatives are similar . Although others have reported that some factors have characteristic spacings with respect to themselves and other factors [The positives are, on average, larger than the negatives , a difference that is significant by the Komogorov-Smirnov (KS) test  would have been successfully identified by such simple comparative methods, positive pCRMs do not collectively appear distinguishable from flanking sequence on the basis of conservation alone. Although positive pCRMs are almost all in highly conserved blocks, there is a surprisingly high amount of non-coding sequence conservation throughout these regions, and most negative pCRMs are also contained in highly conserved blocks. It remains to be seen whether this difference in the conservation landscape of Drosophila non-coding sequences compared to vertebrates reflects a significant difference in the functional organization of non-coding sequences, or simply indicates that there is too little divergence between D. melanogaster and D. pseudoobscura to detect useful differences in the rates of evolution (see Discussion).We first assessed whether positive pCRMs could be distinguished from their flanking sequences based on degree of conservation. In vertebrate comparative genomics, relatively simple methods (such as VISTA ) are comD. melanogaster and D. pseudoobscura. For each pCRM-containing region, we identified orthologous contigs from the D. pseudoobscura assembly and aligned them using the alignment program LAGAN [p = 0.002 by KS test), the distribution of conservation scores for positive and negative pCRMs overlap considerably, and thus conservation alone is not a useful way of distinguishing positive and negative pCRMs  regions in randomly chosen sections of the genome Figure . PositivD. melanogaster and D. pseudoobscura, and that most D. melanogaster enhancers should have functional orthologs in D. pseudoobscura. For those enhancers we seek to identify here - namely those where binding site clustering reflects their function - we expect clustering to be found in both D. melanogaster and D. pseudoobscura. Conversely, clusters that simply occur by chance in either genome but do not reflect the function of the sequence  should not be conserved. Thus, looking for conservation of binding-site clustering should provide a valuable way of distinguishing functional and non-functional binding-site clusters in the D. melanogaster genome.We expect that most genes will have similar expression patterns in D. melanogaster binding site that overlaps a predicted D. pseudoobscura binding site for the same factor in an alignment as an 'aligned' site. We require overlap and not perfect alignment to compensate for alignment ambiguity; the overwhelming majority (85%) of aligned sites are perfectly aligned. Although there is only a subtle difference in the binding-site density in the positive and negative pCRMs in D. melanogaster (22.7 sites/kb compared to 22.2), the density of aligned binding sites in positive pCRMs (13.8 sites/kb) is nearly twice that in negative pCRMs (6.8 sites/kb). This is a highly significant difference (p < 0.001 by KS test) and aligned site density better discriminates positive and negative pCRMs than sequence conservation , and soervation ,26.D. melanogaster to be 'preserved' if it can be matched to a corresponding site in the D. pseudoobscura pCRM . If we consider both aligned and preserved sites to be conserved, then roughly 80% of the sites in positive pCRMs are conserved compared with 40% in negative pCRMs.To characterize the extent of binding site conservation independent of positional conservation, we computed a second measure of binding-site conservation. We consider an unaligned binding site in D. pseudoobscura orthologs of active D. melanogaster CRMs we observe an increase in binding-site density that cannot be explained by the positional conservation of sites found in D. melanogaster or the random occurrence of sites in the genome. Several of the 15 positive CRMs have high densities of these preserved but unaligned sites, but two in particular, runt stripe 3 and hairy stripe 6, stand out from the rest. These two have almost as many preserved sites as strictly aligned sites.The density of preserved but not aligned sites in positive pCRMs (4.3/kb) is considerably higher than in negative pCRMs (2.2/kb) or random sequences (1.8/kb). Thus, in the Aligned plus preserved (conserved) site density Figure  almost pD. melanogaster and D. pseudoobscura successfully distinguishes positive and negative predictions made using the D. melanogaster sequence alone, we incorporated comparative sequence data into our enhancer-prediction algorithm CIS-ANALYST [As the conservation of binding sites and binding-site clusters between -ANALYST . Instead-ANALYST .Using 17 negative pCRMs and an expanded set of 25 positive pCRMs  as are five other known enhancers. One of our negative pCRMs, CE8021, is ranked number 12.As eCIS-ANALYST has markedly better specificity than CIS-ANALYST, we sought to identify BCD, HB, KR, KNI and CAD targets that were missed with the relatively stringent criteria used in our previous analysis. Rather than use a stringent cutoff (15 binding sites per 