Epithelial-mesenchymal transition (EMT) is a cellular process through which epithelial cells transform into mesenchymal cells. EMT-implicated genes initiate and promote cancer metastasis because mesenchymal cells have greater invasive and migration capacities than epithelial cells. In this pan-cancer analysis, we explored the relationship between gene expression changes and copy number variations (CNVs) for EMT-implicated genes. Based on curated 377 EMT-implicated genes from the literature, we identified 212 EMT-implicated genes associated with more frequent copy number gains (CNGs) than copy number losses (CNLs) using data from The Cancer Genome Atlas (TCGA). Then by correlating these CNV data with TCGA gene expression data, we identified 71 EMT-implicated genes with concordant CNGs and gene up-regulation in 20 or more tumor samples. Of those, 14 exhibited such concordance in over 110 tumor samples. These 14 genes were predominantly apoptosis regulators, which may implies that apoptosis is critical during EMT. Moreover, the 71 genes with concordant CNG and up-regulation were largely involved in cellular functions such as phosphorylation cascade signaling. This is the first observation of concordance between CNG and up-regulation of specific genes in hundreds of samples, which may indicate that somatic CNGs activate gene expression by increasing the gene dosage. Epithelial-mesenchymal transition (EMT) is the transformation of an epithelial cell into a mesenchymal cell, the latter of which is a critical cell type for the initiation of cancer metastasis –3. DurinTo explore the molecular mechanism of EMT at a systems-biology level, we developed a literature-based gene resource specific for EMT. In total, 377 human EMT-implicated genes were collected from thousands of published articles, primarily single nucleotide mutation-based studies. Reports of systematic data mining for larger genomic variations related to EMT, such as gene copy number variations (CNVs), are still lacking. As a genomic mutational event, CNV often results in a variable number of repeated DNA fragments with lengths ranging from kilobases to megabases, even encompassing entire genes . Using aCNV is known to correlate with altered gene expression in different cancers . While sTo explore somatic CNV information at the pan-cancer level, we first downloaded 377 human EMT-implicated genes from our literature-based database, dbEMT . Then, wP-value = 2.09E-46), cell proliferation , and cell fate commitment  , stem cell development , and extracellular matrix organization . Intriguingly, these 212 genes are also involved in the responses to wounding  and endogenous stimuli .We first performed a gene ontology analysis to determine the functions of the 212 EMT-implicated genes with frequent CNGs. Not surprisingly, the majority of the functions were related to cell migration  . Cell surface proteoglycans act as either inhibitors or promoters of cancer metastasis, depending on the type of cancer  OR “genome wide association study” [Title/Abstract]) OR )). This search returned 1507 abstracts on 19 December 2013. We manually curated the experimentally verified candidate genes. This information and the related annotations were stored in the dbEMT database and published for public use. In this study, we downloaded all 377 curated human EMT-implicated genes from the dbEMT database in plain text with all the official symbols (oad.cgi) .ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene2refseq.gz). Then, we implemented a Perl script to extract all the genomic coordinates of the 377 EMT-implicated genes from the completed genomic sequences. To provide the most up-to-date information, we used genome version GRCH 38 for accurate genomic locations.To align the EMT-implicated genes with reported CNVs, we first annotated all the genes with precise genomic locations. To this end, we downloaded the RefSeq database from NCBI (https://cancer.sanger.ac.uk/cosmic/files?data=/files/grch38/cosmic/v73/CosmicCompleteCNA.tsv.gz) so that the CNVs with precise gain and loss information could be collected. A number of criteria were used by COSMIC to define copy number loss and gain. For CNGs in the TCGA pan-cancer study, the following parameters were required:  OR . Similarly, the criteria for CNL were:  OR ). In this study, we adopted the same COSMIC criteria and aligned all of the CNV regions with EMT-implicated genes using the GRCH 38 coordinates. We annotated a total of 365 EMT-implicated genes with precise overlapping CNG and CNL information. To cross-validate the data among different cancers, we counted the number of CNG and CNL samples regardless of cancer type. To explore the oncogenic role of EMT-implicated genes, we collected those EMT-implicated genes with more CNGs than CNLs. A threshold was set to collect those genes for which the number of tumor samples with CNGs was at least twice the number of samples with CNLs. Ultimately, 212 EMT-implicated genes with more CNGs than CNLs were collected for the subsequent integrative gene expression analysis.TCGA CNV data were downloaded from COSMIC  . Here, we focused only on those gene expression changes in TCGA samples with matched EMT-related CNGs. For gene expression quantification, the RSEM quantification results from the RNAseq V2 platform in COSMIC were used. The averages and standard deviations of the expression values were calculated based on tumor samples that were diploid for each corresponding gene.P-value calculated using the formula as below:x is the gene expression of a gene in the individual sample; μ is the averaged expression score of a gene across multiple TCGA samples; and σ is the standard deviation of the expression scores of the gene in different individual samples. The threshold of a Z-score ≥ 2 was used to identify EMT-implicated genes of interest among the samples. A Z-score greater than 2 was defined as over-expression, which corresponds to a P-value less than 0.01.The standard Z-score was used to characterize whether an EMT-implicated gene was over- or under-expressed, similar to its implementation in the COSMIC database . Here, ZFor the 71 EMT-implicated genes with concordance between CNG and up-regulation, we further systematically examined the pan-cancer somatic CNV patterns in the TCGA samples using the cBio portal . In addiTo reveal the global gene-gene interaction among genes with frequent CNGs and consistent gene up-regulation, we extracted a sub-network from the human interactome. First, we collected a non-redundant human interactome from the PathwayCommons database , 30 with


Stroke-induced immunodepression is a well characterized complication of acute ischemic stroke. In experimental studies beta-blocker therapy reversed stroke-induced immunodepression, reduced infection rates and mortality. Recent, heterogeneous studies in stroke patients could not provide evidence of a protective effect of beta-blocker therapy. Aim of this study is to investigate the potential preventive effect of beta-blockers in subgroups of patients at high risk for stroke-induced immunodepression.Data from a prospectively derived registry of major stroke patients receiving endovascular therapy between 2011–2017 in a tertiary stroke center  was used. The effect of beta-blocker therapy on pneumonia, urinary tract infection, sepsis and mortality was assessed using multivariate logistic regression analysis.Three hundred six patients with a mean age of 72 ± 13 years and a median NIHSS of 16 (IQR 10.75–20) were included. 158 patients (51.6%) had pre-stroke- and continued beta-blocker therapy. Beta-blocker therapy did not reduce the incidence of pneumonia , urinary tract infections , sepsis  or mortality . Strokes involving the insula and anterio-medial cortex increased the risk for pneumonia  and sepsis , while right hemispheric strokes increased the risk for pneumonia . There was a non-significantly increased risk for urinary tract infections in patients with beta-blocker therapy and insula/anterio-medial cortex strokes  with no effect of beta-blocker therapy on pneumonia, sepsis or mortality in both subgroups.In major ischemic stroke patients, beta-blocker therapy did not lower post-stroke infection rates and was associated with urinary tract infections in a subgroup with insula/anterio-medial strokes. Infections are frequent and decisive complications after a stroke, as they have a significant influence on stroke outcome –3. Up toIn an experimental study, Prass et al. found that the administration of propranolol to middle cerebral artery occlusion (MCAO) mice reinforced the immune response and lowered infection rates . Other ePrevious studies involving humans, however, were inconclusive. Some described a protective effect of beta-blockers regarding infection rates –15, otheThe main idea behind previous experimental and clinical studies was to antagonize the immunodepressive effects caused by an increase in the sympathetic tone with beta receptor antagonists. This theory has not been sufficiently studied in stroke patients at high risk for infections and in patients likely to exhibit the immunodepressive mechanism previously described in several studies. Our aim was to study the possible immunomodulatory effect of beta-blockers in different subgroups of patients with major ischemic strokes.Clinical and neuroradiological data were analyzed from a prospectively derived, monocentric database including neuroradiological and neurological information of patients with endovascular treatment. The database also included detailed information on pre-stroke medication, past medical history and post-stroke complications. Infection related risk factors like the development of dysphagia, naso-gastric tubes and transurethral catheters were obtained retrospectively through a chart review  information system ), in which detailed information on the clinical course of the stroke patients were documented by stroke physicians and therapists. Ethics approval for this study was sought from the Ethics Committee of the University Medicine Göttingen and all patients or next of kin gave informed written consent for the anonymized use of disease-related data on hospitalization.All patients were admitted to a neurointensive care unit directly after endovascular treatment and transferred to a stroke unit at various timepoints depending on their clinical status. Patients receiving beta-blockers before the stroke and continued beta-blocker therapy during their in-hospital stay were considered as beta-blocker positive. Patients without beta-blocker exposure before the stroke or during in-patient stay were considered as beta-blocker negative. Patients were included in the analysis, if all necessary data on pre-stroke history (comorbidities and pre-stroke medication)- and clinical data on post stroke course (e.g. infections or complications) were complete and available, which was the case in 306 (59%) from 518 documented cases in the prospectively derived databank.We defined two high risk subgroups of stroke patients at increased risks for stroke induced immunodepression and post-stroke infections. The first group had insula and anterio-medial cortex infarctions, which have been identified to have an increased sympathetic tone and higher risk for post-stroke infections in other studies ,18. We aFollowing infectious complications were investigated: pneumonia, urinary tract infection and sepsis. For the diagnosis of these infections, the recommendations of the current German guidelines were followed. For the diagnosis of pneumonia, these included a newly diagnosed infiltrate/suspicion of an infiltrate on chest X-ray in presence of at least two clinical signs such as cough, fever (>38.0°C), leukocytosis or leukopaenia  or the detection of a pneumonia-typical pathogen in the sputum, bronchial secretion or blood .3 colony-forming unit (CFU)/ml with symptoms or 105 CFU/ ml without symptoms) or a positive urine culture with bacteria typical of urinary tract infections [Criteria for the diagnosis of an urinary tract infection included a nitrite-positive urine test strip, bacteria in the midstream urine . Characteristics of all patients are shown as mean ± standard deviation (SD), if normally distributed, and as median with interquartile range (IQR), if not . Category variables were given as absolute frequencies and percentages and examined by Pearson Chi-Square test for statistically significant differences between the compared groups. The different groups were examined for significant differences by using independent samples T-test or Mann–Whitney U test, as appropriate. Furthermore, to assess the influence of beta-blocker therapy on post-stroke infections, an uni- and multivariate logistic regression was carried out. Relevant confounders  were included in a multivariate logistic regression together with the predictor beta-blocker therapy. This logistic regression was performed as an inclusion procedure. In all procedures, a p-value of <0.05 was considered as statistically significant.Patients in the beta-blocker positive group were older (p<0.001), were more likely to be female (p<0.001), to have a history of arterial hypertension (p<0.001), coronary heart disease (<0.001), atrial fibrillation (<0.001) and chronic renal failure  developed pneumonia, 80 (26.1%) an urinary tract infection and 40 (13.1%) sepsis. 64 (20.9%) of the patients died during their in-patient stay.As shown in Beta blocker therapy was associated with a significantly increased risk for urinary tract infection in the univariate , but not in the multivariate logistic regression  analysis.Patients with- and without beta-blocker therapy showed no difference in leukocyte count, C-reactive protein or procalcitonin concentration .Patients with strokes affecting the insula and antero-medial cortex showed higher rates of pneumonia , sepsis  and mortality  compared to patients with other stroke localizations. This stroke localization did not influence the risk for urinary tract infections .As shown in There was a non-significant increased risk for pneumonia in patients with right hemispheric strokes , while there was no influence of right sided strokes on urinary tract infection  or sepsis . Right hemispheric strokes were associated with lower mortality .In the subgroup of patients with right sided strokes, there was no difference for pneumonia, urinary tract infections, sepsis or mortality between patients with- and without beta-blocker therapy .In our study, we found no effect of beta-blocker therapy on rates of post-stroke pneumonia, sepsis or mortality in patients at high risk for stroke induced immunodepression and post-stroke infectious complications, with even increased rates for urinary tract infections. These findings were also true for anatomically defined subgroups with insular/ antero-medial cortex and right hemispheric strokes, which have been shown to have increased risks for post-stroke infections and stroke induced immunodepression ,18.One general reason for the incapability of beta-blockers to prevent post-stroke immunodepression may be the fact that nowadays mainly cardioselective (beta-1 receptor selective) beta-blockers are used. Previous studies provided evidence, that immune cells may be modulated primarily via beta 2 receptors (rather via beta-1-receptors) by catecholamines . This coA recent subgroup analysis of the Preventive Antibiotics in Stroke Study (PASS)-study also could not provide evidence for a protective effect of beta blocker therapy on infections, showing even increased pneumonia-rates . HoweverThe increased risk for urinary tract infections associated with beta-blocker therapy could be explained by the fact, that only major stroke patients were studied, and the diagnosis of urinary tract infections was primarily based on bacteriological and urinary status in combination with signs of infections rather than clinical signs attributable to urinary tract infection. These can’t be judged in the majority of cases in major stroke patients, showing aphasia, confusion and decreased conscious state. Therefore, rates of urinary tract infection might be overestimated in our study. The exact mechanism, by which beta-blockers might contribute to an increased rate of urinary tract infections, remains unclear. Given the pleiotropic and complex effects of beta-blockade on the immune system on the one-, and sympathetic nervous system and multitude of organ systems on the other hand, protective effects of beta-blocker therapy might be outweighed by detrimental effects of beta-blocker therapy. However, considering the problem of proper diagnosis and possibility of overestimation of urinary tract infection rates, our study (together with previous studies reporting increased infection rates) provides safety concerns for the use of beta-blockers in patient with ischemic stroke.The strength of the study lies in the fact that we included a homogeneous patient group at high risk for post-stroke infections and stroke-induced immunodepression, which has not been studied before. Moreover, we were able to correct for major confounding factors like naso-gastric tubes, intubation and dysphagia, therefore providing more robust results compared to previous studies . Major lIn conclusion, we could not provide evidence for a protective effect of beta-blockers on post stroke infections and mortality. Data in humans remain inconclusive with studies showing protective effects on pneumonia rates , 14 and Further studies should investigate the possible effect of unselective beta-blockers on post stroke infections using a prospective design to optimize the control for confounders and should not only identify high risk patients for stroke induced immunodepression using radiological data, but also include serological measurements of catecholamines and cortisol as well as continuous measurements of heart rate and blood pressure to identify an increased sympathetic tone.S1 Table(DOCX)Click here for additional data file.


Within caregivers, VLDL particle size (VLDL-Z) was significantly larger at time-point three compared to time-points one (p = .015) and two (p = .048), and VLDL-Z was significantly larger in caregivers than in controls at time point three (p = .012). HPLP-II scores were lower in caregivers than controls at all time-points (p < .01). These findings suggest that caregiving may have a bigger impact on triglycerides than on other lipids, and it is through this pathway that caregivers may be at increased cardiometabolic risk. More sensitive measurement methods, such as NMR lipoprotein particle analyses, may be able to detect early changes in cardiometabolic risk.The purpose of this study was to compare components of cardiometabolic risk and health behaviors of 20 family caregivers of allogeneic hematopoietic stem cell transplant patients to those of age, gender, and race/ethnicity-matched controls. A prospective, repeated measures design was used to compare cardiometabolic risk and health behaviors in caregivers and controls at three time-points: pre-transplantation, discharge, and six weeks post-discharge. Measures included components of metabolic syndrome, Reynolds Risk Score, NMR serum lipoprotein particle analyses, and the Health-Promoting Lifestyle Profile II (HPLP-II). Mixed-model repeated measure analyses were used. There were no between or within group differences in LDL cholesterol, HDL cholesterol, and triglycerides. There was a significant interaction effect between time and role in large VLDL concentration (VLDL-P)  = 4.36,  Exposure to prolonged stress is associated with increased morbidity and mortality, primarily from cardiovascular disease (CVD) . Althougp = .013)), with hypertension making the largest contribution to their CVD risk [p = .48) [The stress and strain of being a caregiver increases one’s all-cause risk of mortality , althougCVD risk . Much ofCVD risk . In addip = .48) .While caregiving appears to increase the risk of morbidity and mortality, particularly from CVD, nearly all of the research has focused on caregivers of individuals with dementia. No published studies were found examining cardiometabolic risk factors in caregivers of patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). In the realm of cancer care, HSCT is one the most intense cancer treatments, potentially leading to serious and sometimes life-threatening treatment toxicities, as well as long-term health issues from chronic graft versus host disease . AccordiWhile the impact of caregiving on psychosocial aspects of caregivers’ health is well described, the effects on health behaviors are less well understood. Evidence shows that HSCT caregivers will prioritize the needs of the patient over their own . Cancer Determining cardiometabolic risk is complex and includes a variety of individual factors that are often used in clinical practice within composite measures. Two common composite measures are metabolic syndrome and the Reynolds Risk Score. Metabolic syndrome, a cluster of symptoms that place individuals at increased risk for CVD , has beeLow-density lipoprotein cholesterol (LDL-C) has also been recognized as an important predictor of cardiometabolic risk, and reducing LDL-C with statins improves morbidity and mortality . RecentlThe purpose of these analyses is to compare over time the cardiometabolic risk and health behaviors of HSCT caregivers to age, gender and race/ethnicity matched controls. The primary research question was: Are there differences in HSCT caregivers compared with controls in measures of cardiometabolic risk , as well as differences in health behaviors that contribute to cardiometabolic risk? A secondary research question was: Do health behaviors predict cardiometabolic risk in HSCT caregivers?This study utilized a prospective repeated measure design to compare changes in components of cardiometabolic risk and health behaviors in 20 family caregivers of adults undergoing HSCT. Data were collected at three time-points: pre-transplantation (pre-HSCT), time of initial hospital discharge (±1 week), and six weeks post-discharge (6wk) (±1 week) compared with age-, gender-, and race/ethnicity matched controls. This study was approved by the National Heart, Lung, and Blood Institute intramural Institutional Review Board and written informed consent was obtained for all participants. The study design, participants and procedures have been described in detail previously in a manuscript that examined biomarkers of stress . Caregiv® Pro 100-400v2 electronic BP machine on either the right or left upper arm of the subject, following a 5-minute rest period in a sitting position; three BP measurements were obtained and the average was used. Blood samples were obtained from an indwelling catheter placed in the subject’s arm and run in a clinical chemistry panel , as well as analyzed for NMR serum lipoprotein particle concentration and size. Clinical chemistry panels were obtained through an enzymatic immunoassay using a Roche Cobas® 6000 Analyzer .At each time-point, caregiver and control subjects were asked to complete a demographic and health behavior questionnaire, as well as a clinical review, including a physical exam, health history, and twelve-hour fasting blood samples. These visits incorporated a determination of Eastern Cooperative Oncology Group (ECOG) performance status  and completion of the Charlson Comorbidity Index , a revieCardiometabolic risk is measured in this study using: components of metabolic syndrome, Reynolds Risk Score, LDL-C, and NMR serum lipoprotein particle concentration and size.The following components of metabolic syndrome were used: SBP, DBP, waist circumference, and fasting levels of serum glucose, triglycerides, HDL-C, and hs-CRP. While related to cardiometabolic risk but not specifically listed as components of metabolic syndrome, an apolipoprotein panel and LDL-C were also obtained, and a Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) was calculated.A Reynolds Risk Score was calculated for each subject . The sco® Clinical Analyzer  at the NIH Clinical Center’s Department of Laboratory Medicine.NMR spectroscopy was used as a technique to quantify both the concentration and size (diameter) of serum lipoproteins. Aliquots of frozen serum samples were used for the NMR analysis. LDL-P, LDL-Z, high-density lipoprotein particle concentration (HDL-P) and size (HDL-Z), and very low-density lipoprotein particle size (VLDL-Z) were calculated from the amplitudes of their spectroscopically unique lipid methyl