700 bp) as we did in , we perfDrosophila embryonic expression patterns [To focus our search for new enhancers on genes likely to be regulated by BCD, HB, KR, KNI and/or CAD, we searched FlyBase  and a dapatterns  and idencis-regulatory modules in Drosophila. Analysis of the in vivo activity of 36 high-density clusters of predicted BCD, HB, KR, KNI and CAD binding sites identified in our previous study [in vivo. An evolutionary analysis of these sequences - based on comparisons of the D. melanogaster and D. pseudoobscura genomes - shows that sequence conservation alone can not reliably discriminate cluster-containing regions that function in vivo from those that do not. However, a new method that combines binding-site clustering and comparative sequence analysis to search for binding-site clusters that are present in multiple species does reliably discriminate active and inactive clusters. Using this method, we make several hundred predictions of new CRMs, a large number of which are located near likely target genes.We performed a large and comprehensive evaluation of the efficacy of computational methods for the identification of functional us study  offers cThe success of relatively simple binding-site clustering methods here and in other work is remarkable given the crudeness of these methods. As our negative predictions demonstrate, the mere presence of a cluster of binding sites is not sufficient to make an active embryonically expressed CRM. Although these 17 sequences have binding-site densities and compositions indistinguishable from their functional cousins, they do not function as enhancers in a simple transgene assay.D. pseudoobscura orthologs of these negative pCRMs have binding-site clusters, and few are near genes with appropriate expression patterns. Thus it is unlikely that many function in their endogenous locations in vivo.It is possible that some of these negative pCRMs may be functional enhancers that respond to the factors used in our screen, perhaps requiring a different promoter or other flanking sequences not used in the transgene. While further experiments could address this possibility, we felt these were a low priority, as few of the cis-regulatory sequences.Both the general activity and, more important, the specific regulatory output of a CRM are a complex, and still poorly understood, function of the specific architecture of its sites. The emerging picture of the ordered multiprotein complexes that mediate enhancer activity suggests constraints on enhancer composition and architecture ,2,47 whoin vivo function related to their observed expression patterns. However, the poor conservation of these elements in D. pseudoobscura suggest that they do not have a regulatory function, and raises the possibility that some 'random' clusters of binding sites  have the necessary characteristics to be active enhancers in the proper genomic environment . That any such sequences exist suggests that the compositional and architectural constraints on binding sites in enhancers may be fairly weak.It is intriguing that three of the clusters we tested direct expression patterns that bear no obvious relationship to the expression of a neighboring gene despite our extensive efforts to identify such genes. We cannot yet exclude the possibility that these pCRMs have an Whatever the nature of these constraints, it is clear that binding-site density is not the sole defining characteristic of functional enhancers. However, it is a surprisingly effective distinguishing one, and the usefulness of this and related methods  suggestsftz and odd. ftz and odd are generally classified as 'secondary' pair-rule genes whose expression is governed by other pair-rule genes rather than by the maternal and gap transcription factors that govern the so-called 'primary' pair-rule genes    . Howeveftz enhancer is an evolutionary relic of the homeotic role played by ftz in primitive insects [D. melanogaster and D. pseudoobscura, it seems unlikely that this element is purely vestigial. In fact, Yu and Pick [ftz gene and show that stripes 1 and 5 appear before other ftz stripes and they postulate the existence of stripe-specific regulatory elements that may exist outside of the characterized zebra and upstream elements such as the one identified and characterized in this study. The conservation of binding sites in both the ftz and odd enhancers suggest that they play an important role in development, and further call into question the distinction between primary and secondary pair-rule genes.It has been suggested that the  insects , a view and Pick  examinednub  and pdm2 are expressed in the anterior and posterior midgut primordium and in neuroblasts. CE8011, found immediately upstream of nub, regulates its early expression, and not its later neuroblast expression. In contrast, CE8012, found in an intron of pdm2 regulates its expression only in neuroblasts and not earlier. While we did not detect a neuroblast enhancer for nub or a blastoderm enhancer for pdm2 in our single-species binding-site cluster search, a number of interesting pdm2 regions were discovered in our eCIS-ANALYST search (two are listed in Table Two of the new enhancers (CE8011 and CE8012) are adjacent to and apparently regulate two linked genes with very similar patterns of embryonic expression. Both Drosophila enhancers have been known to work at distances of up to 100 kb, but most are within 10 kb of their target gene. All of our true-positive predictions were within 10 kb of the known or predicted transcription start site of a gene with a pattern that was known, or plausibly could have been, regulated by the five regulators used in our screen . In contrast, only one of the negative predictions was this close to such a gene - an additional four were within 50 kb. As the comprehensive atlas of embryonic expression patterns is completed [The accuracy of our enhancer predictions would almost certainly be improved if we restricted our search space to genomic regions adjacent to genes known to be regulated by particular transcription factors. ompleted ,53 it wiDrosophila. Even assuming the availability of binding data for all of these factors, it will not be possible to search for targets of all combinations of these factors - there are too many possibilities. This is not just a practical problem - it is a fundamental statistical problem. While the false-positive rate for a single combination of factors is low, if we tried even all pairs of factors, it is likely that every region of the genome would have a high binding-site density for some collection of factors. Sequence data from other Drosophila species may allow us to determine which of these collections are conserved and therefore likely to be functional, but it is unlikely that all aspects of regulation can be inferred from comparative analyses and therefore it is essential that we continue to dissect the regulatory network by traditional means.Comprehensive methods for inferring regulatory interactions where they are not already known will be critical for the widespread application of binding-site clustering methods. In addition to allowing less stringent focused screens, they will also help overcome the combinatorial challenge raised by the existence of up to 700 sequence-specific transcription factors in Drosophila transcription factors. The initial success of methods that use in vitro binding data to predict regulatory targets has prompted the characterization of binding specificities for many additional factors. However, the heterogeneity of approaches used makes it difficult to combine these data in an optimal manner. In addition, most of the available transcription factor binding data consists of a few to several dozen high-affinity sites. While these data are very useful, they do not fully represent the binding capacity of a factor and thus do not permit the identification of intermediate or low-affinity sites which are known to be important in some regulatory systems [A greater current limitation in the widespread application of binding-site clustering methods is the absence of high-quality binding data for most  systems . We have systems  to charaD. melanogaster and D. pseudoobscura was surprising. A major motivation for the National Human Genome Research Institute (NHGRI) support of the D. pseudoobscura genome sequencing was the identification of conserved regions that would guide the annotation of functional sequences in D. melanogaster. D. pseudoobscura was chosen as the second member of this genus to be sequenced in part because it was felt that it had separated from D. melanogaster sufficiently long ago that non-functional sequences would exhibit substantial divergence. However, despite an evolutionary separation that is greater than human and mouse (an average synonymous substitution rate of 1.8-2.6 substitutions/site [The extent of non-coding sequence conservation between ons/site  comparedons/site ), and deons/site .One reason for the limited efficacy of these methods is that they do not recognize the specific patterns of conservation characteristic of different classes of functional sequences. For example, coding sequences can be easily recognized from the characteristic triplet pattern in evolutionary rates where the third (and often synonymous) position of codons tends to evolve at a greater rate than the first two positions ,57. Simiodd stripe enhancer) and PCE8015  that this sequence is actively responding to the presence of these binding sites. The poor conservation of binding sites in PCE8015 (no greater than is found in random regions of genome) suggests either that the BCD, HB, KR, KNI and CAD sites in this region are not functional or that the region is undergoing rapid functional diversification. Of course the absence of binding site conservation does not suggest that the sequence is non-functional, merely that these sequences are unlikely to have the particular function we are studying here.Contrast PCE8010  the probability of finding a site for the same factor in the orthologous region of D. pseudoobscura, even if the site is not in the same  position. Thus, in this set of positive pCRMs, there appears to be selection to maintain binding site composition, but not always the specific order and orientation of sites. This is consistent with models of enhancer plasticity that have been proposed and discussed elsewhere [In addition