group NMR signals. Relative mass percentage was also quantified from methyl group signals, which gave estimations of concentrations and sizes of lipoprotein subclass by size . Prior studies have shown that measurements of total LDL-P and LDL-Z may serve as more accurate indicators of true cardiovascular risk than LDL-C when LDL-C and LDL-P are discordant, especially because cholesterol content of LDL varies greatly among individuals ,31,32. LEach subject was screened for their current and past alcohol and tobacco usage at each clinic visit. Tobacco frequency and duration of consumption was assessed and documented based on the number of tobacco products consumed per day and their lifetime pack year history.The Health-Promoting Lifestyle Profile II (HPLP-II) is a self-administered 52-item instrument that measures the frequency of self-reported healthy behaviors using a 4-point Likert scale . It consThe distributions of all variables at each time-point were examined and appropriate descriptive statistics were computed  for interval level data, median for ordinal level data, frequency and percentage for categorical data). Variables were natural log or square root transformed in the final model if necessary to meet normality assumption for the analyses. Baseline between group differences were evaluated using appropriate parametric or non-parametric tests .p < .05 was considered significant.Mixed model repeated measure analyses were used to determine whether there was a change in all cardiometabolic factors and health behaviors over three study time-points between groups. Visit was treated as a categorical variable. Restricted maximum likelihood (REML) procedure was used for model parameter estimation. Aikake information criterion (AIC) and Bayesian information criterion (BIC) were used to compare and select models. Demographic variables including income and education were tested and added as covariates in the final model if they were significant predictors or contributed to the model. All data analyses were performed using SAS version 9.3 . A p = .02) and education (p = .01) than caregivers at baseline. Otherwise, there were no significant differences in the two groups in demographic variables.Baseline demographic characteristics, cardiometabolic risk factors, and health behaviors for both caregivers and control subjects are detailed in p = 0.169) or CVD (hypertension and/or hypercholesterolemia) (p = 0.159). Approximately one quarter of the caregivers  and 10% (n = 2) of control subjects met the criteria for metabolic syndrome, but the large majority of both the caregivers  and the controls  had Reynold’s Risk Scores that placed them at low risk for CVD. The NMR analysis revealed mean values of HDL-P and large HDL-P placed both caregivers and controls in the higher cardiometabolic risk category, while levels of small LDL-P, large VLDL-P, and VLDL-Z placed both groups at moderate risk [p = .036).At baseline, the majority of subjects in both groups were overweight  or obese  and had at least one chronic health condition . There were no significant group differences in the use of medications for diabetes , with trajectories of large VLDL-P increasing over time in caregivers compared with controls (p = .002) and time-point two (p = .011). Second, when controlling for income, there was a significant interaction effect between time and role in HDL-P  = 3.49, p = .035), with levels of HDL-P increasing in caregivers while HDL-P in controls were stable; time-point three was significantly higher than time-point one in the caregivers (p = .002), and this level was significantly higher than controls (p = .009) (p = .045), with levels of VLDL-Z significantly higher in caregivers than in control subjects at time-point three (p = .012) and within the caregivers, levels of VLDL-Z at time-point three were significantly higher than at time-points one (p = .015) and two (p = .048)  . Finally = .048) .p = .002) (p = .007), interpersonal relationships (p = .005), spirituality (p = .004), stress reduction (p = .004), and health responsibility (p = .004). The trajectories of health behaviors did not change significantly over time in either group, and remained significantly different between the two groups over all time points , nor were there any changes in these behaviors over time in either group. Baseline total scores for the HPLP were significantly lower in the caregivers than controls ( = .002) . With the points . There wThe findings of this study suggest that traditional measures of cardiometabolic risk may not be robust enough to identify at-risk individuals early in the trajectory of caregiving, while NMR lipoprotein particle size and concentration may be sensitive enough to detect such changes. Three of these more sensitive markers of cardiometabolic risk differed between groups. Levels of HDL-P placed both caregivers and non-caregivers at higher cardiometabolic risk at baseline. While levels of HDL-P improved in the caregivers, these levels remained in the higher cardiometabolic risk category. However, the findings with the VLDL assays were more compelling. Prior to the transplant, caregivers’ levels of large VLDL-P and VLDL-Z were similar to the controls, but these levels worsened over time in the caregivers, while levels in the non-caregivers remained unchanged.The increasing levels of large VLDL-P and VLDL-Z seen in the HSCT caregivers in this study may be evidence of very early signs of CVD in this population. Unlike the caregivers in this study, none of whom were in the high-risk category for CVD based on their Reynolds Risk Scores, von Kanel et al  found thAlthough the immediate period around the transplant procedure is often the most stressful, the impact on the patient and caregiver is far from over when they leave the acute care facility. The majority of HSCT recipients survive beyond two years, but many experience lingering health effects and unique complications such as chronic GVHD that lead to chronic physical and psychosocial problems for the survivor as well as the caregiver –38. ThusDespite the increasing levels of large VLDL-P and VLDL-Z along the caregiving trajectory, neither LDL-C nor the NMR lipoprotein measures of LDL-C  explained CVD risk in the caregivers in this study. The Apolipoprotein B/Apolipoprotein A-1 ratio, an important predictor of coronary death that is associated with metabolic syndrome , did notAnother important finding in this study is that participation in all of the health promoting behaviors except nutrition was worse in the caregivers than controls at all time-points. Compared to control subjects, the HSCT caregivers are not exercising, engaging in stress management, seeking routine medical care, or experiencing the benefits of spirituality and social support. Three quarters of the caregivers were overweight or obese and reported having at least one chronic health condition at the start of caregiving. This scenario may be indicative of a “perfect storm,” as caregivers in this study were coming into the caregiving experience in poor health, experiencing high levels of stress related to caregiving , and doiWhile this study adds to the evidence regarding cardiometabolic risk in caregivers, it is not without limitations. The small sample size may have contributed to the inability to identify additional differences between the two groups and to see a significant relationship between health behaviors and cardiometabolic risk. While the researchers carefully matched the caregivers and control subjects in age, gender and race/ethnicity, and they controlled for any significant demographic differences such as income and education, the two groups may have differed in other ways that could have influenced study outcomes. While one of only a few research studies to follow caregivers longitudinally, the average length of time that caregivers were followed in was relatively short (approximately 84 days) , and thi


Medical practitioners are recommending combination therapy in cancer for its various advantages. Combination therapy increases the efficacy of treatment due to its synergistic effects in cancer treatment. In this post-genomic era, microRNAs (miRNAs) are receiving attention for their role in human disease and disease therapy. In this review, we discuss the combination of miRNAs and chemotherapeutic agents for cancer treatment. Moreover, we attempted to portray the role of miRNAs in cancer therapy; outline combination therapy, especially chemo-combination therapy, and discuss the basis for miRNA-based chemo-combination therapies and chemo-combination therapy with miRNA for cancer treatment. Since human genome sequencing and the advancement of the post-genomic era, a new wave of RNomics has been generated, and our understanding about small non-coding RNAs (ncRNAs) has grown exponentially , 2. AmonIt has been noted that miRNAs genes are located in specific key sites. For example, cancer-associated delicate sites, cancer-associated genomic regions or areas those are associated with individual subsets of solid tumors and hematological malignancies , 16. TheWorldwide, cancer is an important cause of death in both economically developed countries as well as in developing countries. According to the WHO , approximately 8.2 million deaths occurred by cancer in 2012, throughout the globe. It was estimated that 14.1 million new cancer cases must have occurred in 2012, worldwide . It is aAt present, chemotherapy remains the primary treatment for cancer, and it is frequently used to treat patients with metastases. It is first line of treatment against cancer, both curable and advanced cancers, and it improves the general survival and quality of life for cancer patients. The age of chemotherapy started with its use during the 1940s using nitrogen mustards and antifolate drugs . HoweverIn this article, we evaluated the combination of miRNAs and chemotherapeutic agents for treating cancer. We also discussed the role of miRNAs in cancer therapy, provided an overview of combination therapy of chemotherapeutic agents, described the reason behind miRNA-based chemo-combination therapies, and detailed the use of combination therapy of miRNA with chemotherapeutic agents for cancer therapy.Several new strategies have been added in the last 5 years, which can increase survival rate and reduces mortality rate; miRNA therapy is one of these strategies , 28. ThemiRNA replacement or restoration therapy employs the reuse of miRNAs that are deleted or downregulated in cancers , 30. SomThe first commercial miRNA restoration therapy is MRX34. This MRX34 is a liposome-based miR-34 mimic that can be intravenously injected. It is currently being investigated in Phase I clinical trials for advanced hepatocellular carcinoma patients .miRNA reduction or inhibition therapy can inactivate those miRNAs that are overexpressed or upregulated in cancers, especially in tumors . SeveralLNA is a nucleic acid analogue. LNA may consist of one or more nucleotide monomers. Anti-miRNA oligonucleotides (AMOs) (2’-O-methyl AMOs and 2’-O-methoxyethyl) are LNA anti-miRs. In this case of LNA, there is an additional methylene bridge linking the 2′-O and 4′-C atoms. This ‘locks’ the ribose ring. In this particular region, a C3’-endo or C2’-endo conformation can be observed . There mSeveral small molecules are reported to inhibit miRNAs. In 2008, Gumi reddy et al., reported on small-molecule inhibitors (SMIRs) of miR-21. In this study, a luciferase-based reporter assay was also developed to recognize large-scale drug screening in which possible miRNA-specific small molecule inhibitors were used . Naro etin vivo. The half-life of RNA increases with miRNA therapy [There are some challenges in developing miRNA-based therapeutics. Low RNA stability has been noted  therapy . Another therapy . However therapy , which f‘Combination therapy’ is used in cancer treatment. The first successful combination chemotherapy could cure acute childhood leukemia and was then extended to treat Hodgkin’s disease and cure advanced cancers . To treaSince the use of drug combinations, the national occurrence and mortality of cancer began to decline in 1990. Cancer mortality has continued to decline annually in spite of the aging U.S. population , 