to this colinear conservation, we also observe that there is an overall enrichment for binding sites in positive pCRMs independent of the conservation of individual sites. Specifically, the presence of a binding site for a factor in a positive lsewhere ,59-61.D. melanogaster and D. pseudoobscura) most binding sites are conserved and found in the same place. Over longer evolutionary distances, individual binding sites are often poorly conserved even as the overall composition and function of a CRM is conserved.The relative importance of binding-site architecture and binding-site composition to maintaining the function of an enhancer over evolutionary time remains unclear. Over relatively short evolutionary distances . The PCR product was digested with AscI and NotI, and inserted in its native orientation into the AscI-NotI site of a modified CaSpeR-AUG-bgal transformation vector [eve basal promoter, starting at -42 bp and continuing through codon 22 fused in-frame with lacZ [w1118 embryos, as described previously [DNA fragments identified as candidate CRMs were amplified from either bacterial artifical chromosome (BAC) or n vector  containiith lacZ . The P-eeviously ,64. Traneviously  from eacin situ RNA hybridizations were performed as previously described [gt), RE14252 (odd), RE34782 (nub), RE49429 (pdm2), and RE47384 (sqz). Exon 1 of the ftz gene was amplified from genomic DNA using forward primer 5' GCGTTGCGTGCACATC and reverse primer 5' ATTCTTCAGCTTCTGCGTCTG. The PCR product was cloned into the TA vector (Invitrogen) and used to generate ftz RNA probe.Embryonic whole-mount escribed . RNA prolacZ RNA probes were labeled with digoxigenin-11-UTP (Roche). Hybridizations were performed as described above with the following modifications: (1) 2 μl of each probe were added to give a final concentration of 1:50; (2) sequential alkaline phosphatase staining was performed first with Sigma Fast red to detect endogenous transcripts, stopped by washing for 30 min in 0.1 M glycine-HCl pH 2.2, 0.1% Tween-20 at room temperature, and then continued as described to detect lacZ expression.RNA probes, using cDNAs or genomic DNA as templates, were labeled with fluorescein-12-UTP while The input to the genome assembly was the set of whole-genome shotgun reads from the Baylor Genome Sequencing Center retrieved from the National Center for Biotechnology Information (NCBI) Trace Archive, consisting of 2,607,525 total sequences. After trimming the sequences to remove vector and low-quality regions, the average read length was 607 bp. Approximately 75% of the reads were from short insert (approximately 2.5-3.0 kb) libraries, with another 25% from longer (6-7 kb) libraries. Another 46,040 reads came from the ends of 40-kb fosmids.j, which measures the probability that a contig is repetitive on the basis of its depth of coverage. The default setting is very conservative: if a contig has more than 50% likelihood of being repetitive, it is marked as such and is set aside during most of the assembly process. For large highly repetitive mammalian genomes this setting may be appropriate, but for D. pseudoobscura we found that setting it to 90% or higher produced considerably better contigs, while apparently causing few if any misassemblies.We ran the Celera Assembler several times, and found that by adjusting one parameter in particular we could produce considerably better assemblies. In particular, the assembler has an arrival rate statistic The overall assembly contained 10,089 scaffolds and 10,329 contigs, containing 165,864,212 bp. The estimated span of the scaffolds, using the gap sizes estimated from clone insert sizes, is 172,362,884. The largest scaffold was 3.05 million base-pairs (Mbp) and the scaffold N50 size was 418,046.  There are 308 scaffolds larger than 100,000 bp, whose total span is 129.5 Mbp. The N50 contig size, using 166 Mbp as the genome size (not counting gaps), was 43,555. Another measure of assembly quality is the number of large contigs: if we define 'large' as 10 kbp, then the assembly contains 3177 large contigs whose total length is 131,067,828 bp.  All of our contigs and scaffolds are freely available by anonymous ftp at [D. melanogaster and D. pseudoobscura in regions containing pCRMs were determined as follows. The D. melanogaster genomic sequence of the region of interest (with known repetitive elements masked) was extracted from a BioPerl genome database [D. pseudoobscura contigs/scaffolds were identified using WU-BLAST 2.0 [D. melanogaster sequence were treated as separate hits. After examining dot-plots of the hits, we noticed a large number of small, local inversions that were found in both our assembly and the assemblies released by the Baylor Human Genome Sequencing Center. We used BLASTZ [D. pseudoobscura sequence. Each D. pseudoobscura sequence was aligned to the D. melanogaster corresponding sequence using LAGAN 1.2 [eve and h loci contain three pCRMs each, and PCE8003 and PCE8004 are within 20 kb of each other). Twenty-eight regions had aligned D. pseudoobscura sequence that spanned all or most of the region. For three regions  we were not able to identify large regions of orthologous sequence; these were excluded from subsequent comparative analyses. Dot-plots of the alignments from all 30 regions are available at [The extent and pattern of conservation between database  containidatabase . PotentiLAST 2.0  using ded BLASTZ ) to autoAGAN 1.2  with deflable at .D. melanogaster bases aligned to the identical base in aligned regions (percent identity).The conservation of a specific genomic segment was scored as the fraction of D. pseudoobscura binding site for the same factor in the LAGAN alignment. Only overlap, and not strict alignment, was required to compensate for small errors in the alignment. A non-aligned binding site was considered 'preserved' if it could be matched to a D. pseudoobscura site for the same factor within the bounds of the pCRM, allowing each D. pseudoobscura site to be the match for only a single D. melanogaster site. The number of aligned plus preserved sites for each factor in a region is thus equal to the minimum number of sites for that factor in the two species.We used two definitions of binding-site conservation. A binding site was considered 'aligned' if it overlaps a predicted D. melanogaster genome (with annotated repetitive elements and transposable elements masked) and the D. pseudoobscura scaffolds described above. NUCmer was run with the command line parameters (-c 36 -g 10 --mum -d 0.3 -l 9). NUCmer generated a collection of short, highly conserved regions of homology ('anchors') spaced on average every 1 kb throughout the D. melanogaster genome. Anchors flanking either side of a D. melanogaster region of interest were used to pull out the corresponding D. pseudoobscura region, and additional flanking anchors were examined to ensure that the region was unambiguously orthologous. The region identified was re-aligned to the melanogaster region with LAGAN 1.2 using default settings.To develop an orthology map for genome-wide searches, we used NUCmer  to alignD. melanogaster genome, sampled uniformly from all non-coding sequence. For 3,300 of these, we could find orthologous regions in D. pseudoobscura, and these were used to calculate the properties of random non-coding sequence shown in Figure To characterize properties of non-coding sequences across the genome, we picked 4,000 1-kb segments of the D. melanogaster genome were determined as described in [Binding-site clusters in the ribed in , where tribed in  using thribed in . In the D. melanogaster cluster, we identified the corresponding D. pseudoobscura region using the homology anchors described above. A pairwise alignment was made using LAGAN 1.2 (default parameters), and the number of aligned and preserved binding sites were determined as described above. The 2-kb flanking either side of the pCRM was included in the alignment to avoid edge effects, and was subsequently removed when calculating pCRM properties.For each potential D. melanogaster sites followed an approximately logarithmic curve . From this observation, we classified a D. melanogaster binding site cluster as conserved if:We examined our functional (positive) and non-functional (negative) pCRMs and noticed that in the positives, the lower bound for the number of conserved sites as a function of NSm is the number of binding sites in the D. melanogaster pCRM and NSc is the number of conserved binding sites. Different values of the logarithmic base b give different behavior. The data shown in Additional data file 3 support values of b between 1.15 and 1.4. We defined a more intuitive parameter, CF (conservation factor), which can range from 0 to 1 where 0 is the least stringent threshold (b = 1.4) and 1 is the most stringent (b = 1.15)where b = 1.4 - (CF * (1.4 - 1.15))     (2)CF values of 0.25, 0.5, 0.55 and 0.75 and manually inspected the results with respect to false-negative and false-positive rates based on our 15 positive and 17 negative pCRMs . While we did not strictly optimize a single metric, we picked the values that gave a reasonable balance between false positives and false negatives, b = 0.25 for aligned sites alone, and b = 0.55 for aligned plus preserved sits.We performed genome searches with D. melanogaster. We merged overlapping clusters from runs 1-3, yielding 929 non-overlapping clusters as described in Results.eCIS-ANALYST genome searches were run with the following parameters: min_sites = 10, wind_size = 700 (run #1), and min_sites = 13, wind_size = 1,100 (run #2). All conserved clusters (with conservation defined as described in Equations 1 and 2 above) were combined. In order to capture weaker clusters, we performed an additional run (run number 3) using min_sites = 9, wind_size = 700. For this low stringency run, we used a non-standard conservation threshold different from the one described above, accepting all clusters with at least four aligned plus preserved sites, independent of the number of sites in Four metrics were then used to rank these 929 pCRMs: the number of aligned binding sites; the density of aligned binding sites; the number of aligned plus preserved binding sites; and the density of aligned plus preserved binding sites. All values were normalized according to background distribution of random non-coding sequences. The four normalized values were then