54. TheDespite considerable success with ‘combination therapy’ in chemotherapy treatment programs, resistance to chemotherapeutic treatment has emerged as a challenge to effective treatment , 57. It Presently, the concept of ‘combination therapy’ with various miRNAs together with chemotherapeutic agents may help cancer patients by providing synergistic effects.One such type of ‘combination therapy’ for treating HCV is being evaluated in a Phase II clinical trial. In this case, one miRNA is anti-miR (Brand name: miravirsen), which is used with antiviral drugs, such as telaprevir and ribavirin, to treat HCV , 60.There is resistance against chemotherapeutic drugs and chemotherapeutic resistance reports are increasing day by day Figure . It has Efflux pumps are present in all cells and they guard the cells from chemical toxicity, especially from organic chemical toxicity. It is an energy-dependent drug efflux pump in prokaryotic and eukaryotic cells , 63. HowThe ATP-binding cassette (ABC) transporter family protein members enable efflux pumps and are present in the plasma membranes of healthy cells. The ABC transporter is part of a normal physiological process. P-glycoprotein efflux pumps are one of the first members of the ABC transporter family protein.The P-glycoprotein-related efflux pumps are usually present on the cell membrane, and they may be found in the nuclear membrane. These P-glycoprotein-related efflux pumps have the capacity to attach to molecules that are positive or neutrally charged . ResearcNon-efflux pump-mediated resistance in chemotherapy is related to drug inactivation, alteration of drug targets, DNA damage repair, cell death inhibition, etc. Housman et al., eloquently described drug inactivation, alteration of drug targets, DNA damage repair, cell death inhibition, etc. .Rueff and Rodrigues described cancer drug resistance from a genetic point of view. They described distorted levels of miRNAs that may act as upstream or downstream effectors . The samUse of different ‘combination therapy’ with several types of miRNA along with the various type of chemotherapeutic agents for the treatment of cancer have been reported Table .Gemcitabine is a novel anticancer nucleoside that is a deoxycytidine analog , 70. ThePaclitaxel (PTX) is also a novel anticancer agent that is used to treat different cancer types. The brand name is Taxol, which is given as an intravenous therapy . Shi et Doxorubicin (DOX) is a chemotherapeutic medicine that is used to treat different cancer types. One of its trade names is Adriamycin . One miRUsing hyaluronic acid-chitosan nanoparticles, Deng et al., performed co-delivery of miR-34a and DOX, which were proficiently capsulated into nanoparticles and delivered to tumor cells or tumor tissues. In this delivery system, DOX-miR-34a co-loaded hyaluronic-chitosan nanoparticles were effectively arranged through the ionotropic gelation method. The combination drug (DOX-miR-34a) was shown to improve the anti-tumor activity by restraining the expression of an anti-apoptosis gene, Bcl-2 .in vitro. On the other hand, same compound  was used to treat the orthotopic mouse breast cancer model in vivo. It was found that the adenoviral constructed miR-145 suppressed the cell growth and motility in both breast cancer cells as well in the breast cancer mouse model. This adenoviral constructed miR-145 was used in combination with 5-fluorouracil which considerably enhanced the antitumor effects in relation to 5-fluorouracil treatment alone [Fluorouracil (5-FU) is also a novel anticancer agent used to treat different cancer types. The brand name is Adrucil, which is given as intravenous therapy . 5-FU rent alone , 81.Docetaxel (DTX) is a unique broad-spectrum chemotherapeutic agent. It is used to treat different cancer types especially, solid cancers , 83. HerThe combination of miRNA and chemotherapeutic agents provides hope as an alternative that can be used to overcome chemo resistance in cancer treatment Figure . Gatheri


Multidrug-resistant tuberculosis (MDR-TB) is a threat for the global TB epidemic control. Despite existing evidence that individualized treatment of MDR-TB is superior to standardized regimens, the latter are recommended in Brazil, mainly because drug-susceptibility tests (DST) are often restricted to first-line drugs in public laboratories. We compared treatment outcomes of MDR-TB patients using standardized versus individualized regimens in Brazil, a high TB-burden, low resistance setting.The 2007–2013 cohort of the national electronic database (SITE-TB), which records all special treatments including drug-resistance, was analysed. Patients classified as MDR-TB in SITE-TB were eligible. Treatment outcomes were classified as successful (cure/treatment completed) or unsuccessful (failure/relapse/death/loss to follow-up). The odds for successful treatment according to type of regimen were controlled for demographic and clinical variables.Out of 4029 registered patients, we included 1972 recorded from 2010 to 2012, who had more complete outcome data. The overall success proportion was 60%. Success was more likely in non-HIV patients, sputum-negative at baseline, with unilateral disease and without prior DR-TB. Adjusted for these variables, those receiving standardized regimens had 2.7-fold odds of success compared to those receiving individualized treatments when failure/relapse were considered, and 1.4-fold odds of success when death was included as an unsuccessful outcome. When loss to follow-up was added, no difference between types of treatment was observed. Patients who used levofloxacin instead of ofloxacin had 1.5-fold odds of success.In this large cohort of MDR-TB patients with a low proportion of successful outcomes, standardized regimens had superior efficacy than individualized regimens, when adjusted for relevant variables. In addition to the limitations of any retrospective observational study, database quality hampered the analyses. Also, decision on the use of standard or individualized regimens was possibly not random, and may have introduced bias. Efforts were made to reduce classification bias and confounding. Until higher-quality evidence is produced, and DST becomes widely available in the country, our findings support the Brazilian recommendation for the use of standardized instead of individualized regimens for MDR-TB, preferably containing levofloxacin. Better quality surveillance data and DST availability across the country are necessary to improve MDR-TB control in Brazil.The online version of this article (10.1186/s12879-017-2810-1) contains supplementary material, which is available to authorized users. Tuberculosis (TB) is still a leading cause of death globally. In 2015, 10 million new cases were reported worldwide . The different standardized regimens are described in Additional file Most patients were treated with standardized regimens [The resistance pattern of second-line and first-line drugs was poorly reported. Among the first-line drugs, besides RIF and INH, 29% of patients were resistant to streptomycin, 15% to pyrazinamide, and 25% to ethambutol  and the use of pyrazinamide, no significant differences were observed considering any combination of unfavorable treatment outcomes  than those recommended by the World Health Organization (WHO) (75%), mainly due to a high proportion of follow-up losses (20%). Not surprisingly, treatment success was more likely in non-HIV patients, in those with a first episode of DR-TB, sputum-negative at baseline and with unilateral disease. Conversely, non-diabetic patients and patients from specific macro-regions of Brazil were less likely to have successful outcomes. Adjusted for these variables, patients who received one of the Brazilian standardized regimens had nearly 3-fold higher odds of success in the efficacy analysis (excluding deaths) and among them, those who used levofloxacin instead of ofloxacin had nearly two-fold higher odds of success, with marginal significance, in the effectiveness analysis, although not in the efficacy analysis.Although comparable to previously reported success rates in MDR-TB patients –10, we mThe effect of the standardized regimens on treatment outcomes should be interpreted with caution. The benefit in efficacy observed in the present study contrasts with three previous aggregate meta-analyses, in which patients who received individualized regimens had higher rates of treatment success , 8, 9. TM. tuberculosis strain was proven to be susceptible to that drug [Unfortunately, analyses of isolated drugs were only possible among patients using standardized regimens. However, we still had a large cohort in this category. Patients who used levofloxacin were 1.7-fold more likely to cure when compared to ofloxacin. The use of later generation quinolones  have indeed been found to be superior,  and by that drug . CurrentFew studies have reported the effect of clinical variables on MDR-TB treatment outcomes. Clinical variables might have a substantial impact in drug- susceptible-TB outcomes. Some of these clinical characteristics, such as indirect signs of advanced disease  and of severity of resistance (previous episodes of DR-TB) were consistently associated with unsuccessful outcomes, no matter which outcomes were included in the analyses. These findings highlight the importance of early diagnosis of MDR-TB. We could not evaluate time elapsed from diagnosis to MDR-TB treatment initiation, due to unreliable information of the date of MDR diagnosis.Non-HIV patients had a nearly three-fold higher chance of successful treatment when death was added to the unsuccessful definition. Poor TB outcomes are expected in HIV-patients, with higher mortality rates –23. TimiThis study has a number of limitations. In addition to the inherent limitations of observational retrospective studies, the available data had a few other problems. Missing information about several variables or not ready-to use information about isolated drugs were important bottlenecks for our analyses, reducing the number of patients included in the multivariate model. However, because we had a large initial dataset, we still had power to find significant associations, the benefit of standardized regimens being the most relevant and original.Another limitation was the reporting of variables such as diabetes, alcohol consumption and smoking as “yes” and “no”; in which missing data were reported as “no”, generating a possible misclassification bias. Moreover, no standard definitions of smoking and alcohol use were available, nor information regarding insulin-dependency in diabetic patients. These co-morbidities are known predictors of poor outcomes in MDR-TB patients –30; thusFinally, nearly half of the cases in our cohort did not have information about culture results during the first six-months of treatment. Thus, the analysis of the six-month culture conversion rate as an intermediate outcome was jeopardized. The identification of predictors of culture conversion is important because it could affect the duration of the intensive phase (i.e. the use of injectable drugs) and could be used as a predictor of cure , 32.Despite these limitations, our inclusion criteria allowed the minimization of outcome biases. In addition, the adjustment of the analyses to relevant clinical variables reduced the risk of confounding, albeit with limitations. Using a large cohort of MDR-TB, we found that adjusting for relevant demographic and clinical variables, standardized regimens were superior to individualized regimens, preferably with levofloxacin.Since randomized trials in MDR-TB patients are scarce, until stronger evidence is produced and DST for first- and second-generation drugs become widely available in the country, our findings support the Brazilian recommendation for the use of standardized regimens for MDR-TB. Levofloxacin rather than ofloxacin should be the quinolone of choice. Better quality surveillance data across the country are needed to improve MDR-TB control in Brazil. Improvement of data reporting in the national database (SITE-TB) is essential, including ART initiation, culture results, more accurate data on diabetes, alcohol abuse, DOT definitions, smoke use and the use of individual drugs.