summed to compute an overall score, which was then renormalized to arrive at a final z-score used to rank pCRMs in Tables The following additional data files are available with the online version of this article.D. melanogaster genome. The blue line plots the cumulative distribution. The colored asterisks show the average values for each class of pCRM. The panel below the histogram shows the values for each pCRM .Additional data file Additional data file D. melanogaster pCRMs to the number of aligned sites (top panel) and aligned plus preserved sites (bottom panel). Blue dots represent the 15 positive pCRMs from the text; green dots the ten known CRMs that were below the threshold used in [Additional data file  used in ; red dotD. melanogaster sequence relative to their D. pseudoobscura ortholog. The location of binding sites in D. melanogaster, binding sites in D. pseudoobscura and aligned binding sites along with the density of sites averaged over 700 bp are shown in the bottom three panels for each region.Additional data file Additional data file The binding site densities (column 1), aligned site densities (column 2), and aligned plus preserved site densities (column 3) for individual transcription factorsClick here for additional data fileExpression patterns of 65 genes adjacent to 122 pCRMs identified by eCIS-ANALYSTClick here for additional data fileD. melanogaster pCRMs to the number of aligned sites (top panel) and aligned plus preserved sites (bottom panel)Discrimination of positive and negative pCRMs. Comparisons of the number of predicted binding sites in Click here for additional data fileNew pCRMsClick here for additional data fileThe primers used to amplify pCRMs for transgenicsClick here for additional data fileAdditional information from Table 2Click here for additional data fileAll new pCRMs from genome-wide eCIS-ANALYST located within 20 kb of annotated transcriptClick here for additional data fileAll new pCRMs from genome-wide eCIS-ANALYST located more than 20 kb from annotated transcriptClick here for additional data fileGenes with anterior-posterior patterns and the source of the informationClick here for additional data fileAll new pCRMs from genome-wide eCIS-ANALYST located within 20 kb of gene with anterior-posterior patternClick here for additional data fileAll new pCRMs from genome-wide eCIS-ANALYST located between 20 kb and 50 kb from gene with anterior-posterior patternClick here for additional data file


Fibrin D-dimer levels have been advocated as an useful clinical marker of thrombogenesis.We hypothesized that i) there is a hyperclotting state after the return of atrial fibrillation to sinus rhythm, ii) the measurement of plasma D-Dimer levels might be a good screening tool of this clotting status, and iii) the duration of arrhythmia influences the haemostasis measured by plasma D-Dimer levels.Forty-two patients with atrial fibrillation undergoing cardioversion were divided into two groups: in Group A  the duration of atrial fibrillation was 72 hours or more (142.7 ± 103.8 hours), in Group B  the duration of atrial fibrillation was less than 72 hours (25 ± 16 hours). Plasma fibrin D-dimer levels were measured by enzyme immunoassay before, and 36 hours after, cardioversion. The change of plasma D-dimer levels 36 hours after cardioversion was calculated as delta-D-dimer.There were no significant differences in demographic, clinical, and echocardiographic data, and the success of cardioversion between the two groups. Compared to the control, the baseline D-dimer levels were significantly higher in both groups. The delta D-dimer levels were significantly higher in Group A than in Group B (p < 0.005). Furthermore, plasma D-dimer levels 36 hours after cardioversion  and delta-D-dimer levels  showed significant correlations with the duration of atrial fibrillation.The longer duration of the atrial fibrillation episode could lead to a more prominent cardiovascular hyperclotting state after cardioversion, and the mean changes of plasma D-Dimer levels could be used as an useful clinical marker of the clotting state after atrial systole return. Atrial fibrillation is the most common sustained arrhythmia in clinical practice. It is associated with an increased risk of thrombus formation, resulting in substantial morbidity, with the augmented risk of stroke being the most serious. This could be explained by haemostasis conditions favouring thrombosis: previous studies have demonstrated that in most patients, AF is a high risk factor for hypercoagulability, irrespective of underlying structural heart diseases or aetiology -8.In addition, it is well known that the direct current cardioversion of atrial fibrillation, especially if persisting >48 h, carries a great risk of thromboembolism, which extends to 5% of cases not receiving anticoagulant therapy. The mechanism and pathogenesis of thromboembolic episodes after restoration of sinus rhythm in these patients is not completely understood.There is some evidence that the prothrombotic state associated with atrial fibrillation might contribute towards the risk of thromboembolism following cardioversion, but reports are not clear. In this context, having a marker of