Patients face increasing insurance restrictions on prescription drugs, including generic-only coverage. There are no generic inhaled corticosteroids (ICS), which are a mainstay of asthma therapy, and patients pay the full price for these drugs under generic-only policies. We examined changes in ICS use following the introduction of generic-only coverage in a Medicare Advantage population from 2003–2004.Subjects were age 65+, with asthma, prior ICS use, and no chronic obstructive pulmonary disorder . In 2004, 74.0% switched from having a $30 brand-copayment plan to a generic-only coverage plan (restricted coverage); 26% had $15–25 brand copayments in 2003–2004 (unrestricted coverage). Using linear difference-in-difference models, we examined annual changes in ICS use (measured by days-of-supply dispensed). There was a lower-cost ICS available within the study setting and we also examined changes in drug choice (higher- vs. lower-cost ICS). In multivariable models we adjusted for socio-demographic, clinical, and asthma characteristics.In 2003 subjects had an average of 188 days of ICS supply. Restricted compared with unrestricted coverage was associated with reductions in ICS use from 2003–2004 : -25.0 to -6.0). Among patients using higher-cost ICS drugs in 2003 (n = 662), more restricted versus unrestricted coverage subjects switched to the lower-cost ICS in 2004 (39.8% vs. 10.3%). Restricted coverage was not associated with decreased ICS use (2003–2004) among patients who switched to the lower-cost ICS , but was among patients who did not switch . In addition, restricted coverage was associated with decreases in ICS use among patients with both higher- and lower-risk asthma .In this elderly population, patients reduced their already low ICS use in response to losing drug coverage. Switching to the lower-cost ICS mitigated reductions in use among patients who previously used higher-cost drugs. Additional work is needed to assess barriers to switching ICS drugs and the clinical effects of these drug use changes. As prescription drug costs continue to rise, public and private payers are increasing insurance restrictions and patient cost-sharing for prescription drugs. Cost-sharing aims to promote cost-effective drug consumption, such as greater use of generic drugs or reduced use of clinically unnecessary drugs, by exposing patients to a greater share of the costs of treatment. For example, in the United States, Medicare Part D drug benefits, which were offered starting in 2006 through a number of private plans, include high levels of cost-sharing. In 2007, about 70% of Part D drug plans included a coverage gap (often referred to as a "doughnut hole") wherein patients lose coverage for all drugs after their total drug spending exceeds $2,400 and until their out-of-pocket costs reach $3,850. About 27% of plans supplement this coverage gap with generic-only coverage, under which patients have coverage for generic drugs, but no coverage for brand-name drugs .Under Medicare Part D cost-sharing arrangements, many beneficiaries may be at risk for losing drug coverage for inhaled corticosteroids (ICS) during the coverage gap, even if they have generic-only coverage supplements, because no generic ICS formulations are available in the U.S. Inhaled corticosteroids are the mainstay of treatment for all forms of asthma, except mild-intermittent  and asthTo gain insight about the impact of brand name drug coverage loss, we examined changes in ICS use among Medicare Advantage (MA) beneficiaries whose plans switched from generic and brand coverage with an annual cap in 2003 to generic-only coverage  in 2004. We compared them with MA enrollees with brand-name coverage in both 2003 and 2004.We used automated data from Kaiser Permanente-Northern California (KPNC), an integrated delivery system that provides comprehensive medical care to over three million members, which included about 280,000 MA beneficiaries. Members could join the KPNC MA plan either by purchasing individual insurance or through plans supplemented by their current or former employers. There was only one MA plan  for individual subscribers, while employers determined KPNC cost-sharing levels for patients with employer-supplemented plans.In 2003 individual MA beneficiaries had $10 generic and $30 brand copayments, and a $1,000 benefit cap . In 2004, the cap was replaced with generic-only coverage – i.e., patients paid full member price for brand drugs, and $10 copayments for generics throughout the year ("restricted coverage"). By contrast, employer-supplemented MA beneficiaries paid $10 generic and $15–25 brand copayments 2003–2004, with no other limits ("unrestricted coverage"). Patients' coverage type was determined by their employment history and enrollees could not switch between the coverage types , at a significantly lower member price compared with other inhaled corticosteroids. Beclomethasone dipropionate was the most frequently prescribed ICS within the health system, comprising 75% of all ICS prescriptions in our study, and was followed by fluticasone propionate (Flovent), which comprised 23% of ICS prescriptions . The average cost increase due to loss of coverage for a canister of beclomethasone dipropionate was about $15, compared with $65 or $110 for fluticasone propionate 110 mcg or 220 mcg, respectively. The KPNC Institutional Review Board approved the study.All subjects included in this study  were continuous MA enrollees , 65+ years old, who met at least one of the following criteria as of January 1, 2003: (1) ≥ 1 inpatient admissions with a primary asthma diagnosis in the previous three years; (2) ≥ 1 ED visits with an asthma diagnosis in the previous two years; or (3) ≥ 2 outpatient visits with an asthma diagnosis in the previous two years. Further, all subjects were dispensed ≥ 30 days-of-supply of an ICS in both 2002 and 2003. We excluded patients with inpatient or outpatient chronic obstructive pulmonary disorder (COPD) diagnoses: ICD-9-CM codes 491 (chronic bronchitis), 492 (emphysema), 493.20 (asthma with COPD), or 496 (COPD) in 2003–2004. We also excluded patients who were dispensed ipratropium bromide (Atrovent), the primary pharmacologic therapy for COPD within KPNC in 2003 or 2004, and patients with Medicaid because they had substantially lower cost-sharing.We measured ICS use based on days-of-supply dispensed. Days-of-supply were calculated for each prescription by multiplying the number of actuations per canister by the number of canisters dispensed and dividing by the prescribed dose (actuations) and frequency of administration. We carried over remaining supply from month-to-month and calculated patients' ICS supply in each month (standardized to a 30-day month) and year. We considered beclomethasone dipropionate as "lower-cost", and all other ICS brands as "higher-cost". We calculated monthly and annual days-of-supply for lower- and higher-cost ICS drugs separately. We also classified patients as either lower- or higher-cost ICS users in each year by the type with the greater days-of-supply.Comparisons between the restricted and unrestricted coverage groups were adjusted for a number of potential confounders including age, gender, and race/ethnicity. Race/ethnicity was determined based on routine member surveys and inpatient records (we included an unknown category with 7.5% of subjects). We constructed a neighborhood socioeconomic status (SES) indicator using patients' residential addresses and information from the 2000 US Census. Neighborhoods were considered low SES if ≥ 20 percent of residents had average household incomes below the federal poverty level, or ≥ 25 percent of residents ≥ 25 years old had less than a high school education ,9. We adTo adjust for asthma severity, we used a number of medication- and diagnosis-related variables in 2002: number of rescue medication canisters dispensed, having a high-dose ICS, any oral steroid dispensed, a high-risk asthma registry flag  approach. The models included indicators for propensity score quintile (2–5), month (2–24), restricted coverage, and interactions between restricted coverage and month. The coefficients of the interactions test the hypothesis that coverage type was associated with different patterns of ICS use over months.To examine annual ICS use associated with restricted coverage we calculated the change in days-of-supply (2004 minus 2003) for each subject and used this difference as the outcome in linear models, adjusted for propensity score quintile. We examined annual changes among all subjects, among users of higher- and lower-cost ICSs in 2003, separately, and by type of ICS use in 2004, separately. We also examined changes in ICS use among patients identified as having high-risk asthma in the health system's disease registry, and those with lower-risk asthma .To examine changes in Lastly, we examined the patient characteristics associated with having no ICS use in 2004 using multivariable logistic regression, and included the covariates described above.Table Figure Figure Table For higher-cost ICS users in 2003, the effect of restricted coverage on ICS use differed by the type of medication used in 2004. Restricted coverage was not associated with decreased ICS use (2003–2004) among patients who switched to the lower-cost ICS in 2004 , but was among patients who did not switch .We also examined changes in ICS use associated with restricted coverage, stratified by asthma disease severity, as defined by having a high-risk asthma flag in the disease registry Table . Table 5In separate analyses (more details available upon request), among all subjects and among higher-cost ICS users in 2003, restricted coverage was not significantly associated with having no ICS use in 2004 . Among all subjects, having a high-dose ICS in 2002 and greater ICS use (days-of-supply) in 2003 were associated with lower odds of no ICS use in 2004 .Inhaled corticosteroids are a critical component of therapy for persistent asthma. In this population of Medicare beneficiaries with asthma, levels of ICS use were low , and switching appeared to mitigate reductions in 2004 ICS use due to loss of drug coverage. However, many patients did not switch to the lower-cost ICS and those with restricted coverage reduced their ICS use by over one month of supply compared with patients with unrestricted coverage.Patients who were taking higher-cost ICS drugs at baseline exhibited greater asthma severity compared with lower-cost ICS users, as indicated by several medication and diagnoses measures shown in Table Encouraging greater substitution of higher-cost with lower-cost ICS drugs could mitigate potential adverse effects of restricted drug coverage policies. In this setting, switching to the lower-cost ICS would not be likely to result in adverse clinical effects as head-to-head trials have not produced definitive conclusions on the comparative efficacy of different inhaled corticosteroids . There iPoor adherence to ICS therapy has been demonstrated in other studies ,23-28. TBecause ours was a non-randomized study it is possible that differential changes in ICS use between the restricted and unrestricted groups are due to unmeasured differences. We were able to adjust for a large a number of socio-demographic and clinical characteristics, including a number of asthma severity measures. Identifying patients with persistent asthma based on administrative data is challenging; we examined other cohort definitions of varying restrictiveness  and found similar results across these definitions. ICS use was measured using pharmacy data. These data do not capture whether patients actually took dispensed medications or took them correctly; we also did not assess the clinical effects of changes in ICS use. The pharmacy data do not include drugs dispensed outside of KPNC; however, patients had a financial incentive to purchase their medications within KPNC because of favorable in-system prices throughout the study period. As a result, in telephone interviews, members report rarely going outside of the system to obtain medications or other services . Lastly,This study highlights the importance of offering a low-cost option within the ICS drug class, particularly during uncovered periods, such as during the Medicare Part D coverage gap. Insurers may also consider maintaining coverage for ICS drugs during otherwise uncovered periods for patients with a clinical need for these drugs. Additional work is needed to understand patient responses to drug cost-sharing, especially for patients who did not switch to the lower-cost option yet reduced ICS use, as well as the clinical effects of restricted drug coverage policies.CI: confidence interval; COPD: chronic obstructive pulmonary disorder; ED: emergency department; ICS: inhaled corticosteroids; KPNC: Kaiser Permanente-Northern California; MA: Medicare Advantage; OR: odds ratio.The authors declare that they have no competing interests.VF made substantial contributions to the study's conception and design, acquisition of data, analysis and interpretation of data, drafted the manuscript, and revised the manuscript critically for important intellectual content. IBT made substantial contributions to the study's design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. RB made substantial contributions to the study's design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. JPN made substantial contributions to the study's design, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. JH made substantial contributions to the study's conception and design, acquisition of data, analysis and interpretation of the data, and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Although acute myocardial infarction is generally associated with obstructive coronary artery disease, myocardial infarction associated with normal coronary arteries is a well-known condition. The overall prevalence rate of myocardial infarction with normal coronary arteries is considered to be low, varying from 1% to12% depending on the definition of "normal" coronary arteries.We describe here a case of a 49-year-old woman with a history of prior myocardial infarction who continued to be asymptomatic after a 10-year follow-up, in the absence of a high-risk profile for adverse outcomes. She was studied with multi-slice coronary computed tomography and whole-body angiography, which showed normal coronary and extra-coronary arteries.This case report raises two important issues. First, the possible role of multi-slice computed tomography/coronary angiography in the risk- and prognosis assessment of patients with known or suspected coronary artery disease. Second, the important role played by long-term pharmacological therapy in patients with prior myocardial infarction and normal coronary arteries. Myocardial infarction (MI) with normal coronary arteries is a medical condition, which has been described in the literature for more than 30 years but is still a challenge in medical practice because of the lack of evidence-based medical data on its prognosis and on secondary prevention. The prevalence of MI with normal coronary arteries has always been considered low, varying between 1% and 12%. However, it actually involves a considerable number of patients, calling for more clinical trials which specifically study this sub-population of patients affected by coronary heart disease (CHD) [We report a case of a young female patient with a previous clinically diagnosed MI, who was asymptomatic in standard care therapy for a 10-year follow-up and has been shown to have normal coronary arteries by coronary multi-slice computed tomography (MSCT) and whole-body angiography.A 49-year-old woman was referred to our department for a cardiological follow-up visit. She had a medical history of hypertension and previous MI, with no history of diabetes mellitus, hypercholesterolemia or premature coronary artery disease in her family. She did not smoke, take recreational drugs and was not on oestrogen replacement therapy.Ten years earlier, she had an inferior MI treated with systemic thrombolysis, unfractioned heparin, aspirin, atenolol and intravenous nitroglycerine. During her hospitalization, the echocardiogram (ECG) revealed akinesis of the posterior-basal wall with an estimated ejection fraction of 50%. Laboratory tests including serum glucose, lipids, blood count, liver-, kidney- and thyroid-function tests were within normal limits. The day before her discharge, she had a stress ECG negative for inducible ischemia. The patient was discharged on the sixth day with a drug regimen of aspirin, atenolol, captopril, simvastatin and isosorbide dinitrate.After the MI and prior to the visit to our department, the patient had been free from chest pain and dyspnea and had a normal exercise tolerance. She had periodical ECGs that showed the presence of akinesis of the posterior-basal wall with a mildly depressed systolic function. She reported a good control of arterial blood pressure with the ongoing therapy, including perindopril 5 mg/day, hydroclhorotyazide 25 mg/day and atenolol 25 mg/day associated with aspirin and simvastatin 20 mg/day.A physical examination showed blood pressure of 135/80 mmHg, heart rate of 64 bpm, BMI of 27 kg/m2, waist circumference of 85 cm and no findings of congestive heart failure. The resting ECG showed a sinus rhythm at 62 bpm and the presence of Q waves in II, III and aVF leads with no other abnormalities.Laboratory tests revealed a total serum cholesterol of 160 mg/dl, HDL cholesterol of 55 mg/dl, triglycerides of 78 mg/dl, LDL cholesterol of 89.6 mg/dl and fasting glycemia of 116 mg/dl, with a normal OGTT and normal plasma homocysteine.On the basis of her medical records concerning the MI, we confirmed the diagnosis of coronary artery disease (CAD) according to the recently published universal definition of MI .For risk stratification, we studied the patient with non-invasive diagnostic tests, including echocardiography and a treadmill exercise test.The ECG confirmed the presence of akinesis of the posterior-basal wall with a mildly depressed systolic function (LVEF 48%) and the treadmill exercise test showed no signs of inducible ischemia.Without any symptoms which suggest cardiac ischemia, the results from the non-invasive tests suggested no high-risk criteria for adverse outcomes for our patient. According to the guidelines by the American Heart Association/American College of Cardiology (AHA/ACC), there was thus no indication for conventional coronary angiography.Consequently, we decided to study her coronary arteries non-invasively. MSCT angiography was performed using a 64-slice computed tomography (CT) scanner . The protocol included a post-coronary phase for whole-body angiography.The MSCT angiographic analysis showed no coronary lesions Figure , but theThe patient was sent to follow-up with a drug regimen of perindopril 5 mg/day and hydrochlorothiazide 25 mg/day. Aspirin, atenolol and simvastatin were discontinued. She was advised to lose weight and implement some lifestyle changes and was asked to repeat laboratory tests for a lipid profile before the next follow-up visit.At the 6- and 12-month follow-up, the patient was free from chest pain, LDL-cholesterol was below 140 mg/dl without statin therapy and blood pressure was stable below 140/90 mmHg.The overall prevalence rate of MI with normal coronary arteries is considered to be low, varying from 1% to 12%, depending on the definition of "normal" coronary arteries, which usually includes no luminal irregularities (strict definition) or arteries with some degree of stenosis  . Thus, sThis case report raises two important issues. First, the possible role of MSCT coronary angiography in risk- and prognosis assessment of patients with known or suspected CAD. Second, the role of long-term pharmacological therapy in patients with prior MI and normal coronary arteries.MSCT provides high-resolution coronary angiograms non-invasively and has been demonstrated to be highly accurate in ruling out coronary atherosclerosis . Its higWe approached our patient according to a diagnostic algorithm based on the current AHA/ACC guidelines. Our patient had been asymptomatic since the MI event and had had no high-risk features for adverse outcomes in routine non-invasive tests. Medical history, clinical features and non-invasive tests did not provide specific diagnosis and prognosis, which allowed us only to continue the current medical therapy. According to the scheme provided by the current guidelines, we should have considered further imaging studies. However, in our patient, routine non-invasive imaging studies, such as nuclear stress testing or stress echocardiography would not have provided any new information for a better risk- and prognosis stratification. We considered the low age and the low cardiovascular risk profile of the patient at the time of acute MI, the absence of symptoms after the event and the absence of inducible ischaemia during the treadmill exercise test and decided that a non-invasive delineation of the presence and severity of coronary atherosclerosis would allow insights for the further management of our patient.MSCT angiography showed absolutely normal coronary arteries. The absence of atherosclerosis lesions in this patient was further confirmed in the extra-coronary arterial tree, thus providing a definitive diagnosis. The high negative predictive value of MSCT coronary angiography allowed us to definitely exclude CAD and the need for further diagnostic tests. Moreover, MSCT provided a more accurate prognosis for our patient for two main reasons. First, long-term prognosis in patients with MI and normal coronary arteries is much better compared to patients with coronary occlusive disease, especially in young and female patients ,5,8. PatThe diagnosis of prior MI with normal coronary arteries raises a second important issue concerning the application of AHA/ACC secondary prevention guidelines for CHD. According to these guidelines, patients who have had MI should receive indefinitely a drug regimen including an antiplatelet agent, a beta-blocker, a statin with an LDL-C goal of < 100 mg/dl or < 70 mg/dl, and an ace-inhibitor with a blood pressure goal < 140/90 mmHg [The estimated annual incidence of MI, new and recurrent in the US is 865 000, and among these, about 350 000 are women (11). Among these women, the above-mentioned 7% to 32% with normal coronary angiography translate into 24 500 to 112 000 women with acute MI and normal coronary arteries annually in the US alone. This sub-population of patients affected by CHD is significant and the lack of randomized clinical trials, comparing therapies for the reduction of adverse cardiac events in patients with MI and normal coronary arteries, makes their management challenging. It is difficult to find a physio-pathological rationale that allows us to transfer secondary prevention guidelines from patients with established coronary artery disease to patients with normal coronary arteries.On the other hand, these patients are young people with good prognoses, which implies that a long-term multi-drug therapy should be carefully considered in terms of cost-benefit analysis. Despite an undemonstrated improvement of the prognosis, a multi-drug regimen including antiplatelet agents, statins, beta-blockers and ace-inhibitors would expose our patient to well-known adverse effects and imply a considerable economic cost.According to the clinical features and the diagnostic test results from our patient, we decided to retain the ace-inhibitor because of arterial hypertension and to discontinue aspirin, statin and beta-blocker because of the absence of coronary atherosclerotic disease, hypercholesterolemia and symptoms reflecting inducible ischaemia.MSCT coronary angiography can have an important role in the risk and prognosis assessment of patients with known or suspected CHD. Large-scale randomized trials need to be conducted to determine an optimal secondary prevention strategy for patients with MI and normal coronary arteries, who constitute a large sub-population of CHD patients.AHA/ACC: American Heart Association/American College of Cardiology; CAD: coronary artery disease; CHD: coronary heart disease; CT: computed tomography; ECG: echocardiogram; MI: myocardial infarction; MSCT: multi-slice computed tomography; OGTT: oral glucose tolerance test.Written informed consent was obtained from the patient for publication of this case report and 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.LI was in charge of the patient clinically and was a major contributor in writing the manuscript. MF collected clinical data and reviewed the literature. AN prepared a CT angiography protocol to study in a single-setting coronary and whole-body arterial system. CC analyzed and interpreted the imaging studies. All authors read and approved the final manuscript.