coagulation activation would be useful in identifying patients at highest thromboembolic risk. Indicators of hypercoagulability, such as D-dimers, which are indicative of a prothrombotic state, might also be indicative of thromboembolic risk . D-dimerWe therefore hypothesized that i) there is still a hyperclotting state after return of sinus rhythm, ii) the measurement of plasma D-dimer levels pre- and post-cardioversion might be a good screening tool of this clotting status, and iii) the duration of arrhythmia could be a good predictor of thromboembolic events after cardioversion, due to the influence on haemostasis measured by plasma D-dimer levels.Over a period of 18 months, we studied 42 consecutive patients, aged between 39–68 years old, with non-valvular atrial fibrillation who underwent successful electrical cardioversion and who remained in sinus rhythm at the one-month visit. Exclusion criteria were other acute causes of atrial fibrillation , acute cardiovascular or cerebro-vascular events  occurring within five months, valvular heart disease, malignancy, connective tissue disease, infectious or inflammatory conditions and chronic renal/hepatic disease. The patients were divided into two groups. In Group A , the duration of atrial fibrillation was 72 hours or more (142.7 ± 103.8 hours). In Group B  the arrhythmia had a duration of less than 72 hours (25 ± 16 hours). We included only patients treated with anticoagulant-coumadin for chronic prophylaxis. There was not a statistically significant difference between the mean duration of anticoagulation treatment between the two groups (14 ± 4 months for group A and 12 ± 6 months for Group B). Prothrombin time to an INR  of 2.5–3 was considered as a necessary inclusion criterion for all patients. The total population had a history of drug refractory atrial fibrillation, with a serious number of arrhythmia episodes. We only analyzed the documented arrhythmia episodes. The total number of documented episodes of paroxysmal atrial fibrillation in Group A was 33, and in Group B the patients had 36 confirmed arrhythmia episodes. There were no significant differences in age, sex, hematocrit, hemoglobin, plasma fibrinogen level, underlying heart disease, success ratio of electrical cardioversion, echocardiographic data, presence of diabetes mellitus or hypertension between the two groups. The clinical characteristics of the patients are shown in Table An intravenous line was placed and blood samples for D-dimer measurement were taken from the patients immediately before cardioversion, and 36 hours after recovery of sinus rhythm. Citrated plasma was obtained from venous blood by centrifugation at 2,500 rpm for 15 min at 4°C. Aliquots were stored at -70°C to allow batch analysis. The plasma D-dimer levels were measured by the enzyme-linked immunosorbent assay method. The measurements were obtained with the use of a quantitative sandwich immunochromatographic technique . For every blood sample, measurements were done twice. The investigators and attending physicians were blinded to the D-dimer test results. Inrta-assay coefficients of variation for assays were <5%, inter-assay variances were 10%. The changes of plasma D-dimer levels 36 hours after cardioversion were calculated as delta- D-dimer.Echocardiographic examinations were performed in all patients, immediately prior to the procedure and 36 hours after successful cardioversion. Transthoracic echocardiographic two-dimensional imaging and guided pulsed wave Doppler recordings were obtained. Transmitral Doppler inflow velocities were recorded from the apical four-chamber view.Peak velocities of early fillings (E) wave and atrial filling (A) wave were determined. The inter- and intra-observer variability was <5% for these measurements. We did not detect the presence of thrombi in the left atrium. However, the absence of thrombus on transthoracic echocardiography does not preclude the real absence of thrombus.Clinical variables are expressed as the mean value ± SD. The effects of duration from the onset of atrial fibrillation on the measured indexes were analyzed by two-way repeated measures analysis of variance (ANOVA). Sequential data pre- and post-cardioversion were analyzed by Friedman's repeated measures analysis of variance. Correlations were performed by Spearman's rank correlation method. Stepwise multiple regression analyses were performed to determine independent predictors for plasma D-dimer levels, using age, sex, left-atrial size, left ventricular dimensions  presence of underlying medical disease, smoking status, and the presence of atrial fibrillation. A p value < 0.05 was considered statistically significant. Analyses were performed with SAS for Windows 8.02  and GraphPad Prism, version 3.00  statistical software packages.The baseline D-dimer levels of each group are shown in Table The patients' sinus rhythm was restored by applying electrical cardioversion in all study patients. Delta D-dimer levels were significantly higher in Group A than in Group B. (p < 0.005).There were no significant differences in plasma D-dimer levels before, and 36 hours after, cardioversion between the two Groups .Two embolic stroke events