Mycoplasma genitalium, Bacillus subtilis, Helicobacter pylori, Haemophilus influenzae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Escherichia coli), and four eukaryotes (Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worm), Drosophila melanogaster (fly), Mus musculus (mouse)).One mechanism to account for robustness against gene knockouts or knockdowns is through buffering by gene duplicates, but the extent and general correlates of this process in organisms is still a matter of debate. To reveal general trends of this process, we provide a comprehensive comparison of gene essentiality, duplication and buffering by duplicates across seven bacteria , but only by up to 13%. Given that duplicates make up to 80% of eukaryotic genomes, the small contribution is surprising and points to dominant roles of other buffering processes, such as alternative metabolic pathways. The buffering capacity of duplicates appears to be independent of the degree of gene essentiality and tends to be higher for genes with high expression levels. For example, buffering capacity increases to 23% amongst highly expressed genes in In sum, the extent of gene essentiality and buffering by duplicates is not conserved across organisms and does not correlate with the organisms' apparent complexity. This heterogeneity goes beyond what would be expected from differences in experimental approaches alone. Buffering by duplicates contributes to robustness in several organisms, but to a small extent – and the relatively large amount of buffering by duplicates observed in yeast and worm may be largely specific to these organisms. Thus, the only common factor of buffering by duplicates between different organisms may be the by-product of duplicate retention due to demands of high dosage. Cells and organisms show a remarkable robustness against loss of one or more genes, which has triggered an ongoing discussion on the factors promoting such robustness ,2. One oFor example, estimates of the role of duplicates as backups for gene loss vary widely within and across organisms. Most yeast genes are non-essential, i.e. dispensable, in rich medium or under standard laboratory conditions >80%, ref. ). A stud0%, ref. Several molecular causes may underlie buffering by duplicates, and their relative contributions are still debated. For example, buffering duplicates lack functional redundancy that would be expected from their backup role. Buffering duplicates in yeast have only partially overlapping expression  and geneDespite the availability of several large-scale datasets on single gene knockouts (KO) or knock-downs (KD) as well as double gene-KOs for all of these organisms, previous studies mainly focused on single organisms like yeast ,11-14, wM. genitalium, H. pylori, H. influenzae, M. tuberculosis, P. aeruginosa, B. subtilis, E. coli, S. cerevisiae (yeast), C. elegans (worm), D. melanogaster (fly), and M. musculus (mouse). To do so, we addressed the above-mentioned challenges in several ways. When selecting essentiality datasets, we aimed to minimize variation in experimental approaches, and, whenever possible, sampled several organisms for a specific technique  is derived from the fraction of genes in a genome which do have one or more duplicates . We also use the probability P(S) which describes for an organism chances of survival upon gene-deletion; P(S) is derived from the fraction of genes identified as dispensable  in large-scale screens. When discussing 'buffering by duplicates' we mean the enrichment of duplicates amongst non-essential genes as inferred from statistical analysis. 'Essentiality/non-essentiality ' is purely based on outcomes of experiments.We define the effective family size P(S) > 0.80), except for M. genitalium, H. influenzae and mouse ); in half (six) of the organisms the difference is significant .Table e Figure . Genes oD ≥ 1) increases from M. genitalium and the other bacteria, to yeast and the three animals  relative to genes with duplicates. In five organisms, there is a significant increase in the fraction of duplicates (D ≥ 1) amongst highly expressed genes compared to other genes ; an exception is B. subtilis in which the trend is inverted. When using Codon Adaptation Index or experimental expression data we obtain similar results /P(S|D = 0) – 1, where P(S|D = 0) is the probability of survival given the gene does not have additional duplicates, i.e. is a singleton. P(S|D ≥ 1) is the probability of survival given the gene has one or more additional duplicates. C is calculated for each organism and quantifies the increase in probability of survival upon gene-KO/KD for genes which have a duplicate in the genome.To assess the contribution of duplicates to survival following gene-KO/KD we define the buffering capacity M. genitalium and mouse in which duplicates appear to decrease chances of KO survival. The extent of buffering by duplicates, i.e. the value of C, does not correlate with the organisms' complexity or genome size. Total C is largest in yeast, worm and H. pylori and smallest in H. influenzae, B. subtilis and fly. While the total number and fraction of genes with duplicates increases from simpler to more complex organisms ; with contributions ranging from 1 to 13% Table . The excs Figure , the proC amongst genes of low and high expression levels . However, only in five organisms , genes of high expression levels and with duplicates have significantly improved chances of survival; with C reaching 23% in E. coli. In M. genitalium and M. tuberculosis, C is positive amongst highly expressed genes when examining experimental expression data  with the number of paralogs present, and ii) their similarity to the mutant protein. We tested these predictions in the eleven organisms.P. aeruginosa, E. coli, and worm, chances of survival correlate significantly  with both the number of duplicates available per gene and with the distance of the gene to the nearest homolog , but not with the number of duplicates per gene. These results do not change even when removing ribosomal genes or gene pairs originating from the whole-genome duplication [D = 1) which buffer for each other ; however, buffering genes have significantly higher sequence identity between the members . If a buffering gene pair in yeast has a single-gene ortholog in another organism , we expect this ortholog to be essential – more often than single-gene orthologs of non-buffering gene pairs. If an ortholog of a buffering two-gene family has paralogs, we do not expect it to be essential. Indeed, buffering gene pairs are enriched for essential single orthologs compared to non-buffering gene pairs, although the trend is very weak and not significant due to small numbers in the dataset  vs. non-buffering gene pairs (eight) compared to worm . This bias holds true even if only regarding the yeast gene pairs identified in large-scale screens: ten buffering and eight non-buffering pairs. Previous work has shown that yeast is enriched for buffering gene pairs which originate from the whole genome duplication . In addiOur study provides a systematic and semi-quantitative assessment of essentiality and gene duplication across eleven prokaryotic and eukaryotic organisms revealing a heterogeneous picture. To the best of our knowledge, this is the first such organism-wide comparison.H. pylori, H. influenzae, and M. tuberculosis vs. P. aeruginosa, B. subtilis and E. coli , i.e. there are only few essential genes Figure . We obseli Table ; howeverThe extremely high chances of survival in fly Figure  can be (not shown); thus the dataset is biased. The lack of buffering by duplicate genes in mouse has been demonstrated recently [The low chances of survival in mouse are likely due to the mouse dataset not originating from a large-scale screen, but from individual experiments that may have preferentially targeted and reported essential genes. For example, the gene targets in the mouse dataset are strongly enriched for orthologs of human disease genes , and similar fractions of duplicate genes, but very different contributions of these duplicates to survival.The degree of gene essentiality  can be influenced by the experimental technique and the definition of essentiality that is used. In contrast, if duplicates contribute to survival upon gene loss, then this effect should be detectable irrespective of the number of essential and non-essential genes identified (provided that the selection is unbiased). In other words, we expect buffering by duplicates to be less dependent on technical differences than essentiality alone. We introduced statistical tests to assess the significance of buffering by duplicates Figure . A smalls Figure , but thes Figure . LikewisH. pylori, M. tuberculosis, P. aeruginosa, E. coli, yeast, worm; Figure Duplicates significantly and positively contribute to survival in nine of the eleven organisms, but have noticeable effects only in six  to be more likely to buffer for loss of function than duplicates that diverged in their sequence. Similarly, we would expect genes with many duplicates (large gene families) to be more likely to be buffered for loss of function than genes of small families. Both expectations are fulfilled in only some of the organisms Table , e.g. inRelated to sequence similarity is function, which is more dissimilar amongst buffering duplicates than naively expected, when measured in terms of expression regulation  and geneM. tuberculosis) and for different measures of expression levels  is expression level. Genes of high expression levels tend to have more duplicates, but these duplicates are also more likely to buffer for loss of the gene's function. (Note the subtle difference between the two observations.) The trend holds true for all organisms with positive buffering capacity ; Caenorhabditis elegans (worm); Drosophila melanogaster (fly); Mus musculus (mouse)) from a collection in the SUPERFAMILY database [. All screens were conducted in rich medium and on whole organisms except for fly (cell line). For mouse, data of ~4,000 individual knockout experiments were obtained from the Mouse Genome Database [We obtained the amino acid sequences for ten genomes  and worm (3.0*10-10). This threshold identified 609 two-gene families in yeast. We tested several other methods of homology prediction including different E-value thresholds, E-value-independent methods and use of InParanoid [We measured similarity between all sequences using a BLAST all-against-all search , and reqParanoid , all witH. pylori. Results for CAI and experimental expression data are in Additional file As a surrogate for gene expression levels, we calculated the Codon Bias Index (CBI) for each gene using the CodonW server , with stbetween the two members of a gene family and characteristics of individual genes, averaged across the whole set. For vector comparisons, we constructed binary vectors  based on networks of functional interactions [In yeast, 50 two-gene families were identified as buffering (SSL phenotype) and eight two-gene families as non-buffering (viable phenotype). The buffering pairs consist of nine pairs identified in the 14 large-scale double-KO screens (see above), and 42 additional pairs identified in small-scale experiments and listed in BioGRID ). The noractions , geneticractions ), and siractions . The simAs a control for the effects of WGD genes, we also compared some characteristics in all 609 yeast two-gene families split into 108 and 501 two-gene families with and without evidence for their origin in the WGD , respectWe used the FunSpec server  and SGD D: effective gene family size ; E-value: expectation value; KD: knockdown; KO: knockout; MIPS: Munich Information Center for Protein Sequences; P(S): probability of survival upon single- or double gene-KO or KD; R2: squared Pearson correlation coefficient; SGA: Synthetic Genetic Array; SSL: synthetic sick or lethal (mutant); SGD: Saccharomyces Genome Database; WGD: whole-genome duplication.CAI: Codon Adaptation Index; CBI: Codon Bias Index; M. genitalium: Mycoplasma genitalium; H. pylori: Helicobacter pylori; H. influenzae: Haemophilus influenzae; M. tuberculosis: Mycobacterium tuberculosis; Paer: Pseudomonas aeruginosa; B. subtilis: Bacillus subtilis; E. coli: Escherichia coli; S. cerevisiae: Saccharomyces cerevisiae (yeast); C. elegans: Caenorhabditis elegans (worm); D. melanogaster: Drosophila melanogaster (fly); M. musculus: Mus musculus (mouse).Organisms: KH conducted the experiments, analyzed results and wrote the paper. EMM provided valuable input and support at all stages of the project. CV initiated and guided the project, conducted some of the experiments, analyzed results and wrote the paper. All authors read and approved the final manuscript.Supplementary Notes. Additional figures and comments on the analyses.Click here for fileSupplementary Data. Data on yeast gene pairs collected during the analyses.Click here for file