were recorded during the six hours after cardioversion in Group A  and none in Group B. The embolic events occurred in two women with an INR of 2.6 and 2.9 respectively. At the onset of the event one patient had D-dimer levels of 112.2 ng/ml while the second patient had D-dimer levels of 109.8 ng/ml. Fortunately, these were transient events. The computerized tomography detected non-extentend thromboembolic areas.In the present study we analyzed D-dimer levels as a screening index of a hyperclotting state after cardioversion of atrial fibrillation. It would be more useful if we used the term abnormal haemostasis, because the fibrin D-dimer is a cross-linked degradation product, resulting from the balance between thrombogenesis and the fibrinolysis process.Fibrin D-dimer levels have been established as a useful clinical marker of thrombogenesis.  The use In "normal" patients, elevated D-dimer levels have been associated with a higher risk of developing cardiovascular disease.  Also, suPatients with long-lasting episodes of atrial fibrillation or chronic atrial fibrillation are characterized by increased levels of plasma D-dimers ,22, platIn the present study, the plasma D-dimer levels decreased in both groups after successful cardioversion, but the mean change in D-dimer levels pre- and post- cardioversion was significantly lower in Group A than in Group B, and D-dimer levels continued to be high, even 36 hours after the procedure in Group A. There were reported embolic events in 8.33% of Group A patients, with the higher delta D-dimer.These data confirm the existence of an abnormal clotting state after the cardioversion of atrial fibrillation, which could be the cause of the thromboembolic events observed after the return to sinus rhythm, even under anticoagulation.The mechanism of thromboembolism is debatable. The results we found could suggest an embolism might be caused by detachment of a formed thrombus during the phase of passage from AF to sinus rhythm, due to being heavily compromised by stunning the mechanical function of the left atrium and left atrium appendage after sinus rhythm restoration. The stunning of the left atrium and left atrium appendage might attenuate the thrombogenetic activation which our result showed.In the present study we also provided evidence that this hypercoaguable state after successful cardioversion of atrial fibrillation related directly to the duration of the arrhythmia episode. It has been accepted that chronic atrial fibrillation is characterized by increased levels of plasmatic D-dimers, with a wide inter-individual variability, depending on the patients and therapeutic characteristics. However, it has not been established if this level could also be characteristic of paroxysmal or persistent atrial fibrillation in patients, and whether it could be a predictive factor for the risk of thromboembolic events after cardioversion to sinus rhythm.Our results show that longer duration of atrial fibrillation could lead to a more prominent cardiovascular hyperclotting state after cardioversion and that the duration of the arrhythmia episode might be a risk factor for the high incidence of post-cardioversion thromboembolic events. Importantly, this hypercoaguable state does not appear to be subject to any other clinical variables, nor is it related to whether or not the patient had a lone atrial fibrillation or not, suggesting that the duration of the arrhythmia episode confers a constant prothrombotic state per se, after cardioversion to sinus rhythm, which was independent of the etiology but dependent on the duration of the arrhythmia. Interestingly enough, this hyperclotting state after cardioversion exists even under appropriate anticoagulative treatment. The relation of the markers of accelerated coagulation to clinical or echocardiographic risk factors for thromboembolism is controversial.Our results clearly demonstrate a positive correlation of the based on the arrhythmia duration clinically predicted embolic risk to the mean change of plasma D-dimer levels 36 hours after successful cardioversion. We could not detect any relation to the echocardiographic risk factors, probably due to our inability to perform a transesophageal echocardiography. The present study suggests that even in the absence of clinical conditions causing increased embolic risk, patients with signs of accelerated coagulation are at risk of thrombus formation in the future.In conclusion, a longer duration of the atrial fibrillation episode could lead to a more prominent cardiovascular hyperclotting state after cardioversion, and the mean changes of plasma D-Dimer levels could be used as an useful clinical marker of the clotting state after atrial systole return.Our study might have practical implications for the management of patients with an episode of atrial fibrillation, regardless of the anticoagulative treatment they are receiving: the episode must be terminated as soon as possible, because the pathogenesis of thrombus formation in atrial fibrillation is very complex and not yet completely defined. Further investigations with a large population are needed to define all the pathophysiologic mechanisms of thrombus formation.