Several authors have suggested that fatty acids found in this propolis might be responsible for its antimicrobial activity; however, so far no evidence concerning this finding has been reported in the literature. The goals of this study were to evaluate the antibacterial activity of the main pure fatty acids in the ethanolic extract and fractions and elucidate the chemical nature of the bioactive compounds isolated from Brazilian propolis type 6.Brazilian propolis type 6  is distinct from the other types of propolis especially due to absence of flavonoids and presence of other non-polar, long chain compounds, but presenting good Staphyloccus aureus ATCC 25923 and Streptococcus mutans Ingbritt 1600 using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).Brazilian propolis type 6 ethanolic extract (EEP), hexane fraction (H-Fr), major fatty acids, and isolated sub-fractions were analyzed using high performance liquid chromatography (HPLC), high resolution gas chromatography with flame ionization detection (HRGC-FID), and gas chromatography-mass spectrometry (GC-MS). Three sub-fractions of H-Fr were obtained through preparative HPLC. Antimicrobial activity of EEP, H-Fr, sub-fractions, and fatty acids were tested against EEP and H-Fr inhibited the growth of the microorganisms tested; nevertheless, no antimicrobial activity was found for the major fatty acids. The three sub-fractions  were isolated from H-Fr by preparative HPLC and only sub-fraction 1 showed antimicrobial activity.a) The major fatty acids tested were not responsible for the antimicrobial activity of propolis type 6; b) Sub-fraction 1, belonging to the benzophenone class, was responsible for the antimicrobial activity observed in the present study. The identification of the bioactive compound will improve the development of more efficient uses of this natural product. Apis mellifera bees from various plant sources, has been reported to have several properties that may confer health benefits to humans since ancient times .,13.12,13®), tested isolatedly or mixed, showed no bacterial activity against Gram-positive bacteria (S. aureus or S. mutans) at concentrations lower than 1600 μg/mL. However, MIC values found for H-Fr were lower than 50 and 100 μg/mL for S. aureus and S. mutans, respectively  for both microorganisms tested, suggesting that sub-fraction 1 might contain an antimicrobial bioactive compound. In addition, since chlorhexidine, a pure mono-drug, and sub-fraction 1, an impure bioactive compound, showed similar MIC results, we might speculate that this compound could be a promising antimicrobial agent, which needs to be isolated and identified. TLC and HPLC analyses of sub-fraction 1 showed a mixture of non-polar compounds; however, when this sub-fraction was analyzed by HRGC-FID, no fatty acid characteristics were found Figure  and thosBenzophenones are among the most important chemical compounds identified and isolated from propolis found in tropical regions -23. TheyNo antimicrobial activity was observed for the main fatty acids identified in Brazilian propolis type 6. Sub-fraction 1 isolated from this type of propolis belongs to the benzophenone class and was responsible for the antimicrobial activity observed in the present study. The identification of this bioactive compound will improve the development of more efficient uses of this natural product.The authors declare that they have no competing interests.This study was conceived, directed, and coordinated by PLR and SMA, who also critically revised the manuscript with the help of MI. MLC participated throughout the study . WR and RCZ worked in the preparation of the extracts, fractions, and analyses of fatty acids. VLR and MAF gave their support in the chromatography laboratory and helped interpret data. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


With the current technological advances in high-throughput biology, the necessity to develop tools that help to analyse the massive amount of data being generated is evident. A powerful method of inspecting large-scale data sets is gene set enrichment analysis (GSEA) and investigation of protein structural features can guide determining the function of individual genes. However, a convenient tool that combines these two features to aid in high-throughput data analysis has not been developed yet. In order to fill this niche, we developed the user-friendly, web-based application, PhenoFam.etc.) without prior pre-filtering or hits-selection steps. PhenoFam utilizes Ensembl databases to link a list of user-provided identifiers with protein features from the InterPro database, and assesses whether results associated with individual domains differ significantly from the overall population. To demonstrate the utility of PhenoFam we analysed a genome-wide RNA interference screen and discovered a novel function of plexins containing the cytoplasmic RasGAP domain. Furthermore, a PhenoFam analysis of breast cancer gene expression profiles revealed a link between breast carcinoma and altered expression of PX domain containing proteins.PhenoFam performs gene set enrichment analysis by employing structural and functional information on families of protein domains as annotation terms. Our tool is designed to analyse complete sets of results from quantitative high-throughput studies (gene expression microarrays, functional RNAi screens, PhenoFam provides a user-friendly, easily accessible web interface to perform GSEA based on high-throughput data sets and structural-functional protein information, and therefore aids in functional annotation of genes. Onto-Express [i.e. level of expression or phenotype strength) are not considered. There are few applications overcoming these limitations by performing gene set enrichment analysis (GSEA) [e.g. GSEA [Analysis of large sets of results derived from high-throughput experiments is a challenging but promising field of study. Enrichment analysis is a very powerful strategy helping researchers in identifying biological processes or pathways related to their studies. Most of the currently available tools  search -Express , KEGG pa-Express  or others (GSEA) . They se.g. GSEA , FatiSca.g. GSEA , GeneTra.g. GSEA ).e.g. a homeodomain fold indicates a transcription factor activity involved in cellular differentiation [et al. demonstrated, that the domain composition of proteins could be used for predicting their pathway membership [etc.). Our tool combines the experimental results with annotations from the databases integrated in InterPro , thereby allowing a streamlined structure/function annotation of proteins. Utilization of information about protein domain families in GSEA is a novel approach that can be used in parallel to other enrichment analysis applications.In recent years, access to high-resolution protein structural information has increased considerably. Many new structures reveal the presence of domains known from other proteins, and the domain composition of a protein can help forming a hypothesis about its biological function (ntiation ). Moreovmbership . There ambership  constitumbership , HAMAP [mbership , Pfam [1mbership , PRINTS mbership , ProDom mbership , SMART [mbership , TIGRFAMmbership , PIRSF [mbership , SUPERFAmbership , Gene3D mbership  and PANTmbership . We havePhenoFam is a Java web application running on a Tomcat 5.5 server. It uses a MySQL database to store mappings between various protein, gene or probe names and identifiers related to member databases of InterPro. This database is an easily updatable compilation of the current releases of the Ensembl database . Client-One of the key features of our application is that it accepts as input a wide range of identifiers used in all genomes integrated in the Ensembl database . Identifi.e. shift in the reading frame). In such cases, a value associated with the user-provided identifier is mapped to all possible protein features that can be associated with the gene  framework. We have designed a simple and user-friendly data management system for storing uploaded data sets and the analysis results. It allows users to investigate and compare multiple data sets at the same time.p-value reported by the Mann-Whitney U-test, a FDR corrected p-value, a ρ statistic and the InterPro identifier. The results associated with one of the selected InterPro member databases are displayed in a pageable table  results. To test the utility of PhenoFam, we analysed a data-set derived from a genome-scale cell cycle progression RNAi screen [z-scores for cell cycle progression phenotypes  for each knockdown.PhenoFam allows many data sets as the starting point, such as results of microarray studies, systematic RNA interference (RNAi) screens, ChIP-Chip/ChIP-Seq experiments or comparative mass-spectrometry (i screen . In thisp < 0.005) for polyploidy phenotypes .To show that PhenoFam is also suitable for analysis of other large-scale data-sets, we examined publicly available gene expression data that compares transcriptomes of human breast carcinoma and healthy tissue . GSEA ofRas GTPases are known to play a role in breast cancer development  and, thei.e. z-score or quantile-based normalization) apply thresholds that can be passed only by genes showing the strongest phenotypes, which often leads to a high false-negatives rate. In case of our GSEA method, a domain may appear to be significantly enriched despite moderate phenotypes of the associated genes. From the potential relationship between the domain and the investigated biological process, genes with moderate phenotypic scores are considered in the list of hits selected from the screen, thereby reducing the false-negative rate.PhenoFam is a computational tool designed to analyse experimental results by integration of functional and structural information about protein families. The distinct features of our application include a user-friendly interface and a broad range of supported genomes and identifiers. It should also be noted that our algorithm, in contrast to existing software, treats the InterPro repository as a collection of partially redundant databases, which improves the power of our testing procedure. Using a specific example, we show that the application can be used as an additional hit selection tool for functional screens. Typical hit selection procedures : Platform independent (web-based application)Programming language: JavaOther requirements: A web browser with JavaScript supportLicense: GNU GPLv3 http://www.gnu.org/licenses/gpl-3.0.htmlAny restrictions to use by non-academics: NoneMPR implemented the application and drafted the manuscript. MS carried out the experiments and contributed in writing the manuscript. MTP contributed in designing the application and in writing the manuscript. FB coordinated the study and contributed in writing the manuscript. All authors read and approved the final manuscript.PhenoFam User's Guide. The file contains a PDF version of the User's Guide provided on the PhenoFam home page.Click here for file